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An adult male Ruwenzori long-haired fruit bat ( Rousettus lanosus) presented for lethargy and unthriftiness. Physical examination revealed cranial alopecia, mandibular ulceration, and dehydration. Supportive care and antibiotic therapy were initiated. The bat was found dead 3 days after presentation. Necropsy revealed alopecia on the head and body, exposed dried bone on the rostral tip of the mandible, and excessive clear pleural fluid. Lungs were congested and contained miliary white foci disseminated randomly throughout the parenchyma. Subcutaneous, intra-thoracic, and intra-abdominal adipose depots were minimal. Histologic examination of skin and lung revealed the presence of algal-like organisms morphologically consistent with Prototheca spp. Polymerase chain reaction amplification revealed >99% sequence identity match with Prototheca zopfii. Protothecosis has been previously reported in a single bat, Lyle's flying fox ( Pteropus lylei), in Switzerland, but definitive protothecal speciation was not possible.

Unlabelled: Current vaccines do not provide sufficient levels of protection against divergent porcine reproductive and respiratory syndrome virus (PRRSV) strains circulating in the field, mainly due to the substantial variation of the viral genome. We describe here a novel approach to generate a PRRSV vaccine candidate that could confer unprecedented levels of heterologous protection against divergent PRRSV isolates. By using a set of 59 nonredundant, full-genome sequences of type 2 PRRSVs, a consensus genome (designated PRRSV-CON) was generated by aligning these 59 PRRSV full-genome sequences, followed by selecting the most common nucleotide found at each position of the alignment. Next, the synthetic PRRSV-CON strain was generated through the use of reverse genetics. PRRSV-CON replicates as efficiently as our prototype PRRSV strain FL12, both in vitro and in vivo. Importantly, when inoculated into pigs, PRRSV-CON confers significantly broader levels of heterologous protection than does wild-type PRRSV. Collectively, our data demonstrate that PRRSV-CON can serve as an excellent candidate for the development of a broadly protective PRRSV vaccine., Importance: The extraordinary genetic variation of RNA viruses poses a monumental challenge for the development of broadly protective vaccines against these viruses. To minimize the genetic dissimilarity between vaccine immunogens and contemporary circulating viruses, computational strategies have been developed for the generation of artificial immunogen sequences (so-called "centralized" sequences) that have equal genetic distances to the circulating viruses. Thus far, the generation of centralized vaccine immunogens has been carried out at the level of individual viral proteins. We expand this concept to PRRSV, a highly variable RNA virus, by creating a synthetic PRRSV strain based on a centralized PRRSV genome sequence. This study provides the first example of centralizing the whole genome of an RNA virus to improve vaccine coverage. This concept may be significant for the development of vaccines against genetically variable viruses that require active viral replication in order to achieve complete immune protection., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The aim of the present study was to determine the distribution and characteristics of microvessels in various histological types of canine renal cell carcinoma (RCC). The study compared microvessel density (MVD) and distribution of blood vessels according to histological type and evaluated the presence of angiogenesis-related proteins. Nine archival samples of canine RCC were studied. MVD was calculated as the mean number of blood vessels per mm(2). The diameter of blood vessels was calculated by determining either the length of the long axis of blood vessels (diameter(max)) or the mean distance from the centre of each blood vessel to the tunica adventia (diameter(mean)). A significant difference in MVD was evident between RCCs and normal kidneys (46.6 ± 28.0 versus 8.4 ± 2.2 microvessels/mm(2)). Diameter(max) in canine RCCs (34.1 ± 14.7 μm) was also significantly different from normal canine kidney (23.2 ± 3.4 μm). Vascular endothelial growth factor (VEGF) was expressed by tumour cells and vascular endothelial cells and tumour necrosis factor (TNF)-α expression was observed in vascular endothelial cells in both neoplastic and normal kidney. Although VEGF is involved in angiogenesis and correlates with tumour stage of development, no correlation was found between VEGF expression and MVD. Tumour-associated macrophages expressing TNF-α and hypoxia inducible factor 1α were identified in peritumoural tissue and may play an important role in angiogenesis., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Inflammatory mammary carcinoma (IMC) is a specific type of rare, very aggressive, and highly metastatic mammary cancer in both human beings and dogs. A 10-year-old female spayed Shih Tzu dog was diagnosed with secondary IMC. At necropsy, brain metastasis of mammary neoplastic cells was observed in tissues of the cerebrum and cerebellum. Metastases were also found in other distant organs such as heart, lung, liver, spleen, and inguinal lymph node. There is limited data about the metastasis of IMC and its pattern. The current report of IMC with brain metastases contributes to the understanding of metastatic IMC.

