Molecular epidemiology of Mycoplasma hyopneumoniae from outbreaks of enzootic pneumonia in domestic pig and the role of wild boar.

Mycoplasma hyopneumoniae is the major cause of enzootic pneumonia (EP) in domestic pigs, a disease with low mortality but high morbidity, having a great economic impact for producers. In Switzerland EP has been successfully eradicated, however, sporadic outbreaks are observed with no obvious source. Besides the possibility of recurrent outbreaks due to persisting M. hyopneumoniae strains within the pig population, there is suspicion that wild boars might introduce M. hyopneumoniae into swine herds. To elucidate possible links between domestic pig and wild boar, epidemiological investigations of recent EP outbreaks were initiated and lung samples of pig and wild boar were tested for the presence of specific genotypes by multilocus sequence typing (MLST). Despite generally different genotypes in wild boar, outbreak strains could be found in geographically linked wild boar lungs after, but so far not before the outbreak. Recurrent outbreaks in a farm were due to the same strain, indicating unsuccessful sanitation rather than reintroduction by wild boar. In another case outbreaks in six different farms were caused by the same strain never found in wild boar, confirming spread between farms due to hypothesized animal transport. Results indicate the presence of identical lineages of wild boar and domestic pig strains, and possible transmission of M. hyopneumoniae between wild boar and pig. However, the role of wild boar might be rather one as a recipient than a transmitter. More important than contact to wild boar for sporadic outbreaks in Switzerland is apparently persistence of M. hyopneumoniae within a farm as well as transmission between farms.

Investigating the role of free-ranging wild boar (Sus scrofa) in the re-emergence of enzootic pneumonia in domestic pig herds: a pathological, prevalence and risk-factor study

Enzootic pneumonia (EP) caused by Mycoplasma hyopneumoniae has a significant economic impact on domestic pig production. A control program carried out from 1999 to 2003 successfully reduced disease occurrence in domestic pigs in Switzerland, but recurrent outbreaks suggested a potential role of free-ranging wild boar (Sus scrofa) as a source of re-infection. Since little is known on the epidemiology of EP in wild boar populations, our aims were: (1) to estimate the prevalence of M. hyopneumoniae infections in wild boar in Switzerland; (2) to identify risk factors for infection in wild boar; and (3) to assess whether infection in wild boar is associated with the same gross and microscopic lesions typical of EP in domestic pigs. Nasal swabs, bronchial swabs and lung samples were collected from 978 wild boar from five study areas in Switzerland between October 2011 and May 2013. Swabs were analyzed by qualitative real time PCR and a histopathological study was conducted on lung tissues. Risk factor analysis was performed using multivariable logistic regression modeling. Overall prevalence in nasal swabs was 26.2% (95% CI 23.3-29.3%) but significant geographical differences were observed. Wild boar density, occurrence of EP outbreaks in domestic pigs and young age were identified as risk factors for infection. There was a significant association between infection and lesions consistent with EP in domestic pigs. We have concluded that M. hyopneumoniae is widespread in the Swiss wild boar population, that the same risk factors for infection of domestic pigs also act as risk factors for infection of wild boar, and that infected wild boar develop lesions similar to those found in domestic pigs. However, based on our data and the outbreak pattern in domestic pigs, we propose that spillover from domestic pigs to wild boar is more likely than transmission from wild boar to pigs.

[Lung and pleural lesions before and after implementation of a national eradication program against enzootic pneumonia and actinobacillosis as well as changes of slaughter carcass organs in slaughter pigs in Switzerland].

In a representative cross-sectional study during 12 months of the years 2008/2009 in four abattoirs in Switzerland, lung and pleura lesions as well as lesions of slaughter carcasses and organs of 34 706 pigs were studied for frequency and type of macroscopic lesions. Of the 24276 examined pigs, 91.2% of the lungs, 94.4% of the heart and 95.5% of the livers showed no macroscopically visible lesions. Pigs that were produced for a label program had significantly less bronchopneumonia and pneumonia residuals, pleuritis and liver lesions due to echinococcosis. Pigs supervised by the Swiss Pig Health Service (SGD), showed significantly less bronchopneumonia and pneumonia residuals, diffuse pleuritis, pleuritis/pericarditis and milkspots compared to the non-SGD supervised farms. Thanks to the national eradication program for enzootic pneumonia (EP) and actinobacillosis, the health-status of lungs has been considerably improved and the prevalence of pleurisy decreased considerably. The results of this study indicate a good herd health in Swiss pig production.

