ASSESSMENT OF THE IMMUNOLOGIC PROPERTY OF Fusarium

graminearium in Mice.
Kedar B.Karki.
Principle Investigator,
College of Veterinary Medicine and Surgery CLUS.
The Philippines
Gemerlyn G. Garcia
College of Veterinary Medicine and Surgery CLUS.
The Philippines.

ABSTRACT
An experiment was conducted to investigate the immunologic property of Fusarium
Graminearum infection. Several groups of mice were randomly selected for the
following groups: (PC, T1and T2 were groups of mice that respectively received a
1:1, 1:100 and 1:100,000 fungal dilution while T3, T4, and T5 were groups of mice that
respectively received the same concentration but each were treated with Diethyl amine
Acetarsol (Acetylarsan). A group of mice was included as a negative control (NC).
Increase in weight of the spleen doubled as early as the second week (from 49 mg to 80
mg) and progressed up to the fourth week (125 mg) where it tapered off in the untreated
group. Similar increase in the weight of the spleen was observed in the treated group mg
to 64 mg) but not as great as that in the untreated group (105 mg). Hematological
findings showed a lymphocyte count of 1.83 that increased to 3.356, monocyte count of
0.47 that increased to 0.981 and neutrophils increased from 0.399 to 1.698 in untreated
groups. Lymphocyte count in the treated group was increased from 1.8 to 3.64,
monocytes increased from 0.068 to 0.325 and neutrophils increased from 0.223 to 1.056.
High incidence of death was observed in animals that did not receive treatment (PC, T1,
and T2) while relatively lower death incidences were exhibited by groups that received
diethyl amine acetarsol (T3, T4 and T5).
Precipitin test showed that F. graminearum stimulated antibody production in untreated
groups detected only from the third to the sixth week post-infection. This was significantly
different (P < 0.01) from the higher detection levels of antibody production elicited in
treated groups which persisted from the second week sustaining peaks until the sixth
week of observation. These findings suggest that F. graminearum is a pathogenic fungi
which can elicit immunity and can be treated with diethyIamine acetarsol.
1
A Masteral thesis by the senor author submitted to the Institute of Graduate Studies,
Central Luzon State University, Graduate student Science City of Muñoz, Nueva
Ecija
2 Assistant
professor Microbiology and Chairman Institute of Graduate Studies faculty of
veterinary.

Introduction.
The genus Fusarium contains important producing species that have been implicated in
several animal.diseases including Degnala disease hemorrhagic,estrogenic, emetic feed
refusal syndromes ,fescue foot moldy sweet potato toxicosis,been hulls poisoning,and

1
Equine leukoencephalomalacia.Many of these mycotoxin producing species have been
also implicated in several human disease such as alimentary toxic aleukia,urov or Kashin-
Beck disease,Akakabi-byo or scabby grain intoxication and esophageal
cancer.(www.mold-help.org)
Importance of the Study
There is insufficient information describing the immunological properties of the fungus
Fusarium in livestock in different parts of the globe. This fungus ad been associated with
Deg Nala disease. This affects largely buffalo's and cattle in India, Pakistan and
Nepal.The precise mechanisms underlying the observed symptoms of Deg Nala disease is
not known. In this study, investigative efforts had been focused on the ability to produce
immune response and efficiency of diethylamine acetarsol as effective therapeutic agent.
Statement of the Problem
Raising buffaloes and cattle in Pakistan, Nepal and India is one way of augmenting the
financial resources of village people.These animals are mainly raised on rice and wheat
straw which are of poor nutritional quality .Rice and wheat plant when infested by fungus
Fusarium causes severe health problem many researcher in this regard has documented.
Infections that may be debilitating in nature can cause significant economic losses as a
result ofDecreased production confounded by reduced growth rate, mortality and poor
animal performance. An effort to improve animal production in the village calls for
suitable control or therapeutic measures of any disease. Experimental evaluation of the
immunologic properties and treatment of F. graminearum infections should be
considered.
Objectives of the Study
The general objective of this study was to determine the immunological characteristics
of F. graminearium infection.
The specific objectives were the following:
1. To evaluate immunologic responses of experimental animals.

