Inhibition of Escherichia coli in cultivated cattle manure1

Z. G. Weinberg,*2 G. Szakacs, Y. Chen,* R. Pinto,* S. Bernstein,* B. Konya, and S. Sela (Saldinger)*

*Department of Food Quality and Safety, Agricultural Research Organization,
The Volcani Center, Bet Dagan 50250, Israel. Department of Applied Biotechnology
and Food Science, Budapest University of Technology and Economics, Gellert ter 4, 1111 Budapest, Hungary

ABSTRACT: A common practice on Israeli dairy barns
comprises daily cultivation of the manure. Cultivation is
a mechanical process used to break up and till the manure
bedding and it results in a drier and aerated bedding and
cleaner cows, which consequently reduces the incidence of
mastitis. Cultivation was associated with a shorter survival
of Escherichia coli in cultivated manure as compared with
noncultivated manure. The objective of the current study
was to elucidate the mechanism responsible for the shorter
survival duration of E. coli in the cultivated manure. We
hypothesized that microorganisms that are antagonistic to
E. coli, developing in the cultivated manure, are respon-
sible for this phenomenon. A cow manure derived E. coli
strain expressing the green fluorescence protein and antibi-
otic resistance markers was used to inoculate cow manure
in 1.5-L jars. Manure treatments included cultivated and
noncultivated manure. Half the jars of each cultivation
treatment were autoclave sterilized at 121C for 1 h on 3
successive days to eliminate from the manure antagonistic
microorganisms. Each cultivationsterilization treatment
was performed in triplicate jars. Following sterilization, E.
coli numbers in the cultivated and noncultivated manure
were comparable, while in the nonsterilized manure the
numbers were lower in the cultivated compared with the
noncultivated manure. Several fungi isolated from the cul-
tivated manure samples displayed inhibition effect on the
tagged E. coli. Antagonistic fungi were also isolated from
large-scale cultivated manure samples collected on several
dairy farms in Israel. These findings support the notion
that manure cultivation might facilitate the development of
microorganisms that are antagonistic to E. coli, thus con-
tributing to the general hygiene of the cattle. Identifying
the mechanisms by which the antagonistic fungi affect
the survival of E. coli in manure could be exploited for
improvement of the animal health and for limiting the
transmission of zoonotic pathogens to food and water.

