An investigation into the transmission and control of pestivirus in sheep in Australia.

Border disease virus (BDV) is a member of the pestivirus genus that primarily affects sheep, causing reproductive losses through abortion, still births and the birth of weak lambs. The key characteristic of this disease is the birth of persistently infected (PI) lambs which, after surviving transplacental infection, are born antibody negative, yet virus positive, and thus shed the virus for their entire life and are the primary source of spread within a flock. The cornerstones of BDV control are detection and elimination of PI animals, biosecurity measures to prevent re-infection, and surveillance programs. Recommendations for the control of BDV in sheep are centred around the approach to bovine viral diarrhoea virus (BVDV), the prominent cattle pestivirus species, due to a lack of specific research into BDV control and elimination. In this study, two aspects of a BDV control program were investigated: the effectiveness of the BVDV vaccine, Pestigard®, and the rate of seroconversion in a flock deliberately exposed to known PI lambs. The vaccine appeared to be safe, and the optimal dose was the full cattle dose (2 mL). While vaccination induced high virus neutralising titres to BVDV when administered as either a quarter, half or full dose registered for cattle, the BDV titres achieved were low and unlikely to prevent transplacental infection. In a second study, after exposure of between 2 and 15 days exposure to two PI lambs in confined conditions, only 3 of 66 previously naïve sheep demonstrated seroconversion. This demonstrated a very low rate of transmission and suggested that deliberate exposure to PI lambs at low-risk times for less than 15 days was not likely to be an effective means of achieving seroconversion throughout a flock and, therefore, not provide protection against BDV challenge during gestation.

Development and evaluation of real-time RT-PCR using ear hair for specific detection of sheep persistently infected with border disease virus (BDV).

The aim of this study was to develop an improved border disease virus (BDV) specific real time RT-PCR and to evaluate its performance on manually plucked hairs from sheep persistently infected with BDV that may act as a non-invasive alternate sample. The BDV real time RT-PCR assay reported here showed a high analytical sensitivity (100.6 TCID50/ml), specificity (no reactivity with BVDV-1, BVDV-2, HoBi-like pestivirus and CSFV) and reproducibility. When the assay was validated on 210 samples from BDV-infected and uninfected sheep, it showed a 100% diagnostic sensitivity and specificity with virus isolation. Further evaluation of the assay on manually plucked hair follicles from ear (mid-lateral, mid-medial) and tail tip from sheep persistently infected with BDV showed that a minimum of 20 hair follicles need to be tested for correct diagnosis of BDV. The BDV load was comparatively higher in hairs from mid-medial ear than those from other tested locations. Evaluation on other samples from PI sheep demonstrated that the test performance was similar to that of pestivirus generic real-time RT-PCR, but improved than the currently available BDV specific real-time RT-PCR. Although more number of PI animals need to be evaluated, the results of the study showed that manually plucked hairs from mid-medial ear pinna is a suitable alternative sample in real-time RT-PCR for detection of BDV persistently infected sheep. Use of the non-invasive ear hair samples and the improved BDV specific real-time RT-PCR reported here may be useful for BDV surveillance in several sheep rearing countries.

Identification and molecular characterization of border disease virus (BDV) from sheep in India.

Pestiviruses isolated from sheep and goats in India thus far have been bovine viral diarrhoea virus 1 (BVDV-1) or BVDV-2. During routine genetic typing of pestiviruses in the years 2009-10, border disease virus (BDV) was detected in eight Indian sheep of a flock showing clinical signs of BD by real time RT-PCR. All the samples yielded positive virus isolates in cell culture but were found negative by a BVDV antigen ELISA. A representative BDV isolate was characterized at genetic and antigenic level. Phylogenetic analysis carried out in 5'-UTR, N(pro) and E2 regions of genome typed the Indian BDV isolate as BDV-3. A more detailed analysis in N(pro) and entire region coding structural proteins showed that the N(pro) (168), C (100 aa), E(rns) (227 aa), E1 (195 aa) and E2 (373 aa) proteins were of size characteristic for BDV reference strain X818. Antigenic differences were evident between the BDV-3 isolate and previously reported BDV-1, BDV-5 and BDV-7 strains. Although origin of BDV-3 in India is not clear, the results reflect probable introduction through trade in sheep between India and other countries or BDV-3 may be more widely distributed. Additionally, this study suggests that for diagnosis of BDV infection, the commercial BVDV Ag-ELISA should be used with caution. This is the first identification of BDV in sheep in India which highlights the need for continued pestivirus surveillance and assessing its impact on sheep and goat production.

