Multiple-locus variable-number tandem-repeat analysis is a suitable tool for differentiation of Mycoplasma hyopneumoniae strains without cultivation.

An assay based on multiple-locus variable-number tandem-repeat analysis allowed differentiating and studying diversity and persistence of Mycoplasma hyopneumoniae strains in pig herds without prior cultivation. The test had a discriminatory index of >0.99 and was applied reliably to porcine bronchoalveolar lavage fluid and tracheal swabs.

Effect of vaccination of pigs against experimental infection with high and low virulence Mycoplasma hyopneumoniae strains.

This study investigated the infection pattern and lung lesion development in pigs caused by a low and highly virulent Mycoplasma hyopneumoniae strain at 4 and 8 weeks (w) post infection (PI). It also determined the efficacy of a commercial inactivated whole-cell vaccine against infection with each one of these M. hyopneumoniae strains. Ninety piglets free of M. hyopneumoniae were selected, and 40 of them were randomly vaccinated during their first week of life. At weaning, all piglets were allocated to 10 different groups and housed in pens with absolute filters. At 4 weeks of age, pigs were inoculated intratracheally with either a highly virulent M. hyopneumoniae strain, a low virulent strain or with sterile culture medium. Half of all animals were euthanized at 4 w PI, while the remaining half was euthanized at 8 w PI. Coughing was assessed daily, and lung lesions, immunofluorescence (IF), bacteriological analysis and nested PCR were assessed after necropsy. It was demonstrated that contrary to the highly virulent strain, the low virulent strain required more than 4 weeks PI (commonly accepted as the standard infection model) to reach maximum clinical symptoms. Vaccination significantly reduced clinical symptoms, macroscopic and microscopic lung lesions in pigs infected with the highly virulent strain. This effect was more pronounced at 4 than at 8 weeks PI. Protective efficacy was also observed in pigs infected with the low virulent strain, but the effect was less pronounced than on the highly virulent strain.

The effect of vaccination on the transmission of Mycoplasma hyopneumoniae in pigs under field conditions.

This study investigated the effect of vaccination against Mycoplasma hyopneumoniae on its transmission in nursery pigs under field conditions. Seventy-two pigs were randomly allocated at weaning into vaccinated (V) and non-vaccinated (NV) groups. Animals in the V group were vaccinated at 3 weeks of age with a commercial M. hyopneumoniae bacterin vaccine. Broncho-alveolar lavage fluid taken at weaning and at the end of the nursery period was assessed for the presence of M. hyopneumoniae by nested PCR, and the reproduction ratio of infection (R(n)) was calculated. The percentage of positive pigs in the V and NV groups was 14% and 36% at weaning, and 31% and 64% at the end of the nursery period, respectively. The R(n)-values for the V and NV groups were 0.71 and 0.56, respectively (P>0.05). The study indicates that vaccination does not significantly reduce the transmission of this respiratory pathogen.

Validation of ATP luminometry for rapid and accurate titration of Mycoplasma hyopneumoniae in Friis medium and a comparison with the color changing units assay.

Limited reports are available on the growth response of Mycoplasma hyopneumoniae in Friis medium and the routinely used color changing units (CCU) assay has not yet been profoundly compared with other titration methods. Firstly, growth kinetics of 7 diverse M. hyopneumoniae isolates were followed by ATP luminometry in five Friis medium batches. Secondly, results of the CCU and ATP assays were compared hereby evaluating the methods. Growth curves of all isolates had log, stationary and senescence phases, and reached similar maximal titres when cultured in the same batch of Friis medium. Doubling times (Tds) of the isolates grown in slowly shaken cultures varied between 4.8 and 7.8 h. Maximal titres, Tds, growth phase in which the phenol red indicator turned from red to yellow due to acidification by mycoplasmal metabolism, and the length of the stationary phase varied depending on the Friis medium batch. The effect of static vs. shaking culture conditions on the Td depended on the isolate. ATP and CCU assays obtained similar growth curves, but when maximal levels were reached the CCU titre dropped earlier than the ATP titre. During log phase, CCU and ATP titres were strongly linearly linked. We developed a model enabling transformation of ATP into CCU titres or vice versa. The calculated amount of ATP per CCU (1.77 amol ATP/ml) indicated that the CCU assay likely underestimates the actual cell concentration. When titres were determined as means of 3 measurements, the ATP assay was 7 times more accurate and had 11-fold lower outliers than the CCU assay. Unlike the CCU assay, luminometry only requires one measurement to obtain sufficient accuracy. It was concluded that the ATP assay constitutes a valuable robust alternative for reproducible real-time titre assessment of freshly grown M. hyopneumoniae cultures. It is faster, more accurate and time, work and cost efficient compared to the CCU assay. The assay is preferred to better standardise and describe M. hyopneumoniae cultures used in various experiments.

In vivo virulence of Mycoplasma hyopneumoniae isolates does not correlate with in vitro adhesion assessed by a microtitre plate adherence assay.

AIMS: Adherence of Mycoplasma hyopneumoniae to the ciliated epithelial cells of the porcine respiratory tract is considered an important first step in the pathogenesis of enzootic pneumonia. It was the aim of this study to verify the usefulness of in vitro adhesion as a virulence marker. METHODS AND RESULTS: Adherence capacity to immobilized cilia from porcine tracheal epithelial cells of three low, two moderately and two highly virulent M. hyopneumoniae field isolates was determined by a microtitre plate adherence assay. CONCLUSIONS: No significant differences between the isolates were demonstrated. SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggest that mechanisms other than adherence might be responsible for the observed differences in virulence of these field isolates or that the in vitro assay does not adequately reproduce in vivo adherence conditions.

