Search

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.

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.

Ehrlichia risticii causes an acute infectious disease in horses called Potomac horse fever. To investigate the biological diversity of E. risticii organisms, nine E. risticii isolates derived from the peripheral blood monocytes of clinically sick horses in Ohio and Kentucky during the summers of 1991 and 1993 were compared with Illinois and Virginia isolates originally obtained from horses in Maryland in 1984. Seven of the nine isolates (081, 606, 380, 679, As, Co, and Ov) formed large morulae (tightly packed inclusions of ehrlichial organisms). The remaining isolates, including 1984 isolates, were individually dispersed or formed small morulae in the cytoplasm of P388D1 cells. In Western blot (immunoblot) analysis with four equine and one rabbit polyclonal anti-E. risticii sera, these recent E. risticii isolates showed patterns of antigenic proteins distinct from those of the 1984 isolates and could be divided into three groups: (i) 081; (ii) 606, 022, 067, 380, and 679; and (iii) As, Co, and Ov. By indirect fluorescent antibody labeling with two panels of murine anti-E. risticii (Illinois and Maryland isolates) monoclonal antibodies, isolate 081 was not labeled with any of 20 monoclonal antibodies tested. The remaining isolates were not labeled with several monoclonal antibodies. The digestion pattern with one of the restriction enzymes, AvaII, of the PCR-amplified partial 16S rRNA gene of E. risticii from all Kentucky isolates (As, Co, and Ov) was different from that of Illinois, Virginia, and six Ohio isolates. These results indicate the presence of distinct variants of E. risticii which vary significantly in morphology, antigenic composition, and the base sequence of the 16S rRNA gene.

Mice that recovered from Ehrlichia risticii infection were immune to a challenge dose of 100 50% lethal doses. Immune or normal mouse serum was passively transferred to mice challenged with E. risticii. Clinical signs of ehrlichiosis were completely prevented in 22 of 24 recipients of immune serum, and the onset of signs of illness was delayed in the remaining two mice compared with the onset of illness in 24 of 24 recipients of nonimmune serum. Purified immunoglobulin G (IgG) was used to passively protect mice from infection with E. risticii. All 15 mice that received IgG from normal serum but none of the 15 mice that received IgG from immune serum developed clinical signs of illness. Antibodies in immune mouse serum immunoprecipitated [35S]methionine metabolically labeled E. risticii proteins with apparent molecular masses ranging from 14 to 90 kDa. The major antigens recognized by dilute immune serum in immunoblot analysis had molecular masses of 62, 53, 40, 33, 27, and 25 kDa, and the 62- and 27-kDa antigens were prominent in immunoprecipitations with dilute antibody. Antigens with molecular masses of 62, 53, 40, 33, and 27 kDa are likely surface exposed, as determined by immunoprecipitation of 125I-labeled organisms with immune mouse serum.

Antibody-dependent cellular cytotoxicity and direct cytotoxicity assays were performed with equine infectious anemia virus-infected target cells, equine leukocytes, and equine anti-equine infectious anemia virus antibody to determine whether these mechanisms play a role in controlling viral replication in equine infectious anemia. Direct cytotoxicity was observed by using peripheral blood mononuclear cells from 7 of 10 infected horses. Antibody-dependent cellular cytotoxicity was not observed. The antibody-dependent cellular cytotoxicity reaction in horses was then studied by using sheep erythrocytes and trinitrophenylated sheep erythrocytes as target cells. Lysis of these target cells was mediated by neutrophils, monocytes, and lymphocytes. The reaction was activated by antibody of the immunoglobulin G class but not by immunoglobulin G(T). Furthermore, immunoglobulin G(T) efficiently inhibited immunoglobulin G in this function.

The ultrastructure of early retrovirus-induced arthritis was studied sequentially in 20 goat kids inoculated with caprine arthritis-encephalitis virus. Synovial lesions began as intercellular edema and collagen fragmentation and continued as progressive mononuclear cell infiltration and lining cell hyperplasia, hypertrophy, and necrosis. At 18 through 45 days after the inoculation, lining cells contained small accumulations of virus-like particles similar to virus seen in infected tissue culture cells. No virus was seen budding from infected lining cell membranes.

Equine infectious anemia virus was found to be comprised of fourteen polypeptides of molecular weight ranging from 10,000 to 79,000. Eighty percent of the virion protein was accounted for by five polypeptides, including two non-glycosylated components (p29 and p13) comprising one-half of the virion protein and three glycoproteins (gp77/79, gp64, and gp40).

