A case of mistaken identity: a systematic review, meta-analysis, and reinvestigation of hemotropic Mycoplasma spp. infection in Ctenocephalides felis (cat flea).

BACKGROUND: Feline-associated hemotropic Mycoplasma (hemoplasmas) are believed to be transmitted by two primary mechanisms: (1) direct transmission via fighting and (2) vector-borne transmission by the cat flea (Ctenocephalides felis). While the efficiency of transmission by C. felis appears low, most manuscripts focus on the prevalence of hemoplasmas in wild-caught fleas and report either a very low (< 3%) or a high (> 26%) prevalence. Therefore, we aimed to assess the influence of sample processing and PCR methods on C. felis hemoplasma infection prevalence. METHODS: A systemic review of PubMed articles identified 13 manuscripts (1,531 fleas/flea pools) that met the inclusion criteria (performed PCR for >1 hemoplasma on C. felis collected from cats). Risk of bias was assessed utilizing the ROBINS-E tool. Meta-analysis performed in R of these manuscripts found that not washing samples and a common set of 16S rRNA primers first published in Jensen et al. 2001 were associated with increased hemoplasma prevalence. To evaluate the influence of washing on newly collected fleas, we assessed the hemoplasma status of 20 pools of 5 C. felis each, half of which were washed and half not washed. RESULTS: Flea washing did not influence the detection of hemoplasma but instead amplified Spiroplasma. To assess non-specific amplification with the Jensen et al. 2001 primers, 67 C. felis samples (34% previously reported hemoplasma infected) were subject to PCR and sequencing. By this method, hemoplasma was detected in only 3% of samples. In the remaining "hemoplasma infected" fleas, PCR amplified Spiroplasma or other bacteria. CONCLUSIONS: Therefore, we concluded that hemoplasma infection in C. felis is rare, and future flea prevalence studies should sequence all positive amplicons to validate PCR specificity. Further investigation of alternative methods of feline-associated hemoplasma transmission and the ability of C. felis to maintain hemoplasma infection is necessary.

The complete genome sequence of unculturable Mycoplasma faucium obtained through clinical metagenomic next-generation sequencing.

Introduction: Diagnosing Mycoplasma faucium poses challenges, and it's unclear if its rare isolation is due to infrequent occurrence or its fastidious nutritional requirements. Methods: This study analyzes the complete genome sequence of M. faucium, obtained directly from the pus of a sternum infection in a lung transplant patient using metagenomic sequencing. Results: Genome analysis revealed limited therapeutic options for the M. faucium infection, primarily susceptibility to tetracyclines. Three classes of mobile genetic elements were identified: two new insertion sequences, a new prophage (phiUMCG-1), and a species-specific variant of a mycoplasma integrative and conjugative element (MICE). Additionally, a Type I Restriction-Modification system was identified, featuring 5'-terminally truncated hsdS pseudogenes with overlapping repeats, indicating the potential for forming alternative hsdS variants through recombination. Conclusion: This study represents the first-ever acquisition of a complete circularized bacterial genome directly from a patient sample obtained from invasive infection of a primary sterile site using culture-independent, PCR-free clinical metagenomics.

Nanopore Sequencing Using the Full-Length 16S rRNA Gene for Detection of Blood-Borne Bacteria in Dogs Reveals a Novel Species of Hemotropic Mycoplasma.

