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 postinfection. 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.( 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 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 .described Fusarium equisiti associated mycotoxin as possible cause of Degnala disease .Swamy 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 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 described that trichothecenes produced by Fusarium can both suppress and stimulate immune function.S.R.Chaudhary concluded that chronic consumption of grains naturally contaminated with Fusarium exerts only minor adverse effect on hematology and some immunological indices of 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. Antigenantibody 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 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, .Haematotoxicity of dietary Fumonisin B1 in growing pigs University of Ibadan,Nigeria. Genevieve by Fungal toxins.Journal of Toxilogy and Environmental Health vol.3, number2/April 1 ,2000 H.V.L.N.Swamy of feeding Blends of Grains naturally Contaminated with Fusarium mycotoxins on growth and immunological parameters of broiler Chickens 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 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 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 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

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