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I): , . n. . . . . l . H . .. , ,. «“6... n ‘. n. n :3 I A . 9.: N cl‘. '4' Flul \ L . a . t . Ir v . . .. TH 5818 Date 07639 l" ‘ “r" ‘— '-—‘\.. 1" ”‘7'? ’ ff .‘ "T"? ' IlllllllllllllllllllUlllllllllillllllUIIIIIHIHHIIIIIIHJI i .. 3 1293 10457 9903 1 if“... 153;; 33' IL I This is to certify that the thesis entitled IMMUNOLOGIC AND EPIDEMIOLOGIC STUDIES ON BOVINE LEUKEMIA VIRUS WITH REGARDS TO PRECOLOSTRAL CALVES presented by Karen Lee Jacobsen, DVM has been accepted towards fulfillment of the requirements for M. S . degree in Large Animal Clinical Sciences Major professor Mayi7. 1982 MSU LIBRARIES a— » RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. M é/szl/o IMMUNOLOGIC AND EPIDEMIOLOGIC STUDIES ON BOVINE LEUKEMIA VIRUS WITH REGARDS TO PRECOLOSTRAL CALVES BY KAREN LEE JACOBSEN A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Large Animal Clinical Sciences 1982 ABSTRACT IMMUNOLOGIC AND EPIDEMIOLOGIC STUDIES ON BOVINE LEUKEMIA VIRUS WITH REGARDS TO PRECOLOSTRAL CALVES BY Karen Lee Jacobsen The prevalence of in utero bovine leukemia virus (BLV) exposure was investigated in 2 Michigan dairy herds with breeding practices designed to increase milk production. Antibodies to BLV were detected by the radioimmunoprecipitation assay utilizing glycoprotein antigen (gp-RIA). Three of 79 calves (3.8%) born to seropositive dams were seropositive prior to colostral ingestion. None of 46 calves born to seronegative dams were seropositive at birth. The blastogenic response of peripheral blood lymphocytes from precolostral calves born to BLV seropositive and seronegative dams was investigated. Lymphocytes were isolated from 5 calves born to gp-RIA positive dams and 5 calves born to gp-RIA negative dams. All of the calves were gp-RIA negative at birth. Significant differences were observed between calves from BLV positive and negative dams in the response to phytohemagglutinin (P <0.001) and pokeweed mitogen (P <0.005) both before and after adjustment for the effect of cort- isol variation. To my mother, Dolores Sefchik Bocher, and my father, Donald James Jacobsen ii ACKNOWLEDGEMENTS I am deeply grateful to Dr. John B. Kaneene and Dr. Robert W. Bull for their excellent guidance and unwavering support during the entire period of my post-graduate research investigations. I am indebted to these men for their dedication to training me in the art of scientific investiga- tion. Their constructive criticism and editorial assistance was inval- uable in the preparation of the thesis. I particularly wish to thank Dr. Bull for his generous provision of laboratory space, equipment, and assis- tance, without which a research project of this magnitude would not have been possible. My sincere appreciation is also extended to my major advisor, Dr. Thomas H. Herdt, for his advice and support throughout the degree program. I also thank Dr. E. Clay Hodgin for serving on my guidance committee, Dr. Raymond F. Nachreiner for assistance with the cortisol study, and Dr. Janice M. Miller for sharing her invaluable knowledge and insight regard- ing bovine leukemia virus. I am particularly grateful to Ms. Peggy Coffman for excellent assis- tance and training in cell culture techniques. Thanks are also due to the following persons at Michigan State University for technical assistance: Ms. S. Gilbert, Mr. J. Harrison, Ms. J. Nicol, Mr. J. Bonasera, and Ms. J. Kivela. I also thank the technical staff of Dr. J.M. Miller at the National Animal Disease Center, Ames, Iowa. Appreciation is also due to the staff of the M.S.U. Dairy and the owners and staff of the commercial dairy that cooperated in the project. I would also like to thank the Michigan Agricultural Experiment Station for providing financial support for the research, and Dr. E.C. Mather and Ms. L. Smith for channeling this funding in my direction. I am particular- ly indebted to Dr. J.R. Welser for obtaining the funds which made comple- tion of the project possible. For assistance with typing, I am sincere- ly grateful to the Steno Pool of the College of Veterinary Medicine, Ms. Rebecca McMahon, Ms. Alvena Lucas, and Ms. Marion Lloyd. Finally, I am indebted to my husband, Dr. Michael Mispagel, for his remarkable assistance and training in computer analysis, but most of all for his love, concern, and understanding throughout the study. TABLE OF CONTENTS Page LIST OF TABLES ........... ... ........ ................... ........... ..vi LIST OF FIGURES .......... ...... . ..................... ...... ........ vii INTRODUCTION ...... . ..................... . ..... . .......... . ...... .....l EXPERIMENT l: ..... ...... ..... ................. .. ......... ... ..... ...4 Transmission of Bovine Leukemia Virus: Prevalence of Antibodies in Precolostral Calves. a) Summary .. ............. ..................................4 b) Introduction .......... .......... ................ ........ 4 c) Materials and Methods .............. ............ ......... 7 d) Results . ....... . ...... ......... ........ . ........ . ...... 10 e) Discussion ............. . .................... .... ....... 12 f) References ....... . ................ .....................l7 EXPERIMENT 2: ................ ............ ..........................19 Effect of Storage at Room Temperature on the Concentration of Cort— isol (Hydrocortisone) in Precolostral Bovine Blood. a) Summary ................................................19 b) Introduction ..... . .............. ... ..... ...... ......... 20 c) Materials and Methods .... ........ .......... ............ 21 d) Results .......... ............... . ...................... 27 e) Discussion ....... ....... .................. ........ .....29 f) References .......................... . ..... . ........ ....31 iv EXPERIMENT 3 : ............................................... . . ..... . 32 Bovine Leukemia Virus: Lymphocyte Blastogenesis and Plasma Cortisol in Precolostral Calves from Antibody Positive and Negative Dams. a) Summary . ......... . ............................ .. ..... ...32 b) Introduction . ............................. ..... ......... 33 c) Materials and Methods .... .................... . .......... 35 d) Results ................................................. 41 e) Discussion ....... ..... . .......................... . ...... 45 f) References ......................................... .....51 EXPERIMENT 4: ............ ......................... ..................54 Comparison of Commercial Agar Gel Immunodiffusion and Radioimmuno— precipitation Assay for Antibodies to Bovine Leukemia Virus. a) Summary ................. ..... .................... ...... .54 b) Introduction ...... ............ ............... ........... 55 c) Materials and Methods ........... . ..................... .-56 d) Results ........ ......... . ....... .. ..... ... ....... .......59 e) Discussion .............. .... ...... .... ................. .61 f) References ............................. ...... ..... . ...... .66 DISCUSSION AND SUMMARY .......................................... .....68 BIBLIOGRAPHY ..... ......... . ...... .... .......... ...... ........ ........72 GENERAL REFERENCES ......... . ...... . .......... . ...... .... ............ .29 LIST OF TABLES Table Page 1.1 Animals Tested for BLV Antibody .. ............... . ........... 8 1.2 Dams-and Calves by BLV Antibody Status ........... . ......... 11 2.1 Specificity of Antiserum for Gamma Coat 125I-Cortisol 2.2 2.3 2.4 3.1 3.2 4.1 4.2 4.3 Radioimmunoassay ............................... .... ..... ...23 Precision of Modified Solid Phase 125I-Cortisol Radio- immunoassay ..... . .................. ..........' ....... .......24 Dilutional Parallelism of Modified Solid Phase 125I-Cortisol Radioimmunoassay .... ........... ............. ...... .........25 Plasma Cortisol Values from Heparinized Calf Blood Stored at Room Temperature (22 C) .................. . ........ .........26 Mitogen-induced blastogenesis of precolostral calf lympho- cytes isolated using 3 RBC lysis techniques .. ........ . ..... 43 Blastogenesis of lymphocytes from precolostral calves born to BLV seronegative and seropositive dams .................. 44 Comparison of Commercial gp ACID with gp RIA ............... 60 Reactions Differing on gp RIA and Commercial gp ACID ....... 62 Reactions with Haze around Test Serum Well . ...... ..........63 vi LIST OF FIGURES Figure Page 2.1 Degradation of cortisol in heparinized calf blood at room temperature (22 C) ................................... 28 3.1 Dose response curves for mitogens ........................ .39 4.1 Interpretation of commercial ACID for antibodies to BLV glycoprotein antigen ...................................... 57 4.2 Interpretation of commercial ACID with haze around test serum wells ..... . ......................................... 58 vii INTRODUCTION Lymphoid tumors in cattle have been recognized by veterinarians since the late 1800's. The condition appeared to be endemic in certain herds and geographic regions, thus gaining the popular terminology of Enzootic Bovine Leukosis. It was observed that adult cattle with lymphosarcoma, as well as many of their non-tumorous herd-mates, often had persistently elevated peripheral blood lymphocyte counts.86 For many years, this persistent lymphocytosis (PL) was the only available diagnostic measure for bovine leukosis. Eradication programs were developed in several European countries based on an index estab- lished by Bendixen, which gave upper limits for lymphocyte counts of cattle, according to age. A cow with lymphocytosis on 2 consecu- tive tests was deemed positive and sent to slaughter. These eradication programs met with only partial success.88 The etiologic agent, BLV, was not discovered until 1969. At that time, Dr. Janice Miller noted C type virus particles in phytohemagglutinin-stimulated lymphocytes from cows with lymphosarcoma, 66 and an explosion of knowledge has ensued. Bovine leukemia virus is an RNA oncornavirus belonging to the family Retraviridae. Like other members of this family, it possesses reverse transcriptase, an enzyme which facilitates the synthesis of DNA from an RNA template. However, unlike many retroviruses, BLV has the ability to induce syncytia formation in vitro.89 Along with the etiologic agents of the leukemia-sarcoma complexes of mice and cats, BLV is classified morphologically as a type C oncornavirus. BLV is a persistent infection in cattle, presumably because the virus genome becomes integrated into 19,20 cellular DNA. The virus naturally infects only cattle, but experimentally sheep goats are also susceptible. The virus is destroyed by pasteurization75 and there is no evidence of transmission to man.84’85 Four types of lymphosarcoma are now recognized in cattle: calf (juvenile), thymic (adolescent), adult, and cutaneous. Only the adult type is enzootic and is associated with BLV infection. The adult type is common, while the other 3 types are rare. The term lymphosarcoma, as used in this thesis, refers to adult bovine lymphosarcoma. The majority of the cattle infected with BLV have no hematologic or clinical abnormalities.78 Less than 5% develop lymphosarcoma and only 30% to 40% develop PL,78 thus explaining the incomplete success of early eradication programs. Many animals in the last 2 categories overlap. It has been hypothesized that PL may be a precancerous stage 92 of infection. However, some animals develop lymphosarcoma without ever having PL, and the majority of animals with PL never develop tumors.78 Some have postulated that genetic factors may influence whether an infected animal develops PL or lymphosarcoma, 78 and recent evidence suggests that the tendency toward BLV infection itself may be hereditary.79 In the United States, the prevalence cf BLV infection ranges from 10% to 48% in dairy cattle and from 1% to 7% in beef cattle, depending on the geographic region surveyed.l For the year 1978, the estimated loss due to slaughter for tumors was $3.4 million, and it was estimated that another $3.4 million was lost due to morbidity and mortality.91 However, even greater economic losses occur when export breeding stock become infected with the virus. A growing number of countries in the world are establishing import restrictions requiring BLV negative status for animals and semen.80 Livestock producers and breed associations are realizing the necessity of control programs to protect valuable breeding stock, and are eager for information on practical control methods.8 Critical to any control program is a thorough understanding of the transmission of the disease in question. Horizontal transmission of BLV by contact is well established,12’13 and insects, hypodermics, and blood transfusions are suspected as mechanisms. ’ Vertical trans— mission of the virus, however, is poorly understood. From studies conducted in an experimental herd that was specifically bred for susceptibility to lymphosarcoma, investigators concluded that 14% to 18% of the calves born to BLV infected dams were infected prior to colostral ingestion.13’15 The results of these studies may not reflect situations in many dairy herds. Information is not available about prenatal transmission of BLV in herds with breeding programs designed to increase milk production. BLV infections in cattle have also been associated with various immunologic