Detection of Mycoplasma hyopneumoniae viability using a PCR-based assay.

Mycoplasma hyopneumoniae detection in clinical specimens is accomplished by PCR targeting bacterial DNA. However, the high stability of DNA and the lack of relationship between bacterial viability and DNA detection by PCR can lead to diagnostic interpretation issues. Bacterial messenger RNA is rapidly degraded after cell death, and consequently, assays targeting mRNA detection can be used for the exclusive detection of viable bacterial cells. Therefore, this study aimed at developing a PCR-based assay for the detection of M. hyopneumoniae mRNA and at validating its applicability to differentiate viable from inert bacteria. Development of the RNA-based PCR encompassed studies to determine its analytical sensitivity, specificity, and repeatability, as well as its diagnostic accuracy. Comparisons between DNA and mRNA detection for the same target gene were performed to evaluate the ability of the RNA-based PCR to detect exclusively viable M. hyopneumoniae after bacterial inactivation using various methods. The RNA-based PCR was also compared to the DNA-based PCR as a tool to monitor the growth of M. hyopneumoniae in vitro. Under the conditions of this study, the developed RNA-based PCR assay detected only viable or very recently inactivated M. hyopneumoniae, while the DNA-based PCR consistently detected cells irrespective of their viability status. Changes in growth activity over time were only observable via RNA-based PCR. This viability PCR assay could be directly applied to evaluate the clearance of M. hyopneumoniae or to determine the viability of the bacterium at late stages of eradication programs.

Characterizing the detection of inactivated Mycoplasma hyopneumoniae DNA in the respiratory tract of pigs.

A positive Mycoplasma hyopneumoniae PCR result in a clinical specimen may eventually represent the mere detection of non-viable bacteria, complicating the diagnostic interpretation. Thus, the objective of this study was to evaluate the PCR detection of non-viable M. hyopneumoniae and its residual cell-free DNA in live pigs. Pigs were inoculated with either active or inactivated M. hyopneumoniae and were sampled for up to 14 days. Mycoplasma hyopneumoniae was not detected by PCR at any timepoint in pigs inoculated with the inactivated bacterium, suggesting that in healthy pigs, the non-viable M. hyopneumoniae DNA was rapidly sensed and cleared.

A diagnostic approach to confirm Mycoplasma hyopneumoniae "Day zero" for pathogen eradication.

Breeding herds in the US are trending toward eradication of Mycoplasma hyopneumoniae (M. hyopneumoniae) due to the positive impact on welfare and downstream production. In an eradication program, "Day 0″ is the time point when the last replacement gilts to enter the herd were exposed to M. hyopneumoniae and marks the beginning of a herd closure. However, no specific diagnostic protocols are available for confirmation of successful exposure to define Day 0. Therefore, the objective of this study was to develop diagnostic guidelines, including sample collection approaches, for two common gilt exposure methods to confirm an entire population has been infected with M. hyopneumoniae following purposeful exposure. Forty gilts, age 21-56 days, were ear-tagged for longitudinal sample collection at five commercial gilt developer units (GDUs) and were exposed to M. hyopneumoniae by natural contact or aerosolization. Study gilts originated from sources known to be negative to major swine pathogens, including M. hyopneumoniae, and were sampled prior to exposure to confirm negative status, then every two weeks. Blood samples were collected for antibody detection, while laryngeal and deep tracheal secretions and pen based oral fluids were collected for PCR, and sampling continued until at least 85% of samples were positive by PCR. Detection of M. hyopneumoniae varied greatly based on sample type. Oral fluids showed the lowest detection in groups of gilts detected positive by other sample types. Detection by PCR in deep tracheal secretions was higher than in laryngeal secretions. Seroconversion to and PCR detection of M. hyopneumoniae on oral fluids were delayed compared to PCR detection at the individual level. By two weeks post-exposure, at least 85% of study gilts in three GDUs exposed by aerosolization were PCR positive in deep tracheal secretions. Natural contact exposure resulted in 22.5% of study gilts becoming PCR positive by two weeks post-initial exposure, 61.5% at four weeks, and 100% at six weeks on deep tracheal secretions. Deep tracheal secretions required the lowest number of gilts to sample for the earliest detection compared to all other samples evaluated. As observed in one of the GDUs using aerosolization, demonstration of failure to expose gilts to M. hyopneumoniae allowed for early intervention in the exposure protocol and delay of Day 0, for accurate timing of the eradication protocol. Sampling guidelines proposed in this study can be used for verification of M. hyopneumoniae infection of gilts following exposure to determine Day 0 of herd closure.

