Bertel Strandbygaard

Appearance of acute PRRS-like symptoms in sow herds after vaccination with a modified live PRRS vaccine

found in the dorsal root ganglia, the ventral horns of the spinal cord or the affected brainstem nuclei. As with other primary dysautonomias, the cause of the dysautonomia in the presented case was not determined. Despite extensive clinical, epizootiological and morphological investigations, mainly on feline and equine dysautonomia, the aetiology of any of these disorders is unknown. A neurotoxic agent is commonly suspected (Pollin and Griffiths 1992).

Full protection in mink against mink enteritis virus with new generation canine parvovirus vaccines based on synthetic peptide or recombinant protein

Two recently developed vaccine--one based on synthetic peptide and one based on recombinant capsid protein--fully protected dogs against heavy experimental canine parvovirus (CPV) infection. The high sequence homology ( > 98%) and antigenic similarity between CPV and mink enteritis virus (MEV), feline panleukopenia virus, and raccoon parvovirus, suggest that both vaccines could protect mink, cats and raccoons against these respective host range variants. This was tested in mink and turned out to be the case. The two vaccines were fully protective and as effective as a conventional commercial vaccine based on inactivated virus. Surprisingly, this protection was obtained after only a single injection. Furthermore, the vaccinal dose of 150 micrograms of conjugated peptide or 3 micrograms of recombinant VP2 particles per animal, are sufficiently low to be cost-effective and applicable on a large scale.

Evaluation of a blocking Elisa for screening of antibodies against porcine reproductive and respiratory syndrome (PRRS) virus

A blocking Elisa was developed for the detection of antibodies against PRRS virus with a view to satisfying the need for examination of blood samples on a large scale. The test was evaluated in comparison with an indirect Elisa and the immunoperoxidase monolayer assay. The blocking Elisa was sensitive and specific. It had a higher capacity and was cheaper to perform than the immunoperoxidase monolayer assay and the indirect Elisa. It was comparable to the immunoperoxidase monolayer assay and better than the indirect Elisa in detecting antibodies formed early after infection, and it was superior to both the immunoperoxidase monolayer assay and the indirect Elisa in detecting antibodies at a late stage of infection.

Blocking ELISA's for the distinction between antibodies against European and American strains of porcine reproductive and respiratory syndrome virus.

A double blocking ELISA was developed in order to satisfy the need for large scale serological screening for PRRS and simultaneous distinction between infection with European and American strains of PRRSV in pig herds. The Immunoperoxidase monolayer assay (IPMA) and the double blocking ELISA enabled distinction on serological basis between infection with European and American strains of PRRSV. The distinction was possible from about day 7 after infection of pigs with PRRSV. The double blocking ELISA enabled the distinction at later stages of infection compared to the IPMA, irrespective of the strain involved.

Experimental Infection of Young Pigs with an Early European Strain of Porcine Epidemic Diarrhoea Virus and a Recent US Strain

Summary Outbreaks of porcine epidemic diarrhoea (PED) were reported across Europe during the 1980s and 1990s, but only sporadic outbreaks occurred in recent years. PED virus (PEDV) spread for the first time into the USA in 2013 and has caused severe economic losses. Retrospectively, it was found that two different strains of PEDV have been introduced into the United States, both are closely related to strains circulating in China where a new wave of the disease occurred from 2010 onwards. Since autumn 2014, new outbreaks of PED have occurred in Europe. In this study, weaned piglets were inoculated with an early European isolate (Br1/87) or faecal/intestinal suspensions derived from pigs infected with a recent European strain of PEDV (from Germany) or a US strain of PEDV. No evidence for infection resulted from inoculation of pigs with the German sample that contained high levels of PEDV RNA; there were no clinical signs, excretion of viral RNA or anti‐PEDV antibody production. In contrast, all the pigs in the other two groups showed evidence of infection. Mild clinical signs of disease, mainly diarrhoea, occurred in piglets inoculated with the Br1/87 and US PEDV strains. PEDV RNA was detected throughout the intestine in euthanized animals at 4 days post‐inoculation. In addition, within these animals, low levels of viral RNA were detected in lungs and livers with higher levels in spleens. Seroconversion against PEDV occurred in all surviving infected animals within 10 days. PEDV RNA excretion occurred for at least 2 weeks. The US PEDV RNA was detected at low levels in serum samples on multiple days. It is apparent that current diagnostic systems can detect infection by the different virus strains.