abnormalities, the economic consequences of which are unknown. Abnormal IgM levels and delayed IgG response to synthetic antigens have been reported in cows with lymphosarcoma.81"83 Abnor- malities of lymphocytes blastogenesis have been reported in adult 47.52954’55 and sera from cows with cattle infected with the virus, tumors inhibits blastogenesis of normal bovine lymphocytes.41 The immune response of calves born to BLV infected dams, however, has not be investigated. The specific objectives of this thesis were: 1) to determine the prevalence of BLV seropositive precolostral calves born to BLV seropositive dams, and 2) to investigate lymphocyte blastogenic responses of calves born to BLV seropositive dams. EXPERIMENT 1: Transmission of Bovine Leukemia Virus: Prevalence of Antibodies in Precolostral Calves SUMMARY A study was undertaken to determine the prevalence of bovine leuke- mia virus (BLV) antibodies in the serum of precolostral calves born to BLV antibody positive dams. Two Michigan dairy herds with breeding programs designed to increase milk production were utilized in this study. The prevalence of seropositive cows was 32% and 94% in herds 1 and 2 respectively, and neither herd had experienced more that 4 cases of lymphosarcoma in 10 years. One hundred and twenty-five calves and their dams were tested for antibody to BLV by the radioimmuno- precipitation assay utilizing glchprotein antigen. Three of 79 calves (3.8%) born to seropositive dams were seropositive prior to colostral ingestion. None of 46 calves born to seronegative dams were seroposi- tive at birth. The percentage of calves born to seropositive dams that were seropositive at birth was 4.7% in herd 1 and 3.4% in herd 2. This is a much lower prevalence of in utero BLV exposure than reported in previous studies utilizing a herd in which cows were bred for suscep- tibility to lymphosarcoma. Therefore, in herds seeking to reduce the prevalence of BLV seropositive animals, it should be possible to retain calves from seropositive dams in greater numbers than previously expected. INTRODUCTION Bovine leukemia virus (BLV) infection is widespread amongst the world's cattle, especially in dairy breeds. Prevalence studies for BLV antibodies in cattle herds have been conducted in the United States‘, numerous countries in EurOpe,2I3 the U.S.S.R.,4 Venezuela,S Japan,6 and the Phillipines,'7 with reports of up to 49% of dairy animals seroposi- tive for BLV (in Japan), and up to 44% of beef cattle (in Venezuela). While losses from clinical tumors are significant individual cattle owners, losses of export and import markets for breeding stock are of greater economic concern to the cattle industry. A growing number of countries in the world are establishing import restrictions requiring BLV negative status for animals or semen.a Livestock producers and breed associations are realizing the economic necessity of control programs to protect valuable breeding stock, and are eager for infor- mation on practical control methods.8 Successful eradication of BLV has been reported by means of trial test and slaughter programs in Europe.9v1° Such methods are prohibi- tively costly and result in the destruction of otherwise healthy livestock and often irreplaceable genetic lines. More conservative test and segregation programs are therefore currently under investigation. The basis for these programs is that horizontal transmission of the virus can be reduced by strict separation of BLV infected and nonin- fected cattle. Preliminary studies suggest that test and segregation is a viable means to decrease and possibly even eliminate BLV infection within herds.11 With this approach, BLV negative replacement animals can be reared from both infected and noninfected groups, thus preserving valuable genetic lines. aScott, RM: Personal communication. USDS-APHIS-Veterinary Services, Lansing, MI Area Office, 1981. Critical to any control program is a thorough understanding of the transmission of the disease in question. Horizontal transmission of BLV by contact is well established,12I13 but vertical transmission of the virus is poorly understood. The prevalence of in utero BLV infections will undoubtedly affect the time required for successful elimination of BLV infection from within a herd. Experimental prenatal transmission of the virus from dam to offspring has been demonstrated. In a recent study by Van Der Maaten et al, an experimentally infected dam gave birth to one live and one dead twin.14 Both had antibody to BLV, and virus was detected in the live twin at one day of age. The prevalence of this occurrence in the on-farm situation, however, has not been carefully investigated. In one study, Perrer et al reported that 14% of calves from a herd with multiple cases of lymphosarcoma were BLV antibody posi- tive prior to colostral ingestion.15 However, not all of the dams of these calves were known to be serOpositive to BLV. The prevalence of in utero transmission from positive dams was, therefore, difficult to accurately determine from this study. In another study, Piper et all reported that 15 of 84 calves (18%) born to BLV infected dams were infected prior to colostral ingestion.13 The dams in both studies, however, were from an experimental herd of approximately 120 Jersey cows specifically bred for susceptibility to lymphosarcoma. By 1979, 69 cases of histOpathologically confirmed leukemia had occurred in the herd through a breeding program in which cattle were bred back to 3 animals from their original genetic stock. Therefore, the results of these stu- dies may not reflect situations in many dairy herds. Information is not available about prenatal transmission of BLV in herds with breeding programs based on production rather than susceptibility to lymphosarcoma. Control programs involving attempts to raise BLV negative replacements from positive dams must involve preco- lostral screening of newborn calves for infections that may have occurred in utero. However, the prevalence of the usual occurrence of such infections has not been investigated. This study was undertaken to determine the prevalence of BLV antibodies in precolostral calves born to BLV antibody positive dams in dairy herds with breeding and culling programs designed to increase milk production. MATERIALS AND METHODS Animals The calves in this study were born to 120 dams in 2 Holstein- Friesian herds in Michigan (Table 1.1). Since S cows had twins, 125 calves were tested: 56 from herd 1 and 69 from herd 2. None of the cows in the study had clinical signs of lymphosarcoma. Herd 1 This commercial dairy had 285 cows of lactating age. The total herd consisted of 550 animals, 30% of which were registered. The herd was tested in the fall of 1980 by the commercial agar gel immunodiffusion testb (AGID-gp) for antibody to BLV glyc0protein antigen. Thirty-two percent of lactating-age cows reacted positively by this test. This prevalence was similar to the average prevalence in Michigan as deter- mined by a current random survey of Michigan dairies.16 In 10 years, only one case of suspected lymphosarcoma had occurred. bLeukassay B, Pitman-Moore, Inc., Washington Crossing, N.J. Table 1.1 Animals Tested for BLV Antibody* Dams Calves Herd 1 54 56tt Herd 2 66 asttt Total 120 125 * as determined by gp-RIA tt 2 sets of twins ttt 3 sets of twins Herd 2 This institutional dairy had 114 cows of lactating age. The total herd consists of 325 animals, all of which were registered. This herd was BLV tested in October of 1978. At that time 94% of lactating-age cows had antibody to BLV by commercial AGID-gp.b Four histopathologically-confirmed cases of lymphosarcoma had occurred in the past 10 years. Breeding Practices All breeding in both herds was by artificial insemination. Sires were selected by predicted difference for milk production and inbreeding was purposely avoided. In herd 2 all replacements were raised on the farm. Herd 1 raised most replacements, but 1 or 2 registered heifers were also purchased annually. In both herds culling was performed on the basis of low production and without regard to BLV status. Calving Procedures Calving in both herds occurred in maternity stalls freshly bedded with straw or in herd 2 occasionally on green pasture. In both herds personnel were on duty at least 21 hours each day, and periparturient dams were closely observed. It was routine procedure in both herds for the calf to be intercepted before nursing because calves were bottle-fed controlled amounts of colostrum. In both herds dams were allowed to lick the calves dry. Collection of Blood All sample collection took place between October, 1980, and October, 1981. From October to December of 1980, all calves born in both herds were included in the study. From January to October of 1981, the only 10 calves sampled were those born to dams that were previously BLV positive by AGID-gp. Blood was drawn from both calf and dam immediately after birth. Samples were not collected if any possibility existed that a calf may have nursed. Collection was by venipuncture into sterile eva- cuated glass tubes. Sera were drawn off and stored at -20 C until sero- logic tests were performed. Serologic Tests The radioimmunOprecipitation assay utilizing BLV glycoprotein anti- gen (gp-RIA) was conducted as previously reported by Miller.17 Sera that precipitated 15% or more of the labeled antigen were regarded as positive. Sera were neither concentrated nor diluted prior to the testing procedure. Serum samples collected from both calves and dams at birth were tested by gp-RIA. The terms BLV positive and negative as used in this study refer to antibody status by gp-RIA. RESULTS All dams that were previously BLV antibody positive by AGID-gp were antibody positive by gp-RIA at the time of calving. None of 46 calves born to BLV negative dams were BLV positive at birth (Table 1.2). Three of 79 calves (3.8%) born to BLV positive dams were BLV positive at birth. The percentage of calves from positive dams that were positive at birth was 4.7% (1 of 21 calves) in herd 1 and 3.4% (2 of 58 calves) in herd 2. Four of the 5 sets of twins were born to positive dams. All 10 twins were BLV negative precolostrally. The 2 cows in herd 2 whose calves were BLV positive at birth had reproductive problems. The first cow was 5 years old and had been treated for postpartum metritis after her last calving. The calf born Table 1.2 Dams and Calves by BLV Antibody Status* 1 (+) calf 20 (+) dams 20 (-) calvest Herd 1 0 (+) calves 34 (-) dams 35 (-) calvest 2 (+) calves 55 (+) dams< 56 (-) calvesttt Herd 2 0 (+) calves 11 (-) dams 11 (-) calves *as determined by gp-RIA t one set of twins ttt three sets of twins 12 BLV positive was a large bull calf which required assistance in deli- very. The cow passed the placenta the afternoon of calving. She con- ceived 60 days after parturition and calved uneventfully the subsequent lactation. The second cow was 3 years old and had delivered a heifer calf the previous year which died shortly after birth. A clinically normal bull calf was born BLV positive during the test period. After this the cow failed to conceive despite functional ovaries. She was culled from the herd 7 months after calving because of repeat breeding. The cow in herd 1 that delivered a BLV positive calf was 7 years old and had no history of reproductive problems. DISCUSSION In this study 3 of 79 calves (3.8%) from BLV antibody positive dams were BLV antibody positive at birth. The presence of BLV antibodies in the serum of precolostral calves most likely indicated in utero exposure to the virus. If infection of calves occurred during the birth process or immediately after birth, there would not have been time for antibo- dies to develop in the serum. In previous studies a close correlation between BLV antibody status and BLV virus infection in precolostral calves has been demonstrated. In a study conducted by Ferrer et al, the majority of 18 antibody posi- tive calves were virus positive prior to colostral ingestion.15 In another study conducted by Piper et a1, all 15 precolostral calves that were antibody positive were also virus positive.13 Therefore, in most cases, the presence of BLV antibodies in precolostral calves represents an active response to virus infection rather than maternal antibodies acquired in utero via a breakdown in the maternal-fetal placental barrier. Antibody positive calves in this study will be referred to as 13 virus exposed with the knowledge that virus infection is a likely possi- bility. In Piper's study, none of 69 antibody negative calves born to BLV infected dams were virus positive.13 Because there have been no reports of antibody negative precolostral calves that were virus positive, it appears that natural in utero exposure to the virus occurs after the fetus develops immunocompetence to BLV. Although it is theoretically possible that a calf could be virus exposed or infected in utero and not produce antibody to the virus, this apparently is not a frequent occurrence. It is likely, therefore, that very few, if any, of the antibody negative calves in this study were virus infected. In adult cattle a close correlation between BLV antibody status and BLV virus infection has also been demonstrated. Ferrer et al demonstrated virus by electron microscopy of phythohemagglutinin- stimulated lymphocyte cultures in 19 of 20 cows that reacted positively by immunofluorescent antibody test.18 Honma et al demonstrated virus by a syncytium induction assay utilizing splenic lymphocytes in 20 of 20 BLV antibody positive cows.6 Seropositive cattle in the study reported here are considered to be virus exposed and highly likely to be virus infected. In our study, utilizing herds with breeding and culling practices based on milk production, less than 4% of calves from BLV positive dams were antibody positive precolostrally. These results are in contrast to previous reports of 14 to 18% in utero viral exposure in an inbred lymphosarcoma-proned herd. Our results suggest that in herds with breeding programs designed to increase milk production almost all of the calves born to naturally infected dams are not eXposed to BLV in utero. 