Dogs across the globe are afflicted by diverse blood- and vector-borne bacteria (VBB), many of which cause severe disease and can be fatal. Diagnosis of VBB infections can be challenging due to the low concentration of bacteria in the blood, the frequent occurrence of coinfections, and the wide range of known, emerging, and potentially novel VBB species encounterable. Therefore, there is a need for diagnostics that address these challenges by being both sensitive and capable of detecting all VBB simultaneously. We detail the first employment of a nanopore-based sequencing methodology conducted on the Oxford Nanopore Technologies (ONT) MinION device to accurately elucidate the "hemobacteriome" from canine blood through sequencing of the full-length 16S rRNA gene. We detected a diverse range of important canine VBB, including Ehrlichia canis, Anaplasma platys, Mycoplasma haemocanis, Bartonella clarridgeiae, "Candidatus Mycoplasma haematoparvum", a novel species of hemotropic mycoplasma, and Wolbachia endosymbionts of filarial worms, indicative of filariasis. Our nanopore-based protocol was equivalent in sensitivity to both quantitative PCR (qPCR) and Illumina sequencing when benchmarked against these methods, achieving high agreement as defined by the kappa statistics (k > 0.81) for three key VBB. Utilizing the ability of the ONT' MinION device to sequence long read lengths provides an excellent alternative diagnostic method by which the hemobacteriome can be accurately characterized to the species level in a way previously unachievable using short reads. We envision our method to be translatable to multiple contexts, such as the detection of VBB in other vertebrate hosts, including humans, while the small size of the MinION device is highly amenable to field use. IMPORTANCE Blood- and vector-borne bacteria (VBB) can cause severe pathology and even be lethal for dogs in many regions across the globe. Accurate characterization of all the bacterial pathogens infecting a canine host is critical, as coinfections are common and emerging and novel pathogens that may go undetected by traditional diagnostics frequently arise. Deep sequencing using devices from Oxford Nanopore Technologies (ONT) provides a solution, as the long read lengths achievable provide species-level taxonomic identification of pathogens that previous short-read technologies could not accomplish. We developed a protocol using ONT' MinION sequencer to accurately detect and classify a wide spectrum of VBB from canine blood at a sensitivity comparable to that of regularly used diagnostics, such as qPCR. This protocol demonstrates great potential for use in biosurveillance and biosecurity operations for the detection of VBB in a range of vertebrate hosts, while the MinION sequencer's portability allows this method to be used easily in the field.

Mycoplasma miroungirhinis sp. nov. and Mycoplasma miroungigenitalium sp. nov., isolated from northern elephant seals (Mirounga angustirostris), Mycoplasma phocoenae sp. nov., isolated from harbour porpoise (Phocoena phocoena), and Mycoplasma phocoeninasale sp. nov., isolated from harbour porpoise and California sea lions (Zalophus californianus).

Seven novel independent strains of Mycoplasma species were isolated from northern elephant seals (ES2806-NAST, ES2806-GENT, ES3157-GEN-MYC and ES3225-GEN-MYC), a harbour porpoise (C264-GENT and C264-NAST), and a California sea lion (CSL7498). These strains were phenotypically and genetically characterized and compared to the known Mycoplasma species. Four strains (C264-GENT, C264-NAST, CSL7498 and ES2806-NAST) hydrolysed arginine but not urea and did not produce acid from carbohydrates. Strains ES2806-GENT, ES3157-GEN-MYC and ES3225-GEN-MYC did not produced acid from carbohydrates and did not hydrolyse arginine or urea; hence, it is assumed that organic acids are used as the energy source for them. All were isolated and propagated in ambient air supplemented with 5±1 % CO2 at +35-37 °C using either SP4 or PPLO medium. Colonies on solid medium showed a typical fried-egg appearance and transmission electron microscopy revealed a typical mycoplasma cellular morphology. The complete genomes were sequenced for all type strains. Average nucleotide and amino acid identity analyses showed that these novel strains were distant from the phylogenetically closely related Mycoplasma species. Based on these data, we propose four novel species of the genus Mycoplasma, for which the name Mycoplasma miroungirhinis sp. nov. is proposed with the type strain ES2806-NAST (=NCTC 14430T=DSM 110945T), Mycoplasma miroungigenitalium sp. nov. is proposed with the type strain ES2806-GENT (=NCTC 14429T=DSM 110944T) and representative strains ES3157-GEN-MYC and ES3225-GEN-MYC, Mycoplasma phocoenae sp. nov. is proposed with the type strain C264-GENT (=NCTC 14344T=DSM 110687T) and Mycoplasma phocoeninasale sp. nov. is proposed with the type strain C264-NAST (=NCTC 14343T=DSM 110688T) and representative strain CSL7498. The genome G+C contents are 24.06, 30.09, 28.49 and 29.05% and the complete genome sizes are 779 550, 815 486, 693 115, and 776 009 bp for strains ES2806-NAST, ES2806-GENT, C264-GENT and C264-NAST, respectively.

Identification of Mycoplasma species and related organisms from ruminants in England and Wales during 2005-2019.

