Exploring Mycoplasma ovipneumoniae NXNK2203 infection in sheep: insights from histopathology and whole genome sequencing.

BACKGROUND: Mycoplasma ovipneumoniae (M. ovipneumoniae) is a significant pathogen causing respiratory infections in goats and sheep. This study focuses on investigating vulnerability of Hu sheep to M. ovipneumoniae infection in the context of late spring's cold weather conditions through detailed autopsy of a severely affected Hu sheep and whole genome sequencing of M. ovipneumoniae. RESULTS: The autopsy findings of the deceased sheep revealed severe pulmonary damage with concentrated tracheal and lung lesions. Histopathological analysis showed tissue degeneration, mucus accumulation, alveolar septum thickening, and cellular necrosis. Immunohistochemistry analysis indicated that M. ovipneumoniae was more in the bronchi compared to the trachea. Genome analysis of M. ovipneumoniae identified a 1,014,835 bp with 686 coding sequences, 3 rRNAs, 30 tRNAs, 6 CRISPRs, 11 genomic islands, 4 prophages, 73 virulence factors, and 20 secreted proteins. CONCLUSION: This study investigates the vulnerability of Hu sheep to M. ovipneumoniae infection during late spring's cold weather conditions. Autopsy findings showed severe pulmonary injury in affected sheep, and whole genome sequencing identified genetic elements associated with pathogenicity and virulence factors of M. ovipneumoniae.

Development of loop-mediated isothermal amplification for detection of Mycoplasma ovipneumoniae in goats, Capra hircus.

Mycoplasma ovipneumoniae is an important pathogen that causes respiratory disease in goats and sheep, leading to significant economic losses in the livestock industry. A quick and robust diagnostic test will aid in early diagnosis and treatment of the disease. Loop-mediated isothermal amplification (LAMP) offers several advantages over traditional PCR, including faster amplification, simpler operation, and lower equipment requirements, making it a promising tool for use in basic livestock units where resources and infrastructure may be limited. The present study reports on developing a LAMP assay to rapidly detect M. ovipneumoniae in goats (Capra hircus) targeting the 16S rRNA gene. LAMP was optimized to perform at 60 °C for 75 min. The result was visualized by a change in colour from deep pink to orange and further confirmed by gel electrophoresis, which gave a typical ladder-like pattern. The detection limit of the assay was as low as 0.04 ng/µl, indicating the high sensitivity of the assay. The test failed to amplify DNA isolated from healthy goat blood, Mycoplasma arginini DNA, and Staphylococcus aureus DNA. The sensitivity, specificity, and accuracy of the assay were 97.73% and 94.83%, and 96.08%, respectively. The study concludes that the developed loop-mediated isothermal amplification assay is a practical and reliable tool for field-level diagnosis of M. ovipneumoniae infections in goats, with high sensitivity and specificity under resource-limited conditions.

The role of Mycoplasma ovipneumoniae and Mycoplasma arginini in the respiratory mycoplasmosis of sheep and goats in Italy: Correlation of molecular data with histopathological features.

Mycoplasma infections are commonly found in the respiratory system of small ruminants; the species most commonly detected are Mycoplasma ovipneumoniae and Mycoplasma arginini, associated with the so-called "atypical non-progressive pneumonia". The pathogenic role of M. ovipneumoniae in pneumonia has been demonstrated in sheep but still needs to be verified in goats; on the other hand, the role of M. arginini in sheep is not well understood, while in goats seems to be of low pathogenic value. The present study aims to investigate the aetiology of pneumonia in sheep and goats that died from respiratory disease using anatomopathological, histopathological, and molecular investigations and to clarify the role of respiratory mycoplasmas by the association of molecular data with histopathological features. First, to better understand which histological changes are actually suggestive of atypical pneumonia in sheep and goats, the study identified the histological lesions significantly associated with Mycoplasma spp. infection. Then, the histological score of lesions considered suggestive of atypical pneumonia was used to estimate the pathogenicity of each mycoplasma detected. The results showed that M. ovipneumoniae and M. arginini (alone or in mixed infections) are pathogenic both in sheep, as well as in goats with similar histology and severity of lesions. Moreover, young animals were statistically more susceptible to M.ovipneumoniae and M. arginini infection than adults. Animals appeared more at risk to the development of M. ovipneumoniae and M. arginini infection in summer.

