Detection of anti-Mycoplasma hyopneumoniae antibodies in backyard pigs in the state of Paraná, Brazil.

Mycoplasma hyopneumoniae is the causative bacterium of porcine enzootic pneumonia and one of the primary etiologic agents of the porcine respiratory disease complex. Most Brazilian commercial pig farms are positive for this pathogen. However, the prevalence of the pathogen in backyard pig farms has not been described, to our knowledge. Therefore, we aimed to determine the prevalence of M. hyopneumoniae in backyard pig farms in the state of Paraná, Brazil. In January-March 2020, we collected 585 serum samples from pigs in 187 non-vaccinated herds. We tested the sera with an indirect ELISA for anti-M. hyopneumoniae antibodies and found that 182 of 585 (31.1%) samples were positive, and were found in 109 of 187 (58.3%) herds assessed.

Shedding reduction and immunity modulation in piglets with an inactivated Mycoplasma hyopneumoniae vaccine encapsulated in nanostructured SBA-15 silica.

Mycoplasma (M.) hyopneumoniae is a primary etiological agent of porcine enzootic pneumonia (PEP), a disease that causes significant economic losses to pig farming worldwide. Current commercial M. hyopneumoniae vaccines induce partial protection, decline in preventing transmission of this pathogen or inducing complete immunity, evidencing the need for improving vaccines against PEP. In our study, we aimed to test the effectiveness of the SBA-15 ordered mesoporous silica nanostructured particles as an immune adjuvant of a vaccine composed of M. hyopneumoniae strain 232 proteins encapsulated in SBA-15 and administered by intramuscular route in piglets to evaluate the immune responses and immune-protection against challenge. Forty-eight 24-day-old M. hyopneumoniae-free piglets were divided into four experimental groups with different protocols, encompassing a commercial vaccine against M. hyopneumoniae, SBA-15 vaccine, SBA-15 adjuvant without antigens and a non-immunized group. All piglets were challenged with the virulent strain 232 of M. hyopneumoniae. Piglets that received the SBA-15 and commercial vaccine presented marked immune responses characterized by anti-M. hyopneumoniae IgA and IgG antibodies in serum, anti-M. hyopneumoniae IgA antibodies in nasal mucosa and showed an upregulation of IL-17 and IL-4 cytokines and downregulation of IFN-γ in lungs 35 days post-infection. Piglets immunized with SBA-15 vaccine presented a reduction of bacterial shedding compared to piglets immunized with a commercial bacterin. In addition, piglets from SBA-15 adjuvant suspension group presented increased IL-17 gene expression in the lungs without involvement of Th1 and Th2 responses after challenge. These results indicated that SBA-15 vaccine induced both humoral and cell-mediated responses in the upper respiratory tract and lungs, first site of replication and provided protection against M. hyopneumoniae infection with a homologous strain with reduction of lung lesions and bacterial shedding. Finally, these results enhance the potential use of new technologies such as nanostructured particles applied in vaccines for the pig farming industry.

A field evaluation of a new porcine circovirus type 2d and Mycoplasma hyopneumoniae bivalent vaccine in herds suffering from subclinical PCV2d infection and enzootic pneumonia.

BACKGROUND: This field efficacy study was designed to determine the efficacy of a new bivalent vaccine containing porcine circovirus type 2d (PCV2d) and Mycoplasma hyopneumoniae at three independent pig farms. METHODS: Three pig farms were selected based on their history of subclinical PCV2 infection and enzootic pneumonia. Each farm housed a total of 40, 18-day-old pigs that were randomly allocated to 1 of 2 treatment groups. Pigs were administered a 2.0 mL dose of the bivalent vaccine intramuscularly at 21 days of age in accordance with the manufacturer's recommendations, whereas unvaccinated pigs were administered a single dose of phosphate-buffered saline at the same age. RESULTS: Clinically, the average daily weight gain of vaccinated groups was significantly higher (p < 0.05) than those of unvaccinated animals during the growing (70-112 days of age), finishing (112-175 days of age) and overall (3-175 days of age) stages of production. Vaccinated animals elicited neutralizing anti-PCV2 antibodies and PCV2d-specific interferon-γ secreting cells (IFN-γ-SC), which reduced the amount of PCV2d genomic copies in blood and reduced lymphoid lesions severity when compared with unvaccinated animals. Similarly, vaccinated animals elicited M. hyopneumoniae-specific IFN-γ-SC, which reduced the amount of M. hyopneumoniae in the larynx and reduced lung lesions severity. CONCLUSIONS: The result of the field trial demonstrated that the bivalent vaccine was efficacious in the protection of swine herds suffering from subclinical PCV2d infection and enzootic pneumonia.

