Mycoplasma hyopneumoniae (M. hyopneumoniae), the primary etiological agent of swine enzootic pneumonia, causes significant economic losses in the pork industry. This fastidious pathogen exhibits extremely slow growth in vitro, complicating its quantification. Several quantification methods, including color-changing units (CCU), colony-forming units (CFU), flow cytometry, and ATP luminometry, are documented in the literature, with CCU being the gold standard. However, the correlation among these techniques has not been thoroughly evaluated. Additionally, viability quantitative polymerase chain reaction (v-qPCR) using propidium monoazide (PMA) or ethidium monoazide (EMA) offers a rapid and sensitive alternative for detecting viable bacteria. This study aims to evaluate and compare different methods for quantifying M. hyopneumoniae, validate an accurate real-time quantitative approach, and develop a tailored v-qPCR assay for this challenging pathogen. The in vitro growth kinetics of three M. hyopneumoniae strains (232, J, and 2010) were evaluated using CCU, CFU, flow cytometry, and ATP luminometry. A confocal laser scanning microscopy (CLSM) protocol was established for direct quantification of M. hyopneumoniae in culture media and used to validate flow cytometry-based quantification under controlled conditions. Finally, a rapid v-qPCR assay was developed and optimized for viable M. hyopneumoniae quantification. The comparison of M. hyopneumoniae growth kinetics across CFU, CCU, flow cytometry, and ATP luminometry demonstrated similar growth dynamics and high assay correlation. Flow cytometry and CLSM quantification showed a strong correlation for strain 232 (r = 0.9973) and a moderate correlation for strain J (r = 0.8933). The detection of live M. hyopneumoniae by v-qPCR correlated strongly with viable cell numbers detected by flow cytometry (R² values: 232: 0.9726; J: 0.8628; 2010: 0.9933, p < 0.05). The limit of detection of the v-qPCR assay for the reference strain 232 was 5 × 10⁴ viable M. hyopneumoniae cells/mL. This study presents the first validated PMA-based v-qPCR assay for mycoplasma species, along with established flow cytometry and CLSM protocols for rapid and accurate differentiation of viable and non-viable M. hyopneumoniae cells. These methods significantly reduce quantification time from the four weeks required by CCU or CFU to just a few hours. Furthermore, upon validation in clinical specimens, v-qPCR can potentially serve as a valuable tool in M. hyopneumoniae eradication programs.

An integrated individual-based model of transmission, clinical outcomes, and economic impact of Mycoplasma hyopneumoniae infection in a commercial pig fattening unit

Swine enzootic pneumonia, caused by Mycoplasma hyopneumoniae (M. hyopneumoniae), remains one of the most economically devastating respiratory diseases in the global swine industry. The emergence of antibiotic resistance in livestock highlights the urgent need for effective, safe, and sustainable alternatives. This study demonstrates that naturally derived protoberberine alkaloids exhibit potent antibacterial activity against M. hyopneumoniae while maintaining low host cytotoxicity and strong anti-inflammatory effects. Among them, jatrorrhizine showed remarkable therapeutic efficacy in infected pigs, comparable with that of florfenicol. These findings provide a scientific basis for developing protoberberine alkaloids as promising natural alternatives to conventional antibiotics for controlling M. hyopneumoniae infections, thereby contributing to improved animal health, reduced antimicrobial resistance, and sustainable swine production.

Interactions between Mycoplasma hyopneumoniae strains and the resident lung microbiota in swine.

AI-based respiratory rate estimation in group-housed pigs infected with Mycoplasma hyopneumoniae under occlusion conditions

Background/Objectives: Porcine Circovirus Type 2 (PCV2) and Mycoplasma hyopneumoniae are primary pathogens causing respiratory disease in pigs. Recently, a Ready-to-Use bivalent PCV2 and M. hyopneumoniae vaccine has been registered in China. The aim of this randomized, side-by-side trial was to evaluate the efficacy and safety of this vaccine under field conditions in a Chinese commercial pig farm. Methods: In total, 938 piglets were allocated to three groups—A (tested vaccine), B, C—and vaccinated according to different schemes. Efficacy was assessed by Average Daily Gain (ADG), pneumonia lesions at slaughter and PCV2 viremia. Systemic reactions were recorded after vaccination to evaluate safety. Results: ADG was higher in group A compared with other vaccination schemes. The prevalence of pneumonia lesions was significantly lower in group A. PCV2 viremia was overall low in all groups, with no reported differences. No severe or moderate systemic reactions were observed after vaccination. Only four pigs showed mild reactions (A: 2/320, B: 2/309; C: 0/309). Conclusions: Under these conditions, the tested vaccine was proved to be efficacious in increasing ADG and reducing pneumonia at slaughter by protecting against both PCV2 and M. hyopneumoniae field infections. It can also be concluded that the Ready-To-Use bivalent vaccine was safe.

