Reduced specificity of Erns antibody ELISAs for samples from piglets with maternally derived antibodies induced by vaccination of sows with classical swine fever marker vaccine CP7_E2alf

Successful implementation of marker vaccines against classical swine fever virus is dependent on a reliable accompanying diagnostic assay that allows differentiation of infected from vaccinated animals (DIVA ) as well as the development of a testing scheme during emergency vaccination. In this context, special attention needs to be paid to breeding farms, because the offspring of marker vaccinated sows possess maternally derived antibodies (MDA s). So far, limited information is available on the influence of MDA s on serological testing in the context of a DIVA strategy. Therefore, two commercially available E^rns antibody ELISA s were compared, using serum samples of piglets with a high‐to‐moderate titre of MDA s against marker vaccine CP 7_E2alf. False‐positive results were detected by both E^rns antibody ELISA s for serum samples of piglets with an age of up to 4 weeks. Interestingly, most samples tested false‐positive in the first E^rns antibody ELISA were identified correctly by the other E^rns antibody ELISA and vice versa. In conclusion, in case of emergency vaccination of sows, the specificity of both ELISA s in newborn piglets younger than 4 weeks may be relatively low. This could be addressed in a testing strategy by either not sampling piglets up to the age of 4 weeks or using both ELISA s in a screening‐confirmation set‐up.     

Development of a Luminex‐Based DIVA Assay for Serological Detection of African Horse Sickness Virus in Horses

African horse sickness (AHS) is considered a fatal re‐emergent vector‐borne disease of horses. In the absence of any effective treatment for AHS, vaccination remains the most effective form of disease control. The new generation of vaccines, such as one based on purified, inactivated AHS virus (AHSV, serotype 4), which does not induce antibodies against non‐structural protein 3 (NS3), enables the development of diagnostic methods that differentiate infected from vaccinated animals (DIVA assays). As detecting AHS in AHSV‐free countries may lead to restrictions on international animal movements and thereby cause significant economic damage, these DIVA assays are crucial for reducing movement restrictions. In this article, we describe a Luminex‐based multiplex assay for DIVA diagnosis of AHS, and we validate it in a duplex format to detect antibodies against structural protein 7 (VP7) and NS3 in serum samples from horses vaccinated with inactivated AHSV4 vaccine or infected with a live virus of the same serotype. Results of the Luminex‐based assay for detecting anti‐NS3 antibodies showed good positive correlation with results from an in‐house enzyme‐linked immunosorbent assay (ELISA). Thus, the Luminex‐based technique described here may allow multiplex DIVA antibody detection in a single sample in less than 2 h, and it may prove adaptable for the development of robust, multiplex serological assays.     

A Review of OIE Country Status Recovery Using Vaccinate‐to‐Live Versus Vaccinate‐to‐Die Foot‐and‐Mouth Disease Response Policies I: Benefits of Higher Potency Vaccines and Associated NSP DIVA Test Systems in Post‐Outbreak Surveillance

To rapidly return to trade, countries with OIE status, FMD‐free country where vaccination is not practised, have destroyed emergency vaccinated animals, raising ethical concerns with respect to social values, the environment, animal welfare and global food security. This two‐part review explores whether science could support eligibility to return to previous OIE status in 3 months irrespective of vaccinate‐to‐live or vaccinate‐to‐die policies. Here, we examine the benefits of higher potency (≥ 6 PD₅₀), high‐purity vaccines formulated from antigen banks for emergency use, their efficacy and performance in differentiating infected from vaccinated animals (DIVA) assays for post‐outbreak surveillance. From an intensive programme of research, we conclude that high‐quality, higher potency vaccines are proven to reduce FMD virus (FMDV) subclinical circulation and the risk of carriers. Broader coverage than predicted by serology suggests the potential to hold a few ‘key’ vaccine strains improving logistics and reducing the financial burden of antigen banks. The OIE should adopt formal definitions for emergency vaccination and emergency vaccines. In terms of supportive tools, we consider that the lack of OIE recognition of DIVA tests other than those of PANAFTOSA in cattle is a shortcoming. There is need for research on maternal antibody interference with DIVA tests and on the use of such tests to establish whether greater purification of vaccines improves performance. We consider that alignment of waiting periods for vaccinate‐to‐live and vaccinate‐to‐die in OIE Code Article 8.5.9 1 b. and c. is feasible until an acceptable level of statistical certainty for surveillance or target probability of freedom is established to substantiate the absence of FMDV infection or circulation. It is surveillance intensity rather than waiting periods that establishes the risk of residual FMDV. EU Directive 2003/85/EC implicitly recognizes this, permitting derogation of the OIE waiting periods.     

