The Occurrence of Mycoplasmas in the Lungs of Swine in Gran Canaria (Spain)

The study was conducted to investigate the mycoplasmal flora in the lungs of pigs with enzootic pneumonia at Gran Canaria (Spain). From 54 pneumonic lungs collected at an abattoir, 85 isolates were cultivated. On the basis of cultural and biochemical characteristics, the isolates were preliminarily identified as Mycoplasma species. Using different species-specific PCRs, 40, 27, 11 and 7 of the isolates were identified as M. hyorhinis, M. hyopneumoniae, M. hyosynoviae and M. flocculare, respectively. Nine of the M. hyopneumoniae cultures were found to be in mixed culture with M. flocculare as demonstrated by PCR. By use of a M. flocculare antiserum it was possible to eliminate M. flocculare from M. hyopneumoniae mixed cultures. This study is the first report on isolation of porcine mycoplasmas at Gran Canaria (Spain).     

A multiplex PCR to identify porcine mycoplasmas present in broth cultures

Mycoplasma hyopneumoniae, Mycoplasma hyorhinis and Mycoplasma flocculare can be present in the lungs of pigs at the same time. These three mycoplasma species all require similar growth conditions and can be recovered from clinical samples using the same media. We have developed a multiplex PCR as a helpful tool for rapid differentiation of these three species in the course of isolation. Based on the 16S ribosomal DNA sequences, three different forward primers and a single reverse primer were selected. Each forward primer was compared to available mycoplasma sequences, showing the primers to be specific. The three amplification products observed of 1129 bp (M. hyorhinis), 1000 bp (M. hyopneumoniae) and 754 bp (M. flocculare) were clearly distinguishable on a 1% agarose gel. In addition, no cross-reaction with Mycoplasma hyosynoviae, another porcine mycoplasma, was noted. This multiplex PCR using the proposed set of primers is the first reported assay that allows the simultaneous identification of the different Mycoplasma species isolated from the lungs of pigs.     

Chronological Development of Mycoplasma hyorhinis and Mycoplasma hyosynoviae Infections in Cultures of a Swine Synovial Cell Strain

The sequential development of Mycroplasma hyorhinis and Mycoplasma hyosynoviae was observed in cultures of a swine synovial fluid cell strain. An early transitory filamentous phase was observed with M. hyorhinis infection followed by the development of cell-associated, relatively large, round structures and some ring forms. Infection with M. hyorhinis was characterized by a generalized distribution of the organism and a severe cytopathic effect.

Infection with M. hyosynoviae was represented by the development of circumscribed foci of small pleomorphic structures and a milder effect on the cells. At a high multiplicity of infection, this organism became associated with the cytoplasmic membranes of the cells.

     

Seroprevalence of Mycoplasma hyopneumoniae in pigs in subtropical southern China

Enzootic pneumonia caused by Mycoplasma hyopneumoniae is a severe disease of pigs, causing significant economic losses to the pig industry worldwide, including the tropical and subtropical regions. In order to obtain the baseline prevalence of M. hyopneumoniae in pigs from intensive farms in southern China, double-sandwich enzyme-linked immunosorbent assay (ELISA) was used to detect M. hyoneumoniae antibodies in 460 pig serum samples collected from 12 administrative cities in China’s southern Guangdong province. According to the proportions of the infected animals, among the 12 intensive farms, only two of them showed no infection of M. hyoneumoniae and the seroprevalence ranged from 0% to 90%, with an averaged prevalence of 45.7%. The highest prevalence was found in breeding boars (68.8%), followed by sows (54.5%). These data showed that the infection of pigs with M. hyopneumoniae is severe, and boars might be more important carriers and transfers of M. hyoneumoniae than sows. Integrated strategies and measures should be taken to control the infection of pigs with M. hyopneumoniae in southern China.     

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.     