CDX-2 is used as a specific cell marker for human intestinal adenocarcinoma. In human studies, HER-3 overexpression predicts poor survival for patients with various cancers including gastric cancer. Gastrointestinal adenocarcinoma is less common in dogs than in man and the expression of immunological markers by the canine tumours has not yet been extensively studied. CDX-2 and HER-3 expression was determined in 18 canine gastrointestinal adenocarcinomas: 13 were of colorectal origin and five were of gastric origin. CDX-2 expression was predominantly observed in the nuclei of normal colonic epithelium and in neoplastic epithelium and neoplastic gastric epithelial cells that which had metastasized to the gastric lymph node. CDX-2 was expressed in 11 of 13 (84.6%) colorectal adenocarcinomas and in all five (100%) gastric adenocarcinomas. HER-3 was consistently expressed in the cytoplasm of neoplastic epithelial cells. HER-3 expression was detected in 12 of 13 (92.3%) colorectal and in all five (100%) gastric adenocarcinomas. CDX-2 and HER-3 may be useful markers for canine gastrointestinal adenocarcinoma., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Distribution and characterization of interlukin-10 (IL-10)-secreting cells in lymphoid tissues of pigs naturally infected with porcine circovirus type 2 (PCV2) were evaluated in accordance with PCV2 antigen detection. After screening a total of 56 pigs showing the symptoms of postweaning multisystemic wasting syndrome (PMWS), 15 pigs were PCV2 positive and 5 pigs, which showed stronger positive signals over multiples tissues were further investigated. This study showed that in PCV2-infected lymphoid tissues, particularly mandibular lymph node, spleen and tonsil, IL-10 expression was mainly localized in T-cell rich areas but rarely in B cell rich areas. IL-10 was highly expressed in bystander cells but rarely in PCV2-infected cells. Elevated IL-10 expression was predominantly associated with T cells, but rarely with B cells or with macrophages. The results of this study provide evidence for the role of IL-10 in chronic PCV2 infection and its relation to PCV2 antigen in affected tissues. Constantly elevated levels of IL-10 lead to immunosuppression in persistent and chronic viral infections. The increased IL-10 expression observed in PCV2 infection in this study suggests that IL-10-mediated immunosuppression may play an important role in the pathogenesis and maintenance of naturally occurring PCV2 infection.

Bovine atypical interstitial pneumonia (AIP) is a multifaceted disease with several known causes or clinical presentations. Multiple causal agents and management practices have been associated with development of the condition. The sporadic incidence and development of disease in a variety of circumstances argues against a common infectious agent, although cases of AIP are often complicated with bacterial, viral, or mycoplasmal organisms. Lesions develop and progress as a basic response of the lung to injury. Metabolic activation of naturally occurring xenobiotic compounds such as 3-methyl indole, perilla ketone, and 4-ipomeanol produce a clinical syndrome that is indistinguishable from naturally occurring AIP. Pulmonary injury is mediated by formation and activation of intermediate electrophilic compounds that covalently bind to cellular proteins and nucleic acids and ultimately cause cell death. Clara cells (nonciliated bronchiolar) and type I alveolar epithelial cells are primarily responsible for metabolism and activation of these naturally occurring xenobiotics., (Copyright 2010 Elsevier Inc. All rights reserved.)

In 2007, two new populations of feral swine were discovered in Nance and Valley counties, Nebraska, USA. Necropsies and serologic testing was done on two individuals from the Nance County herd. Results indicated that a lactating sow had positive antibodies for pseudorabies virus (PRV). Investigations conducted by Nebraska Game and Parks Commission Law Enforcement division confirmed that the infected individual was transported illegally to Nebraska, USA, from Texas, USA. All domestic swine herds located within an 8 km radius of the infected individual tested negative for antibodies to PRV. Our results provide a clear example of how diseases can spread because of anthropogenic activities and highlight the need for disease surveillance and monitoring in the import of invasive species.

Helicobacter hepaticus is a gram-negative, spiral-shaped microaerophilic bacterium associated with chronic intestinal infection leading to hepatitis and colonic and hepatic carcinomas in susceptible strains of mice. In the closely related human pathogen Helicobacter pylori, L-proline is a preferred respiratory substrate and is found at significantly high levels in the gastric juice of infected patients. A previous study of the proline catabolic PutA flavoenzymes from H. pylori and H. hepaticus revealed that Helicobacter PutA generates reactive oxygen species during proline oxidation by transferring electrons from reduced flavin to molecular oxygen. We further explored the preference for proline as a respiratory substrate and the potential impact of proline metabolism on the redox environment in Helicobacter species during host infection by disrupting the putA gene in H. hepaticus. The resulting putA knockout mutant strain was characterized by oxidative stress analysis and mouse infection studies. The putA mutant strain of H. hepaticus exhibited increased proline levels and resistance to oxidative stress relative to that of the wild-type strain, consistent with proline's role as an antioxidant. The significant increase in stress resistance was attributed to higher proline content, as no upregulation of antioxidant genes was observed for the putA mutant strain. The wild-type and putA mutant H. hepaticus strains displayed similar levels of infection in mice, but in mice challenged with the putA mutant strain, significantly reduced inflammation was observed, suggesting a role for proline metabolism in H. hepaticus pathogenicity in vivo.