Synergism between molecules derived from Garcinia brasiliensis and antimicrobial drugs on field isolates of Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae (M. hyopneumoniae) is one of the smallest free-living bacteria found in nature; it has an extremely small genome and lacks a cell wall. It is the main etiological agent of porcine enzootic pneumonia (EP), a chronic respiratory disease with worldwide distribution that causes significant losses in swine production. Due to the great economic impact caused by EP, new strategies for treating and controlling this agent are researched. The objective of this study was to verify the anti-M. hyopneumoniae activity of compounds derived from Garcinia brasiliensis and the synergism with the main antimicrobials used in the treatment of EP; this is the first study assessing the synergism between bioactive molecules and antimicrobial compounds in vitro against isolates of M. hyopneumoniae. The minimum inhibitory concentrations (MICs) of the antimicrobials tiamulin, valnemulin, and enrofloxacin, as well as the bioactive compounds guttiferone-A (Gut-A), 7-epiculsone (7-Epic), copper 7-epiculsone (7-Epic-Cu), and benzophenone, were determined. Subsequently, the interactions of antibiotics with the compounds were evaluated using the checkerboard method. Three field M. hyopneumoniae isolates were used, and the J strain was used as a control. The MIC values of the antimicrobials compared to the field isolates were equal to and lower than those of the reference strain J. Among the compounds used, 7-Epic-Cu showed the lowest MIC value. Synergistic association was observed for Gut-A with tiamulin and valnemulin, whereas 7-Epic and 7-Epic-Cu showed synergistic action with enrofloxacin. No synergistic effect was observed for benzophenone. Despite being an initial study, the results suggest that these combinations hold promise for the treatment of infections caused by M. hyopneumoniae.

Scoring Enzootic Pneumonia-like Lesions in Slaughtered Pigs: Traditional vs. Artificial-Intelligence-Based Methods.

Artificial-intelligence-based methods are regularly used in the biomedical sciences, mainly in the field of diagnostic imaging. Recently, convolutional neural networks have been trained to score pleurisy and pneumonia in slaughtered pigs. The aim of this study is to further evaluate the performance of a convolutional neural network when compared with the gold standard (i.e., scores provided by a skilled operator along the slaughter chain through visual inspection and palpation). In total, 441 lungs (180 healthy and 261 diseased) are included in this study. Each lung was scored according to traditional methods, which represent the gold standard (Madec's and Christensen's grids). Moreover, the same lungs were photographed and thereafter scored by a trained convolutional neural network. Overall, the results reveal that the convolutional neural network is very specific (95.55%) and quite sensitive (85.05%), showing a rather high correlation when compared with the scores provided by a skilled veterinarian (Spearman's coefficient = 0.831, p < 0.01). In summary, this study suggests that convolutional neural networks could be effectively used at slaughterhouses and stimulates further investigation in this field of research.

Efficacy of a novel bivalent vaccine containing porcine circovirus type 2d and Mycoplasma hyopneumoniae against a dual PCV2d and Mycoplasma hyopneumoniae challenge.

