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K3 2:5}! .60 ‘A\\\ THESIS f) .9 3 Cd) Iliili'lillilillllililililillliflill 3 1293 02048 4063 This is to certify that the dissertation entitled PATHOGENESIS OF THE BOVINE VIRAL DIARRHEA VIRUS-INDUCED HEMORRHAGIC DISORDER presented by Paul Harold Walz has been accepted towards fulfillment of the requirements for Ph . D . degree in large Animal Clinical Sciences c. fic/uq Major professor Date 31/ ”S {/0 a MS U is an Affirmative Action/Equal Opportunity Institution 0-12771 LIBRARY Michigan State University PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 11m W,“ PATHOGENESIS O] l" I nl PATHOGENESIS OF THE BOVINE VIRAL DIARRHEA VIRUS-INDUCED HEMORRHAGIC DISORDER By Paul Harold Walz A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Large Animal Clinical Sciences 2000 PATHOGENESIS 0F TI I lip: 11 bovine viral diarr mind a hemorrhagic 51 death The mechanism b) inenhagic smdrome is in BIDV' may be an important mined m'th BVDV infet mechanism of BVDV'~indu ten of spontaneous hem< Whose platelet count was ngeting that altered plat The focus of our first ex Emil of BVDV-induced 'I‘IéIOpment of hemorrha With 10? TCIDV) of ABSTRACT PATHOGENESIS OF THE BOVINE VIRAL DIARRHEA VIRUS-INDUCED HEMORRHAGIC DISORDER By Paul Harold Walz Type II bovine viral diarrhea virus (BVDV) has been associated with severe clinical disease and a hemorrhagic syndrome characterized by thrombocytopenia, bleeding, and death. The mechanism by which type H BVDV causes severe disease and the hemorrhagic syndrome is unknown, but the induction of thrombocytopenia by type H BVDV may be an important factor. Previous experimental studies on thrombocytopenia associated with BVDV infection have been descriptive, but difficult to reproduce, and the mechanism of BVDV-induced thrombocytopenia remains unknown. Moreover, clinical signs of spontaneous hemorrhage have been observed in type H BVDV-infected cattle whose platelet count was not depressed enough to fully account for the hemorrhage, suggesting that altered platelet function may additionally be involved in the pathogenesis. The focus of our first experimental study was to address the need for a reproducible model of BVDV-induced thrombocytopenia and to investigate the discrepancy between development of hemorrhage and platelet count. Calves (n=5) were inoculated on day 3 of age with 107 TCIDso of BVDV 890 (type 11), while sham-inoculated calves served as controls (n=4). Blood cell counts and virus isolation from blood components were performed daily and platelet aggregation tests were performed every other day throughout the study. Calves were euthanatized on day 12 after inoculation, and pathologic, virologic, and irnmunohistochemical examinations were performed. The glaciet counts decreased ar all-es. Bone marrow exam ad etidence of concurren aimed thrombocytopeni innion may eontributet lhe hemorrhagic 5} associated vn'th t}pe ll l to compare platelet hr m-hnombocympem, Ent‘edion. Control c; £34. for each grOUp BVDV 7937 “We l were “Edi-Bled pfi EYUlaIIOn, LeVe manage 35306“ it . mm“ Was onl' platelet counts decreased and platelet ftmction was depressed in type H BVDV—infected calves. Bone marrow examination from infected calves revealed megakaryocyte infection and evidence of concurrent megakaryocyte necrosis and hyperplasia. A model of BVDV- induced thrombocytopenia was characterized, and it was concluded that altered platelet ftmction may contribute to clinical hemorrhage in cattle infected with type II BVDV. The hemorrhagic syndrome and outbreaks of severe disease have been most often associated with type II BVDV, and not type I BVDV. The focus of our second study was to compare platelet function and levels of viremia between a type I BVDV infection, a non-thrombocytopenic type H BVDV infection, and thrombocytopenic type II BVDV infection. Control calves (n=4) were sham inoculated, while calves in infected groups (n=4 for each group) were inoculated with 107 TCIDso of either BVDV 890 (type II), BVDV 7937 (type H), or BVDV TGAN (type I). Platelet function and levels of viremia were evaluated prior to inoculation (day 0) and on days 4, 6, 8, 10, and 12 after inoculation. Level of viremia was determined using the serum viral titers and the percentage association of BVDV with lymphocytes and platelets. Depressed platelet fimction was only observed in type II BVDV-infected calves. A parallel relationship between level of viremia and rectal temperatures, and an inverse relationship between level of viremia and blood cell counts was observed; that is, the greatest elevations in rectal temperature and greatest depressions in blood counts were observed in BVDV 890- infected calves. Isolates of BVDV that induce higher levels of viremia may be more capable of inducing disease. Severe BVDV infections and the hemorrhagic syndrome may occur during infection with isolates of BVDV capable of inducing high levels of viremia. Ct fight by . ’ Pill Huron) Vt L, at Copyright by PAUL HAROLD WALZ 2000 ll gl\'es m. “Old and and encour PerseVerar It gives me great pleasure to dedicate this dissertation to my parents, Lloyd and Marion Walz. In addition to their unfailing love, support, and encouragement, they have instilled in me the virtues of hard work, perseverance, loyalty, and faith. To them, I shall be forever grateful. [would like to extent antlers Drs. John C. B Roger K. Macs for their g m and heartfelt gratit h: gndance. friendship. 2 daemon. lwould also like to II Harold Tt'edten for provi EMF“ lam eSpecia P335 mgarding platelet : Dau- Taylor. Lori 1(ch Dime “mm f0 m5 PIOject. Binding for this pr ACKNOWLEDGMENTS I would like to extend my sincere thanks and gratitude to my graduate committee members, Drs. John C. Baker, Thomas G. Bell, Daniel L. Grooms, Lana Kaiser, and Roger K. Macs for their guidance and assistance. I would especially like to extend my sincere and heartfelt gratitude to Dr. John C. Baker, my mentor and major professor, for his guidance, friendship, and assistance above and beyond the work entailed within this dissertation. I would also like to thank Drs. Barbara Steficek, Janvier Kreihbel, David Bezek, and Harold Tvedten for providing valuable helpful suggestions at various times throughout the project. I am especially grateful to Dr. Steficek who helped and taught me the finer points regarding platelet aggregometry. In addition, I would like to thank Cheri Benson, Dan Taylor, Lori Kunze, Cunquin Han in the Virology section of the Animal Health Diagnostic Laboratory for their assistance in my training in the laboratory methods used in this project. thding for this project was provided by grants through the Michigan Animal Experiment Station, 1433 Animal Health Funds, the American Association of Bovine Practitioners Research Assistantship, the Department of Large Animal Clinical Sciences Graduate Student Grant, and the College of Veterinary Medicine post-DVM Fellowship. vi IN OF TABLES ..... HST OF FIGLRES NRODL’CUON ENTER 1: RE\" IE Mine Viral 1713' The History 0 7718 Clinical . Severe. P6! [We [1 BOY” BOVlne Viral D1; The Bovine Plat Platelet Disorde lilatire I a’III-Ianyf Hog ChOIQr‘ AfTican 311‘ (- Hwnan in?!) ”"9 infe 9)?qu \‘Ir TABLE OF CONTENTS LIST OF TABLES ............................................................................................................. xi LIST OF FIGURES ......................................................................................................... xiii INTRODUCTION ............................................................................................................... 1 CHAPTER I: REVIEW OF THE LITERATURE .............................................................. 4 Bovine Viral Diarrhea Virus .......................................................................................... 5 The History of Bovine Viral Diarrhea Virus ............................................................ 5 The Clinical Manifestations of Bovine Viral Diarrhea Virus: Severe, Peracute Bovine Viral Diarrhea .............................................................. 7 Type II Bovine Viral Diarrhea Virus ....................................................................... 9 Bovine Viral Diarrhea Virus and the Hemorrhagic Syndrome .................................... 13 The Bovine Platelet ...................................................................................................... 24 Platelet Disorders of Cattle .......................................................................................... 3O Qualitative platelet disorders of cattle .................................................................. 31 Quantitative platelet disorders of cattle ................................................................ 36 Some Examples of Virus-induced Thrombocytopenia in Other Species ..................... 4O Hog cholera virus .................................................................................................. 41 African swine fever virus ....................................................................................... 43 Human immunodeficiency virus ............................................................................. 44 Equine infectious anemia virus .............................................................................. 46 Dengue virus .......................................................................................................... 48 References .................................................................................................................... 50 CHAPTER H: JOURNAL ARTICLE: EXPERIMENTAL MODEL OF TYPE 11 BOVINE VIRAL DIARRHEA VIRUS-INDUCED THROMBOCYTOPENIA ................................................................................................. 65 Abstract ........................................................................................................................ 67 Introduction .................................................................................................................. 68 vii Materials and Met? Animals .......... Virus and chal Clinical patho Isolation and l and electror Pathologic en Virologic anti lmmunohisiog detection qr Statistical am- RfiMB ................ Physical exan Clinical pat/2t Transmission Virologic ant; Pathologicfit lmmunohisto. Discussion ......... Sources and Mar .I. O ........ 0.. I . a a .‘ Materials and Methods ................................................................................................. 70 Animals .................................................................................................................. 70 Virus and challenge protocol ................................................................................. 71 Clinical pathologic examination ............................................................................ 71 Isolation and purification of platelets for virus isolation and electron microscopy .................................................................................... 72 Pathologic examination ......................................................................................... 73 Virologic and serologic examination ..................................................................... 74 Immunohistochemical and immunofluorescent antibody detection of B VD V antigen ................................................................................. 75 Statistical analysis ................................................................................................. 76 Results .......................................................................................................................... 76 Physical examination findings ............................................................................... 76 Clinical pathologic findings ................................................................................... 76 Transmission electron microscopic findings ......................................................... 77 Virologic and serologicfindings ............................................................................ 78 Pathologic findings ................................................................................................ 78 Immunohistochemistry and immunofluorescent antibody testing .......................... 80 Discussion .................................................................................................................... 90 Sources and Manufacturers .......................................................................................... 97 Acknowledgments ........................................................................................................ 98 References .................................................................................................................... 98 CHAPTER III: JOURNAL ARTICLE: EFFECT OF EXPERIMENTALLY INDUCED TYPE 11 BOVINE VIRAL DIARRHEA VIRUS INFECTION ON PLATELET FUNCTION IN CALVES .......................................................................................................................... 102 Abstract ...................................................................................................................... 104 Introduction ................................................................................................................ 105 Materials and Methods ............................................................................................... 107 Animals ................................................................................................................ 107 Virus infection protocol ....................................................................................... 108 Collection of platelets .......................................................................................... 108 Platelet aggregation responses ............................................................................ 109 Isolation of B VD V fiom platelets ......................................................................... l 10 Statistical analysis ............................................................................................... l 10 viii Aggregation respom. Aggregation respomt Isolation ofBl 'Dl '_t.r. Discussion..................... Sauces and Manufacture References................... . CHIPIER IV: JOURNAL .' RESPONSES 1WD VIRL'S 1 CAMS EXPERIMENT A L ROWE VIRAL DIARRHE Ahsttact Introduction.............. Materials and Methods Animals ltrus and infection pt Collection of platelet Platelet aggregation . ltrus isolation from ) Statistical analysis . Resdts......... Results ........................................................................................................................ 1 l l Aggregation responses with ADP ........................................................................ 111 Aggregation responses with PAF ......................................................................... 112 Isolation of B VD V fiom platelets ......................................................................... 112 Discussion .................................................................................................................. 129 Sources and Manufacturers ........................................................................................ 132 References .................................................................................................................. 133 CHAPTER IV: JOURNAL ARTICLE: PLATELET AGGREGATION RESPONSES AND VIRUS ISOLATION FROM PLATLETS IN CALVES EXPERIMENTALLY INFECTED WITH TYPE I OR TYPE II BOVINE VIRAL DIARRHEA VIRUS ........................................................................... 136 Abstract ...................................................................................................................... 138 Introduction ................................................................................................................ 139 Materials and Methods ............................................................................................... 140 Animals ................................................................................................................ 140 Virus and infection protocol ................................................................................ 141 Collection of platelets .......................................................................................... 142 Platelet aggregation responses ............................................................................ 142 Virus isolation fiom platelets ............................................................................... 143 Statistical analysis ............................................................................................... 144 Results ........................................................................................................................ 144 Aggregation responses with adenosine diphosphate (A DP) ................................ 144 Aggregation responses with platelet activating factor (PAF) ............................. 146 Isolation of B VD V from platelets ......................................................................... 147 Discussion .................................................................................................................. 161 References .................................................................................................................. 164 CHAPTER V: JOURNAL ARTICLE: RELATIONSHIP BETWEEN LEVEL OF VIREMIA AND DISEASE IN CALVES EXPERIMENTALLY INFECTED WITH BOVINE VIRAL DIARRHEA VIRUS ........................................................................................................ 167 Abstract ...................................................................................................................... 169 ix lntroduction................... Materials and Methods. Animals ................... Viruses and infectim Clinical pathologic I Determination Oflllt l'irologic and serolt Pathologic examinu lmmunolzistochcnztc Statistical anal 1 st; 5 _ Plit‘sical examinatit Clinical pathologic levels of viremia . .. l'irologic and semi Pathologicfinding' lmmunohistochemi Discussion .................. Sources and Manufact Acknowledgments ...... References ...... C“PIERVI: scram References......, Introduction ................................................................................................................ 1 70 Materials and Methods ............................................................................................... 171 Animals ................................................................................................................ 171 Viruses and infection protocol ............................................................................. 172 Clinical pathologic examination .......................................................................... 173 Determination of the levels of viremia ................................................................. 173 Virologic and serologic examination ................................................................... 175 Pathologic examination ....................................................................................... 177 Immunohistochemical examination for BVDV antigen ........................................ 177 Statistical analyses ............................................................................................... 1 78 Results ........................................................................................................................ 179 Physical examination findings ............................................................................. 179 Clinical pathologicfindings ................................................................................. 179 Levels of viremia .................................................................................................. 180 Virologic and serologicfindings .......................................................................... 181 Pathologicfindings .............................................................................................. 182 Immunohistochemistryfindings ........................................................................... 183 Discussion .................................................................................................................. 201 Sources and Manufacturers ........................................................................................ 206 Acknowledgments ...................................................................................................... 207 References .................................................................................................................. 207 CHAPTER VI: SUMMARY AND CONCLUSIONS .................................................... 211 References .................................................................................................................. 217 LIST OF REFERENCES ................................................................................................. 219 M ll Methods for oping, Qualitative platelet Quantitative plarclc Summary ofcliniea 4 uninfected contro Summary of virus i samples for 5 calve Myeloidzemhroid 5 power field for 5 B‘ Virus isolation resu experimentally infe my; 1.1 1.2 1.3 2.1 2.2 2.3 4.1 5.1 5.2 5.3 5.4 LIST OF TABLES Page Methods for typing isolates of bovine viral diarrhea virus ................................. 12 Qualitative platelet disorders of cattle ................................................................ 35 Quantitative platelet disorders of cattle .............................................................. 39 Summary of clinical pathologic findings for 5 BVDV-infected and 4 1minfected control calves before infection and on day 12 afier infection ........ 83 Summary of virus isolation results for BVDV on antemortem samples for 5 calves infected with type H BVDV .............................................. 84 Myeloidzerythroid series ratio and numbers of megakaryocytes per high power field for 5 BVDV-infected and 4 uninfected control calves .................... 85 Virus isolation results for purified platelet samples from calves experimentally infected with type I or type H BVDV ...................................... 160 Manual and automated hematology results for control and BVDV-infected calves before inoculation (day 0) and on day 12 afier inoculation ................... 191 Serum viral titer (level of viremia) results for uninfected control calves, calves infected with the type I isolate BVDV TGAN, calves infected with the type H isolate BVDV 793 7, and calves infected with the type H isolate BVDV 890 ..................................................... 192 Percentage of lymphocytes associated with BVDV (level of viremia) for uninfected control calves, calves infected with the type I isolate BVDV TGAN, calves infected with the type II isolate BVDV 7937, and calves infected with the type H isolate BVDV 890 ............. 193 Percentage of platelets associated with BVDV (level of viremia) for uninfected control calves, calves infected with the type I isolate BVDV TGAN, calves infected with the type H isolate BVDV 7937, and calves infected with the type H isolate BVDV 890 ............. 194 xi no; ii Summary of virus 1 type I BVDV TGA and calves infected Summary of immu calves. calves infec with type II BVDV Table 5.5 5.6 LIST OF TABLES (continued) Page Summary of virus isolation results for calves infected with type I BVDV TGAN, calves infected with type H BVDV 7937, and calves infected with type H BVDV 890 ..................................................... 195 Summary of immunohistochemistry results for uninfected control calves, calves infected with type I BVDV TGAN, calves infected with type H BVDV 7937, and calves infected with type H BVDV 890 ........... 200 xii l? 3.4 u.’ l in Scheme of mega Mean whole blot in control and 8‘ Transmission e1: and an infected ( Histologic sectit after infection (I Aggregometer t' stimulated with Platelet aggrega B: Slope of the diphosphate at : and BVDV-1111‘s Aggregometer ' Stimulated Witt 1.1 2.1 2.2 2.3 3.1 3.2 3.3 3.4 3.5 3.6 LIST OF FIGURES Page Scheme of megakaryocytopoiesis ....................................................................... 26 Mean whole blood platelet counts by manual determination in control and BVDV-infected calves ................................................................. 81 Transmission electron micrograph of platelets from a control (left) and an infected (right) calf at day 12 after infection ........................................... 86 I-Iistologic sections of bone marrow taken on day 12 after infection (postmortem examination) .......................................................... 88 Aggregometer tracings of bovine platelet-rich plasma suspensions stimulated with adenosine diphosphate at a concentration of 10 11M ............... 1 13 Platelet aggregation responses (A: Maximum percent aggregation; B: Slope of the aggregation curve) induced by adenosine diphosphate at a concentration of 1011M for control (n=4) and BVDV-infected (n=5) calves during the experimental time period ........... 115 Aggregometer tracings of bovine platelet-rich plasma suspensions stimulated with adenosine diphosphate at a concentration of 100 M ............. 117 Platelet aggregation responses (A: Maximum percent aggregation; B: Slope of the aggregation curve) induced by adenosine diphosphate at a concentration of 100 11M for control (n=4) and BVDV-infected (n=5) calves during the experimental time period ........... 119 Aggregometer tracings of bovine platelet-rich plasma suspensions stimulated with platelet activating factor at a concentration of 0.1 M ........... 121 Platelet aggregation responses (A: Maximum percent aggregation; B: Slope of the aggregation curve) induced by platelet activating factor at a concentration of 0.1M for control (n=4) and BVDV-infected (n=5) calves during the experimental time period .................. 123 xiii Aaaregometer trac stimulated with pls Platelet aggregatioi B: Slope of the ag factor at a concent: BVDV-infected (n Platelet aggregatir B: Slope of the a; diphosphate at a c Platelet aggregati B: Slope of the a diphosphate at a Aggregometer tr Stimulated with Platelet aggrege B: Slope of the activating factc Ag“ gomeler stimulated “cu . lope of \‘ Rhyming faC P'Iean feQu“ . "es in BVFDV feet 793‘ 4.1 4.2 4.3 4.4 4.5 4.6 5.1 5.2 LIST OF FIGURES (continued) Page Aggregometer tracings of bovine platelet-rich plasma suspensions stimulated with platelet activating factor at a concentration of 1.0 uM ........... 125 Platelet aggregation responses (A: Maximum percent aggregation; B: Slope of the aggregation curve) induced by platelet activating factor at a concentration of 1.0uM for control (n=4) and BVDV-infected (n=5) calves during the experimental time period .................. 127 Platelet aggregation responses (A: Maximum percent aggregation; B: Slope of the aggregation curve) induced by adenosine diphosphate at a concentration of 1011M ........................................................... 148 Platelet aggregation responses (A: Maximum percent aggregation; B: Slope of the aggregation curve) induced by adenosine diphosphate at a concentration of 100 M ......................................................... 150 Aggregometer tracings of bovine platelet-rich plasma suspensions stimulated with adenosine diphosphate at a concentration of 100 11M ............. 152 Platelet aggregation responses (A: Maximum percentage aggregation; B: Slope of the aggregation curve) induced by platelet activating factor at a concentration of 0.1uM ................................................... 154 Aggregometer tracings of bovine platelet-rich plasma suspensions stimulated with platelet activating factor at a concentration of 0. 1 uM ............ 156 Platelet aggregation responses (A: Maximum percentage aggregation; B: Slope of the aggregation curve) induced by platelet activating factor at a concentration of 1.0 uM ................................................... 158 Mean rectal temperature measurements for uninfected control calves, calves infected with type I BVDV TGAN, calves infected with type H BVDV 7937, and calves infected with type H BVDV 890 ............................... 185 Mean whole blood platelet counts by manual determination for uninfected control calves, calves infected with type I BVDV TGAN, calves infected with type H BVDV 7937, and calves infected with type H BVDV 890 ........... 187 xiv Mean platelet volu infected with type BVDV 7937. and Histologic sections infection tpostmo Histologic sections infection tpostmor 5.4 5.5 LIST OF FIGURES (continued) Page Mean platelet volume for uninfected control calves, calves infected with type I BVDV TGAN, calves infected with type II BVDV 7937, and calves infected with type H BVDV 890 ............................... 189 Histologic sections of bone marrow taken on day 12 after infection (postmortem examination) ................................................................. 196 Histologic sections of thymus taken on day 12 after infection (postmortem examination) ................................................................. 198 XV the disease of cattle 1 described in 1946 (Childs relintlantly named bovine litter et al.. 1954). Th: :lirical and pathologic f1 causal disease. immun Elm l5 years. there biology of this virus. Tl E1935; Brovmlie et t EC been sequenced ( ‘39:). In spite of the a INTRODUCTION The disease of cattle that would become known as bovine viral diarrhea was first described in 1946 (Childs, 1946; Olafson et al., 1946). The virus, which would be redundantly named bovine viral diarrhea virus (BVDV), was discovered seven years later (Baker et al., 1954). The 50 years following the first report brought into view other clinical and pathologic features associated with the virus such as persistent infection, mucosal disease, immunosuppression, and reproductive inefficiency (Baker, 1995). In the past 15 years, there has been a vast increase in the knowledge of the molecular biology of this virus. The pathogenesis of mucosal disease has been determined (Bolin et al., 1985; Brownlie et al., 1984), and the complete genomes for several BVDV isolates have been sequenced (Colett et al., 1988; De Moerlooze et al., 1993; Deng & Brock, 1992). In spite of the advances that have been made in our understanding of this complex virus and its associated diseases, BVDV remains an important pathogen of cattle worldwide and control has not been achieved. Economic losses created by infection with BVDV continue to be substantial. Outbreaks of severe clinical disease associated with BVDV infection in cattle immmocompetcnt to BVDV have been reported within the last decade (Carman et a1 ., 1998; David et al., 1994; Drake et al., 1994), and this has reinforced the importance of BVDV as a pathogen. These outbreaks were characterized by high morbidity and mortality rates (Carman et al., 1998; David et al., 1994; Drake et al., 1994). Affected emit developed fever an Dale et al., 1994). Lab 011 some farms, affectec mm at al., 1998). l niections with BVDV re 1995). The BVDV isolatc oiBl‘DV was identified ( A hemorrhagic syndr infection in adult cattle mealed thrombocytopen 1989). Thrombocytopeni; associated with infection “El BVDV infection has iRjdpath, 1993; Corapi 1 ttttious exPerimental stu itnonhagic Syndrome‘ tl fur exatnple, Prefioug ex imitation of the in fectic {Back et al., 1994, C0 ,. r. suites failed to cattle developed fever and diarrhea, and pregnant cattle aborted (Carman et al., 1998; Drake et al., 1994). Laboratory evaluation revealed leukopenia and thrombocytopenia. On some farms, affected cattle died without demonstrating clinical signs of disease (Carman et al., 1998). Prior to these outbreaks, it was generally believed that acute infections with BVDV resulted in mild disease, without considerable mortality (Baker, 1995). The BVDV isolates from these outbreaks were analyzed, and the type H genotype of BVDV was identified (Pellerin et al., 1994; Ridpath et al., 1994). A hemorrhagic syndrome has also been recently reported in association with BVDV infection in adult cattle and calves (Rebhun et al., 1989). Hematologic evaluation revealed thrombocytopenia as the cause of the hemorrhagic syndrome (Rebhun et al., 1989). Thrombocytopenia and the resulting hemorrhagic syndrome have been most often associated with infection of cattle with type H BVDV. Thrombocytopenia associated with BVDV infection has been reported under natural and experimental conditions (Bolin & Ridpath, 1992; Corapi et al., 1990; Corapi et al., 1989; Rebhun et al., 1989). Although previous experimental studies evaluating type H BVDV infection have characterized the hemorrhagic syndrome, they have been descriptive in nature and difficult to reproduce. For example, previous experimental studies used infection protocols that did not allow quantitation of the infectious dose of virus, making reproduction of their study impossible (Bezek et al., 1994; Corapi et al., 1990; Corapi et al., 1989). Furthermore, several studies failed to use controls or failed to use age matched controls (Bolin & Ridpath, 1992; Corapi et al., 1990; Corapi et al., 1989). Control calves are critical in the evaluation of thrombocytopenia because of the variation in the normal platelet count (Nelson et al., 1974; Semrad & Dubielzig, 1993); and age-matching is important as the platelet count varies betvve milking, 1993; Vaugher BlDV isolates that failed 1 tCurapi et al., 1989'. Ellis e: lhe emergence of type at hemorrhage has creat innunocompetent cattle. . innmocompetent cattle LLJOIEJOC)Iopettiat inducti dissertation is to provide he pathophysiology of the platelet count varies between calves of different ages (Nelson et al., 1974; Semrad & Dubielzig, 1993; Vaugher et al., 1973). Finally, some investigations have used type H BVDV isolates that failed to consistently induce thrombocytopenia in all infected calves (Corapi et al., 1989; Ellis et al., 1998). The emergence of type H BVDV isolates capable of inducing severe clinical disease and hemorrhage has created a renewed interest in the study of BVDV infections in immunocompetent cattle. At present, much remains unknown about BVDV infection in immunocompetent cattle and thrombocytopenia, including the mechanism of thrombocytopenia induction. The purpose of the studies described within this dissertation is to provide an understanding of the mechanism of thrombocytopenia and the pathophysiology of the hemorrhagic syndrome caused by BVDV. CHAPTER 1: REVIEW OF THE LITERATURE Bovine viral diarrhea ‘ m L'nited States (Olafson he initial outbreak invoh mtoenteritis with severe minded elevated body tet dehydration. and abortions unml outbreak. Interest Elm" infection appeared present in the gastrointe timosa on postmortem ex betel disease was also r injection of blood collec emulsions fiom deceased c-1946). A very sin: Bovine Viral Diarrhea Virus The History of Bovine Viral Diarrhea Virus Bovine viral diarrhea virus (BVDV) was first recognized and reported in 1946 from the United States (Olafson et al., 1946) and Canada (Childs, 1946). In the United States, the initial outbreak involved 6 dairy herds in New York state with clinical signs of gastroenteritis with severe diarrhea. Other clinical characteristics of affected cattle included elevated body temperatures, salivation, nasal discharge, depression, anorexia, dehydration, and abortions during the outbreak and extending three months beyond the initial outbreak. Interestingly, the first description of hemorrhages associated with BVDV infection appeared in this report (Olafson et al., 1946), with hemorrhages being present in the gastrointestinal tract, subcutaneous tissues, epicardium, and vaginal mucosa on postmortem examination of affected cattle. Experimental reproduction of this novel disease was also reported. Oral inoculation with fecal material, subcutaneous injection of blood collected from febrile cases, and subcutaneous injection of splenic emulsions from deceased cases all proved to be infective to susceptible cattle (Olafson et al., 1946). A very similar disease syndrome, which was termed “X disease,” was reported from Canada during the same year (Childs, 1946). Hemorrhage, in the form of bloody diarrhea, was reported as a clinical sign, in addition to the clinical signs mentioned in an earlier report (Olafson et al., 1946). A viral etiology for this new transmissible disease of cattle was not demonstrated until seven years after the initial reports (Baker et al., 1954). Early descriptions of the virus indicated that two forms existed, one causing cell vacuolation and death and the other producing no obvious pathology in cell monolayers (Gillespie et al., 1960; Lee & magpie. I957)- This mncytopathic BVDV- “1 the virus was sub anning enteric disease termed mucosal disease ( associated with mucosal d not. 1961). This woul election, the pathogenes ta'hogenesis of mucosal 398-1). Cattle immunorol SM1°13 mucosal disease irt‘ection established in ca $6me with the nonC}to tliess etal., 1984; McCl: Gillespie, 1957). This formed the first subclassification of BVDV, cytopathic and noncytopathic BVDV, which later was designated as the biotype of the virus. The virus was subsequently recognized to be associated with a sporadically occurring enteric disease characterized by low morbidity and high mortality that was termed mucosal disease (Ramsey & Chivers, 1953). Bovine viral diarrhea virus isolates associated with mucosal disease were found to belong to the cytopathic biotype (Gillespie et al., 1961). This would set the stage for the most widely studied aspect of BVDV infection, the pathogenesis of mucosal disease. Nearly 35 years passed before the pathogenesis of mucosal disease was determined (Bolin et al., 1985; Brownlie et al., 1984). Cattle immunotolerant to, and persistently infected with noncytopathic BVDV develop mucosal disease after superinfection with cytopathic BVDV. The persistent infection established in cattle was determined to be a result of transplacental infection of the fetus with the noncytopathic biotype of BVDV during the first 100 days of gestation (Liess et al., 1984; McClurkin et al., 1984). This results in the birth of a calf that is immunotolerant to, and persistently infected with noncytopathic BVDV. Within the past decade, outbreaks of severe, peracute BVDV infections in immunocompetent cattle have been reported in the United States, Canada, and the United Kingdom involving beef, dairy, and veal operations (Carman et al., 1998; David et al., 1994; Pellerin et al., 1994). Prior to these outbreaks, the ability of BVDV to cause severe clinical disease following primary acute infections in cattle was debatable (Baker, 1995). Analysis of BVDV isolates from outbreaks in Canada and the United States revealed a distinct group of BVDV (Ridpath et al., 1994). This formed the second subclassification of the virus, termed the genotype. Two distinct genotypes of BVDV, type I and type H BlDV. are currently ICCt were primarily of the l)?