Ultrastructural observation of the airways of recovered and susceptible pigs after inoculation with Mycoplasma hyopneumoniae

To determine the morphological differences in the epithelium of the airways of recovered and susceptible pigs after Mycoplasma hyopneumoniae challenge, twenty-four 4-week-old M. hyopneumoniae-free pigs were intratracheally inoculated with 107ccu/ml of a pure low-passaged culture of the P5722-3 strain of M. hyopneumoniae challenge material. Eight pigs (group I) were challenged at the beginning of the experiment and rechallenged 3 months later. Group II pigs were also challenged at the beginning of the experiment and necropsied 3 months later. Group III pigs were challenged at the same time as the rechallenge of group I pigs. Eight nonchallenged pigs served as controls (group IV). Three days after the second challenge of group I and the first challenge of group III, and every 3 and 4 days thereafter, two pigs from each group were euthanatized by electrocution and necropsied. Samples of bronchi and lung tissue were examined using light and electron microscopy (SEM and TEM). Macroscopic lesions were observed in the lungs of all group III pigs (average = 4.74%) and were characterized by purple-red areas of discoloration and increased firmness affecting the cranioventral aspect of the lungs. Macroscopic lesions of pneumonia in groups I and II were minimal (less than 1%). There were no gross lesions of pneumonia in control (group IV) pigs. Microscopic lesions were characterized by hyperplasia of the peribronchial lymphoid tissue and mild neutrophilic infiltrates in alveoli. Electron microscopy showed patchy areas with loss of cilia and presence of leukocytes and mycoplasmas in bronchi of susceptible pigs (group III). The bronchial epithelium of rechallenged (group I), recovered (group II), and control (group IV) pigs was ultrastructurally similar indicating recovery of the former two groups. Although mycoplasmas were seen among cilia, a second challenge on pigs of group I did not produce another episode of the disease nor did it enhance morphological changes, suggesting that those pigs could become carriers of M. hyopneumoniae.

Étude de la diversité génétique d’isolats québécois de Mycoplasma hyopneumoniae provenant d’infections simples ou mixtes et caractérisation de leur sensibilité aux antimicrobiens

Mycoplasma hyopneumoniae est l’agent causal de la pneumonie enzootique. On le retrouve dans plusieurs élevages de porcs à travers le monde. Même si ce micro-organisme est présent dans plusieurs troupeaux canadiens, peu d’informations sont présentement disponibles sur les isolats québécois. Un total de 160 poumons de porcs possédant des lésions de pneumonie ont été récupérés à l’abattoir, mis en culture et testés par PCR pour M. hyopneumoniae et Mycoplasma hyorhinis. D’autres pathogènes bactériens communs du porc et les virus du syndrome reproducteur et respiratoire porcin (VSRRP), de l’influenza et le circovirus porcin de type 2 (CVP2) ont été également testés. Quatre-vingt-dix pourcent des échantillons étaient positifs pour M. hyopneumoniae et 5.6% l’étaient seulement pour M. hyorhinis. Dans ces échantillons positifs pour M. hyopneumoniae, la concentration de ce mycoplasme variait de 1.17 x 105 à 3.37 x 109 génomes/mL. Vingt-cinq poumons positifs en culture ou par PCR en temps réel pour M. hyopneumoniae ont été sélectionnés, parmi ceux-ci 10 étaient en coinfection avec Pasteurella multocida, 12 avec Streptococcus suis, 9 avec CVP2 et 2 avec le VSRRP. Les analyses des nombres variables de répétitions en tandem à de multiples loci (MLVA) et PCR-polymorphisme de longueur de fragments de restriction (PCR-RFLP) de M. hyopneumoniae ont démontré une forte diversité des isolats de terrain. Par contre, il semble y avoir plus d’homogénéité à l’intérieur d’un même élevage. L’analyse MLVA a également démontré que près de la moitié des isolats possédaient moins de 55% d’homologie avec les souches vaccinales et de référence utilisées dans la présente étude. L’absence d’amplification du locus 1 de M. hyopneumoniae en MLVA a été significativement associée à une baisse de la concentration de bactérie et de la sévérité des lésions. Pour tous les isolats de M. hyopneumoniae, des concentrations minimales inhibitrices (CMI) de faibles à intermédiaires ont été obtenues envers tous les antimicrobiens testés. Les isolats possédant des CMI intermédiaires envers les tétracyclines, les macrolides et les lincosamides ont été testés pour la présence des gènes de résistance tetM, ermB et pour des mutations ponctuelles dans les gènes des protéines L4, L22 et de l’ARNr 23S. Aucun de ces gènes n’a été détecté mais la mutation ponctuelle G2057A a été identifiée. Cette mutation est responsable de la résistance intrinsèque de M. hyopneumoniae face aux macrolides à 14 carbones. Ces résultats indiquent qu’il ne semble pas y avoir de résistance acquise aux antimicrobiens parmi ces isolats. En conclusion, cette recherche a permis d’obtenir de nouvelles données scientifiques sur les isolats québécois de M. hyopneumoniae.

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.