Time and Place of the Study

The study was conducted from October 2002 to January 2003 at the Veterinary
Microbiology Laboratory, College of Veterinary Science and Medicine, Central Luzon
State University, Science City of Munoz, Nueva Ecija, Philippines.
Review of Literature.
Deg Nala disease is a common infection among buffaloes and cattle in Pakistan, India
and Nepal. This has a seasonal occurrence which usually comes during the months of
November to January. It is believed that animals contract the disease when they consume
feedstuffs like rice straw infected with Fusarium that proliferated during storage at
winter.IL12, IL4, 11,10) in increasing antifungal activity of effecter cells has been
investigated (Rodriguez et al., 1998; and Stevens, 1998).The study of Karki (1999)
described the oral and potential use of Arsenic sulfate also termed as Deg Nala Liquor in
the area at 2% and 5% ratio and found to be effective. IL12, IL4, 11,10) in increasing
antifungal activity of effecter cells has been investigated (Rodriguez et al., 1998; and
Stevens, 1998).Deg Nala disease has been known to exist in Western Pakistan for nearly
half a century. The disease got its name because cases in buffalo were first been in the
Deg Nala (river) area. Shirlaw (1939) reported the occurrence of the disease which

2
affected a large number of buffaloes in the various village of Shekhpura and Mudrika,
parts of Deg Nala area during the year 1929-1930. Since then, cases of this disease have
been observed in other parts of Pakistan. The disease is no longer confined to area around
Deg Nala nowadays but is reportedly seen infecting animals a raised in other low-lying
areas where rice is cultivated.Shirlaw (1939) described Deg Nala disease in buffaloes as
associated with Fever, pain in the abdomen, painful gait and anorexia. Kwatra and Singh
(1971) characterized the disease as one that caused necrosis of the tips of the ears, tail
and tongue; and swelling of the extremities with subsequent peeling of the skin leaving
open wounds. The same type of disease has been reported in some parts of the state of
Hariyama from 1969 to 1971 and the state of Punjab, India (Dhillon, 1973)Fusarium-
related Deg Nala disease was described to have a seasonal incidence and sporadic cases
were seen in winter months when rice straw was used as a fodder (Irfan, 1971). During
the same period, sporadic cases were reported by many field veterinarians in many parts
of Nepal with obscure diagnosis and treatment. In the year 1986, Karki reported this
disease in Banke district of Nepal where buffaloes were mostly infected. He also
attempted to initiate treatment which was followed by soother researchers in India with
apparent success. An effort to isolate the fungus from affected straw was undertaken by
the Commonwealth Mycological Laboratory which led to the identification of Fusarium
(Irfan and Maqbool, 1986).Antanio Logrieco et.al has documented the epidemiology of
toxigenic Fungi and their Associated mycotoxin for some Mediterranean crops. There is
now compelling evidence implicating the Fusarium mycotoxin in livestock disorders in
different parts of the world and the risk of continuing exposure has no diminished inspire
of enhanced awareness of its debilitating effects. It is clear that chronic intake of
Fusarium by farm livestock is inevitable. There is sufficient information of specific
conditions positively identified with sufficient data to propose further syndromes arising
from other Fusarium (Irfan and Maqbool, 1986).Kalra et.al .described Fusarium equisiti
associated mycotoxin as possible cause of Degnala disease .Swamy et.al described the
supplementation with GM polymer in contaminated diet nonspecifically increased the
white blood cell count and lymphocyte count, while prevented mycotoxin induced
decreases in B-cell count.He concluded that broiler chickens are susceptible during
extended feeding of grains naturally contaminated with Fusarium.Gbore.et.al described
animal fed with Fusarium contaminated diet is haematotoxic in pigs .There was decrease
in haemoglobin,erythrocytes,MCV,PCV, values were decreased, on the other hand
leukocyte and platelet value was increased. Sharma described the development of
leucopenia and decreased functioning of peripheral lymphocytes in sheep and calves.
There was production of antibodies against mycotoxin conjugates with increase in dose
of Fusarium .Genevieve .et.al described that trichothecenes produced by Fusarium can
both suppress and stimulate immune function.S.R.Chaudhary et.al concluded that chronic
consumption of grains naturally contaminated with Fusarium exerts only minor adverse
effect on hematology and some immunological indices of turkeys.I.P.Oswald.et.al
described mycotoxin induced immunosuppressant is manifested as depressed T-or B-
lymphocyte activity, suppressed antibody production and impaired
macrophage/neutrophil-effector function
Blood and Serum Collection
Mice were sacrificed at predetermined time interval (7, 14, 21, 28 and 35-day post
infection). The animals were sedated in chloroform (Appendix Plate 2) and disinfected

