Genetic heterogeneity of bovine viral diarrhoea virus (BVDV) isolates from Turkey: identification of a new subgroup in BVDV-1

Genetic heterogeneity of Turkish ruminant pestiviruses was investigated by phylogenetic analysis of complete N(pro) encoding nucleotide sequences. A total of 30 virus isolates obtained from 15 provinces around the country between 1997 and 2005 were included in the phylogenetic analysis. Virus isolates mostly originated from cattle with one isolate from sheep. The bovine isolates all belonged to BVDV-1, the sheep isolate to BVDV-2. Fifteen isolates formed a new subgroup within BVDV-1, tentatively named BVDV-1l. The remaining bovine isolates were typed as BVDV-1a (n=4), BVDV-1b (n=4), BVDV-1d (n=3), BVDV-1f (n=2) and BVDV-1h (n=1). The isolates allocated to BVDV-1l originated from various geographical regions in different years. There was no correlation between genetic grouping and locations where isolates were obtained. Viruses originating from one farm in most cases belonged to the same subgroup (n=5). This study indicates that the newly detected subgroup BVDV-1l is predominant and widespread in Turkey. Moreover, an ovine virus isolate was identified as the first member of BVDV-2 reported in Turkey. A serological survey using samples from western Turkey indicated that BVDV-2 is also present in cattle.     

Epitopic characterisation of Turkish bovine viral diarrhoea virus (BVDV) isolates using monoclonal antibodies

In this study, 15 bovine viral diarrhoea viruses (BVDV) isolated from the field in Turkey were characterised for their biotype, cloned and eventually analysed for their epitopic composition in terms of glycoprotein E2. Immunoplaque assay, plaque assay, limiting dilution and streptavidin-biotin-peroxidase techniques were used for biotype characterisation, cloning of cytopathic (cp) and noncytopathic (ncp) biotypes and epitope analysis, respectively. While 14 out of 15 BVDV isolates were distinguished as ncp biotype, 1 isolate was found to be containing both biotypes (cp + ncp). According to the reactivity patterns of isolates with 15 monoclonal antibodies, 4 different antigenic groups could be formed. There were no antigenic differences between the isolates derived from the same animal with various time intervals. On the other hand, biotype clones isolated from the same animal exhibited difference in one epitope. This is the first study describing antigenic characterisation of BVDV field isolates in Turkey.     

Genetic typing of bovine viral diarrhoea virus isolates from India

Thirteen BVDV isolates collected in four geographic regions of India between 2000 and 2002 were typed in 5'-UTR. To confirm results of genetic typing, selected viruses were also analysed in the N(pro) region. Phylogenetic analysis revealed that all Indian BVDV isolates belong to BVDV-1b (Osloss-like group). Despite a long distance between the farms from which the viruses were isolated there was no correlation between the origin of viral isolates and their position in a phylogenetic tree. Higher genetic similarity of Indian BVDV isolates was observed most probably due to the uncontrolled movement of cattle as well as the uncontrolled use of semen from bulls for breeding of local and farm cattle in different states of India.     

Bovine viral diarrhoea virus (BVDV) diversity and vaccination. A review

BVDV is associated with a range of economically important clinical diseases including reproductive disorders and acute fatal haemorrhagic disease in cattle industry. Vaccination is still the most important control strategy for controlling BVDV infections in many countries of the world. The existence of great genetic and antigenic diversity of BVDV isolates is very important concern for BVDV vaccine development and protective efficacy of current vaccines. In this review, the protective efficacies of the selected examples of BVDV vaccines with regard to BVDV diversity and the novel marker vaccine development studies are discussed.     

Apoptosis inhibitors delay the cytopathic effect of bovine viral diarrhoea virus (BVDV)

Based on their action in cell culture, two biotypes of bovine viral diarrhoea virus (BVDV) can be distinguished. The noncytopathic (ncp) BVDV isolated from persistently infected animals cause no visible damage to cultured bovine cells. In contrast, cytopathic (cp) BVDV induces severe damage and apoptosis in cell cultures. Cp BVDV can be isolated from cattle suffering from mucosal disease (MD) and is associated with the severe lesions that primarily affect the gastrointestinal tract. To get an insight into the molecular events during BVDV induced cytopathic effect (CPE), the effect of three chemical reagents (3-aminobenzamide, ascorbic acid and N-acetyl-leucyl-leucyl-methional) with completely different mode of actions in infected cells was analysed. All three substances were able to delay the cytopathic effect induced in permissive bovine cells.     

