Adipokines secretion in feline primary adipose tissue culture in response to dietary fatty acids.

BACKGROUND: Obesity in cats has been associated with alterations in adipokines including: adiponectin, interleukin-6 (IL6), and tumor necrosis factor-α (TNFα). Omega-3 polyunsaturated fatty acids have multiple beneficial effects on obesity-associated disorders, and therefore may alleviate these alterations. This study aimed to determine the effects of body condition, fat depot, troglitazone, and different fatty acids on secretion of adiponectin, IL6 and TNFα from adipose tissue of healthy cats. Subcutaneous and visceral adipose tissue samples were collected from 18 healthy intact female cats, and body condition score (Range 3-7/9) was determined. Concentrations of adiponectin were measured in mature adipocytes cultures and concentrations of IL6 and TNFα were measured in stromovascular cells cultures following treatment with control medium, troglitazone at 10 µM, eicosapentaenoic acid, arachidonic acid, or palmitic acid, at 25, 50, or 100 µM. RESULTS: Stromovascular cells of visceral origin secreted higher concentrations of IL6 than corresponding cells of subcutaneous origin (P = 0.003). Arachidonic acid treatment at 25, 50, and 100 µM increased IL6 secretion in subcutaneous (P = 0.045, P = 0.002, and P < 0.001, respectively) and visceral (P = 0.034, P = 0.001, and P < 0.001, respectively) stromovascular cells. Eicosapentaenoic acid treatment increased TNFα secretion in subcutaneous stromovascular cells at 25, 50, and 100 µM (P = 0.002, P = 0.001, and P = 0.015, respectively) and in visceral stromovascular cells at 50 µM (P < 0.001). No significant effect on medium adiponectin concentration was observed following troglitazone treatment (P = 0.4) or fatty acids treatments at 25 (P = 0.2), 50 (P = 0.8), or 100 (P = 0.7) µM. Body condition score did not have significant effects on medium concentrations of adiponectin (P = 0.4), IL6 (P = 0.1), or TNFα (P = 0.8). CONCLUSIONS: This study demonstrated higher basal secretion of IL6 from visceral compared to subcutaneous adipose tissue, a stimulatory effect of arachidonic acid on secretion of IL6 and a stimulatory effect of eicosapentaenoic acid on TNFα from feline adipose tissue.

Differential secretion of adipokines from subcutaneous and visceral adipose tissue in healthy dogs: Association with body condition and response to troglitazone.

This study aimed to determine the effects of body condition, fat depot, and a peroxisome proliferator-activated receptor γ-agonist (troglitazone) on secretion of adiponectin, interleukin-6 (IL6), and tumor necrosis factor-α (TNFα) from adipose tissue of healthy dogs. Subcutaneous and omental visceral adipose tissue samples were collected from 16 healthy intact female dogs, and body condition score (range 4-8/9) was determined. Concentrations of adiponectin were measured in mature adipocytes cultures and concentrations of IL6 and TNFα were measured in stromovascular cells cultures after 48 h incubation in fresh control medium, or fresh medium containing 10 µM troglitazone. Mature adipocytes and stromovascular cells of subcutaneous origin secreted higher concentrations of adiponectin and lower concentration of IL6 and TNFα, respectively, than corresponding cells of visceral origin, in both the control (P = 0.015, P = 0.004, and P = 0.016, respectively) and troglitazone-treated cultures (P <0.001, P = 0.004, and P = 0.016, respectively). Troglitazone increased adiponectin secretion from mature adipocytes in visceral (P = 0.019), but not in subcutaneous fat cultures (P = 0.4). Troglitazone decreased IL6 and TNFα secretion from stromovascular cells both in visceral (P = 0.047 and P = 0.016, respectively) and subcutaneous (P = 0.047 and P = 0.016, respectively) fat cultures. Higher body condition score was associated with lower secretion of adiponectin from mature adipocytes (P = 0.007), lower secretion of IL6 (P = 0.040) and higher secretion of TNFα (P = 0.040) from stromovascular cells. This study showed differential secretion of adipokines by subcutaneous and visceral fat depots in dogs and association between body condition and adipokine secretion. Activation of PPARγ altered adipokine secretion.

Evaluation of the interferon-γ assay on blood collected at exsanguination of cattle under field conditions for surveillance of bovine tuberculosis.

