Intra-nasal inoculation of American bison (Bison bison) with ovine herpesvirus-2 (OvHV-2) reliably reproduces malignant catarrhal fever.

Sheep-associated malignant catarrhal fever (MCF) due to infection with ovine herpesvirus 2 (OvHV-2) is common in commercial herds of American bison ( Bison bison). Inability to propagate OvHV-2 in vitro has been a constraint on experimental studies of the disease. We sought to establish whether nasal secretions from sheep that shed OvHV-2 might induce the disease in bison and to define a minimum challenge dose. Fourteen bison were nebulized with sheep nasal sections containing 10(3)-10(7) OvHV-2 deoxyribonucleic acid (DNA) copies. Most challenged bison (11/14, 78.6%) developed clinical signs at 29-52 days postnebulization (DPN). The mean incubation time was 42.18 (+/-7.33 SD) DPN. Using real-time polymerase chain reaction, we detected OvHV-2 DNA in peripheral blood leukocytes at 21-31 DPN. All bison that developed MCF had antibodies against the MCF group viruses. Gross and histologic lesions were typical of the acute disease. There was no morphologic evidence of a dose-related difference in the severity or distribution of lesions. This is the first successful reproduction of MCF in bison using a nasal route of exposure. Experimentally challenged bison are more susceptible to MCF, compared with experimentally challenged domestic cattle in a previous experiment. Bison are a pertinent ruminant species in which the pathogenesis of the disease can be investigated.

Malignant catarrhal fever in a bison (Bison bison) feedlot, 1993-2000.

A fatal enteric syndrome was identified in American bison (Bison bison) at a large feedlot in the American Midwest in early 1998. An estimated 150 bison died of the syndrome between January 1998 and December 1999. The syndrome was identified as malignant catarrhal fever (MCF), primarily the alimentary form. Clinical onset was acute, and most affected bison died within 1-3 days; none recovered. Consistent lesions were hemorrhagic cystitis, ulcerative enterotyphlocolitis, and arteritis-phlebitis. Vasculitis was milder and more localized than that in cattle with MCF, and in contrast to the situation in cattle, lymphadenomegaly was minimal. Virtually all affected bison examined were positive for ovine herpesvirus 2 (OvHV-2) by polymerase chain reaction (PCR) assay. A retrospective study of archived tissues established that MCF occurred in the yard as early as 1993. A prospective study was undertaken to establish the importance of MCF relative to other fatal diseases at the feedlot. The fate of a group of 300 healthy male bison in a consignment of 1,101 animals was followed for up to 7 months to slaughter. At entry, 23% (71/300) of bison were seropositive for MCF viruses, and 11% (8/71) of these seropositive bison were PCR positive for OvHV-2. Forty seronegative bison were selected at random from the group, and all were PCR negative for OvHV-2. There was no change in seroprevalence in the group during the investigation. The minimum infection rate for MCF virus was 36.3% (93/256). Twenty-two (7.3%) of the 300 bison in the feedlot died. Of these, 15 had MCF, 4 had acute or chronic pneumonia, and 3 were unexamined. Losses in the entire consignment were higher (98/1,101; 8.8% death loss); 76% of deaths were attributable to MCF. The study failed to reveal a relationship between subclinical infection and development of clinical disease.

A simpler, more sensitive competitive inhibition enzyme-linked immunosorbent assay for detection of antibody to malignant catarrhal fever viruses.

An earlier competitive inhibition enzyme-linked immunosorbent assay (CI-ELISA) was developed for detection of specific antibody against malignant catarrhal fever (MCF) viruses (MCFV) in ruminants. In this study, the indirect CI-ELISA was improved by conjugating the monoclonal antibody 15-A directly with horseradish peroxidase and by developing a method of producing precoated, dried antigen plates. This new test is referred to as a direct CI-ELISA. The reformatted test yielded a significantly improved sensitivity, and the time required was reduced to about one-sixth of the previous time. Of 37 MCF cases in cattle that were confirmed by histopathology and polymerase chain reaction (PCR) assay, 37 (100%) were positive by the new test, whereas the indirect CI-ELISA detected only 23 (62%). The direct CI-ELISA detected antibody to MCFV in 100% of 48 sheep that had been defined as infected with ovine herpesvirus 2 (OvHV-2) by PCR, whereas the indirect CI-ELISA detected only 41 (85%). Comparison of antibody titers measured by the 2 assays for sera collected from OvHV-2-infected sheep and from cattle, bison, and deer with clinical sheep-associated MCF revealed that the direct CI-ELISA offered a 4-fold increase in analytical sensitivity over the indirect format. The number of seropositive animals detected by the direct CI-ELISA among apparently normal cattle and bison was 2-3 times greater than the number detected by the indirect CI-ELISA, indicating that a significant percentage of normal cattle and bison are subclincally infected with MCFV.

