Mycoplasma hyopneumoniae-derived lipid-associated membrane proteins induce inflammation and apoptosis in porcine peripheral blood mononuclear cells in vitro

Mycoplasma hyopneumoniae is the causative agent of swine enzootic pneumonia (EP), a disease that causes considerable economic losss in swine industry. Lipid-associated membrane proteins (LAMPs) of mycoplasma play important roles in causing mycoplasma diseases. The present study explores the pathogenic mechanisms of M. hyopneumoniae LAMPs by elucidating their role in modulating the inflammation, apoptosis, and relevant signaling pathways of peripheral blood mononuclear cells (PBMCs) of pig. LAMP treatment inhibited the growth of PBMCs. Up-regulation of cytokines, such as IL-6 and IL-1β, as well as increased production of nitric oxide (NO) and superoxide anion were all detected in the supernatant of LAMPs-treated PBMCs. Furthermore, flow cytometric analysis using dual staining with annexin-V-FITC and propidium iodide (PI) showed that LAMPs of M. hyopneumoniae induced a time-dependent apoptosis in lymphocyts and monocytes from PBMCs, which was blocked by NOS inhibitor or antioxidant. In addition, LAMPs induced the phosphorylation of p38, the ratio of pro-apoptotic Bax protein to anti-apoptotic Bcl-2, activation of caspase-3 and caspase-8, and poly ADP-ribose polymerase (PARP) cleavage in PBMCs. These findings demonstrated that M. hyopneumoniae LAMPs induced the production of proinflammatory cytokines, NO and reactive oxygen species (ROS), and apoptosis of PBMCs in vitro through p38 MAPK and Bax/Bcl-2 signaling pathways, as well as caspase activation.     

Optimization of culture media of pathogenic Mycoplasma hyopneumoniae by a response surface methodology

Composition of culture medium for mass production of Mycoplasma hyopneumoniae was optimized using a response surface methodology (RSM). Initially, the influence of glucose, thallium acetate, fresh yeast extract, horse serum, and porcine serum on the production of mycoplasmal protein was assessed using a ''one factor at a time'' technique. Next, factors with a significant effect, including fresh yeast extract, and horse and porcine sera, were selected for further optimization using a central composite design (CCD) of RSM. The experimental results were fitted into a second order polynomial model equation. Estimated optimal condition of the factors for maximum production of mycoplasmal protein (i.e., triple-fold increase from 0.8 mg/L produced by basal mycoplasma media to 2.5 mg/L) was 10.9% fresh yeast extract, 15% horse serum, and 31.5% porcine serum (v/v). For the optimized conditions, a 2.96 mg/L experimental result was observed, similar to the estimated optimal conditions result of the CCD.     

Evidence of long distance airborne transport of porcine reproductive and respiratory syndrome virus and Mycoplasma hyopneumoniae

The ability of porcine reproductive and respiratory syndrome virus (PRRSV) and Mycoplasma hyopneumoniae to be transported over long distances via the airborne route was evaluated. A source population of 300 grow-finish pigs was experimentally inoculated with PRRSV MN-184 and M. hyopneumoniae 232 and over a 50-day period, air samples were collected at designated distances from the source herd using a liquid cyclonic collector. Samples were tested for the presence of PRRSV RNA and M. hyopneumoniae DNA by PCR and if positive, further characterized. Of the 306 samples collected, 4 (1.3%) were positive for PRRSV RNA and 6 (1.9%) were positive for M. hyopneumoniae DNA. The PRRSV-positive samples were recovered 4.7 km to the northwest (NW) of the source population. Four of the M. hyopneumoniae-positive samples were obtained at the NW sampling point; 2 samples at approximately 2.3 km and the other 2 samples approximately 4.7 km from the source population. Of the remaining 2 samples, one sample was obtained at the southeast sampling point and the other at the southwest sampling point, with both locations being approximately 4.7 km from the source. The four PRRSV-positive samples contained infectious virus and were ≥ 98.8% homologous to the MN-184 isolate used to inoculate the source population. All 6 of the M. hyopneumoniae-positive samples were 99.9% homologous to M. hyopneumoniae 232. These results support the hypothesis that long distance airborne transport of these important swine pathogens can occur.     