Development of point of concentration (POC) surveillance strategies for bovine tuberculosis (bTB) would facilitate global efforts to eradicate bTB. The interferon-gamma (IFNγ) assay can detect IFNγ responses to Mycobacterium bovis in blood collected at commencement of exsanguination (COE) of experimentally challenged cattle but has not been evaluated under field conditions. The current study was aimed at determining (i) whether blood collected at COE of cattle at slaughter, under field conditions, is practical to obtain and useful for identifying cattle as IFNγ positive for bTB, (ii) whether the results of the IFNγ assay obtained at COE reliably compare with results obtained from live animals in the field, and (iii) whether the identified animal(s) originated from bTB-infected or bTB-exposed herds. Cattle from three risk groups were used: the highest risk group consisted of 49 cattle from 3 bTB-infected herds; the medium risk group consisted of 24 cattle from a potentially exposed herd; and the lowest risk group consisted of 60 cattle from herds with no known history of bTB exposure. The IFNγ assay was performed on blood collected both before stunning and at COE of cattle at slaughter. An enhanced slaughter inspection for gross lesions consistent with bTB was performed on all cattle. In addition, lymph nodes were cultured for M. bovis for cattle that tested positive for bTB via the IFNγ assay and for most cattle that tested negative for bTB. Cattle, both with and without lesions consistent with bTB, were identified as positive for bTB by the IFNγ assay using blood collected at COE, but none of the positive cattle originated from the lowest risk group. The current study demonstrates that blood collected at COE of cattle is both a practical and moderately reliable sample for accessing bTB infection using the IFNγ assay.

Distribution of eastern equine encephalomyelitis viral protein and nucleic acid within central nervous tissue lesions in white-tailed deer (Odocoileus virginianus).

An outbreak of eastern equine encephalomyelitis (EEE) occurred in Michigan free-ranging white-tailed deer (Odocoileus virginianus) during late summer and fall of 2005. Brain tissue from 7 deer with EEE, as confirmed by reverse transcriptase polymerase chain reaction, was studied. Detailed microscopic examination, indirect immunohistochemistry (IHC), and in situ hybridization (ISH) were used to characterize the lesions and distribution of the EEE virus within the brain. The main lesion in all 7 deer was a polioencephalomyelitis with leptomeningitis, which was more prominent within the cerebral cortex, thalamus, hypothalamus, and brainstem. In 3 deer, multifocal microhemorrhages surrounded smaller vessels with or without perivascular cuffing, although vasculitis was not observed. Neuronal necrosis, associated with perineuronal satellitosis and neutrophilic neuronophagia, was most prominent in the thalamus and the brainstem. Positive IHC labeling was mainly observed in the perikaryon, axons, and dendrites of necrotic and intact neurons and, to a much lesser degree, in glial cells, a few neutrophils in the thalamus and the brainstem, and occasionally the cerebral cortex of the 7 deer. There was minimal IHC-based labeling in the cerebellum and hippocampus. ISH labeling was exclusively observed in the cytoplasm of neurons, with a distribution similar to IHC-positive neurons. Neurons positive by IHC and ISH were most prominent in the thalamus and brainstem. The neuropathology of EEE in deer is compared with other species. Based on our findings, EEE has to be considered a differential diagnosis for neurologic disease and meningoencephalitis in white-tailed deer.

Outbreak of neonatal diarrhea in farmed mink kits (Mustella vison) associated with enterotoxigenic Staphylococcus delphini.

An outbreak of diarrhea on a large commercial mink farm affected 5,000 of 36,000 neonatal mink kits, with 2,000 dying within a 2-week period. Affected kits were severely dehydrated, and their furcoats and paws were covered with yellow- to green-tinged mucoid feces. On necropsy, the small intestines of examined animals were markedly distended by serous to mucoid fluid. Microscopically, there was prominent colonization of the intestinal villar epithelium by gram-positive bacterial cocci in the absence of inflammation and morphologic changes in villous enterocytes. The colonizing bacteria were phenotypically identified as belonging to the Staphylococcus intermedius group of bacteria. This was confirmed by nucleic acid sequence analysis of the 16S ribosomal RNA gene. Further nucleic acid sequencing of polymerase chain reaction (PCR) amplicons from the superoxide dismutase gene and the heat shock protein 60 gene differentiated the isolate as Staphylococcus delphini. Production of staphylococcal enterotoxins A and E was demonstrated with a commercial ELISA-based immunoassay. Sequencing of PCR amplicons confirmed the presence of the enterotoxin E gene, but PCR amplification of the enterotoxin A, B, C, or D genes was not successful. Although direct causation was not confirmed in this study, the authors postulate that the observed hypersecretory diarrhea in these mink kits was the result of colonization of the small intestine by S delphini and subsequent production of enterotoxin.

