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Opportunity Um mm: m“ EFFECTS OF T-2 TOXIN ON VACCINAL IMMUNITY AGAINST MAREK’S DISEASE AND ON THE IMMUNE SYSTEM OF WHITE LEGHORN CHICKENS BY Eric Kufuor—Mensah A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Pathology and Center for Environmental Toxicology 1996 ABSTRACT EFFECTS OF T-2 TOXIN ON VACCINAL IMMUNITY AGAINST MAREK'S DISEASE AND ON THE IMMUNE SYSTEM OF WHITE LEGHORN CHICKENS BY Eric Kufuor-Mensah Four trials were conducted to determine the effects of T-2 toxin on vaccinal immunity against Marek’s disease (MD). Day-old, RPRL line 1515}( 71chicks were treated daily for seven days via crOp gavage with T—2 toxin at a subclinical dose of 1.25 mg/kg body weight. Treated and untreated chicks were vaccinated with herpesvirus of turkeys (HVT) at hatch and challenged with JM strain of MD virus (MDV) at 8 days of age. Chickens were tested for HVT and MDV viremia and were observed for the development of MD lesions within 8 weeks of age. T-2 toxin significantly reduced body weight by 7 days after treatment. T-2 toxin shortened the incubation period for the development of MD lesions and mortality, but only in unvaccinated, challenged chickens. T—2 toxin also significantly reduced titres (Hf HVT viremia within.'7 days post—vaccination. However, the percent protection from MD in T-2 toxin-treated, HVT—vaccinated chickens ranged from 82%- 95% and was comparable to that in untreated chickens (89%- 100%). In a second experiment, the effect of T-2 toxin on peripheral blood. and. splenic: B- and. T— lymphocyte subpopulations was evaluated using flow cytometry. Day—old, 1515 x 71 chicks were treated daily for 7 days via crop gavage with T-2 toxin at a subclinical dose of 1.25 mg/kg body weight. Peripheral blood and splenic lymphocytes from T—2 toxin-treated and untreated control chicks were analysed for phenotypic expression of CD4, CD8, CD3 and IgM cell surface markers at 8-9, and 21—22 days of age. The percentages of both peripheral blood and splenic B- lymphocytes in T—2 toxin-treated chickens were significantly lower than that in untreated chickens but only at 8-9 days of age. However, at 21-22 days of age the percentages of B- and T-lymphocytes were comparable to untreated chickens. The data suggest that exposure of chickens to T-2 toxin may influence the development of a) HVT viremia; and b) MD lesions and nmmtality, but (Hug! in unvaccinated chickens. The data also suggest that T-2 toxin may severely deplete B- lymphocytes and relatively increase CD4 anui CD3 T- lymphocytes. Dedicated to the memory of my parents and brother, Nana Twum Barima Apawu II Madam Yaa Tenewaa Dominic Apawu-Mensah. iv ACKNOWLEDGEMENTS Special thanks to Drs. Aly' M. Fadly and. Stuart D. Sleight, my research and academic advisors, for their advice, guidance, support and encouragement during the course of this study. I extend my gratitude to Drs. Willie Reed and James Pestka for their advice and suggestions as members of my guidance committee. My appreciation is extended to the many individuals who contributed their time, expertise and advice to this project: Drs. Jon Patterson, Gregory Fink, Richard Witter, Henry Hunt, Eileen Thacker, Mona Aly, Janet Fulton, Mr. Ralph Common and Ms. Melanie Fleisberg. Special thanks to my sisters and brothers, Mary Apawu- Mensah, Vida Sarpong-Mensah, Mrs. Georgina Kusi-Appouh, Mrs. Christina Mensah—Bunduka, Johannes Twumasi-Mensah, Nana Frimpong-Mensah, and all the members of my extended family for their love and encouragement. Thanks ix: Drs. Sheila. Grimes, IMargo IHolland, Robert Sills, Gary Watson, Soegiarto Soegiarto and Ms. Patricia Lowrie for their support, friendship and encouragement. Finally, I would like to thank my girlfriend, Ms. Hattie Mae King, for her endless love, support, patience and understanding. TABLE OF CONTENTS Page LIST OF TABLES ........................................ Viii LIST OF FIGURES .......................................... X LIST OF ABBREVIATIONS .................................. Xii INTRODUCTION ............................................. 1 LITERATURE REVIEW ........................................ 6 T-2 Toxin ............................................. 6 Historical Background ............................... 6 Natural Occurrence .................................. 7 Chemical Structure ................................. 12 Physical Properties ................................ l4 Mechanisms of Action ............................... 15 Inhibition of Protein and DNA Synthesis ....... 15 Alteration of Cell Membrane Function .......... l8 Lipid Peroxidation of Cell Membranes .......... l9 Altered Intercellular Communication ........... 23 Induction of Apoptosis ........................ 23 Pharmacokinetics of T-2 toxin ...................... 23 Metabolism ........................................ 25 Excretion ......................................... 29 Pathotoxicologic Effects of T-2 toxin in chickens..30 Clinical Signs ................................ 30 Acute Toxicosis .............................. 32 Coagulation Disorders ......................... 34 Estimation of Acute Toxicity .................. 34 Subacute and Chronic Toxicosis ................ 35 Immunologic Effects ................................ 37 Immunosuppression ............................. 37 Immunomodulation .............................. 39 Humoral Immunity .............................. 39 Cell—Mediated Immunity ........................ 44 Delayed-Type Hypersensitivity ............ 44 Graft Rejection .......................... 46 Role of Macrophages ...................... 47 Cell-Mediated Resistance ................. 48 Vi Antiviral Activity ............................... 52 Effects of T—2 toxin on Vaccinal Immunity ........ 53 Marek’s Disease .................................... 55 Etiology ......................................... 55 Pathogenesis ..................................... 57 Clinical Signs ................................... 62 Gross Lesions .................................... 63 Control of Marek's Disease by Vaccination ........ 63 Vaccinal Immunity Against Marek’s Disease ........ 65 Marek’s disease Vaccine Failure .................. 70 Stress Factors Associated with Marek's Disease Vaccine Failure ............................. 7O Infectious Bursal Disease ................... 72 Chicken Anemia Virus ........................ 74 Reticuloendotheliosis Virus and Reovirus....75 Cyclophosphamide and Corticosterone ......... 75 Polychlorinated Biphenyls (PCBs) ............ 77 Aflatoxin ................................... 78 CHAPTER 1: EFFECTS OF T-2 TOXIN ON VACCINAL IMMUNITY AGAINST MAREK’S DISEASE .......................... 80 Abstract .................................... 80 Introduction ................................ 81 Materials and Methods ....................... 85 Results ..................................... 91 Discussion ................................. 104 CHAPTER 2: FLOW CYTOMETRIC ANALYSIS OF PERIPHERAL BLOOD AND SPLENIC B- AND T- LYMPHOCYTE SUBPOPULATIONS IN CHICKENS TREATED WITH T-2 TOXIN ................ 109 Abstract .................................. 109 Introduction .............................. 110 Materials and Methods ..................... 112 Results ................................... 117 Discussion ................................ 125 CONCLUSIONS ......................................... 129 APPENDIX ............................................ 131 Introduction .............................. 131 Vii Trial 1 ................................... 131 Material and Methods ................. 131 Results .............................. 132 Trial 2 ................................... 136 Materials and Methods ................ 136 Results .............................. 138 Discussion ........................... 146 LIST OF REFERENCES .................................. 151 viii LIST OF TABLES Experimental design ........................... Effect of T-2 toxin on body weight of 7-day-old, 1515 X 71chickens unvaccinated or vaccinated with HVT at hatch ............... Effect of T—2 toxin on the development of HVT viremia in 1- and 4-week-old, 15I5 X 71 chickens vaccinated with HVT at hatch, and challenged with MDV at 8 days of age .......... Development of MDV—induced viremia in 4-week-old, T—2 toxin-treated chickens vaccinated with HVT at hatch and challenged with MDV at 8 days of age ..................... Percent MD mortality and lesions in T-2 toxin-treated and untreated chickens within 8 weeks post-challenge with MDV at 8 days of Page ..87 ..92 ..97 ..98 age ............................................ 102 Percent MD protection in T-2 toxin-treated, HVT-vaccinated 1515)('h chickens challenged with MDV at 8 days of age ...................... 103 Experimental design ............................ 114 Effect of T-2 toxin on body weight of 7-day-old, 15I5)<'h chicks treated with toxin for 7 consecutive days after hatch ....... 119 Effect of T-2 toxin on B— and T-lymphocyte subpopulations in peripheral blood of 8-day-old, 15IS)('h chicks treated with toxin for 7 consecutive days after hatch ....... 120 Effect of T-2 toxin on B- and T—lymphocyte subpopulations in peripheral blood of 21-day-old chickens treated with toxin for 7 consecutive days after hatch ............. 122 ix Table Effect of T—2 toxin on B- and T-lymphocyte subpopulations in spleen of 9-day-old chickens treated with toxin for 7 consecutive days after hatch .................. Effect of T-2 toxin on B- and T-lymphocyte subpopulations in spleen of 22-day-old chickens treated with toxin for 7 consecutive days after hatch .................. T-2 toxin-induced mortality in 1515 X 71 chicks within 24—72 hrs after treatment with various doses of toxin at hatch .......... T—2 toxin—induced mortality in 1515 X 71 chicks within 24-48 hrs after treatment with various doses of toxin for 7 consecutive days after hatch .................. Hematocrit values of 15I5 X 71 chicks at various ages after treatment with various doses of T-2 toxin for 7 consecutive days after hatch ................................... Body weight of 15I5 X 71 chicks at various ages after treatment with various doses of T-2 toxin for 7 consecutive days after hatch... Relative weight of bursa of Fabricius of 1515)<'h chicks at various ages after treatent with various doses of T—2 toxin for Page ..123 ..124 ..134 ..139 ..140 .141 7 consecutive days after hatch .................. 142 Relative weight of spleen of 15I5 X 71 chicks at various ages after treatment with various doses of T—2 toxin for 7 consecutive days after hatch ................................ 143 Relative weight of thymus (right) of 15I5)('h chicks at various ages after treatment with various doses of T-2 toxin for 7 consecutive days after hatch ............ ..144 Figure LIST OF FIGURES Page Chemical structure of T-2 toxin ................. 13 Typical plaque morphology of HVT in CEF at 5 days PI with PBMs from 8-day-old, T-2 toxin—treated 1515)('h chicks that had been vaccinated with HVT at hatch .......... 94 Typical plaque morphology of MDV in DEF at 7 days PI with PBMs from 4-week-old, T-2 toxin-treated chickens vaccinated with HVT at hatch and challenged with MDV at 8 days of age ..................................... 95 Typical TEA-stained (using serotype 1 MDV-specific mab) MDV plaque morphology in DEF at 7 days PI with PBMs from 4-week-old, T-2 toxin-treated chickens vaccinated with HVT at hatch and challenged with MDV at 8 days of age .......... y ......................... 96 Photomicrograph of sciatic nerve from a 4-week-old, 1515)('h chicken treated with T-2 toxin (1.25 mg/kg body weight) for 7 consecutive days after hatch and challenged with MDV at 8 days of age. Notice a metastatic lymphoid nodule and neoplastic cellular infiltrates .................................... 100 Photomicrographs of bursa of Fabricius from 2-day-old untreated control chick (A); and from a chick treated at hatch with T-2 toxin at a dose of 4.5 mg/kg body weight (B). Notice the severe extensive acute lymphoid necrosis within follicles and numerous heterophils within interfollicular connective tissue stroma ................................. 135 xi Figure 3.2 Page Photomicrographs of liver from 2-day-old untreated control chick (A); and from a chick treated at hatch with T-2 toxin at a dose of 4.5 mg/kg body weight (B). Notice the severe locally extensive area of hemorrhage with associated hepatocellular necrosis ......................... 137 Photomicrographs of bursa of Fabricius from 8-day-old untreated control chick (A); and from a chick treated with T-2 toxin at a dose of 1.25 mg/kg body weight for 7 consecutive days after hatch (B). Notice the extensive depletion of lymphoid cells within follicles and increased interfollicular connective tissue ............... 145 Photomicrographs of thymus from 8-day-old untreated control chick (A); and from a chick treated with T-2 toxin at a dose of 1.25 mg/kg body weight for 7 consecutive days after hatch (B). Notice the moderate depletion of cortical lymphocytes ............... 147 Photomicrographs of spleen from 8-day-old untreated control chick (A); and from a chick treated with T-2 toxin at a dose of 1.25 mg/kg body weight for 7 consecutive days after hatch (B). Notice moderate depletion of lymphocytes with reticuloendothelial cell hyperplasia ............ 148 Photomicrographs of bone marrow from 8-day-old untreated control chick (A); and from a chick treated with T—2 toxin at a dose of 1.25 mg/kg body weight for 7 consecutive days after hatch (B). Notice absence of loss of hematopoeitic cells and the similar cellularity compared to control chick ....................... . ............ 149 xii KEY TO ABBREVIATIONS T-2 toxin ........... 3d-hydroxy-43,15-diacetoxy-8a-(3- methylbutyryloxy)-12,13-epoxytrichothec-9-ene MD ................................. Marek’s disease MDV ................................ Marek's disease virus PBS ................................ Phosphate buffered saline HVT ................................ Herpesvirus of turkey CEF ................................ Chick embryo fibroblasts DEF ................................ Duck embryo fibroblasts ETOH ............................... Ethanol PC ................................. Post-challenge PV ................................. Post—vaccination PI ................................. Post-inoculation xiii INTRODUCT I ON INTRODUCTION Marek's disease (MD) is a herpesvirus-induced lymphoproliferative neoplastic disease <1f chickens characterized by infiltration of various nerve trunks and/or visceral organs with pleomorphic lymphoid cells (Calnek and Witter, 1991). Until the early 1970s, MD was an economically important disease causing losses of billions of dollars worldwide due to condemnations in broiler chickens and reduced egg production and mortality in layer chickens (Purchase, 1985). Several vaccines have been developed and used in the field but the one most widely used is HVT isolated. by Witter et al., (1970). The introduction of vaccines, and the common practice of vaccinating 1-day-old chicks drastically reduced the economic losses associated with MD (Purchase, 1973). However, the Delmarva region of the United States, (parts of Delaware, Maryland and Virginia) has consistently experienced higher MD losses in broilers than in other regions (Purchase, 1985; Witter, 1989a). The introduction of serotype 2 MD virus (MDV) in bivalent vaccines has markedly reduced losses although data from the late 19805 have shown losses to be somewhat on the rise (Witter, 1989b). Failure of vaccines to provide the expected protection levels (“vaccination breaks”) has occasionally been reported in Asia, and the Middle East (Shieh, 1989; Davidson, 1989). Such vaccination. breaks have been attributed to several factors including the emergence of very highly oncogenic strains of MDV (Eidson et al., 1978, 1981; Witter et al., 1980; Schat et al., 1981), inadequate methods of vaccine production or ndsmanagement of vaccines at hatcheries causing loss of vaccine titres during thawing, reconstitution and use (Thornton et al., 1975; Halvorson and Mitchell, 1979). Increased genetic susceptibility to MD or the presence of maternal antibodies against HVT have also been suggested as possible reasons of vaccination failures (Calnek and Smith, 1972; Spencer et al., 1974. In addition poultry operation practices sudh as nm1ti-age rearing and insufficient cleaning between generations of chickens could also be responsible for early exposure to MDV before vaccinal immunity develops (Good, 1983; Price, 1983; Witter et al., 1984). Infectious disease agents such as infectious bursal disease virus (Giambrone et en”, 1976; Jen and CHE» 1980; Sharma, 1983), chicken anemia virus (Otaki et al., 1988) reticuloendotheliosis virus (Bulow, 1977); reovirus (Rosenberger, 1983); chemical immunosuppressants such as cyclophosphamide anxi corticosterone (Purchase aumi Sharma, 1974; Witter et al., 1976; Payne et al., 1978; Powell and Davidson, 1986); polychlorinated biphenyls (Halouzka and Jurajda, 1992) and mycotoxin such as aflatoxin (Edds et al., 1973; Batra et al., 1992) have been reported to influence the efficacy