Occurrence and severity of lung lesions in slaughter pigs vaccinated against Mycoplasma hyopneumoniae with different strategies

Different vaccination strategies against Mycoplasma hyopneumoniae have been adopted worldwide. Reports from the field indicate varying levels of protection among currently available vaccines. The goal of the present study was to compare the efficacies of three widespread commercial vaccination strategies against M. hyopneumoniae under field conditions. 20 farms were included. 14 farms used different single dose vaccines (vaccine 1 [V1], 8 herds; vaccine 2 [V2], 6 herds); another 6 farms (V3) used a two dose vaccination strategy. Gross lesions of 854 lungs and histopathology from 140 lungs were quantified, and a quantitative PCR was applied to detect M. hyopneumoniae and porcine circovirus 2 (PCV2) DNA in lung tissue (n = 140). In addition, porcine reproductive and respiratory disease virus (PRRSV), swine influenza virus (SIV), Actinobacillus pleuropneumoniae, Haemophilus parasuis and Pasteurella multocida were tested by qualitative PCR. 53% of lungs were positive for M. hyopneumoniae. 55.9% of lungs showed macroscopic enzootic pneumonia (EP)-like lesions. Lung lesion scores (P < 0.001) and M. hyopneumoniae-loads (P < 0.008) differed significantly among the vaccination groups, with the most severe cases and highest amounts occurring in V1. Histological alterations differed (P < 0.001) between V1 and V3. Lung lesion scores and histopathological changes were significantly correlated, with prevalence and load of M. hyopneumoniae indicating that the applied diagnostic tools are valuable in confirming the prevalence and severity of M. hyopneumoniae infections. Comparing different vaccination strategies against M. hyopneumoniae indicates varying levels of protection. M. hyopneumoniae is still a major problem despite the widely applied vaccination.     

A field evaluation of two vaccines against Mycoplasma hyopneumoniae infection in pigs

Background

A field trial was carried out with two Mycoplasma hyopneumoniae vaccines in order to investigate the benefit of vaccination under field conditions in modern Danish pig production facilities with pigs being positive for M. hyopneumoniae. The M. hyopneumoniae infection of the herd was confirmed through blood samples that were positive for antibodies against M. hyopneumoniae combined with gross lesions of the lungs related to M. hyopneumoniae at slaughter and detection of M. hyopneumoniae by polymerace chain reaction in these lesions.

Results

A total of 2,256 pigs from two herds were randomly divided into three groups. Group 1 received 2 mL ThoroVAX^®VET, Group 2 received 1 mL Ingelvac^®MycoFLEX, and Group 3 was a non-vaccinated control group. The vaccination was performed by a person who was not involved in the rest of the trial and vaccination status thereby blinded to the evaluators.The prevalence of lung lesions related to M. hyopneumoniae were significantly lower for pigs vaccinated with ThoroVAX^®VET but not for pigs vaccinated with Ingelvac^®MycoFLEX^®, when compared to non-vaccinated pigs. There was no significant effect of vaccination on growth rate, antibiotic consumption or mortality.

Conclusion

This trial demonstrated that vaccination with Thoro^®VAX VET was effective in reducing the prevalence of lung lesion in pig units infected with M. hyopneumoniae.     

Herd specific risk factors for Mycoplasma hyopneumoniae infections in suckling pigs at the age of weaning

Background

Mycoplasma hyopneumoniae is the etiologic agent of enzootic pneumonia mainly occurring in fattening pigs. It is assumed that horizontal transmission of the pathogen during nursery and growing phase starts with few suckling pigs vertically infected by the sow. The aim of the present study was the exploration of the herd prevalence of M. hyopneumoniae infections in suckling pigs followed by an investigation of various herd specific factors for their potential of influencing the occurrence of this pathogen at the age of weaning.

Results

In this cross-sectional study, 125 breeding herds were examined by taking nasal swabs from 20 suckling pigs in each herd. In total, 3.9% (98/2500) of all nasal swabs were tested positive for M. hyopneumoniae by real-time PCR. Piglets tested positive originated from 46 different herds resulting in an overall herd prevalence of 36.8% (46/125) for M. hyopneumoniae infection in pigs at the age of weaning. While the herds were epidemiologically characterized, the risk for demonstration of M. hyopneumoniae was significantly increased, when the number of purchased gilts per year was more than 120 (OR: 5.8), and when the number of farrowing pens per compartment was higher than 16 (OR: 3.3). In herds with a planned and segregated production, where groups of sows entered previously emptied farrowing units, the risk for demonstration of M. hyopneumoniae in piglets was higher in herds with two or four weeks between batches than in herds with one or three weeks between batches (OR: 2.7).

Conclusions

In this cross-sectional study, several risk factors could be identified enhancing the probability of breeding herds to raise suckling pigs already infected with M. hyopneumoniae at the time of weaning. Interestingly, some factors (farrowing rhythm, gilt acclimatisation issues) were overlapping with those also influencing the seroprevalences among sows or the transmission of the pathogen between older age groups. Taking the multifactorial character of enzootic pneumonia into account, the results of this study substantiate that a comprehensive herd specific prevention programme is a prerequisite to reduce transmission of and disease caused by M. hyopneumoniae.     