A new genotype of border disease virus with implications for molecular diagnostics.

Border disease virus (BDV) is a (+) single-stranded RNA pestivirus affecting mainly sheep and goats worldwide. Genetic typing of BDV has led to the identification of at least seven major genotypes. This study reports the detection of a BDV strain from a goat in northwestern Italy during routine investigations. Sequence analysis revealed mutations in the 5'-UTR of the virus with implications for BDV molecular diagnostics. Moreover, subsequent phylogenetic analysis based on the combined 5'-UTR and Npro/partial C genes, showed divergence from known BDV genotypes, revealing the detection of a novel pestivirus group, for which we propose the name BDV genotype 8.

The two sides of border disease in Pyrenean chamois (Rupicapra pyrenaica): silent persistence and population collapse.

In 2001, border disease virus (BDV) was identified as the cause of a previously unreported disease in Pyrenean chamois (Rupicapra pyrenaica) in Spain. Since then, the disease has caused a dramatic decrease, and in some cases collapse, of chamois populations and has expanded to nearly the entire distribution area in the Pyrenees. Chamois BDV was characterized as BDV-4 genotype and experimental studies confirmed that it was the primary agent of the disease. The infection has become endemic in the Central and Eastern Pyrenees. However, while most Pyrenean chamois populations have been severely affected by the disease, others have not, despite the circulation of BDV in apparently healthy individuals, suggesting the existence of different viral strategies for persisting in the host population. Changes in the interplay of pathogen, host and environmental factors may lead to the formation of different disease patterns. A key factor influencing disease emergence may be pathogen invasiveness through viral mutation. Host factors, such as behavior, immunity at the population level and genetic variability, may also have driven different epidemiological scenarios. Climatic and other ecological factors may have favored secondary infections, such as pneumonia, that under particular circumstances have been major contributing factors in the high mortality observed in some areas.

Development and evaluation of indirect enzyme-linked immunosorbent assay for a screening test to detect antibodies against classical swine fever virus.

An indirect enzyme-linked immunosorbent assay (ELISA) was developed for a screening test to detect antibodies against classical swine fever virus (CSFV). Viral glycoproteins, which were purified from swine kidney cells infected with CSFV ALD/A76 strain by the immunoaffinity purification using monoclonal antibody against E2 protein, were adsorbed on a microtiter plate as the antigen for the antibody detection. Each antibody titer of serum sample was expressed as a sample per positive value calculated with optical absorbance of each sample and that of a positive control. The advantage of this ELISA is its higher sensitivity: most sera containing more than 4 neutralization titers were determined to be positive. This ELISA is unable to discriminate between antibodies against CSFV and those against other ruminant pestiviruses, therefore positive sera in this ELISA should be evaluated by a cross-neutralization test using CSFV, bovine viral diarrhea virus, and border disease virus. Taken together, the indirect ELISA developed in this study is useful screening tool to detect antibodies against CSFV for the large-scale monitoring of classical swine fever.

Short communication: performance of a blocking antibody ELISA bulk-tank milk test for detection of dairy sheep flocks exposed to border disease virus.

The aim of this study was to investigate the test characteristics of a blocking antibody ELISA applied to bulk-tank milk (BTM) samples for the detection of dairy sheep flocks positive for antibodies to border disease virus. In 161 flocks recruited in 2009 and 2010, the antibody inhibition percentage (AIP) in BTM was compared with the prevalence estimate of antibody-positive ewes obtained from an age-representative sample of 45 milking ewes. A strong negative exponential relationship (R(2)=0.89) was found between AIP in BTM and seroprevalence level. Using receiver operating characteristic analysis, the best AIP decision threshold in BTM to discriminate between low (<10%) and high (≥10%) antibody-positive flocks was 65%. Diagnostic performance estimates based on observed seroprevalence levels and Monte Carlo simulations showed that this threshold value was associated with high sensitivity and specificity (91.9±5.5% and 95.9±1.6%, respectively), whereas the 80% decision threshold recommended in dairy cows yielded lower specificity (83.6±2.0%). Results obtained from the same flocks during 2 subsequent milking campaigns showed that the 65% AIP cut-off value was associated with fewer false-positive results and is preferred. Testing of BTM samples could be a powerful tool in inferring border disease virus seroprevalence in a flock and in Pestivirus control schemes in dairy sheep flocks.

