Epigenetic analysis of sheep mycoplasma pneumonia and the effect of FOXF1 on Mycoplasma ovipneumoniae infection

Background: Mycoplasma infections are widespread globally, causing respiratory and extrapulmonary diseases in animals, particularly in sheep and goats. Symptoms include coughing, nasal discharge, respiratory distress, and pneumonia. Several Mycoplasma species like Mycoplasma ovipneumoniae (MO), Mycoplasma capricolum ssp. capripneumoniae (MCCP), Mycoplasma mycoides ssp. capri (MMC), and Mycoplasma arginini (MA) are identified as causes. MA was first identified in sheep with respiratory diseases in 1972, and its presence has been consistently reported. In China, MA isolation from respiratory samples was achieved in 1991, correlating with pulmonary lesions. Sheep and goats are economic resources in China. In recent years, China encourages the sheep and goat breeding model to convert into scale. Nevertheless, sheep and goats farming are threatened by Mycoplasma, which greatly slowed down the production. Currently, there is a scarcity of data and reports regarding Mycoplasma infection in sheep and goat farming in Jiangsu province. This study aims to determine the prevalence and species of Mycoplasma in sheep and goats in Jiangsu province. The results may contribute to the prevention and control of Mycoplasma infection in Jiangsu region, and will also expand our understanding of the distribution of these respiratory diseases in sheep and goats. Materials, Methods & Results: The study collected 920 nasal swab samples from sheep and goats in six cities in Jiangsu province. Following sample processing, DNA extraction was carried out using the lysis boiling method for both clinical samples and reference strains. Subsequently, PCR amplification was conducted utilizing universal and specific primers to detect and identify Mycoplasma species. Finally, the positive PCR products underwent sequencing and were compared for phylogenetic analysis. The results show that through using PCR specific primers to amplify genes associated with the Mycoplasma, 109 (11.85%) were confirmed to be Mycoplasma positive out of 920 samples that were collected from clinically diseased (470) and healthy (450) sheep and goats from Jiangsu province, China. 109 positive samples were obtained and classified as MO in 78.9% and MA in 21.1%. No MCCP and MMC were detected, and there was no evidence of mixed Mycoplasma infection. The phylogenetic analysis of the partial sequences of positive samples based on the MO p113 gene and MA ADI gene showed a high degree of similarity with some sequences of Mycoplasma in GenBank. Discussion: Among clinically healthy animals, 28 out of 450 samples (6.22%) tested positive, while among those with respiratory diseases, 81 out of 470 samples (17.23%) were positive, indicating a higher risk of respiratory disease in infected sheep and goats. Mycoplasma was found even in asymptomatic sheep and goats, suggesting it may not be the sole cause of respiratory symptoms. Immunized animals had lower infection rates, indicating some effectiveness of current vaccines, though overall efficacy remains modest. Regional variations in infection rates may stem from differences in vaccination and hygiene practices. Out of 109 positive samples, 86 were identified as MO infection and 23 as MA infection, while no infections of MCCP and MMC were detected. In this study, MO isolates lacked a central umbilicus in colonies, while MA isolates resembled "fried egg like" morphology. MO P113 gene sequencing revealed lower homology (86.0%) with certain strains compared to ATCC 29419, GZ-QX1, and NCTC 10151. This discrepancy could be ascribed to genetic mutations or disparities in pathogenic strains. MA ADI gene sequencing showed high homology (> 99.1%) with ATCC 23838, NCTC 10129, and HAZ145 strains. This indicates that prevalent Mycoplasma linked to respiratory diseases in Jiangsu province are MO and MA. Keywords: ovine, caprine, small ruminants, Mycoplasma, epidemiology, sequencing, hylogenic analysis.

