Experimentally induced bluetongue virus infection in white-tailed deer: coagulation, clinical pathologic, and gross pathologic changes

Ten yearling white-tailed deer (Odocoileus virginianus) were inoculated with bluetongue virus serotype 17. Two yearling white-tailed deer were inoculated with sonicated heparinized noninfected blood and served as controls. Clinical signs of bluetongue virus infection included increased rectal temperature, erythema, facial edema, coronitis, and stomatitis. By postinoculation day (PID) 8, excessive bleeding and hematoma formation at venipuncture sites, dehydration, and diarrhea developed. At necropsy, the most consistent findings were oral lesions and widespread hemorrhage, which ranged from petechia to massive hematoma formation. Bluetongue virus caused progressive prolongation of activated partial thromboplastin time and prothrombin time, and progressive reduction of Factors VIII and XII plasma activities beginning on PID 6. A progressive decrease in platelet numbers also developed on PID 6. Changes in platelet size were not detected. Mean thrombin time was shortened, but prolongation developed in 1 deer. Mean fibrinogen concentration and Factor V plasma activity initially increased and then decreased, but remained above preinoculation values. Factor V activity was low in a few deer. Results of screening tests for inhibitors of the intrinsic coagulation system were positive in 2 deer. High concentrations of fibrin(ogen) degradation products were first detected between PID 3 and 6. Hematologic changes included leukopenia, lymphopenia, neutrophilia, and low total plasma protein concentration. Differences in PCV, hemoglobin concentration, or RBC counts were not detected between infected and control deer. Serum total bilirubin concentration increased by PID 6, primarily because of increased unconjugated bilirubin concentration. Mild to severe increases in serum aspartate transaminase activity were accompanied by more marked increases in creatine kinase activity. Indirect Coombs test results were negative in all deer.(ABSTRACT TRUNCATED AT 250 WORDS)     

EPIZOOTIOLOGY OF BLUETONGUE: THE SITUATION IN THE UNITED STATES OF AMERICA

Bluetongue was first reported in the United States in 1948 in sheep in Texas. The virus has now been isolated from sheep in 19 States. When the disease first occurs in a flock, the morbidity may reach 50 to 75% and mortality 20 to 50%. In subsequent years, the morbidity may be only 1 to 2% with very few deaths. Difference in breed susceptibility has not been observed. Natural bluetongue infection has not been observed in Angora or dairy goats. Bluetongue virus was first isolated from cattle, in Oregon, in 1959. The virus has now been isolated from cattle in 13 States. In cattle, the disease is usually inapparent but can cause mild to severe clinical disease and neonatal losses. Natural clinical bluetongue has also been reported in bighorn sheep, exotic ruminants in a zoo, mule deer, and white-tailed deer. Serological evidence of exposure to the virus has also been found in other species of ruminants in the wild. Inoculation of virulent bluetongue virus, vaccine virus, or natural disease can cause congenital deformities and neonatal losses in calves, lambs, and white-tailed deer fawns. Culicoides is considered the important insect vector of bluetongue. The virus has also been isolated from sheep keds and cattle lice. U.S. field strains of the virus fit into four serologic groups. No cross reactions were found between bluetongue and epizootic haemorrhagic disease of deer viruses. Cattle are considered significant virus reservoirs. It is necessary to use washed erythrocytes, rather than whole blood, and to inoculate susceptible sheep, rather than embryonated chicken eggs, to detect longer-term viraemia in cattle.     

