Transposon mutagenesis in Mycoplasma hyopneumoniae using a novel mariner-based system for generating random mutations

Mycoplasma hyopneumoniae is the cause of enzootic pneumonia in pigs, a chronic respiratory disease associated with significant economic losses to swine producers worldwide. The molecular pathogenesis of infection is poorly understood due to the lack of genetic tools to allow manipulation of the organism and more generally for the Mycoplasma genus. The objective of this study was to develop a system for generating random transposon insertion mutants in M. hyopneumoniae that could prove a powerful tool in enabling the pathogenesis of infection to be unraveled. A novel delivery vector was constructed containing a hyperactive C9 mutant of the Himar1 transposase along with a mini transposon containing the tetracycline resistance cassette, tetM. M. hyopneumoniae strain 232 was electroporated with the construct and tetM-expressing transformants selected on agar containing tetracycline. Individual transformants contained single transposon insertions that were stable upon serial passages in broth medium. The insertion sites of 44 individual transformants were determined and confirmed disruption of several M. hyopneumoniae genes. A large pool of over 10 000 mutants was generated that should allow saturation of the M. hyopneumoniae strain 232 genome. This is the first time that transposon mutagenesis has been demonstrated in this important pathogen and could be generally applied for other Mycoplasma species that are intractable to genetic manipulation. The ability to generate random mutant libraries is a powerful tool in the further study of the pathogenesis of this important swine pathogen.     

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.     

Adaptation of a latex agglutination tube test for diagnosis of Mycoplasma hyopneumoniae swine pneumonia

A tube latex agglutination test (LAT) for diagnosis of Mycoplasma hyopneumoniae swine pneumonia was developed. In pigs inoculated with M. hyopneumoniae, LAT antibodies were generally detected 2–3 weeks post-inoculation, which was 1 week or more before complement-fixing antibodies were first detected in the corresponding pigs. Correlation of LAT results and gross and microscopic lung lesions of corresponding pigs revealed that LAT titers persisted after the pneumonic lesions had resolved.Latex agglutination titers in pigs exposed by contact with M. hyopneumoniae inoculated pigs were demonstrated 4–12 weeks post-contact.Latex agglutination antibodies were detected up to 48 weeks post-inoculation in pigs inoculated with M. hyopneumoniae and this was similar to the duration of detectable indirect hemagglutination titers. Complement-fixation titers, however, were demonstrated no longer than 28 weeks post-inoculation in corresponding pigs.Evaluation of repeatability of LAT results revealed some variation of LAT titers of sera titered on four different occasions.Sera from pigs inoculated with other swine mycoplasmas, including M. hyosynoviae and M. hyorhinis, did not react in the M. hyopneumoniae LAT. In addition, no detectable titers were demonstrated in the M. hyopneumoniae LAT using sera from pigs infected with Metastrongylus spp., Ascaris suum, or in sera from pigs vaccinated with any of four commonly used swine vaccines.     

The Mycoplasma hyopneumoniae recombinant heat shock protein P42 induces an immune response in pigs under field conditions

Enzootic pneumonia (EP), resulting from Mycoplasma hyopneumoniae infection is one of the most prevalent diseases in pigs and is a major cause of economic losses to the swine industry worldwide. EP is often controlled by vaccination with inactivated, adjuvanted whole-cell bacterin. However, these bacterins provide only partial protection and do not prevent M. hyopneumoniae colonization. Attempts to develop vaccines that are more efficient have made use of the recombinant DNA technology. The objective of this study was to assess the potential of recombinant M. hyopneumoniae heat shock protein P42 in vaccine preparations against EP, using piglets housed under field conditions in a M. hyopneumoniae-positive farm. The cellular and humoral immune responses were elicited after a single intramuscular inoculation of rP42 in an oil-based adjuvant, or in conjunction with whole-cell vaccine preparation. The production of INF-γ and IL-10 cytokines was quantified in the supernatant of the cultured mononuclear cells. The rP42 emulsified in oil-based adjuvant was able to trigger a strong humoral immune response. Further, it induced a cellular immune response, accompanied by the production of antibodies that reacted with the native M. hyopneumoniae protein. The rP42 mediated induction of cellular and humoral immune response in the host suggests that rP42 emulsified in an oil-based adjuvant holds promise as an effective recombinant subunit vaccine against EP.     