BackgroundPneumonia constitutes a major health challenge in sheep, severely compromising growth rates and overall productivity, and resulting in considerable economic losses to the sheep industry. To address this issue, the development of disease-resistant breeding programs based on the identification of genetic markers associated with pneumonia susceptibility is of critical importance. This study investigated a sheep population on a farm where pneumonia was endemic. The purpose was to use multi-omics methods to rapidly identify the principal pathogens responsible for pneumonia outbreaks, and to screen for genetic loci and key genes related to pneumonia resistance, thereby providing a scientific basis for the implementation of targeted breeding strategies for pneumonia resistance.ResultsHere, we assessed the impact of pneumonia on sheep growth by evaluating the pneumonia phenotypes of 912 sheep. High-throughput transcriptome sequencing of 40 lungs was conducted to obtain exogenous RNA fragments for microbial sequence alignment. Additionally, 16S rRNA sequencing was performed on lung tissues from 10 healthy and 10 diseased sheep to identify biomarkers associated with phenotypic differences. Mycoplasma ovipneumoniae was identified as the primary pneumonia pathogen, and its presence was further validated by load quantification and immunohistochemical analysis. Integration of genome-wide association study (GWAS) data from 266 lung pathological scores with transcriptome-based differentially expressed genes analysis enabled the identification of five single nucleotide polymorphisms (SNPs) and three potential candidate genes associated with Mycoplasma pneumonia. Subsequent genotyping and phenotype association analyses confirmed the significance of two SNPs and established a strong association between the FOXF1 gene and resistance to Mycoplasma pneumonia.ConclusionsHigh-throughput sequencing technologies have enabled the rapid and accurate identification of the causative pathogen of sheep pneumonia. By integrating multi-omics data, two genomic loci significantly associated with Mycoplasma pneumonia were screened, as well as an anti-Mycoplasma pneumonia key gene, FOXF1.

Macrophages are considered the first line of defence against Mycoplasma pneumoniae infection in sheep, and their polarization significantly affects lung tissue damage and remodelling. Here, we collected sheep bronchoalveolar lavage fluid (BALF) 28 days after infection with Mycoplasma ovipneumoniae (MO). RNA-seq analysis revealed a significant increase in peptidyl arginine deiminase 2 (PADI2) expression in the MO group, and upregulated genes were significantly enriched in the “FoxO signalling pathway” and “AMPK signalling pathway”. Immunohistofluorescence (IHF) staining revealed a significant increase in the number of macrophages, which were widely distributed in the alveolar cavity, in the MO infection group, and PADI2 was also positively expressed in the macrophages. In bone marrow-derived macrophages (BMDMs) isolated in vitro, inhibiting the expression of PADI2 induced infected macrophages to polarize towards alternatively activated (AAM, M2) macrophages. Moreover, the expression levels of the anti-inflammatory gene IL10 and tissue repair-related genes such as vascular endothelial growth factor alpha (VEGFA), transforming growth factor-β (TGF-β), and hepatocyte growth factor (HGF) significantly increased. In an in vitro coculture system, inhibition of PADI2 expression in BMDMs reduced apoptotic and intracellular reactive oxygen species (ROS) levels in lung fibroblasts. Interestingly, correlation analysis between the tracheal MO load and PADI2 expression in the bronchoalveolar lavage fluid of naturally infected MO sheep revealed that the higher the MO load was, the greater the PADI2 expression. A dual luciferase reporter assay confirmed that this process may be regulated by the transcription factor SP3. These results contribute to the understanding of the polarization state of macrophages after mycoplasma infection and to the preliminary exploration of PADI2 as an effective target to enhance host defence against MO infection. Targeting PADI2 to promote the polarization of M2 macrophages provides a promising approach for treating MO infection or preparing anti-Mycoplasma pneumonia gene-edited sheep.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13567-025-01632-7.

Respiratory disease is common in sheep globally. The ovine respiratory complex (ORC) is a polymicrobial disease of lambs and adult sheep associated with bacterial, mycoplasmal and/or viral infection, exacerbated by a range of host and environmental factors. Much is known about ORC, including aetiology and the pivotal role of Mycoplasma (Mesomycoplasma) ovipneumoniae, risk factors for infection and clinical disease, pathological mechanisms, the immune response to infection and the economic cost of the disease throughout sheep supply chains globally. Despite this, effective options to treat and prevent ORC, in particular M. ovipneumoniae, are still not readily available, perhaps reflecting the silent nature of the disease that downplays its significance and the tendency of domestic sheep to hide signs of weakness, a reflection of evolutionary defence mechanisms to avoid predation. M. ovipneumoniae was first isolated in southern Queensland, Australia in the 1970s from two large sheep flocks that had shown poor growth rates, reduced exercise tolerance and high incidence of respiratory distress in the flock even while resting, causing serious economic losses. This review summarizes the original Australian descriptions of M. ovipneumoniae in sheep, including studies undertaken in Queensland and Victoria, studies that confirmed the role of M. ovipneumoniae in respiratory disease of sheep in other regions of the world, pathogenesis of infection and immune responses, risk factors for infection and the economic cost of infection. A holistic approach to control of ORC in Australian sheep is proposed, that includes monitoring prevalence through abattoir surveillance, improved management to reduce stress throughout the supply chain (farm to feedlot to abattoir), better nutrition, particularly of pregnant ewes and young sheep, culling chronically infected carrier ewes and rams, the introduction of testing programs for stud sheep that attend shows and breeding centres, and careful attention to ram source. This approach offers a solution now and aligns with current industry initiatives, such as Lifetime Ewe Management and Making More from Sheep, providing an opportunity to embed control of ORC within these successful industry programs.