Safety profile of a replication‐deficient human adenovirus‐vectored foot‐and‐mouth disease virus serotype A24 subunit vaccine in cattle

The safety of a replication‐deficient, human adenovirus‐vectored foot‐and‐mouth disease virus (FMDV ) serotype A24 Cruzeiro capsid‐based subunit vaccine (AdtA24) was evaluated in five independent safety studies. The target animal safety studies were designed in compliance with United States (U.S.) regulatory requirements (Title 9, U.S. Code of Federal Regulation [9CFR ]) and international standard guidelines (VICH Topic GL ‐44) for veterinary live vaccines. The first three studies were conducted in a total of 22 vaccinees and demonstrated that the AdtA24 master seed virus (MSV ) was safe, did not revert to virulence and was not shed or spread from vaccinees to susceptible cattle or pigs. The fourth safety study conducted in 10 lactating cows using an AdtA24 vaccine serial showed that the vaccine was completely absent from milk. The fifth safety study was conducted under typical U.S. production field conditions in 500 healthy beef and dairy cattle using two AdtA24 vaccine serials. These results demonstrated that the vaccine was safe when used per the product label recommendations. Additional data collected during these five studies confirmed that AdtA24 vaccinees developed FMDV A24 and the HA d5 vaccine vector serum neutralization antibodies that test negative in a FMDV non‐structural protein antibody test, confirming AdtA24 vaccine's capability to differentiate infected from vaccinated animals (DIVA ). In conclusion, results from this comprehensive set of cattle studies demonstrated the safety of the replication‐deficient AdtA24 vaccine and fulfilled safety‐related requirements for U.S. regulatory requirements.     

GFP tagging of Brucella melitensis Rev1 allows the identification of vaccinated sheep

Brucellosis is a worldwide zoonosis causing important economic loss and a public health problem. Small ruminants are the preferred hosts of Brucella melitensis and thus the main source of human infections. Effective control of sheep and goat brucellosis has been achieved in several countries through vaccination with the live‐attenuated B. melitensis Rev1 vaccine. However, Rev1 induces a long‐lasting serological response that hinders the differentiation between infected and vaccinated animals. A Rev1::gfp strain expressing constitutively the Green Fluorescent Protein (GFP) was built by stable insertion of a mini‐Tn7‐gfp in the glmS‐recG non‐codifying chromosomal region. An associated indirect ELISA‐GFP was developed to identify anti‐GFP antibodies in vaccinated animals. The resulting Rev1::gfp kept the biological properties of the Rev1 reference strain, including residual virulence and efficacy in mice, and was readily distinguished from Rev1 and other Brucella field strains by direct visualization under ultraviolet illumination, fluorescence microscopy and a multiplex PCR‐GFP. The Rev1::gfp strain did not elicit anti‐GFP antibodies itself in lambs but when applied in combination with recombinant GFP induced an intense and long‐lasting (>9 months) anti‐GFP serological response readily detectable by the ELISA‐GFP. Overall, our results confirm that Rev1 GFP‐tagging can be a suitable alternative for identifying vaccinated sheep in infected contexts.     