BACKGROUND: Mycoplasma species have been associated with economically important diseases affecting ruminants worldwide and include contagious bovine pleuropneumonia (CBPP), contagious caprine pleuropneumonia (CCPP) and contagious agalactia, listed by the World Organisation for Animal Health (OIE). The Mycoplasma Team at the Animal and Plant Health Agency provides an identification service for Mycoplasma and Ureaplasma species of veterinary importance to the United Kingdom (UK), supporting the detection of new and emerging pathogens, as well as contributing to the surveillance of endemic, and the OIE listed diseases exotic to the UK. Mycoplasma and other Mollicutes species were identified from diagnostic samples from farmed ruminants in England and Wales using a combination of culture and 16S rRNA gene-based PCR-denaturing gradient gel electrophoresis, submitted between 2005 and 2019. RESULTS: A total of 5578 mollicutes identifications, which include mycoplasmas and the related acholeoplasmas and ureaplasmas, were made from farmed ruminant animals during the study period. Throughout the study period, the pathogen Mycoplasma bovis was consistently the most frequently identified species, accounting for 1411 (32%) of 4447 molecular identifications in cattle, primarily detected in the lungs of pneumonic calves, followed by joints and milk of cattle showing signs of arthritis and mastitis, respectively. M. bovirhinis, M. alkalescens, M. dispar, M. arginini and Ureaplasma diversum, were also common. Mixed species, principally M. bovis with M. alkalescens, M. arginini or M. bovirhinis were also prevalent, particularly from respiratory samples. The non-cultivable blood-borne haemoplasmas Candidatus 'Mycoplasma haemobos' and Mycoplasma wenyonii were identified from cattle, with the latter species most often associated with milk-drop. M. ovipneumoniae was the predominant species identified from sheep and goats experiencing respiratory disease, while M. conjunctivae preponderated in ocular samples. The UK remains free of the ruminant mycoplasmas listed by OIE. CONCLUSIONS: The continued high prevalence of M. bovis identifications confirms its ongoing dominance and importance as a significant pathogen of cattle in England and Wales, particularly in association with respiratory disease. M. ovipneumoniae has seen a general increase in prevalence in recent years, notably in coughing lambs and should therefore be considered as a primary differential diagnosis of respiratory disease in small ruminants.

Occurrence of Mycoplasma spp. in wild birds: phylogenetic analysis and potential factors affecting distribution.

Different Mycoplasma species have been reported in avian hosts. However, the majority of studies focus on one particular species of Mycoplasma or one host. In our research, we screened a total of 1141 wild birds representing 55 species, 26 families, and 15 orders for the presence of mycoplasmas by conventional PCR based on the 16S rRNA gene. Selected PCR products were sequenced to perform the phylogenetic analysis. All mycoplasma-positive samples were tested for M. gallisepticum and M. synoviae, which are considered the major pathogens of commercial poultry. We also verified the influence of ecological characteristics of the tested bird species including feeding habits, habitat types, and movement patterns. The presence of Mycoplasma spp. was confirmed in 498 birds of 29 species, but none of the tested birds were positive for M. gallisepticum or M. synoviae. We found possible associations between the presence of Mycoplasma spp. and all investigated ecological factors. The phylogenetic analysis showed a high variability of Mycoplasma spp.; however, some clustering of sequences was observed regarding particular bird species. We found that wild migratory waterfowl, particularly the white-fronted goose (Anser albifrons) and mallard (Anas platyrhynchos) could be reservoirs and vectors of mycoplasmas pathogenic to commercial waterfowl.

Molecular survey and genetic characterization of 'Candidatus Mycoplasma haemolamae' in llamas (Lama glama) and alpacas (Vicugna pacos) from Southern Chile.

This study aimed to perform a molecular survey and identification of hemotropic Mycoplasma spp. in domestic South American Camelids from Southern Chile. Conventional PCR (cPCR) for hemotropic Mycoplasma spp. based on 16S rRNA gene (620bp fragment) was performed in 87 EDTA-blood samples taken from 48 llamas (Lama glama) and 39 and alpacas (Vicugna pacos) from to Temuco, La Araucanía region and Valdivia, Los Rios region, Southern Chile. 16S rRNA hemotropic Mycoplasma PCR-positive were sequenced for species identification, phylogenetic and haplotype analyses, and further tested by cPCR targeting a fragment (160-210 bp) of the RNaseP (rnpB) gene. Based upon 16S rRNA cPCR results, the overall hemotropic Mycoplasma spp. occurrence in Southern camelids was 9.2% (8/87 [95% CI (4.0-17.3%)]), with five positive alpacas (12.8%; 5/39 [95% CI (4.3-27.4%)]) and three llamas (6.3%; 3/48 [95% CI (1.7-17.2%)]). All 16S rRNA PCR-positive samples were negative for the rnpB gene. Obtained 16S sequences presented high identity (99-100%) by BLASTn analysis to 'Candidatus Mycoplasma haemolamae' from an alpaca in the United Kingdom. Phylogenetic and haplotype analyses of the 16s rRNA gene showed high similarity among 'Candidatus M. haemolamae' sequences of this study and the ones from North America, Europe, and Asia evidencing a low diversity of Chilean samples, with only one haplotype detected (#1). Haplotype #1 from South American Camelids in Chile was worldwide distributed and observed in North America, Europe, and Asia. 'Candidatus M. haemolamae' detected for the first time in South American camelids in Southern Chile had low diversity and was worldwide spread.