Pathogenesis of experimental vesicular stomatitis virus (New Jersey serotype) infection in the deer mouse (Peromyscus maniculatus)

The pathogenesis of vesicular stomatitis virus (VSV) infection has not been investigated previously in native New World rodents that may have a role in the epidemiology of the disease. In the present study, 45 juvenile and 80 adult deer mice (Peromyscus maniculatus) were inoculated intranasally with VSV New Jersey serotype (VSV-NJ) and examined sequentially over a 7-day period. Virus was detected by means of immunohistochemistry and in situ hybridization in all tissues containing histologic lesions. Viral antigen and mRNA were observed initially in olfactory epithelium neurons, followed by olfactory bulbs and more caudal olfactory pathways in the brain. Virus also was detected throughout the ventricular system in the brain and central canal of the spinal cord. These results support both viral retrograde transneuronal transport and viral spread within the ventricular system. Other tissues containing viral antigen included airway epithelium and macrophages in the lungs, cardiac myocytes, and macrophages in cervical lymph nodes. In a second experiment, 15 adult, 20 juvenile, and 16 nestling deer mice were inoculated intradermally with VSV-NJ. Adults were refractory to infection by this route; however, nestlings and juveniles developed disseminated central nervous system infections. Viral antigen also was detected in cardiac myocytes and lymph node macrophages in these animals. Viremia was detected by virus isolation in 35/72 (49%) intranasally inoculated juvenile and adult mice and in 17/36 (47%) intradermally inoculated nestlings and juveniles from day 1 to day 3 postinoculation. The documentation of viremia in these animals suggests that they may have a role in the epidemiology of vector-borne vesicular stomatitis.     

Ultrastructural changes related to the lymph node haemorrhages in acute African swine fever

In order to determine the pathogenic mechanisms involved in lymph node haemorrhages in acute African swine fever ( ), eight pigs were inoculated with virus, strain Malawi'83. Lymph node haemorrhages were observed from three days post infection (dpi) onwards, coinciding with ASF virus replication in monocytes and macrophages adjacent to stimulated endothelial cells, phagocytic stimulation of capillary and small-vessel endothelial cells, increase in the number of fenestrations of endothelial cells, and endothelial cell loss, as well as clusters of blood cells and necrotic material beneath the endothelium. Vascular lumina were blocked by platelet plugs and fibrin microthrombi. These phenomena became more marked as the disease progressed. At five dpi, virus replication was also found in circulating neutrophils. At seven dpi, lesions were more intense and were accompanied by virus replication in sinus and capillary endothelial cells, and in other cell populations including pericytes, fibroblasts, smooth muscle fibres and reticular cells. The results obtained in this study suggest that lymph node haemorrhages are related to endothelial stimulation and the onset of disseminated intravascular coagulation. Virus replication in vessel wall cells occurs only in the final stages of the disease and plays a secondary role.     

Thrombocytopenia Associated With Acute Bovine Virus Diarrhea Infection in Cattle

Thrombocytopenia was observed in 15 of 146 cases of clinically acute bovine viral diarrhea virus (BVDV) infection in adult cattle. Platelet counts ranged from 2,000 to 33,000/microliters. Clinically, a bleeding tendency was manifested by bloody diarrhea, petechial and ecchymotic hemorrhage, epistaxis, and abnormal bleeding from injection sites. Coagulation testing (six cases) gave no evidence of disseminated intravascular coagulation. Bone marrow aspirates were suggestive of active marrow necrosis (two cases) or recent repopulation (three cases). Treatment, when given, was supportive and empirical in nature. Six animals experienced complete clinical recoveries; the others died or were euthanatized. Although the pathogenesis of the thrombocytopenia was not definitively determined, thrombocytopenia associated with acute BVDV infection should be considered in the differential diagnosis for cattle with bleeding disorders.     

