Bovine viral diarrhoea: Pathogenesis and diagnosis

Bovine viral diarrhoea virus (BVDV) is the most prevalent infectious disease of cattle. It causes financial losses from a variety of clinical manifestations and is the subject of a number of mitigation and eradication schemes around the world. The pathogenesis of BVDV infection is complex, with infection pre- and post-gestation leading to different outcomes. Infection of the dam during gestation results in fetal infection, which may lead to embryonic death, teratogenic effects or the birth of persistently infected (PI) calves. PI animals shed BVDV in their excretions and secretions throughout life and are the primary route of transmission of the virus. These animals can usually be readily detected by virus or viral antigen detection assays (RT-PCR, ELISA), except in the immediate post-natal period where colostral antibodies may mask virus presence. PI calves in utero (the ‘Trojan cow’ scenario) currently defy detection with available diagnostic tests, although dams carrying PI calves have been shown to have higher antibody levels than seropositive cows carrying non-PI calves.Acute infection with BVDV results in transient viraemia prior to seroconversion and can lead to reproductive dysfunction and immunosuppression leading to an increased incidence of secondary disease. Antibody assays readily detect virus exposure at the individual level and can also be used in pooled samples (serum and milk) to determine herd exposure or immunity. Diagnostic tests can be used to diagnose clinical cases, establish disease prevalence in groups and detect apparently normal but persistently infected animals. This review outlines the pathogenesis and pathology of BVD viral infection and uses this knowledge to select the best diagnostic tests for clinical diagnosis, monitoring, control and eradication efforts. Test methods, types of samples and problems areas of BVDV diagnosis are discussed.     

Limited BVDV transmission and full protection against CSFV transmission in pigs experimentally infected with BVDV type 1b

Bovine viral diarrhea virus (BVDV) in pigs may interfere with the detection and epidemiology of classical swine fever virus (CSFV). To investigate the importance of BVDV infections in pigs, first we studied the transmission dynamics of a recent BVDV field isolate. Subsequently, the protection of BVD antibodies against transmission and clinical disease of CSF virus was studied. Only limited transmission of BVDV occurred (R = 0.20), while no CSFV transmission occurred in pigs with BVDV antibodies. We concluded that BVDV transmission among pigs is possible, but seems to be limited and thus the virus should disappear from a population if no new introductions occur. Furthermore, the presence of BVD antibodies may completely prevent the transmission of CSFV and therefore could protect pigs against classical swine fever. It was also noticed that double infections with BVDV and CSFV were incorrectly diagnosed using the neutralization peroxidase linked assay (NPLA), which is the golden standard for antibody detection. This might hamper the diagnosis of CSF in herds with a high BVD prevalence.     

Laboratory diagnostic investigations for bovine viral diarrhoea virus infections in cattle

There are no pathognomonic clinical signs of infection with bovine viral diarrhoea virus (BVDV) in cattle. Diagnostic investigations therefore rely on laboratory-based detection of the virus, or of virus-induced antigens or antibodies in submitted samples. In unvaccinated dairy herds, serological testing of bulk milk is a convenient method for BVDV prevalence screening. Alternatively, serological testing of young stock may indicate if BVDV is present in a herd. In BVDV positive herds, animals persistently infected (PI) with BVDV can be identified by combined use of serological and virological tests for examination of blood samples. ELISAs have been used for rapid detection of both BVDV antibodies and antigens in blood, but should preferably be backed up by other methods such as virus neutralization, virus isolation in cell cultures or amplification of viral nucleic acid. Detailed knowledge of the performance of the diagnostic tests in use, as well as of the epidemiology of bovine virus diarrhoea is essential for identification of viremic animals in affected herds.     

Bovine virus diarrhoea (BVD) control programme in an area in the Rome province (Italy)

A BVD control programme based on the identification and removal of persistently infected (PI) animals is being undertaken in an area in the Rome province, where BVD outbreaks had been previously detected. It involves 174 mainly dairy herds, from which blood samples of all bovines older than 1 year are obtained through the national brucellosis and leukosis eradication programme. Samples sufficient to detect the presence of seropositive animals at a prevalence of 5% or more are initially screened for antibodies against BVD virus (BVDV) using an immunoenzymatic assay. Upon identification of seroreagents additional blood samples are tested from the 6-12-month age category not included in the initial samples. Animals are considered immunotolerant if BVDV is demonstrated twice at a minimum 30-day interval. When no seropositive animals are detected during the first serological screening the herd is declared BVD-free if a second testing, preferably carried on the same animals previously tested, confirms the seronegative status of the herd. At present 147 farms have been tested, of which 63 (42.9%) are negative with respect to antibodies against BVDV. Of the 84 remaining herds in which one or more seropositives are detected, 13 are classified as recently infected. In eight of these recently infected herds, 22 PI animals have been identified.     

