In situ hybridization technique for the detection of swine enteric and respiratory coronaviruses, transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV), in formalin-fixed paraffin-embedded tissues.

The in situ hybridization (ISH) technique was developed to detect the swine coronaviruses, transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV), in cell culture and tissue sections from TGEV-or PRCV-infected pigs. The 35S-labeled RNA probes were generated from two plasmids pPSP.FP1 and pPSP.FP2 containing part of the S gene of TGEV. The procedure was first standardized in cell cultures. The radiolabeled pPSP.FP2 probe detected both TGEV and PRCV in virus-inoculated cell cultures, whereas pPSP.FP1 probe detected TGEV but not PRCV. The probe was then used to detect TGEV or PRCV in tissues of pigs experimentally infected with TGEV or PRCV or naturally infected with TGEV. Again, the probes detected TGEV in intestines of experimentally and naturally infected pigs and PRCV in the lungs of experimentally infected pigs. TGEV RNA was detected mainly within the enterocytes at the tips of villi and, less often, within some crypt epithelial cells. PRCV was shown to replicate mainly in the bronchiolar epithelial cells and in lesser amount in type II pneumocytes, type I pneumocytes, alveolar macrophages and bronchial epithelial cells, respectively. ISH has potential applications as a diagnostic test for the detection and differentiation of TGEV and PRCV in tissues and in studies to gain a better understanding of the mechanism of pathogenesis of enteric and respiratory coronavirus infections.

Histological studies of bovine herpesvirus type 4 infection in non-ruminant species.

The pathology of bovine herpesvirus type 4 (BHV-4) infection was studied in cats, rabbits and guinea pigs. Twenty kittens, twenty-two rabbits and ten guinea pigs, some treated with glucocorticoid-were inoculated with a BHV-4 strain of feline origin, via various routes of inoculation (conjunctival, intranasal, peritoneal). Clinical signs were recorded. After euthanizing at different post inoculation days macro- and microscopic changes were observed by necropsy and in hematoxylin-eosin stained histological sections. The presence of the virus in organs was detected by immunohistochemistry and a nested PCR assay. Inclusion bodies and monoclonal antibody-stained cells were found in the conjunctiva, trachea, lungs, spleen and lymph nodes. Most of the lesions were localized to the respiratory and the immune system. The macro- and microscopic lesions and clinical signs were more severe in kittens and guinea pigs. The histological data indicated that cats, especially kittens, were susceptible for BHV-4 and the infection was not confined to the urinary bladder.

An overview of immunological and genetic methods for detecting swine coronaviruses, transmissible gastroenteritis virus, and porcine respiratory coronavirus in tissues.

Transmissible gastroenteritis (TGE) is an enteric disease of swine caused by a coronavirus, designated as transmissible gastroenteritis virus (TGEV). Commonly used methods for TGEV detection include viral isolation and detection of the viral antigen by indirect immunofluorescence (IFA), immunoperoxidase, and immunogold silver staining. Each of these techniques has some advantages and disadvantages. In general IFA and immunohistochemistry are preferred over viral isolation as TGEV isolation is not very reliable because not all field isolates replicate in cell cultures. The diagnosis of TGEV has become more complicated since the emergence of porcine respiratory coronavirus (PRCV). PRCV is believed to be a TGEV mutant, and can not be easily differentiated from TGEV by immunological tests. Nucleic acid probes and polymerase chain reaction (PCR) have successfully been used to detect and differentiate these viruses. These techniques can detect viral nucleic acids in the specimen but do not provide information on the cell types infected by these viruses. Recently we have developed isotopic and nonisotopic in situ hybridization techniques (ISH) for the detection of these viral nucleic acids in formalin-fixed paraffin-embedded tissues. Furthermore, this procedure can differentiate between TGEV- and PRCV-infected cells. By ISH, TGEV is detected in the mature absorptive enterocytes of tissues infected by TGEV and the crypt epithelial cells are also infected but to a lesser extent. For PRCV, the main infected cells are epithelial cells of the bronchioles, type II pneumocytes, and alveolar and septal macrophages. ISH is an excellent tool for studying molecular pathogenesis of these two viruses especially when used in combination with immunohistochemistry.

Pathogenesis of ovine pseudorabies (Aujeszky's disease) following intratracheal inoculation.

