Assessment of the Immunologic Property of Fusarium Graminearium in Mice

8/14/2019 Assessment of the Immunologic Property of Fusarium Graminearium in Mice

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ASSESSMENT OF THE IMMUNOLOGIC PROPERTY OF Fusarium gramineariuminMice.

Kedar B.Karki.Principle Investigator,

College of Veterinary Medicine and Surgery CLUS.

The PhilippinesGemerlyn G. GarciaCollege of Veterinary Medicine and Surgery CLUS.

The Philippines.ABSTRACT

An experiment was conducted to investigate the immunologic property ofFusarium Graminearum infection. Several groups of mice were randomlyselected for the following groups: (PC, T1and T2 were groups of micethat 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 sameconcentration 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 49mg to 80 mg) and progressed up to the fourth week (125 mg) where it taperedoff in the untreated group. Similar increase in the weight of the spleen wasobserved in the treated group mg to 64 mg) but not as great as that in theuntreated group (105 mg). Hematological findings showed a lymphocyte countof 1.83 that increased to 3.356, monocyte count of 0.47 that increased to0.981 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 0.223to 1.056. High incidence of death was observed in animals that did not receive

treatment (PC, T1, and T2) while relatively lower death incidences wereexhibited by groups that received diethyl amine acetarsol (T3, T4 and T5).Precipitin test showed that F. graminearum stimulated antibody production inuntreated groups detected only from the third to the sixth week post-infection. This was significantly different (P < 0.01) from the higher detectionlevels of antibody production elicited in treated groups which persisted fromthe second week sustaining peaks until the sixth week of observation. These

findings suggest that F. graminearum is a pathogenic fungi which can elicitimmunity 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 diseasehemorrhagic,estrogenic, emetic feed refusal syndromes ,fescue foot moldysweet potato toxicosis,been hulls poisoning,and Equineleukoencephalomalacia.Many of these mycotoxin producing species have beenalso implicated in several human disease such as alimentary toxic aleukia,urovor Kashin-Beck disease,Akakabi-byo or scabby grain intoxication and esophagealcancer.(www.mold-help.org)Importance of the Study


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There is insufficient information describing the immunological properties of thefungus Fusarium in livestock in different parts of the globe. This fungus ad beenassociated with Deg Nala disease. This affects largely buffalo's and cattle inIndia, Pakistan and Nepal.The precise mechanisms underlying the observedsymptoms of Deg Nala disease is not known. In this study, investigative efforts

had been focused on the ability to produce immune response and efficiency ofdiethylamine acetarsol as effective therapeutic agent.Statement of the ProblemRaising buffaloes and cattle in Pakistan, Nepal and India is one way ofaugmenting the financial resources of village people.These animals are mainlyraised on rice and wheat straw which are of poor nutritional quality .Rice andwheat plant when infested by fungus Fusarium causes severe health problemmany researcher in this regard has documented. Infections that may bedebilitating in nature can cause significant economic losses as a resultofDecreased production confounded by reduced growth rate, mortality andpoor animal performance. An effort to improve animal production in the village

calls for suitable control or therapeutic measures of any disease. Experimentalevaluation of the immunologic properties and treatment of F. graminearuminfections should be considered.Objectives of the StudyThe 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 StudyThe study was conducted from October 2002 to January 2003 at the VeterinaryMicrobiology 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 themonths of November to January. It is believed that animals contract the diseasewhen they consume feedstuffs like rice straw infected with Fusarium thatproliferated during storage at winter.IL12, IL4, 11,10) in increasing antifungalactivity of effecter cells has been investigated (Rodriguez et al., 1998; andStevens, 1998).The study of Karki (1999) described the oral and potential useof Arsenic sulfate also termed as Deg Nala Liquor in the area at 2% and 5% ratioand 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 nearlyhalf a century. The disease got its name because cases in buffalo were firstbeen in the Deg Nala (river) area. Shirlaw (1939) reported the occurrence ofthe disease which affected a large number of buffaloes in the various village ofShekhpura 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


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reportedly seen infecting animals a raised in other low-lying areas where rice iscultivated.Shirlaw (1939) described Deg Nala disease in buffaloes as associatedwith 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 theears, 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 insome 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 aseasonal incidence and sporadic cases were seen in winter months when ricestraw was used as a fodder (Irfan, 1971). During the same period, sporadiccases were reported by many field veterinarians in many parts of Nepal withobscure diagnosis and treatment. In the year 1986, Karki reported this diseasein Banke district of Nepal where buffaloes were mostly infected. He alsoattempted to initiate treatment which was followed by soother researchers inIndia with apparent success. An effort to isolate the fungus from affected strawwas undertaken by the Commonwealth Mycological Laboratory which led to the

