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Bovine coronaviruses (BoCVs) have been found in respiratory tissues in cattle and frequently associated with bovine respiratory disease (BRD); however, pathogenesis studies in calves are limited. To characterize the pathogenesis and pathogenicity of BoCV isolates, we used 5 different BoCV strains to inoculate colostrum-deprived calves, ~ 2-5 wk of age. Later, to determine if dual viral infection would potentiate pathogenicity of BoCV, calves were inoculated with BoCV alone, bovine viral diarrhea virus (BVDV) alone, or a series of dual-infection (BVDV-BoCV) schemes. A negative control group was included in all studies. Clinical signs and body temperature were monitored during the study and samples collected for lymphocyte counts, virus isolation, and serology. During autopsy, gross lesions were recorded and fixed tissues collected for histopathology and immunohistochemistry; fresh tissues were collected for virus isolation. Results suggest increased pathogenicity for isolate BoCV OK 1776. Increased body temperature was found in all virus-inoculated groups. Lung lesions were present in calves in all dual-infection groups; however, lesions were most pronounced in calves inoculated with BVDV followed by BoCV inoculation 6 d later. Lung lesions were consistent with mild-to-moderate interstitial pneumonia, and immunohistochemistry confirmed the presence of BoCV antigen. Our studies demonstrated that BVDV-BoCV dual infection may play an important role in BRD pathogenesis, and timing between infections seems critical to the severity of lesions.

Bovine viral diarrhea virus (BVDV) 1b was isolated from tissues of a term bovine fetus with petechial hemorrhages noted throughout the body and placenta at autopsy. Fresh lung, kidney, thymus, and liver tissues were examined by direct fluorescent antibody testing and were positive for BVDV antigen and negative for bovine herpesvirus 1 antigen. An organ pool of fresh tissues was positive for noncytopathic (NCP) BVDV-1 by virus isolation. BVDV-1b was identified by sequencing of the 5'-UTR region of the genome. Fixed brain, placenta, thymus, lymph node, lung, kidney, skeletal muscle, liver, and bone marrow were positive for BVDV antigen by immunohistochemistry. Although BVDV hemorrhage and/or thrombocytopenia has been associated historically with NCP strains of BVDV-2, this case adds to more recent reports of BVDV-1 infections and hemorrhage in cattle. This BVDV-1b isolate should be investigated for its potential to cause hemorrhage in postnatal cattle.

Effective control and the eventual eradication of Bovine viral diarrhea virus (BVDV) from cattle populations depend on the accurate identification of infected animals. Although typically a disease agent of cattle, BVDV is known to infect a wide variety of nonbovine species, including sheep. However, validation of serologic tests in these nonbovine species, particularly sheep, is lacking. We analyzed 99 sheep sera (57 samples from Pestivirus-naive sheep, and 42 samples from BVDV-inoculated sheep) in order to investigate 3 serologic tests: the agarose gel immunodiffusion (AGID) and 2 commercial enzyme-linked immunosorbent assays (ELISAs) for detection of BVDV antibodies. At the manufacturer's cutoff thresholds, the AGID performed with 95.2% diagnostic sensitivity; ELISA-A performed with sensitivity of 90.5% and ELISA-B with 69.1%. All 3 tests performed with 100% diagnostic specificity. Two-graph receiver operating characteristic analysis showed that performance characteristics were optimized, such that both diagnostic sensitivity and diagnostic specificity were >95% for both ELISAs, if the thresholds were altered to 34.9% inhibition for ELISA-A and 63.5 signal-to-noise ratio for ELISA-B.

Four species of ruminant pestivirus are currently circulating in the United States: Bovine viral diarrhea virus 1 and 2 (BVDV-1, -2; predominant host: cattle), Border disease virus (BDV; predominant host: sheep), and pronghorn virus (sporadically detected in wild ruminants). A third bovine pestivirus called HoBi-like virus has been detected in cattle and water buffalo in South America, Asia, and Europe. To date, no isolations of HoBi-like viruses from U.S. cattle have been reported. To assess exposure, 2,000 cattle sera, collected between 2014 and 2015 as part of the U.S. brucellosis surveillance program, were tested for antibodies against BVDV-1, BVDV-2, and HoBi-like viruses. In addition, RNA was extracted and tested by reverse transcription-polymerase chain reaction for the presence of pestiviruses; all samples tested negative. The percent of VN-positive samples was 91.3% for BVDV-1, 89.3% for BVDV-2, and 84.9% for HoBi-like viruses. Because the 3 bovine pestiviruses are antigenically cross-reactive, the comparative level of antibody against each pestivirus species was determined. Based on comparative titers, samples were segregated into 6 categories: no titers (7.6%), titers clearly higher against BVDV-1 (22.2%), titers substantially higher against BVDV-2 (9.1%), BVDV-1 and BVDV-2 titers equivalent but substantially higher than HoBi titers (25.7%), titers substantially higher against HoBi-like viruses (0%), and equivocal (35.4%). Titers tended to be higher against BVDV-1 than BVDV-2. However, the overall percentage of animals with titers below levels considered protective against acute bovine pestivirus infection were ~11% for BVDV-1, 12% BVDV-2, and 18% for HoBi-like virus.

