Jan Shivers

Detection of bovine viral diarrhea virus by TaqMan reverse transcription polymerase chain reaction.

Detection and elimination of calves and cows persistently infected with bovine viral diarrhea virus (BVDV) is important for the control of this pathogen. Historically, BVDV detection involved cell culture isolation followed by virus detection through immunofluorescence or immunoperoxidase monolayer assay (IPMA) methods. More recently, immunohistochemistry (IHC) has been added as a routine test for BVDV detection. The detection of BVDV by gel-based reverse transcription polymerase chain reaction (RT-PCR) is more sensitive and rapid than by cell culture isolation, but test results can be compromised by sample contamination during nucleic acid amplification. This study was designed to develop a closed-tube format of BVDV nucleic acid amplification and detection, TaqMan® RT-PCR. The results of this new technique were compared with those obtained with virus isolation, IPMA, and IHC. With TaqMan® RT-PCR, BVDV was detected in many samples negative by IPMA, IHC, and virus isolation with the exception of 1 sample that was positive by IHC. TaqMan® RT-PCR in a closed-tube format offers a rapid, economical, high volume, and sensitive method for BVDV detection without the concerns of amplified cDNA product contamination associated with open-tube gel-based PCR tests.

Porcine reproductive and respiratory syndrome virus infection in neonatal pigs characterised by marked neurovirulence

Neonatal pigs from three herds of pigs were somnolent and inappetent and had microscopic lesions characterised by severe meningoencephalitis, necrotic interstitial pneumonia and gastric muscular inflammation. Porcine reproductive and respiratory syndrome virus (PRRSV) infection was diagnosed and confirmed by virus isolation, fluorescent antibody examination of frozen lung sections, serology, immunohistochemistry and in situ hybridisation. Each herd had a history of PRRSV infection and was using or had used a modified-live vaccine. The isolates from the affected pigs were genetically distinct from the modified-live vaccine strain of the virus when compared by restriction enzyme analysis and nucleotide sequencing of PRRSV open reading frames 5 and 6. The virus was identified in macrophages or microglia of brain lesions by immunohistochemical staining of brain sections with an anti-PRRsv monoclonal antibody and an anti-macrophage antibody. The replication of the virus in the brain was verified by in situ hybridisation. The meningoencephalitis induced by the virus in pigs from each of the herds was unusually severe and the brain lesions were atypical when compared with other descriptions of encephalitis induced by the virus, which should therefore be considered as a possible diagnosis for neonatal pigs with severe meningoencephalitis. In addition, field isolates of the virus which are capable of causing disease can emerge and coexist with modified-live vaccine virus in some pig herds.

Pathologic Findings in Red-Tailed Hawks (Buteo jamaicensis) and Cooper's Hawks (Accipiter cooperi) Naturally Infected with West Nile Virus

Abstract Carcasses of 13 red-tailed hawks (RTHAs) and 11 Coopers hawks (COHAs) were tested for West Nile virus (WNV) using WNV-specific reverse transcriptase–polymerase chain reaction (RT-PCR) on fresh brain tissue and WNV-specific immunohistochemistry (IHC) on various organs. Ten COHAs (91%) and 11 RTHAs (85%) were positive for WNV RNA by RT-PCR. All 11 COHAs (100%) and 10 RTHAs (77%) were positive for WNV antigen by IHC. A triad of inflammatory lesions, including chronic lymphoplasmacytic and histiocytic encephalitis, endophthalmitis, and myocarditis, was common in both species. In COHAs, the heart (54%), cerebrum (50%), and eye (45%) were the organs that most commonly contained WNV antigen. The amount of WNV antigen was usually small. In RTHAs, the kidney (38%), cerebrum (38%), cerebellum (38%), and eye (36%) were the organs most commonly containing WNV antigen. Unlike COHAs, larger amounts of WNV antigen were present in the cerebrum of RTHAs. WNV antigen was detected in similar cell populations in both species, including neurons of brain, spinal cord, and retina, pigmented epithelial cells of the retina, epithelial cells of renal medullary tubules, cardiomyocytes, endothelial cells and smooth muscle cells of arteries, dendritic cells of splenic lymph follicles, exocrine pancreatic cells, adrenal cells, and keratinocytes of the skin. The study presents strong evidence that WNV can cause a chronic fatal disease in RTHAs and COHAs. The lesion distribution of WNV infection in both species is variable, but inflammatory lesions are common, and a triad of lesions including encephalitis, myocarditis, and endophthalmitis is indicative of WNV infection in both species.