Levels of ovine herpesvirus 2 DNA in nasal secretions and blood of sheep: implications for transmission.

A recently developed competitive PCR for ovine herpesvirus 2 (OvHV-2) was used to examine the levels of viral DNA in nasal secretions and peripheral blood leukocytes (PBL) of lambs and adult sheep. Viral DNA first appeared in the PBL of most lambs after about 3 months of age and the levels remained relatively constant thereafter. In most of the lambs (83%, n=12), viral DNA was undetectable by PCR in nasal secretions prior to 5 months of age. A dramatic rise of OvHV-2 DNA levels in the nasal secretions occurred starting at 5-6 months of age, which peaked at approximately 7 months. The highest level recorded in lamb nasal secretions was 7.5x10(8)copies/2microg DNA which were 75,000-100,000-fold higher than the levels in PBL of the same lambs. In adult sheep (n=10), the viral DNA levels in both PBL and nasal secretions were relatively stable over the 13-month period of the study, which included a lambing season. The data strongly suggest that neonatal lambs are not an important source for the transmission of OvHV-2 to clinically susceptible species, and that the nasal cavity is an important portal for shedding of infectious OvHV-2 in sheep. Furthermore, this study failed to identify a seasonal pattern in levels of viral DNA in nasal secretions or PBL of adult sheep that would provide a basis for the traditionally held belief that clinical cases of malignant catarrhal fever are significantly associated with lambing ewes.

Newly recognized herpesvirus causing malignant catarrhal fever in white-tailed deer (Odocoileus virginianus).

Malignant catarrhal fever (MCF) was diagnosed by clinical signs and lesions in five out of six white-tailed deer (Odocoileus virginianus) in a North American zoo. The clinical signs and histopathological lesions in these deer were typical of MCF. Antibody to an epitope conserved among the MCF viruses was detected in the sera collected from the deer. PCR failed to amplify viral sequences from DNA extracted from peripheral blood leukocytes (PBL) and/or spleens of the deer with primers specific for ovine herpesvirus 2 (OHV-2) or specific for alcelaphine herpesvirus 1 (AHV-1). By using degenerate primers targeting a conserved region of a herpesviral DNA polymerase gene, a DNA fragment was amplified from the PBL or spleens of all six deer and sequenced. Alignment of the sequences demonstrated that the virus in the deer belongs to the Gammaherpesvirinae subfamily, exhibiting 82% identity to OHV-2, 71% to AHV-1, and 60% to a newly identified bovine lymphotropic herpesvirus. This virus, which causes classical MCF in white-tailed deer, is a newly recognized agent belonging to the MCF group of gammaherpesviruses. It is the third reported pathogenic MCF virus, genetically distinct but closely related to OHV-2 and AHV-1. The reservoir for the virus has not been identified.

Transmission of ovine herpesvirus 2 among adult sheep.