Background: Information on efficacy of a novel bivalent vaccine containing porcine circovirus type 2d (PCV2d) and Mycoplasma hyopneumoniae. Objective: To evaluate bivalent vaccine for efficacy under experimental conditions. Animals: Clinically healthy 35 weaned piglets at 18 days of age were used. Methods: A 2.0 mL dose of bivalent vaccine was administered intramuscularly to pigs at 21 days of age in accordance with the manufacturer's instructions. The pigs were challenged at 42 days of age either intranasally with PCV2d, or intratracheally with M. hyopneumoniae, or with both. Results: Vaccinated-challenged pigs improved the growth performance compared to pigs that were unvaccinated and then, challenged. Vaccinated-challenged pigs elicited a significant amount of protective immunity for PCV2d-specific neutralizing antibodies and interferon-γ secreting cells (IFN-γ-SC) as well as for M. hyopneumoniae-specific IFN-γ-SC compared to unvaccinated/challenged pigs. Induction of systemic cellular and humoral immune responses from bivalent vaccination reduced the viral and mycoplasmal loads in the blood and larynx. Vaccination and challenge simultaneously reduced both lung and lymphoid lesion severity when compared to unvaccinated-challenged pigs. Discussion: The results of this study demonstrated that the evaluated bivalent PCV2d and M. hyopneumoniae vaccine was efficacious in protecting pigs from the most predominant PCV2d genotype in the field today, as evaluated with a dual PCV2d and M. hyopneumoniae challenge under experimental conditions.

Differential domains and endoproteolytic processing in dominant surface proteins of unknown function from Mycoplasma hyopneumoniae and Mycoplasma flocculare.

Mycoplasma hyopneumoniae causes porcine enzootic pneumonia (PEP), a chronic respiratory disease that leads to severe economic losses in the pig industry. Swine infection and PEP development depend on the adhesion of the pathogen to the swine respiratory tract and the host immune response, but these and other disease determinants are not fully understood. For instance, M. hyopneumoniae has a large repertoire of proteins of unknown function (PUFs) and some of them are abundant in the cell surface, where they likely mediate so far unknown pathogen-host interactions. Moreover, these surface PUFs may undergo endoproteolytic processing to generate larger repertoires of proteoforms to further complicate this scenario. Here, we investigated the five PUFs more represented on the surface of M. hyopneumoniae pathogenic strain 7448 in comparison with their orthologs from the nonpathogenic M. hyopneumoniae J strain and the closely related commensal species Mycoplasma flocculare. Comparative in silico analyses of deduced amino acid sequences and proteomic data identified differential domains, disordered regions and repeated motifs. We also provide evidence of differential endoproteolytic processing and antigenicity. Phylogenetic analyses were also performed with ortholog sequences, showing higher conservation of three of the assessed PUFs among Mycoplasma species related to respiratory diseases. Overall, our data point out to M. hyopneumoniae surface-dominant PUFs likely associated with pathogenicity.

Ureaplasma diversum-Ureaplasma sp. alone or concomitantly with Mycoplasma hyopneumoniae in pig lungs with and without pneumonia: A descriptive exploratory study in abattoirs.

Occurrence of Ureaplasma diversum (U. diversum) has been associated with reproductive failures in cattle and detected in pigs with and without pneumonia. However, its role in the porcine respiratory disease complex (PRDC) is unclear. A cross-sectional study was conducted in abattoirs, inspecting 280 pig lungs from eight herds. All the lungs were inspected, processed and classified according to the histopathological analysis. Moreover, bronchoalveolar lavage (BAL) specimens were collected and processed by PCR for detection of U. diversum and Mycoplasma hyopneumoniae (M. hyopneumoniae). Ureaplasma sp.-U. diversum and M. hyopneumoniae were detected in 17.1% and 29.3% of the analyzed BAL specimens, respectively. The concomitant presence of both microorganisms was detected in 12.5% of the inspected lungs. Both agents were found in lungs with and without pneumonia. M. hyopneumoniae was detected in 31.8% of pig lungs with enzootic pneumonia-like lesions, while Ureaplasma sp.-U. diversum was detected in 27.5% of lungs with these lesions. This descriptive exploratory study provides information for future experimental and field-based studies to better define the pathogenic role of this organism within the PRDC.