Genetic diversity of Mycoplasma hyopneumoniae isolates of abattoir pigs

Mycoplasma hyopneumoniae, the causative agent of porcine enzootic pneumonia, is present in swine herds worldwide. However, there is little information on strains infecting herds in Canada. A total of 160 swine lungs with lesions suggestive of enzootic pneumonia originating from 48 different farms were recovered from two slaughterhouses and submitted for gross pathology. The pneumonic lesion scores ranged from 2% to 84%.Eighty nine percent of the lungs (143/160) were positive for M. hyopneumoniae by real-time PCR whereas 10% (16/160) and 8.8% (14/160) were positive by PCR for M. hyorhinis and M. flocculare, respectively. By culture, only 6% of the samples were positive for M. hyopneumoniae (10/160). Among the selected M. hyopneumoniae-positive lungs (n = 25), 9 lungs were co-infected with M. hyorhinis, 9 lungs with PCV2, 2 lungs with PRRSV, 12 lungs with S. suis and 10 lungs with P. multocida. MLVA and PCR-RFLP clustering of M. hyopneumoniae revealed that analyzed strains were distributed among three and five clusters respectively, regardless of severity of lesions, indicating that no cluster is associated with virulence. However, strains missing a specific MLVA locus showed significantly less severe lesions and lower numbers of bacteria. MLVA and PCR-RFLP analyses also showed a high diversity among field isolates of M. hyopneumoniae with a greater homogeneity within the same herd. Almost half of the field isolates presented less than 55% homology with selected vaccine and reference strains.     

Failure of lungworm-larvae-infected earthworms to transmit mycoplasmal pneumonia to swine

Two herds of swine, believed to be free from mycoplasmal pneumonia of swine (MPS) based upon negative Mycoplasma hyopneumoniae microtiter complement fixation test (CFT) results, subsequently exhibited clinical signs of MPS.Lungworms (Metastrongylus spp.) were common to both herds. The possibility that lungworms could serve as a reservoir for M. hyopneumoniae was investigated. Lungworm-larvae-infected earthworms, collected from each of the farms, were fed to swine free of lungworms and MPS. This procedure resulted in lungworm infection in the recipient pigs, but failed to produce pneumonic lesions or CFT antibody titers against MPS.     

Genotype distribution of Mycoplasma hyopneumoniae in swine herds from different geographical regions

Genetic heterogeneity of Mycoplasma hyopneumoniae in pigs has been reported, however there has been limited reproducibility on the molecular methods employed so far. The aim of this study was to modify and standardize a high-resolution multiple locus variable number tandem repeat analysis (MLVA), to investigate the genetic variability of M. hyopneumoniae circulating in the United States of America (USA), Brazil, Mexico and Spain. The MLVA was standardized on the basis of the number of tandem repeats in two Mycoplasma adhesins, P97 and P146, which are proteins involved in the adherence of the pathogen to cilia. A total of 355 samples obtained from the four countries were analyzed. The Simpson's diversity index for the assay was D = 0.976 when samples from all countries were combined. A large number of MLVA types (n = 139) were identified, suggesting that multiple M. hyopneumoniae variants are circulating in swine. The locus P97 had 17 different types with 2–18 repeats. The P146 locus showed higher heterogeneity, with 34 different types, ranging from 7 to 48 repeats. MLVA types that presented more than 30 repeats in P146 were found in Spain and Brazil, while shorter repeats were observed in the USA and Mexico. This simplified MLVA method proved to be an efficient tool for typing M. hyopneumoniae with a high degree of stability, repeatability, and discriminatory power. In conclusion, M. hyopneumoniae showed a high variable number tandem repeat heterogeneity and this assay can be applied in molecular epidemiology investigations within farms and productions systems.     