Differences in phagocytosis susceptibility in Haemophilus parasuis strains

Haemophilus parasuis is a colonizer of the upper respiratory tract of healthy pigs, but virulent strains can cause a systemic infection characterized by fibrinous polyserositis, commonly known as Glässer’s disease. The variability in virulence that is observed among H. parasuis strains is not completely understood, since the virulence mechanisms of H. parasuis are largely unknown. In the course of infection, H. parasuis has to survive the host pulmonary defences, which include alveolar macrophages, to produce disease. Using strains from different clinical backgrounds, we were able to detect clear differences in susceptibility to phagocytosis. Strains isolated from the nose of healthy animals were efficiently phagocytosed by porcine alveolar macrophages (PAM), while strains isolated from systemic lesions were resistant to this interaction. Phagocytosis of susceptible strains proceeded through mechanisms independent of a specific receptor, which involved actin filaments and microtubules. In all the systemic strains tested in this study, we observed a distinct capsule after interaction with PAM, indicating a role of this surface structure in phagocytosis resistance. However, additional mechanisms of resistance to phagocytosis should be explored, since we detected different effects of microtubule inhibition among systemic strains.     

Experimental evaluation of Mycoplasma hyopneumoniae bacterin against a Korean M. hyopneumoniae challenge

The objective of this study was to evaluate the efficacy of a new Mycoplasma hyopneumoniae bacterin against a Korean M. hyopneumoniae challenge under experimental conditions. Fifteen pigs were allocated randomly into 3 groups (5 pigs per group) that were designated in 1 of 3 ways: vaccinated-challenged, unvaccinated-challenged, or unvaccinated-unchallenged. The pigs in the vaccinated-challenged group were immunized with an M. hyopneumoniae whole-cell bacterin at a 1.0 mL dose-level at 21 d old. At 42 d old (0 d post-challenge), the pigs in the vaccinated-challenged and unvaccinated-challenged groups were inoculated intranasally with a strain of Korean M. hyopneumoniae. Vaccinated-challenged pigs elicited a strong cell-mediated immunity as measured by M. hyopneumoniae-specific interferon-γ secreting cells when compared with unvaccinated-challenged pigs. Vaccination of pigs with this new M. hyopneumoniae bacterin reduced nasal shedding and lung lesions. The evaluated vaccine was therefore considered effective in controlling M. hyopneumoniae infection.     

Individual risk factors for Mycoplasma hyopneumoniae infections in suckling pigs at the age of weaning

Background

In recent years, the occurrence and the relevance of Mycoplasma hyopneumoniae infections in suckling pigs has been examined in several studies. Whereas most of these studies were focused on sole prevalence estimation within different age groups, follow-up of infected piglets or assessment of pathological findings, none of the studies included a detailed analysis of individual and environmental risk factors. Therefore, the aim of the present study was to investigate the frequency of M. hyopneumoniae infections in suckling pigs of endemically infected herds and to identify individual risk factors potentially influencing the infection status of suckling pigs at the age of weaning.

Results

The animal level prevalence of M. hyopneumoniae infections in suckling pigs examined in three conventional pig breeding herds was 3.6% (41/1127) at the time of weaning. A prevalence of 1.2% was found in the same pigs at the end of their nursery period. In a multivariable Poisson regression model it was found that incidence rate ratios (IRR) for suckling pigs are significantly lower than 1 when teeth grinding was conducted (IRR: 0.10). Moreover, high temperatures in the piglet nest during the first two weeks of life (occasionally >40°C) were associated with a decrease of the probability of an infection (IRR: 0.23-0.40). Contrary, the application of PCV2 vaccines to piglets was associated with an increased infection risk (IRR: 9.72).

Conclusions

Since single infected piglets are supposed to act as initiators for the transmission of this pathogen in nursery and fattening pigs, the elimination of the risk factors described in this study should help to reduce the incidence rate of M. hyopneumoniae infections and thereby might contribute to a reduced probability of high prevalences in older pigs.     

Interaction between single-dose Mycoplasma hyopneumoniae and porcine reproductive and respiratory syndrome virus vaccines on dually infected pigs

The objective of this study was to determine the effects of Mycoplasma hyopneumoniae and/or porcine reproductive and respiratory syndrome virus (PRRSV) vaccination on dually infected pigs. In total, 72 pigs were randomly divided into nine groups (eight pigs per group), as follows: five vaccinated and challenged groups, three non-vaccinated and challenged groups, and a negative control group. Single-dose vaccination against M. hyopneumoniae alone decreased the levels of PRRSV viremia and PRRSV-induced pulmonary lesions, whereas single-dose vaccination against PRRSV alone did not decrease nasal shedding of M. hyopneumoniae and mycoplasma-induced pulmonary lesions in the dually infected pigs. The M. hyopneumoniae challenge impaired the protective cell-mediated immunity induced by the PRRSV vaccine, whereas the PRRSV challenge did not impair the protective cell-mediated immunity induced by the M. hyopneumoniae vaccine. The present study provides swine practitioners and producers with efficient vaccination regimes; vaccination against M. hyopneumoniae is the first step in protecting pigs against co-infection with M. hyopneumoniae and PRRSV.