Equine multinodular pulmonary fibrosis: a newly recognized herpesvirus-associated fibrotic lung disease.

Pulmonary fibrosis and interstitial lung disease are poorly understood in horses; the causes of such conditions are rarely identified. Equine herpesvirus 5 (EHV-5) is a gamma-herpesvirus of horses that has not been associated with disease in horses. Pathologic and virologic findings from 24 horses with progressive nodular fibrotic lung disease associated with EHV-5 infection are described and compared with 23 age-matched control animals. Gross lesions consisted of multiple nodules of fibrosis throughout the lungs. Histologically, there was marked interstitial fibrosis, often with preservation of an "alveolar-like" architecture, lined by cuboidal epithelial cells. The airways contained primarily neutrophils and macrophages. Rare macrophages contained large eosinophilic intranuclear viral inclusion bodies; similar inclusion bodies were also found cytologically. The inclusions were identified as herpesviral-like particles by transmission electron microscopy in a single horse. In situ hybridization was used to detect EHV-5 nucleic acids within occasional macrophage nuclei. With polymerase chain reaction (PCR), the herpesviral DNA polymerase gene was detected in 19/24 (79.2%) of affected horses and 2/23 (8.7%) of the control horses. Virus genera-specific PCR was used to detect EHV-5 in all of the affected horses and none of the control horses. EHV-2 was detected in 8/24 (33.3%) of affected horses and 1/9 (11.1%) of the control horses. This disease has not been reported before, and the authors propose that based upon the characteristic gross and histologic findings, the disease be known as equine multinodular pulmonary fibrosis. Further, we propose that this newly described disease develops in association with infection by the equine gamma-herpesvirus, EHV-5.

Abomasitis associated with multiple antibiotic resistant Salmonella enterica serotype Typhimurium phagetype DT104.

Salmonella enterica serotype Typhimurium phagetype DT104 is a multiple antibiotic resistant pathogen that has been purported to be more pathogenic than other Salmonella. In this study, we evaluated the possibility that DT104 is the causative agent of veal calf abomasitis observed in four independent outbreaks of salmonellosis. This study was undertaken to determine if the outbreaks might be due to hypervirulent S. enterica serotype Typhimurium phagetype DT104 (DT104) since Salmonella does not usually cause abomasitis. Tissues and fluids from these calves were subjected to bacteriologic culture. Pure Salmonella cultures were then used in bovine challenge experiments. DT104 was identified as the causative agent of abomasitis in calves. Thus, abomasitis is a potential indicator of infection with multiple antibiotic resistant DT104 and adds credence to the apparent hypervirulence of this pathogen.

Kinetics of porcine circovirus type 2 replication.

The kinetics of porcine circovirus type 2 (PCV2) replication in PK15 cells was examined. During productive infection, viral antigens, RNA transcripts and progeny viruses all increased in a time dependent manner. Viral antigens were observed in a few cells at 18 hour postinfection (h p.i.) and cell-free progeny viruses began to appear at about 30 h p.i. Viral transcripts were detected by 18 h p.i. and the capsid protein RNA of 950 nucleotides (nt) was the most abundant RNA species. Two other RNAs of sizes 750 and 450 nt, derived from the predicted replication associated protein (Rep) gene region, were also detected. These two RNAs share 3' common nucleotide sequences and they are transcribed in the same orientation as the proposed unspliced Rep RNA or the recently described Rep' RNA. The 35 kD capsid protein was observed at 30 h p.i. by Western blot analysis and it appeared to be the most immunodominant protein in swine exposed to PCV2. The capsid proteins of PCV type 1 and PCV2 each contain a nuclear localization signal sequence capable of targeting a reporter protein to the nucleoli of transfected cells when the capsid proteins were expressed as 3' fusion polypeptides. Although previous reports indicated that PCV2 capsid proteins localized predominantly in the nuclei of infected cells, we observed an abundant amount of PCV2 capsid proteins in the cytoplasm of many cells of the infected cultures. The cells that exhibited cytoplasmic capsid proteins also contained virus nucleic acids, indicating that these proteins were synthesized by the infected cells and not through uptake from the culture medium. Elucidation of the changes that affect the localization pattern of PCV2 capsid proteins, nuclear versus cytoplasmic, requires further investigation.