The effect of gilt flow management during acclimation on Mycoplasma hyopneumoniae detection.

The objective of this study was to characterize the Mycoplasma hyopneumoniae (M. hyopneumoniae) detection and seroconversion patterns in recently acclimated gilts to be introduced to endemically infected farms using different types of replacement management. Three gilt developing units (GDUs) belonging to sow farms were included in this investigation: two farms managed gilts in continuous flow, and one farm managed gilts all-in/all-out. Two replicates of 35 gilts each were selected per GDU and sampled approximately every 60 days for a total of four or five samplings, per replicate and per GDU. Detection of M. hyopneumoniae was evaluated by PCR, while antibodies were measured using a commercial ELISA assay. Also, M. hyopneumoniae genetic variability was evaluated using Multiple-Locus Variable number tandem repeat Analysis. Detection of M. hyopneumoniae was similar across GDUs. Although a significant proportion of gilts was detected positive for M. hyopneumoniae after acclimation, an average of 30.3 % of gilts was negative at any point during the study. Detection of M. hyopneumoniae antibodies was similar among GDUs regardless of flow type or vaccination protocol. The genetic variability analysis revealed a limited number of M. hyopneumoniae types within each GDU. Results of this study showed a similar pattern of M. hyopneumoniae detection by PCR and seroconversion by ELISA among GDUs, regardless of the type of flow management strategies applied to gilts.

Improving Mycoplasma hyopneumoniae diagnostic capabilities by harnessing the infection dynamics.

Mycoplasma hyopneumoniae remains one of the most problematic bacterial pathogens for pig production. Despite an abundance of observational and laboratory testing capabilities for this organism, diagnostic interpretation of test results can be challenging and ambiguous. This is partly explained by the chronic nature of M. hyopneumoniae infection and its tropism for lower respiratory tract epithelium, which affects diagnostic sensitivities associated with sampling location and stage of infection. A thorough knowledge of the available tools for routine M. hyopneumoniae diagnostic testing, together with a detailed understanding of infection dynamics, are essential for optimizing sampling strategies and providing confidence in the diagnostic process. This study reviewed known information on sampling and diagnostic tools for M. hyopneumoniae and summarized literature reports of the dynamics of key infection outcomes, including clinical signs, lung lesions, pathogen detection, and humoral immune responses. The information gathethered in this manuscript can facilitate better understanding of the performance of different diagnostic approaches at various stages of infection with Mycoplasma hyopneumoniae.

Effect of testing protocol and within-pen prevalence on the detection of Mycoplasma hyopneumoniae DNA in oral fluid samples.

Combinations of 2 nucleic acid extractions and 3 Mycoplasma hyopneumoniae (MHP) PCRs (namely Protocol 1, 2, 3, and 4) were compared in terms of the probability of detecting DNA in pen-based oral fluid samples as a function of within-pen MHP prevalence. Oral fluid samples were created by randomly assigning 39 7-week old pigs to one of 5 pens, i.e., negative control pen (3 pigs) and 4 pens of 9 pigs each that differed in the proportion of MHP-inoculated pigs (1, 3, 6, or 9). Deep tracheal swabs were collected twice weekly to establish individual pig MHP infection status and derive within-pen prevalence estimation. On DPI 3, tracheal swabs from 15 of 19 inoculated pigs were MHP DNA positive. Oral fluids (n = 320) were collected daily from - 4 to 59 days post inoculation (DPI). Using a piecewise exponential model to account for within-pen transmission dynamics followed by a mixed-effect logistic regression, the probability of detecting MHP DNA in oral fluids was positively associated with within-pen prevalence (P < 0.0001) and differed among test protocols. MHP DNA was detected in 173 oral fluid samples with Protocol 3 versus 148, 134, and 101 with Protocols 4, 2, and 1, respectively. At 100% within-pen prevalence, the probability of detecting MHP DNA in oral fluids was highest using Protocol 3 (95.7%), followed by Protocols 4 (70.1%), 2 (60.1%), and 1 (34.0%). The fact that PCR protocols performed differently suggests that further improvements in extraction methods and MHP PCRs are possible. In the field, the dynamics of MHP infections should be taken into account if using oral fluid samples in surveillance.