Dynamics of viral spread in bluetongue virus infected calves

The kinetics of viremia and sites of viral replication in bluetongue virus (BTV) infected calves were characterized by virus isolation, serology and immunofluorescence staining procedures. In addition, the role of the regional lymph node and lymphatics draining inoculated skin in the pathogenesis of BTV infection was determined by analyzing efferent lymph collected from indwelling cannulas. Viremia persisted for 35 to 42 days after inoculation (DAI) and virus co-circulated with neutralizing antibodies for 23 to 26 days. Virus was first isolated from peripheral blood mononuclear (PBM) cells at 3 DAI, after stimulation of PBM cells with interleukin 2 and mitogen. BTV was frequently isolated from erythrocytes, platelets and stimulated PBM cells but never from granulocytes and rarely from plasma during viremia. Virus was consistently isolated from erythrocytes late in the course of veremia. Interruption of efferent lymph flow by cannulation delayed the onset of viremia to 7 DAI. BTV was infrequently isolated from lymph cells, and few fluorescence positive cells were observed after lymph and PBM cells were labelled with a BTV-specific monoclonal antibody. Virus was isolated from spleen by 4 DAI and most tissues by 6 DAI, whereas virus was isolated from bone marrow only at 10 DAI. Virus was not isolated from any tissue after termination of viremia. It is concluded that primary viral replication occurred in the local lymph node and BTV then was transported in low titer to secondary sites of replication via infected lymph and PBM cells. We speculate that virus replication in spleen resulted in release of virus into the circulation and non-selective infection of blood cells which disseminated BTV to other tissues. Virus association with erythrocytes likely was responsible for prolonged viremia, although infected erythrocytes eventually were cleared from the circulation and persistent BTV infection of calves did not occur.     

Histopathologic and electron microscopic studies on the acute toxicity of ochratoxin A in rats

Ochratoxin A was given by gavage to male rats. Moribund and dead animals were necropsied, and the surviving rats, including the controls, were killed 48 hours after dosing. Many of the principal rats were moribund, or began dying, within 12 to 24 hours after dosing. Lesions suggestive of disseminated intravascular coagulation were seen by light microscopy as early as 12 hours after dosing; fibrin deposits were in the spleen, brain choroid plexus, glomerular capillaries, liver, and heart. Renal tubular nephrosis, hepatic and lymphoid necrosis, and necrotic enteritis with villous atrophy were also seen. Electron microscopy demonstrated fibrin strands mixed with degranulated platelets, necrotic leukocytes, and swollen endothelial cells in glomerular capillaries. Myocardial changes included focal supercontracted sarcomeres adjacent to intercalated disks. Swollen sarcolemma, lysed myofibrils and fragmented Z-bands with interstitial edema, vascular thrombosis, and endothelial damage were also seen. The acute pathologic changes induced by ochratoxin A in the intestine, liver, and lymphoid tissues were more obvious than the tubular nephrosis, and the development of a disseminated intravascular coagulation-like syndrome with myocardial changes was a complicating factor.     

Comparison of slot blot nucleic acid hybridization, immunofluorescence, and virus isolation techniques to detect bluetongue virus in blood mononuclear cells from cattle with experimentally induced infection.

A slot blot hybridization technique was applied for detection of bluetongue virus (BTV) in blood mononuclear cells (BMNC) obtained from cattle with experimentally induced infection. This technique lacked sensitivity to detect the viral nucleic acid directly in clinical specimens. When aliquots of mononuclear cells from these cattle were cultivated in vitro for 10 days to amplify virus titer, only 33.3% of the samples collected during viremia gave a positive signal in the slot blot hybridization format. By contrast, results for 34.3% of noncultured and 63.3% of cultured mononuclear cell samples collected during viremia were positive by immunofluorescence. The average number of infected cells, as detected by immunofluorescence in the noncultured mononuclear cell samples, was 1 to 5/300,000, and was usually > 10/300,000 in the cultured cell samples. Virus was isolated from all postinoculation blood samples obtained from 4 heifers that were seronegative at the time of inoculation, but was not isolated from any of the preinoculation samples, or from any of the postinoculation samples obtained from 2 heifers that were seropositive at the time of inoculation. When virus isolation was attempted from separated mononuclear cells in 2 heifers, 43.7% of the noncultured and 87.5% of the cultured samples had positive results.     