Mesomycoplasma ovipneumoniae from goats with respiratory infection: pathogenic characteristics, population structure, and genomic features.

BACKGROUND: Mycoplasma ovipneumoniae is a critical pathogen that causes respiratory diseases that threaten Caprini health and cause economic damage. A genome-wide study of M. ovipneumoniae will help understand the pathogenic characteristics of this microorganism. RESULTS: Toxicological pathology and whole-genome sequencing of nine M. ovipneumoniae strains isolated from goats were performed using an epidemiological survey. These strains exhibited anterior ventral lung consolidation, typical of bronchopneumonia in goats. Average nucleotide identity and phylogenetic analysis based on whole-genome sequences showed that all M. ovipneumoniae strains clustered into two clades, largely in accordance with their geographical origins. The pan-genome of the 23 M. ovipneumoniae strains contained 5,596 genes, including 385 core, 210 soft core, and 5,001 accessory genes. Among these genes, two protein-coding genes were annotated as cilium adhesion and eight as paralog surface adhesins when annotated to VFDB, and no antibiotic resistance-related genes were predicted. Additionally, 23 strains carried glucosidase-related genes (ycjT and group_1595) and glucosidase-related genes (atpD_2), indicating that M. ovipneumoniae possesses a wide range of glycoside hydrolase activities. CONCLUSIONS: The population structure and genomic features identified in this study will facilitate further investigations into the pathogenesis of M. ovipneumoniae and lay the foundation for the development of preventive and therapeutic methods.

The trehalose glycolipid C18Brar promotes antibody and T-cell immune responses to Mannheimia haemolytica and Mycoplasma ovipneumoniae whole cell antigens in sheep.

Bronchopneumonia is a common respiratory disease in livestock. Mannheimia haemolytica is considered the main causative pathogen leading to lung damage in sheep, with Mycoplasma ovipneumoniae and ParaInfluenza virus type 3, combined with adverse physical and physiological stress, being predisposing factors. A balance of humoral and cellular immunity is thought to be important for protection against developing respiratory disease. In the current study, we compared the ability of the trehalose glycolipid adjuvant C18Brar (C18-alkylated brartemicin analogue) and three commercially available adjuvant systems i.e., Quil-A, Emulsigen-D, and a combination of Quil-A and aluminium hydroxide gel, to stimulate antibody and cellular immune responses to antigens from inactivated whole cells of M. haemolytica and M. ovipneumoniae in sheep. C18Brar and Emulsigen-D induced the strongest antigen-specific antibody responses to both M. haemolytica and M. ovipneumoniae, while C18Brar and Quil-A promoted the strongest antigen-specific IL-17A responses. The expression of genes with known immune functions was determined in antigen-stimulated blood cultures using Nanostring nCounter technology. The expression levels of CD40, IL22, TGFB1, and IL2RA were upregulated in antigen-stimulated blood cultures from animals vaccinated with C18Brar, which is consistent with T-cell activation. Collectively, the results demonstrate that C18Brar can promote both antibody and cellular responses, notably Th17 immune responses in a ruminant species.

The first Mycoplasma ovipneumoniae recovered from a sheep with respiratory disease in Brazil - draft genome and genomic analysis.

