The prevalence of PCV2 viremia in newborn piglets on four endemically infected Dutch sow farms is very low.

Porcine circovirus type 2 (PCV2) systemic disease is currently considered one of the most relevant infectious diseases in swine industry worldwide from an economical point of view. Although piglets generally become diseased between 8 and 16 weeks of age, they can be infected much earlier, even already in utero. However, data on the prevalence of PCV2 infection in newborn piglets are very variable (lower than 40 up to 82%) and most of the studies have been performed in US. In European pig farms, using group-housing systems for gestating sows, a different herd PCV2 infection and immunological status may be expected and was recently reported in Germany. If that is the current scenario in most European farms, strategies to prevent horizontal transmission become essential for the control of the infection. The aim of our study was to determine the PCV2 prevalence in newborn piglets on 4 endemically infected farms in the Netherlands under European conditions. Eleven sows and 8 piglets per litter from 4 farms selected by their assumed PCV2 endemic infection status were sampled. Plasma from piglets was analysed with a PCV2 qPCR and serum from the sows was analysed with a commercial circovirus IgG ELSIA, circovirus IgM ELISA and PCV2 qPCR. In none of the samples from the piglets PCV2 was detected by the qPCR. None of the samples from the sows tested positive in the qPCR and circovirus IgM ELISA. The true- and apparent prevalence of IgG at herd and sow level were 0.75 and 0.81 and, 0.30 and 0.32, respectively, and no statistically significant association with sow parity was observed. These results reveal a very low prevalence of PCV2 in newborn piglets on endemically infected farms in The Netherlands, opening the opportunity of re-evaluation of the control measures applied in these farms.

A Serological Protein Microarray for Detection of Multiple Cross-Reactive Flavivirus Infections in Horses for Veterinary and Public Health Surveillance.

The genus Flavivirus in the family Flaviviridae includes some of the most important examples of emerging zoonotic arboviruses that are rapidly spreading across the globe. Japanese encephalitis virus (JEV), West Nile virus (WNV), St. Louis encephalitis virus (SLEV) and Usutu virus (USUV) are mosquito-borne members of the JEV serological group. Although most infections in humans are asymptomatic or present with mild flu-like symptoms, clinical manifestations of JEV, WNV, SLEV, USUV and tick-borne encephalitis virus (TBEV) can include severe neurological disease and death. In horses, infection with WNV and JEV can lead to severe neurological disease and death, while USUV, SLEV and TBEV infections are mainly asymptomatic, however, and induce antibody responses. Horses often serve as sentinels to monitor active virus circulation in serological surveillance programmes specifically for WNV, USUV and JEV. Here, we developed and validated a NS1-antigen protein microarray for the serological differential diagnosis of flavivirus infections in horses using sera of experimentally and naturally infected symptomatic as well as asymptomatic horses. Using samples from experimentally infected horses, an IgG and IgM specificity of 100% and a sensitivity of 95% for WNV and 100% for JEV was achieved with a cut-off titre of 1 : 20 based on ROC calculation. In field settings, the microarray identified 93-100% of IgG-positive horses with recent WNV infections and 87% of TBEV IgG-positive horses. WNV IgM sensitivity was 80%. Differentiation between closely related flaviviruses by the NS1-antigen protein microarray is possible, even though we identified some instances of cross-reactivity among antibodies. However, the assay is not able to differentiate between naturally infected horses and animals vaccinated with an inactivated WNV whole-virus vaccine. We showed that the NS1-microarray can potentially be used for diagnosing and distinguishing flavivirus infections in horses and for public health purposes within a surveillance setting. This allows for fast, cheap, syndrome-based laboratory testing for multiple viruses simultaneously for veterinary and public health purposes.

Coxiella burnetii in bulk tank milk samples from dairy goat and dairy sheep farms in The Netherlands in 2008.