Efficacy evaluation of a novel oral silica-based vaccine in inducing mucosal immunity against Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae, the main etiological agent of Porcine Enzootic Pneumonia, is widely spread in swine production worldwide. Its prevention is of great interest for the productive system, since its colonization in the lung tissue leads to intense production losses. This study aimed to compare the M. hyopneumoniae shedding and acute-phase response in 30 pigs submitted to different vaccination protocols: an experimental oral vaccine using a nanostructured mesoporous silica (SBA-15) as adjuvant (n = 10); an intramuscular commercially available vaccine at 24 days of age (n = 10); and a control group (n = 10) following experimental challenge with M. hyopneumoniae. Laryngeal and nasal swabs were collected weekly and oral fluids were collected at 7, 10, 14, 17, 23, 28, 35, 42, and 49 days post-infection to monitor pathogen excretion by qPCR. Nasal swabs were also used to detect anti-M. hyopneumoniae IgA by ELISA. Blood samples were collected for monitoring acute phase proteins. The antibody response was observed in both immunized groups seven days after vaccination, while the control group became positive for this immunoglobulin at 4 weeks after challenge. Lung lesion score was similar in the immunized groups, and lower than that observed in the control. SBA-15-adjuvanted oral vaccine provided immunological response, decreased shedding of M. hyopneumoniae and led to mucosal protection confirmed by the reduced pulmonary lesions. This study provides useful data for future development of vaccines against M. hyopneumoniae.

Clinical Efficacy of Two Novel, Differentially Administered (IM, ID) Vaccines against Mycoplasma hyopneumoniae and PCV2 in Swine under Field Conditions.

Enzootic pneumonia (EP) of pigs is caused by Mycoplasma hyopneumoniae (M.hp.), which is, together with the porcine circovirus type 2 (PCV2), among the most prominent inducers of the porcine respiratory disease complex (PRDC). Therefore, vaccination of piglets against M.hp. and PCV2 is crucial in the fight against pulmonary infections. In this field study, we tested the clinical efficacy of two novel vaccines, one delivered IM (Hyogen® + Circovac®) and the other ID (MHyo-Sphere®PCV ID), on a fattening farm in Lower Austria with a history of still ongoing EP. Average daily weight gain, coughing/sneezing index, losses due to morbidity/mortality, and lung scoring data at slaughter by means of CLP (Ceva Lung Program) were recorded for three consecutive fattening cohorts to achieve a powerful number of animals, one half each vaccinated with the IM vaccine and the other half with the ID vaccine (n = 659 in total). No statistically significant differences could be observed between the two vaccination groups for the parameters investigated, but the total median EP score, which categorizes pulmonary lesions due to infection by M.hp. with a theoretical range of 0-28, was lowered from initially 1.9 to 1.0, indicating that both vaccines proved very suitable measures in the fight against EP.

An evaluation of intradermal all-in-one vaccine based on an inactivated recombinant Mycoplasma hyopneumoniae strain expressing porcine circovirus type 2 (PCV2) capsid protein against Korean stains of PCV2d and M. hyopneumoniae challenge.

This study evaluates the efficacy of intradermal all-in-one vaccine (MHYOSPHERE® PCV ID, Laboratorios Hipra S.A. Amer, Spain) based on the strain Nexhyon, an inactivated recombinant M. hyopneumoniae strain with an embedded/integrated PCV2a capsid protein thereof, as the single active substance. Pigs were administered the vaccine intradermally at 21 days of age with 0.2 mL, then challenged at 49 days of age with either M. hyopneumoniae (intratracheal route), PCV2d (intranasal route), or both. Upon dual challenge, growth performance was improved when the intradermal all in one vaccine was administered compared to the unvaccinated group. In pigs receiving single or dual challenge, vaccination increased neutralizing antibodies against PCV2d and specific interferon-γ secreting cells for each pathogen. In contrast, viral load of PCV2d in the blood, M. hyopneumoniae load in the larynx, and the severity of pulmonary and lymphoid lesions were decreased. Vaccination provided good protection against challenge with M. hyopneumoniae and PCV2d.