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.     

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.     

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.     

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.     

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.     

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.     

Mycoplasma hyopneumoniae-derived lipid-associated membrane proteins induce apoptosis in porcine alveolar macrophage via increasing nitric oxide production,oxidative stress,and caspase-3 activation

Mycoplasma hyopneumoniae is the primary etiological agent of enzootic pneumonia in swine. Lipid-associated membrane proteins (LAMP) of mycoplasma are the main pathogenicity factors in mycoplasma diseases. In this study, we investigated the effects of M. hyopneumoniae LAMP on porcine alveolar macrophage (PAM) 3D4/21 cell line. Apoptotic features, such as chromatin condensation and apoptotic bodies, were observed in LAMP-treated PAM 3D4/21 cells. Moreover, LAMP significantly increased the number of TUNEL positive apoptotic cells in PAM 3D4/21 cells compared with the untreated control. In addition, flow cytometric analysis using dual staining with annexin-V-FITC and propidium iodide (PI) showed that LAMP of M. hyopneumoniae induced a time-dependent apoptosis in PAM 3D4/21 cells. Moreover, increased levels of superoxide anion production and activated caspase-3 in PAM 3D4/21 cells were observed after exposure to LAMP. Increased production of nitric oxide (NO) was also confirmed in the cell supernatants. Besides, apoptotic rates increase and caspase-3 activation were suppressed by NOS inhibitor or antioxidant. It is suggested that LAMP of M. hyopneumoniae induced apoptosis in porcine alveolar macrophage via NO production, superoxide anion production, and caspase-3 activation.     

Protein and Antigenic Variability among <Emphasis Type="Italic">Mycoplasma hyopneumoniae</Emphasis> Strains by SDS-PAGE and Immunoblot

Porcine enzootic pneumonia (PEP), with Mycoplasma hyopneumoniae as the primary agent, is a chronic respiratory disease that causes major economic losses to the pig industry worldwide. The aim of this work was to analyse 18 field strains of M. hyopneumoniae isolated in Gran Canaria (Spain) and the reference M. hyopneumoniae strain by SDS-PAGE and immunoblot. A monoclonal antibody (MAb) against the membrane protein p46 reacted with all the strains in this study. In contrast, a purified polyclonal antibody (PAb) against the cytoplasmic protein p36 reacted with this protein in only 10 strains. A MAb against the adhesin protein p97 stained multiple proteins of different sizes and with different intensities. Different antigenic patterns in the same M. hyopneumoniae strains were also observed after different numbers of passages in culture medium. Furthermore, variability in the staining of the 36 kDa protein was observed, depending on whether the p36 PAb or the antiserum against M. hyopneumoniae reference strain was used. It is concluded that local M. hyopneumoniae field isolates in Gran Canaria are characterized by protein diversity.     

The protective effect of a Toxoplasma gondii SAG1 plasmid DNA vaccine in mice is enhanced with IL-18

More effective vaccines against Toxoplasma gondii may contribute to the control of this pathogen that has major veterinary and public health significance. In this study, two recombinant plasmids pcDNA/TgSAG1 and pVAX/mIL-18 containing T. gondii SAG1 (TgSAG1) and murine cytokine interleukin-18 (IL-18) were evaluated for their ability to protect mice against T. gondii challenge. Mice were given two intramuscular immunizations 3 weeks apart, and challenged with T. gondii 3 weeks later. All animals vaccinated with pcDNA/TgSAG1 alone or with pVAX/mIL-18 developed specific anti-TLA (T. gondii lysate antigen) antibodies and specific lymphocyte proliferative responses. Co-injection of pVAX/mIL-18 significantly increased the production of IFN-γ and IL-2. Further, challenge experiments showed that co-immunization with pVAX/mIL-18 significantly (P < 0.05) increased the survival rate (60%), compared with pcDNA/TgSAG1 alone (40%). Therefore, codelivery of the IL-18-secreting plasmid potentiates the induction and maintenance of the type 1 helper T-cell immune response and may be a potent strategy for enhancing the protective efficacy of vaccines against T. gondii.     