Quantitative real-time polymerase chain reaction for detecting Mycoplasma hyosynoviae and Mycoplasma hyorhinis in pen-based oral,tonsillar, and nasal fluids

Mycoplasma (M.) hyorhinis and M. hyosynoviae are pathogens known to cause disease in pigs post-weaning. Due to their fastidious nature, there is increased need for culture-independent diagnostic platforms to detect these microorganisms. Therefore, this study was performed to develop and optimize quantitative real-time PCR (qPCR) assays to rapidly detect M. hyorhinis and M. hyosynoviae in pen-based oral fluids as well as nasal and tonsillar fluids as proxies for samples used in swine herd surveillance. Two methods of genomic DNA extraction, automated versus manual, were used to compare diagnostic test performance. A wean-to-finish longitudinal study was also carried out to demonstrate the reproducibility of using pen-based oral fluids. Overall, pen-based oral and tonsillar fluids were more likely to be positive for both types of bacteria whereas only M. hyorhinis was detected in nasal fluids. DNA extraction protocols were shown to significantly influence test result. Although the initial detection time somewhat differed, both organisms were repeatedly detected in the longitudinal study. Overall, this study evaluated two qPCR methods for rapid and specific detection of either mycoplasma. Results from the present investigation can serve as a foundation for future studies to determine the prevalence of the two microorganisms, environmental load, and effectiveness of veterinary interventions for infection control.     

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.     

A blocking ELISA using a monoclonal antibody for the serological detection of Mycoplasma hyopneumoniae

A blocking ELISA was developed by using a monoclonal antibody (4082-05-344-18) which specifically detected an epitope on the Mycoplasma hyppneumoniae 40 kDa membrane protein without cross-reacting with M flocculare or M hyorhinis. The results obtained with sera from specific pathogen-free pigs inoculated with M flocculare or M hyorhinis confirmed the specificity of the assay. An immunoblotting procedure was used to characterise the antibody response of pigs experimentally infected with M hyopneumoniae. Antibodies to the 40 kDa antigen were detected two weeks after infection and remained as major markers for at least 20 weeks. Cross-reacting antibodies to this antigen were not detected in convalescent sera from piglets infected with M flocculare or M hyorhinis. Sera from experimentally infected pigs were compared by means of the blocking ELISA and an indirect ELISA. The kinetics of ELISA antibodies after experimental inoculation were also studied. The detection of antibody was rather more stable for a longer time with the blocking ELISA than with the indirect ELISA. In an evaluation of more than 1000 sera from the field there was excellent agreement between the two methods.     

Reduced lung lesions in pigs challenged 25 weeks after the administration of a single dose of Mycoplasma hyopneumoniae vaccine at approximately 1 week of age

Two independent studies assessed the duration of immunity of an inactivated adjuvanted Mycoplasma hyopneumoniae vaccine against mycoplasmal pneumonia in seronegative (study A, n = 52) and seropositive (study B, n = 52) pigs. The pigs were allocated randomly to treatment and were then injected with a single dose of either the vaccine or a placebo at approximately 1 week of age. Twenty-five weeks after treatment administration, the pigs were challenged with a virulent strain (LI 36, Strain 232) of M. hyopneumoniae and the extent of lung lesions consistent with mycoplasmal pneumonia was assessed 4 weeks later.In study A, the geometric mean lung lesion score (expressed as least squares mean percentages of lung lesions) was significantly (P = 0.0001) lower in vaccinated (0.3%, n = 20) than in control pigs (5.9%, n = 24) seronegative to M. hyopneumoniae at enrolment; similarly, in study B, the extent of lung lesions was significantly reduced (P = 0.0385) in seropositive vaccinated pigs (2.0%, n = 22) compared to controls (4.5%, n = 26). At the end of the investigation period, 4 weeks after challenge, mean antibody sample-to-positive (S/P) ratios were significantly higher both in seronegative (P = 0.0012) and seropositive (P = 0.0001) vaccinated pigs (mean values = 0.77 and 0.81, respectively) than in controls (mean values = 0.51 and 0.38, respectively).     

Enzootic Pneumonia of Pigs: Identification of a Causative Mycoplasma in Infected Pigs and in Cultures by Immunofluorescent Staining

Immunofluorescent staining has been used to identify Mycoplasma hyopneumoniae in smears of broth cultures, in infected pig testicle cell cultures, and in frozen cut sections of pneumonic lungs from field and experimentally produced cases of enzootic pneumonia. In the pneumonic pig lung, fluorescent staining was limited to the surface of the bronchial and bronchiolar epithelium and to the contained exudate. In a series of trials using experimentally infected pigs fluorescence was not detected until 25 days post-infection and was regularly seen in pigs killed thereafter. Porcine immune globulin precipitated from the serum of experimentally infected pigs and conjugated with fluorescein isothiocyanate was reactive and specific for the detection of M. hyopneumoniae. Immune globulin conjugates prepared from the serum of hyperimmunized rabbits were reactive but in some cases produced a faint non-specific staining of frozen tissue sections. No such non-specific reactions were noted on stained culture smears or cell cultures.