3
with 70% ethanol then they were bled by cardiac puncture. Samples of blood were
collected for the determination of total WBC and differential counts. Blood was allowed
to clot to facilitate serum collection. Serum collected from mice from each treatment
group was pooled as one and stored frozen until use for the precipitin test.
Determination of White Blood Cell Counts and Relative Differential Counts
Blood samples were collected at weekly intervals for six weeks for the enumeration of
white blood cell such as lymphocytes, monocytes and neutrophils. A volume of 20 yl
blood was added to a 180 ~L1 volume of blood previously deposited in a Petri dish. The
mixture was mixed gently before a volume of 20 ml was charged in a haemocytometer
counting chamber.
The total WBC count was calculated by using the following formula:

Total Number of cells counted in 4 corners/4 X 10 X104 cells/ml Replicates of blood

smears were likewise made on clean slides for the differential counts. These were
allowed air-dried and stained. Each individual cell type has been recorded on a tally sheet
until a total of 100 cells had been counted.
The relative differential count was computed by following the formula:
Average cell count X 100% X Total WBC count (at a given time interval)
Determination of Antibody Production (Precipitin Test)
The precipitin test used in the study was based on the methods of Turgeon (1996). The
center wells were filled almost to the brim with antiserum from mice while the peripheral
wells were filled with two suspensions of the fungi. The lid of the plate was replaced
before incubation for two to four days at 37°C. After incubation, the plate was examined
for basic reaction patterns (identity, partial identity and nonidentity) which form at points
of equivalence if precipitating antibodies to Fusarium graminearum are present. Antigen-
antibody relationships were interpreted as scores and were used to categorize responses:
0.0 (Inhibition) - This means that the antigens carry unrelated determinants different
from antibody components.

1.0 (Non-identity pattern) - This is expressed when the precipitation lines cross each
other. This indicates that the antigens in the samples are non-identical meaning they
contain no antigenic determinants in common.

28
3.0 (Reaction of Partial Identity) - This happens when the precipitation lines merge with
spur formation.

5.0 (Identity) - The identity pattern is indicated by a precipitin band forming a single
smooth arc that suggests fusion of antibody in the
antiserum and antigen in the wells. This interaction further indicates ability of the
antibody to precipitate identical antigens in each of two fungal preparations.

Mortality, Morbidity Incidence and Recovery of Fungi

4
Mortality rate of experimental animal was recorded. Signs of splenomegaly were
monitored by taking the weight of the spleen at prescribed time intervals. Tissue samples
from the lungs, liver, and kidney were collected and streaked on Sabouraud's dextrose
agar for fungal recovery.

Analysis of Data
All data in in vivo and in vitro experiments were interpreted mean (± standard deviation)
responses of experimental animals. ` Differences of responses among treatment means
were analyzed using two-way analysis of variance.
RESULTS AND DISCUSSION
.
Splenomegaly
Splenomegaly (Table 5) was observed in the positive control. The
Initial weight of 49 mg increased to 125 mg in the fifth week. The group that was given
lower concentration of pathogen had the lowest spleen weight (43 mg) and reached
weight of 115 mg in the fifth week. On those groups that were treated with antifungal
drug, the lowest weight of the spleen was 60 mg and highest weight was of 200 mg in the
fourth week.
This pattern indicated that in the control group and even in mice that received lower
concentration of fungi, the body system took a longer time to develop the capacity to
build up the body immune systems. These results are similar to the findings of other
authors relating to splenomegaly in human beings diagnosed with systemic infection due
to Fusarium (Guarro and Gene, 1995).
Total, White Blood Cell Count
The hematological findings of this study (Table 6) indicated that there was abrupt
increase of total white blood cell count during the entire study period. In the uositive
control, an initial count of 2.35 x 106 cells/ml was recorded which increased to 6.56 x
106 cells/ml in the sixth week. In the group that was given lower concentration of
Table 5. Weekly weight of spleen graminearum mice
WEEKLY INTERVAL TREATMENT GROUPS
PC T1 T2 Ts Ta Ts NC
0.0499 0.0435 0.0660 0.0395 0.0405 0.0405 0.0380
(0.009)(0.085)(0.006)(0.005)(0.0025) (0.0045) (0.003)
0.073 0.073 0.0775 0.0675 0.126 0.063 0.064
(0.0015) (0.005)(0.005)(0.005)(0.014)(0.0015) (0.002)
0.075 0.165 0.085 0.11 0.165 0.125 0.0415
(0.005)(0.055)(0.005)(0.01) (0.055)(0.005)(0.035)
0.08 0.0825 0.055 0.105 0.2 0.1275 0.045
(0.00?) (0.U025) (0.005)(0.005)(0.01) (0.0075) (0.003)
0.125 0.115 0.0365 0.079 0.175 0.1225 0.0455
(0.005)(0.005)(0.0015) (0.001)(0.005)(0.005)(0.005)
0.0985 0.099 0.035 0.062 0.105 0.099 0.0485
(0.005)(0.001)(0.000)(0.004)(0.005)(0.005)(0.005)