Mycoplasma ovipneumoniae is an important etiological agent of sheep respiratory disease worldwide. Here, we describe the first isolation and draft genome sequence of M. ovipneumoniae strain USP-BR2017 retrieved from tracheobronchial lavage of a sheep showing clinical signs of respiratory disease in the Rio de Janeiro State, Brazil. The culture of tracheobronchial lavage resulted in glucose-fermenting fried egg colonies, which were identified as M. ovipneumoniae by polymerase chain reaction. The genome was sequenced using the Illumina NextSeq 2000 and de novo assembled using SPAdes. The genome of the sequenced organism presented an approximate size of 1,122,253 bp. The annotation revealed 773 coding DNA sequences (CDSs), 806 genes, three rRNAs, and 30 tRNAs. Data analysis revealed M. ovipneumoniae strain USP-BR2017 contains a few virulence genes, including the hemolysing C gene (hlyC). In addition, strain USP-BR2017 showed high identity over the 16S rRNA gene with other sheep isolates from China and United States. This first description of M. ovipneumoniae in diseased Brazilian sheep demonstrates the importance of continuous surveillance and diagnostics of pathogens causing respiratory disease in sheep in Brazil.

Multilocus Sequence Typing of Mycoplasma ovipneumoniae Detected in Dall's Sheep (Ovis dalli dalli) and Caribou (Rangifer tarandus grantii) in Alaska, USA.

In 2018, Mycoplasma ovipneumoniae was detected in free-ranging caribou (Rangifer tarandus grantii) and Dall's sheep (Ovis dalli dalli) in Alaska, US. Evaluation of additional nasal swabs and archived tissues for M. ovipneumoniae suggested that this bacterium was widespread geographically and temporally in populations of both species. Multilocus sequence typing of four loci identified a single, novel, apparently stable strain type of M. ovipneumoniae in 11 Dall's sheep and 15 caribou in multiple populations across Alaska sampled over a period of 15 yr (2004-19). This strain type differs from those detected to date from wild or domestic sheep (Ovis aries) or goats (Capra aegagrus hircus) tested in Alaska or the lower 48 states. Although the population health implications of this strain are unknown, it has not been associated with population-wide mortality events. The presence of this strain does not decrease the potential risk from the introduction of a pathogenic M. ovipneumoniae strain associated with severe disease in other wildlife populations; therefore, continued monitoring for signs of disease and additional strains is important.

Development of a real-time PCR assay for detection and differentiation of Mycoplasma ovipneumoniae and a novel respiratory-associated Mycoplasma species in domestic sheep and goats.

A novel respiratory-associated Mycoplasma species (M. sp. nov.) of unknown clinical significance was recently identified that causes false positive results with multiple published PCR methods reported to specifically detect Mycoplasma ovipneumonaie, a well-known respiratory pathogen in small ruminants. This necessitates our objective to develop a real-time PCR (qPCR) assay for improved specificity and sensitivity, and more rapid detection and differentiation of M. ovipneumoniae and the M. sp. nov. in domestic sheep (DS) and domestic goat (DG) samples, as compared to a conventional PCR and sequencing (cPCR-seq) assay. Primers and probes were designed based on available M. ovipneumoniae 16S rRNA gene sequences in the GenBank database, and partial 16S rRNA gene sequences provided by the United States Department of Agriculture, Agricultural Research Service (USDA-ARS) for M. ovipneumoniae and M. sp. nov. USDA-ARS provided DS (n = 153) and DG (n = 194) nasal swab nucleic acid that previously tested positive for either M. ovipneumoniae (n = 117) or M. sp. nov. (n = 138), or negative for both targets (n = 92) by cPCR-seq. A host 18S rRNA gene was included as an internal control to monitor for the failure of nucleic acid extraction and possible PCR inhibition. For samples positive by cPCR-seq, qPCR agreement was 88.0% (103/117; κ = 0.81) and 89.9% (124/138; κ = 0.84) for M. ovipneumoniae and M. sp. nov., respectively; 12 of 255 (4.7%) cPCR-seq positive samples were qPCR positive for both targets. Of samples negative by cPCR for both mycoplasmas, qPCR detected M. ovipneumoniae and M. sp. nov. in 6.5% (6/92) and 4.3% (4/92), respectively. Samples with discordant results between the cPCR and sequencing assay and the new qPCR were analyzed by target sequencing; successfully sequenced samples had identity matches that confirmed the qPCR result. The increased target specificity of this qPCR is predicted to increase testing accuracy as compared to other published assays.