Coinfection of porcine circovirus type 2 (PCV2) with Mycoplasma hyopneumoniae (M. hyo) causes major worldwide economic losses within the swine industry. This study aimed to assess the safety and efficacy of a single dose of a bivalent vaccine containing PCV2d and M. hyo antigen (Cirbloc® M Hyo) under field conditions. Two studies were performed under the GCP (Good Clinical Practice) requirements on farrow-to-finish farms in Hungary and Cyprus. On both farms, the presence of both PCV2 and M. hyo infection was demonstrated. For both studies, safety parameters were observed and measured from inclusion at 21 (±3) days of age until 14 days after vaccination. Efficacy parameters were observed and measured from inclusion until slaughter. Administration of the vaccine was safe in both studies, as no general, immediate, or local reactions were observed. The efficacy of the vaccine was confirmed in both studies as the following parameters were significantly reduced in the vaccinated groups compared to the control groups: viraemia, faecal shedding, viral load in lungs and in all collected lymphoid tissues, M. hyo-specific lung lesions, and average daily body weight gain. These results collectively support the vaccine’s potential as an effective tool for disease control.

ABSTRACTBackgroundA field efficacy trial was conducted on three farms to evaluate a novel combined vaccine containing porcine circovirus types 2a/d (PCV2a/d), Mycoplasma hyopneumoniae, and M. hyorhinis.MethodsThree farms with a history of subclinical PCV2 infection, enzootic pneumonia, and polyserositis were enrolled. Each farm included 40 piglets (18 days old), which were randomly allocated to vaccinated or unvaccinated groups. Pigs in the vaccinated group received a single 2‐mL intramuscular dose of the combined vaccine at 21 days of age, while unvaccinated pigs received phosphate‐buffered saline.ResultsVaccination significantly improved (p < 0.05) body weight and average daily weight gain on all three farms compared with unvaccinated pigs. Vaccinated pigs mounted protective humoral and cellular immune responses against PCV2, M. hyopneumoniae, and M. hyorhinis. Furthermore, vaccination reduced viral and mycoplasmal loads in serum, laryngeal, and nasal samples and decreased the severity of associated lesions.ConclusionsThe combined vaccine demonstrated strong efficacy under field conditions, providing protection against subclinical PCV2 infection, enzootic pneumonia caused by M. hyopneumoniae, and polyserositis caused by M. hyorhinis. These findings support its potential as an effective intervention for improving both health and productivity in swine herds.

The objective of this study was to prepare polyclonal antibodies (pAb) against the Mycoplasma hyopneumoniae (Mhp) membrane protein Mhp271 and systematically evaluate their immunological characteristics and potential applications. The mhp271 gene (3 156 bp) was amplified by PCR and cloned into the prokaryotic expression vector pMAL-c5x to construct the recombinant plasmid pMAL-c5x-mhp271. After verification by PCR and DNA sequencing, the construct was transformed into Escherichia coli BL21(DE3) competent cells, and the expression of recombinant Mhp271 (rMhp271) was induced with IPTG. Western blotting revealed a specific band at approximately 160 kDa, confirming successful expression of rMhp271. Purified rMhp271 was emulsified and used to immunize BALB/c mice three times. Serum samples were collected one week after the final immunization, and anti-Mhp271 specific pAb was isolated. To assess the reactivity of the anti-Mhp271 pAb, we used porcine alveolar macrophages (PAMs) to establish a cell model of Mhp infection. After Mhp infection for 12 h, Western blotting and indirect immunofluorescence assay (IFA) were employed to assess protein expression. Western blotting results showed a specific band at approximately 118 kDa in the lysate from Mhp-infected PAMs, while no corresponding band was detected in the uninfected control group. IFA demonstrated distinct green fluorescence signals in infected cells, whereas no fluorescence was observed in the uninfected control group. Furthermore, the potential of anti-Mhp271 pAb to inhibit Mhp infection was evaluated through in vitro blocking assays. Mhp was pre-incubated with either 100-fold diluted anti-Mhp271 serum (experimental group) or negative serum (control group) for 30 min before being inoculated into PAMs for 12 h. TaqMan real-time quantitative PCR indicated a significant reduction in Mhp load in the experimental group compared with the control group, which was further confirmed by the weakened fluorescence in IFA. Overall, the prepared anti-Mhp271 pAb demonstrated good reactogenicity and anti-infective activity, being suitable for immunological detection methods such as Western blotting and IFA.