Experimental reproduction of severe disease in CD/CD pigs concurrently infected with type 2 porcine circovirus and porcine reproductive and respiratory syndrome virus.

Three-week-old cesarean-derived colostrum-deprived (CD/CD) pigs were inoculated with porcine circovirus type 2 (PCV2, n = 19), porcine reproductive and respiratory syndrome virus (PRRSV, n = 13), concurrent PCV2 and PRRSV (PCV2/PRRSV, n = 17), or a sham inoculum (n = 12) to compare the independent and combined effects of these agents. Necropsies were performed at 7, 10, 14, 21, 35, and 49 days postinoculation (dpi) or when pigs became moribund. By 10 dpi, PCV2/PRRSV-inoculated pigs had severe dyspnea, lethargy, and occasional icterus; after 10 dpi, mortality in this group was 10/11 (91%), and all PCV2/ PRRSV-inoculated pigs were dead by 20 dpi. PCV2-inoculated pigs developed lethargy and sporadic icterus, and 8/19 (42%) developed exudative epidermitis; mortality was 5/19 (26%). PRRSV-inoculated pigs developed dyspnea and mild lethargy that resolved by 28 dpi. Microscopic lesions consistent with postweaning multisystemic wasting syndrome (PMWS) were present in both PCV2- and PCV2/PRRSV-inoculated pigs and included lymphoid depletion, necrotizing hepatitis, mild necrotizing bronchiolitis, and infiltrates of macrophages that occasionally contained basophilic intracytoplasmic inclusion bodies in lymphoid and other tissues. PCV2/ PRRSV-inoculated pigs also had severe proliferative interstitial pneumonia and more consistent hepatic lesions. The most severe lesions contained the greatest number of PCV2 antigen-containing cells. PRRSV-inoculated pigs had moderate proliferative interstitial pneumonia but did not develop bronchiolar or hepatic lesions or lymphoid depletion. All groups remained seronegative to porcine parvovirus. The results indicate that 1) PCV2 coinfection increases the severity of PRRSV-induced interstitial pneumonia in CD/CD pigs and 2) PCV2 but not PRRSV induces the lymphoid depletion, granulomatous inflammation, and necrotizing hepatitis characteristic of PMWS.

Postweaning multisystemic wasting syndrome induced after experimental inoculation of cesarean-derived, colostrum-deprived piglets with type 2 porcine circovirus.

Cesarean-derived, colostrum-deprived pigs (n = 23) were inoculated intranasally and subcutaneously with a low cell culture passage of type 2 porcine circovirus. In 11 pigs, a persistent fever that lasted 7-17 days began 12-15 days after inoculation with virus. Additional signs of disease in those 11 pigs included depression (11 of 11 pigs), palpable enlargement of inguinal, prefemoral, and popliteal lymph nodes (11 of 11), icterus (6 of 11), and hyperpnea (2 of 11). The remaining 12 pigs had fever that occurred intermittently for 2-4 days between days 12 and 20 postinoculation. Overt signs of disease in those pigs were limited to palpable enlargement of inguinal and popliteal lymph nodes (9 of 12 pigs). When compared with control pigs of similar age, the average daily rate of weight gain for all pigs inoculated with virus was less over a 2-week period that began 2 weeks post inoculation. At postmortem examination, lymph node enlargement was seen in 14 of 14 pigs euthanized between days 20 and 28 postinoculation. Lymph node enlargement was especially prominent in pigs that developed a persistent fever. Microscopic lesions noted in pigs that developed a persistent fever included cellular depletion in lymphoid tissues; hepatic cell necrosis; and lymphogranulomatous inflammation of lymph nodes, Peyer's patches of the intestine, liver, kidney, and heart. Virus was isolated with varying frequency from nasal, rectal, or tonsil swab specimens, buffy coat, serum, urine, and lung lavage fluid obtained antemortem or postmortem. Virus was isolated from or viral DNA was detected in a variety of tissues obtained postmortem up to 125 days postinoculation. Antibody against type 2 porcine circovirus usually was detected in serum between 15 and 20 days postinoculation; however, antibody against virus was not detected in serum from 4 pigs euthanized 20-24 days postinoculation. Direct contact with pigs inoculated with virus 42 days previously resulted in transmission of virus to 3 of 3 control pigs.