Mycoplasma ovipneumoniae induces caspase-8-dependent extrinsic apoptosis and p53- and ROS-dependent intrinsic apoptosis in murine alveolar macrophages.

Mycoplasma ovipneumoniae (MO) is a principle causative agent of chronic respiratory disease in ruminants, including sheep, goats, and deer, posing a great threat to the ruminant industry worldwide. However, the pathogenesis of MO infection still remains not well understood and needs further clarification. Here we report a time-dependent apoptosis in cultured murine alveolar macrophage (MH-S) cell lines in response to MO infection in vitro. Mechanistically, MO infection activated apoptosis in MH-S cells through caspase-8-dependent extrinsic pathway and through tumor protein 53 (p53)- and reactive oxygen species (ROS)-dependent intrinsic mitochondrial pathways. Moreover, MO infection promoted both transcription and translation of proinflammatory cytokine genes including interleukin-1ß (IL-1ß), IL-18, and tumor necrosis factor-α (TNF-α), in a caspase-8-, p53-, and ROS-dependent manner, implying a potential link between MO-induced inflammation and apoptotic cell death. Collectively, our results suggest that MO infection induces the activation of extrinsic and intrinsic apoptotic pathways in cultured MH-S cells, which is related to upregulated expression of proinflammatory cytokines. Our findings will contribute to the elucidation of pathogenesis in MO infection and provide valuable reference for the development of new strategies for controlling MO infection.

Recombinase polymerase amplification assay combined with a dipstick-readout for rapid detection of Mycoplasma ovipneumoniae infections.

Mycoplasma ovipneumoniae infects both sheep and goats causing pneumonia resulting in considerable economic losses worldwide. Current diagnosis methods such as bacteriological culture, serology, and PCR are time consuming and require sophisticated laboratory setups. Here we report the development of two rapid, specific and sensitive assays; an isothermal DNA amplification using recombinase polymerase amplification (RPA) and a real-time PCR for the detection of M. ovipneumoniae. The target for both assays is a specific region of gene WP_069098309.1, which encodes a hypothetical protein and is conserved in the genome sequences of ten publicly available M. ovipneumoniae strains. The RPA assay performed well at 39°C for 20 min and was combined with a lateral flow dipstick (RPA-LFD) for easy visualization of the amplicons. The detection limit of the RPA-LFD assay was nine genome copies of M. ovipneumoniae per reaction and was comparable to sensitivity of the real-time PCR assay. Both assays showed no cross-reaction with 38 other ovine and caprine pathogenic microorganisms and two parasites of ruminants, demonstrating a high degree of specificity. The assays were validated using bronchoalveolar lavage fluid and nasal swab samples collected from sheep. The positive rate of RPA-LFD (97.4%) was higher than the real-time PCR (95.8%) with DNA as a template purified from the clinical samples. The RPA assay was significantly better at detecting M. ovipneumoniae in clinical samples compared to the real-time PCR when DNA extraction was omitted (50% and 34.4% positive rate for RPA-LFD and real-time PCR respectively). The RPA-LFD developed here allows easy and rapid detection of M. ovipneumoniae infection without DNA extraction, suggesting its potential as a point-of-care test for field settings.

Population structure and antimicrobial susceptibility of Mycoplasma ovipneumoniae isolates in France.

