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.

Transmissible gastroenteritis virus induced apoptosis in swine testes cell cultures.

Transmissible gastroenteritis virus (TGEV) is a coronavirus which causes severe gastroenteritis and atrophy of intestinal villous epithelial cells in piglets. However, the mechanism of cell death caused by TGEV is not known. In this study, we report that TGEV induces cell death by apoptosis. TGEV-induced apoptosis was demonstrated by agarose gel electrophoresis, electron microscopy, and terminal deoxytransferase digoxigenin-dUTP nick end labeling (TUNEL). Double labeling experiment confirmed the result from electron microscopy and showed that most of the apoptotic cells were bystander cells as they were negative for TGEV nucleic acids. Results of this study indicate that TGEV induces apoptosis in vitro and that most of the cells undergoing apoptosis are bystander cells, thus amplifying the cytopathic effect of TGEV.

A pneumo-virulent United States isolate of porcine reproductive and respiratory syndrome virus induces apoptosis in bystander cells both in vitro and in vivo.

Evidence of apoptosis was detected for the United States porcine reproductive and respiratory syndrome virus (PRRSV) in ATCC CRL11171 cells inoculated with strain ATCC VR2385 and in the tissues of pigs infected with the same strain. Apoptosis was detected by agarose gel electrophoresis, transmission electron microscopy and terminal deoxytransferase dUTP nick end labelling (TUNEL) techniques. By electron microscopy and double-labelling techniques, apoptosis was detected primarily in uninfected bystander cells in the continuous cell line rather than the PRRSV-infected cells. In the lungs, the apoptotic cells were predominantly alveolar and pulmonary intravascular macrophages, and mononuclear cells in the alveolar septa. In the lymph nodes, the apoptotic cells were predominantly tingible body macrophages and mononuclear cells. The induction of apoptosis in a large number of mononuclear cells in the lungs and lymph nodes appears to be a mechanism of PRRSV pathogenesis and might be an explanation for a dramatic reduction in the number of alveolar macrophages and circulating lymphocytes and monocytes in PRRSV-infected pigs.

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.

Rapid in situ hybridization technique for the detection of ribonucleic acids in tissues using radiolabelled and fluorescein-labelled riboprobes.

In situ hybridization (ISH) is a useful diagnostic and research tool, but is also time consuming. This study was conducted to determine if a rate enhancement hybridization (REH) buffer, developed for membrane hybridization, could be used to decrease hybridization time for ISH. Tissue from swine with an enteric disease produced by a swine coronavirus, transmissible gastroenteritis virus (TGEV), was used as a model to standardize hybridization conditions for a rapid ISH technique. Small intestinal sections from pigs experimentally and naturally infected with TGEV were hybridized for various times at 52 degrees C and 70 degrees C with a radiolabelled or a fluorescein-labelled RNA probe in a standard hybridization or a REH buffer. Viral RNA was detected in intestines from as early as 30 min of hybridization by using both buffers with the radiolabelled probe; however, the signal was stronger with the REH buffer. With the fluorescein-labelled probe, viral RNA was detected in virus-infected cells of the intestines after 30 min of hybridization by using the REH buffer. Signal intensity was greater with the REH buffer than with the standard hybridization buffer when compared at each hybridization time and hybridization temperature using both radiolabelled and fluorescein-labelled probes. With the REH buffer, hybridization signal intensity was greater at 70 degrees C than at 52 degrees C for both probes. The best results were obtained when small intestinal sections were hybridized at 70 degrees C for 2 h using a radiolabelled or a fluorescein-labelled probe diluted in the REH buffer. The fluorescein-labelled RNA probe with REH buffer resulted in a minimal non-specific signal when compared with the radiolabelled probe. These studies demonstrated that the REH buffer can be used to decrease the time of ISH for the detection of viral RNA. This rapid ISH technique should have broad applications in the utilization of probe technology in diagnostics and research for the detection of target ribonucleic acids in situ

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.

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.

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.

Infected cell types in ovine lung following exposure to bovine respiratory syncytial virus.