Lesions and transmission of experimental adenovirus hemorrhagic disease in black-tailed deer fawns

Adenovirus infection was the cause of an epizootic of hemorrhagic disease that is believed to have killed thousands of mule deer (Odocoileus hemionus) in California during the latter half of 1993. A systemic vasculitis with pulmonary edema and hemorrhagic enteropathy or a localized vasculitis associated with necrotizing stomatitis/pharyngitis/glossitis or osteomyelitis of the jaw were common necropsy findings in animals that died during this epizootic. To study transmission of adenovirus infection in deer and susceptibility of black-tailed deer (Odocoileus hemionus columbianus) fawns to adenovirus infection, six 3-6-month-old black-tailed fawns were divided into two treatment groups. One group was inoculated intravenously and the other group was inoculated through the mucous membranes of the eyes, nose and mouth with purified adenovirus. Each treatment group also included two additional fawns (four total) that were not inoculated but were exposed to inoculated animals (contact animals). One fawn served as a negative control. Between 4 and 16 days postinoculation, 8/10 fawns developed systemic or localized infection with lesions identical to lesions seen in animals with natural disease that died during the epizootic. Transmission was by direct contact, and the route of inoculation did not affect the incubation period or the distribution of the virus (systemic or the localized infection). Immunohistochemical analysis using polyclonal antiserum against bovine adenovirus type 5 demonstrated staining in endothelial cells of vessels in numerous tissues in animals with systemic infection and endothelial staining only in vessels subtending necrotic foci in the upper alimentary tract in animals with the localized form of the disease. All inoculated or exposed animals had staining in the tonsillar epithelium. Transmission electron microscopic examination of lung and ileum from two fawns with pulmonary edema and hemorrhagic enteropathy demonstrated endothelial necrosis and adenovirus virions in endothelial cell nuclei. Adenovirus was reisolated in black-tailed deer pulmonary artery endothelial cells using lung homogenate of the first fawn that developed systemic adenovirus infection. Serum virus neutralization test results suggest that this deer adenovirus is a new serotype.     

Presence of bluetongue virus in the marginal zone of the spleen in acute infected sheep

Bluetongue virus, a member of the genus Orbivirus of the family Reoviridae, is the causative agent of bluetongue, which is a non-contagious Culicoides mediated blood-borne disease. The present study characterizes the pathogenicity of a Taiwan prototype BTV2/KM/2003 in Corriedale sheep inoculated subcutaneously into the ear pinna. Histologically, multifocal petechiated hemorrhage, with mild to moderate inflammation and edema, were present in the contralateral ear pinna, tongue, and facial skin, without remarkable lesions in lymphoid organs. By days post-infection (DPI) 7, viral VP7 antigen, detected by immunohistochemistry, presented in the spleen, chiefly located in the outer rim of <3 cell thickness of marginal zone macrophages bordering the marginal zone and red pulp, and T lymphocytes of the red pulp. By DPI 11, viral signals shifted from the marginal zone to macrophages and small lymphocytes within follicles of the spleen. In situ hybridization with VP7 gene probe detected strong signals in the spleen, chiefly spanning the whole width of 5-10 cell thickness of the marginal zone, including the marginal zone macrophages and marginal zone B cells, as well as macrophages of sheathed capillaries in the red pulp. This study demonstrates molecular as well as morphologic evidence of the presence of bluetongue virus in the marginal zone of the spleen, most likely associated with viremia in acute infection, as previously demonstrated by the authors.     

A seroepidemiologic survey of antibody to bluetongue virus in Alabama cattle

Bluetongue (BT) is an insect transmitted viral disease of sheep that often causes mild or inapparent disease but rarely causes severe disease in cattle. Until recently, bluetongue viral infection was believed to be more prevalent in the Western United States, as compared with other regions of the country. However, a national survey for bluetongue antibody and clinical evidence of the disease in the Southeastern United States prompted the present investigation that was designed to determine the serological prevalence of BT virus in Alabama cattle. Results of the study demonstrated that 16% of the samples collected from 1,500 cattle in 64 of the 67 counties were positive. The prevalence of positive cattle in the western part of the State was significantly higher (P less than .001) than the prevalence in the eastern half of the State. On a herd basis, 52% of all herds tested had positive animals. Results of this study suggest that bluetongue infection is more common in the Southeastern United States than previously suspected.     

