Dries Calus

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.

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.

Multiple-Locus Variable-Number Tandem-Repeat Analysis Is a Suitable Tool for Differentiation of Mycoplasma hyopneumoniae Strains without Cultivation

ABSTRACT 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.

Effect of challenge of pigs previously immunised with inactivated vaccines containing homologous and heterologous Mycoplasma hyopneumoniae strains

BackgroundMycoplasma hyopneumoniae is the primary cause of enzootic pneumonia in pigs. Although vaccination is an important control tool, the results observed under field conditions are variable. This may be due to antigenic differences between the strains circulating in pig herds and the vaccine strain. This study compared the protective efficacy of four bacterins against challenge infection with a highly virulent field strain of M. hyopneumoniae.Seventy eight, one-week old piglets were randomly assigned to five treatment groups (A, B, C, D, E), 14 piglets each, and a negative control group (F) consisting of 8 piglets. All pigs were injected at 1 (D7) and 4 weeks of age (D28), with 2 ml of either a placebo or a bacterin based on selected M. hyopneumoniae strains, namely A (F7.2C), B (F20.1L), C (B2V1W20 1A-F), D (J strain), E (placebo; positive control), F (placebo; negative control). At D56, all pigs except those of group F were challenged intratracheally with 7 ml culture medium containing 107 CCU/ml of M. hyopneumoniae strain F7.2C. All pigs were euthanized and necropsied at D84. The severity of coughing and pneumonia lesions were the main parameters. Immunofluorescence (IF) testing, nested PCR testing of bronchoalveolar lavage (BAL) fluid and serology for M. hyopneumoniae were also performed.ResultsThe different bacterins only slightly improved clinical symptoms (average 0.38 in vaccinated groups vs. 0.45 in group E) and histopathological lung lesions (average 3.20 in vaccinated groups vs. 3.45 in group E), but did not improve macroscopic lung lesions (score 4.30 vs. 4.03 in group E). None of the vaccines was significantly and/or consistently better or worse than the other ones. All bacterins evoked a serological response in the vaccinated animals. All pigs, except those from group F, were positive with nPCR in BAL fluid at D84.ConclusionThe bacterins did not induce a clear overall protection against challenge infection, and there were no significant differences in protective efficacy between bacterins containing homologous and heterologous M. hyopneumoniae strains. Further research is necessary to better characterize the antigens involved in protection and to elucidate the protective immunity responses following M. hyopneumoniae vaccination and/or infection.

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.