Effects of intranasal inoculation with Bordetella bronchiseptica, porcine reproductive and respiratory syndrome virus, or a combination of both organisms on subsequent infection with Pasteurella multocida in pigs.

OBJECTIVE: To determine effects of intranasal inoculation with porcine reproductive and respiratory syndrome virus (PRRSV) or Bordetella bronchiseptica on challenge with nontoxigenic Pasteurella multocida in pigs. ANIMALS: Seventy 3-week-old pigs. PROCEDURE: In experiment 1, pigs were not inoculated (n= 10) or were inoculated with PRRSV (10), P. multocida (10), or PRRSV followed by challenge with P. multocida (10). In experiment 2, pigs were not inoculated (n = 10) or were inoculated with B. bronchiseptica (10) or PRRSV and B. bronchiseptica (10); all pigs were challenged with P. multocida. Five pigs from each group were necropsied 14 and 21 days after initial inoculations. RESULTS: Pasteurella multocida was not isolated from tissue specimens of pigs challenged with P. multocida alone or after inoculation with PRRSV. However, in pigs challenged after inoculation with B. bronchiseptica, P. multocida was isolated from specimens of the nasal cavity and tonsil of the soft palate. Number of bacteria isolated increased in pigs challenged after coinoculation with PRRSV and B. bronchiseptica, and all 3 agents were isolated from pneumonic lesions in these pigs. CONCLUSIONS AND CLINICAL RELEVANCE: Infection of pigs with B. bronchiseptica but not PRRSV prior to challenge with P. multocida resulted in colonization of the upper respiratory tract and tonsil of the soft palate with P. multocida. Coinfection with PRRSV and B. bronchiseptica predisposed pigs to infection of the upper respiratory tract and lung with P. multocida. Porcine reproductive and respiratory syndrome virus and B. bronchiseptica may interact to adversely affect respiratory tract defense mechanisms, leaving pigs especially vulnerable to infection with secondary agents such as P. multocida.

A simple and rapid flow cytometric method for detection of porcine cell surface markers.

The objective of this study was to develop a rapid and reliable method for flow cytometric analysis of porcine whole blood cells. Fifty-microliters of heparin- or EDTA-treated whole blood was added to wells of a round-bottom 96-well microtitration plate. Each well contained 10 microl of an appropriate dilution of four different antibodies (40 microl total; two primary monoclonal antibodies and two fluorescent-labeled secondary antibodies). For convenience, the antibody mixture could be added to plates 1-2 days prior to assay and stored at 4 degrees C. Once whole blood was added to wells, plates were mixed gently, placed in a sealed bag and incubated in the dark at room temperature for 20 min. Contents of wells were then transferred to polystyrene tubes containing 2 ml of 1.5% formalin in distilled water and mixed gently. Cells were fixed for a minimum of 30 min and then stored in the dark at 4 degrees C until analysis by flow cytometry. Analysis of cell samples may be done up to 3 days after fixation. Results indicate that the percentages of Class I, Class II, CD3, CD8, CD4, CD45, monocyte, gamma-delta T-cell populations, and total number of granulocytes identified using this method were comparable to standard values or to values obtained following separation of white blood cells from red blood cells. The percentage of labeled B-cells was lower than standard values. Total assay time from receipt of blood to acquisition of data by flow cytometry required less than 2 h. This modified assay was shown to be simple, reliable, and useful for screening large numbers of porcine samples in a minimal period of time.

Morphologic lesions in type 2 BVDV infections experimentally induced by strain BVDV2-1373 recovered from a field case.