Mycoplasma ovipnuemoniae impairs the immune response of sheep and suppresses neutrophil function by inhibiting S100A9.

Mycoplasma pneumoniae (MP) is a common pathogen that can cause respiratory infections. MP pneumonia (MPP) leads to numerous complications, including lung injury and even death. The present study aimed to investigate the protective effects of Baicalin treatment on MP infection-induced lung injury and the molecular mechanism underlying these effects. Briefly, after mice were infected intranasally by MP and treated with Baicalin (80 mg/kg), serum levels of MP-immunoglobulin M (IgM) were detected by ELISA. The expression levels of C-reactive protein (CRP) in lung tissue were detected by immunohistochemistry and the bronchoalveolar lavage fluid (BALF) was examined by ELISA. Inflammatory factors and inflammatory cells in the BALF were assessed. The expression levels of microRNA (miR)-221 in lung tissue were examined by reverse transcription-quantitative PCR and pathological changes in lung tissue were detected by H&E staining. Cell apoptosis was evaluated by TUNEL assay and the protein expression levels of TLR4, MyD88 and NF-κB were detected by western blotting. Baicalin treatment significantly reduced serum levels of MP-IgM and CRP expression in lung tissue during MP infection. In addition, Baicalin decreased the levels of IL-1β, IL-6, IL-18 and TNF-α in the BALF, and the number of inflammatory cells. Baicalin also reduced the inflammatory infiltration in lung tissue induced by MP infection, improved the pathological changes detected in lung tissue, reduced apoptosis, and downregulated the protein expression levels of TLR4, MyD88 and NF-κB. Furthermore, Baicalin treatment downregulated the expression of miR-221 and the protective effects of Baicalin were attenuated by miR-221 overexpression. In conclusion, Baicalin has a therapeutic effect on mice with MP infection-induced lung injury, which may be related to inhibition of miR-221 expression and regulation of the TLR4/NF-κB signaling pathway.

Objective To investigate the changes of the levels of interleukin(IL)-17 in bronchoalveolar lavage fluid (BALF) of BALB/c mice infected by mycoplasma pneumonia (MP).Methods Sixtyfour BALB/c mice were randomly divided into two groups,normal group (n =32) and MP infected group (n =32).The BALF and lung tissue of two groups were collected after MP infection on day 3,7,14,21,each time 8.HE staining was used to observe the pathological changes of the lung tissue.IL-17 content in BALF was detected by ELISA.Results The inflammatory changes of the lung tissue was the most obvious at 7 day,and had begun to subside at 14 day,disappeared at 21 day after MP infection.IL-17 levels in BALF were (53.783 ±2.218) pg/ml(3 day),(65.913 ± 10.693) pg/ml(7 day),(59.915 ± 8.085) pg/ml(14 day),(57.043 ± 11.997) pg/ml(21 day) in MP infection group,and (46.220 ± 3.260) pg/ml in normal group.BALF IL-17 content increased significantly in MP infection group compared with normal group(P ＜ 0.05).IL-17 content reached its peak on day 7 after MP infection (P ＜ 0.05).Conclusion MP infection increased the level of IL-17 in the mice of BALF,reaching the peak on day 7.MP infection may be involved in airway inflammation in asthma through the IL-17 pathway. Key words: Mycoplasma pneumonia ; Bronchial asthma ; Bronchoalveolar lavage fluid; Interleukin-17

