Non-cytocidal infection of keratinocytes by canine distemper virus in the so-called hard pad disease of canine distemper

A late, but not uncommon sequel to canine distemper virus (CDV) infection of dogs is thickening of footpads and nasal planum, the so-called hard pad disease, originally described as vacuolar degeneration of epidermal keratinocytes with inclusion body formation and massive hyperkeratosis. However, in a recent study of footpads of naturally CDV-infected dogs only hyperkeratosis was observed without any of the other changes. Instead, acanthosis was frequently noticed. CDV nucleoprotein was present in the suprabasal keratinocytes and eccrine epithelial glands only. No CDV nucleoprotein was present in basal keratinocytes. This observation in combination with lack of obvious cytocidal changes strongly suggested the possibility of a restricted viral infection with presence of viral mRNA but without protein expression. Therefore, the presence of CDV nucleoprotein mRNA was investigated using in situ hybridization and compared to the localization of the nucleoprotein in footpads of clinically healthy and distemper dogs. Viral nucleoprotein and nucleoprotein mRNA in nearly all cases co-localized to the same compartments and basal keratinocytes did not contain nucleoprotein mRNA. These findings dispute the idea of a restricted viral infection of footpad keratinocytes in dogs with natural CDV infection. Instead, a migration of the virus to the epidermal surface along with the proliferating and differentiating epithelium is the most likely explanation for the lack of virus antigen in basal keratinocytes.     

Immunohistochemical detection of canine distemper virus in haired skin, nasal mucosa, and footpad epithelium: a method for antemortem diagnosis of infection.

A reliable antemortem diagnostic method is needed for determining infection with canine distemper virus (CDV). The utility of immunohistochemical detection of CDV antigen was examined was examined for samples of nasal and footpad epithelium and haired skin in dogs with and without detectable CDV antigen in the lung and/or brain. Tissues from 57 dogs at risk of CDV infection were tested. Viral antigen was found in the lung and/or brain of 28 dogs. Among these dogs, viral antigen was demonstrated in the epithelial cells of the nasal mucosa in 24 of 27 dogs, in the footpad epithelium in 24 of 26 dogs, and in the haired skin of the dorsal neck in 26 of 27 dogs. Among the 29 dogs without CDV antigen in either the lung or brain, 1 dog had positive staining for viral antigen in the skin and nasal mucosa. Biopsies of haired skin of the dorsal neck, which is relatively simple to sample, can be used for immunohistochemical testing for acute and subacute infection with CDV.     

Vesicular stomatitis virus infection and neuropathogenesis in the murine model are associated with apoptosis

This study examines apoptosis and viral neuropathogenesis in a murine model infected with vesicular stomatitis virus (VSV). VSV induces apoptotic cell death in cultured cell lines, raising the possibility that apoptosis of infected neurons and other target cells may contribute to disease and mortality. To determine whether or not VSV induces apoptosis in neural tissues, mice were inoculated intranasally with VSV. At 24, 48, 72, 96, and 120 hours postinfection, brain tissues were assayed for the presence of viral RNA by in situ hybridization and viral antigen by immunohistochemistry. Apoptosis was identified by in situ terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling and electron microscopy. Viral replication and lesions were observed predominantly in central nervous system neurons. Apoptotic cell death was restricted to the same regions of the brain in which infected cells and tissue injury were identified. Results suggest that VSV-induced apoptosis is a mechanism causing cell death, tissue injury, and mortality in VSV-infected mice.     

Distribution of viral antigen and development of lesions after experimental infection with highly virulent bovine viral diarrhea virus type 2 in calves

OBJECTIVE: To correlate tissue distribution with development of lesions after experimental infection with a virulent strain of noncytopathic bovine viral diarrhea virus (BVDV) type 2 in calves. ANIMALS: Ten 14-day-old and two 2-month-old colostrum-deprived calves. PROCEDURE: Calves were intranasally inoculated with BVDV type-2 strain 1373 from an outbreak of clinically severe bovine viral diarrhea (BVD).Two 14-day-old calves served as noninfected controls. Two calves each were euthanatized on postinoculation days 3, 6, and 12, and 1 each on days 8, 9, 13, and 14. Tissues were collected for immunohistologic and histologic examination. RESULTS: Inoculated calves developed nonspecific clinical signs characterized by high fever and decreased numbers of leukocytes and thrombocytes. Viral antigen was detected focally in lymphoid tissues on day 3. On days 6, 8, 9, 12, and 14, viral antigen became increasingly widespread throughout organs and tissues. Viral antigen in lymphoid tissues was associated with severe depletion of all compartments. Lesions in other tissues were not well correlated with distribution of viral antigen. Depletion of lymphoid tissues was observed in a calf on day 13, but viral antigen had been cleared from most tissues and was detected in vascular walls only. CONCLUSIONS AND CLINICAL RELEVANCE: Infection with a virulent BVDV strain resulted in wide dissemination of viral antigen in host tissues. Severe lymphoid depletion developed in lymphoid tissues, whereas viral antigen was generally not associated with lesions in other tissues. Findings suggest that development of lesions in acute BVD is not solely a function of viral replication and is also attributable to host reaction to infection.     