Mycoplasma hyopneumoniae detection by PCR in naturally infected finishing pigs.

The aim of this study was to compare the sensitivity of different in vivo and post-mortem samples collected from finishing pigs under field conditions on Mycoplasma hyopneumoniae detection by PCR. Results suggested that tracheobronchial secretions and bronchial swabs conferred the highest sensitivity in vivo and post-mortem, respectively.

Environmental detection of Mycoplasma hyopneumoniae in breed-to-wean farms.

There is a need to develop cost-effective and non-invasive approaches to sample large populations to evaluate the disease status of breeding herds. In this study we assessed the detection of the M. hyopneumoniae genetic material in environmental surfaces and air of farrowing rooms, and skin (udder, snout and vagina) of lactating sows at weaning, in farms having different M. hyopneumoniae infection status (negative, positive sub-clinically infected and positive clinically affected). Mycoplasma hyopneumoniae was detected in air, air deposition particles, dam and stall surfaces of the positive clinically affected herd. Mycoplasma hyopneumoniae could only be detected in dam and stall surfaces in sub-clinically infected herds. Mycoplasma hyopneumoniae was not detected in all samples collected in the negative herd. The cycle threshold of the positive PCR samples were not statistically different between sample types or farms. However, a significant difference (p < 0.05) was observed in the percentage of positive samples between the positive clinically affected farm and the rest. Likewise, M. hyopneumoniae was detected in the environment and surfaces at weaning in positive breeding herds. Further testing and validation is recommended for environmental and surface samples before they can be employed as part of the M. hyopneumoniae diagnostic process. In addition, results from this study highlight potential sources of M. hyopneumoniae infection for piglets in breeding herds, especially during an outbreak.

Development of an ELISA for distinguishing convalescent sera with Mycoplasma hyopneumoniae infection from hyperimmune sera responses to bacterin vaccination in pigs.

Vaccination with inactivated bacterin is the most popular and practical measure to control enzootic pneumonia. After immunisation with inactivated bacterin, Mycoplasma hyopneumoniae colonised on the respiratory tract and lung stimulates the humoural immune responses and produces IgG and IgA antibodies. ELISA is a widely used serological method to detect M. hyopneumoniae antibodies. However, commercial IgG-ELISA kit cannot distinguish between inactivated bacterin-induced hyperimmune sera and convalescent sera stimulated by natural infection. SIgA-ELISA method needs to collect nasal swabs, but collecting nasal swabs is not easy to operate. Establishment of a discriminative ELISA detecting humoural IgG from convalescent sera but not hyperimmune sera facilitates to evaluate the natural infection of M. hyopneumoniae after inactivated bacterin vaccination. We expressed and purified a recombinant protein named Mhp366-N which contains an epitope recognised by the convalescent sera but not hyperimmune sera. The developed discriminative IgG-ELISA could discriminate between inactivated bacterin-induced hyperimmune sera and convalescent sera and was reproducible, sensitive and specific to M. hyopneumoniae antibody produced by natural infection. Compared to SIgA-ELISA method, discriminative IgG-ELISA was more convenient to detect IgG antibody from sera than IgA from nasal swabs, although it has limited sensitivity in the early stages of infection. Additionally, to some extent, it has a potential to avoid the interference of maternally derived IgG antibodies. The established discriminative IgG-ELISA was efficient to judge the serological IgG antibodies induced from natural infection or inactivated vaccine stimulation and provided a useful method to investigate and evaluate the live organism infection after the application of inactivated bacterin.