Discrepant serological assays for Pneumococcus in renal transplant recipients – a prospective study

Vaccine immunoprotection for Streptococcus pneumoniae is mediated by opsonizing antibodies targeting serotype‐specific capsular polysaccharides. Quantitative antibody levels enzyme‐linked immunosorbent assay (ELISA) and antibody‐mediated opsonophagocytic assays (OPA) measure vaccine‐induced protection; correlation of these assays in transplantation requires investigation. This study examines the laboratory assessment of antibody titers in vaccinated renal recipients. Streptococcus pneumoniae 19A is common in immunocompromised hosts and is represented in protein‐conjugate vaccines (PCV) and polysaccharide vaccines (PSV). Antibodies to 19A in serial sera from 30 vaccinated renal transplant recipients were compared using ELISA and OPA assays. Subject titers were classified as protected or not by ELISA (>0.35 μg/ml) and OPA titer (>1:8). Antibody titers analyzed using McNemar's test indicate that protection measured by the two assays are not the same (P = 0.0078); simple linear regression of within‐subject geometric means of 19A enzyme‐linked immunosorbent assay (ELISA) antibody levels versus 19A opsonophagocytic assays (OPA) titers demonstrates significant correlation between the two assays (P < 0.001). Vaccination is increasingly important given increasing antimicrobial resistance worldwide. OPA and ELISA antibody assays do not correlate well using current values for protective immunity against the Pneumococcus in immunosuppressed transplant recipients. Future studies of vaccination in transplant recipients should evaluate protective antibody levels using both functional antibody assays and standard ELISA antibody titers. (ClinicalTrials.gov: NCT00307125).     

A Review of OIE Country Status Recovery Using Vaccinate‐to‐Live Versus Vaccinate‐to‐Die Foot‐and‐Mouth Disease Response Policies II: Waiting Periods After Emergency Vaccination in FMD Free Countries

For countries with OIE status, FMD free country where vaccination is not practised, vaccinate‐to‐live policies have a significant economic disincentive as the trade restriction waiting period is double that of vaccinate‐to‐die policies. The disposal of healthy vaccinated animals strictly for the purpose of regaining markets with debatable scientific justification is a global concern. The feasibility of aligning the waiting periods to facilitate vaccinate‐to‐live is explored. The first article of this two‐part review (Barnett et al., 2015) explored the qualities of higher potency Foot‐and‐Mouth Disease (FMD) vaccines, performance of differentiating infected from vaccinated animals (DIVA) diagnostic assays particularly in vaccinates and carriers, as well as aspects of current limitations of post‐outbreak surveillance. Here, the history behind the OIE waiting periods for FMD free status is reviewed as well as whether the risk of vaccinated animals and their subsequent products differ appreciably at 3 versus 6 months. It is concluded that alignment is feasible for vaccinate‐to‐live using higher potency FMD vaccines within the current OIE waiting period framework of 3 and 6 months blocks of time. These waiting periods reflect precedence, historical practicalities and considered expert opinion rather than a specific scientific rationale. The future lies in updated epidemiological and diagnostic technology to establish an acceptable level of statistical certainty for surveillance or target probability of freedom of FMDV (infection or circulation) not time restricted waiting periods. The OIE Terrestrial Code limits trade from a FMD free country where vaccination is not practiced to animal products and live non‐vaccinated animals. The risk of FMDV in products derived from higher potency vaccinated animals is appreciably less than for countries with infected FMD status or even from a FMD free country where vaccination is practised for which the Code has Articles with guidelines for safe trade with time restrictions of 3 months or less. All these presume that key requirements in the implementation of emergency vaccination including appropriate vaccine match, vaccine application, susceptible population coverage, etc. are addressed.     

