ACKNOWLEDGEMENTS

First of all thanks to God. It has been my ultimate pleasure to do this research and the
information gathered during this work will hopefully help in future investigations.
The dissertation work was completed in collaboration with the Department of Zoology,
University of Dhaka and Environmental Microbiology Laboratory (EML), Centre for Food
and Waterborne Diseases (CFWD), International Centre for Diarrhoeal Disease Research,
Bangladesh (ICDDR,B).
I pay my profound sense of gratitude to my honorable guide, research supervisors Dr.
Hamida Khanum, Professor, Department of Zoology, University of Dhaka and Dr. Md.
Sirajul Islam, Environmental Microbiologist and emeritus scientist, EML, CFWD, ICDDR,B
for their supervision, guidance, scholarly advice,

creative criticism and inspiration in

completing this dissertation.

I sincerely express my special thanks to research supervisor Dr. Zahid Hayat Mahmud,
Associate Scientist and acting head of EML, CFWD, ICDDR,B for his multifarious
guidelines and logistic supports to make this study up to this standard.
I am also indebted to Dr. Md, Abul Bashar, Professor and Chairman, Department of Zoology,
University of Dhaka for his generous assistances and all other teachers, Department of
Zoology, University of Dhaka.
I am thankful to the Research Team of EML, ICDDR,B; particularly Md. Riadul Haque
Hossainy, Hafij Al Mahmud Md. Rafiqul Islam, Ehtesham, Alvee, Shobnom, Tammi, Faroza
for their all out support.
A special thanks to my friends Sadiq, Maria, Chhanda, Salawat, Rajib, Sumon, Imran,
Mandira, Diponkor, Ssauro, for their suggestions and help throughout the study.
Finally I would like to thank my beloved parents for all the encouragement, guidance and
moral support that they have given to me throughout the years that I worked, as it would have
been difficult to cope without your presence and words of wisdom.

June, 2015

The Author

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ABSTRACT
The present study was performed to find out the optimum time and temperature for
inactivation of bacteria and parasites in the cow dung and pit faecal sludge, a forthcoming
fertilizer. This study also enriched the finding of prevalence and intensity of bacteria and
parasites from cow dung and pit soil.40 cow dung samples were collected from Dohar,
Gopalgonj, Keranigonj and Mymensing and 40 pit soil samples were collected from Dohar,
Gopalgonj, Keranigonj and Hajaribag, 10 from each site. Floatation concentration, formolether sedimentation and centrifugal flotation techniques were initially applied and centrifugal
floatation was found best for quantitative analysis of parasitic contamination. From highest
prevalent samples, 10 gm were heated at an increased rate of 5C from 50C to 80C for 15
mins, 30 mins and 60 mins and examined. In cow dung, total coliforms, feacal coliforms and
Escherichia coli were 100% prevalent but faecal streptococci were 97.5% prevalent. On the
other hand, pit soilswere 85% prevalent for total coliforms, feacal coliforms and E. coli
whereas, faecal streptococci were 82.5% prevalent. About 40% cattle were found infected
with one or more intestinal parasites among which four species were of nematode, namely
Trichostrongylus (32.5%), Strongyloides (15%), Haemonchus (5%) and Toxocara (5%), a
species of trematode Paramphistomum (2.5%) and coccidian (32.5%). In pit soil, the
prevalence of Ascaris lumbricoides was highest (47.5%) followed by Strongyloides
stercoralis (30%), Ancylostoma duodenale (27.5%), Trichuris trichiura (22.5%), Entamoeba
histolytica (10%), Enterobius vermicularis (7.5%) and Hymenolepis nana (7.5%).The
thermal process, ultimate objective of study revealed that at60C for 30 mins ofexposure all
bacteria failed to grow except faecal streptococci that was 65C for 60 mins.Among parasites
found, A.lumbricoides and H.nana were the most resistant, inactivated at 75C within 15
mins. T.trichiura survived at 70C for 15 mins, though inactivated at 65C of 30 mins of
exposure. A. duodenale eggs and larva required 65C (60 mins) and 60C (60 mins) where, S.
stercoralis eggs and larva required 70C (30 mins) and 65C (30 mins) to be killed
respectively. E. histolytica at 60C for 30mins became 100% inactive. In cow dung,
Paramphistomim was the most resistant, became inactivated at 65C for 60mins where,
Haemonchus at 65C for 30 mins. Moreover, Trichostrongylus eggs and larva required 65C
for 30 mins and 60C for 60 mins respectively and Strongyloides eggs and larva required
60C for 60 mins and 60C for 30 mins respectively. Coccidian cysts took least temperature
(60C) and time (15mins) to be inactivated. Finally, it can be concluded that a temperature
more than 750C with a time duration of minimum 15 mins can easily kill all the pathogens of
would be fertilizer making them biologically safe to use.

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CONTENTS
ACKNOWLEDGEMENTSi
ABSTRACT ..................................................................................................................ii
INTRODUCTION...................................................................................................... 15
1.1.

Overview ........................................................................................................... 15

1.2.

Risk Assessment................................................................................................ 16

1.3.

Bacterial isolation ............................................................................................. 17

1.4.

Cattle Pathogens ............................................................................................... 18

1.5.

Human Pathogens ............................................................................................ 21

1.6.

Intestinal Parasitism: a health problem in Bangladesh ................................ 24

1.7.

Faecal Waste management .............................................................................. 24

1.8.

Indicator organisms ......................................................................................... 26

1.9.

Coliform bacteria ............................................................................................. 27

1.10. Helminths .......................................................................................................... 28

1.11. Faecal Waste Use .............................................................................................. 28
1.12. Enumeration Techniques ................................................................................. 29
1.13. Inactivation of Pathogens ................................................................................ 30
1.14. Pathogens Biology ........................................................................................... 32
1.14.1. Bacteria ...................................................................................................... 32
1.14.2. Parasites ..................................................................................................... 35

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1.15. Pathogen Safety Data Sheet ............................................................................. 46

1.15.1. Section I Infectious Agents .................................................................... 46
1.15.2. Section II Exposure Controls / Personal Protection ........................... 46
1.15.3. Section III Handling and Storage ......................................................... 47
1.16. Objectives:......................................................................................................... 47
LITERATURE REVIEW ......................................................................................... 48
2.1.

Human Faecal Parasites .................................................................................. 48

2.2.

Cattle Parasites ................................................................................................. 57

2.3.

Inactivation of Pathogens ................................................................................ 59

MATERIALS & METHODS................................................................................ 64

3.1.

Period of study .................................................................................................. 64

3.2.

Study area ......................................................................................................... 64

3.3.

Consents ............................................................................................................ 64

3.4.

Sample Size ....................................................................................................... 64

3.5.

Collection and transport of samples ............................................................... 64

3.6.

Sample analysis procedures............................................................................. 66

3.6.1.

Instrument and chemicals required ........................................................ 67

3.6.2.

Direct Smear Technique ........................................................................... 67

3.6.3.

Formol-Ether Concentration Technique ................................................ 68

3.6.4.

Floatation Technique:............................................................................... 70

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3.7.

Identification of Parasites ................................................................................ 73

3.8.

Viability testing................................................................................................. 73

3.9.

Bacteriological Testing ..................................................................................... 73

3.9.1.

Serial Dilutions .......................................................................................... 74

3.9.2.

Fecal Coliforms Testing ............................................................................ 76

3.9.3.

Fecal Streptococci Testing ........................................................................ 76

3.9.4.

Isolation of E. coli...................................................................................... 78

3.9.5.

Isolation of Enterococci ............................................................................ 79

3.9.6.

Antimicrobial susceptibility testing ......................................................... 79

3.10. Data analysis ..................................................................................................... 82

3.11. Ecological terms................................................................................................ 82
RESULTS & OBSERVATIONS .............................................................................. 85
4.1.

Bacteriological Observations ........................................................................... 85

4.2.

Bacteria examined ............................................................................................ 85

4.3.

Parasitological Examination ........................................................................... 98

4.4.

Efficacy of Different Parasite Identification Techniques ............................. 99

4.5.

Annihilation of Bacteria................................................................................. 112

4.6.

Annihilation of Parasites ............................................................................... 116

4.7.

Biochemical Identification ............................................................................. 147

4.8.

Antibiotics Susceptibility Testing ................................................................. 148

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DISCUSSION ........................................................................................................... 158

CONCLUSIONS ...................................................................................................... 158
BIBLIOGRAPHY .................................................................................................... 160
APPENDIX ............................................................................................................... 184
PLATES ....................................................................................................................... 1923

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LIST OF TABLES

Table 1:The main pathogenic helminths eliminated in human faeces. Helminths diseases
cycle characteristics. ......................................................................................................... 23
Table 2: Specific Gravity of Parasite Structures Recovered by Flotation Methods ......... 70
Table 3: Specific gravity of commonly recommended flotation solutions ....................... 70
Table 4: Concentrations and diffusion zone breakpoints for resistance against
antimicrobial agents tested for E.coli................................................................................ 81
Table 5: Concentrations and diffusion zone breakpoints for resistance against
antimicrobial agents tested faecal streptococci ................................................................. 82
Table 6: Bacteriological counts of cow dung samples from Dohar .................................. 85
Table 7: Bacteriological counts of cow dung samples from Gopalgonj ........................... 86
Table 8: Bacteriological counts of cow dung samples from Keranigonj .......................... 87
Table 9: Bacteriological counts of cow dung samples from Mymensing ......................... 88
Table 10: Bacteriological counts of pit soil samples from Dohar .................................... 90
Table 11: Bacteriological counts of pit soil samples from Gopalgonj.............................. 91
Table 12: Bacteriological counts of pit soil samples from Keranigonj ............................ 92
Table 13: Bacteriological counts of pit soil samples from Hajaribag ............................... 93
Table 14: Comparative Prevalence of E. coli, faecal streptococci, faecal coliforms and
total coliforms in cow dung .............................................................................................. 94
Table 15: Overall prevalence of bacteria of cow dung ..................................................... 95
Table 16: Comparative Prevalence of E.coli, faecal streptococci, faecal coliforms and
total coliforms of pit soil samples ..................................................................................... 96

vii |

Table 17: Overall prevalence of bacteria of pit faecal sludge .......................................... 97

Table 18: Identified parasites from pit faecal sludge and cow dung ................................ 99
Table 19: Parasites enumerated through different techniques from cow dung samples . 100
Table 20: Prevalence of Parasites in Cow Dung Samples through Different Techniques
......................................................................................................................................... 101
Table 21: Anova (F-test) table for parasite recovery techniques from cow dung .......... 101
Table 22: Comparison of different techniques (correlations) ......................................... 102
Table 23: Parasites enumerated through different techniques from pit soil samples ..... 103
Table 24: Prevalence of parasites of pit soil samples ..................................................... 104
Table 25: Anova (F-test) for parasite recovery techniques from pit faecal sludge ......... 105
Table 26: Comparision of different parasite enumeration techniques ............................ 106
Table 27: Parasitic prevalence in cow dung ................................................................... 107
Table 28: Overall prevalence and intensity of parasites from cow dung ........................ 108
Table 29: Prevalence of parasites of pit soil ................................................................... 109
Table 30: Overall prevalence and intensity of parasites of pit faecal sludge .................. 111
Table 31: Thermal inactivation of bacteria from cow dung sample ............................... 112
Table 32: Time-temperature relationship in bacterial counts ......................................... 113
Table 33: Thermal inactivation of bacteria from pit soil samples .................................. 114
Table 34: Time- temperature correlation in inactivation of bacteria .............................. 115
Table 35: Overall time- temperature required in inactivation of parasites ..................... 116
Table 36: Thermal Inactivation of Paramphistomum ..................................................... 117

viii |

Table 37: Time- temperature correlation in inactivation of Paramphistomum............... 119

Table 38: Thermal Inactivation of Haemonchus spp ...................................................... 119
Table 39 : Time- temperature correlation in inactivation of Haemonchus ..................... 121
Table 40: Thermal Inactivation of Trichostrongylus eggs .............................................. 121
Table 41: Time- temperature correlation in inactivation of Trichostrongylus eggs ....... 123
Table 42: Thermal Inactivation of Trichostrongylus larva ............................................. 123
Table 43: Time- temperature correlation in inactivation of Trichostrongylus larva ...... 125
Table 44: Thermal Inactivation of Strongyloides eggs ................................................... 125
Table 45: Time- temperature correlation in inactivation of Strongyloides eggs ............ 127
Table 46: Thermal Inactivation of Strongyloides larva .................................................. 127
Table 47: Time- temperature correlation in inactivation of Strongyloides larva ............ 129
Table 48: Thermal Inactivation of Coccidian cyst .......................................................... 129
Table 49: Time- temperature correlation in inactivation of coccidian cysts ................. 131
Table 50: Thermal Inactivation of A. lumbricoides ....................................................... 131
Table 51: Time- temperature correlation in inactivation of A. lumbricoides ................ 133
Table 52: Thermal Inactivation of T. trichiura .............................................................. 133
Table 53: Time- temperature correlation in inactivation of T. trichiura ........................ 135
Table 54: Thermal Inactivation of A. duodenale eggs .................................................... 135
Table 55: Time- temperature correlation in inactivation of A. duodenale eggs ............. 137
Table 56: Thermal Inactivation of A. duodenale larva ................................................... 137

ix |

Table 57: Time- temperature correlation in inactivation of A. duodenale larva ............. 139
Table 58: Thermal Inactivation of S. stercoralis eggs .................................................... 139
Table 59: Time- temperature correlation in inactivation of S. stercoralis eggs ............. 141
Table 60: Thermal Inactivation of S. stercoralis larva ................................................... 141
Table 61: Time- temperature correlation in inactivation of S. stercoralis larva............. 143
Table 62: Thermal Inactivation of H. nana .................................................................... 143
Table 63: Time- temperature correlation in inactivation of H. nana .............................. 145
Table 64: Thermal Inactivation of E. histolytica cyst ..................................................... 145
Table 65: Time- temperature correlation in inactivation of E. histolytica cysts ............. 147
Table 66: Biochemical test results .................................................................................. 147
Table 67: Diffusion zone (mm) for resistance against antimicrobial agents tested for E.
coli isolated from cow dung ............................................................................................ 148
Table 68: Diffusion zone (mm) for resistance against antimicrobial agents tested for
faecal streptococci isolated from cow dung .................................................................... 149

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LIST OF FIGURES
Figure 1: Parasitic and bacterial inactivation scheme ....................................................... 65
Figure 2: Formol-Ether concentration technique (after centrifugation) ........................... 68
Figure 3: A schematic view of dilution and plating .......................................................... 75
Figure 4: Diagram showing the design of the experiments .............................................. 77
Figure 5: log10 of average of bacteria in cow dung from Dohar...................................... 86
Figure 6: log10 of average of bacteria in cow dung from Gopalgonj ............................... 87
Figure 7: log10 of average of bacteria in cow dung from Keranigonj.............................. 88
Figure 8: log10 of average of bacteria in cow dung from Mymensing ............................ 89
Figure 9: log10 of average of bacteria in cow dung from study sites ............................... 89
Figure 10: log10 of average of bacteria in pit soil from Dohar ........................................ 90
Figure 11: log10 of average of bacteria in pit soil from Gopalgonj ................................. 91
Figure 12: log10 of average of bacteria in pit soil from Keranigonj ................................ 92
Figure 13: log10 of average of bacteria in pit soil from Hajaribag .................................. 93
Figure 14: log10 of average of bacteria in pit soil samples from study sites .................... 94
Figure 15: Prevalence of Bacteria in cow dung ................................................................ 95
Figure 16: Overall prevalence graph of bacteria of cow dung.......................................... 96
Figure 17: Graphical presentation of prevalence of bacteria in pit samples from study sites
........................................................................................................................................... 97
Figure 18: Overall prevalence graph of bacteria from pit faecal sludge ........................... 98
Figure 19: Prevalence of Parasites in pit soil samples through different techniques...... 104

xi |

Figure 20: Prevalence of parasites in cow dung from study sites ................................... 107
Figure 21: Overall prevalence of cow dung parasites ..................................................... 108
Figure 22: overall intensity of cow dung parasites ......................................................... 109
Figure 23: Prevalence of parasites in pit soil from study sites ....................................... 110
Figure 24: Bar diagram of overall prevalence of parasites from pit faecal sludge ......... 111
Figure 25: Overall intensity graph of parasites of pit faecal sludge ............................... 112
Figure 26: Time - temperature relationship in inactivation of Paramphistomum spp.. .. 118
Figure 27: Fig.: Inactivation rate of Paramphistomum spp. in relation to time and
temperature ..................................................................................................................... 118
Figure 28: Time - temperature relationship in inactivation of Haemonchus .................. 120
Figure 29: Inactivation rate of Haemonchus in relation to time and temperature .......... 120
Figure 30: Time - temperature relationship in inactivation of Trichostrongylus eggs ... 122
Figure 31: Inactivation rate of Trichostrongylus eggs .................................................... 122
Figure 32: Time - temperature relationship in inactivation of Trichostrongylus larva... 124
Figure 33: Inactivation rate of Trichostrongylus larva ................................................... 124
Figure 34: Time - temperature relationship in inactivation of Strongyloides eggs......... 126
Figure 35: Inactivation rate of Strongyloides eggs ......................................................... 126
Figure 36: Time - temperature relationship in inactivation of Strongyloides larva ........ 128
Figure 37: Inactivation rate of Strongyloides larva......................................................... 128
Figure 38: Time - temperature relationship in inactivation of Coccidian cyst ............... 130
Figure 39: Inactivation rate of Coccidian cyst ................................................................ 130

xii |

Figure 40: Time - temperature relationship in inactivation of A.lumbricoides............... 132

Figure 41: Inactivation rate of A. lumbricoides ............................................................. 132
Figure 42: Time - temperature relationship in inactivation of T. trichiura .................... 133
Figure 43: Inactivation rate of T. trichiura .................................................................... 134
Figure 44: Time - temperature relationship in inactivation of A.duodenale ova ............ 136
Figure 45: Inactivation rate of A. duodenale eggs .......................................................... 136
Figure 46: Time - temperature relationship in inactivation of A.duodenale larva .......... 138
Figure 47: Inactivation rate of A. duodenale larva ......................................................... 138
Figure 48: Time - temperature relationship in inactivation of S. stercoralis ova ........... 140
Figure 49: Inactivation rate of S. stercoralis eggs .......................................................... 140
Figure 50: Time - temperature relationship in inactivation of S. stercoralis larva ......... 142
Figure 51: Inactivation rate of S. stercoralis larva ......................................................... 142
Figure 52: Time - temperature relationship in inactivation of H.nana .......................... 144
Figure 53: Inactivation rate of H. nana ........................................................................... 144
Figure 54: Time - temperature relationship in inactivation of E. histolytica cysts ......... 146
Figure 55: Inactivation rate of E. histolytica cysts ......................................................... 146

xiii |

LIST OF PLATES
Plate 1: Instrument .......................................................................................................... 203
Plate 2: Bacterial Isolates ................................................................................................ 204
Plate 3: Biochemical Identification ................................................................................. 205
Plate 4: Anbiotics Susceptibility Test ............................................................................. 206
Plate 5: Parasites ............................................................................................................. 207

xiv |

INTRODUCTION
1.1.Overview
Scientists have researched on agronomic effectiveness of human manure (Mnkeni et al.,
2006; Mnkeni and Austin, 2008) and human urine as an important basis of nutrients for
crop fertilization in countries such as Mexico, Germany, USA, Sweden, Denmark and
Zimbabwe, China, Korea, and Japan (Mnkeni et al., 2008; Steineck et al., 1999). Manures
contribute to the soil fertility by adding organic matters and nutrients, such as nitrogen,
that are trapped by bacteria in the soil. It naturally activates the microorganisms found in
the soil restoring the soils natural fertility and stimulates plant growth. Nearly 200
million farmers in China, India, Vietnam, sub-Saharan Africa and Latin America harvest
grains and vegetables from fields that use untreated human waste as fertilizer
(Eichenseher, 2008). Fecal sludge may contain disease causing microorganisms even after
keeping it undisturbed in the pit for months. Farmers of developing countries tend to use
human feces as fertilizer due to availability and lower cost. Therefore, they ultimately
face the greatest risk of suffering from disease if such fecal sludge is not treated well
before field use. In our country, farmers who deal with these are poor and uneducated.
They can easily get infected by pathogenic bacteria and parasites present in fecal sludge
as they rarely maintain good hygiene practices. On the other hand, due to poor sanitation,
ponds and rivers become contaminated from manures used in irrigation fields. Substantial
amounts of surface water are contained in ponds scattered throughout villages in rural
Bangladesh (Knappett et al., 2011). Contact with pond water has previously been
identified as a driver of diarrheal disease in Bangladesh when people drink, bathe in, or
live near a pond (Emch et al., 2008; Ali et al., 2002).
It has been common practice for thousands of years to dispose of human excreta on land.
Problems presented by such disposal have been exacerbated recently by the
intensification of agriculture, the growth of the human and farm animal population, the
cost of artificial fertilizers, which renders materials such as sewage sludge, previously
regarded as waste, a valuable commodity, and the growth of organic farming methods.
Both animal and human wastes may be used in agriculture following composting.
Composting may also be applied to other waste products to release nutrients and render
them safe to use. This includes green compost composed of waste plant material but
15 |

which may also contain animal waste, human sewage and catering waste and which may
be contaminated with the excreta of farm animals, wild animals and pets such as dogs.
1.2. Risk Assessment
The agricultural or domestic use of compost may possibly increase the risk of disease
transmission by a number of mechanisms. The most obvious is direct contact by humans
handling the material or animals grazing pastures or crops on which the material has been
used as a fertilizer. This could be particularly important for organisms such as
entrohaemorrhagic Escherichia coli O157:H7 which is thought to have a low infectious
dose (possibly as few as 10 cells may initiate an infection in susceptible humans; Tarr,
1995; Anon, 1997). Indeed, direct contact with ruminant faeces on farms and on
contaminated pastures has resulted in outbreaks of E.coli O157:H7 (OBrien et al., 2001;
Ogden et al., 2002; Stevens et al., (2002). Disease, and particularly enteric disease, may
also occur following contamination of food crops. Outbreaks have also resulted from the
consumption of water, unpasteurized apple drinks and vegetables contaminated with
ruminant faeces (Olsen et al., 2002; Cody et al., 1999; Hillborn et al., 1999). The use of
compost could also provide a mechanism for the maintenance of organisms such as
Salmonella, E.coli and Campylobacter in the environment. The main source of these
organisms for the human population is the consumption of contaminated animal products.
The environment is, however, already heavily contaminated with a variety of disease
organisms, some of which, such as C. perfringens and Listeria, occur extensively in soil.
It is doubtful whether the use of compost will create any greater hazard than the use of
human and animal wastes as agricultural fertilizers.
The five major types of human pathogenic (disease causing) organisms (bacteria, viruses,
fungi, protozoa and helminths) all are present in human faeces and in cow dung. The
enteropathogenic bacteria are Escherichia coli, Enterococcus faecalis, Vibrio cholarae,
Salmonella, Shegella etc. cause diarrhoea, cholera and other enteric diseases along with
some enteric virues like rota virus and fungi like Histoplasma capsulatumand
Paracoccidioides brasiliensis (Eduar et al., 2011). E. coli is normal floara of both human
and cattle guts but enteropathogenic E. coli infection might cause noninflammatory
diarrhea and inflammatory diarrhea (Doyle et al., 1996). Vibrio cholarae is one of the
most devastating bacteria that cause cholera and death occurs in 50 to 70% of untreated
patients (Shah et al., 1998).
16 |

Since a large variety of pathogenic organisms may be excreted by infected animals it may
be assumed that a similar number of organisms may be found in wastes or compost at
some time. In addition materials such as green compost may become contaminated by
organisms such as Clostridium perfringens which are common inhabitants of soil but
which may occasionally cause disease in humans or animals.
The EPA estimates that up to 3.5 million people fall ill from swimming in waters
contaminated by sanitary sewer overflows alone ever year. However, the number of
illnesses caused by raw sewage could be much higher than we think. Many people that
get sick from untreated sewage arent aware of the cause of their illness and dont report
it to their doctors or local health officials. A recent study found that up to 1.5 million
people get gastroenteritis at beaches in two counties in California each year. If this is the
case, the number could be much higher.
It has been estimated that several hundred diseases may be transmitted from animal to
animal and that more than one hundred and fifty may be transmitted from animal to man
(Diesch, 1974; Strauch, 1978; Strauch, 1994). The causative organisms of such diseases
are often excreted in the faeces, urine and other fomites of diseased individuals, recovered
and convalescent cases and healthy carriers, even when the disease is systemic rather
than enteric.
The spread of many human diseases such as cholera, typhoid and dysentery has been
controlled by isolating man from human excreta and improvements in personal hygiene
and the use of sewage and water treatment processes. In areas of the world where such
improvements have not been made, or systems are inefficient, such diseases remain
endemic. Although improvements in animal husbandry systems have been made this
isolation has not been possible for the farm animal population.
1.3.Bacterial isolation
While most E. coli are generally harmless, certain strains of E.coli have virulence
properties that may account for life threatening infections. Currently, six E. coli
pathotypes are recognized that can cause diarrhea in humans (Turnerand Henderson IR,
2006): enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC), Shiga toxin-

17 |

producing E. coli (STEC), enteroaggregative E. coli (EAEC), enterotoxigenic E. coli

(ETEC) and diffusely adhering E. coli (DAEC).
Enterococci form part of the bowel flora of humans and other animals, and are known to
be responsible for several clinical infections, including urinary tract infections and
infective endocarditis (Megran 1992; Moellering 1992). In addition, they are increasingly
recognized as major nosocomial pathogens (Korten and Murray 1997; Marcus et al.
1997). Of particular concern is the rapid increase in strains isolated from the hospital
setting that are resistant to vancomycin and other antibiotics (French, 1998). Several
schemes for the identification of enterococci to species level have been published
previously (Facklam and Collins 1989; Facklam and Sahm 1995; Manero and Blanch
1999; Murray et al. 1991). These schemes are time consuming to perform, especially for
the high through put screening of large numbers of isolates required in a clinical setting.
Over 85% of clinical enterococcal isolates are either Enterococcus faecalis or
Enterococcus faecium, with few other species occurring (Facklam and Sahm 1995). As
these are the strains of most clinical significance, the possibility is raised of devising a
simplified identification scheme, i.e. one that comprises a simplified, less time consuming
set of tests based on the existing biochemical schemes.
1.4.Cattle Pathogens
Pell (1997) indicated that there are numerous pathogens in livestock manure which can
infect humans. The principal ones are Crytosporidium parvum, Giardia sp., L.
monocytogenes, E. coli O157:H7, Salmonella sp. and M. paratuberculosis. Healthy cattle
and sheep sporadically carry E.coli O157:H7 in their gastrointestinal tract and shed the
bacteria in their faeces (Chapman et al., 1993; Hancock et al., 194; Kudva et al., 1997;
Kudva et al., 1996). In cattle, E. coli O157:H7 occurs with an overall prevalence of 0.3 to
6.1 per cent and the average time those faeces from an individual animal remains positive
are 30 days (Sanderson et al., 1995; Wells et al., 1991; Kudva et al., 1996; Paiba et al.,
2002). In sheep the prevalence is 0.9 to 31per cent and the organism is shed in faeces for
more than a year (Kudva et al., 1997; Kudva et al., 1996; Zhao et al., 1995).
Contamination of non-ruminant food sources of infection is most often from ruminant
manure (Samadpour et al., 1994; Tarr, 1995; Waters et al., 1994) and vegetables
associated with outbreaks were found to have been grown in soil layered with manure
(USDA, 1997; Cieslak et al., 1993).
18 |

The infection rates are variable depending upon different intrinsic and extrinsic
epidemiological and biological factors. The economic losses may be in the form of
mortality, decreased productivity, reduced weight gain and other means resulting globally
from parasitic diseases. The most important and widely prevalent nematodes are
Ostertagia sp., Trichostrongylus sp., Cooperia sp., Oesophagostomum sp.etc. Toxocara
sp. and Dictyocaulus sp. have the worldwide distribution and the prevalence is higher in
cattle and Strongyliasis is an intestinal infection of man caused by the penetration of skin
by the filariform larvae of Strongyloides stercoralis. Among roundworms of cattle the
commonest are Trichostrongylus sp., hookworms, Ascaris sp., Strongyloides sp. Adult
roundworms can cause anaemia, diarrhea, poor growth and even death. Hookworms like
Ancylostoma sp.show severe symptoms like anemia, laziness and lack of physical and
mental ability. The heavy infection of the worms numbering from 250-400 may lead to
loss of about 268ml blood per day from the host body.
Cestodes found in gut are acquired by eating contaminated food or water found to be
largely affecting the ruminants like cattle and sheep. This group comprises of the genera
Moniezia sp, which is cosmopolitan in distribution and Taenia sp., commonly found in
domesticated and wild carnivores and harbivores.
Trematodes are common in the bile duct of cattle and buffalo or in the small intestine and
may also infect lungs. Their eggs are passed with feces of the host. Trematodes especially
include Fasciola sp., Schistosoma sp., Paramphistomum sp. etc. Trichostrongyliasis is a
disease occurs in gastro-intestinal tract of cattle, sheep, goat and other herbivorous
animals. Schistosoma sp. is the only trematode living in the blood stream of warm
blooded hosts causes schistosomiasis or bilharziasis.
Cooperia and Ostertagia (Strongylida: Trichostrongylidae) are common roundworms that
negatively affect cattle and sheep health and production. A study in Wyoming evaluated
the survival of larvae of both types at 2,225m (7,300) and 3,011m (9,880) elevation for
winter survival (Schwink 1963). Development and survival for both species of
roundworms was greatest at the highest elevation. Larvae could survive for more than a
year if the moisture was above normal. Overwinter survival for both was greater in these
regions with cooler temperatures and higher elevations than in lower and warmer
locations, a result that has been documented in similar roundworm species in Switzerland
19 |

and the Himalaya foothills of Nepal (Eckert et al. 1981, Joshi et al. 1997), although
roundworm survival may be optimal at higher elevations (Schwink 1963).
A study of intestinal parasitic infections among highland and lowland dwellers in
Ethiopia found no elevation difference (Wegayehu et al. 2013). Producers need to be
aware that wetter years may enhance winter survival of roundworms. If animals are
congregated, then close observation and potential intervention could be warranted
(Schwink 1963).
Most cattle population in Bangladesh comes from primitive and low productive breeds.
Most animals are reared in house under the age old traditional husbandry practices. Many
cattle are over worked and most of them under fed or half fed during most of the time of
the years. They are not supplied with adequate balanced ration. As a result the general
nutritional status of most of the cattle is in subnormal level, which greatly increases
susceptibility to parasitic diseases (Blood et al., 1990). The temperature, humidity and
rainfall of the country are highly favorable for parasites. In Bangladesh a limited number
of studies on some epidemiological aspects of different gastro-intestinal parasites have
been carried out (Islam et al., 1971; Rahman and Razzak, 1973; Rahman and Ahmed,
1991) in indigenous breed of cattle.
Gastrointestinal parasite cause impaired digestion and also affect the absorption of
minerals particularly the calcium and phosphorus (Speedy, 1992). Like other diseases,
parasitic infections or concurrently occurring infections cause economic losses in terms of
mortality, stunted growth, loss of body weight gain leading to poor quality of skin,
decreased milk and meat production (Dewan et al.1979; Nooruddin et al. 1987; Ahmed et
al.1994). Gastrointestinal nematodes are also serious problems for ruminants, especially
young animals. Debnath et al., (1995) suggested that 50% calves up to 1 year of age died
due to gastrointestinal parasites that cause digestive disturbances and malnutrition leading
to calf mortality. Different helminth infections are responsible for about 54.22% calf
mortality in Bangladesh.

20 |

1.5.Human Pathogens
Throughout evolutionary history humans have been infected with parasites. Today, it is
estimated that over a third of the world's population, mainly individuals living in the
tropics and sub - tropics, are infected by one or more parasitic helminths (worms) and
protozoans (de Silva et al., 2003; Snow et al., 2005). Infestation can cause morbidity, and
sometimes death, by compromising nutritional status, affecting cognitive processes,
inducing tissue reactions, such as granuloma, and provoking intestinal obstruction or
rectal prolapse (WHO, 2002). Intestinal protozoans and soil transmitted helminthes are
major problems in health worldwide, especially in the tropical and sub-tropical regions
(Savioli et al., 1992; Buddy, 1997). Intestinal parasitic infections (IPIs) are globally
endemic and constitute greatest single worldwide cause of illness and disease (Bethony,
et al.2006, Curtale et al., 1998).
Helminthiasis is particularly common in regions where poverty and poor sanitary
conditions are dominant. There are several kinds of helminthiasis endemic in Africa,
Latin America and the Far East. Even though the mortality rate is low, most of the people
infected are children under 15 years with problems of faltering growth and/or decreased
physical fitness (Jimenez-Cisneros, 2007).
Globally, it is estimated that two billion people are affected by intestinal parasites, of
whom 300 million suffer from associated severe morbidity; however, it is difficult to
estimate the actual burden due basically to underreporting (WHO, 2002; Kosek et al.,
2003), whereas Soil-transmitted helminths (STHs) are a group of nematodes that infect
more than a billion people worldwide (Bethony et al. 2006). Ascaris lumbricoides,
Trichuris trichiura and hookworms are of particular public health concern (Hotez et al.
2004), along with Strongyloides stercoralis (Hotez et al. 2003), responsible for morbidity.
Moreover, intestinal parasites contribute to malnutrition, protein and iron deficiencies, an
increment in health costs, as well as long-term deleterious effects, often forgotten, such as
disability and cognitive impairment (WHO, 2002; Kosek et al., 2003). This is true for
children but also adults and probably especially fertile women from rural communities
who have an increment in their mortality rate and also side effects in their offspring.

21 |

These problems occur even in parasitic infestations of mild intensity, and they are
particularly evident in concomitant infections (Larocque et al., 2005; Ngui et al., 2012).
It is estimated that there are approximately 340 parasite species capable of infecting
humans, with the majority of the 3 billion people currently infected residing in developing
regions of the world (Garcia, 2001). Enteric protozoan parasites remain the most
commonly encountered parasitic diseases and continue to cause significant morbidity and
mortality.
Helminthiasis is particularly common in regions where poverty and poor sanitary
conditions are dominant. There are several kinds of helminthiasis endemic in Africa,
Latin America and the Far East. Even though the mortality rate is low, most of the people
infected are children under 15 years with problems of faltering growth and/or decreased
physical fitness (Jimenez-Cisneros, 2007). Helminthiasis is linked to lack of sanitation,
lack of access to safe water and improper hygiene; therefore they occur wherever there is
poverty. IPIs deprive the poorest of the poor of health, contributing to economic
instability and social marginalization. The poor people of under developed nations
experience a cycle where under nutrition and repeated infections lead to excess morbidity
that can continue from generation to generation. People of all ages are affected by this
cycle of prevalent parasitic infections; however, children are the worst affected (Garzon,
2003).