of vaccination, presumably through depression of cell-mediated and/or humoral immune response. T—2 toxin has received much attention because of its adverse effects on humans and farm animals (Mayer, 1953a, b). T-2 toxin, a type A trichothecene, is a secondary metabolite produced primarily by Fvsarium spp, which grows on cereal grains and contaminates agricultural products usually in temperate regions of North America, Europe and Asia (Scott, 1989; Russell et al., 1991; Wang et al., 1993; Chu and Li, 1994). Exposure to T-2 toxin has been associated with a variety of clinical syndromes in both humans and farm animals. T-2 toxin has been implicated as a major causative factor in fatal alimentary toxic aleukia in humans during World War II (Mayer, 1953a, b; Joffe, 1971; 1978), and red mold disease in humans and animals (Ueno et al., 1972; Saito and Ohtsubo, 1977). Fusariotoxicosis in lactating cows and poultry has also been reported (Bamburg et al., 1968; Wyatt et al., 1972; Hsu et al., 1977; Chi and Mirocha, 1978). In addition, T—2 toxin and its metabolites have been reported as possible constituents of “yellow rain”, a chemical warfare agent used in South East Asia (Haig, 1982; Watson et al., 1984). T-2 toxin, has been reported as ea potent immunotoxin which impairs the immune system through its ability to inhibit protein and DNA synthesis and thereby causing severe damage to actively dividing cells within the bone narrow, lymph nodes, spleen, thymus, bursa of Fabricius, and the gastrointestinal tract (Saito 6%: al., 1969; ‘Ueno 6N: al., 1971; Wyatt et al., 1973; Lutsky et al., 1978; Hayes et al., 1980; Hoerr et al., 1981; LaFarge-Frayssinet et al., 1981; Thompson and Wannemacher, 1990). T-2 toxin has also been found tx> lower E3 and 7? cell. numbers, inhibit. lymphocyte transformation, decrease IgG enmi 1mm antibody levels, and cause lymphocytolysis (Hayes em: al., 1980; Jagadeesan. et al., 1982; Rosenstein and LaFarge-Frayssinet, 1983; Cooray and Jonsson, 1990). Previous studies have shown that repeated exposure to subclinical doses of T-2 toxin may cause immunosuppression and may decrease the resistance of exposed animals to various infectious diseases Ennfii as salmonellosis in chickens (Boonchuvit et al., 1975) and mice (Ziprin and McMurray, 1988; Tai and Pestka, 1988, 1990), tuberculosis (Kanai and Kondo, 1984; Ziprin and McMurray, 1988), listeriosis (Corrier and Ziprin, 1986), herpes simplex infection.i111nice (Friend et en”, 1983), and aspergillosis in rabbits (Niyo en: al., 1988). In addition.tx> decreased host resistance, T-2 toxin may impair host control of tumor cell growth by suppressing tumor defense mechanisms. (Schoental et al., 1979; Schiefer et al., 1987; Corrier and Norman, 1988). The objectives of the present studies were to determine the effects of sublethal doses of T—2 toxin on vaccinal immunity against Marek’s disease and to further characterize its effects on both peripheral blood and splenic B- and T- lymphocyte subpopulations of white leghorn chickens using flow cytometry. LI TERATURE REVIEW LITERATURE REVIEW T-2 Toxin Historical Background Interest in T-2 toxin and other trichothecene mycotoxins began after a widespread outbreak of alimentary toxic aleukia (ATA), or septic angina, in humans and animals in the lhfixni of Soviet Socialist Republics (USSR) anui in Eastern Europe during World War II (Joffe, 1986a). The characteristic symptoms of the disease were fever, necrotic angina, leukopenia, hemorrhage, exhaustion of bone marrow, sepsis, and in) tx> 60% .mortality' (Mayer, 1953a,b; Joffe, 1978, 1986b). The cause of ATA was connected with a trichothecene compound, mainly T-2 toxin, which was isolated from authentic outbreak samples of cereal grains infected by Fusarium poae and Fusarium sporotrichioides (Joffe, 1971, 1986b; Joffe and Yagen, 1977, 1978; Lutsky and Mor 1981a). The causative fungi capable of producing T-2 toxin and related toxic trichothecene-type compounds induced skin inflammation, diarrhea, vomiting, hemorrhages, feed refusal, depression of bone marrow, damage to hematopoietic tissues, leukocytosis, leukopenia and nervous disorders in experimental and farm animals (Ueno et al., 1972; Saito and Ohtsubo 1974; Sato et al., 1975; Lustky and Mor, 1981b; Mirocha, C. (I. 1983; Joffe, 1978; 1986b). (If the trichothecene mycotoxins, T-2 is one of the most highly toxic and has been the major compound studied in this group (Ueno, 1983). (Rue seasonal occurrence of ZUHL its endemic nature, and the composition of the affected population suggested the importance of climatic and ecological factors in producing toxins that were found in field grains naturally infected by Fusarium spp. (Joffe, 1986c). The role of Fusarium strains and their toxins as causative agents in human disorders has acquired considerable significance after evidence that some potent mycotoxins of the trichothecene group (T-2 toxin, deoxynivalenol (DON or vomitoxin) and nivalenol (NIV) may have been used as “Yellow' Rain” in chemical warfare in Southeast Asia (Haig, 1982; Watson et al., 1984) Natural occurrence T-2 toxin is a very potent biologically and chemically active secondary metabolite, produced worldwide in temperate climatic zones by various species of Fusarium, which infects cereal grains, feedstuffs, corn, wheat, oats, barley, and rice in.tju2 fieLd or during storage (Joffe, 1986e; Scott, 1989; Keeler and Tu, 1991). The toxin has been associated with mycotoxicosis in farm animals and humans who ingest moldy agricultural commodities (Bamburg et al., 1969; Hsu et al., 1977; Joffe, 1978, 1986d; Wang et al., 1993; Beardall and Miller, 1994). The trichothecene mycotoxins have affected all parts of the world because they are naturally occurring contaminants of agricultural commodities (Vesonder, 1983; Ichinoe, et al., 1984; Scott, 1989). In any area (M5 the world where cereal grains, forages, or corn are grown under required weather conditions, including Inn: not limited txn ambient humidity, temperature, crop management, and geographical location, a genus or many genera of fungi may infect the plant (Joffe, 1986c). Overwintering conditions under low fluctuating temperatures appeared to promote vaarium mycotoxin production in these grains. Fusariwn sp. generally colonize grains (n: cereals in the field, but most toxin production occurs in storage at cool temperatures, up to 15° C (Joffe and. Yagen, 1977b; Joffe, 1978). Important environmental factors include high moisture or relative humidity and both warm and cool temperatures (Joffe, 1986c). Greater trichothecene production is usually found in years when the autumn is cool and wet, and harvest is delayed. Although the appearance of fungi is not the ultimate factor in determining the presence of trichothecenes, it is the most common indicator since they are responsible for trichothecene production. The toxicosis related to the presence of the naturally occurring trichothecene compounds was characterized as fusariotoxicosis in North America first by investigators at the University of Wisconsin where T—2 toxin produced by F. tricinctum in moldy corn was associated with illness and death in lactating cows (Hsu et al., 1972). Species of Fusarium are common and widespread in nature and cause many important diseases of nan.enni farm animals after consuming cereals that overwintered in time fields. A recent outbreak of human toxicosis caused by moldy rice contaminated with Fusarium and T-2 toxin was associated with heavy rainfall during rice harvest season in China (Wang et al., 1993). The chief symptoms 'were nausea, dizziness, vomiting, chills, abdominal distention enmi pain, thoracic stiffness, and diarrhea. Fusarium .heterosporium and F. graminearum were the predominant fungi isolated from the moldy rice. The highest level of T-2 toxin detected was 420 ppb. Simultaneous occurrences of fumonisin B, (FBl) and other mycotoxins in moldy corn collected in the People's Republic of China from regions with high incidences of esophageal cancer were recently reported. by Chu and Li 10 (1994). Samples contained high levels of FBl (18—155 ppm) and total type A trichothecenes (139-2030 ppb), including T- 2 toxin, HT-2 toxin iso-neosolaniol, and monoacetoxyscirpenol. In. addition, the concentration. of total type B trichothecenes in some of the corn samples ranged from 470 to 5826 ppb. Mold and nwcotoxin contaminatumi of mixed samples of corn is widespread in the midwestern corn belt of the U.S. In a survey of incidences of molds and mycotoxins in commercial animal feed mills in seven midwestern states in 1988-89, Evsarium sp. vfius found to 1x3 predominant. When assayed for mycotoxins, 19.5% of the samples were positive for at least one of the following: aflatoxin, zearalenone, T-2 toxin, or DON (Russell et al., 1991). In another survey in Argentina, examination for Fusarium toxins in corn and milling byproducts revealed that 33% of the samples were contaminated by DON, its amount ranging from traces to 1200 ug/kg; 15% of the samples contained T-2 toxin at concentrations ranging from 900 to 2400 ug/kg (Saubois et al., 1992). Only a few samples (7%) contained cereal grains. El-Maghraby enmi Abdel-Sater (1993) reported the contamination of tobacco and cigarettes in Egypt. DAS, T—2 and Zea were isolated from F. moniliforme infections of banana fruits in which 4 out of 40 samples had aflatoxin Bl (15—20 ug/kg), Zea (5.5 ug/kg) and T-2 toxin at a level of 2.8 ug/kg (Chakrabarti and Ghosal, 1986). Trichothecene mycotoxins, including T—2 toxin, and DAS were detected in unusual locations such as the dust of ventilation systems in office buildings in urban areas of Montreal, Canada, reportedly affected by the “sick building syndrome” (Smoragiewicz et al, 1993). There are toxigenic strains of vaarium indigenous to the warmer regions of the ‘U.S., and. other tropical and semitropical areas of the world. Richardson et al., (1985) isolated Fusarium spp. from plant material grown in the hot, humid climate of North Carolina which tested positive for the production of mycotoxins, T-2 toxin, Zea, and DON. In South Africa, oat grain and barley were contaminated with F. 12 aluminatum which produced T—2 toxin 1J1 large amounts (0.8- 2600 mg/kg at a relatively high (250 C) temperature (Rabie et al., 1986). Samples of moldy maize harvested from farms in Jos district, Nigeria, were contaminated. by Fusarium mycotoxins, NIV, Fusarenon-X, T-2 toxin, and HT-2 toxin (Okoye, 1993). Chemical Structure T-2 toxin is a member of a large group of sesquiterpenoid fUngal metabolites, called trichothecenes, whose common skeleton includes a tetracyclic ring system containing an epoxide group at C-12,13 and a double bond at C-9,10. The skeleton is thus characterized as 12, 13- epoxytrichothec—9-ene (Bamburg et al., 1968). The name for the structure of a tetracyclic skeleton composed of cyclohexane, cyclopentane and 6-membered oxyrane rings, and four methyl groups was proposed as “trichothecane” by Godtfredsen et a1. (1967). The structure of T-2 toxin is shown in Figure 1. All natural trichothecenes have the same stereochemistry: a at C-3 (R1), B at C-4 (R2) and C-15 (R3), a at C-7 (R4), and a at C—8 (R5) for groups A and B. The full systematic chemical name of T-2 toxin, therefore, is 3a-hydroxy-4B,15-diacetoxy- 8d-(3-methylbutyryloxy)—12,13-epoxytrichothec-9-ene. 13 H H3C ---OH 0’ E ---H I e co | CH3 OAc I OAc CH2 | CH(CHfl2 Figure 1: Chemical Structure of T-2 toxin The trichothecenes are. (divided. into simple and macrocyclic trichothecenes, according to their chemical characters depending on the presence of a macrocyclic ring linking at C94 anni C-5 with diesters or triesters, and on the fungi producing the trichothecene (Ueno, 1983). The simple trichothecenes are again divided into four types, namely, type A, B, C and D. Type A trichothecenes include Te 2 toxin, DAS, HT-2 toxin, and others. T-2 toxin, the first to be recognized as a naturally occurring trichothecene, is the most highly toxic among these trichothecene mycotoxins ( Ueno, 1983; Trenholm et al., 1989; Keeler and Tu, 1991; Sharma and Salunkhe, 1991). The type A trichothecenes are primarily produced by F. sporotrichioides, F. tricinctum and F. poae. Type B trichothecenes, characterized by the 14 presence of 23 ketone group at time C—8 position, include nivalenol, fusarenon—X and deoxynivalenol (vomitoxin or DON) and are produced primarily by F. nivale and F. episphaeria. Type C trichothecene is represented by crotocin produced by Cephalosporium contocinigenum and has an epoxide at the 7—8 position” Type I) trichothecenes comprise tin; macrocyclic trichothecenes which bear a bridge of varying length and composition between carbons 4 and 5. Types A and B trichothecenes are the most important. Physical properties T—2 toxin readily crystallizes from purified ethyl acetate extracts as white crystals. Analysis and molecular weight determination verified the molecular formula as .CMHymb (Bamburg et al., 1968). The toxin is very stable in the solid state and freely soluble in. moderately‘ polar organic solvents, such as methanol, ethanol, chloroform, and dimethyl sulfoxide, but ix; only slightly soluble :nu water (Bamburg and Strong, 1971). T-2 toxin is relatively stable in methanol. However, it was transformed to several products after 22 days exposure to methanol (Wei and Chu, 1986). The stability of the toxin in aqueous media decreased. with increasing temperature. The toxin was more stable at 4° C and least stable at 37° C, with formation of HT-2, T-2 triol, and T-2 tetraol (Trusal, 1985). Duffy' and. Reid 15 (1993) estimated tjua half-life CM? T—2 toxin 1J1 deuterated phosphate-buffered saline solutions under quasi- physiological conditions to be about 4 years and concluded that the contribution of nonezymatic degradation to the detoxification of T-2 toxin and its metabolites was negligible. Mechanisms of Action Inhibition of Protein and DNA Synthesis Many in vitro and in vivo studies have been done in various systems in an effort to determine the mechanism of action of T—2 mycotoxin. The general conclusion from these studies was that T-2 toxin blocks cellular protein synthesis by binding to the 608 subunit on the ribosome and thereby inhibits initiation, elongation, and termination of protein synthesis in eukaryotic cells (Ueno et al., 1968; Bamburg, 1972, 1974; Cundliffe et al., 1974; Cannon et al., 1976b; Cundliffe and Davis, 1977). T-2 toxin was also found to interfere with peptidyl transferase activity which is required for elongation and termination at the transcription site in eukaryotic cells (Tate and Caskey, 1973; Wei and McLaughlin, 1974; Mclaughlin. et al., 1977). Ueno et al. (1968, 1973) who originally described inhibition of protein synthesis as a dominant effect of the trichothecenes pointed out that T-2 toxin was a weak inhibitor of protein synthesis 16 in cell—free systems, but was very potent in intact cells. Bamburg (1972) reported there was no direct correlation between. protein. synthesis inhibition anui cell growth. and replication, and suggested other mechanisms of action may be involved. When results of the various trichothecenes’ ability to inhibit protein synthesis in vitro were compared with the results of whole animal lethality, it was shown that several of the trichothecenes were weak inhibitors of protein synthesis in Vero cells and rat spleen lymphocytes in Vitro but were highly toxic in Vivo (Thompson and Wannemacher, 1986). Thus, in vitro cell response of ea given trichothecene is not always an accurate predictor of toxicity in whole animals and protein synthesis inhibition might not be the sole cellular mechanism of action for in vivo toxicity (Bamburg, 1972; Ueno et al., 1973b). The degree of protein synthesis inhibition correlated with the amount but not the rate at which T—2 toxin is taken up by the mitochondria. The rate of uptake of labeled leucine into mitochondrial protein, using isolated rat liver mitochondria supplemented with an S-100 supernatant from rat liver and an external ATP-generating system, was unaffected by the addition of T—2 toxin and was not a rate-limiting step in incorporation. However, 0.02 pig/ml of T-2 toxin 17 decreased the rate of protein synthesis by 50% in isolated mitochondria (Pace et al., 1988). T—2 toxin also interferes with mitochondrial functions, resulting iii the inhibition cu? growth cu? yeast (Schappert and Khachatourians, 1983). A significant amount of [3H]T-2 localized in the mitochondrial fraction within 15 minutes of its addition to an isolated perfused rat liver and inhibited the electron transport chain in mitochondria (Pace, 1983). The effect CM? T—2 toxin (M1 protein and.IDUX synthesis depends on the type of organ involved. Intraperitoneal injection of T-2 toxin in mice caused inhibition of DNA and protein synthesis in bone marrow, spleen, and thymus (Rosenstein and. Lafarge—Frayssinet, 1983). In vitro, T-2 toxin inhibited DNA and protein synthesis of mice spleen cells and rat hepatoma cells stimulated with phytohemagglutinin. Thompson and Wannemacher, (1990) studied the in vivo effects of T—2 toxin on protein and DNA synthesis in serum, liver, heart, kidney, spleen, muscle, and