Widespread outbreaks of severe acute BVDV, some associated with hemorrhagic syndrome (HS), were reported in Quebec and Ontario in 1993. These outbreaks caused significant economic hardship in infected herds. In the Ontario outbreak 150 dairy, 600 beef and 100 milk and grain fed veal herds were affected with losses estimated at $40000-$10000 per herd in lost animals, milk production, abortions and genetics. Fever, pneumonia, diarrhea, and sudden death occurred in all age groups of cattle. Abortions were frequently observed in pregnant cattle. The viruses associated with this outbreak were determined to be noncytopathic BVDV from the type 2 genotype. All BVDV2 associated with these outbreaks were noncytopathic. One of the viruses isolated from the Ontario outbreak, BVDV2-1373, was used to experimentally induce HS in 5-6 weeks old colostrum deprived, seronegative calves. All animals developed leukopenia and thrombocytopenia within 6-10 days with some developing bloody diarrhea and becoming moribund. Animals were killed for necropsy between 6 and 11 days postinfection. Histopathologically lesions were similar, but more severe, to those seen early on (within first 9 days after superinfection) in animals with experimentally induced mucosal disease (MD). There were no erosions and ulcerations present in the upper digestive tract. In hemorrhages in the mucosa, virus antigen (VA) was present in macrophages of both the lamina propria and the submucosa and in basal epithelial cells. Cells containing VA were vacuolated and separated from each other. The most severe lesions observed in the digestive tract were in the Peyers patches and were characterized by depletion of lymphocytes and proliferation of crypt cells resulting in crypthyperplasia. Apoptotic cells were present in crypts and areas of lymph follicles where viral antigen was detected. Out of the six animals, VA was present in four animals in the pancreas, three animals in the pituitary and in two animals in the adrenal glands. The results suggest that the pathology resulting from acute infection with a highly virulent noncytopathic BVDV2 differs from the pathology observed in classic mucosal disease.

Effects of intranasal inoculation of porcine reproductive and respiratory syndrome virus, Bordetella bronchiseptica, or a combination of both organisms in pigs.

OBJECTIVE: To examine effects of co-infection with porcine reproductive and respiratory syndrome virus (PRRSV) and Bordetella bronchiseptica in pigs. ANIMALS: Forty 3-week-old pigs. Procedure-30 pigs (10 pigs/group) were inoculated with PRRSV, B bronchiseptica, or both. Ten noninoculated pigs were control animals. RESULTS: Clinical signs, febrile response, and decreased weight gain were most severe in the group inoculated with both organisms. The PRRSV was isolated from all pigs in both groups inoculated with virus. All pigs in both groups that received PRRSV had gross and microscopic lesions consistent with interstitial pneumonia. Bordetella bronchiseptica was cultured from all pigs in both groups inoculated with that bacterium. Colonization of anatomic sites by B bronchiseptica was comparable between both groups. Pigs in the group that received only B bronchiseptica lacked gross or microscopic lung lesions, and B bronchiseptica was not isolated from lung tissue. In the group inoculated with B bronchiseptica and PRRSV, 3 of 5 pigs 10 days after inoculation and 5 of 5 pigs 21 days after inoculation had gross and microscopic lesions consistent with bacterial bronchopneumonia, and B bronchiseptica was isolated from the lungs of 7 of those 10 pigs. CONCLUSIONS AND CLINICAL RELEVANCE: Clinical disease was exacerbated in co-infected pigs, including an increased febrile response, decreased weight gain, and B bronchiseptica-induced pneumonia. Bordetella bronchiseptica and PRRSV may circulate in a herd and cause subclinical infections. Therefore, co-infection with these organisms may cause clinical respiratory tract disease and leave pigs more susceptible to subsequent infection with opportunistic bacteria.

Bovine viral diarrhea virus cytopathic and noncytopathic biotypes and type 1 and 2 genotypes in diagnostic laboratory accessions: clinical and necropsy samples from cattle.

