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Vaccination programs for viral pathogens in the dairy industry span the full spectrum of possibilities even though few of these have been evaluated in field situations. One such program is the vaccination of fresh cows 30 to 60 days postpartum with modified live viral (MLV) vaccines. The purpose of this study was to evaluate the antibody response to booster vaccinations during this period. The impact of vaccinations on milk production and reproductive performance was also examined. The response of cattle boosted with MLV bovine viral diarrhea virus (BVDV) was greatly enhanced compared with the saline controls and the killed vaccine test group. Similar increases were not seen with the MLV infectious bovine rhinotracheitis virus (IBR) and bovine respiratory syncytial virus (BRSV). Changes in milk production were not detected. There was a positive effect on the rate of conception with the MLV group even though there was no evidence of the presence of the three viruses in the herd at the time of study. Although this was a single field trial, and thus limited in scope and repeatability, the results indicate that the vaccines used had a positive effect.

Cattle immunotolerant to and persistently infected (PI) with bovine viral diarrhea (BVD) virus (BVDV) constitute the mechanism by which BVDV persists in and spreads among cattle herds. Detection and elimination of PI cattle are necessary for control of BVD. Serum is an excellent specimen for BVD PI testing because of high survivability of BVDV in serum and ease of collection, storage, and handling. Currently, microtiter virus isolation (VI) employing serum and sandwich ELISAs (S-ELISA) on tissues or leukocytes are used for BVDV PI screening. This paper evaluates a new S-ELISA kit that uses serum as the diagnostic sample. Cattle sera (n = 408) were tested using VI and the S-ELISA. The VI detected 172 BVDV-positive sera. Of these, 18 were confirmed PI cattle. The S-ELISA was positive on all PI samples. Considering only the PI animals, and using VI as the gold standard, the relative sensitivity of S-ELISA was 100%. The overall relative sensitivity was 93.6% and the agreement quotient (kappa) was 0.94. The relative specificity of the kit, based on 236 VI-negative sera, was 100%. These data indicate that the new kit is very adequate for detection of BVDV PI cattle.

To ascertain the virulence of bovine viral diarrhea virus (BVDV) genotype II, isolate NY-93 was inoculated intranasally into 3 calves, 2 of which were treated with a synthetic glucocorticoid prior to and after virus inoculation. Anorexia, fever (up to 42 C), dyspnea, and hemorrhagic diarrhea developed 6 days after intranasal inoculation with BVDV NY-93. The condition of all calves deteriorated further until the end of the study on day 14 postinoculation. The most significant postmortem macroscopic changes in all calves were limited to the gastrointestinal tract and consisted of moderate to severe congestion of the mucosa with multifocal hemorrhages. Microscopic lesions found in the gastrointestinal tract were similar to those observed in mucosal disease, including degeneration and necrosis of crypt epithelium and necrosis of lymphoid tissue throughout the ileum, colon, and rectum. The basal stratum of the epithelium of tongue, esophagus, and rumen had scattered individual necrotic cells. Spleen and lymph nodes had lymphocytolysis and severe lymphoid depletion. Severe acute fibrinous bronchopneumonia was present in dexamethasone-treated calves. Abundant viral antigen was detected by immunohistochemistry in the squamous epithelium of tongue, esophagus, and forestomachs. BVDV antigen was prominent in cells of the media of small arteries and endothelial cells. The presence of infectious virus in tissues correlated with an absence of circulating neutralizing antibodies. These findings highlight the potential of BVDV genotype II to cause severe disease in normal and stressed cattle.

In 1993, noncytopathic bovine viral diarrhea virus (BVDV) strains with enhanced virulence caused unprecedented outbreaks of severe acute bovine viral diarrhea (BVD) in dairy, beef, and veal herds in Ontario (Canada). Fever, pneumonia, diarrhea, and sudden death occurred in all age groups of cattle. Abortions often occurred in pregnant animals. Gross lesions in the alimentary tract were similar to those associated with mucosal disease, especially in animals >6 months of age. Cattle of all age groups had microscopic lesions in the alimentary tract similar to those seen with mucosal disease. The epidemic peaked in the summer of 1993, with 15% of all bovine accessions from diseased cattle presented to the diagnostic laboratory being associated with BVDV. The virus strains involved in the outbreak were analyzed using monoclonal and polyclonal antibodies and the polymerase chain reaction. The virus isolates from these outbreaks of severe disease were determined to be type 2 BVDV. Type 2 BVDV has been present in Ontario at least since 1981 without causing widespread outbreaks of severe acute BVD, which suggests that type 2 designation in itself does not imply enhanced virulence. Cattle properly vaccinated with type 1 BVDV vaccines appear to be protected from clinical disease.