‘ 1994'). In addition. infe noncytopathic isolates of tj characterized by thrombc hdpath. I992; Corapi er pathogenesis of the hemo with some type II BVDV it The Clinical Manifesto Ii 01' lira! Diarrhea The clinical manifesta l995). Multiple clinical inclining subclinical. cIi EEOEthiC syndrome non +- egame Cattle are sub 3‘ I,. . \Dl mfectrons have be: , ElDll‘ed bCCf, TW 3 d .~ It BVDV, are currently recognized. The BVDV isolates obtained from these outbreaks were primarily of the type H genotype of BVDV (Pellerin et al., 1994; Ridpath et al., 1994). In addition, infection of nonimmune, immunocompetent cattle with some noncytopathic isolates of type H BVDV has been associated with a hemorrhagic disorder characterized by thrombocytopenia and hemorrhage (Bezek et al., 1994; Bolin & Ridpath, 1992; Corapi et al., 1990; Corapi et al., 1989; Rebhun et al., 1989). The pathogenesis of the hemorrhagic syndrome and the mechanism of increased virulence with some type H BVDV isolates are unknown, and are the focus of this dissertation. The Clinical Manifestations of Bovine Viral Diarrhea Virus: Severe, Peracute Bovine Viral Diarrhea The clinical manifestations of BVDV infection in cattle have been reviewed (Baker, 1995). Multiple clinical forms of BVDV infection in immunocompetent cattle exist, including subclinical, clinical bovine viral diarrhea (BVD), severe BVD, and a hemorrhagic syndrome. The majority of BVDV infections in immunocompetent and seronegative cattle are subclinical (Baker, 1995); however, outbreaks of severe, peracute BVDV infections have been reported in immunocompetent cattle (Carman et a1 ., 1998; David et al., 1994; Drake et al., 1994; Pellerin et al., 1994). These outbreaks have involved beef, dairy, and veal operations in the United States, Canada, and the United Kingdom (Carman et al., 1998; David et al., 1994; Drake et al., 1994; Pellerin et al., 1994; Swecker et al., 1997). Both calves and adult cattle have been affected (Carman et al., 1998; Drake et al., 1994; Swecker et al., 1997). In Quebec, mortality due to BVDV in veal operations was estimated at 25% of 143,000 calves for 1993 (Pellerin et al., 19%). he outbreak in (_. mortality of up to 50% in seine. peracute BVD in ulcerations in some case' torment diseases such '. experiencing outbreaks (D death was observed. in \s it died sithin 24 hours of ft“ nitration of affected eat inthe alimentary tract. (Carman et al., 1998; S\\ Analysis of BVDV rattled that they were . ail-.1994; Ridpath er al. 1994). The outbreak in Ontario involved 150 dairy, 600 beef, and 100 veal herds, with mortality of up to 50% in some herds (Carman et al., 1998). Clinical manifestations of severe, peracute BVD include diarrhea, pyrexia, decreased milk production, and oral ulcerations in some cases (Carman et al., 1998; Drake et al., 1994). In addition, concurrent diseases such as pneumonia and abortion were frequently reported in herds experiencing outbreaks (David et al., 1994; Drake et al., 1994). In some herds, sudden death was observed, in which cattle either were found dead without clinical signs or had died within 24 hours of first exhibiting clinical signs (Carman et al., 1998). Postmortem evaluation of affected cattle revealed mucosal-like lesions, including erosions and ulcers in the alimentary tract, necrosis of the intestinal mucosa, and Peyer’s patch necrosis (Carman et al., 1998; Swecker et al., 1997). Analysis of BVDV isolates from outbreaks in Canada and the United States has revealed that they were of the type H genotype of BVDV (Carman et al., 1998; Pellerin et al., 1994; Ridpath et al., 1994). As an example, 61 of the 64 isolates recovered from the outbreaks in Ontario, Canada were type H BVDV (Carman et al., 1998). Prior to these described type II BVDV outbreaks, there was a tendency to depreciate the importance of BVDV as causing clinical disease in cattle immunocompetent to BVDV. The reports of these severe BVDV infections clearly demonstrate that some isolates of BVDV can cause severe life-threatening disease in immunocompetent cattle. The outbreaks of severe BVDV infection have also demonstrated that some isolates of BVDV can induce severe disease and death losses in adult cattle. Trptll Bovine Viral Diar Borine viral diarrhea ' nucleotide sequence. and it the basis for genotyping. dillettni genolflms 0f B\ biology techniques that id monoclonal antibody proll chain reaction of the 5‘ l Ridpath et al., l994). but regions of the BVDV get listinct genotypes of BW r1994). In addition. t)“ hon analysis of the 5' L" Analysis and compai he revealed greater that mmentrated in the re: finenees in nucleic : rePorted to be as great a and beg cholera Virus (I Although ombreak Type II Bovine Viral Diarrhea Virus Bovine viral diarrhea virus isolates may be subdivided based upon diflerences in the nucleotide sequence, and these specific differences in the viral nucleic acid sequences are the basis for genotyping. Several different techniques are available for identifying different genotypes of BVDV. Most typing procedures involve the use of molecular biology techniques that identify sequence differences in specific genome segments, or monoclonal antibody profiles (Table 1.1, page 12). Reverse transcription and polymerase chain reaction of the 5’ UTR have been the most widely used (Fulton et al., 1999; Ridpath et al., 1994), but reverse transcription and polymerase chain reaction of other regions of the BVDV genome are proving acceptable (Bolin & Ridpath, 1996). Two distinct genotypes of BVDV exist, which are classified as type I and type H (Ridpath et al., 1994). In addition, type I BVDV can be further divided into type Ia and type Ib based upon analysis of the 5’ UTR sequence (Ridpath & Bolin, 1998). Analysis and comparison of nucleic acid sequences from type I and type H genotypes has revealed greater than 30% dissimilarity (Ridpath & Bolin, 1995). This dissimilarity is concentrated in the regions of the viral genome encoding for E2 and the 5’ UTR. The difl‘erences in nucleic acid sequences between type I and type H BVDV have been reported to be as great as the differences in nucleic acid sequences between type I BVDV and hog cholera virus (Bolin & Ridpath, 1996). Although outbreaks of severe peracute BVD and the hemorrhagic syndrome have been associated with type H isolates of BVDV, viral virulence does not equate with genotype. Type II BVDV infections have also resulted in subclinical to mild disease under natural (Bolin & Ridpath, 1996) and experimental conditions (Marshall et al., E996). D'tt’fere“Ces in l ifierences bcm'ee" 0pc region of the viral genon normalizing antibody prt‘ intently. most commerc i. nopel. Questions has BlDV to induce protectio HBlDV may escape the elicited by vaccination \ demonstrated when 3 “13¢ calf born to a healthy c attaining ope l BVDV (E The origin of type ll existed prior to its asso montage syndrome. .1 so; , ital We 11 BVDV iso YEW‘CQ' of a type II isol 1996). Differences in BVDV genotype may define antigenic differences, because differences between type I and type H nucleotide sequences are concentrated in the region of the viral genome encoding for E2. The E2 glycoprotein is the target for neutralizing antibody production by the humoral immune system during infection. Currently, most commercial BVDV vaccines contain isolates of BVDV that are classified as type I. Questions have been raised as to the ability of vaccines containing type I BVDV to induce protection to type II BVDV infection. An anti genically divergent type H BVDV may escape the neutralizing humoral immune response to the E2 glycoprotein elicited by vaccination with a type I BVDV-containing vaccine. This has been demonstrated when a type H BVDV isolate was recovered fiom a persistently infected calf born to a healthy cow that had been vaccinated with an inactivated vaccine containing type I BVDV (Bolin et al., 1991). The origin of type H BVDV is unknown, but it is likely that type H BVDV has existed prior to its association with outbreaks of severe peracute disease and the hemorrhagic syndrome. An analysis of BVDV isolates from 1981 was performed, and several type H BVDV isolates were identified (Bolin & Ridpath, 1996). In addition, the recovery of a type H isolate was just as likely in the 1981-1985 period as in the 1991- 1994 period during which outbreaks of severe peracute BVDV were occurring (Carman et al., 1998). An explanation for the emergence of type H BVDV and its association with severe peracute disease and the hemorrhagic syndrome cannot be provided; however, most Canadian herds involved in outbreaks of severe peracute BVDV infections had not been vaccinated against BVDV, or the vaccination protocols were incomplete (Carrnan et al., 1998). Use of vaccines containing type I BVDV provides at least partial protection 10 horn ope ll BVDV. but Ridpath. 1996). For exam rim-sparing etlect t1 thereas unt'accinates de\ t from type H BVDV, but the protections appears to be of limited duration (Bolin & Ridpath, 1996). For example, a modified-live vaccine containing type I BVDV provided a disease-sparing effect following challenge with a virulent isolate of type H BVDV, whereas unvaccinates developed severe BVDV infection (Cortese et al., 1998). ll Table 1.1. Methods for t} ' Method ——7 l ___1 Nested Rl-PCR of S'Ul Nested RI-PCR of E0 RI-PCR of S’L'TR and s. :PCR product RI-PCR of S'UTR follox digestion of PC R product \ RI-PCR amplification on ‘N83 161 bp product M .lntihody-based t}ping r th-lonoelonal antibody p Cross-neutralization exp hot". I I E‘IL-PCR: Reverse tra mili- S’untransl: {3'} Region of] ' " Region of‘. Table 1.1. Methods for typing isolates of bovine viral diarrhea virus Method Reference(s) Nested RT-PCR of S’UTR (el-Kholy et al., 1998; Ridpath et al., 1994; Tajima et al., 1995) Nested RT-PCR of E0 (Fulton et al., 1999; Sullivan & Akkina, 1995) RT-PCR of 5’UTR and sequencing of (Pellerin et al., 1994; Vilcek et al., 1999a; PCR product Vilcek et al., 1999b) RT-PCR of 5’UTR followed by restriction (Harpin et al., 1995; Vilcek et al., 1994) digestion of PCR products RT-PCR amplification and sequencing of (Yousif er al., 1999) N83 161 bp product Antibody-based typing methods (Beer & Wolf, 1999; Dekker et al., 1995; (Monoclonal antibody panel analysis or Deregt et al., 1998; Paton et al., 1995; Cross-neutralization experiments) Pellerin et al., 1994) Key: RT-PCR: Reverse transcription-polymerase chain reaction 5’UTR: 5’ untranslated region of the BVDV genome E0: Region of BVDV genome coding for the structural glycoprotein 0 (gp48) NS3: Region of BVDV genome coding for the nonstructural polypeptide 3 (p80) 12 Boiineliinl Wm“ V lhe original report 07 motion (Olafson et t nioded descriptions of l‘ hesutepicarditun. gall bl .tselinical cases of BVD' decribed As an exam naturally infected with t hemorrhages were desc: orin many cases. es'alu: A bleeding syndrc ado-teed thromboc )‘top im‘estigator did not in chis was the Bovine Viral Diarrhea Virus and the Hemorrhagic Syndrome The original report of BVDV infection in cattle included hemorrhages in its clinical description (Olafson et al., 1946). In addition, the first report on mucosal disease included descriptions of hemorrhages, which were present on pathologic examination in the subepicardium, gall bladder, and gastrointestinal mucosa (Ramsey & Chivers, 1953). As clinical cases of BVDV were being reported throughout the world, hemorrhages were described. As an example, abomasal and renal hemorrhages were observed in calves naturally infected with noncytopathic BVDV in Hungary (Romvary, 1965). Although hemorrhages were described in these early reports, thrombocytopenia was not reported, or in many cases, evaluation of platelet counts was not performed. A bleeding syndrome in veal calves, consistent with later descriptions of BVDV- induced thrombocytopenia, was first reported in 1978 (Harrison, 1978). However, the investigator did not include diagnostics for BVDV in the investigation, so it is uncertain if this was the first report describing BVDV-induced thrombocytopenia. Thrombocytopenia in association with BVDV infection was first reported in New York (Perdrizet et al., 1987; Rebhun et al., 1989). These first descriptions occurred in calves and adult cattle naturally infected with BVDV (Perdrizet et al., 1987; Rebhun et al., 1989). In a retrospective case study, 15 animals ranging in age from 1 to 8 years were diagnosed with BVDV and thrombocytopenia. In these animals, platelet counts ranged from 2,000 platelets/pl to 33,000 platelets/pol. All cattle were treated symptomatically, but only 6 animals survived the thrombocytopenic BVDV infection. In general, the cattle that survived the thrombocytopenic BVDV infection had higher platelet counts than the 13 cattle that died. 0f the platelets’iil. Bloody din: hrombocitopenic B\'D\ episutis petechial hemo sites or insect bites. C ant severe signs of bleeding. partial thromboplastin tim| the results obtained v. thrombocytopenia and intrarascular coagulatior 14 of 15. cattle and nonr hdaneutropenia and l WitOpenia (thrombc W5 Prompted thi WOW necrosis was al.1989). nlrOmboc.“()peri me report describing Tht? plaifllet “him “a cattle that died. Of the 6 survivors, 4 survivors had platelet counts greater that 25,000 platelets/p11. Bloody diarrhea was the most common clinical sign associated with the thrombocytopenic BVDV infections in these cattle. Other clinical signs included epistaxis, petechial hemorrhages, ecchymotic hemorrhages, and bleeding from injection sites or insect bites. Cattle with platelet counts less than 25,000 platelets/pt] had the most severe signs of bleeding. Coagulation testing, consisting of prothrombin time, activated partial thromboplastin time, and fibrinogen determination, was performed on 6 cattle, and the results obtained were normal. This provided the first evidence that the thrombocytopenia and hemorrhages were not likely the result of disseminated intravascular coagulation. An interesting observation was the presence of leukopenia in 14 of 15 cattle and nonregenerative anemia in 3 of the 15 cattle. All 14 leukopenic cattle had a neutropenia and 10 of the 14 leukopenic cattle had a lymphopenia. The presence of pancytopenia (thrombocytopenia, neutropenia, and a nonregenerative anemia) in these animals prompted this group of investigators to speculate that myelosuppression or marrow necrosis was the mechanism of BVDV-induced thrombocytopenia (Rebhun et al., 1989). Thrombocytopenia, anemia, and bone marrow necrosis were also demonstrated in a case report describing 2 beef calves naturally infected with BVDV (Scruggs et al., 1995). The platelet cormt was only performed in 1 of the calves, and was 16,000 platelets/til. A postmortem bone marrow evaluation was performed on both calves. Decreased numbers of megakaryocytes and megakaryocyte degeneration, characterized by pyknotic nuclei, vacuolated pale staining eosinophilic cytoplasm, and poorly defined cytoplasmic borders, were observed. In addition to megakaryocyte necrosis, osteopetrosis of the long bones l4 was aimed as an incidr tlebhun er a1.. 1989,). 1 wow suppression and thrombocytopenic E he original descriptions ‘ .dpath 1992; Corapi 9. reduction in the plate intravenous injection 0 h's‘ttitrrental thrombo 1990; Corapi er al., I “it“? Preparations v Ridpath, 1992; cu: hitheir ability to in ma‘hlfected mor {hkketar,1994 nations Chose int he BWDV isolate mammal in 0Cu CD89 (Corapi er a or! 1998p and NY the We 1] BVDV sears, NADL (r mates failed was observed as an incidental finding. This case report supported the earlier hypothesis (Rebhun et al., 1989), that BVDV-induced thrombocytopenia is likely due to bone marrow suppression and decreased platelet production. Thrombocytopenic BVDV infections were experimentally reproduced shortly after the original descriptions in naturally infected cattle appeared (Bezek et a1 ., 1994; Bolin & Ridpath, 1992; Corapi et al., 1990; Corapi et al., 1989). Thrombocytopenia is defined as a reduction in the platelet count below the minimum level of the species (Jain, 1986). Intravenous injection of blood mononuclear cells from affected cattle was used in early experimental thrombocytopenic BVDV infections (Bezek et al., 1994; Corapi et a1 ., 1990; Corapi .et al., 1989). Intranasal inoculation of cattle with BVDV-infected cell culture preparations was used in later experimental infections (Bezek et al., 1994; Bolin & Ridpath, 1992; Ellis et al., 1998). One study compared these two inoculation methods in their ability to induce thrombocytopenia, and found that intravenous inoculation of BVDV-infected mononuclear cells was more effective in inducing thrombocytopenia (Bezek et al., 1994). In spite of this finding, later investigators using experimental infections chose intranasal routes of inoculation to simulate natural exposure. To date, the BVDV isolates that have been shown to induce thrombocytopenia following experimental inoculation in cattle include CD87 (Bezek et al., 1994; Corapi et al., 1990), CD89 (Corapi et al., 1989), BVDV 890 (Bolin & Ridpath, 1992), BVDV 24515 (Ellis et al., 1998), and NY-l (Marshall et al., 1998). Platelet count has also been evaluated with the type H BVDV isolates 7937 and 126 (Marshall at al., 1996), and the type I BVDV isolates NADL (Bezek et al., 1994) and TGAN (Bolin & Ridpath, 1992), but these isolates failed to induce thrombocytopenia in experimentally infected calves. 15 lntcrtstlngl)‘. the W3 \ it mvthlarshall er al., 19‘ other BVDV isolates aforementioned BVDV i this to induce ““0“le which induce thromboc )1- typell genonpe of BVD With respect to the l platelet count in as obse' platelet count depressic 1994; Bolin & R, Wents monitore la cali'es infected w mm days 10-15 CD87 and CD89, an occur until at least 1 huh-ml CalVes infec. hays after inoculatit CD87 or CD89 inf lCorapi er al., 199 him infection Vt hiked from so [4‘ Interestingly, the type I isolate NY-l has been shown to induce thrombocytopenia in one study (Marshall et al., 1998), but failed to induce it in another study (Bezek et al., 1994). Other BVDV isolates may be capable of inducing thrombocytopenia, but the aforementioned BVDV isolates are the only isolates that have been examined for their ability to induce thrombocytopenia thus far. All of the aforementioned BVDV isolates, which induce thrombocytopenia, are noncytopathic. In addition, all isolates belong to the type H genotype of BVDV, with the exception of NY-l , which is a type I BVDV isolate. With respect to the platelet count following experimental inoculation, a decline in the platelet count was observed to occur as early as 3 days afier inoculation, with maximum platelet count depression occurring between days 9 and 17 following infection (Bezek et al., 1994; Bolin & Ridpath, 1992; Corapi et al., 1990; Corapi et al., 1989). Several experiments monitored recovery of platelet counts following experimental inoculation. In calves infected with BVDV 890, a gradual rise in the platelet count would occur between days 10-15 after inoculation (Bolin & Ridpath, 1992). In calves infected with CD87 and CD89, an elevation in the platelet count from thrombocytopenic levels did not occur until at least 17 days after inoculation (Corapi et al., 1990; Corapi et al., 1989). In several calves infected with CD89 and CD87, the platelet count remained low tmtil 35 days after inoculation. Interestingly, the rise in platelet count in calves recovering from CD87 or CD89 infection corresponded to the presence of serum neutralizing antibody (Corapi et al., 1990). This study was unique in that a prolonged viremia following BVDV infection was observed in some of the calves (Corapi et al., 1989). Virus was isolated from some calves as late as 46 days after inoculation, and subsequently, the appearance of neutralizing antibodies to BVDV was delayed to beyond day 50 after 16 inoculation in those C3l\t after infection (Baker. 1 appearance of neutralizin ; Clinical sigis of he Billh’ infections resultir l993; Corapi er al., 199 pntaneous hemorrhage nay be present if plate studies utilizing the B‘ has only observed in ( "Corset et al., 1990) hihence of hemorrh: 79000 Platelets-’m ; hemorrhage and m « r , . canes mfected w inoculation in those calves. Neutralizing antibodies usually appear within 2 to 4 weeks after infection (Baker, 1995). An explanation for the prolonged viremia and delayed appearance of neutralizing antibodies to BVDV was not determined. Clinical signs of hemorrhage have been observed in all reports of experimental BVDV infections resulting in thrombocytopenia (Bezek et al., 1994; Bolin & Ridpath, 1992; Corapi et al., 1990; Corapi et al., 1989; Ellis et al., 1998). Clinical signs of spontaneous hemorrhage occur when platelet numbers fall below 20,000 platelets/pl, and may be present if platelet numbers are between 20,000 and 50,000/ul (Jain, 1986). In studies utilizing the BVDV isolates CD87 and CD89, clinical evidence of hemorrhage was only observed in cattle whose platelet count decreased to less than 5,000 platelets/pl (Corapi et al., 1990). In contrast, BVDV 890 infection in calves resulted in clinical evidence of hemorrhage in 3 out of 7 calves in which platelet counts failed to drop below 70,000 platelets/pl at the time of death. This discrepancy between the development of hemorrhage and the platelet count prompted our investigations of platelet function in calves infected with BVDV. The reader is referred to Chapters III and IV of this dissertation for discussion of our platelet function studies. The extent and location of hemorrhages in experimentally infected calves are similar to the reports describing BVDV infection in naturally infected calves (Rebhun et al., 1989; Scruggs et al., 1995; Swecker et al., 1997). Hemorrhages have been observed during physical examination of infected calves on the oral mucosa, ventral surface of the tongue, eye (hyphema), sclera and conjunctiva of the eye, and subcutis, especially in areas surrounding the carpus and hock (Corapi et al., 1990). Prolonged bleeding from venipuncture sites was a common finding in BVDV-infected calves (Corapi et al., 1990; 17 Corapi er al., 1989). tiequently than other hemorrhages are most t) ileinn (Bolin & Ridpath hemorrhages were found BVDV 890 and the t}pe 1,998,). Additional heir reticulum, mitten, oma5\ tndtalvaria. diaphragm n: reticulum in one c 34515 (Ellis et al., \99 mhis calf. Coagulation prof resettled no abhor-ma gm findings fiom Corapi et al., 1989). Epistaxis was also observed (Corapi et al., 1990), but less frequently than other antemortem hemorrhages. On postmortem examination, hemorrhages are most typically found in the alimentary tract, especially the rumen and ileum (Bolin & Ridpath, 1992; Corapi et al., 1990; Corapi et al., 1989). Extensive hemorrhages were found in lymph nodes in the calves infected with the type H isolate BVDV 890 and the type I isolate BVDV NY-l (Bolin & Ridpath, 1992; Marshall at al., 1998). Additional hemorrhages were observed in the urinary bladder, abomasum, reticulum, rumen, omasum, adrenal glands, gallbladder, heart, thymus, esophagus, brain and calvaria, diaphragm, and aorta. A suffusive, subserosal hemorrhage in the mucosa of the reticulum in one calf was the only hemorrhage reported using the isolate BVDV 24515 (Ellis et al., 1998). Moreover, the platelet count was less than 10,000 platelets/pl in this calf. Coagulation profile data from an experimental thrombocytopenic BVDV infection revealed no abnormalities (Corapi et al., 1989), which supported the previous coagulation data findings from naturally infected cattle, indicating that disseminated intravascular coagulation is not likely to be the mechanism of BVDV-induced thrombocytopenia. However, coagulation profiles were only performed in a single experimental infection (Corapi et al., 1989). Neutropenia was an additional, consistent laboratory finding in the experimental type II BVDV infections (Bolin & Ridpath, 1992; Corapi et al., 1990; Corapi et al., 1989; Ellis et al., 1998). In calves infected with BVDV 24515, bone marrow examination revealed that granulopoiesis was minimal, with immature granulocytic cells predominating (Ellis et al., 1998). As similar to the thrombocytopenia, the neutropenia 18 maybe the result of b« profuction. An interesting ObSt: demonstration of BVDV 1991'. Corapi et al., 1990 platelets was demonstrat 1992) and immunofluor Corapietal., 1990). Th 17 after inoculation (Be; eta}..l989). Both stuc C087 and CD89 demo ll after inoculation in We aSsoeiated with F am 890 with Na 2%.4 r. may be the result of bone marrow suppression, and a subsequent lack of neutrophil production. An interesting observation in previous experimental BVDV infections was the demonstration of BVDV association with platelets (Bezek et al., 1994; Bolin & Ridpath, 1992; Corapi et al., 1990; Corapi et al., 1989). The association of BVDV with peripheral platelets was demonstrated by virus isolation fiom purified platelets (Bolin & Ridpath, 1992) and immunofluorescent staining of platelet preparations (Bezek et al., 1994; Corapi et al., 1990). This association was determined to occur between days 7 through 17 after inoculation (Bezek et al., 1994) and days 12 through 15 after inoculation (Corapi et al., 1989). Both studies utilized the BVDV isolate CD87. Another study evaluating CD87 and CD89 demonstrated that virus association with platelets was observed on day 11 after inoculation in 9 of 12 calves examined, but the specifics of which viral isolates were associated with platelets were not reported (Corapi et al., 1990). An association of BVDV 890 with platelets was also demonstrated (Bolin & Ridpath, 1992). Virus association with platelets was demonstrated for an average of 5.9 days in 7 infected calves, but the initial and last days after inoculation in which an association could be demonstrated was not reported. Evaluation of BVDV 890 association with platelets was also performed in our experimental infections (See Chapters H, III, IV, and V). To date, virus association with platelets has not been evaluated with the thrombocytopenia- inducing BVDV isolates NY-l and 24515. The origin and nature of the association of BVDV with platelets is unknown. It has been suggested that the platelets may serve as "carriers" of circulating virus (Corapi et al., 1989). This may be important in the pathogenesis of the hemorrhagic syndrome, as platelets may allow for a more rapid and 19 extensive dissemination associates with platelet platelets from an uninf (Corapiet al., l989). A ln addition. we perfomie andBlDV-infected cell platelets (unpublished internalized in the plate tumor-mediated uptak infection with BVDV [Mature of this B\.’D\ Pathologic exam‘1r teenPerformed for at {mm and lOcation ( review. In general, It Elan in lhe reports c extensive dissemination of virus throughout the body. It is unlikely that BVDV associates with platelets by nonspecific adherence. Mixing experiments utilizing platelets from an uninfected normal calf and BVDV-positive serum were performed (Corapi et al., 1989). Afier incubation and washing of platelets, virus was not isolated. In addition, we performed mixing experiments between platelets from an uninfected steer and BVDV-infected cell culture supernatant, and virus was not isolated fi'om the washed platelets (unpublished observation). These findings suggest that BVDV may be internalized in the platelet. Intemalized virus in platelets may occur as a result of receptor-mediated uptake of BVDV by platelets or may be the result of megakaryocyte infection with BVDV. Further study utilizing irnmunoelectron microscopy may clarify the nature of this BVDV-platelet association. Pathologic examination of experimental thrombocytopenic BVDV infections has been performed for all the aforementioned thrombocytopenia-inducing BVDV isolates. Extent and location of hemorrhages have been described previously in this literature review. In general, lesions have been less pronounced in the experimental reproduction than in the reports describing the natural outbreaks of severe peracute BVDV or the hemorrhagic syndrome (Carman et al., 1998; Rebhun et al., 1989). For example, the only pathologic findings in calves infected with BVDV 24515 were hemorrhages and pulmonary consolidation (Ellis et al., 1998). Besides hemorrhages, linear erosions in the spiral colon similar to lesions described for cases of mucosal disease, were observed in calves infected with BVDV 890 (Bolin & Ridpath, 1992). In addition, abomasal edema was observed in calves infected with BVDV 890 (Bolin & Ridpath, 1992), and this was a 20 consistent finding in our pathologic descriptions t1 Histologic evaluatio dnombocuopenic BVD megakaryocyte h)perpla 1989‘). lhe significanCe investigators h)pothesize_ consistent finding in our experimental studies evaluating BVDV 890. In our studies, the pathologic descriptions for BVDV 890-infected calves are found in Chapters 11 and V. Histologic evaluation of bone marrow has also been performed in the experimental thrombocytopenic BVDV infections. In calves infected with CD87, a pronounced megakaryocyte hyperplasia was observed at day 12 after inoculation (Corapi et al., 1989). The significance of this finding was unknown at the time, but this group of investigators hypothesized that decreased platelet production was not the mechanism of BVDV-induced thrombocytopenia because of the presence of megakaryocyte hyperplasia. In contrast, decreased thrombopoiesis was observed at days 10-12 after inoculation in 5 of 6 calves infected with BVDV 24515 (Ellis et al., 1998). Of those 5 calves, overt bone marrow necrosis, characterized by areas of indistinct morphology, karyorrhexis, and phagocytosis of debris by large mononuclear cells, was reported in 2 calves. Megakaryocyte hyperplasia, characterized by increased numbers of immature megakaryocytes was observed in the remaining calf (Ellis et al., 1998). Bone marrow evaluation was not described for BVDV 890-infected calves (Bolin & Ridpath, 1992). In calves infected with the type I isolate NY-l, bone marrow findings were unremarkable (Marshall at al., 1998). Different interpretations of histologic examination of bone marrow likely stem from the fact that marrow was examined at a single, variable point in time. In addition, kinetics of infection may be different for the viral isolates examined. Histologic evaluation of bone marrow from BVDV-infected calves was performed in our experimental infections with BVDV 890 (see Chapters H and V). Distribution of BVDV in tissues of experimentally infected calves has been evaluated using immunofluoresccnt antibody and immunohistochemical staining for 21 3th antigen- The minbocfiOPCnla has b6 antigen in megakmloq mimofluorescent antil line of irnmunohistoel' also teen demonstrate: thromltocfiopenia (Mar determined (Spagnuolo nth the type 1 isolate hit megakaryocyte inf demousmted (Marsha hermined for the th $0“? Of investigator 5* Chapters 11 and ‘ The mechani: BVDV antigen. The most interesting observation relevant to the discussion of thrombocytopenia has been the identification of BVDV antigen in megakaryocytes. Viral antigen in megakaryocytes has been demonstrated in CD87-infected calves by use of immunofluorescent antibody testing (Corapi et al., 1989), and in 24515-infected calves by use of immunohistochemistry (Ellis et al., 1998). Viral antigen in megakaryocytes has also been demonstrated in other studies utilizing isolates that did not induce thrombocytopenia (Marshall et al., 1996) or in which the platelet count was not determined (Spagnuolo et al., 1997; Spagnuolo-Weaver et al., 1997). In calves infected with the type I isolate NY-l , immunohistochemistry was performed on tissue sections, but megakaryocyte infection, as determined by viral antigen in megakaryocytes, was not demonstrated (Marshall et al., 1996). Megakaryocyte infection with BVDV has not been determined for the thrombocytopenia-inducing BVDV isolate CD-89. We are the only group of investigators that has demonstrated BVDV 890 infection of megakaryocytes (see Chapters H and V). The mechanism of BVDV-induced thrombocytopenia is unlmown. Thrombocytopenia, regardless of the underlying etiology, results from only three basic mechanisms: decreased production, accelerated destruction, or abnormal sequestration of platelets (Warkentin & Kelton, 1994). The mechanism of BVDV-induced thrombocytopenia may involve one or a combination of these mechanisms. Coating of platelets with antibodies or deposition of viral antigen-antibody complexes on platelet membranes can trigger immune-mediated destruction of platelets (Shulman & Reid, 1994). Immune-mediated destruction of platelets in the peripheral circulation as the cause of BVDV-induced thrombocytopenia is not likely, based upon a previous study 22 demonstrating that there inCD87 and CD89-inn coagulation also causes (Shulman & Reid, 1994 calves suggest that disst induced thrombocytOpen notlikely the cause of l Was not observed on pc than 1992). 2451 5:1 “CD89 (Corapi er of BlDl" 890-infected c: than 1992), m fimmfion are in“ wow findings of r hmheSis that decre mated mmboc >10 demonstrating that there was no evidence of platelet-associated antibody and complement in CD87 and CD89-infected calves (Corapi et al., 1989). Disseminated intravascular coagulation also causes thrombocytopenia due to an accelerated destruction of platelets (Shulman & Reid, 1994), but normal coagulation profiles in CD87 and CD89-infected calves suggest that disseminated intravascular coagulation is not involved in BVDV- induced thrombocytopenia (Corapi et al., 1989). Abnormal sequestration of platelets is not likely the cause of BVDV-induced thrombocytopenia because hepatosplenomegaly was not observed on postmortem examination in BVDV 890-infected calves (Bolin & Ridpath, 1992), 24515-infected calves (Ellis et al., 1998), and calves infected with CD87 or CD89 (Corapi et al., 1990). In addition, postmortem examination of the spleen in BVDV 890-infected calves revealed them to be smaller than normal in size (Bolin & Ridpath, 1992). The lack of evidence that accelerated destruction and abnormal sequestration are involved in BVDV-induced thrombocytopenia and histologic bone marrow findings of marrow necrosis and viral antigen in megakaryocytes support the hypothesis that decreased production of platelets is the primary mechanism of BVDV- induced thrombocytopenia. 