Conventional Mycoplasma spp. diagnostics involve culture, often considered the gold standard in diagnostic test evaluation. However, culture protocols lack empirical derivation and primarily adhere to National Mastitis Council recommendations, tracing back to initial cultivation of Mycoplasma bovis. Despite a wide range of carbon dioxide (CO2) supplementation reported in literature, specific impacts of CO2 on Mycoplasma spp. growth remain unexplored. Our objective was to assess the effect of CO2 concentration on growth detection rates of 24 Mycoplasma spp. isolates from dairy cows. These isolates, mainly M. bovis, were incubated at 37°C in triplicate and three dilution ranges under three CO2 conditions: ambient air or 5% CO2 or 10% CO2. Bacterial growth was evaluated on incubation days 3, 5, 7, and 10. When cultured using ambient air, log10 cfu/mL was lower on days 3, 5, and 7 of incubation compared with isolates incubated in the recommended 5% or 10% CO2, with less variation observed in ambient air compared with 5% or 10% CO2. However, by 10 days of incubation, no differences in the detection of observable growth were noted among isolates incubated in ambient air, 5% CO2, or 10% CO2. Consequently, Mycoplasma spp. isolated from dairy cattle demonstrated growth after the recommended 7-10 days of culture, even in the absence of supplemental CO2. Given the expected concentration of M. bovis in (sub)clinical samples had similar concentrations to those used in our study, with the majority of isolates being M. bovis, we recommend expanding CO2 concentration ranges in M. bovis culture from 10% CO2 to ambient air when incubating for 10 days. However, the turnaround time could be shortened when incubating with supplemental CO2.
OBJECTIVE: Current Mycoplasma spp. culture protocols lack empirical derivation concerning carbon dioxide (CO2) supplementation and are primarily based on the initial cultivation of Mycoplasma bovis. This study indicates that the suitable range for CO2 supplementation is broader than what is currently recommended by the National Mastitis Council for culturing within the specified 7-10 days. No differences in bacterial growth detection rates were observed among ambient air, 5% CO2, or 10% CO2 supplementation during the 7- and 10-day incubation intervals. These new insights provide evidence supporting the possibility of culturing Mycoplasma spp. under ambient air conditions in a laboratory setting.

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The role of Mycoplasma bovirhinis in the development of pulmonary disease in cattle is controversial and was never evaluated in cattle from Latin America. This study investigated the respiratory infection dynamics associated with M. bovirhinis in suckling calves from 15 dairy cattle herds in Southern Brazil. Nasal swabs were obtained from asymptomatic (n = 102) and calves with clinical manifestations (n = 103) of bovine respiratory disease (BRD) and used in molecular assays to identify the specific genes of viral and bacterial disease pathogens of BRD. Only M. bovirhinis, bovine coronavirus (BCoV), ovine gammaherpesvirus 2 (OvGHV2), Histophilus somni, Pasteurella multocida, and Mannheimia haemolytica were detected. M. bovirhinis was the most frequently diagnosed pathogen in diseased (57.8%; 59/102) and asymptomatic (55.3%; 57/103) calves at all farms. BCoV-related infections were diagnosed in diseased (52%; 53/102) and asymptomatic (51.4%; 53/103) calves and occurred in 93.3% (14/15) of all farms. Similarly, infectious due to OvGHV2 occurred in diseased (37.2%; 38/102) and asymptomatic (27.2%; /28/103) calves and were diagnosed in 80% (12/15) of all farms investigated. Significant statistical differences were not identified when the two groups of calves were compared at most farms, except for infections due to OvGHV2 that affected five calves at one farm. These results demonstrated that the respiratory infection dynamics of M. bovirhinis identified in Southern Brazil are similar to those observed worldwide, suggesting that there is not enough sufficient collected data to consider M. bovirhinis as a pathogen of respiratory infections in cattle. Additionally, the possible roles of BCoV and OvGHV2 in the development of BRD are discussed.