'Candidatus Mycoplasma haematohydrochoerus', a novel hemoplasma species in capybaras (Hydrochoerus hydrochaeris) from Brazil.

Three different species of hemoplasmas have been described in rodents, Mycoplasma coccoides, 'Candidatus Mycoplasma haemomuris' and 'Candidatus Mycoplasma haemosphiggurus'. Additionally, potentially novel hemoplasma species have been detected in wild rodents from Brazil, including capybaras (Hydrochoerus hydrochaeris). Capybaras are the largest rodent in the world and are well adapted to live within close proximity to humans, which increases the risk to spread of zoonotic pathogens. Herein, we investigate the occurrence and genetic diversity of hemoplasmas infecting free-ranging capybaras from southern Brazil. Blood samples and ticks from 17 capybaras were collected. Packed cell volume and total plasma protein were measured, DNA was extracted, and further screened by species-specific and pan-hemoplasma PCR assays targeting the 16S rRNA gene of hemoplasmas. Sixteen out of 17 (94.12%; 95% CI: 73.02-98.95%) were anemic. Only one young female was hypoproteinemic. All capybaras were infested by adults and nymphs of Amblyomma dubitatum ticks. Using the PCR assay targeting the 16S rRNA gene of M. coccoides, 13/17 (76.47%; 95% CI: 52.74-90.44%) capybaras were positive for hemoplasmas. When DNA samples were tested by the pan-hemoplasma PCR, 16/17 (94.12%; 95% CI: 73.02-98.95%) animals were positive. One out of 11 (9.09%) adult ticks salivary glands tested positive for hemoplasma by the pan-hemoplasma PCR assay. Sequencing and phylogenetic analysis of the 16S and 23S rRNA gene fragments confirmed that animals were infected by a novel hemotropic Mycoplasma sp. previously reported in capybaras from Brazil. Additionally, sequencing and phylogenetic analysis of the 23S rRNA gene from three hemoplasma-positive capybaras samples from a previous study performed in midwestern Brazil also confirm our findings. Based on phylogenetic and Neighbor-Net network analysis of the 16S rRNA and 23S rRNA genes, the name 'Candidatus Mycoplasma haematohydrochoerus' is proposed for this novel organism.

Rapid detection of Mycoplasma mycoides subsp. capri and Mycoplasma capricolum subsp. capripneumoniae using high-resolution melting curve analysis.

Mycoplasma capricolum subsp.subsp. capripneumonia (Mccp) and Mycoplasma mycoides subsp.sbusp. capri (Mmc) cause caprine pleuropneumonia (CCPP) and mycoplasmal pneumonia in goats and sheep (MPGS), respectively. These diseases cannot be identified on clinical symptoms alone and it is laborious to distinguish them using biochemical methods. It is therefore important to establish a simple, rapid identification method for Mccp and Mmc. Here, we report a high-resolution melting (HRM) curve analysis using specific primers based on the Mmc 95010 strain MLC_0560 and Mccp F38 strain MCCPF38_00984 gene sequences. The method was highly specific with intra- and inter-batch coefficients of variation < 1%. The lower limit of detection for Mccp and Mmc was 55 copies/µL and 58 copies/µL, respectively. HRM and fluorescence qPCR results were compared using 106 nasal swabs and 47 lung tissue samples from goats (HRM-qPCR coincidence rate 94.8%; 145/153). Mycoplasma isolation and identification was performed on 30 lung tissue samples and 16 nasal swabs (HRM-culturing coincidence rate 87.0%; 40/46). HRM analysis was more sensitive than fluorescence qPCR and Mycoplasma isolation, indicating the practicality of HRM for accurate and rapid identification of Mccp and Mmc, and diagnosis and epidemiology of CCPP and MPGS.

Comparative genomic analysis of Mycoplasma anatis strains.

