Evaluation of the efficacy of a new inactivated vaccine against Staphylococcus aureus, Echerchia coli and Mycoplasma bovis mastitis in cows.

Staphylococcus aureus, Escherichia coli and Mycoplasma bovis are the most commonly isolated mastitis pathogens. The aim of this study was to evaluate the efficacy of a new mixed vaccine against mastitis caused by  Staphylococcus aureus, Escherichia coli, and Mycoplasma bovis. For this purpose, a mixed inactivated vaccine was administered subcutaneously to 24 heifers as one dose (2 mL) on the 45th day before birth and the second dose 21 days later. In 9 heifers, 2 mL of PBS was administered as placebo instead of vaccine. Then, heifers were divided into 3 groups as 7 vaccinated and 3 unvaccinated animals. Staphylococcus aureus, Escherichia coli, and Mycoplasma bovis were administered to the groups through intramammary route. Three vaccinated heifers were considered the common control without bacteria in all groups. The parameters considered to assess the effect of vaccination were clinical findings, bacterial count in milk, somatic cell count, and antibody titers. Clinical signs were observed only in the unvaccinated placebo group. Bacteria count and somatic cell count in milk increased in vaccinated and unvaccinated heifers. However, this increase was less in vaccinated animals and gradually returned to the normal level. In the unvaccinated heifers, it was ever high. Serum antibody titers were measured before and after vaccination. Antibody titers were high in vaccinated heifers after vaccination and were negative in unvaccinated heifers. In conclusion, the mixed vaccine had beneficial effect against Staphylococcus aureus, Escherichia coli, and Mycoplasma bovis mastitis and stimulated the immune response of vaccinated heifers.

Correlation between the Colony Phenotype and Amino Acid Sequence of the Variable Vaa Antigen in Clinical Isolates of Mycoplasma hominis.

A new Mycoplasma hominis phenotype forming mini-colonies (MC) on agar and distinct from the phenotype forming typical colonies (TC) not only in size, but also in morphology, growth rate, and resistance to adverse factors, has been previously identified. In this study, the phenotype of colonies was determined and a comparative analysis of the amino acid sequence of the main variable antigen Vaa of the laboratory strain N-34 and seven clinical isolates of M. hominis was performed. It is demonstrated that the amino acid sequence of Vaa in clinical isolates forming TC (similar to the laboratory strain N-34) is entirely analogous to that of laboratory strain. Clinical isolates forming MC carry amino acid substitutions in the variable C-terminal region of Vaa, which can contribute to adhesion to eukaryotic cells and immune evasion. The connection between colony phenotype and amino acid sequence of Vaa is established.

Clinical and uterine cervix characteristics of women with Mycoplasma and Ureaplasma in genital discharge.

OBJECTIVE: The objective of this study was to assess the clinical and uterine cervix characteristics of patients displaying vaginal discharge with positive results for Mycoplasma sp. and/or Ureaplasma spp. METHODS: An analytical cross-sectional study involving women aged 18-45 years was conducted. Microbiological assessments included Ureaplasma and Mycoplasma cultures, as well as human papillomavirus hybrid capture using ecto and endocervix swabs. All tests were two-tailed, and significance was set at p<0.05. RESULTS: Among 324 women, Ureaplasma prevalence was 17.9%, and Mycoplasma prevalence was 3.1%. The Ureaplasma-positive group exhibited a higher frequency of urinary tract infections (39.1 vs. 19%, p=0.002) and human papillomavirus (39.7 vs. 12.8%, p≤0.001) compared with controls. The Mycoplasma-positive group showed a higher frequency of non-contraceptive use compared with controls (66.2 vs. 30.0%, p=0.036). Abnormal colposcopic findings were more prevalent in the Mycoplasma/Ureaplasma-positive group than in controls (positive: 65% vs. control: 35%, p=0.001). Pap smear findings did not differ between the groups. CONCLUSION: Ureaplasma spp. was associated with urinary tract infections and human papillomavirus, while the presence of Mycoplasma sp. was linked to reduced contraceptive use. When analyzing both pathogens together, a higher frequency of abnormal colposcopic findings was observed, with no difference in cytological findings in the positive group.