Molecular detection of pestiviruses in aborted foetuses from provinces in northern Turkey.

All pestiviruses are important veterinary pathogens causing economic losses in cattle, sheep and pigs. Besides the important economical losses, pestiviruses may compromise the normal immune response to other pathogens and increase the severity of other infections in sheep. In this study, aborted foetuses (cattle and sheep) in either coastal or inland Black Sea region of Turkey were surveyed for the presence of RNA from pestiviruses (bovine viral diarrhoea virus (BVDV), border disease virus (BDV)). The presence of BVDV RNA was found in 6 of 21 aborted calves (28.57%), although BDV RNA was detected in 14 of 21 aborted lambs (66.66%) by reverse transcriptase polymerase chain reaction. This study also investigates the distribution of viral RNA within the brain, liver and lung of aborted foetuses. The viral RNA positivity rates for the organs varied and were as follows: brain 40.47% and liver and lung 38.09%. The results revealed that pestiviruses are important abort pathogen in the provinces of northern Turkey.

Border disease virus shedding and detection in naturally infected Pyrenean chamois (Rupicapra pyrenaica).

Pyrenean chamois (Rupicapra pyrenaica) populations of the central and eastern Pyrenees have been affected by severe outbreaks associated with Border disease virus (BDV) since 2001. Eight Pyrenean chamois (7 males and 1 female) from 1 to 8 years of age with clinical signs consistent with BDV infection were studied. At necropsy, whole blood, tissue samples (skin, brain, prescapular lymph node, thyroid gland, lung, liver, spleen, kidney, small intestine, bone marrow, and testicle), urine, and nasal, oral, and rectal swabs were obtained. The fetus from a pregnant female was also studied. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the virus in all samples, and virus isolation was performed. Sera and tissue samples were positive to RT-PCR, and the virus was isolated from all chamois. The nasal, oral, and rectal swabs and urine samples were RT-PCR positive in 100%, 85.71%, 71.43%, and 100% of chamois, respectively, confirming the excretion of the virus via these 4 routes. In addition, sera were tested for BDV antibodies using enzyme-linked immunosorbent assay and seroneutralization techniques, with negative results. Sequence analysis of the 5' untranslated region in 7 of the chamois confirmed that the virus is grouped into the BDV-4 genotype, the same BDV previously described in Pyrenean chamois. To the authors' knowledge, this is the first study of naturally infected Pyrenean chamois, providing evidence that infected animals shed BDV through nasal, oral, fecal, and urinary excretion routes.

Mucosal lesions in a sheep infected with the Border Disease Virus (BDV).

A 28-week-old sheep was presented at the animal hospital because of chronic emaciation, anemia and slight diarrhea. Due to poor general condition and bad prognosis the animal was euthanized and submitted for postmortem investigation. Multiple erosions and ulcerations were found in the dorsal region of the tongue, the pharynx, the hard palate, in the esophagus and the ruminal pillars. Histologically, these lesions consisted of necrosuppurative inflammation. The animal was tested positive for pestivirus antigen both by immunohistochemical and by virological examination (cell culture, antigen capture ELISA and RT-PCR). A non-cytopathic Border Disease Virus was identified, and sequencing revealed a virus belonging to the BDV-3 cluster. Based on the macroscopical, histological, immunohistological and virological results this case was diagnosed as Border Disease with mucosal lesions. This is the first report of such a case in Switzerland.

Detection of Border disease virus in fetuses, stillbirths, and newborn lambs from natural and experimental infections.