22 |

Table 1: The main pathogenic helminths eliminated in human faeces and helminths
diseases cycle characteristics (Roque, 1997).
Parasites

Disease

Transmission Path

Ancylostoma duodenale

ancylostomiasis

human soil human

Hymenolepis nana

hymenolepiasis

human soil human

Ascaris lumbricoides

Ascaridiasis

human soil human

Necator americanus

Necatorosis

human soil human

Trichuris trichiura

Tricurosis

human soil human

Taenia saginata

Taeniasis

human cattle human

Taenia solium

Taeniasis

human soil - human

Taenia solium

cysticercosis

human pigs human

According to WHO, 25% of the world's hospital beds are occupied by patients with
diseases transmitted by contaminated water. About 1.5 billion people are infected with
Ascaris lumbricoides and 1.3 billion with Ancylostoma spp. infection (Crompton,
1999).The establishment of maximum concentrations of viable eggs of helminths in
sewage sludgehas been the criterion worldwide used to allow the agricultural use of this
material (Capizzi-Banas et al., 2001). Epidemiological studies have shown that the high
frequencyof helminths in human population, the long survival of the eggs in the
environment and thelow infectious dose are risks associated with agricultural use of
sewage sludge (Soccol et al., 1997). In the world 3.5 billion people are infected with
helminths or protozoa and childrenare the most frequently contaminated, resulting in
approximately 60000 deaths associated with Ascaris lumbricoides each year and 70000
deaths with Entamoeba histolytica (WHO, 2000).
23 |

1.6. Intestinal Parasitism: a health problem in Bangladesh

Population in the developing world like Bangladesh is at particular risk for infestation
with intestinal parasitic worms. Risk factors include use of contaminated water for
drinking, cooking, irrigation, washing food, eating uncooked or undercooked food and
walking barefoot on contaminated soil. The prevalence of intestinal parasites in
Bangladesh (East Pakistan) is very high (Muazzem and Ali, 1961) especially adolescents
and children are at high risk (Khanum et al. 2008).
In Bangladesh infestation of intestinal protozoa and soil transmitted helminth (STH)
parasites such as Entamoeba histolytica, Giardia lamblia, Ascaris lumbricoides and
Trichuris trichiura is a major public health problem both in rural and urban areas with a
wide spread endemicity (Dhar,2011). The first five year plan of Bangladesh (1973-78)
reported 64% of the children of Bangladesh suffered from intestinal parasitic infections.
However, the intestinal parasites of human beings have received very little attention from
research workers in Bangladesh. Begum and Rahman (1975) studied intestinal helminthes
and protozoa and reported five species of protozoa (Entamoeba histolytica, Giardia
lamblia, Entamoeba nana and Iodomoeba butchlii) and four species of helminths (Ascaris
lumbricoides, Trichuris trichiura, Enterobius vermicularis and hookworm.
Geohelminth infection bears a large bulk of the problem and helminthiasis is common
throughout the country (Huq et al. 1982). Intestinal worm infestations are widely
prevalent in tropical and subtropical countries and occur where there is poverty and poor
sanitation. Soil-transmitted helminth (STH) infections form the most important group of
intestinal worms affecting two billion people worldwide and the main species which
infect are Ascaris lumbricoides, (roundworms), Trichuris trichiura, (whip worms) and
Necator americanus/Ancylostoma duodenale (hookworms) are of major importance for
public health in tropical and subtropical countries like Bangladesh (Bethonyet al. 2006;
Muttalib, 1976; Huq et al. 1982; Khanum et al. 1997).
1.7. Faecal Waste management
The activities performed daily by humans generate large volumes of waste from various
areas, increasing environmental pollution and public health problems. According to the
recommendations of the World Health Organization (WHO), the reactor effluent
24 |

treatment from domestic sewages can be used in agriculture, since it applied to cultures
that provide little risk of contamination with pathogens (Ayres and Mara, 1996). The
most probable number of coliforms per gram of dried sludge must be less than 1000, and
the parasitic contamination should be less than one viable egg of helminths in four grams
of dried sludge and less than one egg per litre of effluent (WHO, 1989; Fernandes, 2000).
The processes most often employed for the stabilization of sewage include the aerobic
and anaerobic digestion. The application of lime and the composting are also
recommended in some countries like USA, France and Brazil. However, the efficiency of
the stabilization processes depends of the operational quality and of the pathogen
characteristics present in the sewage sludge (Paulino et al., 2001).
The occurrence and survival of pathogens in human sewage and animal wastes and the
subsequent survival and infectivity of organisms when these materials are disposed of on
land has been extensively reviewed (Strauch et al., 1978; Dumontet et al., 1999; Ellis and
McCalla, 1978; Jones, 1980; Jones, 1981; Jones, 1981; Jones, 1982; Jones, 1984; Jones,
1986; Jones, 1992; Jones et al., 1980; Strauch, 1996; Strauch and Ballineri, 1994).
Pathogens are commonly found in animal wastes which could be transmitted to other
animals (Jones, 1982). The main risk organisms are still Salmonella, pathogenic E. coli,
M. tuberculosis and M. bovis. In agricultural use of animal wastes these organisms can be
controlled by codes of practice limiting spreading and grazing by animals (Jones, 1982).
Sewage wastewater discharges are worldwide risk factors for the introduction of human
protozoan enteropathogens into surface waters. The demand for microbiologically safe
reclaimed waters grows exponentially owing to the global demographic rise of the
population. Improvements in reclaimed water quality by lowering faecal coliform counts
are not a sound solution for human protozoan enteropathogens (Hespanhol, 1997).
Several countries have researched alternatives for final disposal of the waste from water
sewage and sludge treatment. The sewage, prior to the stabilization treatment and
disinfection, can contain macro and micronutrients and many pathogenic microorganisms
and parasites. The handling and use of sewage and sludge obtained, without prior
treatment, may promote severe infection to humans and animals (Paulino et al., 2001).
Almost every village family rears cattle as the cattle are used in cultivation, milk
production, meat production and fuel source. Villagers rear cattle in open fields, where
25 |

they excrete and defaecate. People who walk bear foot on the field could be infected.
With the rain the waste washed into nearby water bodies and act as a source of infection.
In urban areas the use of treated wastewater can be considered in irrigation of public
parks and recreational centers, sports fields, school gardens, landscaped areas, residential
gardens, for commercial uses such as car and glass washing, for decorative purposes such
as fountains and waterfalls, for dust control and building projects, to combat fires, in
industrial and commercial constructions including bathroom flushing (United States
Environmental Protection Agecy, 1992).
After bacteria, protozoa are the most important microorganisms in wastewater treatment.
Their high sensibility to the variations of their environment makes them excellent
indicators of the plant state. The absence of detectable indicator bacteria does not
effectively predict the absence of opportunistic organisms such as parasitic protozoan all
of which are more resistant to disinfection (Technical Information Series).
The use of pathogen indicators has been mentioned for sanitary control of wastewater,
and a set of measures is necessary to assure public health safety for multiple uses, given
that waterborne pathogenic infections represent one of the several modalities of infections
(Rowe and Abdel-Magid, 1995). Isolating parasite agents from artifical environments
such as municipal treatment systems represents most of the efforts towards solving the
security of epidemiological studies in this field (Ayres et al. 1992).
1.8. Indicator organisms
When measuring the treatment efficiency of faecal sludge treatment processes, it is too
expensive and labour intensive to measure all types of pathogens. Instead, it is common
practice to select indicators of pathogenic activity which are measured to provide an
indication of the level of pathogen removal during treatment. These indicators can be
either pathogenic, or non-pathogenic, but the organisms need to be carefully selected in
order to provide adequate information on the inactivation of pathogens.
The following requirements should be met when selecting an indicator (Mara, 2004):

be exclusively of faecal origin;

be in numbers greater than those of the pathogens of concern;

26 |

have a removal that mimics, and is close to, that of pathogens of concern; and

be simple, inexpensive, accurate, and reliable to measure

In addition, indicator organisms should have the ability to survive longer than the
pathogen of concern. Some of the typical indicator organisms for wastewater, faecal
sludge and environmental contamination are the coliform bacteria, helminths and
baceteriophage (as an indicator for viruses). Other indicators of faecal contamination that
have been used in pollution control and pathogen die-off studies include faecal
streptococci, Klebsiella, Clostridium perfringens, Bacteroides, enterococci, and Bifi
dobacterium (Feachem et al., 1983; WHO, 1984).
1.9.Coliform bacteria
Coliform bacteria are bacteria that populate the intestinal tract, and are pervasive in
faeces. Their presence in the environment is therefore used as an indicator of faecal
contamination. Escherichia coli (E. coli) is the target organism that has traditionally been
used to identify faecal contamination in the environment (Feachem et al., 1983).
However, there are complicating factors such as bacteria from the genus Escherichia that
can grow in the environment, and the test is therefore not 100% indicative of faecal
contamination. Tests have been developed to enable the quantification of total coliforms,
faecal coliforms, and E. coli. However, since these bacteria are not indicators of viral or
protozoan contamination, and although they are used as indicators of faecal pollution in
the environment, they do not necessarily provide a good indication of pathogen reduction
in faecal sludge treatment processes. Total and faecal coliforms are not good indicators in
tropical and sub-tropical climates.
The standard method for coliforms analysis relies on the production of acid and gas in
medium incubated at a temperature equal to that of the human body (37C). The standard
method of analyzing thermal tolerant faecal coliforms relies on their production of acid
and gas from lactose when incubated at 44C. However, under tropical and sub-tropical
conditions, it has been found that coliforms, which are not necessarily faecal, also grow
and produce acid and gas from lactose at 44C (Mara, 2004). Enzymatic and biochemical
assays have been developed for the detection of E. coli (APHA, 2005), and commercial
kits are also available for total and faecal coliforms and E. coli.

27 |

1.10. Helminths
When analyzing faecal sludge, helminths are most commonly used as an indicator of the
effectiveness of pathogen reduction due to their prevalence in low- and middle-income
countries, and their persistence following treatment. Helminths (parasitic worms) are
eukaryotic parasites, which are prevalent in about one third of the worlds population.
Helminths include nematodes (round worms), cestodes (flat worms) and trematodes
(flukes). These are important pathogens to monitor, as eggs from one infected person can
infect hundreds of people. Ascaris lumbricoides, a type of round worm, is the most
commonly used indicator, as the eggs are one of the pathogens most resistant to
inactivation in treatment processes, and can be identifi ed relatively easily (Feachem et
al., 1983). The ability of Ascaris lumbricoides eggs to remain viable stems from a highly
impermeable eggshell, which is considered to be one of the most resistant biological
structures. The shell allows the passage of essential respiratory gases while protecting the
eggs from a wide array of chemicals and extreme pH conditions (Nordin et al., 2009).
The monitoring of the removal of Ascaris eggs therefore provides an indication that less
resistant pathogens have also been inactivated.
1.11. Faecal Waste Use
Based on epidemiological studies WHO has set a recommended limit of 1 HO L-1 for
the irrigation of crops that are eaten uncooked for both restricted and unrestricted
irrigations.
According to the International Crop Research Institute for Semi Arid Tropics (ICRISAT),
bio-fertilizers are ready to use formulates of such beneficial microorganisms which on
application to seed, root or soil mobilize the availability of nutrients by their biological
activity in particular, and help build up the micro-flora and in turn the soil health in
general.
Cow manure is an excellent fertilizer, but fresh dung has a disagreeable odor, high
ammonia levels that may burn plants, and may contain excess salt. Composting will
create good fertilizer or topdressing without the odor, salt concentration or toxic ammonia
levels found in fresh dung. The manure should be mixed with high carbon material for
rapid aerobic composting. It will heat naturally, killing many microbes and weed seeds.
28 |

1.12. Enumeration Techniques

There is no standardized analytical technique to detect and enumerate helminth eggs, and
as a result few laboratories make these measurements. Those that do so employ different
methods producing results that may be somewhat difficult to compare. Moreover, most of
these laboratories only report the Ascaris content, and not the total concentration of
helminth eggs. The most commonly used techniques are direct methods, in which the
eggs are separated, recovered and concentrated from the sample, and are identified and
counted under a microscope. Among these techniques are the membrane filter, Leeds I
and II, Faust and US-EPA methods, all of which have different egg recovery rates (Ayres,
1989, Faust, 1939, US-EPA, 1992; Maya et al., 2006). Indirect techniques are based on
measuring an alternative property, such as the total solids or particle content (Chavez et
al., 2004) of the samples and relating these to the content of helminth eggs. To measure
the viability of eggs, either incubation at 26C for 3-4 weeks to observe larval
development or staining techniques (De Victorica and Galvan, 1993) are used.
A variety of procedures are available to detect parasite eggs, larvae or oocysts in feces or
fecally contaminated water and soil. Several researchers have evaluated the recovery
efficiency of different methods; however, validating the methods also requires ensuring
that the viability of the eggs is not adversely affected by any of the treatment steps.
Floatation, Concentration and Cellophane Thick Faecal Smear (Kato Katz) techniques are
frequently used in laboratory. Formol Ether concentration technique is used to detect
helminths eggs from samples which are free from contaminating particulate matters. The
methods most frequently used to recover parasite eggs and oocysts are flotation
techniques that rely on the differences in the specific gravity (SG) of the egg(s), fecal
debris, and flotation solution. Cellophane Thick Faecal Smear (Kato Katz) technique is
effective for thick shelled helminths` eggs detection.
The enumeration of viable Helminth eggs employs the coproscopic method, involving
sedimentation, desorption, centrifugation and fl oatation. During development, this
method was estimated to achieve an effi ciency of 30-70% in the enumeration of viable
eggs (Gaspard and Schartzbroad, 1995). However, further work has increased this to
100% based on a sensitivity of 0.4 ppm, by calculating the number of helminth eggs in
the sample, and incorporating the estimated efficiency of the procedure (Malicki et al.,
29 |

2001). In addition, improvements in the method have significantly reduced the number of
replications that are necessary based on previous methods (USEPA, 1994).
For successful control of ascariasis, efficient and reliable diagnosis is mandatory.
However, Kato-Katz and Formol-ether concentration technique are two methods which
are well appropriate to be used in field for the diagnosis of Ascaris lumbricoides so far.
Since poverty-facing and unprivileged children are the prior victim of ascariasis, the
present study will focus on the comparison between the two techniques to reveal the
easier, rapid, cheaper and more effective tool for the field diagnosis of Ascaris
lumbricoides. The motto of the experiment was to determine whether Formol-ether can be
replaced by Kato-Katz technique with a moderate expenditure and reliable calculation.
This method was adopted by WHO for quantitative and qualitative diagnosis Ascaris
lumbricoides.
1.13. Inactivation of Pathogens
In the new version of the World Health Organization (WHO), water reuse guidelines
helminth ova are considered one of the main target pollutants to be removed from
wastewater reuse for agriculture and aquaculture purposes. In spite of this, along with the
fact that helminth ova have been considered the main health risk to wastewater reuse for
agriculture for at least 20 years, relatively little research has been done to control
helminth ova in the wastewater treatment field (WHO, 2005). Helminth eggs remain
viable for 1-2 months in crops and for many months in soil, fresh water, and sewage.
They may remain viable for several years in faeces, night soil and sludge (Feachem et al.,
1989, Nelson et al., 2004; Sanguinetti et al., 2005; Kon et al., 2007). Because of this and
because they are not inactivated - at least at affordably - using chlorine, UV-light or
ozone they are considered highly resistant biological structures. This resistance derives
from the composition of the egg shell. This consists of a variable number of layers (3-4
depending on the species) each providing mechanical resistance or protection from toxic
compounds, such as strong acids, bases, oxidants, reducing agents, detergents and
proteolytic substances. To inactivate helminth eggs it is recommended either to raise the
temperature (above 40C), to reduce moisture (below 5%) or to maintain both of these
conditions for an extended period of time. These conditions are not readily achieved
during wastewater treatment, thus helminth eggs are usually physically removed from
30 |

wastewater to be subsequently inactivated in sludge (Jimnez, 2008). In order to remove

helminth eggs from wastewater, processes which remove particles, such as sedimentation,
filtration or coagulation-flocculation are employed (Mara, 2004, Jimnez et al., 2010).
Not all treatment processes are efficient enough to inactivate the amount of eggs
contained in sludge in developing countries, reliably or affordably (Carrington et al.,
1991, Gantzer et al., 2001, Keller et al., 2004, Capizzi-Banas, et al., 2004; Jimnez and
Wang, 2006; Kon, 2007).
WHO (2006) has published guidelines with regard to the level of treatment required.
These suggest that a value of 1 egg/L in wastewater intended for the irrigation of crops
to be eaten uncooked is safe. For aquaculture the proposal is that no eggs should remain
at all, since trematode eggs (largely Schistosoma spp.) may multiply in an intermediary
host (snails) before infecting fish and humans. For excreta, the recommended value is 1
egg/gTS.
Source separation and reuse of human urine can decrease the environmental pollution of
recipient waters and reduce the need for artificial mineral fertilizers. However, the reuse
of urine introduces another pathogen transmission route that needs to be managed. The
inactivation of enteric pathogens and model organisms by urine storage was studied by
Nordin et al. (2009).
UV disinfection technology is of growing interest in the water industry since it was
demonstrated that UV radiation is very effective against (oo)cysts of Cryptosporidium
and Giardia, two pathogenic micro-organisms of major importance for the safety of
drinking water. Quantitative Microbial Risk Assessment, the new concept for microbial
safety of drinking water and wastewater, requires quantitative data of the inactivation or
removal of pathogenic micro-organisms by water treatment processes. Hijnen, et al.,
(2006) reported effectiveness of UV in drinking water and waste water for several
bacteria but for protozoa study is still required.
Differential scanning calorimetry (DSC) is used to evaluate the thermal stability and
reversibility after heat treatment of transitions associated with various cellular
components of Escherichia coli and Lactobacillus plantarum (Lee and Kaletun, 2002)

31 |

Helminth eggs are the most difficult biological parasites to inactivate in wastewater and
sludge. In developing countries, in particular, they are present in high concentrations and
are the cause of many diseases that impact seriously on the human population. The
considerable differences in inactivation conditions among helminth eggs and a high level
of resistance was confirmed for the eggs of Ascaris lumbricoides and Ascaris suum
(Maya, et al., 2012)
1.14. Pathogens Biology
The five major types of human pathogenic (disease causing) organisms (fungus, bacteria,
viruses, protozoa, and helminths) are categorized.
1.14.1. Bacteria
Bacteria are a major group of pathogens cause several diseases.
Escherichia coli
E.coli is the type species of the genus Escherichia, which contains mostly motile gramnegative bacilli within the family Enterobacteriaceae and the genus Escherichia
(Edwards & Ewing, 1972).
Escherichia coli are in the bacterial family Enterobacteriaceae, which is a rod shaped,
gram-negative,

facultative

anaerobe,

lactose-fermenting,

nonendospore-forming

microorganism. Its cell measures 12 m in length and 0.10.5 m in diameter. Optimal

growth temperature of E.coli is 37C (98.6F).E. coliare commonly motile in liquid by
means of peritrichous flagella.
Role as Normal Flora
E. coli normally first colonizes an infant's gastrointestinal tract within 40 hours of birth,
arriving with food or water or with the individuals handling the child. In the bowel, it
adheres to the mucus of the large intestine. It is the primary facultative organism of the
human gastrointestinal tract (Todar, 2007). As long as these bacteria do not acquire
genetic elements encoding for virulence factors, they remain benign commensals (Evans
et al., 2007).
E.coli is the most common bacteria that inhabit in the small intestine and large intestine of
mammals. They are often the most abundant facultative anaerobes in this environment.
32 |

The human colon maintains a huge microbial density approaching 1012 organisms per
gram of feces and that represents a perfect balanced ecosystem. Human intestine contains
more than 400 species of commensal microorganisms that live in perfect harmony within
it (Hooper & Gordon, 2001). These perform a variety of functions, with many benefits for
the host organism. For example, bacteria produce enzymes capable of digesting many of
the molecules that are indigestible to vertebrates, produce small amounts of vitamins for
absorption into the blood, and help to prevent colonization by toxic bacteria. E. coli also
provides some of these same values for the host organism, assisting with waste
processing, vitamin K production, and food absorption when located in the large intestine.
Healthy individuals can also contain a great diversity of commensal non-pathogenic E.
coli strains belonging to many different serotypes and these can be isolated from the
feces. These strains may contaminate food of animal origin or other foods like vegetables,
fruits and their derivatives when they massively shed in the environment. They may also
contaminate surface and underground water, generally without any adverse effects on
human health.
Infections caused by pathogenic E. coli
Three general clinical syndromes result from infection with inherently pathogenic E.
colistrains: (i) urinary tract infection, (ii) sepsis/meningitis, and (iii) enteric/diarrheal
disease. These conditions depend on a specific array of virulent factors possessed by the
organism.
Transmission of E. coli
Escherichia coli are commonly found in all warm-blooded animals meaning that humans,
domestic livestock, pets, wild animals, and birds are all reservoirs. Faecal-oral route is the
main route of transmission of E. coli (Evans et al., 2007; Gehlbach, 1973). In addition, E.
coli can transmit to humans through a variety of ways including contact with: other
humans or animals, contaminated surfaces (e.g. door handles), manure or sewage, or
contaminated meat/poultry products, vegetables, raw milk, or untreated water
(Kmmerer, 2004; Cohen, 1992; Lipsitch and Samore, 2002; Linton, 1986).
Dairy and beef cattle are primary reservoirs of Shiga toxin-producing E. coli (Bach et al.,
2002) and they can carry it asymptomatically and shed it in their feces (Bach et al., 2002).
33 |

E. coli outbreaks are connected with various food products, these include raw ground beef
(Institute of Medicine of the National Academies, 2002), raw seed sprouts or spinach
(Sabin, 2006), raw milk, unpasteurized juice, and foods contaminated by infected food
workers via faecal-oral route.
Large concentration of bacteria, including E. coli is known to transmit through water and
this amount is large enough to cause illness in humans (Jackson et al., 1998; Schets et al.,
2005). In Canada and the United States, several large outbreaks of E. coli O157:H7 have
implicated drinking water as the source of infection, including a large outbreak in
Walkerton, Ontario in 2000 (Bruce-Grey-Owen Sound Health Unit, 2004). According to
the U.S. Food and Drug Administration, the faecal-oral cycle of transmission can be
disrupted by cooking food properly, preventing cross- contamination, instituting barriers
such as gloves for food workers, instituting health care policies so food industry
employees seek treatment when they are ill, pasteurization of juice or dairy products and
proper hand washing requirements. Shiga toxin-producing E. coli (STEC), specifically
serotype O157:H7, have also been transmitted by flies, (Szalanski et al., 2004; Sela al.,
2005; Alam and Zurek, 2004) as well as direct contact with farm animals, (Rahn et al.,
1998; Trevena et al., 1999) petting zoo animals(Heuvelink et al., 2002), and airborne
particles found in animal-rearing environments (Varma et al., 2003).
Faecal streptococci
Faecal streptococci are gram positive rounded bacteria of family enterococcaceae. They
are facultative anaerobic, catalase-negative Gram- positive cocci, arranged individually,
in pairs, or short chains (Nannin and Murray, 2006). Optimal temperature for growth of
E. faecalis and E. faecium is 35C (Teixeira et al., 2007). E. faecalis and E. faecium are
normal inhabitants of the intestinal tract, female genital tract, and (less commonly) oral
cavity, they are capable of cellular respiration in both oxygen-rich and oxygen-poor
environments (Fischetti et al., 2000).
Normal Flora
Enterococci are part of the normal intestinal flora of humans and animals. They have been
long recognized as important human pathogens and are becoming increasingly so. The
genus Enterococcus includes more than 17 species, although only a few cause clinical
34 |

infections in humans. Since the beginning of the antibiotic era, they have posed major
therapeutic challenges, including the need for synergistic combinations of antibiotics to
successfully treat enterococcal infective endocarditis (IE).
Infections caused by faecal streptococci
Enterococci can cause urinary tract, wound, and soft tissue infections. They are also
associated with bacteremia which can lead to endocarditis in previously damaged cardiac
valves. E. faecalis is the most frequent species isolated from human intestine samples
(80-90%), E. faecium accounts for 5-10% of isolates (Nannin and Murray, 2006).
Transmission of Faecal Streptococci
Faecal streptococcicantransmit to humans through a variety of ways including contact
with: other humans or animals, contaminated surfaces (e.g. door handles), manure or
sewage, or contaminated meat/poultry products, vegetables, raw milk, or untreated water,
raw milk, unpasteurized juice, and foods contaminated by infected food workers via
faecal-oral route.
1.14.2. Parasites
Parasites are broadly categorized as protozoans and helminthes. Helminths are then
subdivided as trematode, cestode and nematode.
Giardia intestinalis
Giardia intestinalis (also known as G. lamblia or G. duodenalis) is a protozoan flagellate
causing giardiasis in the small intestine. It attaches to the mucosa and absorbs nutrients
that it gets from the intestinal wall. In addition to humans, G. intestinalis infects birds,
cows, sheep, deer, dogs and cats. Giardiasis is found worldwide mostly in warm climates.
G. intestinalis lives as active trophozoites in the small intestine. Some trophozoites encyst
into cysts which are released in a bowel movement. The feces might contaminate soil,
water, food or surfaces such as bathroom sinks. The cyst has a protective shell and it can
survive in the environment for many weeks (in cold water many months). You become
infected after accidentally swallowing the microscopic cysts. Each cyst releases two
trophozoites in the small intestine. They remain in the lumen where they can feed freely
35 |

or attached to the mucosa by a ventral sucking disk. After eating enough, they go through
another transformation and multiply by binary fission. The trophozoites encysted as they
move towards the colon. Cysts are found more often in firm stool whereas both
trophozoites and cysts are present in loose stool. Because the cysts become infective
almost instantly after being passed out, the disease can be transmitted during anal-oralsexual intercourse.
Common giardiasis symptoms include:

bloating

bad breath and farts

dehydration

diarrhea or greasy floating stools

fatigue

loss of appetite

nausea

stomach ache

weakness

weight loss

Diarrhea can be fatal, if you do not drink enough water with salt and glucose. Another not
so recognizable effect is the lack of B12-vitamin. This is due to the impaired absorption
(malabsorption) in the damaged intestinal wall. 50 % of giardiasis cases are
asymptomatic. Symptoms begin usually within two weeks after becoming infected. In
healthy individuals the sickness normally persists up to three weeks, but sometimes
longer.
Entamoeba histolytica - Amoebiasis
Entamoeba histolytica is a protozoan parasite responsible for a disease called amoebiasis.
It occurs usually in the large intestine and causes internal inflammation as its name
suggests. 50 million people are infected worldwide, mostly in tropical countries in areas
of poor sanitation. In industrialized countries most of the infected patients are immigrants,
institutionalized people and those who have recently visited developing countries.

36 |

The life cycle of Entamoeba histolytica does not require any intermediate host. Mature
cysts are passed in the feces of an infected human. Another human can get infected by
ingesting them in fecally contaminated water, food or hands. If the cysts survive the
acidic stomach, they transform back into trophozoites in the small intestine. Trophozoites
migrate to the large intestine where they live and multiply by binary fission. Both cysts
and trophozoites are sometimes present in the feces. Cysts are usually found in firm stool,
whereas trophozoites are found in loose stool.
Symptoms that include:

gas (flatulence)

intermittent constipation

loose stools

stomach ache

stomach cramping.

anemia

appendicitis (inflammation of the

liver abscesses (can lead to death,

if not treated)

malnutrition

painful defecation (passage of the

stool)

peritonitis (inflammation of the

peritoneum which is the thin
membrane

that

lines

the

abdominal wall)

appendix)

pleuropulmonary abscesses

bloody diarrhea

stomach ache

fatigue

stomach cramping

fever

toxic megacolon (dilated colon)

genital and skin lesions

weight loss.

intermittent constipation

Hymenolepis nana - Dwarf Tapeworm

Hymenolepis nana is the most common tapeworm in humans. The disease,
hymenolepiasis is found worldwide. In temperate zones children and institutionalized
people are infected more often. The disease is somewhat common in the Eastern Europe.
The life cycle of Hymenolepis nana starts, when microscopic eggs are passed with the
stool of an infected human. They then get ingested either by rodents, humans (definite
hosts) or insects (intermediate hosts). If a person ingests eggs (from contaminated fingers,
water, food or soil), oncospheres (hexacanth larvae) hatch in the small intestine. A larva
37 |

penetrates an intestinal villus and develops into a cysticercoid. A cysticercoid develops to

look more like an adult having a scolex (head) and a neck. Some proglottids degenerate,
resulting in autoinfection. Hymenolepis nana does not necessarily need an intermediate
host to complete its life cycle. Larvae can develop in spite of the high temperature of a
human body. Adults live 46 weeks, but internal autoinfection allows hymenolepiasis to
persist for years.
Hymenolepiasis is usually asymptomatic in adults. But prolonged infection or multiple
tapeworms

especially

in

children

can

cause

more

severe

symptoms.

Hymenolepiasis symptoms sometimes include:

anal itching

diarrhea (can be bloody)

headache

increased appetite or loss of appetite

insomnia

muscle spasms

nausea

seizures

stomach ache

Ascarislumbricoides - Giant Roundworm

Ascaris lumbricoides, giant roundworm, is the most common parasitic worm in humans.
According to some estimates 25 % of humans are infected with the disease, ascariasis.
Ascariasis occurs worldwide, mostly in tropical and subtropical countries. It has highest
prevalence in areas of poor sanitation and where human feces are used as fertilizer.
The life cycle of Ascaris lumbricoides takes about three months. Ascariasis starts,
when Ascaris lumbricoides eggs are accidentally swallowed. They can be acquired from
dirty fingers, water or food that has been contaminated with feces of an infected human.
Larvae hatch from the eggs, penetrate the intestinal wall and enter the bloodstream. They
stop at pulmonary arteries and stay in the lungs for two weeks. They break into the alveoli

38 |

and travel up the respiratory system to the throat to be swallowed again. The migration is
needed for the larvae to develop into adults.
Ascariasis can be asymptomatic, if there are only a few worms. If there are tens or
hundreds of worms, symptoms might include:

diarrhea

fever

nausea

stomach ache

slower growing of a child or a teen

vomiting

weakness.

Unlike many other human roundworms, Ascaris lumbricoides does not usually feed on
blood. When larvae migrate through the lungs, the following pulmonary symptoms may
occur:

breathing difficulty

cough and/or coughing up blood

eosinophilic pneumonitis.

Trichuris trichiura - Whipworm

Whipworm is a parasitic worm infecting 500 million humans in tropical countries. It lives
its adult life in the large intestine. The human whipworm is called Trichuris trichiura and
causes trichuriasis. Whipworm gets its name from its appearance.
Whipworm's life cycle starts, when the female lays eggs in the large intestine of an
infected human. The eggs are carried out in the feces. If landed on warm moist soil, after
23 weeks the eggs have embryonated and are infective ready to be ingested. The eggs
get inside you, if you eat contaminated unwashed and uncooked vegetables, rice or beans.
Larvae hatch in the small intestine and invade the intestinal villi and start growing. After

39 |

a while they move to the large intestine where they penetrate the mucosa and develop into
adults.
The symptoms are alike to ascariasis.
Hookworms
Hookworms are bloodsucking roundworms living in the small intestine. Some common
names for hookworm infections are: ancylostomiasis, necatoriasis, Egyptian chlorosis,
tunnel disease, miners' anemia and brickmaker's anemia. Hookworms are the second most
common human worms (the most common is A.lumbricoides). There are thousands of
hookworm

species

but

only

two

of

them

target

humans. Ancylostoma

duodenale (ancylostomiasis) infect over one billion people around the globe mostly in
tropical and subtropical climates. Necatoriasis predominates in the Americas (North,
Central and South America) and Australia, whereas ancylostomiasis occurs in the Middle
East, southern Europe and North Africa.
The hookworm larvae start the infection as filariform live on warm moist soil that has
been contaminated with infected human feces. Upon touch, a tiny filariform larva attaches
to the skin and penetrates it. It burrows through tissue until it reaches a blood vessel or
lymphatic duct. It travels in the bloodstream to the small pulmonary capillaries. It breaks
into the lung alveoli and is taken towards the bronchus and trachea by the movement of
microvilli. It is coughed up to the throat and swallowed through the esophagus to the
stomach. After passing the stomach it hooks into the intestinal mucosa in the small
intestine and starts sucking blood. Then within a few weeks it develops into an adult and
is ready to mate. The produced eggs exit the body in the feces. Rhabditiform (first stage,
L1) larvae hatch in the feces or in warm, moist, sandy soil within two days. They feed on
organic matter and grow rapidly. They molt twice within 10 days to become filariform
(third stage, L3) larvae that are infective. Filariform larvae can survive up to four weeks
in the right conditions (warmth, moisture, shade).
Hookworms can cause some of the following symptoms:

anemia (pale skin etc.) and protein deficiency caused by blood loss

constipation

congestive heart failure

40 |

decreased rate of growth and mental development in children (caused by protein

and iron deficiency)

diarrhea

dizziness

dyspnea (shortness of breath)

excessive coughing during larvae migration

fatigue (tiredness)

fever

loss of appetite

nausea

rash or sore and itchy feet after larval invasion

stomach or chest pain

vomiting

weight loss

Strongyloides stercoralis
Strongyloides stercoralis causes strongyloidiasis. It is common in tropical and subtropical
areas but also occurs in temperate zones. Unlike most parasitic worms, Strongyloides
stercoralis has a heterogenic life cycle. So in addition to the parasitic life cycle it has a
separate free-living cycle where it lives and reproduces without a host in the
soil. Strongyloides stercoralis can autoinfect the same host over and over without any
intermediate host. This makes strongyloidiasis a very persistent disease. Both rabditiform
and filariform larva can be infective. Transmission is as like hookworms.
Minor infections can be asymptomatic but usually one or more of the following
symptoms occur:

anemia (for example, pale skin)

constipation

cough

diarrhea

eosinophilic pneumonitis (during larvae migration through the lungs)

41 |

nausea

rashes in waist and buttocks

stomach ache

vomiting

weight loss

death

distension

neurological and pulmonary complications

septicemia

shock

Enterobius vermicularis - Pinworm

Human pinworm, Enterobius vermicularis, is the most common parasitic worm infection
in the United States and Western Europe. Pinworms are easily transmitted from human to
human and are particularly common in children. Luckily the disease, Enterobius is,
causes only anal itching.
Enterobius vermicularis does not need an intermediate host to complete its lifecycle.
Humans get infected by accidentally swallowing or inhaling microscopic pinworm eggs.
Once inside the first part of the small intestine, duodenum, pinworm larvae hatch from the
eggs.
Sometimes pinworms lay eggs inside the colon. If the eggs are not taken out in the feces
the larvae might have enough time to hatch. This can only happen in the large intestine or
rectum and only if enough oxygen is present. The larvae migrate back up the intestinal
tract and develop into adults. This is very rare but happens every now and then.
Symptoms are as follows:

anal itching

abdominal pain

nausea

anemia

diarrhea

weight loss

vomiting

42 |

Strongyles:
Trichostrongylus, Strongyloides, Haemonchus are very common parasite and one of the
most pathogenic nematodes of ruminants. Trichostrongylus species are nematodes (round
worms), which are ubiquitous among herbivores worldwide, including cattle, sheep,
donkeys, goats, deer, and rabbits. At least 10 Trichostrongylus species have been
associated with human infections. Infections occur via ingestion of infective larvae from
contaminated vegetables or water. Epidemiological studies indicate a worldwide
distribution of Trichostrongylus infections in humans, with the highest prevalence rates
observed in individuals from regions with poor sanitary conditions, in rural areas, or who
are farmers / herders. Human infections are most prevalent in the Middle East and Asia,
with a worldwide estimated prevalence of 5.5 million people.
The adult female worm can release between 5,000 and 10,000 eggs, which are passed out
in the feces. Eggs then develop in moist conditions in the feces and continue to develop
into the L1 (rhabditiform), and L2 juvenile stages by feeding on bacteria in the dung. The
L1 stage usually occurs within four to six days under the optimal conditions of 2429 C.
The L2 rhabditform sheds its cuticle and then develops into the L3 filiariform infective
larvae. The L3 form has a protective cuticle, but under dry, hot conditions survival is
reduced. Sheep, goats and other ruminants become infected when they graze and ingest
the L3 infecting larvae. The infecting larvae pass through the first three stomachs to reach
the abomasum. There, the L3 shed their cuticles and burrow into the internal layer of the
abomasum, where they develop into L4, usually within 48 hours, or preadult larvae. The
L4 larvae then molt and develop into the L5 adult form. The male and female adults mate
and live in the abomasum, where they feed on blood.
Clinical signs are largely due to blood loss. Sudden death may be the only observation in
acute infection; other common clinical signs include pallor, anemia, oedema, ill thrift,
lethargy and depression. The accumulation of fluid in the submandibular tissue, a
phenomenon commonly called "bottle jaw, may be seen. Growth and production are
significantly reduced.