Cellular insertions in the NS2-3 genome region of cytopathic bovine viral diarrhoea virus (BVDV) isolates

When compared to noncytopathic (ncp) bovine viral diarrhoea virus (BVDV), some cytopathic (cp) BVDV contain additional sequences in the NS2-3 genomic region. One of these insertions, which is 270 nucleotides long and of host origin (cINS), was first described for strain NADL. To find out how frequently this type of insertion occurs in other cp BVDV, 32 cp BVDV field isolates and the BVDV reference cp strain Indiana were screened using RT-PCR which detected cINS in NADL. For most cp viruses an RT-PCR product of 402bp indicated the presence of NS2-3 genes without insertions. In addition, one or two DNA fragments, around 600-850bp in size, were amplified from the genomes of 13 cp viruses indicating the presence of insertions. Sequencing of the PCR products, i.e. 402bp DNA fragment (with no insertion) and longer fragments (with insertion) revealed the location of the insertions in the NS2-3 coding region of eight cp BVDV genomes. All of the insertions were confirmed to be of the cINS type and were located in a very similar position to that found previously in the NADL genome. They were in the same reading frame as the viral polypeptide and they encoded 90-140 amino acids. The 5' and 3' ends of the insertions were different in most of the cp isolates studied. Interestingly, a 14-amino-acid stretch at the 5'-end of the insertion in the cp 5569 isolate as well as 15 amino acids at the 3'-end of the insertion in the cp 5.19516 isolate were not homologous to the cINS sequence. No significant matches for these stretches were found in the EMBL and Swissprot databases.     

Genetic diversity and frequency of bovine viral diarrhea virus (BVDV) detected in cattle in Turkey

The aim of this study was to investigate the frequency and diversity of bovine viral diarrhea viruses (BVDV) infecting cattle in Turkey. A total of 1124 bovine blood samples from 19 farms in 4 different Turkish regions were tested by antigen capture ELISA (ACE). BVDV antigen was found in 26 samples from 13 farms. Only 20 of the 26 initial test positive cattle were available for retesting. Of these, 6 of 20 tested positive for BVDV, by ACE and real-time RT-PCR, one month after initial testing. Phylogenetic analysis, based on comparison of the E2 or the 5'UTR coding regions, from 19 of the 26 initial positive samples, indicated that 17 belonged to the BVDV-1 genotype and 2 to the BVDV-2 genotype. Comparison of 5'UTR sequences segregated 8 BVDV-1 strains (strains 5, 6, 10, 11, 12, 13, 17, and 19) to the BVDV1f, 1 strain (strain 8) to the BVDV1i and 1 strain (strain 14) to the BVDV1d subgenotypes. One strain (strain 4) did not group with other subgenotypes but was closer to the BVDV1f. The remaining 6 BVDV-1 strains (strains 1, 2, 3, 7, 9, and 18) segregated to a novel subgenotype. The E2 sequence comparison results were similar, with the exception that strain 5 grouped with the novel subgenotype rather than BVDV1f subgenotype. It appears that among the diverse BVDV strains in circulation there may be a subgenotype that is unique to Turkey. This should be considered in the design of diagnostics and vaccines to be used in Turkey.     

Effect of the bovine viral diarrhoea virus (BVDV) infection on dairy calf rearing

The aim of this study was to compare the cumulative incidence of mortality, clinical diarrhoea and respiratory disease in calves, during their first six months of age, in herds with different bovine viral diarrhoea virus (BVDV) infection status. Calves’ health indicators were tested by comparing proportions in 101 farms with dissimilar infection condition. The results indicate that there was a significant relationship between the BVDV status (actively infected herd or not) and the cumulative incidence of mortality and respiratory disorders.     