Previous studies from this laboratory have defined the pattern of acquisition of ovine herpesvirus 2 (OHV-2) in lambs under natural flock conditions. This study examined the question of whether OHV-2 could be transmitted between adult sheep. Two potential routes of transmission were examined: (1) direct inoculation of either viable leukocytes or whole blood from OHV-2 positive sheep, and (2) horizontal transmission through natural contact with OHV-2 positive sheep. Two groups of OHV-2 negative adult sheep were inoculated with material from infected sheep, one with 5x10(8) viable peripheral blood leukocytes (PBL), and the other with 100 ml of whole peripheral blood. No PCR signals were detected in any of the three sheep inoculated with the PBL during the 20 weeks following inoculation. In the group of five sheep inoculated with whole blood, two became PCR-positive at 7 and 8 weeks post-inoculation, respectively, and the remaining three sheep maintained their negative status until termination of the experiment at 20 weeks post-inoculation. In two experiments conducted in different flocks, a total of 20 adult sheep were used to examine horizontal transmission by contact; all animals became PCR-positive within 12 months of mixing the uninfected and infected animals. The results of these experiments support two conclusions. First, the susceptibility to OHV-2 is not limited to young lambs; adult sheep remain fully susceptible. Second, the fact that whole blood, but not PBL, from infected sheep was able to transmit the infection to only two of five inoculated sheep suggests that the infection in peripheral blood cells may be largely non-productive.

Sheep-associated malignant catarrhal fever in a petting zoo.

In a privately owned petting zoo in Arizona, 17 deer from five different species, white-tailed deer (Odocoileus virginianus), Reeve's muntjac (Muntiacus reevesi), mule deer (Odocoileus hemionus), reindeer (Rangifer tarandus), and axis deer (Axis axis), died of suspected malignant catarrhal fever (MCF) over a period from late 1992 to early 1995. A PCR assay specific for ovine herpesvirus 2, the putative causative agent of sheep-associated MCF, and a competitive-inhibition enzyme-linked immunosorbent assay based on a monoclonal antibody specific to an epitope conserved among all known MCF viral isolates were used to investigate the outbreak. Ovine herpesvirus 2 DNA sequences were detected by PCR from fresh-frozen and/or formalin-fixed, paraffin-embedded tissue samples in seven deer out of eight available animals previously suspected as cases by histopathology. A high seroprevalence to the virus was found among mouflon (Ovis musimon, 80%) and pygmy goats (Capra hircus, 61%), both of which were present on the farm during the outbreak. Sixteen percent of fallow deer (Dama dama) were also seropositive to the virus. After removal of the mouflon and positive pygmy goats, no further MCF cases occurred on the farm, confirming the importance of careful management to avoid mixing clinically susceptible species with carrier species. Until better control measures are available, adherence to this practice is necessary if MCF is to be prevented in intense exposure environments such as zoos and densely populated animal parks.

Analysis of the 16S rRNA gene of micro-organism WSU 86-1044 from an aborted bovine foetus reveals that it is a member of the order Chlamydiales: proposal of Waddliaceae fam. nov., Waddlia chondrophila gen. nov., sp. nov.

The structural gene encoding the 16S rRNA of the new obligate intracellular organism presently designated WSU 86-1044T was sequenced and analysed to establish its phylogenetic relationships. The 16S rDNA sequence was most closely related to those of chlamydial species, having 84.7-85.3% sequence similarity, while it had 72.4-73.2% similarity with rickettsia-like organisms. When the sequences of the four species of chlamydiae (Chlamydophila psittaci, Chlamydia trachomatis, Chlamydophila pneumoniae and Chlamydophila pecorum) were compared, they had > 93% sequence similarity indicating that WSU 86-1044T was not close enough to be in the same family as current Chlamydiaceae members. However, based on the 84.7-85.3% 16S rDNA sequence similarity of WSU 86-1044T and other previously described characteristics, WSU 86-1044T belongs to a novel family within the order Chlamydiales; hence, the proposal of Waddliaceae fam. nov., Waddlia chondrophila gen. nov., sp. nov.

Diagnosis of malignant catarrhal fever by PCR using formalin-fixed, paraffin-embedded tissues.