14 Therefore, control programs aimed at raising BLV negative calves from positive dams should meet with greater success than previous reports had indicated. Our results do not differ greatly from a study recently reported by Van Der Maaten et al in which most of the calves tested were born to experimentally infected dams.14 In that study 18 calves from BLV infected dams were not infected or seropositive at birth. In herd 2 of our study, in which 94% of the dams were BLV positive, only 2 of 58 calves (3.8%) from these dams were seropositive at birth. It is not uncommon to encounter dairy herds with over 90% of cattle antibody positive in BLV endemic areas.16 Such herds could eventually convert to BLV negative status by means of test and segregation programs involving careful screening of precolostral calves. The mechanisms by which some calves are infected with BLV in utero and not others is poorly understood. Prenatal transmission of the virus theoretically occurs either genetically or by exposure of the fetus to the dam's infected lymphocytes. The dosage of virus and the virulence of a particular BLV strain are other factors that may influence the pre- valence of in utero BLV transmission. There is no evidence to date to support genetic or chromosomal transmission of the BLV genome via the gametes. To the contrary, there is evidence that BLV, unlike many retroviruses, is an exogenous virus and as such does not infect gamete cells.19v20 Prenatal transmission of the virus, therefore, most likely occurs epigenetically via exposure of the fetus to the dam's infected lymphocytes. It is conceivable that viral transmission across the placenta occurs more readily during periods of inflammation or disease. The 2 cows in herd 2 that delivered BLV positive calves had reproductive problems. One cow had postpartum 15 metritis prior to conception of the positive calf and another cow failed to conceive after parturition despite functional ovaries. However, the number of dams that delivered positive calves was too small for a valid comparison of their reproductive status with that of the rest of the herd. Ferrer et al demonstrated that cows with clinical leukemia (lymphosarcoma) have higher antibody titers than BLV infected cows without tumors.18 He theorized that higher antibody titers may reflect higher doses of virus, and that the level of virus infection may be an important determinant in the develoPment of clinical leukemia. He also noted that in the multiple-case BF herd there was a positive correlation between antibody titer and leukemia risk. This was the same lymphosarcoma-proned herd that was subsequently used in studies on the prenatal transmission of BLV. If Ferrer's hypothesis is correct, it may account for the greater prevalence of in utero BLV transmission in the BF herd than we observed in herds 1 and 2 in our study. It would seem logical that a calf exposed to a higher dose of virus in utero would be more likely to become infected with BLV. Many questions remain unanswered about the prenatal transmission of BLV, leaving several possible areas for future investigation. Histopathologic studies of the placenta and uterus of dams delivering BLV positive calves would be useful to further investigate the possibi- lity that increased transmission of the virus occurs during inflam- mation. A comparison of antibody titers of dams delivering infected and non-infected calves would help answer the questions of whether virus dose affects in utero transmission. Quantitation of the virus in these dams, although difficult, would be ideal. 16 In summary, we report a much lower prevalence of in utero BLV expo- sure in herds in which breeding and culling are based on milk production rather than susceptibility to lymphosarcoma. Therefore, it appears that a greater number of calves from BLV positive dams than previously expected can be retained within herds seeking to achieve BLV negative status.j Based on this information, test and segregation control programs should meet with greater success, and will undoubtedly be more readily accepted by cattle owners. 17 REFERENCES 1. Burridge MJ, Puhr DM, Hennemann JM: Prevalence of bovine leukemia virus infection in Florida. J Am Vet Med Assoc 179: 704-707, 1981. 2. DeVries G: Leucosis in cattle in the Netherlands. Ann Rech Vet 9: 903-907, 1978. 3. Markson LM, Roberts DH, and Bradley R: Studies on bovine leukosis as it is seen in England and Wales. Ann Rech Vet 9: 899-902, 1978. 4. Valikhov AF: Studies on the morphology and on some immunologi- cal properties of bovine leukemia virus, in The Serological Diagnosis of Enzootic Bovine Leukosis. Luxembourg, Commission of the European Comminities, 1978, pp 172-187. 5. Marin, C, de Lopez NM, Alvarez, et a1: Epidemiology of bovine leukemia Venezuela. Ann Rech Vet 9: 743-746, 1978. 6. Honma T, Onuma M, Mikami T, et al: Bovine leukemia virus infection in Japan: antibody and virus detection in cattle. Jpn J Vet Sci 42: 58, 1980. 7. Masangkay JS, Lieberman 0: A serological survey for the pre- sence of bovine leucosis (lymphosarcoma) in Central Luzon, Philippines. Philipp J Vet Med 18: 92-101, 1979. 8. Mix M: Export significance and industry observations. Proc Bov Leukosis Symposium of the USDA, May 22-23, College Park, MD, 166-172, 183-186, 1979. 9. Mammerickx M, Cormann A, Burnry A, et al: Eradication of enzootic bovine leukosis based on detection of the disease by the GP immunodiffusion test. Ann Rech Vet 9: 885-898, 1978. 10. Straub 0C: Preliminary results of a new sanitation program for the eradication of enzootic bovine leukosis. Ann Rech Vet 9: 895-898, 1978. ~ 11. Van Der Maaten MJ, Miller JM: Appraisal of control measures for Bovine leukosis. J Am Vet Med Assoc 175(12): 1287-1290, 1979. 12. Straub OC: Herizontal transmission studies on enzootic bovine leukosis. Ann Rech Vet 9: 809-814, 1978. 13. Piper C, Ferrer J, Abt D, et al: Postnatal and prenatal transmission of the bovine leukemia virus under natural conditions. J Natl Cancer Inst 62: 165-168, 1979. 18 14. Van Der Maaten MJ, Miller JM, Schmerr MJF: In utero transmission of bovine leukemia virus. Am J Vet Res 42: 1052-1054, 1981. 15. Ferrer JP, Piper CE, Abt DA, et al: Natural mode of transmission of the bovine C-type leukemia virus (BLV). Bibl Haemat 43: 235-237, 1976. 16. Kaneene JM, Nicol J: Prevalence of antibody to bovine leukemia virus in Michigan dairy herds. Submitted for publication to Am J Vet Res, 1982. 17. Miller JM, Schmerr MJF, Van Der Maaten MJ: Comparison of four serologic tests for the detection of antibodies to bovine leukemia virus. Am J vet Res 42: 5-8, 1981. 18. Ferrer JF, Abt DA, Bhatt DA, et al: Studies on the relationship between infection with bovine C-type virus, leukemia, and persistent lymphocytosis in cattle. Cancer Res. 34: 893-900, 1974. 19. Kettman R, Portetelle D, Mammerickx M, et al: Bovine leukemia virus: an exogenous RNA oncogenic virus. Proc Nat Acad Sci USA. 73: 20. Callahan R, Lieber MM, Todaro GJ, et al: Bovine leukemia virus DNA of leukemic cattle. Science 192: 1005-1007, 1976. EXPERIMENT 2: Effect of Storage at Room Temperature on the Concentration of Cortisol (Hydrocortisone) in Precolostral Bovine Blood SUMMARY A study was undertaken to determine the effect of storage at room temperature on the concentration of cortisol in neonatal bovine blood. Heparinized blood samples were collected from 7 Holstein-Friesian calves within 15 minutes of birth and prior to colostral ingestion. Samples from each of the calves were divided into aliquots and stored undisturbed at room temperature (22 C) until centrifugation. The first aliquot from each calf was centrifuged one hour from sample collection. Additional aliquots were centrifuged at varying intervals up to 72 hours from the time of sample collection. After centrifugation, plasma was immediately collected and frozen until assayed for cortisol. Cortisol was determined by a modified commercial 125I-radioimmunoassay. A least squares linear regression of percent degradation of cortisol on storage time was performed. The initial mean plasma cortisol value for the 7 precolostral calves (109 :_6 ng/ml) agreed well with reports in the literature. As blood storage time at room temperature increased, plasma cortisol values for each calf steadily declined. The degradation of cortisol in heparinized blood at room temperature was demonstrated by the regression equation, Y = 9.534 + 0.832 X, where Y = percent cortisol degradation and x = hours from first cortisol determination. The regression coefficient (b) of 0.832 was significantly different from 0 (p < 0.001). The coefficient of determination (r2) was 0.77. Predicted values for percent 19 20 degradation of cortisol at 12, 24, and 48 hours were 20, 30, and 50 per- cent, respectively. The regression derived from our data can be used to calculate adjusted plasma cortisol values when neonatal calf blood samples are handled similarly. INTRODUCTION Bovine fetal plasma cortisol concentration gradually rises during the last week of gestation, with an abrupt twofold increase immediately after birth.1 Elevated glucocorticoid levels in fetal calves may play a role in the induction of parturition in this species.“2 High glucocor- ticoid levels in newborn calves have been associated with depressed lymphocyte mitogen responses3 and hematologic changes such as neutrophi- lia, lymphopenia, and eosinopenia.4 In one study, calves which later develOped diarrhea had significantly higher plasma cortisol levels in the first 4 days of life than did calves which remained healthy.5 Thus, decreased immune responses associated with elevated cortisol con- centrations were postulated to lessen a calf's resistance to infection.3 It has also been hypothesized that increases in cortisol concentration following colostral ingestion in calves may be involved in the regulation of intestinal closure to macromolecular absorption.6 It is therefore evident that plasma cortisol determinations in perinatal calves are useful in many fields of investigation. When samples for plasma cortisol determination are collected from calves on the farm, immediate centrifugation is often not possible. Thus, blood samples must often be tranSported. Refrigeration is often impractical and is sometimes not desirable when additional use is to be made of a heparinized blood sample. In lymphocyte blastogenesis 21 studies, for example, refrigeration of bovine blood samples results in decreased lymphocyte function, whereas storage at room temperature up to 48 hours has no significant effect.7 Philip and Marotta demonstrated the uptake of exogenous cortisol by erythrocytes in canine blood incubated at 37 C for 15 minutes.8 Nachreiner found significant degradation of endogenous cortisol in canine plasma samples stored 2 days at 37 C and in samples stored 4 days at either 4 or 27 C.9 Olson et al also noted significant degradation in canine serum stored 5 days at room temperature, but only in samples with large initial concentrations of cortisol.10 We found no reports regarding the effect of storage of heparinized whole blood at room tem- perature on plasma cortisol values. This study was undertaken to deter- mine the effect of storage at room temperature on the concentration of cortisol in neonatal bovine blood. MATERIALS AND METHODS Animals - Seven newborn Holstein-Freisian calves were used, 2 of which were twins. Precolostral calves were used because cortisol levels are highest immediately after birth. This allowed the greatest change in values before reaching levels that would no longer be detectable by the radioimmunoassay technique. Sample Collection - All samples were collected within 15 minutes of birth and prior to colostral ingestion. Fifteen m1 of blood were collected from each calf by jugular venipuncture into evacuated glass tubes containing heparin.a aVacutainer tubes, Becton, Dickinson & Co, Rutherford, NJ. 22 Experimental Procedure - Heparinized blood from each calf was thoroughly mixed prior to division into aliquots. The blood was stored at room temperature (22 C) until centrifuged. Sedimentation of erythro- cytes occurred because aliquots were left undisturbed until centrifuga- tion. For each calf, aliquot A (Table 2.4) was centrifuged one hour from the time of sample collection. Additional aliquots were centrifuged at varying intervals up to 71 hours from the time of centrifugation of aliquot A as shown in Table 2.4. After centrifugation, plasma was immediately collected and frozen. The plasma was stored at -20 C until assayed for cortisol. Assay Procedure - Cortisol radioimmunoassay (RIA) was performed using a commercial 125I-Cortisol RIA kit.b The kit was modified for use with bovine plasma by using larger sample volumes (20 U1), adding an additional 0.2 mg 8-anilino-l-naphthalene sulfonic acid per sample tube, and incubating at 37 C for 2 hours before decanting tubes. Specificity of the antiserum used, as reported by the commercial company, is shown in Table 2.1. Validation of the modified assay included measurement of sensitivity, precision and dilutional paralle- lism using lyOphilized aliquots of pooled bovine serum diluted with cortisol-free serum. Sensitivity, as calculated from the standard curve at 90% of trace binding, was 3.8 mg/ml. Precision and dilutional parallelism are shown in Tables 2.2 and 2.3, respectively. The coef- ficients of variation were calculated using data from within one lot number of the assay. When new lots were included, the interassay variation was much higher. bClinical Assays, Division of Travenol Laboratories, Inc, Cambridge, MA. 23 TABLE 2.1 - Specificity of Agtiserum for Gamma Coat 125I-Cortisol Radioimmunoassay Compound Cross reactivity (%) Cortisol 100 Prednisolone 87.5 11-Deoxycortisol 6.3 Cortisone 4.5 Prednisone 3.9 Corticosterone 2.5 Deoxycorticosterone 2.1 17-Hydroxyprogesterone 0.13 Aldosterone < 0.06 Dexamethasone < 0.06 Progesterone < 0.06 Spironolactone < 0.06 The results were determined using substance concentrations which resulted in 50% inhibition of maximum binding. 