43 |

Paramphistomum
Paramphistomum is a genus of parasiticflat worm belonging to the digenetic trematodes.
It includes tiny flukes which are mostly parasitisinglivestockruminants, as well as some
wild mammals. They are responsible for a serious disease called paramphistomiasis (or
classically amphistomosis), especially in cattle and sheep.
The life cycle is indirect, requiring a definitive host such as ruminants, an intermediate
host such as snail, and a free-living of external phases in water and plants. The sexually
mature monoeciousself-fertilises in the mammalian rumen, and release the eggs along
with faeces. Eggs hatch in water into ciliated miracidia. The miracidia then enters the
body of an intermediate host, which are snails. Then the miracidia lost their cilia to
become sporocysts. After a few days they develop up to 8 rediae, which are rapidly
liberated. Each redia contains about 15-30 cercariae. Mature cercariae are possessed by
two eyespots and a long slender tail, by which they find aquatic plants or other suitable
substrata, to which they get attached and encysted to become metacercariae. The
mammalian hosts ingest the infective larvae. Once inside the duodenum and jejunum,
their cysts are removed. They penetrate the intestinal wall by actively destroying the
mucosa, and then migrate to the rumen, where they grow into adult.
Paramphistomiasis causes enteritis and anemia in livestocks mammals and result in
substantial production and economic losses. Pathological symptoms are produced by
immature flukes. When the young flukes start to gather in the intestine, there is watery
and fetid diarrhoea which is often associated with high mortality (even up to 80-90%) in
ruminants. At a given time, as many as 30,000 flukes may accumulate, fervently attacking
the duodenal mucosa to induce acute enteritis. Adult flukes are relatively harmless. Liver
tissue is generally damaged extensively, indicated by swelling, haemorrhage,
discolouration, necrosis, bile duct hyperplasia, and fibrosis.
Coccidians
Coccidians are apicomplexan parasites that include various species capable of causing the
disease coccidiosis in cattle and poultry, among other animals. The most prevalent species
of Eimeria that cause coccidiosis in cattle would be E. bovis, E. zuernii, and E.
auburnensis.
44 |

Symptoms of coccidiosis infection include bloody diarrhea due to intestinal epithelium

dying off when a large number of oocysts and merozoites burst out of the cells. The
severity of the disease is directly dependent on the number of infective Eimeria oocysts
that are ingested by the bovine. In light infections the damage to the gut might only be
minimal and be rapidly repaired as cells are rapidly replaced by the body. However, in
heavy infections it may only take two weeks for many intestinal epithelial cells to be
infected with either meronts or gametocytes. These cause the epithelial cells to burst,
which causes significant damage to the intestine epithelial layer, and releases blood, fluid,
and electrolytes into the intestine.
Toxocara
Toxocara causes toxocariasis. Humans are accidental hosts of Toxocara, yet toxocariasis
is seen throughout the world. Most cases of toxocariasis are seen in people under the age
of twenty. Seroprevalence is higher in developing countries, but can be considerable in
first world countries, as well. In Bali, St. Lucia, Nepal and other countries, seroprevalence
is over fifty percent. Previous to 2007, the U.S. seroprevalence was thought to be around
5% in children. However, Won et al. discovered that U.S. seroprevalence is actually 14%
for the population at large. In many countries, toxocariasis is considered very rare.
Approximately 10,000 clinical cases are seen a year in the U.S., with ten percent being
OLM. Permanent vision loss occurs in 700 of these cases.
Cats, dogs and foxes can become infected with Toxocara through the ingestion of eggs or
by transmission of the larvae from a mother to her offspring. Transmission to cats and
dogs can also occur by ingestion of infected accidental hosts, such as earthworms,
cockroaches, rodents, rabbits, chickens, or sheep Eggs hatch as second stage larvae in the
intestines of the cat, dog or fox host (for consistency, this article will assume that second
stage larvae emerge from Toxocara eggs, although there is debate as to whether larvae are
truly in their second or third stage of development Larvae enter the bloodstream and
migrate to the lungs, where they are coughed up and swallowed. The larvae mature into
adults within the small intestine of a cat, dog or fox, where mating and egg lying occurs.
Eggs are passed in the feces and only become infective after several weeks outside of a
host. During this incubation period, molting from first to second (and possibly third) stage
larva takes place within the egg. In most adult dogs, cats and foxes, the full lifecycle does
45 |

not occur, but instead second stage larvae encysted after a period of migration through the
body. Reactivation of the larvae is common only in pregnant or lactating cats, dogs and
foxes. The full lifecycle usually only occurs in these females and their offspring.
Second stage larvae will also hatch in the small intestine of an accidental host, such as a
human, after ingestion of infective eggs. The larvae will then migrate through the organs
and tissues of the accidental host, most commonly the lungs, liver, eyes, and brain. Since
L2 larvae cannot mature in accidental hosts, after this period of migration, Toxocara
larvae will encyst as second stage larvae.
1.15. Pathogen Safety Data Sheet
According to recommendations of Public Health Agency of Canada (2011), the following
safety and security measures were followed in the Environmental Microbiology
Laboratory, icddr,b during the study period to ensure the biosafety and biosecurity.
1.15.1. Section I Infectious Agents
Names: Ascaris spp, Balantidium coli, Entamoeba histolytica, Fasciola hepatica,
Fasciola gigantic, Giardia lamblia, Taenia spp, Toxocara canis, Toxocara cati,
Toxoplasma gondii, Trichuris trichiura, Hymenolepis nana, H. diminuta, Ancylostoma
duodenale, Strongyloides stercoralis, Enterobius vermicularis, Paramphistomum,
Strongyles, E.coli, E. faecalis and other coliforms.
1.15.2. Section II Exposure Controls / Personal Protection
Risk Group Classification: Risk Group 2. This risk group applies to the genus as a
whole, and may not apply to every species within the genus.
Containment Requirements: Containment Level 2 facilities, equipment, and operational
practices for work involving infectious or potentially infectious material. These
containment requirements apply to the genus as a whole, and may not apply to each
species within the genus.
Protective Clothing: Lab coat. Gloves when direct skin contact with infected materials
or animals is unavoidable. Eye protection must be used where there is a known or
potential risk of exposure to splashes.
46 |

Other Precautions: All procedures that may produce aerosols, or involve high
concentrations or large volumes should be conducted in a biological safety cabinet (BSC).
Even if the eggs of Ascaris spp. require an extrinsic maturation period to become
infective, preserved specimens should be handled with care. Ascaris spp. eggs are sticky,
and contaminated laboratory surfaces and equipment must be thoroughly cleaned.
1.15.3. Section III Handling and Storage
Spills: Allow aerosols to settle and, wearing protective clothing, gently cover the spill
with paper towels and apply an appropriate disinfectant, starting at the perimeter and
working towards the centre. Allow sufficient contact time before clean up.
Disposal: Decontaminate all wastes that contain or have come in contact with the
infectious organism before disposing by autoclave, chemical disinfection, gamma
irradiation, or incineration.
Storage: The infectious agent should be stored in leak-proof containers that are
appropriately labeled.
1.16. Objectives:
The study was conducted aiming at following objectives:
o

To identify the risk associated with the use of raw or under treated biofertilizer.

To discover the optimum time-temperature for inactivation of bacteria and

parasite in cow dung and human faecal sludge.

To recommend safe use and disposal of cattle dung and human faecal
sludge.

To find out the prevalence and intensity of parasites in the four different
areas of Bangladesh.

To compare parasites recovery techniques commonly used.

Isolation and identification (phenotypic and biochemical) of bacteria and

parasites.

To detect antimicrobial susceptibility of E.coliand faecal streptococci to

commonly used antibiotics.
47 |

LITERATURE REVIEW

2.1.Human Faecal Parasites

Craig (1934) lists five species of amoebae found in the intestinal tract of man. Only one,
Entamoeba histolytica, is definitely known to be disease producing. The most important
modes of transmission of the organism are considered to be contaminated water supplies,
foodstuffs grown on contaminated soil or handled by " carriers ''and flies.
Hamlin (1946) reported the following conclusions from the sampling of sew age in five
Johannesburg, South Africa, sewage treatment plants. Cysts of E. histolytica, as well as
eggs of parasitic helminths, were frequently present in screened and settled sewage and in
trickling filter, humus tank, and activated sludge effluents. However, effluents of
secondary sand filters or of under drained land areas showed no cysts or eggs.
The environmental condition of Bangladesh especially the soil texture, temperature,
moisture, humidity, and rain fall, etc. serve as suitable preconditions for the hatching of
the fertilized eggs of Ascaris lumbricoides, Trichuris trichiura and other intestinal
parasites. In northern Vietnam, it was common practice to fertilize rice fields with fresh
excreta. As this was a dangerous practice, in 1956 the health authorities started campaigns
to construct double vault dry toilet. The campaigns were followed by long and persistent
health-education programs. The objective of the new toilet design was to kill pathogens
before the feces were spread on the fields. A precursor to the Vietnamese system was
developed around 1950 at the Kanagawa Prefectural Public Health Laboratory in
Yokohama (Kodama et al. 1955).
Kuntz (1960) examined the stools of the students of Tejgaon Polytechnic High School,
Mirpur High School and Demra Secondary High School. Muazzem and Ali (1968) also
detected prevalence rate in East Pakistan (now Bangladesh) among the children and it
was 25.6% for Ascaris lumbricoides, 16.4%for hookworms, 9.3% for Giardia sp. and
4.06% for T. trichiura.

48 |

Muazzem et al. (1961) conducted a study among the children. It was shown that the
prevalence of intestinal parasites was 25.6% for A. lumbricoides, 16.4% for hookworms,
9.3% for G.lamblia and 4.06% for T. trichiura.
Muttalib (1970), studied different parasitic infestation in the stool samples which were
collected from certain private clinic in the city. He reported that the infestation of Ascaris
lumbricoides as 40%, A. duodenale as 8% and T. trichiura as 18% cases. They also
reported that there were monoparasitism in 89% and polyparasitism in 59% cases.
Hla (1970), reported from the survey on school children of Rangpur area, with the use of
concentration method in the diagnosis of ova in stool, where he found 98.6% positive
cases. From his record he reported 87.7% infestation of A. lumbricoides, 87.4% of T.
trichiura, 21.1% of Giardia and 5.7% infestation of E. histolytica.
Tu et al. (1970), from a village of Burma reported parasitism in 90% of their study
population. Out of 90%, 59% were positive for A. lumbricoides, 30% for A. duodenale
and only 3% were positive for Giardia infestation.
Pay (1970), studied on the population of a village of Burma and reported that 70%
infestation was with Ascaris lumbricoides, 15% infestation with E. histolytica and 3%
infestation with Giardia.
From two zones of Malaysia Joe, (1971), reported about the occurrence of parasitic
infestation. From one area, he reported 82% infestation of A. lumbricoides, 84%
infestation of T. trichiura and 50% infestation of Hookworm. In another area, 33%
infestation A. lumbricoides, 31% infestation of T. trichiura and 24% infestation of
hookworm. Obviously there were certain mixed infestations.
Nuruzzaman and Huda (1974) studied in hospitals. The incidence rate was over 70%.
They reported the individual prevalence rate which was 50% for hookworms, 41% for A.
lumbricoides, 13% for E. histolytica, 12.3% for Oxyris sp, and 12% for Giardia sp.
Muttalib (1976) carried out a study. He collected 933 stool samples of newly admitted
students of the University of Dhaka. Those students were from different villages of
Bangladesh. He observed 40.6% infestation of A. lumbricoides, 16.6% infestation of T.
49 |

trichiura, 7.1% infestation of A. duodenale, 32% infestation of S. stercoralis, 11.07%

infestation of E. histolytica and 1.87% infestation of Giardia. He reported the occurrence
of 57.3% parasitism in his study.
Muttalib et al. (1976) carried out another extensive study. Their study was intended to see
the prevalence of intestinal parasite. They studied children from 13 different villages.
Those villages were of different environments. By single stool examination they observed
a gross variation of different infestations from village to village, but high incidence of
Ascaris and Trichuris infestations were common in all the villages. In the observation
they found 92.9% of the rural children of Bangladesh infected with A. lumbricoides,
52.46% with T. trichiura, 9.9% with A. duodenale and 0.1% with H. nana (Muttalib et.
al. 1976). In Bangladesh, the prevalence of intestinal parasites is alarmingly high both in
rural and urban areas as the environment fulfills the criteria for transmission of infection
from host to host.
Ahmed et al. (1979) studied in Mirpur, Nilkhet, Tejgaon and Medical College Staff
Quarter and found the prevalence of A. lumbricoides were always higher than T.
trichiura. It might be due to higher rate of egg production (200,000egg per day) by the
female A. lumbricoides. The eggs with highly resistant shell remain viable for longer
periods compared to other parasites. The chief source of transmission of infections of
these worms is the contaminated soil. Because of promiscuous defecation of slum
children and earthen-floor houses, the infective eggs with embryo might chiefly
transmitted by hand to mouth, raw vegetables, contaminated foods and water (Ahmed,
1986).
In 1979, the prevalence of the geohelminthes was as follows: 23.18% for A. lumbricoides,
10% for T. trichiura, 6.2% for hookworm, 0.28% for E. vermicularis and 10.5% for
multiple infections of different types of geohelminth in the community of the Dhaka city
(Chowdhury, 1979).
Shahidullah (1981) examined 807 stool samples of the patient treated in Modernized
Sadar Hospitals from February to July, 1982. Out of 807 stool samples, 534 (66.18%)
common people were suffering from parasitic infestation. He did not find any remarkable
seasonal variation during the six months.
50 |

Elkins and David (1983) conducted a survey in different social communities in Madras,
among poor community. He found Ascaris 94%, T. trichiura 97%, hookworm 37% and
H. nana in 16% cases.
Khaled (1983), in a laboratory finding with stool specimens of 500 persons of Bangladesh
Rifles, 205 (41%) were positive for helminth, 27 (5.4%) were positive for E. histolytica, 2
(0.4%) were positive for Giardia and 75 (45%) were positive for mixed (both protozoa
and helminthes) infestation. Ascariasis was found to have the highest frequency (35.4%)
next T. trichiura and then others. Hossain et al. (1983) reported that prevalence in urban
hospital patients, aged 5-9 years was 21%. According to Gilmen et al. (1985), prevalence
was higher in 5-10 years old village children. It is apparent that the infection of G.
lamblia was acquired early in life.
Ahmed (1986) reported the factors related to prevalence of ascariasis and malnutrition
were identified as low socio-economic status, illiteracy of mother, poor environmental
sanitation, overcrowding, unhygienic housing and in sanitary disposal of refuses and
human excreta. The prevalence of A. lumbricoides was 61.5%, 17.5% for T. trichiura,
7.41% for Ancylostoma duodenale and 0.7% Enterobius vermicularis were observed.
In 1987, a study was carried out by Begum and Ahmed among the people of three
different restaurant of Dhaka City. Stool examination revealed that, out of 153
respondents 59.80% had one or more type of parasitic infection and E. histolytica positive
cases were 4.59%. Majority of the cases respondents do not have the habit of hand
washing with soap or ash after defecation.
Holland et al. (1988) carried out a cross sectional investigation on intestinal helminthiasis
in relation to the socio-economic condition of Panamanian children. Strong associations
were established between the socio-economic status of the children and infection with
Ascaris lumbricoides, T, trichiura and hookworm infestation. In general the prevalence of
single and multiple helminth infestations was significantly higher in children living in
houses made of wood or bamboo than in those living in houses made of concrete blocks.
The same pattern applied to levels of sanitation. Evidence was also obtained to indicate
that the intensity of intestinal infections was greater in the children from the poorer
environment.
51 |

Solid organ transplant recipients can experience serious disease and death from infection
due to the parasitic roundworm Strongyloides stercoralis. This parasite lives in soil
contaminated with human feces. Domestic dogs and cats may be another reservoir.
Transplant recipients are at highest risk during the first 3 months post transplant (Stone et
al., 1990).
Islam (1990) showed that there is a significant sex variation in the prevalence of intestinal
parasites among the children of Pathokoli School in Dhaka.
In 1993, Lynch et al. worked on a group of urban slum children in Caracas of Venezuela
and demonstrated the relationship that exists between poverty and condition of hygiene.
The occurrence of parasitic infection directly related to the degree of poverty and lack of
sanitary facilities.
Rowsan (1993) studied with under 5 years children. In that study the prevalence of A.
lumbricoides was 41.2%, A. duodenale 2.4%, T. trichiura 4.7% and E. vermicularis was
0.2%. She also showed the occurrence of helminthiasis with relation to different variables
such as age, income of the parents and education status of the mother. Baktaet al. (1993)
stated that the prevalence and intensity of infection increased with age.
In 1994, Ferreira et al. studied among 407 samples of a slum area of Sao Paulo, SouthEastern Brazil. Among all of them, the most prevalent parasites were Ascaris
lumbricoides (23.8%), T. Trichiura (17.2%), only 4.2% harbored G. duodenalis and 1.5%
harbored E. histolytica.
In 1994, Rahman observed that the prevalence and intensity of Soil Transmitted Helminth
(STH) were low in 0-1 age group and above 50 years age groups when compared with
other age groups. The prevalence and intensity of infection in five villages were quite
similar due to the same socio-economic status. The highest intensity as observed for
Ascaris lumbricoides followed by hookworm.
Uqa et al. (1995) examined soil contaminated by parasite eggs in Surabaya Indonesia.
Surveys were carried out on three occassion; July, 1993 (dry season), March, 1994 (rainy
season), and August, 1994 (dry season). Throughout the study, five species of nematode
eggs (Ascaris lumbricoides, Toxocara cati, Trichuris trichiura, Physaloptera sp,
52 |

Capillaria sp), two species of cestode eggs (Hymenolepis diminuta, Spirometra erinacei),
and one species of protozoa oocyst (Isospora felis) were detected. The contamination rate
and number of species found from the soil
In 1997, Khanum et al. studied the prevalence and reported Ascaris lumbricoides 45%, T.
trichiura 23.55% and their mixed infection (15.25%) among 400 children of four slum
areas (Tejgaon, Nilkhat, Mirpur and fourth class employer quarter of Dhaka Medical
College) in Dhaka city. From June 1995 to April 1996, a total of 400 cases were studied.
Ascaris lumbricoides (52.58%) and T. trichiura (27.23%) were more prevalent in male
than in female children. Comparing the highest prevalence of nematode species in
different age groups, it was found that A. lumbricoides showed 60.86% in 2.1-4 years and
T. trichiura showed 32.43% in 4.1-6 years age groups.
Khanum et al. (1998) studied the prevalence of intestinal protozoan parasite, Giardia
intestinalis among the children of three rural areas. A total of 450 samples were examined
during August, 1994 in Mirzapur, October in Bhaluka and November in Kaliganj. Among
150 samples from each area, stool examination showed that 16.7% of them were infected
by Giardia intestinalis from Mirzapur, 3.8% from Bhaluka and 13.9% from Kaliganj.
The wide distribution of cysts and eggs in human and animal populations indicates that
soil is being contaminated with protozoan cysts and geohelminthesthrough fecal
deposition, irrigation and sewage treatment practices (EPA, 1998). One potential route of
transmission is the soil. Soil can be an important vehicle through which cysts and eggs
can travel into water sources (Smith and Rose, 1998).
Khanum et al. (2000) observed a total of 180 samples of 10 types of vegetables to
determine the prevalence of geohelminth ova occurrence. Simple washing method with
water was applied to the vegetables. Three species of geohelminth ova (A. lumbricoides,
T. trichiura and hookworms) were found in the collected vegetables. Maximum
infestations were found in Spinach (28.58%) and Cauliflower (20%), Mint (21.43%) and
Letuce (20%) were moderately infected. Ova of A. lumbricoides showed the highest
(55.55%) prevalence, next the T. trichiura (25.92%).

53 |

Khanum (2000), observed the prevalence of hookworm infection among children in

Mirzapur (30%), Bhaluka (26.67%) and Kaliganj (30%) were more or less similar. Out of
157 positive stool samples, a total of 136 cases were found to develop from eggs to larvae
of which N. americanus was 65.44%, A. duodenale was 33.09% and a mixed infection of
A. duodenale and N. americanus was 1.47%.
Barwick et al. (2003) studied the soil samples of 37 different dairy farms and concluded
that-herd prevalence, vegetation; moisture content and cattle access were significantly
associated with the likelihood of detecting of Giardia Spp. in the soil.
In 2003, Habbari et al. studied the prevalence of ascariasis in Morocco, was found to be
approximately five times higher among children in wastewater impacted regions
compared to control regions. Trichiuris rates did not show a significant difference
between the waste water impacted and the control regions.
In 2004-05, The Bangladesh Academy for Rural Development (BARD), Comilla and
Japan Association of Drainage and Environment (JADE), Japan jointly initiated a pilot
project at four Comprehensive Village Development Programme (CVDP) villages with
the financial assistance of Japan Fund for Global Environment (JFGE). The pilot testing
15 toilets were constructed in four CVDP villages and these toilets are totally different
than any other toilet using by the villagers. Earlier people were not accustomed to use this
type of toilets and they were not familiar with the use of human excreta and urine as
fertilizer for farming. The toilet owners are using human excreta (urine and feces) in their
farm practices and they found positive results. Many people are interested to have the
eco-toilets in their own homestead areas as it increased soil fertility, productivity and
decreased health risks.
In 2005, Rahman et al. showed the overall prevalence of infestation was 24.73%, E.
histolytica 3.95%, G. intestinalis 6.31%, A. lumbricoides 11.84% and T. trichiura 2.63%.
The prevalence of A. lumbricoides was found highest (11.84%) than other parasites
infected.
In 2005, Khanum et al. observed children of 2-12 years old in two different rural areas
(Gazirchat, Savar, Dhaka, Kutumbopur, Comilla) of Bangladesh. It was found that
54 |

prevalence of hookworm infection was higher in Kutumbopur (30%) than Gazirchat

(26.7%). The prevalence was higher in males than females in both the study areas, but it
was significant in Kutumbopur (x = 2.38, P > 0.05).Most of the factors showed positive
association with the hookworm infection. The prevalence of the hookworm infection was
the highest among the age group of 6.1-8 (34.28%) in both the areas and the lowest
among the age group 10.1-12 (7.14%).
Khanum et al. (2007) conducted a study to confirm the relations between the socioeconomic condition and the prevalence of infection of the common intestinal nematodes.
A number of studies regarding the same aim have so far been conducted in slum areas and
other parts of Dhaka city where the socio-economic condition is miserable. This study
highlights on the prevalence of infection of Ascaris lumbricoides among the children (0-5
years) admitted to ICDDR,B hospital during January 2006 to March 2006.Of 2285 stool
samples, 27 were found infected with A. lumbricoides. The overall prevalence of A.
lumbricoides was only 1.18%.
Low socioeconomic condition, poor sanitary practices, inadequate sanitation facilities,
ignorance, poverty, low literacy, congested living and most important is lack of health
education are related to wide prevalence of the intestinal parasitic infection of human in
developing countries but resources available for their control are very limited (Azam et
al., 2007).
Parvin et al. (2010) studied the prevalence of geohelminthes in a lake of Dhaka city and
A. lumbricoides, T. trichiura, S. stercoralis and hookworms were present.
Khanum et al. (2010) found a good number of geohelminth infectious stages in the nail
contents of the street inhabitants of Dhaka city. The prevalence of soil transmitted
nematodes in the left hand of children in the age group 2-4 years had the highest
prevalence (80%).
Khanum et al. (2010) was investigated the prevalence of intestinal parasitic infestation
among the outdoor patients including teacher, student and staff of the Dhaka University
treated at Dhaka University Medical Centre. A total four species of intestinal parasites
were identified, two protozoans (E. histolytica and Giardia intestinalis) and two
55 |

nematodes (A. lumbricoides and T. trichiura). The prevalence of parasitic infestation was
24.73%. The prevalence of E. histolytica was 3.95%, G. intestinalis 6.31%, A.
lumbricoides 11.84% and T. trichiura 2.63%.
Khanum et al. (2011) observed occurrence of Giardia in the effluents of a Pagla Sewage
Treatment Plant (PSTP) in Dhaka. Total 72 raw and treated samples were collected from
PSTP throughout the year 2008. The protozoan parasite Giardia was abundant (2.23
1.44105 cyst/1) in the sewage water dominating the sampling sites - Grit chamber
(44%), Measuring chamber (34%) and Outlet lagoon (38%). A low abundance of Giardia
in the PSTP starting from the first point to the last one indicates the waste water treatment
efficiency for removal of the pathogen. A significant correlation was found for log
(number of Giardia +1) with turbidity

(r = 0.729) and TDS (r = 0.536) at 0.01 level.

Dhar (2011) studied on soil parasites in Haor Basin Area of Bangladesh. He found 20%
soil specimen contaminated with parasites where prevalence of Giardia spp. was 13.3%
and in both Ascaris lumbricoides and Trichuris trichiura prevalence was 7.5%. Children
of weaning food stage were found most vulnerable, both for intestinal infection
(76.10%) and for soil contamination (85.72%). Intestinal infection and yard soil
contamination was higher in non-sanitary latrine user compared to sanitary latrine user
group.
Khanum et al. (2012) again carried out a study to detect protozoan parasites in different
stages of the treatment plant to check its efficacy. Wastewaters were collected from three
points of the Pagla Sewage Treatment Plant (PSTP) of Dhaka, Bangladesh, throughout
the year, 2007-08 at fortnight intervals. Giardia spp, Entamoeba spp, Endolimax nana,
Idoamoeba butschlii and Balantidium coli were detected at different times in different
stages of the treatment plant.
Tavall et al. (2012) conducted a study to determine the prevalence of all parasitic forms
(eggs, larvae, cysts and oocyst) in soil of public places and childrens playgrounds in
Tehran City, Capital of Iran. During 2008 to 2009, 150 soil samples were collected from
various sites and the prevalence of soil parasites was as follows: Toxocara spp. eggs in
sodium nitrate flotation (38.7%) and in sucrose flotation method (33%), Isospora spp. in
sodium nitrate flotation (10.7%) and in sucrose flotation method (18.7%), nematode
56 |

larvae in sodium nitrate flotation (40.7%) and in sucrose flotation method (24%), Eimeria
spp. in sodium nitrate flotation (8.7%) and in sucrose flotation method (24.7%),
Coccidian oocyst and Sarcocystis spp. in sodium nitrate flotation (27%) and in sucrose
flotation method (42%), Dicrocoelium dendriticum in sodium nitrate flotation (2.7%) and
in sucrose flotation method (2%), Geohelminths in sodium nitrate flotation (6.7%) and in
sucrose

flotation

method

(3.4%).

Furthermore,

following

sucrose

flotation

method performance, modified Ziehl-Neelsen staining technique was done and oocysts of
Cryptosporidium spp. was detected in 15 (10%) of soil samples.
2.2.Cattle Parasites
Melo and Bianachin (1977) examined two groups (treated and untreated) of calves and
assessed infestation rate by faecal and post-mortem examination in Brazil. The common
nematodes were Cooperia spp. (71%) and Haemonchus spp. (20%). Faecal eggs counts
showed two peaks, one at the beginning and the other in the middle of the rainy season
(September/October and January/February).
Barboosa et al. (1979) examined faecal samples from 755 dairy cattle in Brazil and
recorded gastro-intestinal helminths in 433 (59%) cattle of which 79% of young cattle
and 46% of adults were infected. The following genera were identified: Haemonchus
spp., Cooperia spp., Trichostrongyloides spp., Oesophagostomum spp., Bunostomum
spp., Strongyloides spp. and Neoascaris spp.
Le Richle et al. (1982) carried out a study in nine farms in Argentina and reported that
Haemonchus spp. and Trichostrongylus axei were the most frequently occurring
helminths.
Assoku (1983) examined faeces of 570 cattle of both sexes and different ages in Ghanna
and showed in high incidence (45.6%) of helminths infection of which 96.92% was the
result of single helminths infection. Seven nematodes, two trematodes, two cestodes and
one protozoan were identified.
Al-Dulaimi et al. (1985) demonstrated that the rate of infection of some parasites such as
Haemonchus contorchus increased towards spring. In contrast, rate of infection of some

57 |

other parasites such as Ostertagia and Nematodirus filicollis were found to be increased
towards autumn.
Omra- Opyene (1985) examined 2227 cattle in Northern Kenya during dry and rainy
seasons and reported that strongyles were serious in young stock especially in post
weaning age and gastro-intestinal parasitism was a predominant problem in rainy season.
Ambrosi et al. (1986) by a faecal examination of 88 cattle in Italy, reported strongylosis,
dicrocoliasis to be wide spread, while fasioliasis, Moniezia infection, strongyloidosis,
trichuriasis and ascaridiasis were less common. Capillariasis occurred rarely and
dicotycaulosis was absent. Vegetation of the area was considered to favour the spread of
endoparasites.
Nakazawa (1986) described a modification of the Wiscosin sugar centrifugal floatation
technique for faecal examination of nematodes eggs and intestinal nematodes were
detected in the abomasums and upper small intestine of 56% of 150 cattle examined in
Hokkaido, Japan. Ostertagia ostertagi was most prevalent (47%) followed by
Mecistocirrus digitatus (29%), Ostertagia punctata (1%), Haemonchus placet (1%) and
Nematodirus helvetinus (1%). In future survey 74% of 231 cattle were found to be
infected with Ostertagia sp. (62.7%), Oesophagastomum sp. (32.2%), Trichurus sp.
(17.3%), Mecistocirrus sp. (3.95), Trichostrongylus sp. (3.5%), Cooperia sp. (1.2%),
Moniezia eggs, Eimeriaocysts and eggs of Toxocara sp. were present in 1.7%, 59.7% and
41.1% of faeces, respectively.
Marnu et al. (1987) conducted a study to observe the monthly and seasonal fluctuation in
abomasal nematodes by examining 191abomasiifrom different abattoirs of Austria during
October, 1980 to September, 1981 and reported that pastured animal showed a higher
intensity of infection than stable animal.
Eslami and Hoseini (1989) investigated faecal materials from 360 cattle and 96 calves
and the following helminths were reported to be present. Nematodirus spp., Trichuris spp.
and Fasciola hepatica.
Besier and Dunsmore (1993) investigated the seasonal pattern of development of H.
contortus eggs to infective larvae in Western Australia. The maximum recoveries
58 |

occurred in late autumn and in late spring when adequate moisture coincided with warm
temperature. Larval development was low and sporadic over the hot and dry summer
period and depressed during winter.
Crespo (1998) surveyed for nematodes during 2 periods in the dry season in 130 cattle.
Faecal examination revealed eggs of Stronyloides spp. in 37 cattle. Following the
necropsy of 20 cattle, Haemonchus placet (32%), Cooperia punctata (52%), and
Oesophagostomum radiatum (60%) were indentified in the abomasums, small intestine
and caecum respectively.
Leo Poldino et al. (1999) collected faecal samples from 486 cattle in Brazil and were
examined for helminths. 69.3% cattle were found positive for helminths eggs. Larva of
seven genera also detected during the study.
Holland et al. (2000) observed faecal samples and identified Toxocara vitulorum,
Cooperia punctata, C. Pectinata, Oesophagostomum radiatum, Trichostrongylus axei,
Haemonchus spp., Fasciola spp., and Paramphistomum.
Keyyu et al. (2003) determined the prevalence and burden of gastrointestinal nematode
infection in indigenous zebu cattle in Tanzania. They found 97.2% animals were infected.
2.3.Inactivation of Pathogens
Galli-Valerio (1915) found that Ascaris eggs could develop to the embryo stage in 50 per
cent sulfuric, hydrochloric, nitric, or acetic acids; saturated solutions of copper sulfate,
ferrous sulfate, and copper acetate; and in 50 per cent commercial formalin or 50 per cent
antifoaming.
Wenyon (1917) found that 100 p.p.m. of chlorine in water had no effect on E. histolytica
cysts, even after several hours' exposure (eosin staining technique).
Martin (1922) found that the eggs of the closely related pig Ascaris will resist freezing
and remain viable in Nebraska soil over the winter.
Augustine et al. (1922) and Cort Cort (1925) worked separately and among their findings
were the following: 1. the infectious larvae were generally limited to a life in the soil of 6
59 |

to 12 weeks under favorable conditions. 2. Larvae died out in 3 weeks at 350 C, in

somewhat over 9 weeks at 270C, in 10 to 12 weeks at 150 C, but in only 1 week at 00 C. 3.
Direct sunlight caused almost complete drying out in 5 days, whereas shady soil was
more favorable to the larvae. 4. Soils covered with vegetation were more favorable for
larval development. 5. Water-soaked soils produced a rapid death rate. 6. Larvae could be
carried about on soil adhering to shoes. 7. The larvae crept up vegetation and decayed
wood as far as moisture films extended, but were rarely found on leaves of green plants.
Mills et al. (1925) concluded that cysts, like bacteria, would not penetrate the stem end of
fresh vegetables to any great extent. The most effective method of killing protozoan cysts
and helminth eggs, as well as pathogenic bacteria, on fruits and vegetables was dipping
into boiling water for 10 sec. The eosin staining method was used for cyst work.
Faust (1924) discussed the relationship between night soil disposal and the propagation of
helminthic disease in China. Foodstuff may become contaminated with eggs or larvae
either through careless handling or the use of night soil as fertilizer. Sterilization of night
soil was most likely to reduce these diseases, but it had to wait upon development of a
cheap potent agent with possible fertilizer property before it could fit into Chinese
economy. The possibility was suggested of composting night soil for a period of time
determined to be sufficient to allow the most resistant types of larvae or eggs to die off.
Hookworm disease in China in relation to vegetable cultivation and viability in soil and
night soil is significant. Experimental turnip crops were fertilized with a mixture of one
part of heavily infested feces and ten or more parts of water. Only a few eggs hatched into
larvae, the high death rate being attributed to rapid surface drying. Hoeing gave greater
larval development. These workers concluded that the use of human feces on vegetables
constituted only a slight source of infestation, but this might become considerable when
fertilization was done in wet weather on shaded plots, or where broken soil surfaces
existed (Cort et al. 1926).
Yoshida (1926) found that mature eggs of A. lumbricoides were still viable after five to
six months under layers of soil in winter. Immature eggs subjected, in addition, to seven
to eight months in the laboratory were still viable. He also found Yoshida (1919) that

60 |

exposure to strong sunlight checked egg development and eventually killed them. Ascaris
eggs are extremely resistant to many chemicals which are ordinarily lethal.
Bolduc (1935) found that cysts of the dysentery amoeba may live in water for months, but
are killed in 5 min. at 650 C and in 10 min. by drying room temperature. To kill the cysts
with 500 ppm of chlorine required 10 min. Hirst (1932) found as many as 250 hookworm
eggs per gram of silt in the main sewers of a Ceylon system. Larvae were infrequent.
Septic tank sludge averaged 28 hookworm eggs per gram in addition to Ascaris eggs. As
many as 330 eggs per liter of sewage had been encountered. In a small treatment plant
with three Imhoff tanks in series, the final effluent contained 30 eggs per liter (15,500 per
liter in the influent), over 98 per cent being removed in the first tank.
Beaver et al. (1949) reported that E. histolytica cysts survive from 6 to 8 days in moist
soil.
In northern Vietnam it used to be common practice to fertilize rice fields with fresh
excreta. As this was a dangerous practice, in 1956 the health authorities started campaigns
to construct double-vault dry toilets. The campaigns were followed by long and persistent
health- education programs. The objective of the new toilet design was to kill pathogens
before the faeces were spread on the fields. A precursor to the Vietnamese system was
developed around 1950 at the Kanagawa Prefectural Public Health Laboratory in Yokohama. (Kodama et al., 1955)
Storey and Phillips (1985) conducted an experiment for observing the survival rate of
parasite eggs throughout the soil profile by using lysimeters suggested that the eggs
of Taenia saginata and Ascaris lumbricoides survive for only a short time when applied
to pasture in sewage sludge. However, a subsequent experiment which followed the
survival of eggs throughout the soil profile demonstrated that some T. saginata eggs
could still be found at 200 days on the soil surface, and that survival increased down the
profile. Rainfall is shown to be able to wash eggs into the soil where they may be
afforded protection from radiation and desiccation; this may have little epidemiological
significance.