Implementation of two-step vaccination in the control of bovine viral diarrhoea (BVD)

Bovine viral diarrhoea (BVD) control/eradication programmes based on the test and removal of persistently infected cattle without use of vaccination were first introduced by the Scandinavian countries in the early 1990s. Within the last 10 years the programmes have proven to be very successful and have served as a blueprint for several other European regions. However, in areas with high cattle densities, intense animal trade and high BVD prevalence this control approach is risky, because there is a high probability that herds, which have been cleared of persistently infected (PI) animals and have become partly or fully susceptible to reintroduction of the virus, will come in contact with a BVD virus (BVDV) infected animal. A combination of the test and removal strategy with subsequent systematic vaccination of cattle could overcome this problem. The goals of vaccination in such a programme is protection against reintroduction of BVDV into herds free from PI cattle and foetal protection of pregnant animals accidentally exposed to the virus. Two-step vaccination is based on the use of inactivated BVDV-1 vaccine for priming followed by a live attenuated vaccine booster 4 weeks later. The immune response elicited by such a vaccination scheme has proven to be long lasting and foetal infection after challenge with BVDV-1 and BVDV-2 was prevented in pregnant animals 5 months after vaccination. These findings suggest that the implementation of a two-step vaccination in the initial phase of control programmes in addition to test and removal of PI animals in areas with high cattle densities and endemic BVD is practical and efficacious.     

Evaluation of two commercial enzyme-linked immunosorbent assays for detection of bovine viral diarrhoea virus in serum and skin biopsies of cattle

AIM: To assess the ability of two commercial bovine viral diarrhoea (BVD) virus (BVDV) antigen-capture enzyme-linked immunosorbent assays (ELISAs) to detect virus in serum and skin biopsies. METHODS: Thirty cattle persistently infected (PI) with BVDV were identified using routine diagnostic laboratory testing. Additional ear-notch skin biopsies and blood samples were collected from these animals to confirm the diagnosis, and from 246 cohorts, to determine their BVDV status. Skin biopsies were soaked overnight in buffer and the eluate collected. All sera and eluate were tested using two commercially available ELISAs for detecting BVDV antigen, and a subsample of positive and negative sera was tested using a polymerase chain reaction (PCR) test. A study was also performed to ascertain the risk of cross contamination occurring during the collection and processing of skin biopsies. RESULTS: Both serum and skin samples tested using either ELISA resulted in the detection of all cattle identified as PI and no non-infected cattle were incorrectly classified as infected using either method. Agreement between all assays (ELISAs, whether performed on serum or skin, and PCR) was 100%. No cross-contamination of skin samples between animals was evident using routine biopsy methods. CONCLUSIONS: Viraemic cattle infected with BVDV were accurately identified using either of the two commercial ELISAs evaluated on either serum or skin samples. CLINICAL RELEVANCE: Either skin biopsies or serum samples can be collected from cattle to determine their BVDV status. This should overcome problems in accurately identifying the infection status of young calves in which colostral antibodies might interfere with the antigen-capture ELISA.     

Mental notes in tracking BVDV carriers

The detection and elimination of animals persistently infected with bovine viral diarrhoea virus (BVDV) is the key to BVD control. This method has proven to be very efficient in eradicating BVDV in a herd. Several pitfalls in the detection procedure can make that some persistently infected (PI) animals do not get identified or are removed too late, supporting the assumption that circulation of the virus could be possible in absence of PI animals. Furthermore the risk of reintroduction is high since the prevalence of BVD is high in the Netherlands and Belgium. Based on both practical experience and literature, here we review critical control points in order to minimise the risk of a false negative BVDV screening.     