Pooled-sample testing for detection of Mycoplasma hyopneumoniae during late experimental infection as a diagnostic tool for a herd eradication program.

Early and accurate detection of Mycoplasma hyopneumoniae infection in live pigs is a critical component to measure the success of disease eradication strategies. However, the imperfect sensitivity of in vivo diagnostic tools, change in sensitivity over the course of infection, and expected low prevalence level at the end of an eradication program create a challenging diagnostic scenario. Here, the individual and pool sensitivities for detection of M. hyopneumoniae during the chronic phase of infection was determined using deep tracheal catheter samples, the in vivo sample type with the highest reported diagnostic sensitivity. Fifty samples from known infected pigs collected at 113 days post-M. hyopneumoniae intra-tracheal inoculation, were diluted in known negative samples to form pools of 1:3 and 1:5. Samples were tested for M. hyopneumoniae by a species-specific PCR. Ninety-eight percent (49/50) of individual samples, 84 % (42/50) of pools of 1:3, and 82 % (41/50) of 1:5 were detected positive for M. hyopneumoniae. To apply the sensitivity estimates for detection of M. hyopneumoniae in a low prevalence scenario, sample sizes with associated sample collection costs were calculated for individual and pooled testing using algorithms within the program EpiTools One-Stage Freedom Analyses. Assumptions included a ≥95 % population sensitivity, infinite population size, prevalence levels of ≥0.5 %, ≥1 %, ≥2 %, ≥3 %, ≥4 %, or ≥5 %, 100 % specificity, along with the mean and lower confidence limit of the individual or pool sensitivity for each pool size, when appropriate. For instance, following completion of a herd eradication program, if a low risk approach is targeted, sample size estimates for ≥2 % prevalence using the lower limit of the diagnostic or pool sensitivity 95 %CI may be followed. If samples were to be tested individually, 167 individuals would be sampled at a cost of 6,012 USD. If pooled by 3, 213 would be sampled (testing cost 3,266 USD), and for pools of 5, 220 individuals would be sampled (testing cost 2,464 USD). Population sensitivity was also calculated for a range of testing scenarios. Our study indicated that pooling samples by 3 or 5 was a cost-effective method for M. hyopneumoniae detection in low prevalence scenarios. Cost-effective detection was evidenced despite the increased sample collection costs associated with large sample sizes in order to offset decreased testing sensitivity attributable to pooling. The post-eradication sample collection scheme, combined with pooling, suggested lower cost options than individual sampling for testing to be applied at the end of an eradication program, without significantly compromising the likelihood of detection.

Mycoplasma hyopneumoniae Surveillance in Pig Populations: Establishing Sampling Guidelines for Detection in Growing Pigs.

Antemortem detection of Mycoplasma hyopneumoniae infection in swine production systems has relied on antibody testing, but the availability of tests based on DNA detection and novel diagnostic specimens, e.g., tracheal swabs and oral fluids, has the potential to improve M. hyopneumoniae surveillance. A field study was performed over a 14-week period during which 10 pigs in one pen at the center of a room with 1,250 6-week-old pigs housed in 46 pens were intratracheally inoculated with M. hyopneumoniae Thereafter, one tracheal sample, four serum samples, and one oral fluid sample were collected from every pen at 2-week intervals. Tracheal and oral fluid samples were tested for M. hyopneumoniae DNA and serum samples for M. hyopneumoniae antibody. Test results were modeled using a hierarchical Bayesian model, based on a latent spatial piecewise exponential survival model, to estimate the probability of detection by within-pen prevalence, number of positive pens in the barn, sample allocation, sample size, and sample type over time. Analysis showed that tracheal samples provided the earliest detection, especially at large sample sizes. While serum samples are more commonly collected and are less expensive to test, high probability of detection estimates were only obtained 30 days postexposure at large sample sizes. In all scenarios, probability of detection estimates for oral fluids within 30 days were significantly lower than those for tracheal and serum samples. Ultimately, the choice of specimen type, sample number, and assay will depend on testing objectives and economics, but the estimates provided here will assist in the design of M. hyopneumoniae surveillance and monitoring programs for different situations.