23 The Bovine Platelet Platelets are the 5‘1 Johnstone, 1988). Plate anucleate. platelets par“ including maintenance trombosis. tumor meta: play a pivotal role in the aith endothelial cells. It adhere to exposed sube | activated. By adhering Platelets form hemostat: Platelets contain intrac )1 ml Serotonin (Gentrv aggregation and vasecon The Bovine Platelet Platelets are the smallest and most numerous elements in blood (Gentry, 1992; Johnstone, 1988). Platelets circulate as anucleate, discoid cells. Although small and anucleate, platelets participate in numerous physiologic and pathologic processes including maintenance of vascular integrity, coagulation, fibrinolysis, inflammation, thrombosis, tumor metastasis, neovascularization, and repair (Gentry, 1992). Platelets play a pivotal role in the maintenance of normal hemostasis, via their close interaction with endothelial cells. Injury to blood vessels triggers the hemostatic process. Platelets adhere to exposed subendothelial collagen or damaged endothelial cells and become activated. By adhering to injured blood vessels, and then each other (aggregation), platelets form hemostatic aggregates that contribute to the cessation of hemorrhage. Platelets contain intracytoplasmic granules that contain adenosine diphosphate, calcium, and serotonin (Gentry, 1992). When secreted, these substances promote platelet aggregation and vasoconstriction, which further enhances hemostasis. In addition to their function during blood vessel injury, the interaction between platelets and endothelial cells promotes wound healing after tissue damage, as a result of soluble substances released from platelets, including platelet-derived growth factor, epidermal growth factor, and transforming growth factor-8 (Gentry, 1992). Platelets also modulate the inflammatory response through cellular interactions with other cellular components such as neutrophils. Platelets are derived from their hematopoietic precursor cell, the megakaryocytes (Wright, 1906). Although megakaryocytes comprise only 0.05% of the nucleated cells in the bone marrow (Harker, 1968), they are responsible for maintaining the large 24 initiating platelet popt Ct“ that is also commor Megalantocytes underg progressive increase in Cram, W95). Prolif , numerous soluble C)10l\‘l considerable attention i lnonn stimulator of e Kanshansky, 1995'. l mammocyte colon ) many 35 tw0 thirds QTOkines act SETtet. combination of inter megakarFocyte grc lamentation of n 1976). Initially, 1h "he demarcation m lit-e megakary-OC 1“ hemmed fiOm .1 circulating platelet population. The megakaryocyte is derived from a pluripotent stem cell that is also common to the myeloid and erythroid cell lines (Zauli & Catani, 1995). Megakaryocytes undergo endoreduplication, which is a complex process involving a progressive increase in nuclear ploidy (from 2N to 64N) during proliferation (Zauli & Catani, 1995). Proliferation and maturation of megakaryocytes are regulated by numerous soluble cytokines (Figure 1.1, page 26), of which thrombopoietin has received considerable attention in recent years. Thrombopoietin is considered the most potent known stimulator of endoreduplication and polyploidy in me gakaryocytes (Broudy & Kaushansky, 1995; Kaushansky, 1998). In addition, thrombopoietin is a potent megakaryocyte colony stimulating factor, capable of inducing colony formation from as many as two thirds of all megakaryocyte progenitors (Kaushansky, 1998). Many cytokines act synergistically in megakaryocytopoiesis. Thrombopoietin and the combination of interleukin-3, interleukin-11, and steel factor (stem cell factor) influence megakaryocyte growth (Broudy et al., 1995). Platelets are produced by the fragmentation of the cytoplasm of mature megakaryocytes (Breton-Gorius & Reyes, 1976). Initially, the maturation of megakaryocytes is associated with the appearance of the demarcation membrane system, which is contiguous with the surface membrane of the megakaryocyte (Breton-Gorius & Reyes, 1976). Platelets and proplatelets are fragmented from individual areas in the demarcation membrane system. 25 o / .. ,x .. / . .t .. .r: assist m.....<-:..£ O I \ EBBEL egocgguo—Z 355903 41m #2 Amado. Jpn-33.0 ye ..-::N 2.05% EOuQSVn—v amaOmOQCtADOKLQXRNOFZk0 Duh-020$. u . — 9531?.— r cage 328 58a =8 82m Haom 88am b.5322 288304 #5 882 maumfigum .328 8.32555 ”$0-0 daemonofiubm "0mm 882 warm—=85 .328 owfimobmgoioe—zg ” “50-20 32:28:: ”d o$oo§w022§ wanton .3200 “mos—-20 35233825. "on: onobaxmwozég wfincou “mam "mo—2-3mm Sov— :a 6.4m .mkfl .Omm .Omh. 6.882 coup—Ba: m5 .mom .550.sz .mmUAv 471: 6-1: £5: .de .Omh. 6.888 gagemzpfl / \ s . t c (a 3 .\s\\ /, T J lt\.\ . s 0 es «1\ ' .....s. \\O\ D s s. \ fl- . f... . .. , \ a. 4‘ N. h- \ s! \I’... a. a, c}. A” a, , U ‘. n . at... U \f.‘ I. .(t. 1 ./ .., ., .., . I . ‘ . . .. s. .. I , .. \L a? r. s... .5: .11: . .\ ssesi c)! . ss- N... H “‘10,“... 1. ti... . It... Xena. . /. , . ,-.\\ .../t.... \ mos—5&0 32-3mm 30 3n _ E nicogmuz “83°32“on A33 3330 a. BEN 82m 3:563 mmmomomowhoobmxamofi mo oEonom Z 2.2... 26 The primary funct irritation platelets unof change. secretion. and a receptors. signal transdr degranulation, and the other cellular elements. frnction of bovine plate and feline platelets. T Ween bovine platele Platelet structure l divided into 4 Compa 1098., the organelle Zr mmfionnected 01 WEI-bane WhOSQ f1} exChange of sigms malicular system, file “85) In hUm mules “ith rele Emma 1969)_ The primary function of platelets is maintenance of vascular integrity. Upon activation, platelets undergo a complex series of events described as adherence, shape change, secretion, and aggregation. The mechanisms of platelet activation, including the receptors, signal transduction pathways, the soluble substances released during platelet degranulation, and the interactions between platelets and the coagulation system and other cellular elements, have been reviewed (Gentry, 1992). In general, the structure and function of bovine platelets has not been characterized to the extent of human, canine, and feline platelets. The focus of the remainder of this section will be on differences between bovine platelets and those of other species, and platelet disorders of cattle. Platelet structure has been reviewed (White & Gerrard, 1994). The platelet can be divided into 4 compartments or structural domains: the membrane system, the sol- gel zone, the organelle zone, and the peripheral zone. As part of the membrane system, the surface-connected open canalicular system is a series of invaginations in the plasma membrane whose function is to increase surface area for the presence of receptors and exchange of signals (White & Gerrard, 1994). Bovine platelets do not possess an open canalicular system, which is found in all other mammalian platelets (Zucker-Franklin et al., 1985). In human platelets, the release reaction is accompanied by a centralization of granules with release of granule contents into the open canalicular system (Zucker- Franklin, 1969). In contrast, the granules remain distributed peripherally in bovine platelets, and the release of granule contents occurs on the cell surface of bovine platelets. The significance of this observation in terms of frmction is unknown, as bovine platelets function similarly in hemostasis as human platelets (Zucker-Franklin et al., 1985). 27 following activatio long. Pom‘ed ”endow endothelial cell and 8. causes the platelets to pseudopods. The bovirr other species, the devel bovine platelets do not 1 al., 1990). In additior resistant to deforrnatior these findings are unknr Differences exist 1 Mill 0f agonists to i Hminc platelets, in ge than platelets of most “39mm 5 to 10 times $151“ from sheep. dattlsed Sensitivity 1. MUG” became boy 031111}! 1989). In add 0? Following activation, platelets change shape. Platelets become irregular and extend long, pointed pseudopods, which provide surface receptors for the adhesive platelet- endothelial cell and aggregative platelet-platelet reactions. Continuous stimulation causes the platelets to develop into spread forms in which cytoplasm extends into the pseudopods. The bovine platelet is also able to change shape, but unlike platelets from other species, the development of the cytoplasm-filled pseudopods does not occur and bovine platelets do not possess the ability to spread over an exposed surface (Grouse et al., 1990). In addition, once shape change has occurred, bovine platelets are more resistant to defamation than human platelets (Grouse et al., 1990). The significance of these findings are unknown (Gentry, 1992; Zucker-Franklin et al., 1985). Difl‘erences exist between bovine platelets and platelets from other species in the ability of agonists to induce platelet aggregation (Gentry, 1992; Marzec et al., 1975). Bovine platelets, in general, are less receptive to stimulation by adenosine diphosphate than platelets of most other mammalian species (Marzec et al., 1975). Bovine platelets require 5 to 10 times more adenosine diphosphate to elicit maximal aggregation than platelets from sheep, horses, humans, and primates (Bondy & Gentry, 1989). This decreased sensitivity to adenosine diphosphate observed in vitro may be due to impaired secretion because bovine platelets do not possess an open canalicular system (Bondy & Gentry, 1989). In addition, bovine platelets are not responsive to either arachidonic acid or thromboxane A2, in spite of the fact that large amounts of thromboxane A2 are released from these platelets in response to agonists such as collagen and adenosine diphosphate (Meyers et al., 1980). In fact, there is no correlation between thromboxane A2 production and release and the extent of platelet aggregation in bovine platelets (Bondy & Gentry, 28 not. the steam“ arachidonic acid path“ pathways such as 3 Ph‘ 2989; Gentry et al.. cyclooxygenase inhibitt tattle (Gentry er al., 198 Variation exists in Dubielzig, 1993; V’augl calves ranging in age fr. ”L 1974)- In their stur 175.000; however, the tithose Holstein calx dwtlrer Study TCport e c iEe to be 841,000 plat- etuI.,1973). In 2mm: as 841,000 platelets] 1989). The significance of this finding is that bovine platelets have a poorly developed arachidonic acid pathway, and platelet aggregation in cattle is dependent upon other pathways such as a phospholipid pathway and calcium mobilization (Bondy & Gentry, 1989; Gentry et al., 1989). This observation is supported by the inability of cyclooxygenase inhibitors such as aspirin to have an effect on platelet aggregation in cattle (Gentry et al., 1989). Variation exists in the normal platelet count in cattle (Nelson et al., 1974; Semrad & Dubielzig, 1993; Vaugher et al., 1973). Platelet counts on 32 normal male Holstein calves ranging in age from 9 to 15 weeks were 192,000 to 892,000 platelets/pl (Nelson et al., 1974). In their study, the mean :t 2 standard deviations of the mean was 542,000 :r: 175,000; however, the actual observed minimum and maximum platelet counts observed in those Holstein calves was 274,000 and 836,000 platelets/til (Nelson et al., 1974). Another study reported the mean platelet count in Holstein calves less than 2 months of age to be 841,000 platelets/pl with a range of 480,000 to 1,200,000 platelets/pl (V augher et al., 1973). In another 4 calves less than 2 months of age, the average platelet count was 841,000 platelets/pl (Winqvist, 1954). The mean platelet count in 14 Holstein calves weighing between 115 to 300 pounds was 632,000 platelets/pl (Marzec et al., 1975). Only one study has evaluated the platelet count in neonatal calves (Semrad & Dubielzig, 1993). At birth, the mean platelet count i standard error of the mean for 5 Holstein calves was 212,050 3: 73,945 platelets/pl. The platelet count increased progressively as the calves aged with a mean platelet count of 325,500 :1: 55,672 on day l of age, 447,750 :t 48,120 on day 2 of age, 502,000 i 75,182 on day 3 of age, and 645,750 :t 54,571 on day 4 of age (Semrad & Dubielzig, 1993). Platelet counts were not continued beyond 29 day 4 of age in that st likely due to the differ. calves used in the varior Platelet life span in 1975). Platelet life spar Whflique of ex vim vslr implanted with an intrai lnornral male Jersey ca 0161 days for a male 198). Another study u platelet life span in l- detcrrnined for 5 calves a". 1975). In the earlie 10 k 9.7 days in fema mm back into th to9.7 days in cattle. day 4 of age in that study. Reasons for the reported variability in platelet counts are likely due to the different methods of assessing platelet count and the different-aged calves used in the various studies. Platelet life span in cattle has also been determined (Baker et al., 1998; Marzec et al., 1975). Platelet life span ranges from 4.9 to 10 days. The most recent study assessed the technique of ex vivo whole blood biotinylation for evaluating platelet life span in calves implanted with an intraventricular assist device (Baker et al., 1998). Platelet life span in 1 normal male Jersey calf was calculated to be 4.9 days, compared to a platelet life span of 6.1 days for a male Jersey calf with an intraventn'cular assist device (Baker et al., 1998). Another study utilized disappearance of radioactivity as a method for determining platelet life span in Holstein calves (Marzec et al., 1975). Platelet life span was determined for 5 calves and averaged 6.22 days with a range of 5 to 10 days (Marzec et al., 1975). In the earliest study on platelet life in calves, platelet life span was determined to be 9.7 days in female Holstein calves using platelets radiolabeled in vitro, and then transfused back into the autologous calf. In summary, platelet life span ranged from 4.9 to 9.7 days in cattle. Platelet Disorders of Cattle Platelet disorders can be broadly categorized as qualitative or quantitative, and inherited or acquired. Qualitative platelet disorders are also known as thrombocytopathies and involve impairment of platelet function. Quantitative platelet disorders may involve a decrease in the number of circulating platelets 3O (rhormobocstopenia) or (thrombocytosis). Ihrt quantitative platelet dlSi In general. platelet . leatrnes. as compared it Petechiae are common nith platelet disorders. membranes, and prolo contrast abnormalities bleeding, and bleeding rare nith coagulation . “the bleedmg maV St QWIIatiVCPIQIeIet d A Summary Of 1 to. - . merited disorders 1 hmmbopathy an (thormobocytopenia) or may involve an increase in the number of circulating platelets (thrombocytosis). Thrombocytosis has not been reported in cattle, so the discussion on quantitative platelet disorders will be limited to thrombocytopenia. In general, platelet abnormalities often present with a characteristic group of clinical features, as compared to other abnormalities of the coagulation system (J ohnstone, 1988). Petechiae are common clinical signs and hematomas are rare clinical signs associated with platelet disorders. In addition, bleeding at multiple sites and often involving mucous membranes, and prolonged bleeding from cuts are typical of platelet disorders. In contrast, abnormalities in the coagulation system often present with hematomas, localized bleeding, and bleeding into body cavities, muscles, and joints. Moreover, petechiae are rare with coagulation disorders, and the bleeding may be delayed at onset, then profuse, or the bleeding may stop and start again (J ohnstone, 1988). Qualitative platelet disorders of cattle A summary of qualitative disorders of cattle is presented in Table 1.2 (page 35). Inherited disorders have been the best characterized and include Simmental hereditary thrombopathy and the Chédiak-Higashi syndrome. Acquired qualitative platelet disorders are less characterized in cattle. The most common acquired qualitative platelet disorders in dogs and cats are drug-induced (Ruiz de Gopegui & Feldman, 1998); however, drug-induced platelet disorders have not been described in cattle. Qualitative platelet disorders are characterized by an increased tendency to bleed or a prolonged bleeding time in the presence of a normal or increased platelet count (Jain, 1986). Diagnosis of qualitative bleeding disorders involves evaluation of the bleeding time and 31 platelet function studie of the platelet count coagulation profiles to r Chedialr-Higashi s‘ reported in hereford (l (Ayers etaI., 1988). ar 1999; Shiraishi er al., humans. cats. rats. mic Gopegui & Feldman. mode of inheritance ( albinos with genera lel’aflsse Black cattlr 1997.“, Ruiz the 60p. tame (Bell et al., iauegatiom Platele an], 1976‘. Ogaw Chédiak‘mgashj s platelet function studies, specifically platelet aggregation testing. In addition, evaluation of the platelet count to rule out quantitative platelet disorders and evaluation of coagulation profiles to rule out disseminated intravascular coagulation are necessary. Chédiak-Higashi syndrome is an inherited disease of platelet function that has been reported in Hereford (Bell et al., 1976; Burns et al., 1984; Padgett et al., 1967), Brangus (Ayers et al., 1988), and Japanese Black cattle (Akuzawa et al., 1991; Fushuku et al., 1999; Shiraishi et al., 1998). Chédiak-Higashi syndrome has also been reported in humans, cats, rats, mice, foxes, mink, and killer whales (Prieur & Collier, 1978; Ruiz de Gopegui & Feldman, 1998). Chédiak-Higashi syndrome has an autosomal recessive mode of inheritance (Padgett et al., 1964). In general, affected animals are incomplete albinos with generalized oculocutaneous hypopigrnentation, with the exception of Japanese Black cattle in which cutaneous ablinism is an infiequent finding (Ogawa et a1 ., 1997; Ruiz de Gopegui & Feldman, 1998). Bleeding times are prolonged in affected cattle (Bell et al., 1976; Ogawa et al., 1997). In studies evaluating in vitro platelet aggregation, platelets from afi‘ected cattle fail to aggregate in response to collagen (Bell et al., 1976; Ogawa et al., 1997). Ultrastructurally, platelets from cattle affected with Chédiak-Higashi syndrome are characterized by a deficiency of platelet.dense granules (Bell et al., 1976; Ogawa et al., 1997). Decreased granule contents and decreased secretion of ATP, ADP, and serotonin are characteristic of platelets from cattle with Chédiak-Higashi syndrome. These findings have characterized Chédiak-Higashi syndrome as a storage pool disease (Bell et al., 1976; Ogawa et al., 1997; Shiraishi et al., 1998). Recent evidence suggests that decreased granule content is not the only mechanism of decreased platelet function, and impaired cytosolic calcium mobilization 32 may be involved (Shir. been hypothesized to Japanese Black cattle . release of endogenous 199%). Simmental herediti 199.7; Searcy et al., 19' l993h). Mode of inhei n autosomal recessive exhibit recurrent episr result of minor traur itshitting hematolog hicchemistry profiles reference ranges and no]. 1993a), In f? e g 1 on re5130115 its. muse to platelet , v35 decmased Or it may be involved (Shiraishi et al., 1998). Impairment of calcium mobilization has now been hypothesized to be the major mechanism of decreased platelet aggregation in Japanese Black cattle affected with Chédiak-Higashi syndrome, rather than a decreased release of endogenous substances such as ADP, ATP, and serotonin (Shiraishi et a1 ., 1998) Simmental hereditary thrombopathy has been described in Canada (Gentry et a1 ., 1997; Searcy et al., 1990) and the United States (Steficek et al., 1993a; Steficek et al., 1993b). Mode of inheritance is unknown, but the distribution in affected cattle supports an autosomal recessive mode of inheritance (Steficek, 1994). Affected cattle commonly exhibit recurrent episodes of epistaxis and hematuria, and they bleed excessively as a result of minor trauma, during surgery, and at parturition. Laboratory evaluation, including hematologic assessment and platelet counts, coagulation profiles, and biochemistry profiles has been performed, and all values were comparable to normal reference ranges and normal matched Simmental cattle (Searcy & Petrie, 1990; Steficek et al., 1993a). In platelet function studies, platelets from affected cattle had no aggregation response to adenosine diphosphate or the calcium ionophore A23187. In response to platelet activating factor and thrombin, platelet aggregation, although present, was decreased or took longer to complete when compared to control bovine platelets (Steficek et al., 1993a). Platelet shape change was demonstrated with all agonists examined, which indicated the presence of functional receptors (Steficek et al., 1993a). Ultrastructurally, platelets from affected Simmental cattle appeared normal. At present, the impairment in platelet fimction observed in affected Simmental cattle appears to be altered mobilization or utilization of external calcium (Steficek, 1994). 33 infection of cattlt ,l—lannheimia, has been al., 1998). Platelet fur Mannheimia hemolytic platelet agonists sucli ; al.1998). In additior leulotoxin in an in \‘it 3995”- The findings associated with Mann tattle infected with .‘l; onplatelets, and the , Pemolyn'ca, BVDV i ll'. The trichothece ills rate and extem I987). The mom < bowie platelets w Infection of cattle with Pasteurella hemolytica, recently moved to the genus Mannheimia, has been associated with an acquired qualitative platelet defect (Cheryk et al., 1998). Platelet function was assessed before and after intrabronchial challenge with Mannheimia hemolytz'ca, and platelets became more reactive to stimulation with known platelet agonists such as adenosine diphosphate and platelet activating factor (Cheryk et al., 1998). In addition, direct exposure of bovine platelets to Mannheimia hemolytica leukotoxin in an in vitro setting resulted in increased platelet reactivity (Cheryk et al., 1998). The findings of this study may partially explain the respiratory tract lesions associated with Mannheimia hemolytica infection in cattle. Thrombosis in the lungs of cattle infected with Mannheimia hemolytica may be the result of the action of leukotoxin on platelets, and the subsequent increased stimulus for aggregation. Unlike Mannheimia hemolytica, BVDV induces decreased platelet function as discussed in Chapters 1]] and IV. The trichothecene mycotoxin, T—2 mycotoxin, has been demonstrated to impair both the rate and extent of platelet aggregate formation (Bondy et al., 1989; Gentry et al., 1987). The most dramatic change following in vitro exposure of the T-2 mycotoxin to bovine platelets was the relative instability of the platelet aggregates that formed in the presence of the toxin (Bondy et al., 1989). The biological significance of these findings is unknown, as these studies were conducted because bovine platelets may be used as a model cell system to evaluate the effects of T-2 toxin on cell fimction in vitro (Bondy et al., 1989; Gentry et al., 1987). 34 III Table 1.2. Qualitative platelet disorders of cattle Inherited qualitative platelet disorders of cattle Disorder Reference(s) Simmental hereditary thrombopathy (Gentry et al., 1997; Steficek et a1 ., 1993a; Steficek et al., 1993b) Chédiak—Higashi syndrome (Bell et al., 1976; Kim et al., 1985; Menard et al., 1990; Meyers & Seachord, 1990; Meyers et al., 1982; Prieur et al., (Akuzawa et al., 1991; Fushuku et al., 1999; Fushuku et al., 1998; Shiraishi et aL,l998: Acquired qualitative platelet disorders of cattle Disorder Reference(s) Bovine viral diarrhea virus (Walz et al., 1999b) Pasteurella haemolytica A1 (Mannheimia (Cheryk et al., 1998) haemolytica) Mycotoxicosis (T-2 toxin) (Bondy etal., 1989) 35 Quantitative platelet d Asununao' 0W“ 39). Thrombocytope“ below the minimum no spontaneous hemorrha; 30.000 plateletsEuL (11 cattle when the platele ham hemorrhage. commonly seen wher platelets'uL. Regardless of the he induction of thrt abnormal sequestrati 5330016 mechanism mmbocllopenia c Uéorders of platelet cruel-708B. In C0“ Penpheral circulatir D“Seminated . 131112 ( Jain, 1986), Quantitative platelet disorders of cattle A summary of quantitative platelet disorders of cattle is presented in Table 1.3 (page 39). Thrombocytopenia is defined as a reduction in the number of circulating platelets below the minimum normal level for the species (Jain, 1986). In general, clinical signs of spontaneous hemorrhage rarely occur unless the platelet count has decreased to less than 20,000 platelets/pl. (Jain, 1986). However, bleeding has sometimes been observed in cattle when the platelet count is between 20,000 and 50,000 platelets/pL (Jain, 1986). Purpura, hemorrhage, prolonged bleeding times, and failure of clot retraction are commonly seen when the circulating platelet count decreases to less than 10,000 platelets/uL. Regardless of the underlying etiology, only three basic mechanisms are involved in the induction of thrombocytopenia: decreased production, accelerated destruction, or abnormal sequestration of platelets (Warkentin & Kelton, 1994). In certain diseases, multiple mechanisms may be contributing to the thrombocytopenia. The mechanisms of thrombocytopenia can often be differentiated by examination of the bone marrow. Disorders of platelet production often have bone marrow findings consistent with aplasia or necrosis. In contrast, bone marrow findings in cases of accelerated destruction in the peripheral circulation often demonstrate increased thrombopoiesis. Disseminated intravascular coagulation is a common cause of thrombocytopenia in cattle (Jain, 1986). Disseminated intravascular coagulation is a disease process that may have many different causes, of which thrombocytopenia is one of its clinical features. Disseminated intravascular coagulation may be initiated by a single cause or by multiple causes occurring sequentially or simultaneously (Levi & Ten Cate, 1999). Regardless of 36 cause. the common tl‘ hypercoagulable state r causes thrombocytope Kelton l994). Thrr coagulation has been 1" Kilrovic, 1989; Kiper é endotoxemia and $6135 eadotoxemia are thé IT cattle, and disseminate calves administered en Several parasitic « hematological finding 3:158le bigemr‘na, B Dalgliesh et al., 1976 T’lPdnosoma congo. Mule, “ith Trj'pa lFOTSbefg el 0]. 19_ mmmwytopeni a a cause, the common thread is the activation of the coagulation pathways leading to a hypercoagulable state (Levi & Ten Cate, 1999). Disseminated intravascular coagulation causes thrombocytopenia due to an increased consumption of platelets (W arkentin & Kelton, 1994). Thrombocytopenia as a component of disseminated intravascular coagulation has been observed in cattle with toxic mastitis (Buntain, 1980; Heuwieser & Kikovic, 1989; Kiper & Paulsen, 1988), metritis (Braun et al., 1990; Buntain, 1980), and endotoxemia and sepsis (Bowersock et al., 1990; Thomson et al., 1974). Sepsis and endotoxemia are the most common causes of disseminated intravascular coagulation in cattle, and disseminated intravascular coagulation has been experimentally reproduced in calves administered endotoxin intravenously (N agaraja et al., 1979). Several parasitic diseases of cattle have been associated with thrombocytopenia as a hematological finding. Thrombocytopenia has been observed with the blood parasites Babesia bigemina, Babesia argentina, and Babesia bovis (Brun-Hansen et al., 1998a; Dalgliesh et al., 1976; Smith et al., 1980). Trypanosomes, including Trypanosoma vivax, Trypanosoma congolense, and T rypanosoma brucei, may also induce thrombocytopenia in cattle, with Twanosoma vivax inducing the greatest depressions in the platelet count (Forsberg et al., 1979; Wellde et al., 1989a; Wellde et al., 1989b). The mechanism of thrombocytopenia associated with these blood-bome parasites is thought to be the result of disseminated intravascular coagulation (Wellde et al., 1989a). Pancytopenia is frequently observed in cattle infected with Trypanosoma vivax (Anosa et al., 1992), and bone marrow suppression may be partly responsible for the depressions in blood counts observed (Anosa et al., 1992). A recent study evaluating the bone marrow in cattle infected with T. vivax revealed a dyserythropoiesis and dysgranulocytopoiesis (Anosa et 37 al. W92). With res hmased and some neutmphils. and 1m; ulnoun but likely in mi. lheileria part". which infection in cat Daugschies er al.. 19¢. mi and T. pan-a i: “Jule the mechanism Decreased prod; thmmbocimpenra in Bone marrow aplas myeloid and erfihrc mecomPound re3p liming in Cattle marrow al’lEiSia am we also been obs Hayashj 9, al., 19 033011 8; Reed3 \ demonstrate lemonhage. al., 1992). With respect to the platelet precursors, the megakaryocyte volume was increased and some megakaryocytes showed emperipolesis of red blood cells, neutrophils, and lymphocytes. The significance of these megakaryocyte findings is unknown, but likely indicates megakaryocyte activation (Anosa et al., 1992). Sarcocystis cruzei, Theileria parva, and Ehrlichia phagocytophila are three additional parasites in which infection in cattle may result in thrombocytopenia (Brun-Hansen et al., 1998b; Daugschies et al., 1998; Kimeto, 1976). The mechanism of thrombocytopenia for S. cruzei and T. parva is considered to be due to disseminated intravascular coagulation, while the mechanism of thrombocytopenia for E. phagocytophila is unknown. Decreased production of platelets has been proposed as the mechanism of thrombocytopenia in cattle with bracken fern toxicity (Evans, 1964; Hirono et al., 1984). Bone marrow aplasia and pancytopenia may be observed due to suppression of the myeloid and erythroid cell lines, as well as the megakaryocyte cell line (Evans, 1964). The compound responsible for the bone marrow suppression associated with bracken fern poisoning in cattle has been determined to be ptaquiloside (Hirono et al., 1984). Bone marrow aplasia and subsequent anemia, thrombocytopenia, and leukopenia (neutropenia) have also been observed in cattle fed soybean meal from which the oil had been extracted by trichlorethylene. Prolonged administration of furazolidone to calves (Buck, 1975; Hayashi et al., 1976; Taylor et al., 1991) and mycotoxins such as vomitoxin or citrinin (Dyson & Reed, 1977; Griffiths & Done, 1991; Jeffers & Lenghaus, 1986), have also been demonstrated to result in pancytopenia, bone marrow aplasia, and clinical signs of hemorrhage. 38 We 13. Quamlml‘ Diso i (Babesiosis (Babesia r higemina) Box-inc viral diarrhea l l \\ Bracken fem toxrcosi Disseminated intrava (sepns, endometritis \ East Coast Fever ( T. infection) l Ehrlichia phagocm \ iWOllClone p0 i SO “,Llfnphosarcoma (1 Mycotoxicosis S» . . l amigos“ (Sat Table 1.3. Quantitative platelet disorders of cattle fl Qauntltave ptlaelet disrders of cale Disorder Reference(s) Babesiosis (Babesia bovis and Babesia bigemina) (Brun-Hansen et al., 1998a; Dalgliesh et al., 1976; Smith et al., 1980) Bovine viral diarrhea virus (Bezek et al., 1994; Bolin & Ridpath, 1992; Corapi et al., 1990; Corapi et al., 1989; Rebhun et al., 1989; Walz et al., 199929 Bracken fern toxicosis (Evans, 1964; Hirono et al., 1984) Disseminated intravascular coagulation (sepsis, endometritis, mastitis) (Bowersock et al., 1990; Braun et al., 1990; Buntain, 1980; Heuwieser & Kikovic, 1989; Kiper & Paulsen, 1988; Thomson et al., 1974) East Coast Fever (Theileria parva infection) (Burridge, 1973; Kimeto, 1976) Ehrlichia phagocytophila infection (Bum-Hansen et al., 1998b) Furazolidone poisoning (Buck, 1975; Hayashi et al., 1976; Taylor et al., 1991) Lymphosarcoma (bone marrow infiltration by neoplastic lymphocytes) (McLaughlin, 1989) Mycotoxicosis (Dyson & Reed, 1977; Griffiths & Done, 1991; Jeffers & Lenghaus, 1986; Nicholls et al., 1985) Sarcocystosis (Sarcocystis cruzei) (Daugschies et al., 1998; Frelier & Lewis, 1984) Trichloroethylene-extracted soybean oil meal (Pritchard et al., 1956; Strafuss & Zimmermann, 1967) Trypanosomiasis (T rypanosoma vivax, Trypanosoma congolense, and Tmanosoma brucez) (Forsberg et al., 1979; Wellde et al., 1983; Wellde et al., 1989a; Wellde et al., 1989b) 39 SomeEnmples of VI Thrombocytopeni hemorrhagic consequ review will briefly exa ofhumans and animal. result from only three mom sequestratic limmbocimpenia in mm “his“ that in mECham'sm. Althr CharacteriZed, the rr understood. Since grollping Viruses by ‘ Many VimSes h Maine, and to humbocnopenia i hr mm Virus was 1 I C of the genus Pesti‘.‘ Some Examples of Virus-induced Thrombocytopenia in Other Species Thrombocytopenia can accompany viral infections with potentially fatal hemorrhagic consequences (Zucker-Franklin, 1994). The remainder of this literature review will briefly examine the mechanisms of thrombocytopenia in other viral infections of humans and animals. As previously stated, thrombocytopenia, regardless of cause, can result from only three basic mechanisms: increased destruction, decreased production, or abnormal sequestration of platelets (Warkentin & Kelton, 1994). Virus-induced thrombocytopenia in both humans and animals can occur by all three mechanisms, but most viruses that induce thrombocytopenia do so by invoking more than a single mechanism. Although immune-mediated destruction of platelets is the best characterized, the mechanisms of many virus-induced thrombocytopenias are poorly understood. Since most viruses induce thrombocytopenia by multiple mechanisms, grouping viruses by their mechanism of thrombocytopenia induction is not possible. Many viruses have been associated with thrombocytopenia in human and veterinary medicine, and to list and discuss all viruses that have been associated with thrombocytopenia is beyond the scope of this review. Therefore, 5 viruses relevant to human and veterinary medicine have been selected for a more detailed discussion. Hog cholera virus was chosen for discussion because of its relationship to BVDV as a member of the genus Pestivirus within the family Flaviviridae (Wengler, 1991). Like hog cholera virus, Afi'ican swine fever virus also causes a hemorrhagic syndrome in pigs (Rodriguez et al., 1996). The Afiican swine fever virus was selected for review because of reports demonstrating and characterizing megakaryocyte infection with this virus (Perez et al., 40 1991; 110000131 9‘ “ anemia rims were chi due to the AIDS (: immunodeficiency \‘i hmily Retroviridae. ‘ platelets may be into Cmuford et al., 1996 BlDV in the family ' l‘h'BSlaWay & Blok. 90100136 of an in mmbocylopenia is Peripheral circulatio H03 Cholera Virus Hog Cholera \ Within the family 1 may result in mu 1 1997; Rodriguez et al., 1996). Human immunodeficiency virus and equine infectious anemia virus were chosen due to the vast amount of research generated in recent years due to the AIDS (Acquired Immunodeficiency Sydrome) epidemic. Both human immunodeficiency virus and equine infectious anemia viruses are RNA viruses in the family Retroviridae, and recent reports have suggested that decreased production of platelets may be involved in the induction of thrombocytopenia (Ballem et al., 1992; Crawford et al., 1996). Finally, dengue virus was chosen because of its relationship to BVDV in the family Flaviviridae, although dengue virus belongs in the genus F lavivirus (W estaway & Blok, 1997). In addition, dengue virus has long been considered the prototype of an immune-mediated hemorrhagic response to a viral infection, and thrombocytopenia is thought to occur through accelerated destruction of platelets in the peripheral circulation (Gubler, 1998). Hog cholera virus Hog cholera virus is related to BVDV, and is a member of the genus Pestivirus within the family Flaviviridae (Wengler, 1991). Infection of pigs with hog cholera virus may result in multiple clinical forms including acute, subacute, chronic, subclinical, or delayed-onset forms (Dahle & Liess, 1992; Terpstra, 1991). Like BVDV, isolates of hog cholera virus vary widely in virulence, and some isolates are associated with severe clinical disease in pigs (Dahle & Liess, 1992; Terpstra, 1991). Clinically, the acute form of hog cholera virus infection may result in a severe and generalized disease in which hemorrhages on the skin and multiple internal tissues are a characteristic finding (Dahle & Liess, 1992). Laboratory evaluation has demonstrated 41 thrombOCllOl’mla an cholera virus (Dahle thrombocytopenia 3‘“ mg hog cholera Vi conjunction with 5ch cholera Virus-induced consumption caused . disseminated intrava- tnegaharyocytes (Dahl Inadequate platele of hog cholera virus—i degeneration have bee infection of megakaq l9’98b). Pigs were i' Wimoltismchemica thrombocytopenia and leukopenia in pigs infected with highly virulent isolates of hog cholera virus (Dahle & Liess, 1992). The mechanism of hog cholera virus-induced thrombocytopenia and hemorrhage is multifactorial. The multiple hemorrhages seen in acute hog cholera virus infections are caused by degeneration of endothelial cells in conjunction with severe thrombocytopenia (Dahle & Liess, 1992; Terpstra, 1991). Hog cholera virus-induced thrombocytopenia is believed to be due to combinations of platelet consumption caused by direct damage to the platelets, virus-induced endothelial lesions, disseminated intravascular coagulation, and inadequate platelet production by the megakaryocytes (Dahle & Liess, 1992; Weiss et al., 1973). Inadequate platelet production has received the least consideration as the mechanism of hog cholera virus-induced thrombocytopenia; however, megakaryocyte necrosis and degeneration have been reported (Hoffmann et al., 1971). In addition, hog cholera virus infection of megakaryocytes has been recently described (Gomez-Villamandos et al., 1998b). Pigs were inoculated with a highly virulent strain of hog cholera virus, and immunohistochemical and ultrastructural examination revealed hog cholera virus infection in approximately 2.5-9.0% of megakaryocytes from days 2 through 9 after inoculation (Gomez-Villamandos et al., 1998b). Viral antigen was demonstrated in platelets, and megakaryocyte infection with hog cholera virus was proposed as the origin of viral antigen in platelets. In addition, it was hypothesized that virus association with platelets may represent a passive vehicle for spreading the virus in the pig. Like BVDV, decreased production of platelets as a result of megakaryocyte infection may be important in the pathogenesis of hog cholera virus-induced thrombocytopenia. African swine fever virus 42 African swine t differentiated from ac~ hemorrhages on the sl inintemal organs ar snine fever virus, Thm"lbOCyTOpeni infection. and has be: of. 19851); Rodriguez thrombocytopenia is demotion, accelerat coagulation. and imi mechanism in differe “i 1997; Rodriguez he dependent upon t highly Virulent isola‘ through the consur k‘eminated intrav; menonuclear phaQO African swine fever virus is associated with clinical signs that cannot be differentiated from acute hog cholera virus infection (Rodriguez et al., 1996). Petechial hemorrhages on the skin, especially the ears and on flanks, and widespread hemorrhages in internal organs are typical findings associated with infection of pigs with African swine fever virus. Thrombocytopenia is a characteristic finding during Afi'ican swine fever virus infection, and has been experimentally reproduced (Edwards et al., 1985a; Edwards et al., 1985b; Rodriguez et al., 1996). The mechanism of Afiican swine fever virus-induced thrombocytopenia is multifactorial and incompletely understood. Immune-mediated destruction, accelerated platelet aggregation as a result of disseminated intravascular coagulation, and impaired thrombopoiesis have all been considered as the primary mechanism in different reports (Edwards et al., 1985a; Edwards et al., 1985b; Perez et al., 1997; Rodriguez et al., 1996). However, involvement of a primary mechanism may be dependent upon the virulence of the isolate (Rodriguez et al., 1996). For example, highly virulent isolates of African swine fever virus primarily induce thrombocytopenia through the consumption of platelets in the peripheral circulation as a result of disseminated intravascular coagulation. Infection and mass destruction of cells of the mononuclear phagocyte system leads to the systemic release of active products such as enzymes, monokines, complement factors, and products of arachidonic acid metabolism (Levi & Ten Cate, 1999). These factors have a significant effect on hemostasis and endothelial cells (Levi & Ten Cate, 1999) and are important in the pathogenesis of thrombocytopenia in pigs infected with highly virulent isolates (Rodriguez et al., 1996). In pigs infected with moderately virulent isolates of African swine fever virus, inhibition 43 ofmmrnbopoiesis as and bone marrow nc number of megakary finding coincided wit 1996). Megakaryoc infection of pigs with eta]. 