Evaluation of the effects of long-term storage of bovine ear notch samples on the ability of 2 diagnostic assays to identify calves persistently infected with bovine viral diarrhoea virus

Research aimed at optimising diagnostic laboratory procedures is central to the development of effective bovine viral diarrhoea virus (BVDV) control programmes. BVDV is a single-stranded RNA virus that crosses the placenta to infect foetuses, resulting in reproductive losses due to foetal death or persistently infected calves that die early in life. Persistently infected animals are widely accepted to be the primary reservoir of BVDV and the largest source of infection. This poses important challenges to overall animal/herd health and can cause major losses to the cattle industry. Long-term storage of bovine ear notch samples from calves persistently infected with BVDV may adversely affect the ability of diagnostic assays to detect the virus efficiently. In order to test this hypothesis, ear notch samples from 7 animals were divided into 2 groups. One set was subjected to prompt formalin fixation and the other set stored either as fresh samples without preservatives at -2 degrees C, or soaked overnight in phosphate buffered saline followed by freezing of the supernatant fluid at -2 degrees C. Frozen ear notches and ear notch supernatant yielded positive results with an antigen-capture, enzyme linked immunosorbent assay (AC-ELISA) for the duration of the study (6 months) and optical density (OD) values remained significantly within range. There was no significant difference between storing fresh ear notch samples or PBS at -2 degrees C. However, positive immunohistochemistry (IHC) staining on formalin fixed ear notches started to fade between Day 17 and Day 29 when stored at room temperature. It was concluded that fresh ear notches could safely be stored at -2 degrees C for a period of 6 months prior to testing for BVD viral antigens.     

Genetic diversity and BVD virus.

The various measures of genetic variation of BVD virus was reviewed with emphasis on the implications for future control of virus-induced disease and diagnosis. While experimental data does not support unique serotypes for BVDV, there is substantial antigenic variation among the isolates examined. This variation may permit fetal infections even in animals assumed to be well vaccinated. The genetic differences between cytopathic and noncytopathic strains of BVDV are expressed in infected cells by the production of a p80 protein by cytopathic strains. In addition, cellular gene inserts have been detected in cytopathic strains. Monoclonal antibodies have demonstrated a high degree of diversity with the pestivirus population. Grouping of BVDV isolates by monoclonal antibody analysis is suggestive at best. The use of nucleic acid probes as diagnostic reagents has been compromised by the nucleic acid sequence variation found in the BVDV isolates tested.     

A survey of bovine viral diarrhea virus testing in diagnostic laboratories in the United States from 2004 to 2005.

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.     

Diagnosis of bovine viral diarrhea virus infection using monoclonal antibodies

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.     

ELISA detection of bovine viral diarrhoea virus specific antibodies using recombinant antigen and monoclonal antibodies

A panel of monoclonal antibodies was prepared by immunization of BALB/c mice with Moredun (BD) virus strains. These antibodies were characterized by immunofluorescence and seroneutralization against BD, BVD and hog cholera (HC) virus strains, and radioimmunoprecipitation of BVD-infected cells extracts. The MAbs reacting with the majority of the Pestivirus strains recognize the 80 kDa antigen of the BVD cytophathic strains. The 80 kDa antigen of the BVD/Osloss virus strain has been cloned and expressed in E. coli as a fusion protein with beta-galactosidase. The fusion protein has been purified from inclusion bodies and used successfully as an antigen for ELISA detection of BVDV specific antibodies in bovine sera. A competitive ELISA using MAbs is more specific than a direct assay. These results compare well with the ones obtained with antigen extracted from BVDV-infected cells.     

新疆部分规模奶牛场牛病毒性腹泻病的流行情况调查与防控措施

为了解新疆地区部分规模奶牛场牛病毒性腹泻病（BVD）的流行情况,优化防控措施,以达到建设防控净化场的目的,自2020年11月到2021年7月累计采集新疆5 个地区16 个奶牛场共计26 997 份血清、3 843 份犊牛耳组织进行全群普检。通过使用IDEXX公司牛病毒性腹泻病毒（BVDV）抗原检测试剂盒检测及RT-PCR复检结合测序等方法,淘汰阳性牛,同源性分析流行毒株情况。BVDV血清抗原检测结果为：沙湾某奶牛场BVD阳性率为1.63%（38/2 326）;乌鲁木齐某奶牛场BVD阳性率为0.35%（4/1 132）;其余奶牛场均为阴性。犊牛耳组织抗原检测结果为：乌鲁木齐某牛场BVDV阳性率为1.17%（4/342）;其余奶牛场均为阴性。研究结果揭示,奶牛场通过淘汰BVDV阳性牛,调整免疫程序、制定消毒程序等方法,可有效净化BVD。     

Prevalence of ruminant pestivirus infections in Namibia.