A previously described polymerase chain reaction (PCR) assay (amplification of a 238-bp fragment of ovine herpesvirus 2 [OHV-2] genomic DNA) for diagnosis of sheep-associated malignant catarrhal fever (MCF) was adapted for use on formalin-fixed, paraffin-embedded tissues. Variables affecting its use were examined. Archived tissues from cattle, white-tailed deer (Odocoileus virginianus), and bison (Bison bison) diagnosed with MCF by clinical signs or histologic lesions were obtained from 2 veterinary diagnostic laboratories. Tissues from healthy animals and from animals diagnosed with other common bovine viral diseases were examined as controls. A total of 86 blocks from 37 suspect MCF cases were examined. Forty-one blocks from healthy animals and animals with unrelated viral diseases were examined as controls. The assay was specific for sheep-associated MCF and did not yield false-positive signals from healthy animals or from cases of infectious bovine rhinotracheitis, bovine virus diarrhea, mucosal disease, or parainfluenza-3 virus infection. A wide variety of tissues were suitable substrates, including spleen, lymph node, intestine, brain, lung, and kidney. Extracted DNA provided a more suitable target than did unextracted tissue lysate. The highest levels of viral DNA were present in lymphoid organs and intestine, but the data indicate that in acute clinical cases, most organs contain sufficient viral DNA to serve as a suitable diagnostic specimen. Fixation of 0.5-cm3 blocks of tissue in 10% neutral buffered formalin was deleterious to the target DNA, and PCR signals progressively diminished after fixation for >45 days. Detection of genomic DNA of OHV-2 by PCR was successful for archived tissues that were 15 years old.

Quantitation of sheep-associated malignant catarrhal fever viral DNA by competitive polymerase chain reaction.

A single-step, competitive polymerase chain reaction technique was developed to quantitate sheep-associated malignant catarrhal fever (SA-MCF) viral DNA. The assay employed coamplification of a fixed quantity of target DNA with graded amounts of a competitor, generated by truncation of the target sequence lying between the 2 primer binding sites. The assay yielded a linear response (r = 0.98) for DNA measurement within the range of 30-300,000 copies. Amplification efficiency analysis by coamplification of target and competitor in equal copy numbers for various numbers of cycles showed that the relative abundance of the coamplified products remained constant with increasing cycle numbers up to 40. Reproducibility was assessed by repetitively assaying a set of blind-coded samples from a variety of animals and tissues. Results indicated that the assay is reliable and reproducible for quantitation of SA-MCF viral DNA in samples from asymptomatically infected sheep and from animals with clinical SA-MCF.

Production of malignant catarrhal fever virus-free sheep.

The study was designed to better define the variables affecting the success of the establishment of ovine herpesvirus 2 (OHV-2)-free sheep flocks. A total of 38 lambs born to OHV-2-positive ewes was selected and divided into four groups. Three groups of 10 lambs each were separated from the positive ewes at 2, 2.5 and 3 months of age, respectively, and maintained in isolation facilities. One group of eight remained in the positive flock as controls. Peripheral blood samples from each lamb were examined regularly by PCR for OHV-2 DNA. All lambs (100%) that were weaned and maintained in isolation from the ages of 2, 2.5 and 3 months remained negative until the termination of the experiment at 1 year of age. One lamb was discovered to be PCR-positive on the day of isolation at 2.5 months of age, and was promptly removed from the isolation group. In contrast, all lambs (100%) that remained with the flock became PCR-positive by 6 months of age. The data confirmed that, with rare exceptions, separation of lambs from OHV-2 infected animals at around 2 months of age reliably yields OHV-2-free sheep. Appropriate PCR monitoring will enable the rare exceptions to be removed from the group, and is recommended as a safety measure.

Equine infectious anemia virus is found in tissue macrophages during subclinical infection.

The equine infectious anemia virus (EIAV) often results in lifelong subclinical infection following early episodes of clinical disease. To identify the cellular reservoirs of EIAV during subclinical infection, horses were infected with EIAV and allowed to develop subclinical infections. Horses with acute disease served as a basis for comparison. The tissue distribution, replication status, location of infected cells, and viral load were characterized by PCR for proviral DNA and reverse transcriptase PCR for viral RNA, in situ hybridization, and in situ PCR. Proviral DNA was widespread in tissues regardless of disease status. Viral gag and env RNAs were also detected in tissues of all horses regardless of disease status. Plasma viral RNA (viremia) could be detected in some, but not all, horses with subclinical infections. In situ assays determined that a primary cellular reservoir and site of viral replication during subclinical infection is the macrophage. During subclinical infection, viral load was decreased 4- to 733-fold and there was decreased viral RNA expression within infected cells. These data indicate that viral replication continues at all times, even in horses that are clinically quiescent. Moreover, restricted viral replication at the cellular level is associated with clinical remission.