Summary The mechanisms involved in the interaction of complement components with sensitized equine arteritis virus (EAV) were investigated. Virus neutralization and virolysis depended on both the concentration of the complement components and the concentration of the sensitizing antibody. High concentrations of C4, 2 and 3, with an optimal concentration of C1, were sufficient for neutralizing virus infectivity in the presence of excess antibody. The addition of the remaining five components (C5 to C9) of the complement system induced lysis of the previously neutralized virus particle. Lysis was initiated by C8 and was augmented by C9. Components C5 to C9 did not enhance neutralization produced by excess antibody and limiting concentrations of complement components. In contrast, addition of components C5 to C9 enhanced neutralization by means of lysis of the virus particle under conditions of low antibody concentration.

Sequential changes induced by an equine adenovirus in cultured fetal equine kidney cells were studied by electron microscopy. The first morphological change was the appearance of type I inclusions. These inclusions developed to type II inclusions which appeared as ring forms. Type III inclusions were formed within the central part of type II inclusions and finally filled up most of the nuclear space. As the infection proceeded, type IV inclusions which appeared as dense dark-staining spheres were formed at the center of the type III inclusions and also inside the cytoplasm. These dark-staining spheres developed and their center was filled with light-staining material and virus particles which eventually resulted in the formation of type V inclusions. Autoradiography study showed that types I, II, and III were composed of nucleoprotein and type IV was composed of protein.

A previously unreported afebrile, infectious leukoencephalomyelitis of oneto four-month-old dairy goats was studied. The disease was characterized by pleocytosis and posterior ataxia that progressed to complete paralysis within two weeks of onset. Dense perivenous accumulations of lymphoreticular cells in white matter and variable myelinoclasis were the essential neuropathologic lesions. These were accompanied by mild interstitial pneumonia and hyperplasia of pulmonary lymphoid tissue. Epizootiologic observations suggest that goats become infected either in utero or immediately after birth. The disease was transmitted to newborn goats by intracerebral and intraperitoneal inoculation of a 220-nm millipore filtrate of nervous tissue from a naturally infected goat. This observation and the failure to isolate bacteria or mycoplasma suggest that the causative agent is a virus, which thus far appears to be serologically unrelated to other animal viruses. The nature of the neuropathologic changes suggests that this disease may share pathogenic mechanisms with postinfectious encephalitis of man.

The sera of horses with persistent infection by equine infectious anemia virus were examined for the presence of infectious virus-antibody complexes, Virus-containing sera were treated with rabbit anti-equine gamma globulin, rabbit anti-equine albumin, and normal rabbit serum. Infectivity remaining in the supernates was titrated in primary equine leukocyte cultures. In 2 of 3 sera anti-gamma globulin precipitated at least 2 log10 of viral infectivity using the anti-albumin and normal rabbit serum treatments as controls. This demonstrates that in some sera EIA virus circulates as an immune complex with antibody, possibly enhancing its ability to persist in the host by providing a continued source of virus for reinfection.

A system for detecting Equine infections anemia viral antigen by direct immunofluorescence and the characteristics of the fluorescent antigen in horse peripheral leukocyte cultures are described. Serum from chronically infected horses yielded conjugates which gave bright specific staining of EIA antigen. The antigen was present by the second day after inoculation of the cultures. It appeared as discrete cytoplasmic dots, irregular cytoplasmic masses and accumulations along the cell membrane. The percentage of infected cells increased from 10–15% on the second day to 80% on the fifth day after inoculation.

Immunoglobulin A (IgA) was demonstrated in equine serum and secretions. This immunoglobulin had a molecular weight extending from 150,000 to 700,000 and reacted with specific antihuman alpha-chain antiserum. Antigenic determinants specific for secretory IgA were demonstrated and found to be absent on serum IgA. Antigen binding activity was detected in IgA from tears. Purified IgA was antigenically distinct from equine IgG, IgM, IgG(T), and aggregating immunoglobulin. Quantitative studies demonstrated that IgA was the predominant immunoglobulin in tears and milk but not in colostrum. The electrophoretic mobility, size, presence of secretory component, and reaction with specific antihuman alpha-chain antiserum demonstrates that this immunoglobulin is the equine homologue of human IgA.

A virus was isolated from an adult goat with chronic arthritis and shown to belong to the retrovirus group by electron microscopy and biochemical methods. Inoculation of the virus into cesarean-derived specific-pathogen-free goats' kids produced arthritic lesions similar to those in the spontaneous disease. Vrus was reisolated from the experimentally induced lesions.