Economic evaluation alongside a clinical trial of near-to-patient testing for sexually transmitted infections.

BACKGROUND: Current clinical care for common bacterial STIs (Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG) and Mycoplasma genitalium (MG)) involves empiric antimicrobial therapy when clients are symptomatic, or if asymptomatic, waiting for laboratory testing and recall if indicated. Near-to-patient testing (NPT) can improve pathogen-specific prescribing and reduce unnecessary or inappropriate antibiotic use in treating sexually transmitted infections (STI) by providing same-day delivery of results and treatment. METHODS: We compared the economic cost of NPT to current clinic practice for managing clients with suspected proctitis, non-gonococcal urethritis (NGU), or as an STI contact, from a health provider's perspective. With a microsimulation of 1000 clients, we calculated the cost per client tested and per STI- and pathogen- detected for each testing strategy. Sensitivity analyses were conducted to assess the robustness of the main outcomes. Costs are reported as Australian dollars (2023). RESULTS: In the standard care arm, cost per client tested for proctitis, NGU in men who have sex with men (MSM) and heterosexual men were the highest at $247.96 (95% Prediction Interval (PI): 246.77-249.15), $204.23 (95% PI: 202.70-205.75) and $195.01 (95% PI: 193.81-196.21) respectively. Comparatively, in the NPT arm, it costs $162.36 (95% PI: 161.43-163.28), $158.39 (95% PI: 157.62-159.15) and $149.17 (95% PI: 148.62-149.73), respectively. Using NPT resulted in cost savings of 34.52%, 22.45% and 23.51%, respectively. Among all the testing strategies, substantial difference in cost per client tested between the standard care arm and the NPT arm was observed for contacts of CT or NG, varying from 27.37% to 35.28%. CONCLUSION: We found that NPT is cost-saving compared with standard clinical care for individuals with STI symptoms and sexual contacts of CT, NG, and MG.

Evaluation of endothelial glycocalyx injury biomarkers in feline hemotropic mycoplasmosis.

The present study aimed to investigate endothelial glycocalyx (eGCx) damage in cats with feline hemotropic mycoplasmosis caused by Mycoplasma haemofelis using selected biomarkers and to determine the diagnostic and prognostic significance of these biomarkers. The study included 25 cats with feline hemotropic mycoplasmosis and 10 healthy cats. Clinical examination, blood gas analysis, complete blood count, and biochemical analysis were performed. Hemotropic mycoplasmosis diagnosed by microscopic examination and molecularly confirmed by PCR targeting the Mycoplasma haemofelis 16s rRNA gene. To evaluate endothelial glycocalyx damage, syndecan-1, endothelin-1 (ET-1), asymmetric dimethylarginine (ADMA), and vascular endothelial growth factor-A (VEGF-A) concentrations were measured using cat-specific commercial ELISA kits. Of the cats with feline hemotropic mycoplasmosis, 14 (56%) survived and 11 (44%) died. While syndecan-1 and ET-1 concentrations were significantly higher in cats with hemotropic mycoplasmosis compared to the control group (p < 0.001), no statistically significant difference was found for ADMA and VEGF-A concentrations (p > 0.05). Endothelial glycocalyx biomarkers showed significant correlations with each other and with hematological parameters (p < 0.01). The results of the ROC analysis showed that ET-1 with area under the curve (AUC) of 0.821 (p < 0.01) and VEGF-A with AUC of 0.805 (p < 0.010) were found to be significant prognostic indicators. In conclusion, this study demonstrated that serum syndecan-1 and ET-1 can be used as diagnostic and serum ET-1 and VEGF-A as prognostic biomarkers in cats with hemotropic mycoplasmosis. Our results indicate the development of eGCx damage in feline hemotropic mycoplasmosis and suggest that glycocalyx disruption may contribute to the pathogenesis of the disease.