Infection with a low virulent Mycoplasma hyopneumoniae isolate does not protect piglets against subsequent infection with a highly virulent M. hyopneumoniae isolate.

The study aimed to evaluate the effect of an infection with low virulent isolates of M. hyopneumoniae (LV1 and LV2) on the subsequent infection with a highly virulent isolate (HV). Fifty-five, 3-week-old piglets free of M. hyopneumoniae were randomly allocated to 6 different groups. At 4 weeks of age (D0), groups LV1-HV and LV1 were intratracheally inoculated with LV1, groups LV2-HV and LV2 with LV2, and group HV with sterile culture medium. Four weeks later (D28), the pigs of these different groups were either intratracheally inoculated with the highly virulent isolate (groups LV1-HV, LV2-HV, HV) or with sterile culture medium (groups LV1 and LV2). A negative control group consisted of pigs inoculated with sterile culture medium on D0 and D28. All animals were necropsied at 28 days after the second inoculation (D56). Clinical symptoms were evaluated daily using a respiratory disease score (RDS). After necropsy, macroscopic and histopathological lung lesions were quantified and immunofluorescence (IF) testing on lung tissue and nested PCR on BAL fluid were performed for the detection of M. hyopneumoniae. Disease signs and lung lesions were not observed in pigs of the negative control group. In the other groups, there were no or only very mild clinical symptoms from D0 until D28. A significant increase in the average RDS values was, however, observed during D28-D56, especially in groups LV1-HV (1.48) and LV2-HV (1.49), in group HV (0.79), and to a lesser extent in groups LV1 (0.50) and LV2 (0.65) (P<0.05). The clinical symptoms during D28-D56, the lung lesions and intensity of IF staining were more pronounced in groups LV1-HV, LV2-HV and HV compared to groups LV1 and LV2. All pigs, except those from the negative control group, were positive on IF testing and PCR at D56. The present study demonstrates that pigs inoculated with low virulent isolates of M. hyopneumoniae are not protected against a subsequent infection with a highly virulent isolate 4 weeks later and may even develop more severe disease signs. This indicates that subsequent infections with different M. hyopneumoniae isolates may lead to more severe clinical disease in a pig herd.

Protein variability among Mycoplasma hyopneumoniae isolates.

Sodium-dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was used to study the protein variability of Mycoplasma hyopneumoniae isolates. Fifty-six M. hyopneumoniae isolates from 6 different countries and 37 different herds were used. From eight herds, more than one isolate was available. All SDS-PAGE patterns of isolates originating from different herds were clearly divergent. Intra-species protein variability was quantified using the reference strain J and seven field strains all obtained from different herds and classified according to virulence. Between the field strains, a variability of 25% was found, while the culture-adapted strain J was clearly divergent and showed 30% variability with the field strains. No clustering according to virulence was obtained, but a protein band of about 181kDa was present in the two highly virulent isolates whereas this protein band was absent in the moderately and low virulent isolates. Protein patterns of isolates derived from different animals from the same herd, were identical or differed in only a few protein bands. This study clearly indicates that, in agreement with previous studies on genomic diversity of M. hyopneumoniae isolates, proteomic variability within the species is high. Our study did not find clear evidence that more than one M. hyopneumoniae isolate circulates within a herd at a specific time point. The minor differences found between M. hyopneumoniae isolates from the same herd might reflect the organism's ability to alter its proteomic expression profile under field conditions.

Interactions of highly and low virulent Mycoplasma hyopneumoniae isolates with the respiratory tract of pigs.

Mycoplasma hyopneumoniae is the etiological agent of swine enzootic pneumonia, a chronic nonfatal disease affecting pigs of all ages. To obtain better insight in the mechanisms responsible for differences in virulence between highly and low virulent M. hyopneumoniae isolates, 23 caesarean-derived, colostrum-deprived piglets were randomly assigned to three groups. Groups 1 and 2 consisted of nine animals each, which were intratracheally inoculated at 1 week of age with a highly or a low virulent isolate of M. hyopneumoniae, respectively. The remaining five animals were inoculated with sterile culture medium. Animals were euthanized at 5, 10, 15 and 28 days post-inoculation (DPI). Animals inoculated with the highly virulent isolate had more neutrophils in BAL fluid at 10, 15 and 28DPI compared to the other groups. At 10 and 15DPI, animals in the highly virulent group had significantly higher concentrations of TNF-alpha in BAL fluid. IL-1beta concentration in this group was higher at 5 and 28DPI compared to the other groups. From 10DPI onwards, significantly higher titres of M. hyopneumoniae were detected in the BAL fluid of animals inoculated with the highly virulent isolate compared to animals inoculated with the low virulent isolate. Additionally, the in vitro generation time of the highly virulent M. hyopneumoniae isolate was significantly shorter than that of the low virulent isolate. The present study indicates that the difference in pathogenicity between the highly and low virulent isolates is associated with a faster in vitro growth, a higher capacity to multiply in the lungs and the induction of a more severe inflammation process by the highly virulent isolate.