Antigen enzyme-linked immunosorbent assay (ELISA) is used for the detection of Bovine viral diarrhea virus persistently infected (BVDV PI) cattle; however, colostrum-derived antibodies may interfere with antigen detection in serum from young PI calves. Our study aimed to assess serum pretreatment methods for reducing such interference. Dilution of PI serum with serum containing specific antibody showed that antibody levels equivalent to those observed in colostrum-fed calves were able to eliminate all antigen signals in a serum sample. Serum was treated with ethylenediamine tetra-acetic acid at pH 4.5, 5.5, 6.5, and 7.5, then boiled, centrifuged, and the supernatant-recovered. BVDV antibody was undetectable by ELISA in supernatants from treated samples, and the antigen ELISA signal was improved. Maximum antigen signal recovery of >90% was achieved at pH 5 ± 0.5. When this optimal treatment method was applied to field samples from 3 PI calves (which were negative in the antigen-capture ELISA without treatment), the antigen signal improved and gave a positive result in each case. Pretreatment may provide an improvement in the detection of young PI calves., (© 2016 The Author(s).)

Bovine viral diarrhea virus (BVDV) continues to be of economic significance to the livestock industry in terms of acute disease and fetal loss. Many of the lesions relating to BVDV infection have been well described previously. The virus is perpetuated in herds through the presence of calves that are persistently infected. Relationships between various species and biotypes of BVDV and host defenses are increasingly understood. Understanding of the host defense mechanisms of innate immunity and adaptive immunity continues to improve, and the effects of the virus on these immune mechanisms are being used to explain how persistent infection develops. The noncytopathic biotype of BVDV plays the major role in its effects on the host defenses by inhibiting various aspects of the innate immune system and creation of immunotolerance in the fetus during early gestation. Recent advances have allowed for development of affordable test strategies to identify and remove persistently infected animals. With these improved tests and removal strategies, the livestock industry can begin more widespread effective control programs.

Detection of antibodies against Bovine viral diarrhea virus (BVDV) in serum and milk by enzyme-linked immunosorbent assay (ELISA) is a crucial part of all ongoing national schemes to eradicate this important cattle pathogen. Serum and milk are regarded as equally suited for antibody measurement. However, when retesting a seropositive cow 1 day after calving, the serum was negative in 6 out of 9 different ELISAs. To further investigate this diagnostic gap around parturition, pre- and postcalving serum and milk samples of 5 cows were analyzed by BVDV antibody ELISA and serum neutralization test (SNT). By ELISA, 3 out of the 5 animals showed a diagnostic gap in the serum for up to 12 days around calving but all animals remained positive in SNT. In milk, the ELISA was strongly positive after birth but antibody levels decreased considerably within the next few days. Because of the immunoglobulin G (IgG)1-specific transport of serum antibodies into the mammary gland for colostrum production, the IgG subclass specificity of the total and the BVDV-specific antibodies were determined. Although all 5 animals showed a clear decrease in the total and BVDV-specific IgG1 antibody levels at parturition, the precalving IgG1-to-IgG2 ratios of the BVDV-specific antibodies were considerably lower in animals that showed the diagnostic gap. Results showed that BVDV seropositive cows may become "false" negative in several ELISAs in the periparturient period and suggest that the occurrence of this diagnostic gap is influenced by the BVDV-specific IgG subclass response of the individual animal.

The objective of the current study was to investigate if the presence of colostral-derived immunoglobulin G (IgG) in blood is an inhibitor of diagnostic polymerase chain reaction (PCR) for detection of Bovine viral diarrhea virus (BVDV). Eleven precolostral and 11 postcolostral blood samples in ethylenediamine tetra-acetic acid (EDTA) anticoagulant as well as serum samples were collected from 11 Holstein bull calves. Calves were fed 3 liters of colostrum once, by oroesophageal tubing. Postcolostral, blood, and serum samples were collected at 48 hr of age. Serum IgG concentrations were determined in the precolostral and postcolostral serum samples using radial immunodiffusion. The blood samples (precolostral and postcolostral) were spiked with BVDV, and 2 diagnostic PCR extraction methods were applied to each sample. The extraction and amplification efficiencies of the 2 PCR methods on the precolostral and postcolostral EDTA blood samples were evaluated. Two of the 11 calves had inadequate passive transfer of colostral immunoglobulins at 48 hr of age based on the serum IgG concentrations. All blood samples from calves were negative for BVDV prior to the spiking with the virus. Evaluation of the 2 different methods among 3 different virus concentrations demonstrated that there was no difference in extraction or amplification efficiency in precolostral and postcolostral samples. The results of this study suggest that bovine IgG is not an inhibitor of PCR used for detection of BVDV in cattle. The methods used in the current study are acceptable for PCR detection of BVDV in cattle.