One hundred three bovine samples submitted to the Oklahoma Animal Disease Diagnostic Laboratory (OADDL) that were positive for bovine viral diarrhea virus (BVDV) were typed by a nested reverse transcription-polymerase chain reaction for BVDV genotypes. These BVDV samples included supernatants from virus isolation (79), serums (17), and buffy coats (7). The biotype, cytopathic (CP) or noncytopathic (NCP), was determined by cell culture virus isolation. Twenty-eight of 103 samples were submitted for herd screening for BVDV, 32 from OADDL necropsy cases, and 43 from live cattle with varied clinical conditions. Two samples contained 2 bands indicating presence of both BVDV types 1 and 2. Of the 105 BVDV samples, 26 were type 1 CP strains (24.8%), 38 were type 1 NCP strains (36.2%), 10 were type 2 CP strains (9.5%), and 31 were type 2 NCP strains (29.5%). From the 105 BVDV isolates, NCP biotypes were isolated more frequently (69, 65.7%) than CP biotypes (36, 34.3%), and type 1 genotypes were more frequently isolated (64, 61.00%) than type 2 genotypes (41, 39.0%). The NCP strains were more common than CP in herd screening samples. Cattle with respiratory disease history at time of sampling had more NCP than CP biotypes and more type 1 than type 2 genotypes. Of the necropsy cases, more were type 1 than type 2 genotypes for the respiratory cases with fibrinous pneumonia, more were type 1 than type 2 genotypes in cattle with enteritis/colitis without systemic lesions, and more were CP than NCP biotypes in cattle with enteritis/colitis with systemic lesions. No CP biotype was isolated from serum samples.

Nested reverse transcriptase-polymerase chain reaction (RT-PCR) for typing ruminant pestiviruses: bovine viral diarrhea viruses and border disease virus.

A nested reverse transcription (RT) polymerase chain reaction (PCR) assay was evaluated for differentiating reference bovine viral diarrhea virus (BVDV) strains, BVDV from diagnostic accessions, modified-live virus (MLV) BVDV strains in bovine viral vaccines, and a reference border disease virus (BDV). The detection level of this assay was compared to viral infection in cell culture. The PCR assay was used to distinguish 3 ruminant pestiviruses, types 1 and 2 BVDV, and type 3 BDV. The consensus (first) PCR assay detected all 3 ruminant pestiviruses, a result of the shared sequence homology. The consensus PCR product was subjected to a second (nested) PCR which used type-specific primers. The nested PCR was able to differentiate the 3 ruminant pestiviruses. Viral stocks of BVDV were diluted 10-fold and processed for the 2-step PCR assay. The sensitivity of this 2-step PCR assay was compared to viral infectivity in cell culture based on identical volumes of the system tested (cell culture assay and processing for RNA). The RT-PCR type-specific assay differentiated BVDV laboratory reference strains (12), diagnostic laboratory isolates (15), 2 MLV BVDV vaccine strains, and a BDV strain. The 30 ruminant pestiviruses typed included: (1) 27 reference strains and diagnostic laboratory isolates; 18 cytopathic (CP) type 1 strains, 3 CP type 2 strains, 3 noncytopathic (NCP) type 1 strains, and 3 NCP type 2 strains; (2) 2 MLV strains, type 1; and (3) 1 CP BDV type 3. The PCR assay had a detection limit of 10 TCID50/0.025 mL of virus when 3 separate BVDV were tested. This 2 step RT-PCR assay would be useful for the typing of ruminant pestiviruses, particularly BVDV isolates from the diagnostic laboratory.

Bovine viral diarrhea virus isolated from fetal calf serum enhances pathogenicity of attenuated transmissible gastroenteritis virus in neonatal pigs.

A bovine viral diarrhea virus (BVDV-C) was isolated from swine tissue culture cells used to attenuate the transmissible gastroenteritis virus (TGEV) after 68 passes. Piglets given a pure culture of BVDV-C developed clinical signs similar to those of a mild TGEV infection and recovered by 10 days postexposure. Villous blunting and fusion was observed in the small intestine, and a lymphocyte depletion was observed in Peyer's patches in the ileum. Piglets given a combination of BVDV-C and attenuated TGEV developed clinical signs similar to those of a virulent TGEV infection and were euthanized. The combined infection induced a generalized lymphocyte depletion throughout the lymphatic system and villous atrophy in the intestinal tract. Piglets exposed to a another type I strain of BVDV (NY-1) either alone or in combination with the attenuated TGEV had mild clinical signs similar to those of a TGEV infection. Moderate villous atrophy in the ileum and a lymphocyte depletion in the mesenteric lymph node were observed in these piglets postmortem. The data indicate a potential problem for diagnostic laboratories in relation to a diagnosis of virulent TGEV infections and in the field for young piglets exposed to a BVDV-contaminated TGEV vaccine.