Frequent infection of wild boar with atypical porcine pestivirus (APPV)

The recently identified atypical porcine pestivirus (APPV ) was demonstrated to be the causative agent of the neurological disorder “congenital tremor” in newborn piglets. Despite its relevance and wide distribution in domestic pigs, so far nothing is known about the situation in wild boar, representing an important wild animal reservoir for the related classical swine fever virus. In this study, 456 wild boar serum samples obtained from northern Germany were investigated for the presence of APPV genomes and virus‐specific antibodies. Results of real‐time RT ‐PCR analyses revealed a genome detection rate of 19%. Subsequent genetic characterization of APPV (n  = 12) from different hunting areas demonstrated close genetic relationship and, with exception of APPV from one location, displayed less than 3.3% differences in the analysed partial NS 3 encoding region. Furthermore, indirect E^rns ELISA revealed an antibody detection rate of approx. 52%, being in line with the high number of viremic wild boar. Analysis of fifteen wild boar samples from the Republic of Serbia by E^rns antibody ELISA provided evidence that APPV is also abundant in wild boar populations outside Germany. High number of genome and seropositive animals suggest that wild boar may serve as an important virus reservoir for APPV .     

Genetic evolution of classical swine fever virus under immune environments conditioned by genotype 1‐based modified live virus vaccine

Modified live vaccines (MLVs) based on genotype 1 strains, particularly C‐strain, have been used to prevent and control classical swine fever virus (CSFV) worldwide. Nevertheless, a shift in the predominant CSFV strains circulating in the field from genotype 1 or 3 to genotype 2 is seen. Genotype 2 is genetically distant from the vaccine strains and was recently reported during outbreaks after vaccine failure; this has raised concerns that vaccination has influenced viral evolution. In Korea in 2016, there was an unexpected CSF outbreak in a MLV‐vaccinated commercial pig herd. The causative CSFV strain was genetically distinct from previously isolated Korean strains but similar to recent Chinese strains exhibiting enhanced capacity to escape neutralization; this suggests the need for global cooperative research on the evolution of CSFV. We analysed global E2 sequences, using bioinformatics tools, revealing the evolutionary pathways of CSFV. Classical swine fever virus genotypes 1 and 2 experienced different degrees and patterns of evolutionary growth. Whereas genotype 1 stayed relatively conserved over time, the genetic diversity of genotype 2 has progressively expanded, with few fluctuations. It was determined that genotype 2 evolved under lower immune pressures and at a higher evolutionary rate than genotype 1. Further, several selected codons, under diversifying selection in genotype 1 but under purifying selection in genotype 2, correspond to antigenic determinants, which could lead to evasion of vaccine‐induced immunity. Our findings provide evidence that evolutionary changes in CSFV are the result of the disproportionate usage of the CSF MLVs in endemic areas; this underscores the need to develop mitigation strategies to minimize the substantial risk associated with the emergence of vaccine‐escaping mutants.     

Development of a chemiluminescence immunoassay using recombinant non‐structural epitope‐based proteins to accurately differentiate foot‐and‐mouth disease virus‐infected and vaccinated bovines

The contamination of inactivated vaccine with non‐structural proteins (NSP s) leads to a high false‐positive rate, which is a substantial barrier to accurately differentiate foot‐and‐mouth disease virus (FMDV )‐infected animals from vaccinated animals. To address this problem, a new chemiluminescence immunoassay (CLIA ) method was developed to detect antibodies targeting the two recombinant epitope‐based proteins located in 3A and 3B. The 3Aepitp‐3Bepitp CLIA exhibited a diagnostic sensitivity of 94.0% and a diagnostic specificity of 97.5% for the detection of serum samples (naïve bovines, n  = 52, vaccinated bovines, n  = 422, infected bovines, n  = 116) from animals with known status. The CLIA method also had a concordance rate of 88.1% with the PrioCHECK FMDV NSP ELISA based on the detection of 270 serum samples from the field. Importantly, the 3Aepitp‐3Bepitp CLIA produced no false‐positives when used to detect FMDV in samples from bovines that had been vaccinated up to five times, and it was demonstrated a low false‐positive rate when the bovines had been vaccinated up to ten (2.15%) and fifteen times (5.93%). Therefore, the 3Aepitp‐3Bepitp CLIA detects FMDV in samples from frequently vaccinated bovines with high accuracy and represents an alternative method to differentiate FMDV ‐infected and vaccinated bovines.     

