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.     

猪肺炎支原体的免疫原蛋白及疫苗的研究进展

猪肺炎支原体是引起猪支原体肺炎的主要病原,严重危害着养猪业的发展。本文综合国内外猪肺炎支原体主要免疫原及疫苗的研究进展,从而为猪肺炎支原体致病机理的进一步研究及该病的防治提供新思路。     

Risk factors for the reinfection of specific pathogen-free pig breeding herds with enzootic pneumonia.

A case control study was designed to determine the risk factors for the reinfection of Swiss specific pathogen-free (SPF) pig herds with enzootic pneumonia. Detailed housing, management and environmental data were collected from 42 case farms and 50 control farms by means of a questionnaire. Factors with a significantly asymmetrical frequency distribution among the two groups were considered to be possibly associated with reinfection; they included the distance to the nearest non-SPF pig herd, the size of that herd, the density of the pig population in the area, the distance to the next road regularly carrying pig transporters and differences in topography. The results tended to support the hypothesis of the airborne transmission of enzootic pneumonia. Using a formula considering the main risk factors, it was possible to classify farms as high or low risk with an 84 per cent specificity and 74 per cent sensitivity.     

Enzootic pneumonia of pigs--a diagnostic dilemma

In an enzootic pneumonia-free Australian pig herd, an outbreak of a severe respiratory disease in the grow-out herd was initially diagnosed as acute tracheitis and pneumonia precipitated by the dusty environment, with a superimposed mixed infection of Pasteurella multocida and Arcanobacterium pyogenes. Culture for Mycoplasma hyopneumoniae, Salmonella sp and fungi was negative. The outbreak persisted. Subsequently, gross lesions consistent with enzootic pneumonia occurred, but histological lesions were equivocal and definitive tests for M hyopneumoniae remained negative. Eighteen months after the initial outbreak, gross and histological lesions were consistent with enzootic pneumonia but serological tests were still negative. Almost 2 years later, one of four nasal swabs was positive by the polymerase chain reaction test for M hyopneumoniae, and then lung samples were sporadically positive. The pneumonic disease became endemic in the herd. Gross lesions consistent with enzootic pneumonia occurred in another herd belonging to the same company nearly 2 years after the initial outbreak. Again, results of laboratory tests were inconsistent. Despite sporadic positive polymerase chain reaction tests for M hyopneumoniae, the respiratory disease resolved within 4 months and there has been no clinical evidence of enzootic pneumonia during the subsequent 4 years. These cases raise important questions about the role of the diagnostic tests and their interpretation, and the ecology of M hyopneumoniae and its role in enzootic pneumonia.     

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.     

一株猪肺炎支原体的分离鉴定

从全国部分猪场采集到疑似猪支原体肺炎肺组织病料12份,提取DNA进行猪肺炎支原体PCR和多重PCR检测,将病料研磨后分离猪肺炎支原体,最终分离到1株疑似猪肺炎支原体;通过测序分析、形态观察、生化试验、血清学试验证实其为猪肺炎支原体。该菌株能适应人工培养基的培养,且传代生长良好,液体培养基中培养活菌滴度达109CCU/m L;菌株有一定的致病性,免疫原性好,可作为疫苗备用菌株,该菌株的分离鉴定为研制猪支原体肺炎疫苗奠定了基础。     

Eradication of Mycoplasma hyopneumoniae from infected swine herds joining the LSO 2000 health class