Recently, confirmed occurrences of persistent bovine viral diarrhea virus (BVDV) infection in North American alpacas have raised concerns about the role of persistently infected (PI) alpacas in transmission of virus among herds, yet only limited pathological descriptions of persistent infections in alpacas have been reported. The objective of this study was to characterize BVDV antigen distribution in 10 PI alpacas of varying age and to compare viral antigen distribution and localization in tissues of PI alpacas with 5 PI calves of varying age. Ocular dysplasia was evident in 1 PI alpaca, constituting the first reported congenital ocular lesion in PI alpacas. Viral antigen was widely distributed in alpaca tissues and was prominent in neurons, endothelial cells, and vascular tunica media myocytes but had limited distribution in lymphoid tissues and moderate distribution in epithelium of several organ systems of alpacas. Macrophages in the alpaca gastrointestinal system submucosa and lymph node medullary sinuses often had prominent labeling. In addition, only 1 alpaca had antigen labeling in the bone marrow in contrast to PI cattle. Labeled cells in calf tissues were more widely distributed, occurring prominently in lymphoid and epithelial tissues. Common features of the 2 host species were widespread antigen labeling and absence of lymphoid depletion.

The genus Pestivirus is composed of 4 important pathogens of livestock: Bovine viral diarrhea virus 1 and 2 (BVDV-1 and BVDV-2), Classical swine fever virus (CSFV), and Border disease virus of sheep (BDV). BVDV are major pathogens of cattle, and infection results in significant economic loss worldwide. A new putative pestivirus species, tentatively called "HoBi-like," "BVDV-3," or "atypical pestiviruses," was first identified in Europe in fetal bovine serum (FBS) imported from Brazil. HoBi-like viruses are related to BVDV at the genetic and antigenic levels. Further, the disease caused by these new viruses resembles clinical presentations historically associated with BVDV infection, including growth retardation, reduced milk production, respiratory disease, reduced reproductive performance, and increased mortality among young stock. Current BVDV diagnostic tests may fail to detect HoBi-like viruses or to differentiate between BVDV and HoBi-like viruses. Further, commercial tests for BVDV exposure, based on serological response, do not reliably detect HoBi-like virus exposure, and cross protection against HoBi-like viruses conferred by current BVDV vaccines is likely limited. As many HoBi-like viruses, characterized to date, were isolated from FBS originating from Brazil, it is assumed that the agent is probably widespread in Brazilian herds. Nevertheless, reports of natural infection in Southeast Asia and Europe demonstrate that these viruses are not restricted to South America. Increased demand for FBS has led to widespread distribution of FBS originating in HoBi-like virus endemic regions. The contamination of such FBS with HoBi-like viruses may lead to spread of this virus to other regions.

A multiplex enzyme-linked immunosorbent assay (ELISA) to detect antibodies to Bovine viral diarrhea virus (BVDV), Bovine herpesvirus 1 (BHV-1), Bovine parainfluenza virus 3 (BPV-3), and Bovine respiratory syncytial virus (BRSV) was developed on the commercially available Meso Scale Discovery (MSD) platform. The multiplex ELISA used the same antigens (relatively crude lysates of cultured virus) that are used in the current in-house separate ELISAs. Testing a panel of field samples demonstrated the multiplex assay could simultaneously detect the presence of several antibodies in a single assay and that there was good agreement between the multiplex ELISA and the in-house ELISAs. The use of a multiplex ELISA could offer significant benefits for routine serological testing through reduced staff time, reduced reagent cost, and streamlined laboratory operation.

Bovine viral diarrhea viruses are economically important pathogens of cattle. Most infections in susceptible animals are acquired from animals persistently infected with the virus. Surveillance programs rely on skin biopsies of persistently infected (PI) cattle to detect the infection. The purpose of this study was to compare antigen capture enzyme-linked immunosorbent assay (ACE) testing results using different types of samples from PI animals. The intent was to determine comparative detection rates in types of samples that are frequently submitted to diagnostic laboratories for evaluation of cases of unknown etiology or samples that could be easily collected for Bovine viral diarrhea virus (BVDV) screening. Eight types of samples were collected from 40 PI animals. The sample types were ear notches, serum, nasal swabs, conjunctival swabs, oral swabs, rectal swabs, vaginal/preputial swabs, and a tail skin fold biopsy. Each type of sample (n  =  8) for each animal (n  =  40) was evaluated with a commercial ACE kit. When using ACE, tail-skin fold and nasal swab samples were 100% sensitive compared with results using ear notches. Sensitivity using other samples was as follows: serum and vaginal/preputial swabs, 92%; conjunctival swabs, 64%; rectal swabs, 10%; oral swabs, 8%. Testing of tail skin fold biopsies, nasal swabs, and ear notch samples resulted in reliable results. In contrast, other sample types were unreliable for diagnosis of persistent infection in calves.