Canine mast cell tumours (MCTs) may be graded microscopically for prognostic purposes. Grade I (well-differentiated) and grade II (intermediate differentiation) tumours have an abundance of metachromatic granules within the cytoplasm; however, grade III (poorly differentiated) MCTs may be difficult to diagnose as they frequently have fewer discernable granules. Herein we report that a cross-reactive anti-human CD1a monoclonal antibody (clone O10) may be used in immunohistochemistry to identify canine MCTs of all grades. The antibody was applied to tissue sections from 48 canine MCTs of different histological grades. Serial sections from each tumour were stained with toluidine blue and safranin O to compare diagnostic sensitivity. All MCTs were labelled positively by the CD1a antibody, but histochemical staining was often equivocal and identification of mast cells was extremely difficult in some cases. This antibody did not label neoplastic cells in cases of canine histiocytoma, plasmacytoma or amelanotic melanoma; therefore, the reagent may be a valuable marker for the diagnosis of canine MCTs, especially those tumours of histological grade III.

An 8-month-old, intact male Golden Retriever with a history of left forelimb lameness for 2 months was presented to the Veterinary Medical Teaching Hospital of Konkuk University (Seoul, Korea). Results of a physical examination revealed a mass in the left axillary region. A thoracic radiography showed an osteolytic lesion in the scapula and the presence of a soft tissue density from the thoracic wall to the scapula. A computerized tomography revealed a mass invading into the scapula, and small nodules in the lung that suggested metastasis. At necropsy, a pale-yellow, irregular, firm, 8 x 10 x 5 cm mass extended from axillary region and destroyed the scapular. In addition, small nodules were noted in the lung. On microscopic examination, the mass consisted of round-to-oval cells, with eccentrically located hyperchromatic nuclei and eosinophilic cytoplasm in fibromyxoid stroma. Tumor cells were observed in blood vessels in the primary mass. Tumor cells strongly expressed vimentin, desmin, and myoglobin. In phosphotungstic acid-hematoxylin staining, cross-striations were detected in rhabdomyoblasts. In periodic acid-Schiff reaction, only a few cells were detected. The diagnosis was primary rhabdomyosarcoma of the appendicular muscle of a young dog. The tumor presumably originated in the skeletal muscle of the limb, invaded into the adjacent scapular bone, and metastasized to the lung.

We describe a 10-month-old, intact female American Cocker Spaniel with pulmonary lymphomatoid granulomatosis (PLG). On clinical examination, this dog presented with nonproductive dry cough, serous nasal discharge, dyspnea, and lack of appetite. Radiography showed a consolidated lesion in the left cranial lung lobe. Histopathologic examination showed a mixed population of atypical lymphoid cells that had infiltrated into the pulmonary blood vessels angiocentrically. The lymphocytes were CD3 positive, consistent with a pan-T-cell phenotype. The lymphoid cells in the lesion were also positive for CD20cy and CD79a, indicative of the presence of B cells. We also observed large Reed-Sternberg-like cells that were positive for CD15 and CD30, similar to observations in human pulmonary Hodgkin's disease (PHD). In conclusion, canine PLG in this Cocker Spaniel was associated with B and T cells, which is first identified in a case of canine PLG. It was histopathologically similar to human lymphomatoid granulomatosis and immunophenotypically similar to human PHD.

The clinical, hematological, and histopathologic features of megakaryoblastic leukemia (M7) were investigated in a 10-year-old female Shih-Tzu dog. Megakaryoblastic leukemia was diagnosed using anti-human platelet glycoprotein (GP IIIa) and anti-human von Willebrand factor (vWF) antibodies. The expression of CD antigen on megakaryoblasts was also assessed using a CD79a monoclonal antibody. Immunological markers allowed visualization of neoplastic megakaryocytes. Antibodies against platelet GP IIIa were demonstrated to be the most useful for the diagnosis of megakaryoblastic leukemia of paraffin-embedded canine tissues. Hematological and histological data coupled with immunohistochemical reactivity for platelet GP IIIa, vWF, and CD79a antigen in blast cells confirmed a diagnosis of M7 megakaryoblastic leukemia.