1997). The rel isunknoun, but was| thrombopoiesis or a s. factor-a and interlet lpoptosis may be : Olsen'ed with Africa Hum" Wunodej mombOCfiopg in (HIV) infeCti< 011992). mom ad is considered thrombocbtopenia impll‘matic HI\ Emu} “Operti a of thrombopoiesis as a result of me gakaryocyte infection, megakaryocyte degeneration, and bone marrow necrosis may be of primary importance. Infection of a substantial number of megakaryocytes by Afiican swine fever virus has been observed, and this finding coincided with considerable reductions in the platelet count (Rodriguez et al., 1996). Megakaryocyte apoptosis has also been observed following experimental infection of pigs with Afiican swine fever virus (Gomez-Villamandos et al., 1998a; Perez et al., 1997). The relationship between megakaryocyte apoptosis and thrombocytopenia is unknown, but was thought to occur as the result of an overwhelming stimulus for thrombopoiesis or a secondary effect of the release of cytokines including tumor necrosis factor-or and interleukins. Indirect damage to megakaryocytes and megakaryocyte apoptosis may be an additional contributing mechanism for the thrombocytopenia observed with Afi'ican swine fever virus infection. Human immunodeficiency virus Thrombocytopenia has been reported in association with human immunodeficiency virus (HIV) infection and the acquired immunodeficiency syndrome (AIDS) (Kaplan et al., 1992). Thrombocytopenia can be an early manifestation in asymptomatic patients, and is considered one of the criteria of the AIDS-related complex. The incidence of thrombocytopenia is 0-10% in asymptomatic HIV patients, and may be up to 40% in symptomatic HIV patients (Rossi et al., 1990). The mechanisms leading to thrombocytopenia in HIV-infected patients are multiple. Immune-mediated destruction of platelets in the peripheral circulation has been implicated as a mechanism of HIV- induced thrombocytopenia due to the presence of platelet-associated irnmunoglobulins, 44 specific antiplatelet . platelet membranes (h antigens has also b thrombocytopenia (Ol that recognizes the glycoprotein gpIIIa (g m1’00“ for an immu associated antibodief ammombocstopenic Direct involvemfi‘ the pathogenesis of kalm1994). Vii patients (Louache e! 1990). In addit megakaq'ocyte nuclt Patients (Zucker-Fra HIV" can productive that unpaired thror ' b0631013etti21. Mean platelet i ilNIhesis that HIV at can p latelet \ 5111': 1982) . gem specific antiplatelet autoantibodies, and circulating immune complexes adsorbing to platelet membranes (Kaplan et al., 1992). Molecular mimicry between HIV and platelet antigens has also been implicated as an immune-mediated cause of HIV -induced thrombocytopenia (Oksenhendler & Seligmann, 1990). An antibody has been identified that recognizes the HIV envelope glycoprotein gp120 and the platelet membrane glycoprotein gpIIIa (Oksenhendler & Seligmann, 1990). However, there is lack of support for an immune-mediated mechanism, which includes evidence that platelet- associated antibodies have been demonstrated in both thrombocytopenic and nonthrombocytopenic HIV-infected patients (Walsh et al., 1984). Direct involvement of progenitor cells has also been considered to be important in the pathogenesis of HIV -induced thrombocytopenia (Ballem et al., 1992; Zucker- Franklin, 1994). Viral RNA has been identified in megakaryocytes from HIV -infected patients (Louache et al., 1991; Zucker-Franklin & Cao, 1989; Zucker-Franklin et al., 1990). ' In addition, ultrastructurally aberrant me gakaryocytes and denuded megakaryocyte nuclei can be found in bone marrow specimens of most HIV-infected patients (Zucker-Franklin et al., 1989). This finding, in combination with the fact that HIV can productively infect megakaryocyte cell lines in vitro, supports the hypothesis that impaired thrombocytopoiesis is at least partially responsible for HIV-induced thrombocytopenia. Mean platelet volume measurements from HIV-infected patients also support the hypothesis that HIV -induced thrombocytopenia may be due to impaired thrombopoiesis. The mean platelet volume has been used to help classify thrombocytopenia (Bessman et al., 1982). In general, platelets recently released into the bloodstream from the bone 45 marrow are large“ thrombocytopenia is platelets is often asso Conversely. aged pla‘, thrombocytopenia is low mean platelet voll decreased platelet pro In spite of confl decreased platelet prr induced thrombocymp ”Indium-labelled pl lBaIlem er al., 199: lDdicating that both ) . ~l:I'llclIOI'l Of platele “Electa ble horseS 1n marrow are larger, and therefore, the mean platelet volume is elevated when the thrombocytopenia is due to increased destruction of platelets. Peripheral destruction of platelets is often associated with an increased megakaryocytopoiesis and thrombopoiesis. Conversely, aged platelets are smaller and mean platelet volume is decreased when the thrombocytopenia is due to decreased marrow production (Bessman et al., 1982). The low mean platelet volume observed in HIV-infected patients supports the hypothesis that decreased platelet production is involved (Koenig et al., 1991). In spite of conflicting evidence suggesting that accelerated platelet destruction or decreased platelet production is the predominant mechanism, the pathogenesis of HIV- induced thrombocytopenia involves both mechanisms. A study evaluating the survival of 111Indium-labelled platelets demonstrated a moderate reduction in platelet survival (Ballem et al., 1992). In addition, mean platelet production was decreased, thus indicating that both accelerated platelet destruction and decreased platelet production contribute to HIV -induced thrombocytopenia (Ballem et al., 1992) Equine infectious anemia virus Thrombocytopenia is a common finding in horses infected with equine infectious anemia virus. The mechanism of thrombocytopenia in horses infected with equine infectious anemia virus was originally believed to be due to immune-mediated destruction of platelets, because platelet-associated irnmunoglobulins G and M were detectable in horses infected with equine infectious anemia virus (Clabougb et al., 1991). However, the development of severe anemia and thrombocytopenia in experimentally infected severe combined immunodeficient foals, which lack fimctional B and T 46 lymphocytes. promp important and may bc Suppression of p mechanism of thro= tCrauford et al., 190 radioactive label. wa anemia Virus (Crawfc 0f investigators be] independent of the i normal foals and Sew anemia tirus (Crawfc Megakarh’ocme i infectious anemia V'lr 0f equine infection mechanism 0f Suppl‘ lymphocytes, prompted the hypothesis that non-immune—mediated mechanisms are important and may be predominant (Crawford et al., 1996; Perryman et al., 1988). Suppression of platelet production has been recently proposed to be the primary mechanism of thrombocytopenia associated with equine infectious anemia virus (Crawford et al., 1996). Platelet production, as measured by metabolic incorporation of radioactive label, was reduced in foals experimentally infected with equine infectious anemia virus (Crawford et al., 1996). Platelet life span was also reduced, but this group of investigators believed that platelet removal in the peripheral circulation was independent of the immune system, as shortened platelet life span was observed in normal foals and severe combined immunodeficient foals infected with equine infectious anemia virus (Crawford et al., 1996). Megakaryocyte infection has not been demonstrated in horses infected with equine infectious anemia virus (Clabougb et al., 1991; Crawford et al., 1996). An indirect effect of equine infectious anemia virus on megakaryocytes has been proposed as the mechanism of suppressed thrombopoiesis (Tomquist & Crawford, 1997). Plasma from horses infected with equine infectious anemia virus suppressed megakaryocyte colony growth in vitro (Tomquist & Crawford, 1997). In addition, the suppression was partially reversed by the addition of neutralizing antibodies to tumor necrosis factor-or and transforming growth factor-l3 (Tomquist & Crawford, 1997). The levels of those cytokines, as well as the cytokine interferon-or, were demonstrated to be elevated in the serum and bone marrow of foals infected with equine infectious anemia virus (Tomquist et al., 1997). Elevations in these cytokines during infection with equine infectious 47 anemia virus may be direct effect of the Vin Dengue virus Dengue virus is c dengue virus belongs infection may result ' mild febrile illness nomenclature describ confining, with two hemorrhagic fever (H. ml“ occur in adults a Nfihiae. epistaxis, 1 been described with turns in a13Proximat Sclfilimmng and rare Dengue hemo” Sl'fldrome MSW 1982). D. ’ Occurs p1 . ‘9 anemia virus may be responsible for the suppression of thrombopoiesis, rather than a direct effect of the virus on megakaryocytes (Tomquist et al., 1997). Dengue virus Dengue virus is classified in the same family as BVDV, the family Flaviviridae, but dengue virus belongs in the genus Flavivirus (Westaway & Blok, 1997). Dengue virus infection may result in a wide spectrum of clinical disease ranging from inapparent or mild febrile illness to severe and fatal hemorrhagic disease (Gubler, 1998). The nomenclature describing the clinical syndromes associated with dengue virus is rather confusing, with two distinct syndromes being described: dengue fever and dengue hemorrhagic fever (Halstead, 1982). Dengue fever is considered the less severe and may occur in adults and children (Halstead, 1982). Evidence of hemorrhage, including petechiae, epistaxis, gum bleeding, gastrointestinal hemorrhage, or purpuric lesions, has been described with dengue fever (Gubler, 1998; Halstead, 1982). Thrombocytopenia occurs in approximately 30% of patients with dengue fever, but the infection is usually self-limiting and rarely fatal (Gubler, 1998) Dengue hemorrhagic fever, which is sometimes referred to as dengue shock syndrome, occurs predominantly in children less than 15 years of age (Gubler, 1998; Halstead, 1982). Dengue hemorrhagic fever occurs in areas in which multiple dengue viruses are simultaneously endemic (Halstead, 1982). Hemorrhages are also common with dengue hemorrhagic fever, especially skin hemorrhages such as petechiae, purpuric lesions, and ecchymoses on the face and extremities (Gubler, 1998). Thrombocytopenia is a constant finding in cases of dengue hemorrhagic fever, and a platelet count of less 48 than l00,000 platelet I998). The pathogenesi mechanisms. The m mombOCE‘OI-‘enla m l platelets in the pei mechanism in patient (Finahara 8101,, 198. hemorrhagic fever us unis serotype afier l dengue vims serotyp the previous exposu {01111 MacrOphage mbOdy is heterolo; Ibis is referred to 3 m1 (Halstead, m‘fimaiEly 1 In addition t0 06c . “18 M a result C mJbOdy‘enhEmced ch‘I't has 3150 Get ’ 3 than 100,000 platelets/mm3 is usually found between days 3 and 8 of illness (Gubler, 1998) The pathogenesis of dengue virus-induced thrombocytopenia involves multiple mechanisms. The majority of studies have dealt with the mechanism of virus-induced thrombocytopenia in patients with dengue hemorrhagic fever. The predominant mechanism in patients with dengue hemorrhagic fever is immune-mediated destruction of platelets in the peripheral circulation and disseminated intravascular coagulation (Funahara et al., 1987a; Funahara et al., 1987b; Gubler, 1998; Halstead, 1982). Dengue hemorrhagic fever usually occurs in people who are exposed to a heterologous dengue virus serotype afier having developed an immune response to a previous and different dengue virus serotype (Gubler, 1998). Pre-existing, heterologous dengue antibody fi'om the previous exposure recognizes the infecting virus and antigen antibody complexes form. Macrophages phagocytize the antigen antibody complexes, and because the antibody is heterologous, it is not neutralized and the virus replicates in the macrophage. This is referred to as antibody-dependent enhancement, which has been studied in great detail (Halstead, 1982). The macrophages produce vasoactive mediators, which ultimately results in disseminated intravascular coagulation and a consumptive thrombocytopenia (Gubler, 1998). In addition to disseminated intravascular coagulation, platelet consumption also occurs as a result of antigen antibody complex deposition on the surface of platelets by antibody-enhanced binding of dengue virus to platelets (Wang et al., 1995). A recent report has also demonstrated an antibody-enhanced binding of dengue virus to platelets (Wang et al., 1995). Dengue virus can also bind to the surface of platelets but at very 49 low levels when c Immune-mediated t disseminated intrave I982). Disseminated ir characteristic of den patients during the decreased thrombon 199(1)- During the hmocellular and fig, of bone marrow stro been reverted and m “W: M» Mo Perslgtem hem filflcnon and la inosa, v.0, L09 ”:1:f05copic st .panOsOma \ Ayers, J_ ’ Lei Chediak‘HiQI: g“ ., Yor Res’ 15. 5:352: low levels when compared to antibody-enhanced binding (Funahara et al., 1987a). Immme-mediated platelet removal subsequently occurs even without evidence of disseminated intravascular coagulation (F unahara et al., 1987a; Gubler, 1998; Halstead, 1982) Disseminated intravascular coagulation and consumptive thrombocytopenia are not characteristic of dengue fever (Gubler, 1998). The mechanism of thrombocytopenia in patients during the initial dengue infection is currently unknown. A possible role of decreased thrombopoiesis in dengue fever has recently been proposed (Rothwell et al., 1996). 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Baker Journal of Veterinary Diagnostic Investigation, 1999: 11(6): 505-514 Running Title: Type II BVDV-induced thrombocytopenia From the Department of Large Animal Clinical Sciences (Walz, Baker, Kaiser), the Animal Health Diagnostic Laboratory (Steficek, Macs), the Department of Microbiology (Macs), and the Department of Pathology (Bell), College of Veterinary Medicine, Michigan State University, East Lansing, MI, USA 48824-1314. Reprinted from a copyrighted article in the Journal of Veterinary Diagnostic Investigation, with kind permission of Arthur A. Bickford, VMD, PhD, Secretary/Treasurer, American Association of Veterinary Laboratory Diagnosticians, Inc. 66 Abstract ThrombOC} (BVDV) infecti purpose of the 1 type H BVDV- obtained immed; plasma transfusi control (n=4) or instillation on da; While control calt “the bl00d cells Which time. all e immuIthistochem control calves rem characteristic of t) Calves, and 3 Sta tis mfected calves Wa meal] platelet vOl U Abstract Thrombocytopenia has been associated with type II bovine viral diarrhea virus (BVDV) infection in immunocompetent cattle, but the mechanism is unknown. The purpose of the present study was to develop and characterize a reproducible model of type II BVDV-induced thrombocytopenia. Colostrum-deprived Holstein calves were obtained immediately after birth, given a BVDV negative and BVDV antibody negative plasma transfusion, housed in an isolation facility, and randomly assigned to either control (n=4) or infected (n=5) groups. Infected calves were inoculated by intranasal instillation on day 3 of age with 107 TCIDso of the prototype type H isolate, BVDV 890, while control calves were sham inoculated. Blood counts and virus isolation from serum, white blood cells, and platelets were performed daily until day 12 after infection, at which time, all experimental calves were euthanatized and pathologic, virologic, and immunohistochemical examinations were performed. On physical examination, the control calves remained normal, but the infected calves developed pyrexia and diarrhea characteristic of type II BVDV infection. The platelet count decreased in all infected calves, and a statistically significant difference in the platelet count between control and infected calves was observed between days 7 through 12 after infection. In addition, the mean platelet volume and white blood cell counts also decreased. Examination of the bone marrow from the infected calves revealed immunohistochemical staining for BVDV antigen in megakaryocytes and evidence of concurrent megakaryocyte necrosis and hyperplasia. In conclusion, we have developed and characterized a reproducible model of type II BVDV-induced thrombocytopenia. This model can be used to elucidate the 67 mechanism of tilt why some type ll lntroduction Bovine viral worldwide econor calves were repoi Drake et al., 199: and veal Operatj thrombocytopenia infections With E 1995). The th ofBVDV Was id Although twe 1 dame from su thrombocnopeu mechanism of the thrombocytopenia associated with BVDV and potentially determine why some type II isolates are more virulent than other BVDV isolates. Introduction Bovine viral diarrhea virus (BVDV) is an important pathogen of cattle, resulting in worldwide economic losses. Outbreaks of severe, peracute disease in adult cattle and calves were reported in 1993 in the United States and Canada (Carman et al., 1998; Drake et al., 1994; Pellerin et al., 1994). These outbreaks, which involved beef, dairy, and veal operations, were characterized by fever, diarrhea, abortion, leukopenia, thrombocytopenia, and death. Prior to 1993, it was generally believed that acute infections with BVDV resulted in mild disease, without considerable mortality (Baker, 1995). The BVDV isolates from these outbreaks were analyzed, and the type H genotype of BVDV was identified (Carman et a1 ., 1998; Pellerin et al., 1994; Ridpath et al., 1994). Although type I and type H BVDV can be involved in the entire spectrum of clinical disease from subclinical to severe disease, the hemorrhagic disorder characterized by thrombocytopenia has been only observed with type H isolates of BVDV (S. R. Bolin, personal communication). Thrombocytopenia associated with type H BVDV infection has been reported under natural (Rebhun et al., 1989) and experimental conditions (Bezek et al., 1994; Bolin & Ridpath, 1992; Corapi et al., 1990; Corapi et al., 1989; Ellis et al., 1998). Although previous experimental studies on type H BVDV infection have characterized the hemorrhagic syndrome, they have been descriptive in nature, and the mechanism of the BVDV-induced thrombocytopenia remains unknown. Several of the previous studies on 68 BVDV-induced quantitation 0f 0 difficult (Bezek seteral studies 1' Ridpath, 1992; C t evaluation of thr (Nelson et al., 19 platelet count va Dubielzig, 1993; BVDV isolates th (Corapi et al., 19 there is no mode 11136 ll BVDV inl The mechan Cmently unknoxx and death loss is Slllflrorne. The virOIOgic, and pa Calves and to de 1,. LII‘Dmbocytopeni BVDV-induced thrombocytopenia used an infection protocol that did not allow quantitation of the infectious dose of virus, which makes reproduction of their model difficult (Bezek et al., 1994; Corapi et al., 1990; Corapi et al., 1989). Furthermore, several studies failed to use controls or failed to use age matched controls (Bolin & Ridpath, 1992; Corapi et al., 1990; Corapi et al., 1989). Control calves are critical in the evaluation of thrombocytopenia because of the variation in the normal platelet count (Nelson et al., 1974; Semrad & Dubielzig, 1993); and age-matching is important as the platelet count varies between calves of different ages (Nelson et al., 1974; Semrad & Dubielzig, 1993; Vaugher et al., 1973). Finally, some investigations have used type H BVDV isolates that failed to consistently induce thrombocytopenia in all infected calves (Corapi et al., 1989; Ellis et al., 1998). Since it is difficult to reproduce these studies, there is no model of type II BVDV available to study the thrombocytopenic aspect of type H BVDV infection. The mechanism of increased virulence with some type H isolates of BVDV is currently unknown; however, the ability of type H BVDV to cause severe clinical disease and death loss is due, at least in part, to thrombocytopenia and the resulting hemorrhagic syndrome. The purpose of this study was to characterize the clinical, hematologic, virologic, and pathologic features of the thrombocytopenic BVDV infection in neonatal calves and to develop a model to study the pathogenesis of type H BVDV-induced thrombocytopenia. 69 Materials and M Animals Colostrum-dc birth from local allocated to contr calves were give Obtained for virt neutralization. A levels to BVDV 3 intravenous admi antibOdy negative A and D,“ Vitamir again“ E- Coli K5 label direCIlOns at Materials and Methods Animals Colostrum-deprived newborn male Holstein calves were obtained immediately after birth from local dairy farms. Calves were moved to an isolation facility, randomly allocated to control or treatment groups, and individually housed in separate rooms. All calves were given a physical examination, and both serum and whole blood were obtained for virus isolation and serum was obtained for BVDV antibody by virus neutralization. All calves positive for BVDV by virus isolation or with serum antibody levels to BVDV greater that 16 were excluded from study. The calves were given, by intravenous administration, 400 ml of fresh frozen plasma obtained from a BVDV antibody negative and virus negative donor cow. Calves were given parenteral vitamins A and D,a vitamin E and selenium,b a commercial oral monoclonal antibody preparation against E. cali K99 pilus antigen,b and fed a nonmedicated milk replacer according to label directions at 12% of body weight twice daily. Each calf was examined twice daily, and the rectal temperature and the physical examination findings recorded. Prior to initiation of the study, criteria for euthanasia were established. Calves demonstrating any one or a combination of the following were euthanatized: complete anorexia, recumbency with inability to rise, dehydration in excess of 12% of body weight, and hemorrhagic diathesis. No calves met these criteria, and therefore, no calves were euthanatized prior to the completion of the study. 70 Virus and challe The noncytor induce thromboo experimental inft turbinate cells in equine senrm (l? llOOpg’ml). Afti lIlIC‘ClCd gIOLlp (n BVDV 890 isolat Sham inoculum. v Clinical Path 010g Manual plate Inantral Counts Automated Com] LLSmg an Electro. Memo“, but ir. (Ml-Vale“ part a fibfinogm) we ex . perUTIEnt a 3 t v . CIErmary Tea Virus and challenge protocol The noncytopathic type H BVDV isolate 890,° which has been previously shown to induce thrombocytopenia in calves (Bolin & Ridpath, 1992), was used for the experimental infection of calves. The BVDV isolate 890 was propagated in bovine turbinate cells in Eagle’s minimum essential medium (EMEM),d containing 10% fetal equine serum (FES),° L-glutamine,f penicillin (3° (100 units/ml), and streptomycin" (100ug/ml). After a three day adjustment period in the isolation facility, calves in the infected group (n=5) were inoculated by intranasal instillation with 107 TCIDso of the BVDV 890 isolate. Control calves (n=4) were inoculated by intranasal instillation with a sham inoculum, which was prepared from an uninfected cell culture lysate. Clinical pathologic examination Manual platelet and leukocyte counts were performed daily on whole blood. The manual counts were performed using the Unopette Systemg and a hemocytometer. Automated complete blood counts, including the mean platelet volume, were performed using an electronic particle counter.h Automated counts were done at various times after infection, but included days 0 and 12 after infection on all calves. Coagulation profiles (activated partial thromboplastin time, prothrombin time, fibrin degradation products, and fibrinogen) were performed using whole blood on days -2, 4, 10, and 12 of the experiment, according to standard procedure in the Clinical Pathology Section, Veterinary Teaching Hospital, Michigan State University. 71 balm" (”'de Whole blOOd containing 1‘0 m Plasma was han’ of platelets “'35 was added to 1 minutes. The P] Hank's Balanced mM Nang’Oa. ar Sepharose 4B col and a manual plat concentration of Z antibiotics, subjec Kg. The supema P018 diameter of BVDV. For transmissi Isolation and purification of platelets for virus isolation and electron microscopy. Whole blood was collected every other day from the jugular vein into plastic syringes containing 1.0 ml of 3.8% w/v sodium citrate for each 9.0 ml of blood. Platelet rich plasma was harvested by difl‘erential centrifugation (Steficek et al., 1993). Purification of platelets was achieved through gel filtration. Briefly, 1 ul of 1 uM prostaglandin Ele was added to 1 ml of platelet rich plasma prior to centrifugation at 800 x g for 15 minutes. The plasma was removed and the platelet pellet was resuspended in 1 ml Hank’s Balanced Salt Solution (136 mM NaCl, 5.4 mM KCL, 0.44 mM KHzPO4, 0.34 mM NazHPO4, and 5.5 mM dextrose, pH 7.4). Platelets were then passed over a 10 ml Sepharose 4B column.° The flow-through fraction containing the platelets was collected and a manual platelet count was performed. The gel-filtered platelets were adjusted to a concentration of 200,000 platelets/pl in EMEM containing 10% FES, L-glutamine, and antibiotics, subjected to three freeze/thaw cycles, and centrifuged for 10 minutes at 1,500 x g. The supernatant was extracted and filtered through a sterile filter with an average pore diameter of 0.45 um,i and stored at -80 C for later use in procedures to isolate BVDV. For transmission electron microscopic examination of platelets, platelet rich plasma was harvested (Steficek et al., 1993). Platelet rich plasma suspensions were divided into 0.5 ml aliquots in glass cuvettes, held at room temperature for 30 minutes, and then warmed for 5 minutes at 37 C. A teflon-coated magnetic stir bar was added to the sample, and the cuvette was then placed into an aggregometer.j The platelets were allowed to equilibrate in the aggregometer at a stir speed of 900 rpm for 30 seconds. At that time, 0.5 ml of 0.1% glutaraldehyde in 0.1 M sodimn cacodylate buffer (pH 7.4) was 72 added and the $3 cuvette were “an g. The suP‘imata bulier was added the Supernatant \i sampleS were the Procedure (Marts Pathologic era m ‘ On day 11 barbimrate.k A po were recorded. 7 lung, heart, small cord, mesenteric 2 10% neutral bufftl 35 fixative.l Fol sectioned at 6 un‘ PTOCCdUIe. To as “filtration of the l added and the sample allowed to continue stirring for one minute. The contents of the cuvette were transferred to an Eppendorf tube and centrifuged for 20 seconds at 12,800 x g. The supernatant was discarded and 1.0 ml of 3.0% glutaraldehyde in 0.1 M cacodylate buffer was added. The platelet pellet was fixed for 6 hours in this solution, at which time the supernatant was discarded and 1.0 ml of 0.1 M cacodylate buffer was added. The samples were then processed for transmission electron microscopy according to standard procedure (Mattson et al., 1977). Pathologic aaminatian On day 12 after infection, all calves were euthanatized with a lethal injection of barbiturate.k A postmortem examination was performed and all gross postmortem lesions were recorded. Tissues collected for histopathologic examination included liver, spleen, lung, heart, small and large intestine, adrenal gland, kidney, thymus, tonsil, brain, spinal cord, mesenteric and bronchial lymph nodes, and bone marrow. All tissues were fixed in 10% neutral buffered formalin, with the exception of bone marrow, which was fixed in B-5 fixative.l Following fixation, the tissues were trimmed, embedded in paraffin, sectioned at 6 um, and stained with hematoxylin and eosin (HE) according to standard procedure. To assess the numbers and morphology of the cell constituents, a detailed evaluation of the bone marrow was performed, including calculations of the numbers of megakaryocytes per high power field and the myeloid:erythroid series ratio (M:E ratio) (Harvey, 1984). Five different high power field areas were evaluated for each calf. Images in this dissertation are presented in color. 73 Virologic and set Serum an blood was proce: cells (Carbrey et EMEM containin cycles, the white The supernatant 1 diameter of 0.45 To detenninc bone marrow. th cerebellum, spin Postmortem. Ap FES’ L‘glmamin Virologic and seralagic mminatian Serum and whole blood were collected daily for detection of BVDV. The whole blood was processed by hypotonic lysis of the red blood cells to yield the white blood cells (Carbrey et al., 1972). The isolated white blood cells were then resuspended in EMEM containing 10% FES, L-glutamine, and antibiotics. Following three freeze/thaw cycles, the white blood cell preparations were centrifuged for 10 minutes at 1,500 x g. The supernatant was extracted and filtered through a sterile filter with an average pore diameter of 0.45 pun,i and stored at -80 C. To determine the tissue distribution of the virus, virus isolation was performed on bone marrow, thymus, spleen, bronchial and mesenteric lymph nodes, tonsil, ileum, cerebellum, spinal cord, lung, esophagus, liver, and pulmonary artery collected postmortem. Approximately 0.5 gram of tissue was added to 4 ml of EMEM with 10% FES, L-glutamine, and antibiotics in a sterile mortar. Sterile sand was added and the tissue was homogenized by grinding with a pestle. The homogenized tissue sample was transferred into conical tubes and centrifirged for 30 minutes at 2,300 x g. The supernatant was removed and filtered through a filter with an average pore diameter of 0.45 urn.i Following processing, tissue preparations were stored at -80 C. The serum, white blood cell preparations, gel-filtered platelet preparations, and tissue preparations were thawed, and 25 pl of each sample was inoculated in duplicate into wells on 96-well nricrotiter plates containing monolayers of bovine turbinate cells in EMEM with 10% FES, L-glutamine and antibiotics. After 3 days of incubation at 37 C 74 in humidified air antigen by an inn Serology fo (Carbrey et 01.. experimental cal inactivated for 31' 1:4 to 1:4,096. \ amount of virus L AHDL #cp10806 0i15,000 cells p and negative ser comlllete inhibit? IMMOhistoch ImmurIOhisti embedded secri monoclonal anti 35 Previously de for BVDV am. PulmOnary arteI in humidified air containing 5% C02, the bovine turbinate cells were stained for BVDV antigen by an immunoperoxidase monolayer assay (Meyling, 1988). Serology for BVDV was performed by a microtiter virus neutralization procedure (Carbrey et al., 1972). Sera obtained on days 0 and 12 afier infection from all experimental calves were tested for neutralizing antibodies to type H BVDV. Sera were inactivated for 30 minutes in a 56 C waterbath. Serial two-fold dilutions, ranging from 1:4 to 1:4,096, were made for each serum sample in a 96-well microtiter plate. The amount of virus used per test well was 500 TCIDso of the type H cytopathic isolate MSU- AIHDL #cp1080626.m Bovine turbinate cells were used as the indicator cells at a dilution of 15,000 cells per well. Each test included a back titration of the virus and a positive and negative serum control. The antibody titer was read as the highest dilution with complete inhibition of cytopathic effect. Immunohistochemical and immunafluarescent antibody detection of B VDV antigen Immunohistochemical staining for BVDV antigen was performed on paraffin- embedded sections of bone marrow, pulmonary artery, lung, and spleen using the monoclonal antibody, 15C5, and an avidin-biotin-complex immunoperoxidase technique as previously described (Haines et al., 1992). The direct immunofluorescent antibody test for BVDV antigen was performed on fresh frozen tissue sections of bone marrow, pulmonary artery, lung, and spleen according to standard procedure in the Animal Health Diagnostic Laboratory, Michigan State University, using an anti-BVDV polyclonal antiserum conjugated to fluorescein isothiocyanate.