Bovine viral diarrhea virus (BVDV) is divided into 2 different species within the Pestivirus genus, BVDV type 1 (BVDV-1) and BVDV type 2 (BVDV-2). Further phylogenetic analysis has revealed subgenotype groupings within the 2 types. Thus far, 12 BVDV-1 subgenotypes (a-l) and 2 BVDV-2 subgenotypes (a and b) have been identified. The purpose of the current study was to determine the prevalence of BVDV subgenotypes in the United States and Australia and to determine if there are detectable antigenic differences between the prevalent subgenotypes. To determine prevalence, phylogenetic analysis was performed on 2 blinded panels of isolates consisting of 351 viral isolates provided by the Elizabeth Macarthur Laboratory, New South Wales, and 514 viral isolates provided by Oklahoma State University. Differences were observed in the prevalence of BVDV subgenotypes between the United States (BVDV-1b most prevalent subgenotype) and Australia (BVDV-1c most prevalent subgenotype). To examine antigenic differences between the subgenotypes identified in samples from the United States and Australia, polyclonal antisera was produced in goats by exposing them at 3-week intervals to 2 noncytopathic and 1 cytopathic strain of either BVDV-1a, BVDV-1b, BVDV-1c, BVDV-2a, or Border disease virus (BDV). Virus neutralization (VN) assays were then performed against 3 viruses from each of the 5 subgenotypes. Comparison of VN results suggests that there are antigenic differences between BVDV strains belonging to different subgenotypes. The present study establishes a foundation for further studies examining whether vaccine protection can be improved by basing vaccines on the BVDV subgenotypes prevalent in the region in which the vaccine is to be used.

The BVDV envelope glycoprotein E(rns)/gp48 and the C terminal 79 amino acids of the capsid protein coding region were expressed in a baculovirus system and antigenically characterized. Western blot assay was used to detect recombinant E(rns) (r-E(rns)) in infected insect cells using specific monoclonal antibodies. The r-E(rns) was then used in an indirect ELISA to detect BVDV specific antibodies in a panel of 540 well-characterized sera. Results of the r-E(rns) ELISA were compared to those obtained with a commercially available competitive ELISA targeting anti-NS2/3 antibodies. A good correlation was observed between the 2 ELISA (kappa = 0.916, 95% C.I.: 0.876, 0.956). Using the commercial NS2/3 ELISA as the reference test, the relative sensitivity of r-E(rns) ELISA was 97.5% (95% C.I.: 94.3%, 99.1%) and the relative specificity was 93.9% (95% C.I.: 89.4%, 96.9%), while relative specificity was 100% (95% C.I.: 97%, 100%) using true negative sera (derived from a negative herd). All but 1 antigen positive animals (n = 36) tested negative in the r-E(rns) ELISA; among them all 22 confirmed PI animals were negative by r-E(rns) ELISA. The ability of r-E(rns) ELISA to identify cattle immunized with inactivated vaccine was also demonstrated in a small group of cattle, compared to an NS2/3 antibody ELISA. Results suggest that r-E(rns) ELISA represents an alternative test for antibody generated by natural infection or BVDV vaccination.

Bovine viral diarrhea virus (BVDV) has a great economic impact on the United States cattle industry. The Academy of Veterinary Consultants, the American Association of Bovine Practitioners, and the National Cattlemen's Beef Association have called for the goal of BVDV control and eventual eradication in the U.S.A. One of the key factors in such efforts will be the detection of BVDV infections, particularly targeting persistently infected animals. To assess current BVDV detection methods in the U.S.A., 26 veterinary diagnostic laboratories in 23 states were surveyed. Survey questions related to the types of tests currently offered, the number of tests performed, the reasons for test requests, the type of samples used, whether sample pooling was performed, and whether follow-up testing or information regarding bovine viral diarrhea (BVD) management was provided after positive tests. There was no clear consensus on an individual BVDV testing method, the pooling of samples or the retesting of positive animals. Ear-notch antigen capture enzyme-linked immunosorbent assay (ACE) was the test most frequently performed based on the absolute number of tests. However, when the data were adjusted to reflect individual laboratory choices, the number of ACE and immunohistochemistry tests performed on ear notches was nearly equal. Only 55% of diagnostic laboratories provided BVD management information to producers or veterinarians who submitted positive samples. There was no significant difference in the number of positive tests in laboratories that received the majority of their samples for screening purposes versus laboratories that received the majority of their samples because BVDV was suspected based on clinical signs in a herd.

To reduce the cost of whole herd screening for bovine viral diarrhea virus persistently infected animals, the sensitivity and specificity of an antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) and a microtiter virus isolation ELISA using saline from ear notch samples or pooled serum was determined. Pooled saline from ear notch samples, assayed by AC-ELISA, gave a sensitivity and specificity of 98% and 94%, respectively, for pools containing 2 samples and 72% and 100%, respectively, for pools of 5. The sensitivity of pooled ear notch or serum samples for bovine viral diarrhea virus detection by microtiter virus isolation (sensitivity < 5%) or serum samples for detection by AC-ELISA (sensitivity < 15%) is too low to be used for whole herd screening. Pooling saline from ear notch samples from 2 animals tested by AC-ELISA, however, could provide a less expensive, reliable method for whole herd screening for bovine viral diarrhea virus.