Transmission of ovine herpesvirus 2 in lambs.

The pattern of acquisition of ovine herpesvirus 2 (OHV-2) infection in lambs was examined by a competitive-inhibition enzyme-linked immunosorbent assay and PCR. Newborn lambs (n = 118) did not exhibit antibody at birth. Viral DNA in peripheral blood leukocytes was detected in only 3% (n = 77) of newborn lambs before suckling. After nursing, viral DNA was sporadically present in about 10 to 20% of lambs until about 3 months of age. Thereafter, strong DNA signals began to appear in increasing numbers of lambs, reaching 100% by 5.5 months of age. Viral DNA in nasal secretions began to be detectable in about 30% of lambs at 5.5 months of age, achieved significant levels in 88% of lambs by 7.5 months of age, and then declined. The kinetics of the humoral immune response in lambs paralleled those of viral DNA in nasal secretions but did not parallel its presence in blood leukocytes. In the experiment to define the time of infection of OHV-2 in lambs, all five lambs separated from the flock at 2.5 months of age remained uninfected until the termination of the experiment at 1 year of age. In contrast, lambs weaned at 2.5 months of age and returned to the flock had become infected at 3.5 months of age. Weaning and separation from the flock at 3.5 months of age did not prevent infection. The study showed that OHV-2 infection does not commonly occur in perinatal lambs and that OHV-2-free sheep can be established by separation of lambs at the proper time, which has important implications for potential control measures.

Suppression of megakaryocyte colony growth by plasma from foals infected with equine infectious anemia virus.

Foals infected with equine infectious anemia virus become thrombocytopenic 7 to 20 days after virus inoculation, and within a few days following the onset of detectable viremia. The thrombocytopenia is associated with suppression of platelet production. Possible mediators of suppression of thrombopoiesis include tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-beta), cytokines that are released during inflammation. To assess effects of plasma or serum from infected foals on megakaryocyte (MK) growth and maturation in vitro, equine low-density bone marrow cells were cultured for clonogenic and ploidy assays. Neutralizing antibodies to TNF-alpha and TGF-beta were added to cultures to determine the contribution of these cytokines to suppression of thrombopoiesis. Plasma from the immediately pre-thrombocytopenia (Pre-Tp) period significantly reduced MK colony numbers. This suppression was partially reversed upon antibody neutralization of plasma TNF-alpha, TGF-beta, or both. There were no differences in ploidy distribution of MK grown in the presence of preinfection serum compared with those grown in the presence of Pre-Tp serum. These results indicate that TNF-alpha and TGF-beta may contribute to suppression of MK proliferation and represent likely factors in the pathogenesis of thrombocytopenia.

Chronic and recovered cases of sheep-associated malignant catarrhal fever in cattle.

Malignant catarrhal fever (MCF) is traditionally regarded as a disease with a short clinical course, low morbidity and high case fatality rate. Owing to the limitations of the assays used for laboratory diagnosis. It was difficult in characterise the clinical spectrum of sheep-associated MCF, particularly when the cattle recovered from an MCF-like clinical syndrome. Over a period of three years, 11 cattle that survived MCF for up to two-and-a-half years were identified on four premises. A clinical diagnosis of MCF was confirmed by the detection of ovine herpesvirus-2 DNA in peripheral blood leucocytes using a polymerase chain reaction (PCR) assay that detects a specific 238 base-pair fragment of viral genomic DNA. Of the 11 cattle examined, six recovered clinically with the exception of bilateral corneal oedema with stromal keratitis (four animals) and unilateral perforating keratitis (one animal). The 10 animals available for postmortem examination had disseminated subacute to chronic arteriopathy. Recovery was associated with the resolution of the acute lymphoid panarteritis that characterises the acute phase of MCF, and with the development of generalised chronic obliterative arteriosclerosis. Bilateral leucomata were due in part to the focal destruction of corneal endothelium secondary to acute endothelialitis. Formalin-fixed tissues and/or unfixed lymphoid cells from all 11 cattle were positive for sheep-associated MCF by PCR. These observations indicate that recovery and chronic disease are a significant part of the clinical spectrum of MCF and that such cases occur with some frequency in the area studied. The affected cattle remain persistently infected by the putative sheep-associated MCF gammaherpesvirus.