Chronic non-progressive pneumonia in small ruminants caused by Mycoplasma (M.) ovipneumoniae is mainly controlled by chemotherapy. In France, during the last decade, a rise in M. ovipneumoniae cases was recorded in both sheep and goats, suggesting a possible emergence. Whether this rise is associated with antimicrobial resistance, as observed in other ruminant Mycoplasma species, has yet to be examined. The aim of the study was to characterize the diversity of M. ovipneumoniae strains circulating in France and assess their antimicrobial resistance, together with the underlying mechanisms, to help find an explanation for the increase in reported cases. The genetic diversity of 56 strains isolated between 2007 and 2018 from sheep and goats was assessed using different subtyping methods. Their susceptibility to six antimicrobial classes was profiled by estimating Minimum Inhibitory Concentrations (MICs) using an optimised agar dilution method. Resistance mechanisms were explored by sequence analysis of rRNA targets. A high genetic diversity of strains was evidenced, with consistent, marked animal-host clustering in the Hsp70 gene and whole genome sequence phylogeny. No clonal evolution could thus account for putative emergence. Apart from florfenicol, MICs were low except for a few isolates with increased values for tetracyclines, macrolides and lincosamides. Hotspot mutations in the target ribosomal gene could explain increased tetracycline MICs. Other mechanisms are suspected for macrolide-lincosamide and florfenicol resistance. The emergence of M. ovipneumoniae is thus not related to any increase in resistance or to a clonal spread. Explanations may lie in breeding practices.

Development and validation of the isothermal recombinase polymerase amplification assays for rapid detection of Mycoplasma ovipneumoniae in sheep.

BACKGROUND: Mycoplasmal pneumonia is an important infectious disease that threatens sheep and goat production worldwide, and Mycoplasma ovipneumoniae is one of major etiological agent causing mycoplasmal pneumonia. Recombinase polymerase amplification (RPA) is an isothermal nucleic acid amplification technique, and RPA-based diagnostic assays have been described for the detection of different types of pathogens. RESULTS: The RPA assays using real-time fluorescence detection (real-time RPA) and lateral flow strip detection (LFS RPA) were developed to detect M. ovipneumoniae targeting a conserved region of the 16S rRNA gene. Real-time RPA was performed in a portable florescence scanner at 39 °C for 20 min. LFS RPA was performed in a portable metal bath incubator at 39 °C for 15 min, and the amplicons were visualized with the naked eyes within 5 min on the lateral flow strip. Both assays were highly specific for M. ovipneumoniae, as there were no cross-reactions with other microorganisms tested, especially the pathogens involved in respiratory complex and other mycoplasmas frequently identified in ruminants. The limit of detection of LFS RPA assay was 1.0 × 101 copies per reaction using a recombinant plasmid containing target gene as template, which is 10 times lower than the limit of detection of the real-time RPA and real-time PCR assays. The RPA assays were further validated on 111 clinical sheep nasal swab and fresh lung samples, and M. ovipneumoniae DNA was detected in 29 samples in the real-time RPA, 31 samples in the LFS RPA and 32 samples in the real-time PCR assay. Compared to real-time PCR, the real-time RPA and LFS RPA showed diagnostic specificity of 100 and 98.73%, diagnostic sensitivity of 90.63 and 93.75%, and a kappa coefficient of 0.932 and 0.934, respectively. CONCLUSIONS: The developed real-time RPA and LFS RPA assays provide the attractive and promising tools for rapid, convenient and reliable detection of M. ovipneumoniae in sheep, especially in resource-limited settings. However, the effectiveness of the developed RPA assays in the detection of M. ovipneumoniae in goats needs to be further validated.

Genetic structure of Mycoplasma ovipneumoniae informs pathogen spillover dynamics between domestic and wild Caprinae in the western United States.

Spillover diseases have significant consequences for human and animal health, as well as wildlife conservation. We examined spillover and transmission of the pneumonia-associated bacterium Mycoplasma ovipneumoniae in domestic sheep, domestic goats, bighorn sheep, and mountain goats across the western United States using 594 isolates, collected from 1984 to 2017. Our results indicate high genetic diversity of M. ovipneumoniae strains within domestic sheep, whereas only one or a few strains tend to circulate in most populations of bighorn sheep or mountain goats. These data suggest domestic sheep are a reservoir, while the few spillovers to bighorn sheep and mountain goats can persist for extended periods. Domestic goat strains form a distinct clade from those in domestic sheep, and strains from both clades are found in bighorn sheep. The genetic structure of domestic sheep strains could not be explained by geography, whereas some strains are spatially clustered and shared among proximate bighorn sheep populations, supporting pathogen establishment and spread following spillover. These data suggest that the ability to predict M. ovipneumoniae spillover into wildlife populations may remain a challenge given the high strain diversity in domestic sheep and need for more comprehensive pathogen surveillance.