BACKGROUND: The Gram-negative intracellular bacterium Mycoplasma anatis is a pathogen of respiratory infectious diseases in ducks and has caused significant economic losses in the poultry industry. OBJECTIVE: This study, as the first report of the structure and function of the pan-genome of Mycoplasma anatis, may provide a valuable genetic basis for many aspects of future research on the pathogens of waterfowl. METHODS: We sequenced the whole genomes of 15 Mycoplasma anatis isolated from ducks in China. Draft genome sequencing was carried out and whole-genome sequencing was performed by the sequencers of the PacBio Sequel and an IonTorrent Personal Genome Machine (PGM). Then the common genic elements of protein-coding genes, tRNAs, and rRNAs of Mycoplasma anatis genomes were predicted by using the pipeline Prokka v1.13.7. To investigate homologous protein clusters across Mycoplasma anatis genomes, we adopted Roary v3.13.0 to cluster orthologous genes (OGs) based on the following criteria. RESULTS: We obtained one complete genome and 14 genome sketches. Microbial mobile genetic element analysis revealed the distribution of insertion sequences (IS30, IS3, and IS1634), prophage regions, and CRISPR arrays in the genome of Mycoplasma anatis. Comparative genomic analysis decoded the genetic components and functional classification of the pan-genome of Mycoplasma anatis that comprised 646 core genes, 231 dispensable genes and among them 110 was strain-specific. Virulence-related gene profiles of Mycoplasma anatis were systematically identified, and the products of these genes included bacterial ABC transporter systems, iron transport proteins, toxins, and secretion systems. CONCLUSION: A complete virulence-related gene profile of Mycoplasma anatis has been identified, most of the genes are highly conserved in all strains. Sequencing results are relevant to the molecular mechanisms of drug resistance, adaptive evolution of pathogens, population structure, and vaccine development.

Co-infections by Mycoplasma hyopneumoniae, Mycoplasma hyorhinis and Mycoplasma flocculare in macroscopic lesions of lung consolidation of pigs at slaughter.

Infections with Mycoplasma hyopneumoniae (Mhyo), Mycoplasma hyorhinis (Mhr) and Mycoplasma flocculare (Mfloc) are common in swine. However, the degree of co-infections and the correlations between these mycoplasma co-infection and the severity of macroscopic lung consolidation lesions (MLCL) have not yet been explored in Brazil.The objectives were to quantify Mhyo, Mhr, and Mfloc in MLCL of slaughter pigs in Brazil, and to assess correlations with the degree of MLCL in slaughter pigs. To this end, five groups of lungs were made based on severity of lung lesions, and 80 lungs were collected for each group (400 lungs in total). The Mycoplasmas were quantified using a multiplex qPCR. Statistical differences and comparison between the groups were evaluated, respectively, by the Kruskal-Wallis test (p < 0.05) and Dunn's test (p < 0.05), and the correlation between the data was performed by Spearman's method (p < 0.05). The results revealed that the extent of MLCL showed a positive correlation with the Mhyo estimate (rho = 0.26; p < 0.05), a negative correlation with the Mfloc estimate (rho= -0.15; p < 0.05), and no significant correlation with the Mhr estimate (p = 0, 12). The extension of MLCL showed a positive correlation with the co-infection by Mfloc and Mhr (rho = 0.17; p < 0.05), and no significant correlation with Mhyo and Mhr (p = 0.87), and a negative correlation with Mhyo and Mfloc (rho= -0.28; p < 0.05). This study allowed to infer that, regarding the extension of MLCL, Mhr and Mfloc did not present opportunistic activity in relation to primary infection by Mhyo, but revealed some potential aggravation of these lesions. In addition, Mhyo expressed inhibitory behavior towards Mfloc, suggesting that one can compete with the other's presence.

Evaluation of 5 commercial assays for the detection of Mycoplasma genitalium and other Urogenital Mycoplasmas.

The focus on urogenital mycoplasmas as the possible etiologic agents of urogenital infections and syndromes, has increased in the last decade. Of these, Mycoplasma genitalium is proven to be pathogenic and sexually transmitted. We compared five commercially available assays for the detection of these organisms in urogenital mycoplasma culture specimen remnants. Stored specimen remnants were tested on Aptima Mycoplasma genitalium, Allplex™ STI Essential and CGMT, ResitancePlus®MG and Allplex™ MG & AziR Assays. All positive M. genitalium specimens and culture negative, nucleic acid positive Ureaplasmas were sent to the National Microbiology Laboratory for confirmation. The Aptima Mycoplasma genitalium assay detected 7 M. genitalium infections, the Allplex™ STI-EA and the Allplex™ CGMT detected 6 M. genitalium positives, and the Allplex™MG and AziR and SpeeDx ResistancePlus® MG detected 5 M. genitalium positives, four with macrolide resistant genes. The Allplex™ STI Essential assay was 100% sensitive and specific for Mycoplasma hominis and Ureaplasma targets. As seen in other studies, the Aptima Mycoplasma genitalium assay was 100% sensitive and specific for the detection of M. genitalium. The multiplex assays had lower sensitivities for M. genitalium detection (Allplex™ STI Essential and CGMT sensitivity of 85.71%; Allplex™ MG & AziR and SpeeDx ResistancePlus® MG sensitivity of 71.43%) with high specificities of 100%. Assays tested have high sensitivities and specificities for the detection of urogenital mycoplasmas especially M. genitalium macrolide resistance markers. All labs wanting to perform onsite detection of these organisms will find an assay to easily fit into their workflow.