The study was conducted in order to determine if eradication of swine enzootic pneumoniae (SEP) had succeeded with different variants of partial depopulation during the eradication programme on swine farrowing farms joining a health class, LSO 2000. The farms in the health class need to be free from swine enzootic pneumoniae, swine dysentery, sarcoptic mange and atrophic rhinitis. Twenty-one eradication attempts for M. hyopneumoniae were carried out using different variants based on separating adult animals for 2 weeks from infected young pigs which were not returned to the herd. The infected young pigs were kept in the same building (variant 1) in 4 herds and on the same compound (variant 2) as disease-free pigs in 12 herds. The infected young pigs were finally all sold. In 5 herds only adult animals were present during the eradication (variant 3). The eradication attempt succeeded in 81% and failed or remained uncertain in 19% of the herds. The result was confirmed with 1) frequent clinical follow-up of the health status in the herds (both the farrowing and the finishing units) joining the LSO 2000 health class 2) milk and/or blood serology. Possible causes of the failure of the eradication attempt were described: a short distance between infected and uninfected animals, the time period between diagnosis of SEP and initiation of the programme, the age of the youngest animal kept on the farm, the period of time when animals with different status were reared close to each other, the medications used, the cleaning of the facilities during the programme and the season. Further, a good cooperation between the farmer, the local veterinarian and the animal health service of the slaughterhouse was an essential part of the initiation and the follow-up of the programme. The secondary aim of the study was to collect information about the expenses during the programme. Only 57% of the farmers gave some estimates for the expenses on their farms. For variants 1, 2 and 3 the expenses were 879, 1110 and 1274 FIM per sow (1 USD = 5.5 FIM), respectively (p > 0.1).     

ISOLATION OF MYCOPLASMA HYOPNEUMONIAE AND ITS ASSOCIATION WITH PNEUMONIA OF PIGS IN AUSTRALIA

Nine strains of mycoplasmas were isolated from the lungs of 5 pigs with clinical signs of naturally acquired enzootic pneumonia. Mycoplasma hyopneumoniae was isolated from the lungs of 1 pig and M. hyorhinis from the lungs of 4. An unidentified mycoplasma, which utilized arginine, grew rapidly in broth culture and produced centred colonies on solid medium, was isolated from the lungs of 4 pigs. The pathogenicity of the isolated strain of M. hyopneumoniae was determined by inoculation of pigs from an enzootic pneumonia-free herd. Enzootic pneumonia was produced in the lungs of all 5 pigs inoculated intranasally and intratracheally with broth cultures of the organism isolatied by limit dilution techniques. Enzootic pneumonia was produced in 3 of 6 pigs inoculated intranasally and intratracheally with M. hyopneumoniae purified by the passage of colonies on agar blocks. M. hyopneumoniae was isolated in pure culture from the lungs of all pigs with induced pneumonic lesions.     

Immunological characterization of Mycoplasma hyopneumoniae recombinant proteins

Mycoplasma hyopneumoniae, the primary pathogen of enzootic pneumonia, is highly prevalent worldwide and causes major economic losses to the pig industry. Commercial vaccines are widely used in the control of this disease, however, they provide only partial protection. The aim of this study was to evaluate 34 recombinant proteins of M. hyopneumoniae expressed in Escherichia coli. Antigenic and immunogenic properties of these proteins were analyzed. For this, the proteins were tested against hyperimmune and convalescent pig sera through ELISA and Western blot. Immunogenicity of the recombinant proteins was evaluated in BALB/c mice following intramuscular inoculation. Most antigens were able to induce a strong immune response and sera from inoculated mice were able to recognize native proteins by cell ELISA and Western blot. Several recombinant proteins were specifically recognized by convalescent pig sera, indicating they are expressed during infection. These data may help to develop more efficacious vaccines against M. hyopneumoniae.     

A COMPLEMENT-FIXATION TEST FOR ENZOOTIC PNEUMONIA OF PIGS USING A COMPLEMENT DILUTION METHOD

Complement-fixing antibody to Mycoplasma hyopneumoniae in the serums of pigs experimentally infected with enzootic pneumonia was demonstrated by comparing the haemolytic titre of guinea-pig complement titrated in the presence of heated test serum, M. hyopneumoniae antigen and unheated normal pig serum with the titre obtained when the antigen was omitted. The haemolytic titres against sensitised sheep erythrocytes were determined after a fixation period of 16 to 18 hours at 5°C. When serums, collected at intervals of 3 to 7 days, from 43 pigs exposed to pigs experimentally infected with enzootic pneumonia were tested, 4.6 or more complement units were first fixed 14 to 44 (mean 23.4) days after contact began. Serums collected subsequently fixed from 4.6 to more than 31 complement units. This positive reaction usually persisted until the pigs were killed 4 to 35 weeks after contact began. Thirty-three had gross enzootic pneumonia lesions and 9 had lung lesions detected microscopically. Serum antibody was not detected in 73 weaned pigs aged 7 weeks in a pneumonia-free herd but serums from 9 of 15 unweaned piglets aged 9 to 14 days in the same herd, fixed between 3 and 7 complement units.     