Pathogenesis of type 1 (European genotype) porcine reproductive and respiratory syndrome virus in male gonads of infected boar

The objective of this study was to determine the pathogenesis of experimental infection with a type 1 porcine reproductive and respiratory syndrome virus (PRRSV) by defining the sites of viral replication and apoptosis in male gonads from infected boars for a period of 21 days after intranasal inoculation. Microscopically, hypospermatogenesis and abundant germ cell depletion and death were observed in the testes. Such germ cell death occurs by apoptosis, as determined by a characteristic histological patterns and evidence of massive DNA fragment detected in situ terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) reaction. PRRSV was detected in the testicular tissue of infected boars only. Viral nucleic acid was localized in spermatogonia, spermatocytes and spermatids but not in the vesicular and bulbourethral gland. In serial sections, PRRSV-positive cells did not co-localized with apoptotic cells. TUNEL-positive apoptotic cells were more numerous than PRRSV-positive cells in testicular sections. The present study demonstrated that type 1 PRRSV infects the spermatogonia and their progeny, and induces apoptosis in these germ cells.     

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.     

Localization of classical swine fever virus from chronically infected pigs by in situ hybridization and immunohistochemistry

Classical swine fever (CSF) virus (CSFV) nucleic acid and antigen were detected in 15 pigs with naturally occurring chronic CSF by in situ hybridization and immunohistochemistry. The most consistent and prominent microscopic lesions were perivascular mononuclear cell infiltration and gliosis in the central nervous system of pigs with chronic CSF. Positive cells typically exhibited a dark brown (in situ hybridization) or red (immunohistochemistry) reaction product in the cytoplasm without background staining. A positive signal for both in situ hybridization and immunohistochemistry was detected in mononuclear cells and lymphocytes of lymphoid tissues. Viral nucleic acid was detected in some tissue sections in the absence of viral antigen. The in situ hybridization technique developed in this study was useful for the detection of CSFV RNA in tissues taken from chronically infected pigs and may be a valuable technique for studying the pathogenesis of chronic CSFV infection.     

Virological and serological studies on the role of PI-3 virus, BRSV, BVDV and BHV-1 on respiratory infections of cattle. I. The detection of etiological agents by direct immunofluorescence technique

Nasal cells extracted from nasal swabs obtained from 95 cattle with signs of respiratory disease, out of eleven different herds, were tested for BHV-1, PI-3 virus, BRSV and BVDV using direct immunofluorescence technique. Viral antigen positive samples were detected in seven out of eleven herds examined. Of the 95 individual diseased cattle, 19 were found positive for at least one viral antigen. It was found that especially BHV-1 and PI-3 virus are important causative agents in cattle respiratory disease, both or in combination with other pathogenic agents. Multiple infection in virologically positive herds were observed in six (9.8%) of 61 animals tested. The findings reveal that single or multiple infections of selected viruses may be present in an important range in cattle and that direct immunofluorescence technique as a rapid method, based on the detection of viral antigen in nasal swab samples, is useful to establish the viral aetiology of acute bovine respiratory disease caused by these viruses, particularly in the diagnosis of mixed viral infections.     