Principles for eradication of bovine viral diarrhoea virus (BVDV) infections in cattle populations

Systematic eradication of BVDV without vaccination started in Scandinavia in 1993. In principle, the schemes include; (1) identification of non-infected and infected herds using different combinations of serological herd tests such as bulk milk tests and spot tests (sample of animals in a certain age), (2) monitoring/certification of non-infected herds by repeated sampling, applying one of the above-mentioned methods and (3) virus clearance in infected herds aimed at removing persistently infected (PI) animals in a cost- and time-efficient manner. In the virus clearance protocol described, an initial test is performed on all animals with subsequent follow-up of calves born as well as of dams seronegative in the initial test. It is generally recommended to perform an initial antibody test on all samples. This should be done not only to screen for seronegative animals on which virus isolation should be attempted (i.e. possible PI animals), but more in order to identify non-immune animals in reproductive age, that is, the key animals in herd-level persistence of infection. In Sweden, a common finding has been self-clearance, where the infection ceases without any other intervention than controlled introduction of new animals. Other epidemiological observations concern the course of events following virus introduction. Important risk factors for spreading BVDV are discussed, where livestock trade is perceived as the most central to control. Live vaccines, imported semen and embryos constitute special hazards, since they may act as vehicles for the introduction of new BVDV strains. The importance of making farmers aware of herd biosecurity and their own responsibility for it is stressed, and in order to maintain a favourable situation after a scheme has been concluded, effort must be put into establishing such a persisting attitude in the farming community.     

Fatal congenital anaplasmosis associated with bovine viral diarrhoea virus (BVDV) infection in a crossbred calf

Clinical disease resulting from the vertical transmission of Anaplasma marginale has only been reported on 5 occasions despite studies demonstrating successful in utero transmission. During the reported experimental induction of congenital anaplasmosis in calves, the outcome was variable but mostly led to inapparent or mild infection. There are previous case reports of fatal congenital anaplasmosis following natural infection. The clinical findings in a 2-day-old calf presented to the Onderstepoort Veterinary Academic Hospital with clinical signs of congenital anaplasmosis, which was unresponsive to treatment, are described. Subsequent post mortem diagnostic tests revealed that this calf was co-infected with bovine viral diarrhoea virus (BVDV). It is postulated that immunosuppression resulting from BVDV infection predisposed to severe, fatal anaplasmosis in this calf.     

Evaluation of long-term antibody responses to two inactivated bovine viral diarrhoea virus (BVDV) vaccines

The aim of the present study was to determine the serological response of heifers after vaccination with two inactivated bovine viral diarrhoea virus (BVDV) vaccines by means of various ELISA tests. Three dairy farms were selected from the Galicia region of Spain. In each herd, a batch of heifers to be vaccinated for the first time was selected and followed for 15 months. Heifers from farm 1 (n = 25) were vaccinated with Vaccine A, whereas heifers from farm 2 (n = 16) were vaccinated with Vaccine B. Heifers from farm 3 (n = 17), where no BVDV vaccines were used, acted as controls. Blood samples were analyzed periodically for BVDV antibodies, using five commercial ELISAs, based on BVDV p80 antigen or whole virus.At the end of the study, none of the animals vaccinated with Vaccine A seroconverted according to p80 antibody status, whereas up to 80% tested positive by ELISA against whole virus antigen. For the animals vaccinated with Vaccine B, 2/16 animals seroconverted according to p80 antibody ELISAs, whereas all had seroconverted according to the ELISA against whole virus antigen. In most cases, based on the use of ELISAs to detect specific antibodies against the p80 protein, at 15 months post-vaccination with inactivated BVDV vaccines the responses did not seem to interfere with detection of antibody to BVDV infection. However, the finding of a small proportion of vaccinated animals seropositive against BVDV p80 antigen suggests that antibodies that interfere with diagnosis of BVDV infection within the herd could exist, even when using p80 ELISAs.     

Serological relationships among subgroups in bovine viral diarrhea virus genotype 1 (BVDV-1)