The purpose of the present study was to evaluate the use of enzyme-linked immunosorbent assay (ELISA) antigen detection in blood or fetal fluids and reverse transcription polymerase chain reaction (RT-PCR) amplification in tissues for routine laboratory diagnosis of Border disease virus (BDV) infection. Samples from 67 fetuses, 6 stillbirths, and 11 lambs from 25 commercial flocks with suspicion of BDV abortion and 3 fetuses, 7 stillbirths, and 15 lambs obtained from an experimental infection with a local isolate (BDV genotype 4) were investigated. Presence of BDV was detected by RT-PCR in 7.9% of fetuses, 50% of stillbirths, and 50% of lambs from the commercial flocks analyzed, corresponding to 8 of the 25 farms (32%). A similar percentage of the lambs and stillbirths from the experimental infection were positive by RT-PCR of tissue samples (54.5%), and the highest positivity was detected in lymph node, thyroid gland, and kidney. The current study revealed that RT-PCR analysis of stillbirths and lambs with clinical symptoms is more suitable than the analysis of fetuses to confirm the presence of BDV in a flock. Pestiviral antigen was detected by antigen ELISA in a high proportion of fetuses (24/58) and stillbirths (3/4) from commercial flocks, but in lambs, the presence of colostral antibodies masked the detection of the antigen by ELISA. Nevertheless, in lambs from the experimental infection that were not fed colostrum, antigen ELISA was less efficient than RT-PCR in detecting viral presence in stillbirths and lambs. Antigen ELISA is therefore recommended for fetuses with advanced autolysis that can adversely affect RNA integrity.

Border disease virus among chamois, Spain.

Approximately 3,000 Pyrenean chamois (Rupicapra pyrenaica pyrenaica) died in northeastern Spain during 2005-2007. Border disease virus infection was identified by reverse transcription-PCR and sequencing analysis. These results implicate this virus as the primary cause of death, similar to findings in the previous epizootic in 2001.

[Seroepidemiological survey of sheep in Carinthia for the dissemination of ruminant pestiviruses]. / Seroepidemiologische Untersuchung bei Schafen in Kärnten zur Verbreitung von ruminanten Pestiviren.

In this study serological investigations were performed to determine the prevalence of pestiviral infections in sheep in one Federal State of Austria, namely Carinthia. 1527 blood samples from sheep in 147 flocks were collected and tested by Enzyme-linked immunosorbent assay and virus-neutralisation tests for antibodies to ruminant pestiviruses. The estimated flock prevalence was 47.6%, the individual prevalence 16.3%. Significant geographical variations in the flock as well in the individual prevalence were found. The highest prevalence in sheep and in sheep flocks was established in the region Spittal/Drau with 25.9% and 69.7%.The individual and the flock prevalence was significantly higher on farms where cattle or sheep from other farms were present than on farms with no cattle (p < 0.017). All Enzyme-linked immunosorbent assay positive sera were tested for Bovine viral diarrhea virus-1 (strain NADL), Bovine viral diarrhea virus-2 (strain 125) and for Border disease virus (strain MOREDUN) by virus neutralisation tests. Seventy out of 249 positive samples revealed the highest titres (> or = two-fold) to Bovine viral diarrhea virus-1 and 25 to Border disease virus. The remaining positive samples did not show clear results because of cross reactions.

Development of a real time RT-PCR to detect and type ovine pestiviruses.

A real time one-step RT-PCR was designed to detect and type border disease virus (BDV), bovine viral diarrhea virus (BVDV) type 1 and BVDV type 2 in ovine samples. The real time RT-PCR was shown to behave in a linear manner and had limits of detection of 100-1000 copies of viral RNA as judged by in vitro transcribed RNA. The real time RT-PCR was validated on 50 clinical samples from UK flocks and was more sensitive than a virus isolation and a classical nested RT-PCR (nRT-PCR). The results of real time RT-PCR virus typing agreed completely with sequencing. The majority of ovine isolates were BDV; a small proportion were BVDV type 1. BVDV type 2 was not detected in any sample. This test appears reliable and can be used for the typing of ovine pestiviruses in the UK.

[Differential diagnosis of classical swine fever and border disease: seroepidemiological investigation of a pestivirus infection on a mixed sheep and swine farm]. / Zur Differentialdiagnostik Klassische Schweinepest/Border Disease: Seroepidemiologische Untersuchung einer Pestivirusinfektion auf einem schaf- und schweinehaltenden Betrieb.