Bacterial pneumonia in captive wild boars in southern Brazil: etiological and pathological causes

Respiratory diseases are one of the major health issues described in intensive pig production, causing important economic losses. However, there is little information on the prevalence, etiology and clinical-pathological presentation of these diseases in wild boars. For this reason, this work investigated the presence in captive wild boars of pneumonic lesions and bacterial pathogens commonly detected and associated with respiratory diseases in domestic pigs. A total of 226 captive wild boar lungs from two farms were examined in a slaughterhouse in Southern Brazil. The pneumonic lesions were classified as cranioventral, dorsocaudal, and disseminated, and the quantification of lesions was calculated. From the total of 226 lungs, 121 were collected for laboratory examination. Lungs with macroscopic lesions suggestive of pneumonia were collected for histological, bacteriological and molecular analysis. The molecular analysis was performed to detect the presence of Actinobacillus (A.) pleuropneumoniae, Glaesserella (G.) parasuis, Mycoplasma (M.) hyopneumoniae, Mycoplasma (M.) hyorhinis and Streptococcus (S.) suis serotype 2. The percentages of histological lesions and bacterial agents and their association were calculated. Cranioventral consolidation (75.2%) was the most prevalent macroscopic lung lesion, followed by disseminated (21.5%) and dorsocaudal (3.3%) distribution. Microscopically, chronic lesions were the most prevalent, representing 70.2% of the lungs. Moreover, BALT hyperplasia was present in 86.5% of the lungs, suppurative bronchopneumonia in 65.7%, and alveoli infiltrate in 46.8%. Six bacterial pathogens commonly described as agents of pig pneumonia were identified by bacterial or molecular methods: Pasteurella (P.) multocida, S. suis, M. hyopneumoniae, A. pleuropneumoniae, G. parasuis and M. hyorhinis. Twenty-eight different combinations of pathogens were identified in 84 samples (69.4%). The most common combinations were: M. hyopneumoniae and A. pleuropneumoniae (13.1%), M. hyopneumoniae, G. parasuis and M. hyorhinis (10.7%), and M. hyopneumoniae, A. pleuropneumoniae and G. parasuis (8.3%). Additionally, M. hyopneumoniae was the most frequent pathogen detected in this study, representing 58.7% of the samples. The detection of M. hyopneumoniae and M. hyorhinis by PCR was associated with the presence of BALT hyperplasia (P < 0.05) and there was also an association between the detection of M. hyopneumoniae by PCR and suppurative bronchopneumonia (P < 0.05). In addition, a significant association (P < 0.05) between the detection of M. hyopneumoniae and A. pleuropneumoniae by PCR and the histological classification (acute, subacute or chronic lesions) was observed. The results of this study were similar to those observed in slaughtered domestic pigs, although, the detection of opportunist pathogens was less frequent than that usually described in pig pneumonia. The high prevalence of pneumonia in captive wild boars at slaughter and the similar characteristics of pneumonia in captive wild boars and domestic pigs suggest that the close phylogenetic relationship between pigs and wild boars could influence the susceptibility of both species to the colonization of the same pathogens, indicating that captive wild boars raised in confined conditions could be predisposed to respiratory diseases, similar to domestic pigs.(AU)

Performance of Commercial Mycoplasma hyopneumoniae Serum Enzyme-Linked Immunosorbent Assays under Experimental and Field Conditions.