Ear-notch samples from 3,599 yearling heifers were collected to detect persistently infected (PI) animals with suspect bovine viral diarrhea virus (BVDV). Individual immunohistochemistry (IHC), individual antigen-capture enzyme-linked immmunosorbent assay (AC-ELISA), and reverse transcription-polymerase chain reaction (RT-PCR) tests with pooled ear-notch supernatants were compared with samples from 3,016 heifers, whereas RT-PCR ear-notch pools and individual AC-ELISA tests were compared with samples from all 3,599 heifers. Four heifers were identified positive by both IHC and AC-ELISA, whereas the remaining heifers were identified negative by both tests. When supernatant from ear notches from 100 animals was randomly pooled and RT-PCR was accomplished on each pool, RT-PCR identified 2 pools that contained 1 positive AC-ELISA sample and 1 pool that contained 2 positive AC-ELISA samples. Further evaluation of the pooled RT-PCR ear-notch supernatant detected 100% (n = 36) samples spiked with supernatant from a single randomly selected positive AC-ELISA ear notch. Although follow-up confirmatory tests were not completed, all 3 methods correlated 100% in detecting suspect PI animals, with a kappa value of 1. The use of RT-PCR on pooled ear-notch supernatant could provide an initial, rapid, cost-effective method of screening cattle herds for BVDV PI animals. Subsequent serial testing with an AC-ELISA to evaluate individual samples included in the positive pool could minimize the length of time other animals are exposed to the virus.

This study determined the prevalence of diseases and pathogens associated with mortality or severe morbidity in 72 Ontario beef feedlots in calves that died or were euthanized within 60 days after arrival. Routine pathologic and microbiologic investigations, as well as immunohistochemical staining for detection of bovine viral diarrhea virus (BVDV) antigen, were performed on 99 calves that died or were euthanized within 60 days after arrival. Major disease conditions identified included fibrinosuppurative bronchopneumonia (49%), caseonecrotic bronchopneumonia or arthritis (or both) caused by Mycoplasma bovis (36%), viral respiratory disease (19%), BVDV-related diseases (21%), Histophilus somni myocarditis (8%), ruminal bloat (2%), and miscellaneous diseases (8%). Viral infections identified were BVDV (35%), bovine respiratory syncytial virus (9%), bovine herpesvirus-1 (6%), parainfluenza-3 virus (3%), and bovine coronavirus (2%). Bacteria isolated from the lungs included M. bovis (82%), Mycoplasma arginini (72%), Ureaplasma diversum (25%), Mannheimia haemolytica (27%), Pasteurella multocida (19%), H. somni (14%), and Arcanobacterium pyogenes (19%). Pneumonia was the most frequent cause of mortality of beef calves during the first 2 months after arrival in feedlots, representing 69% of total deaths. The prevalence of caseonecrotic bronchopneumonia caused by M. bovis was similar to that of fibrinosuppurative bronchopneumonia, and together, these diseases were the most common causes of pneumonia and death. M. bovis pneumonia and polyarthritis has emerged as an important cause of mortality in Ontario beef feedlots.

Two techniques performed on skin biopsy samples (ear notches), immunohistochemistry (IHC) and antigen-capture ELISA (AgELISA), were compared for detection of bovine viral diarrhea virus (BVDV) persistent infection (PI) in 559 Angus calves between the ages of 1 and 5 months. The calves also were tested for BVDV infection using virus isolation (VI) and reverse transcription (RT)-PCR on buffy coat samples and for antibodies to BVDV types la and 2 by serum neutralization (SN). Sixty-seven of 559 (12.0%) calves tested positive at initial screening by IHC, AgELISA, or VI, and all 67 were kept for a minimum of 3 months and retested monthly by IHC, AgELISA, VI, RT-PCR, and SN. Of the calves positive at initial screening, 59/67 (88.1%) were determined PI and 8/67 (11.9%) were determined acutely infected. Both IHC and AgELISA detected 100% of PI calves; however, IHC and AgELISA also detected 6 and 8 acutely infected calves, respectively, at initial screening. Furthermore, IHC and AgELISA continued to detect 3 and 4 acutely infected calves, respectively, 3 months after initial screening. Three acutely infected calves had IHC staining indistinguishable from PI calves at initial screening. Both IHC and AgELISA are accurate at detecting BVDV-infected calves, but veterinarians and producers should be advised that both tests detect some calves acutely infected with BVDV in addition to PI animals. Repeat testing using VI or RT-PCR on buffy coat samples should be performed at 30 days after initial screening to conclusively discriminate between acute and PI.

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

Serum samples were collected at slaughter from 226 24-30-month-old American bison (Bison bison) bulls from Kansas, Minnesota, North Dakota, and Manitoba and assayed for antibodies to ovine herpesvirus type-2 (OHV-2), bovine viral diarrhea virus (BVDV), bovine herpesvirus type-1 (BHV-1), and bovine respiratory syncytial virus (BRSV). Antibodies were detected by serum neutralization for BVDV, BHV-1, and BRSV, while antibodies to OHV-2 were detected by competitive inhibition-ELISA (CI-ELISA). Detectable antibodies were found against all viruses: 10 of 226 (4.40%) against OHV-2, 125 of 226 (55.3%) against BVDV, 99 of 226 (43.8%) against BHV-1, and 208 of 226 (92.0%) against BRSV. Titers from 93.6% of the BVDV-positive animals, 79.8% of the BHV-1-positive animals, and 98.1% of the BRSV-positive animals were > or = 1.25. These data indicate that a low percentage of clinically normal bison are seropositive for OHV-2 while a high percentage of bison sampled are seropositive for BVDV, BHV-1, and BRSV.