One hundred pigs from the NE Index Line (NEI) and 100 Hampshire-Duroc cross pigs (HD) were inoculated intranasally with porcine respiratory and reproductive syndrome virus (PRRSV 97-7895 strain) at 26 d of age to determine whether genetic variation in response to PRRSV exists. An uninfected littermate to each infected pig served as a control. Pigs were from 163 dams and 83 sires. Body weight and rectal temperature were recorded, and blood samples were drawn from each pig on d 0 before inoculation and on d 4, 7, and 14 after inoculation. Pigs were sacrificed on d 14. Lung and bronchial lymph nodes were collected, placed in optimal cutting temperature compound, and frozen at -80 degrees C. The presence of PRRSV in serum and in lung tissue and bronchial lymph nodes was determined by isolation in cell culture. The presence of antibodies in serum collected on d 14 was determined by a commercial ELISA test. Lung tissue was examined microscopically and scored for incidence and severity of lesions (score of 1 to 3; 1 = no or few lesions, and 3 = severe interstitial pneumonia). Data were analyzed with a mixed model that included random sire and dam effects. The interaction of line x treatment was significant (P < 0.001) for weight change and rectal temperature. Un-infected HD pigs gained 0.67 kg more from d 0 to 14 and averaged 0.32 degrees C higher rectal temperature than uninfected NEI pigs (P < 0.001), whereas infected NEI pigs gained 0.34 kg more and had -0.54 degrees C lower temperature than infected HD pigs (P < 0.001). Viremic titer (cell culture infectious dose 50%/mL) was greater (P < 0.05) in HD than NEI at d 4 (10(4.52) vs. 10(4.22)), 7 (10(4.47) vs. 10(3.99)), and 14 (10(3.49) vs. 10(3.23)). Viral titer loads in lung (P = 0.11) and bronchial lymph nodes tended (P = 0.07) to be greater in HD than NEI pigs. Antibody signal-to-positive (S/P) ELISA ratios in infected pigs ranged from 0.18 to 3.38, and 88% had levels > or = 0.40, which is the positive threshold for this ELISA. The S/P range in uninfected pigs was 0 to 1.11, and 99% had levels < or = 0.40. Mean S/P ratio for infected pigs was 0.23 units higher in HD than in NEI (P < 0.001). The HD pigs had a greater incidence of interstitial pneumonia and 0.65 higher mean lesion scores than NEI pigs (P < 0.001). In summary, responses of pigs of the two lines to infection with PRRSV differed, indicating that underlying genetic variation existed.

Pigs were immunised with recombinant BCG (rBCG) expressing a truncated form of GP5 (lacking the first 30 NH(2)-terminal residues) (rBCGGP5) and M protein (rBCGM) of porcine reproductive and respiratory syndrome virus (PRRSV). At 30 days post-inoculation (dpi), pigs inoculated with rBCGGP5 and rBCGM developed a specific humoral immune response against the viral proteins, as detected by commercial ELISA and Western blot tests, and at 60 dpi, three out of five animals developed neutralizing antibodies with titers ranging from 1:4 to 1:8. At 67 dpi, an IFN-gamma response against BCG antigens, but not against the viral proteins, was detected by ELISPOT in inoculated pigs. Following challenge with a pathogenic strain of PRRSV, pigs inoculated with rBCG showed lower (P<0.05) temperature, viremia and virus load in bronchial lymph nodes than control animals, suggesting the establishment of partial protection against PRRSV infection.

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.

Understanding the dynamics of porcine reproductive and respiratory syndrome virus (PRRSV) persistence in individual pigs is essential to the development of successful control programs. The objectives of this study were to investigate the proportion of inoculated pigs that become persistently infected with PRRSV and the duration of their infection. Additionally, different diagnostic techniques that detect persistent infections were compared. Twenty-eight 35-day-old pigs were inoculated with PRRSV. Serum and tonsil biopsy samples were collected on days 0, 7, 14, and 28 and then approximately monthly thereafter until day 251 postinoculation (p.i.). Tonsil, lymph node, and lung samples were collected following euthanasia on day 251 p.i. Virus was isolated from serum and tonsil biopsy samples that had been collected through days 28 and 56 p.i., respectively. Viral RNA was detected by reverse transcription (RT)-PCR in serum and tonsil biopsy samples that had been collected through day 251 p.i., although no serum samples collected from days 84 to 196 p.i. were positive and the presence of infectious PRRSV was not detected by swine bioassay of tissue samples collected at necropsy. The results confirmed that RT-PCR is more sensitive than virus isolation in identifying PRRSV-infected pigs. Six pigs that were persistently infected through days 225 or 251 p.i. remained seropositive throughout the study, although one pig had an enzyme-linked immunosorbent assay sample-to-positive ratio that was only slightly above the cutoff value of 0.40. Twenty of 28 tonsil biopsy samples collected on day 84 p.i. were positive by RT-PCR compared to only 1 positive biopsy sample out of 28 collected on day 119 p.i. The study's results indicate that most pigs clear PRRSV within 3 to 4 months, but that some may remain persistently infected for several months.