In 2007, a human Q fever epidemic started, mainly in the south eastern part of The Netherlands with a suspected indirect relation to dairy goats, and, to a lesser degree, to dairy sheep. This article describes the Q fever prevalences in Dutch dairy goat and dairy sheep bulk tank milk (BTM) samples, using a real-time (RT) PCR and ELISA. Results of BTM PCR and ELISA were compared with the serological status of individual animals, and correlations with a history of Q fever abortion were determined. When compared with ELISA results, the optimal cut-off value for the RT-PCR was 100 bacteria/ml. In 2008, there were 392 farms with more than 200 dairy goats, of which 292 submitted a BTM sample. Of these samples, 96 (32.9 per cent) were PCR positive and 87 (29.8 per cent) were ELISA positive. All farms with a history of Q fever abortion (n=17) were ELISA positive, 16 out of 17 were also PCR positive. BTM PCR or ELISA positive farms had significantly higher within-herd seroprevalences than BTM negative farms. In the south eastern provinces, the area where the human Q fever outbreak started in 2007, a significantly larger proportion of the BTM samples was PCR and ELISA positive compared to the rest of The Netherlands. None of the BTM samples from dairy sheep farms (n=16) were PCR positive but three of these farms were ELISA positive. The higher percentage of BTM positive farms in the area where the human Q fever outbreak started, supports the suspected relation between human cases and infected dairy goat farms.

[Infectious bovine keratoconjunctivitis (IBK) in cows, clinical and lab review at four farms]. / Infectieuze keratoconjunctivitis bij runderen, overzicht aan de hand van kliniek- en laboratoriumonderzoek op vier bedrijven.

After several reports to the GD (Dutch Animal Health Service) from practitioners in The Netherlands concerning serious Infectious Bovine Keratoconjunctivitis (IBK) in dairy herds during summer and autumn 2003, the GD has carried out a pilot-study to determine the most responsible agent. This pilot was thought to be important because of the painfulness of the illness and problems like, (for the farmer) an intensive and difficult therapy. Also the report of a Chlamydophilae infection causing IBK in a dairy herd in the UK prompted to this study. The most frequently isolated infectious agent in our study was Moraxella, probably M. bovis. For the presence of Chlamydophila, mycoplasmata or BHV1 viruses were no indications.

The results of using faecal culture as confirmation test of paratuberculosis-seropositive dairy cattle.

A total of 15,822 cattle aged 3 years and older, belonging to 378 randomly selected herds, were tested for paratuberculosis using an absorbed enzyme-linked immunosorbent assay (ELISA); 3.3% tested positive. This percentage was lowest for the group of cattle aged 3-4 years (2.3%) and highest for cattle with the age of 5-6 years (4.5%). The mean Sample to Positive (S/P) ratio of seropositive cattle vaccinated against paratuberculosis was higher (0.75 +/- 0.33) than that of seropositive, non-vaccinated cattle (0.58 +/- 0.26). Faecal samples of 422 ELISA-positive cattle were cultured for the presence of Mycobacterium avium subsp. paratuberculosis, 12% of these were contaminated. The percentage of non-contaminated samples with positive culture results was 17.3%, with a substantial difference between vaccinated (1.7%) and non-vaccinated cattle (20.2%). Of the positive cultures, the number of colonies varied from 1-10 (22% of cultures), 11-100 (22%), to more than 100 (55%). The percentage of ELISA-positive, non-vaccinated cattle tested culture-positive was positively correlated with the magnitude of the S/P ratio. This percentage varied from 12% (S/P ratio 0.3-0.5) to 58% (S/P ratio > 1.1), a result that might have implications for interpretation of the test. In this study, the percentage of ELISA-positive cattle with positive faecal culture results was limited and these individuals were mostly moderate to heavy shedders.

Prevalence and regional distribution of paratuberculosis in dairy herds in The Netherlands.