Fluorescence was not seen in known positive preparations stained with non-immune pig globulin conjugates or in preparations from uninoculated cell cultures or pigs, stained with non-immune or immune globulin conjugates.

Mycoplasma hyorhinis was detected by immunofluorescent staining with homologous conjugates, in smears of broth cultures and in tissue sections from pigs with polyserositis.

Immunofluorescent staining was found to be species specific and useful for the early species identification of mycoplasma isolated from pigs.

     

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.     

Evaluation of serovar-independent ELISA antigens of Actinobacillus pleuropneumoniae in pigs, following experimental challenge with A. pleuropneumoniae, Mycoplasma hyopneumoniae and Pasteurella multocida

Objective To compare the sensitivity and specificity of six serological enzyme‐linked immunosorbent assays (ELISAs) based on serovar‐independent antigens of Actinobacillus pleuropneumoniae (App) and investigate cross‐reactivity in disease‐free pigs challenged with Mycoplasma hyopneumoniae and Pasteurella multocida. Design Five experimental pig trials using direct challenge with App serovars 1, 7 or 15 or direct challenge with M. hyopneumoniae and/or various dose rates of P. multocida. Procedure A 39‐kDa outer membrane protein antigen and five recombinant antigens from the apxIVA gene of App were evaluated. The latter were derived from the ApxIVA N‐terminus (ApxIVA‐N, ApxIVA‐NP, ApxIVA‐NPS) or C‐terminus (ApxIVA‐C, ApxIVA‐CP). Pigs were sampled after challenge and clinical and necropsy findings evaluated. Results The 39‐kDa ELISA had high sensitivity but lacked specificity, with significantly increased cross‐reactivity following P. multocida challenge. ELISAs based on ApxIVA N‐terminus antigens were significantly more sensitive than C‐terminus antigens for the detection of App‐induced disease. Although ApxIVA‐N and ApxIVA‐NP ELISAs had increased reactivity following P. multocida challenge, they retained high specificity for App‐induced disease (90–93%). Affinity purified ApxIVA‐NP antigen had marginally better specificity than ApxIVA‐N, without reduced sensitivity. Mycoplasma hyopneumoniae did not affect serological cross‐reactivity. In disease‐free pigs, the specificity of the ApxIVA‐NPS ELISA may be adversely affected by nasal carriage of apparently low‐virulence App strains. Conclusions ApxIVA‐N‐based ELISAs can be used for evaluating App status in commercial herds, but some appear limited by high carriage rates of low‐virulence App. The 39‐kDa antigen is only of merit in exclusion of App disease by negative serology.     

Optimized protocol for multiplex nested polymerase chain reaction to detect and differentiate Haemophilus parasuis,Streptococcus suis,and Mycoplasma hyorhinis in formalin-fixed,paraffin-embedded tissues from pigs with polyserositis

An optimized protocol was developed for the simultaneous detection and differentiation of Haemophilus parasuis, Streptococcus suis, and Mycoplasma hyorhinis in formalin-fixed, paraffin-embedded (FFPE) tissues with multiplex nested polymerase chain reaction (PCR). This method also determines the prevalence of these bacteria in pigs with polyserositis. DNA extraction with a combination of a commercial reagent and proteinase K resulted in more frequent detection of the pathogens than DNA extraction with proteinase K alone. Among FFPE tissue samples from 312 cases of polyserositis in which at least 1 bacterial species was detected, multiplex nested PCR detected H. parasuis in 239 (77%), S. suis in 124 (40%), and M. hyorhinis in 40 (13%). The disease was caused by a single pathogen in 224 (72%) of the cases and multiple pathogens in 88 (28%). Among the pigs positive for H. parasuis, S. suis, and M. hyorhinis by multiplex nested PCR, the pathogen was isolated from only 11%, 35%, and 28%, respectively. Therefore, the PCR protocol developed in this study is a useful diagnostic method when samples are negative after isolation methods and even for samples in which only 1 pathogen was isolated.     

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.     

