Apparent prevalence of hemotropic mycoplasma in dairy calves and replacement heifers on Michigan farms.

The bovine hemoplasmas include Mycoplasma wenyonii and Candidatus Mycoplasma haemobos, which are increasingly recognized as infecting cattle throughout the world. Infection with hemotropic mycoplasma has been reported to be widespread in mature dairy cows, but little is known about its prevalence in calves and heifers. The objective of this study was to investigate the prevalence and dynamics of infection with M. wenyonii and C. M. haemobos in calves and replacement heifers on Michigan dairy farms and assess the potential associations between infection status and hematological values. The study was designed as a prospective cross-sectional study with a longitudinal component. A convenience sample of 11 farms agreed to participate and were visited twice between March and September 2022. During the first farm visit, researchers collected blood samples from up to 94 animals per farm distributed among newborn and preweaning calves (n ≤ 31), weaned calves (n = 21), pre-breeding heifers (n = 21), and pregnant heifers (n = 21). During the first visit, blood samples (n = 174) were also collected from a convenience sample of mature cows to confirm the herd infection status. The same calves and heifers were sampled again ∼95 d (±3.0) later. During the first visit, blood samples were collected from 797 calves and replacement heifers, whereas 675 samples were collected during the second visit due to the inability to locate some animals. Detection of M. wenyonii and C. M. haemobos was based on results of real-time PCR. The hematocrit was determined using microcentrifugation, and the concentration of leukocytes using an automated cell counter. In all herds, most mature cows that were sampled tested positive for infection. The within-herd apparent prevalence of hemoplasma in calves and replacement heifers was 100% for both M. wenyonii and C. M. haemobos. The apparent prevalence of hemoplasma in youngstock was associated with age. In calves that were 1 to 6 mo old, the prevalence of infection was 6% to 8% but sharply increased to 31% by 8 mo of age. In older animals, the prevalence remained high, and was almost 100% in animals greater than 17 mo of age. Based on calves and heifers sampled twice, the cumulative incidence varied widely among herds, ranging from 3.7% to 96.0%, and increased with the age of the animals. We found no difference in hematocrit or number of lymphocytes, monocytes, neutrophils, or total leukocytes based on infection status. The number of eosinophils was greater in infected animals. This is the first study to report the prevalence of hemoplasmas in calves and replacement heifers in the United States. It indicates that young calves can be infected with hemoplasmas, but the rate of infection is low. The likelihood of infection increases as animals age, with a notable rise in the proportion of infected heifers occurring by 8 mo old, and the prevalence eventually reaching nearly 100% in older animals. Once infected, heifers appear to remain chronic carriers. Hemoplasma infection alone does not usually lead to the development of clinical signs, and most of the animals remain apparently healthy.

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.

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.

Apparent prevalence of Mycoplasma wenyonii, Candidatus Mycoplasma haemobos, and bovine leukemia virus in Wisconsin and Michigan dairy cattle herds.

Mycoplasma wenyonii (formerly Eperythrozoon wenyonii) is a hemotrophic, epicellular bacterial parasite of cattle that has been associated with clinical disorders, including hemolytic anemia, decreased milk yield, and peripheral edema. Mycoplasma wenyonii and a related organism, Candidatus Mycoplasma haemobos, have been detected in both ill and apparently healthy cattle, but little is known about their prevalence in US dairy cattle. The objective of this prospective, cross-sectional study was to determine herd-level apparent prevalence of M. wenyonii and C. M. haemobos in dairy cattle located in Wisconsin and Michigan compared with seroprevalence of bovine leukemia virus (BLV) in the same herds. In summer 2018, researchers collected blood samples from 30 lactating cows per herd from randomly recruited farms in selected dairy-intensive counties in each state. During the farm visit, a brief survey was used to collect herd management information. Detection of M. wenyonii and C. M. haemobos were based on PCR testing, and ELISA was used to test for antibodies to BLV. Blood samples were collected from lactating cows located in 64 Wisconsin herds (n = 1,930 samples) and 18 Michigan herds (n = 591 samples). Herd-level apparent prevalence was 100% for both M. wenyonii and C. M. haemobos. Herd-level seroprevalence for BLV was 83 and 100% for Wisconsin and Michigan herds, respectively. Estimated within-herd apparent prevalence of M. wenyonii was 71.7% ± 1.0% (ranging from 23.3 to 93.5%) and for C. M. haemobos was 77.3% ± 1.0% (ranging from 16.7 to 100%). Within-herd prevalence of BLV positive samples was 39.8% ± 1.0% and ranged from 0 to 86.7%. About 22% of cows were concurrently positive for all 3 organisms. Parity and stage of lactation were recorded for 2,317 cows. Prevalence of positive cows for parity groups 1, 2, and ≥3 were 72.0, 73.8, and 67.7% for M. wenyonii; 80.9, 76.8, and 74.9% for C. M. haemobos; and 25.3, 39.7, and 55.5% for BLV, respectively. None or only minor differences in apparent prevalence were observed based on stage of lactation. This is the first report of the prevalence of hemotrophic mycoplasmas in Wisconsin and Michigan dairy herds and indicates that infection with these organisms is endemic. The impact of infection on cattle health and productivity remains unknown, and risk factors associated with infection warrant further study.