Mycobacterium bovis BCG was used to express a truncated form of GP5 (lacking the first 30 NH(2)-terminal residues) and M protein of porcine reproductive and respiratory syndrome virus (PRRSV). The PRRSV proteins were expressed in BCG under control of the mycobacterial hsp60 gene promoter either in the mycobacterial cytoplasm (BCGGP5cyt and BCGMcyt) or as MT19-fusion proteins on the mycobacterial surface (BCGGP5surf and BCGMsurf). Mice inoculated with BCGGP5surf and BCGMsurf developed antibodies against the viral proteins at 30 days post-inoculation (dpi) as detected by ELISA and Western blot. By 60 dpi, the animals developed titer of neutralizing antibodies of 8. A PRRSV-specific gamma interferon response was also detected in splenocytes of recombinant BCG-inoculated mice at 60 and 90 dpi. These results indicate that BCG was able to express antigens of PRRSV and elicit an immune response against the viral proteins in mice.

The pathogenesis of porcine reproductive and respiratory syndrome virus (PRRSV) infection in ovary was studied in sexually mature, cycling, nonsynchronized gilts infected with the PRRSV 16244B, a virulent field strain. Previous studies have shown that PRRSV can be isolated from ovaries and is transplacentally passed from gilts to the fetuses. The cause of infertility following PRRSV infection is not known. In this study, we identified the tropism of PRRSV in ovarian tissue from experimentally infected gilts in samples collected between 7 and 21 days postinfection (DPI). Tissues were collected and examined by virus isolation, in situ hybridization (ISH), immunohistochemistry (IHC), and double labeling to identify PRRSV-infected cell types. PRRSV was isolated in ovarian follicles at 7 days DPI. The IHC and ISH indicated that PRRSV-positive cells in ovaries were predominantly macrophages, which were numerous in atretic follicles. No evidence of infection and/or perpetuation of PRRSV in ova was observed, indicating that the female gonad is an unlikely site of persistence. No alteration of the normal ovarian architecture that would support a possible role of PRRSV infection in porcine female infertility was observed.

Ulceration of the pars esophagea in swine develops from a complex interaction of dietary particle size, gastric fluidity, dietary carbohydrate content, and presence of certain species of commensal gastric organisms capable of fermenting dietary carbohydrates. Unlike in humans, the significance of the role of Helicobacter sp. in development of porcine gastric ulcers is yet undefined. Management practices that limit the incidence and severity of gastric ulceration without interfering with growth performance appear to be the best option for control.

The sensitivity and specificity of recombinant open reading frame 5 products used in the Western blotting assay for confirmation of porcine reproductive and respiratory syndrome virus (PRRSV) serologic status were evaluated. The recombinant antigen-based assays were specifically compared with a commercial enzyme-linked immunosorbent assay (ELISA) for PRRSV antibodies using 1) PRRSV antibody-negative reference sera (n = 30), 2) naturally infected pig sera (n = 40), 3) sequential sera obtained from 24 experimentally infected pigs, and 4) sera submitted to 3 state diagnostic laboratories (n = 200). The recombinant antigen assay yielded an average increased sensitivity of 10% over the commercial PRRSV ELISA. The negative controls (group 1 sera) showed no difference between the 2 assays. This comparison confirmed that the recombinant antigen-specific assay was more sensitive than the commercial ELISA and is well suited for routine confirmation of the presence of PRRSV antibodies.

Although North American and European serotypes of porcine reproductive and respiratory syndrome virus (PRRSV) are recognized, only the genome of the European Lelystad strain (LV) has been sequenced completely. Here, the genome of the pathogenic North American PRRSV isolate 16244B has been sequenced and compared with LV. The genomic organization of 16244B was the same as LV but with only 63.4% nucleotide identity. The 189 nucleotide 5' non-coding region (NCR) of 16244B was distinct from the LV NCR, with good conservation (83%) only over a 43 base region immediately upstream of open reading frame (ORF) 1a. Major differences were found in the region encoding the non-structural part of the ORF1a polyprotein, which shared only 47% amino acid identity over 2503 residues of the six non-structural proteins (Nsps) encoded. Nsp2, thought to have a species-specific function, showed the greatest divergence, sharing only 32% amino acid identity with LV and containing 120 additional amino acids in the central region. Nsps encoded by the 5'-proximal and central regions of ORF1b had from 66 to 75% amino acid identity; however, the carboxy-terminal protein CP4 was distinct (42% identity). The ORF 1a-1b frameshift region of 16244B had 98% nucleotide identity with LV. Consistent with previous reports for North American isolates, the six structural proteins encoded were 58 to 79% identical to LV proteins. The 3' NCR (150 nucleotides) was 76% identical between isolates. These genomic differences confirm the presence of distinct North American and European PRRSV genotypes.