Mycoplasma hyopneumoniae is an economically significant pathogen of swine. M. hyopneumoniae serum antibody detection via commercial enzyme-linked immunosorbent assays (ELISAs) is widely used for routine surveillance in commercial swine production systems. Samples from two studies were used to evaluate assay performance. In study 1, 6 commercial M. hyopneumoniae ELISAs were compared using serum samples from 8-week-old cesarean-derived, colostrum-deprived (CDCD) pigs allocated to the following 5 inoculation groups of 10 pigs each: (i) negative control, (ii) Mycoplasma flocculare (strain 27399), (iii) Mycoplasma hyorhinis (strain 38983), (iv) Mycoplasma hyosynoviae (strain 34428), and (v) M. hyopneumoniae (strain 232). Weekly serum and daily oral fluid samples were collected through 56 days postinoculation (dpi). The true status of pigs was established by PCR testing on oral fluids samples over the course of the observation period. Analysis of ELISA performance at various cutoffs found that the manufacturers' recommended cutoffs were diagnostically specific, i.e., produced no false positives, with the exceptions of 2 ELISAs. An analysis based on overall misclassification error rates found that 4 ELISAs performed similarly, although one assay produced more false positives. In study 2, the 3 best-performing ELISAs from study 1 were compared using serum samples generated under field conditions. Ten 8-week-old pigs were intratracheally inoculated with M. hyopneumoniae Matched serum and tracheal samples (to establish the true pig M. hyopneumoniae status) were collected at 7- to 14-day intervals through 98 dpi. Analyses of sensitivity and specificity showed similar performance among these 3 ELISAs. Overall, this study provides an assessment of the performance of current M. hyopneumoniae ELISAs and an understanding of their use in surveillance.

Natural transmission and detection of Mycoplasma hyopneumoniae in a naïve gilt population.

Mycoplasma hyopneumoniae (M. hyopneumoniae) continues to be a prevalent and economically important swine respiratory pathogen. For M. hyopneumoniae surveillance, blood samples and/or oral fluids are commonly collected from incoming replacement gilts prior to entering sow farms. However, limitations to this approach exist, particularly due to low sensitivity during acute stages of natural infection, leading to diagnostic uncertainty. Therefore, the objective of this study was to evaluate the natural transmission and detection of M. hyopneumoniae based on the introduction of one infected gilt to a naïve population. Twenty-nine naïve gilts were housed with one M. hyopneumoniae naturally exposed gilt for 8 weeks. Deep tracheal catheters, laryngeal swabs, and blood samples were individually collected from each gilt at 0, 1, 2, 4, 6, and 8 weeks post-contact (wpc), along with one pen-based oral fluid sample. Blood samples were assayed by ELISA, while all other samples were tested by real-time PCR. The transmission rate of M. hyopneumoniae (ꞵ) was estimated using a Bayesian mixed-effects generalized linear model. At 8 wpc, 27 % (8/29) of the naïve gilts had become infected (ꞵ = 0.73 new infected gilts/gilt-week). Seroconversion was detected in 3% of contact gilts at 8 wpc. Oral fluids were negative for M. hyopneumoniae at all samplings. In this study, the natural transmission of M. hyopneumoniae was slow and detection varied based on sample type and timing. Thus, M. hyopneumoniae surveillance protocols should include lower respiratory tract samples that are tested by real-time PCR to avoid the introduction of potentially infected gilts into naïve sow farms.

Comparison of the sensitivity of laryngeal swabs and deep tracheal catheters for detection of Mycoplasma hyopneumoniae in experimentally and naturally infected pigs early and late after infection.

Detection of Mycoplasma hyopneumoniae infection in live pigs is a critical component to measure the success of disease control or elimination strategies. However, in vivo diagnosis of M. hyopneumoniae is difficult and the imperfect sensitivity of diagnostic tools has been deemed as one of the main challenges. Here, the sensitivity of laryngeal swabs and deep tracheal catheters for detection of M. hyopneumoniae early and late after infection was determined using inoculation status as a gold standard in experimentally infected pigs and a Bayesian approach in naturally infected pigs. Three-hundred and twenty 8-week old seeder pigs were intra-tracheally inoculated with M. hyopneumoniae strain 232 and immediately placed with 1920 contact pigs to achieve a 1:6 seeder-to-contact ratio. A subset of seeders and contacts were longitudinally sampled at 7, 28, 97, and 113 days post-inoculation (dpi) and at 28, 56, 84, and 113 days post-exposure (dpe), respectively, using laryngeal swabs and deep tracheal catheters. Samples were tested for M. hyopneumoniae by a species-specific real-time PCR. The sensitivity of deep tracheal catheters was higher than the one obtained in laryngeal swabs at all samplings (seeders: 36% higher than laryngeal swabs at 7 dpi, 29% higher at 97 dpi, and 44% higher at 113 dpi; contacts: 51% higher at 56 dpe, 42% higher at 84 dpe, and 32% higher at 113 dpe). Our study indicates that deep tracheal catheters were a more sensitive sample than laryngeal swabs. The sensitivity of both sample types varied over time and by exposure method, and these factors should be considered when designing diagnostic strategies.