The reproductive organs and fetuses of seven Norwegian Red heifers were investigated for the presence of bovine viral diarrhea virus (BVDV) antigen during the time of initial transplacental transmission of the virus. The heifers were inoculated with a noncytopathogenic BVDV at day 85/86 of gestation and were slaughtered at day 7, 10, 14, 18, or 22 postinoculation (pi). Cryostat sections of uterus, ovaries, placentomes, intercotyledonary fetal membranes, and fetal organs were examined using immunohistochemical techniques. A double immunofluorescence technique was used to identify cells that showed staining with antibodies against the leukocyte common antigen CD45 or the intermediate filament vimentin and BVDV antigens. The earliest stage of infection at which BVDV antigen could be detected in the fetuses was 14 days pi. At this stage, BVDV antigen was detected in cells of mesenchymal origin in the lungs and in large cells that morphologically resembled immature megakaryocytes in the liver. In the intercotyledonary fetal membranes and in the placentomes, BVDV antigen was not detected until 18 and 22 days pi, respectively. BVDV antigen was not detected in maternal tissue from any of the heifers. The present results indicate that fetal infection with BVDV can take place without preceding or simultaneous high concentrations of BVDV in uterus or placenta of acutely infected heifers.

Bovine viral diarrhea virus (BVDV) is a single-stranded RNA virus responsible for enteric disease and reproductive failure in cattle. The virus can pass vertically from cow to fetus, causing abortion, birth of malformed calves, and calves born with persistent and life-long infections. In this study, we investigated the tropism of BVDV in ovarian tissue from persistently infected animals. Three heifers persistently infected with BVDV were euthanatized and their ovaries were recovered. A specimen of each ovary was taken (n = 6) for virus isolation, and the remaining ovarian tissue was stored at -70 C. Cryosections (6 microm) cut from each ovary were analyzed for the presence of BVDV antigens by indirect immunofluorescence. The immunofluorescent analysis employed two monoclonal antibodies, WB103 and WB162, previously raised against the nonstructural protein NS3 and the envelop glycoprotein E2, respectively. High titers (6.97 +/- 0.17 log10 tissue culture infective dose50/ml) of BVDV were recovered from 6/6 ovarian samples; NS3 and E2 were widely distributed within the ovarian stroma, the cumulus cell population, and the oocytes maturing in primordial, primary, and secondary follicles. Overall, 362/1,939 (18.7%) of the oocytes contained BVDV antigens, and there was no significant (P > 0.05) difference in the proportion of BVDV-infected oocytes recorded within the primordial (227/1,247, 18.2%), primary (122/630, 19.4%), and secondary (13/62, 21.0%) follicle populations. Although the developmental potential of the infected oocytes could not be established in the present study, we conclude that bovine oocyte and the cumulus cells are susceptible to BVDV infection.

Commercial enzyme-linked immunosorbent assays (ELISAs) for detection of serum antibodies to bovine viral diarrhea virus (BVDV), parainfluenza-3 virus (PI3V), respiratory syncytial virus (RSV), and infectious bovine rhinotracheitis virus (IBRV) were standardized to give a quantitative result when testing was performed at a single optimum dilution. For each test, serum samples were titrated and their end point titers calculated by an algebraic method directly from a plot of each titration series and also from a regression line fitted to this plot. The corrected optical density (COD) of each sample when tested at dilutions of 1/25, 1/50, and 1/100 was expressed as a percentage of the COD of a positive reference serum included on each plate, this value was the sample/positive (S/P) ratio. For each test, the linear relationship between the S/P ratio obtained at a dilution of 1/25, 1/50, and 1/100 and the end point titer calculated by each method was determined. In each case, the best linear relationship existed when samples were tested at a dilution of 1/100 (r = 0.973 for BVDV, 0.962 for PI3V, 0.961 for RSV, 0.947 for IBRV). From the equation of these lines, an increase in the S/P ratio between acute and convalescent serum samples of 31%, 23%, 21%, and 35% would correspond to a 4-fold rise in ELISA titer to BVDV, PI3V, RSV, and IBRV, respectively. ELISA titers calculated from S/P ratios at 1/100 were significantly related to virus neutralization titers to BVDV, RSV, and IBRV and to hemagglutination inhibition titers to PI3V (P < < 0.001 in all cases). Samples with low S/P ratios had the greatest intraassay and interassay variation. Intraassay reproducibility ranged from 3.5% to 22.3% (coefficient of variation), with a median value of 9.5%. Interassay reproducibility was lower, ranging from 6.0% to 50.6%, with a median of 17.4%.