Chemotherapeutic Strategies with Valnemulin, Tilmicosin, and Tulathromycin to Control Mycoplasma hyopneumoniae Infection in Pigs.

Mycoplasma hyopneumoniae is the primary agent of Swine Enzootic Pneumonia (SEP). Vaccines reduce the clinical manifestation of the disease but do not prevent infection. The present study aimed to evaluate the use of antimicrobial drugs to minimize the impact of M. hyopneumoniae. For this, 32 pregnant female pigs and their litters were selected and then followed from birth to slaughter. The study involved three experimental groups that received metaphylactic treatment with different protocols involving tilmicosin, valnemulin, tulathromycin, and a control group to compare the effect of treatments against M. hyopneumoniae infection throughout the phases. Performance data were recorded, and the piglets were evaluated for the occurrence of cough. Nasal swab and blood collection was conducted periodically to detect M. hyopneumoniae shedding and anti-M. hyopneumoniae IgG, respectively. At slaughter, the lungs of animals from all groups were evaluated, and samples were collected for histopathological examination and qPCR for M. hyopneumoniae detection. All protocols promoted a reduction in consolidation lung lesions when compared to the control group. Individuals treated with valnemulin showed increased performance results, lower mortality, and low bacterial load in the lung. The results are promising and may indicate an alternative in the strategic control of M. hyopneumoniae infection in pigs.

Characteristics of Mycoplasma hyopneumoniae Strain ES-2 Isolated From Chinese Native Black Pig Lungs.

Mycoplasma hyopneumoniae is the primary pathogen of swine enzootic pneumonia and causes great economic losses to the swine industry worldwide. In China, M. hyopneumoniae seriously hinders the healthy development of the native black pigs. To prevent and treat porcine respiratory disease caused by M. hyopneumoniae, the characteristics of M. hyopneumoniae strain ES-2 isolated from Chinese native black pig lungs with gross lesions at post-mortem were studied for the first time in this study. Strain ES-2 cell was round or oval cells and most sensitive to kanamycin. The diameters of most strain ES-2 cells ranged from 0.4 to 1.0 µm with maximum viability of 1010 CCU/ml. Experimental challenge of animals with strain ES-2 showed respiratory disease could be reproduced, with pneumonic lung lesions evident. Comparative genomics analysis identified that 2 genes are specific to pathogenic M. hyopneumoniae strains, which may be predicted to be a molecular marker. These findings suggest that the study on the characteristics of M. hyopneumoniae strain ES-2 will guide the rapid and accurate drug use in the clinic, and develop a theoretical foundation for accurately diagnosing and treating the infection caused by pathogenic M. hyopneumoniae.

Direct and indirect pathways for the spread of African swine fever and other porcine infectious diseases: An application of the mental models approach.

In this study, we investigated the occurrence of direct and indirect infectious disease transmission pathways among pig farms in Switzerland, as well as their specific relevance for the spread of African swine fever, porcine reproductive and respiratory syndrome (PRRS), and enzootic pneumonia. Data were collected using an adapted mental models approach, involving initial interviews with experts in the field of pig health and logistics, semi-structured interviews with pig farmers, and a final expert workshop, during which all identified pathways were graded by their predicted frequency of occurrence, their likelihood of spread of the three diseases of interest, and their overall relevance considering both parameters. As many as 24 disease pathways were identified in four areas: pig trade, farmer encounters, external collaborators, and environmental or other pathways. Two thirds of the pathways were expected to occur with moderate-to-high frequency. While both direct and indirect pig trade transmission routes were highly relevant for the spread of the three pathogens, pathways from the remaining areas were especially important for PRRS due to higher spread potential via aerosols and fomites. In addition, we identified factors modifying the relevance of disease pathways, such as farm production type and affiliation with trader companies. During the interviews, we found varying levels of risk perception among farmers concerning some of the pathways, which affected adherence to biosecurity measures and were often linked to the degree of trust that farmers had towards their colleagues and external collaborators. Our findings highlight the importance of integrating indirect disease pathways into existing surveillance and control strategies and in disease modelling efforts. We also propose that biosecurity training aimed at professionals and risk communication campaigns targeting farmers should be considered to mitigate the risk of disease spread through the identified pathways.