Characterization of a Novel Chimeric Swine Enteric Coronavirus from Diseased Pigs in Central Eastern Europe in 2016

Summary During a severe outbreak of diarrhoea and vomiting in a pig herd in Central Eastern Europe, faecal samples were tested positive for porcine epidemic diarrhoea virus (PEDV) and negative for transmissible gastroenteritis virus (TGEV) using a commercial RT‐qPCR assay that can detect both of these coronaviruses. However, further analyses, using other TGEV‐ and PEDV‐specific RT‐qPCR assays, provided results inconsistent with infection by either of these viruses. Sequencing of an amplicon (ca. 1.6 kb), generated by an RT‐PCR specific for the PEDV S‐gene, indicated a very close similarity (ca. 99% identity) to recently described chimeric viruses termed swine enteric coronaviruses (SeCoVs). These viruses (with an RNA genome of ca. 28 kb) were first identified in Italy in samples from 2009 but have not been detected there since 2012. A closely related virus was detected in archived samples in Germany from 2012, but has not been detected subsequently. Building on the initial sequence data, further amplicons were generated and over 9 kb of sequence corresponding to the 3′‐terminus of the new SeCoV genome was determined. Sequence comparisons showed that the three known SeCoVs are ≥98% identical across this region and contain the S‐gene and 3a sequences from PEDV within a backbone of TGEV, but the viruses are clearly distinct from each other. It is demonstrated, for the first time, that pigs from within the SeCoV‐infected herd seroconverted against PEDV but tested negative in a TGEV‐specific ELISA that detects antibodies against the S protein. These results indicate that SeCoV is continuing to circulate in Europe and suggest it can cause a disease that is very similar to PED. Specific detection of the chimeric SeCoVs either requires development of a new diagnostic RT‐qPCR assay or the combined use of assays targeting the PEDV S‐gene and another part of the TGEV genome.

First international collaborative study to evaluate rabies antibody detection method for use in monitoring the effectiveness of oral vaccination programmes in fox and raccoon dog in Europe

The most effective and sustainable method to control and eliminate rabies in wildlife is the oral rabies vaccination (ORV) of target species, namely foxes and raccoon dogs in Europe. According to WHO and OIE, the effectiveness of oral vaccination campaigns should be regularly assessed via disease surveillance and ORV antibody monitoring. Rabies antibodies are generally screened for in field animal cadavers, whose body fluids are often of poor quality. Therefore, the use of alternative methods such as the enzyme-linked immunosorbent assay (ELISA) has been proposed to improve reliability of serological results obtained on wildlife samples. We undertook an international collaborative study to determine if the commercial BioPro ELISA Rabies Ab kit is a reliable and reproducible tool for rabies serological testing. Our results reveal that the overall specificity evaluated on naive samples reached 96.7%, and the coefficients of concordance obtained for fox and raccoon dog samples were 97.2% and 97.5%, respectively. The overall agreement values obtained for the four marketed oral vaccines used in Europe were all equal to or greater than 95%. The coefficients of concordance obtained by laboratories ranged from 87.2% to 100%. The results of this collaborative study show good robustness and reproducibility of the BioPro ELISA Rabies Ab kit.