Identification of Porcine Reproductive and Respiratory Syndrome Virus in Semen and Tissues from Vasectomized and Nonvasectomized Boars

Previous studies have indicated that porcine reproductive and respiratory syndrome virus (PRRSV) can be identified in and transmitted through boar semen. However, the site(s) of replication indicating the origin of PRRSV in semen has not been identified. To determine how PRRSV enters boar semen, five vasectomized and two nonvasectomized PRRSV-seronegative boars were intranasally inoculated with PRRSV isolate VR-2332. Semen was collected three times weekly from each boar and separated into cellular and cell-free (seminal plasma) fractions. Both fractions were evaluated by reverse transcriptase nested polymerase chain reaction (RT-nPCR) for the presence of PRRSV RNA. Viremia and serostatus were evaluated once weekly, and boars were euthanatized 21 days postinoculation (DPI). Tissues were collected and evaluated by RT-nPCR, virus isolation (VI), and immunohistochemistry to identify PRRSV RNA, infectious virus, or viral antigen, respectively. PRRSV RNA was identified in semen from all vasectomized and nonvasectomized boars and was most consistently found in the cell fraction, within cells identified with a macrophage marker. Viral replication as determined by VI was predominately found within lymphoid tissue. However, PRRSV RNA was widely disseminated throughout many tissues, including the reproductive tract at 21 DPI. These results indicate that PRRSV can enter semen independent of testicular or epididymal tissues, and the source of PRRSV in semen is virus-infected monocytes/macrophages or non-cell-associated virus in serum. PRRSV-infected macrophages in semen may result from infection of local tissue macrophages or may originate from PRRSV-infected circulating monocytes or macrophages.

Pathologic and Immunohistochemical Findings in Goshawks (Accipiter gentilis) and Great Horned Owls (Bubo virginianus) Naturally Infected with West Nile Virus

Abstract The carcasses of 25 great horned owls and 12 goshawks were investigated for West Nile virus (WNV) infection by immunohistochemistry (IHC) performed on various organs, including brain, spinal cord, heart, kidney, eye, bone marrow, spleen, liver, lungs, pancreas, intestine, and proventriculus, using a WNV-antigen–specific monoclonal antibody and by WNV-specific reverse transcriptase-polymerase chain reaction (RT-PCR), performed on fresh brain tissue only. WNV infection was diagnosed by IHC in all owls and all goshawks. WNV-specific RT-PCR amplified WNV-RNA in the brain of all goshawks but only 12 owls (48%). Cachexia was a common macroscopic finding associated with WNV infection in owls (76%). Myocarditis was occasionally macroscopically evident in goshawks (33%). Microscopically, inflammatory lesions, including lymphoplasmacytic and histiocytic encephalitis, myocarditis, endophthalmitis, and pancreatitis were present in both species but were more common and more severe in goshawks than in owls. The most characteristic brain lesion in owls was the formation of glial nodules, in particular in the molecular layer of the cerebellum, while encephalitis affecting the periventricular parenchyma of the cerebral cortex was common in the goshawks. In owls, WNV-antigen–positive cells were present usually only in very small numbers per organ. Kidney (80%), heart (39%), and cerebellum (37%) were the organs that most commonly contained WNV antigen in owls. WNV antigen was frequently widely distributed in the organs of infected goshawks, with increased amounts of WNV antigen in the heart and the cerebrum. Spleen (75%), cerebellum (66%), heart (58%), cerebrum (58%), and eye (50%) were often WNV-antigen positive in goshawks. In contrast with the goshawks, WNV antigen was not present in cerebral and retinal neurons of owls. WNV infection appears to be capable of causing fatal disease in great horned owls and goshawks. However, the distribution and severity of histologic lesions, the antigen distribution in the various organs, and the amount of antigen varied among both species. Therefore, the diagnostician may choose organs for histology and immunohistochemistry as well as RT-PCR depending on the investigated species in order to avoid false-negative results.