Seroprevalence of Mycoplasma hyopneumoniae in sows fifteen years after implementation of a control programme for enzootic pneumonia in Switzerland.

Mycoplasma hyopneumoniae is the etiological agent of enzootic pneumonia (EP), an economically important chronic respiratory disease in pigs. M. hyopneumoniae impacts the mucociliary clearance system by disrupting the cilia and modulates the immune response, resulting in intermittent dry non-productive cough. For progressive control of EP in Switzerland, a corresponding programme was fully implemented in 2004. It is based on total depopulation strategies of affected fattening farms as well as partial depopulation in breeding farms. Surveillance of EP status in Switzerland is mainly based on real-time PCR of nasal swabs from coughing animals or suspicious lungs and thereby sporadic cases are still observed every year. In order to obtain information on the seroprevalence, serum samples of 5021 sows from 968 farms collected in 2018 at eight different slaughterhouses were analyzed for the presence of M. hyopneumoniae-specific antibodies using a commercial ELISA kit. The overall seroprevalence was low with 0.98% of sows testing positive and these seropositive animals could be allocated to 3.92% of farms tested. Most seropositive farms presented weakly positive singleton reactors and only one farm showed several strongly seropositive animals. In conclusion, the serological status mirrors the successful progressive control of M. hyopneumoniae in the Swiss domestic pig population over the years. The current study underlines the added value of serological testing in the surveillance of EP in a country with low prevalence and confirms the sustained benefit of strategic control programmes.

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.

Identification of extracellular vesicles from J strain and wild isolate of Mycoplasma hyopneumoniae.

Respiratory diseases constitute a major health challenge for the worldwide pork industry. Porcine enzootic pneumonia (PES) is caused by Mycoplasma hyopneumoniae (Mhyo). Mycoplasmas have the ability to produce extracellular vesicles (EVs), which can be useful for pathogenicity studies and as delivery systems for vaccines. The aim of this study was to demonstrate and compare, under laboratory conditions, EVs produced by Mhyo strain J and wild isolate in stressed and non-stressed in vitro conditions. Using differential centrifugation, density gradient ultracentrifugation, and transmission electron microscopy, the ability of Mhyo strains to produce EVs was demonstrated under favorable and unfavorable conditions.

Development of ELISA Using Recombinant Proteins for the Diagnosis of Mycoplasma hyopneumoniae Infection.

In order to develop a more sensitive and reliable method for detection of serum antibodies against Mycoplasma hyopneumoniae infection in pigs, six recombinant proteins of M. hyopneumoniae (P102, P95, P46, P97 like, Lppt, and hypothetical P987) were used for the standardization of an indirect enzyme-linked immunosorbent assay (ELISA). The proteins were evaluated against 50 sera of the specific pathogen-free and 50 sera of pigs with lesions suggestive of infection. The sensitivity was 88%, 86%, 78%, 74%, 66%, and 60% for the proteins P102, P95, P46, P97 like, Lppt, and hypothetical protein P987, respectively. Moreover, the proteins were used to establish the seroprevalence in two different commercial herds (254 sera pigs from farm considered free of M. hyopneumoniae and 246 from farm with clinical signs of enzootic pneumonia and positive serology for M. hyopneumoniae) and the positive rate was 65.2% for P95, 54.6% for P102, 40.2% for P46, 37.2% for P97 like, 17.4% for the hypothetical P987, and 14% for Lppt protein. In addition, the ELISA with six recombinant proteins was compared to commercial HerdCheck kit using 118 random pig sera samples and the results showed that ELISA with recombinant proteins were more sensitive than the commercial test. These data show that the recombinant proteins P95 and P102 are potential targets to be used in diagnostic tests to detect antibodies against M. hyopneumoniae. Although more studies are necessary, this study provides insights that these recombinant proteins can be useful in epidemiological investigations and as potential biomarkers in differentiating infected animals from those vaccinated.