Twelve chemicals and commercial disinfectants were tested for inactivation of equine infectious anemia virus. In the presence of 10% bovine serum, all chemicals inactivated 4 log10 (based on 0.1 ml) of the virus within 5 minutes at 23 C. A reduction of at least 4 log10 was observed when the virus was exposed for 1 minute to substituted phenolic disinfectants (3 commercial preparations and sodium orthophenylphenate), halogen derivatives (iodophor and sodium hypochlorite), chlorhexidine, and 70% ethanol. Sodium hydroxide (5%), 2% formalin, and 2% glutaraldehyde were slower to inactivate the virus, but achieved 4 log10 reduction in titer by 5 minutes' contact time. The susceptibility of the equine infectious anemia virus to chemical disinfectants is similar to that of other enveloped viruses.

Experiments were designed to correlate morphologic lesions with the presence of caprine arthritis-encephalitis virus (CAEV). Twenty-one cesarean-derived goat kids were infected with 10(6) to 10(7) TCID50 of virus, killed sequentially, and examined for viral antigens by immunofluorescence, viral infectivity by isolation and titration, and morphologic changes by light microscopy. Fluorescent viral antigens were detected from 1 to 10 days postinoculation (DPI) and only in synovial cells. Virus was reisolated from several joints and from brain 0.5 to 79 DPI. Increases in synovial fluid cell counts were noted by 1 DPI, and morphologic changes in synovial membranes were present from 3 to 45 DPI. Joint lesions progressed from mild synovial cell hyperplasia and perivascular mononuclear cell infiltration to severe synovial cell hyperplasia and mononuclear cell infiltration with villous hypertrophy. Lesions elsewhere were mild, consisting only of perivascular mononuclear cell infiltrates. Eleven cesarean-derived control goats were negative for viral antigens, virus, and morphologic lesions.

Equine dermal fibroblasts persistently infected with equine infectious anemia virus (EIAV) show no alterations in cell morphology or growth kinetics when compared to uninfected cells. The percentage of cells immunofluorescent positive for viral proteins fluctuated, depending upon the stage of the cell cycle, while production of extracellular virus was uniform throughout the cell cycle, increasing only as the cell number increased. This was shown in log versus stationary phase cultures as well as in cultures synchronized by sterum starvation. The establishment of productive infection did not require host cell DNA synthesis. Normal levels of progeny virus were produced in cultures pretreated with mitomycin C and placed in serum-containing medium. Serum-starved cultures, however, did not support EIAV replication as well as other cultures, presumably because synthesis of provirus was inhibited.

Eight cesarean-derived goat kids were inoculated with caprine arthritis-encephalitis virus (CAEV), and proliferative responses of their peripheral blood mononuclear cells to mitogens and CAEV antigen were monitored for 9 months. Antibody specific for CAEV was measured by an enzyme-linked immunosorbent assay. Five cesarean-derived noninfected goats were tested simultaneously. Significant differences between the infected and control mononuclear cell proliferation reactions to CAEV began 14 days post-inoculation and continued in a fluctuant manner until 134 days post-inoculation. The magnitude of the proliferative reaction steadily increased in infected goats until the end of the experiment at 271 days post-inoculation. Responses to mitogens were not significantly different between infected and control goats. Virus-inoculated goats produced CAEV-specific antibody that reached a maximum level between 49 and 77 days post-inoculation and then declined to lower levels through 271 days post-inoculation. The virus-inoculated goats developed mild but characteristic clinical evidence of caprine arthritis-encephalitis, and CAEV was reisolated from four goats at 286 days post-inoculation. The five control goats developed neither an anti-CAEV immune response nor clinical disease, and CAEV could not be reisolated from them.

Sixty-six cases of combined immunodeficiency (CID) in foals were studied to determine the most prevalent causes of infection and death. Lesions of the respiratory system were observed in 59 of the foals and were attributable to infection with equine adenovirus. Pneumocystis carinii, and bacteria. Significant lesions were also observed in liver, pancreas, intestines, heart, and kidneys. Maintenance of foals with CID for experimental purposes is directed at the prevention and control of these secondary infections. Adenovirus can be controlled by administration of horse plasma containing high titers of antiadenovirus antibody. Bacteria are controlled by appropriate antibiotic therapy. Pneumocystis carinii infection remains a significant problem in the maintenance of foals with CID.