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.

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.

Sarcocystis spp. Infection in two Red Panda Cubs (Ailurus fulgens).

Two neonatal male red panda (Ailurus fulgens) littermates were submitted for necropsy examination. One animal was found dead with no prior signs of illness; the other had a brief history of laboured breathing. Post-mortem examination revealed disseminated protozoal infection. To further characterize the causative agent, transmission electron microscopy (TEM), immunohistochemistry (IHC), polymerase chain reaction (PCR) and amplification and nucleic acid sequencing were performed. IHC was negative for Toxoplasma gondii and Neospora caninum, but was positive for a Sarcocystis spp. TEM of cardiac muscle and lung revealed numerous intracellular apicomplexan protozoa within parasitophorous vacuoles. PCR and nucleic acid sequencing of partial 18S rRNA and the internal transcribed spacer (ITS)-1 region confirmed a Sarcocystis spp. that shared 99% sequence homology to Sarcocystis neurona and Sarcocystis dasypi. This represents the first report of sarcocystosis in red pandas. The histopathological, immunohistochemical, molecular and ultrastructural findings are supportive of vertical transmission resulting in fatal disseminated disease.

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.

Comparison of the complete genomic sequence of the border disease virus, BD31, to other pestiviruses.

The genus Pestivirus is composed of hog cholera virus (HCV) [also known as classical swine fever virus (CSFV)], bovine viral diarrhea virus (BVDV), and border disease virus (BDV). Complete sequences have been published for HCV (or CSFV) and the two genotypes of BVDV (BVDV1 and BVDV2). In this study the complete sequence of the border disease virus (BDV), BD31, was determined. BD31 was isolated from a lamb with hairy shaker syndrome and is the BDV type virus offered by ATCC (ATCC VR-996). The genome was 12268 nucleotides long and had a single large open reading frame (ORF) beginning at nucleotide 357 and ending at nucleotide 12045. The sequence identity of the predicted amino acid sequence of BD31 and other published pestivirus sequences varied from 71% to 78%. Phylogenetic analysis of available complete genomic sequences segregated pestiviruses into two branches. One branch contained BD31 and HCV (or CSFV) isolates while the other branch contained BVDV1 and BVDV2 isolates. Pestiviruses from the same branch were similar in the length of the 5' and 3' untranslated regions (UTR). When complete genomic sequences were compared among BD31, HCV (or CSFV), BVDV1 and BVDV2, the highest sequence identity was observed in the 5' UTR. Within the ORF, the highest sequence identity was observed in the genomic region coding for the nonstructural viral polypeptide p80.

Mapping of a type 1-specific and a type-common epitope on the E2 (gp53) protein of bovine viral diarrhea virus with neutralization escape mutants.