Pseudorabies virus was inoculated intratracheally into sheep to investigate the pathogenesis of pseudorabies virus infection. Clinical signs of pyrexia, depression, frequent swallowing, facial fasciculations, chorea, excessive salivation, mild tympanites, labored breathing and focal pruritus were followed by death Macroscopic lesions were severe focal facial trauma, petechiae in cervicothoracic ganglia and dilated esophaguses. The medulla oblongata and the trigeminal, cranial cervical, cervicothoracic and parabronchial ganglia contained pseudorabies virus and pronounced nonsuppurative inflammatory changes. The neural distribution of lesions and virus suggests that the virus travelled from the respiratory mucosa to the central and sympathetic nervous system by two routes: 1) in the vagus and glossopharyngeal nerves to the medulla oblongata and 2) in the postganglionic fibers to the sympathetic ganglia. The presence of virus in the nasal mucus indicated that horizontal transmission of pseudorabies virus may occur among sheep.

In vitro lymphocyte proliferative responses and gamma-interferon production as measures of cell-mediated immunity of cattle exposed to Pasteurella haemolytica.

Cell-mediated immune mechanisms may play a role in the pathogenesis and prevention of pneumonia in cattle caused by Pasteurella haemolytica serotype A1. To determine the circumstances required to stimulate and identify cell-mediated immune responses, calves were vaccinated with a commercial P. haemolytica bacterin or a live commercial P. haemolytica vaccine, or were infected intratracheally with virulent P. haemolytica. All calves were challenge-exposed intratracheally with P. haemolytica 31 d after vaccination or prior infection. Peripheral blood mononuclear cells and mediastinal and superficial cervical lymph node cells were stimulated with antigens prepared from P. haemolytica to evaluate in vitro proliferative responses and gamma-interferon production as measures of cell-mediated immunity. Strong proliferative responses and gamma-interferon production were detected in lymph node cells from calves vaccinated with the live vaccine and from infected calves, especially in response to stimulation with an outer membrane protein preparation from P. haemolytica. Greater proliferative responses and gamma-interferon production were associated with the lymph node nearer the site of bacterin administration (superficial cervical lymph node) or the site of infection (mediastinal lymph node), whereas greater proliferative responses and gamma-interferon production were associated with the more distant lymph node (mediastinal lymph node) in calves vaccinated with the live vaccine. Neither proliferative responses nor gamma-interferon production were detected in peripheral blood mononuclear cells from calves that were vaccinated for or infected with P. haemolytica. Antileukotoxin antibody titers were determined by a serum neutralization assay, and protection against pneumonic lesions was more closely correlated with antileukotoxin antibody responses than with lymphocyte proliferation or gamma-interferon responses.

Attempts to produce salivary cysts in the dog.

Attempts were made to create salivary cysts by ligation of the mandibular salivary duct at the angle of the mandible and near the frenulum of the tongue, by rupture of the mandibular duct at the angle of the mandible and near the frenulum of the tongue, and by direct trauma to the mandibular salivary gland. Cysts were not formed as a result of the experimental procedures, but observations are made on the results obtained.

Pathologic effects of intrauterine deposition of pseudorabies virus on the reproductive tract of swine in early pregnancy.

Pseudorabies virus was inoculated into the uterus of 15 gilts within 6 hours after natural breeding, and gilts were necropsied at postbreeding days (PBD) 3, 6, 10, 14, and 28; 3 control gilts were treated similarly, except for inoculation with pseudorabies virus and were necropsied at PBD 6, 10, and 14. Tissues were collected for virus isolation, fluorescent antibody staining, and histopathologic examination. Pseudorabies virus was isolated from the reproductive tract up to day 14. Lesions in the reproductive tract consisted of multifocal to diffuse lymphohistiocytic vaginitis and endometritis, and lymphoplasmacytic aggregates in the corpora lutea. Multiple ulcers were seen in the vagina or endometrium of several gilts at PBD 3, 6, and 10. Corpora lutea of 1 gilt were necrotic at PBD 14 and contained large numbers of inflammatory cells. Focal aggregates of lymphocytes and plasma cells were seen in vagina and endometrium of 3 gilts and in the ovary of 1 gilt at day 28.