Use of polymerase chain reaction to simultaneously detect and type bovine viral diarrhoea viruses isolated from clinical specimens.

The techniques of indirect immunofluorescence (IF), immuno-peroxidase (IP) staining and the one-step reverse transcriptase polymerase chain reaction (RT-PCR) were compared for detection of 102 isolates of bovine viral diarrhoea virus (BVDV) in infected cell cultures. The BVDV was obtained from bovine clinical specimens, including sera, buffy coats and tissues, submitted from farms located in the States of Iowa and Wisconsin, United States of America. The IF technique detected 88/102 (86.3%) of the viral isolates, whereas IP staining detected an additional 4 isolates (92/102; 90%). The one-step RT-PCR using primers derived from the 5' untranslated region of the BVDV genome detected 102/102 (100%) of the BVDV isolates. A second-round PCR utilising another pair of PCR primers from the 5' untranslated region, allowed rapid genotyping of BVDV. The procedure used showed that the PCR assay based on the 5' untranslated region of the virus genome is the most sensitive indicator for BVDV detection in cell culture, and is also of considerable epidemiological importance since it allowed rapid genotyping of BVDV isolated from clinical specimens. In addition to detection and genotyping of BVDV isolated from clinical specimens, the RT-PCR procedure can be used for routine screening of locally produced and imported biologicals for BVDV contamination. However, the procedure requires further refinement to enable direct application on the clinical specimen.

Acute pulmonary lesions in sheep experimentally infected with bovine viral diarrhoea virus.

Six sheep, aged 6-8 months and seronegative for pestivirus, were inoculated intranasally, through the tracheal wall, and intrabronchially with a non-cytopathogenic isolate of bovine viral diarrhoea virus (ncpBVDV). Infected sheep were killed in pairs on post-inoculation day (PID) 2, 4 and 6. They all exhibited transient leucopenia or lymphopenia, or both. Platelet counts decreased but remained within normal limits. BVDV was isolated from buffy coats and tissues of all sheep inoculated with ncpBVDV but not from two uninfected control animals. Pulmonary lesions, evident in ncpBVDV-inoculated sheep, consisted of moderate oedema with multifocal alveolar septal necrosis and haemorrhage, infiltrates of mononuclear inflammatory cells, and degenerative changes in alveolar epithelium, endothelium and pulmonary intravascular macrophages. Additionally, there was morphological evidence of platelet activation and pulmonary intravascular macrophage stimulation. Lesions were not observed in the two control sheep.

Protection of pregnant cattle and their fetuses against infection with bovine viral diarrhea virus type 1 by use of a modified-live virus vaccine.

OBJECTIVE: To determine efficacy of a vaccine containing modified-live bovine viral diarrhea virus (BVDV) type 1 for protecting pregnant cows and their fetuses against virulent heterologous BVDV type 1. DESIGN: Randomized controlled cohort study. ANIMALS: 18 yearling beef heifers seronegative for BVDV and negative when tested for BVDV by virus isolation. PROCEDURE: Cattle were randomly assigned to control (unvaccinated; n = 6) or vaccinated (12) groups. Vaccinated heifers were given a combination vaccine containing modified-live BVDV type 1 comprising a cytopathic (NADL) strain. All 18 heifers were then bred and challenge-exposed between 70 and 75 days of gestation with BVDV type 1, administered intranasally. Cattle were monitored, and infection status of offspring was determined after parturition. Antibody concentrations of vaccinated and control heifers were also monitored. RESULTS: All 6 calves from control heifers had positive results on multiple virus isolation tests and were considered persistently infected. In comparison, only 2 calves from vaccinated cows had positive results on virus isolation tests and were considered persistently infected. One vaccinated heifer aborted, but the fetus was not persistently infected, and the abortion was not attributed to BVDV infection. CLINICAL IMPLICATIONS: Analysis of these data indicated that a single dose of a modified-live NADL-derived BVDV type 1 vaccine will confer protection to dams and their fetuses against challenge-exposure to heterologous BVDV type 1 organisms.