Challenges for Serology‐Based Characterization of Foot‐and‐Mouth Disease Outbreaks in Endemic Areas; Identification of Two Separate Lineages of Serotype O FMDV in Uganda in 2011

Control of foot‐and‐mouth disease (FMD) in Uganda by ring vaccination largely depends on costly trivalent vaccines, and use of monovalent vaccines could improve the cost effectiveness. This, however, requires application of highly specific diagnostic tests. This study investigated outbreaks of FMD in seven Ugandan districts, during 2011, using the PrioCHECK^® FMDV NS ELISA, solid‐phase blocking ELISAs (SPBEs) and virus neutralization tests (VNTs), together with virological analyses for characterization of the responsible viruses. Two hundred and eighteen (218) cattle and 23 goat sera as well as 82 oropharyngeal fluid/epithelial tissue samples were collected. Some 50% of the cattle and 17% of the goat sera were positive by the PrioCHECK^® FMDV NS ELISA, while SPBEs identified titres ≥80 for antibodies against serotype O FMD virus (FMDV) in 51% of the anti‐NSP positive cattle sera. However, 35% of the anti‐NSP positive cattle sera had SPBE titres ≥80 against multiple serotypes, primarily against serotypes O, SAT 1 and SAT 3. Comparison of SPBEs and VNTs for the detection of antibodies against serotypes O, SAT 1 and SAT 3 in 72 NSP positive cattle sera showed comparable results against serotype O (P = 0.181), while VNTs detected significantly fewer samples positive for antibodies against SAT 1 and SAT 3 than the SPBEs (P < 0.001). Detection of antibodies against serotype O was consistent with the isolation of serotype O FMDVs from 13 samples. Four of these viruses were sequenced and belonged to two distinct lineages within the East Africa‐2 (EA‐2) topotype, each differing from the currently used vaccine strain (EA‐1 topotype). The relationships of these lineages to other serotype O viruses in the Eastern Africa region are discussed. To enhance the control of FMD in Uganda, there is need to improve the specificity of the SAT‐SPBEs, perform vaccine matching and implement improved regional FMD control.     

Diagnostic specificity of the African swine fever virus antibody detection enzyme‐linked immunosorbent assay in feral and domestic pigs in the United States

African swine fever (ASF) is a highly contagious haemorrhagic disease of pigs that has the potential to cause mortality nearing 100% in naïve animals. While an outbreak of ASF in the United States’ pig population (domestic and feral) has never been reported, an introduction of the disease has the potential to cause devastation to the pork industry and food security. During the recovery phase of an outbreak, an antibody detection diagnostic assay would be required to prove freedom of disease within the previously infected zone and eventually nationwide. Animals surviving an ASF infection would be considered carriers and could be identified through the persistence of ASF viral antibodies. These antibodies would demonstrate exposure to the disease and not vaccination, as there is no ASF vaccine available. A well‐established commercial enzyme‐linked immunosorbent assay (ELISA) detects antibodies against ASF virus (ASFV), but the diagnostic specificity of the assay had not been determined using serum samples from the pig population of the United States. This study describes an evaluation of the World Organization for Animal Health (OIE)‐recommended Ingezim PPA COMPAC ELISA using a comprehensive cohort (n = 1791) of samples collected in the United States. The diagnostic specificity of the assay was determined to be 99.4% (95% confidence interval (CI): [98.9, 99.7]). The result of this study fills a gap in understanding the performance of the Ingezim PPA COMPAC ELISA in the ASF naïve pig population of the United States.     