24 TABLE 2.2 - Precision of Modified Solid Phase 125I-Cortisol Radioimmunoassay Intra assay Inter assay Mean No. of Coefficient of No. of Coefficient of (ng/ml) samples variation (%) samples variation (%) 13.3 9 6.0 6 7.7 22.8 8 3.2 8 5.6 57.0 2 2.5 ND ND 25 TABLE 2.3 - Dilutional Parallelism of Modified Solid Phase 125I-Cortisol Radioimmunoassay % of expected concentration (ng/ml) 1:2 dilution 1 :4 dilution 19.1 134 119 1a .8 75 9° 40.0 107 89 isms Ease 26 news» a. .mo>Hmo Has ca scauooHHoo panama scum use: p oommoooum mm: 4 mosvaac + .oomsmauucoo uosvaac umuwu moses ousumuomEou Soon as mono: a c._e e.ec e.em m.- e.m e.e e.~ e.e as: me em on me on no no mo Hohaseoo ..s e._s e.se e.em m.- e.m e.c e.~ e.e as: me .m mm as on Pm me ee. Homssooo ..e e.cm e.~m e.ep m.~. e.m e.e ass on me mm so me. on. someones m m.ev e.c~ m.m. e.m. e.e cos Fe me he «a we. Homsuuoo e e.mc e.e~ e.- m.~ e.e use mm mm em on em Hoesusoo m e.me e.m~ e.m~ e.c e.e use am so so me so. someones m m.op o.o «mu: .e. m.. iasxcc. accesses . m o m m a U m +4 .02 Mano cooHn ocuacwummmn mo muosmdac AU NNV ousumuomsoa Boom um oououm occam maco oonucfiummo: Scum mosam> Homwuuoo mamcam .v.N mamma 27 Analysis of Data - The time of centrifugation for aliquot A (one hour from sample collection) was designated time zero and the plasma cortisol value was designated 0% degradation. For each calf, the per- cent degradation of cortisol for subsequent aliquots was calculated by comparison to aliquot A. Thus, 39 pairs of cortisol vs. time data were available for linear regression analysis (Table 2.4). A least squares linear regression11 of percent degradation of cortisol on storage time was performed. The Shapiro-Wilk test for normality12 was used to con- firm that the residual values for the regression were normally distri- buted. Student's t was computed to determine if the lepe of the regression line was significantly different from zero. Values for the percent degradation of cortisol at 12, 24, and 48 hours were predicted from the regression equation. RESULTS Plasma cortisol determinations and centrifugation times of each ali- quot are recorded in Table 2.4. The mean plasma cortisol value of ali- quot A for the 7 calves was 109 ng/ml (i 6 S.D.). As blood storage time at room temperature increased, cortisol values for each calf steadily declined. In calves 6 and 7 (twins) aliquots A through H were processed simultaneously. Cortisol determinations for the twins were similar for each storage time. The degradation of cortisol in heparinized blood at room temperature is described by the least squares linear regression of Figure 2.1. The equation for this regression is Y = 9.534 + 0.832 x, where Y = % cor- tisol degradation and x 8 hours from first cortisol determination. The regression coefficient (b) of 0.832 is significantly different from 0 28 C 2-1 Y = 9.534 + 0.832 X 1’ = 0.77 g— % CORTISOL DEGRADATION a r n °o 24 .6 ' 73 HOURS FROM FIRST CORTISOL DETERMINATION Figure 2.1 - Degradation of cortisol in heparinized calf blood at room temperature (22 C). 29 (p < 0.001). The coefficient of determination (r2) was 0.77. Predicted values for percent degradation of cortisol at 12, 24, and 48 hours was 20, 30, and 50 percent, respectively. DISCUSSION The results of this study indicate that the concentration of cor- tisol in plasma harvested from heparinized calf blood is inversely related to storage time of blood at room temperature. The r2 value of 0.77 for the least squares regression reported here suggests that 77% of the variation in plasma cortisol could be associated with differences in storage time of heparinized blood at room temperature. Our results indicate that cortisol values for plasma that is not harvested imme- diately after sample collection will be inaccurate if this effect is not taken into account. The equation provided in this report can be uti- lized to derive adjusted cortisol values in such instances. The mean initial plasma cortisol value determined one hour from the time of sample collection is consistent with previous reports for normal plasma cortisol values of calves at birth of 99 ng/ml,4 112 ng/ml,6 and 116 ng/ml.5 Hudson et al demonstrated that plasma cortisol levels in newborn calves decline to less than 1/5 of their initial level by 12 days of age.5 In the study of Olson et al, the initial concentration of cortisol in a serum sample appeared to affect whether significant degra- dation occurred at room temperature.10 Therefore, the results of this study cannot necessarily be applied to older calves or adults. Because erythrocyte sedimentation was permitted in the aliquots of heparinized blood prior to centrifugation, only a small interface existed between erythrocytes and plasma for the majority of the storage time. This 30 suggests that factors other than erythrocyte uptake are primarily responsible for the decline in cortisol values. The reports of Nachreiner9 and Olson10 in which significant degradation of cortisol occurred in plasma and serum samples, respectively, would support this hypothesis. In conclusion, we report that plasma cortisol values of precolostral calves steadily declined with increasing storage time of heparinized blood at room temperature. The regression equation derived from our data can be used to calculate adjusted plasma cortisol values when neonatal calf blood samples are handled similarly. 31 REFERENCES 1. Comline RS, Hall LW, Lavelle RB, et al: Parturition in the cow: Endocrine changes in animals with chronically implanted catheters in the foetal and maternal circulations. J Endocrinol 63: 451-472, 1974. 2. Holm LW, Parker HR, Galligan SJ: Adrenal insufficiency in post- mature Holstein calves. Am J Obstet Gynec 81: 1000-1008, 1961. 3. Osburn BI, Stabenfeldt GH, Ardans AA, et al: Perinatal immunity in calves. J Am Vet Med Assoc 164: 295-298, 1974. 4. LaMotte G, Eberhart R: Blood leukocytes, neutrophil phagocyto- sis, and plasma corticosteroids in colostrum-fed and colostrum-deprived calves. Am J Vet Res 27: 1189-1193, 1976. 5. Hudson S, Mullord M, Whittlestone WG, et al: Plasma corticoid levels in healthy and diarrheoic calves from birth to 20 days of age. Br Vet J 132: 551-556, 1976. 6. Nightengale GT, Stott GH: Adrenal response of the newborn calf to acute inanition and colostral feeding. J Dairy Sci 64(1): 236-240, 1981. 7. Senogles DR, Musc0plat CC, Kaneene JM, et al: In vitro stimula- tion of bovine peripheral blood lymphocytes: effect of short-term storage of blood prior to lymphocyte culture. Am J Vet Res 39: 341-344, 1978. 8. Philip ELI, Marotta SF: Cellular variation in the uptake and metabolism of cortisol by canine erythrocytes. Acta Endocrinol 68: 771-778, 1971. 9. Nachreiner RF: Radioimmunoassay and therapeutic monitoring, in Proceedings Am Anim Hosp Assoc 45th Annu Meet, 1978, pp 55-65 10. Olson PN, Bowen RA, Husted PW, Nett TM: Effect of storage on concentration of hydrocortisone (cortisol) in canine serum and plasma. Am J Vet Res 42: 1618-1620, 1981. 11. Draper NR, Smith H: Fitting a straight line by least squares, in Applied Regression Analysis. New York, John Wiley and Sons, Inc, 1966, pp 1-43. 12. Gill JL: Design and Analysis of Experiments in the Animal and Medical Sciences. Ames, IA, Iowa State Univ Press, 1978, pp 154-155 (Vol 1) and pp 85-88 (Vol 2). EXPERIMENT 3: Bovine Leukemia Virus: Lymphocyte Blastogenesis and Plasma Cortisol in Precolostral Calves from Antibody Positive and Negative Dams SUMMARY A study was undertaken to investigate the blastogenic response of peripheral blood lymphocytes from calves born to bovine leukemia virus (BLV) seropositive and seronegative dams. The relationship of plasma cortisol to lymphocyte blastogenesis was also investigated in the calves. Because erythrocyte (RBC) contamination was a problem when neonatal calf lymphocytes were isolated using ficoll-diatrizoate gradients, 3 methods of RBC lysis were also investigated. Precolostral calves born to dams in 2 Holstein-Friesian herds were utilized. BLV serologic status of both dam and calf at parturition was confirmed by radioimmunoprecipitation assay utilizing BLV glycoprotein antigen. All of the calves were BLV seronegative prior to colostral ingestion. Precolostral calves born to BLV seropositive dams had signi- ficantly lower lymphocyte blastogenic response to pokeweed mitogen even when the results were adjusted for variation in plasma cortisol. Calves born to positive dams also had a significantly lower lymphocyte response to phytohemagglutinin prior to the adjustment for cortisol. After this adjustment, the difference was significant when calves from both herds were included in the comparison but not for calves from a single herd. No significant difference was found in the blastogenic reaponse of lymphocytes cultured in the absence of mitogens for calves born to BLV positive and negative dams. Mitogen-stimulated lymphocyte blastogenesis 32 33 inversely correlated with plasma cortisol concentration for calves within a herd. Of 3 RBC lysis techniques investigated, 15 second hypo- tonic lysis in H20 at 22 C resulted in optimum 3H-thymidine incorporated by precolostral calf lymphocytes. INTRODUCTION Adult cattle infected with bovine leukemia virus (BLV) may have no hematologic or clinical abnormalities or they may develop persistent lymphocytosis (PL) or lymphosarcoma. Abnormalities of in vitro lympho- cyte blastogenesis have been reported for peripheral blood lymphocytes (pbl) isolated from cattle with and without PL and lymphosarcoma. When lymphocytes were cultured with phytohemagglutinin (PHA) and pokeweed mitogen (PWM), the stimulation index (counts per minute of mitogen- stimulated cultures divided by counts per minute of unstimulated control cultures) was markedly lower for cows with PL than for normal cows.1 In the same study, it was reported that unstimulated lymphocytes from cows with PL spontaneously incorporated approximately 5 times as much 3H-thymidine as did control cultures from normal animals. Weiland and Straub found that lymphocytes from cows with PL had lower responses to concanavalin A (Con A) but higher levels of spontaneous blastogenesis than cells from cattle without PL.2 They observed decreases in both counts per minute (cpm) and stimulation index (SI) and noted that cows with the highest number of lymphocytes in peripheral blood had the lowest response to Con A. Jacobs et al found no significant difference when the PEA-stimulated lymphocyte reSponse of BLV seropositive cattle without PL was compared to that of BLV seronegative cattle.3 These results were not in accordance with those of Takashima and Olson, who 34 examined the blastogenic response to PHA, PWM, and Con A of lymphocytes from 3 BLV-infected cows without PL and 8 uninfected cows.4 They found that the lymphocytes from the infected animals had a lower SI and spon- taneously incorporated higher levels of thymidine than the uninfected animals. In the same study, lymphocytes from a steer with both PL and tumorous lymph nodes had extremely low response to PHA and PWM, whether expressed as cpm or SI. It is well-recognized that mitogen stimulation of lymphocyte blasto- genesis in man inversely correlates with plasma cortisol values.5'7 Although species heterogeneity has been demonstrated in the response of lymphoid cells to corticosteroids,8 little is known about the response of bovine lymphocytes to these substances. Muscoplat et al reported that in vivo administration of dexamethasone to calves resulted in a marked, but transient, decrease in the in vitro response of lymphocytes to PHA.9 Even less is known about the interaction of glucocorticoids with lymphocytes from BLV-infected cattle, and only one study has addressed this subject. Bloom et al examined the in vitro effects of increasing doses of cortisol on blastogenesis of lymphocytes from cows with PL. Although the PHA response was relatively unaffected, spon- taneous and PWM-stimulated thymidine