Molecular detection and genetic characterization of Mycoplasma gallisepticum and Mycoplasma synoviae in selected chicken breeds in South Africa.

BACKGROUND: The impact of chickens on maintaining the economy and livelihood of rural communities cannot be overemphasized. In recent years, mycoplasmosis has become one of the diseases that affect the success of South African chicken production. Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) are the most prevalent strains of Mycoplasma in South Africa. MG and MS are significant respiratory pathogens affecting the productivity of chickens. The present study aimed to molecularly detect using qPCR and characterize the presence of MG and MS using phylogenetic analysis. The phylogenetic analysis was utilized to clarify general evolutionary relationships between related taxa of different MG and MS observed in tracheal swabs from South African chicken breeds. METHODS: Forty-five tracheal swabs of the Lohmann Brown (n = 9), Rhode Island Red (n = 9), Ovambo (n = 9), Venda (n = 9), and Potchefstroom Koekoek (n = 9) breeds were collected from symptomatic chickens present in the commercial farm. To detect MG and MS, DNA was extracted from tracheal swabs and faecal samples, and qPCR was performed with a 16 s rRNA (310 bp) and vlhA (400 bp) gene fragment. Following the sequencing of all the amplicons, MG, and MS dendrograms showing the evolutionary relationships among the five South African chicken breeds and the GeneBank reference population were constructed. RESULTS: The qPCR revealed the presence of MG and MS in 22% (2/9) of the tracheal swab samples tested for MS only in Rhode Island Red breeds; 66.6% (6/9) and 33% (3/9) of the tested samples in Ovambo breeds; and 11.1% (1/9) and 44.4% (4/9) of the tested samples in Venda breeds. No MG or MS were detected in the Lohmann Brown or Potchefstroom Koekoek breed. Furthermore, qPCR revealed the presence of MG in pooled faecal samples from Lohmann Brown and Ovambo breeds. Eight different bacterial isolates were recognized from both samples. Four isolates were of the 16 s ribosomal ribonucleic acid (rRNA) gene (named PT/MG51/ck/00, PT/MG48/ck/00, PT/MG41/ck/00 and PT/MG71/ck/00) gene of Mycoplasma gallisepticum, and the other was Mycoplasma Synoviae variable lipoprotein hemagglutinin A (vlhA) gene (named PT/MSA22/ck/01, PT/MS41/ck/01, PT/MS74/ck/01 and PT/MS46/ck/01) which were available in GenBank. These isolates were successfully sequenced with 95-100% similarity to the isolates from the gene bank. CONCLUSION: The study revealed the presence of both MG and MS in the chicken breeds sampled. Furthermore, the different breeds of chicken were found to be susceptible to infection under the intensive or commercial management system. Therefore, continuous surveillance is encouraged to prevent the spread and outbreak of MG and MS in the poultry industry in South Africa.

Cost-effectiveness of resistance-guided therapy for Mycoplasma genitalium in Australia.

The recommended first-line treatment for Mycoplasma genitalium infections is azithromycin. However, the prevalence of macrolide resistance for M. genitalium has increased to more than 50% worldwide. In 2013, Australia introduced a resistance-guided therapy (RGT) strategy to manage M. genitalium infections. This study assesses the cost-effectiveness of the RGT approach compared to no RGT (i.e., without macrolide resistance profile test) in women, men who have sex with men (MSM), and men who have sex with women (MSW) in Australia. We constructed dynamic transmission models of M. genitalium infections in women, MSM, and MSW in Australia, each with a population of 100,000. These models compared the costs and quality-adjusted life-years (QALYs) gained between RGT and no RGT scenarios from a healthcare perspective over ten years. All costs are reported in 2022 Australian dollars (Australian $). In our model, RGT is cost saving in women and MSM, with the incremental net monetary benefit of $1.3 million and $17.9 million, respectively. In MSW, the RGT approach is not cost-effective, with an incremental cost-effectiveness ratio of -$106.96 per QALY gained. RGT is cost saving compared to no RGT for M. genitalium infections in women and MSM, supporting its adoption as the national management strategy for these two population groups.