“ 75 Statitfical analy- DifferenceS l rectal temperatu I using the student using the Fisher~ Results Physical examin None of I infected calves, mucosal casts. v on day 5 afier developed diarr' 39-4 C) Was 01 we“ compare LemDentures o: (P3005), No c Statistical analysis Differences in platelet and white blood cell counts, coagulation profile data, and rectal temperature measurements between control and infected calves were evaluated using the student’s t-test. The virus isolation and virus neutralization data were analyzed using the Fisher’s exact test. A P value < 0.05 was considered statistically significant. Results Physical examination findings None of the calves met the criteria for euthanasia during the study period. In the infected calves, appetite remained normal, but diarrhea, characterized by blood and mucosal casts, was a consistent finding in all infected calves. Diarrhea was first noticed on day 5 after infection (2 calves), and by day 7 after infection, all infected calves developed diarrhea that continued throughout the study. Pyrexia (rectal temperature > 39.4 C) was observed in the infected calves fiom days 2 through 12 after infection. When compared to the control calves, there was a significant elevation in rectal temperatures of the infected calves on day 2 and days 4 through 12 after infection (PS0.05). No clinical signs of disease were noted in the control calves. Clinical pathologic findings The platelet count decreased to less than 200,000/pl in all infected calves, and a platelet count of less than 100,000 platelets/u] was observed in 3 of 5 infected calves during the study. The manual platelet count reached the lowest point in the infected 76 calves on day l (range: 98.000-1 platelets’ul (rar (Figure 2.1). decreased manua study in the infec the manual coun was significantly 2.1). The white t difference in dail was Observed on differential in ir Primarily to a prc onthe white bloc Cahes. N0 Sigllifica; activated Partial til fibfinogen of the ( Tr . calves on day 11 after infection, with a mean platelet count of 139,300 platelets/pl (range: 98,000-178,000 platelets/pl). This compares to a mean platelet count of 801,125 platelets/pl (range: 642,000-825,000 platelets/til) in the age-matched control calves (Figure 2.1). When compared to the age-matched control calves, a significantly decreased manual platelet count was observed from day 7 after infection to the end of study in the infected calves (Figure 2.1). The automated platelet counts closely followed the manual counts (Table 2.1). Platelet size, as measured by the mean platelet volume, was significantly reduced in the infected calves as the study proceeded over time (Table 2.1). The white blood cell counts decreased in the infected calves, and a significant difference in daily manual white blood cell counts between infected and control calves was observed on day 6 and days 8 through 12 after infection. The white blood cell diflerential in infected calves demonstrated that the observed leukopenia was due primarily to a profound neutropenia (Table 2.1). The numbers of all the other cell types on the white blood cell differential were within normal limits and similar to the control calves. No significant difference was noted between control and infected calves in the activated partial thromboplastin time, prothrombin time, fibrin degradation products, and fibrinogen of the coagulation profile (Table 2.1). Transmission electron microscopic findings Ultrastructural evaluation of platelets demonstrated a population of platelets that was heterogeneous in size. Compared to the control calves, there was an overall 77 decreased platel observed (Figure in platelets from li'rologic and 5e Bovine \ samples collecte blood cells, plate rims was demor scnrrn. Bovine infected calves, infected calves), infected Calves) All control dal‘ of in0cu1 ati nlfection in all < the antibody Illt decreased platelet size in the infected calves, but several very large platelets were observed (Figure 2.2). Vesiculation of the platelet plasma membrane was also observed in platelets fi'om calves infected with type H BVDV. Virologic and seralagic findings Bovine viral diarrhea virus was not isolated from antemortem or postmortem samples collected from the control calves. In contrast, BVDV was isolated from white blood cells, platelets, and serum of all infected calves (Table 2.2). Bovine viral diarrhea virus was demonstrated earliest in white blood cells, followed by platelets, and then serum. Bovine viral diarrhea virus was isolated from all the tissues examined in all infected calves, with the exception of bone marrow (BVDV was isolated from 4 of 5 infected calves), and the liver, cerebellum, and spinal cord (BVDV was isolated in 3 of 5 infected calves). All control and infected calves had BVDV-neutralizing antibody titers of $8 on the day of inoculation. The BVDV-neutralizing antibody titers remained $8 on day 12 after infection in all control and infected calves, with the exception of 1 infected calf, in which the antibody titer was 16 at day 12 after infection. Pathologic findings Petechial hemorrhages were present in all infected calves, with the hemorrhages being most prominent in the rumen, abomasum, and/or the endocardial surface of the heart. Oral erosions in the buccal mucosa were present in 3 of 5 infected calves. Abomasal edema, which was most prominent in the spiral folds of the fundus, was 78 present in 4 0 postmortem exal Histologic cl infected calves. (MK‘hpf) than megakaryocytes half of the meg; necrosis as evidL this dissertation Was increased in I~)'Tttphoid d in all infected ca fasciculata of the sulDerficial erosi vacuolizatiOn Wit infected Calve S. the SUIface of sve- stem. pulmonary edema fluid Was 1 Present in all mfe. present in 4 of 5 infected calves. No significant lesions were present on gross postmortem examination of control calves. Histologic evaluation of the bone marrow revealed differences between control and infected calves. Infected calves had more megakaryocytes per high power field (MK/hpf) than the control calves (Table 2.3). In the infected calves, many of the megakaryocytes did not have uniform nuclear size or chromatin pattern. Approximately half of the megakaryocytes in infected calves were either immature, or had undergone necrosis as evidenced by the loss of, or pyknosis of the nuclei (Figure 2.3B). Images in this dissertation are presented in color. The myeloid:erythroid series ratio (M:E ratio) was increased in the infected calves when compared to the control calves (Table 2.3). Lymphoid depletion and reduced follicular size were present in the ileum and spleen in all infected calves. An occasional focus of necrosis of individual cells within the zona fasciculata of the adrenal gland was present in 4- of 5 infected calves and 1 control calf. Superficial erosions of the tongue with epithelial parakcratotic change, basal cell vacuolization with apoptosis, and mild submucosal inflammation was present in 4 of 5 infected calves. A mild angiopathy, characterized by deposits of eosinophilic protein on the surface of swollen endothelial cells, was observed in the cerebellum, cerebrum, brain stem, pulmonary artery, heart, and lung in 3 of 5 infected calves. In the lung, pulmonary edema fluid was present which distended the interlobular spaces. This lesion, which was present in all infected calves, was not associated with any significant inflammation. 79 Immunohistoch In the ct. immunohistoche immunohistocht sections collectt immunohistochel revealed the prc 2.3D). Immuno of blood \"€SS€l.\| the bone manor contained BVDr Immunah istachemistry and immunafluarescent antibody testing In the control calves, there was no evidence of BVDV antigen as detected by immunohistochemical or immunofluoresccnt antibody staining. However, there was immunohistochcnrical staining for BVDV antigen in all paraffin-embedded tissue sections collected for evaluation from all infected calves. In 3 of 5 infected calves, immunohistochemical and immunofluorcsccnt antibody staining of bone marrow revealed the presence of BVDV-specific antigen in megakaryocytes (Figures 2.3C and 2.3D). Immunohistochemical staining for BVDV was also present in the smooth muscle of blood vessels located in the spleen, lung, and the bone marrow. Mononuclcar cells in the bone marrow, spleen, and lung and focal areas of lymphoid cells in the spleen also contained BVDV antigen. 80 1000000 800000 . . 7 Figure 2.1. Mean whole blood platelet counts by manual determination in control and 00000 BVDV-infected calves. Calves were infected at day 0. Standard error bars are shown. 600000 f A statistical difference (p<0.05, t-test) in platelet counts between the control and infected 500000 .0. i calves was detected from day 7 through day 12 after infection. 000 300000 Moan Platelet Count. (par microtiter) 200000 _ 100000 81 Mean Platelet Counts (per microliter) Figure 2.1 7.00mi 1‘ ._ ‘ Control group 600000 / 400000 300000 T Infected group 200000 IT .00... i 0 —~ +«—+* + «w If . é :- . - + —— + “9-47 1' -2-10123456789101112 Days after Infection 82 l 5.5.0:... 8:.“ m. 5% EEUfiE 23.3: 242.23 :esuutz 5.x .2 >3. 5:03.: 82.3 32.5 3202.: $023 3:55 cote 2.52me an 555 05 an hummenxo 0.5 335» 592035 .80? N4 Rah :0 Use 50:09.:: 950.9: 292:9 3.8260 $3002.}... V 3:: 3200.2-7>Q>3 w. as; zwcmfizu U~m0~0£ual ~fi02~=0kc KLEZZSA .~ .N 2a.: .9888 58888 cowoqtnanmmm 6:5 mama—nonfiohu Evan 88>c8utm< 68: Enaohaoafl: H .cocmaoofi 2 Sta mama N 880808 083 8an nous—swag ”Agata—:85 .«o ~33 nov8m£ 8% o >3 so 8888a 083 $58 coo—m ._. .258? 8% Q .96 8 828 8803 23 3980 80389 8088b? “5055mm 38:38..” 888n— _. Swansea 2a 3858 83033.. 5.3332 535 5855 av 2v 2v 2v AER: v an: $5033 3.33.3. 3.38% 3.??? 8:88 hr? 8. 38.2 8.33.5 S. 5:. a 8.503 @883 2 H0503 coun—$80 o «.38 :02 o 228580 80”: 235 3.03 6:80 8:888 $3.8m aging; $333 30min 852%.». m 3;. $32 c 882 53: 85083... .. 3302 NS. 3.8.» 833%.» «8033 $282.8 832: 588% um? ._ 835. 5338.: $3.38.: «2.52% =58 83 8.323 .. 5i 5. Emits £3832 22025 =58 83 382 1383 2.33 2.39% 2.39% av use? .2an =82 _. Sqwméomefi 83083? 5.23350 58.2.33” 3 “58 Ease Emacs... .. 3.808%: ammuaomge 83938.3 $83889. :58 022% 3:82 .8885 8cm 2 >3 838%: 2809 :38? 8d“ 2 x3 +8383 Bowen 033:3 828 @883 828 15:8 coho 9353 n" 508 05 8 83058 08 823/ .88an 8% Q m3 :0 98 8:8.«5 283 838 35:8 “088.33: v 33 8»oo.«£->fl>m m 8m mwfivam ammo—053 Roam—o mo ggm AN 03.2. 83 Table 2.2. Sur calves infected f‘ Day after infec Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 \ +:umel‘zuor: # of c . YOI D0ne Table 2.2. Summary of virus isolation results for BVDV on antemortem samples for 5 calves infected with type II BVDV. Day after infection Gel filtered White blood cells Serum platelets Day O 0/5* 0/5 0/5 Day 1 NDT 0/5 0/5 Day 2 0/5 0/5 0/5 Day 3 ND 1/5 0/5 Day 4 1/5 2/5 0/5 Day 5 ND 3/5 1/5 Day 6 4/5 5/5 3/5 Day 7 ND 5/5 4/5 Day 8 5/5 5/5 4/5 Day 9 ND 4/5 5/5 Day 10 5/5 4/5 3/5 Day 11 ND 4/5 3/5 Day 12 4/5 4/5 3/5 " numerator: # of calves which were virus positive; denominator: # of calves in infected group 1' ND—Not Done 84 l Table 2.3. My5 field for5 BVE #‘ MzE series ram MK hpf‘l ____________. ' M:E=I\lyeloid:er * Mliihpf=megak cattle. Table 2.3. Myeloidzerythroid series ratio and numbers of megakaryocytes per high power field for 5 BVDV-infected and 4 uninfected control calves. control calves infected calves mean range mean range M:E series ratio* 0.5 0.39-0.80 0.68 0.40-0.93 MK/hpf‘l' 5 1-10 10 4-21 "' M:E=Myeloid:erythroid series ratio.Normal M:E ratio (Winqvist, 1954): mean: 0.46; range 0.40-0.52 1' MK/hpf=megakaryocytes per high power field. Normal MK/hpf values have not been established for cattle. 85 Figure 2.2. Transmission electron micrograph of platelets from a control (left) and an infected (right) calf at day 12 afier infection. In addition to observing a heterogeneity of size, most platelets from the infected calf were decreased in size. The surface membranes of platelets from infected calves are more frequently marked by adherent debris and membrane fragmentation. Bar = 1.0 pm. 86 ‘ . . i" . a . d... . 550‘“ - if -. U” ' ' 2"!" up» 0‘, M‘“ "O O 87 Figure 2.3. Histologic sections of bone marrow taken on day 12 after infection (postmortem examination). Bar = 100 pm. A. Histologic section of bone marrow fi'om control calf. B. Histologic section of bone marrow fiom calf infected with type II BVDV. Note the degenerating megakaryocytes undergoing pyknosis (depicted by the arrows) and an immature megakaryocyte (depicted by the arrowheads). C. Positive immunofluorescent antibody staining for BVDV antigen in a megakaryocyte. D. Positive immunohistochemical staining for BVDV antigen in megakaryocytes (note reddish-brown staining depicted by arrow). Images in this dissertation are presented in color. 88 Figure 2.3 . ’\Y~." ‘ . V '37 five, ‘TW ,. "I "K 5'“ K; .1; ..§; ,9'.‘~ 9": '., .. :‘A R.,.fi‘igk. ‘ -- iv; ‘3‘ 9:1,, .‘b B42338 ‘ I ‘.' tu‘figar? ' ." ‘-‘.T$¢"Z"¢§-F ' . ‘ § "0435‘.“ r.‘ J :5?" ?p;. 6‘ “iv“ 89 Discussior We thrombocyl model over of a decrea demonstrate infected ca standardized negative pla Because a pl calves acqui] calves frOm could study t Other iIlfectio Pan cause 1h“ 1993) An ad mommflo “My until its A declin the infected Circulating pl 15 00"Siderabl Discussion We have developed a reproducible model of the type H BVDV-induced thrombocytopenia using newborn male Holstein calves. The principal advantages of our model over previous studies on BVDV-induced thrombocytopenia are the demonstration of a decreased platelet count in all infected calves, the use of age-matched controls to demonstrate statistically significant differences in platelet counts between control and infected calves, an infection protocol which simulates natural infection, and a standardized infectious dose of virus. The use of a virus negative and BVDV antibody negative plasma transfusion should be considered an additional advantage of this model. Because a plasma donor was vaccinated for the common neonatal infectious diseases, the calves acquired adequate immunity against these agents, and thus, prevented the loss of calves from the study due to sepsis, respiratory disease, and enteritis. Therefore, we could study thrombocytopenia in neonatal calves without the confounding influence of other infectious diseases. This is particularly important as both sepsis and endotoxemia can cause thrombocytopenia in neonatal calves (Deldar et al., 1984; Semrad & Dubielzig, 1993). An additional advantage in our model is that infection with BVDV 890 resulted in thrombocytopenia, but the infected calves did not become moribund and remained in the study until its completion. A decline in the platelet count was the most prominent hematologic abnormality in the infected calves. Thrombocytopenia is defined as a reduction in the number of circulating platelets below the minimum normal level for the species (Jain, 1986). There is considerable variation in the literature concerning the platelet count in normal male 90 Holstein C plateletsiu platelets) l1 literature i matched c thrombocfl count berm previous sn (Bezek e! t inoculation 1 ages and bre 1992; Corapi have demons 33¢ more I'm. infected Calve use Of age-ma the Platelet Co the Platelet c0 isdifficult to i This Change Dubiehig. 19g Plateletstm at l Holstein calves, with one group of investigators reporting a range of 192,000-892,000 platelets\ul (Nelson et al., 1974), and another reporting a range of 480,000-1,200,000 platelets\ul (V augher et al., 1973). Therefore, defining thrombocytopenia based upon the literature is difiicult. To circumvent these problems, we used age-, breed-, and sex- matched control calves in order to minimize the variation in the definition of thrombocytopenia, and to demonstrate statistically significant differences in the platelet count between calves infected with type H BVDV and their matched controls. Although previous studies have also demonstrated thrombocytopenia using different viral isolates (Bezek et al., 1994; Corapi et al., 1990; Corapi et al., 1989), different routes of inoculation (Bezek et al., 1994; Corapi et al., 1990; Corapi et al., 1989), and different ages and breeds of calves infected with BVDV (Bezek et al., 1994; Bolin & Ridpath, 1992; Corapi et al., 1990; Corapi et al., 1989), they did not use matched controls. We have demonstrated both thrombocytopenia (platelet count less than 192,000 platelets\ul) and, more importantly, a significant difference in the platelet count between control and infected calves from days 7 through 12 afier infection. Additional justification for the use of age-matched controls is the fact that there appears to be an age-related change in the platelet count in neonatal calves as demonstrated by the findings in this study. Since the platelet count in our age-matched control calves actually increased during the study, it is dificult to interpret a platelet count in an infected calf without the appropriate control. This change in platelet count has been alluded to in a previous study (Semrad & Dubielzig, 1993), in which the platelet count gradually increased from 212,050i73,945 platelets\u1 at birth to 645,000i54,571 platelets\ul by 4 days after birth. 91 In the I decreased 3 evaluation platelets we help classifj released intc platelet vol u platelets. C4 when the tlr 1982). The decreased pr. Virus infectic volume has al infection (ho In the present study, the mean platelet volume in infected calves was significantly decreased at day 12 after infection when compared to control calves. Ultrastructural evaluation of platelets by transmission electron microscopy also demonstrated that platelets were smaller in the infected calves. The mean platelet volume can be used to help classify thrombocytopenia (Bessman et al., 1982). In general, platelets recently released into the bloodstream from the bone marrow are larger, and therefore, the mean platelet volume is elevated when the thrombocytopenia is due to increased destruction of platelets. Conversely, aged platelets are smaller and mean platelet volmne is decreased when the thrombocytopenia is due to decreased marrow production (Bessman et al., 1982). The low mean platelet volume in BVDV-induced thrombocytopenia suggests decreased production of platelets similar to that seen with human immunodeficiency virus infections and myelosuppressive disorders (Koenig et al., 1991 ). The platelet volume has also been reported as decreased in pigs undergoing classical swine fever virus infection (hog cholera virus), which is closely related to BVDV; however, the significance of that finding was unknown at the time (Weiss et al., 1973). Other antemortem characteristics of this model of BVDV-induced thrombocytopenia which warrant discussion include the physical examination findings, the decreased white blood cell counts in the infected calves, and the normal coagulation profiles in infected calves. The physical examination findings of pyrexia and diarrhea are consistent with previous reports regarding type II BVDV infection in calves (Bezek et al., 1994; Bolin & Ridpath, 1992; Corapi et al., 1990; Corapi et al., 1989). A decreased white blood cell count characterized by neutropenia has also been previously described with type II BVDV infection in immunocompetent calves (Corapi et al., 1989; Ellis et al., 1998). 92 Lymphopenia and anemia, observed by some investigators (Corapi et al., 1989; Ellis et al., 1998), were not seen in our model. The myeloid:erythroid series ratio was increased in our infected calves, which indicated the bone marrow responded by increasing the production of leukocytes. Coagulation profiles were within normal limits in our study, and normal findings were reported in a previous study on type II BVDV infection in calves (Corapi et al., 1989). The latter findings and the results of this study support the hypothesis that type H BVDV-induced thrombocytopenia is not likely to be due to disseminated intravascular coagulation. We have demonstrated an association of BVDV with platelets in the peripheral circulation through the isolation of BVDV in purified platelet preparations, and this virus-platelet association has been described by others using immunofluorescent antibody testing (Bezek et al., 1994; Corapi et al., 1990) and virus isolation (Bolin & Ridpath, 1992). It has been suggested that the platelets may serve as "carriers" of circulating virus (Corapi et al., 1990). This virus-platelet association may be responsible for the higher level of virulence seen with some type H isolates of BVDV, as platelets may participate in a rapid dissemination of virus throughout the body. We have demonstrated megakaryocyte infection with BVDV, which may be the origin of the BVDV-platelet association rather than through an extra-medullary asssociation in the peripheral blood. We have demonstrated BVDV antigen in megakaryocytes by immunohistochemistry. This confirms previous studies which used immunohistochemistry to define the tissue distribution of BVDV (Ellis et al., 1998; Marshall et al., 1996; Spagnuolo et al., 1997). In addition to BVDV, several other viruses important in veterinary and human medicine can infect megakaryocytes (Axthelm & Krakowka, 1987; Ballem et al., 1992; Gomez- 93 Villamandos et al., 1998; Louache et al., 1991; Perez et al., 1997). Megakaryocyte infection is responsible, in part, for the virus-induced thrombocytopenia seen with human immunodeficiency virus infection (Ballem et al., 1992; Louache et al., 1991), canine distemper virus infection (Axthelm & Krakowka, 1987), and Afiican swine fever virus infection (Gomez-Villamandos et al., 1998; Perez et al., 1997). The megakaryocyte is the hematopoietic precursor cell for platelets (Wright, 1906). Although megakaryocytes comprise only 0.05% of the nucleated cells in the bone marrow (Harker, 1968), they are responsible for maintaining the large circulating platelet population. Therefore, a decrease in the number of megakaryocytes during the early phase of type H BVDV infection, or disturbance in the maturation of megakaryocytes by a type H BVDV infection, could have a direct impact on the number of circulating platelets. The contribution of megakaryocyte infection to the development of thrombocytopenia with type H BVDV infection is unknown. The virus could directly influence platelet production by infecting megakaryocytes or the effect could be indirect, via inhibitory cytokines or infection of cells which produce thrombopoietic stimulatory factors. Although viral antigen has been demonstrated in megakaryocytes, the number of megakaryocytes containing BVDV antigen were relatively few. In addition, we were only able to demonstrate BVDV antigen in megakaryocytes in 3 of 5 infected calves, yet all the calves became thrombocytopenic. This suggests that the thrombocytopenia may be primarily a result of indirect influences on the megakaryocyte caused by type H BVDV. However, we only examined the bone marrow and megakaryocytes at day 12 after infection, and this does not preclude the possibility that the majority of megakaryocytes become infected earlier in the course of the infection. BVDV-induced 94 thrombocytopenia may be due to both direct and indirect effects on the me gakaryocytes. Afi'ican swine fever virus causes thrombocytopenia through a combination of direct infection of megakaryocytes and indirect damage to megakaryocytes by inducing megakaryocyte apoptosis (Gomez-Villamandos et al., 1998; Perez et al., 1997). Previous studies on the histologic examination of bone marrow fiom type H BVDV- thrombocytopenic infections have been contradictory. In calves naturally infected with BVDV, Scruggs et al. reported megakaryocyte necrosis (Scruggs et al., 1995), while Rebhun et al. reported bone marrow necrosis or adequate to increased numbers of megakaryocytes (Rebhun et al., 1989). Under experimental conditions, megakaryocyte hyperplasia has been reported 12 days after infection with a thrombocytopenia-inducing isolate of BVDV (Corapi et al., 1989). Another experimental study on type H BVDV demonstrated bone marrow necrosis in 5 of 6 calves; and, in the remaining calf, megakaryocyte hyperplasia was observed in which immature megakaryocytes were present in relatively high ntunbers (Ellis et al., 1998). We observed both megakaryocyte degeneration and megakaryocyte hyperplasia. The number of immature megakaryocytes was increased, likely in response to the thrombocytopenia, and yet there were also numerous megakaryocytes undergoing pyknosis and degeneration. We speculate that viral infection of the bone marrow occurs during the initial stages of viremia, leading to a transient suppression of platelet production by the megakaryocytes due to the combined direct result of viral infection and an undetermined indirect result of megakaryocyte degeneration. Thrombocytopenia, regardless of the underlying etiology, results from only three basic mechanisms: decreased production, accelerated destruction, or abnormal 95 sequestration of platelets (Warkentin & Kelton, 1994). Viruses have been associated with thrombocytopenia in humans and animals (Kaplan et al., 1992; Zucker-Franklin, 1994), but the underlying mechanisms are complex, multifactorial, and incompletely understood. Classical swine fever virus (hog cholera virus) induces thrombocytopenia and hemorrhage by multiple mechanisms including accelerated platelet destruction caused by direct damage to the platelets, virus-induced endothelial lesions, and disseminated intravascular coagulation (Weiss et al., 1973); however, megakaryocyte necrosis and degeneration have also been reported (Hoffmann et al., 1971). The mechanism of BVDV-induced thrombocytopenia may, like classical swine fever virus, involve a combination of two or more factors. However, in contrast to classical swine fever virus, we suspect that decreased production of platelets as a result of megakaryocyte infection may be the predominant mechanism. Accelerated platelet destruction and abnormal sequestration of platelets as the cause of type H BVDV-induced thrombocytopenia are less likely based upon previous studies (Bezek et al., 1994; Bolin & Ridpath, 1992; Corapi et al., 1989), and the findings of this study. Coating of platelets with antibodies or deposition of viral antigen-antibody complexes on platelet membranes can trigger immune-mediated destruction of platelets (Shulman & Reid, 1994); however, a previous study has demonstrated that there was no evidence of platelet-associated antibody and complement in BVDV-induced thrombocytopenia (Corapi et al., 1990). Disseminated intravascular coagulation also causes thrombocytopenia due to an accelerated destruction of platelets (Shulman & Reid, 1994). The presence of normal coagulation profiles in this study and a previous study 96 (Corapi et al., 1990) suggests that disseminated intravascular coagulation is not involved in BVDV-induced thrombocytopenia. In summary, we have developed a reproducible model of type H BVDV-induced thrombocytopenia. It appears likely that the thrombocytopenia associated with type H BVDV infection is due primarily to a decreased production of platelets. Our model of thrombocytopenia may prove useful for elucidating the pathophysiology of the hemorrhagic syndrome associated with type H BVDV infection and help us understand what makes some type H BVDV isolates more virulent than others in immunocompetent cattle. Sources and Manufacturers a. Butler Company, Dublin, OH b. Schering-Plough Animal Health Corporation, Kenilworth, NJ c. Dr. Steve Bolin, NADC, Ames, IA d. IRH Biosciences, Lenexa, KS Sigma Chemical Co., St. Louis, MO 9 f. Gibco BRL, Life Technologies, Grand Island, NY g' Becton Dickinson, Franklin, NJ h. Technicon H-l analyzer, Tehnicon Instrumental Corp., Tarrytown, NY i. Millipore Corporation, Bedford, MA j. Chronolog Corporation, Havertown, PA k. Vortech Pharmaceuticals, Dearbom, MI 1. Columbia Diagnostics, Inc., Springfield, VA 97 m. Animal Health Diagnostic Laboratory, Virology Section, E. Lansing, MI n. VMRD, Inc., Pullman, WA Acknowledgments Research support was provided by 1433 Animal Health Funds, the Michigan Agricultural Experiment Station, and the American Association of Bovine Practitioners Research Assistantship. We acknowledge Dr. Deborah Haines (Western College of Veterinary Medicine, University of Saskatchewan) and Mrs. Cherie Benson (Animal Health Diagnostic Laboratory, Michigan State University) for performing the immunohistochemical and immunofluorescent antibody testing, respectively. In addition, we thank Drs. David Bezek, Thomas Mullaney, J anver Krehbiel, and Harold Tvedten for helpful suggestions, Dan Taylor for expert technical assistance, and Ralph Common for photographic support. References Axthelrn, M.K. & Krakowka, S. (1987). Canine distemper virus-induced thrombocytopenia. Am J Vet Res, 48, 1269-75. Baker, J .C. (1995). The clinical manifestations of bovine viral diarrhea virus infection. In The Veterinary Clinics of North America (Food Animal Practice)., Baker, J.C. & Houe, H. (eds), Vol. 11. pp. 425-445. W.B. Saunders: Philadelphia. Ballem, P.J., Belzberg, A., Devine, D.V., Lyster, D., Spruston, 8., Chambers, H., Doubrofl‘, P. & Mikulash, K. (1992). Kinetic studies of the mechanism of thrombocytopenia in patients with human immunodeficiency virus infection [see comments]. N Engl J Med, 327, 1779-84. Bessman, J.D., Williams, L.J. & Gilmer, P.R., Jr. (1982). Platelet size in health and hematologic disease. Am J Clin Pathol, 78, 150-3. 98 Bezek, D.M., Grohn, Y.T. & Dubovi, E.J. 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In Thrombosis and hemostasis: basic principles and clinical practice, Colman, R., Hirsh, J ., Marder, V. & Salzman, E. (eds) pp. 414. J .B. Lippincott Co.: Philadelphia, PA. Spagnuolo, M., Kennedy, 8., Foster, J .C., Moffett, D.A. & Adair, B.M. (1997). Bovine viral diarrhoea virus infection in bone marrow of experimentally infected calves. J Comp Pathol, 116, 97-100. Steficek, B.A., Thomas, J.S., Baker, J.C. & Bell, T.G. (1993). Hemorrhagic diathesis associated with a hereditary platelet disorder in Simmental cattle. J Vet Diagn Invest, 5, 202-7. Vaugher, J ., Pearson, B., Blatt, S. & Kaye, M. (1973). Biochemical and hematologic values in male Holstein-Freisian calves. Am J Vet Res, 34, 273-277. Warkentin, T. & Kelton, J. (1994). Management of thrombocytopenia. In Thrombosis and hemostasis: basic principles and clinical practice, Colman, R., Hirsh, J ., Marder, V. & Salzman, E. (eds) pp. 469. J .B. Lippincott Co.: Philadelphia, PA. Weiss, E., Teredesai, A., Hoffmann, R. & Hoffmann-Fezer, G. (1973). Volume distribution and ultrastructure of platelets in acute hog cholera. Thromb Diath Haemorrh, 30, 371-80. Winqvist, G. (1954). Morphology of the blood and hemopoietic organs in cattle under normal and some experimental conditions. Acta Anatomy Supplement, 21, 1-15. Wright, J. (1906). The origin and nature of blood platelets. Boston Medicine and Surgery Journal, 154-163. Zucker-Franklin, D. (1994). The effect of viral infections on platelets and megakaryocytes. Semin Hematol, 31, 329-37. 101 CHAPTER III: JOURNAL ARTICLE: EFFECT OF EXPERIMENTALLY INDUCED TYPE II BOVINE VIRAL DIARRHEA VIRUS INFECTION ON PLATELET FUNCTION IN CALVES 102 CHAPTER III: JOURNAL ARTICLE: EFFECT OF EXPERIMENTALLY INDUCED TYPE H BOVINE VH{AL DIARRHEA VH{US INFECTION ON PLATELET FUNCTION IN CALVES Paul H. Walz, DVM, MS; Barbara A. Steficek, DVM, PhD; John C. Baker, DVM, PhD; Lana Kaiser, MD, DVM; Thomas G. Bell, DVM, PhD American Journal of Veterinary Research, 1999: 60(11): 1396-1401 From the Department of Large Animal Clinical Sciences (Walz, Baker, Kaiser), the Animal Health Diagnostic Laboratory (Steficek, Bell), and the Department of Veterinary Pathology (Bell), College of Veterinary Medicine, Michigan State University, East Lansing, MI, USA 48824-1314. Reprinted from a copyrighted article in the American Journal of Veterinary Research, with kind permission of Dr. Kurt Matushek, Associate Editor, American Journal of Veterinary Research. 103 Abstract Objective—To evaluate platelet aggregation responses in calves experimentally infected with a thrombocytopenia-inducing type H bovine viral diarrhea virus (BVDV) isolate (BVDV 890). Animals—9 neonatal male Holstein calves. Procedure—Five calves were inoculated with BVDV 890, and 4 were used as controls. Platelet aggregation studies and attempts to isolate BVDV from platelets were performed 2 days before, the day of, and every 2 days for 12 days after inoculation. Platelet function was assessed by means of optical aggregometry, using adenosine diphosphate and platelet activating factor as agonists. Bovine viral diarrhea virus was identified from purified platelet preparations by use of an immunoperoxidase monolayer assay. Results—Maximum percentage aggregation and slope of the aggregation curve decreased over time in calves infected with BVDV. Bovine viral diarrhea virus was not isolated fi'om platelets from control calves, but was isolated from infected calves from 4 through 12 days after inoculation. Conclusions—Results suggest that platelet function may be depressed in calves infected with type H BVDV. Although the mechanism for altered platelet function was not determined, it likely involved an increase in the percentage of aged platelets in the circulation, a direct virus-platelet interaction, or an indirect virus-platelet interaction. Clinical Relevance—Platelet dysfunction, in addition to thrombocytopenia, may contribute to the hemorrhagic syndrome associated with acute type H BVDV infection in calves. 104 Introduction Bovine viral diarrhea virus (BVDV) infection is an economically important viral infection of cattle that can result in a wide spectrum of clinical manifestations, ranging from subclinical infections to severe fatal disease (Baker, 1995). The causative organism is an RNA virus and a member of the genus Pestivirus within the family Flaviviridae (Wengler, 1991). Isolates of BVDV can be classified into 2 genotypes, type I and type H, on the basis of differences in RNA sequences (Ridpath et al., 1994). Infection of nonimmune cattle with some isolates of BVDV has been associated with a clinical disorder characterized by thrombocytopenia (Bolin & Ridpath, 1992; Corapi et al., 1990; Rebhun et al., 1989). To date, only infection with the type H genotype of BVDV has been associated with a hemorrhagic syndrome.a Thrombocytopenia has been documented in adult cattle and calves with naturally occurring (Rebhun et al., 1989) or experimentally induced BVDV infection (Bezek et al., 1994; Bolin & Ridpath, 1992; Corapi et al., 1990). Clinical signs reported for cattle with the hemorrhagic syndrome have included epistaxis, leukopenia, pyrexia, hemorrhage in multiple organ systems, bleeding fiom injection sites or insect bites, bloody diarrhea, and death (Bezek et al., 1994; Bolin & Ridpath, 1992; Corapi et al., 1990; Rebhun et al., 1989). The cause of hemorrhage in cattle with acute BVDV infection has been attributed solely to thrombocytopenia (Corapi et al., 1990; Rebhun et al., 1989), even though the documented decrease in platelet number is often insufficient to result in spontaneous bleeding. Platelets are small anucleate cells in the circulation that play a pivotal role in hemostasis and thrombosis (Day & Rao, 1986). Platelets circulate in a disc-like resting 105 state, but become activated following blood vessel injury. With injury to a blood vessel, platelets are exposed to subendothelial components, triggering a cascade of events that ultimately result in formation of a thrombus. Quantitative platelet disorders, in which circulating numbers of platelets are low, and qualitative platelet defects, in which platelets have altered ftmctional characteristics, will result in an impaired hemostatic response, and hemorrhage may result (Jain, 1986). Clinical signs of spontaneous hemorrhage occur when platelet counts are < 20,000 platelets/pl, and may develop if platelet counts are between 20,000 and 50,000 platelets/u] (Jain, 1986). Several investigations have determined that the bleeding tendency in cattle infected with type H BVDV only becomes clinically apparent when platelet counts in the circulation are less than these critical limits (Corapi et al., 1990; Rebhun et al., 1989). In a retrospective study of 15 cattle with acute BVDV-induced thrombocytopenia (Rebhun et al., 1989), petechial and ecchymotic hemorrhages were reported in association with platelet counts ranging from 2,000 to 33,000 platelets/pl. In a separate study (Corapi et al., 1990), calves with experimentally induced BVDV infection had clinical evidence of hemorrhage only in association with platelet counts < 5,000 platelets/pl. In contrast, experimentally induced type H BVDV infection resulted in hemorrhage and death in 3 of 7 calves in which platelet counts were not < 70,000 platelets/u] at the time of death (Bolin & Ridpath, 1992). This discrepancy between development of hemorrhage and platelet count has suggested that platelet function may be altered in cattle with type H BVDV infection. Because previous studies have not examined platelet function in infected cattle, the purpose of the study reported here was to evaluate platelet aggregation responses in calves experimentally infected with a 106 thrombocytopenia-inducing type H BVDV isolate (BVDV 890). The study was performed to determine whether altered platelet function, in addition to low platelet counts, may contribute to the hemorrhagic diathesis associated with acute type H BVDV infection in cattle. Materials and Methods Animals Nine colostrum-deprived newborn male Holstein calves were obtained immediately after birth from local dairy farms. Calves were moved to an isolation facility, randomly allocated to control (n = 4) or treatment (5) groups, and individually housed in separate rooms. All calves were given a physical examination, and blood and serum were obtained for virus isolation procedures and determination of virus neutralization antibody titers to BVDV. Calves were then given 400 m1 of plasma, IV. Plasma had been obtained from a donor cow that was negative for BVDV antibodies, as determined by means of virus neutralization, and for virus, as determined by means of virus isolation procedures (tests were performed on 3 serial samples obtained prior to collection of plasma). Calves were also given injections of vitamins A and Db and vitamin E and selenium,° as well as a commercial oral monoclonal antibody preparation against Escherichia coli K99 pilus antigen.° Calves were fed a nonmedicated milk replacer according to label directions at a rate of 12% of body weight twice daily throughout the study period. 107 Virus infection protocol A noncytopathic type H BVDV isolate (BVDV 890) that has been shown to induce thrombocytopenia in calves (Bolin & Ridpath, 1992), was used for the experimental infection of calves in the treatment group. The virus was propagated in bovine turbinate cells in Eagle’s minimum essential medium (EMEM)d containing 10% fetal equine serum (FES),° L-glutamine,f penicillin G,e and streptomycin.