Detection of Mycoplasma hyopneumoniae in nasal and laryngeal swab specimens in endemically infected pig herds.

BACKGROUND: Apparently, laryngeal swabs (LS) are more sensitive than nasal swabs (NS) and allow earlier detection of Mycoplasma hyopneumoniae by PCR. However, antecedents about the compared detection of M hyopneumoniae with NS and LS in growing pigs, from naturally infected herds, are lacking in the literature. Thus, this study compared the PCR detection of M hyopneumoniae from NS and LS in pigs of various ages. METHODS: A longitudinal study was performed at two farms where NS and LS were collected from three consecutive groups of 20 pigs at 3, 6, 10, 16 and 22 weeks of age. All samples were analysed by nested PCR for M hyopneumoniae detection. RESULTS: The probability of PCR detection of M hyopneumoniae was higher in LS for pigs of all ages (odds ratio (OR)=1.87; 95 per cent confidence interval (CI) 1.31-2.67) and in 22-week-old pigs (OR=4.87; 95 per cent CI 2.86-8.30). The agreement between both sample types was low to moderate (kappa 0.087-0.508), highlighting that M hyopneumoniae does not appear to colonise the respiratory tract in a generalised and consistent fashion. CONCLUSIONS: The results suggest that LS could be employed at different ages to achieve greater bacterial detection. Considering that LS is a minimally invasive, highly sensitive sample compared with the traditional NS, it could be suggested to employ this sample type for M hyopneumoniae detection in naturally infected pigs.

Occurrence of Mycoplasma hyopneumoniae in slaughter pigs from Southern Brazil / Ocorrência de Mycoplasma hyopneumoniae em amostras de pulmão de suínos obtidas de frigorífico do sul do Brasil

Mycoplasma hyopneumoniae is the causative agent of enzootic pneumonia (EP), a disease that is highly prevalent and globally distributed, causing significant economic losses to the swine industry. Disease progression is characterized by reduced feed conversion and the development of lung lesions. Considering the limited information about the epidemiology of EP in Southern Brazil, the main objective of this study was to determine the occurrence of M. hyopneumoniae in swine lung samples and to evaluate the scores of lung lesions caused by local strains. A total of 120 samples was randomly collected and processed. DNA was extracted from lung tissue to perform nested-PCR and lungs were inspected to evaluate the presence of the pneumonia-like gross lesions of M. hyopneumoniae. The results showed 95.8% positive samples, while the lung lesion score analysis showed suggestive lesions in 60% of samples. The detection of positive samples in nested-PCR was associated with the presence of pneumonia-like gross lesions (P < 0.01). The results demonstrate a high occurrence of EP in slaughter pigs from southern Brazil.(AU)

O Mycoplasma hyopneumoniae é o agente causador da Pneumonia Enzoótica Suína (PES), doença altamente prevalente e mundialmente distribuída, causando grandes perdas econômicas para a indústria suinícola. A progressão da doença é caracterizada pela redução das taxas de conversão alimentar e o desenvolvimento de lesões pulmonares. Visto que há informação limitada sobre a epidemiologia da PES no sul do Brasil, o objetivo do presente trabalho foi determinar a prevalência de M. hyopneumoniae em amostras de pulmão suíno e avaliar o score de lesões pulmonares causadas pelas cepas locais. Um total de 120 amostras foram coletadas aleatoriamente, processadas e analisadas. O DNA foi extraído do tecido pulmonar para realização de Nested-PCR e os pulmões foram inspecionados para presença de lesões macroscópicas sugestivas de M. hyopneumoniae. Os resultados demonstraram 95,8% das amostras positivas para o patógeno. A análise do score pulmonar mostrou lesões sugestivas da PES em 60% das amostras. A detecção de amostras positivas no Nested-PCR foi associada com a presença de lesões sugestivas (P < 0.01). Os dados obtidos neste trabalho demonstram a alta prevalência da PES em granjas do RS.(AU)

Application of a sIgA-ELISA method for differentiation of Mycoplasma hyopneumoniae infected from vaccinated pigs.