intestine of rats and concluded that although the levels of [”C]leucine and [3H]thymidine incorporation in each of the tissues of normal rats were consistent, a significant degree of variability existed in the rates of incorporation among the various tissues. Muscle and heart had the lowest index of leucine incorporation, spleen, intestine, and l8 kidney had intermediate levels, whereas liver had the highest index of protein synthesis , over 10 times that of the muscle. In most tissues recovery was seen with time. DNA synthesis indices were highest in the spleen and intestine. The muscle, heart, kidney, and liver incorporated a 3- to 10-fold lower level of thymidine per mg protein (Thompson and Wannemacher, 1990). Alteration of cell membrane function Studies on cytotoxicity have shown that T-2 toxin exerts its toxic effect by intervening at time subcellular level and inhibiting biochemical functions such as DNA, RNA, and protein syntheses (Ueno et al., 1968; Bamburg 1972; Wei et al., 1974; Suneja et al., 1983, 1987; Pace et al., 1988). However, this does not account for all the cytotoxic effects of T-2 toxin because some studies suggest that T—2 toxin interacts with cell membranes and alters membrane function. Since T—2 toxin is an amphipathic molecule, interactions with various kinds of membranes are expected (Gyongyossy-Issa et al., 1984). The plasma membrane plays a significant role in the uptake and interaction of T-2 toxin with yeast cells (Schappert and Khachatourians, 1984). Membrane-modulating agents such as ethanol, cetyltrimethylammonium bromide and heat were found to increase the sensitivity of the yeast, Saccharomyces sp., 19 towards T-2 toxin. However, a yeast mutant with a reduced plasma membrane permeability was resistant to T-2 concentrations of up to 50 ug/ml. Bunner and Morris (1988) proposed alteration of multiple cell membrane functions as an important mechanism of action and reported that T-2 toxin had multiple effects on cell membrane functions at very low concentrations (0.4 pg/ml to 4 ng/ml) which were independent of" protein synthesis. Uptake of calcium, rubidium. (KW glucose, leucine and tyrosine was reduced within 10 minutes of exposure of L-6 myoblasts to T-2 toxin. Such reduced uptake included either direct or indirect effects of T-2 toxin on amino acid, nucleotide, and glucose transporters, as well as calcium and potassium channel activities (Bunner and Morris, 1988). Lipid peroxidation of cell membranes Free radical-mediated phospholipid peroxidation of membranes by T-2 toxin has been proposed by some workers. Tsuchida et al. , (1984) measured the presence of lipid peroxides (malonaldehyde formation) using the 2- thiobarbituric acid (TBA) nethod 1J1 various tissues including the liver in rats treated with up to 4 mg/kg body weight of T-2 toxin. The TBA values increased in the liver 24 and 48 hours after administration of T—2 toxin, reaching values approximately 6 and 5 times higher than those of 20 time-matched controls. However, no changes were observed :hi the TBA values in time kidney, thymus, and jejunum, 24 hours after toxin administration, and increases of only 20— 50% over the respective control values were recorded after 48 hours. Vitamin E, a free radical scavenger possesses protective activity against the stimulation of hepatic peroxidation induced by T—2 toxin (Tsuchida et al., 1984). Rats were pretreated for 2 weeks with diets containing different amounts (n3 DL—a—tocopheryl acetate (vitamin ED. In the pretreated rats not given T-2 toxin, the hepatic TBA values in the vdtamin E—deficient animals were about 188% above those in the vitamin E—supplemented rats (300 mg/kg of diet), whereas in the T—2 toxin-treated, vitamin E-deficient animals, the TBA values rose about 400% as compared with those in the supplemented animals (Tsuchida et al., 1984). T—2 toxin had consistently produced depressed concentrations of Vitamin E in plasma of chicks. The addition of micelle- promoting compounds such as taurocholic and oleic acids alleviated depresshmi in both plasma vitamin E enni growth (Coffin and Combs, 1981). An increase in nuclear lipid peroxidation induced in rat liver by T-2 toxin administered orally for 5 days resulted in an increase in TBA and a decrease in activity of liver glutathione-S-transferase (Ahmed and Ram, 1986). Chang and Mar (1988) reported an 21 increase 1J1 conjugated Chifik? formation :hi liver, spleen, kidney, thymus, and bone marrow when rats were treated with 3-6 mg/kg body weight of T-2 toxin, indicating an increased level of lipid peroxidation in these tissues. In contrast, Schuster et al., (1987) measured the formation of TBA reactive material in isolated hepatocytes and liver homogenates from rats, then also determined ethane exhalation in vivo after oral administration of T-2 toxin. No significant difference from the controls in these parameters was noted and the authors concluded that lipid peroxidation did not play a major role in the toxicity of T- 2 toxin. This contradictory conclusion Vfifis explained by Schuster et al., (1987) as associated with the different methods used tx> assay TBA reactive material III the liver homogenates. A free radical mechanism inducing lipid peroxidation was first reported by Segal et al., 1983 as a cause of hemolysis. Even though the amount of malonaldehyde production was not significant, specific free radical scavengers, including vitamin EL :mannitol, enmi histidine, inhibited hemolysis caused. by T-2 toxin. Rizzo et al., (1992) recently reported the involvement of the lipoperoxidation mechanism in the hemolytic activity of T-2 toxin (n1 rat erythrocytes. The authors proposed that T¥2 toxin exerts its toxicity on procaryotic cells in 3 22 different ways: by penetrating the phospholipid bilayers and acting at the subcellular level, by interacting with the cellular membranes, and by free radical-mediated phospholipid peroxidathmu. But most likely, more than cme mechanism operates at the same time. The T-2 toxin—induced hemolysis had several characteristics, all of which include an initial time- dependent, and long lag period before hemolysis starts. The characteristic lag period and the hemolytic activity of T-2 toxin on human and guinea pig erythrocytes were observed by Gyongyossy-Issa et al., (1985, 1986a,b) In contrast, cattle erythrocytes were not susceptible (DeLoach et al., 1987). Subsequently, a species-specific hemolysis of mammalian erythrocytes caused by T-2 toxin has been proposed by Deloach et al., (1989). Pig, man, rabbit, guinea pig, horse, dog, rat, and mouse erythrocytes were all lysed to a varying' degree, but cow, sheep, goat, buffalo, and. deer erythrocytes were all resistant to hemolysis by T-2 toxin. Since erythrocytes from ruminant animals contain little or no phosphatidylcholine, perhaps the presence of phosphatidylcholine in the membranes is required for the hemolytic action of T—2 toxin (Deloach et al., 1989). 23 Altered Intercellular Communication T-2 toxin, other trichothecenes, and other inhibitors of protein synthesis, regulate intercellular communication through superinduction of cellular proteins such as interleukin-1 (IL—1) production by macrophages and IL-2 synthesis by lymphocytes (Miller and Atkinson, 1987; Holt et al., 1988). Either the interference with rapid turnover of proteins that limit the translation of mRNA for interleukin synthesis or the impaired degradation of intracellular interleukins has been suggested as the mechanism of the superinduction (Miller and Atkinson, 1987). Cardiovascular lesions induced 1J1 rats after passive transfer (n3 splenic cells from syngeneic rats treated with T—2 toxin may be mediated by IL production (Sherman et al., 1987). Induction of apoptosis T-2 toxin has recently been reported to induce apoptosis, morphological changes of nuclei characterized by fragmentation and the formation of an apoptotic nuclear body, in HL—60 human promyelocytic leukemia cells (Ueno et al., 1995). The toxin induced apoptosis at 10 ng/ml within 2-6 hr without significant cytotoxicity. Pharmacokinetics In inhn) metabolic studies conducted 1J1 chicks administered tritium-labeled T-2 toxin by crop gavage have 24 shown. that T-2 toxin anni its :metabolites were :primarily excreted into the intestine through the bile, and that the liver was a major organ for the metabolism and excretion of the toxin and its metabolites into the feces via the bile KHU_ et al., 1978a; YoShizawa et en”, 1980; Visconti and Mirocha, 1985; Giroir et al., 1991). The chicks excreted 60.5, and 81.6% of the recovered radioactivity at 24, and 48 hours, respectively, and the gastrointestinal (GI) tract contained 26.9, and 10. % of the recovered radioactivity at 24, enmi 48 hours, respectively (Chi en: al., 1978a). The abdominal fat and heart contained the least amount of radioactivity among the tissues analyzed. The radioactivity of 3H-labeled T-2 toxin reached.aa maximum concentration 4 hours after dosing in most tissues except muscle, skin, and bile, in which the maximum radioactivity was attained at 12 hours. The specific radioactivity III the bdood, nmscle, skin, and heart was similar throughout the 48-hour period. The bile, including the gallbladder, contained the highest specific radioactivity among organs and tissues (except the GI tract) during the 48—hour period Chi et al., 1978a). The edible portions of the carcass contained only 0.06 and 0.04 ppm of T—2 or its metabolites at 24 and 48 hours, respectively, after dosing with 0.5 mg of T—2 toxin per kg body weight. This suggests that humans would be unlikely to be affected by consuming the muscle from chickens fed diets 25 containing concentrations (n5 T—2 toxin likely txa occur in natural outbreaks. The transmission of radioactivity into eggs from laying white leghorn hens administered single or multiple doses of tritium-labeled T-2 toxin Via gastric-intubation. was reported i11 a separate study thCHu_ et al., (1978b). In single dosed birds, the maximum radioactivity in eggs occurred at 24 hours after dosing; the yolk and egg white contained 0.04 and 0.13% of the administered radioactivity, respectively. In nmltiple-dosed birds given 8 consecutive daily doses, the radioactivity in.tju2 yolk increased with each dose, whereas the radioactivity in the egg white increased rapidly until the third dose, and thereafter remained constant. Therefore, in both the single and multiple dosed birds the specific radioactivity of the egg white was greater than that of the yolk. The amount of residue transmitted into au1 egg 1J1 birds intubated daily with 1 mg/kg T—2 toxin for 8 consecutive days (equivalent to 1.6 ppm dietary T-2 toxin) was about 0.9 1.19, suggesting little danger, if any, to public health from residues (Chi et al., 1978b). Metabolism The initial in vitro metabolism studies using hepatic homogenates indicated that T-2 toxin was rapidly transformed 26 by ester hydrolysis catalysed by esterases at C—4 to several products, including' HT—2 toxin, 4-deacetylneosolaniol, neosolaniol and T-2 tetraol (Ellison and Kotsonis, 1974; Ohta et al., 1977, 1978; Yoshizawa et al., 1980a). Two oxidation products, 3'-hydroxy HT-2 toxin and 3'—hydroxy T-2 toxin, were further identified 1J1 liver homogenates supplemented with an NADPH-generating system from mice and monkeys (Yoshizawa et al., 1984) and from pigs and rats (Wei and Chu, 1985). This suggests that cytochrome P-450 catalyzes the hydroxylation reaction at the C-3’ position of T-2 and HT—2 toxin. In phenobarbital-induced rat liver microsomes, T-2 txndxl is rapidly metabolized tx> 33hydroxy T-2 toxin, 3'éhydroxy HT-2 toxin, HT—2 toxin, and T-2 triol (Knupp et al., 1984). The metabolic profiles of T-2 toxin incubated with inducers and inhibitors of the cytochrome P- 450-dependent monoxygenase system in rats, mice, rabbits, chickens, guinea pigs, cows, and pigs were studied by Knupp et al., (1987) and Kobayashi et al., (1987). The hepatic S-9 and microsomal fractions from various species hydroxylated T-2 toxin and HT-2 toxin, a deacetylated metabolite of T-2 toxin formed by reactions involving microsomal esterases, to form 3'—hydroxy T—2 toxin and 3’-hydroxy HT—2 toxin, respectively. This two hydroxylation reactions were catalyzed by the cytochrome P-450-dependent monoxygenase system. Species comparisons indicated that the rate of 27 hydroxylation reaction was highest in the hepatic microsomes of guinea pigs and mice. Microsomal fractions from chickens had a low activity in the hydrolysis and hydroxylation reactions. The major metabolite in microsomal preparations from control and phenobarbital-induced rats, rabbits, and mice was HT—2 toxin. In ndcrosomes isolated from phenobarbital—treated chickens, 3'-hydroxy T-2 toxin was the major metabolite, and 30 and 79% of the added T-2 toxin remained unmetabolized at 60 minutes in incubations from phenobarbital—induced and control birds, respectively. The percentage of hydroxylated metabolites formed in the microsomal preparations of the species studied. was significantly increased after phenobarbital treatment compared with the nontreated controls (Knupp et al., 1987). Treatment cu? rats with phenobarbital induced esterase and mixed function oxidase activity, the latter being increased tx> a greater extent. Two ‘previously ‘unidentified metabolites detected in chicken, rat, and mouse microsomal preparations were tentatively classified as isomers of 3’— hydroxy T-2 toxin. Some cultured cell lines possess enzyme systems capable of limited metabolism of T-2 toxin to a variety of known and some yet unknown metabolites. Chinese hamster ovary cells (CH0), and African green monkey kidney cells (VERO) metabolized T-2 toxin to a greater degree and to a wider 28 variety of metabolites than the human fibroblasts and mouse connective tissue cells (L-929) (Trusal, 1986). In CHO, fibroblasts, and L—929 cells, the major metabolite was HT-2 toxin, whereas in VERO cells an unknown metabolite, more polar than T-2 toxin, was the major metabolite. Cell and media extracts of CH0 and VERO cells contained small amounts of T—2 triol, T-2 tetraol, and several unknown metabolites. The sensitivity of lymphoid cells to the uptake, metabolism and cytotoxic effects of T—2 toxin varies according tx> their‘ degree CHE differentiation (Porcher et al., 1988). Of the human lymphoid cell lines studied, susceptible Dandi (B-cell lymphoma) cells, and resistant REH (non-differentiated non-B non—T cell leukemia) and KE37 (T- cell leukemia) cells took up 20 and 3% of the T—2 present in the medium, respectively, when the cells were incubated with [3H]T-2 toxin. Metabolites recovered from the culture medium and cells included T-2 tetraol, T-2 triol, HT-2 toxin, neosolaniol, and T—2 toxin. Studies on the survival rates of various lymphoid cells upon exposure to 400 um T—2 toxin per 1 x 107 cells per ml revealed that thymocytes and cells collected by peritoneal lavage were exquisitely sensitive to time cytotoxic effects of the toxin, with no survival after 5 tx> 6 hours exposure (DiNinno et al., 1985). Spleen cells had an intermediate 29 survival rate with a rapid decline in viability. Bone marrow cells were relatively resistant, with only' a small but significant decline in viability. A large proportion of a heterogeneous population of bone marrow cells was resistant to T-2 toxin and had a 40-50% survival rate, suggesting that these cells would allow the lymphoid organs to be repopulated, even upon prolonged exposure to T—2 toxin. Excretion About 80% of orally administered 3H—labeled T-2 toxin in broiler chickens was rapidly metabolimai to more polar derivatives such as HT-2 toxin, neosolaniol, 4— deacetylneosolaniol, T-2 tetraol, T-2 toxin, and several unknown derivatives were eliminated in the excreta within 48 hours after administration (Yoshizawa et al., 1980). In a subsequent study, ‘Visconti and. Mirocha (1985), used gas chromatography—mass spectrometry to identify several of the unknown T—2 metabolites detected in the chicken excreta. Namely, 35%hydroxy HT—2 toxin, the major metabolite present in excreta and organs, Ef—hydroxy T-2 toxin, 8-acetoxy and 15-acetoxy T—2 tetraol (also called 4—deacetyl neosolaniol) in addition to another monoacetylated isomer of T—2 tetraol were identified. Although most of time T—2 metabolites were found in the excreta, considerable amounts were also found in the liver. However, no trichothecenes were detected in 30 the heart and kidneys, and only trace amounts were detected in the lungs. Pathotoxicologic effects of T—2 toxin in chickens Clinical signs Wyatt et al., (1972a) described a disease syndrome in 6—week—old broiler birds 1J1 several commercial flocks and fancy' pigeons. The syndrome was characterized. by raised proliferative caseous, yellowish-white, plaque-like lesions in the oral cavity. Morbidity varied from 10% to 25%, with up to 10% mortality and depressed growth rates in the remaining chicks. Later, Wyatt EH: al., (1972b) reproduced identical oral lesions :hi the chickens fed control feed which had been spiked with 4 and 16 ppm concentrations of T- 2 toxin for ‘three ‘weeks. Chi et (al. (1977a, 1978) also reported that similar oral lesions developed 1J1 birds fed