Human and ovine lentiviral infections compared

Maedi-visna virus (MVV) of sheep was the first lentivirus to be isolated. The genomic organization of MVV is very similar to that of human immunodeficiency virus (HIV) with several genes regulating the expression of the viral genome. Viral replication is severely restricted in the host and some cells apparently contain the genetic information in a DNA provirus form with little or no expression of viral antigens. This seems to be a major factor in causing the “slowness” of lentiviral infections and the persistence of the virus in the host since the immune system may not recognize the provirus-containing cells. The target cells for HIV and MVV are similar although T4 lymphocytes are not specifically destroyed in maedi-visna. There are also certain similarities in the pathological changes in both diseases, both in the central nervous system, the lungs and the lymphatic system. Although the severe final immunodeficiency state characteristic of AIDS has not been observed in maedi-visna, the basic biological features of the MVV and its interaction with host cells are so similar to HIV infection, that we consider ovine maedi-visna useful animal model for the human lentivirus infections.     

Classical Swine Fever: pathology of bone marrow

Twenty pigs were inoculated with a virulent isolate (Quillota strain) of classical swine fever (CSF) virus to determine the chronological development of lesions in bone marrow. Histopathologic, ultrastructural and immunohistochemical (detection of viral antigen gp55, myeloid-histiocyte antigen, CD3 antigen, and FVIII-rag), and morphometric techniques were employed. Viral antigen was detected from 2 days postinfection (dpi) in stromal and haematopoitic cells, and severe atrophy related to apoptosis of haematopoitic cells was observed. Megakaryocytes (MKs) did not show significant changes in number, but there were important qualitative changes including 1) increased numbers of cloud-nuclei MKs, microMKs, apoptotic MKs, and atypical nucleated MKs and 2) decreased number of typical nucleated MKs. Morphometric study of these cells showed a decrease in cytoplasmic area. MK infection was detected from 2 dpi, but in a small percentage of cells. Myeloid cells showed quantitative changes, with an increase in granulocyte numbers. Apoptosis of lymphocytes and viral infection of erythroblasts were also observed. The main changes in stroma were depletion of T lymphocytes in the middle phase of the experiment and macrophages. Viral infection was also observed in these cells. MK lesions suggest dysmegakaryocytopoiesis, which would aggravate the thrombocytopenia already present and could be responsible for it. Granulocyte changes would lead to the appearance of circulating immature forms, whereas lymphocyte apoptosis in bone marrow would contribute to lymphopenia.     

Pulmonary lesions induced by 3-methylindole and bovine respiratory syncytial virus in calves

Our objectives were to describe the ultrastructural morphogenesis of pulmonary lesions induced by 3-methylindole in 30- to 45-day-old Holstein calves and to determine whether toxic exposure to 3-methylindole exacerbates pulmonary lesions induced by bovine respiratory syncytial virus. Administration of 3-methylindole (0.25 g/kg) to calves resulted in interstitial edema and ultrastructural swelling of type-I alveolar epithelial cells and nonciliated bronchiolar epithelial cells as early as 4 to 6 hours after intraruminal administration. More severe alveolar edema containing protein was associated with swelling of capillary endothelial cells at 2 days after administration. Proliferation of type-II alveolar epithelial cells was first observed at 2 days after 3-methylindole administration, and marked hyperplasia of type-II epithelial cells and nonciliated bronchiolar epithelial cells was evident by 4 days after administration. Pulmonary cytochrome P-450 monooxygenase concentrations decreased significantly (P less than 0.001) by 12 hours after administration and did not increase significantly again by 8 days after administration. Calves were inoculated with bovine respiratory syncytial virus 3 days after administration of 3-methylindole, and pulmonary lesions were assessed 5 days after viral inoculation. Viral replication was demonstrated by fluorescence microscopy for viral antigen or by transmission electron microscopy in ciliated and nonciliated airway epithelial cells. Viral antigen was identified infrequently in alveolar macrophages and in type-II alveolar epithelial cells. 3-Methylindole exposure in calves did not result in more widespread distribution of viral antigen in alveolar tissue of respiratory syncytial virus-inoculated calves or in significant enhancement of viral pneumonia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sites of Persistence of Lumpy Skin Disease Virus in the Genital Tract of Experimentally Infected Bulls