We studied apoptosis caused by porcine reproductive and respiratory syndrome virus (PRRSV) in vivo, focusing on the tissues that constitute the main targets for infection: lung and lymphoid tissues. Previous investigators have shown that the PRRSV glycoprotein p25, encoded by PRRSV open reading frame 5, induces apoptosis when expressed in COS-1 cells. Results of studies conducted in our laboratory indicate the simultaneous occurrence of PRRSV-induced alterations of spermatogenesis and apoptotic death of germinal epithelial cells in the testicle. In this study, the goal was to determine whether virus-induced apoptosis is a direct mechanism of cell death caused by PRRSV in infected pigs. Eight 3-week-old pigs were intranasally inoculated with PRRSV 16244B, a highly virulent field strain. Lung, tonsil, bronchial lymph node, spleen, and heart were assessed histologically at 4 and 7 days postinfection. To characterize PRRSV-infected cells and apoptotic cell death, we used immunohistochemical methods for detection of viral antigen, DNA electrophoresis for detection of DNA fragmentation, the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-fluorescein nick end labeling method for in situ detection of DNA strand breaks, and electron microscopy for ultrastructural morphologic studies. PRRSV infection resulted in widespread apoptosis in the lungs and lymphoid tissues of infected pigs. Virus infection-induced apoptotic cells were more abundant than PRRSV-infected cells in all tissues. DNA laddering was detected in lung and lymphoid tissues. However, double-labeling experiments demonstrated that the majority of apoptotic cells did not colocalize with PRRSV-infected cells. Our findings suggest the presence of an indirect mechanism in the induction of apoptosis for PRRSV.

Like other arteriviruses, porcine reproductive and respiratory syndrome virus (PRRSV) is shed in semen, a feature that is critical for the venereal transmission of this group of viruses. In spite of its epidemiological importance, little is known of the association of PRRSV or other arteriviruses with gonadal tissues. We experimentally infected a group of boars with PRRSV 12068-96, a virulent field strain. By combined use of in situ hybridization and immunohistochemistry, we detected infection by PRRSV in the testes of these boars. The PRRSV testicular replication in testis centers on two types of cells: (i) epithelial germ cells of the seminiferous tubules, primarily spermatids and spermatocytes, and (ii) macrophages, which are located in the interstitium of the testis. Histopathologically, hypospermatogenesis, formation of multinucleated giant cells (MGCs), and abundant germ cell depletion and death were observed. We obtained evidence that such germ cell death occurs by apoptosis, as determined by a characteristic histologic pattern and evidence of massive DNA fragmentation detected in situ (TUNEL [terminal deoxynucleotidyltransferase-mediated digoxigenin-UTP nick end labeling] assay). Simultaneously with these testicular alterations, we observed that there is a significant increase in the number of immature sperm cells (mainly MGCs, spermatids, and spermatocytes) in the ejaculates of the PRRSV-inoculated boars and that these cells are infected with PRRSV. Our results indicate that PRRSV may infect target cells other than macrophages, that these infected cells can be primarily responsible for the excretion of infectious PRRSV in semen, and that PRRSV induces apoptosis in these germ cells in vivo.

An improved method for the diagnosis of canine parvovirus using in situ hybridization in standard formalin-fixed, paraffin-embedded tissue sections was developed. A digoxigenin-labeled probe complementary to DNA sequences that code for the entire sequence of the capsid protein VP-1 and the middle part of the sequence of the capsid protein VP-2 was designed. Specific histologic localization of canine parvovirus-infected cells was demonstrated in small intestine, tonsil, lymph node, thymus, spleen, heart, liver, and kidney from dogs diagnosed at necropsy with canine parvovirus infection. The in situ hybridization accurately pinpointed the specific sites of viral infection. The detection of canine parvovirus in liver, kidney, and heart tissues together in the same pups could represent an enhanced virulence of this strain of canine parvovirus and suggests a broadened tissue tropism not seen before in Korean strains of canine parvovirus.

We studied the distribution of porcine reproductive and respiratory syndrome virus (PRRSV) RNA in tissues by in situ hybridization at different times postinfection (p.i.). The probe used for in situ hybridization was prepared by reverse transcription of PRRSV RNA, followed by PCR amplification of the cDNA. The sequence amplified corresponded to 433 bp from PRRSV open reading frame 7, which is contained in the nucleocapsid protein gene and which is highly conserved in both European and American strains (H. Mardassi, L. Wilson, S. Mounir, and S. Dea, J. Clin. Microbiol. 32:2197-2203, 1994). An immunohistochemical technique was used to detect PRRSV antigen in tissue from virus-infected animals by using a monoclonal antibody specific for the PRRSV nucleocapsid protein (E.A. Nelson, J. Christopher-Hennings, T. Drew, G. Wensvoort, J.E. Collins, and D.A. Benfield, J. Clin. Microbiol. 31:3184-3189, 1993). The detection of PRRSV RNA was conducted in tissues of 6-week-old pigs that had been infected with one of three different field PRRSV isolates and collected at times ranging from 4 to 42 days p.i. Hybridization signals specific for PRRSV RNA were detected in lung, lymphoid tissues, alveolar macrophages (obtained by lavage at the time of necropsy), Peyer's patches, and kidney. The PRRSV-positive cells in these tissues appeared to be predominantly macrophages. In lung tissue we also obtained evidence suggesting the involvement of type II pneumocytes in the replication of PRRSV. During the acute period of infection there was a close correlation between the detection of RNA and the detection of nucleocapsid protein in individual cells. At later times p.i. (28 and 42 days p.i.), instead, more cells containing only PRRSV RNA than those containing PRRSV RNA and also expressing PRRSV nucleocapsid protein were detected. These results suggest that PRRSV RNA might persist in the tissues of infected animals for a longer time than PRRSV antigen expression.