Pretransplant human leukocyte antigen antibodies detected by single‐antigen bead assay are a risk factor for long‐term kidney graft loss even in the absence of donor‐specific antibodies

Clinical relevance of ELISA‐ and single‐antigen bead assay (SAB)‐detected pretransplant HLA antibodies (SAB‐HLA‐Ab) for kidney graft survival was evaluated retrospectively in 197 patients transplanted between 2002 and 2009 at the University Clinic Frankfurt. Having adjusted for retransplantation and delayed graft function, a significantly increased risk for death‐censored graft loss was found in patients with pretransplant SAB‐HLA‐Ab [HR: 4.46; 95% confidence interval (CI): 1.47–13.48; P = 0.008]. The risk for increased graft loss was also significant in patients with pretransplant SAB‐HLA‐Ab but without SAB‐detected donor‐specific Ab (SAB‐DSA) (HR: 4.91; 95% CI of 1.43–16.991; P = 0.012). ELISA was not sufficient to identify pretransplant immunized patients with an increased risk for graft loss. In immunized patients, graft loss was predominantly present in patients who received transplants with a mismatch on the HLA‐DR locus. In conclusion, even if our study is limited due to small sample size, the results show an increased risk for long‐term graft loss in patients with pretransplant SAB‐HLA, even in the absence of DSA. SAB‐HLA‐Ab‐positive patients, being negative in ELISA or CDC assay, might profit from a well‐HLA‐DR‐matched graft and intensified immunosuppression.     

Development of a Microsphere‐based Immunoassay for Serological Detection of African Horse Sickness Virus and Comparison with Other Diagnostic Techniques

African horse sickness (AHS) is a viral disease that causes high morbidity and mortality rates in susceptible Equidae and therefore significant economic losses. More rapid, sensitive and specific assays are required by diagnostic laboratories to support effective surveillance programmes. A novel microsphere‐based immunoassay (Luminex assay) in which beads are coated with recombinant AHS virus (AHSV) structural protein 7 (VP7) has been developed for serological detection of antibodies against VP7 of any AHSV serotype. The performance of this assay was compared with that of a commercial enzyme‐linked immunosorbent assay (ELISA) and commercial lateral flow assay (LFA) on a large panel of serum samples from uninfected horses (n = 92), from a reference library of all AHSV serotypes (n = 9), on samples from horses experimentally infected with AHSV (n = 114), and on samples from West African horses suspected of having AHS (n = 85). The Luminex assay gave the same negative results as ELISA when used to test the samples from uninfected horses. Both assays detected antibodies to all nine AHSV serotypes. In contrast, the Luminex assay detected a higher rate of anti‐VP7 positivity in the West African field samples than did ELISA or LFA. The Luminex assay detected anti‐VP7 positivity in experimentally infected horses at 7 days post‐infection, compared to 13 days for ELISA. This novel immunoassay provides a platform for developing multiplex assays, in which the presence of antibodies against multiple ASHV antigens can be detected simultaneously. This would be useful for serotyping or for differentiating infected from vaccinated animals.     

Posttransplant De Novo Donor‐Specific HLA Antibodies Identify Pediatric Kidney Recipients at Risk for Late Antibody‐Mediated Rejection

The emerging role of humoral immunity in the pathogenesis of chronic allograft damage has prompted research aimed at assessing the role of anti‐HLA antibody (Ab) monitoring as a tool to predict allograft outcome. Data on the natural history of allografts in children developing de novo Ab after transplantation are limited. Utilizing sera collected pretransplant, and serially posttransplant, we retrospectively evaluated 82 consecutive primary pediatric kidney recipients, without pretransplant donor‐specific antibodies (DSA), for de novo Ab occurrence, and compared results with clinical–pathologic data. At 4.3‐year follow up, 19 patients (23%) developed de novo DSA whereas 24 had de novo non‐DSA (NDSA, 29%). DSA appeared at a median time of 24 months after transplantation and were mostly directed to HLA‐DQ antigens. Among the 82 patients, eight developed late/chronic active C4d+ antibody‐mediated rejection (AMR), and four C4d‐negative AMR. Late AMR correlated with DSA (p < 0.01), whose development preceded AMR by 1‐year median time. Patients with DSA had a median serum creatinine of 1.44 mg/dL at follow up, significantly higher than NDSA and Ab‐negative patients (p < 0.005). In our pediatric cohort, DSA identify patients at risk of renal dysfunction, AMR and graft loss; treatment started at Ab emergence might prevent AMR occurrence and/or progression to graft failure.     