contaminated diet: Speers et al. (1977) also reported the results of a 21—day feeding trial in which laying hens were fed purified T-2 toxin at 4, 8, and 16 ppm or corn invaded by F. tricinctum. Mouth lesions developed and their severity was proportional to the amount of toxin in the feed. These observations by Wyatt et al. (1972a,b); Chi et al. (1977a, 1978) and. Speers et al. (1977) showed. that appearance (n3 oral lesions are excellent diagnostic indicators of T-2 intoxication in chickens. Large numbers of 31 bacteria, namely Staphylococcus aureus, S. epidermitis, and Escherichia coli, were isolated from the oral lesions (Hamilton et al., 1971; Wyatt et al., 1972a). Neurological. disturbances (Observed 1J1 chickens after ingestion of dietary T-2 toxin include abnormal positioning of the wings, hysteriod seizures, and an impaired righting reflex (Wyatt et al., 1973). Changes in brain catecholamine concentrations could be responsible for altered motor activity. Chi et al., (1981) administered T-2 toxin to 4— week-old chickens in a single dose of 2.5 mg/kg body weight and then determined the brain accumulations of dopamine, norepinephrine and serotonin over the next 48 hours. Although serotonin concentrations were not altered from those of controls, brain dopamine levels were almost double the control, while norepinephrine levels decreased by 25% in 25 hours after T—2 toxin administration. Since dopamine is a precursor to norepinephrine, it is possible that the toxin has some direct inhibitory effect (Hi this conversion. The observed effects of T-2 toxin on brain monoamines and the resulting neurochemical imbalance may account for the physiological manifestation of trichothecene intoxication (MacDonald et al., 1988; Wang et al., 1993). T—2 toxin acting as an emetic factor in moldy corn has occasionally been reported in ducklings (Ueno et al., 1974) and pigeons (Fairhurst et al., 1987; Ellison and Katsonis, 32 1973) but not in chickens. T-2 toxin at oral and intravenous sublethal doses of 0.72 and 0.15 :mg/kg' body weight, respectively, induced vomition jiijpigeons (Ellison and Katsonis, 1973). In an acute toxicity study in pigeons, Fairhurst et al., (1987) administered T-2 toxin sublingually in doses ranging from 0.2 to 10 mg/kg to pigeons. Vomiting began at 20-120 minutes and persisted for 3-4 hours, during which each pigeon vomited several times. The birds then became subdued and tremorous with marked ataxia; in some birds the wings were held in an abnormally low position, and most deaths occurred within 24 hours. Acute Toxicosis Saito et EH”, (1969) described time acute histological and hematological findings, both characterizing the “radiomimetic” effects of the toxic metabolites of F. nivale on proliferating cells when mice were injected subcutaneously or intraperitoneally, or given orally. Histological examination revealed marked cytotoxic changes in tissues with actively dividing cells, including the crypt cells and Paneth cells of small intestine, especially the duodenum, germ center of the lymphoid follicles in the spleen and lymph node, cortex of the thymus, and the hematopoietic cells of the bone marrow. The affected cells had degenerative changes, atypical mitosis, pyknosis, 33 hyperchromatosis of the nuclear membrane and fragmentation or karyorrhexis of the nuclei. It is now acknowledged that the trichothecenes affect multiple tissue target sites, with predominant lesions occurring in the gastrointestinal tract, lymphoid tissues, and the hematopoietic tissue of the bone marrow (Ueno, 1977; Hayes et al., 1980; Hoerr et al., 1981b; Bamburg, 1983; Otokawa, 1983). Experimental administration of either culture preparations of Ensarium or purified T—2 toxin, by various routes, produced the clinical picture of intoxication in rabbits (Gentry and Cooper, 1981), cats (Sato et al., 1975; Lutsky et al., 1978), rats and mice (Ueno, 1977; Schoental et al., 1979; Hayes et al., 1980), monkeys (Rukimini et en”, 1980; Jagadeesan en: al., 1982) chickens, ducks and pigeons (Wyatt et al., 1973, 1975; Chi et al., 1977; Joffe and Yagen, 1978; Hoerr 6N: al., 1981, 1982a,b; Fairhurst et al., 1987), pheasants (Huff et a1., 1992), bobwhite and Japanese quail (Ruff et al., 1992), and guinea pigs (DeNicola et al., 1978). Hoerr et al., (1981b) reported the “radiomimetic” effects of T-2 toxin when 7-day-old broiler chicks were administered T-2 toxin via crop gavage, at a single dose of 2.0-2.5 mg/kg body ‘weight. Severe, acute necrosis and depletion of lymphoid tissue and bone marrow were observed within 24 hours after treatment. Cell repletion with partial or complete restoration occurred by 72 to 168 hours 34 in the more severely necrotic tissues. Necrosis of tips of villi and crypts in duodenum, multiple foci of hepatocellular coagulative necrosis, and necrosis of feather and follicular epithelium were described. Coagulation disorders T-2 toxin is known to cause hematological disorders and produce serious coagulopathies as- measured by various clotting assays in chickens and guinea pigs (Doerr et al., 1974, 1981; Cosgriff et al., 1984). Growth inhibition doses of T—2 toxin caused deficiency of coagulation factors VII, X, prothrombin and fibrinogen which led to prolonged prothrombin. and (activated.;partial thromboplastin. clotting times. However pretreatment of animals with vitamin K had no effect , indicating that T—2 toxin does not act as a vitamin K antagonist. Platelet aggregation in whole blood was depressed in response to adenosine diphosphate and collagen. A sublethal dose of T—2 toxin affected the kallikrein-kinin system by depletion of prekallikrein, which indicated increased bradykinin levels in plasma (Johnsen et al., 1988). Estimation of acute toxicity The 10-day median lethal dose for 1-day-old broiler chicks ranged from 5.03 i 0.25 to 5.25 i 0.21 mg/kg body weight (Chi et al., 1977). In addition, the 10-day median 35 lethal dose for 8-week-old broiler chicks and laying hens was 4.97 i 0.25 and 6.27 i 0.42 mg/kg body weight, respectively. Within 4 hours after dosing, birds developed asthenia, inappetence, diarrhea, and panting. Death occurred within 48 hours after T-2 toxin administration. Sublethal doses of T—2 toxin resulted :hi El decrease in weight gain and feed consumption. The abdominal cavities of birds given lethal doses contained a white, chalk-like material, which covered the viscera. No salient pathologic or clinical signs were observed at 10 and 20 days after administration of T-2 toxin. Hoerr and Carlton, (1981a) estimated the 72-hour single oral dose LDSO and 14 daily oral doses LD50 of T-2 toxin administered to 7-day-old broiler chicks as 4.0 and 2.90 mg/kg body weight, respectively. Subacute and chronic effects T-2 toxin caused losses in weight gain when fed either as commercial feed contaminated with Fusarium poae or Fvsarium sporotrichioides or as pure T-2 toxin added to a balanced diet. Doses as low as 4 ppm of pure T-2 toxin in feed caused reduced feed consumption and decreased weight gains in broiler chicks (Wyatt et al., 1973; Chi et al., 1977a). Sublethal doses of T-2 toxin decreased feed consumption and weight gain proportionately with the amounts 36 of toxins administered (Wyatt et al., 1973; Chi et al., 1977a, 1978). The most marked effects occurred within the first 10 days of the treatment during a 30-day experiment. Other toxins that have been shown to have adverse effects on weight gains include HT-2 toxin, T-2 tetraol and deacetyl HT-2 toxin, all of which are metabolic byproducts of T-2 toxin (Chi et al., 1978). T-2 toxin not only affects weight gain but often reduces egg production and quality. In subacute studies, single comb white leghorn laying hens fed T-2 toxin at a dose of In) to 63 ppm significantly decreased feed consumption, egg production, and egg shell thickness (Wyatt et al., 1975; Chi et al., 1977). The fertility and progeny performance were not depressed by feeding T-2 toxin, but the hatchability of fertile eggs of hens fed 2 and 8 ppm was significantly lowered. Speers et al., (1977) also observed loss of body weight and decreased egg production when laying hens were fed purified T—2 toxin consumed at the rate of 16 ppm. Egg production of 8-month-old laying hens decreased precipitously during a 5-day period after the birds began to consume feed containing T-2 toxin and HT-2 toxin at 3.5 and 0.7 mg/kg, respectively (Shlosberg et al., 1984). However, when these hens were given uncontaminated feed, production returned almost to the expected value within 12 days. 37 In broiler chicks, T-2 toxin administered at the rate of 14 daily doses ranging from 1.5 to 3.0 mg/kg body weight/day caused emaciation, decreased body weight and hematocrit, malformed feathers, pale yellow beaks and legs and death (Hoerr et al., (1982). Lymphoid organs were atrophic, bone marrow was pale red or yellow, the liver was discolored yellow, and the crop mucosa was ulcerated. During the first three weeks of a six—week trial, mice fed a balanced semipurified diet containing 20 ppm T-2 toxin had hypoplasia of lymhoid tissues, bone marrow, and splenic red pulp, resulting in anemia, lymphopenia, and eosinophilia (Hayes et al., 1980). After continued. exposure to 'T—2 toxin, hematopoietic cells regenerated in.lxnua marrow and splenic red. pulp and. became hyperplastic by six weeks. However, all lymphoid tissues remained atrophic. Granulopoiesis and thrombopoiesis occurred before erythropoiesis. The suppression. of Ihematopoiesis was transient and did not lead to hematopoietic failure (Hayes et al., 1980). Immunologic effects Immunosuppression The broad inmmnosuppressive effects of Chi? toxin and other trichothecenes on humoral and cell mediated immunity have been well documented (Otokawa, 1983; Taylor et al., 38 1989; Corrier, 1991; Sharma, 1993; Pestka and Bondy, 1994a,b). Exposure to trichothecene mycotoxins has been reported to cause severe damage to actively dividing cells within the ibone :marrow, lymph. nodes, spleen, thymus, bursa of Fabricius, and time gastrointestinal tract (Saito en: al., 1969; Ueno et al., 1971; Wyatt et al., 1973; Lutsky et al., 1978; Hayes et EH”, 1980; Hoerr et en”, 1981; LaFarge- Frayssinet et al., 1981). Repeated exposure of animals to T-2 toxin has been shown to cause immunosuppression and may decrease their resistance to various infectious diseases such as salmonellosis in chickens (Boonchuvit, et al., 1975) and mice (Ziprin and McMurray, 1988; Tai and Pestka, 1988, 1990), tuberculosis (Kanai and Kondo, 1984; Ziprin and McMurray, 1988), listeriosis (Corrier and Ziprin, 1986), and herpes simplex virus infection in nUrxa (Friend et al., 1983), and aspergillosis in rabbits (Niyo et al., 1988). The increased susceptibility of T—2 toxin-exposed animals to gram—negative bacterial infections may be due to increased endotoxin sensitivity associated with increased absorption (M? the endotoxin (Taylor et en”, 1991). Acute simultaneous eXposure tx> T-2 toxin (per Cfifl and endotoxin (intraperitoneal) in mice resulted in increased mortality, hypothermia, TNF-alpha production and thymic atrophy 39 compared to treatment with either T-2 toxin or endotoxin alone. It has also been shown that T-2 toxin toxemia is associated with elevated plasma levels of eicosanoids through its effect on the cyclooxygenase pathway of arachidonic acid vnifli increased release (Hf prostaglandin, PGEZ and thromboxane (Shohami and Feuerstein, 1986) which play an important role in the pathophysiology of bacterial endotoxemia (Feuerstein et al., 1981; Slotman et al., 1985). In addition to decreased host resistance, T-2 toxin may also suppress tumor defense mechanisms and impair host control of tumor cell growth (Schoental et al., 1979; Schiefer et al., 1987; Corrier and Norman, 1988). Immunomodulation The mechanisms by which T-2 toxin and other trichothecenes exert their specific effect on immunological function have been best characterized in the mouse model (Taylor et al., 1989). T—2 toxin and the other trichothecenes may induce other immunomodulating effects in addition to immunosuppression (Taylor et al., 1989; Pestka and Bondy, 1994). Humoral immunity Humoral immunity Can be both stimulated or inhibited by T-2 toxin and the other trichothecenes. The effect of T-2 4O toxin on antibody production appeared dependent on the type and dose of antigen, and the frequency of toxin administration. Chronic exposure of mice to T-2 toxin enhanced a dose—dependent antibody response to T-cell- independent antigens (polyvinylpyrrolidone and dinitrophenyl-ficoll), but cmmmessed antibody response to T-cell-dependent antigen, sheep red. blood cells, (SRBC) (Otokawa en: al., 1979, 1983; Lafarge-Frayssinet et EH”, 1979; Rosenstein et EH”, 1979, 1981). However, Holt aumi DeLoach (1988) reported that T-2 toxin suppressed the antibody response to both T-cell-dependent antigen, (SRBC), and also the T-cell—independent antigen, TNP- Lipopolysaccharide. 131 further contrast, dietary exposure of CD-1 mice to T-2 toxin did not alter T-cell-independent antibody responses to DNP—Ficoll or Escherichia coli lipopolysaccharide (LPS), but at 10 ppm, T-2 toxin enhanced the T-cell-dependent responses (Tomar en: al., 1988a). In total, these findings showed that T-2 toxin can modulate immune response and that this modulation is attributable to the direct toxic effects of the toxin on the cells of the immune system. Mice sensitized to SRBC and given intraperitoneal injection of a single dose of T-2 toxin (3 mg/kg body weight), 2-3 days after antigen (SRBC) sensitization developed higher SRBC titres than controls (Masuko et al., 41 1977). The titres of antibodies to SRBC measured 8 or 15 days after SRBC sensitization were also elevated in mice intraperitoneally injected with T—2 toxin at a dose of 3 mg/kg body weight, either 2 days before or on the same day as the SRBC sensitization. T-2 toxin may enhance or depress in vitro lymphoblastogenic response of B- and T-lymphocytes to various mitogens (Lafarge-Frayssinet et add” 1979; Buening et al., 1982; Friend et al., 1983; Holt et al., 1988b; Tomar et al., 1988a). T-2 toxin given at very low dose (0.05-1 ng/ml), increased mitogenic responses of splenic B- and T-lymphocytes to phytohemagglutinin (PTH) and LPS. However, at higher doses (2—10 ng/ml) of T-2 toxin, mitogenic responses of splenic B- and T-lymphocytes from animals stimulated by PTH, LPS and concanavalin A (Con A) mitogens were depressed. These results suggested that both B- and 'T—lymphocytes ‘were sensitive 11> T-2 toxin“ The effect on lymphoblastogenic response was transient when CD- 1 mice were repeatedly exposed to low levels of T—2 toxin (Taylor et al, 1985). Tomar et al., (1988b) reported that various lymphocyte subpopulations of human peripheral blood have different susceptibilities to T-2 toxin. Mitogenic response was inhibited at a lower concentration (1.6 ng/ml) of Con A. mitogen as compared to PTH (2.4 ng/ml) and pokeweed mitogens (PWM). The effects of T-2 toxin on 42 mitogenic responses of murine splenic cells in vitro appeared to be dependent on the type of mitogen used in the lymphoblastogenesis assay (Taylor et al., 1987). Murine splenic cells cultured with various mitogens and exposed to T-2 toxin (10'11 to 10‘10M) after 24 hr increased 3H- thymidine uptake by splenic cells. Stimulation by PWM increased dramatically while the response to LPS was increased to a lesser extent. Conversely, exposure to T-2 toxin decreased T-cell responses to both PHA and Con A (Taylor et al., 1987). T-2 toxin affected the levels of specific classes of immunoglobulins when various experimental and domestic animals were repeatedly exposed to low levels of T-2 toxin. A chronic dietary exposure of mice to T-2 toxin caused a significant dose-dependent increase in the number of spontaneous antibody-secreting cells in the spleen as detected by the protein A plaque assay (Cooray and Lindahl- Kiessling, 1987; Pestka and Bondy, 1990; Schiefer et al., 1987). The reduction 1J1 the suppression cu? B-cell growth and stimulation of B-cells caused. by erythropoiesis or activated macrophages could be responsible for the increase in antibody production (Cooray and Lindahl—Kiessling, 1987). In contrast, repeated exposure of mice to T—2 toxin administered orally or injected intraperitoneally at a dose of 2.5 mg/kg body weight caused a decrease in titres of 43 antibodies to SRBC and plaque forming cells (Rosenstein et al., 1979; Taylor et al., 1985). Dose-dependent antibody production assessed by the number of plaque-forming cells, was not affected by T-2 toxin up to an in Vivo dose of 0.5 mg/kg body weight (Taylor et al., 1985). However, the number of plaques per 106 splenic cells was depressed at a dose of 2.5 mg/kg body weight. T-2 toxin Inns been. shown tx> decrease immunoglobulin synthesis and antibody response in domestic and laboratory animals. Long term exposure to dietary T—2 toxin decreased antibodies to SRBC (IgG and IgM levels) in CD—1 mice (Taylor et al., 1985) and in monkeys (Jagadeesan et al., 1982). The levels of immunoglobulin G, M, and A were significantly' lower‘ in T-2 toxin-treated. calves than in untreated controls (Mann et al., 1982, 1983, 1984; Osweiler et al., 1981). Using' peanut agglutinin (PNA) receptors and surface immunoglobulin (SIG) as surface markers for bovine T- and B-cells respectivelyn Mann. et al., (1984) reported. that subclinical levels of T-2 toxin at 0.3 and 0.5 mg/kg/day for 56 and 18 days, respectively, caused a slight increase in (SIG+) B-cells. However, T—cell (PNAI) numbers were not affected at the low dose or transiently reduced in calves treated at 0.5 mg/kg T—2 toxin fed orally for 28 days. Antibody synthesis is also regulated by T cells. 