The objectives of this work were to determine the site of persistence of lumpy skin disease virus (LSDV) in bulls shedding the virus in semen for a period longer than 28 days, to determine if the virus is present in all fractions of semen and to study lesions that developed in the genital tract. Six serologically negative postpubertal bulls were experimentally infected with a virulent field isolate of LSDV. The polymerase chain reaction (PCR) was performed on sheath washes, vesicular fluid, supernatant and cell‐rich fractions of semen from day 10 to day 26 postinfection (p.i.). Bulls that were positive by PCR on the whole semen sample collected on day 28 p.i. were slaughtered and tissue samples from their genital tracts submitted for histopathological evaluation, immunoperoxidase staining, virus isolation and PCR. Two of the bulls developed severe lumpy skin disease (LSD) and were found to be shedding viral DNA in their semen on day 28 p.i. Viral DNA was identified in all semen fractions from all bulls, but mostly from the cell‐rich fraction and from the severely affected bulls. The PCR assay was positive on postmortem samples of testes and epididymides from the two severely affected bulls. Virus could be recovered from the testes of these two bulls and from the epididymis of one of them. Immunoperoxidase staining was positive for LSDV staining in sections of testes and epididymides exhibiting necrosis. This study suggests that the testis and epididymis are sites of persistence of LSDV in bulls shedding virus in semen for prolonged periods and revealed that viral DNA is present in all fractions of the ejaculate.     

Bovine viral diarrhea virus type 2-induced meningoencephalitis in a heifer

The brain from a 15-month-old, black female Angus, with a 48-hour history of central nervous system disease, was submitted to the Oklahoma Animal Disease Diagnostic Laboratory. Microscopic findings consisted of acute, multifocal meningoencephalitis, with neuronal degeneration and necrosis and gliosis. Viral isolation yielded noncytopathic bovine viral diarrhea virus (BVDV). Virus genotyping classified the virus as BVDV type 2. Immunohistochemical labeling for BVDV antigens with BVD MAb 3.12F1 clone was prominent in the cytoplasm of neurons, glial cells, ependymal epithelium, perivascular macrophages and spindle cells, smooth muscle cells, and intravascular monocytes of the cerebrum and brain stem. Laboratory results support that tissue alterations occurred as a result of BVDV type 2 infection. In the absence of other clinical signs related to BVDV infection and using the microscopic and laboratory evidence presented, we propose that the BVDV type 2 isolated from this case may represent a neurovirulent strain of the virus. To the best of our knowledge, this is the first report of brain lesions and neuronal viral antigen localization in BVDV genotype 2 viral infection, acquired either congenitally or postnatally.     

Prevalence of equine herpesvirus type 2 (EHV-2) DNA in ocular swabs and its cell tropism in equine conjunctiva

Equine herpes virus 2 (EHV-2), a gamma(2)-herpesvirus, is common in horses of all ages. Its role as a primary pathogen is unclear but there is an association between EHV-2, respiratory disease and keratoconjunctivitis. The purpose of this study was to gain more information on the prevalence of EHV-2 DNA in conjunctival swabs from horses with and without ocular disease and to define the anatomical site and cell type harbouring viral genome or antigen. By polymerase chain reaction (PCR) 22 out of 77 (28.6%) ocular swabs of clinically healthy and only 4 out of 48 (8.3%) samples from diseased horses were positive. To define the main virus reservoir ocular tissue from 13 randomly selected horses without pathological evidence of ocular disease were analysed by nested PCR. In two horses optic nerve, lacrimal gland and conjunctiva, in further two cases lacrimal gland and conjunctiva and in four horses the conjunctiva only were EHV-2 PCR positive. For specifying the target cell we focused on conjunctivae and selected 3 out of 15 clinically healthy slaughterhouse horses positive for EHV-2 by PCR. In situ hybridisation on sections of these paraffin embedded conjunctivae localized viral genome in histiocyte-like cells of the submucosa. Immunohistochemical staining with an EHV-2 or S100 specific polyclonal antiserum demonstrated that Langerhans cells were co-localized in the same region of the sample section where virus positive cells were detected. Furthermore, we concluded that detection of viral antigen revealed a productive virus infection.     

Experimental vesicular stomatitis virus infection of swine: Extent of infection and immunological response