61 |

Mackey et al. (1991) described the biochemical and molecular basis of thermal
inactivation of E. coli.
After 1 week in a mesophilic anaerobic digester, 95% of A. suum eggs produced two-cell
larvae into vitro, with 86% progressing to motile larvae while after 5 weeks in the
digester 51% progressed to motile larvae. Between 42% and 49% of eggs stored in a
sludge lagoon for 29 weeks were viable and able to develop motile larvae. In the case of
eggs that were embryonated before treatment, > 98% survived up to 5 weeks in the
digester and was able to develop motile larvae. More than 90% of embryonated eggs
survived for 29 weeks in the sludge lagoon and were able to develop motile larvae
(Johnson et al., 1998).
Differential scanning calorimetry (DSC) is used to evaluate the thermal stability and
reversibility after heat treatment of transitions associated with various cellular
components of Escherichia coli and Lactobacillus plantarum. The reversibility and the
change in the thermal stability of individual transitions are evaluated by a second
temperature scan after preheating in the DSC to various temperatures between 40 and
130C. The viability of bacteria after a heat treatment between 55 and 70C in the DSC is
determined by both plate count and calorimetric data. The fractional viability values
based on calorimetric and plate count data show a linear relationship. Viability loss and
the irreversible change in DSC thermo grams of pretreated whole cells are highly
correlated between 55 and 70C. Comparison of DSC scans for isolated ribosomes shows
that the thermal stability of E. coli ribosomes is greater than that of L. plantarum
ribosomes, consistent with the greater thermal tolerance of E. coli observed from viability
loss and DSC scans of whole cells (Lee and Kaletun, 2002).
Higher UV fluencies are required to obtain the same level of inactivation. Hence, for
bacteria and spores, a correction factor of 2 and 4 was included in the MIC calculation,
respectively, whereas some wastewater studies suggest that a correction of a factor of 7 is
needed under these conditions. For phages and viruses this phenomenon appears to be of
little significance and for protozoan (oo) cysts this aspect needs further investigation. For
UV systems that are primarily dedicated to inactivate the more sensitive pathogens
(Cryptosporidium, Giardia, and pathogenic bacteria), additional model organisms are
needed to serve as biodosimeter. (Hijnen et al. 2006)
62 |

B. Jimenez (2007) addresses (1) characteristics of helminth ova and differences with
microorganisms; (2) the most frequent helminth ova genus found in wastewater; (3)
helminth ova content in developed and developing countries wastewater; (4) reasons why
conventional disinfection methods cannot be applied; (5) main removal mechanisms; and
(6) processes that in practice have effectively removed or inactivated helminth ova.
Source separation and reuse of human urine can decrease the environmental pollution of
recipient waters and reduce the need for artificial mineral fertilizers. However, the reuse
of urine introduces another pathogen transmission route that needs to be managed. The
inactivation of enteric pathogens and model organisms by urine storage was studied by
Nordin et al (2008). They recommended storage time for urine of 6 months at 20 C or
higher is safe for unrestricted use and could probably be shortened, especially for
undiluted urine.
Maya et al. (2012) investigated to inactivate six species of larval and non-larval helminth
eggs of medical importance in developing countries under controlled conditions of
temperature, pH, dryness and contact time and showed considerable differences in
inactivation conditions among helminth eggs and a high level of resistance was confirmed
for the eggs of Ascaris lumbricoides and Ascaris suum. The appropriate conditions for
inactivation of all types of eggs were found by applying combinations of pH, temperature
and dryness. At 45 C it was possible to inactivate all species with a pH of 5.3 and 90%
dryness within 6 days. If alkalization was applied, a pH of 12.7 was sufficient over 19
days at the same conditions of dryness and temperature. From these results it is proposed
that both Ascaris spp. and Taenia solium may be used as indicators of biological
contamination in wastewater and sludge.

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MATERIALS & METHODS

3.1.Period of study
The study was conducted from 1stMay, 2014 to 31st May, 2015.
3.2.Study area
For the present study, samples were collected from different districts of Bangladesh. Cow
dung were collected from Dhaka, Mymensing and Gopalgonj, and the pit samples were
collected from Dhaka (Dohar, Hajaribag and Keranigonj) and Gopalgonj.
3.3.Consents
Approval for this study was obtained from the Parasitology branch, Department of
Zoology, at the University of Dhaka and the Environmental Microbiology Laboratory of
ICDDR,B provided facilities, laboratory space and equipment.
3.4.Sample Size
A total of 40 cow-dung samples and 40 pit faecal sludge were examined and 1 sample
with the highest contamination from each category were then heated for inactivation of
bacteria and parasites.
3.5.Collection and transport of samples
Approximately 200 to 250 grams of fecal sludge and fresh cow dung werecollected and
stored in sterile plastic containers. These were kept in an insulated foam box with ice
packs and transported to the laboratory maintaining temperature around 4-8 C. Collected
samples were processed within 24 hours to avoid disintegration.
Sample collection procedure:

Samples were collected in a clean, leak proof, transparent alcohol (70%) washed
container. No antiseptic was used.

No de-worming medication was given before taking these samples.

Different sterile spoon were used for collection of samples.

Identification number labels were put on the container.

64 |

Collection date, time and place were put on the containers.

In case of cow-dung, age groups (calves and old) were written on the containers.

In case of pit soil, sample types (dry, moist or liquid) were written on the
containers.

The approach followed to develop a standard Inactivation Temperature:

Sample
Collection
Stored in ice box and transported to lab within 24hr

Sample
Processing

Microscopic observation

50C

55C

15 mins

60C

30 mins

65C

70C

75C

80C

60 mins

Figure 1: Parasitic and bacterial inactivation scheme

65 |

The current investigation was a laboratory based inactivation of parasites and bacteria
aiming to produce bio-fertilizer from night soil/ faecal sludge and cow dung. A series of
time-temperature framework was developed to assess the optimum temperature and time
required to kill a particular bacterium or parasite.

3.6. Sample analysis procedures

Salt floatation, formol-ether concentration and centrifugal flotation/sedimentation
techniques are those most frequently used to detect intestinal parasites (Cheesbrough
(2004).Salt floatation, centrifugal floatation and formol-ether concentration techniques
were applied in the current study.
10 grams sample was taken in 4 oz. Temperature was increased at a rate of 5C from
50C to 80C. 3 bottles containing a particular sample were kept at each temperature. All
three samples were removed subsequently after exposing a fixed temperature for 15
minutes, 30 minutes and 60 minutes and observed under microscope..
Threedifferent quantitative techniques were initially tested for faecal sludge and cow
dung. Among them the best one was used for quantitative analysis of parasitic
contamination of the samples.

Methods used (For Parasites):

Direct Smear Technique

Formol-Ether Concentration Technique

Salt Floatation Technique
Floatation Concentration Technique
Centrifugal Floatation Technique

Methods used (For Bacteria):

Phenotypic examination
Drop plate technique

Biochemical tests
66 |

KIA
MIU
Citrate
Catalase
Oxidase
Glucose
Sucrose
Lysine
Arginine
Ornithine

Antimicrobial susceptibility pattern test

3.6.1. Instrument and chemicals required

Falcon tube (15ml)

Light microscope

Centrifugal machine

Vortex machine

Slide

Coverslip and

Pipette

Chemicals:

Formalin

Dyethayl ether

ZnSO4 solution (specific gravity 1.2)

Normal saline (0.85%)

3.6.2. Direct Smear Technique

Microscopic examination was done by Olympus (CH) light microscope. In microscopic
examination samples were taken into slide and examined whether it is positive or not. In
case of solid samples, were diluted into normal saline and taken into slide and examined
whether it is positive or not. Microscopic examination must be performed on unpreserved
specimens.
67 |

(a): Steps of Microscopy (Direct Smear Technique):

Take a small amount of sample on a glass slide.

Put a drop of normal saline (0.85%) on the slide.

Mix well and the mixture must provide a uniform suspension under a 22 by 22mm
cover slip (be careful the suspension must not be thick).

The entire cover slip must be systematically examined with low power objectives
(10X) and low intensity light.

Any suspicious structure then is examined with high dry objectives (40X).

3.6.3. Formol-Ether Concentration Technique

If the number of parasites in the specimens is low, examination of a direct wet mount may
not detect them, hence the samples should be concentrated. Eggs, cysts and larvae are
recovered after concentration procedures whereas trophozoites get destroyed during the
procedure. Formol-Ether Concentration technique is the most commonly used and
recommended by WHO (2006),

Figure 2: Formol-Ether concentration

technique (after centrifugation)

68 |

(a):Procedure
The procedure was used according to Cheesbrough (2004) with some modifications
described below;
1. 1 ml /1 g of sample was taken into a 15 ml falcon tube.
2. Mix thoroughly with the help of a vortex machine.
3. Strain through a tea strainer into a small beaker (50ml).
4. Pour the strained suspension into a falcon tube.
5. Centrifuged for 2 minutes at 3000 rpm.
6. Discardthe supernatant.
7. The sediment was re-dissolved in 7 ml of Formol-Water.
8. 3 ml of ether was added and mixed well by vortex machine.
9. The tube was then centrifuged at 3000 rpm for 5 minutes.
10. Four layers were become visible; the top layer consists of ether, second was a plug of
debris, third was a clear layer of formalin and the fourth was the sediment containing
parasitic stages.
11. The top three layers were discarded leaving a small amount of formal-water for
suspension of the sediment.
12. With a pipette, the sediment was removed and transferred to an eppendorf tube.
13. The preparation was stored at 4C until microscopic examination.
(a):Calculation
The number of eggs or cysts per gram of soil was calculated by the drop count from the
following equation:
N= (SS P) / TD
Here, SS= Total drops of the sub sample
P = Number of parasite (egg or cyst) observed
TD= Total number of drops examined

69 |

3.6.4. Floatation Technique:

The specific gravity of salt solution helps to float the eggs, larvae and cysts which have
lower specific gravity.

Table 2: Specific Gravity of Parasite Structures Recovered by Flotation Methods(M.

W. Dryden et al., 2005)
Parasite Species

Specific Gravity

Ancylostoma sp.

1.06

Toxocara sp.

1.09-1.10

Trichuris vulpis

1.15

Taenia sp.

1.22

Isospora sp./Toxoplasma gondii

1.11

Giardia sp.

1.05

Table 3: Specific gravity of commonly recommended flotation solutions (M. W.

Dryden et al., 2005)

Solutions

Specific gravity

Composition (for 1litre)

Zinc sulfate

1.18-1.2

Sodium nitrate saturated

1.18-1.2

Sodium chloride saturated

1.18-1.2

Magnesium sulfate

1.2

331 g ZnSO4 dissolve into1,000 ml warm

tap water
338 g NaNO3 dissolve into 1,000 ml tap
water
350 g NaCl dissolve into 1,000 ml tap
water
450 g MgSO4 dissolve into 1,000 ml tap
water

saturated

70 |

(a):Floatation concentration technique(Cheesbrough, 2004)

1. Measure 1g of sample into a 15 ml falcon tube.

2. About one quarter fill the tube with the floatation solution (ZnSO4, specific gravity
1.2).
3. Vortex and mix well.
4. Fill the tube with floatation solution and mix well.
5. Stand the tube in a completely vertical position in a rack.
6. Using micropipette add further solution to ensure that tube is filled to the brim.
7. Carefully place cover glass on top of the tube, avoiding trapping any air bubbles.
8. Leave undisturbed for 30-40 minutes to give time for the cysts, larvae and egg to
float.
9. Carefully lift the cover glass from the tube by a straight pull upward
10. Place the cover glass face downwards on a slide.
11. Examine microscopically by 10X and 40X magnifications.
Note: After 60 minutes eggs will sink.
(a): Calculation:
Total number of cysts, larva and eggs found was the count in 1g of sample.

(b): Centrifugal floatation technique (Cheesbrough, 2004)

1. To concentrate the helminth ova from the fecal sludge, one gram of the collected
sample is suspended into 5 ml sterile normal saline and homogenized using a
vortex machine.
2. The large particulates are removed by filtering the homogenized mixture using a
160 um diameter sieved strainer.
3. The filtrate isthen centrifuged for 3.5 minutes at 1800 rpm.
4. The supernatant is discarded leaving a small amount of fluid just above the
sediment.
5. 5 ml salt (ZnSO4, MgSO4 and NaNO3) solution (S.G-1.20) is added to re-suspend
the sediment.
71 |

6. The suspension is then homogenized again using vortex machine.

7. Homogenized sample is centrifuged for 1.5 minutes at 1500 rpm.
8. The flotation procedure yields a surface layer that contains parasite eggs.
9.

1ml of surface layer is transferred into an eppendorf tube.

Examine under light microscope.

(a): Calculation
The amount of ova, larvae and cyst retrieved is expressed in ova per gram of dry solids.
The percent total solids were determined by drying 10 g of the remaining sample in an
oven until no change in weight is observed. The weight of the sample after drying was
recorded.
After calculating the percentage of total solids, the amounts of ova per gram of dry solids
were computed using the equation below:
/ =

() ()
() () ()

Where:
NO= no. of ova
FV= final volume in ml
TV= tested volume in ml
SP= sample processed in ml or gm
TS= % total solids

The percentage of total solids was computed using the formula below:
% =

Where:
W= weight of stool after drying in an oven
W = weight of stool before drying in an oven

72 |

3.7. Identification of Parasites

Preparations of the concentrated samples were examined for different parasitic stages
under the electronic compound microscope. Eggs, larvae and cysts as observed under the
microscope were identified by following the descriptions and pictures published by
Chatterjee (1980), Schimidth and Roberts (1989), Cheesbrough (2004) and Hambridge
(2004). Bench Aids of World Health Organization(WHO) and Center for Disease Control
and Prevention (CDC).
3.8.Viability testing
1. Add a drop of eosin methylene blue for determination of viability.
2. Ependorfwas kept 3-5minutes and homogenized using a vortex machine in case of
modified floatation technique.
3. In case of formol-ether sedimentation technique dye was used after discarding
upper three layers.
4. Examined microscopically to enumerate the viable and non-viableova. Viableova,
larvae and cyst weremade confirm since they failed to retain the dye.
Note:If the dye penetrates the egg through rupturing fragile or vulnerable egg shell
then it was considered as nonviable. The physical condition of egg shell demonstrates
its ability to infection or further propagation.
3.9. Bacteriological Testing
In the present study, four different types of bacteria were investigated to reveal their
presence in the feacal sludge. They are namely total coliform, feacalcoliforms, E. coli and
faecal streptococci. These all are under the coliform group and their presence in a sample
indicates the presence of other pathogens in that particular sample. In terms of sludge
sample, a commonly used method to enumerate bacterial colonies is the serial dilution
method because investigating a raw sample usually gives higher bacterial load
implicating the complication in counting the colony forming units. Hence, serial dilution
method was adopted in the present study.

73 |

3.9.1. Serial Dilutions

All three bacterial plate count methods described in lab require to serially dilutingthe
samples until have 30-300 colony forming units (CFU) on the plate. Plates with more
than 300 CFU are very difficult to count. Plates with less than 30 CFU are not statistically
reliable.

(a): Materials needed for serial dilutions:

Sterile Normal saline

15 ml falcon tube

1.5 ml eppendorf

Micropipettes

Sterile tips

(b): Procedure
The tricky part of doing serial dilutions is determining the correct dilution to get 30300 CFUs per plate.
1. Label a falcon tube with 10-1 and 3 eppendorfs as 10-2, 10-3 and 10-4.
2. Weight 1g of sample into a falcon tube.
3. Using a sterile 1mL pipette, add 9mL of sterile normal saline into the falcon tube.
4. Vortex well to mix.
5. Using a sterile micropipette, aliquot 900L of sterile normal saline into each
eppendorf.
6. Using a 100L micropipette and sterile tip, transfer 100L from the falcon tube of 10 1

dilution into 10-2 labeled eppendorf.

7. Cap the tube. Vortex to mix well.

8. Using a new sterile tip, transfer 100L from the eppendorf of 10-2 dilution into 10-3
labeled eppendorf.
9. Repeat the tube mixing procedure.
10. Using a new sterile tip, transfer 100L from the eppendorf of 10-3 dilution into 10-4
labeled eppendorf.
11. Repeat the tube mixing procedure to avail lower dilution as required.
74 |

(c): Plating
Since we do not know which of your dilutions will yield countable results, I plate from
each dilution.
1. 1. Dry the plates with a drier to leave the moisture from agar.
2. Label particular plates for particular bacteria as 10-1, 10-2, 10-3 and 10-4.
3. Drop 25l from each dilution into respective labels of dilution, using sterile tips
and micropipette.
4. Vortex well before taking samples to drop.

Do NOT invert the plates until all the liquid has absorbed into the surface of the
agar.

Incubate the plates for the appropriate time and temperature.

Figure 3: A schematic view of dilution and plating

(d): Counting:

The typical colonies of a particular bacteriumare counted after the incubation and the
CFU for all four dilutions were recorded.

75 |

(e): Calculations:

The original number of colony forming unit (CFU) per ml of solution was calculated by
using following formula.

/ =

()

3.9.2. Fecal Coliforms Testing

Thermotolerant total coliforms bacteria (Escherichia coli, Citrobacter freundii and
Klebsiella pneumoniae) are capable of growth with acid and gas production at 44.5 0.2
C. The determination of feecal coliforms bacteria in any sample suggests that the sample
has been contaminated with faeces (Technical Information SeriesBooklet No. 13).
3.9.3. Fecal Streptococci Testing
Streptococci from the feces of humans and warm-blooded animals have been referred to as
enterococci, fecal streptococci, and group D streptococci. These bacteria were recognized
as being indicative of dangerous pollution and characteristic of sewage and animal fecal
wastes (Technical Information SeriesBooklet No. 13).

76 |

Testing Procedure of Indicator Bacteria:

Sample collection

Bacterial isolation:
Drop plate (after dilution)

mTEC agar

Drop plate (after dilution)

SB agar

Drop plate (after dilution)

Purple colony(E.coli suspected)

brick colony(Faecal streptococcisuspected)

mFCagar

blue colony(coliforms)

Phenotypic confirmation of E.coli&faecal streptococci

Colony culture confirmation:

Mackonkey agar (Gummy pink colony

EA Plate (Black with mass glow colony
streptococci)

E.coli)
Faecal

Biochemical confirmation: KIA,

MIU, Citrate, Catalase, Oxidase,
Glucose, Sucrose, Lysine,
Arginine, Ornithine

Antimicrobial
susceptibility
testing
Figure 4: Diagram showing the design of the experiments

77 |

All the samples were tested for fecal coliforms and faecal streptococci by drop plate
method. The procedures were as follow:
1. In a test tube containing 9 ml of Normal Saline 1 gm of sample were mixed using a
vortex mixture. This is 10 times dilution (10-1dialution).
2. From the test tube 100 l of sample was taken into an eppendorf which has 900l
sterile normal saline. This will be 100 times dilution (10-2 dilution).
3. Vortex thoroughly to mix well.
4. From the eppndorf with 10-2 dilution take 100 l of sample into another eppendorf
which has sterile normal saline. This is 1000 times dilution (10-3 dilution).
5. Again do the same for 10000 times (10-4) and 100000 times (10-5) dilution.
6. From each dilution take 25 l with the help of a micro pipette and drop into mFC
Agar medium for faecal coliforms and total coliforms testing and for faecal
streptococci (Faecal streptococci) testing use SBA plates. For E. colitesting use
mTEC agar plates.
7. Incubate mFC Agar plates for faecal coliforms for 18-22 hours at 44.5 0.2 C and
mFC Agar plates for total coliforms at 37 C for 18-22 hours.
8. For thermotolarentE. coli, incubate mTEC ager plates at 37 C for 2 hours and then at
44.5 0.2 C for 18-22 hours.
9. After incubation, specific colonies were visible showing distinct colouration which
was considered as positive.
10. All the positive colonies were counted to determine the magnitude of contamination.
The indicator bacteria test results revealed that all the collected soil samples were highly
contaminated with human feces as well as animal feces. This result of indicator bacteria
also reveals that other pathogens including parasites may be present in the collected
samples.
3.9.4. Isolation of E. coli
Samples are at first diluted and each diluted samples were droppedon mTEC agar plate
(Difco, MD, USA). Plates were incubated at 37C for the initial 2 hours, and then at
44.5C for 1824 hours. Purple colored colonies were selected and suspected as E.coliand
for further confirmation; suspected colonies were sub cultured on MacConkey agar plate
and incubated at 37C for 18-24h. From each plate, at least 5 pink colonies with typical E.
colicolony morphology were taken and again sub cultured on MacConkey plates to obtain
78 |

pure culture. Isolates identified on the culture plates were further tested by using short
biochemical tests according to standard methods described in manual for laboratory
investigation of acute enteric infections (WHO, 1999). The following biochemical tests
were performed: Kligers Iron Agar (KIA) test, Motility Indole Urease (MIU), Citrate
utilization test, Catalase and Oxidase test. In case of E. coli, butt and slant were turned into
yellow color with formation of gas in KIA test. Isolates those were positive for citrate,
indole, catalase tests and negative for urease, oxidase were considered to be E. coliand
were stored at -70C in tryptic soy broth supplemented with 30% (vol/vol) glycerol. In
each biochemical tests, E. coli ATCC 25922 and Klebsiella strain ATCC 33495 were used
as positive and negative controls, respectively.
3.9.5. Isolation of Enterococci
Samples are at first diluted and each diluted samples were plated on Slanetz and Bartley
Agar (SBA) (Slanetz and Bartley, 1957) platesby the method of drop plate. Plates were
incubated at 37C for 48 hours. Dark brown colored colonies were selected and suspected
as faecal streptococci and for further confirmation suspected colonies were sub cultured on
faecal streptococci agar plate and incubated at 44C for 2 hours. From each plate, at least 5
colonies with typical faecal streptococci colony morphology were taken and again sub
cultured on SB agar plates to obtain pure culture. Isolates identified on the culture plates
were further tested by using short biochemical tests according to standard methods
described in manual for laboratory investigation of acute enteric infections (WHO, 1999).
The following biochemical tests were performed: KIA, MIU, Citrate, Catalase, Oxidase,
Glucose, Sucrose, Lysine, Arginine, Ornithine.
3.9.6. Antimicrobial susceptibility testing
We followed standard Kirby-Bauer disk diffusion method to determine the antimicrobial
susceptibility patterns of the E. coliand enterococciisolates. We interpreted the
susceptibility results for 10 antimicrobial agents according to the guidelines recommended
by the Clinical and Laboratory Standards Institute (1997, 1999) for E.coliand enterococci.
These antimicrobial agents were chosen on the basis of their importance in treating human
or animalinfections and their use as feed additives to promote growth in animals and on
the basis of their ability to provide diversity for representation of different antimicrobial
agent classes (Krumperman P H, 1983). Commercially available discs (Oxoid Limited,
79 |

Hampshire, England) were used for the test. The antibiotics used in this study (with their
potency) were Ampicillin (10 g), Cefixime (5 g), Ceftriaxone(30 g),Ciprofloxacin (5
g), Nalidixic Acid (30 g), Sulfamethoxazole/ Trimethoprime(25 g), Gentamycin (10
g), Chloramphenicol (30 g), Tetracycline (30 g) and PolymixinB (300g) for E.coli.
Ampicillin (10 g), Ciprofloxacin (5 g), PolymixinB (300g), Sulfamethoxazole/
Trimethoprime (25 g), Gentamycin (10 g), Meropenem (10 g), Erythromycin (15 g),
Penicillin G (10 g), Tetracycline (30 g) and Streptomycin (10 g) were used for faecal
streptococci.
Following the standard method of inoculation, an inoculating needle was touched to a
single well-isolated colony, and inoculated into 2 ml of Mueller Hinton broth. The broth
culture was then allowed to incubate at 37C for 4 h to obtain the young culture. The
turbidity of the actively growing broth cultures was then adjusted to a MacFarland 0.5
standard (3108 CFU/ml). To inoculate the agar medium, a sterile non-toxic cotton swab
was dipped into the standardized suspension. Excess broth was purged by pressing and
rotating the swab firmly against the inside wall of the tube above the fluid level. The swab
was then streaked evenly in three directions over the entire surface of the agar plates to
obtain a uniform inoculum. A final sweep was made of the agar rim with the cotton swab.
This plate was then allowed to dry for 3 to 5 minutes before the discs were applied.
Antibiotic discs were then applied to the surface of the inoculated plates using a sterile
forceps.
Usually three different antibiotic discs were place in a single plate. All discs were gently
pressed down on the agar with sterile forceps to ensure complete contact with agar surface.
Within 15 minutes of application of the discs, the plates were inverted and placed in an
incubator at 37C. After 16 to 18 h of incubation, the plates were examined, and the
diameters (in millimeters) of the clear zones of growth inhibition or no inhibition pattern
around a particularantimicrobial discs, including the 6-mm disc diameter, were measured
(Clinical and Laboratory Standards Institute, 1997, 1999).
The zone diameter for individual antimicrobial agents was then translated into susceptible,
intermediate or resistant categories according to the interpretation guideline provided by
CLSI (CLSI, 2010 [CLSI (2009) Performance Standards for Antimicrobial Disk
Susceptibility Test; Approved Standard. Wayne, PA: Clinical and Laboratory Standard
Institute.].
80 |

Table 4: Concentrations and diffusion zone breakpoints for resistance against

antimicrobial agents tested for E. coli (CLSI 2010).

Antimicrobial
agent

Drug
code

Diffusion zone breakpoint (mm)

Disc drug
concentration
(g)
Resistant Intermediate Susceptible

Ampicillin

AMP-10

10

13

14-17

17

Cefixime

CFM-5

15

16-18

19

Ceftriaxone

CRO-30

30

13

14-20

21

Ciprofloxacin

CIP-5

15

16-20

21

Chloramphenicol

C-30

30

12

13-17

18

Gentamycin

CN-10

10

12

13-14

15

Nalidixic Acid

NA-30

30

13

14-18

19

Tetracycline

TE-30

30

11

12-14

15

Trimethoprim/
Sulfamethoxazole SXT-25

25

10

11-15

16

Polymixin B

300U

12

N/A

17

PB

81 |

Table 5: Concentrations and diffusion zone breakpoints for resistance against

antimicrobial agents tested faecal streptococci (CLSI 2010).

Antimicrobial
agent

Drug
code

Disc drug
concentration
(g)

Polymixin B

PB

300

Tetracycline

TE-30

Trimethoprim/
Sulfamethoxazole

Diffusion zone breakpoint (mm)

Resistant

Intermediate

Susceptible

30

14

15-18

19

SXT-25

25

50

80

Erythromycin

E-15

15

13

14-22

23

Penicillin G

PG-10

10

14

16

Gentamycin

CN-10

10

0.5

0.58-0.75

0.83

Ampicillin

AMP-10

10

16

17

Meropenem

MEM-10

10

Streptomycin

S-10

10

0.2

0.23-0.30

0.33

Ciprofloxacin

CIP-5

15

16-20

21

3.10. Data analysis

All the findings were recorded in the respective data sheet and transferred into the
computer. In the computer the data were analyzed using Microsoft Excel 2007 and SPSS
v20.0 software.
3.11. Ecological terms
All the ecological terms considered in this study were according to Margolis et al., 1982
and Bush et al., 1997.
Prevalence =

Number of infected sample

100
Total number of infected hosts

Intensity =

Total number of parasites

Total number of infected hosts
82 |

3.12. Statistical Formulas

(a). Standard deviation:
( )2
=

Here,
= Standarddeviation
x = Theindividualvalues
x = Thearithmaticmean
n = Numberofobservation
(b). Co-efficient of correlation:
=

( ) ( )
( )2 ( )2

Here,
= Co efficient of correlation
= The values of the first variable
= The values of the second variable
= The mean of x variable
= The mean of y variable
The values of r by itself are not always sufficient to reveal the significance of r
between a given pair of variables.

83 |

(c). Anova (F test):

=

Where,
F = Anova Coefficient
MST = Mean sum of squares due to treatment
MSE = Mean sum of squares due to error.
Formula for MST is given below
=

)2
= (
Where,
SST = Sum of squares due to treatment
p = Total number of populations
n = Total number of samples in a population.

Formula for MSE is given below:

=

= ( )

Where,
SSE = Sum of squares due to error
S = Standard deviation of the samples
N = Total number of observations.

84 |

RESULTS & OBSERVATIONS

The sampling sites were randomly visited to select possible sampling locations. A total 80
samples (40 cow dung and 40 pit soil) were collected from the study sites. Cow dung
samples were collected from Dohar, Gopalgonj, Keranigonj and Mymensing. Pit soil
samples were collected from Dohar, Gopalgonj, Keranigonj and Hajaribag, 10 samples
from each site.
4.1.Bacteriological Observations
Initially, thermotolarent E.coli, faecal streptococci (faecal streptococci) faecal coliforms
and total coliforms were counted from each sample. Out of 40 cow dung samples 39 were
positive forfaecal streptococci where all were positive for E.coli, faecal coliforms and total
coliforms. A total of 6 samples were negative for all examined bacteria.
4.2.Bacteria examined
1. Escherichia coli
2. Faecal streptococci
3. Faecal coliforms
4. Total coliforms
Table 6: Bacteriological counts of cow dung samples from Dohar
Sample ID

CD001
CD002
CD003
CD004
CD005
CD006
CD007
CD008
CD009
CD010

Faecal
streptococci
1.44105
1.04105
6.40104
1.08105
1.16105
1.20104
1.60104
1.20104
1.20104
2.40104

Bacteriological counts CFU/g

E.coli
Faecal
coliforms
7.20106
1.36107
7.20106
2.20107
2.00106
3.60106
7
1.5210
2.60107
1.44107
2.24107
5
7.2010
8.00106
8.40106
1.84107
6
1.6010
3.20106
6.80107
4.80107
4.40106
1.84107

Total coliforms
1.90107
3.10107
8.80106
2.68107
2.60107
1.60106
1.90107
3.60106
8.40107
2.24107

All the samples of Dohar were contaminated with faecal streptococci, E.coli, faecal
coliforms and total coliforms.

85 |

6.781759442

7.14166023

7.18714095

Faecal coliforms

Total coliforms

Avarage at log10

8
4.590133251
6
4
2
0

Faecal streptococci

E.coli

Bacteria

Figure 5: log10 of average of bacteria in cow dung from Dohar

The log10 value of average of total coliforms is the highest among all (7.19), faecal
coliforms showed the second highest (7.14), E. coli third highest (6.78) and faecal
streptococcigave the lowest (4.59) average at Dohar.
Table 7: Bacteriological counts of cow dung samples from Gopalgonj
Sample ID

CD011
CD012
CD013
CD014
CD015
CD016
CD017
CD018
CD019
CD020

Bacteriological counts CFU/g

Faecal
E.coli
streptococci
1.20104
4.40106
4.00103
3.60106
1.60104
1.20106
8.00104
1.32107
2.80104
8.00106
8.00106
1.20107
2.80104
2.08106
5.20104
2.48107
4.40104
2.20107
4.00103
1.56106

Faecal
coliforms
1.12107
4.40106
2.80106
1.52107
2.00107
1.601-07
1.60106
1.52107
2.40107
1.80106

Total coliforms
1.20107
5.20106
4.40106
2.00107
2.40107
1.20107
4.40106
2.00107
2.60107
2.08106

All the samples of Dohar were contaminated with faecal streptococci, E. coli, faecal
coliforms and total coliforms.

86 |

6.884826961

6.762984388

6.98765791

Avarage at log10

6
5

4.554737327

4
3
2
1
0

Faecal streptococci

E.coli

Faecal coliforms

Total coliforms

Bacteria

Figure 6: log10 of average of bacteria in cow dung from Gopalgonj

The log10 value of average of total coliforms is the highest (6.99), following faecal
coliforms (6.88), E. coli (6.76) and faecal streptococci showed the lowest (4.55) average at
Gopalgonj.
Table 8: Bacteriological counts of cow dung samples from Keranigonj
Sample ID

CD021
CD022
CD023
CD024
CD025
CD026
CD027
CD028
CD029
CD030

Faecal
streptococci
4.00103
9.60104
0
3.20104
2.40104
4.40104
1.50105
8.00105
4.00103
1.16105

Bacteriological counts CFU/g

E.coli
Faecal
coliforms
3.20107
3.60107
4.36106
2.80106
4.80106
5.20106
5
2.4010
1.60107
3.64107
3.20107
2.68107
2.30107
2.12107
2.56107
3.16107
3.28107
9.2105
1.12106
1.72107
4.00107

Total coliforms
3.68107
3.00106
6.00106
1.92107
3.16107
2.32107
2.44107
3.20107
1.60106
4.80107

All samples are contaminated with E. coli, faecal coliforms and total coliforms. All but
one was negative for faecal streptococci.

87 |

Avarage at log10

7.13658535

6.922066434

7.174749764

4.1858844

4
2
0
Faecal streptococci

E.coli

Faecal coliforms

Total coliforms

Bacteria

Figure 7: log10 of average of bacteria in cow dung from Keranigonj

Fig. 2: log10 of average of bacteria in cow dung from Keranigonj
The log10 value of average of total coliforms is highest (7.14), following faecal coliforms
(7.13), E. coli (6.92) and faecal streptococcigave the lowest (4.18) average at Keranigonj.
Table 9: Bacteriological counts of cow dung samples from Mymensing
Sample ID

CD031

Bacterial counts CFU/g

Faecal streptococci
E.coli
Faecal
coliforms
9.00105
9.54107
1.40108

1.92108

CD032

1.02105

1.32107

2.0107

2.71107

CD033

8.0104

5.71106

8.2106

1.02107

CD034

5.2104

1.56107

8.0107

8.28107

CD035

4.0105

7.8107

1.2108

3.75108

CD036

8.72105

3.24107

5.2107

5.78107

CD037

2.0106

1.02108

2.8108

2.88108

CD038

1.85104

6.2105

4.2105

5.8105

CD039

2.78104

2.0107

5.32107

5.56107

CD040

4.4104

2.56106

8.52106

8.80106

Total coliforms

All samples are contaminated with E.coli, faecal coliforms, total coliforms and faecal
streptococci.

88 |

7.589123808

7.478600501

7.196278445

Avarage at log10

8
5.178021168
6
4
2
0
Faecal streptococci

E.coli

Faecal coliforms

Total coliforms

Bacteria

Figure 8: log10 of average of bacteria in cow dung from Mymensing

The log10 value of average of total coliforms is highest (7.58), following faecal coliforms
(7.47), E. coli (7.19) and faecal streptococcishowed the lowest (5.18) average at
Mymensing.