5
Data are mean (±) weights of spleen (g) from animals experimentally infected with the
fungal pathogen determined at the indicated time intervals.
4j
Table 6. Weekly total white blood infected with F. grarninearum
count x 106)

TREATMENT GROUPS
INTERVAL P,` TZ ,I,3 T4 TS NC
2.35 2.37 2.26 2.32 2.28 2.23 1.16
1
(0.05) (0.125)(0.01) (0.04) (0.02) (0.03)
4.55 4.69 4.45 4.65 4.36 4.35 2.95
(0.02) (0.01) (0.85) (0.90) (0.16) (O.1S) (0.01)
3 5.38 5.37 4.48 5.45 5.33 5.25 3.55
(0.02) (0.02) - (0.15) (0.20) (0.03) (0.25) (0.03)
4 5.62 5.08 5.82 4.87 4.78 4.05 3.92
Data are mean (±) weights of spleen (g) from animals experimentally infected with the
fungal pathogen determined at the indicated time intervals, the pathogen, initial count of
2.37 x 10c) cells/ml and an increase of up to 5.28 x 106 cells/ml on the sixth week were
recorded. In the group which received the antifungal drug, an initial count of 2.28 x 106
cells/ml increased up to 6.32 x 106 cells/ml up to the sixth week. In the negative control,
initially it was recorded 1.16 x 106 cells/ml to the highest count of 3.'?5 x 106 cells/ml in
the last of observation. These data suggest that white blood cell responses in this type of
infection is activated for defense mechanism but takes longer time in both treated and
untreated r-troups.
Relative Differential Counts
Data on relative WBC differential counts (Tables 7a to c) expressed in the cellular
component of the blood. The number of lymphocytes was 1.83 which increased to 3.35,
for rnonocytes it increased from 0.47 to 0.981 and
in x 106 cells/ml revealed changes
Tablr 7a. Weekly lymphocyte counts (106 cells/ml) of mice infected with F. graminearum
WEEKLY
INTERVAL PC Ti TREATMENT
T2 Ts GROUPS
T4 Ts NC
1 1.88 1.835 1.859 1.879 1.938 1.917 1.06
2 3.04 3.00 3.204 4.407 3.488 3.48 2.65
3 3.44 3.436 2.867 3.706 3.997 3.83 3.266
4 3.48 3.351 3.433 3.068 3.641 2.754 3.606
5 2.76 2.733 3.095 2.713 3.641 2.274 3.249
6 1.973 1.836 2.196 2.062 2.11 2.288 3.203
47
Table 7b. Weekly monocyte counts (106 cells/ml) of mice infected with F.
graminearum
WEEKLY
INTERVAL PC Ti TREATMENT