Loop-mediated isothermal amplification-lateral-flow dipstick (LAMP-LFD) to detect Mycoplasma ovipneumoniae.

In order to establish a rapid detection method for Mycoplasma ovipneumoniae, this study used the loop-mediated isothermal amplification (LAMP) technique to carry out nucleic acid amplification and chromatographic visualization via a lateral flow dipstick (LFD) assay. The M. ovipneumoniae elongation factor TU gene (EF-TU) was detected using a set of specific primers designed for the EF-TU gene, and the EF-TU FIP was detected by biotin labeling, which was used in the LAMP amplification reaction. The digoxin-labeled probe specifically hybridized with LAMP products, which were visually detected by LFD. Here, we established the M. ovipneumoniae LAMP-LFD rapid detection method and tested the specificity, sensitivity, and clinical application of this method. Results showed that the optimized LAMP performed at 60 °C for 60 min, and LFD can specifically and visually detect M. ovipneumoniae with a minimum detectable concentration at 1.0 × 102 CFU/mL. The sensitivity of LAMP-LFD was 1000 times that of the conventional PCR detection methods, and the clinical lung tissue detection rate was 86% of 50 suspected sheep infected with M. ovipneumoniae. In conclusion, LAMP-LFD was established in this study to detect M. ovipneumoniae, a method that was highly specific, sensitive, and easy to operate, and provides a new method for the prevention and diagnosis of M. ovipneumoniae infection.

Icelandic ovine Mycoplasma ovipneumoniae are variable bacteria that induce limited immune responses in vitro and in vivo.

PURPOSE: Mycoplasma ovipneumoniae is a pathogen that causes atypical pneumoniae in sheep and goats. While infection of lambs can induce strong immune responses, typically measured as serum antibodies, experimental vaccines appear to induce lower antibody titres. The purpose of this study was to better understand the bacterium and its interaction with the host, in order to improve the vaccination strategy. METHODOLOGY: We designed primers to compare seven M. ovipneumoniae gene sequences, in addition to the 16S sequence typically used, to estimate the variability between isolates. In addition, we labelled bacteria with a two-step process to examine whether bacteria could be intracellular as well as on the host surface in vitro. Finally, we vaccinated sheep four times and examined the induction of humoral and cellular responses. RESULTS: We were able to reliably amplify the seven housekeeping gene sequences to examine variability of the different isolates, and the bacteria could be found intracellularly, as well as on the host cell surface. Four vaccinations of sheep produced only modest humoral and cellular responses in this study, likely due to previous exposure of the animals to mycoplasmas. CONCLUSIONS: The moderate immune responses seen in this study indicate that previous exposure to mycoplasmas is a challenge for vaccination of lambs against M. ovipneumoniae. However, an alternative vaccination strategy, e.g. utilizing a recombinant vaccine, may overcome this vaccination hurdle in endemic regions and we suggest a possible vaccine candidate.

Assessing respiratory pathogen communities in bighorn sheep populations: Sampling realities, challenges, and improvements.