In the available literature on this subject there are many studies which describe the effects of sexually transmitted infections on pregnancy and fertility of women. Because of the frequency of the infections with the atypical bacteria of the Ureaplasma Spp., Mycoplasma Spp., Chlamydia Trachomatis, as well as HPV infections in women of reproductive age, it is easy to underestimate their importance when establishing the basis of the genital health of women of reproductive age. In this prospective analysis, conducted from 2014 to 2018 in the laboratory for HPV and Molecular diagnostics at the University Clinic of Gynaecology and Obstetrics in Skopje, North Macedonia, we analysed the results of 10,387 patients of all ages, of which 973 patients were of reproductive age. A Panel analysis was also conducted (including the above-mentioned pathogens). An HPV analysis was also conducted on 643 patients in this group. Within the group of 643 patients, there was a positive result for HPV in 26.7% of them, while in 40.9% there was a positive result for one or more pathogens on the Panel analysis of bacterial pathogens. The statistical analysis of the results showed that the most frequent of all bacterial pathogens within the Macedonian population of women of reproductive age is Ureaplasma Spp, with an incidence of 33%, followed by Mycoplasma Spp., with 7.8%, while Chlamydia Trachomatis was present in 6.4% of the cases. We should highlight that a co-infection with HPV was present in 18.5% of all the patients where there was analysis of both diagnostic procedures. The analysis of the results in the patients co-infected with HPV and at least one bacterial pathogen on the Panel Analysis, showed a very high statistical correlation (p<001).

BACKGROUND: Bacteria belonging to the genus Mycoplasma are small-sized, have no cell walls and small genomes. They commonly cause respiratory disorders in their animal hosts. Three species have been found in the respiratory tract of horses worldwide, that is., Mycoplasma (M.) equirhinis, M. pulmonis and M. felis, but their role in clinical cases remains unclear.
OBJECTIVE: The aim of this study was to i) develop and validate tools to detect, isolate and identify different Mycoplasma spp. strains in clinical equine respiratory-tract specimens and ii) subsequently define the prevalence of the three species in France depending on sample types and horse characteristics (age, breed, sex).
METHODS: Validation of a workflow for mycoplasma diagnosis and subsequent prevalence study.
METHODS: Mycoplasma-free tracheal wash samples spiked with numerated strains and DNA dilutions were used to validate the culture methods and real-time PCR (rt-PCR) assay. Isolated strains were identified by 16S rRNA gene sequencing. Prevalences were determined on a population of 616 horses with respiratory disorders, sampled in France in 2020.
RESULTS: In total, 104 horses (16.9%) were found to be positive for Mycoplasma spp. by at least one method. M. equirhinis was the predominant circulating species, accounting for 85% of the rt-PCR-positive samples and 98% of the 40 cultured strains.
CONCLUSIONS: The proposed pre-enrichment procedure improves the sensitivity of detection but hinders the quantification of the initial mycoplasma load in the clinical specimens.
CONCLUSIONS: Prevalence of mycoplasma varied with age, breed, and type of sample.
UNASSIGNED: Bakterien, die zur Gattung Mycoplasma gehören, sind klein, haben keine Zellwände und ein kleines Genom. Sie verursachen bei ihren tierischen Wirten häufig Erkrankungen der Atemwege. Drei Spezies wurden weltweit in den Atemwegen von Pferden gefunden, nämlich Mycoplasma (M.) equirhinis, M. pulmonis und M. felis, aber ihre Rolle in klinischen Fällen bleibt unklar.
UNASSIGNED: Ziel dieser Studie war es, i) Instrumente zum Nachweis, zur Isolierung und zur Identifizierung verschiedener Mycoplasma spp. in klinischen Atemwegsproben von Pferden zu entwickeln und zu validieren und ii) anschließend die Prävalenz der drei Spezies in Frankreich in Abhängigkeit von Probentypen und Pferdemerkmalen (Alter, Rasse, Geschlecht) zu bestimmen.
METHODS: Validierung eines Arbeitsablaufs für die Mykoplasmen-Diagnose und anschließende Prävalenzstudie.
METHODS: Mykoplasmenfreie Trachealspülungen, die mit nummerierten Stämmen und DNA-Verdünnungen versetzt waren, wurden zur Validierung der Kulturmethoden und des real-time PCR-Tests (rt-PCR) verwendet. Isolierte Stämme wurden durch Sequenzierung des 16S rRNA-Gens identifiziert. Die Prävalenzen wurden in einer Population von 616 Pferden mit Atemwegserkrankungen bestimmt, die 2020 in Frankreich beprobt wurden.
UNASSIGNED: Insgesamt wurden 104 Pferde (16.9%) mit mindestens einer Methode positiv auf Mycoplasma spp. getestet. M. equirhinis war die vorherrschende zirkulierende Spezies und machte 85% der rt-PCR-positiven Proben und 98% der 40 kultivierten Stämme aus.
UNASSIGNED: Das vorgeschlagene Voranreicherungsverfahren verbessert die Nachweisempfindlichkeit, erschwert aber die Quantifizierung der ursprünglichen Mykoplasmenbelastung in den klinischen Proben.
UNASSIGNED: Die Prävalenz von Mykoplasmen variierte je nach Alter, Status des Rennpferdes und Art der Probe.

