Evaluation of the immunogenicity of the P97R1 adhesin of Mycoplasma hyopneumoniae as a mucosal vaccine in mice.

The immunogenicity of P97 adhesin repeat region R1 (P97R1) of Mycoplasma hyopneumoniae, an important pathogenesis-associated region of P97, was evaluated in mice as a mucosal vaccine. Mice were immunized orally with attenuated Salmonella typhimurium aroA strain CS332 harbouring a eukaryotic or prokaryotic expression vector encoding IP97R1. Local and systemic immune responses were analysed by ELISA on mouse sera, lung washes and splenocyte supernatants following splenocyte stimulation with specific antigens in vitro. Although no P97R1-specific antibody responses were detected in serum and lung washes, significant gamma interferon was produced by P97R1-stimulated splenocytes from mice immunized orally with S. typhimurium aroA harbouring either expression system, indicating induction of a cell-mediated immune response. These results suggested that live bacterial vectors carrying DNA vaccines or expressing heterologous antigens preferentially induce a Th1 response. Surprisingly, however, mice immunized with the vaccine carrier S. typhimurium aroA CS332 induced serum IgG, but not mucosal IgA, against P97R1 or S. typhimurium aroA CS332 whole-cell lysate, emphasizing the importance of assessing the suitability of attenuated S. typhimurium antigen-carrier delivery vectors in the mouse model prior to their evaluation as potential vaccines in the target species, which in this instance was pigs.

COMPARATIVE-ANALYSIS OF GLYCOPROTEIN AND GLYCOLIPID COMPOSITION OF VIRULENT AND AVIRULENT STRAIN MEMBRANES OF MYCOPLASMA-HYOPNEUMONIAE

The glycoproteins and glycolipids from membranes of virulent strain Z and avirulent strain M of Mycoplasma hyopneumoniae have been compared. The proteins and the glycoproteins were identified by SDS-polyacrylamide gel electrophoresis and concanavalin A-biotin labeling, respectively. The membrane preparation contained approximately 34 protein bands with molecular weights between 20 KD and 100 KD. The concanavalin A-biotin system reacted with a glycoprotein of a molecular weight of approximately 28,000 from avirulent strain M and did not react with the correspondent band from virulent strain Z. The membrane glycolipids of both strains consisted of monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), and the percentages of 16:0, 18:0, and 18:1 fatty acids comprised more than 80% of the total fatty acids of membrane glycolipids. The 18:0 fatty acid of MGDG in avirulent strain M was twofold higher than that of virulent strain Z.

Application of flow cytometry for the determination of minimal inhibitory concentration of several antibacterial agents on Mycoplasma hyopneumoniae

P. Assun??o, N.T. Antunes, R.S. Rosales, C. Poveda, C. de la Fe, J.B. Poveda and H.M. Davey (2007). Application of flow cytometry for the determination of minimal inhibitory concentration of several antibacterial agents on Mycoplasma hyopneumoniae. Journal of Applied Microbiology, 102(4), 1132-1137. Sponsorship: Gobierno de Canarias (IDT-LP-04/016) RAE2008

Evaluation of Mycoplasma hyopneumoniae bacterins for porcine torque teno virus DNAs

Objective-TO determine whether commercial Mycoplasma hyopneumoniae bacterins sold for use in swine contain porcine torque teno virus (TTV).

MHJ_0125 is an M42 glutamyl aminopeptidase that moonlights as a multifunctional adhesin on the surface of Mycoplasma hyopneumoniae

Bacterial aminopeptidases play important roles in pathogenesis by providing a source of amino acids from exogenous proteins, destroying host immunological effector peptides and executing posttranslational modification of bacterial and host proteins. We show that MHJ_0125 from the swine respiratory pathogen Mycoplasma hyopneumoniae represents a new member of the M42 class of bacterial aminopeptidases. Despite lacking a recognizable signal sequence, MHJ_0125 is detectable on the cell surface by fluorescence microscopy and LC-MS/MS of (i) biotinylated surface proteins captured by avidin chromatography and (ii) peptides released by mild trypsin shaving. Furthermore, surface-associated glutamyl aminopeptidase activity was detected by incubation of live M. hyopneumoniae cells with the diagnostic substrate H-Glu-AMC. MHJ_0125 moonlights as a multifunctional adhesin, binding to both heparin and plasminogen. Native proteomics and comparative modelling studies suggest MHJ_0125 forms a dodecameric, homopolymeric structure and provide insight into the positions of key residues that are predicted to interact with heparin and plasminogen. MHJ_0125 is the first aminopeptidase shown to both bind plasminogen and facilitate its activation by tissue plasminogen activator. Plasmin cleaves host extracellular matrix proteins and activates matrix metalloproteases, generating peptide substrates for MHJ_0125 and a source of amino acids for growth of M. hyopneumoniae. This unique interaction represents a new paradigm in microbial pathogenesis.

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.