7.20
6.92
6.78
6.76

Avarage at log10

7
6
5
4

7.48
7.14 7.14
6.88

7.59
7.197.17
6.99

5.18
4.55

Avarage Dohar

4.59

Avarage Gopalgonj

4.19

Avarage Keranigonj
3

Avarage Mymensing

2
1
0

Faecal streptococci

E.coli

Faecal coliform

Total coliforms

Bacteria

Figure 9: log10 of average of bacteria in cow dung from study sites

The log10 value of average of total coliforms and faecal streptococci is the highest in
Mymensing (7.59) followed by Gopalgonj (7.19), Keranigonj (7.17) and Dohar (6.99).
Faecal coliforms showed the highest value in Mymensing (7, 48) followed by Gopalgonj

89 |

(7.14), Keranigonj (7.4) and Dohar (6.88). E. coligave the highest value in Mymensing
(7.20) followed by Keranigonj (6.92), Gopalgonj (6.78), and Dohar (6.76). Faecal
streptococcialso showed the highest average in Mymensing (5.18) followed by Gopalgonj
(4.59), Dohar (4.55) and Keranigonj (4.19), respectively.
Table 10: Bacteriological counts of pit soil samples from Dohar
Sample ID

PS001
PS002
PS003
PS004
PS005
PS006
PS007
PS008
PS009
PS010

Bacterial counts CFU/g

Faecal
E.coli
streptococci
1.6103
0
3
6.410
2.0103
3
9.210
5.8105
2.6104
8.0103
3
4.410
4.8104
3.6105
1.8105
3
4.010
8.0105
4.8105
1.56105
7.28105
2.0104
6
3.210
6.4104

Faecal
coliforms
0
2.8103
1.08103
1.76105
2.28105
1.3105
2.4104
1.9105
3.6104
2.0104

Total coliforms
0
8.0103
4.2106
3.20106
3.20106
1.32105
2.4104
2.02104
2.92104
1.52105

The entire pit samples from Dohar were contaminated with faecal streptococci but one was
free from E.coli, faecal coliforms and total coliforms.

4.699000222

4.62393989

log10 of average

4.8
4.6

4.310748627

4.4
4.071426999

4.2
4
3.8
3.6

Faecal streptococci

E.coli

Faecal coliforms

Total coliforms

Bacteria

Figure 10: log10 of average of bacteria in pit soil from Dohar

Here total coliforms in pit soils showed the highest average at log10 (4.70) followed by
faecal streptococci (4.62), E.coli (4.31) and faecal coliforms (4.07) in Dohar.
90 |

Table 11: Bacteriological counts of pit soil samples from Gopalgonj

Sample ID
Faecal
streptococci
9.8103
0
4.0103
9.2104
0
4.28103
5.2103
1.64104
1.12105
0

PS011
PS012
PS013
PS014
PS015
PS016
PS017
PS018
PS019
PS020

Bacterial counts CFU/g

E.coli
Faecal
coliforms
1.6105
2.8105
0
0
4
4.810
7.2104
8.8105
1.04106
0
0
1.52105
9.50104
2.08106
2.18106
5
4.210
3.92105
2.6106
3.48106
3
2.010
6.0103

Total coliforms
2.28105
0
7.6104
1.36106
0
1.9105
2.81106
2.08106
4.16106
4.8103

Out of 10 pit samples 7 were found contaminated withfaecal streptococci,E.coli, faecal

coliforms and faetal coliforms.

log10 of average

5
4

3.977256928

4.103690393

Faecal coliforms

Total coliforms

3.936797345
2.916858288

3
2
1
0
Faecal streptococci

E.coli

Bacteria

Figure 11: log10 of average of bacteria in pit soil from Gopalgonj

Here total coliforms in pit soils from Gopalgonj showed the highest average at log10 (4.10)
followed by faecal coliforms (3.97), E.coli (3.93) and faecal streptococci (2.91).

91 |

Table 12: Bacteriological counts of pit soil samples from Keranigonj

Sample ID

PS021
PS022
PS023
PS024

4.4105
0
8.0102
2.06104

Bacterial counts CFU/g

E.coli
Faecal
coliforms
2.62107
8.0107
0
0
2.28103
2.42103
5.58106
1.08107

PS025
PS026
PS027
PS028
PS029
PS030

4.4103
0
8.0102
6.2103
4.8104
2.07104

6.8106
0
4.2103
4.4105
3.34106
2.0107

Faecal streptococci

7.2106
0
5.8103
6.8105
2.52106
4.5107

Total coliforms
8.81107
0
5.2103
1.73107
7.89106
0
6.6103
1.02106
2.89106
8.9107

All but one was not contaminated with faecal streptococci,E. coli, faecal coliforms and
total coliforms.

4.744685768

4.882821157

E.coli

Faecal coliforms

5.003453432

log10 of average

5
4

3.219655582

3
2
1
0
Faecal streptococci

Total coliforms

Bacteria

Figure 12: log10 of average of bacteria in pit soil from Keranigonj

Here, total coliforms in pit soils showed the highest average at log10 (4.70) followed by
faecal streptococci (4.62), E.coli (4.31) and faecal coliforms (4.07) in Dohar.

92 |

Table 13: Bacteriological counts of pit soil samples from Hajaribag

Sample ID
Faecal
streptococci
5.6104
3.6104
8.0102
1.64103
3.4104
5.2104
2.28103
5.52105
0
0

PS031
PS032
PS033
PS034
PS035
PS036
PS037
PS038
PS039
PS040

Bacterial counts CFU/g

E.coli
Faecal
coliforms
1.52106
1.68106
9.6105
1.32106
4.4104
7.2104
3.34105
4.4105
1.92106
2.68106
4.4106
1.02107
5.08105
1.18106
2.8107
3.89107
0
0
0
0

Total coliforms
2.32106
1.4106
6.8104
4.60105
8.02106
3.56107
1.20106
4.4107
0
0

All but one is not contaminated with faecal streptococci, E. coli, faecal coliforms and total
coliforms.

6
4.841108961

4.994523496

5.118514105

Faecal coliforms

Total coliforms

5
4

3.376978055

3
2
1
0

Faecal streptococci

E.coli

Bacteria

Figure 13: log10 of average of bacteria in pit soil from Hajaribag

Here total coliforms in pit soils from Hajaribag showed the highest average at log 10 (5.11)
followed by faecal coliforms (4.99), E.coli (4.84) and faecal streptococci (3.37).

93 |

6
4.62

Average at log10

5
4

3.38
3.22
2.92

5.12
5.00 4.70

4.99
4.88

4.84
4.74

4.07
3.98

4.31
3.94

4.10
Avarage Hajaribag
Avarage Keranigonj

Avarage Gopalgonj
Avarage Dohar

1
0

Faecal streptococci

E.coli

Faecal coliforms

Total coliforms

Bacteria

Figure 14: log10 of average of bacteria in pit soil samples from study sites
The average log10 value of faecal streptococci is highest in Dohar (6.62) followed by
Hajaribag (3.38) Keranigonj (3.22) and Gopalgonj (2.92). E. coliis at highest average in
Hajaribag (4.84) followed by Keranigonj (4.74), Dohar (4.31) and Gopalgonj (3.94).
Faecal coliforms and total coliforms showed the highest value in Hajaribag (4.99, 5.12)
followed by Keranigonj (4.88, 5.00), Dohar (4.07, 4.70) and Gopalgonj (3.98, 4.10).
Table 14: Comparative Prevalence of E. coli, faecal streptococci, faecal coliforms and
total coliforms in cow dung
Sampling sites

Prevalence
Faecal

E.coli

streptococci

Faecal

Total

coliforms

Coliforms

Dohar

100

100

100

100

Gopalgonj

100

100

100

100

Keranigonj

90

100

100

100

Mymensing

100

100

100

100

All study sites had 100% prevalence for all of four bacteria in cow dung except
Keranigonj for faecal streptococci (90%).

94 |

100%

100%

Prevalence

100%

100%
90%

90%

Prevalence Faecal
streptococci

80%

Prevalence E.coli

70%
Prevalence Faecal
coliforms

60%
50%

Prevalence Total
Coliforms

40%

30%
20%
Dohar

Gopalgonj

Keranigonj

Mymensing

Study Site

Figure 15: Comperative prevalence of bacteria in cow dung

Faecal streptococci, E. coli, faecal coliforms and total coliforms had 100% prevalence in
cow dung from all four study sites except faecal streptococci from Keranigonj, which had
90% of prevalence.
Table 15: Overall prevalence of bacteria of cow dung
Bacteria

Overall prevalence

Faecal streptococci

97.5%

E. coli

100%

Faecal coliforms

100%

Total coliforms

100%

E. coli, faecal coliforms and total coliforms were at 100% prevalence where faecal
streptococci was at 97.5%.

95 |

97.50%

100%

100%

100%

Overall
prevalence

Faecal streptococci

E.coli

Faecal coliforms Total coliforms

Bacteria

Figure 16: Overall prevalence graph of bacteria of cow dung

Total coliforms, faecal coliforms and E.coli were 100% prevalent whereas, faecal
streptococci were 97.5% prevalent in cow dung.

Table 16: Comparative Prevalence of E. coli, faecal streptococci, faecal coliforms and
total coliforms of pit soil samples
Sampling sites

Prevalence
E.coli

Dohar

Faecal
streptococci
100

Gopalgonj

100

Faecal
coliforms
100

Total
Coliforms
100

70

80

80

80

Keranigonj

80

80

80

80

Hajaribag

80

80

80

80

Faecal streptococci, E. coli, faecal coliforms and total coliforms had high prevalence for
all the study sites.

96 |

100%
100%

90%
Prevalence

80%

80%

80%

Prevalence Faecal
streptococci

80%
Prevalence E.coli
70%
70%
Prevalence Faecal
coliforms
60%
Prevalence Total
Coliforms
50%
Dohar

Gopalgonj

Keranigonj

Hajaribag

Study Sites

Figure 17: Graphical presentation of comperative prevalence of bacteria in pit

samples from study sites
Samples from Dohar had 100% prevalence for all four bacterial groups, where Keranigonj
had and Hajaribag had 80%. Prevalence of faecal streptococciof pit soils from Gopalgonj
is lowest (70%); others had same result of 80%.
Table 17: Overall prevalence of bacteria of pit faecal sludge
Bacteria

Overall prevalence

Faecal streptococci

82.5%

E. coli

85%

Faecal coliforms

85%

Total coliforms

85%

97 |

Prevalence

100.00%
90.00%
80.00%
70.00%
60.00%
50.00%
40.00%
30.00%
20.00%

85%

82.50%

85%

85%

Overall
prevalence

Faecal
streptococci

E. coli

Faecal
coliforms
Bacteria

Total
coliforms

Figure 18: Overall prevalence graph of bacteria from pit faecal sludge
Faecal streptococci had 82.50% of overall prevalence where E. coli, faecal coliforms and
total coliforms had 85%.
Initially bacteria were counted to find out the best positive sample to be heated, which is
the main purpose of this study. The positive samples gave high count, made the study
easier to choose a sample to be heated.
4.3.Parasitological Examination
In the present study, a total 7 different parasite species were found from pit soil samples,
among them 1 species of protozoan, 1 species of cestode and 5 species of nematodes were
recognized whereas, from cow dung samples, a total of 6 different parasite species were
found from cow dung, accounting 4 nematodes, 1 trematode and 1 protozoan (Table-18).

98 |

Table 18: Identified parasites from pit faecal sludge and cow dung
Sample
Cow dung

Group of Parasites
Protozoa
Trematoda
Nematoda

Name of Parasites
Coccidian cyst
Paramphistomum
Toxocara
Trichostrongylus
Strongyloides
Haemonchus

Pit soil

Protozoa

Entamoeba histolytica

Cestoda

Hymenolepis nana

Nematoda

Ascaris lumbricoides
Trichuris trichiura
Enterobias vermicularis
Ancylostoma duodenalae
Strongyloides stercoralis

4.4. Efficacy of Different Parasite Identification Techniques

The initially collected ten cow dung and ten pit faecal sludge samples were analyzed
through direct smear, formol ether sedimentation, floatation concentration and centrifugal
floatation techniques to investigate the efficacy of different methods for future study and
to apply in the present study. Direct smear shows qualitative results and others show both
qualitative and quantitative results (Table-19 and Table-20).

99 |

Table 19: Parasites enumerated through different techniques from cow dung samples
Sample
ID

Absent
Present

Formol
ether
(epg)
Absent
153

Trichostrongylus
(larva))

Present

22

17

69

Strongyloides (egg)

present

13

31

Strongyloides
(larva)
Coccidian cyst

Absent

Present

41

58

217

Trichostrongylus
(egg)

Absent

14

Trichostrongylus
(larva)
Coccidian cyst

Absent

14

Absent

12

23

46

CD004

Absent

Absent

CD005

Toxocara

present

11

31

CD006

Absent

Absent

CD007

Trichostrongylus
(egg)

Present

14

Trichostrongylus
(larva)

Present

24

31

76

Coccidian cyst

Present

104

153

169

CD008

Coccidian cyst

Present

12

31

CD009

Absent

Absent

CD010

Absent

CD001
CD002

CD003

Parasite

Trichostrongylus
(egg)

Direct
smear

Floatation
concentratio
n(epg)
Absent
53

Centrifugal
floatation
(epg)
Absent
155

100 |

Table 20: Prevalence of Parasites in Cow Dung Samples through Different

Techniques
Parasites
Direct
smear
Trichostrongylus 20
Strongyloides
10
Toxocara
0
Coccidian
30

Prevalence (%)
Formol
Floatation
ether
concentration
20
30
10
10
10
10
40
40

Centrifugal
floatation
30
10
10
40

The coccidian cysts were recovered at highest rate (40%) from formol ether sedimentation,
floatation concentration and centrifugal floatation whereas direct smear showed 30%. For
Trichostrongylus, 30% recovery rate was observed from both floatation concentration and
centrifugal floatation whereas 20% from both formol ether sedimentation and direct smear
techniques. For Strongylus, 10% rate revealed through all four techniques and 10 % for
Toxocara through formol ether sedimentation, floatation concentration and centrifugal
floatation but 0% through direct smear.
Table 21: Anova (F-test) table for parasite recovery techniques from cow dung
Sum of Squares

df

Mean

Square
Between

3985.609

797.122

Within Groups

21644.083

3092.012

Total

25629.692

12

9793.583

1958.717

Within Groups

11305.333

1884.222

Total

21098.917

11

16393.558

3278.712

Within Groups

40694.750

5813.536

Total

57088.308

12

Formol Ether *

Groups

Parasites

Floatation
Concentration *
Parasites

Between
Groups

Between
Centrifugal
Floatation *
Parasites

Groups

(Combined)

(Combined)

(Combined)

.258

.923

1.040

.472

.564

.727

The P value for formol ether sedimentation, floatation concentration and centrifugal
floatation techniques were 0.923, 0.472 and 0.727 which is insignificant at 5% of error
levels.
101 |

Table 22: Comparison of different techniques (correlations)

Parasites

Formol Ether

Mean
Trichostrongylus (egg)

N
Std. Deviation
Variance
% of Total Sum
Mean

Trichostrongylus (larva)

N
Std. Deviation

18.33

61.00

86.927

30.039

81.406

7556.333

902.333

6627.000

40.3%

15.6%

20.9%

15.33

16.33

53.00

3
33.956
1153.000

11.7%

13.9%

18.2%

13.00

9.00

31.00

Std. Deviation

Variance

3.3%

2.5%

3.5%

3.00

.00

7.00

Std. Deviation

Variance

% of Total Sum

0.8%

0.0%

0.8%

Mean

40.25

78.00

115.75

45.375

67.268

91.533

2058.917

4525.000

8378.250

41.1%

66.3%

53.0%

11.00

6.00

31.00

Std. Deviation

Variance

% of Total Sum

2.8%

1.7%

3.5%

Mean

30.15

29.42

67.23

13

12

13

46.215

43.796

68.974

2135.808

1918.083

4757.359

100.0%

100.0%

100.0%

Mean

N
Std. Deviation
Variance
% of Total Sum
Mean

N
Total

52.67

15.011

% of Total Sum

Toxocara

Floatation

225.333

Mean

Coccidian cyst

Concentration

13.317

% of Total Sum

Strongyloides (larva)

Centrifugal

177.333

Variance

Strongyloides (egg)

Floatation

Std. Deviation
Variance
% of Total Sum

102 |

For every parasites of cow dung centrifugal floatation technique gave the highest intensity
and percentage of recovery except coccidian cyst which was highest in floatation
concentration technique.
Table 23: Parasites enumerated through different techniques from pit soil samples
Sample
ID

Parasite

Direct
smear

PS001

A. lumbricoides
T.trichiura
E.vermicularis
H.nana
A. lumbricoides
T.trichiura
E.vermicularis
H.nana
E.histolytica
A. lumbricoides
T.trichiura
E.vermicularis
H.nana
S.stercoralis (egg)
S.stercoralis (larva)
A.duodenale (egg)
A.duodenale (larva)
E.histolytica
A. lumbricoides
T.trichiura
E.vermicularis
E.histolytica
Absent
Absent
A. lumbricoides
S.stercoralis (egg)
S.stercoralis (larva)
A. lumbricoides
A. lumbricoides
T.trichiura

PS002

PS003

PS004

PS005
PS006
PS007
PS008
PS009
PS010

Present
Present
Absent
Absent
Present
Present
Absent
Absent
Absent
Present
Present
Absent
Absent
Present
Present
Present
Present
Absent
Present

Formol
ether
(epg)
456
188
0
0
14
18
0
0
2
14
9
1
0
156
24
680
341
9
46

Floatation
concentratio
n(epg)
76
9
0
0
50
2
0
0
0
4
0
0
1
106
23
440
210
0
20

Centrifuga
l floatation
(epg)
594
86
4
2
143
2
4
2
1
80
3
0
0
155
17
932
762
3
49

Absent
Absent
Absent
Absent
Absent
Absent
absent
absent
Present
Present
Present

8
0
0
0
2
2
4
25
93
46

2
0
1
0
0
0
1
7
41
22

4
1
0
0
13
13
13
46
155
31

Centrifugal floatation technique recovered highest number of cysts, larva and eggs except
T. trichiura eggs which was best recovered by formol ether sedimentation technique.

103 |

Table 24: Prevalence of parasites of pit soil samples

Parasites

A. lumbricoides
T. trichiura
S.stercoralis
A.duodenale
E.vermicularis
E.histolytica
H.nana

Direct
smear

Prevalence
Formol
ether

Floatation
concentration

Centrifugal
floatation

60
40
10
10
0
0
0

70
50
20
10
10
20
0

60
40
20
10
0
10
10

70
50
20
10
30
20
20

70 70
70

60
50

60 60
50 50
40 40

40
30
20

30
20 20
10

10

20 20
1010 10

10
0

20

10
0

10
0 0

Prevalance Direct smear

Prevalance Floatation concentration

Prevalance Formol ether

Prevalance Centrifugal floatation

Figure 19: Prevalence of Parasites in pit soil samples through different techniques

Among the four techniques Formol ether sedimentation and centrifugal floatation reviled
highest prevalence for A.lumbricoides (70%), T. trichiura (50%), E.histolytica (20%) and
S.stercoralis (20%), where direct smear reviled 60% A.lumbricoides, 40% T. trichiura,
10% S.stercoralis and A.duodenale, 0% for E.vermicularis and H.nana.For A.duodenale
all four techniques showed same prevalence (10%). To enumerate E.vermicularis (30%)
and H.nana (20%) centrifugal floatation reviled highest prevalence.

104 |

Table 25: Anova (F-test) for parasite recovery techniques from pit faecal sludge
Sum of

df

Mean

Squares
Between
Groups
FE *
Parasites

(Combined)

74
698098.4

Total

Groups
FC *
Parasites

29
(Combined)

CF *
Parasites

Within Groups

Total

62
223669.2

Total

Groups

99877.98

123791.2

Within Groups

Between

55
469493.5

Within Groups

Between

228604.8

50
(Combined)

594429.9
38
949779.3
12
1544209.
250

Sig.

Square
8 28575.607 1.156

.373

19 24710.188

27

8 12484.749 1.916

19

.117

6515.330

27

8 74303.742 1.486

.227

19 49988.385

27

Note: FE = Formol ether sedimentation; FC =Floatation concentration and

CF=Centrifugal Floatation
The P value for formol ether sedimentation, floatation concentration and centrifugal
floatation techniques are 0.373, 0.117 and 0.227 which is insignificant at 5% of error
levels.

105 |

Table 26: Comparision of different parasite enumeration techniques

Parasites

A. lumbricoides

T. trichiura

E.vermicularis

H.nana

E.histolytica

S.stercoralis (egg)

S.stercoralis (larva)

A.duodenale (egg)

A.duodenale (larva)

Total

Mean
N
Std. Deviation
% of Total Sum
Mean
N
Std. Deviation
% of Total Sum
Mean
N
Std. Deviation
% of Total Sum
Mean
N
Std. Deviation
% of Total Sum
Mean
N
Std. Deviation
% of Total Sum
Mean
N
Std. Deviation
% of Total Sum
Mean
N
Std. Deviation
% of Total Sum
Mean
N
Std. Deviation
% of Total Sum
Mean
N
Std. Deviation
% of Total Sum
Mean
N
Std. Deviation
% of Total Sum

Formol Ether
sedimentation
92.86
7
162.975
30.4%
53.80
5
76.578
12.6%
.25
4
.500
0.0%
.00
3
.000
0.0%
3.67
3
4.726
0.5%
156.00
1
.
7.3%
24.00
1
.
1.1%
341.00
2
479.418
31.9%
172.50
2
238.295
16.1%
76.36
28
160.796
100.0%

Floatation
concentration
28.29
7
28.347
19.5%
7.00
5
9.055
3.4%
.00
4
.000
0.0%
.33
3
.577
0.1%
.33
3
.577
0.1%
106.00
1
.
10.4%
23.00
1
.
2.3%
220.00
2
311.127
43.3%
105.50
2
147.785
20.8%
36.25
28
91.017
100.0%

Centrifugal
Floatation
154.29
7
200.735
34.7%
25.20
5
36.093
4.0%
2.25
4
2.062
0.3%
1.33
3
1.155
0.1%
1.33
3
1.528
0.1%
155.00
1
.
5.0%
17.00
1
.
0.5%
472.50
2
649.831
30.3%
387.50
2
529.623
24.9%
111.25
28
239.150
100.0%

For every parasites of pit faecal sludge centrifugal floatation technique gave the highest
intensity and percentage of recovery except T. trichiura which was highest in formol ether
sedimentation technique.

106 |

From the above results of recovery rates through different parasite (cysts, eggs and larva)
recovery techniques, it was clear that centrifugal floatation recovers highest amount of
parasites. This technique was used for further study of recovery of parasites at diffenent
temperatures.
Table 27: Parasitic prevalence in cow dung
Parasites
Trichostrongylus
Strongyloides
Paramphistomum
Haemonchus
Toxocara
Coccidian

Prevalence (%)
Dohar
Gopalgonj
30
40
10
10
0
10
0
10
10
0
50
20

Keranigonj
40
10
0
0
0
30

Mymensing
30
30
0
10
10
30

Trichostrongylus, Strongyloides and Coccidians found in all four study sites.

Pramphistomum found only in Gopalgonj. Haemonchus found in Gopalgonj and
Mymensing were Toxocara found in Dohar and Mymensing.

60

Prevalence

50
40

30

Dohar

20

Gopalgonj

10

Keranigonj
Mymensing

Parasites

Figure 20: Prevalence of parasites in cow dung from study sites

Highest prevalence of Trichostrongylus found both in Gopalgonj and Keranigonj (40%)
followed by Dohar (30%) and Mymensing (30%). Strongyloides had 10% prevalence for
Dohar, Gopalgonj and Keranigonj, and 30% for Mymensing.

107 |

Table 28: Overall prevalence and intensity of parasites from cow dung
Parasites
Paramphistomum
Haemonchus
Toxocara
Trichostrongylus
Strongyloides
Coccidian

Overall prevalence
2.5%
5%
5%
32.5%
15%
30%

Overall intensity
30
62
13.5
88.08
57
127.17

Trichostrongylus (32.5%) had highest prevalence where Paramphistomum had the lowest
(5%). Overall intensity was highest for coccidian cysts (127.17) and lowest for Toxocara
(13.5).

Overall prevalence

32.50%

30%

15%

2.50%

5%

5%

Figure 21: Overall prevalence of cow dung parasites

Highest peak was produced by Trichostrongylus where lowest peak was produced by
Paramphistomum.

108 |

127.17

140
120
88.08

100
80

62

57

Overall
intensity

60
40

30
13.5

20
0

Figure 22: overall intensity of cow dung parasites

Overall intensity was highest for coccidian cysts (127.17) and lowest for Toxocara (13.5).
Table 29: Prevalence of parasites of pit soil
Parasites

Prevalence (%)

A. lumbricoides

Dohar
80

Gopalgonj
30

Keranigonj
40

Hajaribag
50

T. trichiura

50

10

10

20

S.stercoralis

20

10

40

50

A.duodenale

10

20

30

60

E.vermicularis

30

E.histolytica

10

10

10

H.nana

20

10

109 |

100
90
80
80
70
60
60
50 50

50

50
40

Dohar

40

Gopalgonj

40
30

30

30

Keranigonj

30
20 20

20

20

Hajaribag

20
1010

10

10

10 1010

10

10
000

00

Figure 23: Prevalence of parasites in pit soil from study sites

All the seven parasite species were found in Dohar. Samples from Gopalgonj were
positive for A. lumbricoides, T. trichiura, S.stercoralis and A.duodenale. . Samples from
Keranigonj were positive for A. lumbricoides, T. trichiura, S.stercoralis, A.duodenale and
E.histolytica where pit samples from Hajaribag is negative only for E.vermicularis,
positive for others.
Highest prevalence for A. lumbricoides found in Dohar (80%) followed by Hajaribag
(50%), Keranigonj (40%) and Gopalgonj (30%). Highest prevalence for T. trichiura found
in Dohar (50%) followed by Hajaribag (20%), Keranigonj (10%) and Gopalgonj (10%).
Highest prevalence for S. stercoralis found in Hajaribag (50%) followed by Keranigonj
(40%), Dohar (20%) and Gopalgonj (10%). Highest prevalence for S. stercoralis found in
Hajaribag (60%) followed by Keranigonj (30%), Gopalgonj (20%) and Dohar (10%). E.
vermicularis found only in Dohar at 30% prevalence. E.histolytica had 10% prevalence for
Dohar, Keranigonj and Hajaribag, and 0% for Gopalgonj. H. nana had 20% prevalence in
Dohar, 10% in Hajaribag but absent for others.

110 |

Table 30: Overall prevalence and intensity of parasites of pit faecal sludge
Parasites
A. lumbricoides

Overall prevalence
47.5%

Overall intensity
230.68

T. trichiura
E. vermicularis
A.duodenale
S. stercoralis
H. nana
E. histolytica

22.5%
7.5%
27.5%
30%
7.5%
10%

27.33
3
350.45
302.42
10.33
14.25

A. lumbricoides was at highest prevalence (47.5%) followed by S. stercoralis (30%), A.

duodenale (27.5%), T. trichiura (22.5%), E. histolytica (10%), E. vermicularis (7.5%)
and H. nana (7.5%). The highest intensity was observed for A.duodenale followed by S.
stercoralis, A. lumbricoides, T. trichiura, E. histolytica, H. nana and E. vermicularis.

Overall prevalence

47.50%

27.50%

30%

22.50%

7.50%

7.50%

10%

Figure 24: Bar diagram of overall prevalence of parasites from pit faecal sludge
A.lumbricoides showed the highest peak (47.5%) and E. vermicularis (7.5%) and H. nana
(7.5%) showed the lowest peak among the parasites.

111 |

400

350.45

350
300
250

302.42
230.68

200
150

Overall intensity

100
27.33

50

14.25

10.33

Figure 25: Overall intensity graph of parasites of pit faecal sludge

A.duodenale (350.45) gave the highest value and E. vermicularis (3.00) gave the lowest
value.
4.5. Annihilation of Bacteria

Table 31: Thermal inactivation of bacteria from cow dung sample

Sample
ID

CD019

Temperature
(C)

50

55

60

65

70

75

80

Time

15
30
60
15
30
60
15
30
60
15
30
60
15
30
60
15
30
60
15
30
60

Bacteria
Faecal
streptococci
4.54104
3.73104
8.68103
9.54104
6.64104
4.73103
5.11103
2.16103
5.68102
2.27103
9.09102
0
0
0
0
0
0
0
0
0
0

E.coli
5.45106
7.27106
1.82106
9.54106
4.32106
7.27104
2.16106
0
0
0
0
0
0
0
0
0
0
0
0
0
0

Faecal
coliforms
6.36107
1.14107
1.95106
8.18106
3.98106
1.36105
2.32106
0
0
0
0
0
0
0
0
0
0
0
0
0
0

Total
coliforms
2.27107
4.54106
1.82106
9.09106
4.73106
1.27105
2.49106
0
0
0
0
0
0
0
0
0
0
0
0
0
0

112 |

In cow dung faecal streptococci were killed at 65C for 60 of heat treatment, whereas
others were killed at 60C for 30 mins of exposure.
Table 32: Time-temperature relationship in bacterial counts
Correlations
Faecal
streptoc
occi

E.coli

FC

Pearson
1 .928**
.444*
Faecal
Correlation
streptococc
Sig. (2-tailed)
.000
.044
i
N
21
21
21
Pearson
.928**
1
.515*
Correlation
E.coli
Sig. (2-tailed)
.000
.017
N
21
21
21
Pearson
.444*
.515*
1
Correlation
FC
Sig. (2-tailed)
.044
.017
N
21
21
21
Pearson
.667** .704** .958**
Correlation
TC
Sig. (2-tailed)
.001
.000
.000
N
21
21
21
Pearson
-.575** -.644** -.452*
Temperatur Correlation
e
Sig. (2-tailed)
.006
.002
.040
N
21
21
21
Pearson
-.315
-.331
-.283
Correlation
Time
Sig. (2-tailed)
.164
.143
.215
N
21
21
21
**. Correlation is significant at the 0.01 level (2-tailed).
*. Correlation is significant at the 0.05 level (2-tailed).

TC

Temperat
ure

Time

.667**

-.575**

-.315

.001

.006

.164

21

21

21

.704**

-.644**

-.331

.000
21

.002
21

.143
21

.958**

-.452*

-.283

.000
21

.040
21

.215
21

-.546*

-.339

21

.010
21

.132
21

-.546*

.000

.010
21

21

1.000
21

-.339

.000

.132
21

1.000
21

21

Note : FC= faecal coliforms. TC= total coliforms

Temperature and time had negative correlation for all types of bacteria studied but
significant only for temperature at 0.05 level of significance.

113 |

Table 33: Thermal inactivation of bacteria from pit soil samples

Sample

temperature

time

Bacteria

id

Faecal

E.coli

streptococci
PS004

50

55

60

65

70

75

80

Faecal

Total

coliforms

coliforms

15

1.16106

2.32106

9.30106

4.65107

30

6.05105

5.58106

1.12106

1.12106

60

8.37104

6.51104

4.65104

3.26104

15

7.44105

1.30106

7.44106

2.09107

30

3.86104

6.47105

1.25105

1.10106

60

4.65103

2.35104

4.42104

1.02105

15

1.63104

2.09104

8.84104

9.30104

30

5.12103

60

1.8610

15

5.12103

30

9.30102

60

15

30

60

15

30

60

15

30

60

In pit soil faecal streptococci were killed at 65C for 60 of heat treatment, whereas others
were killed at 60C for 30 mins of exposure.

114 |

Table 34: Time- temperature correlation in inactivation of bacteria

Correlations
Temperatur
e

Time

Faecal
streptoco
cci

Pearson
1
.000
Temperatu Correlation
re
Sig. (2-tailed)
1.000
N
21
21
Pearson
.000
1
Correlation
Time
Sig. (2-tailed)
1.000
N
21
21
Pearson
Faecal
-.560**
-.331
Correlation
streptococ
Sig. (2-tailed)
.008
.143
ci
N
21
21
Pearson
-.517*
-.199
Correlation
E.coli
Sig. (2-tailed)
.016
.387
N
21
21
Pearson
-.451*
-.352
Faecal
Correlation
coliforms Sig. (2-tailed)
.040
.118
N
21
21
Pearson
-.420
-.325
Correlation
Total
colidorms Sig. (2-tailed)
.058
.150
N
21
21
**. Correlation is significant at the 0.01 level (2-tailed).

E.coli

Faecal
colifor
ms

Total
colidorms

-.560**

-.517*

-.451*

-.420

.008
21

.016
21

.040
21

.058
21

-.331

-.199

-.352

-.325

.143
21

.387
21

.118
21

.150
21

.705**

.936**

.906**

21

.000
21

.000
21

.000
21

.705**

.426

.369

.000
21

21

.054
21

.100
21

.936**

.426

.963**

.000
21

.054
21

21

.000
21

.906**

.369

.963**

.000
21

.100
21

.000
21

21

*. Correlation is significant at the 0.05 level (2-tailed).

Temperature and time had negative correlation for all types of bacteria studied but
significant only for temperatureat 0.05 level of significance.
Along with the bacterial inactivation, parasitic inactivation was performed and samples
from same model temperatures and times were examined.

115 |

4.6. Annihilation of Parasites

Table 35: Overall time- temperature required in inactivation of parasites
Sample

Parasites

Inactivation

Inactivation

Temperature (C)

Time (mins)

Coccidian cyst

60

15

Paramphistomum

65

60

Trichostrongylus egg

65

30

Trichostrongylus larva

60

60

Strongyloides egg

60

60

Strongyloides larva

60

30

Haemonchus

65

30

Pit Soil E. histolytica

60

60

H. nana

75

15

A. lumbricoides

75

15

T. trichiura

65

30

A. duodenalae egg

65

60

A. duodenalae larva

60

60

S. stercoralis egg

70

30

S. stercoralis larva

60

60

Cow Dung

In case of cow dung Paramphistomum survived longer (65C, 60 mins) and coccidian
cysts survived lesser (60C, 15 mins). On the other hand, A. lumbricoides and H. nana
from pit soil became nonviable/inactivated at 75C for 15 mins of heat treatment, whereas
E. histolytica, A. duodenalae larva and S. stercoralis larva at 60C for 60 mins.

116 |

Table 36: Thermal Inactivation of Paramphistomum

Sample CD019 (Dry Weight) (Paramphistomum EPG)
Temperature

50C

55C

60C

65C

70C

75C

80C

Inactivation rate (%)

Time

15

0.00%

30

0.00%

60

35.29%

15

32.35%

30

44.11%

60

64.70%

15

50.00%

30

67.64%

60

85.29%

15

61.76%

30

91.17%

60

100%

15

100%

30

100%

60

100%

15

100%

30

100%

60

100%

15

100%

30

100%

60

100%

117 |

Eggs /gram of dry weight

50
45
40
35
30
25

15 mins

20

30 mins

15

60 mins

10
5
0
25C

50C

55C

60C
65C
Temperature

70C

75C

80C

Figure 26: Time - temperature relationship in inactivation of Paramphistomum

The number of eggs of Paramphistomum decreases with the increasing temperature. It also
decreases with increasing time at each temperature and become 0 at 65C for 60 minutes
but remains still viable for 15 and 30 minutes. At 70C it becomes 0 for 15 minutes.

120.00%
100.00%
80.00%
60.00%
inactivation rate

40.00%
20.00%
0.00%
15 30 60 15 30 60 15 30 60 15 30 60 15 30 60 15 30 60 15 30 60
50C

55C

60C

65C

70C

75C

80C

Figure 27: Fig.: Inactivation rate of Paramphistomum spp. in relation to time and
temperature
Paramphistomum 100% inactivation rate was performed at 70C for each time interval.

118 |

Table 37: Time- temperature correlation in inactivation of Paramphistomum

Correlations
Paramphistomu
m
Pearson Correlation

Temperature

Paramphistomum Sig. (2-tailed)

N
Pearson Correlation
Temperature
Sig. (2-tailed)
N
Pearson Correlation
Time
Sig. (2-tailed)

-.834**

-.265

.000
21
1
21
.000

.246
21
.000
1.000
21
1

1.000
21

21

21
-.834**
.000
21
-.265

.246
N
21
**. Correlation is significant at the 0.01 level (2-tailed).