IntroductionDevelopment of vaccines to preserve and improve human and animal health requires effective protective antigens, delivery platforms, and adjuvants. The immunostimulant based on heat-inactivated Mycobacterium bovis (IV) was developed to boost protective immune response in different animal species against pathogen infection and tick infestations.MethodsIn this study, a serum proteomics approach was used with functional annotations and enrichment network analysis for the characterization of immune pathways and biomarkers associated with parenteral administration of one, two, or three IV doses in the wild boar (Sus scrofa) animal model. An independent False Discovery Rate (FDR) analysis with the target-decoy approach provided by ProteinPilot™ was used, and positive identifications were considered when identified proteins reached a 1% FDR. Furthermore, pathogen surveillance was also performed to evaluate the IV treatment effect.ResultsThe proteomics analysis identified a total of 205 proteins, of which 97 displayed significant differential representation with 64 and 33 over (e.g., C4a, C5, C6, C7, and C9) and underrepresented (e.g., C3), respectively, in response to treatment. Results showed that IV administration activated both innate and adaptive immune responses through humoral immunity, regulation of the actin cytoskeleton pathway, coagulation cascade, and complement system. A single or two doses of IV significantly increased the activities of the classical, alternative, and lectin complement pathways. Moreover, a tendency was observed towards reducing seroprevalence in IV-treated wild boar over time for the causative agents of tuberculosis (Mycobacterium tuberculosis complex), pneumonia (Mycoplasma hyopneumoniae), and Aujeszky’s disease (porcine herpesvirus type 1).DiscussionThese results support a role for IV in stimulating immune and anti-inflammatory responses with possible application in different vaccine formulations for the control of infectious diseases.

Evaluation of pro-inflammatory activity and immunogenicity of an inactivated SBA-15 silica vaccine against Mycoplasma hyopneumoniae in piglets.

Antimicrobial resistance (AMR) is a significant global health concern, and the use of antibiotics in livestock, including swine production, is a major contributor. Vaccines offer a promising alternative for controlling bacterial infections in pigs, but their widespread use is often hindered by biological, economic, and practical challenges. This study surveyed U.S. swine veterinarians to identify which bacterial diseases require better vaccines and to understand the barriers to their adoption. Nineteen veterinarians with an average of 24.7 years of experience were surveyed across 21 states. The results identified Streptococcus suis, Escherichia coli, Mycoplasma hyopneumoniae, and Glaesserella parasuis as the most critical pathogens needing improved vaccines. Veterinarians anticipated significant improvements in vaccine efficacy for S. suis and E. coli during the nursery stage and expressed a willingness to pay 1.8 and 1.9 times their current prices, respectively. While expectations for M. hyo vaccine improvements were not significant, veterinarians expressed the highest willingness to pay (4.2 times the current price), citing the potential for disease eradication. This research highlights that developing effective vaccines for S. suis and E. coli should be the most urgent priority due to their significant economic impact and rising AMR concerns. However, M. hyo vaccine development holds the most economic potential due to the possibility of eradication. Our research provides a roadmap for future efforts to combat AMR in the swine industry, emphasizing key economic, policy, and educational considerations for successful vaccine implementation.

Prevalence of porcine circoviruses (PCV2 and PCV3) in slaughtered pigs with different pleurisy lesions score: Coinfections with Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae, and Pasteurella multocida.

Economic impact of productivity losses attributable to porcine reproductive and respiratory syndrome virus in United States pork production, 2016-2020.