Pathological and Immunohistochemical Findings in American Crows (Corvus Brachyrhynchos) Naturally Infected with West Nile Virus

Twenty-one American crows were identified as being West Nile virus (WNV) infected by WNV-specific reverse transcriptase-polymerase chain reaction (RT-PCR) performed on fresh brain tissue (cerebrum and cerebellum of 16 crows) or by WNV-specific immunohistochemistry of various organs (21 crows). Consistent gross lesions attributable to WNV infection were not detected. Common histological lesions included necrosis of spleen and bone marrow. West Nile virus antigen was consistently detected in heart and kidney (100%). In addition, bone marrow (92%), duodenum (89%), proventriculus (87%), liver (86%), lung (85%), spleen (80%), pancreas (61%), and brain (45%) contained WNV antigen-positive cells. Infected cells included cardiomyocytes; neurons; endothelial cells and vascular smooth muscle cells; hematopoietic cells of bone marrow; and macrophages of spleen, liver (Kupffer cells), and lungs. Epithelial cells of renal tubules, duodenum, pancreas, and proventriculus were also infected. The diagnostic histopathologist should consider WNV infection in crows in the absence of any inflammatory lesions. Immunohistochemistry of heart and kidney is as reliable in detecting WNV infection in American crows as RT-PCR of fresh brain tissue.

Antigen tissue distribution of Avian bornavirus (ABV) in psittacine birds with natural spontaneous proventricular dilatation disease and ABV genotype 1 infection

Tissues of 10 psittacines from aviary 1 (“case birds”) and 5 psittacines from different aviaries were investigated for the presence of Avian bornavirus (ABV) antigen by immunohistochemistry using a polyclonal serum specific for the viral nucleocapsid (N) protein. Seven of 10 case birds had clinical signs, and necropsy findings consistent with proventricular dilatation disease (PDD) while 3 case birds and the 5 birds from other aviaries did not exhibit signs and lesions of this disease. In birds with clinical signs of PDD, ABV antigen was largely limited to neuroectodermal cells including neurons, astroglia, and ependymal cells of the central nervous system, neurons of the peripheral nervous system, and adrenal cells. ABV antigen was present in the nuclei and cytoplasm of infected cells. In 2 case birds that lacked signs and lesions of PDD, viral antigen had a more widespread distribution and was present in nuclei and cytoplasm of epithelial cells of the alimentary and urogenital tract, retina, heart, skeletal muscle, and skin in addition to the mentioned neuroectodermal cells. ABV RNA was identified by reverse transcription polymerase chain reaction (RT-PCR) in tissues of all 7 case birds available for testing from aviary 1, including 4 birds with PDD lesions and the 3 birds without PDD lesions. Sequencing and phylogenetic analysis indicated the presence of ABV genotype 1 in all cases. Findings further substantiate a role of ABV in PDD of psittacine bird species.

Acid Phosphatase Activity of Chondroclasts from Fusarium-Induced Tibial Dyschondroplastic Cartilage

Tibial dyschondroplasia was induced in broiler chickens by oral administration of fusarochromanone, the toxic component of Fusarium equiseti. In two experiments, the activity of acid phosphatase in chondroclasts was assessed histochemically. Chicks were examined at 7, 14, 21, and 28 days of treatment in Expt. 1 and at 2, 4, and 6 days of treatment in Expt. 2. The staining for acid phosphatase was consistently lower in fusarochromanone-treated chicks after 2 days of treatment than in age-matched controls, and the onset of this difference corresponded to the onset of lesions. However, the decrease in acid phosphatase staining intensity was significant only at day 21 in Expt. 1 and at day 6 in Expt. 2. The deficiency of acid phosphatase in chondroclasts was judged to be of insufficient magnitude to account for the accumulation of growth plate cartilage that characterizes tibial dyschondroplasia.