Time

Number of parasites had negative correlation with temperature and time but significant for
temperature not for time at 0.01 significant levels forParamphistomum.
Table 38: Thermal Inactivation of Haemonchus spp
Sample CD019 (Dry Weight) (Haemonchus EPG)
Temperature

50C

55C
60C

65C

70C
75C

80C

Time

Inactivation rate (%)

15
30
60
15
30
60
15
30
60
15
30
60
15
30
60
15
30
60
15
30
60

7.38%
13.11%
37.70%
20.49%
45.08%
54.92%
76.23%
87.0%
95.08%
98.36%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%

119 |

140

Eggs per gram

120
100
80

15mins

60

30mins

40

60mins

20
0
25C

50C

55C

60C
65C
Temperature

70C

75C

80C

Figure 28: Time - temperature relationship in inactivation of Haemonchus

The number of eggs ofHaemonchus decreases with the increasing temperature. It also
decreases with increasing time at each temperature and become 0 at 65C for 30 minutes
but remains still viable for 15 minutes. At 70C, 75C and 80C it becomes 0 for every
time of exposure.

120.00%
100.00%
80.00%

60.00%
40.00%
20.00%
0.00%
15 30 60 15 30 60 15 30 60 15 30 60 15 30 60 15 30 60 15 30 60
50C

55C

60C

65C

70C

75C

80C

Inactivation Rate of Haemonchus

Figure 29: Inactivation rate of Haemonchus in relation to time and temperature

100% inactivation rate was found from 65C for 30 minutes to all others.

120 |

Table 39 : Time- temperature correlation in inactivation of Haemonchus

Correlations
Parasites
Pearson Correlation
Parasites

Sig. (2-tailed)
N
Pearson Correlation
Sig. (2-tailed)
N
Pearson Correlation

Temperature

Temperature

21
-.830**
.000
21
-.151

Time

Sig. (2-tailed)
.514
N
21
**. Correlation is significant at the 0.01 level (2-tailed).

Time

-.830**

-.151

.000
21
1
21
.000

.514
21
.000
1.000
21
1

1.000
21

21

Number of parasites had negative correlation with temperature and time but significant for
temperature not for time at 0.01 significant levels for Haemonchus.

Table 40: Thermal Inactivation of Trichostrongylus eggs

Sample CD019 (Dry Weight) (Trichostrongylus EPG)
Temperature
Time

50C

55C

60C

65C

70C

75C

80C

15
30
60
15
30
60
15
30
60
15
30
60
15
30
60
15
30
60
15
30
60

Inactivation rate (%)

16.59%
19.91%
54.98%
46.45%
58.77%
75.36%
55.45%
91.94%
100%
99.05%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
121 |

250

200

Eggs per gram

150
15 mins

100

30 mins
60 mins

50

0
25C

50C

55C

60C

65C

70C

75C

80C

Temperature

Figure 30: Time - temperature relationship in inactivation of Trichostrongylus eggs

At 60C for 60 mins Trichostrongylus eggs were became inactive.
120.00%
100.00%
80.00%
60.00%
Inactivation Rate of
Trichostrongyloides

40.00%
20.00%
0.00%
15 30 60 15 30 60 15 30 60 15 30 60 15 30 60 15 30 60 15 30 60
50C

55C

60C

65C

70C

75C

80C

Figure 31: Inactivation rate of Trichostrongylus eggs

100% inactivation was performed at 60C for 60 mins and all other temperatures for every
time intervals.

122 |

Table 41: Time- temperature correlation in inactivation of Trichostrongylus eggs

Correlations
Parasites

Temperature

Pearson
1
Correlation
Parasites
Sig. (2-tailed)
N
21
Pearson
-.799**
Correlation
Temperature
Sig. (2-tailed)
.000
N
21
Pearson
-.238
Correlation
Time
Sig. (2-tailed)
.299
N
21
**. Correlation is significant at the 0.01 level (2-tailed).

Time

-.799**

-.238

.000
21

.299
21

.000

21

1.000
21

.000

1.000
21

21

Number of parasites had negative correlation with temperature and time but significant for
temperature not for time at 0.01 significant levels for Trichostrongylus eggs.
Table 42: Thermal Inactivation of Trichostrongylus larva
Sample CD019(Dry Weight) (Trichostrongylus LPG)
Temperature

50C

55C

60C

65C

70C

75C

80C

Time

Inactivation rate (%)

15
30
60
15
30
60
15
30
60
15
30
60
15
30
60
15
30
60
15
30
60

18.93%
38.80%
52.37%
31.55%
67.82%
79.81%
92.11%
97.16%
100%
99.05%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
123 |

350
300
250

15 mins

200

30 mins

150

60 mins

100
50
0
25C

50C

55C

60C

65C

70C

75C

80C

Figure 32: Time - temperature relationship in inactivation of Trichostrongylus larva

Within 60 mins at 60C Trichostrongylus larva were inactivated, but few sill remained
viable at 70C for 15 mins and killed for 30 mins.

120.00%
100.00%
80.00%

Inactivation Rate of
Trichostrongyloides
larva

60.00%
40.00%
20.00%
0.00%
15 30 60 15 30 60 15 30 60 15 30 60 15 30 60 15 30 60 15 30 60
50C

55C

60C

65C

70C

75C

80C

Figure 33: Inactivation rate of Trichostrongylus larva

100% inactivation was performed at 60C for 60 mins and all other temperatures for every
time intervals.

124 |

Table 43: Time- temperature correlation in inactivation of Trichostrongylus larva

Correlations
Parasites
Pearson
1
Correlation
Parasites
Sig. (2-tailed)
N
21
Pearson
-.763**
Correlation
Temperature
Sig. (2-tailed)
.000
N
21
Pearson
-.194
Correlation
Time
Sig. (2-tailed)
.400
N
21
**. Correlation is significant at the 0.01 level (2-tailed).

Temperature

Time

-.763**

-.194

.000
21

.400
21

.000

21

1.000
21

.000

1.000
21

21

Number of parasites had negative correlation with temperature and time but significant for
temperature not for time at 0.01 significant levels for Trichostrongylus larva
Table 44: Thermal Inactivation of Strongyloides eggs
Sample CD019(Dry Weight) (Strongyloides EPG)
Temperature

Time

Inactivation rate (%)

15
30
60
15
30
60
15
30
60
15
30
60
15
30
60
15
30
60
15
30
60

0.00%
12.86%
31.43%
24.29%
52.86%
70.00%
84.29%
97.14%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%

Initial count
50C

55C

60C

65C

70C

75C

80C

125 |

80
70
60
50

15 mins

40

30 mins
60 mins

30
20
10
0
25C

50C

55C

60C

65C

70C

75C

80C

Figure 34: Time - temperature relationship in inactivation of Strongyloides eggs

60C temperatue for 60 mins of exposure was found lethal for Strongyloides eggs, where
full inactivation was performed.

120.00%
100.00%
80.00%
60.00%
40.00%
20.00%
0.00%
15 30 60 15 30 60 15 30 60 15 30 60 15 30 60 15 30 60 15 30 60
50C

55C

60C

65C

70C

75C

80C

Figure 35: Inactivation rate of Strongyloides eggs

100% inactivation was performed at 60C for 30 mins and all other temperatures for every
time intervals.

126 |

Table 45: Time- temperature correlation in inactivation of Strongyloides eggs

Correlations
Parasites
Pearson Correlation
1
Parasites
Sig. (2-tailed)
N
21
Pearson Correlation
-.784**
Temperature
Sig. (2-tailed)
.000
N
21
Pearson Correlation
-.167
Time
Sig. (2-tailed)
.470
N
21
**. Correlation is significant at the 0.01 level (2-tailed).

Temperature
-.784**
.000
21
1
21
.000
1.000
21

Time
-.167
.470
21
.000
1.000
21
1
21

Number of parasites had negative correlation with temperature and time but significant for
temperature not for time at 0.01 significant levels for Strongyloides eggs.
Table 46: Thermal Inactivation of Strongyloides larva
Sample CD019 (Dry Weight) (Strongyloides LPG)
Temperature

Time

Inactivation rate (%)

15
30
60
15
30
60
15
30
60
15
30
60
15
30
60
15
30
60
15
30
60

0.00%
5.77%
32.69%
32.69%
55.77%
75.00%
84.62%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%

Initial count
50C

55C

60C

65C

70C

75C

80C

127 |

60
50
15 mins

40

30 mins

30

60 mins

20
10
0
25C

50C

55C

60C

65C

70C

75C

80C

Figure 36: Time - temperature relationship in inactivation of Strongyloides larva

At 60C within 60 mins whole inactivation of Strongyloides larva was performed.
120.00%
100.00%
80.00%
60.00%
40.00%
20.00%
0.00%
15 30 60 15 30 60 15 30 60 15 30 60 15 30 60 15 30 60 15 30 60
50C

55C

60C

65C

70C

75C

80C

Figure 37: Inactivation rate of Strongyloides larva

100% inactivation was performed at 60C for 30 mins and all other temperatures for every
time intervals.

128 |

Table 47: Time- temperature correlation in inactivation of Strongyloides larva

Correlations
Parasites

Temperature

Time

Pearson Correlation
1
-.772**
-.167
Parasites
Sig. (2-tailed)
.000
.469
N
21
21
21
**
Pearson Correlation
-.772
1
.000
Temperature
Sig. (2-tailed)
.000
1.000
N
21
21
21
Pearson Correlation
-.167
.000
1
Time
Sig. (2-tailed)
.469
1.000
N
21
21
21
**. Correlation is significant at the 0.01 level (2-tailed).
Number of parasites decreases with the increasing temperature and time but significant for
temperature not for time at 0.01 significant levels for Strongyloides larva.
Table 48: Thermal Inactivation of Coccidian cyst
Sample CD019(Dry Weight) (Coccidian cyst CPG)
Temperature

50C

55C

60C

65C

70C

75C

80C

Time

Inactivation rate (%)

15
30
60
15
30
60
15
30
60
15
30
60
15
30
60
15
30
60
15
30
60

16.35%
42.96%
53.57%
50.27%
64.52%
87.83%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%

129 |

800
700
600
15 mins

500

30 mins

400

60 mins

300
200
100
0

25C

50C

55C

60C

65C

70C

75C

80C

Figure 38: Time - temperature relationship in inactivation of Coccidian cyst

At 60C for 15 mins all the coccicdan cyst became inactive.

120.00%
100.00%
80.00%
60.00%
Inactivation rate of
Coccidian cysts

40.00%
20.00%
0.00%
15 30 60 15 30 60 15 30 60 15 30 60 15 30 60 15 30 60 15 30 60
50C

55C

60C

65C

70C

75C

80C

Figure 39: Inactivation rate of Coccidian cyst

100% inactivation was performed at 60C for 15 mins and all other temperatures for every
time intervals.

130 |

Table 49: Time- temperature correlation in inactivation of coccidian cysts

Correlations
Parasites

Temperature

Pearson
1
Correlation
Parasites
Sig. (2-tailed)
N
21
Pearson
-.740**
Correlation
Temperature
Sig. (2-tailed)
.000
N
21
Pearson
-.174
Correlation
Time
Sig. (2-tailed)
.450
N
21
**. Correlation is significant at the 0.01 level (2-tailed).

Time

-.740**

-.174

.000
21

.450
21

.000

21

1.000
21

.000

1.000
21

21

Number of parasites had negative correlation with temperature and time but significant for
temperature not for time at 0.01 significant levels for coccidian cysts.
Table 50: Thermal Inactivation of A. lumbricoides
Sample PS004 (Dry Weight) (A. lumbricoides EPG)
Temperature
Initial count
50C

55C
60C
65C

70C

75C

80C

Time

Inactivation rate (%)

4650

15
30
60
15
30
60

0.62%
2.45%
19.03%
1.35%
17.16%
35.76%

15
30
60

9.20%
24.90%
31.74%

15
30
60
15
30
60
15
30
60
15
30
60

22.86%
45.40%
50.17%
68.26%
81.91%
88.75%
100%
100%
100%
100%
100%
100%
131 |

5000
4500
4000
3500

15 mins

3000

30 mins

2500

60 mins

2000
1500
1000
500
0
25C

50C

55C

60C

65C

70C

75C

80C

Figure 40: Time - temperature relationship in inactivation of A.lumbricoides

A.lumbricoides became inactive at 75C within 15 mins.

120.00%
100.00%
80.00%
60.00%
Inactivation rate of A.
lumbricoides
40.00%
20.00%
0.00%
153060153060153060153060153060153060153060
50C

55C 60C

65C

70C

75C

80C

Figure 41: Inactivation rate of A. lumbricoides

A.lumbricoides at 75C temperature 100% inactivation was performed.

132 |

Table 51: Time- temperature correlation in inactivation of A.lumbricoides

Correlations
Parasites

Temperature

Time

Pearson Correlation
1
-.937**
-.183
Parasites
Sig. (2-tailed)
.000
.428
N
21
21
21
**
Pearson Correlation
-.937
1
.000
Temperature
Sig. (2-tailed)
.000
1.000
N
21
21
21
Pearson Correlation
-.183
.000
1
Time
Sig. (2-tailed)
.428
1.000
N
21
21
21
**. Correlation is significant at the 0.01 level (2-tailed).
Number of parasites had negative correlation with temperature and time but significant for
temperature not for time at 0.01 significant levels for A. lumbricoides.

Table 52: Thermal Inactivation of T. trichiura

Sample PS034 (Dry Weight) (T. trichiura EPG)
Temperature

Time
46

Initial count
50C

55C
60C

65C

70C

75C

80C

Inactivation rate (%)

15
30
60
15
30
60
15
30
60
15
30
60
15
30
60
15
30
60
15
30
60

6.52%
15.22%
32.61%
15.22%
43.48%
65.21%
26.09%
76.08%
95.65%
91.30%
100%
100%
86.95%
100%
100%
100%
100%
100%
100%
100%
100%

133 |

50
45
40
35
30
25
20
15
10
5
0

15 mins
30 mins
60 mins

25C

50C

55C

60C

65C

70C

75C

80C

Figure 42: Time - temperature relationship in inactivation of T. trichiura

At 65C for 30 mins the eggs of T.trichiura became inactive, but at 70C for 15 minutes
they remained viable, though the count was low.

120.00%
100.00%
80.00%

60.00%

Inactivation rate of
T. trichuta

40.00%
20.00%

0.00%
15 30 60 15 30 60 15 30 60 15 30 60 15 30 60 15 30 60 15 30 60
50C

55C 60C

65C

70C

75C

80C

Figure 43: Inactivation rate of T. trichiura

100% inactivation was performed at 65C for 30 mins and all other temperatures for every
time intervals except at 70C for 15 minutes, which inactivated 86.95% of T. trichiura
eggs.

134 |

Table 53: Time- temperature correlation in inactivation of T. trichiura

Correlations
Parasites

Temperature

Pearson
1
Correlation
Parasites
Sig. (2-tailed)
N
21
Pearson
-.827**
Correlation
Temperature
Sig. (2-tailed)
.000
N
21
Pearson
-.273
Correlation
Time
Sig. (2-tailed)
.231
N
21
**. Correlation is significant at the 0.01 level (2-tailed).

Time

-.827**

-.273

.000
21

.231
21

.000

21

1.000
21

.000

1.000
21

21

Number of parasites had negative correlation with temperature and time but significant for
temperature not for time at 0.01 significant levels for T.trichiura.
Table 54: Thermal Inactivation of A.duodenale eggs
Sample PS034 (Dry Weight) (A. duodenale EPG)
Temperature
Time
Initial count
15
50C
30
60
15
30
55C
60
15
60C
30
60
15
65C
30
60
15
70C
30
60
15
75C
30
60
15
80C
30
60

Inactivation rate (%)

1032
5.04%
18.02%
30.23%
16.96%
13.47%
33.72%
34.69%
58.33%
69.86%
51.07%
89.05%
100%
98.84%
100%
100%
100%
100%
100%
100%
100%
100%
135 |

1200
1000

800

15 mins

600

30 mins
60 mins

400
200

0
25C

50C

55C

60C

65C

70C

75C

80C

Figure 44: Time - temperature relationship in inactivation of A.duodenale ova

At 65C for 60 mins A.duodenale ova were fully inactivated but at and at 70C for 15
mins few eggs remained viable.

120.00%
100.00%
80.00%
60.00%
40.00%
20.00%
0.00%
15 30 60 15 30 60 15 30 60 15 30 60 15 30 60 15 30 60 15 30 60
50C

55C

60C

65C

70C

75C

80C

Figure 45: Inactivation rate of A. duodenale eggs

100% inactivation was performed at 65C for 60 mins and all other temperatures for every
time intervals but at 70C for 15 mins 98.84% killing was carried out in case of
A.duodenale ova.

136 |

Table 55: Time- temperature correlation in inactivation of A. duodenale eggs

Correlations
Parasites

Temperature

Time

Pearson
1
-.899**
-.201
Correlation
Parasites
Sig. (2-tailed)
.000
.382
N
21
21
21
Pearson
-.899**
1
.000
Correlation
Temperature
Sig. (2-tailed)
.000
1.000
N
21
21
21
Pearson
-.201
.000
1
Correlation
Time
Sig. (2-tailed)
.382
1.000
N
21
21
21
**. Correlation is significant at the 0.01 level (2-tailed).
Number of parasites had negative correlation with temperature and time but significant for
temperature not for time at 0.01 significant levels for A. duodenale eggs.
Table 56: Thermal Inactivation of A. duodenale larva
Sample PS034 (Dry Weight) (A. duodenale LPG)
Temperature

446

Initial count
50C

55C
60C

65C

70C

75C

80C

Inactivation rate (%)

Time
15
30
60
15
30
60
15
30
60
15
30
60
15
30
60
15
30
60
15
30
60

31.39%
41.03%
57.85%
37.67%
54.71%
68.16%
62.11%
83.86%
100%
96.19%
99.55%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
137 |

500

450
400
350

15 mins

300

30 mins

250

60 mins

200

150
100
50

0
25C

50C

55C

60C

65C

70C

75C

80C

Figure 46: Time - temperature relationship in inactivation of A.duodenale larva

At 60C for 60 mins, all the larva of A.duodenalewere killed but the larva survived at
65C for 15 mins and 30 mins of heat exposure.

120.00%
100.00%
80.00%
60.00%
40.00%
20.00%
0.00%
15 30 60 15 30 60 15 30 60 15 30 60 15 30 60 15 30 60 15 30 60
50C

55C

60C

65C

70C

75C

80C

Figure 47: Inactivation rate of A. duodenale larva

100% inactivation of A. duodenale larva was performed at 60C for 60 mins whereas,
65C killed 99.55% and 96.19% for 15 mins and 30 mins of heat treatment respectively.

138 |

Table 57: Time- temperature correlation in inactivation of A. duodenale larva

Correlations
Parasites

Temperature

Time

Pearson
1
-.832**
-.233
Correlation
Parasites
Sig. (2-tailed)
.000
.309
N
21
21
21
Pearson
-.832**
1
.000
Correlation
Temperature
Sig. (2-tailed)
.000
1.000
N
21
21
21
Pearson
-.233
.000
1
Correlation
Time
Sig. (2-tailed)
.309
1.000
N
21
21
21
**. Correlation is significant at the 0.01 level (2-tailed).
Number of parasites had negative correlation with temperature and time but significant for
temperature not for time at 0.01 significant levels for A. duodenale larva.
Table 58: Thermal Inactivation of S. stercoralis eggs
Sample PS034(Dry Weight) (S. stercoralis EPG)
Temperature
Time
Initial count
15
50C
30
60
15
30
55C
60
15
60C
30
60
15
65C
30
60
15
70C
30
60
15
75C
30
60
15
80C
30
60

Inactivation rate (%)

780
5.00%
16.03%
40.51%
10.64%
35.26%
58.59%
41.67%
70.00%
77.44%
71.92%
88.59%
96.54%
84.49%
100%
100%
100%
100%
100%
100%
100%
100%
139 |

900
800
700
600
500
400
300
200
100
0

15 mins
30 mins
60 mins

25C

50C

55C

60C

65C

70C

75C

80C

Figure 48: Time - temperature relationship in inactivation of S. stercoralis ova

At 70C for 30 mins S. stercoralis ova were fully inactivated.

120.00%
100.00%
80.00%
60.00%
40.00%
20.00%
0.00%
15 30 60 15 30 60 15 30 60 15 30 60 15 30 60 15 30 60 15 30 60
50C

55C

60C

65C

70C

75C

80C

Figure 49: Inactivation rate of S. stercoralis eggs

84.49% of inactivation of S. stercoralis eggs were performed at 70C for 15 mins of

treatment and 100% inactivation was performed at 70C for 30 mins.

140 |

Table 59: Time- temperature correlation in inactivation of S. stercoralis eggs

Correlations
Parasites

Temperature

Time

Pearson
1
-.881**
-.275
Correlation
Parasites
Sig. (2-tailed)
.000
.228
N
21
21
21
Pearson
-.881**
1
.000
Correlation
Temperature
Sig. (2-tailed)
.000
1.000
N
21
21
21
Pearson
-.275
.000
1
Correlation
Time
Sig. (2-tailed)
.228
1.000
N
21
21
21
**. Correlation is significant at the 0.01 level (2-tailed).
Number of parasites had negative correlation with temperature and time but significant for
temperature not for time at 0.01 significant levels for S. stercoralis eggs.
Table 60: Thermal Inactivation of S. stercoralis larva
Sample PS034 (Dry Weight) (S. stercoralis LPG)
Temperature

Time
262

Initial count
50C

55C
60C

65C

70C

75C

80C

Inactivation rate (%)

15
30
60
15
30
60
15
30
60
15
30
60
15
30
60
15
30
60
15
30
60

24.43%
44.66%
65.65%
34.35%
66.79%
77.86%
70.61%
91.22%
100%
98.85%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
141 |

300
250
200

15 mins

150

30 mins
60 mins

100
50
0
25C

50C

55C

60C

65C

70C

75C

80C

Figure 50: Time - temperature relationship in inactivation of S. stercoralis larva

S. stercoralis larva became inactivated at 60C for 60 mins of heat exposure.

120.00%
100.00%
80.00%
60.00%
40.00%
20.00%
0.00%
15 30 60 15 30 60 15 30 60 15 30 60 15 30 60 15 30 60 15 30 60
50C

55C

60C

65C

70C

75C

80C

Figure 51: Inactivation rate of S. stercoralis larva

100% inactivation was performed at 60C for 30 mins and all other above temperatures for
every time intervals.

142 |

Table 61: Time- temperature correlation in inactivation of S. stercoralis larva

Correlations
Parasites

Temperature

Time

Pearson Correlation
1
-.781**
-.270
Parasites
Sig. (2-tailed)
.000
.236
N
21
21
21
Pearson Correlation
-.781**
1
.000
Temperature
Sig. (2-tailed)
.000
1.000
N
21
21
21
Pearson Correlation
-.270
.000
1
Time
Sig. (2-tailed)
.236
1.000
N
21
21
21
**. Correlation is significant at the 0.01 level (2-tailed).
Number of parasites had negative correlation with temperature and time but significant for
temperature not for time at 0.01 significant levels for S. stercoralis eggs

Table 62: Thermal Inactivation of H.nana

Sample PS034(Dry Weight) (H.nana EPG)
Temperature
Initial count
50C

55C
60C

65C

70C

75C

80C

Time

Inactivation rate (%)

31

15
30
60
15
30
60
15
30
60
15
30
60
15
30
60
15
30
60
15
30
60

0.00%
16.13%
12.90%
19.35%
38.71%
41.94%
29.03%
48.39%
58.06%
38.71%
54.84%
70.97%
58.06%
74.19%
90.32%
100%
100%
100%
100%
100%
100%

143 |

35
30
25

15 mins

20

30 mins

15

60 mins

10
5
0
25C

50C

55C

60C

65C

70C

75C

80C

Figure 52: Time - temperature relationship in inactivation of H.nana

H. nana was fullyinactivated at 75C for 15 mins of heat exposure.

120.00%
100.00%
80.00%
Inactivation
rate of H. nana

60.00%
40.00%

20.00%
0.00%
15 30 60 15 30 60 15 30 60 15 30 60 15 30 60 15 30 60 15 30 60
50C

55C 60C

65C

70C

75C

80C

Figure 53: Inactivation rate of H. nana

100% H. nana were inactivated at 75C for 15 mins.

144 |

Table 63: Time- temperature correlation in inactivation of H. nana

Correlations
Parasites

Temperature
-.942**
.000
21
1

Pearson Correlation
1
Parasites
Sig. (2-tailed)
N
21
Pearson Correlation
-.942**
Temperature
Sig. (2-tailed)
.000
N
21
Pearson Correlation
-.220
Time
Sig. (2-tailed)
.339
N
21
**. Correlation is significant at the 0.01 level (2-tailed).

21
.000
1.000
21

Time
-.220
.339
21
.000
1.000
21
1
21

Number of parasites had negative correlation with temperature and time but significant for
temperature not for time at 0.01 significant levels for H. nana.
Table 64: Thermal Inactivation of E. histolytica cyst
Sample PS034 (Dry Weight) (E. histolytica CPG)
Temperature
Initial count
50C

55C
60C

65C

70C

75C

80C

Time

Inactivation rate (%)

46

15
30
60
15
30
60
15
30
60
15
30
60
15
30
60
15
30
60
15
30
60

17.39%
13.04%
19.57%
21.74%
36.96%
97.83%
95.65%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%

145 |

50
45
40
35
30
25
20
15
10
5
0

15 mins
30 mins
60 mins

25C

50C

55C

60C

65C

70C

75C

80C

Figure 54: Time - temperature relationship in inactivation of E. histolytica cysts

No E. histolytica cysts were found from 60C for 30 mins to all other above temperature.

120.00%
100.00%
80.00%
60.00%

Inactivation rate
of E. histolytica
cysts

40.00%
20.00%
0.00%
15 30 60 15 30 60 15 30 60 15 30 60 15 30 60 15 30 60 15 30 60
50C

55C 60C

65C

70C

75C

80C

Figure 55: Inactivation rate of E. histolytica cysts

100% inactivation of E. histolytica cysts was performed at 60C for 30 mins.

146 |

Table 65: Time- temperature correlation in inactivation of E. histolytica cysts

Correlations
Parasites
Pearson
1
Correlation
Parasites
Sig. (2-tailed)
N
21
Pearson
-.742**
Correlation
Temperature
Sig. (2-tailed)
.000
N
21
Pearson
-.152
Correlation
Time
Sig. (2-tailed)
.512
N
21
**. Correlation is significant at the 0.01 level (2-tailed).

Temperature

Time

-.742**

-.152

.000
21

.512
21

.000

21

1.000
21

.000

1.000
21

21

Number of parasites had negative correlation with temperature and time but significant for
temperature not for time at 0.01 significant levels for E. histolytica.
4.7.Biochemical Identification

Table 66: Biochemical test results

E. coli
Organism control

Isolated
E. coli
from cow
dung

Isolated
E. coli
from pit
soil

+
+
+

+
+
+

+
+
+
A
K

+
+
+
A
K

Tests
Gram stain
Dextose
Sucrose
Ornithine
Arginine
Lysine
Citrate
Urease
Indole
production
H2S production
Motility
Citrate
Catalase
Oxidase
Slant
Butt

+
+
+

Faecal
Isolated
streptococci Faecal
control
streptococci
from cow
dung
+
+
+
+
+
+
+
+

Isolated
Faecal
streptococci
from pit
soil
+
+
+
+

+
+
+
A
A

+
+
A
K

+
+
A
A

+
+
A
K

Note: A= acidic; K= alkaline

147 |

Suspected E. colifrom both sources was negative at gram staining. It also gave negative
results for Ornithine, Arginine,Lysine, Citrate, Urease, H2S production and Citrate, where
positive for Catalase, Oxidase, Sucrose, Dextose and Indole production which is similar
to that of E. colicontrol starin. In KIA media slant became acidic and butt became alkaline.
They also showed motility.
Faecal streptococci were suspected and phenotypically identified in different media. The
above biochemical test confirmed that the isolates were faecal streptococci.
4.8.Antibiotics Susceptibility Testing

Table 67: Diffusion zone (mm) for resistance against antimicrobial agents tested for
E. coli isolated from cow dung
E. colicow dung

E. colifrom pit soil

Antibiotics

Diffusion
zone

Sensitivity

Diffusion
zone

Sensitivity

Ampicillin

16

Cefixime

17

19

Ceftriaxone

27

25

Ciprofloxacin

21

18

Chloramphenicol

23

21

Gentamycin

14

16

Nalidixic Acid

21

17

Tetracycline

17

Trimethoprim/
Sulfamethoxazole

23

Polymixin B

13

12

Note: I= Intermediate, R= Resistant and S = Sensitive

Tetracycline, Sulfamethoxazole and Polymixin Bfrom two different isolates, E. coli of
cow dung showed resistance to Polymixin B and sensitivity to Ceftriaxone, Ciprofloxacin,
148 |

Chloramphenicol, Nalidixic Acid, Tetracycline, Sulfamethoxazole whereas isolates from

pit samples showed resistance to Ampicillin, Tetracycline, Sulfamethoxazole and
Polymixin B and sensitivity to Gentamycin, Cefixime, Ceftriaxone and Choloramphenicol.
Table 68: Diffusion zone (mm) for resistance against antimicrobial agents tested for
faecal streptococci isolated from cow dung
Antibiotics

Faecal streptococci from cow

dung
Diffusion
zone (mm)

Sensitivity

Faecal streptococci from pit soil

Diffusion zone

Sensitivity

Polymixin B

Tetracycline

19

19

Trimethoprim/
Sulfamethoxazole 19

26

Erythromycin

19

20

Penicillin G

23

18

Gentamycin

16

Ampicillin

24

23

Meropenem

20

12

Streptomycin

12

13

Ciprofloxacin

16

21

Note: I= Intermediate, R= Resistant and S = Sensitive

Enterococci of both isolates showed resistance to only Sulfamethoxazole and sensitivity to
Ampicillin, Penicillin G, Erythromycin, Meropenem, Streptomycin, Gentamycin,
Tetracycline and Polymixin B. Besides, Enterococcus of pit soil showed sensitivity to
Ciprofloxacin.

149 |

DISCUSSION
The present research was carried out to establish a model approach of killing or
inactivating pathogens (bacteria and parasites) through an optimum time and temperature
from cow dung and human pit, a usual object used in agriculture as fertilizer in
Bangladesh. This study provided the information about the optimum temperature and time
required to annihilate a pathogen from cow dung and human pits, a forthcoming fertilizer,
which is the first laboratory based experiment. The study showed different time and
temperature scale for different pathogens, where only bacteria and parasites taken into
consideration as pathogens for the investigation. While revealing optimum inactivation
time temperature scale, study also enriched the finding with giving information about the
prevalence and intensity of bacteria and parasites in cow dung and pit faecal sludge from
four different areas of Bangladesh.
Use of bio-fertilizer is increasing day by day with the increasing population to meet the
vast demand of crops and vegetable. Use of chemical fertilizers to the greater extent is
detrimental to health and environment. On the other hand, use of bio-fertilizer is
environmental friendly and increases the soil fertility. The present study was aimed to
produce a biologically safe fertilizer with the inactivation of pathogens present in cow
dung and faecal sludge as they represent common means of bio-fertilizer containing full
of organic nutrients. Because the term biosafety implicates the use of raw or under treated
cow dung and human pit as fertilizer, it accentuates their treatment to produce a
biologically safe manure, to be protected from possible health and environmental hazard
caused by raw cow dung or human sludge.
Moreover, there is a practice of using dried cow dung as fuel in many areas of Indian
subcontinent and also is a practice of disposal of cattle dung and sewage sludge in the
open place. There are several process to make the sludge become pathogen free, but
almost all of them use different chemicals like lime (CaO), soda lime (NaOH) amonea
(NH3) and so on (Espinoza et al., 2012; Nordin et al., 2009; Yilmaz and Iceme, 2014).
Those chemicals are either acidic or basic, so after treatment the PH changes and become
useless whereas the present study was conducted based only heat treatment, mostly safe
than others.