Sixteen adult sheep (ten females, six males obtained from a closed flock at National Animal Disease Center, Ames, IA) were experimentally infected with bovine respiratory syncytial virus strain 375 (BRSV), and lung tissues were stained for viral antigen. Two infected sheep were euthanatized at each of the following post-inoculation times: 12, 24, 36, 48, 72, 96, 144, and 192 hours. Lung, nasal turbinates, trachea, right cranial bronchial and mediastinal lymph nodes, liver, and spleen were collected for histologic evaluation. An indirect immunoperoxidase technique was performed on routine paraffin-embedded sections of lung tissue, trachea, turbinates, and bronchial and mediastinal lymph nodes to determine the location of the BRSV antigen. For lung tissue from each sheep 400 light microscopic fields at 160x magnification were examined for staining for BRSV antigen. Lung tissue was also collected for virus and bacterial isolation. Daily serum samples were taken for determination of anti-BRSV titers. Severe respiratory disease was not produced in any sheep. Bovine respiratory syncytial virus was isolated from lung tissue collected from all sheep up through 144 hours post-inoculation. At 12 hours post-inoculation (case No. 2) respiratory syncytial virus antigen was detected in bronchiolar epithelium and a mononuclear cell within an alveolar space. Lung tissue from the sheep necropsied between 24 and 144 hours post-inoculation (case Nos. 3-14) contained BRSV antigen in bronchiolar epithelium, type I pneumocytes, type II pneumocytes, alveolar macrophages, and mononuclear cells within alveolar spaces. Macrophages staining for viral antigen were rare. Bronchiolar and type I epithelial cells comprised the majority of infected cells.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

Experimental primary postnatal bovine viral diarrhea viral infections in six-month-old calves.

Eight clinically healthy calves were inoculated intranasally, four with either noncytopathic or four with cytopathic bovine viral diarrhea virus, and were necropsied 5 or 12 days post-inoculation. The most frequent gross lesion associated with noncytopathic or cytopathic viral infection was proximal colonic mural edema. Consistent microscopic findings were acute to subacute tracheitis, mild enterocolitis with edema, petechial hemorrhages of mesenteric lymph nodes with mild follicular lymphocytic depletion, and paracortical lymphocytic hyperplasia. At necropsy, cytopathic virus was recovered from 4/4 calves and noncytopathic virus was isolated from 2/4 calves. Neutralizing antibodies to noncytopathic bovine viral diarrhea virus were detected in the two calves from which noncytopathic virus was not recovered. Immunohistochemical analysis of lymphoid tissues demonstrated a small, randomly distributed population of mononuclear cells that contained bovine viral diarrhea viral antigen in 7/8 calves.

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.

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.

Testicular changes observed in boars following experimental inoculation with pseudorabies (Aujeszky's) virus.

Four boars were inoculated intranasally with pseudorabies virus to determine if microscopic testicular changes occurred as a result of infection. Testicular biopsies and semen samples were taken at two, four and six weeks postinoculation and the boars were castrated immediately after the last sample collection. Testicular samples and semen were cultured to determine if the virus was present. Pseudorabies virus was not isolated from the semen or testicular tissue. Virus was isolated from trigeminal ganglia at necropsy and from nasal swabs taken one day after castration. Consequently, a time of high risk for shed of the virus from clinically normal carrier animals is immediately following castration. Gross changes were not observed in testicular tissues and microscopic changes in the testicles were the result of biopsy. Lesions consistent with pseudorabies virus infection were observed in the central nervous system of all inoculated boars. Temporary lowered fertility may result from the effects of elevated body temperature on spermatogenesis during acute clinical disease. However, it appears that the strain of pseudorabies virus used, lacked the ability to infect and/or replicate in the boars' reproductive tracts.

The effect of pseudorabies (Aujeszky's) virus infection on young mature boars and boar fertility.

This study was designed to determine the effects of experimental inoculation with pseudorabies virus on the reproductive tracts of young adult boars. Pseudorabies virus was inoculated intranasally into 12 boars and intrapreputially into four boars. All animals seroconverted after nasal or preputial inoculation. Semen abnormalities were observed 21 days postinoculation with partial recovery by 50 days postinoculation. Virus was isolated from the preputial sheath of two intrapreputially inoculated boars 12 days postinoculation. It was concluded that pseudorabies virus infection can be established via preputial inoculation and that decreased spermatogenesis and infertility can result.

The effects of challenge on the humoral and cellular immune responses in pseudorabies vaccinated swine.

The effects of challenge exposure on the humoral and cellular immune responses in pseudorabies vaccinated swine were studied in 84 barrows. The pigs were divided into seven groups and challenge exposed to a virulent strain of pseudorabies virus on months 1, 3, 5, 8, 10, 12 and 14 after vaccination. The pigs were vaccinated with commercial attenuated and inactivated pseudorabies virus vaccines. The protection conferred by vaccination was equally effective with both types of vaccines. The levels of cellular and humoral immunity after challenge exposure in pigs vaccinated with either type of vaccine were similar. The cell-mediated immune response can be effectively used for the early detection of pigs exposed to pseudorabies virus. Virus isolation attempts from the brain and spleen in most of the vaccinated pigs were unsuccessful.