Low CD4/CD8 ratio in classical swine fever postnatal persistent infection generated at 3 weeks after birth

Classical swine fever virus (CSFV) is one of the most important pathogens affecting swine. After infection with a moderate virulence strain at 8 hours after birth, CSFV is able to induce viral persistence. These animals may appear clinically healthy or showed unspecific clinical signs despite the permanent viremia and high viral shedding, in absence of immune response to the virus. Given the role played by this infection in disease control, we aimed to evaluate the capacity of CSFV to induce postnatal persistent infection at 3 weeks after birth. Nine pigs were CSFV infected and sampled weekly during 6 weeks and viral, clinical, pathological and immunological tests were carried out. Also, the CD4/CD8 ratio was calculated with the purpose to relate this marker with the CSFV persistent infection. The IFN‐α response was detected mainly 1 week after infection, being similar in all the infected animals. However, 44.4% of animals were CSFV persistently infected, 33.3% died and 22.2% developed specific antibody response. Interestingly, in persistently infected pigs, the T‐CD8 population was increased, the T‐CD4 subset was decreased and lower CD4/CD8 ratios were detected. This is the first report of CSFV capacity to confer postnatal persistent infection in pigs infected at 3 weeks after birth, an age in which the weaning could be carried out in some swine production systems. This type of infected animals shed high amounts of virus and are difficult to evaluate from the clinical and anatomopathological point of view. Therefore, the detection of this type of infection and its elimination in endemic areas will be relevant for global CSF eradication. Finally, the low CD4/CD8 ratios found in persistently infected animals may be implicated in maintaining high CSFV replication during persistence and further studies will be performed to decipher the role of these cells in CSFV immunopathogenesis.     

Distinction Between Persistent and Transient Infection in a Bovine Viral Diarrhoea (BVD) Control Programme: Appropriate Interpretation of Real‐Time RT‐PCR and Antigen‐ELISA Test Results

Control of bovine viral diarrhoea (BVD) in Belgium is currently implemented on a voluntary basis at herd level and mainly relies on detection and culling of persistently infected (PI) animals. The present field study was conducted during the winter of 2010/2011 to assess the performances of diagnostic assays used in the testing scheme for BVD as proposed by the two Belgian regional laboratories. Individual blood samples were collected from 4972 animals, and individual samples from the same herd were pooled (maximum of 30 individual samples per pool) and screened for the presence of Bovine Viral Diarrhoea Virus (BVDV)‐specific RNA using a commercial real‐time RT‐PCR test (ADIAGENE). Individual samples from positive pools were then tested in parallel with the same RT‐PCR test and with an antigen‐capture ELISA test (IDEXX) to detect viremic animals. This study demonstrated that individual results differed according to the type of assay used (P < 0.001): 140 animals (2.8%) were positive by RT‐PCR and 72 (1.4%) by antigen‐ELISA. A second blood sample was taken 40 days later from 74 PCR positive animals to detect persistent viremia: 17 (23%) of these were still PCR positive and considered to be PI and the 57 that no longer tested positive were assumed to be transiently infected (TI) animals. All PI animals were positive also by antigen‐ELISA at both time points. Among TI animals, 10 (16%) were positive by antigen‐ELISA at the first but none at the second sampling. A highly significant difference in cycle threshold (C_t) values obtained by RT‐PCR was observed between PI and TI animals. ROC analysis was performed to establish thresholds to confirm with high probability that an animal is PI, based on the result of RT‐PCR test performed on a single individual blood sample.