° The viral titer of the inoculum was determined according to described methods (Lennette & Schmidt, 1969). After a 3-day adjustment period in the isolation facility, calves in the treatment group were inoculated by intranasal instillation with 107 median tissue culture infective doses of BVDV 890 in 5 ml of EMEM without serum (2.5 ml per nostril). Control calves were inoculated with a similar amount of an uninfected cell culture preparation. This dose of BVDV 890 was chosen because intranasal administration of 106 median tissue culture infective doses of BVDV 890 did not consistently induce thrombocytopenia in a previous study (Bolin & Ridpath, 1992). Collection of platelets Blood samples were collected for platelet aggregation studies and for isolation of BVDV fiom platelets 2 days before, the day of, and every 2 days for 12 days after inoculation of calves. Blood was collected by means of jugular venipuncture, using an 18-gauge needle, into plastic syringes containing 1 ml of 3.8% trisodiurn citrate for each 9 ml of blood. Platelet-rich plasma was obtained by means of 2 centrifugation steps at 1,324 x g for 60 seconds and 30 seconds, with platelet-rich plasma being removed after each centrifugation step. Platelet-poor plasma was obtained by means of centrifugation 108 of the remaining blood at 1,324 x g for 13 minutes. Platelets were manually counted, using a hemocytometer.g Platelet aggregation responses Aggregation studies were carried out in a dual-channel aggregometer.h Aggregation agonists used for this study were adenosine diphosphate (ADP)," at concentrations of 10 and 100 Ml, and platelet activating factor (PAF),i at concentrations of 0.1 and 1.0 uM. Platelet-rich plasma was adjusted to 300,000 platelets/pl with homologous platelet-poor plasma. Adjusted platelet-rich plasma suspensions were then divided into 0.5 ml aliquots in glass cuvettes and held at room temperature (approx 20 C) for 30 minutes. The platelet-rich plasma suspensions were then warmed for 5 minutes at 37 C. A magnetic stir bar was added to the sample, and the cuvette was placed into the aggregometer. For each experiment, the aggregometer was calibrated using nonaggregated platelet-rich plasma to establish the 0% aggregation limit and platelet-poor plasma to establish the 100% aggregation limit. The sample was allowed to stir at 900 rpm for approximately 30 seconds, at which time 20 ul of agonist was added to the platelet suspensions, and change in light transmission recorded. Maximum percentage aggregation and slope of the aggregation curve were measured for each aggregation test. All aggregation studies were completed within 5 hours after collection of blood samples. All studies were done at least in duplicate, and the mean value was obtained. 109 Isolation of BVD V from platelets Platelets were gel-filtered to remove plasma constituents. One microliter of 1 uM prostaglandin Ele was added per ml of platelet-rich plasma, and samples were centrifuged at 800 x g for 15 minutes. The supernatant was removed with a sterile pipette, and the platelet pellet was resuspended in 1 ml of Hank’s balanced salt solution (136 mM NaCl, 5.4 mM KCl, 0.44 mM KH2P04, 0.34 mMNazHPO4, and 5.5 mM dextrose; pH 7.4). The platelet suspension was then transferred to a 10 ml polystyrene column containing Sepharose 4B.° The eluate was collected, and platelet count was determined manually. Gel-filtered platelet suspensions were adjusted to a count of 200,000 platelets/pl, using EMEM containing 10% FES, L-glutamine, and antibiotics. Suspensions were subjected to 3 freeze-thaw cycles and then centrifuged for 10 minutes at 1,500 x g. The supernatant was removed and filtered through a sterile filter with an average pore diameter of 0.45 um.j Platelet preparations were stored at -80 C until used in virus isolation procedures. To isolate BVDV from platelets, platelet preparations were thawed, and 25 ul of each sample was inoculated in duplicate into wells on 96-well microtitration plates containing monolayers of bovine turbinate cells in EMEM with 10% FES, L-glutamine, and antibiotics. After 3 days of incubation at 37 C in humidified air containing 5% C02, the bovine turbinate cells were stained for BVDV antigen by use of an immunoperoxidase monolayer assay (Meyling, 1988). Statistical analysis For each data collection period, maximum percentage aggregation and slope of the aggregation curve were compared between infected and control calves by use of 110 Student’s t-test. Results of virus isolation from platelets were compared between groups by use of Fisher’s exact test. A value of P < 0.05 was considered significant. Results Aggregation responses with ADP Throughout the study, platelets from control calves displayed the expected shape change and aggregation in response to 10 M ADP (Figure 3.1). In BVDV-infected calves, the shape change appeared normal throughout the study, but maximum percentage aggregation and slope of the aggregation curve became progressively decreased over time (Figure 3.2). When 10 M ADP was used as the agonist, zero aggregation (ie, a failure to record an increase in light transmission, compared with baseline transmission, when the sample was allowed to stir in the aggregometer for 5 minutes after addition of the agonist) was observed for 2 of 5 infected calves on days 6 and 8 after inoculation, in all infected calves on day 10, and in 4 of 5 infected calves on day 12. Throughout the study, 100 M ADP induced the expected shape change and aggregation responses in platelets from control calves (Figure 3.3). A shape change preceded the aggregation response in platelets from the BVDV-infected calves; however, maximum percentage aggregation and slope of the aggregation curve decreased over time in the infected calves (Figure 3.4). When 100 M ADP was used as the agonist, zero aggregation was also observed for 2 of 5 BVDV-infected calves on day 12 after inoculation. 111 Aggregation responses with PAF Throughout the study, platelets from control calves displayed expected aggregation responses when mixed with 0.1 uM PAF (Figure 3.5). For platelets fi'om infected calves, maximum percentage aggregation and slope of the aggregation curve were progressively decreased over time (Figure 3.6). Similarly, 1.0 uM PAF induced expected responses in platelets from control calves (Figure 3.7), but maximum percentage aggregation and slope of the aggregation curve were progressively decreased over time for platelets from infected calves (Figure 3.8). Isolation of B VD V from platelets Bovine viral diarrhea virus was not isolated from platelets fi'om control calves, but was isolated from platelets from 1 BVDV-infected calf 4 days after inoculation, from 4 of 5 infected calves 6 and 12 days after inoculation, and from all infected calves 8 and 10 days after inoculation. 112 Figure 3.1. Aggregometer tracings of bovine platelet-rich plasma suspensions stimulated with adenosine diphosphate at a concentration of 10 uM. The top two tracings represent the aggregation response curves of bovine platelets obtained from the same control calf on days 0 and 12 after infection. The bottom two tracings represent the aggregation response curves of bovine platelets obtained from the same BVDV-infected calf on days 0 and day 12 after infection. In the infected calf at day 12 after infection, the lower percent change in light transmission, compared to the other aggregation tracings, represents a depressed aggregation response. 113 Figure 3.1 Control calf (day 0) Control calf (day 12) BVDV-infected calf (day 0) % change in light transmission BVDV-infected calf (day 12) 1 minute 114 Figure 3.2. Platelet aggregation responses (A: Maximum percent aggregation; B: Slope of the aggregation curve) induced by adenosine diphosphate at a concentration of IOuM for control (n=4) and BVDV-infected (n=5) calves during the experimental time period. Results are expressed as the mean i standard error. * Indicates values that are significantly (P<0.05, t-test) different between control and BVDV-infected calves at the same collection time. 115 Figure 3.2 I Infected group T L q. T I+-l L t 1+ t-ttl-lrtl_ 2 o 8 6 4 2 o 1 1 53.523. E022. :55.an 12 10 Days after Infection 1 l DControl group I Infected group 1.6 - I. Illii-li+tll+lltt I111 1w 1.4 1 8. 0 one.» 6. O 4. 0 2. 0 Days after Infection 116 Figure 3.3. Aggregometer tracings of bovine platelet-rich plasma suspensions stimulated with adenosine diphosphate at a concentration of 100 M. The top two tracings represent the aggregation response curves of bovine platelets obtained from the same control calf on days 0 and 12 after infection. The bottom two tracings represent the aggregation response curves of bovine platelets obtained from the same BVDV-infected calf on days 0 and 12 after infection. In the infected calf at day 12 after infection, the lower percent change in light transmission, compared to the other aggregation tracings, represents a depressed aggregation response. 117 Figure 3.3 % change in light transmission 1 minute Control calf (day 0) Control calf (day 12) BVDV-infected calf (day 0) BVDV-infected calf (day 12) 118 Figure 3.4. Platelet aggregation responses (A: Maximum percent aggregation; B: Slope of the aggregation curve) induced by adenosine diphosphate at a concentration of 100pM for control (n=4) and BVDV-infected (n=5) calves during the experimental time period. Results are expressed as the mean i standard error. * Indicates values that are significantly (P< 0. 05, t-test) different between control and BVDV-infected calves at the same collection time. 119 ‘ A El Control group Figure 3.4 I hfected group 90 . * * * * |_|_. 8 8 8 8 5‘ AAfl-a-I’ I4 N O t-A-w Maximum percent aggregation .5 O O -2 0 2 4 6 8 10 B E] Control group I hfected group 2.5 . * * Slope Days after Infection 120 Figure 3.5. Aggregometer tracings of bovine platelet-rich plasma suspensions stimulated with platelet activating factor at a concentration of 0.1 M. The t0p two tracings represent the aggregation response curves of bovine platelets obtained from the same control calf on days 0 and 12 after infection. The bottom two tracings represent the aggregation response curves of bovine platelets obtained from the same BVDV-infected calf on days 0 and 12 after infection. In the infected calf at day 12 after infection, the lower percent change in light transmission, compared to the other aggregation tracings, represents a depressed aggregation response. 121 Figure 3.5 % change in light transmission Control calf (day 0) Control calf (day 12) BVDV-infected calf (day 0) BVDV-infected calf (day 12) _ 1 minute 122 Figure 3.6. Platelet aggregation responses (A: Maximum percent aggregation; B: Slope of the aggregation curve) induced by platelet activating factor at a concentration of 0.1uM for control (n=4) and BVDV-infected (n=5) calves during the experimental time period. Results are expressed as the mean i standard error. * Indicates values that are significantly (P<0.05, t-test) different between control and BVDV-infected calves at the same collection time. 123 Figure 3.6 [3 Control group 1 A l hfected group * * 90 1 801 Maximum percent aggregation 2 4 6 Days after infection B [1 Control group I hfected group 2 3 .5 j . 21 l i 1.5 ~ ‘ l 1: 0.5 1 i 0 + » k 1 -2 0 2 4 Slope 1r 6 Days after infection 124 70¢ sol 50:» 40T 30.1» 201 y mi 01 .k A .m ,,_l .L -2 o 8 8 .1 10 10 Figure 3.7. Aggregometer tracings of bovine platelet-rich plasma suspensions stimulated with platelet activating factor at a concentration of 1.0 M. The top two tracings represent the aggregation response cm'ves of bovine platelets obtained from the same control calf on days 0 and 12 after infection. The bottom two tracings represent the aggregation response curves of bovine platelets obtained from the same BVDV-infected calf on days 0 and 12 after infection. In the infected calf at day 12 after infection, the lower percent change in light transmission, compared to the other aggregation tracings, represents a depressed aggregation response. 125 Figure 3.7 Control calf 1 (day 0) Control calf (day 12) fl .2 .2 E a BVDV-infected g calf (day 0) H u: .20 .5 = fl .3 U BVDV-infected 32 calf (day 12) 1 minute 126 Figure 3.8. Platelet aggregation responses (A: Maximum percent aggregation; B: Slope of the aggregation curve) induced by platelet activating factor at a concentration of 1.0M for control (n=4) and BVDV-infected (n=5) calves during the experimental time period. Results are expressed as the mean i standard error. * Indicates values that are significantly (P< 0.05, t-test) different between control and BVDV-infected calves at the same collection time. 127 Maximum percent aggregation l hfected group 90 - l 80 1 70 60 D Control group I hfected group Figure 3.8 4 6 4 6 Days after infection 128 Discussion Platelets play an important role in hemostasis, and altered platelet function may lead to hemorrhage (Jain, 1986). Results of the present study indicate that platelets from calves infected with BVDV 890, a type H BVDV isolate, display abnormal aggregation responses. It has been reported that BVDV infection is associated with thrombocytopenia and hemorrhage (Bezek et al., 1994; Bolin & Ridpath, 1992; Corapi et al., 1990; Rebhun et al., 1989), yet in some instances, the decrease in platelet count has been insufficient to be completely responsible for clinical evidence of hemorrhage. Findings of this study suggest that altered platelet function, in addition to thrombocytopenia, may contribute to the hemorrhage observed in some calves with type H BVDV infection. Although the mechanism responsible for diminished platelet responses in association with BVDV 890 infection is unknown, it is likely to involve an alteration in the population of platelets in the circulation resulting in an increase in the percentage of aged platelets, a direct virus-platelet interaction, an indirect virus-platelet interaction involving intermediate factors, or some combination of these mechanisms. Megakaryocytes are the precursor cells for platelets (Wright, 1906), and infection of megakaryocytes with BVDV has been demonstrated through immunohistochemical and immunofluorescent antibody techniques (Corapi et al., 1989; Marshall et al., 1996; Spagnuolo-Weaver et al., 1997). Bovine viral diarrhea virus infection of megakaryocytes may decrease production of platelets, resulting in thrombocytopenia and increasing the percentage of older platelets in the circulation. In dogs, humans, and rats, platelets in the circulation become smaller and less sensitive to aggregatory stimuli as they age (Carty & 129 Gear, 1986; Peng et al., 1994). Younger platelets are larger and substantially more sensitive than aged platelets to aggregation agonists, such as ADP and thrombin. In the present study, aggregation studies were performed on platelet-rich plasma suspensions, rather than blood, to control for changes in platelet count in calves infected with BVDV. Because platelet aggregation studies were performed on platelet-rich plasma suspensions adjusted to counts of 300,000 platelets/pl, decreased aggregation responses for platelets from infected calves could have been a result of an increase in the percentage of dysfunctional platelets, compared with control calves. These dysfunctional platelets may have been aged platelets in which aggregation responses were impaired as a result of decreased granule content of secretory factors, impaired signal transduction pathways, or alterations in the cell surface receptors for agonists. In this study, we identified a direct virus-platelet interaction by isolating BVDV from purified platelet preparations. Previous studies have also identified an association between platelets and BVDV, using virus isolation procedures (Bolin & Ridpath, 1992) and immunofluorescent antibody testing (Bezek et al., 1994; Corapi et al., 1989). We attempted to isolate BVDV from purified platelet preparations at the same time aggregation studies were performed to determine whether a direct virus-platelet interaction could be responsible for platelet dysfunction in infected calves. We found that a significant decrease in maximum percentage aggregation in response to ADP or PAF only became apparent when virus could be isolated from platelets (ie, 4 through 12 days after inoculation). Therefore, it is possible that a direct virus-platelet interaction was responsible for the aggregation defects in infected calves. However, in a previous study (Bolin & Ridpath, 1992), the percentage of platelets from which BVDV could be isolated 130 was very low (approx 0.1%). Thus, it seems likely that direct virus-platelet interaction was not the sole cause of platelet dysfunction. In addition, it has been demonstrated that feline infectious peritonitis virus causes hyperaggregability, probably as a result of a direct virus-platelet interaction (Boudreaux et al., 1990), not the hypoaggregability detected in the present study. Prostacyclin (prostaglandin 12), prostaglandin E, nitric oxide, circulating immune complexes, and a variety of other circulating substances have been demonstrated to inhibit platelet aggregation responses (Rao & Carvalho, 1994), and an increase in the concentration of 1 or more of these intermediate soluble factors in platelet-rich plasma suspensions could alter aggregation responses of platelets. Decreased aggregation responses have been demonstrated in human patients infected with Lassa fever virus, a hemorrhagic fever of humans (Cummins et al., 1989). The platelet defect in Lassa fever virus infection has been determined to be a result of an inhibitory factor in plasma that does not appear to be a viral protein or virus antibody (Cummins et al., 1989). In pigs infected with Afiican swine fever virus, high plasma concentrations of the platelet aggregation inhibitor prostaglandin E2 have been demonstrated (Anderson et al., 1987). High concentrations of nitric oxide and prostaglandin B have been demonstrated in bone marrow macrophages (Adler et al., 1994) and alveolar macrophages (Van Reeth & Adair, 1997), respectively, from cattle with BVDV infection, but what impact these high concentrations have on platelet function is not known. Adenosine diphosphate and PAF were used as aggregation agonists in this study because of their predictable aggregation response in cattle (Bondy & Gentry, 1989) and ease in use. In addition, they use different receptors and signal transduction pathways to 131 induce platelet aggregation (Hawiger et al., 1994). The shape changes induced by ADP throughout the study indicated that platelets had a fimctional ADP receptor. The observation of a shape change without aggregation (zero aggregation) in response to 10 W and 100 M ADP in some BVDV-infected calves suggested that although the receptor was functionally present, the enzyme pathway or secretion of additional ADP by platelets may have been impaired. When PAF was used, platelets from control and infected calves underwent a shape change and aggregated, but the aggregation response was diminished in platelets from the infected calves. Detection of fimctional ADP and PAF receptors suggests that the defect in function of platelets from infected calves reflects deficiencies in the signal transduction pathways, the release reaction of the granule constituents, or the secretory substances themselves. In summary, hemorrhagic syndrome associated with BVDV 890 infection in neonatal calves can be attributable to thrombocytopenia and altered platelet function. At present, it is not known whether other type I or type H BVDV isolates also induce qualitative platelet defects. Further study is required to define the pathophysiologic mechanism of altered platelet function in infected calves and to identify the role that altered platelet fitnction may have, particularly in relation to the increased virulence of some type H BVDV isolates. Sources and Manufacturers a. Bolin SR, National Animal Disease Center, Ames, IA: Personal communication, 1997. b. Butler Company, Dublin, Ohio. c. Schering-Plough Animal Health Corporation, Kenilworth, NJ. 132 d. JRH Biosciences, Lenexa, Kan. e. Sigma Chemical Co, St. Louis, Mo. f. Gibco BRL, Life Technologies, Grand Island, NY. g. Becton Dickinson, Franklin, NJ. h. Chronolog Corporation, Havertown, Pa. i. Calbiochem Biochemicals, San Diego, Calif. j. Millipore Corporation, Bedford, Mass. References Adler, H., Frech, B., Meier, P., Jungi, T.W. & Peterhans, E. (1994). Noncytopathic strains of bovine viral diarrhea virus prime bovine bone marrow-derived macrophages for enhanced generation of nitric oxide. Biochem Biophys Res Commun, 202, 1562-8. Anderson, E.C., Williams, S.M., Fisher-Hoch, S.P. & Wilkinson, P.J. (1987). Arachidonic acid metabolites in the pathophysiology of thrombocytopenia and haemorrhage in acute Afiican swine fever. Res Vet Sci, 42, 387-94. Baker, J .C. (1995). The clinical manifestations of bovine viral diarrhea virus infection. In The Veterinary Clinics of North America (Food Animal Practice). Baker, J.C. & Houe, H. (eds), Vol. 11. pp. 425-445. W.B. Saunders: Philadelphia. Bezek, D.M., Grohn, Y.T. & Dubovi, E.J. (1994). Effect of acute infection with noncytopathic or cytopathic bovine viral diarrhea virus isolates on bovine platelets. Am J Vet Res, 55, 1115-9. Bolin, S.R. & Ridpath, J .F . (1992). Differences in virulence between two noncytopathic bovine viral diarrhea viruses in calves. Am J Vet Res, 53, 2157-63. Bondy, G.S. & Gentry, P.A. (1989). Characterization of the normal bovine platelet aggregation response. Comp Biochem Physiol C, 92, 67-72. Boudreaux, M.K., Weiss, R.C., Toivio-Kinnucan, M., Cox, N. & Spano, J.S. (1990). Enhanced platelet reactivity in cats experimentally infected with feline infectious peritonitis virus. Vet Pathol, 27, 269-73. 133 Carty, D.J. & Gear, AR. (1986). Fractionation of platelets according to size: functional and biochemical characteristics. Am J Hematol, 21, 1-14. Corapi, W.V., Elliott, R.D., French, T.W., Arthur, D.G., Bezek, D.M. & Dubovi, E.J. (1990). Thrombocytopenia and hemorrhages in veal calves infected with bovine viral diarrhea virus. J Am Vet Med Assoc, 196, 590-6. Corapi, W.V., French, T.W. & Dubovi, E.J. (1989). Severe thrombocytopenia in young calves experimentally infected with noncytopathic bovine viral diarrhea virus. J Virol, 63, 3934-43. Cummins, D., Fisher-Hoch, S.P., Walshe, K.J., Mackie, I.J., McCormick, J .B., Bennett, D., Perez, G., Farrar, B. & Machin, SJ. (1989). A plasma inhibitor of platelet aggregation in patients with Lassa fever. Br J Haematol, 72, 543-8. Day, H.J. & Rao, AK. (1986). Evaluation of platelet function. Semin Hematol, 23, 89- 101. Hawiger, J ., Brass, L. & Salzman, E. (1994). Signal transduction and intracellular regulatory processes in platelets. In Thrombosis and hemostasis: basic principles and clinical practice, Colman, R., Hirsh, J ., Marder, V. & Salzman, E. (eds) pp. 603- 628. J .B. Lippincott Co.: Philadelphia, PA. Jain, N. (1986). Qualitative and quantitative disorders of platelets. In Schalm's veterinary hematology, Jain, N. (ed) pp. 466—486. Lea & F ebiger: Philadelphia, PA. Lennette, E. & Schmidt, N. (1969). Diagnostic procedures for viral and richettsia] infections. American Public Health Association Incorporated: New York, NY. Marshall, D.J., Moxley, R.A. & Kelling, CL. (1996). Distribution of virus and viral antigen in specific pathogen-free calves following inoculation with noncytopathic bovine viral diarrhea virus. Vet Pathol, 33, 311-8. Meyling, A. (1988). Detection of BVD virus in viremic cattle by an indirect immunoperoxidase technique. In Recent advances in virus diagnosis, McNulty, M. & MacFerran, J. (eds) pp. 37-46. Martinus Nijhoff Publishing: Boston, MA. Peng, J ., Friese, P., Heilrnann, E., George, J .N., Burstein, S.A. & Dale, G.L. (1994). Aged platelets have an impaired response to thrombin as quantitated by P-selectin expression. Blood, 83, 161-6. Rao, A. & Carvalho, A. (1994). Acquired qualitative platelet defects. In Thrombosis and hemostasis: basic principles and clinical practice, Colman, R., Hirsh, J ., Marder, V. & Salzman, E. (eds) pp. 685-704. J .B. Lippincott Co.: Philadelphia, PA. 134 Rebhun, W.C., French, T.W., Perdrizet, J .A., Dubovi, E.J., Dill, S.G. & Karcher, L.F. (1989). Thrombocytopenia associated with acute bovine virus diarrhea infection in cattle. J Vet Intern Med, 3, 42-6. Ridpath, J.F., Bolin, S.R. & Dubovi, E.J. (1994). Segregation of bovine viral diarrhea virus into genotypes. Virology, 205, 66-74. Spagnuolo-Weaver, M., Allan, G.M., Kennedy, 8., Foster, J.C. & Adair, B.M. (1997). Distribution of cytopathic and noncytopathic bovine viral diarrhea virus antigens in tissues of calves following acute experimental infection. J Vet Diagn Invest, 9, 287- 97. Van Reeth, K. & Adair, B. (1997). Macrophages and respiratory viruses. Pathol Biol (Paris), 45, 184-92. Wengler, G. (1991). Family F Iaviviridae. In Classification and nomenclature of viruses. International committee on taxonomy of viruses, Francki, R.I.B., F auquet, C.M., Knudsen, D.L. & Brown, F. (eds) pp. 223-233. Springer Verlag: Berlin. Wright, J. (1906). The origin and nature of blood platelets. Boston Medicine and Surgery Journal, 154-163. 135 CHAPTER IV: JOURNAL ARTICLE: PLATELET AGGREGATION RESPONSES AND VIRUS ISOLATION FROM PLATELETS IN CALVES EXPERIMENTALLY INFECTED WITH TYPE I OR TYPE II BOVINE VIRAL DIARRHEA VIRUS 136 CHAPTER IV: JOURNAL ARTICLE: PLATELET AGGREGATION RESPONSES AND VIRUS ISOLATION FROM PLATELETS IN CALVES EXPERIMENTALLY INFECTED WITH TYPE I OR TYPE H BOVINE VIRAL DIARRHEA VIRUS Paul H. Walz, Thomas G. Bell, Daniel L. Grooms, Lana Kaiser, Roger K. Maes, and John C. Baker From the Department of Large Animal Clinical Sciences (W alz, Grooms, Kaiser, Baker), the Animal Health Diagnostic Laboratory (Bell, Maes), the Department of Microbiology (Macs), and the Department of Veterinary Pathology (Bell), College of Veterinary Medicine, Michigan State University, East Lansing, MI, USA 48824-1314. 137 Abstract Altered platelet function has been reported in calves experimentally infected with type H bovine viral diarrhea virus (BVDV). The purpose of the present study was to further evaluate the ability of BVDV isolates to alter platelet function and to specifically determine if platelet dysfunction only occurs following challenge with type H BVDV. Colostrum-deprived Holstein calves were obtained immediately after birth, housed in isolation, and assigned to one of four groups (1 control and 3 treatment groups). Control calves (n=4) were sham inoculated, while calves in the infected groups (n=4 for each group) were inoculated by intranasal instillation with 107 TCH)50 of either BVDV 890 (type H), BVDV 7937 (type H), or BVDV TGAN (type 1). Whole blood was collected prior to inoculation (day 0) and on days 4, 6, 8, 10, and 12 after inoculation for platelet function testing by optical aggregometry using the aggregation agonists adenosine diphosphate (10 and 100 uM) and platelet activating factor (0.1 and 1.0 uM). Virus isolation procedures from platelets were performed on the same days as platelet fimction with the exception of day 10 after inoculation. The maximum percentage aggregation and the slope of the aggregation curve decreased over time in BVDV-infected calves; however statistically significant differences (Freidman Repeated Measures AN OVA on Ranks, p<0.05) were only observed in calves infected with the type H BVDV isolates. Bovine viral diarrhea virus was not isolated from control calves, but was isolated from all calves infected with both type H BVDV isolates from days 4 through 12 after inoculation. In calves infected with type I BVDV, the virus was isolated from 1 of 4 calves on days 4 and 12 after inoculation and from all calves on days 6 and 8 afier inoculation. Altered platelet function was observed in calves infected with both type H BVDV isolates, while 138 altered platelet function was not observed in calves infected with type I BVDV. Altered platelet function may be an important difference in virulence between type I and type II BVDV infections. Introduction Circulating platelets play an important role in hemostasis and thrombosis. Impairment of the hemostatic response may occur with quantitative platelet disorders (thrombocytopenia), in which circulating numbers of platelets are low, or qualitative platelet defects (thrombocytopathy), in which platelets have altered functional characteristics (Jain, 1986). Impairment of the hemostatic response can result in clinical signs of hemorrhage. Bovine viral diarrhea virus (BVDV) has been associated with outbreaks of severe peracute disease (Carman et al., 1998) and a hemorrhagic syndrome characterized by thrombocytopenia, bleeding, and death (Bolin & Ridpath, 1992; Corapi et al., 1989; Rebhun et al., 1989). Thrombocytopenia associated with type H BVDV infection has been reported in adult cattle and calves under natural conditions (Rebhun et al., 1989), and has been experimentally reproduced (Bolin & Ridpath, 1992; Corapi et al., 1990; Walz et al., 1999a). Although most cases of BVDV-induced thrombocytopenia have been associated with type H BVDV, a recent study demonstrated that a type I isolate induced thrombocytopenia in experimentally infected calves (Marshall et al., 1996). In addition to thrombocytopenia, altered platelet function has been reported following experimental type H BVDV infection in calves (Walz et al., 1999b); however, a single type H BVDV isolate was examined, and it is currently unknown if type I BVDV 139 isolates or other type H BVDV isolates are capable of altering platelet function. Altered platelet function may be an important contributing factor for the hemorrhagic syndrome and for the increased virulence observed with some type H BVDV isolates. Therefore, this study was designed to further evaluate the ability of BVDV isolates to alter platelet function, and to determine if platelet dysfunction occurs exclusively following challenge with type H BVDV. Materials and Methods Animals Colostrum-deprived newborn male Holstein calves (n=16) were obtained immediately after birth fiom local dairy farms. Calves were moved to an isolation facility, allocated to one of four groups (1 control and 3 treatment groups), and individually housed in separate rooms. All calves were given a physical examination, and serum and whole blood were taken for virus isolation procedures and determination of antibody levels to BVDV by virus neutralization. The calves were administered an intravenous plasma transfusion, consisting of 400 ml of plasma obtained from a donor cow. The plasma donor was BVDV antibody negative by virus neutralization and virus negative by virus isolation procedures on three serial serum samples obtained prior to the collection of plasma. In addition, calves were given injections of vitamins A and D (Butler Company, Dublin, OH) and vitamin E and selenium (Schering-Plough Animal Health Corporation, Kenilworth, NJ), as well as a commercial oral monoclonal antibody preparation against E. coli K99 pilus antigen (Schering-Plough Animal Health 140 Corporation, Kenilworth, NJ). Calves were fed a nonmedicated milk replacer according to label directions at 12% of body weight twice daily throughout the study period. The experiment was performed with approval and under the guidelines of the Michigan State University All University Committee on Animal Use and Care. Virus and infection protocol The following three noncytopathic BVDV isolates were used for the experimental infection of calves: BVDV 890 (Dr. Steve Bolin, National Animal Disease Center, Ames, IA), BVDV 7937 (Dr. Clayton Kelling, University of Nebraska, Lincoln, NE), and BVDV TGAN (Dr. Steve Bolin, National Animal Disease Center, Ames, IA). The BVDV 890 is a type H isolate that has been previously shown to induce thrombocytopenia and platelet dysfunction in experimentally infected calves (Bolin & Ridpath, 1992; Walz et al., 1999a; Walz et al., 1999b). The BVDV 7937 (type H) and BVDV TGAN (type I) are BVDV isolates that have been reported not to induce thrombocytopenia (Bolin & Ridpath, 1992; Marshall et al., 1996). The viral isolates were propagated in bovine turbinate cells in Eagle’s minimum essential medium (EMEM; JRH Biosciences, Lenexa, KS) containing 10% fetal equine serum (FES; Sigma Chemical Co., St. Louis, MO), L-glutamine (Gibco BRL, Life Technologies, Grand Island, NY), penicillin G (Sigma Chemical Co.), and streptomycin (Sigma Chemical Co.). The viral titer of the inoculum was determined according to previously described methods (Lennette & Schmidt, 1969). After a three-day adjustment period in the isolation facility, all calves were inoculated. Calves in the 3 infected groups (n=4 for each group) were inoculated by 141 intranasal instillation with 107 TCIDso of the respective viral isolate in 5 ml of EMEM without serum (2.5 ml per nostril). Control calves (n=4) were inoculated by intranasal instillation with a sham inoculum, consisting of a BVDV-uninfected cell culture preparation. Collection of platelets Whole blood was collected for platelet aggregation studies on the day of inoculation (pre-inoculation), and then on days 4, 6, 8, 10, and 12 afier inoculation. In addition, whole blood was collected for virus isolation from platelets on the day of inoculation, and then on days 4, 6, 8, and 12 after inoculation. Blood was collected by jugular venipuncture through an 18-gauge needle into plastic syringes containing 1.0 ml of 3.8% trisodium citrate for each 9.0 ml of blood. Platelet-rich plasma was obtained by two centrifugation steps at 1,324 x g for 60 seconds and 30 seconds, with the platelet-rich plasma being removed after each centrifugation. Platelet-poor plasma was obtained by centrifugation of the remaining blood at 1,324 x g for 13 minutes. Platelets were manually counted using a white blood cell microcollection system (Becton Dickinson, Franklin, NJ) and a hemocytometer. Platelet aggregation responses Aggregation studies were performed in an aggregometer (Chronolog Corporation, Havertown, PA) by optical aggregometry. Aggregation agonists used for this study were adenosine diphosphate (ADP; Sigma Chemical Co.), at concentrations of 10 and 100 M, and platelet activating factor (PAF; Calbiochem Biochemicals, San Diego, CA), at 142 concentrations of 0.1 and 1.0 uM. Platelet-rich plasma was adjusted to 300,000 platelets/pl with homologous platelet-poor plasma. Adjusted platelet-rich plasma suspensions were then divided into 0.5 ml aliquots in glass cuvettes and held at room temperature for 30 minutes. The platelet-rich plasma suspensions were warmed for 5 minutes at 37°C. A magnetic stir bar was added to the sample, and the cuvette was then placed into the aggregometer. For each experiment, the aggregometer was calibrated using nonaggregated platelet-rich plasma to establish the 0% aggregation limit and platelet-poor plasma to establish the 100% aggregation limit. The sample was allowed to stir at 900 rpm for approximately 30 seconds, at which time 20 pl of agonist was added to the platelet suspensions, and the changes in light transmission recorded. The maximum percentage aggregation and the slope of the aggregation curve were measured for each aggregation run. All aggregation studies were completed within 5 hours of collection of blood samples. All studies were done in duplicate, and the mean value was obtained. Virus isolation from platelets Platelets were gel-filtered and washed to remove plasma constituents. Platelet-rich plasma was collected as described above and 1 ul of 1 uM prostaglandin E. (Sigma Chemical Co.) was added per 1 ml platelet-rich plasma prior to centrifugation at 800 x g for 15 minutes. Plasma supernatant was removed with a sterile pipette, and the platelet pellet was resuspended in 1 rrrl of Hank’s Balanced Salt Solution (136 mM NaCl, 5.4 mM KCL, 0.44 mM KH2P04, 0.34 mM Nazl-IPO4, and 5.5 mM dextrose, pH 7.4). The platelet suspension was transferred to a 10 m1 polystyrene column containing, Sepharose 4B (Sigma Chemical Co.). The eluate was collected, washed three times in 10 ml of 143 Hank’s Balanced Salt Solution, resuspended in Hank’s Balanced Salt Solution, and counted manually. Approximately 107 platelets were inoculated into wells on 96-well microtiter plates containing monolayers of bovine turbinate cells in EMEM with 10% FES, L-glutamine and antibiotics. Afler 5 days of incubation at 37°C in humidified air containing 5% C02, 50 u] of supernatant was transferred to 96-well rrricrotiter plates freshly seeded with bovine turbinate cells. After an additional 5 days of incubation at 37°C in humidified air containing 5% C02, the bovine turbinate cells were stained for BVDV antigen by an immunoperoxidase monolayer assay (Meyling, 1988). Statistical analysis Comparisons of the maximum percentage aggregation and the slope of the aggregation curve among groups were made using the F reidman repeated measures analysis of variance on ranks. When significant (P<0.05) differences were determined, post-hoe comparisons were made between the day 0 sample period (pre-inoculation) to days 4, 6, 8, 10, and 12 sample periods (after inoculation) by use of Dunn’s multiple comparisons test. Results Aggregation responses with adenosine diph osphate (ADP) The aggregation results for the control and BVDV-infected groups of calves in response to 10 MM ADP are presented in Figure 4.1. Throughout the study, platelets fi'om control calves displayed an expected shape change and aggregation response to 10 uM ADP. A normal shape change was also observed in the BVDV-infected calves in 144 response to 10 M ADP. However, platelet function was depressed in the two groups of calves inoculated with type H BVDV, with the greatest depression occurring in the calves inoculated with the type H isolate BVDV 890 (Figure 4.1). Zero aggregation, which is defined as a failure to record an increase in light transmission above the zero baseline after the addition of agonist, was observed using 10 M ADP in all calves infected with the type H isolate BVDV 890 on day 10 after infection. In calves infected with the type I isolate BVDV TGAN, a difference in platelet function was not observed between pre- and after inoculation samples in response to 10 M ADP. The aggregation results for the control and infected groups of calves in response to 100 M ADP are presented in Figure 4.2. In the control calves, ADP at a concentration of 100 M induced the expected shape change and aggregation response throughout the study. A shape change also preceded the aggregation response in the BVDV-infected calves; however, a decrease in the maximum percentage aggregation and the slope of the aggregation curve was observed in calves infected with both type H BVDV isolates as the experiment proceeded overtime (Figure 4.2). The greatest depression in platelet function occurred on day 10 after inoculation, and aggregation tracings fiom a representative calf in each of the 4 groups are presented in Figure 4.3. Zero aggregation, as previously defined, was observed in response to 100 M ADP in 2 of 5 calves infected with the type H isolate BVDV 890 on day 10 after inoculation. Although a statistically significant difference was not detected between pre— and post-inoculation sample periods in calves infected with the type I isolate BVDV TGAN, there was a mild depression in platelet function as the experiment proceeded over time (Figures 4.2 and 4.3). 