6
TZ T3 GROUPS
T4 TS NC
1 0.047 0.123 1.568 0.928 0.068 0.089 0.023
2 0.546 0.449 0.578 2.260 0.305 0.176 0.029
3 0.699 0.859 0.716 0.654 0.400 0.368 0.040
4 0.899 0.624 0.640
a 0.889 0.533 0.284 0.078
5 0.865 1.125 0.712 0.399 0.405 0.361 0.036
6 0.918 1.147 1.464 0.562 0.325 0.176 0.141
Table 7r. Weekly neutrophil counts (106 cells/ml) of mice infected with F.
grantinearum
WEEKLY TREATMENTGROUPS
INTERVAL PC T1 T2 Ts T4 Ts NC
1 0.399 0.041 0.226 0.348 0.2736 0.223 0.070
2 0.455 '.030 0.667 1.017 0.567 0.609 0.266
3 1.237 1.074 0.896 1.090 1.333 1.050 0.284
4 1.292 1.700 1.746 1.412 1.439 1.013 0.235
5 1.202 1.340 1.629 0.877 1.734 0.976 0.325
6 1.698 1.848 1.569 1.125 0.813 1.056 0.176
for rieutrophils it increased from 0.399 to 1.698 for untreated groups. Lymphocytes in the
treated groups increased from 1.8 to 3.64, rnonocvtes increased from 0.068 to 0.325) and
neutrophils increased from 0.2)23 t0 1.056.
Data indicate that with the introduction of infection, all three cell types (lymphocytes,
monocytes and neutrophils) were activated simultaneously. The tapering of the
lymphocyte count on the fifth week shows that it has a low capacity to counteract
infection. On the other hand, continuous and sustained increment of macrophage and
neutrophil counts throughout the study period indicates that these were main cellular
components that responded to this type of infection.
Antigen-Antibody Reactions
On the data on antigen antibody reaction measured in terms of scores on precipitin lines
exhibited, it took some time for antibody against high levels of Fusarium infection to
build up (Table 8). Only in induced infection with relatively lower density of Fusarium
(T2) was antibody production detected at an earlier onset (third week). Groups of mice
that received the antifungal diethylamine acetarsol ((T5) began to rxhibit antigen
antibody responses as early as in the first week. All trc:atrci groups manifested reactions
indicative of partial identity (3.0
'i'~thlc 8. Week 1), antigen
antibody
reaction of
49
mice infected with F.
grantinearum -._-

WE' E:}iLY TREATMENTGROUPS

IN"I'I:RVAL PC T1 T2 T3 Ta Ts NC
1 Mean 0.0 0.0 0.0 0.0 0.0 2.0 0.0

7
 SD 0.00 0.00 0.00 0.00 0.00 1.41 0.0
2 Mean 0.00 0.00 0.00 3.0 3.67 4.33 0.00
SD 0.00 0.00 0.00 0.00 0.943 0.943 0.0
3 Mean 1.0 1.0 2.0 3.67 4.33 4.33 0.0
SD 1.41 1.41 1.41 0.943 0.943 0.943 0.00
4 Mean 2.0 3.0 3.67 4.33 5.0 5.0 0.0
SD 1.41 0.00 0.943 0.943 0.00 0.00 0.00
5 Mean 3.0 3.0 3.67 5.0 5.0 5.0 0.0
SD 0.00 0.0 0.943 0.00 0.00 000 0.00
6 Mean 2.33 3.0 3.67 5.0 5.0 5.0 0.00
SD 0.943 0.00 0.943 0.00 0.00 0.00 0.00
scores) from second to third week. Reaction scores suggestive of strong identity to the
antibody specific to the antigen were observed from fourth week onwards. This was not
at all observed in groups not treated with Observed reactions merely suggested partial
the antifungal drug.
identity towards the antigen as evidenced by a majority of 3.0 score.
Highly significant differences in responses between treatments as well as responses
elicited in each time interval indicated were noted (P < 0.01). These findings are
preliminary reports on the establishment of antigen antibody reactions related to F.
graminearum-induced infections.
Summary Conclusion and Recommendation.
Based on this findings gathered in this study,F.graminearum is a pathological agent which
has the ability to effect vital organs of the body which could cause impairment of organ
functions.This pathogen posses the ability to digest elastin and collagen present in body
tissue which could attribute the manifestation of disease .This study also indicated that
F.graminearum caused substantial pathological damage to liver,lung,spleen.The pathogen
was found to induce leukocytosis and marked increase of
lymphocytes,neutrophil,macrophage.In this study it was found that when infection was
induced the mortality ranges from20% in first week and decline to7.69% in the third
week in positive control group and untreated group.While in treated group mortality was
only 5% from first to third week .Moreover simultaneous use of antifungal(Diethylamine
acetarsol,Acetylarson,Antidegnala liquior) induced development of immunity and was
proven to be effective against infection.Taking the result of this study it is recommended
that effort should be directed toward the prevention of F.graminearum infection in
animals. Moreover further study should be conducted to confirm the involvement of this
fungus in other animal and poultry diseases.Finaly the application of Diethylamine
Acetarsol or its depravities(anti-degnala liquior) other immunomodulator for treatment of
F.graminearum infection in domestic animals should be looked into.
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