Lesions and localization of viral antigen in tissues of cattle with experimentally induced or naturally acquired mucosal disease, or with naturally acquired chronic bovine viral diarrhea.

Tissues from cattle that died of experimentally induced mucosal disease (n = 3), naturally acquired mucosal disease (n = 6), or naturally acquired chronic bovine viral diarrhea (n = 4) were examined. Consistent findings were lymphocytic depletion of lymphoid tissues, degeneration of myenteric ganglion cells, and mild adrenalitis. Intracytoplasmic viral antigen was detected in myenteric ganglia and in endocrine glandular cells. Noncytopathic virus was isolated from all cattle, and cytopathic virus was isolated from 12 of 13 cattle.

Effects of vaccination on lesion development in pseudorabies virus-challenged swine.

The effects of vaccination against pseudorabies virus (PRV) infection were studied in 93 barrows. Commercial attenuated and inactivated PRV vaccines were used. The protection provided was evaluated by the comparison of clinical signs, gross lesions, and microscopic lesions among vaccinated and nonvaccinated pigs after challenge exposure with a sublethal dose of PRV. Histopathologic changes are described for the CNS and other tissues. Neither the modified live- nor the inactivated-PRV vaccines prevented clinical signs or microscopic lesions in pigs after challenge exposure. However, both vaccines diminished the severity of clinical signs and microscopic lesions in the animals for a period of up to 8 months after they were vaccinated. Protection against clinical signs and microscopic lesions was absent in the pigs 10 months after the vaccination.

Experimental infection of lambs with bovine respiratory syncytial virus and Pasteurella haemolytica: clinical and microbiologic studies.

Four-week-old lambs were inoculated transtracheally with respiratory syncytial virus (RSV), Pasteurella haemolytica, or RSV and P haemolytica. When given in combination, RSV administration preceded P haemolytica by 3 or 5 days. Lambs inoculated with P haemolytica or RSV developed a mild respiratory tract disease accompanied by a transient pyrexia in a few lambs. By 24 hours after inoculation of bacteria, all lambs inoculated with RSV and P haemolytica were listless, reluctant to move, and exhibited hyperpnea and dyspnea. Most lambs had pyrexia and a few coughed and had serous nasal discharge. These clinical signs persisted for 3 to 4 days and were more pronounced in those inoculated with P haemolytica 5 days after RSV than in those inoculated with P haemolytica 3 days after RSV. Respiratory syncytial virus was isolated from 8 of 15 inoculated lambs and P haemolytica was isolated from 12 of 15 inoculated lambs. All lambs responded serologically to RSV, but none responded to P haemolytica.

Duck plague in gnotobiotic ducks.

Four-week-old gnotobiotic and conventional ducks were inoculated orally with duck plague virus. Both groups of ducks died on the third and fourth day after inoculation. Gross and microscopic lesions of duck plague were similar in gnotobiotic and conventional ducks, indicating the synergistic action of species of Salmonella and Pasteurella was not essential for development of lesions.

Lawsonia intracellularis-like organism infection in a miniature foal.

A 7-month-old foal was admitted to the hospital with a history of lethargy, weight loss, mild diarrhea, and anorexia. A diagnosis of proliferative enteritis caused by Lawsonia intracellularis-like organisms was made after necropsy and histologic examination of the small intestine. Although infection with L intracellularis-like organisms is a rare cause of enteritis in foals, it should be considered in the differential diagnosis, especially if the foal was housed in the proximity of pigs or pig feces. Antemortem diagnosis remains challenging because isolation of the organism in fecal material requires cell culture, and histologic evaluation of intestinal biopsy specimens may be unrewarding because of the lack of information regarding the frequency and distribution of lesions in horses. Alternatively, use of immunochemical stain, dot-blot technique, and polymerase chain reaction provide specific diagnostic tests that can be performed on fecal material. Postmortem diagnosis relies on histologic examination of infected tissues and use of immunofluorescence and polymerase chain reaction.

Hungary remains free of scrapie and bovine spongiform encephalopathy (BSE).

Brains from 44 sheep and 43 cattle with CNS clinical signs not due to rabies virus infection were collected from diagnostic institutes throughout Hungary. The brains were examined for histological lesions diagnostic of scrapie/bovine spongiform encephalopathy (BSE) and all were found to be negative. These findings confirm that Hungary remains free of scrapie and BSE.