The prevalence of bovine viral diarrhea virus (BVDV) infection was examined in a population of 5129 recently weaned steer calves entering a large feedlot in central Saskatchewan from September to December 1991. Serum samples were collected within 24 h of arrival at the feedlot from every fifth calf processed and again 96 d postarrival. A microtiter virus isolation test was used to determine the prevalence of calves viremic with BVDV on entry to the feedlot. An enzyme-linked immunosorbent assay (ELISA) which detects antibody against glycoprotein 53 of the BVDV was used on paired sera to determine the seroconversion risk during the first 96 d in the feedlot. A virus neutralization (VN) test for BVDV was conducted on a sub-sample of paired sera to measure agreement in determination of seroconversion risk with the ELISA. A polymerase chain reaction (PCR) test which detects BVDV was used to determine if cattle were acutely viremic when treated for disease. The estimated prevalence of persistently infected calves in this population was < 0.1%. The seroconversion risk for BVDV was 27% (236/864) according to the ELISA and it varied from 0 to 63% among the 20 pens sampled. According to the VN test, the seroconversion risk for BVDV was 40% (132/327) and it varied from 0 to 100% among the 11 pens tested. The agreement between the ELISA and VN tests in seroconversion risk to BVDV was very poor (kappa = 0.15 +/- 0.039 SE). The prevalence of acute viremia in calves treated at the feedlot hospital was low at 4% (6/149).(ABSTRACT TRUNCATED AT 250 WORDS)

The major economic impact of BVDV infections is in producing reproductive dysfunctions. The virus can have a detrimental impact on the developing fetus at all stages, but the most severe consequences occur early in gestation. The virus is maintained in the bovine population through the production of PI offspring. Only the NCB biotype of the virus can produce PI calves. The NCB biotypes is the one that produces the vast majority of BVDV infections and, as a consequence, the greatest economic losses. Biotype and antigenic variation are two independent variables and vaccines should emphasise antigenic composition, not biotype. A BVDV control program that does not emphasize heifers is doomed to failure.

A total of 21 calves were inoculated i.v. with 1 of the following isolates of bovine viral diarrhea virus (BVDV); CD87 (n = 10), NY-1 (n = 3), NADL (n = 5), or were sham-inoculated with virus-free medium (n = 3). Subsequent to inoculation, platelet counts were monitored to detect differences between noncytopathic (CD87, NY-1) and cytopathic (NADL) isolates in their ability to induce thrombocytopenia. Platelet count decrease throughout infection was statistically analyzed by comparing the slope of the line drawn from the count on the day of infection to the lowest count achieved by that calf. Significant difference was observed in the CD87-inoculated calves and in the NY-1-inoculated calves, compared with those of the same control group. Significant difference was not observed in the slope of platelet count decrease between the cytopathic NADL-infected calves and control-group calves. The data indicate that noncytopathic BVDV isolates may more easily induce thrombocytopenia than do cytopathic isolates in immune-naive, immunocompetent calves; acute infection with 1 cytopathic BVDV isolate (NADL) did not induce thrombocytopenia. In addition, although each calf seroconverted, virus was rarely isolated from mononuclear cells obtained from calves with cytopathic infections. At some point after infection, virus was always isolated from each of the calves undergoing noncytopathic infections, and occasionally, transient association of noncytopathic BVDV antigen with platelets was observed during these infections.

This study describes the epidemiological investigation of an outbreak of mucosal disease that occurred on a ranch in southwestern Saskatchewan. Over a six month period during the fall and winter of 1991-1992,in a herd of 515 beef cattle and 96 bison, 20 yearling cattle from a group of 105 housed in one feedlot pen died from mucosal disease. A further eight yearlings were slaughtered for salvage because they were at risk of dying from mucosal disease. Mucosal disease mortalities were the first observed evidence of fetal infections with bovine viral diarrhea virus in this herd. Animals that died from mucosal disease exhibited signs of ill thrift prior to death. Deaths from mucosal disease were confined to the progeny of one herd of beef cows. Following an outbreak of fetal infection with bovine viral diarrhea virus during 1989-1990, at least 28 (22%) of the 128 calves born from this herd of cows in the spring of 1990 were persistently infected with bovine viral diarrhea virus. However, only one calf born from this herd in 1991, and five calves born from all herds in 1992 were persistently infected. Of the five persistently infected calves born in 1992, three were born to persistently infected replacement heifers born in 1990. These heifers calved without assistance in 1992, but only one of their calves survived past three days of age, and it was persistently infected. In January 1992, 82% of the total herd had reciprocal antibody titers to bovine viral diarrhea virus of >/=1024 which suggested a high level of herd immunity to bovine viral diarrhea virus. Thus, following the outbreak of fetal infection with bovine viral diarrhea virus in 1989-1990, herd immunity to bovine viral diarrhea virus had developed rapidly in the breeding cows and heifers. Subsequently, in the next two years, there was a dramatic decline in the number of calves born persistently infected with bovine viral diarrhea virus.