Key words: cattle manure, cultivation, Escherichia coli, fungi

2014 American Society of Animal Science. All rights reserved. J. Anim. Sci. 2014.92:23362341
doi:10.2527/jas2013-7285

INTRODUCTION
Cattle may be a source of pathogens that might
infect cattle and contaminate the food supply. A sig-
nificant number of recent foodborne disease outbreaks
were attributed to Escherichia coli O157:H7 and
Salmonella spp., which are commonly found in bovine
manure and slurry (Pell, 1997; Semenov et al., 2009).
In Israel the animals in all dairy barns are loose
with no stalls. The wet slurry is removed daily from the
1Contribution from the Agricultural Research Organization, The
Volcani Center, Bet Dagan, Israel, number 668/13-E, 2013 series.
This study was supported by the Israeli Milk Board.
2Corresponding author: zgw@volcani.agri.gov.il
Received October 22, 2013.
Accepted February 23, 2014.
2336
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feeding alley to a storage basin and used along with the
drier manure for composting. According to a new man-
agement practice, the wet slurry is added daily to the
solid manure at the barn followed by daily cultivation to
a depth of 50 to 60 cm (Friedman, 2008). Cultivation is
implemented by a duck feet cultivator (Fig. 1) drawn
by a tractor, the shovels of which break up, loosen, and
turn the bedding. Recently, the feeding alleys were re-
placed by feeding tubs, which are placed in the center
of the barn. In this way the slurry is constantly mixed
with the manure, which continues to be cultivated daily.
This management saves the need to shovel the slurry
from the feeding alleys by a front load bucket tractor
and pour it over the roofed bedding. It was suggested
that daily cultivation of the manure results in overall
aerated and drier bedding, which favors the welfare of
 Escherichia coli in cultivated cattle manure
Figure 1. Duck-feet cultivator. Curtsy Yoram Kalgrad, Kibbutz Harduf,
Israel. See online version for figure in color.
the animals and lowers the incidence of mastitis. This
notion was supported by Menis et al. (2011) who found
drier bedding, cleaner cows, lower incidence of masti-
tis, and lower numbers of various pathogenic bacteria
in cultivated manure as compared with noncultivated
manure on the same dairy farm of kibbutz Netzer-Sireni,
Israel. This type of management has gained much popu-
larity on dairy farms all over Israel.
Weinberg et al. (2011) used an E. coli strain that was
isolated from cow manure and tagged with green fluo-
rescence protein (GFP) and antibiotic resistance mark-
ers to study the effect of cultivation on the fate of E. coli.
The cultivated manure had a higher DM content and
contained lower numbers of the tagged E. coli, which
could be detected for shorter duration time as compared
with the noncultivated manure. There might be several
explanations for the observed effect on E. coli. The ob-
jective of the current study was to test the hypothesis
that microorganisms that are antagonistic to E. coli de-
velop in the cultivated manure and limit its survival.
MATERIALS AND METHODS
Experimental
Experiment 1 (2012). Untreated dairy cow manure
from the Volcani Center dairy research farm (Bet Dagan,
Israel) was brought to the laboratory. Part of it was cul-
tivated daily for 1 mo with a small manual garden cul-
tivator (Fig. 2) in a 40 by 30 cm plastic tub. Then, the
noncultivated and cultivated manure were placed each
in six 1.5-L glass jars, 1 kg per jar, which were covered
with aluminum foil. Three jars from each manure type
were autoclave sterilized 3 times at 121C for 1 h on 3
successive days. This sterilization process was applied
to eliminate from the manure also heat-resistant spores,
such as those of Actinomycetes and Bacillus spp. Four
manure treatments were included: cultivated, cultivated
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2337
Figure 2. The garden cultivator used in the experiments. See online ver-
sion for figure in color.
sterilized, noncultivated, and noncultivatedsterilized.
There were triplicate jars per treatment. The efficiency
of the sterilization process was tested by total aerobic
counts on plate count agar (Scharlau Microbiology,
Barcelona, Spain) and on actinomycete isolation agar
(Difco, Becton, Dickinson and Company, Sparks, MD).
All the jars were then inoculated with a cow manure de-
rived E. coli strain ZP1 tagged with GFP and antibiotic
resistant markers (Weinberg et al., 2011). Inoculation
was performed by emptying the contents of each jar into
a tub and adding 20 mL of the E. coli suspension fol-
lowed by thorough mixing. The inoculated manure was
then returned to the jars.
The inoculation rate (cfu/g) was determined from
the number of the E. coli in the growth suspension in
Luria-Bertani broth. The number of the tagged E. coli in
each jar was determined on Days 0, 1, 2, 3, 4, 7, and 9
after inoculation. A composite 4-g sample from each jar
was resuspended with 36 mL of sterile saline solution
in 50-mL sterile conical polypropylene tubes equipped
with a lid (Miniplast, Ein-Shemer, Israel). The bacteria
were extracted by vigorous vortexing for 1 min followed
by serial 10-fold dilutions in saline. Samples were plated
on Tryptic soy agar (Difco) supplemented with kanamy-
cin and streptomycin at 50 and 100 g/mL, which en-
abled visualization of GFP fluorescence under UV light.
All plates were incubated at 39C for 24 h.
Experiment 2 (2013). A similar experiment was per-
formed that served as a repetition of Exp. 1. In this ex-
periment, 30 mL of the tagged E. coli suspension was
added to 750 g manure in each jar. In this experiment ad-
ditional 2 treatments were added: cultivated manure from
the Harduf dairy farm and sterilization of this manure as
described above. Each treatment comprised 3 jars.
In both experiments, no colonies were found on
plate count agar or on actinomycete isolation agar after
sterilization of the manure.
Additional Analyses
The following analyses were performed on manure
from each of the triplicate jars at the end of Exp. 1 and
2. Dry matter content of the manure was determined by
2338 Weinberg et al.
oven drying at 105C for 24 h. The pH value was deter-
mined with a pH meter (Mettler-Toledo, Schwerzenbach,
Switzerland). Total volatile base nitrogen (TVBN) was
determined only in Exp. 2 on Day 3. Total volatile base
nitrogen was determined by the Kjeldahl method with-
out the digestion step, according to Anderson (2008).
Isolation of Microorganisms with Inhibitory Activity
against Escherichia coli by Isolated Cultivated Manure
Mesophilic actinomycetes and filamentous fungi
have been isolated from dried samples of the cultivated
cattle manure by conventional isolation methods in pe-
tri plates at 30C. These microorganisms were isolated
both from cultivated manure of laboratory Exp. 2 and
from cultivated manure of 3 commercial dairy farms in
Israel. For isolation of actinomycetes, medium ATCC-
5 (sporulation agar) and Bacto actinomycete isolation
agar (Difco Manual, 10th ed., 1984, p. 66, Olson, per-
sonal communication) were used. Fungi were isolated
on potato dextrose agar (PDA) medium supplemented
after sterilization with 100 g/mL doxycycline hyclate
(Sigma-Aldrich, St. Louis, MO) to suppress bacterial
growth. Primary fungal colonies were further purified
by streaking on PDA medium supplemented with 0.5 g/L
Triton X-100 (colony growth restricting agent; Sigma-
Aldrich) to isolate single colonies. Pure actinomycetes
and fungal cultures were freeze-dried for long-term
storage and deposited at the Technical University of
Budapest culture collection (www.tub-collection.com or
www.tub-collection.hu).
From manure samples cultivated in Exp. 2, alto-
gether 16 different actinomycetes and 12 fungal strains
have been isolated. Additional strains were obtained and
freeze-dried from the large-scale cultivated cattle ma-
nure materials collected on the Harduf (Lower Galilee,
northern Israel) and Netzer-Sireni (central coastal plain)
kibbutz dairy farms. From the Harduf farm samples 8
actinomycetes and 27 fungi were isolated. Sampling at
the Netzer-Sireni farm resulted in 7 actinomycetes and
16 fungal isolates. All these microorganisms were tested
in a later step against the tagged E. coli and other bacte-
ria for antagonistic activity.
Inhibition Assay. Agar cross-streak method was
used for testing the antimicrobial activity of the isolated
actinomycetes and fungi against E. coli and other bac-
teria . The isolates were streaked centrally on Grove-
Randall number 1 (GR-1) and ATCC-5 sporulation
agar media in petri plates. These 2 media were selected
because production of antibiotics depends on the quan-
tity of carbon source, for example, glucose. In medium
GR-1 the glucose concentration is low while in medium
ATCC-5 the concentration of sugar is high. The com-
position of these 2 media is as follows: GR-1, 1.5 g/L
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meat extract, 3.0 g/L yeast extract, 4.0 g/L peptone from
casein, 6.0 g/L peptone from meat, 1.0 g/L glucose, and
18.0 g/L agar, pH 6.5 (before sterilization); and medium
ATCC-5 (sporulation agar), 1.0 g/L yeast extract, 1.0
g/L beef extract, 2.0 g/L tryptose, 0.05 g/L FeSO4, 10.0
g/L glucose, and 19.0 g/L agar, pH 7.2 (before steriliza-
tion). The isolated actinomycetes or fungi were inocu-
lated as streaks on GR-1 and ATCC-5 agar media in petri
plates. After 2 d of incubation at 30C, they were cross-
streaked with overnight broth cultures of 7 test bacte-
ria (Fig. 5). These included Bacillus subtilis American
Type Culture Collection (ATCC) 7972, Bacillus subti-
lis ATCC 14593, E. coli ZP1 GFP tagged, Micrococcus
luteus ATCC 10240, Pseudomonas fluorescens ATCC
13525, Pseudomonas fluorescens Loc (local isolate in
Hungary), and Micrococcus luteus (Sarcina subflava)
ATCC 7468. Bacillus subtilis and M. luteus strains are
gram positive while E. coli and P. fluorescens are gram
negative. Except for the manure derived E. coli strain,
the strains were originated from ATCC (American Type
Culture Collection, Manassas, VA) or Loc (local isolate
in Hungary). The plates were then further incubated at
37C for additional 1 d. Growth inhibition was observed
as a clearing zone near the central streaks of actinomy-
cetes or fungal isolates.
Statistical Analysis
The experiments were performed in a randomized
block design to determine significant differences be-
tween the applied treatments within a sampling date of
each experiment. Such design is also consistent with our
previous work (Weinberg et al., 2011). Therefore, 1-way
ANOVA was applied to the results of each experiment
and day of sampling separately in the General Linear
Model of SAS (SAS Inst. Inc., Cary, NC). All manure
treatments were included in the analysis. Significant
differences between means of the dependent variables
(log10 cfu/g and pH, DM content, and TVBN) were
identified by using the Tukeys studentized range test in
which P < 0.05 was declared as significant. In addition, a
2-way ANOVA was also performed to determine the sig-
nificance of the effects of cultivation, sterilization, and
their interactions, which were included in the statistical
model.
RESULTS
Table 1 gives the results of the chemical analysis of
the manure of the various treatments at the end of Exp.
1 and 2. This analysis was performed to follow changes
in the manure incurred by both cultivation and steriliza-
tion treatments, which could have affected the survival
of the added E. coli strain. In Exp. 1, DM content varied
 Escherichia coli in cultivated cattle manure 2339