Co-infections of avian species with different RNA viruses and pathogenic bacteria are often misdiagnosed or incompletely characterized using targeted diagnostic methods, which could affect the accurate management of clinical disease. A non-targeted sequencing approach with rapid and precise characterization of pathogens should help respiratory disease management by providing a comprehensive view of the causes of disease. Long-read portable sequencers have significant potential advantages over established short-read sequencers due to portability, speed, and lower cost. The applicability of short reads random sequencing for direct detection of pathogens in clinical poultry samples has been previously demonstrated. Here we demonstrate the feasibility of long read random sequencing approaches to identify disease agents in clinical samples. Experimental oropharyngeal swab samples (n = 12) from chickens infected with infectious bronchitis virus (IBV), avian influenza virus (AIV) and Mycoplasma synoviae (MS) and field-collected clinical oropharyngeal swab samples (n = 11) from Kenyan live bird markets previously testing positive for Newcastle disease virus (NDV) were randomly sequenced on the MinION platform and results validated by comparing to real time PCR and short read random sequencing in the Illumina MiSeq platform. In the swabs from experimental infections, each of three agents in every RT-qPCR-positive sample (Ct range 19-34) was detectable within 1 h on the MinION platform, except for AIV one agent in one sample (Ct = 36.21). Nine of 12 IBV-positive samples were assigned genotypes within 1 h, as were five of 11 AIV-positive samples. MinION relative abundances of the test agent (AIV, IBV and MS) were highly correlated with RT-qPCR Ct values (R range-0.82 to-0.98). In field-collected clinical swab samples, NDV (Ct range 12-37) was detected in all eleven samples within 1 h of MinION sequencing, with 10 of 11 samples accurately genotyped within 1 h. All NDV-positive field samples were found to be co-infected with one or more additional respiratory agents. These results demonstrate that MinION sequencing can provide rapid, and sensitive non-targeted detection and genetic characterization of co-existing respiratory pathogens in clinical samples with similar performance to the Illumina MiSeq.

UNASSIGNED: Hyperammonemic encephalopathy caused by Ureaplasma spp. and Mycoplasma hominis infection has been reported in immunocompromised patients undergoing lung transplant, but data are scarce in patients with hematological malignancies.
UNASSIGNED: We describe the cases of 3 female patients aged 11-16 years old, developing initially mild neurologic symptoms, rapidly evolving to coma and associated with very high ammonia levels, while undergoing intensive treatment for acute leukemia (chemotherapy: 2 and hematopoietic stem cell transplant: 1). Brain imaging displayed cerebral edema and/or microbleeding. Electroencephalograms showed diffuse slowing patterns. One patient had moderate renal failure. Extensive liver and metabolic functions were all normal. Ureaplasma spp. and M. hominis were detected by PCR and specific culture in two patients, resulting in prompt initiation of combined antibiotics therapy by fluoroquinolones and macrolides. For these 2 patients, the improvement of the neurological status and ammonia levels were observed within 96 h, without any long-term sequelae. M. hominis was detected post-mortem in vagina, using 16S rRNA PCR for the third patient who died of cerebral edema.
UNASSIGNED: Hyperammonemic encephalopathy linked to Ureaplasma spp. and M. hominis is a rare complication encountered in immunocompromised patients treated for acute leukemia, which can lead to death if unrecognized. Combining our experience with the few published cases (n=4), we observed a strong trend among female patients and very high levels of ammonia, consistently uncontrolled by classical measures (ammonia-scavenging agents and/or continuous kidney replacement therapy). The reversibility of the encephalopathy without sequelae is possible with prompt diagnosis and adequate combined specific antibiotherapy. Any neurological symptoms in an immunocompromised host should lead to the measurement of ammonia levels. If increased, and in the absence of an obvious cause, it should prompt to perform a search for Ureaplasma spp. and M. hominis by PCR as well as an immediate empirical initiation of combined specific antibiotherapy.