Background: Mycoplasmal pneumonia causes severe economic loss in goat farms due to loss of production performance and mortality. Mycoplasma ovipneumoniae is an important pathogen causing mycoplasmal pneumonia in small ruminants. The present research work was carried out to study the pathomorphological alterations associated with M. ovipneumoniae infection in an organized goat farm. Methods: The research work was carried out in an organized goat farm having history of respiratory illness with mortality and suspected for mycoplasmal pneumonia. Samples from ailing animals and dead animals were collected for investigation. Tissue samples from lungs, trachea, mediastinal lymph nodes and swabs from lungs, trachea and pleural fluid were collected during postmortem examination and subjected to laboratory examination. Result: The samples were confirmed to be positive for M. ovipneumoniae infection by PCR using specific primers. The clinical signs of M. ovipneumoniae infection observed were anorexia, mucopurulent nasal discharge, coughing, increased body temperature, respiratory distress and death in severely affected animals. In the present study, 10 animals exhibited clinical signs and 3 animals died out of 10 animals. Necropsy lesions observed in lungs were fibrinous adhesions, severe consolidation of left lung and blackish discoloration of right lung. Histopathologically, lesions in lungs were characterized by widespread congestion, presence of inflammatory cells, widened interlobular septa with fibrin and inflammatory cells. Congestion of bronchioles and presence of pinkish fibrinous exudate within the bronchiolar lumen were observed. From the present study it is concluded that M. ovipneumoniae infection causes severe pathology of respiratory organs and mortality in goats under stressful conditions like long transport.

Mycoplasma ovipneumoniae is recognized as an important pathogen associated with respiratory disease in sheep, contributing to considerable economic losses in the livestock sector. The present investigation aimed to determine the prevalence of respiratory infections in sheep from the Hassan district and adjoining regions, and to characterize the related pathological changes while confirming the involvement of Mycoplasma ovipneumoniae through molecular analysis. A total of one hundred sheep carcasses were examined, and tissue samples including lungs, trachea, bronchi, bronchioles, and mediastinal lymph nodes were collected for detailed gross, microscopic, and polymerase chain reaction evaluation. Respiratory lesions were observed in seventy-one animals. Molecular detection targeting the GPO3 gene of the Mycoplasma genus revealed infection in 32.39% of samples, and further amplification using species-specific primers for the 16S ribosomal ribonucleic acid gene confirmed Mycoplasma ovipneumoniae in 9.85% of cases. Prominent gross lesions in affected animals included consolidation of the lungs predominantly in cranio-ventral lobes, pulmonary congestion, edema, frothy exudate in the tracheal lumen, and mild fibrinous inflammation of the pericardium. Microscopic examination of pulmonary tissues revealed bronchopneumonia, interstitial pneumonia, and broncho-interstitial pneumonia. Additionally, fibrinous inflammation of the pericardium and depletion of lymphoid tissue in the lymph nodes, indicated systemic involvement associated with respiratory infection. The outcomes of this study confirm the circulation of Mycoplasma ovipneumoniae in sheep within the Hassan district and surrounding areas, highlighting its significant impact on the respiratory system and associated organs.

Long-term pathological and micro-biological progress in sheep of experimental disease resembling atypical pneumonia

In order to investigate the role played by platelet derived growth factor-BB (PDGF-BB) in the pathogenesis of pulmonary interstitial fibrosis in rats repeatedly infected with mycoplasma pneumoniae (MP), a rat MP infection model was developed by infecting rats with MP for 9 times during a period of 24 weeks with a technique of ultrasonic nebulizing inhalation. Then in situ hybridization was performed with PDGF-B chain cDNA probe and the results were quantitatively analyzed to measure the changes in PDGF-B chain mRNA expression in the lung tissue. The results showed that: (1) MP polymerase chain reaction (PCR) tests showed positive results in the bronchoalveolar lavage fluid (BALF) from all of the MP-infected rats (n = 4) while they were all negative in BALF from the control animals (n = 4, P < 0.05) and in BALF from those rats both infected with MP and, at the same time, treated with erythromycin (n = 4, P < 0.05). Bacterial cultures of the bronchial and lung tissue were negative in all three groups. The observation under a transmission electron microscope indicated that the interalveolar septa were widened with increased amount of collagen in the MP-infected rats while there were no obvious abnormalities in the other two groups. (2) Strong positive expression of PDGF-B chain mRNA was found in the plasma of monocytes and macrophages located in the locally widened interalveolar septa and alveolar spaces in the lung tissue from the MP-infected animals with the integral optical densities being 37.42 +/- 9.05 (n = 4) which was significantly higher than the values of control group (0.42 +/- 0.08, n = 4, P < 0.01) and of the group with MP-infection plus erythromycin treatment (1.62 +/- 0.40, n = 4, P < 0.01). These results suggest that PDGF-BB may be involved in the process of the development of pulmonary interstitial fibrosis caused by the repeated MP-infection. It may be an important growth factor for mediating the roles of monocytes and macrophages to promote the aggregation and proliferation of fibroblasts which can then secrete collagen in large quantity in the pulmonary interstitium.