Table 1. Analytical results of the cattle manure at the treatment. The 2-way ANOVA revealed that on Days 7
end of Exp. 1 and 2 and 9 the noncultivated manure had significantly higher
Experiment Treatment1 pH DM, g/kg TVBN2 numbers of the tagged E. coli than the cultivated manure.
1 CULT 9.4 587ab The interactions cultivation sterilization were nonsig-
CULT-STE 9.3 575b nificant at all sampling times.
NOCULT 9.4 639a Similar results were obtained in Exp. 2 (Fig. 4). On
NOCULT-STE 9.4 637ab Days 1 through 4 the noncultivated manure tended to
SEM 0.07 19.5
have higher E. coli counts than the cultivated manure;
2 CULT 9.0ab 838a 13.9a
on Days 1 through 7 the sterilized manure had higher
CULT-STE 8.6b 758a 15.7a
counts than the nonsterilized manure. The interactions
NOCULT 9.1a 497b 3.0b
NOCULT-STE 8.7ab 487b 14.3a
cultivation sterilization were nonsignificant at all sam-
HARDUF 9.2a 594b 1.5b pling times. Also the cultivatedsterilized manure from
HARDUF-STE 8.9ab 564b 16.8a Harduf had significantly higher E. coli counts compared
SEM 0.17 5.5 3.14 with the nonsterilized manure.
a,bFor each experiment, means within the same column followed by differ- The majority of fungi isolated from the manure were
ent superscripts differ significantly (P < 0.05). Aspergillus spp. and of actinomycetes were Streptomyces
1CULT = cultivated; NOCULT = noncultivated; STE = sterilized; spp. All isolates (actinomycetes and fungi) were tested
HARDUF = cultivated manure from Kibbutz harduf. first against the tagged E. coli on GR-1 and ATCC-5
2TVBN = total volatile base nitrogen. media. Strains with anti-E. coli activity were retested
against the 7 bacterial species indicated above. The re-
between 575 and 639 g/kg and pH values were around sults show that none of the 31 actinomycetes isolates
9.4. In Exp. 2, the DM content varied between 487 (for (laboratory and farm isolates) had inhibitory activity
noncultivated manure) and 838 g/kg (for cultivated ma- against E. coli ZP1 strain. However, 10 out of 55 fungal
nure) and the pH values varied between 8.6 and 9.2. In isolates inhibited the growth of E. coli and they were
Exp. 2, cultivation tended to increase DM content and subsequently tested against 7 other bacterial species.
sterilization tended to decrease pH values. The effect of Four selected fungi with activity against the different
sterilization on TVBN values was inconsistent among bacteria are shown in Fig. 5.
cultivation treatments.
Figure 3 gives the log10 cfu per gram of the added DISCUSSION
E. coli in the various manure treatments of Exp. 1. The
counts on Day 0 agreed with the calculated inoculation Cattle manure and slurry are accumulated at large
rate mentioned above. The numbers of the tagged E. coli quantities in rural areas and may potentially contami-
tended to decrease with time, however, to a smaller ex- nate soil and groundwater. Pathogens that exist in the
tent in the noncultivated sterilized manure. From Day 1 manure bedding may cause mastitis, which results in re-
and on the sterilized manure contained significantly (P< duction in milk yield and quality (Barkema et al., 2009;
0.05) higher counts of the tagged E. coli as compared Burnevich et al., 2003; Vangroenweghe et al., 2005).
with the nonsterilized manure of the same cultivation