Elevation of cytokines associated with the thrombocytopenia of equine infectious anaemia.

Thrombocytopenia is a common finding in infection with equine infectious anaemia virus (EIAV), a lentivirus with some homology to human immunodeficiency virus (HIV). The thrombocytopenia of EIA, like that in some HIV patients, appears to have a multifactorial pathogenesis. To investigate the decreased platelet production seen in experimental EIA, the levels of three potential negative regulators of platelet production--tumour necrosis factor-alpha (TNF-alpha), transforming growth factor-beta (TGF-beta) and interferon-alpha (IFN-alpha)--were measured in serum and bone marrow of six severe combined immunodeficient (SCID) foals and ten immunocompetent EIAV-infected foals. Levels of cytokines in pre-infection foal sera and bone marrow were compared with levels observed during clinical EIA. Mean serum levels of TNF-alpha and IFN-alpha were significantly higher (P < 0.05) on days -4 to 0 of thrombocytopenia than before infection. Serum TGF-beta was significantly elevated on all days except day -1 of thrombocytopenia. Bone marrow TNF-alpha levels were significantly increased in infected foals just before clinical thrombocytopenia. TGF-beta activity was not different in pre-infection and pre-thrombocytopenia bone marrows, but levels of TGF-beta protein as determined by immunohistochemical staining were significantly higher in pre-thrombocytopenia bone marrow. IFN-alpha activity in bone marrow increased just before thrombocytopenia, but the difference was not significant at P < 0.05. Serum TNF-alpha levels were 2-2.5 times higher in SCID foals on three of the days prior to thrombocytopenia than in immunocompetent foals. No significant differences were found between the levels in SCID and immunocompetent foals of serum and bone marrow TGF-beta or IFN-alpha at any of the times examined.

A primary production deficit in the thrombocytopenia of equine infectious anemia.

The purpose of this study was to identify the mechanisms responsible for the thrombocytopenia that develops following infection of horses by the lentivirus equine infectious anemia virus (EIAV). Immunocompetent Arabian foals and Arabian foals with severe combined immunodeficiency (SCID), which lack functional B and T lymphocytes, were experimentally infected with EIAV. Levels of viremia and a number of clinical and hematologic parameters were examined prior to and following infection. Thrombocytopenia was not dependent on the immune response: SCID foals were affected as severely as immunocompetent foals. Production of platelets, measured by metabolic incorporation of radioactive label, was significantly reduced. The decrease ranged from 35 to 89% in three SCID and two immunocompetent foals examined. Platelet survival, measured by 51Cr labeling, also declined following infection in both SCID and immunocompetent foals: 51 and 68%, respectively, relative to the preinfection life spans. The difference between immunocompetent and immunodeficient foals was not statistically significant. The number of megakaryocytes (MK) per square millimeter of bone marrow, determined by digitizing morphometry, was not significantly altered in either SCID or immunocompetent thrombocytopenic foals. Numbers of denuded MK nuclei per unit area increased, but the elevation was not statistically significant. No evidence for viral replication in MK was found. Three different parameters of intravascular coagulation (activated prothombin time, fibrin degradation products, and one-step prothombin time) remained normal until after platelet numbers had declined significantly, arguing against an important role for disseminated intravascular coagulation. The findings indicate that EIAV induces thrombocytopenia principally through an indirect, noncytocidal suppressive effect on platelet production, the mechanism of which is unknown. A shortening of platelet life span apparently contributes moderately to the platelet deficit as well. The shortening of platelet life span is multifactorial in origin, including both mechanisms that depend on an active immune response and those that do not.

Prevalence of antibody to malignant catarrhal fever virus in wild and domestic ruminants by competitive-inhibition ELISA.