incorporation were reduced in the presence of cortisol.10 Although studies have investigated pbl function in adult BLV-infected cattle, the lymphocyte response of calves born to these dams has not been investigated. The objective of the present study was to determine if the blastogenic response of pbl from calves of BLV seropositive dams differed from that of calves from BLV seronegative dams. The rela- tionship of plasma cortisol to lymphocyte blastogenesis in these calves 35 was also investigated. Precolostral calves were utilized so that BLV serologic status of the calves could be ascertained prior to the absorp- tion of colostral antibodies. The influence of colostral ingestion on calf lymphocyte function11 was thereby avoided. Because we found that erythrocyte (RBC) contamination was a problem when neonatal calf lympho- cytes were isolated using ficoll-diatrizoate gradients, 3 methods of RBC lysis were also investigated. MATERIALS AND PETE-{ODS Animals The calves in this study were born to dams in 2 Holstein-Friesian herds in Michigan. Characteristics of these herds are described in detail elsewhere.12 Briefly, breeding practices were designed to increase milk production without regard to BLV status of dam or sire. The percentage of BLV seropositive lactating-age cows was 32% in Herd 1 and 94% in Herd 2 as previously determined by agar gel immunodiffusion test (Leukassay B, Pitman-Moore, Washington Crossing, N.J.). None of the dams in this study had clinical signs of lymphosarcoma. Collection of Blood All sample collection took place during October and November of 1980. Blood was drawn from both calf and dam immediately after birth, but neither sample was collected if any possibility existed that a calf may have nursed. Ten ml of blood were collected from calf and dam by venipuncture into sterile evacuated glass tubes (Vacutainer, Becton, Dickinson & Co., Rutherford, N.J.). An additional 30 ml were collected from each calf into heparinized Vacutainer tubes. The non-heparinized samples were allowed to clot and sera were drawn off and stored at -20 C 36 until serologic tests were performed. Room temperature (22 C) storage times of heparinized samples prior to lymphocyte isolation are listed in Table 1. Serologic Tests BLV serologic status of dam and calf at calving was confirmed by radioimmunoprecipitation assay utilizing BLV glycoprotein antigen (gp-RIA), conducted as previously reported by Miller.13 Sera that pre- cipitated 15% or more of the labeled antigen were regarded as positive. Sera were neither concentrated nor diluted prior to the testing proce- dure. The terms BLV positive and negative as used in this study refer to antibody status by gp-RIA unless otherwise stated. Lymphocyte Isolation Procedures Thirty ml of heparinized blood were diluted to 120 ml with sterile 0.9% NaCl solution. A ficoll-diatrizoate solution (F-D) was prepared by adding 114 ml of 75% diatrizoate (Hypaque-M, Winthrop, New York, N.Y.) to a solution consisting of 54 g ficoll (Sigma Chem., St. Louis, Mo.) dissolved in 500 m1 H20. Density of the final F-D solution was adjusted to 1.077 gm/cm3. Nine ml of diluted blood were layered over 3 ml of F-D in plastic tubes (Falcon 2057 tubes, Becton, Dickinson & Co., Oxnard, Cal.) and centrifuged at 600 x g for 12 minutes at room temperature. Cells at the opaque interface were washed once with RPMI 1640 tissue culture media (MA BiOproducts, Walkersville, Md.) that was supplemented with amphotericin B (1.5 pg/ml), gentamycin (16—ug/ml), and Hepes buffer (11.5 mM, pH adjusted to 7.1 with NaOH). This supplemented solution is henceforth denoted as RPMI. The sedimented cells were subjected to 1 of the 3 RBC lysis techniques described below. After RBC lysis the 37 resultant cell suspension was centrifuged at 800 x g for 5 minutes. The pellet was gently resuspended in RPMI and allowed to stand 2 minutes at room temperature to permit granulocyte sedimentation. The supernatant was aspirated and recentrifuged at 150 x g for 10 minutes, causing lymphocytes to sediment but platelets to remain suspended. The lympho- cyte pelllet was resuspended in RPMI + 10% pooled bovine serum. (This serum pool consisted of sera from 5 BLV seronegative beef steers that were approximately 6 months of age. The sera were inactivated at 56 C for 30 minutes and were noncytotoxic by the complement dependent microdroplet lymphocytotoxicity assay. The sera did not suppress blastogenesis of normal bovine lymphocytes cultured with PHA, PWM, and Con A.) Cell viability as assessed by eosin dye exclusion under oil regularly exceeded 90% for techniques 1 and 2 and ranged from 60% to 90% for technique 3. Concentration of the final lymphocyte preparation was adjusted 1 x 106 viable cells per ml. RBC Lysis Techniques Samples from 17 calves born to BLV negative dams in Herd 1 were uti- lized in the comparison of RBC lysis techniques. All 17 calves were BLV negative prior to colostral ingestion. Peripheral blood lymphocytes from these calves were isolated as described above. The cell prepara- tions from 5, 9, and 3 calves were subjected to the first, second, or third RBC lysis technique reSpectively. Technique 1 consisted of resuspension of the sedimented cells in distilled water for 15 seconds at 22 C. Technique 2 was resuspension for 30 seconds at 22 C, and tech- nique 3 was resuspension for 20 minutes at 37 C in a solution consisting of 0.8% NH4C1, 0.1% EDTA, 0.01% KH2P04 (pH adjusted to 7.0 with NaOH). For techniques 1 and 2 an equal volume of double isotonic strength 38 saline (1.8% NaCl, 0.7% EDTA, 0.2% KH2P04, pH 7.3) was immediately added to the cell suspension to prevent lymphocyte lysis. For technique 3, the cells in NH4Cl suspension were resedimented and washed twice in RPMI before proceeding. Isolation of Lymphocytes from Calves of BLV Positive and Negative Dams Samples from 10 calves were utilized in the comparison of lymphocyte blastogenic response of calves from BLV positive and negative dams. All 10 precolostral calves were BLV negative by gp-RIA. Five calves were from negative and 5 were from positive dams. The 5 calves born to nega- tive dams were from Herd 1, as were 2 calves born to positive dams. Three calves born to positive dams were from Herd 2. Peripheral blood lymphocytes from these calves were isolated as described in preceding paragraph, utilizing RBC lysis technique 1. Mitggen-induced Lymphocyte Blastogenesis One hundred ul of the final lymphocyte suspension were added to each of 12 wells in a 96-well round-bottomed microtiter plate, yielding 1 x 105 cells per well. Three wells were controls, 3 wells each contained 11.25 pg PHA (Welcome, Beckenham, England), 3 contained 0.05 ug PWM (Gibco, Grand Island, N.Y.), and 3 contained 0.01 pg Con A (Sigma Chem., St. Louis, Mo.). Dose response curves (Figure 3.1) were used to deter- mine the concentration of mitogen which gave the Optimum stimulation of neonatal calf peripheral blood lymphocytes in this culture system. The plates were covered with sterile plastic sealers (Dynatech, Alexandria, Va.) and incubated at 37 C in a 5% C02 incubator. After 72 hours, 1 uCi of 3H-thymidine was added to each well. After 16-20 additional hours of incubation, cultures were terminated by cooling to 4 C. Cells were 39 300 .. -..: I C P M x 1000 200 \‘A :00 4 l/ o . E..— 0 I I o r I I I 0.: 0.7 1.: 2.0 is 11.: 22.: «1.0 90.0 PHA (pg per Culture) W 250 _L4 200 C P Mx 1000 ‘1" cummmwmmoauanw PWM (pg per Culture) .. /\ -. \ \ 3 < \ / ° - - - . I t l 7 0 can new M» 0m :00 Tax CON A (pg per' Culture) 200 CPMXIOOO Figure 3.1 - Dose response curves for mitogens. Counts per minute were determined from the mean of triplicate cultures of a newborn calf's lymphocytes. 40 harvested using a semiautomatic harvestor (Ilacon, England). This pro- cess included aspirating the lymphocytes onto glass fiber filter discs and washing with 0.9% NaCl, 5.0% trichloroacetic acid, and anhydrous methyl alchohol. The discs were dried and then transferred to 6 ml vials with 100 ul soluene (Soluene-100, Packard Instrument Co., Downer's Grove, Ill.) and 5 ml scintillation fluid. The scintillation fluid contained 0.04 g dimethylpop (Research Products International Corp., Elk Grove Village, 111.), 22.74 g diphenyloxazole (PPO, New England Nuclear, Boston, Mass.), per 1 gallon toluene. The vials were counted for 10 minutes in a scintillation spectrometer (Beckman LS-230, Beckman Instruments, Palo Alto, Cal.). Cortisol Assay Procedure Plasma cortisol values were determined for the precolostral calves from BLV positive and negative dams. Two ml of heparinized blood were centrifuged immediately prior to lymphocyte isolation. The plasma was stored at -20° until cortisol was determined by radioimmunoassay as described by Jacobsen et al.14 Because plasma cortisol declines in a linear fashion when precolostral calf blood is stored at room tem- perature,14 storage times prior to centrifugation were used to calculate adjusted cortisol values. Statistical Methods Mitogen stimulation of lymphocyte blastogenesis as reflected by lymphocyte incorporation of 3H-thymidine was expressed and analyzed in 3 data forms: mean cpm of triplicate cultures, mean cpm of stimulated cultures minus mean cpm of control cultures (A cpm), and SI. Because scintillation counts often do not meet normality requirements of 41 parametric statistics, log10 transformation was utilized for all blasa- togenesis data prior to analysis. Unless otherwise stated, data were analyzed by the computer programs of Nie et al.15 Data for the comparison of RBC lysis techniques was analyzed by one way analysis of variance (ANOVA). Differences identified by the ANOVA were further investigated by the least significant difference test. For comparison of Con A-stimulated cultures with controls, the Student's T test for paired observations16 was performed. Plasma cortisol values for positive and negative dams were analyzed by one-way ANOVA, and a least squares linear regressionr7 of mitogen- stimulated blastogenesis on plasma cortisol was performed. The lympho- cyte blastogenic response of calves from BLV positive and negative dams was analyzed by one-way ANOVA and by analysis of covariance with plasma cortisol as the covariate. For all of the above tests, lack of signifi- cant difference was concluded for P >0.05. RESULTS RBC Lysis Techniques Precolostral calf lymphocytes isolated using RBC lysis technique 1 had significantly greater (P (0.001) blastogenic response to PHA and PWM than lymphocytes isolated using techniques 2 or 3 (Table 3.1). The latter 2 techniques did not differ significantly from one another. Lymphocyte response to Con A did not differ among the 3 RBC lysis tech- niques. For the majority of calves, the reaponse to Con A was low. Blastogenic response of Con A stimulated cultures was not significantly different from that of controls for 12 of the 17 calves. For control 42 cultures without mitogen, technique 1 differed from technique 2 (P (0.05), but both did not differ from technique 3. Lymphocyte Blastogenesis and Plasma Cortisol of Calves from BLV Negative and Positive Dams Lymphocyte blastogenesis and plasma cortisol values for calves of BLV negative and BLV positive dams are reported in Table 3.2. Plasma cortisol differed significantly (P (0.01) between calves of positive and negative dams within Herd 1, but the difference was not significant when calves from both herds were compared. When PHA or PWM blastogenesis results were regressed on cortisol values for calves from Herd 1, the regression coefficients were significantly different from 0 (P (0.05). 3H-thymidine incorporation by lymphocytes was lowest in calves with high cortisol values, and vice versa. Coefficients of determination (r2) for the PHA regressions were 0.83, 0.76, and 0.95 for cpm, A Cpm, and SI, respectively. For PWM, r2 values were 0.97, 0.97, and 0.86 for the same data forms. The regressions were not significant for Con A using calves from Herd 1.'~ When similar regressions were performed using calves from both herds, the regression coefficient was not significantly different from 0 for any mitogen. When analyzed by one-way ANOVA, the lymphocyte response of calves born to positive dams was significantly less than that of calves born to negative dams for mitogens PHA (P (0.005) and PWM (P (0.025). The dif- ference was significant at these or higher probability levels whether calves from both herds (n=10) or calves from Herd 1 alone (n=7) were compared and whether data was expressed as cpm, A cpm, or SI. The Con A stimulated response of lymphocytes from calves of BLV positive and nega- tive dams did not differ significantly. Table 3.1 - Mitogen-induced blastogenesis of precolostral calf lymphocytes isolated using 3 RBC lysis techniques TECHNIQUE 1* 2* 3* DATA FORM (n = 5) (n = 9) (n = 3) 2,89017153 924115110 1353:4115“b 146,765:45,259a 351511999b 4,359:3,27sb cpm 164,397:17,0036 1, 069128813 2,7571: ,sszb 20,693:17,1353 2,066:1,382a 6,665:3,131a 143 ,885:44,800a 2,591isssb 3.10513 ,204b Acpu 161,517117,171a 14512391) 1504113633 17,813:17,0593 1,14211,349*El 5,41232,731a 51.9_+_10.9a 3310.7b 3.512.:b 91 95.614343 1.1:0.2b 2.410.910 6.81-5.33 1.931.231 4.311.761 a'bValues reported are iiSEM. Letters differing within a row indicate significant differences in techniques (P (0.001 for PHA and PWM; P (0.05 for Control). *Technique 1 = 15 seconds, H20, 22 C Technique 2 = 30 seconds, H20, 22 C Technique 3 = 20 minutes, NH4Cl, 37 C 44 m? m: m... m... e.c~ S e... 8 Rm". 