Cytokine modulation of the interaction between bluetongue virus and endothelial cells in vitro.

An in vitro model was developed to examine the interaction between endothelial cells and the host inflammatory response in bluetongue virus (BTV) infections. Whole cell enzyme-linked immunosorbent assays, a tritiated thymidine uptake assay, and a colorimetric assay of mitochondrial function were used to assess how four cytokines (interleukin-1, interleukin-2, interferon-gamma, and tumor necrosis factor-alpha) affect endothelial cell metabolism and susceptibility to BTV infection. Concurrent alterations in major histocompatibility complex (MHC) antigen expression were also examined. BTV infection suppressed target cell mitochondrial function and DNA synthesis and enhanced MHC class I expression. Interferon-gamma and tumor necrosis factor alpha suppressed viral antigen expression and were synergistic early in the infection. Interferon gamma enhanced MHC class I and induced MHC class II antigen expression in both BTV infected and uninfected endothelial cells. The other cytokines had minimal effect on endothelial cell surface antigen expression, although interleukin-1 (IL-1) did inhibit cell growth. Infected endothelial cell cultures produced interferon at 20 hours and 40 hours after infection. Electron microscopic analysis confirmed previous findings in other cell lines regarding BTV morphogenesis in endothelial cells, the putative target cell population in vivo.     

Histopathologic and immunohistochemical characterization of Nipah virus infection in the guinea pig

Mortality rate in humans infected with Nipah virus (NiV) has been reported as high as 92%. Humans infected with NiV show a widespread multisystemic vasculitis with most severe clinical and pathologic manifestations in the brain, lungs, and spleen. The purpose of this study was to study pathologic and immunohistochemical findings in guinea pigs infected with NiV. Of 28 animals inoculated intraperitoneally, only 2 survived the infection, and most died between 4 and 8 days postinoculation (dpi). Viral antigen with minimal pathologic changes was first detected 2 dpi in lymph nodes and spleen. More severe changes were noted in these organs 4-8 dpi, where pathologic damage had a vasocentric distribution and viral antigen was abundant in vascular endothelium, tunica media, adventitia, as well as in macrophages lining sinuses. The urinary bladder, uterus, and ovaries were also affected with necrosis and acute inflammation. In these organs, immunohistochemical positive staining was intense in blood vessels, epithelial cells, and ovarian follicles. Approximately 50% of the animals that died or were euthanized in extremis had evidence of viral antigen and histopathologic changes in brain, especially involving meninges and ependymal cells, with lesser changes in the neural parenchyma. A unifying feature of the damage for all affected tissues was necrosis and inflammation of the vasculature, chiefly in arterioles, capillaries, and venules. Inoculation of guinea pigs intraperitoneally with NiV produces a disease with considerable resemblance to the disease in humans, but with reduced pulmonary involvement and marked infection of urinary bladder and the female reproductive tract.     

Antibodies to bluetongue viruses in animals imported into United States zoological gardens.

Three hundred forty-five serum samples from 30 zoological animal species which had been imported into the United States were examined retrospectively for the presence of antibody to bluetongue viruses. Ninety eight (28.4%) were positive for antibody to bluetongue group antigen by the bluetongue agar gel immunodiffusion test. Bluetongue antibodies, most of which were against serotypes exotic to the United States, were detected in 13 animal species from Africa not previously reported to be infected by bluetongue virus. The lack of virus neutralizing antibody to any of the 20 known bluetongue virus types in four of the 28 positive serums studied may indicate the existence of new bluetongue virus serotypes, cross reactions with other orbiviruses or a more rapid decline of neutralizing than precipitating antibody. The possibility of recrudescence of bluetongue virus infection from some inapparently infected zoological animals and existence of a known bluetongue vector (Culicoides variipennis) in the United States would suggest that further assessment of bluetongue in zoological animals be made.     