Respiratory disease has been a persistent problem for the recovery of bighorn sheep (Ovis canadensis), but has uncertain etiology. The disease has been attributed to several bacterial pathogens including Mycoplasma ovipneumoniae and Pasteurellaceae pathogens belonging to the Mannheimia, Bibersteinia, and Pasteurella genera. We estimated detection probability for these pathogens using protocols with diagnostic tests offered by a fee-for-service laboratory and not offered by a fee-for-service laboratory. We conducted 2861 diagnostic tests on swab samples collected from 476 bighorn sheep captured across Montana and Wyoming to gain inferences regarding detection probability, pathogen prevalence, and the power of different sampling methodologies to detect pathogens in bighorn sheep populations. Estimated detection probability using fee-for-service protocols was less than 0.50 for all Pasteurellaceae and 0.73 for Mycoplasma ovipneumoniae. Non-fee-for-service Pasteurellaceae protocols had higher detection probabilities, but no single protocol increased detection probability of all Pasteurellaceae pathogens to greater than 0.50. At least one protocol resulted in an estimated detection probability of 0.80 for each pathogen except Mannheimia haemolytica, for which the highest detection probability was 0.45. In general, the power to detect Pasteurellaceae pathogens at low prevalence in populations was low unless many animals were sampled or replicate samples were collected per animal. Imperfect detection also resulted in low precision when estimating prevalence for any pathogen. Low and variable detection probabilities for respiratory pathogens using live-sampling protocols may lead to inaccurate conclusions regarding pathogen community dynamics and causes of bighorn sheep respiratory disease epizootics. We recommend that agencies collect multiples samples per animal for Pasteurellaceae detection, and one sample for Mycoplasma ovipneumoniae detection from at least 30 individuals to reliably detect both Pasteurellaceae and Mycoplasma ovipneumoniae at the population-level. Availability of PCR diagnostic tests to wildlife management agencies would improve the ability to reliably detect Pasteurellaceae in bighorn sheep populations.

Comparison of phenotypic and genotypic profiles among caprine and ovine Mycoplasma ovipneumoniae strains.

Mycoplasma ovipneumoniae (Movp) is considered to be one of the most important mycoplasmas causing respiratory disease in small ruminants. Most epidemiologic and characterisation studies have been conducted on strains collected from sheep. Information on the presence and characteristics of Movp in healthy and pneumonic goats is limited. Phenotypic or genotypic differences between sheep and goat isolates have never been studied. The objective of our study was to characterise and compare the similarities and differences between caprine and ovine Movp strains isolated from affected and asymptomatic animals in order to elucidate phenotypic and genotypic variability. Four different techniques were used on a set of 23 Movp isolates. These included SDS-PAGE, Western blotting, random amplified polymorphic DNA and the heat shock protein 70 gene sequence-based method. A high degree of phenotypic and genotypic heterogeneity among Movp strains was demonstrated in this study. Our results demonstrated differences between goat and sheep strains, revealing not only a link between strains and host ruminant species, but by geographical origin as well. However, the finding of immunodominant antigens of molecular masses 36, 38, 40 and 70 kDa (±3 kDa) in Movp isolates from sheep and goats foretells their potential use in the development of serological diagnostic tests and vaccines.

Serological and molecular survey of sheep infected with Mycoplasma ovipneumoniae in Xinjiang, China.

Mycoplasma pneumonia is one of the most important infectious diseases that threaten sheep production. In order to investigate the epidemic status of Mycoplasma ovipneumoniae infection in sheep, indirect hemagglutination assay was used to analyze 1679 serum samples collected from four different breeds of sheep (Kazak sheep, Hu sheep, Merino sheep, and Duolang sheep) in six regions in Xinjiang between 2012 and 2014. One thousand one hundred sixty-nine sheep nasal swabs and 180 lungs were PCR analyzed. The results showed that the average positive rates of the serum samples were 17.75 %. The positive rates were between 9.76 and 30.61 % in the four breeds. Among them, the Hu sheep had a significantly higher rate than other breeds (P < 0.05). The average positive rates of nasal swabs and lungs were 10.18 and 28.89 %, respectively. Based on the phylogenetic trees of 16S RNA gene, the isolates were closest to those strains isolated from inland areas of China, indicating that these epidemic isolates came from the trans-province introductions. Our survey suggests that quarantine is necessary for sheep imported from inland, and effective immunization should be implemented in sheep susceptible to M. ovipneumoniae in Xinjiang, China.