Bovine infectious infertility represents a problem due to the high impact on animal production and, in many cases, in public health. A lack of information on the characteristics of the bacterial population of the bovine reproductive system can hamper a comprehensive understanding of reproductive pathologies and the role that the microbiome could play. A metagenomic study based on the V3-V4 hypervariable region of the bacterial 16S rRNA gene was performed in 1029 preputial samples from bulls raised in an extensive regimen in Spain (944 from herds with low fertility rates -case group-, and 85 samples from reproductively healthy herds -control group-). The most representative phyla as well as the most 10 abundant bacterial families and their abundance did not show significant differences in both case and control groups. Similarly, the (alpha and beta) diversity of the bacterial populations was similar in both type of herds: the Shannon and Simpson indices show a high diversity of species, while the Bray-Curtis dissimilarity index did not show relevant differences in the bacterial communities. A deeper analysis of the operational taxonomic units showed the presence of one genera, Mycoplasma spp. significantly associated with fertility problems. Our study highlights the promising potential that the application of sequencing techniques (e.g. 16S rRNA-based metagenomics) possesses in examining bovine infertility, as they are able to reveal different pathogens that could go unnoticed using diagnostic approaches for only the main known pathogens.

Mycoplasma infections have been found in different species of waterfowl worldwide. However, the question of how the pathogens have been transmitted and dispersed is still poorly understood. Samples collected from clinically healthy greater white-fronted geese (Anser albifrons) (N = 12), graylag geese (Anser anser) (N = 6), taiga bean geese (Anser fabalis) (N = 10), and barnacle geese (Branta leucopsis) (N = 1) were tested for Mycoplasma spp. All Mycoplasma-positive samples were specified by species-specific PCR for Mycoplasma anserisalpingitidis (formerly known as Mycoplasma sp. 1220), M. anseris, M. anatis, and M. cloacale. The presence of Mycoplasma spp. was confirmed in 22 of 29 sampled birds (75.9%). Mycoplasma anserisalpingitidis was the most frequently detected species (15 of 22, 68.2%). However, we did not detect any of the other Mycoplasma spp. typical for geese, among which are M. anatis, M. anseris, and M. cloacale. Phylogenetic analysis revealed that Polish sequences of M. anserisalpingitidis formed a distinct branch, along with 2 Hungarian isolates obtained from domestic geese. Eight of the samples identified as Mycoplasma spp.-positive were negative for the aforementioned Mycoplasma species. A phylogenetic tree constructed based on partial 16S rRNA gene analysis showed that Mycoplasma spp. sequences collected from Polish wild geese represent a distinct phylogenetic group with Mycoplasma sp. strain 2445 isolated from a domestic goose from Austria. The results of our study showed that wild geese could be a reservoir and vector of different species of the Mycoplasma genus that can cause significant economic losses in the domestic goose industry.

There is scarce information about the frequency and epidemiological and clinical features associated with the presence of Mycoplasma spp. in Argentine dairy herds. The objectives of this study were to develop a multiplex PCR for identifying M.bovis and M.canadense and to describe the frequency of Mycoplasma spp. isolated from clinical samples submitted to a diagnostic laboratory. Of a total of 1548 samples from intramammary infections, bulk tank milk and biological fluids, 38 Mycoplasma isolates were obtained. M. bovis, M. canadense, M.californicum and M.leachii were detected by using two multiplex PCRs, confirming their presence in clinical conditions in dairy cattle. The techniques used in the present study can be useful to broaden the knowledge about Mycoplasma infections in cattle, since the search for these organisms is not usually included in routine diagnoses.