Four canine weakly beta-hemolytic intestinal spirochetes associated with intestinal spirochetosis (IS-associated WBHIS) were compared with IS-associated human and porcine WBHIS and the type species for Serpulina hyodysenteriae and S. innocens by using phenotypic and genotypic parameters. The IS-associated canine, human, and porcine WBHIS belonged to a phyletic group distinct from but related to previously described Serpulina type species.

A 2-phase study was conducted to evaluate the ability of the NEB-1 strain of porcine reproductive and respiratory syndrome virus (PRRSV) to potentiate common bacterial pathogens of swine. In phase I, 25 of 50 4-5-week-old specific-pathogen-free (SPF) pigs were exposed to NEB-1 PRRSV (day 0). Seven days after virus inoculation, 8 groups received 1 of 4 bacterial pathogens: Haemophilus parasuis, Streptococcus suis, Salmonella cholerasuis, and Pasteurella multocida. The ability of NEB-1 PRRSV to produce clinical disease, viremia, neutralizing antibody, gross and microscopic lesions and to potentiate bacterial pathogens was assessed. Response to NEB-1 PRRSV was similar among inoculated pigs; prolonged hyperthermia, lethargy, mild to moderate dyspnea, and cutaneous erythema were consistent clinical signs. No clinical differences were observed in groups after bacterial challenge. Virus was isolated from serum at weekly intervals through the end of the study, and all PRRSV-inoculated pigs had seroconverted by study termination. Two of 5 pigs died in non-PRRSV-inoculated groups challenged with H. parasuis and Streptococcus suis. Mortality in PRRSV-infected pigs was limited to 1 of 5 pigs from the Salmonella cholerasuis-challenged group. Gross lesions were seen in pigs dying after inoculation in H. parasuis- and Streptococcus suis-inoculated groups, in Salmonella cholerasuis- and P. multocida-challenged pigs, and in 1 non-PRRSV-inoculated control pig. Microscopic lesions consisted of mild to moderate proliferative interstitial pneumonia, nonsuppurative myocarditis, lymphoid hyperplasia, and nonsuppurative encephalitis in PRRSV-inoculated pigs. Findings in phase I indicated that NEB-1 PRRSV does not potentiate bacterial disease while inducing consistent clinical signs, viremia, seroconversion, and microscopic lesions.(ABSTRACT TRUNCATED AT 250 WORDS)

The infectivity and pathogenicity of selected bovine viral diarrhea virus (BVDV) isolates were determined in gnotobiotic, colostrum-deprived neonatal lambs. Five-day-old cesarean-derived gnotobiotic lambs were exposed to 1 of 10 BVDV isolates via aerosol suspension. These isolates were from tissues or secretions of calves or lambs affected with respiratory tract disease, weak neonatal calves, aborted bovine fetuses, or reference Singer or Draper BVDV. The pathogenicity of each isolate, relative to the others, was evaluated in lambs by measurement of the neutralizing antibody response, virus isolation from nasal secretions or tissues, and postmortem lesions. The BVDV isolates varied in their infectivity and pathogenicity. Singer, the cytopathic reference strain, was the most lymphotrophic isolate and stimulated the greatest neutralizing antibody response. Encephalitis was the most consistent lesion observed and was used as the final determinant of relative pathogenicity of the viruses. The most neuropathogenic isolates were the 2 viruses originating from lambs affected with respiratory tract disease, the 2 weak neonatal calf isolates, and 1 isolate from an aborted bovine fetus. The least pathogenic isolates were the 2 reference isolates, Draper and Singer; the 2 mucosal disease isolates; and 1 isolate originating from an aborted bovine fetus.