Following several clinical cases of suspected bovine virus diarrhoea (BVD) on three Namibian cattle farms, a serological survey was conducted on bovine, ovine, caprine and wild ruminant sera originating from different regions of the country. Neutralizing antibodies to BVD virus (BVDV) were detected in 58% of 1,014 cattle sera, 14% of 618 sheep sera and 4.6% of 1,118 goat sera. Sera from seven of ten wildlife species were positive with kudu, eland and giraffe having prevalence rates greater than 40%. BVDV was isolated from six clinically affected bovines and three healthy heifers persistently infected with BVDV. The survey demonstrated that pestivirus infections are widespread in Namibia in both domestic and wild ruminants.     

Diagnostic assays applied in BVDV control in The Netherlands

In The Netherlands, a voluntary bovine virus diarrhoea virus (BVDV)-free certification programme was started in 1997. After an intake procedure in which all cattle are tested for the presence of BVD virus, a herd obtains the status "BVD-virus-free". To maintain this status a monitoring procedure is executed to verify absence of BVDV circulation in the herd. Several diagnostic tests are used: RT-PCR in bulk milk and pooled blood samples, antigen-ELISA (Ag-ELISA) and antibody ELISA in individual blood samples. Sensitivity and specificity of these tests are discussed. In addition, a diagnostic quick scan has been introduced, consisting of a combination of bulk milk tests for virus and antibody, and antibody tests in samples from young stock. Preliminary results are presented.     

牦牛病毒性腹泻病研究进展

牦牛感染牛病毒性腹泻病毒(Bovine viral diarrhea virus,BVDV)后常发生胃肠炎等消化系统疾患和呼吸系统疾患、繁殖障碍,造成机体损伤,给牦牛产业发展造成一定影响。基于5"-UTR的差异将BVDV分为BVDV-I和BVDV-II两种基因型,牦牛源BVDV的主要基因型为BVDV-1型;根据BVDV对宿主细胞的致病性,分为致细胞病变型(Cy-topathogenic,CP)和非致细胞病变型(Non-cytopathogenic,NCP)两个生物型,牦牛源BVDV既有CP型又有NCP型,且这两种生物型能相互转化。牦牛生活的高原地区风、鸟类的迁徙等因素可能都会导致牦牛BVD传播范围广泛,牛群中大量的BVDV携带者和牦牛特殊而又复杂的生态系统导致了BVDV种间传播的巨大潜力,影响着牦牛产业的健康发展和养殖经济收益。针对本病在牦牛群中的感染流行等情况未发现相关详细的研究报道,笔者就牦牛BVD病原学、流行病学调查、实验室诊断、综合防治等方面研究进展情况进行整理,供同仁在研究和防控中参考借鉴。     

Virus Neutralizing Antibodies Against a Panel of 18 BVDV Isolates in Calves Vaccinated with Rispoval™ RS‐BVD

Seven of nine colostrum‐deprived calves, free from infection with bovine virus diarrhoea virus (BVDV), were vaccinated with Rispoval^? RS‐BVD on two occasions, 21 days apart, while the other two were kept as BVDV infection controls. The virus neutralizing (VN) serum antibodies induced by vaccination were tested for their ability to neutralize 18 European BVDV isolates, including laboratory reference strains and recent field isolates, both cytopathic and non‐cytopathic biotypes as well as genotypes I and II. The strains were isolated in Belgium, France, Germany and the United Kingdom. While there were large variations in the vaccine‐induced VN titres of the individual calves against all the strains, e.g. the titres against Osloss NCP, the European reference strain ranged from 1.7 to 6.7 (1 : log₂), serum from each animal was capable of neutralizing between nine and all 18 of the strains tested. Nevertheless, from the results of this study, it can be concluded that in colostrum‐deprived BVDV seronegative calves, Rispoval^? RS‐BVD can stimulate the production of VN antibodies capable of neutralizing a wide range of antigenically diverse European isolates of BVDV, including genotypes I and II.     