During recent years neutralizing antibodies against Border Disease Virus (BDV) were found repeatedly in German pig herds. Consequently there was a demand for a differential diagnostic system. A permanent sheep cell line and BDV reference strain Moredun were chosen and were applied in a could be used case study. A pestivirus could be isolated from piglets on a mixed farm and was characterised as 'non-Classical Swine Fever' (CSF) by using monoclonal antibodies. Due to a CSF suspicion the pig herd was destroyed immediately. Serum samples of sheep from the same farm were used for further characterisation of the new virus isolate. A neutralization test of the sheep sera was performed against different pestiviruses and the new isolate. Neutralizing antibody titres against the new virus pig isolate were significantly higher than against all other pestiviruses. BDV strain Moredun recognised the antibodies clearly, whereas CSF viral strain Alfort 187 and several isolates of bovine viral diarrhoea virus (BVDV) strains scored the lowest cross reaction.

[Detection of border disease virus in a sheep flock in Saxony]. / Nachweis von Border Disease Virus in einem Schafbestand in Sachsen.

A pestivirus has been isolated from brain samples of two lambs suffering from clinical signs of border disease. The two identical isolates were allocated to the "true" border disease virus group concerning their reaction pattern with monoclonal antibodies and the 5'UTR sequence data. Nevertheless, alterations of phenotype and genotype in comparison with references of both BDV-subgroups have been shown. The existence of an infection with border disease virus in the flock has been confirmed by serological studies.

A RT-PCR assay for the rapid recognition of border disease virus.

A reverse transcription--polymerase chain reaction (RT-PCR) method was developed for the specific detection of border disease virus (BDV), using the primers PBD1 and PBD2 flanking a 225 bp DNA fragment, selected from the 5'noncoding region of the pestivirus genome. In tests on 70 pestiviruses it was shown to be BDV-specific. A closed, one-tube nested RT-PCR method employing general pestivirus outer primers (324 and 326), and the same BDV-specific inner primers (PBD1 and PBD2) in conjunction with a BDV-specific fluorogenic TaqMan probe also detected only BDV and was more sensitive. BDV-specific RT-PCR was used in combination with a PCR specific for bovine viral diarrhoea virus type 2 (BVDV2) to ascertain whether virus stocks contained mixtures of BDV and BVDV2. It was shown that the ovine pestivirus strains 175375 and 59386 were originally BDV, but after subculture had become contaminated with BVDV2. This explains a previously reported discrepancy in the genetic typing of 59386. Although the BDV-specific RT-PCR can also detect BDV in clinical samples, the assay is likely to be most useful for the rapid typing of laboratory pestivirus strains.

Laboratory decision-making during the classical swine fever epidemic of 1997-1998 in The Netherlands.

The National Reference Laboratory for classical swine fever (CSF) virus in The Netherlands examined more than two million samples for CSF virus or serum antibody during the CSF epizootic of 1997-1998. The immense amount of samples and the prevalence of border disease (BD) virus and bovine viral diarrhoea (BVD) virus infections in Dutch pig herds necessitated the diagnostic efforts of the laboratory to be focused on generating CSF specific test results throughout the eradication campaign. Detection of 82% of the 429 outbreaks was achieved through the combined use of a direct immunofluorescence and peroxidase assay (FAT/IPA) with samples (tonsils) collected from clinically-suspected pigs. This suggests that in the majority of the outbreaks, the pigs had clinical signs that were recognised by the farmer and/or veterinarians, indicating the presence of CSF virus in a pig herd. A positive diagnosis of 74% of all the tissue samples (tonsils) collected at infected pig holdings was established by FAT. More than 140,000 heparinised blood samples were examined by virus isolation, resulting in the detection of 4.5% of the infected herds. CSF virus was isolated in approximately 29% of all the blood samples collected from pigs at infected or suspected farms. Several serological surveys--each done within a different framework--led to the detection of 13.5% of the total number of outbreaks. The detection of CSF virus antibody in serum was carried out by semi-automated blocking ELISA. Approximately 28.5% of the sera which reacted in the ELISA were classified as CSF virus-neutralising antibody positive and 26.5% as positive for other pestiviruses following the virus neutralisation test (VNT). We concluded that two of the CSF laboratory diagnostic methods described were determinative in the eradication campaign: first, the FAT for the screening of diseased pigs; and second, the ELISA and VNT when millions of predominantly healthy pigs needed to be screened for the presence of CSF serum antibody. Decision-making on the basis of results generated by either method can, however, be seriously hindered when samples are examined from pig herds with a high prevalence of non-CSF pestiviruses.