Bovine viral diarrhoea viruses (BVDV) have recently been segregated into two genotypes, BVDV 1 and BVDV 2. However, the antigenic differences and similarities of BVDV 1 and BVDV 2 remain poorly defined. In this study, the E2 epitopes of two neutralizing monoclonal antibodies (mAbs) produced against an isolate of BVDV 1 were mapped. The mAb 157, previously determined to be broadly cross-reactive to BVDV, was discovered to be BVDV 1-specific, whereas mAb 348 bound to and neutralized BVDV 2. Both mAbs bound to epitopes within the first 192 amino acids of the E2 protein as determined by reactions with a C-terminally truncated E2. To identify critical amino acids affecting these epitopes, mAb escape mutants were selected for sequencing from BVDV 1 and BVDV 2 strains with different (wild-type) mAb binding phenotypes. In addition, the E2 gene of several BVDV were sequenced and the sequences were compared with amino acid changes in mutant viruses. Single nucleotide changes in escape mutants selected with mAb 157 resulted in deduced amino acid changes at E2 positions 9, 32 or 72. Amino acid changes at position 72 also affected the epitope of mAb 348. Alignment of E2 nucleotide sequences revealed that BVDV 2 are missing six nucleotides encoding the equivalent of amino acids 31 and 32 of BVDV 1 and thus, this difference can account for the BVDV 1-specificity of mAb 157. Single nucleotide mutations in mAb 348 escape mutants of BVDV 1 and BVDV 2 resulted in changes in 3 amino acids in the previously described immunodominant 71-74 region (Virology 190, 763-772). A fourth amino acid change observed in a mutant of BVDV 2 extended this region to position 77. Thus, the amino acid changes affecting the conserved epitope of mAb 348 occurred in a short spatial array over only seven amino acids, unlike the described composite epitopes previously mapped to this region.

Natural recombination in bovine viral diarrhea viruses.

BVDV isolates exist as two biotypes differentiated at the molecular level by production of a p80 polypeptide. Insertions consisting of host cell sequences and/or duplicated and rearranged viral sequences have been observed in the portion of the genome coding for the p80 polypeptide in some, but not all, cytopathic BVDV. The significance of these insertions to biotypic expression has yet to be demonstrated. It has been hypothesized that recombination results in the production of the p80 polypeptide by introduction of a cleavage site into a precursor polypeptide or the introduction of a second copy of the p80 gene. Because inserts have not been identified in all cytopathic BVDV examined, it appears that recombination may not be the only mechanism involved in biotypic determination.

Survey of cell lines in the American Type Culture Collection for bovine viral diarrhea virus.

Cell lines originating from cattle, sheep, goat, deer, bison, swine, rabbit, hamster, cat, dog, monkey, human, and mosquito were obtained from the American Type Culture Collection and tested for contamination with bovine viral diarrhea virus (BVDV). Immunocytochemical procedures and polymerase chain reaction (PCR) amplification were used to detect viral antigen or viral RNA in 13 of 41 cell lines. The results of these procedures correlated exactly. Cell lines derived from cattle, sheep, goat, deer, bison, rabbit, and domestic cat were found contaminated with BVDV. Attempts were made to experimentally infect 14 swine, rabbit, hamster, cat, dog, monkey, and human cell lines that had been found free of virus. All swine cell lines, and most rabbit and cat cell lines, became infected with BVDV. Hamster, human, dog, and certain rabbit and cat cells were refractory to BVDV infection. Experimental infection of monkey cells produced variable results.

Immunogens of bovine viral diarrhea virus.

Bovine viral diarrhea virus (BVDV) is a ubiquitous pathogen of cattle that induces economically important diseases affecting multiple organ systems. In the United States, over 150 biological products are licensed for control of BVDV. These products contain live or killed BVDV, and many products contain other viruses or bacteria. Potency tests for these vaccines are based on animal inoculation and serology. For live virus vaccines, titration of viral infectivity in cell culture is an accepted alternative to animal inoculation. The immunogens in a killed virus vaccine may be measured by enzyme linked immunoabsorbent assay. Immunogens of BVDV that stimulate a protective immune response have not been conclusively identified. Epitopes on a putative viral envelope glycoprotein, gp53, are involved in viral neutralization. Other viral glycoproteins, gp48 and gp25, are immunogenic but epitopes on these proteins do not stimulate production of antibodies that efficiently neutralize virus. Progress in developing meaningful in vitro assays for quantitation of BVDV immunogens awaits identification of viral proteins that stimulate a protective immunity.

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.

Efficiency of various cloned DNA probes for detection of bovine viral diarrhea viruses.