identification of Fusarium (Irfan and Maqbool, 1986).Antanio Logrieco et.al hasdocumented the epidemiology of toxigenic Fungi and their Associatedmycotoxin for some Mediterranean crops. There is now compelling evidenceimplicating the Fusarium mycotoxin in livestock disorders in different parts ofthe world and the risk of continuing exposure has no diminished inspire ofenhanced awareness of its debilitating effects. It is clear that chronic intake ofFusarium by farm livestock is inevitable. There is sufficient information ofspecific conditions positively identified with sufficient data to propose furthersyndromes arising from other Fusarium (Irfan and Maqbool, 1986).Kalra et.al.described Fusarium equisiti associated mycotoxin as possible cause of Degnaladisease .Swamy et.al described the supplementation with GM polymer in

contaminated diet nonspecifically increased the white blood cell count andlymphocyte count, while prevented mycotoxin induced decreases in B-cellcount.He concluded that broiler chickens are susceptible during extendedfeeding of grains naturally contaminated with Fusarium.Gbore.et.al describedanimal fed with Fusarium contaminated diet is haematotoxic in pigs .There wasdecrease in haemoglobin,erythrocytes,MCV,PCV, values were decreased, on theother hand leukocyte and platelet value was increased. Sharma described thedevelopment of leucopenia and decreased functioning of peripherallymphocytes in sheep and calves. There was production of antibodies againstmycotoxin conjugates with increase in dose of Fusarium .Genevieve .et.aldescribed that trichothecenes produced by Fusarium can both suppress and

stimulate immune function.S.R.Chaudhary et.al concluded that chronicconsumption of grains naturally contaminated with Fusarium exerts only minoradverse effect on hematology and some immunological indices ofturkeys.I.P.Oswald.et.al described mycotoxin induced immunosuppressant ismanifested as depressed T-or B-lymphocyte activity, suppressed antibodyproduction and impaired macrophage/neutrophil-effector functionBlood and Serum Collection


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Mice were sacrificed at predetermined time interval (7, 14, 21, 28 and 35-daypost infection). The animals were sedated in chloroform (Appendix Plate 2) anddisinfected with 70% ethanol then they were bled by cardiac puncture. Samplesof blood were collected for the determination of total WBC and differentialcounts. Blood was allowed to clot to facilitate serum collection. Serum

collected from mice from each treatment group was pooled as one and storedfrozen until use for the precipitin test.Determination of White Blood Cell Counts and Relative Differential CountsBlood samples were collected at weekly intervals for six weeks for theenumeration of white blood cell such as lymphocytes, monocytes andneutrophils. A volume of 20 yl blood was added to a 180 ~L1 volume of bloodpreviously deposited in a Petri dish. The mixture was mixed gently before avolume 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 ofblood smears were likewise made on clean slides for the differential counts.

These were allowed air-dried and stained. Each individual cell type has beenrecorded 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 frommice 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 at37C. 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 categorizeresponses:0.0 (Inhibition) - This means that the antigens carry unrelated determinantsdifferent from antibody components.1.0 (Non-identity pattern) - This is expressed when the precipitation lines crosseach other. This indicates that the antigens in the samples are non-identicalmeaning they contain no antigenic determinants in common.283.0 (Reaction of Partial Identity) - This happens when the precipitation linesmerge with spur formation.

5.0 (Identity) - The identity pattern is indicated by a precipitin band forming asingle smooth arc that suggests fusion of antibody in theantiserum and antigen in the wells. This interaction further indicates ability ofthe antibody to precipitate identical antigens in each of two fungalpreparations.Mortality, Morbidity Incidence and Recovery of FungiMortality rate of experimental animal was recorded. Signs of splenomegalywere monitored by taking the weight of the spleen at prescribed time


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intervals. Tissue samples from the lungs, liver, and kidney were collected andstreaked on Sabouraud's dextrose agar for fungal recovery.Analysis of DataAll data in in vivo and in vitro experiments were interpreted mean ( standarddeviation) responses of experimental animals. ` Differences of responses

among treatment means were analyzed using two-way analysis of variance.RESULTS AND DISCUSSION.SplenomegalySplenomegaly (Table 5) was observed in the positive control. TheInitial weight of 49 mg increased to 125 mg in the fifth week. The group thatwas given lower concentration of pathogen had the lowest spleen weight (43mg) and reached weight of 115 mg in the fifth week. On those groups that weretreated with antifungal drug, the lowest weight of the spleen was 60 mg andhighest 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 developthe capacity to build up the body immune systems. These results are similar tothe findings of other authors relating to splenomegaly in human beingsdiagnosed with systemic infection due to Fusarium (Guarro and Gene, 1995).Total, White Blood Cell CountThe hematological findings of this study (Table 6) indicated that there wasabrupt 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 recordedwhich increased to 6.56 x 106 cells/ml in the sixth week. In the group that wasgiven lower concentration ofTable 5. Weekly weight of spleen graminearum mice

