Detection of Actinobacillus pleuropneumoniae in pigs by real-time quantitative PCR for the apxIVA gene.

A real-time quantitative PCR (qPCR) for detection of the apxIVA gene of Actinobacillus pleuropneumoniae was validated using pure cultures of A. pleuropneumoniae and tonsillar and nasal swabs from experimentally inoculated Caesarean-derived/colostrum-deprived piglets and naturally infected conventional pigs. The analytical sensitivity was 5colony forming units/reaction. In comparison with selective bacterial examination using tonsillar samples from inoculated animals, the diagnostic sensitivity of the qPCR was 0.98 and the diagnostic specificity was 1.0. The qPCR showed consistent results in repeatedly sampled conventional pigs. Tonsillar brush samples and apxIVA qPCR analysis may be useful for further epidemiological studies and monitoring for A. pleuropneumoniae.

Implementing a probabilistic definition of freedom from infection to facilitate trade of livestock: putting theory into praxis for the example of bovine herpes virus-1.

International trade of livestock and livestock products poses a significant potential threat for spread of diseases, and importing countries therefore often require that imported animals and products are free from certain pathogens. However, absolute freedom from infection cannot be documented, since all test protocols are imperfect and can lead to false-negative results. It is possible instead to estimate the "probability of freedom from infection" and its opposite, the probability of infection despite having a negative test result. These probabilities can be estimated based on a pre-defined target prevalence, known surveillance efforts in the target population and known test characteristics of any pre-export test. Here, calculations are demonstrated using the example of bovine herpes virus-1 (BoHV-1). In a population that recently became free of BoHV-1 without using vaccination, the probability of being infected of an animal randomly selected for trade is 800 per 1 million and this probability is reduced to 64 (95% probability interval PI 6-161) per 1 million when this animal is tested negatively prior to export with a gB-ELISA. In a population that recently became free of BoHV-1 using vaccination, the probability of being infected of an animal randomly selected for trade is 200 per 1 million, and this probability can be reduced to 63 (95% PI 42-87) when this animal is tested negatively prior to export with a gE-ELISA. Similar estimations can be made on a herd level when assumptions are made about the herd size and the intensity of the surveillance efforts. Subsequently, the overall probability for an importing country of importing at least 1 infected animal can be assessed by taking into account the trade volume. Definition of the acceptable level of risk, including the probability of false-negative results to occur, is part of risk management. Internationally harmonized target prevalence levels for the declaration of freedom from infection from selected pathogens provide a significant contribution to the facilitation of international trade of livestock and livestock products by allowing exporting countries to design tailor-made output-based surveillance programs, while providing equivalent guarantees regarding the probability of freedom from infection of the population. Combining this with an approach to assess the overall probability of introducing at least 1 infected animal into an importing country during a defined time interval will help importing countries to achieve their desired level of acceptable risk and will help to assess the equivalence of animal health and food safety standards between trading partners.

Homologous whole bacterin vaccination is not able to reduce Streptococcus suis serotype 9 strain 7997 transmission among pigs or colonization.

Streptococcus suis (S. suis) is an important porcine pathogen worldwide, and antibiotics are often applied to treat or prevent clinical signs. Vaccination could be an alternative measure to reduce the abundant use of antimicrobials. The aim of this study was to determine the effect of vaccination with homologues whole bacterin vaccine containing S. suis serotype 9 strain 7997 on transmission of this serotype among pigs and on mucosal colonization. Caesarean derived, colostrum deprived pigs (N=50) were housed pair wise. Thirteen pairs were vaccinated intramuscularly with 2-3×10(9) colony forming units (CFU) inactivated S. suis serotype 9 per dose and α-tocopherolactetaat as adjuvant at 3 and 5 weeks of age; twelve pairs served as non-vaccinated controls. At 7 weeks of age, one pig of each pair was intranasally inoculated with 1-2×10(9)CFU of the homologues strain, whereas the other pig of each pair was contact-exposed. Tonsil brushings and saliva swabs were collected for 4 weeks, and tested for the presence of S. suis by bacteriological culture. No differences in number of S. suis in the tonsils or saliva samples or in clinical signs were observed between vaccinated and control pigs. In all pairs, transmission between inoculated and contact exposed pigs occurred, and no difference was observed in rate at which this occurred. The estimated transmission rate parameter ß between vaccinated pigs was ß(v)=5.27/day, and for non-vaccinated pigs ß(nv)=2.77/day (P=0.18). It was concluded that vaccination against S. suis serotype 9 did not reduce transmission, nor colonization and that there were no indications that protection against clinical signs was induced.

Vitamin E supplementation during the dry period in dairy cattle. Part I: adverse effect on incidence of mastitis postpartum in a double-blind randomized field trial.