Swine, a natural host species for infection by vesicular stomatitis virus (VSV), were infected with VSV-New Jersey (VSV-NJ) serotype virus obtained from a recent field isolate. Tissues collected from the infected pigs were examined for the presence of infective virus, for viral antigens, and/or for viral nucleic acid. Infective virus could be recovered from tissues near the site of infection for as long as 6 days after the primary infection with VSV. However, no infective virus was recovered following hypothermia induced 11 weeks after infection, or following a secondary challenge with virus 22 weeks after initial infection. Immunofluorescence tests for viral antigens and nucleic acid hybridization assays failed to detect viral antigens or nucleic acids in tissues from which no infective virus could be recovered. Titers of serum-neutralizing antibody peaked 3–5 weeks after infection and then fell slightly until the secondary infection which caused a rapid anamnestic response. Peripheral blood mononuclear cells (PBM) tested 3, 5, 8 or 18 weeks after primary infection all produced readily detectable antigen-specific proliferative responses when cultured with VSV. Thus, although direct tests failed to demonstrate persistence of virus after infection, the humoral and cellular immune response remained elevated for months. Infective VSV was not required to stimulate the proliferative response since UV-inactivated VSV was immunogenic in these in vitro tests. Following primary infection, antigen-specific proliferative responses could be stimulated by several strains of VSV-NJ, but not by VSV-Indiana (VSV-Ind) serotype virus. Secondary infection had relatively little effect on the proliferative response to VSV-NJ strains, but it did cause the PBM to gain responsiveness to VSV-Ind.     

Lesions in clinically healthy cattle persistently infected with the virus of bovine viral diarrhea--glomerulonephritis and encephalitis

Four clinically healthy cattle persistently infected with the virus of bovine viral diarrhea were examined for viral antigen and lesions. Antigen was seen by direct immunofluorescence in cytoplasm of the neurons of the brain and cervical part of the spinal cord, cells and basement membrane of renal glomeruli, reticular cells of lymph nodes and spleen, epithelial cells of small intestinal crypts and renal and testicular tubules, and endothelial cells of blood vessels. Infected neurons were pyknotic and surrounded by astrocytes and macrophages. A few blood vessels in the brains were cuffed with mononuclear cells. Basement membranes of renal glomeruli were irregularly thick with eosinophilic material, and mesangial cells in the glomeruli were plentiful. The virus had a direct effect on some tissues, but was restricted in its cytopathogenicity and was not eliminated by defense mechanisms of the host. Renal glomerular lesions were believed to have an immunologic basis.     

Herpetiform genital lesions in a heifer with mucosal disease

A 14-month-old heifer with a 17-day history of unresponsive bloody diarrhea was necropsied. There were focal, pink-red erosions of the nares and hard palate; ulcers and fissures of the tongue; and multiple ulcerative lesions of the alimentary canal. Interdigital skin of both rear limbs was ulcerated and bleeding; and the margins of the vulva contained punctiform red ulcers. The gross lesions were consistent with mucosal disease. Histopathology and laboratory testing ruled out rinderpest, foot-and-mouth disease, and vesicular stomatitis, and identified bovine virus diarrhea virus to be the cause of this disease. Lesions of the vulva similar to those seen in some stages of infectious pustular vulvovaginitis were negative for bovine herpesvirus-1 and tested positive for bovine viral diarrhea virus antigen by immunohistochemistry.     

Distribution of viral antigen and tissue lesions in persistent and acute infection with the homologous strain of noncytopathic bovine viral diarrhea virus.

Viral distribution and lesions were compared between calves born with persistent infection (PI) and calves acutely infected with the same bovine viral diarrhea virus (BVDV) isolate. Two PI calves from 1 dairy herd were necropsied. The PI viruses from these calves were isolated, characterized by sequencing, and found to be identical. This virus strain, designated BVDV2-RS886, was characterized as a noncytopathic (ncp) type 2 BVDV. To establish acute infections, BVDV2-RS886 was used to inoculate clinically healthy, seronegative calves which were 3 weeks to 3 months old. Nine calves received 10(6)-10(7) tissue culture infective dose of BVDV2-RS886 intranasally. Four additional age-matched animals served as noninfected controls. Infected calves were necropsied at 3, 6, 9, or 13 days postinoculation (dpi). Viral antigen was detected by immunohistochemistry in frozen sections, and lesions were evaluated in hematoxylin eosin-stained paraplast sections. In the PI calves, a wide distribution of viral antigen was found in all tissues and was not associated with lesions. In the acutely infected calves, viral antigen was widespread in lymphoid tissues at 6 dpi but had been mostly eliminated at 9 and 13 dpi. Depletion of lymphoid tissues was seen at 6, 9, and 13 dpi and repopulation at 9 and 13 dpi. In 1 of the calves at 13 dpi, severe arteritis was present in lymph nodes and myocardium. This comparison shows that an ncp BVDV strain that causes no lesions in PI animals is able to induce marked depletion of lymphoid tissues in calves with acute infection. Therefore, the failure to eliminate PI cattle from a herd causes problems not only in pregnant cattle but may also affect other age groups.