Potential use of a recombinant replication-defective adenovirus vector carrying the C-terminal portion of the P97 adhesin protein as a vaccine against Mycoplasma hyopneumoniae in swine.

Mycoplasma hyopneumoniae causes severe economic losses to the swine industry worldwide and the prevention of its related disease, enzootic porcine pneumonia, remains a challenge. The P97 adhesin protein of M. hyopneumoniae should be a good candidate for the development of a subunit vaccine because antibodies produced against P97 could prevent the adhesion of the pathogen to the respiratory epithelial cells in vitro. In the present study, a P97 recombinant replication-defective adenovirus (rAdP97c) subunit vaccine efficiency was evaluated in pigs. The rAdP97c vaccine was found to induce both strong P97 specific humoral and cellular immune responses. The rAdP97c vaccinated pigs developed a lower amount of macroscopic lung lesions (18.5 ± 9.6%) compared to the unvaccinated and challenged animals (45.8 ± 11.5%). rAdP97c vaccine reduced significantly the severity of inflammatory response and the amount of M. hyopneumoniae in the respiratory tract. Furthermore, the average daily weight gain was slightly improved in the rAdP97c vaccinated pigs (0.672 ± 0.068 kg/day) compared to the unvaccinated and challenged animals (0.568 ± 0.104 kg/day). A bacterin-based commercial vaccine (Suvaxyn® MH-one) was more efficient to induce a protective immune response than rAdP97c even if it did not evoke a P97 specific immune response. These results suggest that immunodominant antigens other than P97 adhesin are also important in the induction of a protective immune response and should be taken into account in the future development of M. hyopneumoniae subunit vaccines.

Detection of Mycoplasma hyopneumoniae by polymerase chain reaction in swine presenting respiratory problems

A Nested-PCR (N-PCR) tem como objetivo melhorar a sensibilidade do diagnóstico direto da Pneumonia Enzoótica Suína, pois o isolamento do Mycoplasma hyopneumoniae é trabalhoso tornando-se inviável na rotina. Neste trabalho, foi realizado um projeto piloto para a otimização da técnica de N-PCR, utilizando três variáveis: tipo de amostra biológica, meio de transporte da amostra e método de extração do DNA, utilizando oito animais. Os resultados obtidos foram empregados no segundo experimento para a validação do teste utilizando 40 animais. Os resultados obtidos, pela otimização da N-PCR, neste trabalho, permite sugerir esta prova como método de diagnóstico de rotina no monitoramento das infecções por Mycoplasma hyopneumoniae em granjas de suínos.

Soropositividade para Mycoplasma hyopneumoniae em suínos abatidos em frigoríficos da região central do estado de São Paulo

Mycoplasma hyopneumoniae is the etiologic agent of enzootic pneumonia in pigs and causes large economic losses in the swine industry. There is little data on the positivity of this disease in Brazil. The objective of this study was to evaluate the seropositivity for this agent in 200 serum samples collected from pigs in a slaughterhouse located in the central region of São Paulo. A high percentage (52%) of positivity was found indicating the presence of the agent and the need to implement control measures.

Genotyping of Mycoplasma hyopneumoniae in wild boar lung samples

Mycoplasma hyopneumoniae is the etiologic agent of enzootic pneumonia (EP), an important cause of disease-associated losses in swine production and a role of wild boar in recurrent infections can be supposed. Genotypes of M. hyopneumoniae from wild boar are unknown but could indicate its role as a potential reservoir. Therefore, 34 lung samples being PCR-positive for M. hyopneumoniae from wild boar from the Geneva region in Switzerland were assayed by genotyping using the p146 and multi-locus sequence typing (MLST) approaches and compared to data from outbreak cases from domestic swine in Switzerland. Successful genotyping was dependent on a sufficiently high concentration of M. hyopneumoniae DNA in the samples as assessed by different real-time PCR assays. The p146 genotyping was more successful with 24 samples (70.5%) being typeable whereas only 6 samples (17.6%) could be genotyped using the MLST approach. Variability of genotypes was high but identical types were found in geographically related animals. Genotypes from wild boar showed phylogenetic relatedness to those from domestic pigs but no matching types could be identified. Results show that direct genotyping from wild boar lung samples is possible and provides a promising approach to investigate future EP outbreak related samples from wild boar. (C) 2011 Elsevier B.V. All rights reserved.