Cough associated with the detection of Mycoplasma hyopneumoniae DNA in clinical and environmental specimens under controlled conditions.

BACKGROUND: The association of cough with Mycoplasma hyopneumoniae (MHP) DNA detection in specimens was evaluated under conditions in which the MHP status of inoculated and contact-infected pen mates was closely monitored for 59 days post-inoculation (DPI). METHODS: Seven-week-old pigs (n = 39) were allocated to five rooms (with one pen). Rooms contained 9 pigs each, with 1, 3, 6, or 9 MHP-inoculated pigs, respectively, except Room 5 (three sham-inoculated pigs). Cough data (2 × week) and specimens, tracheal swabs (2 × week), oral fluids (daily), drinker wipes (~ 1 × week), and air samples (3 × week) were collected. At 59 DPI, pigs were euthanized, and lung and trachea were evaluated for gross and microscopic lesions. Predictive cough value to MHP DNA detection in drinker and oral fluid samples were estimated using mixed logistic regression. RESULTS: Following inoculation, MHP DNA was first detected in tracheal swabs from inoculated pigs (DPI 3), then oral fluids (DPI 8), air samples (DPI 10), and drinker wipes (21 DPI). MHP DNA was detected in oral fluids in 17 of 59 (Room 1) to 43 of 59 (Room 3) samples, drinker wipes in 4 of 8 (Rooms 2 and 3) to 5 of 8 (Rooms 1 and 4) samples, and air samples in 5 of 26 (Room 2) or 3 of 26 (Room 4) samples. Logistic regression showed that the frequency of coughing pigs in a pen was associated with the probability of MHP DNA detection in oral fluids (P < 0.01) and nearly associated with drinker wipes (P = 0.08). Pathology data revealed an association between the period when infection was first detected and the severity of gross lung lesions. CONCLUSIONS: Dry, non-productive coughs suggest the presence of MHP, but laboratory testing and MHP DNA detection is required for confirmation. Based on the data from this study, oral fluids and drinker wipes may provide a convenient alternative for MHP DNA detection at the pen level when cough is present. This information may help practitioners in specimen selection for MHP surveillance.

Reannotation of Mycoplasma hyopneumoniae hypothetical proteins revealed novel potential virulence factors.

Mycoplasma hyopneumoniae is a bacterium that inhabits the swine respiratory tract, causing porcine enzootic pneumonia, which generates significant economic losses to the swine industry worldwide. The knowledge on M. hyopneumoniae biology and virulence have been significantly increased by genomics studies. However, around 30% of the predicted proteins remained of unknown function so far. According to the original annotation, the genome of M. hyopneumoniae 7448, a Brazilian pathogenic strain, had 693 coding DNA sequences, 244 of which were annotated as coding for hypothetical or uncharacterized proteins. Among them, there may be still several genes coding for unknown virulence factors. Therefore, this study aimed to functionally reannotate the whole set of 244 M. hyopneumoniae 7448 proteins of unknown function based on currently available database and bioinformatic tools, in order to predict novel potential virulence factors. Predictions of physicochemical properties, subcellular localization, function, overall association to virulence and antigenicity are provided. With that, 159 out of the set of 244 proteins of unknown function had a putative function associated to them, allowing identification of novel enzymes, membrane transporters, lipoproteins, DNA-binding proteins and adhesins. Furthermore, 139 proteins were generally associated to virulence, 14 of which had a function assigned and were differentially expressed between pathogenic and non-pathogenic strains of M. hyopneumoniae. Moreover, all extracellular or cytoplasmic membrane predicted proteins had putative epitopes identified. Overall, these analyses improved the functional annotation of M. hyopneumoniae 7448 genome from 65% to 87% and allowed the identification of new potential virulence factors.