44 Cell-mediated immunity Delayed-type hypersensitivity Masuko et al., (1977) and Otokawa et al., (1979), reported an enhancing effect (Hf T-2 toxin on the development of delayed—type hypersensitivity (DTH) to SRBC cells in mice. The dose, timing, and frequency of T—2 toxin administration were crucial factors in the enhancing effect of T-2 toxin. When mice received 3 mg/kg T-2 toxin 2 or 3 days before sensitization with subcutaneous injection of SRBC, no appreciable effect was observed on the DTH response (footpad. swelling) (Masuko et .al., 1977). Sensitization of mice with the toxin administered 3 hours before antigen injection did run: cause significant depression in DTH responses by 7 and 14 days later. However, when the toxin was injected 2 days after sensitization, marked enhancement of DTH response was observed. In mice treated with the toxin 3 days after sensitization, DTH responses were also enhanced but t1) a lower degree than those treated on day 2. The optimal time for toxin administration was 2 days after antigen stimulation. A single dose of T—2 toxin larger than 3 mg/kg body weight was required to induce significant enhancement of DTH response (Masuko et al., 1977). The administrathmd of the 45 toxin only slightly affected or caused no appreciable suppressive effect on the hemagglutinating antibody response at the time of the DTH response (Masuko et al., 1977). These findings led. to the hypothesis that T—2 toxin seems to preferentially inhibit proliferation of a subset of T lymphocytes which have a suppressor function. When spleen cells from mice, which had received a tolerogenic high dose of SRBC 14 days earlier, were transferred to unsensitized mice, DTH response of the recipients was significantly suppressed on day 7, while spleen cells from mice further treated with T-2 toxin 2 days later showed significantly less suppressive activity in the recipients (Otokawa et al., 1979). Results of these spleen cell transfer experiments showed that suppressor cells in the DTH response were produced by an intravenous injection of a large dose of SRBC, but decreased by T—2 toxin treatment. The decreased suppressor activity in the spleen seems to explain the enhancement of DTH response observed in mice treated with T— 2 toxin after the intravenous injection (Otokawa et al., 1979). T-2 toxin could not completely inhibit the development of suppressor cells for DTH since there was partial recovery of tolerance by T-2 toxin. The inhibition of tolerance induction by T-2 toxin may likely be due to the 46 susceptibility of the suppressor cells or their precursors to T-2 toxin (Otokawa et al., 1979). T-2 toxin probably has an inhibitory effect on the generation of suppressor cells by inhibiting development of their precursors, since T-2 toxin was effective only when administered within a few days after antigen stimulation (Otokawa et al., 1979). In contrast, Taylor et en”, (1985) reported 51 dose-dependent depression in DTH when low levels of T—2 toxin (0.02-5.0 mg/kg body weight) was fed intermittently to CD-1 mice sensitized intraperitoneally on day 14 and challenged 5 days later. Topical application of T—2 toxin inhibits the contact hypersensitivity response in mice (Blaylock et al., 1993). T-2 toxin. reduced time ear-swelling'.response 11> oxazolone challenge when the toxin was applied topically at or within 1 hr. after challenge. The toxin significantly reduced MHC class 11(1A) expression and antigen presentation. One mechanism of action of T—2 toxin in reducing the contact hypersensitivity response is via inhibition of protein synthesis and effective antigen presentation by epidermal Langerhans cells (Blaylock et al., 1993). Graft rejection T—2 toxin caused delayed graft rejection when mice were given daily intraperitoneal injections of the toxin at 0.75 47 mg/kg body weight for 7 days prior to receiving allografts and for 2 to 4 times a week for 20 days after the graft (Rosenstein et al., 1979). Role of Macrophages T-2 toxin administered as a single dose that caused marked lymphoid depletion, and suppressed (n: enhanced in vivo macrophage phagocytic activity 1J1 antigenically sensitized mice (Corrier et al., 1987a). Enhancement or suppression of phagocytosis was a function of the time T—2 toxin was administered in relation to antigenic stimulation or challenge. T—2 toxin had no effect on the viability or phagocytic activity of resident peritoneal macrophages to sRBC in nonsensitized mice (Corrier et al., 1987a). Thus, resident peritoneal macrophages are resistant to toxic effects cu? T—2 toxin _N7 vivo. However, 51 significant increase in phagocytic activity occurred in cells from mice treated with toxin and subsequently sensitized with SRBC. In contrast, phagocytosis of SRBC was significantly suppressed in cells from mice treated with T-2 toxin after sensitization (Corrier et al, 1987a). In vitro viability studies have shown that eXposure of alveolar macrophages to submicromolar concentrations of T-2 toxin for 20 In: influenced alveolar macrophage viability, cell number, and viability index (Gerberick and Sorenson, 1983). In addition, T-2 toxin is toxic to alveolar 48 macrophages in nmmolayer cultures and inhibits macrophage phagocytic activity in vitro (Gerberick et al., 1984). Both single-dose and successive treatments of mice with T-2 toxin by oral gavage enhanced the respiratory burst activity of macrophages and pre—inoculation treatment with T-2 toxin also caused a significant increase in the number of peritoneal cells (Cooray and Jonsson, 1990). Cell-mediated resistance Treatment of mice with T-2 toxin after Listeria challenge suppressed resistance to listeriosis, leading to rapid growth of Listeria and significant increases in mortality? (Corrier and Ziprin, 1986b; Corrier et al., 1987b). Conversely, pretreatment of mice with T-2 toxin prior‘ to challenge with Listeria :markedly' enhanced resistance to listeriosis, as shown by significant reduction in Listeria-induced mortality in T-2 toxin- treated mice (Corrier and Ziprin, 1986a). The incidence of Listeria—induced mortality was dependent on T—2 toxin dose, and progressively decreased with increasing dose. Despite the fact that T-2 toxin caused significantly reduced thymus and spleen weights, bone marrow cellularity, the total number of circulating leukocytes, lymphocytes, and neutrophils, the enhanced resistance to listeriosis was accompanied. by a .significant increase in the influx of 49 macrophages into Listeria-elicited. jperitoneal exudates (Corrier et al., 1987b). In addition, in vivo phagocytosis of SRBC by peritoneal macrophages was significantly increased 1J1 T-2 toxin-treated inice tflmn: were sensitized with SRBC. The enhancement of resistance to listeriosis in mice pretreated with T-2 toxin was associated with increased migration or activation of macrophage effector cells (Corrier et al., 1987b). Increased concentration of serum amyloid P-component, an acute-phase reactant protein that increases macrophage listericidal activity have also been associated vniji enhanced. resistance (Singh 6N: al., 1986; Ziprin et al., 1987). In contrast, T-2 toxin had no effect on either the course of infection or serum amyloid P- component concentration.:h1 mice challenged vniji Salmonella typhimurium (Ziprin et al., 1987). In another study, Cooray and Jonsson, 1990, reported that T—2 toxin had a modulatory effect on the cell—mediated immune system. Pretreatment of mice with a single dose of T-2 toxin by oral gavage caused an enhanced resistance to common mastitis pathogens, E. coli or S. aureus. This enhanced resistance was associated with migration and activation of macrophages into sites of bacterial infection. In addition, T-2 toxin reduced the virulence of both pathogens. The exact mechanism of action involved in T-2 toxin- enhanced resistance to listeriosis is not known. One theory 50 that had been proposed to explain resistance to listeriosis in thymus-deprived mice (Cheers anui Waller, 1975; Newborg and North, 1980) and in mice treated with cytotoxic drug Cyclophosphamide (Tripathy and Mackaness, 1969; Luster et al., 1981) was attributed to the removal of a radiosensitive population (Hf regulatory T? cells. Suppressor CF cells are short-lived lymphocytes that are more susceptible to cytotoxic agents than are other subpopulations of lymphocytes (Mitsuoka et al., 1976; Chan et al., 1977). Therefore, enhanced host resistance in mice treated with T-2 toxin prior to Listeria-challenge may have been caused by depletion or impaired function of a regulatory T suppressor cell population and a subsequent enhancement of macrophage activity. Exposure to T-2 toxin may also inhibit cell-mediated immune function by causing defects at multiple hematopoietic sites or compartments. Mice exposed to T—2 toxin had an apparent dose-dependent delay' in thymocyte :maturation or differentiation, characterized in! significant increases in the percentage of double-negative (CD4’8‘) thymocytes and significant decreases 1J1 the percentage (n3 double-positive (CD4W?) thymocytes (Smith et al., 1994). The effects of T- 2 toxin on multiple hematopoietic compartments involved in the production of T—lymphocytes may contribute to the peripheral T-cell lymphocytopenia and T—cell mediated 51 immunosuppression produced by the T—2 toxin. Thymic atrophy was characterized. by significant reduction in the total number of cells within all phenotypes defined by CD4 and CD8 cell surface antigen expression. The bone marrow from T=2 toxin—treated mice had a highly significant hypocellularity, indicating that this hematopoietic compartment may also be a target. A non-significant reduction in overall splenic celularity was observed. However, there was ea significant decrease in the total number of both B- and T‘lymphocytes present within the spleen (Smith et al., 1994). Prenatal exposure of mice to T-2 toxin resulted in significant fetal thymic atrophy and a significant reduction in CD44 and CD45 fetal liver prolymphoid cell subpopulations (Lafarge-Frayssinet et en”, 1990; Holladay et EH”, 1993). These results showed tjmn: T-2 toxin easily' passes the placental barrier and that lymphocyte progenitors, compared with thymocytes, represent highly sensitive targets for T-2 toxin, and are therefore responsible for thymic atrophy (Holladay et al 1993). The authors concluded that direct cytotoxic effects of T-2 toxin had limited effect on thymic atrophy, as evident by expression of CD4, CD8, and TCR cell- surface antigens. T-2 toxin has also been shown to impair migration— chemotaxis and phagocytosis of neutrophils in humans, cattle 52 and. rats (Buening' et (al., 1982; Gerberick: and. Sorenson, 1983; Yarom et al., 1984) and rabbits (Niyo et al., 1988). Antiviral Activity T-2 toxin and the other trichothecene mycotoxins (diacetoxyscirpenol and neosolaniol) inhibit herpes simplex virus type 2 (HSV—2) replication in human epidermoid carcinoma No. 2 (HEp-2) cells by blocking viral protein syntheses and not by inhibiting adsorption and penetration of virions into the host cells (Okazaki et al., 1988a). The addition of these toxins within 4 hours after HSV-2 infection was necessary for the inhibition of Virus replication. Viral polypeptides synthesized in HSV-2- infected cells treated with the toxins was analyzed by immunoblotting using rabbit antiserum to HSV-2. The results showed that syntheses of early viral proteins were greatly inhibited when the toxins were added 1 In: after infection. Late viral proteins were also inhibited by the addition of T-2 toxin and the other trichothecenes, 4 to 6 hr after infection. The toxins added after the completion of the late viral protein syntheses did not significantly' affect the HSV-2 replication. However, viral RNA. synthesis was not inhibited when the toxins were added 1 hour after infection (Okazaki et al., 1988a). The inhibition of HSV-2 replication by the trichothecenes is closely correlated with blocking of viral protein synthesis (Okazaki et al., 1988b). In time 53 course studies of virus growth and viral protein synthesis in HSV—2—infected HEp-2 cells, the presence of T—2 toxin and other trichothecenes completely inhibited viral plaque formation. In HSV-2—infected cells treated with the toxins, neither ‘virus replication. nor ‘virus-specific: polypeptides was noted although two major nonspecific polypeptides were detected in treated cells as wells as in mock-treated cells. Okazaki et al. (1992) reported that both hydrolysis at the C-4 position, hydroxylation at time C-3’ position, and hydrolysis at the C-8 position of T—2 toxin are responsible for the reduction in antiviral activity. The metabolic conversion of T—2 toxin to 3'-hydroxy HT-2 toxin or to T-2 tetraol decreases the antiviral activity. In plaque- reduction tests, the HSV-2 plaque formation was inhibited by the trichothecenes in a dose-dependent manner. Effect of T—2 toxin on vaccinal immunity Despite the fact of the broad immunosuppressive effects of T-2 toxin in various domestic animals, poultry and laboratory animals, there are very few studies on the effect of the toxin on vaccinal immunity. Repeated exposure of mice to T-2 toxin prior to vaccination with BCG (Mycobacterium bovis) against hwcobacterium tuberculosis resulted in depressed vaccinal immunity, as evidenced by increased multiplication with larger viable counts of organisms 54 recovered from the lungs and spleen as compared to the non- treated. BCG group (Kanai and. Kondo, 1984). In contrast, Ziprin and McMurray, (1988) reported a consistent and significant influence of BCG in reducing the number of virulent tubercle bacilli present in the lungs, but T-2 toxin treatment did not influence the efficacy of the BCG vaccine. The number of organisms found in the lungs of mice that received T—2 toxin without subsequent BCG vaccination was not significantly different from the number present in untreated mice. Similarly, the number of organisms found in the lungs of T-2 toxin-treated vaccinated. mice did not differ‘ significantly' from time number' found 1J1 vaccinated mice that had not been treated with toxin. In another study, a single oral treatment with T—2 toxin 7 days before intraperitoneal challenge with Mycobacterium bovis decreased resistance to infection (Ziprin and..McMurray, 1989). In addition, T-2 toxin treatment had no effect on BCG infection when this was initiated by inhalation of microbial aerosol. No apparent effect of T—2 toxin. on either antibody production or the size of the thymus, spleen or the bursa of Fabricius in chickens immunized. with. a bacterin against Pasteurella multocida and treated with 10 gmxnoo oumocmum com mcmozm .Amo.ovmv ucmumuuae sauamufluacsam mum mumuuma uoflnomnoQSW ucoummwflp >n omonHow QEDHoo m chuHB monam> smmza coumc Houmm m>mp o>Husoomcoo co>mm How pcofloz >oon mx\oEmN.H wo mmoo >Hfimo m um co>Ho mm: saxou NIB no©.v H om.mm um.m H om.ow evo.m H mm.o® amm.w H o>.mm I I oom.m H om.bv 0H.m H om.mw onv.v H om.>m nvv.m H mo.mm + I now.m H om.mm am.© H OH.©v .tmm.m H n©.mv mmm.v H ev.mv + + aom.v H om.hm .%©.m H ov.mv new.v H ma.wv mHo.w H em.ov I + moo.m H om.mv mm.m H om.Hm mwv.v H vo.om mmo.m H vo.mv I + v m m H e>m conu NIH AEGVmfiuconz >oon cmoz II .02 Hmflue oucoEumwue .coumc um e>m cuflz omumcfloom> no Umumcfloom>co mcoxoflco rumemH .UHOIsmUIs no Dream; seen so cHxOL NIB no bomuum .N.H manna 93 Effects of T-2 toxin on development of HVT and MDV Viremia. Typical cytopathic effects (plaques) of HVT and MDV were detected in monolayer cultures of CEF and DEF, respectively (Figures 1.1, 1.2). Indirect immunofluorescent staining of DEF monolayer cultures, was used to differentiate between HVT and MDV plaques using MDV serotype 1-specific monoclonal antibody (Lee em: al., 1983). Figure 1.3. shows a typical immunofluorescent MDV plaque. The effect of T-2 toxin on the development .of HVT viremia in chickens vaccinated with HVT at hatch and challenged with MDV at 8 days of age is shown in Table 1.3. At 8 days of age, in trials 2, 3, and 4, but not in trial 1, the titre of HVT viremia in T-2 toxin-treated chickens was significantly lower than that in untreated chickens. By 4 weeks of age the titres of HVT viremia in T-2 toxin-treated chickens was comparable to that in untreated chickens, regardless of challenge with MDV. The effect of T-2 toxin or HVT on the development of MDV-induced viremia in chickens challenged with MDV at 8 days of age is shown in Table 1.4. The titres of MDV viremia in. HVT-vaccinated, MDV-challenged. chickens that had. been treated with T-2 toxin was comparable to that in the untreated group. The titres of'lflfi/ in T—2 toxin-treated, MDV-challenged chickens was consistently higher than that in 94 Figure 1.1. Typical plaque morphology of HVT in CEF at 5 days P1 with PBMs from 8-day—old, T-2 toxin-treated, 15I5 X 71 chicks that had been vaccinated with HVT at hatch (X 354). 