Cytopathogenic bovine viral diarrhea virus (BVDV) arises by RNA recombination in animals persistently infected with noncytopathogenic BVDV. Such animals develop fatal mucosal disease. In this report, the genome of a cytopathogenic BVDV isolate, termed CP9, is characterized. CP9-infected cells contained not only viral genomic RNA of 12.3 kb but also a BVDV-specific RNA of 8 kb. cDNA cloning and sequencing revealed that the 8-kb RNA is a BVDV genome with an internal deletion of 4.3 kb. The 8-kb RNA represents the genome of a typical defective interfering particle (DI), since its replication was strictly dependent on the presence of a helper virus and strongly interfered with the replication of the helper. Cell culture experiments demonstrated that the CP9 virus stock contains two viruses, namely, a helper virus and DI9. While the helper virus alone was noncytopathogenic, the presence of the DI conferred cytopathogenicity. Expression experiments demonstrated that p80, the marker protein of cytopathogenic BVDV, is translated from the defective genome. The occurrence of this cytopathogenic DI is linked to a fatal disease in cattle.

To characterize the immune response of cattle to bovine viral diarrhea virus (BVDV) glycoprotein gp48, we have produced a large amount of recombinant glutathione-s-transferase-gp48 (GST-gp48) fusion protein in Escherichia coli. Antibodies to gp48 were present in cattle vaccinated with killed or modified-live virus vaccination, or following natural infection. These results were in agreement with results of serum neutralization (SN) test which detected gp53 of BVDV.

Bovine viral diarrhoea virus (BVDV) belongs to the pestivirus group, a genus within the Flaviviridae family. It possesses a positive-sense ssRNA genome with a single large open reading frame (ORF) encoding about 4000 amino acids. Here we report the continuation of our studies of pestivirus protein biogenesis, involving expression from the viral non-structural protein-encoding region. The 3'-terminal 60% of the BVDV ORF was cloned into a plasmid transfer vector which was then used to construct a recombinant baculovirus. Infection of Spodoptera frugiperda Sf9 cells with this recombinant virus resulted in the production of the expected mature viral proteins. Polyprotein processing by the BVDV p80 proteinase appeared to be nearly identical to that observed in authentic BVDV-infected bovine cells, and as previously shown to occur when expression of the same region was studied in a mammalian cell transient expression system. However, one viral proteolytic cleavage did not occur in the baculovirus-infected insect cells; the viral p80 proteinase failed to act at its own N terminus. This recombinant baculovirus/insect cell expression system provides an abundant source of BVDV non-structural proteins. Therefore we explored the utility of the proteins produced in this system for the detection of anti-BVDV antibodies in bovine sera. In preliminary experiments using these antigens in an ELISA we found a positive correlation between the presence of ELISA-reactive antibody and virus-neutralizing activity.

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.

The relationship between bovine viral diarrhea virus (BVDV) infection and thrombocytopenia was studied in 18 veal calves experimentally infected with BVDV. All calves were free of BVDV, and 13 calves were free of serum neutralizing antibodies to BVDV before virus inoculation. Calves were inoculated at approximately 10 days of age, and platelet counts were monitored over a period of several weeks. Ten additional calves housed in close proximity were kept as uninoculated controls. A profound decrease in platelet counts by 3 to 11 days after inoculation was seen in all calves that had neutralizing antibody titers less than 1:32 before infection. Severe thrombocytopenia (less than 5,000 platelets/microliter) was seen in 12 calves, 11 of which also developed hemorrhages. Necropsy findings in 3 severely thrombocytopenic calves that died included multiple hemorrhages throughout the body. Calves that recovered had increased platelet counts, and in most instances, a corresponding increase in neutralizing antibody titers to BVDV. At 11 days after inoculation, BVDV was detected on platelets by use of immunofluorescence, but evidence of surface-bound immunoglobulin was not found. The results suggest that a nonimmunoglobulin-mediated method of platelet destruction or sequestration develops as a sequela to BVDV infection.

A herd problem of abortions and the birth of malformed calves developed subsequent to a transient febrile illness in adult cows. The herd was not vaccinated against bovine viral diarrhea (BVD). Bovine viral diarrhea virus was isolated from blood of one calf. The problem continued for 8 months. The entire herd was vaccinated with a killed BVD virus product. No further problems developed. This herd problem illustrates one of the many recognized clinical manifestations of BVD.