Multilocus sequence typing of the goose pathogen Mycoplasma anserisalpingitidis.

Mycoplasma anserisalpingitidis infection is associated with the inflammation of the genital tract and cloaca, embryo lethality, and decreased egg production in geese, leading to serious economic losses. M. anserisalpingitidis has been detected mainly in Central and Eastern Europe, especially in Hungary, but the pathogen was identified recently in China, predicting it's worldwide occurrence. In this study, a novel multilocus sequence typing (MLST) scheme was developed to analyse phylogenetic relationships between M. anserisalpingitidis field isolates and clinical specimens originating from different geographical locations. Five loci (atpG, fusA, pgiB, plsY, and uvrA) were selected for the final MLST study. The examined 89 M. anserisalpingitidis samples yielded 76 unique sequence types with a 0.994 Simpson's index of diversity. The samples were originated from Hungary, Poland, Ukraine, China, and Vietnam. Phylogenetic analysis revealed the existence of three distinct clades (A-C) and six subclades within clade C. Generally, samples originating from the same geographical locations or livestock integration clustered together. Isolates in clade A showed the closest relationships to the M. anatis outgroup due to sequence similarity of the plsY locus. The highest genetic distance was observed in 5C among the subclades of clade C, containing the Asian and some Hungarian field isolates. The developed MLST assay revealed high diversity of the investigated M. anserisalpingitidis samples. The method proved to be a valuable and cost-effective tool for sequence typing of this waterfowl Mycoplasma species, enabling the better understanding of its phylogeny and providing a robust assay for future molecular epidemiological investigations.

'Candidatus Mycoplasma haemoalbiventris', a novel hemoplasma species in white-eared opossums (Didelphis albiventris) from Brazil.

Opossums of the genus Didelphis are considered synanthropic animals due to their close contact with human beings. Previously, two species of hemotropic mycoplasmas (hemoplasmas) have been detected in opossums: 'Candidatus Mycoplasma haemodidelphidis' in the North American opossum (Didelphis virginiana) and a potentially novel hemotropic Mycoplasma sp. in the white-eared opossums (Didelphis albiventris) from Brazil. Accordingly, the aims of this study were as follows: (a) to determine the prevalence of hemotropic Mycoplasma spp. in free-ranging opossums, (b) to characterize molecularly the hemotropic Mycoplasma sp. infecting opossums and (c) to determine factors associated with hemoplasma infection in opossums from Canoinhas municipality, Santa Catarina State, southern Brazil. For this purpose, 50 white-eared opossums (33 captured and 17 road-killed animals) were evaluated by a pan-hemoplasma PCR assay based on 16S rRNA. Six out of 50 (12%; 95% CI: 5.6%-23.8%) opossums were infested by Ctenocephalides felis fleas. Twenty out of 50 (40%; 95% CI: 26.41%-54.82%) opossums tested positive for hemotropic Mycoplasma sp. by PCR. Sequencing and phylogenetic analysis of the 16S and 23S rRNA gene fragments confirmed that animals were infected by a potentially novel hemotropic Mycoplasma sp. previously reported in white-eared opossums from Brazil. No significant association was found between gender (p = .7759), trap area (p = .0887) or presence of fleas (p = .3811) and positivity for hemoplasmas. The potentially novel hemoplasma species seems to be highly prevalent in white-eared opossums from the states of Paraná, Santa Catarina and Mato Grosso do Sul. Based on the phylogenetic analyses of the 16S rRNA and 23S rRNA genes along with epidemiological data, the name 'Candidatus Mycoplasma haemoalbiventris' is proposed for this novel organism.

The first isolation and molecular characterization of Mycoplasma capricolum subsp. capripneumoniae Pakistan strain: A causative agent of contagious caprine pleuropneumonia.