Diversity of Mycoplasma hyopneumoniae in pig farms revealed by direct molecular typing of clinical material

Mycoplasma hyopneumoniae is the etiological agent of enzootic pneumonia in swine. Various reports indicate that different strains are circulating in the swine population. We investigated the variety of M. hyopneumoniae strains by a newly developed genetic typing method based on the polyserine repeat motif of the LppS homolog P146. PCR amplification using M. hyopneumoniae specific, conserved primers flanking the region encoding the repeat motif, followed by sequencing and cluster analysis was carried out. The study included strains isolated from different geographic regions as well as lysates from lung swabs from a series of pig farms in Switzerland. High diversity of M. hyopneumoniae was observed but farms being in close geographic or operative contact generally seemed to be affected by the same strains. Moreover, analysis of multiple samples from single pig farms indicated that these harbored the same, farm-specific strain. The results indicate that multiple strains of M. hyopneumoniae are found in the swine population but that specific strains or clones are responsible for local outbreaks. The method presented is a highly reproducible epidemiologic tool allowing direct typing of M. hyopneumoniae from clinical material without prior isolation and cultivation of strains.

Multilocus sequence typing (MLST) of Mycoplasma hyopneumoniae: a diverse pathogen with limited clonality

A multilocus sequence typing (MLST) scheme was established and evaluated for Mycoplasma hyopneumoniae, the etiologic agent of enzootic pneumonia in swine with the aim of defining strains. Putative target genes were selected by genome sequence comparisons. Out of 12 housekeeping genes chosen and experimentally validated, the 7 genes efp, metG, pgiB, recA, adk, rpoB, and tpiA were finally used to establish the MLST scheme. Their usefulness was assessed individually and in combination using a set of well-defined field samples and strains of M. hyopneumoniae. A reduction to the three targets showing highest variation (adk, rpoB, and tpiA) was possible resulting in the same number of sequence types as using the seven targets. The established MLST approach was compared with the recently described typing method using the serine-rich repeat motif-encoding region of the p146 gene. There was coherence between the two methods, but MLST resulted in a slightly higher resolution. Farms recognized to be affected by enzootic pneumonia were always associated with a single M. hyopneumoniae clone, which in most cases differed from farm to farm. However, farms in close geographic or operational contact showed identical clones as defined by MLST typing. Population analysis showed that recombination in M. hyopneumoniae occurs and that strains are very diverse with only limited clonality observed. Elaborate classical MLST schemes using multiple targets for M. hyopneumoniae might therefore be of limited value. In contrast, MLST typing of M. hyopneumoniae using the three genes adk, rpoB, and tpiA seems to be sufficient for epidemiological investigations by direct amplification of target genes from lysate of clinical material without prior cultivation.

[Field validation of a real-time PCR test for the detection of Mycoplasma hyopneumoniae in nasal swabs of live pigs]

Enzootic pneumonia (EP) of pigs, caused by Mycoplasma hyopneumoniae has been a notifiable disease in Switzerland since May 2003. The diagnosis of EP has been based on multiple methods, including clinical, bacteriological and epidemiological findings as well as pathological examination of lungs (mosaic diagnosis). With the recent development of a real-time PCR (rtPCR) assay with 2 target sequences a new detection method for M. hyopneumoniae became available. This assay was tested for its applicability to nasal swab material from live animals. Pigs from 74 herds (average 10 pigs per herd) were tested. Using the mosaic diagnosis, 22 herds were classified as EP positive and 52 as EP negative. From the 730 collected swab samples we were able to demonstrate that the rtPCR test was 100% specific. In cases of cough the sensitivity on herd level of the rtPCR is 100%. On single animal level and in herds without cough the sensitivity was lower. In such cases, only a positive result would be proof for an infection with M. hyopneumoniae. Our study shows that the rtPCR on nasal swabs from live pigs allows a fast and accurate diagnosis in cases of suspected EP.

Development of two real-time PCR assays for the detection of Mycoplasma hyopneumoniae in clinical samples

In order to improve the diagnosis of enzootic pneumonia (EP) in pigs two real-time polymerase chain reaction (rtPCR) assays for the detection of Mycoplasma hyopneumoniae in bronchial swabs from lung necropsies were established and validated in parallel. As a gold standard, the current "mosaic diagnosis" was taken, including epidemiological tracing, clinical signs, macro- and histopathological lesions of the lungs and immunofluorescence. One rtPCR is targeting a repeated DNA element of the M. hyopneumoniae genome (REP assay), the other a putative ABC transporter gene (ABC assay). Both assays were shown to be specific for M. hyopneumoniae and did not cross react with other bacteria and mollicutes from pig. With material from pigs of defined EP-negative farms the two assays showed to be 100% specific. When testing lungs from pig farms with EP, the REP assay detected 50% and the ABC assay 90% of the farms as positive. Both tests together detected all positive farms. Within a positive herd the two assays tested similarly with on average over 90% of the lung samples analysed from a single farm showing positive scores. A series of samples with suspicion of EP and samples from pigs with diseases other than respiratory taken from current routine diagnostic was assayed. None of the assays showed false positive results. The sensitivities in this sample group were 50% for the REP and 70% for the ABC assays and for both assays together 85%. The two assays run in parallel are therefore a valuable tool for the improvement of the current diagnosis of EP.

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.