145 Aggregation responses with platelet activating factor (PAF) The aggregation results for the control and infected groups of calves in response to 0.1 uM PAF are presented in Figure 4.4. The platelets from control calves displayed a normal aggregation response to 0.1 uM PAF throughout the study period, while platelets from the calves infected with the type H isolate BVDV 890 exhibited a significantly depressed maximum percentage aggregation and slope on day 10 after inoculation. Although a statistically significant depression in platelet function was not detected between pre- and after inoculation samples in calves infected with the type H isolate BVDV 7937, there was a depression in the maximum percentage aggregation and the slope of the aggregation curve as the experiment proceeded over time. The greatest depression in platelet function in the type H BVDV-infected calves occurred on day 10 after inoculation, and aggregation tracings from a representative calf in each of the 4 groups are presented in Figure 4.5. A statistically significant difl‘erence was not detected between pre- and post-inoculation samples in calves infected with the type I isolate BVDV TGAN, but there was a mild depression in the maximum percentage aggregation and the slope of the aggregation curve as the experiment proceeded over time (Figures 4.4 and 4.5). The aggregation results for the control and infected groups of calves in response to 1.0 uM PAF are presented in Figure 4.6. The maximum percentage aggregation and slope of the aggregation curve in calves infected with the type H isolate BVDV 890 were significantly depressed on day 10 and days 8, 10, and 12 after inoculation, respectively, when compared to day 0 in response to 1.0 uM PAF. The trend of a depressed maximum percentage aggregation and slope of the aggregation curve was also observed in calves 146 infected with the type H isolate BVDV 7937 as the experiment proceeded over time, but statistically significant differences were only observed with respect to the slope of the aggregation curve. There was a mild depression in the maximum percentage aggregation in calves infected with the type I isolate BVDV TGAN, but the differences were not statistically significant when compared to the day 0 sample. Isolation of B VD V from platelets Bovine viral diarrhea virus was not isolated from platelet preparations from control calves. Bovine viral diarrhea virus was isolated from platelets from all post-inoculation time periods in calves that were infected with the type H isolates (Table 4.1). Bovine viral diarrhea virus was also isolated from all calves infected with the type I isolate BVDV TGAN, but was not isolated from all the calves at all post-inoculation time periods (Table 4.1). 147 Figure 4.1. Platelet aggregation responses (A: Maximum percent aggregation; B: Slope of the aggregation curve) induced by adenosine diphosphate at a concentration of lOuM for uninfected control calves, calves infected with the type I isolate BVDV TGAN, calves infected with the type H isolate BVDV 793 7, and calves infected with the type H isolate BVDV 890. Calves were infected at day 0. Results are expressed as the mean i standard error. Statistically significant differences (Freidman RM ANOVA on ranks, p < 0.05, Dunn’s test) are denoted as follows: "‘ = BVDV 890 afler inoculation vs. before inoculation (day 0); # = BVDV 7937 after inoculation vs. before inoculation (day 0). 148 Figure 4.1 smmm W W W W LLLLLLLLLLLL: D I I I a“, LLLLLLLLLLLL: LLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLL_L_L_L___ L LLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLL _. 1-1H11W11 . . ii 1.1 L. coaaueuua‘ 38:09.0."— :58.an Day after Infection DContmi I BVDV TGAN -- LLLL.L..LLLL up 3 BVDV 7937 I BVDV 890 #,* w- #,* .- LL:LLLLLLL:25:22:25.: LLLLLLLLL:L::::1:_.:::::L L======E=LLLL _L__.==._.._._._..:L.___EL LLLLLLLLLLLLLLLLLLLLLL.LLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLL L:.W\$I~« .Rt..mdc~.l.0\.i...# LLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLL iQwrk‘UE.S!1-lfiwtlz 12 1O Dayaafterlnfectlon 149 Figure 4.2. Platelet aggregation responses (A: Maximum percent aggregation; B: Slope of the aggregation curve) induced by adenosine diphosphate at a concentration of lOOuM for uninfected control calves, calves infected with the type I isolate BVDV TGAN, calves infected with the type II isolate BVDV 7937, and calves infected with the type H isolate BVDV 890. Calves were infected at day 0. Results are expressed as the mean i standard error. Statistically significant differences (Freidman RM ANOVA on ranks, p < 0.05, Dunn’s test) are denoted as follows: * = BVDV 890 after inoculation vs. before inoculation (day 0); # = BVDV 7937 after inoculation vs. before inoculation (day 0). 150 Figure 4.2 Maximum Percentage Aggregation lllllllllllllllllllllIllllllllIlllll|llllllllilllllllllllllllllll Dy after Infection [3 Control I BVDV TGAN a BVDV 7937 I BVW 890 6 8 Day after Infection El Control I BVDV TGAN I BVDV 7937 I BVDV 890 #,* 151 Figure 4.3. Aggregometer tracings of bovine platelet-rich plasma suspensions stimulated with adenosine diphosphate at a concentration of 100 M. The four tracings represent the aggregation response curves of bovine platelets obtained from a calf in each group on day 10 after inoculation. In the BVDV-infected calves, the lower percent change in light transmission, compared to the other aggregation tracing from the control calf, represents a depressed aggregation response. 152 Figure 4.3 W BVDV TGAN BVDV 7937 :0.nn.£e:fl.5 spa: :— Gnclso .x. BVDV 890 153 Figure 4.4. Platelet aggregation responses (A: Maximtun percentage aggregation; B: Slope of the aggregation curve) induced by platelet activating factor at a concentration of 0.1M for uninfected control calves, calves infected with the type I isolate BVDV TGAN, calves infected with the type H isolate BVDV 7937, and calves infected with the type II isolate BVDV 890. Calves were infected at day 0. Results are expressed as the mean 1: standard error. Statistically significant differences (Freidman RM ANOVA on ranks, p < 0.05, Dunn’s test) are denoted as follows: * = BVDV 890 after inoculation vs. before inoculation (day 0); 'i' = Control post-sham inoculation vs. pre-sham inoculation (day 0). 154 Figure 4.4 Maximum Percentage Aggregation 8 8 8 20 Day after Infection El Control I BVDV TGAN I BVDV 7937 I BVDV 890 lllllllllillllll'lllllllllllllllllllllilllllllllllllllllllllllllllllllillllllllll El Control I BVDV TGAN E BVDV 7937 I BVDV 890 Figure 4.5. Aggregometer tracings of bovine platelet-rich plasma suspensions stimulated with platelet activating factor at a concentration of 0.1uM. The four tracings represent the aggregation response curves of bovine platelets obtained from a calf in each group on day 10 after inoculation. In the BVDV-infected calves, the lower percent change in light transmission, compared to the other aggregation tracing from the control calf, represents a depressed aggregation response. 156 Figure 4.5 BVDV TGAN a n m m m I I coin—£25.: Eu: :. once—.0 .x. 157 Figure 4.6. Platelet aggregation responses (A: Maximum percentage aggregation; B: Slope of the aggregation curve) induced by platelet activating factor at a concentration of 1.0M for uninfected control calves, calves infected with the type I isolate BVDV TGAN, calves infected with the type II isolate BVDV 7937, and calves infected with the type H isolate BVDV 890. Calves were infected at day 0. Results are expressed as the mean i standard error. Statistically significant differences (Freidman RM ANOVA on ranks, p < 0.05, Dunn’s test) are denoted as follows: * = BVDV 890 after inoculation vs. before inoculation (day 0); # = BVDV 7937 after inoculation vs. before inoculation (day 0). 158 Figure 4.6 Maximum Percentage Aggregation 8 8 8 8 5‘ 8 8 3 lliillllllllllllllllllllllillllllllllllllllllllllllllllilllllll llilllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllll l I l l l illlllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllillllllll lllllllllll llllllll El Control B I BVDV TGAN aavov 7937 #3 I evov 890 2.5 1 Table 4.1. Virus isolation results for purified platelet samples from calves experimentally infected with type I or type H BVDV ’ Day after Control BVDV TGAN BVDV 7937 BVDV 890 infection (Uninfected) (Type 1) (Type II) (Type H) DayO 0/415 0/4 0/4 0/4 Day4 0/4 1/4 4/4 4/4 Day6 0/4 4/4 4/4 4/4 Day8 0/4 4/4 4/4 4/4 Day12 0/4 1/4 4/4 4/4 a Control calves were inoculated with an uninfected tissue culture preparation, while infected calves received 107 TCIDso of the respective viral isolate b Cells under groups: numerator: # of calves that were virus positive; denominator: # of calves in group 160 Discussion Platelets are essential for normal hemostasis, and alterations in their function may lead to hemorrhage (Jain, 1986). Depression of platelet function has been previously demonstrated in calves experimentally infected with the type H isolate BVDV 890 (Walz et al., 1999b), and this altered platelet function likely contributes to the pathogenesis of the hemorrhagic syndrome, as clinical signs of hemorrhage were observed in the infected calves (Walz et al., 1999a). The findings of this study further support the ability of the type H isolate BVDV 890 to alter platelet function in experimentally infected calves. In comparison to the previous BVDV 890 infection study (Walz et al., 1999b), the mean values for maximum percentage aggregation and slope of the aggregation curve in response to both ADP and PAF were slightly lower, thus indicating a greater depression in platelet function observed in this study. An additional type H BVDV isolate was tested in this study to determine if altered platelet function occurs only in calves infected with the type H isolate BVDV 890. The type H isolate BVDV 7937 was utilized in this study because it was an isolate that has been reported to not induce thrombocytopenia (Marshall et al., 1996), and as such, would allow a comparison between two type H isolates that do and do not induce thrombocytopenia. Platelet ftmction was depressed in calves infected with the type H isolate BVDV 7937, although the depression observed was less than that observed in calves infected with the type H isolate BVDV 890. Type H BVDV infection results in altered platelet function. In addition, type H BVDV isolates that also induce 161 thrombocytopenia may be more adept at depressing platelet function than type H BVDV isolates that do not induce thrombocytopenia, as observed by the results in this study. Prior to the report describing thrombocytopenia in calves experimentally infected with a type I BVDV isolate, it was believed that only type H isolates were capable of inducing thrombocytopenia. We examined the type I isolate BVDV TGAN because it was reported to not induce thrombocytopenia (Bolin & Ridpath, 1992). This would allow a comparison between a thrombocytopenia-inducing type H BVDV isolate (BVDV 890) with a type I isolate that does not induce thrombocytopenia. Our study, which examined a single type I isolate that does not induce thrombocytopenia, also demonstrates that this type I isolate did not induce platelet dysfunction. This does not rule out the potential for other type I BVDV isolates to alter platelet function. Our studies were initiated prior to the report describing the ability of the type I BVDV isolate NY-l to induce thrombocytopenia (Marshall et al., 1996). Platelet ftmction testing in calves infected with the type I isolate BVDV NY-l may further characterize type I BVDV infections and alterations in platelet function. Variability in platelet aggregation responses, as evidenced by the large standard error, and a nrild depression in platelet function was observed in control calves in response to ADP IOuM, as the calves aged. A similar depression in platelet function in response to ADP lOuM was observed in an earlier study as well (Walz et al., 1999b). This may be an age-related effect that is obscured in the BVDV-infected calves as a result of infection. In addition to hypoaggregability in response to ADP 10 M in the control calves, a relative thrombocytosis peaking at approximately 7 days of age was also observed in a previous study (Walz et al., 1999a). These results reinforce the importance 162 of age-matched control calves in studying BVDV infection and platelet disorders in neonatal calves. Another observation that requires discussion is the depression in platelet function in response to ADP 10 M observed prior to inoculation in the calves infected with BVDV TGAN. No explanation can be afforded for this depression, although analysis of variance testing revealed no difference between any of the groups at the day 0 sample period. Although the exact mechanism responsible for the diminished platelet response associated with type H BVDV infection is unknown, the isolation of BVDV from platelets may be an important factor. The association of platelets and BVDV is supported by previous studies that have also demonstrated an association using virus isolation procedures (Bolin & Ridpath, 1992) and immunofluorescent antibody testing (Bezek et al., 1994; Corapi et al., 1989). Platelets are small, anucleate cytoplasmic fiagments that are derived from precursor cells in the bone marrow, the megakaryocytes (Wright, 1906). Bovine viral diarrhea virus infection of megakaryocytes has been demonstrated through immunohistochemical and immunofluorescent antibody techniques identifying BVDV antigen (Corapi et al., 1989; Ellis et al., 1998; Marshall et al., 1996; Spagnuolo-Weaver et al., 1997). The infection of megakaryocytes with BVDV may result in BVDV association with platelets, but more importantly, the virus infection of megakaryocytes may be responsible for altered platelet function. Bovine viral diarrhea virus infection in megakaryocytes may alter thrombopoiesis and/or megakaryocytopoiesis, thus resulting in thrombocytopenia and a population of predominantly older platelets in the peripheral circulation. As platelets age in the peripheral circulation, they become less sensitive to aggregatory stimuli (Carty & Gear, 1986; Peng et al., 1994). In contrast, younger 163 platelets are significantly more sensitive than aged platelets to the aggregation agonists, ADP and thrombin (Carty & Gear, 1986). A population of predominantly aged platelets in the peripheral circulation may explain our observations of platelet dysfunction in calves experimentally infected with type H BVDV. Further studies are required to associate the roles of aged platelets, BVDV association with platelets, and type H BVDV infection of megakaryocytes in the mechanism of altered platelet fimction associated with type H BVDV infection. In summary, we have demonstrated altered platelet function using two different type H BVDV isolates. Altered platelet function was not observed in the single type I BVDV isolate examined. Circulating platelets participate in many diverse physiologic and pathologic processes, including inflammation, coagulation, maintenance of vascular integrity, and vascular repair. Alteration of platelet function in cattle infected with type H BVDV likely contributes to the pathogenesis of the hemorrhagic syndrome, but type H BVDV infection may also be important for other platelet-related functions. In addition, altered platelet function may be an important factor for differences in virulence between type I and type H BVDV infections. References Bezek, D.M., Grohn, Y.T. & Dubovi, E.J. (1994). Efiect of acute infection with noncytopathic or cytopathic bovine viral diarrhea virus isolates on bovine platelets. Am J Vet Res, 55, 1115-9. Bolin, S.R. & Ridpath, J .F. (1992). Differences in virulence between two noncytopathic bovine viral diarrhea viruses in calves. Am J Vet Res, 53, 2157-63. Carman, S., van Dreumel, T., Ridpath, J ., Hazlett, M., Alves, D., Dubovi, E., Tremblay, R., Bolin, S., Godkin, A. & Anderson, N. (1998). Severe acute bovine viral diarrhea in Ontario, 1993-1995. J Vet Diagn Invest, 10, 27-35. 164 II _— Carty, D.J. & Gear, AR. (1986). Fractionation of platelets according to size: functional and biochemical characteristics. Am J Hematol, 21, 1-14. Corapi, W.V., Elliott, R.D., French, T.W., Arthur, D.G., Bezek, D.M. & Dubovi, E.J. (1990). Thrombocytopenia and hemorrhages in veal calves infected with bovine viral diarrhea virus. J Am Vet Med Assoc, 196, 590-6. Corapi, W.V., French, T.W. & Dubovi, E.J. (1989). Severe thrombocytopenia in young calves experimentally infected with noncytopathic bovine viral diarrhea virus. J Virol, 63, 3934-43. Ellis, J .A., West, K.H., Cortese, V.S., Myers, S.L., Carman, S., Martin, K.M. & Haines, D.M. (1998). Lesions and distribution of viral antigen following an experimental infection of young seronegative calves with virulent bovine virus diarrhea virus-type H. Can J Vet Res, 62, 161-9. Jain, N. (1986). Qualitative and quantitative disorders of platelets. In Schalm's veterinary hematology, Jain, N. (ed) pp. 466—486. Lea & F ebiger: Philadelphia, PA. Lennette, E. & Schmidt, N. (1969). Diagnostic procedures for viral and richettsia] infections. American Public Health Association Incorporated: New York, NY. Marshall, D.J., Moxley, R.A. & Kelling, CL. (1996). Distribution of virus and viral antigen in specific pathogen-free calves following inoculation with noncytopathic bovine viral diarrhea virus. Vet Pathol, 33, 311-8. Meyling, A. (1988). Detection of BVD virus in viremic cattle by an indirect immunoperoxidase technique. In Recent advances in virus diagnosis, McNulty,M & MacFerran, J. (eds) pp. 37-46. Martinus Nijhoff Publishing: Boston, MA. Peng, J ., Friese, P., Heilmann, E., George, J .N., Burstein, S.A. & Dale, G.L. (1994). Aged platelets have an impaired response to thrombin as quantitated by P-selectin expression. Blood, 83, 161-6. Rebhun, W.C., French, T.W., Perdrizet, J.A., Dubovi, E.J., Dill, S.G. & Karcher, L.F. (1989). Thrombocytopenia associated with acute bovine virus diarrhea infection in cattle. J Vet Intern Med, 3, 42-6. Spagnuolo-Weaver, M., Allan, G.M., Kennedy, 8., Foster, J.C. & Adair, B.M. (1997). Distribution of cytopathic and noncytopathic bovine viral diarrhea virus antigens in tissues of calves following acute experimental infection. J Vet Diagn Invest, 9, 287- 97. 165 Walz, P.H., Bell, T.G., Steficek, B.A., Kaiser, L., Maes, R.K. & Baker, J.C. (1999a). Experimental model of type H bovine viral diarrhea virus-induced thrombocytopenia in neonatal calves. J Vet Diagn Invest, 11, 505-514. Walz, P.H., Steficek, B.A., Baker, J.C., Kaiser, L. & Bell, T.G. (1999b). Effect of experimentally induced type H bovine viral diarrhea virus infection on platelet function in calves. Am J Vet Res, 60, 1396-401. Wright, J. (1906). The origin and nature of blood platelets. Boston Medicine and Surgery Journal, 154-163. 166 CHAPTER V: JOURNAL ARTICLE: RELATIONSHIP BETWEEN LEVEL OF VIREMIA AND DISEASE IN CALVES EXPERIMENT ALLY INFECTED WITH BOVINE VIRAL DIARRHEA VIRUS 167 CHAPTER V: JOURNAL ARTICLE: RELATIONSHIP BETWEEN LEVEL OF VIREMIA AND DISEASE IN CALVES EXPERIMENTALLY NFECTED WITH BOVINE VIRAL DIARRHEA VIRUS Paul H. Walz, Thomas G. Bell, Jennifer L. Wells, Daniel L. Grooms, Lana Kaiser, Roger K. Macs, and John C. Baker From the Department of Large Animal Clinical Sciences (Walz, Wells, Grooms, Kaiser, Baker), the Animal Health Diagnostic Laboratory (Bell, Macs), the Department of Microbiology (Macs), and the Department of Veterinary Pathology (Bell), College of Veterinary Medicine, Michigan State University, East Lansing, MI, USA 48824-1314. 168 Abstract Objective—To compare levels of viremia and clinical, hematologic, virologic, immunohistochemical, and pathologic findings during type I and type H bovine viral diarrhea virus (BVDV) infection in calves. Animals —1 6 neonatal male Holstein calves. Procedure—Colostrum-deprived calves were obtained immediately after birth, housed in isolation, and assigned to one of four groups (1 control and 3 treatment groups). Control calves (n=4) were sham inoculated, while calves in the infected groups (n=4 for each group) were inoculated by intranasal instillation with 107 TCH)50 of either BVDV 890 (type II), BVDV 7937 (type H), or BVDV TGAN (type I). Blood cell counts and virus isolation fi'om serum and leukocytes were performed daily, while levels of viremia (serum viral titer and percentages of lymphocytes and platelet associated with BVDV) were determined on day 0 (before inoculation) and days 4, 6, 8, and 12 after inoculation. All experimental calves were euthanatized on day 12 after inoculation, and pathologic, virologic, and immunohistochemical examinations were performed. Results—Type H BVDV 890 induced the highest level of viremia, followed by type II BVDV 7937, and then type I BVDV TGAN. In addition, BVDV was isolated from serum and leukocytes more frequently and for a longer duration in calves infected with BVDV 890, followed by BVDV 7937, and then BVDV TGAN. A parallel relationship between the level of viremia and rectal temperatures and an inverse relationship between the level of viremia and blood cell counts was observed, that is the greatest elevations in rectal temperature and greatest depressions in platelet and leukocyte counts were observed in calves infected with BVDV 890. Pathologic and immunohistochemical 169 examinations revealed more pronounced lesions and more extensive viral antigen distribution in the type H BVDV-infected calves as compared to the type I BVDV- infected calves and control calves. Conclusions—The level of viremia induced during BVDV infection is positively correlated to the development of disease in calves experimentally infected with type I or type H BVDV. In addition, the level of viremia induced during infection may explain the differences in virulence between isolates of BVDV. Clinical Relevance—Isolates of BVDV, which induce higher levels of viremia, may be more capable of inducing clinical signs of disease. Severe peracute BVD and the hemorrhagic syndrome may occur as a result of infection with isolates of BVDV capable of inducing high levels of viremia. Strategies such as vaccination that reduce viremia may be adequate to control clinical signs associated with acute infection with BVDV. Introduction Bovine viral diarrhea virus (BVDV) is an important infectious disease in cattle, resulting in economic losses throughout the world (Baker, 1995). Bovine viral diarrhea virus is an RNA virus and a member of the genus Pestivirus within the family Flaviviridae (Wengler, 1991). Isolates of BVDV can be classified into two biotypes, noncytopathic and cytopathic, and two genotypes, type I and type H (Ridpath et al., 1994). Bovine viral diarrhea virus infection may result in a wide spectrum of clinical manifestations, with the majority of BVDV infections occurring without clinical signs of infection (Baker, 1995). However, outbreaks of severe peracute disease in cattle have been reported in association with type H BVDV (Carman et al., 1998; Pellerin et al., 170 1994). In addition, infection with some type H BVDV isolates has been associated with a hemorrhagic disorder (Bolin & Ridpath, 1992; Corapi et al., 1990; Corapi et al., 1989; Rebhun et al., 1989). These infections are characterized by thrombocytopenia, bloody diarrhea, epistaxis, petechial and ecchymotic hemorrhages on mucosal membranes, hyphema, bleeding fiom injection sites, pyrexia, leukopenia, and death (Bolin & Ridpath, 1992; Corapi et al., 1990; Rebhun et al., 1989). Thrombocytopenia associated with BVDV has been reported from outbreaks in adult cattle and calves, and has also been experimentally reproduced (Bezek et al., 1994; Bolin & Ridpath, 1992; Corapi et al., 1990; Ellis et al., 1998; Rebhun et al., 1989; Walz et al., 1999a). The differences in virulence between type I and type H BVDV and the mechanisms by which some type H BVDV isolates cause severe peracute disease and the hemorrhagic syndrome are unknown. A difference in the level of viremia induced during infection has been demonstrated between a type I and a type H BVDV isolate (Bolin & Ridpath, 1992), and this higher level of viral replication with type H BVDV may be important in the pathogenesis of severe peracute BVDV and the hemorrhagic syndrome. The purpose of the present study was to characterize the level of viremia induced during BVDV infection, and correlate levels of virerrria with severity of clinical disease. Materials and Methods Animals Colostrum-deprived newborn male Holstein calves (n=16) were obtained immediately after birth from local dairy farms. Calves were moved to an isolation 171 facility, allocated to one of four groups (1 control and 3 treatment groups), and individually housed in separate rooms. All calves were given a physical examination, and serum and whole blood were obtained for virus isolation procedures and determination of antibody levels to BVDV by virus neutralization. Calves were then administered 400 ml of plasma obtained from a donor cow that was BVDV antibody negative by virus neutralization and virus negative by virus isolation procedures on three serial serum samples obtained prior to collection of plasma. In addition, calves were b as well as an oral given injections of vitamins A and D” and vitamin E and selenium, monoclonal antibody preparation against E. coli K99 pilus antigen.b Calves were fed a nonmedicated milk replacer according to label directions. Physical examinations, including rectal temperature measurements, were made on all calves twice daily throughout the study period. Prior to initiation of the study, criteria for euthanasia were established. Calves demonstrating any one or a combination of the following were euthanatized: complete anorexia, recumbency with inability to rise, dehydration in excess of 12% of body weight, and hemorrhagic diathesis. Viruses and infection protocol Three noncytopathic BVDV isolates were used for the experimental infection of calves: BVDV 890,c BVDV 7937,d and BVDV TGAN.c The BVDV 890 is a type II isolate that has been previously shown to induce thrombocytopenia and platelet dysfunction in experimentally infected calves (Bolin & Ridpath, 1992; Walz et al., 1999b). The BVDV 7937 (type H) and BVDV TGAN (type I) are BVDV isolates that have not been associated with thrombocytopenia (Bolin & Ridpatir, 1992; Marshall et al., 172 1996). The viral isolates were propagated in bovine turbinate cells in Eagle’s minimum essential mediume (EMEM) containing 10% fetal equine serumf (FES), L-glutamine,g f After a three-day adjustment period in the isolation penicillin G,f and streptomycin. facility, calves in the 3 infected groups (n=4 for each group) were inoculated by intranasal instillation with 107 TCH)50 of the respective viral isolate in 5 ml of EMEM without serum (2.5 ml per nostril). Control calves (n=4) were inoculated by intranasal instillation with an uninfected cell culture supemate. Clinical pathologic examination Manual platelet and leukocyte counts were performed daily on whole blood, using a white blood cell microcollection systemh and a hemocytometer. Automated blood cell counts, which included the mean platelet volume, were performed on day 0 (before inoculation) and day 12 after inoculation using an electronic particle counter.i Determination of the levels of viremia Serum virus titer and the percentages of lymphocytes and platelets carrying virus were used to assess the level of virerrria (Bolin & Ridpath, 1992). The titer of virus in serum was determined by making serial 5-fold dilutions of serum in EMEM. The serial dilutions were inoculated in 96-well rrricrotiter plates containing monolayers of bovine turbinate cells in EMEM with 10% FES, L-glutamine, and antibiotics. After 3 days of incubation at 37°C in humidified air containing 5% CO2, the bovine turbinate cells were stained for BVDV antigen by an immunoperoxidase monolayer assay and the viral titer calculated (Meyling, 1988). 173 A limiting dilution method was used for estimating the frequency of association of BVDV with platelets and lymphocytes (Bolin & Ridpath, 1992). Whole blood was collected for virus isolation from platelets and lymphocytes on the day of challenge, and then on days 4, 6, 8, and 12 after challenge. Blood was collected by jugular venipuncture through an 18-gauge needle into plastic syringes containing 1.0 ml of 3.8% trisodium citrate for each 9.0 ml of blood. Platelet-rich plasma was obtained by two centrifugation steps at 1,324 x g for 60 seconds and 30 seconds, with the platelet-rich plasma being removed after each centrifugation. Prostaglandin E1f(l ul of 1 uM prostaglandin per ml of platelet-rich plasma) was added to platelet-rich plasma prior to centrifugation at 800 x g for 15 minutes. The plasma supernatant was removed with a sterile pipette, and the platelet pellet was resuspended in 1 ml of Hank’s Balanced Salt Solution (HBSS; 136 mM NaCl, 5.4 mM KCL, 0.44 mM KH2PO4, 0.34 mM Na2HPO4, and 5.5 mM dextrose, pH 7.4). The platelet suspension was transferred to a 10 ml polystyrene column containing Sepharose 4B.f The eluate was collected, washed three times in 10 rrrl of HBSS, and a manual platelet count was determined. Approximately 107 platelets were inoculated into the top left-hand well of a 96-well nricrotiter plate containing EMEM. Serial twofold dilutions were made down the left-hand column of the plate, then serial twofold dilutions were made across the plate, beginning at the left-hand column of wells. Bovine turbinate cells (9 x 104 cells/ml) suspended in EMEM containing 10% FES, L- glutamine, and antibiotics were added to each well. After 5 days incubation at 37°C in humidified air containing 5% CO2, 50p] of supernatant from each well was transferred to a new 96-well microtiter plate. Bovine turbinate cells suspended in EMEM containing 10% FES, L-glutamine, and antibiotics were added to each well. After another 5 days of 174 incubation at 37°C in humidified air containing 5% CO2, the bovine turbinate cells were stained for BVDV antigen by an immunoperoxidase monolayer assay (Meyling, 1988), and the percentage of platelets associated with BVDV was calculated. For virus isolation from lymphocytes, citrated whole blood that had the platelet-rich plasma previously removed was diluted in HBSS, and the suspension was layered on Ficoll-sodium diatrizoate medium.j After centrifugation at 600 x g for 40 minutes at 18°C, the lymphocytes were harvested and resuspended in 10 ml I-IBSS. Contaminating red blood cells were removed by hypotonic lysis to yield lymphocytes and residual platelets (Carbrey et al., 1972). In order to remove contaminating platelets, centrifugation at 600 x g for 40 minutes at 18°C in a 15% sucrose gradient layered over Ficoll-sodium diatrizoate medium was used to purify lymphocytes. This procedure successfully removed contaminating platelets, and lymphocytes were collected below the sucrose at the interface of the F icoll sodium diatrizoate medium, washed three times in HBSS, and manually counted. Approximately 10‘5 lymphocytes were inoculated into the top left-hand well of a 96-well microtiter plate containing EMEM. Serial twofold dilutions were made as described for platelets. Detection of virus association with lymphocytes was performed as described for platelets. Virologic and serologic examination Serum and whole blood were collected daily for detection of BVDV to determine the frequency of virus isolation over the experimental time period. Whole blood was processed by hypotonic lysis of the red blood cells to yield the white blood cells (Carbrey et al., 1972). Isolated white blood cells were then resuspended in EMEM containing 175 10% FES, L-glutamine, and antibiotics. Following three freeze/thaw cycles, white blood cell preparations were centrifuged for 10 minutes at 1,500 x g. The supernatant was extracted and stored at —80°C. Virus isolation was also performed on bone marrow, thymus, spleen, bronchial and mesenteric lymph nodes, and ileum collected postmortem. Approximately 0.5 gram of tissue was added to 4 ml of EMEM with 10% FES, L-glutamine, and antibiotics in a sterile mortar. Sterile sand was added and the tissue was homogenized by grinding with a pestle. The homogenized tissue sample was transferred into conical tubes and centrifuged for 30 minutes at 2,300 x g. The supernatant was removed and then stored at —80°C. The serum, white blood cell preparations, and tissue preparations were thawed, and 25 ul of each sample was inoculated in duplicate into wells on 96-well microtiter plates containing monolayers of bovine turbinate cells in EMEM with 10% FES, L-glutamine, and antibiotics. After 3 days of incubation at 37°C in humidified air containing 5% CO2, the bovine turbinate cells were stained for BVDV antigen by an immunoperoxidase monolayer assay (Meyling, 1988). Serology for BVDV was performed by a microtiter virus neutralization procedure (Carbrey et al., 1972). Sera obtained on days 0 and 12 after infection from all experimental calves were tested for neutralizing antibodies to type I and type H BVDV. Serial two-fold dilutions, ranging from 1:4 to 1:4,096, were made for each sertun sample in a 96-well microtiter plate. The amount of virus used per test well was 500 TCH)50 of k the type H cytopathic isolate 125k or the type I cytopathic isolate Singer. Bovine turbinate cells were used as the indicator cells at a concentration of 15,000 cells per well. 176 Each test included a back titration of the virus and a positive and negative serum control. The antibody titer was read as the reverse of the highest dilution with complete inhibition of cytopathic effect. Pathologic examination On day 12 after infection, all calves were euthanatized with a lethal injection of barbiturate.l A postmortem examination was performed and all gross postmortem lesions were recorded. Tissues collected for histopathologic examination included liver, spleen, lung, heart, small and large intestine, adrenal gland, kidney, thymus, tonsil, brain, spinal cord, mesenteric and bronchial lymph nodes, and bone marrow. All tissues were fixed in 10% neutral buffered formalin, with the exception of bone marrow, which was also fixed in B-5 fixative.m Following fixation, the tissues were trimmed, embedded in paraffin, sectioned at 6 pm, and stained with hematoxylin and eosin (HE) according to standard procedure. Numbers of megakaryocytes per high power field were calculated by averaging the number of megakaryocytes under ten high power fields for each calf. Images in this dissertation are presented in color. Immunoh istochemica] examination for B VD V antigen Immunohistochemical staining for BVDV antigen was performed on paraffin- embedded sections of bone marrow, lung, thymus, lymph node, ileum, and spleen, as previously described (Grooms et al., 1996). Tissue sections were cut at 5 um on silane- coated slides and dried. Sections were then deparaffinized and rehydrated by sequential immersion of the slides in a lirnonene-based solvent,“ followed by graded concentrations 177 of ethanol, then tap water. Inactivation of tissue peroxidases, digestion with protease, and blocking of nonspecific antibody binding were performed prior to the application of the primary monoclonal antibody, 15C5.o Following incubation with primary antibody, BVDV antigen was detected using an indirect avidin-biotin-peroxidase detection system,p and the chromagen, 3-amino-9-ethyl carbazole.f After immunohistochemical staining, the slides were counterstained with Mayer’s hematoxylin and coverslipped using an aqueous mounting medium. To detect nonspecific binding, the primary antibody was replaced with a sirrrilar concentration of an antibody of the same isotype directed against infectious bursal disease virus.