A randomized, controlled field trial with dairy cows demonstrated an adverse effect of vitamin E supplementation during the dry period on mastitis incidence in early lactation. This study was conducted on farms with historically high rates of mastitis to investigate the benefit of vitamin E supplementation on udder health; however, the outcome showed an adverse effect. The aim of the study was to evaluate whether daily supplementation of 3,000 IU of vitamin E to dairy cows during the dry period could improve udder health in commercial herds with a high incidence of mastitis. On 5 dairy farms, dry cows were randomly divided into 2 experimental groups: a high and a low group. Both groups received a dry cow mineral mix providing 3,000 or 135 IU of vitamin E/cow per day, respectively, between dry-off and calving for a mean period of 8 wk. Providing 3,000 IU of vitamin E exceeds NRC standards, but this amount has been used in previous studies. The experiment, as well as the majority of the statistical analysis, were carried out blinded. Blood was sampled 3 times before calving and on calving day. Serum was analyzed for vitamin E and cholesterol. Vitamin E and the vitamin E:cholesterol ratio were analyzed as dependent variables in mixed models and Student's t-tests to study trends in time and differences between groups. Relative risk calculation and survival analysis were used to study the effect of supplementation on mastitis incidence in the first 3 mo of lactation. The results showed that vitamin E supplements increased both absolute vitamin E and the ratio of vitamin E to cholesterol in blood. In the high group, significantly more subclinical and clinical cases occurred, showing the same trend on all farms. In this study, an initial vitamin E level at dry off above 14.5 µmol/L was a risk factor for clinical mastitis, suggesting that the vitamin E status at the start of the dry period is important. It is recommended to work out exactly at what threshold vitamin E is harmful for udder health before new trials with high dosages of vitamin E are started. Additionally, further research is required to investigate the mechanism by which vitamin E affects udder health.

Establishing the change in antibiotic resistance of Enterococcus faecium strains isolated from Dutch broilers by logistic regression and survival analysis.

In this study, we investigated the change in the resistance of Enterococcus faecium strains isolated from Dutch broilers against erythromycin and virginiamycin in 1998, 1999 and 2001 by logistic regression analysis and survival analysis. The E. faecium strains were isolated from caecal samples that had been randomly collected from six slaughterhouses. Moreover, between the sample collection in 1998 and the sample collection in 1999, virginiamycin and the macrolide antibiotics (of which erythromycin is a member) have been banned in The Netherlands from use in broiler feeds as growth promoter. In the logistic regression analysis we used the internationally accepted cut-off values to determine whether bacteria were resistant or not. In the survival analysis, inhibition of bacterial growth was the event and time to event was replaced by concentration of antibiotic to event. As a consequence, changes in the growth of bacteria can be tested over an entire range of concentrations and no cut-off value for resistance has to be determined. We performed the survival analysis by use of a Cox logistic model with an odds ratio (OR) for the increase of the odds of the basic hazard rate as outcome. Both the logistic regression and the survival analyses showed that resistance to erythromycin and virginiamycin decreased during the study period. In the logistic regression model the ORs associated with the fraction of bacteria inhibited by the antibiotics in 2001 as compared to 1998 were 3.76 (2.57-5.49) for erythromycin and 11.65 (7.68-17.66) for virginiamycin. The corresponding ORs from the survival analysis were lower; 2.88 (2.21-3.76) and 2.11 (1.80-2.49), respectively. The reason for the differences between the ORs of the survival analysis and the logistic regression analysis is probably because most changes in resistance included the cut-off value and logistic regression specifically examines those changes.

Assessment of the quality of tests for the detection of antibodies to Aujeszky's disease virus glycoprotein gE in a target population by the use of receiver operating characteristic curves.

The performance of tests for the detection of antibodies to Aujeszky's disease virus glycoprotein E (gE) in a target population was evaluated by constructing and analysing receiver operating characteristic (ROC) curves. These curves assess the discriminating ability of a test over the entire range of test signals. The advantages of applying the analysis to a sample of the target population (all commercial pigs in the Netherlands), as compared to using a panel of test sera, are that the estimates of sensitivity and specificity, the comparisons between tests and the choices of the cut-off values are all relevant for the target population. The results of a gE-ELISA in colostrum (test A) and in a single droplet of whole blood (test B) were compared with the results obtained with the same ELISA in serum (gold standard). The area under the ROC curve, which is a quantitative measure of test performance, was significantly (P < 0.01) smaller with test A than test B or the gold standard, indicating that test B performed better than test A. No significant difference was observed between test B and the gold standard.

Use of colostrum to detect antibodies against glycoprotein I of Aujeszky's disease virus.

Serum and colostrum samples taken from 499 sows from five herds of pigs endemically infected and vaccinated against Aujeszky's disease virus, were used to investigate whether colostrum could be used to detect antibodies against glycoprotein I (gI) of the virus. Using serum as the reference, the test applied to colostrum had a sensitivity of 97 per cent and a specificity of 88 per cent. When samples were taken from 50 sows from a gI seronegative vaccinated herd, one colostrum sample was gI-positive, giving a specificity for the test of 98 per cent. The mean gI antibody titres in colostrum were about six times higher than in serum. Samples of colostrum were also taken from 132 sows from eight unvaccinated herds free of Aujeszky's disease virus. All these samples were gI-negative, giving a specificity of 100 per cent. Colostrum samples can be stored for at least six weeks at -20 degrees C without compromising the test results, and the repeatability and reproducibility of the test applied to colostrum were good.