Figure 4. Change in green fluorescence protein Escherichia coli during in-

Figure 3. Change in green fluorescence protein Escherichia coli during in- cubation in cattle manure (Exp. 2). CULT = cultivated; NOCULT = not cultivated;
cubation in cattle manure (Exp. 1). Cult = cultivated; nocult = not cultivated; ste STE = sterilized; HARDUF = cultivated manure from Kibbutz harduf.. a,bWithin a
= sterilized. a,bWithin a day, columns accompanied by different letters are signifi- day, columns accompanied by different letters are significantly different (P< 0.05).
cantly different (P < 0.05). See online version for figure in color. See online version for figure in color.

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2340 Weinberg et al.

Figure 5. Inhibition of growth of 7 test bacteria by selected 4 antagonistic fungi from cultivated cattle manure on medium Grove-Randall number 1 and
medium ATCC-5. Test bacteria are a) Bacillus subtilis American Type Culture Collection (ATCC) 7972, b) Bacillus subtilis ATCC 14593, c) Escherichia coli
green fluorescence protein tagged, d) Micrococcus luteus ATCC 10240, e) Pseudomonas fluorescens ATCC 13525, f) Pseudomonas fluorescens Loc (local isolate
in Hungary); and g) Micrococcus luteus (= Sarcina subflava) ATCC 7468. Med. = medium. See online version for figure in color.