Laboratory-colonized mosquitoes, Culex tarsalis, aedes aegypti, Culiseta inornata, and Anopheles free-borni, and the biting gnat, Culicoides variipennis, were exposed to equine infectious anemia virus. Exposure to the virus was by intrathoracic inoculation for mosquitoes and by oral ingestion of an infective blood meal through a membrane for C variipennis. After various intervals, groups of 15 to 20 insects were homogenized and inoculated into susceptible ponies. Positive immunodiffusion test results were used as criterion for equine infectious anemia infection in ponies. Virus was not detected in the 4 species of mosquitoes at 3, 6, 12, and 18 days after inoculation, or in C variipennis at 6, 8, 12, 14, 21, and 26 days after oral exposure to the virus.

A retrovirus isolated from synovial membrane explants of arthritic joints of goats has been characterized with respect to buoyant density, RNA-dependent DNA polymerase, genome structure and protein composition.

The morphologic changes of viral leukoencephalomyelitis of goats (VLG), an afebrile paralytic disease of 2–4 month old kids, were studied in 13 naturally infected goats: 11 during the first 5–22 days of clinical disease and two that survived 8 months and 3 years after onset of clinical signs. Lesions in the early clinical phase of the disease included interstitial pneumonia and widely disseminated myelinoclastic perivascular lesions in the brain and spinal cord. In the central nervous system, hypertrophied and hyperplastic reticulin fibers of the vascular sheath encompassed an inflammatory cell infiltrate composed of lymphocytes, plasma cells, and macrophages. Occasionally, malacia occurred, and several animals had transverse myelitis. Neuronal necrosis and neuronophagia were not features of the disease. Lesions were most frequent in the subpia and subependyma. Pulmonary lesions were not present in goats that survived the initial clinical phase of the disease, but myelin had disappeared from large areas of the spinal cord and from small foci in the brain. Nodular accumulations of mononuclear cells and small perivascular cuffs were also present in the central nervous system. VLG resembles visna in the topographic distribution and myelinoclastic nature of its lesions. Like visna, it bears some resemblance to post-infectious encephalitis of man.

Features of caprine arthritis-encephalitis, a retrovirus disease of domestic goats, were studied in 60 goats over a 10-year period. The rate of progression and the severity of the disease process were highly variable within and among animals, but the most salient features were chronically swollen joints and bursae, lameness, weight loss, poor coat, mineralization of soft tissue, and death. Of 1,160 goat serum samples from 24 states tested by the immunodiffusion technique, 81% were positive for antibody to caprine arthritis-encephalitis virus antigens.

Four methods of detecting and quantitating mink antibody against Aleutian disease (AD) virus were compared. Counterelectrophoresis, modified, counterelectrophoresis, immunofluorescence, and complement fixation were performed blindly on 274 serum samples. All four methods were reliably specific for AD antibody. Immunofluorescence was less reproducible than the other systems. Immunofluorescence complement fixation were 4- to 8-fold more sensitive than regular or modified counterelectrophoresis, but were limited by background staining and anti-complementary activity, respectively, when used to detect small amounts of antibody in undiluted sera.

Although acute vasculitis is an important part of a number of human and animal diseases, the etiology and pathophysiological consequences of these lesions are not well understood. More knowledge about the incitors and effects of acute damage to blood vessels is needed to facilitate the understanding, prevention, and treatment of peripheral vascular disease. Recent work has begun to reveal some of the stimuli which damage blood vessels and the ways in which the vessels respond to them. We have been studying the pathogenesis of equine viral arteritis (EVA), a viral disease of the horse which causes a fulminant generalized vasculitis (1, 2). Results of these studies show how the virus induces injury to the vessels and shed some light on the pathophysiologic effects of acute vascular damage in the horse.

A number of proposals have been suggested to explain the mechanism of plasmacytosis and hypergammaglobulinemia in mink infected with Aleutian disease (AD) virus. That the virus induces a myeloma-like proliferation of plasma cells and the production of immunoglobulins not directed toward specific antigens was suggested by several workers (1–5). The transition from polyclonal to monoclonal gammopathies in 10% to 20% of the infected mink with genotypes Aa and AA (non-Aleutian mink) supports this suggestion (2). However, detection of anti-virus antibody in the form of infectious virus-antibody complexes (6) and by direct and indirect immunofluorescence on infected tissue (7; J. B. Henson and T. B. Crawford, manuscript in preparation) demonstrates that some of the hypergammaglobulinemia represents antibody to AD viral antigens. A previous attempt by other investigators to demonstrate complement-fixation (CF) between AD-affected sera and infectious organ homogenates was unsuccessful (1).