Abstract Mycoplasma hyopneumoniae (M. hyopneumoniae) is the primary causative agent of enzootic pneumonia, a highly prevalent respiratory disease affecting pigs in the late grow-finish period.1 Infection with this bacterium is associated with reduced animal welfare, performance, and decreased production efficiency. Mycoplasma hyopneumoniae is a contributor to the Porcine Respiratory Disease Complex, along with other agents like porcine reproductive and respiratory syndrome virus (PRRSV).1 Infectious diseases decrease production efficiency and can compromise the sustainability of pork production.2 Using literature on pig performance from M. hyopneumoniae experimental infections and lifecycle impact estimates, the environmental impact of M. hyopneumoniae infection was calculated, which increased as days on feed did.3 However, it is unknown if similar performance is observed in commercial conditions. Therefore, the objective of this study was to assess production performance metrics in M. hyopneumoniae infected pigs under commercial conditions to inform estimates of sustainability in pork production. This study utilized data from three conveniently selected pig flows (A, B, and C) in a US production system. Eight years of historical data were evaluated for each flow. During six of the years in the study, Flow A was positive for M. hyopneumoniae at the sow farm. Flows B and C were negative for M. hyopneumoniae infection and served as controls. All flows were sourced from PRRSV positive sow farms. A retrospective comparison using exploratory data and time series analyses was conducted to identify differences in flows based on M. hyopneumoniae infection and co-infection with PRRSV. Variables related to feed utilization, medication, mortality, carcass weight, and sales were assessed to identify trends. The time series analysis showed the peak mortality was 2.3 times higher in flow A compared to flows B and C. The lowest ADG in flow A was 1.5 times lower than that in the control flows. The peak disruption in multiple performance metrics for flow A, was observed approximately nine months after a Mycoplasma hyopneumoniae outbreak. Metrics displaying peak disruption on average included: mortality, total medication cost, average daily feed intake, gain to feed, substandard sales, average carcass weight, average daily gain, and days first market (Table 1). In this dataset, infection with M. hyopneumoniae resulted in increased medication cost, decreased growth rate and carcass weight, leading to reduced production efficiency, which can ultimately compromise pork production sustainability. A synergistic effect of co-infection of M. hyopneumoniae and PRRSV was observed. The timing of peak disruption in production performance parameters was evidenced several months post the initial M. hyopneumoniae outbreak. Results from this study suggest that the impact of swine diseases on sustainability of pork production requires analysis of commercial farm data as production dynamics are not usually captured in the scientific literature. 1Pieters M., Maes D. (2019). In: Diseases of Swine. 11th Ed. Blackwell Pub. J. Wiley &amp; Sons, Inc. 2Capper, J. (2023). One Health Outlook, 5(1). 3Krebs, S. et al. (2024). Proc. of ASAS, Calgary, Canada.

Background: The efficacy of a novel combined vaccine targeting porcine circovirus types 2a/d (PCV2a/d), Mycoplasma hyopneumoniae, and M. hyorhinis was evaluated in a controlled challenge study. Methods: A total of 45 pigs were randomly allocated into nine groups (five pigs per group). Vaccinated groups received a single 2 mL intramuscular dose of the combined vaccine and were subsequently challenged with PCV2a, PCV2d, M. hyopneumoniae, and M. hyorhinis. Unvaccinated groups received a single 2 mL intramuscular dose of phosphate-buffered saline (0.01 M, pH 7.4). Growth performance, systemic adaptive immune (humoral and cellular) responses, viremia, laryngeal and nasal mycoplasma loads, and histopathological lesions were assessed. Results: Vaccinated pigs exhibited enhanced growth performance and elicited systemic immune responses, including both humoral and cellular immunity, against all four pathogens. Vaccination also significantly reduced viremia, mycoplasmal loads in laryngeal and nasal swabs, and the severity of associated lesions compared with unvaccinated controls. Conclusions: These results indicated that the combined vaccine was efficacious and conferred protection against PCV2a, PCV2d, M. hyopneumoniae, and M. hyorhinis challenge under experimental conditions. This combined vaccine represented an effective strategy to enhance growth performance and control complex co-infection in swine populations.

This is the first systematic review and meta-analysis on the prevalence and risk factors of M. hyopneumoniae infection in swine farms across mainland China from 2003 to 2024. A total of 54 eligible cross-sectional studies were analyzed by stratifying farms as subclinically or clinically infected. The overall pooled prevalence of M. hyopneumoniae was estimated as 33.4%, with clinical infection farms showing a significantly higher prevalence (52.9%) than subclinical farms (11.5%). Subgroup analyses revealed significant variations in infection rates based on age, sampling year, geographic region, farming scale, season, sampling type, and diagnostic method. Small-scale farms, farms with breeding swine, and farms in the Northwest region showed the highest infection rates. Diagnostic methods and sampling types also significantly influenced detection rates. Sensitivity analyses confirmed the robustness of the results, while publication bias was addressed using the Trim-and-Fill method. To effectively reduce the burden of M. hyopneumoniae in the swine industry in mainland China, future efforts should prioritize enhanced biosecurity, improved diagnostic accuracy, and region-specific vaccination and management strategies.