WEEKLY INTERVAL TREATMENT GROUPSPC T1 T2 Ts Ta Ts NC0.04990.04350.06600.03950.04050.04050.0380(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


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(0.005) (0.001) (0.000) (0.004) (0.005) (0.005)(0.005)

Data are mean () weights of spleen (g) from animals experimentally infectedwith the fungal pathogen determined at the indicated time intervals.4j

Table 6. Weekly total white blood infected with F. grarninearumcount x 106)TREATMENT GROUPSINTERVAL P,` TZ ,I,3 T4 TS NC

2.35 2.37 2.26 2.32 2.28 2.23 1.161

(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 3.92Data are mean () weights of spleen (g) from animals experimentally infectedwith the fungal pathogen determined at the indicated time intervals, thepathogen, initial count of 2.37 x 10c) cells/ml and an increase of up to 5.28 x106 cells/ml on the sixth week were recorded. In the group which received theantifungal drug, an initial count of 2.28 x 106 cells/ml increased up to 6.32 x106 cells/ml up to the sixth week. In the negative control, initially it wasrecorded 1.16 x 106 cells/ml to the highest count of 3.'?5 x 106 cells/ml in thelast of observation. These data suggest that white blood cell responses in thistype of infection is activated for defense mechanism but takes longer time inboth treated and untreated r-troups.

Relative Differential CountsData on relative WBC differential counts (Tables 7a to c) expressed in thecellular component of the blood. The number of lymphocytes was 1.83 whichincreased to 3.35, for rnonocytes it increased from 0.47 to 0.981 andin x 106 cells/ml revealed changesTablr 7a. Weekly lymphocyte counts (106 cells/ml) of mice infected with F.graminearumWEEKLYINTERVAL PC Ti TREATMENTT2 Ts GROUPST4 Ts NC

1 1.88 1.835 1.859 1.879 1.938 1.917 1.062 3.04 3.00 3.204 4.407 3.488 3.48 2.653 3.44 3.436 2.867 3.706 3.997 3.83 3.2664 3.48 3.351 3.433 3.068 3.641 2.754 3.6065 2.76 2.733 3.095 2.713 3.641 2.274 3.2496 1.973 1.836 2.196 2.062 2.11 2.288 3.20347


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Table 7b. Weekly monocyte counts (106 cells/ml) of mice infected with F.graminearumWEEKLYINTERVAL PC Ti TREATMENTTZ T3 GROUPS

T4 TS NC1 0.047 0.123 1.568 0.928 0.068 0.089 0.0232 0.546 0.449 0.578 2.260 0.305 0.176 0.0293 0.699 0.859 0.716 0.654 0.400 0.368 0.0404 0.899 0.624 0.640a 0.889 0.533 0.284 0.0785 0.865 1.125 0.712 0.399 0.405 0.361 0.0366 0.918 1.147 1.464 0.562 0.325 0.176 0.141Table 7r. Weekly neutrophil counts (106 cells/ml) of mice infected with F.grantinearumWEEKLY TREATMENT GROUPS

INTERVAL PC T1 T2 Ts T4 Ts NC1 0.399 0.041 0.226 0.348 0.27360.223 0.0702 0.455 '.030 0.667 1.017 0.567 0.609 0.2663 1.237 1.074 0.896 1.090 1.333 1.050 0.2844 1.292 1.700 1.746 1.412 1.439 1.013 0.2355 1.202 1.340 1.629 0.877 1.734 0.976 0.3256 1.698 1.848 1.569 1.125 0.813 1.056 0.176for rieutrophils it increased from 0.399 to 1.698 for untreated groups.Lymphocytes in the treated groups increased from 1.8 to 3.64, rnonocvtesincreased 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. Thetapering of the lymphocyte count on the fifth week shows that it has a lowcapacity to counteract infection. On the other hand, continuous and sustainedincrement of macrophage and neutrophil counts throughout the study periodindicates that these were main cellular components that responded to this typeof infection.Antigen-Antibody ReactionsOn the data on antigen antibody reaction measured in terms of scores onprecipitin lines exhibited, it took some time for antibody against high levels ofFusarium infection to build up (Table 8). Only in induced infection withrelatively lower density of Fusarium (T2) was antibody production detected at

an earlier onset (third week). Groups of mice that received the antifungaldiethylamine acetarsol ((T5) began to rxhibit antigen antibody responses asearly as in the first week. All trc:atrci groups manifested reactions indicative ofpartial identity (3.0'i'~thlc 8. Week 1), antigenantibodyreaction of49


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mice infected with F.grantinearum -._-