Protective effects of an oral microencapsulated Mycoplasma hyopneumoniae vaccine against experimental infection in pigs.

Oral microencapsulated Mycoplasma hyopneumoniae vaccines were tested for their ability to prevent mycoplasmal pneumonia in pigs. Eighteen four-week-old specific pathogen free pigs were divided into six groups of three. Each pig of groups 1 to 4 was inoculated intramuscularly with formalin-inactivated M hyopneumoniae in adjuvant and boosted orally 14 days later with four different microencapsulated vaccine microspheres. Group 5 was used as a positive control. All 15 pigs of groups 1 to 5 were challenged at 28 days after the first vaccination by an intratracheal inoculation of pneumonic lung suspension. The three pigs of group 6 were used as a negative control. All four vaccinated groups showed some protection when challenged, but the protection was more solid in pigs boosted with vaccine D (group 4) which contained less porcine serum in the microsphere. The study indicates that oral vaccination with M hyopneumoniae could play a role in prevention and eradication of mycoplasmal pneumonia in the pig.     

An abattoir survey of pneumonia and pleuritis in slaughter weight swine from 9 selected herds. III. Serological findings and their relationship to pathomorphological and microbiological findings

Blood samples from 777 pigs, originating from 9 different herds, were collected at slaughter and examined for antibodies to Mycoplasma hyopneumoniae and Actinobacillus (Haemophilus) pleuropneumoniae by the indirect hemagglutination assay (IHA) and the complement fixation (CF) test, respectively. Results were compared to pathological and microbiological findings. Antibodies to M. hyopneumoniae in positive titers of 1/80 or higher were found in 62% of the samples. The relationship between positive IHA titers to M. hyopneumoniae and gross findings indicative of enzootic pneumonia of pigs (EPP), histological findings indicative of EPP, the isolation of M. hyopneumoniae and the demonstration of M. hyopneumoniae by indirect immunofluorescent testing ranged from 64% to 68%. No correlation was noted between positive IHA titers and the isolation of Mycoplasma flocculare. Positive antibody titers to A. pleuropneumoniae of 1/10 or higher were detected in 5% to 85% of the samples from individual herds. Positive titers to A. pleuropneumoniae serotype 2 were found in 71% to 79% of the sampled animals from herds with high frequencies of pneumonic lesions indicative of pleuropneumonia. In herds with low frequencies of pleuropneumonia, positive titers were recorded in from 0 to 4% of the tested pigs. However, no statistical association was found between pleuropneumonia and positive titers to A. pleuropneumoniae serotype 2 in individual animals. Twenty-one per cent of samples with positive CF titers to A. pleuropneumoniae showed antibodies to more than one serotype.     

Efficacy of in-feed medication with tylosin for the treatment and control of Mycoplasma hyopneumoniae infections

The efficacy of in-feed medication with tylosin for the treatment of enzootic pneumonia was examined in an experimental Mycoplasma hyopneumoniae infection model. One group of 10 conventional M. hyopneumoniae-free pigs was inoculated intratracheally with a highly virulent field isolate of M. hyopneumoniae; a second group of 10 pigs was inoculated in the same way and after 12 days was given tylosin at 100 mg/kg feed for 21 days; a third group of 10 pigs was inoculated with sterile culture medium, and these pigs were not given tylosin. The pigs were examined daily for clinical signs and each pig was given a respiratory disease score. Thirty-three days after they had been infected the pigs were euthanased, the lung lesions were quantified and samples of lung were processed for immunofluorescence testing for M. hyopneumoniae. The mean (sd) respiratory disease and lung lesion scores were significantly higher (P<0.05) in both the infected groups than in the uninfected group. Between 23 and 33 days after infection the mean respiratory disease score of the pigs treated with tylosin was 0.54 (0.22), significantly (P<0.05) lower than that of the infected pigs which were left untreated, 1.54 (0.46); similarly, their average lung lesion score, 1.72 (1.20), was significantly lower than that of the untreated pigs, 5.27 (3.85).     