8. ed om. 4H 5 ea MW e4. em .08 so 9: 8 ...: 28.: m.e+ m.~H men e.~ Rm 6.. mm .31 mucl 5 mm... a... we me To Em TN me m; as we Tm...“ SSH céfi me HR. ER as ..N QR S 6.3. ..8 TN TR «.9 9— when 38 ..mm TE QR «9: n8. ....Hom “ 9H8 833m Newman <5 :3 SE cocEsBmc acct 3.53 8003 com c3882 e35 coflflomfi 2H 8 HOE 833093 soon on mg... was + PERM NEH new See can can e8 EN. on} «no.8 5... m8..- mm? «me... SE... 8... 2A.. es. Ema ct... Res.“ .25..“ e864.“ ~85 ERG. coma: m8..- «3.5 No.8. m: E62 @968 SR $.ch once: on... ERR. ERR. Scam Swim mare <50 55 SE 28¢ .meao 034.303qu use «53% mum on Eon 9233 1583093 ecu“ EH 90 3850825 «8 H Than Rm M 3m H 48.. SEA Rm; Fm; Em not... do as: N e. as...” 28.8 min N8; m m .8 m8; c2. ems m m Sim one; 8... Sm . a. one exam Ems 8c a o RFSH 822.,” can...“ 9% 898 STE. mead: 8e.~ 83.. 2&8. mmmaem Rea . m 2.3 Scam. e358 Sin . 4 ~26 Redo. comes SAN . m on... 9TB. EAR Rm P N 3.8 81.8 9.4.8 2....” F F 2.8 as am 3380 i. a coma odd Eu sum Secs l>< at 3m ”Erna 45 With the exception of calf #1, the response to Con A was low. For 6 of the 10 calves, the response of Con A stimulated cultures did not differ significantly from that of controls. Spontaneous 3H-thymidine incor- poration in the absence of mitogen did not differ significantly in calves born to BLV positive and negative dams. When blastogenesis results were adjusted for the effect of cortisol by the use of the analysis of covariance, significant differences were observed in the response to PHA (P (0.001) and PWM (P (0.005) when calves from both herds were included in the comparison. However, for calves from calves from Herd 1, a significant difference was observed only in the response to PWM (P (0.05). DISCUSSION While adult bovine lymphocytes may be isolated using ficoll-diatrizoate gradients with minimal RBC contamination, the similar density of RBC's and lymphocytes of neonatal calves prevents complete separation of the two cell populations. Ammonium chloride RBC lysis is a technique routinely used in human lymphocyte isolation procedures.18I19 Its use has been reported for .isolation of calf lymphocytes,zor21 but the effect of NH4C1 on bovine lymphocyte function has not been previously investigated. Resuspension for 30 seconds in H20 reportedly caused death of a signifi- cant prOportion of human lymphocytes and resulted in diminished response to PHA.22 However, sheep lymphocytes subjected to 7 seconds in H20 remained viable and responded well to PHA23 and canine lymphocytes sub- jected to 30 seconds in H20 remained 99% viable and responded well to PHA and PWM.24 Of the 3 RBC lysis techniques investigated in this study, technique 1 was clearly optimum. Resuspension for 15 seconds in 46 H20 at 22 C (technique 1) resulted in greater PHA-and PWM-stimulated blastogenesis than either 30 seconds in H20 at 22 C (technique 2) or 20 minutes in NH4Cl at 37 C (technique 3). Therefore, for comparison for the blastogenic response of BLV positive and negative dams, pbl were isolated using RBC lysis technique 1. It has been reported that approximately 5% of the calves born to BLV seropositive dams are BLV seropositive prior to colostral ingestion.12 In the present study all of the calves were BLV seronegative at birth. In studies which examined precolostral calves born to BLV-infected dams, a close correlation was demonstrated between the presence or absence of BLV antibody in calf serum and the presence or absence of virus.25‘27 In these reports none of the antibody negative precolostral calves were virus positive. Although it is theoretically possible that a calf could be virus exposed or infected in utero and not produce antibody to the virus, this apparently is not a frequent occurrence. It is likely, therefore, that very few, if any, of the BLV seronegative calves in this study were virus infected. The lymphocyte blastogenesis data for calves of BLV positive and negative dams was analyzed both with and without adjustment for the effect of cortisol on lymphocyte blastogenesis. When analyzed by one- way ANOVA, the lymphocyte response to PHA and PWM was markedly less for calves of positive dams than for calves of negative dams. The dif- ference was significant whether calves from one or both herds were com- pared. The high r2 values for the regressions of blastogenesis data on plasma cortisol suggested a strong influence of cortisol on lymphocyte response to mitogens for calves from Herd 1. The same influence could not be demonstrated when calves from both herds were included in the 47 regression analysis, possibly due to variation in cortisol values bet- ween the herds. Analysis of covariance permitted comparison of blasto- genesis data for calves of positive and negative dams after adjusting for the effect of cortisol. Lymphocyte response to PWM remained lower for calves of BLV positive dams than for calves of BLV negative dams whether calves from one or both herds were compared. The difference in lymphocyte response to PHA was only significant when calves from both herds were compared. Thus, while calves from positive dams in Herd 1 had a significantly lower response to PHA prior to the adjustment for cortisol, this was no longer the case when the cortisol effect was fac- tored out. The PHA results could be explained by several alternative hypothe- ses. It is possible that, after adjustment for cortisol, a real dif- ference in lymphocyte response to PHA was not detected for calves in Herd 1 due to the small sample number. Because the analysis of covariance permitted fewer error degrees of freedom, the chance of finding signifiance was reduced. A second possibility is that the dif- ference in response to PHA was entirely due to variation in plasma cor- tisol. It could be argued that variation in lymphocyte response between the herds accounted for the significant difference in reSponse to PHA when calves from both herds were compared by analysis of covariance. Because there were significant differences in plasma cortisol between calves of BLV positive and negative dams, a third possibility exists. If BLV status of the dam influenced cortisol values in the calf, fac- toring out the effect of cortisol would also factor out some of the dif- ference due to BLV status of the dam. 48 Although it is conceivable that BLV infection of adult cows could result in high endogenous cortisol levels in their calves, this study provides no clues as to the possible mechanism of such an occurrence. If BLV status of the dam influences blastogenesis of calf lymphocytes independently of cortisol, decreased mitogen responsiveness in calves of BLV positive dams might be due to a funtional abnormality of the lympho- cytes or to an inhibitory substance acting upon the lymphocytes. It is possible that BLV positive dams could pass on to their calves genetic information coding for a functional abnormality of lymphocytes. Another possibility is that an inhibitory factor is present in the serum of calves born to BLV positive dams. If this were the case, such a factor might have been produced within the calf or it may have crossed the pla- centa from the dam's serum. It is also conceivable that mitogens could stimulate certain lymphocyte subpopulations to produce inhibitory fac- tors in the in vitro culture system. If these suprpulations were pre- sent in greater numbers in calves of positive dams, this could account for decreased blastogenesis in the presence of mitogens. Significant differences in lymphocyte reSponse to Con A were not found between calves of positive and negative dams, nor were they found between different RBC lysis techniques. Also, the regression of Con A blastogenesis data on cortisol was not significant. For the majority of calves in this study, 3H-thymidine incorporation of lymphocytes cultured with Con A did not differ significantly from that of lymphocytes cultured without Con A. The Con A dosage used was one hundredth to one thousandth the dosage reported by others for stimulation of blastogene- sis in calf lymphocytes.21l28 It therefore appears likely that the low responses we observed with this mitogen were due to insufficient dosage. 49 Several investigators have reported elevated levels of spontaneous 3H-thymidine incorporation by lymphocytes from BLV positive cattle.1vzr4r29 In the present study, spontaneous lymphocyte blastoge- nesis did not differ between calves of BLV positive dams and calves of BLV negative dams, even when the results were adjusted for the effect of cortisol. Although mitogen-stimulated blastogenesis was markedly different for lymphocytes from calves of BLV positive and negative dams in this study, the results must be considered preliminary due to the small number of calves examined. Before definitive conclusions can be drawn, the study should be repeated with larger numbers of calves in both groups. Many questions remain unanswered, leaving several possible areas for future investigation. To investigate the possibility that lymphocyte inhibi- tory factors are present in the sera of calves from positive dams, it would be useful to culture lymphocytes from calves of BLV negative dams in the presence of this sera. Studies involving surface markers for B and T lymphocytes and separation of these cells and their subsets would help answer the question of whether decreased lymphocyte responsiveness was due to mitogen stimulation of suppressor lymphocyte subp0pulations. To shed light on the hypothesis that calves of BLV positive dams have a hereditary tendency for decreased lymphocyte response to mitogens, a more thorough investigation of lymphocyte mitogen responsiveness in the dams themselves would be useful. Although abnormalities of lymphocyte blastogenesis have been observed in adult BLV-infected cattle, reports of decreased mitogen responsiveness are conflicting. Also of further interest would be the investigation of lymphocyte blastogenesis in 50 calves that are themselves BLV seropositive prior to colostral ingestion. Although plasma cortisol values are seldom reported in bovine blastogenesis studies, the results of this investigation demonstrate a significant influence of endogenous cortisol on the response of calf lymphocytes to mitogens. By the use of analysis of covariance, the effect of cortisol can be held constant when examining for differences in treatment group means. Our results suggest that bovine blastogenesis data might be more meaningful if plasma cortisol values are reported and analyzed this way in future scientific reports. In conclusion, the present data indicate that techniques of erythro- cyte lysis used in isolation of peripheral blood lymphocytes can signi- ficantly influence lymphocyte blastogenic response. Of the 3 RBC lysis techniques investigated, 15 second hypotonic lysis in H20 at 22°C resulted in Optimum 3H-thymidine incorporation by calf lymphocytes. It was also demonstrated that mitogen-stimulated lymphocyte blastogenesis inversely correlated with plasma cortisol concentration for precolostral calves within a herd. Precolostral calves born to BLV seropositive dams had significantly lower lymphocyte blastogenic response to PWM, even when results were adjusted for variation in cortisol. Calves born to positive dams also had a significantly lower lymphocyte response to PHA prior to adjustment for cortisol. After this adjustment, the difference was significant for calves of 2 herds, but not for calves of a single herd. 51 REFERENCES 1. Musc0plat CC, Alhaji I, Johnson DW, Pomeroy KA, Olson JM, Larson VL, Stevens JB, Sorensen DK: Characteristics of lymphocyte responses to phytomitogens: comparison of responses of lymphocytes from normal and lymphocytotic cows. Am J Vet Res 35: 1053-1055, 1974. 2. Weiland F, Straub OC: Differences in the in vitro response of lymphocytes from leukotic and normal cattle to concanavalin A. Res Vet Sci 20: 340-341, 1976. 3. Jacobs R, Valli V, Wilkie B: Inhibition of lymphocyte blasto- genesis by sera from cows with lymphoma. Am J Vet Res 41: 372-376, 1980. 4. Takashima I, Olson C: Effect of mitogens and anti-bovine leukosis virus serums on DNA systhesis of lymphocytes from cattle. Europ J Cancer 16: 639-645, 1980. 5. Zeman GO, Cohen G, Budrys M, Williams GC, Javor H: The effect of plasma cortisol levels on the lymphocyte transformation test. J Allergy Clin Immunol 49(1): 10-15, 1972. 6. Rissling M, Speck B, Goselink H: Effects of hydrocortisone on lymphocytes stimulated by phytohaemagglutinin and pokeweed mitogen. Vox Sang 23: 344-349, 1972. 7. Heilman DH, Gambrill MR, Leichner JP: The effect of hydrocor- tisone on the incorporation of tritiated thymidine by human blood lymphocytes cultured with phytohaemagglutinin and pokeweed mitogen. Clin Exp Immuno 15: 203-212, 1973. 8. 'Claman HN: Corticosteroids and lymphoid cells. N Engl J Med 287: 388-397, 1972. 9. MuscOplat CC, Shope RE, Chen AW, Johnson DW: Effects of cor- ticosteroids on responses with phytOperipheral blood lymphocytes cultured with phytohemagglutinin. Am J Vet Res 36: 1243-1244, 1975. 10. Bloom JC, Kenyon SJ, Gabuzda TG: Glucocorticoid effects on peripheral blood lymphocytes in cows infected with bovine leukemia virus. Blood 53(5): 899-912, 1979. 11. Clover C, Zarkower A: Immunologic responses in colostrum-fed and colostrum-deprived calves. Am J Vet Res 41: 1002-1007, 1980. 