The specificity of the humoral IgG response of cattle naturally or experimentally infected with bovine viral diarrhea virus (BVDV) was studied by radioimmunoprecipitation. Serum samples were tested against radiolabeled lysates of cells infected with cytopathic and noncytopathic biotypes of BVDV. A biotype-specific serologic marker was not detected. The specificity of the IgG induced in cows naturally or experimentally infected with either BVDV biotype was essentially the same. A strong IgG response to the 2 glycoproteins (56 to 58 kilodaltons, [kD] and 48 kD) of both biotypes and to the major polypeptides was induced in infected cells: 118 kD and 80 kD by cytopathic BVDV and only 118 kD by noncytopathic BVDV. The most consistent difference among cattle was the presence of IgG specific for the 37-kD polypeptide. Sequential serum sample collection after spontaneous and induced infections with either BVDV biotype did not indicate specific IgG markers for determining infection history. Sera from cattle with a confirmed diagnosis of mucosal disease and lacking neutralizing antibodies to BVDV usually lacked (greater than 80%) nonneutralizing BVDV-specific IgG. One animal had substantial amounts of IgG to the 80-kD polypeptide. Other cattle had less readily detectable amounts of IgG specific for 80-kD or 37-kD viral polypeptides.

A panel of monoclonal antibodies that recognize the two major glycoproteins of bovine viral diarrhea virus (BDV) was used to evaluate the antigenic relationship between cytopathic (CP) and noncytopathic (NCP) viruses isolated from cattle dead or dying from fatal BDV infections. Various unrelated BDV isolates were initially screened by indirect immunofluorescence with monoclonal antibodies directed against the 56- to 58- and 48-kilodalton glycoproteins of the virus. A wide spectrum of reactivity that was independent of biotype was found. Biological clones of the same isolate showed only minor variations from the parental isolate, as did isolates taken from different animals located on the same farm. A similar analysis was repeated with pairs of CP and NCP viruses isolated from 16 unrelated clinical cases of BDV infection resulting in fatal disease. The reactivity patterns within individual pairs of isolates taken from the same animals were in most instances very similar and in some cases indistinguishable from one another. The results demonstrate that antigenic similarity between biotypes is a consistent finding in animals dying from fatal BDV infections. In view of the wide degree between biotypes is a consistent finding in animals dying from fatal BDV infections. In view of the wide degree of variation in reactivity patterns between unrelated BDV isolates, the close antigenic similarity of CP BDV to the homologous NCP BDV of a given pair strongly suggests that CP BDV arises by mutation from NCP BDV.

Seven calves between 1 week and 2 months of age were infected with a noncytopathic field isolate of bovine viral diarrhea virus (BDV) in order to evaluate the effect of BDV infection on the concentration of circulating platelets in the blood. All calves were determined to be free of BDV and neutralizing antibodies to BDV before infection. Platelet counts were performed on a daily basis over a 30-day period beginning at the time of infection. By 2 weeks postinfection, all calves showed a significant drop in the number of circulating platelets and a marked hyperplasia of megakaryocytes in the bone marrow. In three of the seven calves, thrombocytopenia was severe (less than or equal to 5,000/microliters) for 1 to 6 days. In two of these three animals, extensive petechial and ecchymotic hemorrhages were observed on all mucosal surfaces and on various internal organs during the period of severe thrombocytopenia. BDV was consistently isolated from the platelets during the early phases of the infection, and viral antigen was occasionally detected on platelets by a fluorescent-antibody assay. The results demonstrate that BDV infection is associated with decreases in platelet numbers and suggest that platelets may serve as carriers of circulating virus.

Thrombocytopenia was observed in 15 of 146 cases of clinically acute bovine viral diarrhea virus (BVDV) infection in adult cattle. Platelet counts ranged from 2,000 to 33,000/microliters. Clinically, a bleeding tendency was manifested by bloody diarrhea, petechial and ecchymotic hemorrhage, epistaxis, and abnormal bleeding from injection sites. Coagulation testing (six cases) gave no evidence of disseminated intravascular coagulation. Bone marrow aspirates were suggestive of active marrow necrosis (two cases) or recent repopulation (three cases). Treatment, when given, was supportive and empirical in nature. Six animals experienced complete clinical recoveries; the others died or were euthanatized. Although the pathogenesis of the thrombocytopenia was not definitively determined, thrombocytopenia associated with acute BVDV infection should be considered in the differential diagnosis for cattle with bleeding disorders.

Bovine Virus Diarrhea (BVDV) was diagnosed as the major etiological agent in 10 dairy herd disease outbreaks. The outbreaks varied between herds in time of onset in individual cattle, signs, morbidity, and clinical course. A definitive diagnosis was obtained by viral isolation from whole blood samples in each herd. Each herd problem and its clinical course was described in detail. A discussion of current knowledge pertaining to BVD--the disease and the virus--follows. The importance of strain variation relative to disease production and prophylaxis was highlighted.