Current perspectives on the diagnosis and epidemiology of Mycoplasma hyopneumoniae infection

Mycoplasma hyopneumoniae is the principal aetiological agent of enzootic pneumonia (EP), a chronic respiratory disease that affects mainly finishing pigs. Although major efforts to control M. hyopneumoniae infection and its detrimental effects have been made, significant economic losses in pig production worldwide due to EP continue. M. hyopneumoniae is typically introduced into pig herds by the purchase of subclinically infected animals or, less frequently, through airborne transmission over short distances. Once in the herd, M. hyopneumoniae may be transmitted by direct contact from infected sows to their offspring or between pen mates.The ‘gold standard’ technique used to diagnose M. hyopneumoniae infection, bacteriological culture, is laborious and is seldom used routinely. Enzyme-linked immunosorbent assay and polymerase chain reaction detection methods, in addition to post-mortem inspection in the form of abattoir surveillance or field necropsy, are the techniques most frequently used to investigate the potential involvement of M. hyopneumoniae in porcine respiratory disease. Such techniques have been used to monitor the incidence of M. hyopneumoniae infection in herds both clinically and subclinically affected by EP, in vaccinated and non-vaccinated herds and under different production and management conditions. Differences in the clinical course of EP at farm level and in the efficacy of M. hyopneumoniae vaccination suggest that the transmission and virulence characteristics of different field isolates of M. hyopneumoniae may vary. This paper reviews the current state of knowledge of the epidemiology of M. hyopneumoniae infection including its transmission, infection and seroconversion dynamics and also compares the various epidemiological tools used to monitor EP.     

Mycoplasma hyopneumoniae infection in pigs: Duration of the disease and evaluation of four diagnostic assays

200 SPF pigs were infected by aerosol with Mycoplasma hyopneumoniae and the development of clinical signs, serological and pathological reactions were studied. Mean time to onset of coughing was 13 days. A mean delay of 9 days was observed from onset of coughing until seroconversion against M. hyopneumoniae as measured by ELISA. At an individual level, the sensitivity for this ELISA was estimated to 98–100% and the specificity to 93–100%. Pasteurella multocida was isolated from the majority of the lungs 4 weeks post inoculation with M. hyopneumoniae and the lung lesions in pigs were significantly larger when P. multocida was present as compared to pigs with M. hyopneumoniae alone. An evaluation of cultivation, immunofluorescence, ELISA and polymerase chain reaction for demonstration of M. hyopneumoniae in lungs showed that all four methods have a high sensitivity in the acute stages of pneumonia. In the later stages the sensitivity of cultivation was superior to the other methods. No differences in specificity were observed between the methods. The antigen-ELISA OD values and the immunofluorescence scores revealed a strong positive correlation. Nasal swabs were additionally used for demonstration of M. hyopneumoniae and the polymerase chain reaction was found superior to the other methods.     

Characterization of Mycoplasma hyosynoviae Strains by Amplified Fragment Length Polymorphism Analysis,Pulsed‐Field Gel Electrophoresis and 16S Ribosomal DNA Sequencing

Mycoplasma hyosynoviae strains from Denmark, Germany, Japan, Sweden, the Netherlands and the UK were examined for variations in the genomic DNA and within the 16S ribosomal RNA (rRNA) gene. Variations in the chromosomal DNA among 57 isolates recovered from the respiratory tract and joints of pigs, were investigated by analysis of amplified fragment length polymorphisms of the BglII and MfeI restriction sites and by pulsed‐field gel electrophoresis of a BssHII digest of chromosomal DNA. Both methods allowed unambiguous differentiation of the analysed strains and showed similar discriminatory potential for the differentiation of M. hyosynoviae isolates. Concordant results obtained with the two whole‐genome fingerprinting techniques evidence the considerable intraspecies genetic heterogeneity of M. hyosynoviae. Sixteen field strains of M. hyosynoviae and the type strain S16^T were further examined for variation within the 16S rRNA gene. Ten field strains possessed the 16S rDNA sequences identical to the type strain, while the remaining six strains had sequences that differed by one to two nucleotides from that obtained from the type strain.     

Associations among Haemophilus parasuis,Mycoplasma hyorhinis,and porcine reproductive and respiratory syndrome virus infections in pigs with polyserositis

The objective of this study was to determine the associations among Haemophilus parasuis, Mycoplasma hyorhinis, and porcine reproductive and respiratory syndrome (PRRS) virus (EU-field strain) infections in 95 pigs with polyserositis. A significant association between H. parasuis and M. hyorhinis was identified. H. parasuis and M. hyorhinis were significantly more often detected in PRRS virus positive pigs.     

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

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

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.