95 Figure 1.2. Typical plaque morphology of MDV in DEF at 7 days PI with PBMs from 4—week—old, T-2 toxin-treated chickens vaccinated with HVT at hatch and challenged with MDV at 8 days of age (X 354) . 96 Figure 1.3. Typical IPA—stained (using serotype 1 MDV— specific mab) MDV plaque morphology in DEF at 7 days P1 with PBMs from 4—week—old, T—2 toxin—treated chickens vaccinated with HVT at hatch and challenged with MDV at 8 days of age (X 360). 97 oocHEuouop uocuoz .omumou dsoum ucoEumonu Hod mxoflco mHINan .Amo.ovmv ucmumuuae sauamuauaaoam msfim> cams. .AcoHumEHowmcmuu uoou mumsom umumm mcoHumH>oo pumpcmum Ucm mammav muHc: mcHEnow msomama .coumc Houwm mxmp m>Husommcoo co>om mom ucoflmz >oon ox\oEmN.H mo mmoo >Hamp m um co>Ho mm: conu NIH mm.o H mm.m mm.o H mm.m Dz m©.o H ow.m v + + I Nv.m H mm.m nm.m H mm.v oz mN.H H mv.m v + + + nv.m H mm.m mH.m H mm.v He.m H v>.© vw.H H mm.m H I + I *Hm.o H mm.H *mw.a H mo.m avo.a H ©H.m MH.H H mH.N H I + + v m m H mxooz >02 e>m NIH CH med Asmmv muufleamflaoufi> e>m II .02 Hmflue ucoEumoue .oom mo m>mo m um >QE cuflz oomcmaamco pom .coumc um e>m cuHB owumcfioom> mcwxoflco :.VamHmH .UHOIxmosz can IH CH mHEmHH> e>m mo ucoEQon>oo co Conu NIB wo powwmm .m.H magma 98 omcHEHmuop pocnoz omumou gnome ucmEumonu Hod mxoflco mHINan .omcmaamcqumOQ mxz w an owummu mHEouH> >026 .imo.ovmc Hamnmuan ancmuHuHcaHm mum mnmuuma uofluomquSm ucoHoMMHc >n ooonHOw canaoo m chqu monam> cmmzm .AcoHumEHoumcmuu uoou mumsom umuwm mcoflumfl>mo Unmocmpm paw mammav mafia: OQHEHOM msomama .coumc umuwm mxmo o>Husommcoo co>om How ucmflmz >oon ox\oEmN.H wo omoo >Hflmo m um so>Ho mmz conu NIB .mo.e H as.mfi .mm.m H Hm.NH a2 ‘ .mm.e H HH.m + I I .so.m H mm.om .Hs.s H Hm.efi Hz .mm.m H sm.m + I + sm.fi H ms.H .m©.H H mm.m oz aae.o H Hs.m + I I 10H.H H we.fi .Na.m H mH.m oz .Hm.H H Hm.m + I + as m N H >oz e>m NIH Lanai mHHHH.aaHEmHH> >az II .02 HHHHH Hcmaummue .mmm uo m>mo m um >92 LDHZ ommcmaamco cam coumc um e>m cuflz ooumcHoom> mcoxoflco ooummquconu NIB .UHOIxmszv CH MHEmHH> omosocflI>oz Ho ucmEdoHo>mo .v.H manme 99 untreated chickens however, the difference was not statistically significant. At 23 weeks post-challenge, the MDV viremia titre in vaccinated, challenged chickens was significantly lower than that in unvaccinated challenged chickens, regardless of treatment with T-2 toxin (Table 1.4). Necropsy Findings. Chickens that died or were moribund following challenge ‘with THAI were necropsied” The gross changes commonly noted were the classical MD lesions including enlarged or edematous peripheral nerves involving the vagus, sciatic, and femoral nerves, and the brachial plexus with loss of cross-striations. The enlargements were segmental or uniformly diffuse along the length of the affected nerves. The bursa of Fabricius and the thymus were atrophied in affected chickens. Single or multiple greyish- white lymphoma nodules were present in one or several visceral organs including gonads, lungs, proventriculus, liver, heart, spleen adrenal glands and the bursa of Fabricius. The lymphoma nodules or infiltrates consisted of pleomorphic neoplastic lymphoid cells, plasma cells and mature lymphocytes (Figure 1.4). Effect of T-2 toxin on Incidence of MD-Induced Mortality and Lesions in HVT-vaccinated and unvaccinated chickens . At 100 Figure 1.4. Photomicrograph of sciatic nerve from a 4-week- old, 15I5 X 71 chicken treated with T-2 toxin (1.25 mg/kg body weight) for 7 consecutive days after hatch and challenged with MDV at 8 days of age. Notice a metastatic lymphoma nodule and neoplastic cellular infiltrates (H&E X 242). 101 4 weeks PC in 3 of 4 trials, the incidence of MD-induced mortality and lesions in MDV-challenged chickens was significantly' higher iii T-2 toxin-treated. chickens (56%, 32%, 67%) than in untreated chickens (10%, 16%, 35%) (Table 1.5). However, the incidence of MD-induced mortality and lesions was comparable in all groups vaccinated with HVT, regardless of treatment with T-2 toxin» In: 8 weeks PC, the percent MD mortality and lesions in T-2 toxin-treated chickens (94%) was comparable to that in untreated chickens (100%) (Table 1.5). The incidence of MD mortality and lesions in HVT vaccinated group ranged from 0%-18%, regardless of treatment with T-2 toxin. Percent Protection by HVT. At termination of the trials (8 weeks PC), the percent protection. in all IHVT vaccinated groups ranged from 82%-100% regardless of treatment with T-2 toxin (Table 1.6). By 8 weeks PC, the incidence of MD in vaccinated groups varied from 94%-100%, regardless of treatment with T-2 toxin. 102 gnome ucmEomouu mom monco HNIONHQ omHmoonomc com mxooz N\HIm pm muoumHomH CH commoowmnw >HHmucmoHoom monco Ho OOOIBOU .mam Ho msme O Hm >oz anz omOQoHHmco ocm .coumc Hmuwm m>mp m>Husowmcoo H How ucOHmz >oon Ox\OEmN.H mo omoo NHHHmo m um conu NIH cqu commouu .coumc um e>m cqu omumcHoom> mum? moncom Amo.ovmv HamHmHuHu ancmoHuHcaHm.. *Lr O O O O O O O O I I I O O 0O O O O HH O + + I m O 0v v O O OH O + + + OOH mm OOH OH mm OH OOH OH + I I vm «no . OOH «mm OOH m OOH How + I + O 0 00 O o o o o I I + mxz O mxz v mxz O mxz v mxz O mxz v mxz O mm: v v m N H >QE B>m NIH amconmH\>uHHmuuoz u:ooumm...um mxomz ...oz HmHue ucoEumoue .mom mo mmmc O on >oz cqu oOcoHHmcqumOQ mxmmz O chqu mcoonco commouucs cam omummHuIconu NIB CH mconmH cam >uHHmuHoE oz Homonwm .m.H mHnme 103 .om mxomz O um coHuomuond oz unmouoma coumc um e>m cqu owumcHoom> can .coumc Houwm m>mp m>Husommcoo h now ucOHmz >oon OH\OEON.H mo omoo HHHmo m um cm>HO mmz conu NIH O O O O I I I OOH OOH OOH OO + + I mm mm Om NO + + + v m N H >QZ H>m NIH acoHuomuonm ucooumm II .02 HmHHe ucmEumoHe .mam no msme a La >o2 anHz emoamHHmzo mcmquao .e x .HOH omumcHoom>Ie>m .oopmoquconu NIB CH coHuomuoue OZ unwoumm .O.H mHnme 104 Discussion Data from this study suggest that exposure of newly hatched chicks to T-2 toxin may lower the titre of HVT viremia at 1 week PV at hatch. The data also suggest that T- 2 toxin may increase the incidence of MD lesions and mortality within 4 weeks of age, but only in unvaccinated chickens. The data confirmed-the negative effects of T—2 toxin on body weight gain. Analysis of viremia results revealed that exposure of newly hatched chicks to T-2 toxin may lower titres of HVT viremia at 1 week, but not at 3 weeks PV. These results are similar to those reported by Witter et al. (1976) in which treatment of HVT—vaccinated chickens with cyclophosphamide (Cy) at a dose of 4 mg per day for the first 4 days after hatch reduced HVT viremia titres at 8 and 15, but not at 22 days of age. In contrast, Sharma et al., 1980, reported that immunosuppression of HVT—vaccinated chickens by neonatal surgical thymectomy and gamma-irradiation increased the titre of HVT. Previous studies have shown a relationship between the level of HVT viremia and vaccinal immunity against MD. A correlation between HVT viremia and protection of chickens against MD was reported by Patrascu et al. (1972). Okazaki et al. (1973) indicated that an early establishment of HVT 105 viremia in vaccinated chickens was necessary for protection against MD iii the field. Cho et en” (1976) and Riddell et al. (1978) reported in surveys that the incidence of detectable HVT viremia in vaccinated chickens was significantly lower in the MD-affected than in the healthy penmates. It is generally accepted that cell—mediated antiviral immunity generated by HVT vaccination plays a major' role III the vaccinal immunity against bfl) (Gupta et al., 1982; Purchase and Sharma, 1974; Payne et al., 1978). In the present study, although T-2 toxin caused moderate to severe lymphoid depletion in the thymus, spleen and bursa of Fabricius, chickens treated with T-2 toxin were still able to mount antiviral immunity leading to protection against MD challenge. The reduced titres of HVT viremia in T-2 toxin-treated chicks in the present study could be explained by the antiviral effect of T-2 toxin on HVT. Okazaki et al. (1988b) reported that the inhibition of HSV-2 replication in vitro was closely correlated with T-2 toxin-induced blocking of early and late viral protein synthesis. The finding that by 3 weeks PV, the level of HVT. viremia in T-2 toxin-treated chickens was comparable to that of untreated chickens could be due to the lack of the antiviral effect of T-2 toxin as the toxin concentration wanes within 1 week after treatment and the immune system regenerates. The metabolic conversion 106 of T-2 toxin ti) its by-products decreases the antiviral activity of T-2 toxin in the HSV—2 plaque formation assay (Okazaki et al., 1992). Analysis of MDV viremia results revealed a consistent increase, although statistically not significant, in titres of MDV at 3 weeks PC, but only in chickens treated with T-2 toxin. Interestingly, the higher titres of MDV viremia were accompanied by EH1 increased incidence of DH) mortality and lesions within 4 weeks PC, but only in unvaccinated groups. This finding its consistent with previous studies :hi which immunosuppressed chickens developed high levels of MDV viremia titres (Sharma et al., 1977; Powell and Davison, 1986). High levels of MDV viremia have been shown to be closely related to lesion formation and MD mortality (Witter et al., 1971). As expected, titres of MDV were significantly lower in HVT-vaccinated chickens than in nonvaccinated controls as has been. previously‘ shown. by ‘Witter et al. (1976). MDV infection is known to initially cause severe depletion of lymphocytes in the thymus, bursa of Fabricius, and spleen resulting in the suppression of both humoral and CMI responses to antigenic stimulation (Payne and Rennie, 1973; Burg et al., 1971; Evans and Patterson, 1971; Jakowaki et al., 1973; Purchase et al., 1968). This MDV-induced immunosuppression combined with the immunosuppression caused 107 by T-2 toxin could explain the early development of MD lesions and mortality in unvaccinated T-2 treated chickens. Furthermore, T—2 toxin has been shown to cause immunosuppression in chickens and other animals making them more susceptible 11) secondary bacterial and viral infections (Boonchuvit et EH”, 1975; Corrier anmi Ziprin, 1986; Friend 6%: al., 1983). Data from time present study suggest that although a significant decrease in HVT viremia was noted in T-2 toxin-treated chickens, the percent MD protection was comparable to that in untreated chicks. This finding is consistent with those of Witter and Offenbecker (1978) who reported good immunity in vaccinated chickens which lacked readily detectable HVT viremia. In centrast, however, Okazaki et al. (1973) and Cho et al. (1976) reported low HVT titres in birds that subsequently developed MD in the field suggesting that immunity to MD depends on maintenance of high levels of HVT viremia. Analysis of data on body weight confirmed the negative effects of T-2 toxin on body weight gain. T-2 toxin has been shown to reduce body weight gains of white leghorn and broiler chickens (Wyatt et al., 1972, 1973; Chi et al., 1977; Hoerr et al., 1982). Furthermore, the weight gain and feed consumption are usually' more dramatically’ decreased during the first 3 week period during T—2 toxin treatment and were associated with feed refusal or reduced feed intake 108 (Chi et al., 1977). The decreased. weight gain. and feed consumption caused by T-2 toxin is usually proportional to the amount of the toxin given. Commercial broiler chicken flocks in several countries around the world including Europe are not vaccinated against MD, as losses from MD during the growing period are usually 0.1% or less (Purchase, 1985). Data from the present study should have some practical implications as subchronic exposure to T-2 toxin enhanced the development of MD lesions and may be very important in inducing early incidence of mortality within 4 weeks of age, but only in unvaccinated chickens. However, in other countries where such commercial broiler chickens are routinely vaccinated against MD, subchronic exposure to T-2 toxin may not pose such a problem. CHAPTER 2 FLOW CYTOMETRIC ANALYSIS OF PERIPHERAL BLOOD AND SPLENIC B- AND T-LYMPHOCYTE SUBPOPULATIONS IN CHICKENS TREATED WITH T-2 TOXIN CHAPTER 2 FLOW CYTOMETRIC ANALYSIS OF PERIPHERAL BLOOD AND SPLENIC B- AND T-LYMPHOCYTE SUBPOPULATIONS IN CHICKENS TREATED WITH T-2 TOXIN Abstract Four trials were conducted to determine the effects of T-2 toxin on peripheral blood and splenic B- and T- lymphocyte subpopulations in white leghorn chickens. Day-old chicks of Regional Poultry Research Laboratory (RPRL) line 1515 X 71 were treated with T-2 toxin daily for seven days via crop gavage at a sublethal dose of 1.25 mg/kg body weight. At 8—9 and 21—22 days of age, single-cell suspensions of pooled samples of blood and splenic lymphocytes from T-2 toxin- treated and untreated control chickens were analysed for the presence of CD4, CD8, CD3 and IgM cell surface markers using monoclonal antibodies and flow cytometry. T—2 toxin significantly reduced the body weight of treated chickens. The percentage of both peripheral blood and splenic B- lymphocytes in T-2 toxin-treated chickens was significantly lower than that in untreated chickens, but only at 8-9 days 109 110 of age. An increase in the percentage of CD4 T-lymphocytes in both peripheral blood and spleen, and CD3 T-lymphocytes in spleen was noted in T-2 toxin-treated chickens, but only at 8—9 days of age. The data suggest that exposure of chickens to T-2 toxin may cause a severe depletion of B- lymphocytes and a .relative increase in CD4 and CD3 T- lymphocytes. Introduction T-2 toxin, a trichothecene mycotoxin, has been shown to cause health problems in humans and animals (Mayer, 1953a, b; Joffe, 1986; Watson et al., 1984; Wang et al., 1993). The toxin is a secondary metabolite produced primarily by Fusarium spp. which grows on cereal grains and contaminates agricultural products in temperate regions of North America, Europe and Asia (Saito and Ohtsubo, 1974; Ueno, 1983; Vesonder, 1983; Scott, 1989; Wang et al., 1993; Beardall and Miller, 1994). The cytotoxic effects of T—2 toxin have been shown to be associated with inhibition of protein and DNA synthesis due ii) the effect cm? the toxin (n1 peptidyl transferase (Ueno et al., 1968; Bamburg, 1974; Cundliffe et al., 1974; Cundliffe and Davis, 1977; Wei and McLaughling, 1974; Pace et al., 1988). T-2 toxin has been shown to have a “radiomimetic” effect on rapidly proliferating cells such as 111 lymphoid organs, hematopoietic cells, and the gastrointestinal epithelium (Saito et al., 1969; Ueno, 1977; Hoerr et al., 1981b; Mirocha, 1983). Although T—2 toxin is known to inhibit synthesis of macromolecules such as proteins and DNA, its effect on the immune system has been shown to be either immunostimulatory or immunosuppressive. The immunotoxic effects of T-2 toxin have been reviewed by many authors (Bamburg, 1983; Otokawa, 1983; Taylor et al., 1989; Sharma and Kim, 1991; Sharma, 1993). Acute and subacute exposure of chickens to T-2 toxin have been shown to cause necrosis and depletion of lymphocytes in thymus, spleen, bursa of Fabricius and cecal tonsils. (Wyatt et al., 1973; Richard et al., 1978; Hoerr et al., 1981, 1982a,b). Low concentrations of T—2 toxin have been shown to increase mitogen-induced B—cell stimulation, but not T-cell responses (Taylor et al., 1985, 1987). T-2 toxin has also been shown to enhance delayed-type hypersensitivity and allograft rejection. time responses (MasukcI et al., 1977; Otokawa et al., 1979; Rosenstein et al., 1979). The effect of T-2 toxin on antibody production has been shown to be inconsistent and difficult to interpret. Repeated lower doses of T—2 toxin decreased antibody production in response to T-dependent antigens (Rosenstein et al., 1979, 1981) and enhanced antibody responses to T- 112 independent antigens (Rosenstein em: al., 1981; Cooray and Kiessling, 1987). Thus, T-2 toxin appears to selectively inhibit specific immune functions that involve T-cell regulation. It is worth noting that the regulation of immune responses is accomplished by the coordinated participation of various types of cells in the lymphoid tissues. Therfore in order to further investigate the mechanism of action of T—2 toxin on immune responses, it is essential to evaluate the cytotoxicity of T—2 toxin to various types of cells participating in immune responses. Selective depletion of B- lymphocytes from peripheral blood and spleen by T-2 toxin may impair antibody production without the involvement of cell-mediated immune responses. Since CD4 T-lymphocyte depletion. may reduce antibody' production. whereas CD8 T- lymphocyte depletion may cause an increase in antibody production, it is important to determine the effect of T-2 toxin on specific subpopulation(s) of T-lymphocytes in lymphoid tissues. The objective of this study was to determine effects of T-2 toxin on peripheral blood and splenic B- and T- lymphocyte subpopulations. Materials and Methods Chickens. Chickens were F3 progeny of RPRL line 15I5 males and 71 females. The breeder chickens are known to be free 113 of antibodies to MDV, herpesvirus of turkeys, avian leukosis virus, REV and other common poultry pathogens. Treated and untreated chickens were maintained iii separate isolators provided with negative pressure for the duration of the trials. T-2 Toxin. T-2 toxin, a trichothecene secondary metabolite of several Fusarium six ‘was obtained iii crystalline fOrm from Sigma Chemical Company (St. Louis, Missouri). The T-2 toxin stock was dissolved in 100% ethanol at a concentration of 2.5 mg per 1 ml, stored in the freezer at —20W3, and used within one week. Control untreated chickens were treated with ethanolzPBS mixture. Experimental design. In each of the four trials conducted, 132-187 day—old chicks were allotted into 2 groups as shown in Table 2.1. Chicks in group 1 were divided into 2 lots, each containing 36—EES chicks. Chicks iii lot 1A were treated with ethanol-PBS vehicle and maintained as untreated controls in separate isolators. Chicks in lot 1B were treated with T—2 toxin at hatch for 7 consecutive days via crop gavage at a dose of 1.25 mg/kg body weight. T-2 toxin dose was based on the average weight of the chicks used in each treatment group. Chicks in group 2 were divided into 2 lots, 2A, 2B. The protocol used to treat chicks in group 2 114 Table 2.1. Experimental design Group # Lot # T-2 toxin Age at Analysis (days) Blood Spleen 1 1A — 8 9 1B + 8 9 2 2A — 21 22 ZB + 21 22 Day—old chicks were divided into 2 groups containing 2 lots each. Chicks in group 1, lot 1A were treated with ethanol/PBS vehicle via crop gavage and maintained as untreated controls :nI separate isolator. Chicks iii lot 1B were treated with T—2 toxin at a dose of 1.25 mg/kg body weight for 7 consecutive days after hatch. At 8 aumi 9 days of age, single-cell suspensions of PBMs and spleen respectively were prepared from pooled samples of peripheral blood and spleen from T-2 toxin-treated and untreated chicks for single color flow cytometric analysis. Chicks in group 2 were treated as shown in group 1, maintained in 2 isolators until 21 and 22 days of age when pooled samples of PBMs and spleen, respectively were prepared for analysis. 