We have evaluated 24 cytopathic (CP) and 37 noncytopathic (NCP) strains of bovine viral diarrhea virus (BVDV) with a dot blot assay using four different genome segments of the NADL strain as hybridization probes (p80, p54, gp53, and gp62). The p80 and p54 probes hybridized to 23/24 (96%) and 22/24 (92%), respectively, of CP strains examined. In contrast, these same two probes only detected 16/37 (43%) and 5/37 (13%), respectively, of the NCP strains examined. The gp53 probe detected 18/24 (75%) and the gp62 probe detected 19/24 (79%) of the CP strains. In contrast, these latter two probes only detected 9/37 (24%) and 7/37 (20%), respectively, of NCP strains. This low detection rate of NCP strains suggests a need for developing a probe based on NCP sequences for identification of NCP strains.

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.

Prevalence of bovine viral diarrhea virus genotypes and antibody against those viral genotypes in fetal bovine serum.

One thousand lots of pooled fetal bovine serum (FBS) were tested for contamination with bovine viral diarrhea virus (BVDV) and/or for contamination with neutralizing antibody against BVDV. Noncytopathic or cytopathic BVDV was isolated from 203 lots of FBS. Analysis of the viral isolates identified 115 type 1 and 65 type 2 BVDV isolates. An additional 23 virus isolates were mixtures of > or = 2 BVDV isolates and were not classified to viral genotype. Further characterization of the type 1 viruses identified 51 subgenotype 1a and 64 subgenotype 1b BVDV isolates. Viral neutralizing antibody was detected in 113 lots of FBS. Differential viral neutralization indicated that type 1 BVDV induced the antibody detected in 48 lots of FBS and type 2 BVDV induced the antibody detected in 16 lots of FBS.

Changes in levels of viremia in cattle persistently infected with bovine viral diarrhea virus.

Virus isolation and serum neutralizing antibody titers were determined over a period of time from samples collected from animals persistently infected with bovine viral diarrhea virus (BVDV). To evaluate over time the ability to detect BVDV by virus isolation from serum or white blood cell preparations, 4 persistently infected calves were monitored from birth until 70 days of age. In 3 of 4 persistently infected calves, virus isolation from serum and white blood cells was negative until approximately 42 days of age, when colostral antibody had declined. The level of viremia in 7 adult (> 12 months) persistently infected animals decreased by 1 10-fold dilution over at least a 2-year period. The level of viremia became undetectable by virus isolation from serum in 1 of the 7 animals examined. This decline was associated with the development of virus neutralizing antibody. Although the level of viremia is fairly stable within persistently infected animals, the presence of specific neutralizing antibody may affect the ability to isolate BVDV. These findings are important when considering diagnostic testing to identify persistently infected animals by virus isolation.

Methods for detection and frequency of contamination of fetal calf serum with bovine viral diarrhea virus and antibodies against bovine viral diarrhea virus.

Methods used by the National Animal Disease Center to test fetal calf serum for contamination with bovine viral diarrhea virus (BVDV) and antibodies against BVDV are described. Using those methods, virus was isolated from 332 of 1,608 (20.6%) lots of raw fetal calf serum obtained specifically for the Center and 93 of 190 (49%) lots of commercially available fetal calf serum. Virus neutralization and immunoperoxidase staining tests were used to detect antibodies against BVDV in 224 of the 1,608 (13.9%) lots of raw fetal calf serum. Both BVDV and antibodies against BVDV were detected in 50 lots of raw serum. The molecular specificity of antibodies against BVDV was determined by radioimmunoprecipitation. Lots of fetal calf serum that contained BVDV-specific antibodies that did not neutralize virus were identified.

Comparison of cytopathic and noncytopathic isolates of bovine viral diarrhea virus by oligonucleotide fingerprinting.

The molecular technique of RNA fingerprinting was used to characterize the genomes of 5 isolates of bovine viral diarrhea virus (BVDV): 2 viral pairs from the same animal, BVD-ILN/BVD-ILC and BVD-TGAN/BVD-TGAC, and the cytopathic viral prototype, BVD-NADL. Oligonucleotide patterns from the viruses were compared, and unique and overlapping oligonucleotides were identified. A comparison of the fingerprints indicated that the genome of each virus was distinguishable by the T1 RNase oligonucleotide fingerprinting technique. The greatest similarity observed was between oligonucleotides from BVD-ILC and BVD-ILN. Eighteen large oligonucleotides were conserved in all 5 BVDV isolates studied. We found that within a pair of BVDV, the cytopathic fingerprint was different from the noncytopathic fingerprint, indicating that cytopathic and noncytopathic BVDV may be distinct viruses.