Immunohistochemical Detection of Lymphocyte Subpopulations in the Tarsal Joints of Chickens with Experimental Viral Arthritis

We characterized the lymphocytes in the tarsal joint synovium of chickens inoculated with an arthrotropic strain of avian reovirus. Cryostat sections of whole joints taken from 2 days to 35 days postinoculation were analyzed using monoclonal antibodies directed against B lymphocytes, T lymphocytes, and chicken Ia antigen. Plasma cells were morphologically identified using stained sections of whole joints. Time-dependent changes were found in the type and number of positively staining cells. Synoviocytes and cells with a dendritic morphology stained positive for Ia in normal joint sections. T cells, mostly CD8 positive, were present in low numbers in acute phase arthritis (2-6 days postinfection) in the perivascular and superficial regions of the synovium. Subacute arthritis (8-14 days postinfection) was characterized by increased numbers of CD4 and CD8 T cells in the perivascular and superficial regions. The perivascular T cells began to organize into aggregates, with IgM-positive B cells and plasma cells on the periphery of these aggregates. Some CD8-positive cells were detected on the surface of the articular cartilage. Cells staining positively for Ia were not lymphocytes. Chronic arthritis (> 14 days postinfection) was characterized by large numbers of T cells in the perivascular and superficial regions, with the CD4-positive T cells found primarly in the lymphoid aggregates of the perivascular regions. IgM-positive B cells were fewer, but more plasma cells, few of which stained positive for IgM, were present. Lymphocytes in chronic arthritis stained positively for Ia. These data suggest that the types, numbers, and activation level of lymphocytes present in the tarsal joints are similar but not identical to those seen in rheumatoid arthritis.

Major Histocompatibility Complex I and II Expression and Lymphocytic Subtypes in Muscle of Horses with Immune-Mediated Myositis

Background Major histocompatibility complex (MHC) I and II expression is not normally detected on sarcolemma, but is detected with lymphocytic infiltrates in immune‐mediated myositis (IMM) of humans and dogs and in dysferlin‐deficient muscular dystrophy. Hypothesis/Objectives To determine if sarcolemmal MHC is expressed in active IMM in horses, if MHC expression is associated with lymphocytic subtype, and if dysferlin is expressed in IMM. Animals Twenty‐one IMM horses of Quarter Horse‐related breeds, 3 healthy and 6 disease controls (3 pasture myopathy, 3 amylase‐resistant polysaccharide storage myopathy [PSSM]). Methods Immunohistochemical staining for MHC I, II, and CD4+, CD8+, CD20+ lymphocytes was performed on archived muscle of IMM and control horses. Scores were given for MHC I, II, and lymphocytic subtypes. Immunofluorescent staining for dysferlin, dystrophin, and a‐sarcoglycan was performed. Results Sarcolemmal MHC I and II expression was detected in 17/21 and 15/21 of IMM horses, respectively, and in specific fibers of PSSM horses, but not healthy or pasture myopathy controls. The CD4+, CD8+, and CD20+ cells were present in 20/21 IMM muscles with CD4+ predominance in 10/21 and CD8+ predominance in 6/21 of IMM horses. Dysferlin, dystrophin, and a‐sarcoglycan staining were similar in IMM and control muscles. Conclusions and clinical importance Deficiencies of dysferlin, dystrophin, and a‐sarcoglycan are not associated with IMM. Sarcolemmal MHC I and II expression in a proportion of myofibers of IMM horses in conjunction with lymphocytic infiltration supports an immune‐mediated etiology for IMM. The MHC expression also occured in specific myofibers in PSSM horses in the absence of lymphocytic infiltrates.