115 with T-2 toxin was identical to that used in group 1. The chicks were maintained in different isolators until 3 weeks of age. At 8 days of age (end of the T-2 toxin treatment), 2—3 chicks from each of treated and untreated lots in group 1 were selected at random and euthanatized for histopathologic evaluation of the lymphoid organs. Blood was also collected from 30 chicks from each of treated and immieated groups using Alsever’s solution. Single cell suspensions of the peripheral blood were prepared from pools of 5 chicks per sample. At 9 days of age, chicks that were bled were euthanatized and spleens were aseptically collected. Five spleens per sample were pooled for analysis. At 21 days and 22 days of age, PBMs and splenic lymphocytes, respectiveiy were obtained from 1i3 chicks (3 chicks per pool) from each of treated and untreated lots in group 2. Except in trial 1, PBMs (collected at 8 and 21 days of age) and splenic lymphocytes (collected at 9 and 22 days of age) were tested. Chicks used in trial 1 were tested only at 21 and 22 days of age. Preparation of single—cell suspensions. Peripheral blood mononuclear cells (PBMs). Multiple pooled blood samples (3—5 chicks per pool) were obtained from the right jugular vein in Alsever’s solution and placed on ice. 116 Five millilitres of pooled blood samples were layered over 6 ml of Ficoll-Hypaque (Sigma Chemical Co., St. Louis, Missouri) and centrifuged at 2000 rpm for 20 minutes. The buffy coat interface was removed with a Pasteur pipette and washed three times with RPMI culture medium. Spleen. Spleens were aseptically collected (3-5 spleens per pooled sample) in centrifuge tubes containing RPMI and placed on ice. The spleens were homogenized using 40umash, and the single cell suspension obtained was sieved through cotton gauze and cleared on Ficoll—Hypaque at 2000 rpm for 20 minutes. The buffy coat interface was collected using a Pasteur pipette, and washed three times in RPMI culture medium. The viability of cell suspensions was determined using erythrosin. Immunofluorescent Staining. Monoclonal antibodies (mab) for CT4, CT8 and CT3, the avian homologues of mammalian CD4, CD8 and CD3, respectively (Southern Biotechnology, Birmingham, Alabama) were used. Fluorescein isothiocyanate- conjugated goat anti-mouse (FITC-GAM) for CT4, CT8, CT3 T- lymphocyte suprpulations, and EITC-goat anti-chick (FITC- GAC (IgM) for B—lymphocytes were used as secondary 117 antibodies. .A single—color staining procedure was performed, as described by Chan et al., 1988. Briefly, 2-3 )( 106 PBMs (n: splenic lymphocytes per sample were incubated with the respective appropriate dilutions (CD3: 1:50, CD4 and CD8: 1:100) of mab for 45 minutes in microtitre plates on ice. The cells were washed three times with sodium azide-bovine serum albumin solution and then incubated with FITC-GAM (1:2000) or FITC-GAC (1:500) antibodies for 30 minutes. To facilitate the exclusion of dead cells after the final wash, the samples were transferred into test tubes and propidium iodide was added to the cell suspensions immediately before analysis. Percentages of B— lymphocytes and T-lymphocyte subpopulations were determined from 104 cells per sample by analysis in a fluorescence-activated. cell sorter (Becton Dickinson, Mountain View, California). Statistical Analysis. Data for calculated means for individual samples were analysed with Student's t-test with significance determined at p30.05. Results Effect of T—2 toxin on body weight. In all trials, the mean body weight of chicks treated with T—2 toxin at hatch 118 for 7 consecutive days was significantly different from that of untreated chicks (Table 2.2). Effect of T—2 toxin on lymphoid organs. Histologically, mild to moderate lymphoid depletion of the thymus and spleen, and severe lymphoid depletion of the bursa of Fabricius were noted at 8 days of age. In the thymus lymphoid depletion was primarily noted in the cortex. In addition, the spleen had reticuloendothelial cell hyperplasia. The bursa of Fabricius had lymphoid depletion of follicles with increased proliferation of interfollicular connective tissue. At 21 days of age, the histologic appearance of the lymphoid organs from treated chickens were comparable to that of untreated control chickens. Effect of T-2 toxin on B- and T-lymphocytes in peripheral blood. At 8 days of age, the percentage of B lymphocytes in peripheral blood was significantly lower in T—2 toxin— treated chickens than in untreated control chickens (Table 2.3). In contrast, the percentage of CD4 T lymphocytes was significantly higher in T-2 toxin-treated chickens than in untreated chickens. As for CD8 and CD3 T lymphocytes, no consistent effect of T—2 toxin was noted. At 2 weeks post-treatment (21 days of age), except in trial 3, time percentage of 13 lymphocytes iii T-2 toxin- 119 mmemndsono unmaumonp Hod monco Ho .02 AOO.OWQO QSOHO Honucoo Bone uanoHHHo >HucmonHcon osHm> cmoaa «mo.m H OH.mv Hmw.o H >>.Ov *mm.O H Ow.mv H>>.v H Om.mq + mm O0.0 H mm.Om Om.m H mm.mO Om.m H Om.mm ,N>.v H PO.HO I Hm N «O0.0 H O>.vv Imm.m H VH.nv Hmm.m H O0.0v HOH.m H O0.0v + mH mm.> H nm.wm HO.v H m>.HO Ov.m H O0.00 OH.m H O>.NO I «H H m m conu NIB x HOH x msouw “Sac bcaHmz Heom cams....oz HHHHH meHco an x mHmH .coumc Hmowm m>mc o>Husoomcoo u now conu NIH cqu common» .UHOIHHUIH Ho naaHmz such so conu NIH no Homwum .N.N mHan 120 QDOHO Hcoebmmuu Hoe coHumH>oo pumpcmum cam osHm> cmmsa HmoHdEmm ooHoom Ov monco omnc AOO.OWQV QSOHO Houucoo EOHH HcmHonHp mHucmonHcOHm onHm> cmoEH HON.O H mv.O NH.N H OO.NN HHO.H H nm.m HmH.N H mv.OH + O0.0 H Om.m Hm.H H mm.Hm >0.0 H O0.0 O0.0 H O0.0H I v IHN.O H ON.H Hmm.m H OO.mN O0.0 H mm.O *vN.N H Om.OH + OH.O H «O.m vO.m H m0.0N m>.O H H0.0 Om.H H ON.HH I m HHO.O H mw.O OH.v H VH.ON O0.0 H OO.> nm.m H n0.0H + OH.O H mm.m OO.N H OH.NN OO.H H NH.H OO.H H mm.OH I m moo OOO woo conu NIH .oz HmHHe mou>ooch>HIm mou>ooch>HIe HOV HmcoHumHSQOQQSO ouxooch>H o>Hunommcoo n How conu cqu pmummuu monco $.VHmHmH CH mcoHumHSQOQQSO ou>ooch>HIe cam Im .coumc Houmm mzmp .UH0I>moIO Ho pooHQ HmHocdHHwQ co conu NIB Ho uoowmm .m.N mHQme 121 treated chickens was comparable to that in untreated control chickens (Table 2.4). Similarly, except in trial 3, the percentage of CD4, CD8 and CD3 T lymphocytes in T-2 toxin-treated chickens was comparable to that in untreated control chickens (Table 2.4). Effect of T—2 toxin on B- and T-lymphocytes in spleen. At 9 days of age, the percentage of B lymphocytes in T-2 toxin- treated chickens was significantly lower than iii untreated controls chickens (Table 2.5). mee percentage of CD4 and CD3 T lymphocytes in T—2 toxin-treated chickens was significantly higher than in untreated controls. In trial 3, the percentage of CD8 T-lymphocytes was significantly higher in T-2 toxin-treated chicks than in untreated control chicks. At 2 weeks post-treatment (22 days of age), the percentage of 13 lymphocytes iii T-2 toxin-treated chickens was comparable to timn: of untreated chickens (Table 2.6). Except in trial 2, the percentages of CD4 and CD8 T- lymphocytes in the T-2 toxin-treated chickens were comparable to that in untreated chicks. No consistent effect of T-2 toxin on the percentage of CD3 T lymphocytes was noted. 122 QSOHO ucwEHmoHH Hod AmoHQEmm ooHoom Oi monao OHIc AOO.OWQV gnome Houucoo Eoum HcoHoHHHo >HucmonHcOHm msHm> Emcee m>.O H O0.0 OO.H H OO.vN N>.O H O0.0 OO.H H Ov.mH + ON.O H mm.> OO.¢ H Nv.mm mm.H H em.m Om.H H vo.vH I v HHO.O H ON.O HHO.H H OO.QN HOO.O H Om.> PH.H H OO.NH + Hm.O H NN.O em.H H >>.Nm O>.O H nm.m H0.0 H Ov.NH I m Om.H H OO.m HH.N H OH.mN OO.H H vN.O OO.H H mv.NH + OO.H H >N.O O>.N H O>.qm vw.O H NH.> mm.H H OO.NH I m O0.0 H m0.0 OH.N H O0.0H v>.O H O0.0 Ov.H H mv.OH + OO.N H OH.n HH.v H H0.0N NO.H H O0.0 O>.H H O0.0H I H moo OOO woo conu NIH .oz HmHHe mmo>ooch>HIm mou>oocQE>HIe av mcoHumHSQOQnsm opmoocoE>H .euoma Hanna mHmo o>Husommcoo b How conu cqu ooummuu mcoonco oHoI>moIHm Ho UOOHQ HmnmcdHumd CH mcoHumHSQOQQSm mu>ooch>HIe ocm Im so conu NIB Ho Hommwm .v.N wHQme 123 QSOHO Hcosummuu Hod AmmHQEmm omHOOQ OO monco omnc AOO.OWQO QDOHO Houucoo Eouw ucmeHHHo >HucmonHcOHm wSHm> cmoEI IHO.H H O>.v *mm.H H ON.O> H0.0 H wm.mH HO>.H H ON.HN + vw.v H OO.NN O0.0 H mn.OO OH.N H PN.HH mv.H H Om.mH I v *vm.H H Om.m HOH.v H OO.HH HHN.N H mv.mm HOO.O H O0.0N + mm.v H OO.HO HN.O H mm.Om NN.N H >>.vm NO.H H Om.mm I m IHO.N H H0.0 HOO.N H v0.0> NH.O H OO.HO HOO.H H vo.mm + HN.N H OH.ON HH.N H NO.v> O0.0 H Om.mm O0.0 H vo.mm I N moo OOO woo conu NIB .oz HmHHe mouaooch>HIm mou>oocQE>HIe HOV mcoHumHSQOQQSO ou>ooch>H .coumc Hmuwm m>mo m>Husommcoo w HOH conu cHHz commowu mcmonco UHOIHHUIO Ho somHom CH mcoHpmHSQOQQSm muHUOcQEHHIe cam Im so conu NIH mo Hommwm .m.m oHnme 124 QSOHO ucmEHmoHH Hoe AmoHQSmm pmHOOQ OO mHoHco Oan AmO.Owev QSOHO Houucoo Eouw uconHpr HHucmonHcOHm osHm> smogI mO.N H Om.mm OO.H H mv.mO OH.N H O0.00 Om.O H mO.>H + m0.0 H H0.0N bv.v H nm.mO H0.0 H bO.mm Ov.H H Ov.>H I v OO.H H HN.ON HHO.N H OO.H> OO.N H OH.OO nv.H H Hm.mH + vO.v H >O.mm nO.N H OH.HO mm.m H O>.Om O0.0 H OP.>H I m Om.m H OO.>N O>.v H O0.0> OO.H H mv.Om HHO.H H O0.0H + O0.0 H O0.0N Om.m H OO.>O ON.O H H0.0N OO.H H O0.0H I m ON.N H Om.Om Imn.m H OO.VO HOv.O H nm.mm O0.0 H ON.OH + Ov.m H Om.>m O>.v H HO.H> OH.m H N>.mm H0.0 H ON.OH I H moo OOO woo cHxOH NIH .oz HmHHe mmu>ooch>HIm mmu>oocdS>HIe vamcoHumHSQOQQSO ou>ooch>H .coumc umuum mmmp o>Husommcoo b How conu cqu omummuu mcoonco UHOIHMUINN Ho cmmHQm CH mcoHumHSQOQQSm muhoocQEHHIe one Im so conu NIH Ho uomwmm .O.N mHDme 125 Discussion Data from this study indicate that exposure of day—old chicks to T-2 toxin may significantly reduce the percentage of both peripheral blood and splenic B-lymphocytes but only within 7 (mum; of treatment. The data clearly suggest that repeated sublethal doses of T-2 toxin severely depleted B- lymphocytes, but not CD4, CD8 and CD3 T-lymphocytes. However, at 3 weeks of age (two weeks post-treatment), the gross appearance of the lymphoid organs, and the percentages of B- and T—lymphocytes in peripheral blood and spleen were comparable to that of untreated control chickens. Repeated exposure of farm and laboratory animals to low levels of T-2 toxin has been reported to produce immunosuppression characterized by inhibition of both humoral and cell- mediated immunity (Masuko et al., 1977; Iafarge-Frayssinet et al., 1979; Otokawa et al., 1979; Rosenstein et al., 1979, 1981; Mann et al., 1982, 1983; Taylor et al., 1985). In this study, T-2 toxin significantly reduced the body weight of chicks within 7 days of treatment. This finding is consistent with results of subchronic and chronic exposure of chickens to T-2 toxin reported by Chi et al. (1977); Wyatt et al. (1972, 1973) and Hoerr et al. (1982a,b). The decrease in body weight associated with exposure to T-2 toxin has been attributed to reductions in feed consumption and weight gain. The decreased body weight observed in this 126 study was accompanied by atrophy of the bursa of Fabricius, spleen and thymus. This finding is also consistent with the findings previously reported by Hoerr et al. (1982a). The present study also confirms that the effect of T-2 toxin on lymphoid organs is transient with rapid regeneration and repopulation. of the lymphoid. organs upon termination of treatment of animals with the toxin. Hoerr et al. (1981) found that a single dose of T-2 toxin administered via crop gavage to 7—day-old chicks caused acute lymphoid necrosis and depletion followed.kny rapid repletion III the severely affected lymphoid organs by 3-7 days post-treatment. Results from flow cytometric analysis conducted in the present study revealed that at 1 week post-treatment, depletion of both peripheral blood and splenic B—lymphocytes and aa relative increase iii CD4, CD3, T-lymphocyte subpopulations, suggesting that B-lymphocytes are highly susceptible to the toxic effects of the toxin. In contrast, Smith et al. (1994) reported the percentage of CD45R+ B cells in the spleen of T-2 toxin—treated adult mice was not statistically different from untreated controls. However, a significant decrease in the total number of both B- and T- lymphocytes was noted. The depletion of B—lymphocytes in chickens treated with T-2 toxin is probably due to a direct effect (Hf T-2 toxin (n1 B-lymphocytes. .Alternatively, T-2 toxin may have a cytotoxic effect on the progenitors of B 127 lymphocytes. Holladay et al. (1993) reported that unlike thymocytes, T-lymphocyte progenitors are highly sensitive targets for cytotoxic effects of T—2 toxin and are likely responsible for the thymic atrophy reported in mice. Further, Smith et al. (1994) reported an increase in percentages of double negative cells (CD4'8') and decrease in percentage of double positive (CD43?) cells in the thymus of T-2 toxin—treated mice, suggesting that T-2 toxin may inhibit thymocyte maturation and differentiation. Results from the present study indicate that at 21-22 days of age, the percentages of both peripheral blood and splenic I&- and. T-lymphocytes (if T¥2' toxin-treated. chickens were comparable to that of untreated controls. This finding correlated well with the findings of the gross examination of the lymphoid organs. Analysis of data on both peripheral blood and splenic B-lymphocytes also confirmed that T-2 toxin may be associated with depression of humoral immunity (Jagadeesan et al. 1982). The reason for the increased proportions of CD3 and CD4 T-lymphocytes in the T—2 toxin-treated chickens or the inconsistent effect of the toxin on CD8 T-lymphocytes reported in the present study was not determined. Since the absolute numbers of B- and T-lymphocytes per unit sample was not determined, it is likely that T-2 toxin induced mild to moderate reductions in the T—lymphocyte subpopulations, as 128 suggested by mild to moderate lymphoid depletion of the thymus and T-lymphocyte regions of the spleen. It is worth noting that the percentages of both peripheral blood and splenic B- and T-lymphocytes in untreated chickens were generally lower than those that had previously been reported for normal chickens (Chan et al., 1988; Th1 et al., 1993; Rodenberg' et Ial., 1994). These discrepancies in percentages of lymphocytes in normal chickens may be explained by experimental protocol, age of chicks, treatment as imatl as genetic factors. Hala et an” (1991, 1992) reported differences in percentages of CD4 and CD8 T— lymphocytes in both peripheral blood and spleen of inbred lines of congenic chickens that differ only in the MHC. It is therefore likely that ontogenetic or physiological development of individual chickens may influence the proportions of T-lymphocyte subpopulations. Flow' cytometric analysis of the tflrmmi and. splenic lymphocytes suggests that treatment of chickens with T-2 toxin may result in significant depletion of B-lymphocytes within 7 days after treatment. Such depletion of B- lymphocytes may lead to reductions in the amount of immunoglobulins (IgG, IgM) which may adversely affect the immunologic and defense mechanisms of animals exposed to subclinical levels of T-2 toxin in the feed (Jagadeesan et al., 1982; Mann et al., 1983; Taylor et al., 1985). CONCLUSIONS The results from the research presented in this dissertation indicate that: 1) Treatment. of EUHU; line 1515 X 71 white leghorn chickens with T-2 toxin at sublethal doses of 0.75, 1.0 and 1.25 mg/kg body weight for seven days after hatch may cause a significant. dose—dependent. decrease iii body"weight, as well as in relative weight of lymphoid organs, thymus, spleen, and bursa of Fabricius. However, at 1 enmi 2 weeks after the last treatment, the body weight and the relative weight of lymphoid organs were comparable to that of untreated controls. The decrease in relative weights of lymphoid organs correlated well vniii a dose-dependent mild to moderate or severe lymphoid depletion of the organs. 