Epizootic pneumonia of bighorn sheep following experimental exposure to Mycoplasma ovipneumoniae.

BACKGROUND: Bronchopneumonia is a population limiting disease of bighorn sheep (Ovis canadensis). The cause of this disease has been a subject of debate. Leukotoxin expressing Mannheimia haemolytica and Bibersteinia trehalosi produce acute pneumonia after experimental challenge but are infrequently isolated from animals in natural outbreaks. Mycoplasma ovipneumoniae, epidemiologically implicated in naturally occurring outbreaks, has received little experimental evaluation as a primary agent of bighorn sheep pneumonia. METHODOLOGY/PRINCIPAL FINDINGS: In two experiments, bighorn sheep housed in multiple pens 7.6 to 12 m apart were exposed to M. ovipneumoniae by introduction of a single infected or challenged animal to a single pen. Respiratory disease was monitored by observation of clinical signs and confirmed by necropsy. Bacterial involvement in the pneumonic lungs was evaluated by conventional aerobic bacteriology and by culture-independent methods. In both experiments the challenge strain of M. ovipneumoniae was transmitted to all animals both within and between pens and all infected bighorn sheep developed bronchopneumonia. In six bighorn sheep in which the disease was allowed to run its course, three died with bronchopneumonia 34, 65, and 109 days after M. ovipneumoniae introduction. Diverse bacterial populations, predominantly including multiple obligate anaerobic species, were present in pneumonic lung tissues at necropsy. CONCLUSIONS/SIGNIFICANCE: Exposure to a single M. ovipneumoniae infected animal resulted in transmission of infection to all bighorn sheep both within the pen and in adjacent pens, and all infected sheep developed bronchopneumonia. The epidemiologic, pathologic and microbiologic findings in these experimental animals resembled those seen in naturally occurring pneumonia outbreaks in free ranging bighorn sheep.

Mycoplasma ovipneumoniae--a primary cause of severe pneumonia epizootics in the Norwegian Muskox (Ovibos moschatus) population.

The Norwegian muskox (Ovibos moschatus) population lives on the high mountain plateau of Dovre and originates from animals introduced from Greenland. In the late summers of 2006 and 2012, severe outbreaks of pneumonia with mortality rates of 25-30% occurred. During the 2012 epidemic high quality samples from culled sick animals were obtained for microbiological and pathological examinations. High throughput sequencing (pyrosequencing) of pneumonic lung tissue revealed high concentrations of Mycoplasma ovipneumoniae in all six animals examined by this method and Pasteurella multocida subsp. multocida in four animals, whereas no virus sequences could be identified. Mycoplasma ovipneumoniae and P. multocida multocida were also isolated by culture. Using real time PCR on lung swabs, M. ovipneumoniae was detected in all of the 19 pneumonic lungs examined. Gross pathological examination revealed heavy consolidations primarily in the cranial parts of the lungs and it also identified one case of otitis media. Histologically, lung lesions were characterized as acute to subacute mixed exudative and moderately proliferative bronchoalveolar pneumonia. Immunohistochemical (IHC) examination revealed high load of M. ovipneumoniae antigens within lung lesions, with particularly intensive staining in the neutrophils. Similar IHC finding were observed in archived lung tissue blocks from animals examined during the 2006 epidemic. An M. ovipneumoniae specific ELISA was applied on bio-banked muskox sera from stray muskoxen killed in the period 2004-2013 and sick muskoxen culled, as well as sera from wild reindeer (Rangifer tarandus tarandus) on Dovre and muskoxen from Greenland. Serology and mycoplasma culturing was also carried out on sheep that had been on pasture in the muskox area during the outbreak in 2012. Our findings indicated separate introductions of M. ovipneumoniae infection in 2006 and 2012 from infected co-grazing sheep. Salt licks shared by the two species were a possible route of transmitting infection.