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.     

Effect of enzootic pneumonia of pigs on growth performance

Growing pigs were naturally infected with a field strain of Mycoplasma hyopneumoniae to assess the effect of enzootic pneumonia on production. Both the initial ("breakdown") and endemic stages of infection were evaluated. The pigs were reared under environmental and management conditions commonly found on commercial piggeries in South Australia. Growth rate of pigs held in-contact with inoculated pigs was reduced by 12.7% (p less than 0.01) between 50 to 85 kg bodyweight. In the second trial inoculated gilts were used to naturally infect piglets during suckling. Growth rate of infected pigs was reduced by 15.9% (p less than 0.001) between 8 to 85 kg bodyweight, while feed conversion was depressed by 13.8% (p less than 0.05) between 10 to 25 kg bodyweight. At current feed and production costs this reduced performance added approximately $2.80 to the cost of every pig produced. These losses were recorded in groups of pigs in which enzootic pneumonia was present. At slaughter, 40% of lungs contained gross lesions of enzootic pneumonia which were free of significant secondary bacteria. The nature of the infection was established by gross and microscopic pathology and confirmed by the detection of specific complement fixing antibody in infected pigs and the demonstration of M. hyopneumoniae by direct immunofluorescent staining of lung sections.     

Apparent reinfection of enzootic-pneumonia-free pig herds: early signs and incubation period

Since 1959, the Pig Health Control Association (PHCA) has run a national health-control scheme for pig herds believed to be free from enzootic pneumonia. During this time, many herds developed this disease without a simple explanation. From 1968, 55 such unexplained breakdowns have been studied in detail. The first signs in 50 breakdowns were either coughing in growing pigs (52 per cent of outbreaks), illness in adult stock (34 per cent of outbreaks) or pneumonia in routinely slaughtered pigs (14 per cent of outbreaks). In some outbreaks, enzootic pneumonia appeared to grow out of a pre-existing respiratory infection, which was not identified as enzootic pneumonia, in suckling pigs, suggesting that either Mycoplasma hyopneumoniae was already present in a latent state, or it more readily seeded damaged respiratory tracts from outside. In three outbreaks of this type, where pathological material was collected during the transition period, no laboratory evidence was obtained for the presence of M hyopneumoniae in the primary respiratory disease. Analysis of breakdowns in two national testing stations indicated that clinical/pathological signs might not develop until three to five months after the introduction of an infected group of weaners. It is possible, therefore, that a pig herd might not show obvious signs of the disease until up to six months or more after initial infection. There was little evidence to indicate that unexplained breakdowns arose from long term latent infection in other herds from which stock had been imported. There was considerable evidence, however, to suggest that breakdowns arose from extraneous sources.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

An abattoir survey of pneumonia and pleuritis in slaughter weight swine from 9 selected herds. II. Enzootic pneumonia of pigs: microbiological findings and their relationship to pathomorphology