2) Treatment of chickens with T—2 toxin at a sublethal dose of 1.25 mg/kg. body weight for seven days after hatch may: a) cause a reduction in HVT titres in vaccinated chicks within 7 days after hatch; b) shorten the incubation period for MD mortality in unvaccinated chickens. 3) Using the regimen described in this dissertation, treatment of chicks with T—2 toxin at a dose of 1.25 mg/kg 129 130 body weight for seven days did not influence the incidence of MD lesions/mortality in HVT-vaccinated chickens. 4) Using flow cytometric analysis, T-2 toxin at a sublethal dose of 1.25 mg/kg body weight administered to chicks for seven consecutive days after hatch may: a) cause severe depletion of B—lymphocytes, and b) a relative increase in CD4 and CD3 T- lymphocytes in peripheral blood and spleen at 8-9 days of age. However, at 21-22 days of age, the percentage of B- and T—lymphocytes was comparable to untreated chickens. The findings from flow cytometric analysis correlated with lesions noted in microscopic examination of the lymphoid organs. APPPENDIX APPENDIX Introduction Information regarding the toxicity of multiple doses of T-2 toxin in RPRL line lfflg x 71 chickens is generally not available. The LDw values for single (72—hr) and multiple (14 days) dose of T—2 toxin administered to 7-day-old male broiler chicks have been shown to kme 4.0 anmi 2.90 mg/kg body weight, respectively (Hoerr et al. 1981). Therefore, in order to study the effects of T-2 toxin on vaccinal immunity to Marek’s dissease and on B- and T-lymphocytes in 1-day-old white leghorn chicks, as described in chapters 1 and 2, respectively, ii: was necessary ti) conduct tn“) preliminary trials to determine the lethal and sublethal (maximum tolerated) doses of T-2 toxin in white leghorn chickens treated with multiple doses of the toxin at hatch. Trial 1 The objective of this trial is to determine the lethal dose of T—2 toxin in white leghorn chickens treated with single or multiple doses of the toxin at hatch. Materials and Methods T—2 toxin was obtained in crystalline form from Sigma Chemical Company (St. Louis, MO) and stored at -%MW F until ready for use. F3 progeny of line 1515 males and 71 females 131 132 were used. In Trial 1, a total of 26, day-old chicks were divided into 2 replicates of 13 chicks each. Each replicate was divided into 4 treament groups. In each of groups 1, 2 and 3, three chicks were treated with T-2 toxin at a dose of 0.5, 1.5 and 4.5 mg/kg body weight. Four chicks in group 4 were maintained as untreated controls. Chicks were wing- banded and weighed everyday and the average weight for each group was used to calculate the dosages of T-2 toxin. T-2 toxin-treated and untreated chicks were housed in separate isolators. T-2 toxin stock solution was prepared by adding 2.5 m1 of 100% ethanol to a 25 mg of purified extract of toxin and stored in the freezer until ready for use. Phosphate buffered saline (PBS) was used as a diluent for T-2 toxin stock solution. For each dose group, T-2 toxin was added to the required volume of PBS and administered to day-old chicks via crop gavage. Untreated controls were treated with ethanol in PBS. Results Clinical signs and mortality The severity of the clinical signs exhibited by chicks treated Math T-2 toxin imms dose-related. Chicks treated with T—2 toxin at a dose of 4.5 mg/kg body weight were depressed and. developed.:moderate to severe straw-colored 133 watery' diarrhea. .All chicks died. within 24-48 hrs after treatment (Table 3.1). Chicks treated with T-2 toxin at a dose 1.5 mg/kg body weight had mild to moderate straw- colored diarrhea; 83% of treated chicks died within 48-72 hrs after treatment. Although 33% of chicks treated with T- 2 toxin at a dose of 0.5 mg/kg body weight died within 48-72 hrs after treatment, no clinical signs were noted. No clinical signs were noted in untreated control groups, but 13% of chicks died from unknown reasons. Necropsy findings Chicks treated with T-2 toxin.em:ea dose of 4.5 mg/kg body weight developed moderate to severe subcutaneous edema around the crop and ventral abdomen which extended to the thighs. A large amount of straw-colored fluid with flecks of chalky-white material was present in the body cavities. The liver had severe multifocal to locally extensive subcapsular hematomas which extended in some areas into the parenchyma. Severe acute necrosis of lymphocytes characterized by pyknosis and karyorrhexis with sparing of the cortical lymphocytes within the medulla of the follicles of bursa of Fabricius was noted. Within (the necrotic areas, numerous heterophils infiltrated the interfollicular connective tissue stroma (Figure 3.1). Severe Iacute random. necrosis with moderate heterophilic infiltrate was noted in the cortex of thymus. Mild to moderate acute lymphocyte necrosis 134 .oouoc oumz mCOHmoH oHQoomonoHE Ho mmOHO HomoHHHCOHm oz.T IOOHO e\e m\m m\m m.e Immc ©\m m\m m\m . m.H Immv ©\m m\o .m\m m.o ImHv m\H e\o IH\H Houucoo Lav Hmuoe m mHHUHHamm H momuHHcmm IncsHmz seen ax\aav omom HHHHmoHoE oooochIconu NIH .coumc Hm conu Ho mmmoo mSOHHm> cqu ucmEHmoHu Hmuwm mun NvaN moncofin xaHmH cH HHHHmuHoE ooosocHIcHxOH NIB .H.m oHnme 135 Figure 3.1. Photomicrographs of bursa of Fabricius from 2— day—old untreated control chick (A); and from a chick treated at hatch with T—2 toxin at a dose of 4.5 mg/kg body weight (B). Notice the severe extensive acute lymphoid necrosis within follicles and numerous heterophils within interfollicular connective tissue stroma (H&E X 242). 136 was noted randomly in the spleen. Severe extensive foci of hemorrhage with associated hepatocellular necrosis was noted in liver (Figure 3.2). Chicks treated with T—2 toxin at a dose of 1.5 mg/kg body ‘weight had. mild. to :moderate acute necrosis of the lymphoid organs and liver had. mild to moderate focally extensive hemorrhages. No significant gross or macroscopic lesions were seen in either chicks treated with T-2 toxin at a dose of 0.5 mg/kg body or in untreated control chicks. Trial 2 The objective of this trial was ‘Uo determine the maximum (sublethal) tolerated dose. Materials and Methods Eighty, day-old. chicks were ‘wing-banded. and «divided into 2 replicates of 40 chicks each. Chickens in each replicate were divided into 4 treatment groups, each consisting of 10 chicks. In groups 1, 2 and 3, chicks were treated with T-2 toxin at hatch via crop gavage at a dose of 0.75, 1.00, 1.25 :mg/kg' body ‘weight, respectively, for 7 consecutive days. T-2 toxin dose was based on the average weight of the chicks used in each treatment group. Chicks in group 4 were maintained as untreated controls. At 8, 15 ahd 22 days of age, 3 chicks from each group in replicate 1 were 137 I .1 a"? I y 1 . Figure 3.2. Photomicrographs of liver from 2-day-old untreated control chick (A); and frmn a chick treated at hatch with T—2 toxin at a dose of 4.5 mg/kg body weight (B). Notice the severe locally extensive area of hemorrhage with associated hepatocellar necrosis (H&E X 242). 138 weighed. and sacrificed. The lymphoid. organs were aseptically collected, weighed and placed in 10% neutral buffered formalin to be processed for histopathological evaluation. lit addition, (3 chicks fawn various 'treatment groups in replicate 2 were bled at 8, 15, and 22 days of age for the determination of hematocrit value. For statistical analysis ANOVA and SNK was done to determine the effects of the toxin on body weight, and on lymphoid organ weights using body weight as a covariate. Results Mortality of chicks within 24-48 hrs after treament is shown in Table 3.2. These deaths probably were due to nonspecific causes or accidental drowning from the treatment. At 7 days of age, (the end of the dosing period) there was a significant dose-related decrease in hematocrit, body weight, and the relative weights of the bursa of Fabricius, spleen and thymus (Table 3.3—3.7). However, at 15 amd 22 days of age, body weights, hematocrit and relative weights of lymphoid organ in treated chicks were comparable to those in untreated chicks. Examination of the bursa of Fabricius revealed moderate to severe depletion of lymphocytes within the medulla of the bursal follicles. The bursal follicles were reduced in size and there was an increased interfollicular fibrosis (Figure 3.3a,b). IMild ti) moderate depletion. of lymphocytes Were 139 AOHV ON\N OH\O OH\N mN.H Amy ON\H OH\O OH\H OO.H AmHO ON\m OH\H OH\N mn.O HOV ON\H OH\H OH\O Houucoo HOV Hmuoe m oumoHHQom H mumoHHQmm AucOHoz moon ax\sal omoo >DHHmHHoE omosocHIconH NIH .cobmc Houwm mama o>Husoomcoo H HoH conu Ho momoo msoHHm> csz ucoaumwuu Hmumm mun OvIvm chqu maano.e x.HmH 2H HLHHmnHos amusecHIconu NIH .N.m mHan 140 .CoHumH>mo pumpcmum pcm mcmozm .Hmo.ovmv HcmHmHHHn HHHcmoHHHcaHm mum HmHHmH HQHHomeQSm ucmHmHHHp >Q ooonHow QESHoo m chqu mmsHm> mamoza hm.v H O0.0N OO.H H OO.Hm chm.H H O0.0N O ON.H OO.m H Om.Nm Hm.H H OO.Hm ohm.H H O0.0N O OO.H OO.NH H mm.ON OO.H H O0.0m mOO.H H mO.Hm O m>.O OO.H H O0.0N OO.H H O0.0N mmv.H H M0.0m O Houucoo AbnaHmz Heon msme mm mHme mH msma O z aH\asc omoo EpHuooumEom II monco Ho mom .coumc Houwm m>mp o>Husommcoo H H0O conu NIH Ho momop msoHHm> cHHz ucmfipmonu Hmuwm mmom mnoHHm> um moncoHn meOH HO mwsHm> DHHooumEom .m.m mHnme 141 GOHHmH>mo Unmocmum cam mcmozm Hmo.ovmv HcmHmHHHn HHHcmoHHHaon mum HoHHmH HQHHomHmQSm HQonHHHp >9 omonHow GESHoo m chqu mosHm> mammza m>.ON H mO.mOH hv.O H M0.0NH mmm.> H Om.mm m ON.H O0.0 H OH.NOH OO.HH H M0.0NH .fimm.O H hv.mm m OO.H O0.0H H OO.HOH NO.HH H OM.ONH 8m>.N H Ov.vO m O0.0 OH.ON H mH.NOH ON.Om H ON.OHH umO.m H OO.VO m Houucou HHeonz NHeon mama Hm mama HH mHme H z sH\an omoo eHHsHmz seem II mHuHao Ho mam .aonmn Houwm m>mo o>Husommcoo b How conu NIB Ho mmmop mSoHHm> cqu ucofiumwnu Houwm mmmm msoHHm> Hm moncoHn meOH HO HcOHmB >oom .v.m mHnme 142 .QOHumH>mp pumocmum pcm HucOHmz Hpon How pmumsmpmv mammzm Hmo.ovmv HcmHmHHHe HHHamoHHHaon mum HmHHmH HQHHomHmQSm uconHHHo HQ ooonHom casHoo m chqu mdem> cmmza HN.O H O0.0 HH.O H O0.0 QvoO H HN.O m OO.H O0.0 H OH.H OH.O H O0.0 HOO.O H ON.O m OO.H mm.O H HH.H VH.O H m>.O mm0.0 H ON.O m O>.O ON.O H OO.H O0.0 H OH.O UOO.O H mv.O m Houucou HHaaHmz seen msme Hm mHmu HH mama e z sx\oav whoa mmusm Ho fichHmz o>HumHmm II monco Ho mod .copmc Houwm mace m>HHsoomcoo b How conu NIH Ho momoc msoHHm> cqu unmaummuu Houwm mmom msoHHm> um moncoHn memH Ho msHoHHomm Ho mmHsn Ho HcOHmz m>HHmHom .m.m mHnme 143 .coHumH>mp oumocmum pcm HucOHmz Hpon How pmumnnpmv mammzm Hmo.ovmv HcmHmHHHa HHHcmoHHHcaHm mum HmuuoH HQHHomHmQSm ucmeHHHo >n omZOHHom CEdHoo m chqu mosHm> cmmza O0.0 H Om.O N0.0 H ON.O “ENO.O H OH.O m OO.H O0.0 H OH.O V0.0 H N0.0 .%H0.0 H HH.O m OO.H O0.0 H OH.O O0.0 H O0.0 mH0.0 H OH.O m OH.O OH.O H vv.O O0.0 H mm.O HUNO.O H NN.O m . Houucoo HHeaHmz Heon is. Hm £8 2 was H z 9:95 omoo cooHQm HomaucOHmz o>HumHom II monco Ho oOm .coumc Hmuwm mHmo m>Husoomcoo b How conu NIH Ho momoo msoHHm> cqu bcmaummuu Hmuwm meow mnoHHm> um moncoHn xHHmH Ho cmoHdm Ho ucOHoz o>HHmHmm .O.O mHnme 144 .coHumH>op pumpcmum cam HucOHmz >pon How omumSOUmO mummzm Hmo.ovmv HcmHmHHHe HHHcmoHHHcaHm mum mumuumH HQHHomHoQSm ucmHmHHHo me omonHoH QESHoo m chqu mosHm> cmoza HH.O H V0.0 AnmNO.O H H0.0 nO0.0 H HN.O m ON.H «H.O H V0.0 an.O H O0.0 an0.0 H mm.O m OO.H OH.O H N>.O .fimo.O H mm.O mm0.0 H ON.O m OH.O HH.O H mm.O mm0.0 H O0.0 omo.O H mv.O m Honucou HHHonz NHeon m>mp Hm m>mo VH m>mo n z Ox\OEO omoo HHeonc msszae Ho enesHmz m>HHmHmm II mHanu Ho mam .coumc Hmuwm m>mp m>Husoomcoo b How conu NIH Ho momoc msoHHm> cHHz Hcoaumwuu Houwm moem mooHHm> um moncon meOH HO HucOHHV OSEch Ho ucOHmz m>HumHmm .n.m mHnme Figure 3.3. Photomicrographs of bursa of Fabricius from 8- day—old untreated control chick (A); and from a chick treated with T—2 toxin at a dose of 1.25 mg/kg body weight for 7 consecutive days after hatch (B). Notice the extensive depletion of lymphoid cells within follicles and increased interfollicular connective tissue (H&E X 242). 146 noted within the cortex of the thymus (Figure 3.4a,b) and white pulp of the spleen (Figure 3.5a,b). The cellularity of the bone marrow from chicks treated with T-2 toxin at a dose of 1.25 mg/kg body weight was comparable to that of untreated controls (Figure 3.6a,b). Discussion Data from these trials indicate treatment of day-old white leghorn chicks with T-2 toxin at a dose of 1.5 and 4.5 mg/kg body weight may cause up to 100% mortality within 72 hrs after treatment. In contrast, the tolerated doses of 0.75 and 1.25 mg/kg body weight reduced both body weight and relative weights of the lymphoid organs, but only at 8 days of age. This is consistent with the findings by Wyatt et al. (1973), Boonchuvit et al. (1975). The data also suggest that of all lymphoid organs examined histologically, the bursa of Fabricius was the most severely affected. This finding is consistent with studies by Hoerr et al., 1981, 1982a,b. 'Analysis of data obtained from examination of bone marrow suggests that the hematopoeitic cells of the bone marrow were not affected by the sublethal dose of T-2 toxin, compared with the negative effects of the toxin on the lymphoid organs (Chi et al., 1977a,b). While specific identification of the heterogeneous population of bone marrow cells was not done, the data suggest that these cells 147 Figure 3.4. Photomicrographs of thymus from 8-day-old untreated control chick (A); and from a chick treated with T—2 toxin at a dose of 1.25 mg/kg body weight for 7 consecutive days after hatch (B). Notice the moderate depletion of cortical lymphocytes (H&E X 242). Figure 3.5. Photomicrographs of spleen from 8-day-old untreated control chick (A); and from a chick treated with T-2 toxin at a dose of 1.25 mg/kg body weight for 7 consecutive days after hatch (B). Notice moderate depletion of lymphocytes with reticoloendothelial cell hyperplasia (H&E X 242). 149 Figure 3.6. Photomicrographs of bone marrow from 8-day-old untreated control chick (A); and from a chick treated with T—2 toxin at a dose of 1.25 mg/kg body weight for 7 consecutive days after hatch (B). Note absence of loss of hematopoeitic cells and the similar cellularity compared to control chick (H&E X 242) 150 may allow the lymphoid organs to be repopulated within one week after termination of treatment with T-2 toxin. In addition, it appears that the sublethal dose of 1.25 mg/kg body weight used in this study was immunotoxic but probably not myelotoxic in chickens. Treatment with T-2 toxin at a dose of 1.25 mg/kg body weight did not affect the liver and kidney' when examined. histologically suggesting that this dose vdmrit is clearly immunotoxic, probably (fiti not have other subclinical effects. These adverse effects of T-2 toxin have been shown to be associated vniii the inhibitory effects (if T-2 toxin CH1 protein synthesis (Ueno et al., 1968; Bamburg, 1974; Cundliffe and Davis, 1977). On the basis of the findings from these two trials, a maximum tolerated dose of 1.25 mg/kg body weight was selected for the studies described in chapters 1 and 2. LIST OF REFERENCES REFERENCES Adldinger, H. K. and Calnek, B. W. (1973). Pathogenesis of Marek’s disease: Early distribution of virus and viral antigens in infected chickens. J. Nat. Cancer Inst. 50:1287-1298. Ahmed, N. and Ram, G. C. (1988). Nuclear lipid peroxidation induced in rat liver by T—2 toxin. Toxicon. 24:947-949. ApSimon, J. W., Blackwell, B., Grenhalgh, R., Meier, R. M., Miller, D., Pare, J. R. J. and Taylor, A. (1986). Secondary metabolites produced by some Fusarium species. In: Bioactive Molecules, IN S. Steyns and It. Vleggar (Eds.). Elsevier, Amsterdam, pp. 125-137. Bamburg, J. R. (1972). Biological activity and detection of naturally 12, 13-epoxy-9-trichothecenes. Clin. Toxicol. 5:495-515. Bamburg, J. R. (1974). Chemical and biochemical studies of the trichothecene mycotoxins. Adv. Chem. 149:144-162. Bamburg, J. R. (1983). BiologiCal and biochemical actions of trichothecene mycotoxins. Progress in Molecular and Subcellular Biology 8:42-110. Bamburg, J. ft. and. Strong, F. TL. (1971). 12,13-Epoxy- trichothecenes. In: Microbial Toxins, A comprehensive treatise, algal and fungal toxin. Kadis, S., Ciegler, A., Ajl S. J. (eds.), Vol. 7, Academic Press, New York. pp. 207- 292. Bamburg, J. R., Riggs, N. V. and Strong, F. M. (1968). The structures of toxins from. two strains of Fusarium tricinctum. Tetrahedron 24:3329-3336. Batra, P., Pruthi. A. K. and Sadana, J. R. (1991). Effect of aflatoxin B1