PURPOSE: Mycoplasma capricolum subsp. capripneumoniae (Mccp) causes a severe, usually fatal disease in goats known as Contagious Caprine Pleuropneumonia (CCPP). CCPP is listed by OIE as a notifiable animal diseases, causing economic losses in terms of high morbidity and mortality. Thus far, very limited information is available on the molecular characterization of the unique Mccp strains prevalent in Pakistan. The study was aimed to isolate Mccp local strain for the development of diagnostics and vaccines. METHODS: Samples were collected during November 2017-December 2018 at Northern areas of Pakistan from 10 goat flocks each in Gilgit-Baltistan, Chitral, Swat, Buner, and Hazara. 900 samples were collected; nasal swabs (n = 400), tracheal swabs (n = 150) from naturally infected goats showing clinical signs of CCPP, and lungs tissue (n = 200), pleural fluid (n = 150) from goats at necropsy. RESULTS: The clinical signs recorded were mucopurulent nasal discharges, cough, abdominal respiration and hyperthermia. The post-mortem revealed, pulmonary consolidation, fibrinous pleuropneumonia, and accumulation pleural fluid. The fried egg like growth was observed on agar in 16 (4%), 11 (7.3%), 38 (19%), and 24 (16%) nasal swab, tracheal swabs, lungs and pleural fluid samples, respectively. PCR targeting 16S rRNA gene revealed isolates, belongs to Mycoplasma mycoides cluster, in 72 (8%) samples. Forty one (4.5%) isolates were Mccp by specie specific PCR generating an amplicon of 316 bp. CONCLUSIONS: We successfully isolated local strain of Mccp for the first time in Pakistan. This Mccp strain could be further utilized for the development of diagnostics and control measures against Mccp infection in goats.

'Candidatus Mycoplasma haemosphiggurus' a novel haemoplasma species in orange-spined hairy dwarf porcupines (Sphiggurus villosus) from Southern Brazil.

In Brazil, the orange-spined hairy dwarf porcupine (Sphiggurus villosus) is widely distributed in the Atlantic Rainforest biome being amongst the most frequently road-killed animal. Porcupines may also be commonly found on forest borders and occasionally, near urban areas where human and domestic dogs injuries caused by its spines may occur. Therefore, the aims of this study were (a) to screen porcupines for TBD pathogens and haemoplasmas and (b) to identify the tick species parasitizing these rodents in Paraná State, southern Brazil. Blood and/or spleen samples were collected from nine orange-spined hairy dwarf porcupines. A total of 275 ticks (34 males, 11 females, 7 nymphs and 223 larvae) were collected from eight porcupines: Amblyomma longirostre, A. parkeri and Amblyomma spp. larvae. Two out of nine (22%; 95% CI: 3%-60%) porcupines were PCR-positive for haemoplasmas. All animals tested negative for Theileria/Babesia spp. and Ehrlichia/Anaplasma spp. by PCR. Phylogenetic and network analysis of the 16S and 23S rRNA gene fragments confirmed that animals were infected by a potentially novel haemotropic Mycoplasma sp. The name 'Candidatus Mycoplasma haemosphiggurus' is proposed for this novel organism that should be further fully characterized.

Genetic diversity of emerging hemotropic mycoplasmas in domestic pigs from Brazil.

Mycoplasma suis and Mycoplasma parvum bind strongly to erythrocytes and may cause clinical hemoplasmosis in swine, affecting several age groups. Mycoplasma spp. infected animals may be asymptomatic carriers and/or show nonspecific clinical signs. In Brazil, information on genetic diversity associated with porcine hemoplasmas (PH) has not been described yet. Therefore, this study has aimed to detect, quantify and characterize the genetic diversity of PH in finishing pigs from technified farms in the state of Goiás, central-western Brazil. Ethylenediaminetetraacetic acid-blood samples from 450 swine belonging to 30 different farms from Goiás state were collected at the slaughterhouse. Quantitative real-time PCR (qPCR) assays were performed for the molecular detection and quantification of PH 16S rRNA gene fragments. Cloning and sequencing of 16S and 23S rRNA amplicons were performed to evaluate the genetic diversity. Moreover, a questionnaire was applied to each farm manager to obtain epidemiological information about the herd. The results on qPCR showed herd occurrence of 68.89% for PH. Quantification values (starting quantity [SQ]) ranged from 8.43 × 10-1 to 4.69 × 106 copies/µl, and 52.71% of the samples presented SQ values equal or lower than 1 × 103 copies/µl. Risk factors were not evaluated once all farms had at least one positive animal. However, Spearman's coefficient test revealed that the occurrence of PH was inversely associated with the number of farrows per week, weaned piglets per week, and weight at slaughter. Phylogenetic analysis based on maximum likelihood and Bayesian methods showed that the 16S rRNA and 23S rRNA gene sequences obtained from five samples formed a single cluster closely related to M. parvum. Genotype analysis using DNASP software confirmed seven and four different 16S and 23S rRNA genotypes among the cloned amplicons, indicating that there are several genotypes of M. parvum circulating in individual pigs and among pig farms in central-western Brazil.