150 |

The abundance of parasites is more common in tropics where combination of moist, hot
climate and poor sanitary environment favor transmission in the community (Eng- Lam et
al., 1976). Bangladesh is a tropical country. Dohar, Keranigonj and Hajaribag are the
highly populated slam area. Many of them use faecal compost as fertilizer without any risk
assessment to produce vegetables and food grains which might provide a great source of
infection. So the community living here is surely vulnerable to pathogens especially when
they use the untreated fertilizer and during eating of uncooked vegetable produced
(Khanum et al., 2000).
During study period, the samples from different area were primarily analyzed to find out
the prevalence of parasites and bacteria in both cow dung and faecal sludge, latter one
highly positive sample from each type was accounted for a series of heat treatment. For
cow dung, a sample from Gopalgonj found highly positive and for faecal sludge, a sample
from Dohar was at highest number for A. lumbricoides and one from Hajaribag gave the
best positive result for other parasites. These highly contaminated samples were then
undergone with heat treatments. For bacteria, same samples were used for inactivation
because almost all the samples from both groups had greatest number of bacterial
community for all four bacterial groups.
Prior to heat treatment process centrifugal floatation technique was found as the best
parasite recovery technique among the quantitative techniques studied Cheesbrough
(2004) reported that formol ether is more effective in case of stool samples but floatation
is more appropriate for soil sample that adduces the current study.
While investigating the prevalence of different parasites individuals among two types, a
total six species of parasites were found in cow dung, three of them were nematodes
(Trichostrongyloides,

Strongyloides,

Haemonchus,

and

Toxocara),

trematode

(Paramphistomum) and a protozoan (coccidian). Crespo (1998) reported Strongyloides,

Haemonchus placet, Cooperia punctata, Trichuris and Oesophagostomum radiatum
Guinea Bissau. Leo Paldino et al. (1999) reported Strongyloides, Moniezia, Cooperia,
Trichuris, Trichostrongyloides, Haemonchus, Ostertagia, Oesophagostomum and
Dictyocaulus in Brazil. Hollad et al. (2000) found Toxocara vitulorum, Cooperia
punctata,

C.

pectinata;

Trichostrongyloides,

Oesophagostomum,

Fasciola

and

Paramphistomum in Vietnam. Landim et al. (2001), Borkovcova and Chlakdek (2002),

Keyyu et al. (2003) and several other authors reported helminthiasis in cattle in different
151 |

part of the world. The present study indicated 40% cattle were found infected with one or
more intestinal parasites where four species of nematode, namely Trichostrongyloides
(32.5%), Strongyloides (15%), Haemonchus (5%) and Toxocara (5%), one species of
trematode Paramphistomum (2.5%) and coccidian cysts (32.5%).
The current result resembles the reports of a number of different authors such as Hirani et
al. (2006) in Gujarat, Assuko (1983) in Ghana, Afzal et al. (1984) in Pakistan, Thilakan
and Satianesan (1997) in India and Hirani et al. (1999) in India reported 45.8%, 45.6%,
41.9%, 38.3% and 38.9% of cattle were infested by parasites, respectively. But higher
prevalence was reported by several authors, Keyyu et al. (2003) in Tanzania, FikruRegassa et al. (2006) in Oromia, Leo Poldino et al. (1999) in Brazil and Barbosa et al.
(1979) in Brazil who reported 97.2%, 69.6%, 69.3% and 59% cattle were infested with
helminth parasites, respectively. Moreover, in Bangladesh, Mutaleb (1996) reported 47%
of helminthiasis in buffalo calves.
On the other hand, a total seven parasite species were found from pit soil samples. Among
them one species of protozoan (Entamoeaba histolytica), one species of cestode
(Hymenolepis nana) and five species of nematodes (Ascaris lumbricoides, Trichuris
trichiura, Enterobius vermicularis, Ancylostoma duodenale and Strongyloides stercoralis)
were found. Begum and Rahman (1975) found five species of protozoa (E. histolytica, E.
coli, Endolimex nana, Iodamoeba butschii, G. lamblia) and four species of helminth (A.
lumbricoides, A. duodenale, T. trichiura and E. vermicularis) predominant in Bangladesh.
In another study, Muttalib et al. (1976) detected Entamoeba histolytica, Giardia
intestinalis, Ascaris lumbricoides, Ancylostoma duodenale, Trichiuris trichiura,
Fasciolopsis buski, Strongyloides stercoralis, Hymenolepis nana, and Oxyris vermicularis
from rural children of Bangladesh. Khanum et al. (2010, 2013) examined stool samples
collected from teacher, student, and stuff of the Dhaka University treated at Dhaka
University Medical Centre and found two protozoans (Entamoeba histolytica and Giardia
intestinalis) and tow nematodes (Ascaris lumbricoides and Trichuris trichiura).
From current study it was observed that the prevalence of A. lumbricoides was at highest
(47.5%) followed by S. stercoralis (30%), A.duodenale (27.5%), T. trichiura (22.5%), E.
histolytica (10%), E. vermicularis (7.5%) and H. nana (7.5%). Muttalib (1970) reported
that the infestation of Ascaris lumbricoides as 40%, A. duodenale as 8% and T. trichiura
as 18% cases. Hla (1970) reported 87.7% infestation of A. lumbricoides, 87.4% of T.
152 |

trichiura, 21.1% of Giardia and 5.7% infestation of E. histolytica. Tu et al. (1970), from a
village of Burma reported parasitism in 90% of their study population. Out of 90%, 59%
were positive for A. lumbricoides, 30% for A. duodenale and only 3% were positive for
Giardia infestation. Lower prevalence of Ascaris (11.84%) was observed by Reinthaler et
al. (1988) in Southwest Nigeria.
In 1994, Rahman observed the highest intensity for Ascaris lumbricoides followed by
hookworm. But in the current study, A.duodenale gave the highest intensity and E.
vermicularis gave the lowest in pit faecal sludge.
Dhar (2011) found 15% prevalence of Giardia spp in soil sample of Haor Basin as the
highest and Entamoeba spp as the lowest (2.5%) with E. vermicularis and S. stercoralis, a
prevalence of 2.5% each. In Joypur of Comilla, Jotirmoyee (2010) observed highest
percentage (23.33%) of Ascaris lumbricoides followed by Entamoeba histolytica
(18.06%), Taenia Spp. (14.89%), Strongyloides stercoralis (10.35%), Enterobius
vermicularis (9.24%), Ancylostoma duodenale (7.60%), Giardia lamblia (6.07%) and
lowest for Trichomonas hominis (2.37%).
The prevalence of all parasites in this study was higher than earlier studies except E.
vermicularis (7.5%) and E. histolytica (10%). No Giardia was found among the samples
examined. This fact might be due to different climate conditions or diagnostic methods
that were used in this study. Taren et al. (1988) found strong associations between the
socio-economic status of the children and infection with Ascaris lumbricoides, T. trichiura
and hook worm infestation in Panamanian. Ahmed (1986) reported the factors related to
prevalence of ascariasis and malnutrition were identified as low socio-economic status,
illiteracy of mother, poor environmental sanitation, overcrowding, unhygienic housing and
in sanitary disposal of refuses and human excreta. This can be assumed that the high
prevalence of parasites from three slum areas (Dohar, Keranigonj and Hajaribag indicate
low hygiene condition and poor socio-economic condition.
During investigating bacterial fauna in cow dung was found 100% prevalent for, total
coliforms, feacal coliforms and E.coli but faecal streptococci were 97.5% prevalent. On
the other hand, pit soil was 85% prevalent for Total coliforms, Feacal coliforms and E.coli
whereas, faecal streptococci were 82.5% prevalent. Mymensing was provided by highest
average for all studied bacteria. In case of pit samples total colioforms, faecal coliforms
153 |

and E. coli highest average were found in Hajaribag, where faecal streptococci in Dohar.
All the bacteria were isolated and identified through application of ISO methods
respective to each bacterium.
Biochemical tests were applied to further confirmation. Suspected E. coli from both
sources was negative at gram staining. It also gave negative results for Ornithine,
Arginine, Lysine, Citrate, Urease, H2S production and Citrate, where positive for Catalase,
Oxidase, Sucrose, Dextose and Indole production which were similar to that of E. coli
ATCC2922. In KIA media slant became acidic and butt became alkaline. They also
showed motility.
Faecal streptococci were found gram positive in gram stain and non motile. Others tests
showed the same results as at E.coli.
As the use of untreated bio-fertilizer implicates the bacterial infection, antimicrobial
susceptibility pattern tests were adopted to investigate their resistance or susceptibility to
some commonly used antibiotics. This investigation was aimed to asses their drug
resistance if a person is infected with any of the bacteria.
Antibiotics applied for susceptibility pattern test were namely Ampicillin, Cefixime,
Ceftriaxone, Ciprofloxacin, Chloramphenicol, Gentamycin, Nalidixic Acid, Tetracycline,
Sulfamethoxazole and Polymixin B. From two different isolates, E. coli of cow dung
showed resistance to Polymixin B and sensitivity to Ceftriaxone, Ciprofloxacin,
Chloramphenicol, Nalidixic Acid, Tetracycline, Sulfamethoxazole whereas isolates from
pit samples showed resistance to Ampicillin, Tetracycline, Sulfamethoxazole and
Polymixin B and sensitivity to Gentamycin, Cefixime, Ceftriaxone and Choloramphenicol.
Kibret and Abera (2011) reported E. coli high resistance rates to amoxicillin and
tetracycline and significantly high degree of sensitivity rates to gentamicin and
ciprofloxacin. Tadesse et al. (2012) recommended that resistance to gentamicin has
increased among animal E. coli isolates but present work indicated sensitive for both
isolates.
In case of enterococci, applied antibiotics were Ampicillin, Penicillin G, Erythromycin,
Meropenem, Ciprofloxacin, Streptomycin, Gentamycin, Tetracycline, Sulfamethoxazole
and Polymixin B. Enterococci of both isolates showed resistance to only
154 |

Sulfamethoxazole and sensitivity to Ampicillin, Penicillin G, Erythromycin, Meropenem,

Streptomycin, Gentamycin, Tetracycline and Polymixin B. Besides, enterococci of pit soil
showed sensitivity to Ciprofloxacin. Miskeen and Deodhar, (2002) demonstrated the
magnitude of resistance to both penicillin and ampicillin among the Enterococcus spp. was
23.13%, and that to ciprofloxacin was 55.78%. High-level aminoglycoside resistance
among strains of enterococci for streptomycin and gentamicin was found to be 33.84%
and 36.92%, respectively. All the strains were susceptible to the glycopeptide antibiotics
tested. Faecal streptococci isolates showed high-level resistance to gentamicin (Hllgren
et al., 2001).
The ultimate goal of the current study was to bring the bacterial and parasitic fauna under
heat treatment to annihilate them and making the cow dung and human pits fit for
agricultural use. Here, biological safety and security was considered as the concern if they
are used untreated in agricultural land. While investigating optimum time temperature for
killing the pathogens, study found different time temperature scale for bacteria and
parasites, independently. From both cow dung and pit soil, all the bacteria failed to grow
at 60C with an exposure of 30 minutes except faecal streptococci that was 65C for 60
minutes. Hence, a temperature more than 65C and a time duration more than 60 minutes
is enough efficient to kill the enteric pathogenic bacteria.
Claire Turner (2002) recommended that temperatures in excess of 55 C for 2 h are
required for inactivation of Escherichia coli 11943. Enterococcus faecalis were the most
heat resistant of the bacteria studied by Anthony et al. (2006), followed by the pathogenic
Escherichia coli O157:H7 and the non-pathogenic E. coli O3:H6. Klebsiella pneumoniae
displayed minimal heat resistance capacities. They also reported that at 65 C, little
thermal resistance was demonstrated by any species, with log reductions in concentration
occurring within seconds. They suggested that the temperature range from 55C to 65 C
was critical for effective elimination of enteric/pathogenic bacteria components. Thermal
inactivation of working suspension of Enterococcus faecium was performed between
temperature range 60C to 65C ( pelina et al., 2007), that support current study.
Current work showed a remarkable decrease of number in parasites and bacteria with the
increase in temperature and time. Both time and temperature were negatively correlated
with the number of parasites and bacteria. Temperature is highly significant in the
155 |

inactivation of parasites and bacteria. Among the parasites investigated, A. lumbricoides

and H. nana were the most resistant. They survived at 70C for 60 mins but at 75C
became 100% inactive within 15 mins. T. trichiura survived at maximum temperature of
70C for 15 minutes and after 30 minutes at same temperature it became 100% inactive.
100% inactivation rate was found for A. duodenale eggs at 65C in 60 mins but survived
at 70C for 15 mins. In case of A. duodenale larva 60C for 60 mins was required to
inactivate. S. stercoralis eggs survived at 70C for 15 minutes but 100% inactivation was
achieved within 30 mins, where S. stercoralis larva required 65C for 30 mins of
exposure. Moreover, E. histolytica took 60C for 30mins to become 100% inactive.
Storey and Phillips (1985) conducted an experiment for observing the survival rate of
parasite eggs throughout the soil profile by using lysimeters suggested that the eggs
of Taenia saginata and Ascaris lumbricoides survive for only a short time when applied to
pasture in sewage sludge.
The thermal death time for spiked A.lumbricoides ova was reported when a suspension of
1.7105 viable ova per ml was held at 60C was 60 mins or 30 mins at 65C (Wiley and
Westerbarg. 1969). To make the compost pathogen free it was required greater than 55 C
for at least 3 days (Wichuk and McCartney, 2007). Gotaas, (1956) reported that Ascaris
lumbricoides eggs, Taenia saginata, Necator americanus and Entamoeba histolytica cysts
were killed within 1 hour at temps over 50C, within a few minutes at 55C, within 50
minutes at 45C and within a few minutes at 45C respectively which is quite different
from the current findings because, in the current study faecal sludge were heated directly,
whereas the above studies used spiked parasites.
Among the parasites of cow dung, Paramphistomim and Haemonchus showed 100%
inactivation at 65C for 60mins and 30 mins. Trichostrongylus eggs and larva were fully
inactivated at 65C for 30 mins and 60C for 60 mins, respectively. 100% inactivation rate
was achieved at 60C for 60 mins and 60C for 30 mins in case of Strongyloides eggs and
larva respectively. Coccidian cysts took least temperature (60C) and time (15mins) to
inactivate.
Olsen and Nansen (1987) estimated by in vitro cultivation and baermanization, eggs of
Cooperia oncophora lost viability within 2 to 5 days at 35C and in less than 15 min at
53C. Dictyocaulus viviparus larvae did not survive 1 week at 35C. Eimeria oocysts were
156 |

morphologically unaffected after 50 days at 35C, 20C, and 4C and after 24 h at 53C.
Bruce et al., (1988) demonstrated that by pasteurisation intestinal parasite eggs such as
Ascaris suum and Taenia saginata which completely lost viability in 20 minutes at 70C
(Strauch and Berg, 1980). Exposure of Taenia saginata eggs to treatments of 60C for 15
minutes and 70C for five minutes eliminated their infectivity (Shafai, 1975). Pike and
Carrington (1985) observed the relationship between temperature and treatment time on
the treatment effect. A longer treatment time of three hours was required for pasteurisation
of sewage sludge at 55C to reduce infectivity of Taenia saginata eggs by 98.6%. Eggs of
helminths including Ascaris spp., Trichuris spp. and hookworms need a period of time,
outside the host body to develop and attain infective stage. Presence of these parasites in
the environment can be a public health indicator (Saathoff et al., 2002). The present study
provided the data about inactivation of nonspore forming bacteria and parasites in faecal
sludge and cow dung aiming to use as bio-fertilizer or disposal in a secured way.
Finally, 75C was found lethal for all the pathogens studied. Within 15 mins at 75C all
the parasites and bacteria were found to be dead.

157 |

CONCLUSIONS
Cow and humanure is a valuable resource suitable for agricultural purposes and has been
recycled for such purposes by large segments of the world's human population for
thousands of years. However, they contain the potential for harboring human pathogens,
including bacteria, viruses, protozoa and parasitic worms or their eggs, and thereby can
contribute to the spread of disease when improperly managed or when discarded as a
waste material. When pathogenic raw manure is applied to soil, pathogenic bacteria may
continue to survive in the soil for over a year, and round-worm eggs may survive for many
years, thereby maintaining the possibility of human reinfection for lengthy periods of time.
Earlier studies used chemicals along with temperature, which increases or decreases the PH
of the manure. But this study set up a new dimension of viable processing of human and
cow dung. However, when the manure is heated, pathogens are destroyed and the manure
is thereby converted into a hygienically safe form suitable for soil applications for the
purpose of human food production.
Thermal processing produces no waste, no pollutants and no toxic by-products and dont
requires any chemicals, so that the PH remains same. Thermal inactivation process has no
stress on our ecosystems, any unnecessary consumption of resources and any garbage or
sludge for our landfills. A large number of pathogenic viruses, bacteria, protozoa and
parasites may gain access to waste materials including those destined for composting. It is
possible to compile extensive lists of organisms which may contaminate wastes and
present a risk of human or animal infection when they are utilized for most the risk is
theoretical.
The current study also showed centrifugal floatation technique was best to recover
helminths from faecal sludge, though it may differ with floatation solutions and sample
types. Extensive studies are required in these parasites recovery techniques.
Extensive studies on large scale inactivation scheme and their field implication is required.

158 |

To minimize the possible health risk following recommendations should be considered:

1. Treatment is required before use of faecal matter.
2. Proper isolation and identification is essential.
3. Regular monitoring is required.
4. Row manure should be avoided to use.
5. People must know about the possible routes of transmission of parasitic diseases
from various sources.
6. Dumping of domestic and faecal waste should be managed in secuired way.
7. Waste of domestic and firm cattle should be dispose in hygienic way.
8. Every home should be provided with sanitary toilet and proper disposal of human
feces.
9. People should not walk on bear foot.
10. Row vegetables eating should be avoided.
11. Biosafety and biosecurty rules should be followed.

159 |

BIBLIOGRAPHY
Afzal, M., Shafique, M., Hossain, A. and Saeed, M., 1984.A study of helminthes in cattle
and buffaloes in Lahore.Pakistan Journal of Science. 33: 14-20.
Ahmed, N.U., Mostofa. M., Awal, M., Awal, A. and Alam, M.N. 1994. Comparative
efficacy of modern anthelmintics with that of neem seeds against gastrointestinal
nematodiasis in sheep. Bangladesh Veterinary Journal. 28: 21-23.
Alam, M. J., and Zurek, L. 2004. Association of Escherichia coli O157: H7 with
houseflies on a cattle farm. Applied and Environmental Microbiology.70(12):75787580.
Al-Dulaimi, S.S., Jassin, B.A. and Molan, A.L. 1985.A survey on some gestro-intestinal
helminth parasites in sheep of Arbil (in Iraq).Zanco (Iraq). 3: 73-80.
Ali, M., Emch, M., Donnay, J.P., Yunus, M. and Sack, R.B.2002.The spatial epidemiology
of cholera in an endemic area of Bangladesh.Soc Sci Med. 55:101524.
Ambrosi, M. , Adorisio, P.L. and Grelloni, V. 1986. Endoparasites of cattle in areas of
Appenines; Preliminary studies in Umbira and Marche regions.Atti della Society
Italiana of Buiatria. 18:571-577.
Anon, 1997.Interim advice from the Health and Safety Executive on laboratory work with
vero toxin-producing Escherichia coli.Communicable Disease Report.
APHA/AWWA/WEF, 2005.Standard Methods for the examination of water and
wastewater.21st Edition.American Public Health Association, American Water Works
Association, and Water and Environment Federation Publication. Washington D.C.,
USA.
Assoku, R. R.G. 1983. Gastro-intestinal parasites of cattle in Ghana.Bulletin of Animal
Health and Production in Africa. 31: 223-230.
Ayres R. 1989. A Practical Guide for the Enumeration of Intestinal Helminths in Raw
Wastewater and Effluent from Stabilization Ponds.Leeds University Department of
Civil Engineering.
160 |

Ayres R. and Mara, D. 1996. Analysis of wastewater for use in agriculture.A laboratory
manual of parasitological and bacteriological techniques. Geneva. :31.
Ayres, R., Alabaster, G., Mara, D. and Lee, D.1992. A Design Equation for Human
Intestinal Nematode Egg Removal.In: Waste Stabilization Ponds.Water Research.
26(6): 863-866.
Bach, S. J., McAllister, T. A., Veira, D. M., Gannon, V. P. J., and Holley, R. A. 2002.
Transmission and control of Escherichia coli O157: H7-A review. Canadian journal
of animal science. 82(4):475-490.
Barbosa, C.S., Nonato, G.R., Santigo, G.E. and Casta, M.J.L.D.A., 1979. Study of
helminthes in dairy cattle in the region of Fortaleza Ceara.Revista do centro de
ciencias Rurais. 4:387-390.
Beaier, R. B. and Dunsmore, J.D. 1993. The ecology of Haemonchus contortus in a winter
rainfall region in Australia: the development of eggs to infective larva. Veterinary
Parasitology.45 (3-4): 275-292.
Begum, N. N. and Rahman, K. M. 1975.Comparative finding of helminth and protozoa by
various techniques.Bang. Med. J. 4: 8-12.
Bethony, J.; Brooker, S.; Albonico, M.; Geiger, S. M. and Loukas, A. 2006. Soil
transmitted helminth infections: ascariasis, trichuriasis, and hookworm. The Lancet
Infectious Disease.367: 15211532.
Blood, D.C., Radostits, O.M., Henderson, J.A., Arundel, J.H. and Gay, C.C. 1990.
th

Veterinary Medicine. 7

edn.,The English Language Book Society and Bailliere

Tindall.
Bruce, A.M. and Davis, R.D. 1983.Utilisation of sewage sludge in agriculture.
Maximizing benefits and minimizing risks. In Biological Reclamation and Land
Utilisation of Urban Wastes Ed. Zucconi, F., De Bertoldi, M. and Coppola, S.
Proceedings of the International Symposium, Naples. October (11-14):121-142.
Bundy DAP, 1997. This wormy world then and now.Parasitology Today.13:407408
161 |

Carrington E., Pike E., Autry D. and Morris R. 1991. Destruction of faecal bacteria,
enteroviruses and ova of parasites in wastewater sludge by aerobic thermophilic and
anaerobic mesophilic digestion.Water Science and Technology. 24(2): 377-380.
Capizzi-Banas, S., Deloge, M., Remy, M. and Schwartzbrod, J. 2001. Liming as an
advanced treatment for sludge sanitization: Helminth eggs elimination Ascaris eggs
as model.Water Research. 38:3251-3258.
Capizzi-Banas, S., Deloge, M., Remy, M. and Schwartzbrod, J. 2004. Liming as an
advanced treatment for sludge sanitization: helminth eggs elimination - Ascaris eggs
as model.Water Research. 38(14-15):3251-3258.
Cappello M. 2004. Global health impact of soil-transmitted nematodes.Pediatr Infect Dis
J. 23:663664.
Chapman, P.A., Siddons, C.A., Wright, D.J., Norman, P., Fox, N.J. and Crick, E.
1993.Cattle as a possible source of verocytotoxin-producing Escherichia coli O157
infections in man.Epidemiology and Infection.111:439-447.
Chavez A., Jimnez B. and Maya C. 2004.Particle size distribution as a useful tool for
microbial detection Water Science and Technology. 50(2):179-186.
Cheesbrough, M. 1998, Diseases In: District Laboratory Practice in Tropical Countries
Low Price Edition. Cheesbrough M (Edition). Cambridge University Press, United
Kingdom. :185-300.
Cieslak, P.R., Barrett, T.J., Griffin, P.M., Gensheimer, K.F., Beckett, G., Buffington, J.
and Smith, M.G. 1993. Escherichia coli O157:H7 infection from a manured garden.
Lancet.342:8867.
Cody, S.H., Glynn, M.K. and Farrar, J.A. 1999. An outbreak of Escherichia coli infection
from unpasteurized commercial apple juice.Annals of Internal Medicine.130:202-209.
Cohen, M. L. 1992. Epidemiology of drug resistance: Implications for a post
Antimicrobial era. Science. 257(5073):1050-1055.

162 |

Crespo, M. V. M. M. 1998. A preliminary study of the nematode cattle from the Republic
of Guinea Bissau.Gareia de Ona Serie de Zoologia. 22: 63-67.
Crompton, D. W. T. 1999. How much human helminthiasis is there in the world? J.
Parasitol.85:379403.
Curtale, F.; Pezzotti, P.; Sharbini, A. L., Al Maadat, H. and Ingrosso, P. 1998.
Knowledge, perceptions and behaviour of mothers toward intestinal helminths in
Upper Egypt: implications for control. Health Policy Plan. 13: 423432.
Debnath, N.C., Taimur, M.J.F.A., Saha, A.K., Ersaduzaman, M., Heleluddin, M., Rahman,
M.L., Roy, D.K. and Islam, M.A. 1995.A retrospective study of calf losses on the
central dairy cattle breeding station in Bangladesh.Preventive Veterinary Medicine.
24: 4353.
de Silva, N.R., Brooker, S., Hotez, P.J., Montressor, A., Engels, D. And Savioli, L. 2003.
Soil-transmitted helminth infections: updating the global picture. Trend Parasitol. 19:
54751.
De Victorica, J. and Galvan, M. 2003. Preliminary testing of a rapid coupled methodology
for quantization/viability determination of helminth eggs in raw and treated
wastewater. Water Research. 37(6):1278-87.
Dewan, M.L., Hossain, M.I., Baki, M.A. and Uddin, M. 1979. Observation on the visceral
larval migration of Toxocara(Neoascaris) vitulorumin buffalo cow. Bangladesh
Veterinary journal. 13(2-4): 35-37
Dhar, R. C. 2011. Faecal contamination of soil: Probable source of Geohelminth and
Giardiasis among children in Haor areas of Bangladesh. 98 pp.
Diesch, S.L. 1974) cited by Strauch (1978)
Dumontet, S., Dinel, H. and Baloda, S.B. 1999. Pathogen reduction in sewage sludge by
composting and other biological treatments: A review. Biological Agriculture and
Horticulture.16:409-430.

163 |

EckertJ., PerlR. and InderbitzinF..1981. Significance of nematodiasis in cattle grazing on

alpine pasture. InEpidemiology and Control of Nematodiasis in Cattle.Springer,
Netherlands.:177191.
Eduar A. Bravo, Arturo J. Zegarra, Alejandro Piscoya, Jose L. Pinto, Ra ul E. de los
Rios,Ricardo A. Prochazka, Jorge L. Huerta-Mercado, Jaime Cok, andMartin Tagle,
2011. Dimorphic Fungal Co-infection as a Cause of Chronic Diarrhea and
Pancolitis.Case Reports in Medicine.
Edwards, P. R. and Ewing, W. H. 1972.Identification of Enterobacteriaceae. Minneapolis:
Burgess Publish- ing Co.
Eichenseher, T. 2008. Human Waste Used by 200 Million Farmers, Study Says. National
Geographic News, Stockholm, Sweden.
Ellis, J.R. and McCalla, T.M. 1978. Fate of pathogens in soils receiving animal wastes A
review.Transactions of the ASAE.21(2):309-319.
Emch.M., Ali, M. and Yunus, M. 2008.Risk areas and neighborhood-level risk factors for
Shigella dysenteriae 1 and Shigella fexneri.Health Place.14:96115.
Epstein, E. 2002. Human pathogens: hazards, controls and precautions in compost. In:
Compost utilization in horticultural cropping systems. Lewis Publishers, Boca
Raton, USA.: 361-380.
Erdman, D.D. 1981. Clinical Comparison of Ethyl Acetate and Diethyl Ether in the
Formalin-Ether Sedimentation Technique, Journal of Clinical Microbiology. 14:483485
Eslami, A. and Hoseini, S. 1989. Investigation and review of the cattle parasitic infections
in farms around Tehran.Journal of Veterinary faculty(Iran Islamic Republic). 44: 3541.

164 |

Evans, Jr. D.J. and Evans.D.G. 1996.Chapter 25: Escherichia Coli in Diarrheal Disease.
Medical Microbiology.4th edition.
Evans Jr, D. J., Dolores, G. E. and Evans, G. 2007. Escherichia coli, Medical
Microbiology.
Facklam, R.R. and Collins, M.D. 1989. Identification of Enterococcus species isolated
from human infections by a conventional test scheme. Journal of Clinical
Microbiology. 27:731734.
Facklam, R.R. and Sahm, D.F. 1995. Enterococcus .In Manual of Clinical Microbiology ,
6th edn. eds Murray, P.R., Baron, E.J., Pfaller, M.A., Tenover, F.C. and Yolken, R.H.
ASM Press. Washington DC: 308314.
Faruque, S. M., Albert, M. J. and Mekalanos, J. J., 1998.Epidemiology, Genetics, and
Ecology of Toxigenic Vibrio cholerae.Microbiology and Molecular Biology
Reviews.62(4): 13011314.
Faust. E. C. 1924. Some Facts Regarding the Relation Between Nightsoil Disposal in
China and the Propagation of Helminthic Diseases. Am. Jour. Trop. M ed. 4:487.
Faust E. C., Sawitz W., Tobie J., Odem V. and Peres C. 1939.Comparative efficiency of
various techniques for the diagnosis of protozoa and helminths in faeces.Journal
Parasitology. 25: 241-262.
Feachem, R.G., Bradley, D.J., Garelick, H., Mara, D.D. 1983. Sanitation and
Disease.Health aspects of excreta and wastewater management. World Bank studies
in water supply and sanitation. John Wiley and Sons. New York.
Feachem, R., Bradley, D., Garelick, H., Mara, D. 1983. Sanitation and Disease: Health
Aspects of Excreta and Wastewater Management. John Wiley and Sons.New York,
NY.:349-356
Fischetti, V.A., Novick, R.P., Ferretti, J.J., Portnoy, D.A. and Rood, J.I. 2000. GramPositive Pathogens.ASM Press.ISBN 1-55581-166-3.

165 |

French, G.L. 1998. Enterococci and vancomycin resistance.Clinical Infectious Disease.

27(Suppl.),S75S83.
Gantzer, C., Gaspard, P., Galvez, L., Huyard, A., Dumouthier N. and Schwartzbrod, J.
2001. Monitoring of bacterial and parasitological contamination during various
treatment of sludge.Water Research. 35(16):3763-3770.
Garcia, L. S. 2001. Diagnostic medical parasitology.ASM Press,Washington, DC. :872.
Garzon, M. 2003. Parasites- A Holistic Approach. In: Associates NIH, editor, Capital
University of Integrated Medicine.: 527.
Gaspard, P.G., Wiart, J. and Schwartzbrod, J. 1995. Urban sludge reuse in agriculture:
waste treatment and parasitological risk. Bioresource Technology. 52:37-40.
Gehlbach, S. H., Maccormack, J. N., Drake, B. M., and Thompson, W. V. 1973. Spread of
disease by fecal-oral route in day nurseries. Health services reports: 88(4):320.
Gotaas, H. B. 1956. Composting - Sanitary Disposal and Reclamation of Organic Wastes.
World Health Organization, Monograph Series Number 31. . p.81. Geneva.
Hllgrena, A., Abednazarib, H., Ekdahlb, C.,

Hanbergerb, H.,

Nilssona, M.,

Samuelssona, A., Svenssona, E., Nilssona L.E. and the Swedish ICU Study Group,
2001. Antimicrobial susceptibility patterns of enterococci in intensive care units in
Sweden evaluated by different MIC breakpoint systems, J. Antimicrob. Chemother.48
(1): 53-62.
Hancock, D.D., Besser, T.E. and Rice, D.H. 1993. Macro- and micro-epidemiology of E.
th

coli O157:H7 prospects for pre-harvest control. In: 97 Annual meeting of the US
Animal Health Association.
Hespanhol, I. 1997. Waste water as a resource. In: Water pollution control. A Guide to the
use of Water Quality Management Principles (Eds. Heimer, R. and Hespanhol, I.E.
and Spon, London on behaif of UNESCO, WHO and UNEP).

166 |

Heuvelink, A. E., Van Heerwaarden, C., Zwartkruis-Nahuis, J. T. M., Van Oosterom, R.,
Edink, K., Van Duynhoven, Y. T. H. P., & De Boer, E. 2002. Escherichia coli O157
infection associated with a petting zoo. Epidemiology and infection. 129(02):295-302.
Hijnen, W.A.M., Beerendonk, E.F. andMedema, G.J. 2006. Inactivation credit of UV
radiation for viruses, bacteria and protozoan (oo) cysts in water: A review, Water
Research. 40(1):322
Hilborn, E.D., Mermin, J.H., Mshar, P.A., Hadler, J.L., Voetsch, A., Wojtkunski, C.,
Swartz, M., Mshar, R., Lambert-Fair, M.A., Farrar, J.A., Glynn, M.K. and Slutsker, L.
1999. A multistate outbreak of Escherichia coli O157:H7 infections associated with
consumption of mesclun lettuce. Arch Intern Med. 159:17581764.
Hirani, N. D., Katariya, M. A., Abdullah- Patel, Hasnani, J. J., Kathiria, L. G. and Patel, P.
V., 1999. Prevalence of gastro-intestinal parasitic infections in cattle and buffaloes of
Kheda district of Gujrat.Journal of VeterinaryParasitology. 13: 147-149.
Hirani, N. D., Patel, P. V. and Joshi, R. S., 2006.Prevalence of gastro-intestinal parasites
in cows of Gaushalas in and around Anand.Gujrat- a coprological survey.Indian
Journal of Field Veterinarians.1 : 23-25.
Holland, W.G., Luong, T.T., Nguen, L.A. Do, T.T. and Vercruysse, J. 2007. The
epidemiology of nematode and fluke infections in cattle in the Red River Delta in
Vietnum.Veterinary Parasitology. 93: 141-147.
Hooper, L.V. and

Gordon, J.I., 2001. Commensal host-bacterial relationships in the

gut.Science. 292(5519):1115-8.
Hotez, P.J., Brooker, S., Bethony, J.M., Bottazzi, M.E., Loukas, A. And Xiao, S. 2004.
Hookworm Infection.N Engl J Med. 351(8): 799-807.
Hotez, J.P., De Silva N., Brooker, S. And Bethony, J. 2003. Soil-Transmitted Infections:
The Nature, Causes and Burden of the Condition. Working paper No.3, Disease
Control Priorities Project Bethesda, Maryland: Fogarty International Center, National
Institute of Health.

167 |

Hotez, J.P., Molyneux, D.H., Fenwick, A., Ottesen, E., Sachs, S.E. and Sachs, J.D. 2006.
Incorporating a rapid-impact package for neglected tropical diseases with programs
for HIV/AIDs, Tuberculosis and Malaria.PLoS Medicine. 3(5):001-009.
Hotez, P.J., Bethony, J., Bottazzi, M.E., Brooker.S., Diemert, D. and Loukas, A.
2006.New technologies for the control of human hookworm infection.Trends in
Parasitology.22: 327-331.
Hotez, P.J., Brooker, S., Bethony, J.M., Bottazzi, M.E., Loukas, A. and Xiao, S. 2004.
Hookworm infection.New England Journal of Medicine.351: 799-807.
Huq, F., Hamid, A., Ali, S., Asaduzzaman, M. And Yesmin, M. 1982. Epidemiological
study and comparison of pyrantel and levamisol in the treatment of round worm and
hook worm infections. Bangladesh Medical Research Council Bull. 8(1): 1-6.
Institute of Medicine of the National Academies, 2002
Islam, K.S., Shaikh, H. and Soliman, K.N. 1971. Occurrence of visceral Schistosomiasis
of cattle and goats in East Pakistan.Proc. Pakistan Science Conference, University of
Peshwar.:16.
Jimnez B. and Wang L. 2006. Sludge Treatment and Management, Chapter 10, pp 237292 in Municipal Wastewater Management in Developing Countries: Principles and
Engineering, Ujang Z. and Henze M. Editors. International Water Association
Publishing. London, U.K.
Jimenez, B. 2007.Helminth ova removal from wastewater for agriculture and aquaculture
reuse.Water Science & Technology. 55(1-2):485493.
Jimenez-Cisneros, B. E. 2007.

Helminth Ova control in wastewater and sludge for

agricultural reuse, In Water and Health, [Ed.W.O.K. Grabow], In Encyclopedia of

Life Support Systems (Eolss), Developed under the auspices of the Unesco, Eolss
Publishers, Oxford ,UK.
Jimnez, B. 2008.Helminth Ova Control in Wastewater and Sludge for Agricultural Reuse.
In: W.O.K. Grabow (ed.) Water reuse new paradigm towards integrated water

168 |

resources management in Encyclopedia of Biological, Physiological and Health

Sciences, Water and Health. 2:429-449.
Jimnez, B., Mara D., Carr, R. and Brissaud, F. 2010. Wastewater treatment for pathogen
removal and nutrient recovery: Suitable systems for use in developing countries,
Chapter 8 in: Wastewater Irrigation and Health: Assessing and Mitigating Risks In
Low-Income Countries, Dreschel and Scott Editors, Earthscan Press.London.:149170.
Jones, P.W. 1976. The effect of temperature, solids content and pH in the survival of
salmonellas in cattle slurry.British Veterinary Journal.132:284-293.
Jones, P.W., Rennison, Lynne M., Lewin, V.H. and Redhead, D.L. 1980(a). The
occurrence and significance to animal health of salmonellas in sewage and sewage
sludge. Journal of Hygiene.84:47-62.
Jones, P.W., Rennison, Lynne M., Matthews, P.R.J., Collins, P. and Brown, Anne
1980(b). The occurrence and significance to animal health of Leptospira,
Mycobacterium, Eschericha coli, Brucella abortus and Bacillus anthracis in sewage
and sewage sludge. Journal of Hygiene, Cambridge.86:129-137.
Jones, P.W. 1980. Health hazards associated with the handling of animal wastes.
Veterinary Record.106: 4-7.
Jones, P.W. 1981. A short survey of the most important pathogens found in farm animal
wastes. In: Communicable diseases resulting from storage, handling, transport and
land spreading of manures Ed. Walton and White. Luxembourg CEC, EUR 7627
EN.:139-147.
Jones, P.W. 1982. Waste and animal health.The Public Health Engineer.10:35-39.
Jones, P.W. 1984. The survival and infectivity for cattle of salmonellas on grassland. In:
Processing and Use of Sewage Sludge, Ed P. lHermite and H. Ott. D. Reidel
Publishing Co., Dordrecht, Holland: 178-190.

169 |

Jones, P.W. 1986. Sewage sludge as a vector of salmonellosis. In: Epidemiological studies
of risks associated with the agricultural use of sewage sludge: Knowledge and needs.
Ed: Block, J.C., Havelaar, A.H. and Hermite, P. Elsevier, London. :21-33.
Jones, P.W. 1992. Pathogenic bacteria in animal wastes and hazards for any other animals
and humans from handling animal wastes.Montava, Italy, EU.
Joshi,B. R.,Gibbons,L.M. and Jacobs,D. E.1997. Ostertagia nianqingtanggulaensis K'ung
& Li, 1965 (Nematoda: Trichostrongyloidea) from sheep and goats at high altitudes in
Nepal. J. Helminthol. 71: 2128.
Jotirmoyee. 2010. Presence of intestinal parasites (eggs and larvae) in soil at Joypur under
Comilla district, Bangladesh.: 130
Knappett, P.S.K, Escamilla, V., Layton, A., McKay. L.D., Emch, M., Williams, D.E.,
Huq, M. R., Alam, M.J., Farhana, L., Mailloux, B.J., Ferguson, A., Sayler, G.S.,
Ahmed, K.M. and van Geen, A. 2011a. The impact of sanitation on fecal bacteria and
pathogens in village ponds in Bangladesh.Sci. Tot. Environ. 409:31743182.
Keller, R., Passamani, F., Cassini, S. and Goncalves, F. 2004.Disinfection of sludge using
lime stabilization and pasteurization in a small wastewater treatment plant.Water
Science and Technology. 50(1): 13-17.
Keyyu,

J.D.,

Kassiiku,

A.A.,

Kyvsgaard,

N.C.

and

Willingham,

A.

L.