Feral populations of aoudad (Ammotragus lervia) occur in Texas bighorn sheep (Ovis canadensis) habitat and pose several conceptual ecological threats to bighorn sheep re-establishment efforts. The potential threat of disease transmission from aoudad to bighorn sheep may exacerbate these issues, but the host competency of aoudad and subsequent pathophysiology and transmissibility of pneumonic pathogens involved in the bighorn sheep respiratory disease complex is largely unknown. Because the largest population-limiting diseases of bighorn sheep involve pathogens causing bronchopneumonia, we evaluated the host competency of aoudad for Mycoplasma ovipneumoniae and leukotoxigenic Pasteurellaceae. Specifically, we described the shedding dynamics, pathogen carriage, seroconversion, clinical patterns, and pathological effects of experimental infection among wild aoudad held in captivity. We found that aoudad are competent hosts capable of maintaining and intraspecifically transmitting Mycoplasma ovipneumoniae and Pasteurellaceae and can shed the bacteria for 53 days after exposure. Aoudad developed limited clinical signs and pathological findings ranged from mild chronic lymphohistiocytic bronchointerstitial pneumonia to severe and acute suppurative pneumonia, similarly, observed in bighorn sheep infected with Mycoplasma spp. and Pasteurellaceae bacteria, respectively. Furthermore, as expected, clinical signs and lesions were often more severe in aoudad inoculated with a combination of Mycoplasma ovipneumoniae and Pasteurellaceae as compared to aoudad inoculated with only Mycoplasma ovipneumoniae. There may be evidence of interindividual susceptibility, pathogenicity, and/or transmissibility, indicated by individual aoudad maintaining varying severities of chronic infection who may be carriers continuously shedding pathogens. This is the first study to date to demonstrate that aoudad are a conceptual disease transmission threat to sympatric bighorn sheep populations due to their host competency and intraspecific transmission capabilities.

Bighorn sheep pneumonia: Sorting out the cause of a polymicrobial disease

<p id="C3">Bacterial blight caused by <italic>Xanthomonas oryzae</italic> pv. <italic>oryzae</italic> is the most destructive bacterial disease in rice production, and cultivating new disease-resistance variety by mining and utilizing the novel disease resistance genes is one of the most effective ways to control this disease. In our study, a population of 139 recombinant inbred lines (RILs) derived from super hybrid rice Xieyou 9308, between the parents of which there was genetic difference in resistance to bacterial blight, were inoculated with four <italic>Xoo</italic> strains. A genome-wide association study (GWAS) was carried out using 476,505 SNPs for the lesion length as a quantitative phenotype. The result revealed that a total of 109 significant SNPs including two genes encoding NBS-LRR containing proteins were detected to be significantly difference at <italic>P </italic>&lt; 1×10^-4 for four <italic>Xoo</italic> strains-treated lesions, accounting for phenotype variation of 59.78%-63.29%, respectively. Furthermore, 25 SNPs located in/nearby 19 candidate genes were identified by the <italic>CR4 </italic>inoculation, accounting for 61.00%. Relative expression analysis of two selected candidate genes, LOC_Os11g43420 and LOC_Os11g45930, demonstrated their higher expression levels in the resistant cultivar Zhonghui 9308 than in the susceptible variety Xieqingzao B, suggesting that these two genes might positively regulated bacterial blight resistance. Phylogenetic tree analysis indicated that these two genes were different from the previous cloned resistant genes in evolution, suggesting they were novel disease resistance genes. These results lays a theoretical basis and provides genetic resources for future resistant breeding in rice.

Mycoplasma ovipneumoniae has long been associated with respiratory disease in young sheep and goats. Here, the authors present evidence that it may be a cause of a coughing syndrome and...