12. Jacobsen KL, Bull RW, Miller JM, Herdt TH, Raneene JB: Transmission of bovine leukemia virus: prevalence of antibodies in pre- colostral calves. Prev Vet Med, in press. 13. Miller JM, Schmerr MJF, Van Der Maaten MJ: Comparison of four serologic tests for the detection of antibodies to bovine leukemia virus. Am J Vet Res 42: 5-8, 1981. 52 14. Jacobsen KL, Nachreiner RF, Kaneene JB, Bull RW: Effect of storage at room temperature on the concentration of cortisol (hydrocortisone) in precolostral bovine blood. Submitted for publica- tion to Am J Vet Res. 15. Nie NH, Hull CH, Jenkins JG, Steinbrenner K, Bent DH: Statistical Package for the Social Sciences. McGraw-Hill Book Co, New York, pp. 398-433, 1975. 16. Steel RGD, Torrie JH: Principles and Procedures of Statistics. McGraw-Hill Book Co, New York, pp. 102-104, 1980. 17. Draper NR, Smith H: Applied Regression Analysis. John Wiley and Sons, Inc, New York, pp 1-43, 1966. 18. Gutterman JU, Mavligit GM, Hunter CY, Hersh EM: Lymphocyte transformation against human tumor antigens. In: BR Bloom and JR David (Editors), In Vitro Methods in Cell-Mediated and Tumor Immunity. Academic Press, New York, pp. 587-596, 1976. 19. Denman AM: Methods of separating human blood lymphoid cell populations. J Immunol Meth 2: 331-351, 1973. 20. Woodard L, Renshaw H, Burger D: Cell-mediated immunity in neonatal calves: delayed-type hypersensitivity and lymphocyte blastoge- nesis following immunization with a mycobacterial immunopotentiating glycolipid and tuberculoproteins of Mycobacterium bovis. Am J Vet Res 39: 579-584, 1978. 21. Kelley KW, Osborne CA, Evermann JF, Parish SM, Hinrich DJ: Whole blood leukocyte vs. separated mononuclear cell blastogenesis in calves: time-dependent changes after shipping. Can J Com Med 45: 249-258, 1981. 22. Thomson AER, Bull JM, Robinson MA: A procedure for separating viable lymphocytes from human blood and some studies on their suscep- tibility to hypotonic shocks. Br J Haemat 12: 433-446, 1966. 23. Dain AR, Hall JG: A method for the isolation of white cells from the blood of sheep by differential lysis with hypotonic saline solution. Vox Sang 13: 281-284, 1967. 24. Whitacre CC, Lang RW: A technique for separation of canine lymphocytes and their use in the lymphocytotoxic, blastogenic, and rosette assays. Transfusion 15(4): 346-350, 1975. 25. Piper C, Ferrer J, Abt D, Marshak R: Postnatal and prenatal transmission of the bovine leukemia virus under natural conditions. J Natl Cancer Inst 62: 165-168, 1979. 26. Ferrer JF, Piper CE, Abt DA, Marshak RR, Bhatt DM: Natural mode of transmission of the bovine C type leukemia virus (BLV). Bibl Haemat 43: 235-237, 1976. 53 27. Van Der Maaten MJ, Miller JM, Schmerr MJF: In utero transmission of bovine leukemia virus. Am J Vet Res 42: 1052-1054, 1981. 28. Renshaw HW, Eckblad WP, Everson DO, Tassinari PD, Amos D: Ontogeny of immunocompetence in cattle: evaluation of phytomitogen- induced in vitro bovine fetal lymphocyte blastogenesis, using a whole blood culture technique. Am J Vet Res 38: 1141-1150, 1977. 29. Thorn RM, Gupta P, Kenyon SJ, Ferrer JF: Evidence that the spontaneous blastogenesis of lymphocytes from bovine leukemia virus-infected cattle is viral antigen specific. Infection and Immunity 34: 84-89, 1981. EXPERIMENT 4 : Comparison of Commercial Agar Gel Immunodiffusion and RadioimmunOprecipitation Assay for Antibodies to Bovine Leukemia Virus SUMMARY In a double-blind study, the commercial agar gel immunodiffusion test (AGID) was compared to a radioimmunoprecipitation assay with gly- caprotein antigen (gp RIA) for detection of antibodies to bovine leuke- mia virus (BLV). Of 240 sera tested, 115 were from adult cows and 125 were from precolostral calves. The majority of the adult animals were tested within one week of parturition. Sera from 74 animals were positive and sera from 166 animals were negative by gp RIA. The sensitivity of the AGID as compared to gp RIA was 85.1% when the test was read at 48 hours and 94.6% when read at 72 hours. Specificity increased from 92.2% at 48 hours to 96.4% at 72 hours. Reading the AGID again at 72 hours also permitted clarification of the majority of reactions that were unclear at 48 hours due to a haze around the test serum well. Of 3 RIA positive precolostral calf sera, 2 were AGID negative and one resulted in an unclear reaction by AGID at 48 hours. Of 5 RIA posi- tive sera that were AGID negative at 48 hours, 2 were precolostral calves and 3 were cows tested at parturition. Of 166 RIA negative reac- tions, none were falsely positive by AGID at either 48 or 72 hours. 54 55 INTRODUCTION Lymphoid neoplasia in cattle has been recognized since the late 1800's.1 However, the etiologic agent, bovine leukemia virus (BLV) was only identified in 19692v3, and serologic detection of BLV has only been possible in the last decade.3I4 Since BLV infections are persistent, the presence or absence of serum antibodies correlates closely with the presence or absence of viral infection.5r6 In 1972, an agar gel immunodiffusion test (AGID) was introduced which detected antibodies to the major internal virion antigen, p24.3 A second AGID test, develOped in 1976, detected antibodies to the gly- cOprotein antigen (gp) of the virion envelope.9 This test proved to be more sensitive than other serologic methods including complement fixa- tion and p24 AGID.9"12 Although radioimmunoprecipitation assay (RIA) performed with gp antigen has since emerged as the most sensitive sero- logic test,13'14 the gp AGID has several practical advantages. Unlike RIA, it is simple to perform and does not require expensive laboratory equipment. The gp AGID was recommended for field use by the Commission of European Communities15 and has been successfully used in eradication programs for the control of BLV infection.15r17 An AGID test kit for detection of BLV antibody is available from a single commercial supplier.a The kit antigen contains both gp and p24 antigens, but since the control serum precipitates only gp, it is func- tionally a gp AGID test. Although the kit has been licensed by the United States Department of Agriculture and is widely used by diagnostic laboratories in this country, both the sensitivity and specificity of aLeukassay B, Pitman-Moore, Inc, Washington Crossing, NJ. 56 the test have recently been questi.oned.18"20 Therefore, the present study was undertaken to compare the commercial gp AGID with the gp RIA. MATERIALS AND METHODS Animals - The 240 Holstein Friesian cattle used in this study were part of various BLV survey and transmission experiments at our labora- tory. One hundred and fifteen were adult cows of calving age and 125 were precolostral calves. All BLV infections occurred naturally and none of the animals had clinical signs of lymphosarcoma. These animals were from 2 Michigan dairies, the characteristics of which are described in detail elsewhere.21 Sample Collection - The majority of the cows in this study were sampled within one week of parturition. Blood was collected by veni- puncture into sterile evaculated glass tubes.b Sera were drawn off and stored at -20C until serologic tests were performed. Sera were neither concentrated nor diluted prior to testing. Radioimmunoprecipitation Assay - The RIA for antibodies to BLV gp was conducted as reported by Miller.23 Sera that precipitated 15% or more of the labled antigen were regarded as positive. Agar Gel Immunodiffusion - The AGID test for antibodies to BLV gp was conducted following the instructions of the commercial test kit,a with the exception that the test was read at both 48 and 72 hours. Reactions of the gp AGID were recorded as positive, weak positive, or negative (Figure 4.1). When a haze around the test serum well precluded identification of a possible weak positive reaction, the reaction was recorded as questionable (Figure 4.2). When possible, the AGID was bVacutainer tubes, Becton, Dickinson & Co, Rutherford, NJ. 57 .comaucm cdououmooaam >qm cu moaoonducc new QHum Hmaowoesoo mo sodumumumumucH I p.v musmam 53.3% 33:00 538 «no... 530m 30k 0222:. 02:08 3003 Eaton Esaom 32:00 .2230 E30.» «no... 023qu 58 omHH03 ghmm ONT“ “VGA—OHM 0N0: £0fl3 QHUfl HMHUHOEEOO NO GOfiUTUGh—QHQUCH I No? Gugfih Eseom .2230 humuuflfi» .. one: 5.3 :53 «no 2530a w cincomzoowo ’ Eseom Eseom 32:00 .2500 one: 5.3 532.... So... 639.2325 59 repeated on these samples. Only precipitation lines that were con- tinuous with the control line were recorded. The sensitivity and specificity of the commercial gp AGID were calculated according to the formulae of Wilson and Jungnerzz: Sensitivity a # of infected animals with (+) test # of infected animals in sample p0pulation Specificity = # of uninfected animals with (-) test # of uninfected animals in sample pOpulation For this study, these values were calculated as follows: Sensitivity of = # of gp RIA (+) samples that were gp AGID (+) gp AGID 1* of 9p RIA (+) samples Specificity of a # Of 9P RIA (-) samples that were gp AGID (-) 9p AGID # 0f 9P RIA (-) samples Weak positive AGID reactions were included in the total gp AGID positive reactions. Experimental Design - This study was designed to permit a double- blind comparison of the 2 serologic tests. Technicians performing gp AGID had no knowledge of gp RIA results, and vice versa. RESULTS The results of the comparison of commercial gp AGID with gp RIA are shown in Table 4.1. Sera from 74 animals were positive by gp RIA. By the 48 hour AGID reading, 63 were positive or weak positive, 6 were questionable, and 5 were negative. When the AGID was read at 72 hours, 70 were positive or weak positive, 2 were questionable, and 2 were nega- tive. Thus, the sensitivity of the ACID as compared to gp RIA was 85.1% 60 TABLE 4.1 - Comparison of Commercial gp AGID with gp RIA gp AGID No. of No. of 48hr 72hr Cows Calves Total gp RIA POSITIVE + + 49 0 49 W + 9 0 9 W W 5 0 5 7 W 5 1 6 - W 0 1 1 - ? 2 0 2 - - _1 .1 .3 71 3 74 48hr Sensitivity a 63/74 = 85.1% 72hr Sensitivity a 70/74 t 94.6% gp RIA NEGATIVE - - 41 109 150 - 7 1 2 3 ? - 2 8 10 ? 7 .9 ...3. ..3. 44 122 166 48hr Specificity = 153/166 8 92.2% 72hr Specificity = 160/166 - 96.4% W a weak positive reaction ? = questionable reaction due to haze around test serum well 61 when read at 48 hours and 94.6% when read at 72 hours. Sera from 166 animals were negative by gp RIA. Of these, 153 were negative and 13 were questionable by 48 hours AGID. In contrast, 160 were negative and only 6 were questionable by 72 hour AGID. The Specificity of the AGID was 92.2% when read at 48 hours and 96.4% when read at 72 hours. Three of the gp RIA positive sera were from precolostral calves. Detailed results are shown for calves 72 and 74 in Table 4.2 and for calf 73 in Table 4.3. None of the 3 reactions were positive or weak positive at the 48 hour AGID reading, but 2 were weak positive at 72 hours. Detailed results of the 5 false negative reactions by the 48 hour AGID reading are presented in Table 4.2. The 3 cows in this group (cows 8, 25, and 43) were tested at parturition. One of these sera yielded a weak positive reaction when retested by AGID. There were no false posi- tive AGID reactions at either 48 or 72 hours. Detailed results of the reactions with a haze around the AGID test serum well (questionable reactions) at either 48 or 72 hours are shown in Table 4.3. Of 19 sera that were questionable at 48 hours, 10 became clearly negative and 6 became clearly weak positive at 72 hours. These readings were in agreement with gp RIA results. Three of the sera that were questionable at 48 hours remained so at 72 hours. All 3 of these sera reacted negatively by gp RIA. Five sera were read as negative at 48 hours and questionable at 72 hours by AGID. Of these, 3 were gp RIA positive and 2 were gp RIA negative. DISCUSSION In the present comparison of 240 sera, the commercial AGID compared favorably with gp RIA. Reading the agar gel plates again at 72 hours TABLE 4.2 - Reactions Falsely Negative at 48 Hour AGID Reading Repeat Animal Cow or gp AGID g2 AGID No. Calf gp RIA 48hr 72hr 48hr 72hr 72 calf + - W 8 cow + - 7 - - 25 cow + - 7 W W 43 cow + - - - 7 74 calf + - - W a weak positive reaction 7 = questionable reaction due to haze around test serum well 63 TABLE 4.3 - Reactions with Haze around AGID Test Serum Well Repeat Animal Cow or 32 AGID gp AGID No. Calf gp RIA 48hr 72hr 48hr 72hr 19 cow - 7 - 20 calf - 7 - 63 calf - 7 - - - 111 calf - 7 - 126 calf - 7 - 132 calf - 7 - - - 148 calf - 7 - 150 calf - 7 - 157 calf - 7 - 166 calf - 7 - - - = 10 6O calf - 7 7 - - 61 calf - 7 7 - - 71 calf - 7 7 - - = 3 2 cow + 7 W W W 9 cow + 7 W + + 11 cow + 7 W - W 41 cow + 7 W 66 cow + 7 W W + 73 calf + 7 W = 6 23 cow - - 7 66 calf - - 7 - - 97 calf - - 7 8 cow + - 7 - - 25 cow + - 7 W W = 5 W a weak positive reaction 7 a questionable reaction due to haze around test serum well 64 increased both the sensitivity and the specificity of the test. Of 3 RIA positive precolostral calf sera, none were AGID positive at 48 hours and 2 were only weak positive at 72 hours. This would suggest that the commercial AGID may not be the serologic test of choice for detection of BLV antibodies in precolostral calves. It is possible that calves infected in utero have lower antibody titers to BLV gp than adult animals, resulting in delayed and weaker antigen-antibody precipitation lines on AGID. Of the 5 RIA positive sera that were