The etiology of abomasal ulcers/tympany was investigated in 48 animals from 36 ranches in Wyoming and Nebraska. Results indicate that subclinical trace mineral deficiencies of copper and/or selenium exist in the range cattle in west central Nebraska and Wyoming. Etiological agents most frequently incriminated by bacteriologic cultures and/or histopathic examination were Clostridium perfringens and Campylobacter species. Histopathologic evaluation of abomasums revealed 31 of 38 cases contained abundant gram-positive bacteria associated with the damaged abomasal mucosa. Campylobacter-like organisms were demonstrated in 9 of 38 cases using the modified Dieterle stain. Clostridium perfringens was isolated in 14 of 38 cases, and Campylobacter jejuni was recovered from 5 of 38 cases.

A multicentric liposarcoma was identified in various skeletal muscles of a mature Canada goose. The light and electron microscopic appearance of the tumor is discussed in relation to the appearance of liposarcoma in other species.

Infection with Trichosporon spp was diagnosed in 2 cats. In one cat, infection consisted of a granulomatous dermatitis and was concurrent with disseminated lymphoblastic lymphosarcoma. In another cat, urinary cystitis caused by T beigelii was diagnosed.

Cattle given intraruminal administration of 4-ipomeanol, a furanoterpenoid originally obtained from sweet potatoes infected with Fusarium solani (F. javanicum), developed a respiratory syndrome clinically and histologically indistinguishable from atypical interstitial pneumonia. There were edema and emphysema in the lungs and mediastinum. The maximum nonlethal oral dose of 4-ipomeanol was estimated to be between 7.5 and 9 mg/kg of body weight.

Streptococcus suis was isolated from swine with localized and diffuse infections. The isolates (n = 121) were unable to grow in broth containing 6.5% NaCl. Their abilities to hydrolyze esculin were variable, as were their fermentative reactions in lactose, trehalose, raffinose, and inulin. Sorbitol was not attacked by any strain. Serologic reactions were obtained between the isolates and coagglutination reagents prepared from commercially available antisera to streptococcal groups D, R, or S.

Repeated intraperitoneal injections of 4-ipomeanol in rats resulted in extensive degeneration and necrosis of nonciliated (Clara) bronchiolar epithelial cells. Subsequently there was necrosis and detachment of ciliated cells from the bronchiolar basal lamina. The remaining nonciliated cells divided and differentiated into ciliated epithelial cells and mature Clara cells. The ability of Clara cells to metabolize certain xenobiotics suggests that they may play an important role in the pathogenesis of pulmonary disease and neoplasia.

An indirect immunoperoxidase staining technique was evaluated for detection of Mycoplasma hyopneumoniae in formalin-fixed, paraffin-embedded porcine lung. Lungs from swine with induced (n = 4) or naturally occurring M hyopneumoniae infection (n = 31) were examined grossly, by light and immunofluorescent microscopy, and by an indirect immunoperoxidase test, using antibody raised in swine against M hyopneumoniae as the primary antibody. Organisms stained by the indirect immunoperoxidase method were identified in tissue sections as pleomorphic brown-staining structures corresponding to those observed with immunofluorescence. Mycoplasma hyosynoviae, M hyorhinis, and Acholeplasma laidlawii did not stain with the indirect immunoperoxidase method, using antibody raised against M hyopneumoniae.

A total of 62 gnotobiotic pigs were inoculated with Bordetella bronchiseptica at 7 days of age. There were 25 noninoculated controls. Thirteen of the inoculated pigs were killed and 6 pigs which died were examined between 3 and 5 weeks after inoculations were done (group A). Of these 19 pigs, 16 had severe pneumonia and B bronchiseptica was isolated from the lungs of 17. The turbinates in most of the pigs appeared to have some atrophy. Four other pigs died of pneumonia after they were transferred to the floor in isolation rooms, and 9 inoculated pigs were examined at necropsy at about 112 days of age (group B). Bordetella bronchiseptica was isolated from 8 of the latter 9 pigs, and 7 of the pigs had pneumonic lesions. The turbinates all appeared normal. Thirty inoculated pigs (group C) were killed at 180 days of age. Of these, 22 had pneumonic lesions. Bordetella bronchiseptica was reisolated from the turbinates of 2 of 30 pigs, although the turbinates in all appeared normal. The noninoculated controls examined at necropsy at 3 to 5 weeks, 112 days, and 180 days of age did not have any pulmonic lesions, and their turbinates appeared normal. The inoculated pigs killed at 112 days of age gained an average of 0.25 kg/day, and the control pigs gained 0.31 kg. At 180 days of age, the inoculated pigs had gained 0.41 kg/day and the controls, 0.49 kg. Bordetella bronchiseptica infection in susceptible pigs can cause a severe pneumonia which can persist and reduce the growth rate.

A seminoma and parathyroid adenoma were diagnosed in an aged snow leopard. The ultrastructural appearance of the seminoma was similar to that described in the dog and in man. The lack of significant amounts of rough endoplasmic reticulum, Golgi complexes and free ribosomes in the parathyroid adenoma suggested that it was non-functional. Parathyroid adenoma has not been previously described in a large wild feline.