Escherichia coli is 1 of several etiological agents caus-
ing bovine mastitis (e.g., Bradley and Green, 2000;
Blum and Leithner, 2013).
The Harduf practice developed several years ago in
Israel comprises daily deep cultivation of the manure
bedding along with the wet slurry. Klaas et al. (2010)
and Menis et al. (2011) showed that such management
results in drier manure, keeps the cows cleaner, decreas-
es the microbial load, and decreases the incidence of
mastitis. In a previous study (Weinberg et al., 2011) we
tested the effect of cultivation on the survival of a ma-
nure-derived E. coli strain in a model system. Cultivation
of cattle manure resulted in drier and aerated manure
as compared with noncultivated manure. Concurrently,
the viable numbers of the inoculated E. coli decreased
much faster in cultivated than in the noncultivated ma-
nure. This result was attributed to the dryness of the cul-
tivated manure, which could have been less favorable
for the survival of E. coli. In the current study we have
tested the hypothesis that the conditions in the culti-
vated manure favor the development of microorganisms
that are antagonistic to E. coli. Such microorganisms
could be fungi and actinomycetes since both groups are
well known for their capacity to produce antimicrobial
substances (Berdy, 2005; Demain and Sanchez, 2009;
Monaghan and Barrett, 2006; Pell, 1997). The idea was
that sterilization would eliminate from the manure all
microorganisms including those that inhibit E. coli, and
therefore, the survival of the added E. coli would be
comparable in both cultivated and noncultivated ma-
nure. Although sterilization resulted in some changes in
the manure composition (Table 1), in both Exp. 1 and
2 the numbers of the tagged E. coli were significantly
higher in sterilized manure as compared with nonsteril-
ized manure. This included also the sterilized cultivated
manure, in spite of its high DM content (Table 1). These

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 Escherichia coli in cultivated cattle manure
findings are concurrent with the hypothesis that the sur-
vival of E. coli in the manure is affected by the presence
of antagonistic microorganisms.
The chemical analyses (Table 1) ware performed
to follow possible changes in the manure incurred by
both cultivation and sterilization, which could have also
affected the survival of the inoculated E. coli strain. The
relatively high TVBN value in the laboratory-cultivated
versus noncultivated manure might be related to increased
aerobic microbial activity in the former. It is possible that
sterilization has also resulted in nitrogen release in all
samples as a result to the heat treatment. The low TVBN
concentration in the sample from Harduf may be due to
unknown intrinsic factors. Further studies are needed to
negate possible nonbiological effects of the sterilization
process on the survival of E. coli. The E. coli counts were
similar or slightly higher in noncultivated manure than in
cultivated manure, which is generally in agreement with
our previous findings (Weinberg et al., 2011). Further
research is required to confirm the effect of cultivation
on mastitis-related strains of E. coli and other pathogens,
such as Staphylococcus aureus and streptococci.
Two fungal strains isolated from the laboratory culti-
vated manure exhibited activity against the tagged E. coli
strain. Additional 8 fungal strains have been found in the
large-scale (farm) cultivated samples. Interestingly, none
of the actinomycetes isolated from the laboratory and farm
samples were active against the tagged E. coli. This was
somewhat surprising because actinomycetes are known
to produce antibacterial compounds against gram-nega-
tive bacteria. The reason might be that the GFP-tagged
E. coli carries genes for streptomycin and kanamycin re-
sistance. Another reason might be that almost all these
isolated strains belong to the group of white-sporulating
actinomycetes, and therefore they represent only a nar-
row spectrum of the actinomycetes group.
Since both in the laboratory and farm-scale experi-
ments only some fungi proved to be antagonistic against
E. coli, the DM content of the manure during cultivation
may be involved. It should be optimal for fungal growth
in large-scale technology. Another important parameter
is the occasional mixing of the material to provide suf-
ficient amount of air for metabolism, while anaerobic or
semi-anaerobic conditions should be avoided.
Further studies are needed to elucidate the role of
antagonistic fungi in inhibiting E. coli and potentially
other bacterial pathogens in cultivated cattle manure. In
case such a role is found, it would be possible to develop
inoculants for cultivated cattle manure comprising such
fungi, which could inhibit E. coli and perhaps other det-
rimental bacteria at the barn.
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2341
Conclusions
The autochthonous microbial flora in cattle manure
consists of microorganisms, which affect the survival of
E. coli in the manure environment. It is possible that the
shorter survival duration of E. coli in cultivated manure
as compared with noncultivated manure is related to the
establishment of microbial populations with anti-E. coli
activity. Further studies are required to support this con-
clusion in commercial cow barns.
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