WE' E:}iLY TREATMENT GROUPSIN"I'I:RVAL PC T1 T2 T3 Ta Ts NC

1 Mean 0.0 0.0 0.0 0.0 0.0 2.0 0.0SD 0.00 0.00 0.00 0.00 0.00 1.41 0.02 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 0.03 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 0.004 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 0.005 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 0.006 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 0.00scores) from second to third week. Reaction scores suggestive of strong identityto the antibody specific to the antigen were observed from fourth weekonwards. This was not at all observed in groups not treated with Observedreactions merely suggested partialthe antifungal drug.identity towards the antigen as evidenced by a majority of 3.0 score.Highly significant differences in responses between treatments as well asresponses elicited in each time interval indicated were noted (P < 0.01). Thesefindings are preliminary reports on the establishment of antigen antibodyreactions related to F. graminearum-induced infections.

Summary Conclusion and Recommendation.Based on this findings gathered in this study,F.graminearum is a pathologicalagent which has the ability to effect vital organs of the body which could causeimpairment of organ functions.This pathogen posses the ability to digest elastinand collagen present in body tissue which could attribute the manifestation ofdisease .This study also indicated that F.graminearum caused substantialpathological damage to liver,lung,spleen.The pathogen was found to induceleukocytosis and marked increase of lymphocytes,neutrophil,macrophage.Inthis study it was found that when infection was induced the mortality rangesfrom20% in first week and decline to7.69% in the third week in positive controlgroup and untreated group.While in treated group mortality was only 5% from

first to third week .Moreover simultaneous use of antifungal(Diethylamineacetarsol,Acetylarson,Antidegnala liquior) induced development of immunityand was proven to be effective against infection.Taking the result of this studyit is recommended that effort should be directed toward the prevention ofF.graminearum infection in animals. Moreover further study should beconducted to confirm the involvement of this fungus in other animal andpoultry diseases.Finaly the application of Diethylamine Acetarsol or its


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depravities(anti-degnala liquior) other immunomodulator for treatment ofF.graminearum infection in domestic animals should be looked into.Literature Cited:Antonio Logreco:et.al:Epidemiology of Toxigenic Fungi and their associatedmycotoxins for some Mediterranean Crops:Europiean Journal of Plant Pathology

Volume 109, number 7/September,2003Dhillon,K.S. 1973 preliminory observation on the treatment of Degnala diseasein Buffaloes.Indian VET.J. 50:5,482-484.Gbore,F.A.et.al .Haematotoxicity of dietary Fumonisin B1 in growing pigsUniversity of Ibadan,Nigeria.Genevieve S.Bondy.et.al:Immunomodulation by Fungal toxins.Journal ofToxilogy and Environmental Health vol.3, number2/April 1 ,2000H.V.L.N.Swamy et.al.Effects of feeding Blends of Grains naturallyContaminated with Fusarium mycotoxins on growth and immunologicalparameters of broiler Chickenshttp://www.poultryscience.org/ps/paperpdfs/04/po44o533.pdf

Ifran,M 1971 The clinical picture and pathology of Degnala diseasesin buffaloesand cattle in West Pakistan.Vet.Rec.88:422-424.Ifran,M.and A.Maqbool.1986.Studies on Degnala disease in cattle andbuffaloes.Pakistan .Vet.J.6:87-93.I.P.Oswald et.al. Immunotoxicological risk of mycotoxins for domesticanimals:Food Additives and Contaminants vol:22 number 4/April2005 pp354-360Karki,K.1999.Degnala disease in Nepal.District Livestock Survices Banke NepalAnnual report.(Unpublished).Kalra ,D.S.,Bhatia,K.C.Degnala disease in buffalo and cattle:Epidemiologicalinvestigation.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):745-52P.E.Nelson et.alTaxonomy,biology,andclinical aspects of Fusariumspecies.Deparment of plant pathology,Pennsylvania State University,Universitypark 16802Rodriguez-Adrian,L.J.,M.L.Grazzuiutti,J.H.Rex and E.J. Anaissie 1998 Thepotential role of cytokine therapy for fungal infections in patients withcancer:Is recovery from neutropenia all that is needed?CID.26 1270-1278.Sarah I.Phillips et.al. The mycoflora and incidence of aflatoxin zearalenine andsterigmatocystin in dairy feed and forage samples from eastern India andBangladesh.amaycopathologia.Biomedical and LifeScience.vol133.number1/January,1996

S.R.Chowdhary et.al:Effect of Feed-Borne Fusarium Mycotoxins on Hematologyand 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 andessential pathology.Indian.Vet.Sci.Animal.Husb.9853-864.Turgeon,M.L 1996 PrecipitationMethods :Immunology and Serology inLaboratoryMedicine 2nd Ed.Mosby.Missouri USA PP.131-133

Assessment of the Immunologic Property of Fusarium Graminearium in Mice

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