Gross and microscopic lesions and distribution of virus were studied in specific pathogen-free calves (SPF) 10 days post-inoculation (PI) with bovine viral diarrhea virus (BVDV). To investigate possible differences in tissue tropism between BCDV isolates, two clinically and antigenically different noncytopathic (ncp) isolates of BVDV were compared in the study. Four calves were exposed to noncytopathic (ncp) BVDV 7937, and four to ncp-BVDV 126. Two additional calves that were not exposed to virus served as controls. Both ncp-BVDV 7937 and ncp-BVDV 126 induced mild disease characterized by variable fever and anorexia. Lymphoid depletion was evident in Peyer's patch of four calves and the thymus of two calves exposed to BVDV. Differences between these isolates in the distribution of BVDV or BVDV antigen in tissues of inoculated calves were not found. High concentrations of BVDV and BVDV-specific antigen were detected in the thymus, Peyer's patch, and mesenteric lymph node of all exposed calves. BVDV was shown to infect cells of the bone marrow without causing microscopic lesions. High concentrations of BVDV were recovered from the bone marrow of all calves exposed to BVDV and BVDV-specific antigen was demonstrated at this location in six of these calves. Platelet counts of calves exposed to BVDV were significantly reduced during infection, which resulted in thrombocytopenia in one calf. Focal areas of necrosis were observed in squamous epithelial cells of the tonsil and ruminal mucosa. BVDV-specific antigen was found in and adjacent to these foci. Calves exposed to ncp-BVDV 7937 had slightly more severe clinical signs than those exposed to ncp-BVDV 126.

Formalin-fixed paraffin-embedded tissues from 50 spontaneous cases (39 bovine, nine ovine, two caprine) of bovine viral diarrhea virus (BVDV) infection diagnosed by virus isolation were retrospectively examined for BVDV antigen by immunohistochemistry using anti-BVDV gp-43 monoclonal antibody (Mab 15C5). The cases were separated into enteric disease syndrome, respiratory disease syndrome, and abortion/weak calf syndrome based upon clinical disease. The purposes of the study were to 1) compare routine virus isolation with immunohistochemistry in determining BVDV infection and 2) define tissue and cellular distribution of BVDV in various clinical manifestations of infection. In bovids, there was 100% concordance of virus isolation and immunohistochemistry using Mab 15C5 in cases of enteric disease (mucosal disease, acute and chronic diarrhea, neonatal diarrhea), respiratory disease, and abortion. When laboratory tests were restricted to gastrointestinal tissue and/or feces, virus isolation detected BVDV in only 65% of cattle, whereas immunohistochemistry detected BVDV antigen in 100% of cattle. Immunohistochemical detection of pestivirus was poor in cases of ovine abortion, ovine hairy shaker syndrome, and caprine abortion. The tissue distribution of BVDV antigen was widespread in individual cattle with all clinical forms of BVDV infection. Viral antigen accumulation was spatially correlated with tissue lesions (in the absence of other pathogens) only in the gastrointestinal tract, lymphoid tissue, lung, placenta, and eye. This study demonstrates the utility of immunohistochemistry using Mab 15C5 to diagnose BVDV infections in cattle with a broad spectrum of clinical disease.

Bovine viral diarrhoea (BVD) virus is a cosmopolitan pestivirus of animals which is associated with diarrhoea, immunosuppression and synergy with other pathogens. This study was conducted to establish the prevalence of anti-BVD virus antibodies in healthy Zambian adults and those with asymptomatic and symptomatic HIV disease. Sera from 1159 adults were tested for anti-BVD virus antibodies using the indirect immunofluorescence test and the confirmatory Western blot. Of the 1159 sera examined, 180 (15.5%) showed significantly elevated titres of anti-BVD antibodies. These included 70 out of 477 (14.7%) HIV-negative healthy adults; 73 out of 480 (15.2%) of HIV-positive asymptomatic individuals; 23 out of 129 (17.8%) HIV-seropositive patients with associated illnesses excluding diarrhoea; and 14 out of 73 (19.2%) of HIV-seropositive patients with chronic diarrhoea. HIV-seropositive patients with chronic diarrhoea or associated illnesses appear to have significantly increased seroprevalence of anti-BVD virus antibodies (P = > 0.01). The mechanism of interaction between BVD virus and HIV infections and the synergistic effects with other opportunistic pathogens in humans requires definition.

Thirty-two monoclonal antibodies directed against epitopes on bovine viral diarrhea virus proteins and glycoproteins were tested for immunohistochemical reactivity with bovine viral diarrhea virus in formalin-fixed and paraffin-embedded tissues from 45 cases of bovine viral diarrhea virus-associated mucosal disease. Only one antibody, designated 15C5, which reacts with the 48-kD glycoprotein of bovine viral diarrhea virus, detected an epitope preserved in these specimens. Monoclonal antibody 15C5 and a polyclonal antibody to bovine viral diarrhea virus successfully detected bovine viral diarrhea viral antigens in 44/45 cases of mucosal disease and did not react with formalin-fixed tissues from 30 uninfected cattle. Monoclonal antibody-based immunohistochemical detection of bovine viral diarrhea virus is routinely fixed tissue specimens has advantages over other currently available techniques in terms of the convenience of specimen submission, the relative ease of method standardization, and the rapidity of the test, and by enabling identification of the virus in association with specific tissues, cell types, and histologic lesions.

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.