Endothelial cells as active participants in veterinary infections and inflammatory disorders

Endothelial cells were once viewed as relatively inert cells lining the vasculature. They are now recognized as active and responsive regulators of coagulation, platelet adhesion, fluid homeostasis, wound healing, leukocyte extravasation and vascular tone. Endothelial cells play a key role in the host response to infectious agents by regulating leukocyte trafficking, producing inflammatory cytokines and presenting antigen in association with major histocompatibility class II (MHC II) molecules. A number of infectious agents have a tropism for endothelial cells. Infection of endothelial cells can promote thrombosis, vascular leakage, and increased adherence and emigration of leukocytes. Furthermore, activation of a systemic inflammatory response, in the absence of direct endothelial cell infection, can also lead to endothelial cell dysfunction. The purpose of this review is to highlight the interactions between endothelial cells and infectious or inflammatory agents that contribute to coagulation disturbances, vasculitis and edema. A select group of viral and bacterial pathogens will be used as examples to demonstrate how endothelial cell dysfunction contributes to the pathogenesis of infectious and inflammatory disorders.     

The impact of bluetongue virus on reproduction

Bluetongue virus has been recognized as an important noncontagious, arthropodborne infectious viral disease of ruminants. 24 different serotypes of virus have been recognized world-wide. The most severe clinical disease has been associated with severe clinical disease in sheep and some free ranging wild ruminants. A number of reports have implicated the viruses as causing reproductive disorders in both males and females. The bluetongue related reproductive disorders include early embryonic deaths, abortions, malformed fetal calves or lambs, transient infertility in bulls and rams, and shedding of virus in semen. Recently, bluetongue virus contamination of modified live commercial canine vaccine was associated with abortion and acute death of pregnant bitches. The pathogenesis of these various aspects of reproductive failure are discussed herein.     

Experimental Bluetongue Disease in White-Tailed Deer

Nine white-tailed deer and six sheep were experimentally exposed to the California BTV-8 strain of bluetongue virus. The infections were fatal for seven of the nine deer. An additional deer died from exposure to an isolate of bluetongue virus from bighorn sheep. Clinical signs and lesions of bluetongue in deer were described. The incubation period, signs and lesions of bluetongue and epizootic hemorrhagic disease of deer appear to be similar. Virus isolations were made from the blood and a variety of tissues of exposed deer and identified as bluetongue virus. Neutralizing antibodies were detected in all of the convalescent sera.     

Susceptibility of in vitro produced hatched bovine blastocysts to infection with bluetongue virus serotype 8

ABSTRACT: Bluetongue virus serotype 8 (BTV-8), which caused an epidemic in ruminants in central Western Europe in 2006 and 2007, seems to differ from other bluetongue serotypes in that it can spread transplacentally and has been associated with an increased incidence of abortion and other reproductive problems. For these reasons, and also because BTV-8 is threatening to spread to other parts of the world, there is a need for more information on the consequences of infection during pregnancy. The aim of the present study was to investigate whether hatched (i.e. zona pellucida-free) in vitro produced bovine blastocysts at 8-9 days post insemination are susceptible to BTV-8 and whether such infection induces cell death as indicated by apoptosis. Exposure of hatched in vitro produced bovine blastocysts for 1 h to a medium containing 103.8 or 104.9 TCID50 of the virus resulted in active viral replication in between 25 and 100% of the cells at 72 h post exposure. The infected blastocysts also showed growth arrest as evidenced by lower total cell numbers and a significant level of cellular apoptosis. We conclude from this in vitro study that some of the reproductive problems that are reported when cattle herds are infected with BTV-8 may be attributed to direct infection of blastocysts and other early-stage embryos in utero.