2003.Gastrointestinal nematodes in indigenous, zebra cattle under pastoral

andnormadic management system in the lower plain of the southern highlands of
Tanzania, Veterinary Research Communication. 27: 351-380.
Khakhria, R. 1998. Escherichia coli O157 [ratio] H7 diarrhoea associated with well water
and infected cattle on an Ontario farm. Epidemiology and Infection.120(1):17-20.
Khanum, H., Alam, M.S. 2005. Infection of Ascaris lumbricoides and Trichuris trichiura
among the children of two slum area in Dhaka city.Bangladesh J. Zool. 33(1): 89-95.

170 |

Khanum, H., Ahmed, S., Uddin, M. H., Rahman. A. B. M. M., Dey, R. R. and Farhana, R.
2008. Prevalence of intestinal parasites and anaemia among the slum male children in
Dhaka city.Dhaka Univ. J. Biol. Sci. 17(2): 137-145.
Khanum, H.; Islam, N. and Dhar, T. 1997.Prevalence of Ascaris lumbricoides and
Trichuris trichiura among the children of four slum areas of Dhaka city.Univ. J. of
Zool. 16: 89-94.
Khanum, H. Islam, M. R. and Parvin, S. 2010. Occurrences of eggs and larvae of
gastrointestinal nematodes in nails of street inhabitants in Dhaka City.J. Life Earth
Sci. 5: 75-79.
Khanum, H. Rahman, F. and Zaman, R. F. 2013.Occurrence of intestinal parasites among
the teachers, students and staffs of Dhaka University.J. Asiat. Soc. Bangladesh, Sci.
39(2):239-246.
Kibret, M. and Abera, B.2011.Antimicrobial susceptibility patterns of E. coli from clinical
sources in northeast Ethiopia, Afr Health Sci. 1:4045.
Kloos, H., Lemma, A., Kibru, B., Gebre, A., Mazengia, B., Feleke, G. and DeSole, G.
1980. Intestinal Parasitism in migrant farm labour populations in the Awash Valley,
Ethiopia, and in major labour source areas. Ethiop. Med .J. 18: 52-61.
Kon, D. and Strauss, M. 2004. Low-cost Options for Treating Faecal Sludges (FS) in
Developing Countries Challenges and Performance. Paper presented to the 9th
International IWA Specialist group conference on wetlands systems for water
pollution control; and to the 6th International IWA Specialist Group Conference on
Waste Stabilisation Ponds, Avignon, France, 27th Sept. - 1st Oct. 2004
Kone, D., Cofie, O., Zurbru, C., Gallizzi, K., Moser, D., Drescher, S. and Strauss, M.
2007. Helminth eggs inactivation efficiency by faecal sludge dewatering and cocomposting in tropical climates. Water Resarch.41(19): 4397-4402.
Korten, V. and Murray, B.E. 1997.Enterococcus. In Principles and Practice of Clinical
Bacteriology eds Emmerson, A.M., Hawkey, P.M. and Gillespie, S.H. John Wiley &
Sons Ltd.: 93-107
171 |

Kosek, M., Bern, C. and Guerrant, R.L. 2003. The global burden of diarrhoeal disease, as
estimated from studies published between 1992 and 2000. Bulletin of the World
Health Organization. 81: 197-204.
Kudva, I.T., Hatfield, P.G. and Hovde, C.J. 1996. Escherichia coli O157:H7 in microbial
flora of sheep. Journal of Clinical Microbiology.34:431-433.
Kudva, I.T., Hatfield, P.G. and Hovde, C.J. 1997. Characterization of Escherichia coli
O157:H7 and other Shiga toxin-producing E. coli isolated from sheep. Journal of
Clinical Microbiology.35:892-899.
Kmmerer, K. 2004. Resistance in the environment. Journal of Antimicrobial
Chemotherapy.54(2):311-320.
Larocque. R., Casapia, M., Gotuzzo, E.and Gyorkos, T.W.2005.Relationship between
intensity

of

soil-transmitted

helminth

infections

and

anemia

during

pregnancy.American Journal of Tropical Medicine and Hygiene.73:783789.

Lee, J. and Kaletun, G. 2002. Evaluation of the Heat Inactivation of Escherichia coli and
Lactobacillus plantarum by Differential Scanning Calorimetry.Appl Environ
Microbiol. 68(11): 53795386.
Leo Poldino, M. V., mundim, M. J.S. Mundum , A. V. , Cabral, D.D., Barbosa, F.C. and
Nolasco, R.M. 1999. Endoparasites in naturally infected cattle from the Triangulo
Mineiro region, Brazil.Veterinary Noticias. 5: 41-46.
Le Riche, P.D., Kuhne, G. I.and Dwinger, R.H. 1982. An epidemiological study of
helminthiasis in cattle in subtropical Argentina.Tropical Animal Helth and
Production. 14: 207-215.
Levecke B, Behnke JM, Ajjampur SSR, Albonico M, Ame SM, et al. 2011. A comparison
of the sensitivity and fecal egg counts of the McMaster egg counting and Kato-Katz
thick smear methods for soil-transmitted helminthes. PLoS Negl Trop Dis. 5(6):1201.
Lipsitch, M., and Samore, M. H. 2002. Antimicrobial use and antimicrobial resistance: a
population perspective. Emerging Infectious Diseases.8(4):347-354.

172 |

Malicki, J., Montusiewicz, A. and Bieganowski, A. 2001. Improvement of counting

helminthes eggs with internal standard. Water Research. 35(9):2333-2335.
Mara, D. D. 2004.Domestic wastewater treatment in developing countries.Earthscan UK
and US.ISBN 1-84407-019-0.
Marcus, N., Peled, N. and Yagupsky, P. 1997. Rapid increase in the prevalence of
antimicrobial drug resistance among enterococcal blood isolates in southern Israel
.European Journal of Clinical Microbiology and Infectious Diseases. 16:913-915.
Markell, E.K. and Voge, M. 1971. Medical Parasitology.3rd Ed. W.B. Sounders Company.
Philadelphia London.Toronto.: 171-173.
Marnu, W., Wintersteller, E. and Prosl, H. 1987. Monthly and seasonal fluctuations in
abomasal nematode worm burden of normally infected cattle in Austria. Veterinary
Parasitology. 23: 237-248.
Maya, C., Jimnez, B. and Schwartzbord, J. 2006. Comparison of Techniques for the
Detection of Helminth Ova in drinking water and Wastewater.Water Environment
Research. 78(2): 118-124.
Maya, C., ,Torner-Morales, F.J., Lucario, E.S., Hernndez, E. and Jimnez, B. 2012.
Viability of six species of larval and non-larval helminth eggs for different conditions
of temperature, pH and dryness. Water Research.46(15):47704782.
Megran, D.W. 1992. Enterococcal endocarditis.Clinical Infectious Disease.15:63-71.
Melo, H.J.H. and Bianachin, I. 1977. Epidemiological study of gastrointestinal nematode
infections of beef cattle reared in the savanna area of the state of Moto Grosso.
Pesquisa-Agropecuiria-Brasileria.12:205-216;57.
Miskeen, P.A.and Deodhar, L. 2002. Antimicrobial susceptibility pattern of Enterococcus
species from urinary tract infections, J Assoc Physicians India. 50:378-81.
Mnkeni, P.N.S., Austin, L.M. and Kutu, F.R. 2005. Preliminary studies on the evaluation
of human urine as a source of nutrients for vegetables in the Eastern Cape Province,

173 |

South Africa. Proc. 3rd Int. Ecological Sanitation Conference. 23-26 May 2005 at the
International Convention Centre Durban, South Africa.:418-426.
Mnkeni, P.N.S., Cisneros, B.J., Pasha, M.C. and Austin, L.M. 2006. Use of Human
Excreta from Urine-Diversion Toilets in Food Gardens: Agronomic and Health
Aspects. WRC Report No. 1439/3/06. Water Research Commission, Pretoria, South
Africa.
Mnkeni, P.N.S and Austin, L.M. 2008.Effectiveness of human manure from ecological
sanitation toilets as a source of nutrients for cabbage. In: Haneklaus S, Hera C, Rietz
R-M and Schnug E (eds.) Proc. 15th Int. Symp. Int. Sci. Centre for Fertilisers:
Fertiliser and Fertilization for Sustainability in Agriculture: The First World meets
the Third World- Challenges for the Future. 27-30 Sept., 2004, Pretoria, South
Africa.:379-392.
Mnkeni, P.N.S and Austin, L.M. 2009.Fertilizer value of human manure from pilot urinediversion toilets. Water S.A. 35 (1):133- 138.
Moellering, R.C., Jr 1992. Emergence of Enterococcus as a significant pathogen.Clinical
Infectious Disease. 14:1173-1178.
Muazzem, M. G. and Ali, A. T. 1961.Incidence of intestinal parasites among children East
Pakistan.Medicus. 25: 21-215.
Muttalib, M. A. 1976. Intestinal parasites among University of Dhaka students.J. Trop.
Med. Hyp. 78: 10-11.
Muttalib, L. and Islam, S. 1976. Prevalence of parasites in rural children of
Bangladesh.Bang. Med. J. 4(1): 11-26.
Murray, B.E., Evans Patterson, J., Zervos, M.J. et al. 1991.Evidence for clonal spread of a
single strain of b-lactamase producing Enterococcus (Streptococcus) faecalis to six
hospitals in five states.Journal of Infectious Disease. 163:780-785.
Muznebin, F. Khanum, H. and Hossain, A. 2007. Incidence of nematode

infections

among the children brought To ICDDR, B Hospital, Dhaka, Bangladesh. J. bio-sci.

15: 159-164.
174 |

Nakazawa, M. 1986. Prasitological survey and the antihelminthic effect of ivermectin on

gastrointestinal nematodes in cattle of Hpkkaido, Japan.Japanese Journal of
Veterinary Research.
Nannin, E. C., & Murray, B. E. 2006.Enterococcus spp. In S. H. Gillespie, & P. M.
Hawkey (Eds.), Principles and Practice of Clinical Bacteriology (2nd ed). West Sussex
UK: John Wiley & Sons, Ltd.:59-71.
Nelson, K., J-Cisneros, B., Tchobanoglous, G. and Darby, J. 2004. Sludge accumulation,
characteristics, and pathogen inactivation in four primary waste stabilization ponds in
central Mexico. Water Research. 38(1):111-127.
Ngui, R., Lim, Y.A., Chong Kin, L., Sek Chuen, C. & Jaffar, S. 2012. Association
between anaemia, iron deficiency anaemia, neglected parasitic infections and
socioeconomic factors in rural children of West Malaysia. PLoS Neglected Tropical
Diseases. 6:1550.
Nooruddin, M., Baki, M.A. and Das, J.G. 1987. Clinico-pathological studies of an
outbreak of trichuriasis in cow calves. Indian Journal of Veterinary Medicine. 7: 116119
Nordin, A., Nyberg, K. and Vinners, B. 2009. Inactivation of Ascaris Eggs in SourceSeparated Urine and Feces by Ammonia at Ambient Temperatures, Appl Environ
Microbiol. 75(3): 662667.
OBrien, S.J., Adak, G.K. and Gilham, C. 2001. Contact with farming environment as a
major risk factor for Shiga toxin (verocytotoxin)-producing Escherichia coli O157
infection in humans. Emerging Infectious Diseases.7:1049-1051.
Ogden, I.D., Hepburn, N.F., MacRae, M., Strachan, N.J., Fenlon, D.R., Rushbridge, S.M.
and Pennington, T.H. 2002. Long-tern survival of Escheichia coli O157 on pasture
following an outbreak associated with sheep at a scout camp. Letters in Applied
Microbiology.34:100-104.

175 |

Okeke, I.N., Laxminarayan, R., Bhutta, Z.A., Duse, A.G., Jenkins, P. and OBrien, T.F.,
2005. Antimicrobial resistance in developing countries. Part I: recent trends and
current status. Lancet Infect Dis. 5:481493.
Olsen, J. E. and Nansen, P. 1987. Inactivation of some parasites by anaerobic digestion of
cattle slurry.Biological Wastes. 22(2):107-114.
Olsen, J. E, Miller, G., Kennedy, M., Higgins, C., Walford, J., McKee, G., Fox, K., Bibb,
W. and Mead, P. 2002. A water-borne outbreak of Eschericha coli O157:H7
infections and haemolytic uremic syndrome: implications for rural water systems.
Emerging Infectious Diseases. 8:370-375.
Omra-Opyene, A.I. 1985.A survey of gestro-intestinal parasitism in cattle under normadic
management in Marsabit district of Northern Kenya.Bulletin Animal Heltha and
Production , Africa. 33: 107-112
Paiba, G.A., Gibbens, J.C., Pascoe, S.J., Wilesmith, J.W., Kidd, S.A., Byrne, C., Ryan,
J.B., Smith, R.P., McLaren, M., Futter, R.J., Kay, A.C., Jones, Y.E., Chappell, S.A.,
Willshaw, G.A. and Cheasty, T. 2002. Faecal carriage of verocytotoxin-producing
Escherichia coli O157 in cattle and sheep at slaughter in Great Britain.Vet
Rec.150:593598.
Paulino, R., Castro, E. and Thomaz-Soccol, V. 2001.Helminth eggs and protozoan cysts in
sludge obtained by anaerobic digestion process.Revista de la Sociedad Brasilea de
Medicina Tropical. 34(5): 421-428.
PCT

Databank.2010

Geneva,

World

Health

Organization,

http://www.who.int/neglected_diseases/preventive_chemotherapy/databank/en/,
Pell, A.N. 1997. Manure and microbes: Public and animal health problem? Journal of
Dairy Science.80:2673-2681.
Pike and Carrington, 1985, Processing and use of organic sludge and liquid
agriculturalwastes.Reidal Publishing Co., Dordrecht, 198-209.

176 |

Rahman, M.H. and Ahmed, Z. 1991. Pilot project for the control of parasitic disease of
animals in Bangladesh, Bangladesh Livestock Research Institute, Dhaka.
Rahman, M.H. and Razzak, A. 1973.Incidence of helminth parasites infecting cattle in the
Kotowali Thana of Comilla.First Bangladesh Veterinary Conference. BAU,
Mymensingh. P-25.
Rahn, K., Renwick, S. A., Johnson, R. P., Wilson, J. B., Clarke, R. C., Alves, D., and
Spika, J. 1998. Follow-up study of verocytotoxigenic Escherichia coli infection in
dairy farm families. Journal of Infectious Diseases.177(4):1139-1140.
Roque, O.C.C. 1997.Sistemas Alternativas de Tratamento de Esgotos Aplicveis as
Condies Brasileiras.Tese de Doutorado em Sade Pblica. Fiocruz Rio de
Janeiro.:153.
Rozendaal, J .R. 1997.Vector Control Methods for use by Individuals and
Communities.Geneva, WHO.337-356.
Rowe, D. and AbdelMagid, I.M.1995.Handbook of Wastewater Reclamation and Reuse,
CRC Press, Boca Raton, FL.
Saathoff, E., Olsen, A., Kvalsvig, J.D. and Geissler, W.P., 2002. Geophagy and its
association with geohelminth infection in rural school children from northern
KwaZulu-Natal, South Africa.Trans.R Soc.Trop.Med.Hyg.96(5): 485-490.
Sabin, R., 2006. Spinach E. coli linked to cattle manure on pasture had same strain as
bacteria in outbreak San Francisco Chronicle.
Samadpour, M., Ongerth, J.E., Liston, J., Tran, N., Nyugen, D., Whittam, T.S., Wilson,
R.A. and Tarr, P.I., 1994.Occurrence of Shiga-like toxin-producing Escherichia coli
in retail fresh seafood, beef, lamb, pork and poultry from grocery stores in Seattle,
Washington.Applied and Environmental Microbiology.60:1038-1040.
Sanderson, M.W., Gay, J.M., Besser, T.E., Hancock, D.D., Fox, L.K. and Gay, C.C., 1995.
Sensitivity of bacteriological culture for detection of Escherichia coli O157:H7 in
bovine faeces. Journal of Clinical Microbiology.33:2616-2619.

177 |

Sanguinetti G., Tortul C., . Garca M, Ferrer V., Montangero V. and Strauss M., 2005.
Investigating helminth eggs and Salmonella sp. in stabilization ponds treating septage,
Water Science & Technology. 51 (12): 239247.
Savioli, L., Bundy D.A.P. and Tomkins, A. 1992. Intestinal parasitic infections: a soluble
public health problem. Trans R SocTrop Med Hyg. 86: 3534.
Savioli,

L.

and

Albonico,

M.,

2004.Soil-transmitted

helminthiasis.Nat

Rev

Microbiol.2:6189.
Schets, F. M., During, M., Italiaander, R., Heijnen, L., Rutjes, S. A., Van der Zwaluw, W.
K., and de Roda Husman, A. M., 2005. Escherichia coli O157: H7 in drinking water
from private water supplies in the Netherlands. Water Research.39(18), 4485-4493.
Schwink, T. M., 1963. Development and survival of some roundworm larvae of cattle at
high altitudes in Wyoming.Helminthol. Soc. 30: 1518.
Sela, S., Nestel, D., Pinto, R., Nemny-Lavy, E., and Bar-Joseph, M., 2005. Mediterranean
fruit fly as a potential vector of bacterial pathogens. Applied and environmental
microbiology.71(7), 4052-4056.
Shafai, 1975.Studies on some aspects of bovine Cystercerus bovis, University of Dublin.
330-349
Shah M. F., John, M. A. and Mekalanos, J. 1998. Epidemiology, Genetics, and Ecology of
Toxigenic Vibrio cholera.Microbiol Mol Biol Rev. 62(4): 13011314.
Slanetz L. W. and Bartley C. H., 1957.J. Bact. 74:591-595.
Snow, R.W., Guerra, C.A. Noor, A.M., Myint, H.Y. and Hay, S.I., 2005.The global
distribution of clinical episodes of Plasmodium falciparum malaria.Nature. 434:214
217.
Speedy, A.W., 1992. Progress in sheep and goat research, C.A.B. International,
Wallingford, Oxon, U.K.:179-188

178 |

Soren L. Becker, Laurent K. Lohourignon,Benjamin Speich,Laura Rinaldi, Stefanie

Knopp, Eliezer K. NGoran, Giuseppe Cringoli, and Jurg Utzinger 2011.
Comparison of the Flotac-400 Dual Technique and the Formalin-Ether Concentration
Technique for Diagnosis of Human Intestinal Protozoon Infection.Journal of clinical
microbiology. 49:21832190
Steineck, S., Stintzing, R., Rodhe, L., Elmquist, H. and Jakobsson, C. 1999. Plant nutrients
in human urine and food refuse. Use of municipal organic waste.Proceedings of NJF
seminar no. 292, November 2325, 1998 Agricultural Research Centre, Jokioinen,
Finland. DIAS report Plant Production no. 13, June 1999. 2:125130.
Strauch, D. 1978. Animal and human health hazards associated with the utilization of
animal effluents. Ed. W R Kelly; CEC EUR 6009 EN.
Strauch, D. and Ballarini, G., 1994. Hygienic aspects of the production and agricultural
use of animal wastes.Journal of Veterinary Medicine.41:176.
Strauch, D., 1996. Occurrence of microorganisms pathogenic for man and animals in
source separated biowaste and compost importance, controls, limits, epidemiology.
In The Science of Composting Ed. de Bertoldi, M., Sequi, P., Lemmes, B. and
Tizano Papi.CEC, Blackie Academic and Professional. London: 224-232.
Stevens, M.P., van Diemen, P.M., Dziva, F., Jones, P.W. and Wallis, T.S., 2002. Options
for

the

control

of

enterohaemorrhagic

Escheichia

coli

in

ruminants.Micobiology.148:3767-3778.
Storey, G. W. and Phillips, R. A. 1985. The survival of parasite eggs throughout the soil
profile.Parasitology. 91(3):585-590.
Suwansaksri, J., Nithiuthai, S., Wiwanitkit, V., Soogarun, S. and Palatho, P. 2002. The
formol-ether concentration technique for intestinal parasites: comparing 0.1 n sodium
hydroxide with normal saline preparations. Southeast Asian J Trop Med Public
Health.33 (3).

179 |

Szalanski, A. L., 2004. Detection of Campylobacter and Escherichia coli O157: H7 from
filth flies by polymerase chain reaction. Medical and veterinary entomology.18(3):
241-246.

Tadesse DA, Zhao S, Tong E, Ayers S, Singh A, Bartholomew MJ, 2012. Antimicrobial
drug resistance in Escherichia coli from humans and food animals, United States,
19502002. Emerg Infect Dis [serial on the Internet].
Tarr, P.I. 1995. Escherichia coli O157:H7: clinical, diagnostic, and epidemiological
aspects of human infection. Clinical Infectious Diseases.20:1-10.
Teixeira, L. M., Carvalho, Maria da Gloria Siqueira, and

Facklam, R. R., 2007.

Enterococcus.In P. R. Murray (Ed.), Manual of Clinical Microbiology.ASM: (9th ed.,

pp. 430-442). Washington D.C.
Thunegard, E., 1975. On the persistence of bacteria in manure, a field and experimental
study with special reference to Salmonella in liquid manure. Acta Veterinaria
Scandinavica, Supplementum.56:5-86.
Todar, K., 2007. Pathogenic E. coli. Online Textbook of Bacteriology.34-67.
Trevena, W. B., Willshaw, G. A., Cheasty, T., Domingue, G., and Wray, C., 1999.
Transmission of Vero cytotoxin producing Escherichia coli O157 infection from farm
animals to humans in Cornwall and west Devon.Communicable disease and public
health/PHLS. 2(4), 263-268.
Turner, C., Williams, S.M., Burton, C.H., Cumby, T.R., Wilkinson, P.J. and Farrent, J.W.,
1999. Pilot scale thermal treatment of pig slurry for the inactivation of animal virus
pathogens. Journal of Environmental Science and Health.34 (6), 989-1007
Turner, C., 2002 .The thermal inactivation of E. coli in straw and pig manure. Bioresource
Technology. 84(1): 5761
Uddin, M.H., Rahman, M.M. and Khanum, H., 2005. Hemoglobin level among adolescent
girls and its relation to intestinal parasites. Bangladesh J. Zool. 33(2): 183-187.
180 |

United States Environmental Protection Agency (USEPA), 1992. Method for the detection
and enumeration of Helminth eggs.147-152.
United States Environmental Protection Agency (USEPA), 1992. Guideline for waste
reuse.United states environmental protection agency. Washington DC, EPA/625/R92/004.
United States Environmental Protection Agency (USEPA), 1992. Control of Pathogens
and Vector Attraction in Sewage Sludge EPA/625/R-92-004. Washington, D.C.
United States Environmental Protection Agency (USEPA), 1994. Guide to Septage
Treatment and Disposal. Document EP/625/R-94/002. Washington D.C. 20460.
USDA, 1997.US Department of Agriculture Animal and Plant Health Inspection Service.
An update: Escherichia coli O157:H7 in humans and cattle. 1-28 In: Report from
Centers for Epidemiology and Animal Health, Fort Collins, Columbia.
Varma, J. K., Greene, K. D., Reller, M. E., DeLong, S. M., Trottier, J., Nowicki, S. F. and
Mead, P. S. 2003. An outbreak of Escherichia coli O157 infection following exposure
to a contaminated building. Jama. 290(20): 2709-2712.
Waters, J.R., Sharp, J.C.M. and Dev, V.J. 1994. Infection caused by Escherichia coli
O157: H7 in Alberta, Canada and in Scotland: a five-year review, 1987-1991. Clinical
Infectious Diseases.19: 834-843.
Weber, J. T., and Courvalin, P. 2005. An emptying quiver: antimicrobial drugs and
resistance. Emerging Infectious Diseases. 11(6):791-793.
Wegayehu, T., Tsalla, T., Seifu, B.and Teklu, T., 2013.Prevalence of intestinal parasitic
infections among highland and lowland dwellers in Gamo area, South Ethiopia.BMC
Public Health.13:151
Wells, J.G., Shipman, L.D., Greene, K.D., Sowers, E.G., Green, J.H., Cameron, D.N.,
Downes, P.F., Martin, M.L., Griffin, P.M., Ostroff, S.M., Potter, M.E., Tauxe, R.V.
and Wachsmuth, I.K. 1991. Isolation of Escherichia coli serotypes O157:H7 and other

181 |

Shiga-like toxin-producing E. coli from dairy cattle. Journal of Clinical

Microbiology.29:985-989.
Wichuk, K. M., and McCartney, D. 2007.A review of the effectiveness of current time
temperature regulations on pathogen inactivation during composting.Journal of
Environmental Engineering and Science. 6(5): 573-586.
World Health Organization (WHO), 1984. Guidelines for drinking water quality, volume
2: Health criteria and other supporting information.
World Health Organization (WHO), 1989.Health guidelines for the use of wastewater in
agriculture and aquaculture.Technical Report Series.No. 778, World Health
Organization, Geneva.
World Health Organization (WHO), 1994.Report of the WHO informal consultation on
hookworm infection and anaemias in girls and women. WHO/CTD/STP/961: 1- 46.
World Health Organization (WHO), /UNICEF, 2000.Global Water Supply and Sanitation
Assessment, 2000. World Health Organization, Geneva, and the United Nations
Childrens Fund, New York, Joint Monitoring Programme for Water Supply and
Sanitation.
World Health Organization (WHO), 2002. Prevention and Control of Schistosomiasis and
Soil Transmitted Helminthiasis, Technical Report Series 912.WHO, Geneva,
Switzerland.
WHO, 2002.Disease trends. Available from: www.who.int/ctd/intpara/disease.php
World Health Organization (WHO), 2004. The world health report 2004: changing
history, WHO, Geneva.
World Health Organization. 2006. Guidelines on Standard Operating Procedures for
Microbiology. Geneva: World Health Organization.
World Health Organization, 2006.Software for assessing growth and development of the
worlds children.2005-beta ed. Geneva.

182 |

World Health Organization (WHO), 2015.Soil-transmitted helminth infections.Fact sheet

N366.
Yilmaz, V. and Icemer, G.T. 2014. Sludge Characteristic and Pathogen Inactivation of
Two Different Wastewater Treatment Plants in Antalya.The International Journal Of
Engineering And Science. 3(3):63-67.
Zhao, T., Doyle, M.P., Shere, J. and Garber, L. 1995. Prevalence of enterohaemorrhagic
Escherichia coli O157:H7 in a survey of dairy herds. Applied and Environmental
Microbiology.61, 1290-1293.

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APPENDIX
Preparation of chemicals
The following chemicals are prepared according to W. Dryden et al.( 2005) (a-d)
Preparation of ZnSO4 Solution (SP. GR. 1.18-1.20)
A specific gravity of ZnSO4 solution is made by adding 331 grams of ZnSO4 to 1 liter of
water. The mixture is checked with a hydrometer and adjusted to 1.18. The ZnSO4
solution is always stored in an airtight sterile bottle to prevent evaporation and the possible
change in the specific gravity of the solution.
Preparation of MgSO4 Solution (SP. GR. 1.20)
To prepare MgSO4 Solution, dissolve 450 g MgSO4 into 1000ml water. Same conditions
applied here as in ZnSO4.
Preparation of NaNO3 Solution (SP. GR. 1.18-1.20)
NaNO3solution is made by adding 338 grams of NaNO3to 1 liter of water. Conditions
applied here as in ZnSO4.
Preparation of NaCl Solution (SP. GR. 1.20)
350 g NaCl dissolve into 1,000 ml tap water and mix well. Conditions applied here as in
ZnSO4.
Preparation of Normal Saline (0.85%)
Dissolve 8.5gm NaCl into 1000ml of distilled water, autoclave and store at room
temperature.
Media
mTEC agar
A medium for the detection of E.coli

Typical formula

gm/litre

Protease peptone

3 5.0 g

Yeast extracts

3.0 g
184 |

Lactose

10.0 g

Sodium chloride

7.5 g

Dipotassium phosphate (K2HPO4)

3.3 g

Monopotassium phosphate (KH2PO4)

1.0 g

Sodium lauryl sulfate

0.2 g

Sodium desoxycholate

0.1 g

Bromcresol purple

0.08 g

Bromphenol red

0.08 g

Agar

15.0 g

pH

7.3 0.2

* Adjusted as required to meet performance standards

Directions
Add reagents to 1 L of reagent-grade water, mix thoroughly, and heat to dissolve.
Autoclave at 121C (15 PSI) for 15 minutes, and cool in a 50C water bath. Pour the
medium into each 9 50 mm culture dish to a 4-5 mm depth (approximately 4-6 mL), and
allow to solidify.

Description
This medium is used for E.coli isolation and identification.

Incubation and Colonial characteristics

It is incubated at 35 2C for 2 0.5 hours and then at elevated temperatures 44.5C
0.2C for 22 2 hours. All pink depressed colonies may be accepted as presumptive E.coli
(US-EPA, 2010)

Storage conditions and Shelf life

Store the dehydrated medium at 10-30C and use before the expiry date on the label.

Appearance
Dehydrated medium: Straw coloured, free-flowing powder
Prepared medium: Straw-brown coloured gel

185 |

MACCONKEY AGAR (MAC)

It is a differential medium for the isolation of coliforms and intestinal pathogens in water,
dairy products and biological specimens.
Typical Formula* gm/litre
Peptone

20.0

Lactose

10.0

Bile salts

5.0

Sodium chloride

5.0

Neutral red

0.075

Agar

12.0

pH 7.4 0.2
* Adjusted as required to meet performance standards

Directions
Suspend 52g in 1 litre of distilled water. Bring to the boil to dissolve completely. Sterilise
by autoclaving at 121C for 15 minutes. Dry the surface of the gel before inoculation.

Description
This is a differential medium for the detection, isolation and enumeration of coliforms and
intestinal pathogens in water, dairy products and biological specimens. MacConkey Agar
corresponds to the medium recommended by the World Health Organization1, the Dept. of
Health2 and by Windle Taylor3 for the bacteriological examination of water.
Although principally used for coliforms, this medium may also be employed for the
differentiation of other enteric bacteria (including pathogens) and is suitable for the
differentiation of Pasteurella species4.

Incubation and Colonial characteristics

186 |

The plate is incubated for 24 hours at 35-37C. After 24 hours at 35-37C typical
E.colicolonies are red in colour and non mucoid.

Storage conditions and Shelf life

Store the dehydrated medium at 10-30C and use before the expiry date on the label. Store
the prepared plates of medium at 2-8C.

Appearance
Dehydrated Medium: Straw pink coloured, free-flowing powder.
Prepared medium: Dark red coloured gel.
m-FC AGAR
A medium for the detection of coliforms
Typical Formula

gm/ liter

Enzymatic Digest of Casein

10.0g

Enzymatic Digest of Animal Tissue

5.0g

Yeast Extract

3.0g

Sodium Chloride

5.0g

Lactose

12.5 g

Bile Salts

1.5 g

Aniline Blue

0.1 g

Agar

15.0g

Final pH

7.4 0.2 at 25C

Supplement
1% Rosolic Acid, 10mL

* Adjusted as required to meet performance standards

Directions
Suspend 52 g of the medium in 1Lof purified water containing 10 mL of 1% Rosolic Acid
in 0.2 N NaOH.If necessary, adjust pH to 7.4 with 1N HCl.Heat with frequent agitation
and boil for one minute to completely dissolve the medium. Cool to 45 -50C and pour
plates.DO NOT AUTOCLAVE.
187 |

Rosolic Acid:
Dissolve 1 g in 100 mL of 0.2 N NaOH to prepare a 1% solution

Description
Geldreich et al. formulated a medium to enumerate fecal coliforms (FC) using the
membrane filter (m) technique without prior enrichment (Geldreich et al.,1965). Fecal
coliforms, i.e., those found in feces of warm-blooded animals, are differentiated from
environmental coliforms by their ability to grow at 44.5 0.5C (Eaton et al., 1999).

Incubation and colony morphology

Incubate for 22 -24 hours at 44.5 0.5C. The coliforms appear as blue-grey to deep blue
colonies.

Storage conditions and Shelf life

Store sealed bottle containing the dehydrated medium at 2 -30C. Once opened and
recapped, place container in a low humidity environment at the same storage temperature.
Protect from moisture and light.

Appearance
Dehydrated medium: Straw coloured, free-flowing powder
Prepared medium: maroon coloured gel
Slanetz And Bartley Medium (SBA)
A medium for the detection of enterococci.

Typical Formula*

gm/liter

Tryptose

20.0

Yeast extract

5.0

Glucose

2.0

Di-potassium hydrogen phosphate 4.0

Sodium azide

0.4

Tetrazolium chloride

0.1
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Agar

10.0

pH 7.2 0.2 @ 25C

* Adjusted as required to meet performance standards

Directions
Suspend 42g in 1 litre of distilled water and bring to the boil to dissolve the agar
completely.
Excessive heating must be avoided. Dispense into Petri dishes and allow solidifying. It
should not be remelted. The medium may be used with membrane filters or by spreading
dilutions of the sample over the surface of the agar with a glass rod.

Description
Slanetz & Bartley (Slanetz and Bartley, 1957) originally devised this medium to detect
and enumerate enterococci by the technique of membrane filtration, but it has also proved
useful as a direct plating medium( Burkwall and Hartman,1964; Nordic Committee on
Food Analysis,1968)

Incubation and colony morphology

The medium is very selective for enterococci and, when it is incubated at elevated
temperatures (44-45C), all red or maroon colonies may be accepted as presumptive
enterococci (Taylor and Burman, 1964; Mead, 1966).

Storage conditions and Shelf life

Store the dehydrated medium at 10-30C and use before the expiry date on the label. Store
the prepared medium at 2-8C away from light.

Appearance
Dehydrated medium: Straw coloured, free-flowing powder
Prepared medium: Straw coloured gel
Enterococcosal Agar (EA)
It is a differential medium for the isolation and presumptive identification of enterococci /
Group D streptococci.
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Typical Formula*

gm/litre

Peptone

14.0

Bile salts

15.0

Ferric citrate

0.5

Aesculin

1.0

Agar

14.0

pH 7.1 0.2 @ 25C

* Adjusted as required to meet performance standards

Directions
Suspend 44.5g in 1 litre of distilled water and bring gently to the boil to dissolve
completely. Sterilise by autoclaving at 121C for 15 minutes.

Description
The major use of Bile Aesculin Agar is to differentiate between enterococci / Group D
streptococci and non Group D streptococci. It may also be used for the presumptive
identification of other groups of organisms.
Enterococci / Group D streptococci hydrolyse aesculin to form aesculetin and dextrose.
Aesculetin combines with ferric citrate in the medium to form a dark brown or black
complex which is indicative of a positive result. Bile salts will inhibit Gram-positive
bacteria other than enterococci / Group D streptococci.
The value of bile tolerance together with hydrolysis of aesculin as a means of
presumptively identifying enterococci / Group D streptococci is widely recognized
(Facklam and Moody, 1970; Senberg et al., 1970; Sabbaj et al., 1971; Facklam, 1972 and
Facklam, 1974).

Incubation and colonsal carecteristics

Using a sterile loop inoculate the medium with 4-5 colonies and incubate at 37C for 1824 hours. The result is positive for bile salt tolerance and aesculin hydrolysis if blackening
of the medium occurs.

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Storage conditions
Store the dehydrated medium at 10-30C and use before the expiry date on the label.

Appearance
Dehydrated medium: Straw coloured, free-flowing powder
Prepared medium: Gel like.

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PLATES

Plate-1: Instrument

Vortex Machine

Water Bath

Centrifuge Machine
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Plate-2: Bacterial Isolates

Faecal Streptococci

E. coli

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Plate-3: Biochemical Identification

Control

From Pit

From Cow Dung

Faecal Streptococci

E. coli

Indole Production

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Plate-4: Antibiotics Susceptibility Test

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Plate-5: Parasites

Hookworm egg

Hookworm larvae

E. vermicularis

H. nana

A. lumbricoides

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S. stercoralis

T. trichiura

Trichostrongylus

E. histolytica

Coccidian cysts

Strongyloides
Haemonchus

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