In the Netherlands a survey was conducted to estimate the prevalence of paratuberculosis in dairy herds. In total 15822 cows of at least 3 years of age, belonging to 378 herds were tested using an absorbed ELISA. Of these herds, 55% (n=207) had one or more serologically positive cows. Of the positive non-vaccinated herds, most had one (n=98) or two (n=49) positive cows. The percentage positive cows per herd was 2.5+/-3.2%.The true prevalences on cow and herd levels, based on a test sensitivity that ranged from 0.3 to 0.4 and a specificity that ranged from 0.985 and 0.995, were estimated at 2. 7-6.9% and 31-71%. Seven herds had been vaccinated against paratuberculosis and these herds had a significantly higher percentage of serologically positive cows (23%) than the non-vaccinated herds (2.5%). In conclusion, a small percentage of the dairy cows and a high percentage of the dairy herds in the Netherlands is serologically positive. The percentages true infected cows and herds are difficult to estimate precisely due to uncertainties in test sensitivity and specificity.

A comparison of four serological tests for the diagnosis of caseous lymphadenitis in sheep and goats.

A double antibody sandwich ELISA (ELISA A) developed for the detection of Corynebacterium pseudotuberculosis infection in sheep and goats was modified to improve its sensitivity. To establish the sensitivity and specificity of this modified ELISA (ELISA B), sera from 183 sheep and 186 goats were tested using ELISAs A and B. Comparison was also made with two further ELISAs (C and D) developed in Australia that, respectively, detect antibodies to cell wall antigens or toxin.ELISA B had the best performance of the four tests. Its specificity was 98+/-1% for goats and 99+/-1% sheep. Its sensitivity was 94+/-3% for goats and 79+/-5% for sheep. ELISA B will now be tested for use in caseous lymphadenitis eradication and control programmes in The Netherlands. It will also be used in experimental studies of CL in Scotland.

Proliferative lymphocyte responses to foot-and-mouth disease virus and three FMDV peptides after vaccination or immunization with these peptides in cattle.

We studied proliferative responses of bovine T lymphocytes to foot-and-mouth disease virus (FMDV) serotypes A, O and C as well as to three peptides including the two major B-cell epitopes of FMDV (VP1[141-156] and VP1[200-213]). Peripheral blood mononuclear cells (PBMC) from cattle previously vaccinated with monovalent vaccine responded to both homotypic and heterotypic virus strains. Of 14 FMDV-specific bovine T-cell clones, which were prepared from PBMC of an animal vaccinated with the trivalent vaccine, 11 reacted to each of the three serotypes A, O and C. This indicates that several T-cell epitopes might be conserved among these serotypes. PBMC from one of two cattle immunized with VP1[141-156]KLH, one of two cattle immunized with VP1[200-213]KLH and two of three cattle immunized with CC-VP1[200-213]-PPS-VP1[141-156]-PCG responded to the homotypic virus strain. After immunizations with VP1[200-213]KLH also heterotypic responses were found. Thus, it appears that these two B-cell sites include T-cell determinants that are recognized by some cattle. However, when proliferative responses of PBMC from an animal vaccinated with the trivalent vaccine were tested, no responses were found to VP1[141-156] and VP1[200-213], whereas the response was very poor to CC-VP1[200-213]-PPS-VP1[141-156]-PCG. These results suggest that these sequences do not represent dominant T-cell epitopes and/or that T-cell reactivity towards these synthetic peptides does not completely cover the T-cell reactivity towards the fragments present after processing of the whole virus.

Protection of guinea-pigs against foot-and-mouth disease virus by immunization with a PhoE-FMDV hybrid protein.

A hybrid protein was constructed containing two antigenic determinants of the structural protein VP1 of foot-and-mouth disease virus, inserted in a cell surface-exposed region of Escherichia coli outer membrane protein PhoE. Immunization of guinea-pigs with partially purified protein resulted in high levels of neutralizing antibodies and complete protection against challenge with the virus.