Serological evidence for exposure to bovine viral diarrhoea virus in sheep co-grazed with beef cattle in New Zealand

ABSTRACT

Aims: To determine whether sheep that co-grazed with cattle that were suspected to be positive for bovine viral diarrhoea (BVD) virus had serological evidence of exposure to the virus.

Methods: Eighteen commercial farms that routinely co-grazed cattle and sheep in the same paddocks were recruited through purposive sampling. The recruiting veterinarians identified nine farms with cattle herds that were known or highly suspected to be positive for BVD and nine farms that were considered to be free of BVD. Blood samples were taken from 15 ewes aged 1 year on each farm and samples were submitted to a commercial diagnostic laboratory to test for antibodies against pestiviruses using an ELISA. All samples that were positive were then tested using a virus neutralisation test (VNT)for antibodies against BVD virus.

Results: Of the 270 blood samples, 17 were positive for pestivirus antibodies by ELISA and these originated from two farms that were known or suspected to have BVD virus-positive cattle. None of the samples from the nine flocks co-grazed with cattle herds that were known or suspected to be BVD virus-negative were positive for pestivirus antibodies. Within the two positive farms, 2/15 samples from the first farm and 15/15 samples from the second farm were antibody-positive. When the 17 positive blood samples were submitted for VNT, all 15 samples from the second farm tested positive for BVD virus antibodies with the highest titre being 1:512.

Conclusions and clinical relevance: In this small sample of New Zealand sheep and beef farms with suspected BVD infection in cattle, there was evidence of pestivirus exposure in co-grazed sheep. Although we were unable to confirm the origin of the exposure in these sheep, these findings highlight that farmers who are trying to eradicate BVD from their cattle should be mindful that the infection may also be circulating in sheep, and both populations should be considered a possible risk to each other for generating transient and persistent infections. Further work is needed to estimate the true prevalence of New Zealand sheep flocks that are affected by BVD and the associated economic impacts.     

Identification of hog cholera viral isolates by use of monoclonal antibodies to pestiviruses

A collection of 90 field isolates of hog cholera virus (HCV) was used to test the specificity of four hybridoma cell lines secreting monoclonal antibodies against pestiviruses. Reaction of virus isolates and monoclonal antibodies was controlled by an indirect immunofluorescence assay (IFA). Two monoclonal antibodies which had been generated against HC virus strain "Alfort 187" were reactive only with HCV field isolates and an HCV reference strain but not with bovine viral diarrhoea virus (BVDV) reference strains. Two other monoclonal antibodies (generated against BVDV, strain NADL) reacted only with BVDV reference strains but not with HCV field isolates, although with 3 of these strains focal reactions involving only a few cells were detected. The ability to discriminate between both viruses is a diagnostic need which may be fulfilled by these monoclonal antibodies.     

Ten years of bovine virus diarrhoea virus (BVDV) control in Norway: a cost-benefit analysis

A retrospective cost-benefit analysis was carried out on the Norwegian bovine virus diarrhoea (BVD) control and eradication strategy, for the years 1993-2003. Information regarding the control cost input parameters was gathered from the cattle industry (TINE Norwegian Dairies, GENO Breeding and AI association, and GILDE Norwegian Meat), The National Animal Health Authorities and The Veterinary Institute. We accounted for variable costs (both direct costs associated with the control, and those costs carried by the farmers as a consequence of the control program). The benefit was estimated as the difference between the assumed losses without control - represented overall as 10% increase of the observed 1993 BVD virus infection level - and the observed losses during the control period. An estimate of the financial losses associated with the BVD virus (BVDV) infection was based on studies of the herd level effects on health, reproduction, and production in dairy herds with evidence of recent BVDV infection. We used a stochastic simulation model to account for the total uncertainty in both the control cost and financial loss estimates. The annual net benefits over the 10 years of BVD control were discounted to a 1993 net present value (NPV). The median NPV of the BVD control, nationally, was estimated at 130 million NOK with a distribution of the NPV ranging from +51 to +201 million NOK (5th and 95th percentiles, respectively). Out of the total control costs the farmers and the farmer-owned industries (the co-operatives) had carried about 62% of these costs; however, the farmers were also the main beneficiaries. The Norwegian experience shows a robust cost-efficiency for a BVDV eradication strategy; this stands in sharp contrast to earlier studies where the results were not supportive. Even though every cattle population and country is unique, the Norwegian findings and experiences should have wider implications.