AGID negative at 48 hours, 2 were precolostral calves, and 3 were cows that were tested at par- turition. It has been reported that BLV AGID reactions are sometimes falsely negative in periparturient cows,23'24 presumably due to the transfer of serum BLV antibodies to colostrum. One investigator found that BLV antibody titers in cattle were decreased from 4 weeks prior to parturition to as much as 4 weeks afterwards.23 Since the majority of adult animals in this study were sampled within one week of parturition, one might expect even higher sensitivity if the test were not used on periparturient dams. One disadvantage of the commercial AGID was that 19 of 240 sera (7.9%) could not be interpreted at 48 hours due to a haze around the test serum well. In the instructions for the commercial test kit, it is suggested that the haze may be due to lipids in the serum and it is recommended that these reactions be interpreted as negative.3 In this study, 16 of the 19 reactions could be correctly interpreted by rereading the test at 72 hours. Furthermore, 6 sera yielded weak posi- tive reactions at this reading, all of which reacted positively by gp 65 RIA. Therefore, rereading the agar plates at 72 hours would appear to be desirable. In previous publications, it has been suggested that impurities of the commercial AGID test kit antigen may result in numerous false posi- tive reactions.18‘20 Since no false positive reactions occurred in this study, it would appear that if such reactions occur, they are uncommon to rare. In summary, the commercial AGID test kit for detection of antibodies to BLV gp compared favorably with gp RIA. Both the sensitivity and spe- cificity of the test were increased by reading the test at both 48 and 72 hours. This also permitted clarification of the majority of the reactions that were unclear at 48 hours due to a haze around the test serum well. The AGID read at 48 hours did not detect antibodies to BLV in 3 precolostral calves, suggesting that this may not be the serologic test of choice for detection of in utero exposure to BLV. That no false positives occurred in this study would lend support to the conclusion that the commercial AGID is a suitable test for field use in adult cattle. 66 REFERENCES 1. Siedamgrotzky O: Ueber Leukamie bei den Haustieren. Pflugs Vortrage fur Tierarzte 10: 393-401, 1878 (cited from Bendixen HJ: Bovine enzootic leukosis. Adv Vet Sci Comp Med 10: 129-204, 1965). 2. Miller JM, Miller LD, Olson C, Gillette KG: Virus-like par- ticles in phytohemagglutinin-stimulated lymphocyte cultures with reference to bovine lymphosarcoma. J Natl Cancer Inst 43(6): 1297-1305, 1969. 3. Miller JM, Olson C: Precipitiating antibody to an internal antigen of the C-type virus associated with bovine lymphosarcoma. J Natl Cancer Inst 49: 1459-1462, 1972. 4. Ferrer JF, Avila L, Stock ND: Serological detection of type C viruses found in bovine cultures. Cancer Res 32: 1864-1870, 1972. 5. Ferrer JF, Abt DA, Bhatt DA, Marshak RR: Studies on the rela- tionship between infection with bovine C-type virus, leukemia, and per- sistent lymphocytosis in cattle. Cancer Res 34: 893-900, 1974. 6. Honma T, Onuma M, Mikami T, Izawa H: Bovine leukemia virus infection in Japan: Antibody and virus detection in cattle. Jpn J vet Sci 42: 5-8, 1980. 7. Ferrer JF, Piper CE, Abt DA, Marshak RR, Bhatt DM: Natural mode of transmission of the bovine C type leukemia virus (BLV). Bibl Haemat 43: 235-237, 1976. . 8. Piper C, Ferrer J, Abt D, Marshak R: Postnatal and prenatal transmission of the bovine leukemia virus under natural conditions. J Natl Cancer Inst 62: 165-168, 1979. 9. Miller JM, Van Der Maaten MJ: Serologic detection of bovine leukemia virus infection. Vet Microbiol 1: 195-202, 1976. 10. Miller JM, Van Der Maaten MJ: Use of glycoprotein antigen in the immunodiffusion test for bovine leukemia virus antibodies. Eur J Cancer 13: 1369-1375, 1977. 11. Mammerickx M, Burny A, Dekegel D, Ghysdael J, Kettmann R, Portetelle D: Comparative study of four diagnostic methods of enzootic bovine leukosis. Zbl Vet Med 8 24: 733-740, 1977. 12. Straub 0C: Diagnosis of enzootic bovine leukosis: a com- parison of haematological and immunodiffusion tests. Res Vet Sci 25: 13-15, 1978. 13. Miller JM, Schmerr MJF, Van Der Maaten MJ: Comparison of four serologic tests for the detection of antibodies to bovine leukemia virus. Am J Vet Res 42(1): 5-8, 1981. 67 14. Gupta P, Ferrer JF: Comparison of various serological and direct methods for the diagnosis of BLV infection in cattle. Int J Cancer 28: 179-184, 1981. 15. Ressang AA (ed): The serological diagnosis of enzootic bovine leukosis. A workshop in the European Economic Communities program of coordination of research on bovine leukosis, Aug 29-30, 1977, Rotterdam. Commission of the European Communities, Luxembourg, 1978. 16. Mammerickx M, Cormann A, Burny A, Dekegel D, Portetelle D: Eradication of enzootic bovine leukosis based on detection of the disease by the GP immunodiffusion test. Ann Rech Vet 9: 885-898, 1978. 17. Straub OC: Preliminary results of a new sanitation program for the eradication of enzootic bovine leukosis. Ann Rech Vet 9: 895-898, 1978. 18. Ferrer JF: Bovine lymphosarcoma. Compendium on Continuing Education 2(11): 236-242, 1980. 19. Ferrer JF: Bovine lymphosarcoma. Adv Vet Sci Comp Med 24: 1-68, 1980. 20. Ferrer JF: Bovine leukemia virus: detection, transmission, eradication and public health implications. Vet Cancer Soc Newsletter 3(4): 1979. 21. Jacobsen KL, Bull RW, Miller JM, Herdt TH, Kaneene JB: Transmission of bovine leukemia virus: prevalence of antibodies in pre- colostral calves. Submitted for publication to Prev Vet Med. 22. Wilson JMG, Jungner G: Principles and practice of screening for disease. Geneva, Switzerland, Wbrld Health Organization Publications : 20-22, 1968. 23. Bause I, Mass-Inderwiesen F, Schmidt FW: Results of an epide- miological survey of enzootic bovine leukosis in the northern part of lower saxony and preliminary communication of an examination into rela- tionship between BLV-antibody development and calving. Ann Rech Vet 9: 765-770, 1978. . 24. Burridge, MJ, Thurmond MC, Miller JM, et al: Fall in antibody titer to bovine leukemia virus in the periparturient period. Can J Comp Med, in press. DISCUSSION AND SUMMARY The prevalence of bovine leukemia virus antibodies in the serum of precolostral calves born to BLV antibody positive dams was investigated. Two Michigan dairy herds with breeding and culling practices designed to increase milk production were used. One hundred and twenty-five calves and their dams were tested for antibody to BLV by the radioimmunoprecipitation assay utilizing glycoprotein antigen. Three of 79 calves (3.8%) born to seropositive dams were seropositive prior to colostral ingestion. None of 46 calves born to seronegative dams were seropositive at birth. The percentage of calves born to seropositive dams that were seropositive at birth was 4.7% in herd 1 and 3.4% in herd 2. These results are in contrast to previous reports of 14 to 18% in utero viral exposure in an inbred lymphosarcoma-proned herd.13’15 This would suggest that in herds with breeding programs designed to increase milk production almost all of the calves born to naturally infected dams are not exposed to BLV in utero. Therefore, control programs aimed at raising BLV negative calves from positive dams should meet with greater success than previous reports had indicated. These results do not differ greatly from a study recently reported by Van Der Maaten et al in which most of the calves tested were born to exper- imentally infected dams.14 In that study, 18 calves from BLV infected dams were not infected or seropositive at birth. The effect of storage at room temperature on the concentration of cortisol in neonatal bovine blood was investigated. Heparinized blood samples were collected from 7 Holstein-Freisian calves within 68 69 15 minutes of birth and prior to colostral ingestion. Cortisol was determined by a modified commercial 125I-radioimmunoassay. A least squares linear regression of percent degradation of cortisol on storage time was performed. The initial mean plasma cortisol value for the 7 calves (109 t 6 ng/ml) agreed well with reports in the literature.24’25’26 As blood storage time increased, plasma cortisol values for each calf steadily declined. The degradation of cortisol in heparinized blood at room temperature was demonstrated by the regression equation, Y = 9.534 + 0.832 X, where Y = percent cortisol degradation and X 8 hours from first cortisol determination. Predicted values for percent degradation of cortisol at 12, 24, and 48 hours were 20, 30 and 50 percent, respectively. The regression equation derived from this data can be used to calculate adjusted plasma cortisol values for neonatal calf blood samples. Lymphocyte blastogenic response of calves born to BLV seroposi- tive and seronegative dams was investigated. Peripheral blood lympho- cytes were isolated from 10 precolostral calves, 5 of which were born to positive dams and 5 of which were born to negative dams. The 5 calves born to positive dams were from herd 1, as were 2 calves born to positive dams. Three calves born to positive dams were from herd 2. All of the calves were BLV seronegative precolostrally. Plasma cortisol was determined by radioimmunoassay from the heparinized samples collected for lymphocyte isolation. Because these samples had been stored at room temperature, the regression equation described above was used to calculate adjusted cortisol values. The lymphocyte blasto- genic response to PWM was significantly lower for calves born to BLV positive dams, even when results were adjusted for the effect of 7O cortisol. Calves born to positive dams also had a significantly lower lymphocyte blastogenic response to PHA prior to adjustment for cortisol. After this adjustment, the difference was significant when calves from both herds were compared, but not for calves from herd 1 alone. Significant differences were not found in the spontan- eous blastogenic response of lymphocytes cultured without mitogens between calves from BLV positive and negative dams. Decreased mitogen responsiveness in calves of BLV positive dams might be due to a functional abnormality of the lymphocytes or to an inhibitory substance acting upon the lymphocytes. It is possible that BLV positive dams could pass on to their calves genetic inform- ation coding for a functional abnormality of lymphocytes. Another possibility is that an inhibitory factor is present in the serum of calves born to BLV positive dams. Such a factor might have been produced within the calf or it may have crossed the placenta from the dams's serum. It is also conceivable that mitogens could stim- ulate certain lymphocyte subpopulations to produce inhibitory factors in the in vitro culture system. If these subpopulations were present in greater numbers in calves of positive dams, this could account for the decreased blastogenesis in the presence of mitogens. In a double blind study, the commercial agar gel immunodiffusion test for antibodies to BLV glycoprotein antigen was compared to the radioimmunoprecipitation assay for antibodies to gp antigen (gp RIA). Sera from 115 adult cows of calving age and 125 precolostral calves were compared. The animals were from herds 1 and 2 described above. The commercial AGID test kit for detection of antibodies to BLV gp 71 compared favorably with the gp RIA. Both the sensitivity and speci- ficity of the test were increased by reading the test at both 48 and 72 hours. This also permitted clarification of many reactions that were unclear at 48 hours due to a haze around the test serum well. The AGID read at 48 hours did not detect antibodies to BLV in 3 pre- colostral calves, suggesting that this may not be the serologic test of choice for detection of in utero exposure to BLV. Many questions remain unanswered about the prenatal transmission of BLV and about the immune response of calves born to BLV seroposi- tive dams, leaving several possible areas for future investigation. The mechanism of in utero transmission of BLV remains a mystery. His- topathologic studies of the placenta and uterus of dams delivering BLV positive calves would be useful to further investigate the possi- bility that increased transmission of the virus occurs during inflam- mation. A comparison of antibody titers of dams delivering infected and non-infected calves would help answer the question of whether virus dose affects in utero transmission. Results of the comparison of lymphocyte blastogenesis values for calves of BLV positive and negative dams presented in this thesis must be considered prelim- inary due to the small numbers of calves compared. Before definitive comclusions can be drawn, the study should be repeated with higher numbers of calves in both groups. Adjustment for the effect of cortisol on lymphocyte blastogenic response would appear to be man- datory in such a study, and will undoubtedly be enlightening in future studies on bovine lymphocyte function. BIBLIOGRAPHY BIBLIOGRAPHY l. Burridge MJ, Puhr DM, Hennemann JM: Prevalence of bovine leukemia virus infection in florida. J Am Vet Med Assoc 179: 704-707, 1981. 2. DeVries G: Leucosis in cattle in the Netherlands. Ann Rech Vet 9: 903-907, 1978. 3. 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