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Historically, the MISTEACHING (microbiome, immunity, sex, temperature, environment, age, chance, history, inoculum, nutrition, genetics) framework to describe the outcome of host−pathogen interaction, has been applied to human pathogens. Here, we show, using Actinobacillus pleuropneumoniae as an exemplar, that the MISTEACHING framework can be applied to a strict veterinary pathogen, enabling the identification of major research gaps, the formulation of hypotheses whose study will lead to a greater understanding of pathogenic mechanisms, and/or improved prevention/therapeutic measures. We also suggest that the MISTEACHING framework should be extended with the inclusion of a ‘strain’ category, to become MISTEACHINGS. We conclude that the MISTEACHINGS framework can be applied to veterinary pathogens, whether they be bacteria, fungi, viruses, or parasites, and hope to stimulate others to use it to identify research gaps and to formulate hypotheses worthy of study with their own pathogens.

A long-term research programme has been underway in Ireland to evaluate the usefulness of badger vaccination as part of the national bTB (bovine tuberculosis) control strategy. This culminated in a field trial which commenced in county Kilkenny in 2009 to determine the effects of badger vaccination on Mycobacterium bovis transmission in badgers under field conditions. In the present study, we sought to optimise the characteristics of a multiplex chemiluminescent assay for detection of M. bovis infection in live badgers. Our goal was to maximise specificity, and therefore statistical power, during evaluation of the badger vaccine trial data. In addition, we also aimed to explore the effects of vaccination on test characteristics. For the test optimisation, we ran a stepwise logistic regression with analytical weights on the converted Relative Light Units (RLU) obtained from testing blood samples from 215 badgers captured as part of culling operations by the national Department of Agriculture, Food and the Marine (DAFM). The optimised test was applied to two other datasets obtained from two captive badger studies (Study 1 and Study 2), and the sensitivity and specificity of the test was attained separately for vaccinated and non-vaccinated badgers. During optimisation, test sensitivity was maximised (30.77%), while retaining specificity at 99.99%. When the optimised test was then applied to the captive badger studies data, we observed that test characteristics did not vary greatly between vaccinated and non-vaccinated badgers. However, a different time lag between infection and a positive test result was observed in vaccinated and non-vaccinated badgers. We propose that the optimized multiplex immunoassay be used to analyse the vaccine trial data. In relation to the difference in the time lag observed for vaccinated and non-vaccinated badgers, we also present a strategy to enable the test to be used during trial evaluation.

The aim of this study was to estimate the power, using simulation techniques, of a group randomized vaccine field trial designed to assess the effect of vaccination on Mycobacterium bovis transmission in badgers. The effects of sample size (recapture percentage), initial prevalence, sensitivity and specificity of the diagnostic test, transmission rate between unvaccinated badgers, Vaccine Efficacy for Susceptibility (VES) and Vaccine Efficacy for Infectiousness (VEI), on study power were determined. Sample size had a small effect on power. Study power increased with increasing transmission rate between non-vaccinated badgers. Changes in VES had a higher impact on power than changes in VEI. However, the largest effect on study power was associated with changes in the specificity of the diagnostic test, within the range of input values that were used for all other modelled parameters. Specificity values below 99.4% yielded a study power below 50% even when sensitivity was 100% and, VEI and VES were both equal to 80%. The effect of changes in sensitivity on study power was much lower. The results from our study are in line with previous studies, as study power was dependent not only on sample size but on many other variables. In this study, additional variables were studied, i.e. test sensitivity and specificity. In the current vaccine trial, power was highly dependent on the specificity of the diagnostic test. Therefore, it is critical that the diagnostic test used in the badger vaccine trial is optimized to maximize test specificity.

Decomposition products of ingested garlic are to a certain extent excreted via the lungs. If the supposed health-supporting capacities associated with garlic extend to these exhaled sulfurous compounds, they could have an effect on the course of pneumonia. In this study, the garlic-derived volatile allyl methyl sulfide (AMS) as a lead compound of volatile garlic metabolites was shown to exhibit an antibacterial effect against the pig pathogen Actinobacillus pleuropneumoniae serotype 9. AMS caused a delay in the appearance of the optical density-monitored growth of A. pleuropneumoniae in medium when compared to unaffected growth curves, yet without lowering the stationary phase yield at the concentration range tested. At 1.1mM, AMS impaired the in vitro growth rate of A. pleuropneumoniae serotype 9 by 8% compared to unimpeded growth. In an animal trial, a garlic-fed group of 15 pigs that received a diet with 5% garlic feed component and a control group of 15 pigs that received a diet without garlic were infected with A. pleuropneumoniae serotype 2 via an aerosol and subsequently followed for 4 days. At the day of the challenge, blood AMS in the garlic-fed group amounted to 0.32 ± 0.13 μM. A beneficial, alleviating effect of garlic on the course and severity of an A. pleuropneumoniae infection in pigs was indicated by the reduced occurrence of characteristic pleuropneumonia lesions (27% of the lungs affected in the garlic-fed group vs. 47% in the control group) and a near to significant (p=0.06) lower relative lung weight post mortem in the garlic-fed group.

Most serotypes of A. pleuropneumoniae produce more than one toxin in vivo. To determine the value of the production of more than one toxin in the development of disease, we tested the pathogenicity of isogenic strains of A. pleuropneumoniae serotype 1 that are mutated in the toxin genes apxIA and/or apxIIA or in the transport genes apxIBD. Bacteria mutated in both apxIA and apxIIA, or in apxIBD, were unable to induce pathological lesions, thereby confirming the conclusion that ApxI and ApxII are essential for the pathogenesis of pleuropneumonia. Infection with isogenic strains lacking either ApxI or ApxII did not consistently lead to pleuropneumonia unlike the parent strain S4074. ApxII seemed at least as important as ApxI for the development of clinical and pathological symptoms. Only one of the four pigs inoculated with a mutant strain unable to produce ApxII developed mild pneumonia whereas two out of the three pigs inoculated with a mutant strain unable to produce ApxI developed more severe lesions. The results indicate that both ApxI and ApxII of A. pleuropneumoniae serotype 1 are necessary for full virulence.

This paper describes a methodology to quantify the transmission of Actinobacillus (A.) pleuropneumoniae from subclinically infected carrier pigs to susceptible contact pigs, and to test the effect of possible interventions on the transmission. The methodology includes the design of a transmission experiment, and a method with which A. pleuropneumoniae transmission can be quantified and with which the effect of an intervention on the transmission can be tested. The experimental design consists of two parts. First, subclinically infected carrier pigs are created by contact exposure of specific-pathogen-free pigs to endobronchially inoculated pigs. Second, transmission is observed from the group of carrier pigs to a second group of susceptible contact pigs after replacing the inoculated pigs by new contact pigs. The presented analytical method is a generalised linear model (GLM) with which the effect of an intervention on the susceptibility and infectivity can be tested separately, if the transmission is observed in heterogeneous populations. The concept of the experimental transmission model is illustrated by describing an A. pleuropneumoniae transmission experiment in which the effect of vaccination on the susceptibility is quantified. Although it could not be demonstrated that vaccination has an effect on the susceptibility of pigs, it was demonstrated that nasal excretion of A. pleuropneumoniae is related to the infectivity of pigs. (C) 2003 Elsevier Science B.V. All rights reserved.