Lungs from 191 slaughter pigs with gross lesions indicative of enzootic pneumonia of pigs (EPP) and 80 grossly normal lungs, all originating from 9 different herds, were subjected to microbiological and pathological examinations. The microbiological studies included both bacterial and mycoplasmal culture and also testing for Mycoplasma hyopneumoniae antigen in tissue by indirect immunofluorescent technique. M. hyopneumoniae, Pasteurella multocida and Mycoplasma hyorhinis were detected in 83%, 43% and 37% of the pneumonic lungs, respectively. Mycoplasma flocculare was the most frequently isolated organism in the non-pneumonic lungs. The greatest amounts of macroscopic pneumonia (25.2%) were recorded in lungs with all the three agents M. hyopneumoniae, P. multocida and M. hyorhinis present. The amounts of pneumonia in lungs with M. hyopneumoniae alone and in concurrence with P. multocida, were 9.3% and 15.6%, respectively. M. hyorhinis was also, in this study, associated with higher frequency of diffuse pleuritis. These findings indicate that M. hyorhinis might be involved in the pathogenesis of pneumonia in slaughter pigs. Ninety-six per cent of the isolates of P. multocida from pneumonic lungs could be characterized as type A. In the herds which had the most severe pneumonia problems, toxin production was detected in 83% of the P. multocida strains while only 28% were toxigenic in herds with subclinical to moderate pneumonia problems.     

A nested PCR assay for the detection of Mycoplasma hyopneumoniae in tracheobronchiolar washings from pigs

A nested polymerase chain reaction (PCR) was developed for the detection of Mycoplasma hyopneumoniae, the etiological agent of enzootic pneumonia, in tracheobronchiolar washings from live pigs. Two nested pairs of oligonucleotide primers were designed from the sequence of a specific DNA probe (I 141; accession number U02537). The primer combination was Hp1/Hp3 for the first step PCR while the nested primers (Hp4/Hp6) allowed amplification of a 706 bp fragment. All strains of M. hyopneumoniae tested in this study could be detected by the nested PCR. DNA from other bacterial species isolated from the respiratory tract of pigs or from other mycoplasmal species were not amplified. The detection limit was estimated to be 1 fg, corresponding approximately to one organism, while in the one step PCR previously described 4 x 10(2) organisms were required. The nested PCR was evaluated on 362 tracheobronchiolar lavages collected from pigs at 2, 4 and 6 months of age in eight herds chronically infected with M. hyopneumoniae. The nested PCR was compared to a blocking ELISA performed with sera collected from the same pigs at the same ages, and to an immunofluorescence test at slaughter on 65 lungs from 6-month old pigs. The comparison indicated that the nested PCR was significantly (p<0.05) more sensitive (157 positive results of 362 samples) than ELISA (118 positive results of 362 samples) for detection of M. hyopneumoniae infection. Nested PCR was also significantly more sensitive (54 positive results of 65 samples) than immunofluorescence (29 positive results of 65 samples) for detection of M. hyopneumoniae in pig lungs at slaughter. Moreover, the nested PCR was used to confirm the absence of the mollicute in a pig herd without any history of M. hyopneumoniae infection. Thus, nested PCR appears to be a useful test to assess M. hyopneumoniae infection on pig farms.     

Enzootic pneumonia (EP) in Swiss swine herds after area-wide eradication: epidemiological analysis of outbreaks occurring between 1999 and 2003

In a retrospective analysis, infections of swine herds with Enzootic Pneumonia (EP), occurring after the regional eradication programme had been completed, were evaluated and described according to epidemiological criteria. The aim of this study was to obtain interim results about status, progress and trends of the area-wide eradication in Switzerland over all regions involved in the programme. The population comprised pig farms with eradication of EP completed by the end of 2002. Incidence of EP infection was calculated for the years 2000-2003. Seasonal effects, influences from production-type, herd size, and pig density in the surrounding area on incidences of EP outbreaks were investigated. In 2000-2002, annual incidences were steadily decreasing from 3.1% to 2.0%, and they showed great regional variation. In 2003, contrary to the long-term trend, a minor increase of overall incidence from 2.0% to 2.1% was observed while the incidence for breeding-only farms continued to recede. Possible explanations for this are discussed. Previous observations on EP infections accumulating in the cold season could not be confirmed. Large farms, finishing farms, and farms located in densely populated pig areas as well as farms being part of multisite production rings had a higher risk of infection. In spite of occurrence of EP outbreaks in eradicated areas, the current status of the area-wide eradication is promising, and a further decline in incidence of EP infection can be expected in the future.