In order to evaluate the sIgA-ELISA method reported previously for differentiating Mycoplasma hyopneumoniae (M. hyopneumoniae) infected from vaccinated pigs, dynamics of anti-M. hyopneumoniae secretory IgA (sIgA) antibody secretion in nasal mucus and IgG antibodies in serum from 10 pigs experimentally infected with M. hyopneumoniae or vaccinated with an inactivated vaccine were examined using sIgA-ELISA and a commercial M. hyopneumoniae antibody detection kit (IgG-ELISA), respectively. In addition, nasal swabs and serum samples from 2368 pigs of different ages originating from 10 pig farms with different M. hyopneumoniae infection and vaccination status were examined using the two ELISA. In the experimental model, anti-M. hyopneumoniae IgG antibodies were detected in both, the challenge group and the vaccine group. Anti-M. hyopneumoniae sIgA antibodies were detected in the challenge group from 7 days post challenge onwards, but not in the vaccine group. According to the data obtained from pig farms maintaining administration of inactivated vaccine, the prevalence of anti-M. hyopneumoniae sIgA antibody positive pigs was significantly lower than that of IgG antibody positive pigs. In non-vaccinating herds, the prevalence of sIgA antibodies was correlated with the severity of clinical symptoms typical for porcine enzootic pneumonia. In all suckling pigs, no matter vaccinated or not, the prevalence of anti-M. hyopneumoniae sIgA antibody positives was significantly lower than that of IgG antibody positives. These results prove that the sIgA-ELISA is a valuable method enabling the surveillance of M. hyopneumoniae infections in pig herds without interference due to maternally derived antibodies or antibodies induced by administration of inactivated vaccines.

Update on Mycoplasma hyopneumoniae infections in pigs: Knowledge gaps for improved disease control.

Mycoplasma hyopneumoniae (M. hyopneumoniae) is the primary pathogen of enzootic pneumonia, a chronic respiratory disease in pigs. Infections occur worldwide and cause major economic losses to the pig industry. The present paper reviews the current knowledge on M. hyopneumoniae infections, with emphasis on identification and analysis of knowledge gaps for optimizing control of the disease. Close contact between infected and susceptible pigs is the main route of M. hyopneumoniae transmission. Management and housing conditions predisposing for infection or disease are known, but further research is needed to better understand M. hyopneumoniae transmission patterns in modern pig production systems, and to assess the importance of the breeding population for downstream disease control. The organism is primarily found on the mucosal surface of the trachea, bronchi and bronchioles. Different adhesins and lipoproteins are involved in the adherence process. However, a clear picture of the virulence and pathogenicity of M. hyopneumoniae is still missing. The role of glycerol metabolism, myoinositol metabolism and the Mycoplasma Ig binding protein-Mycoplasma Ig protease system should be further investigated for their contribution to virulence. The destruction of the mucociliary apparatus, together with modulating the immune response, enhances the susceptibility of infected pigs to secondary pathogens. Clinical signs and severity of lesions depend on different factors, such as management, environmental conditions and likely also M. hyopneumoniae strain. The potential impact of strain variability on disease severity is not well defined. Diagnostics could be improved by developing tests that may detect virulent strains, by improving sampling in live animals and by designing ELISAs allowing discrimination between infected and vaccinated pigs. The currently available vaccines are often cost-efficient, but the ongoing research on developing new vaccines that confer protective immunity and reduce transmission should be continued, as well as optimization of protocols to eliminate M. hyopneumoniae from pig herds.