Multilocus sequence typing of pathogenic Mycoplasma mycoides subsp. capri reveals the predominance of a novel clonal complex among isolates from goats in India.

Mycoplasma mycoides subsp. capri (Mmc) typically causes pneumonia, mastitis, arthritis, keratitis and septicaemia in goats. Mortality associated with Mmc in goat flocks is lower compared to Mycoplasma capricolum subsp. capripneumoniae-associated respiratory infections. Case fatality rates associated with Mmc ranged from 9.8 to 26.8% among several states in India. Molecular epidemiology approaches aimed at genotyping help to identify the diversity of isolates involved in a disease. Ten clinical pathogenic Mmc isolates were analysed by multilocus sequence typing (MLST) for studying genotypic relationships with 50 isolates available from public databases. The MLST analysis indicates high genetic diversity among Mmc isolates. From a total number of 60 isolates, 43 six sequence types (STs) were recognized comprising of six STs from India and 37 STs from other geographical regions. MLST profiles of isolates revealed none of the STs observed in Indian isolates were shared with global isolates. Some of the STs representing Indian isolates (four STs) were clustered into a novel clonal complex 1 (CC1). Maintenance of genetically related STs forming CCs among the goat population in India for longer periods indicates disease causing potentiality of these isolates. Based on various recombination analysis, weak clonal relationship among Mmc isolates were identified. The present study has enlightened further steps in disease investigations and to design future control measures by employing prevalent genotypes as vaccine candidates against Mmc infections.

Challenges in gene-oriented approaches for pangenome content discovery.

Given a group of genomes, represented as the sets of genes that belong to them, the discovery of the pangenomic content is based on the search of genetic homology among the genes for clustering them into families. Thus, pangenomic analyses investigate the membership of the families to the given genomes. This approach is referred to as the gene-oriented approach in contrast to other definitions of the problem that takes into account different genomic features. In the past years, several tools have been developed to discover and analyse pangenomic contents. Because of the hardness of the problem, each tool applies a different strategy for discovering the pangenomic content. This results in a differentiation of the performance of each tool that depends on the composition of the input genomes. This review reports the main analysis instruments provided by the current state of the art tools for the discovery of pangenomic contents. Moreover, unlike previous works, the presented study compares pangenomic tools from a methodological perspective, analysing the causes that lead a given methodology to outperform other tools. The analysis is performed by taking into account different bacterial populations, which are synthetically generated by changing evolutionary parameters. The benchmarks used to compare the pangenomic tools, in addition to the computational pipeline developed for this purpose, are available at https://github.com/InfOmics/pangenes-review. Contact: V. Bonnici, R. Giugno Supplementary information: Supplementary data are available at Briefings in Bioinformatics online.

Molecular Diagnosis of Mycoplasma Species Infection in Camels Using Semi-nested PCR.

BACKGROUND AND OBJECTIVE: Bacteriological isolation and identification of Mycoplasma species is difficult and time-consuming, therefore, molecular identification of Mycoplasma using PCR targeting specific genes is considered a specific and sensitive method for identification. The aim of current study was to isolate, characterize Mycoplasma infection in dromedary camels in Saudi Arabia. MATERIALS AND METHODS: Nasal swabs were randomly collected from 93 camels and tested for Mycoplasma by sequencing of their 16S rRNA genes using universal primers. RESULTS: The 93 samples, 24 were positive for Mycoplasma. However, no positive results were obtained using species-specific primers for Mycoplasma arginine, M. bovis or M. mycoides subsp. mycoides, thus, 16S rDNA sequencing methods and semi-nested PCR were employed. Sequences were matched to those in GenBank and phylogenetic analysis was performed. Mycoplasma edwardii (77-84% similarity with Mycoplasma edwardii ATCC 23462) and one isolate of Mycoplasma yeastsii (100% similarity with M. yeastsii GM274B) were identified. Further, some Mycoplasma species were identified as previously uncultured. The incidence of Mycoplasma infection in camels in Taif city, Saudi Arabia, was approximately 26%. CONCLUSION: This study provides insights into the accuracy and efficiency of PCR and universal primers for the detection and identification of Mycoplasma, thereby circumventing conventional culturing methods that require several days to complete and exhibit low accuracy.