The monoclonal antibody (MAb) D89 against bovine viral diarrhea virus (BVDV) was used in conjunction with fluorescein-conjugated anti-mouse immunoglobulin in an indirect fluorescent antibody (IFA) procedure on frozen tissue sections and cell culture. During the 2-year study, BVDV was isolated from specimens submitted in 460 cases. The D89 Mab detected all but 2 BVDV isolates, both cytopathic. In 316 of the cases in which BVD virus was detected by IFA, specimens were inoculated on bovine turbinate cells and examined for BVDV antigens at 3-5, 10, and 20 days postinoculation. The BVDV was detected in 238/316 cases (75%) after 3-5 days incubation. The remainder were not detected until 10 or 20 days postinoculation. Virus isolation was enhanced in the early test if plates were centrifuged at the time of inoculation. Results suggest that D89 monoclonal antibody is a suitable diagnostic reagent for the detection of BVDV isolated from diagnostic specimens. The D89 MAb can be used for the detection of BVDV in both cell culture and tissues. Combination of D89 with another BVDV MAb (C17) did not improve the ability to detect BVDV in tissues compared to using D89 only, and the combined Mab's resulted in an increase in nonspecific fluorescence when used on tissues. Although pooling of different BVDV monoclonal antibodies may be necessary to detect all strains of BVDV in cell culture, pooling should be used with caution on tissues. Early detection of BVDV in cell culture by this IFA procedure permits faster confirmation of BVDV diagnosis when compared to the usual routine testing for noncytopathic BVDV at termination of first passage in cell culture.

A prospective serologic investigation was undertaken on 3 California dairies (herds 1, 3, and 4, as previously designated in a report on abortion surveillance) to determine if fetal loss was associated with infectious disease agents in cows. The diagnostic problem in these herds was typical of many dairies in that abortions were not discovered for several months and aborted fetuses were seldom recovered. Blood from approximately 100 pregnant cows in each herd was sampled at monthly intervals, beginning when the cows were palpated at approximately 40 days gestation. Sera were tested for antibodies to infectious bovine rhinotracheitis virus and bovine viral diarrhea virus (BVDV) and to the Leptospira serovars pomona, hardjo, grippotyphosa, icterohemorrhagiae, canicola, and szwajizak. Logn antibody titers were examined for an association with fetal loss, using multivariate methods of logistic regression and survival analysis. Of the 325 cows followed, 37 aborted, and no fetuses were recovered. Statistical analyses indicated that significant fetal loss was associated with high titers to L. hardjo and with low titers to L. szwajizak (herds 1 and 4) and BVDV (herd 1). Results for herd 3 revealed a connection between abortion and L. icterohemorrhagiae (P = 0.036) and L. canicola (P = 0.050) and possible vaccinal protection against abortion caused by L. grippotyphosa (P = 0.027 and 0.015). For herd 4, there was a marginally significant tendency for the first vaccination of the gestation against leptospirosis to have protected against fetal death (P = 0.077). Advantages of the diagnostic design were that it permitted comparison of titers of aborted cows with those of nonaborted cows and it considered vaccination-induced titers in analyses.(ABSTRACT TRUNCATED AT 250 WORDS)

Delayed-type hypersensitivity responses to Mycobacterium paratuberculosis purified protein derivative (PPD) were decreased in cows experimentally exposed to M. paratuberculosis 7 days after exposure to a modified-live bovine viral diarrhea virus (ML-BVDV) vaccine. In vitro lymphocyte blastogenic responses to phytohemagglutinin were decreased in each of 3 cows 7 days after exposure to ML-BVDV vaccine. Also, decreased lymphocyte blastogenic responses to M. paratuberculosis PPD were observed in cultures of 2 of 3 cows 7 days after exposure to ML-BVDV vaccine. No significant differences in enzyme-linked immunosorbent assay reactions were detected in sera of M. paratuberculosis-infected cattle collected before and at 4 and 12 weeks after exposure to ML-BVDV vaccine.

Bovine viral diarrhea (BVD) virus infection was diagnosed in neonatal calves with enteritis. Successful diagnostic procedures included direct immunofluorescence of frozen tissue sections, histopathology, and virus isolation. Virus isolation from buffy coats and serum was successful in detecting infected animals, whereas direct immunofluorescence of buffy coat samples was found to be less reliable. Virus was not isolated from any fecal samples. Booster vaccinations and the culling of animals shedding virus resulted in improved calf viability in this herd. It is suggested that procedures for the diagnosis of BVD virus infection should always be included in the diagnosis of neonatal calf enteritis.

Distribution of viral antigens in the central nervous system of 25 cattle with a persistent bovine viral diarrhea virus (BVDV) infection was studied. Using a polyclonal antiserum produced in pigs and the direct immunofluorescence and immunoperoxidase technique, BVDV antigen was located exclusively in neurons. Predilection sites for viral persistence were cerebral cortex and hippocampus; in other areas of brain and spinal cord, viral antigens were in single neurons or small groups of neurons. There was no morphological evidence of cellular alteration due to viral persistence. Perivascular lymphocytic infiltrations were in affected nervous tissue. It is concluded that the central nervous system is an important location for persistence of BVDV.