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.     

ISOLATION OF MYCOPLASMA HYOPNEUMONIAE FROM LESIONS IN EXPERIMENTALLY INFECTED PIGS

Pigs aged 6 to 9 weeks from enzootic pneumonia-free herds were inoculated intranasally with a suspension of pneumonic lung containing Mycoplasma hyopneumoniae or were placed in contact with such inoculated pigs. All the inoculated pigs had gross lesions of enzootic pneumonia when killed 27 to 42 days after inoculation. The culture methods described enabled M. hyopneumoniae to be isolated from all 29 inoculated pigs. Of 45 pigs in contact with inoculated pigs 35 had gross lesions of enzootic pneumonia when killed 28 to 71 days later and M. hyopneumoniae was isolated from 33. Another 9 had lesions, detected only microscopically, and M. hyopneumoniae was recovered from 3 of these when killed 75 to 98 days after contact began. In a separate experiment M. hyopneumoniae isolated from experimentally infected pigs, and adapted to the culture medium after 6 passages, caused gross lesions of enzootic pneumonia in 1 of 4 pigs inoculated intranasally.     

Enhancement of the increase in intracellular calcium concentration in stimulated neutrophils byMycoplasma hyopneumoniae

Neutrophils isolated from the peripheral blood of pigs free of infection withMycoplasma hyopneumoniae were loaded with a fluorescent indicator (Fura-2) for detection of cytosolic free calcium concentration. The kinetics of the intracellular calcium flux were examined after incubation with or without a pathogenic or a non-pathogenic strain ofM. hyopneumoniae. The basal intracellular calcium concentration was not altered by incubation withM. hyopneumoniae. However, the relative increase in cytoplasmic calcium concentration caused by the addition of opsonized zymosan was significantly (p<0.05) higher in neutrophils incubated withM. hyopneumoniae as compared to neutrophils not incubated withM. hyopneumoniae. Additionally, after zymosan stimulation, the intracellular calcium concentration was greater in neutrophils incubated with a pathogenic strain ofM. hyopneumoniae than in those incubated with a non-pathogenic strain. This suggests thatM. hyopneumoniae alters the signal transduction mechanisms in neutrophils and that this alteration may be related to virulence.Abbreviations [Ca]_i intracellular concentration of calcium - CCU colour changing units/ml - Fura-2/AM pentaacetoxymethyl ester - PBS phosphate-buffered saline, pH 7.2 - TNF tumour necrosis factor     

Dynamics of <Emphasis Type="Italic">Mycoplasma hyopneumoniae</Emphasis> seroconversion and infection in pigs in the three main production systems

In this study, we investigated the dynamics of Mycoplasma hyopneumoniae infections in 66 pig farms, with different production systems (one-, two-, and three-site systems), and considered different risk factors. Serological assay was used to detect serum antibodies against M. hyopneumoniae and real time polymerase chain reaction (RT–PCR) was performed to detect M. hyopneumoniae DNA in tracheobronchial swabs. Results demonstrated that M. hyopneumoniae infection status was predominantly influenced by the age of the animals and the type of production system. Infection rates were higher in older animals and the prevalence was higher in the one- and two-site systems than in the three-site systems. Dynamics of infection by RT-PCR showed that earlier M. hyopneumoniae infection on one-site farms occurs earlier, while on two- and three-site farms occurs later but spreads faster, suggesting that contact between animals of different age favors the transmission.