Bluetongue in Denmark during 2008

Following the first ever case of bluetongue in Denmark during late 2007, further outbreaks were observed in Denmark during 2008, despite vaccination against bluetongue virus (BTV) serotype 8 (BTV-8) in the southern part of the country. In total, 15 separate outbreaks of infection were identified, mostly as a result of clinical suspicions but also because of surveillance of bulk milk samples. These outbreaks led to extensions of the original vaccination zone planned for 2008. Blood samples from clinical suspects were analysed using ELISA and real-time RT-PCR assays for the presence of anti-BTV antibodies and viral RNA, respectively. A newly infected calf from the primary outbreak in 2008 was studied for a period of three months, during which time it seroconverted to BTV, but the presence of viral RNA in its blood was maintained throughout this time. Each outbreak was caused by BTV-8, as determined by a serotype-specific real-time RT-PCR assay. Furthermore, the nucleotide sequence of a portion of segment 2 of the viral RNA (encoding the outer capsid protein VP2) from the samples analysed was identical to the BTV-8 segment 2 that circulated in the Netherlands during 2006.

Inter-laboratory study to characterize the detection of serum antibodies against porcine epidemic diarrhoea virus

Abstract Porcine epidemic diarrhea virus (PEDV) has caused extensive economic losses to pig producers in many countries. It was recently introduced, for the first time, into North America and outbreaks have occurred again in multiple countries within Europe as well. To assess the properties of various diagnostic assays for the detection of PEDV infection, multiple panels of porcine sera have been shared and tested for the presence of antibodies against PEDV in an inter-laboratory ring trial. Different laboratories have used a variety of “in house” ELISAs and also one commercial assay. The sensitivity and specificity of each assay has been estimated using a Bayesian analysis applied to the ring trial results obtained with the different assays in the absence of a gold standard. Although different characteristics were found, it can be concluded that each of the assays used can detect infection of pigs at a herd level by either the early European strains of PEDV or the recently circulating strains (INDEL and non-INDEL). However, not all the assays seem suitable for demonstrating freedom from disease in a country. The results from individual animals, especially when the infection has occurred within an experimental situation, show more variation.

Diagnostic evaluation of assays for detection of antibodies against porcine epidemic diarrhea virus (PEDV) in pigs exposed to different PEDV strains

Abstract Porcine epidemic diarrhea virus (PEDV) has caused economic losses in the Americas, Asia and Europe in recent years. Reliable serological assays are essential for epidemiological studies and vaccine evaluation. The objective of this study was to compare the ability of five enzyme-linked immunosorbent assays (ELISAs) to detect antibodies against different PEDV strains in pig serum. A total of 732 serum samples from North American or European pigs were tested. Samples included experimental samples from pigs infected with classical (G1a PEDV) or variant genogroup 1 PEDV (G1b PEDV), pandemic genogroup 2 PEDV (G2b PEDV) or non-infected controls. Field samples from herds with confirmed or unknown PEDV exposure were also used. Three indirect ELISAs based on G2b antigens (ELISAs 1, 2 and 3), a competitive ELISA based on the G2b antigen (ELISA 4) and a competitive ELISA based on the G1a antigen (ELISA 5) were compared. Overall, the tests had a moderate agreement (κ=0.61). G1a PEDV infected pigs were earliest detected by ELISA 3, G1b PEDV infected pigs were earliest detected by ELISAs 4 and 5 and the performance of all tests was similar for the G2b PEDV group. ELISA 1 showed the overall lowest detection on experimentally and field derived samples. Diagnostic sensitivity and specificity with a 95% probability interval were estimated to be 68.2% (62.1–74.4%) and 97.5% (95.2–99.0%) for ELISA 1, 73.7% (71.5–79.6%) and 98.4% (96.6–99.5%) for ELISA 2, 86.2% (81.1–90.6%) and 91.6% (87.7–94.8%) for ELISA 3, 78.3% (72.8–83.5%) and 99.7% (98.2–100%) for ELISA 4, and 93.5% (90.3–96.0%) and 91.2% (83.8–97.9%) for ELISA 5. Differences in detection among assays seem to be more related to intrinsic factors of an assay than to the PEDV antigen used.