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.
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 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 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 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
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
0.04990.04350.06600.03950.04050.04050.0380
(0.009) (0.085) (0.006) (0.005) (0.0025) (0.0045)
(0.003)
0.073 0.073 0.07750.06750.126 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 0.08250.055 0.105 0.2 0.12750.045
(0.00?) (0.U025) (0.005) (0.005) (0.01) (0.0075)
(0.003)
0.125 0.115 0.03650.079 0.175 0.12250.0455
(0.005) (0.005) (0.0015) (0.001) (0.005) (0.005)
(0.005)
0.09850.099 0.035 0.062 0.105 0.099 0.0485
(0.005) (0.001) (0.000) (0.004) (0.005) (0.005)
(0.005)
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
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 TREATMENT GROUPS
INTERVAL PC T1 T2 Ts T4 Ts NC
1 0.399 0.041 0.226 0.348 0.27360.223 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 TREATMENT GROUPS

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
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.
Literature Cited:
Antonio Logreco:et.al:Epidemiology of Toxigenic Fungi and their associated
mycotoxins for some Mediterranean Crops:Europiean Journal of Plant Pathology
Volume 109, number 7/September,2003
Dhillon,K.S. 1973 preliminory observation on the treatment of Degnala disease
in Buffaloes.Indian VET.J. 50:5,482-484.
Gbore,F.A.et.al .Haematotoxicity of dietary Fumonisin B1 in growing pigs
University of Ibadan,Nigeria.
Genevieve S.Bondy.et.al:Immunomodulation by Fungal toxins.Journal of
Toxilogy and Environmental Health vol.3, number2/April 1 ,2000
H.V.L.N.Swamy et.al.Effects of feeding Blends of Grains naturally
Contaminated with Fusarium mycotoxins on growth and immunological
parameters of broiler Chickens
http://www.poultryscience.org/ps/paperpdfs/04/po44o533.pdf
Ifran,M 1971 The clinical picture and pathology of Degnala diseasesin buffaloes
and cattle in West Pakistan.Vet.Rec.88:422-424.
Ifran,M.and A.Maqbool.1986.Studies on Degnala disease in cattle and
buffaloes.Pakistan .Vet.J.6:87-93.
I.P.Oswald et.al. Immunotoxicological risk of mycotoxins for domestic
animals:Food Additives and Contaminants vol:22 number 4/April2005 pp354-360
Karki,K.1999.Degnala disease in Nepal.District Livestock Survices Banke Nepal
Annual report.(Unpublished).
Kalra ,D.S.,Bhatia,K.C.Degnala disease in buffalo and cattle:Epidemiological
investigation.J.Environ.Pathol Toxicol ONCOL 1990 May-JUNE:10(3):132-135.
Kalra ,D.S.,Bhatia,K.C.:Fusarium eqiseti associated mycotoxins as possible
cause of Degnala disease:Ann.Nutr.Aliment.1977;31(4-6):745-52
P.E.Nelson et.alTaxonomy,biology,andclinical aspects of Fusarium
species.Deparment of plant pathology,Pennsylvania State University,University
park 16802
Rodriguez-Adrian,L.J.,M.L.Grazzuiutti,J.H.Rex and E.J. Anaissie 1998 The
potential role of cytokine therapy for fungal infections in patients with
cancer:Is recovery from neutropenia all that is needed?CID.26 1270-1278.
Sarah I.Phillips et.al. The mycoflora and incidence of aflatoxin zearalenine and
sterigmatocystin in dairy feed and forage samples from eastern India and
Bangladesh.amaycopathologia.Biomedical and Life
Science.vol133.number1/January,1996
S.R.Chowdhary et.al:Effect of Feed-Borne Fusarium Mycotoxins on Hematology
and Immunology of Turkeys;Poultry Science –vol84 November 2005 number11.
Sharma .P:Immunotoxicity of Mycotoxins:1993.J.Dairy.Sci.76:892-897.
Shirlow,J.E.1939 Degnala Disease of Buffaloes:an account of the lesions and
essential pathology.Indian.Vet.Sci.Animal.Husb.9853-864.
Turgeon,M.L 1996 PrecipitationMethods :Immunology and Serology inLaboratory
Medicine 2nd Ed.Mosby.Missouri USA PP.131-133