IntroductionRespiratory diseases have a substantial impact on swine production worldwide. Understanding the relationship between gross lung lesions and the presence of infectious agents is crucial for developing effective disease control strategies that target both primary and secondary pathogens.Material and MethodsA cross-sectional study was conducted on 22 pig farms in western Poland. Cranioventral pulmonary consolidation (CVPC) in slaughtered pigs was assessed, and 20 lung tissue samples were collected from each herd. The presence of common bacterial and viral respiratory pathogens was identified using PCR-based methods.ResultsThe disorder was observed in 79.3% (95% confidence interval 75.3–82.8) of slaughtered pigs across all examined herds. The most frequently detected pathogens at both the herd and individual animal levels were Glaesserella parasuis, Mycoplasma hyopneumoniae and porcine circovirus 2. Co-infections involving two or more respiratory pathogens were prevalent, occurring in 100% of herds and 87.7% of individual pigs. Mean CVPC scores were significantly higher in pigs infected with Mycoplasma hyopneumoniae, Mycoplasma hyorhinis and porcine reproductive and respiratory syndrome virus 1.ConclusionThese findings highlight the multifactorial nature of respiratory infections in pigs. Effective control measures should consider the high prevalence of co-infections and their impact on lung lesion severity to improve overall herd health and productivity.

Respiratory infections are a major concern in pig farming as they negatively impact animal health and productivity. Coughing is a key symptom of respiratory disease and can be classified as productive or non-productive, but human assessment often leads to inconsistencies. This study aimed to use a machine learning model to classify pig coughs and investigate their association with respiratory infections. Cough sounds from 49 fattening pigs were recorded and analyzed using a Python-based machine learning system. The model's accuracy in detecting coughs was 0.72, compared to 0.69 for farmers. For classification of non-productive coughs, the machine learning results showed strong agreement with infection status by Mycoplasma hyopneumoniae, with a Spearman's correlation of 0.80 and a Cohen's Kappa of 0.79. However, the association with Porcine Circovirus type 2 was weak, with correlation and Kappa values of 0.05 and 0.037, respectively. These findings indicate that machine learning can classify pig coughs more accurately than human evaluators and that non-productive coughs are strongly linked to Mycoplasma infection but not to PCV2. This suggests the potential use of machine learning for more reliable disease monitoring and early detection in swine production.

Increasing antibiotic resistance in Mycoplasma hyopneumoniae (Mhp)necessitates alternative treatments. Piper sarmentosum extracts (PSE) andguava extracts (GE) show promise due to their antimicrobial and antioxidant properties.This study evaluates the combined in vitro effects of PSE and GE againstMhp and their antioxidant activities. Anti-Mhp activity was assessed using microdilutionminimum inhibitory concentration (MIC) and synergy via fractional inhibitory concentrationindex (FICI), while antioxidant capacity was measured with 2,2’-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and ferric reducing antioxidant power(FRAP) assays for individual extracts, and a 1:1 mixture (based on the results of FICI).Phytochemical profiling used high performance liquid chromatography (HPLC) to quantifymain six bioactive compounds. HPLC showed PSE containedvitexin-2-O-rhamnoside (8.58 mg/g) and pellitorine (3.31 mg/g); GE hadquercetin-3-O-glucuronide (6.85 mg/g), quercetin (3.72 mg/g),isoquercitrin (0.51 mg/g), and avicularin (1.37 mg/g). PSE (MIC 0.78 mg/mL) and GE (MIC0.39 mg/mL) exhibited anti-Mhp effects, with their 1:1 combination being additive(FICI=0.75) and reducing MIC to 0.195 mg/mL. This mix also enhanced antioxidant activityby 9.0% in ABTS and 27.0% in FRAP assays. The 1:1 combination of PSE and GE demonstratedsuperior anti-Mhp activity (additive effect) and enhanced antioxidant properties(synergistic effect) compared to the individual extracts. These findings highlight thepotential of these extracts as phytopharmaceutical agents.