‘1 Tissue sections fi'om a persistently infected calf and a calf not infected with BVDV as determined by virus isolation and serology were used as positive and negative controls, respectively. Statistical analyses Comparisons of rectal temperatures and manual and automated platelet and leukocyte counts were performed by one-way AN OVA. When differences in group means were statistically significant (P < 0.05), the Bonferroni t-test was used for pairwise comparisons of control versus infected group means. Mean platelet volume measurements and data describing the levels of virerrria (serum viral titer and virus association with lymphocytes and platelets) were analyzed using the Kruskal-Wallis one- way ANOVA on ranks. When differences in mean platelet volume values were statistically significant (P < 0.05), the Dunn's method was used for comparisons of control versus infected group means. When differences in the means in the data 178 describing the level of viremia were statistically significant (P < 0.05), the Student- Newman-Keuls method was used for all pairwise comparisons of group means. Results Physical examination findings None of the calves met the predetermined criteria for euthanasia during the study period. Appetite remained normal in all control and infected calves throughout the study. All calves infected with type H BVDV developed diarrhea, while 3 of 4 calves infected with type I BVDV developed diarrhea during the study. In calves infected with type H BVDV 890, the diarrhea was longer in duration than calves infected with the other type II isolate or the type I isolate, and was characterized by hemorrhage and mucosal casts. Pyrexia (rectal temperature > 39.4°C) was only observed in BVDV-infected calves. Statistically significant differences in the mean rectal temperature measurements were observed between control calves and BVDV-infected calves from days 6 through 12 after inoculation, with the longest duration of an elevated temperature being observed in calves infected with the type H isolate BVDV 890 (Figure 5.1). Clinical pathologic findings The greatest depression in platelet count occurred in calves infected with the type II isolate BVDV 890 (Figure 5.2). The platelet count decreased to less than 200,000/pl in 2 of 4 calves infected with BVDV 890 and 1 of 4 calves infected with BVDV 7937. The platelet count did not decrease to less than 100,000 platelets/pl in any calves, and did not decrease to less than 200,000/pl in any control calves or calves infected with BVDV 179 TGAN. When compared to control calves, a significantly decreased manual platelet count was observed from day 7 after inoculation to the end of study in the BVDV- infected calves (Figure 5.2). The automated platelet counts closely followed the manual counts (Table 5 .1). Platelet size, as measured by the mean platelet volume, was significantly reduced in the BVDV-infected calves on day 12 after inoculation when compared to the control calves (Figure 5.3). The automated white blood cell counts decreased in the infected calves, but statistically significant differences were only observed in calves infected with type H BVDV, when compared to control calves (Table 5.1). With respect to manual white blood cell counts, statistically significant differences were observed between the control group and all BVDV-infected groups. The only statistically significant difference observed between calves infected with BVDV TGAN and control calves occurred on day 3 after inoculation, and a significant difference was only observed on days 3 and 12 after inoculation in the BVDV 7937-infected calves. A statistically significant difference in the manual white blood cell count was observed on days 3-5, 7, and 10-12 after inoculation between control calves and calves infected with BVDV 890. Levels of viremia A clear difference in the level of viremia induced during infection was observed among the 3 groups of BVDV-infected calves, with the highest serum viral titer and percentages of lymphocytes and platelets associated with BVDV being observed in the calves infected with BVDV 890 (Tables 5.2, 5.3, 5.4). The highest serum viral titer in calves infected with BVDV 890 was observed on day 12 after inoculation (Table 5.2, 180 mean log titer: 5.4771; range:4.1021-6.1021). In contrast, the viral titer was highest on day 8 after inoculation in the BVDV 7937-infected calves (mean log titer: 3.5187; range: 27687-42687) and the BVDV TGAN-infected calves (mean log titer: 1.9771; range: 1.4354-3.4354). With respect to the association of BVDV with lymphocytes, a similar trend was observed in the 3 groups of calves, with the percentage association highest on day 12 after inoculation in the BVDV 890-infected calves (Table 5.3, mean maximal percentage association: 7.2648; range: 3.225 8-12.50). The highest percentage association in the BVDV 7937-infected calves (mean maximal percentage association: 2.2814; range: 00498-6667) and the BVDV TGAN-infected calves (mean maximal percentage association: 0.1098; range: 0.0004-0.4098) was observed on day 8 after inoculation. Finally, the percentage of platelets associated with BVDV was highest on day 8 after inoculation in all BVDV-infected groups of calves, with the BVDV 890-infected calves highest (Table 5.4, mean maximal percentage association: 0.2457; range: 0.1638-0.3277). The percentage of platelets associated with BVDV was lower for the BVDV 7937- irrfected calves (mean maximal percentage association: 0.0320; range: 0.0051-0.0820), and the BVDV TGAN-infected calves (mean maximal percentage association: 0.0030; range: 0.0008-0.0103). Virologic and serologic findings Bovine viral diarrhea virus was not isolated from antemortem or postmortem samples collected from the control calves. In the infected groups of calves, BVDV was isolated from serum and white blood cells more frequently and for a longer duration in calves infected with BVDV 890, followed by BVDV 7937, and then BVDV TGAN 181 (Table 5.5). Bovine viral diarrhea virus was isolated from all tissues from all calves infected with either BVDV 890 or BVDV 7937, as compared to calves infected with BVDV TGAN, in which the thymus was the only tissue that BVDV was isolated in all calves (Table 5.5). All control and BVDV-infected calves had BVDV-neutralizing antibody titers of $8 on day 0 (before inoculation) and day 12 after inoculation. Pathologic findings Petechial hemorrhages were present on gross postmortem examination in all calves infected with BVDV 890 and BVDV 7937, and in 2 of 4 calves infected with BVDV TGAN. The hemorrhages were most prominent in calves infected with BVDV 890, and were located primarily on the mucosal surfaces of the urinary bladder, ileum, colon, and rumen. Hemorrhages were only observed on the colonic mucosa in the calves infected with BVDV TGAN. Abomasal edema, which was most prominent in the spiral folds of the ftmdus, was present in all calves infected with BVDV 890, in 3 of 4 calves infected with BVDV 793 7, and in 1 of 4 calves infected with BVDV TGAN. No significant lesions were present on gross postmortem examination of control calves. Histologic evaluation of the bone marrow revealed differences between the 4 groups of calves (Figure 5.4A-D). Images in this dissertation are presented in color. Sections of bone marrow in calves infected with BVDV 890 were hypercellular, when compared to the other groups of calves. In addition, calves infected with BVDV 890 had the most megakaryocytes per high power field (MK/hpf; mean: 8.85 MK/hpf; range: 7.62-10.45), followed by calves infected with BVDV 7937 (mean: 5.98 MK/hpf; range: 5.09-6.76), 182 calves infected with BVDV TGAN (mean: 5.24 MK/hpf; range: 3.62-6.56), then the control calves (mean: 3.60 MK/hpf; range: 1.84-5.32). In the calves infected with BVDV 890, many of the megakaryocytes did not have uniform nuclear size or chromatin pattern. Approximately 30% of the megakaryocytes in BVDV 890-infected calves were immature, as characterized by a small nuclear:cytoplasmic ratio and nonsegmented nucleus. Immature megakaryocytes were also observed in the other groups, but less fiequently (calves infected with BVDV 7937: 18%; calves infected with BVDV TGAN: 5%; and controls: 3%). Approximately 7.4% of megakaryocytes in the BVDV 890- infected calves had undergone necrosis, as evidenced by the loss of, or pyknosis of the nuclei. However, more pyknotic megakaryocytes were observed in calves infected with BVDV 7937 (approximately 9.5%) and calves infected with BVDV TGAN (approximately 9.3%), when compared to calves infected with BVDV 890 and control calves (approximately 3.9%). Lymphoid depletion and reduced follicular size were present in the thymus (Figure 5.5), ileum and spleen in all BVDV-infected calves. Images in this dissertation are presented in color. The greatest lymphoid depletion was observed in calves infected with BVDV 890 (Figure 5.5). Immunoh istoch emistry findings In the control calves, there was no evidence of BVDV antigen as detected by immunohistochemical antibody staining. However, there was immunohistochemical staining for BVDV antigen in all paraffin-embedded tissue sections collected for evaluation from all BVDV-infected calves, but viral antigen was more prevalent in the calves infected with type II BVDV than calves infected with BVDV TGAN (Table 5.6). 183 In 3 of 4 calves infected with BVDV 7937 and in 2 of 4 calves infected with BVDV 890, immunohistochemical staining of bone marrow revealed the presence of BVDV-specific antigen in megakaryocytes (Figure 5.4D and 5B). In addition, BVDV-specific antigen was present in myeloid precursors in the bone marrow (Figure 5.4D). Immunohistochemical staining for BVDV was also present in the muscular trabeculae and smooth muscle of blood vessels located in the spleen of the type H BVDV-infected calves. Mononuclear cells in the medullary sinus of the lymph node, bone marrow, red pulp of the spleen, and ileum also contained BVDV antigen. In the ileum, viral antigen was present in lymphoid cells in Peyer’s patch follicles and in perifollicular connective tissue, and viral antigen was also present in lymphoid and mononuclear cells in the cortex and medulla of the thymus. 184 Figure 5.1. Mean rectal temperature measurements for uninfected control calves, calves infected with the type I isolate BVDV TGAN, calves infected with the type H isolate BVDV 7937, and calves infected with the type H isolate BVDV 890. Calves were infected at day 0. Standard error bars are shown. Statistically significant differences (ANOVA, p < 0.05, Bonferroni t-test) are denoted as follows: * = BVDV 890 vs. Control; + = BVDV 7937 vs. Control; # = BVDV TGAN vs. Control. 185 Figure 5.1 Mean Rectal Temperature (C) 40.5- g "0“ Control at #,+,1i1 1: 0 1i: +,a +,e --O-~BVDVTGAN .. --A--BVDV 7937 +BVDV890 4o. / . ' p" 39.54 ,-' \ ._ , H‘A‘HA’ -3- ” I I i. ‘P I. I '\. —— _ Ill \ I \-‘ J, . ‘ . g ..... ..- 2 «I . ‘5<-- ' I” Y T 39‘ . ’ I \ a" I. I. / ‘ ---A ’ ." \' - . \ ‘ . .. / . ‘ \ . [.1 . _‘ “no I \ I .'~ .' o . a ’ I \ I ‘.§ .- ./ A § 0" 38.5- 7" A 38 . 37.5 i 1 U U I I I I I V fir I I fir -2 -1 0 1 2 3 4 5 6 7 8 9 1O 11 12 Days after infection 186 Figure 5.2. Mean whole blood platelet counts by manual determination in uninfected control calves, calves infected with the type I isolate BVDV TGAN, calves infected with the type II isolate BVDV 7937, and calves infected with the type H isolate BVDV 890. Calves were infected at day 0. Standard error bars are shown. Statistically significant differences (ANOVA, p < 0.05, Bonferroni t-test) are denoted as follows: * = BVDV 890 vs. Control; + = BVDV 7937 vs. Control; # = BVDV TGAN vs. Control. 187 Figure 5.2 Platelets per microliter g g g g 1600000 - a .+ a .+ a- - '0- Control —0- -BVDV TGAN —A— BVDV 7937 +BVDV 890 - - 2 % 1400000 - 1200000-i A A r 200000‘ -2 -1 0 1 2 3 4 5 6 7 8 9 1O 11 12 Day after Infection 188 Figure 5.3. 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Histologic section of bone marrow from calf infected with BVDV 890 (type II). E. Positive immunohistochemical staining for BVDV antigen in megakaryocytes and myeloid and erythroid precursors from calf infected with BVDV 7937 (note reddish-brown staining depicted by arrow). F. Positive immunohistochemical staining for BVDV antigen in a megakaryocyte from calf infected with BVDV 890. Images in this dissertation are presented in color. 196 Figure 5.4 . A ‘ '\c-§.‘ 11 >30 HIV/VAL ,0, 197 Figure 5.5 Histologic sections of thymus taken on day 12 after infection (postmortem examination). Bar = 100m. A. Histologic section of thymus from control calf. B. Histologic section of thymus from calf infected with BVDV TGAN (type I). C. Histologic section of thymus from calf infected with BVDV 7937 (type II). D. Histologic section of thymus from calf infected with BVDV 890 (type II). Images in this dissertation are presented in color. 198 199 Table 5.6. Summary of immunohistochemistry results for uninfected control calves, calves infected with the type I isolate BVDV TGAN, calves infected with the type II isolate BVDV 7937, and calves infected with the type II isolate BVDV 890. Tissues were collected at day 12 after inoculation. Tissue Control BVDV TGAN BVDV 7937 BVDV 890 (type I) (type 11) (type 11) Thymus O/4"' 2/4 4/4 4/4 Spleen 0/4 1/4 2/4 4/4 Bonemarrow 0/4 1/4 4/4 4/4 (+ megakaryocytes) (O / 4) (0 / 4) (3 / 4) (2 / 4) Mesenteric Lymph Node 0 / 4 3 l 4 4 / 4 4 / 4 Ileum 0/4 4/4 3/4 4/4 Lung 0 / 4 O / 4 1 /4 3 /4 * numerator: # of calves that were positive for viral antigen by immunohistochemistry; denominator: # of calves in infected group 200 Discussion A clear difference in the level of viremia induced during infection was demonstrated in the 3 groups of BVDV-infected calves in this study. In addition, a parallel relationship between the level of viremia and rectal temperatures, and an inverse relationship between the level of viremia and blood counts was observed, with the greatest elevations in rectal temperature and greatest depressions in platelet and leukocyte counts being observed in calves infected with BVDV 890. The results of this study, indicating that the level of viremia is correlated to disease manifestations, support the hypothesis that the level of viremia induced during BVDV infection is an important virulence factor, and may be a defining factor for differences in virulence observed between BVDV isolates. The three BVDV isolates utilized in this study were chosen because they would allow a comparison among BVDV isolates of predetermined and different virulence. The type H isolate BVDV 890 was considered the most virulent, as it had been previously demonstrated to induce pyrexia, diarrhea, thrombocytopenia, leukopenia, altered platelet function, and death in experimentally infected calves (Bolin & Ridpath, 1992; Walz et al., 1999a; Walz et al., 1999b). The other type H isolate, BVDV 7937, was considered less virulent than BVDV 890, as it had previously been shown to induce pyrexia and a depression in the platelet count, but did not induce diarrhea, leukopenia, thrombocytopenia, or death in experimentally infected calves (Marshall et al., 1996). The BVDV TGAN was chosen because it was a type I isolate that did induce pyrexia, but did not induce leukopenia, thrombocytopenia, or death in experimentally infected calves (Bolin & Ridpath, 1992). The ability of these 3 isolates to induce different disease manifestations allowed for comparisons with the level of viremia. The clinical signs 201 associated with BVDV infection in this study are similar to previous reports for each of the 3 isolates, with BVDV 890 inducing the more severe clinical signs of disease when compared to BVDV 7937 and BVDV TGAN. Pyrexia (rectal temperature > 39.4 C) and diarrhea observed in the BVDV 890-infected calves are similar to previous reports (Bolin & Ridpath, 1992; Walz et al., 1999a), and are typical of a virulent type H BVDV infection. Pyrexia was also observed in the BVDV 7937-infected calves, but in contrast to a previous study (Marshall et al., 1996), diarrhea was observed in 3 of 4 calves in this study. The reason for this difference is tmknown, but may be due to the use of younger calves in the present study. Similar to a previous report (Bolin & Ridpath, 1992), pyrexia was observed in all BVDV TGAN-infected calves. A statistically significant difference in platelet counts was observed between the control calves and all BVDV-infected groups of calves, including calves infected with the type I isolate BVDV TGAN; however, thrombocytopenia was only observed in 2 of 4 calves infected with BVDV 890. Thrombocytopenia is defined as a reduction in the number of circulating platelets below the minimum normal level for the species (Jain, 1986), but defining thombocytopenia is difficult because there is considerable variation in the literature concerning the platelet count in normal male Holstein calves (Nelson et al., 1974; Vaugher et al., 1973). We used age-, breed-, and sex-matched control calves in order to demonstrate statistically significant differences in the platelet count between control calves and BVDV-infected calves. This was important, because our results and the results fi'om previous studies (Semrad & Dubielzig, 1993; Walz et al., 1999b) demonstrate a relative thrombocytosis peaking at approximately 8 days of age in newborn Holstein calves. In this study, the age-related relative thrombocytosis is likely obscured 202 in the BVDV-infected calves as a result of infection, and may explain the inability to induce thrombocytopenia in all BVDV 890-infected calves. In addition, the absence of the relative thrombocytosis in the BVDV TGAN-infected calves may be the result of infection, and may indicate that greater depressions in the platelet count following infection with BVDV TGAN might have been observed if older calves had been used in our experimental infection. These results reinforce the importance of age-matched control calves in studying BVDV infection and platelet disorders in neonatal calves. In the present study, the mean platelet volume was significantly decreased at day 12 after infection in all BVDV-infected groups of calves when compared to control calves. The mean platelet volume can be used to help classify the mechanism of thrombocytopenia (Bessman et al., 1982). Platelets recently released into the bloodstream from the bone marrow are larger and, consequently, the mean platelet volume is elevated when thrombocytopenia is due to increased destruction of platelets in the peripheral circulation. Alternatively, older platelets are smaller and the mean platelet volume is decreased when the thrombocytopenia is due to decreased marrow production (Bessman et al., 1982). Platelet size, as measured by the mean platelet volume, was smallest in the BVDV 890-infected calves, which supports a previous study demonstrating low mean platelet volume in BVDV 890-infected calves (Walz et al., 1999a). In addition, the concept that decreased production of platelets is the cause of BVDV-induced thrombocytopenia is supported by these findings. Interestingly, although the mean platelet volume was smaller in the BVDV TGAN-infected calves than in calves infected with the other type H isolate, BVDV 7937, the platelet count was lower in the BVDV 7937-infected calves. The reason for this discrepancy is unknown, but 203 megakaryocyte degeneration was demonstrated in the BVDV TGAN-infected calves, and a lack of platelet production by the bone marrow in these calves cannot be ruled out. An increased cellularity of the bone marrow and megakaryocyte hyperplasia were observed in the BVDV 890-infected calves in this study and a previous study (Walz et al., 1999a), and likely occurs in response to the decreased peripheral platelet and leukocyte counts, respectively. Previous studies evaluating the bone marrow histology from type H BVDV-thrombocytopenic infections have reported both megakaryocyte necrosis (Ellis et al., 1998; Scruggs et al., 1995), and adequate to increased numbers of megakaryocytes (Corapi et al., 1989; Ellis et al., 1998; Rebhun et al., 1989). Similar to those previous reports examining different BVDV isolates, we observed both megakaryocyte degeneration and megakaryocyte hyperplasia. Me gakaryocyte hyperplasia was greatest in the BVDV 890-infected calves. Megakaryocyte degeneration was also observed in the BVDV 890-infected calves, but the numbers of megakaryocytes undergoing degeneration, as evidenced by the loss of, or pyknosis of the nucleus were greater in calves infected with BVDV 7937 and BVDV TGAN. These differences may be due to different levels of virerrria induced during infection or may be related to different infection kinetics with BVDV 890, BVDV 7937, and BVDV TGAN. We speculate that viral infection of the bone marrow in the BVDV 890-infected calves occurs earlier in the course of infection and to a greater extent than in calves infected with BVDV 7937 or BVDV TGAN. This is supported by our virus isolation data from antemortem samples and the level of viremia data that demonstrate earlier isolation of virus from blood components and higher levels of viremia, respectively. In turn, this earlier and higher level of bone marrow infection may lead to suppression of platelet 204 production by the megakaryocytes, and greater depression in the peripheral platelet count. At day 12 after inoculation, bone marrow megakaryocyte hyperplasia was observed in response to the thrombocytopenia in the BVDV 890-infected calves. Conversely, bone marrow infection may occur later and to a lower level in the BVDV TGAN-infected calves. At day 12 after inoculation, extensive megakaryocyte hyperplasia is not observed in the BVDV TGAN-infected calves because thrombocytopenia is not observed. In addition, we speculate that megakaryocyte degeneration is observed in the BVDV TGAN-infected calves as a result of bone marrow suppression occurring later during the course of BVDV infection. Virus isolation fiom tissues and immunohistochemical staining for BVDV antigen in tissues were performed to compare the virus distribution among the 3 BVDV-infected groups. Identification of BVDV antigen and isolation of BVDV in the thymus, lymph node, Peyer’s patches of the ileum, bone marrow, and spleen are similar to results from previous studies (Ellis et al., 1998; Marshall et al., 1996; Spagnuolo-Weaver et al., 1997). However, the results of this study demonstrate considerable differences in the ability to isolate BVDV and demonstrate BVDV antigen by immunohistochemistry between calves experimentally infected with type I or type H BVDV. Isolation of BVDV from tissues and demonstration of viral antigen in tissues was more frequent in the type H BVDV-infected calves, as compared to BVDV TGAN-infected calves. This observation is compatible with the level of viremia results demonstrating higher levels of viremia in the type H BVDV-infected calves. In addition, the implications from this finding may be significant from a diagnostic standpoint, as it may be difficult to successfully identify calves acutely infected with type I BVDV. 205 The mechanism allowing for the type H isolates to achieve higher levels of viremia than the type I isolate BVDV TGAN is unknown, but may be the result of differences in RNA sequence. Comparison of nucleic acid sequences from type I and type H BVDV revealed a homology of less than 70% (Ridpath et al., 1994). This dissimilarity is concentrated in the regions of the BVDV genome encoding for the structural glycoprotein E2 and the S’untranslated region. The E2 glycoprotein is responsible for the viral attachment to cells (Xue & Minocha, 1993), while the 5’untranslated region contains the internal ribosome entry site element, which is responsible for the initiation of viral RNA translation (Poole et al., 1995). Differences between type I and type H BVDV in the RNA sequence coding for the E2 glycoprotein may enable type H isolates to possess increased ability for cell attachment. Differences in the RNA sequence corresponding to the 5’untranslated region may give type H isolates an advantage in viral RNA translation. A higher level of viremia may be observed with either situation. In srunmary, we have demonstrated a positive correlation between levels of viremia and manifestations of disease in cattle experimentally infected with type I and type H BVDV. In addition, severe peracute BVDV and the hemorrhagic syndrome may occur as a result of infection with isolates of BVDV capable of inducing high levels of viremia. Sources and Manufacturers a. Butler Company, Dublin, OH b. Schering-Plough Animal Health Corporation, Kenilworth, NJ c. Dr. Steve Bolin, National Animal Disease Center, Ames, IA (1. Dr. Clayton Kelling, University of Nebraska, Lincoln, NE 206 e. JRH Biosciences, Lenexa, KS f. Sigma Chemical Co., St. Louis, MO g. Gibco BRL, Life Technologies, Grand Island, NY h. Becton Dickinson, Franklin, NJ i. Technicon H-l analyzer, Technicon Instrumental Corp., Tarrytown, NY j. Amersham Pharmacia Biotech USA, Piscataway, NJ k. National Veterinary Services Laboratory, Ames IA 1. Vortech Pharmaceuticals, Dearbom, MI m. Columbia Diagnostics, Inc., Springfield, VA 11. Fischer Scientific, Pittsburgh, PA. 0. Dr. Ed Dubovi, Cornell University, Ithaca, NY p. Vector Laboratories, San Francisco, CA q. Dr. Y. M. Saif, Ohio Agricultural Research and Development Center, Wooster, OH. Acknowledgments This research project was supported by 1433 Animal Health Funds (Unites States Department of Agriculture), Michigan Agricultural Experiment Station, the Department of Large Animal Clinical Sciences at Michigan State University, and an American Association of Bovine Practitioners Research Assistantship. References Baker, J .C. (1995). The clinical manifestations of bovine viral diarrhea virus infection. In The Veterinary Clinics of North America (Food Animal Practice). Baker, J .C. & Houe, H. (eds), Vol. 11. pp. 425-445. W.B. Saunders: Philadelphia. 207 Bessman, J .D., Williams, L.J. & Gilrner, P.R., Jr. (1982). Platelet size in health and hematologic disease. Am J Clin Pathol, 78, 150-3. Bezek, D.M., Grohn, Y.T. & Dubovi, E.J. (1994). Effect of acute infection with noncytopathic or cytopathic bovine viral diarrhea virus isolates on bovine platelets. Am J Vet Res, 55, 1115-9. Bolin, S.R. & Ridpath, J .F . (1992). Differences in virulence between two noncytopathic bovine viral diarrhea viruses in calves. Am J Vet Res, 53, 2157-63. Carbrey, E., Brown, L., & Chow, T. (1972). Recommended standard laboratory techniques for diagnosing infectious bovine rhinotracheitis, bovine virus diarrhea and shipping fever (parainfluenza—3). Proceedings of the annual meeting of US animal health association, 75, 629-648. Carman, S., van Drerunel, T., Ridpath, J ., Hazlett, M., Alves, D., Dubovi, E., Tremblay, R., Bolin, S., Godkin, A. & Anderson, N. (1998). Severe acute bovine viral diarrhea in Ontario, 1993-1995. J Vet Diagn Invest, 10, 27—35. Corapi, W.V., Elliott, R.D., French, T.W., Arthur, D.G., Bezek, D.M. & Dubovi, E.J. (1990). Thrombocytopenia and hemorrhages in veal calves infected with bovine viral diarrhea virus. 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J Gen Virol, 74, 73-9. 210 CHAPTER VI: SUIVIMARY AND CONCLUSIONS 211 Some isolates of BVDV have been associated with severe clinical disease and a hemorrhagic syndrome characterized by thrombocytopenia. The experimental studies described in this dissertation were designed and conducted to provide insight into the mechanism of BVDV-induced thrombocytopenia and the pathogenesis of the hemorrhagic syndrome associated with BVDV infection. Thrombocytopenia associated with type H BVDV infection has been reported under natural (Rebhun et al., 1989) and experimental conditions (Bezek et al., 1994; Bolin & Ridpath, 1992; Corapi et al., 1990; Corapi et al., 1989; Ellis et al., 1998). Previous experimental studies on BVDV-induced thrombocytopenia considered principally the clinical and pathologic findings associated with BVDV-induced thrombocytopenia, but these previous studies failed to define the following: 1. What is/are the mechanism(s) of type H BVDV-induced thrombocytopenia? 2. Why are hemorrhages observed in experimentally infected calves, yet the platelet count is not decreased to a magnitude consistent with the occurrence of spontaneous hemorrhage? 3. Why are thrombocytopenia and severe clinical disease most often associated with infection of cattle with type H BVDV, and not type I BVDV. That is, what are the differences between a type I and type II BVDV that are responsible for the increased virulence observed during type H BVDV infections in cattle? The focus of our first experimental study was to address the need for a reproducible model of BVDV-induced thrombocytopenia. This was important, as our subsequent pathogenesis studies needed to be based upon a reproducible model of type H BVDV- 212 ll induced thrombocytopenia. Previous studies failed to generate a reproducible experimental model because of several limitations. Several of the previous studies on BVDV-induced thrombocytopenia used an infection protocol that did not allow quantitation of the infectious dose of virus, which makes reproduction of their study difficult (Bezek et al., 1994; Corapi et al., 1990; Corapi et al., 1989). Furthermore, several studies failed to use controls or failed to use age matched controls (Bolin & Ridpath, 1992; Corapi et al., 1990; Corapi et al., 1989). Control calves are critical in the evaluation of thrombocytopenia because of the variation in the normal platelet count (Nelson et al., 1974; Semrad & Dubielzig, 1993); and age-matching is important as the platelet count varies between calves of different ages (Nelson et al., 1974; Semrad & Dubielzig, 1993; Vaugher et al., 1973). Finally, some investigations used type H BVDV isolates that failed to consistently induce thrombocytopenia in all infected calves (Corapi et al., 1989; Ellis et al., 1998). In addition to developing a model, we also wanted to characterize the clinical, hematologic, virologic, and pathologic features of the thrombocytopenic BVDV infection in neonatal calves. Characterization of the above parameters in an experimental thrombocytopenic BVDV infection would provide information into the mechanism of BVDV-induced thrombocytopenia. Decreased mean platelet volume, which was reported for the first time in BVDV-infected calves in this study, and the presence of viral antigen in megakaryocytes provide support to the hypothesis that BVDV-induced thrombocytopenia is a result of decreased platelet production. In addition, normal coagulation profiles, which rules out disseminated intravascular coagulation, and an early induction of thrombocytopenia prior to the presence of neutralizing antibodies provide 213 ll evidence that BVDV-induced thrombocytopenia is not due to increased destruction of platelets in the peripheral circulation by an immune-mediated mechanism. Sequestration of platelets as the cause of BVDV-induced thrombocytopenia is not likely because splenomegaly and hepatomegaly were not observed on postmortem or histologic examination in BVDV-infected calves. Further study evaluating platelet turnover in the peripheral circulation and/or reticulated platelet assays are necessary to fully evaluate the decreased platelet production hypothesis. The discrepancy between development of hemorrhage and platelet count suggested that platelet frmction might be altered in cattle infected with type H BVDV. Evaluation of platelet function in calves experimentally infected with the type H isolate BVDV 890 was the focus of the second experiment. Platelet function was depressed in calves experimentally infected with the type H isolate BVDV 890, when compared to sham- infected control calves. This was the first report describing altered platelet function in calves infected with BVDV. In addition, the depressed platelet function likely contributed to hemorrhaging, as bloody diarrhea was observed antemortem and hemorrhages were observed on postmortem examination in those BVDV 890-infected calves whose platelet function was assessed. The hemorrhagic syndrome and outbreaks of severe disease have been associated with type H isolates of BVDV, and not type I BVDV. Altered platelet function may be an important contributing factor for the hemorrhagic syndrome and for the increased virulence observed with some type H BVDV isolates. The third experiment of this dissertation was designed to further evaluate the ability of BVDV isolates to alter platelet function, and to determine if platelet dysfunction occurs exclusively following challenge 214 I l with type H BVDV, as compared to type I BVDV. Platelet dysfunction was only observed in the type H BVDV-infected calves. Another important observation from this third experiment was that the effect of BVDV infection on platelet function was reproducible. Like the second experiment, similar depressions in platelet fimction were observed in this study utilizing a different group of BVDV 890-infected calves. Depressed platelet function may be an important factor for differences in virulence between type I and type H BVDV; however, further study evaluating other type I and type H isolates is necessary to determine if this observation remains valid. Differences in virulence between type I and type H BVDV may be the result of differences in the level of viral replication. A difference in the level of viremia has been previously demonstrated between a type I and a type H BVDV infection (Bolin & Ridpath, 1992). This higher level of viral replication with type H BVDV may be important in the pathogenesis of severe peracute BVDV and the hemorrhagic syndrome. The purpose of the fourth and final experiment was to characterize the level of viremia induced during BVDV infection, and correlate levels of viremia with severity of clinical disease. A clear difference in the level of viremia induced during infection was demonstrated in calves infected with type I or type H BVDV. The level of viremia was also correlated to severity of clinical disease and pathology. The type I isolate BVDV TGAN induced the lowest level of viremia, and clinical signs of disease were less severe and depressions in platelet and leukocyte counts were milder when compared to the type H isolates, BVDV 7937 and BVDV 890. In addition, a parallel relationship between the level of viremia and rectal temperatures, and an inverse relationship between the level of viremia and platelet and leukocyte counts was observed, with the greatest elevations in 215 rectal temperature and greatest depressions in platelet and leukocyte counts being observed in calves infected with BVDV 890. The results of this study, indicating the level of viremia is correlated to disease manifestations, support the hypothesis that the level of viremia induced during BVDV infection is an important factor for differences in virulence among BVDV isolates. Isolates of BVDV that induce higher levels of viremia may be more capable of inducing clinical signs of disease. Severe peracute BVD and the hemorrhagic syndrome may occur as a result of infection with isolates of BVDV capable of inducing high levels of viremia. Future studies in the area of BVDV-induced thrombocytopenia may now be initiated as a result of the findings described in this dissertation. Platelet turnover and quantification of reticulated platelets may determine the exact mechanism of type H BVDV-induced thrombocytopenia. The mechanism of altered platelet fimction in calves infected with type H BVDV may be determined through further evaluation of aggregation responses utilizing other agonists, or evaluating platelet secretion. Future studies can also be directed at other related aspects of BVDV infection. Uncovering the molecular basis for the differences in the level of viremia between type I and type H BVDV isolates is an important future study. In addition, evaluating strategies, such as vaccination, that reduce viremia are essential, and these same strategies may be important in reducing clinical signs associated with BVDV infection. Moreover, limiting the level of viremia during acute BVDV infection may limit reproductive consequences of BVDV infection. This is critical because strategies designed to limit the level of viremia may also be important for preventing fetal infection with BVDV, and thus, eliminating the birth of persistently infected calves. 216 In summary, the studies encompassed by this dissertation contribute information on the pathogenesis of acute BVDV infections in general and BVDV-induced thrombocytopenia specifically. Three major findings have emerged from the studies described in this dissertation. First, decreased production of platelets may be the primary mechanism associated with BVDV-induced thrombocytopenia. Second, altered platelet function is important in the pathogenesis of the hemorrhagic syndrome. Finally, the ability to induce a high level of viremia is an important factor for differences in virulence between type I and type H BVDV. References Bezek, D.M., Grohn, Y.T. & Dubovi, E.J. (1994). 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