Bovine viral diarrhea virus (BVDV) has various economic impacts associated with diarrhea, poor performance, an increase in the frequency of other infections and lethal outcomes. Both genotypes, namely BVDV-1 and BVDV-2, as well as different subgroups within these genotypes have been reported worldwide. Understanding the serological differences among the BVDV subgroups is important for disease epidemiology and prevention as well as vaccination programs. The aim of this study was to determine the serological relatedness among the subgroups in BVDV-1. For that purpose, sheep hyperimmune sera were collected against representative strains from 6 of the subgroups of BVDV-1 (BVDV-1a, -1b, -1d, -1f, -1h and -1l). The serum samples that gave the peak antibody titer to the homologous strains were used to perform cross neutralization assays. The highest homologous antibody titer (1:5160) was obtained against BVDV-1h. Regarding the cross neutralizing (heterologous) antibodies, the lowest titer (1:20) was produced by the BVDV-1f antiserum against the BVDV-1a and BVDV1-b viruses. The highest cross neutralizing titer (1:2580) achieved by the BVDV-1h antiserum was against the BVDV-1b strain. The cross neutralization results indicated particular serological differences between the recently described subgroup (BVDV-1l) and BVDV-1a/-1b, which are widely used in commercial vaccines. Considering the cross neutralization titers, it is concluded that selected BVDV-1l and BVDV-1h strains can be used for the development of diagnostic and control tools.     

Natural changes in the spread of bovine viral diarrhoea virus (BVDV) among Estonian cattle

The results of a survey conducted during 1993-2000 to study the spread of bovine viral diarrhoeal virus (BVDV) among Estonian cattle are presented. The BVDV infection status of a representative random sample of cattle herds housing 20 or more dairy cows was established to estimate the prevalence of herds with active BVDV infection [potentially having persistently infected (PI) cattle--suspect PI herds]. The herds investigated comprised approximately 70% of all Estonian dairy cows. The BVDV infection status was established in 315-350 herds (making the sampling fraction about 20%) during three sampling periods: 1993-95, 1997-98, 1999-2000. BVDV antibodies were detected in herd bulk milk samples and/or sera from young stock by a liquid-phase-blocking enzyme-linked immunosorbent assay developed in the Danish Veterinary Institute for Virus Research. The results of the survey demonstrate the reduction in the prevalence of herds with active BVDV infection in the studied fraction of the Estonian cattle population. During the first sampling period (1993-95) a prevalence of 46% (+/- 5%) for suspect PI herds was observed, during the second sampling period this prevalence was 16% (+/- 3%) and in the third period it was 18% (+/- 3%). As there is no control programme for BVDV in Estonia, the observed changes reflect the natural course of the infection in the study population. A possible cause for these changes is the decreased trade in breeding animals as a result of the economic difficulties present in cattle farming during the study period. The farming practices (most large herds are managed as closed herds) and the low density of cattle farms have obviously facilitated the self-clearance of herds from the BVDV infection, diminishing the new introduction of infection into the herds.     

Genetic typing of bovine viral diarrhoea virus: most Slovenian isolates are of genotypes 1d and 1f

A selection of 43 bovine viral diarrhoea viruses isolated from mainly persistently infected cattle on 23 Slovenian farms between 1997 and 2001 were characterised genetically. Viral RNA was extracted from infected cell cultures, reverse transcribed and amplified by PCR with primers targeting the 5'-UTR and the N(pro) gene, followed by direct sequencing of purified PCR products obtained for both genomic regions. The N(pro) sequences provided the best genetic resolution, and gave also higher statistical support for phylogenetic classification of the viruses. Thirty-eight of the Slovenian isolates were of genetic subtypes 1d and 1f, four were 1b, and one subtype 1g. No BVDV type 2 viruses were found. This genetic prevalence matched those previously reported for neighbouring countries, as opposed to findings reported for more distant European countries, e.g. France, Spain and the UK. From eight cattle herds several virus isolates were analysed; with one exception all isolates from each herd were of the same genetic group. Extended sequencing of the N(pro) and part of the C gene of virus isolates with identical 5'-UTR sequences allowed differentiation between isolates obtained at different times from one herd.     

Survivorship of animals persistently infected with bovine virus diarrhoea virus (BVDV)

The survivorship of animals persistently infected with bovine virus diarrhoea virus was studied in ten Danish dairy herds. The ages of 34 persistently infected animals were compared with the ages of non-persistently infected animals in the herds in a cross-sectional study in which the risk rate of removal of animals in the two groups within 1 year was estimated from an exponential probability function of the age. In a cohort study, the 34 persistently infected animals were followed from the date of the initial blood test and onwards and the risk rates of dying or being slaughtered due to unthriftiness or either of these events were calculated.

The attributable risk of leaving the herds within 1 year was 0.35 among persistently infected animals. The risk rate of dying or being slaughtered due to unthriftiness within 1 year was 0.28 and 0.31, respectively; the risk rate of either of these events was 0.5.     

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.