A competitive-inhibition ELISA (CI-ELISA), based on a monoclonal antibody to an epitope conserved among malignant catarrhal fever virus (MCFV) strains of both wildebeest and sheep origin, was used to determine the prevalence of antibody to MCFV in selected domestic and wild ruminants, both free-ranging and captive, from the USA. We evaluated 2528 sera from 14 species between 1990 and 1995, including 80 pronghorn antelope (Antilocapra americana), 339 bighorn sheep (Ovis canadensis), 103 biston (Bison bison), 17 black-tailed deer (Odocoileus hemionus columbianus), 395 domestic cattle (Bos taurus), 291 domestic goats (Capra hircus), 680 domestic sheep (Ovis ammon), 323 elk (Cervus elaphus), 41 llamas (Lama glama), 21 mouflon sheep (Ovis musimon), 54 mountain goats (Oreamnos americanus), 101 mule deer (Odocoileus hemionus), 20 muskox (Ovibos moschatus), and 63 white-tailed deer (Odocoileus virginianus). A high seroprevalence (37 to 62%) was observed in domestic sheep, domestic goats, muskox, and some bighorn sheep populations. Seroprevalence in these species was generally age-related: a very low seroprevalence was present in these animals under one year of age. A low seroprevalence (2% to 13%) was found in clinically-susceptible species such as domestic cattle, deer, elk and bison, supporting the concept that significant numbers of non-lethal infections occur among clinically susceptible ruminants.

Malignant catarrhal fever virus. Characterization of a United States isolate and development of diagnostic assays.

Malignant catarrhal fever (MCF) is a severe lymphoproliferative disease of certain domestic and wild ruminants. Two distinct but closely related viruses cause clinically indistinguishable syndromes in susceptible ruminant species: wildebeest-associated MCF virus (WA-MCFV) and sheep-associated MCF virus (SA-MCFV). Neither the pathogenesis nor the epidemiology of SA-MCF is understood, primarily because of a lack of adequate detection methods for the etiologic agent or antibody against that agent. Work designed to develop these tests has been under way in our laboratory. To obtain basic information about the virus, the in vitro growth properties of a US isolate of MCF virus were studied and its major viral proteins identified and characterized by a panel of monoclonal antibodies generated against the isolate. A monoclonal antibody to a broadly conserved epitope of MCF virus was identified, and a competitive-inhibition ELISA (CI-ELISA) was developed for detection of anti-MCF antibody in sheep and other ruminants. The monoclonal antibody (15-A) reacted with an epitope located on a glycoprotein complex, which was present in all isolates of MCF virus examined. Antibody from a wide variety of ruminants infected with MCF virus of both sheep and wildebeest origin competed with the monoclonal antibody 15-A for the epitope, which was not present on 14 other common ruminant viruses. The assay detected antibody in inapparently infected sheep, and in cattle, deer, and bison with clinical MCF. A PCR assay for DNA of the sheep-associated virus was developed, based on previously reported primers. Comparative studies demonstrated that the CI-ELISA was specific for MCFV antibody and that the PCR was more reliable for diagnosis of clinical MCF.

A morphometric study of bone marrow megakaryocytes in foals infected with equine infectious anemia virus.

Morphometric evaluation of bone marrow core biopsies was used to determine megakaryocyte (MK) numbers and MK size in nine foals with equine infectious anemia virus (EIAV)-induced thrombocytopenia. Both immunocompetent normal foals and foals with severe combined immunodeficiency (SCID) were used. Platelet counts were made three times weekly following viral infection. Bone marrow core biopsies were taken from the ilium of each foal prior to experimental infection, immediately after the onset of thrombocytopenia, and at necropsy. All foals developed thrombocytopenia by 23 days postinfection. The bone marrow MK density did not change in response to the thrombocytopenia. MK area did not change significantly; however, the MK nuclear area at necropsy was significantly higher than that preinfection. The presence of thrombocytopenia in the SCID foals showed that immune-specific responses were not required for the production of EIAV-induced thrombocytopenia. Furthermore, the lack of a compensatory megakaryocytopoiesis in both SCID and normal foals was consistent with the theory that altered platelet production plays a role in the development of this thrombocytopenia.