Isolation and characterization of Chlamydia felis and its pathogenesis in cats.

Feline upper respiratory tract disease (URTD) is a common but complicated disease that occurs in domestic cats, worldwide. 396 cats in Guangxi Province, China were screened for URTD-associated pathogens from March 2022 to August 2023. Mycoplasma felis was found to be the most prevalent infectious agent with a positivity rate of 24.75 %, followed by feline calicivirus (FCV), Chlamydia felis, feline herpesvirus 1 (FHV-1) and feline influenza A virus (FeIAV) with rates of 15.91, 11.62, 5.56 and 1.52 %, respectively. In particular, C. felis and M. felis were found in 13 of 55 co-infected cats. Of the 46 C. felis-positive samples, one strain, named as GXNN36, was successfully isolated using chicken embryos and it was characterized both in vivo and in vitro. For the cat studies, both high- and low-dose challenged groups showed severe conjunctivitis, accompanied by transient fever and respiratory symptoms. C. felis replicated well in turbinate, trachea and lung tissues with high copy numbers and the infection subsequently spread to the livers, spleens, pancreas, kidneys, hearts and intestines. These findings will help our understanding of the role of C. felis in feline URTD and provide a valuable model to evaluate the efficacy of vaccines and therapeutic remedies in the future.

Effect of co-infection with Newcastle disease virus on Mycoplasma gallisepticum pathogenesis in vivo and in vitro.

The co-infection of Newcastle disease virus (NDV) and Mycoplasma gallisepticum (MG) has a detrimental effect on chicken production performance, exerts a deleterious impact on poultry production performance, resulting in substantial economic losses. However, the exact impact and underlying mechanisms remain ambiguous. In this study, co-infection models were established both in vivo and in vitro. Through these models, it was found that the co-infection facilitated the replication of MG and NDV, as well as MG induced pathogenesis. The administration of lentogenic NDV resulted in the suppression of the innate immune response in vivo. At cellular level, co-infection promoted MG induced apoptosis through caspase-dependent mitochondrial endogenous pathway and suppressed the inflammatory secretion. This research contributes novel insights in co-infection.

Evaluation of commercial, customized microdilution plates for Ureaplasma parvum, Ureaplasma urealyticum, and Mycoplasma hominis antimicrobial susceptibility testing and determination of antimicrobial resistance prevalence in France.

Antimicrobial susceptibility testing (AST) of human mycoplasmas using microdilution is time-consuming. In this study, we compared the performance of MICRONAUT-S plates (Biocentric-Bruker) designed for AST of Ureaplasma parvum, Ureaplasma urealyticum, and Mycoplasma hominis with the results using the Clinical & Laboratory Standards Institute (CLSI) reference method. Then, we investigated the prevalence and mechanisms of resistance to tetracyclines, fluoroquinolones, and macrolides in France in 2020 and 2021. The two methods were compared using 60 strains. For the resistance prevalence study, U. parvum-, U. urealyticum-, and M. hominis-positive clinical specimens were collected for 1 month each year in 22 French diagnostic laboratories. MICs were determined using the MICRONAUT-S plates. The tet(M) gene was screened using PCR, and fluoroquinolone resistance-associated mutations were screened using PCR and Sanger sequencing. Comparing the methods, 99.5% (679/680) MICs obtained using the MICRONAUT-S plates concurred with those obtained using the CLSI reference method. For 90 M. hominis isolates, the tetracycline, levofloxacin, and moxifloxacin resistance rates were 11.1%, 2.2%, and 2.2%, respectively, with no clindamycin resistance. For 248 U. parvum isolates, the levofloxacin and moxifloxacin resistance rates were 5.2% and 0.8%, respectively; they were 2.9% and 1.5% in 68 U. urealyticum isolates. Tetracycline resistance in U. urealyticum (11.8%) was significantly (P < 0.001) higher than in U. parvum (1.2%). No macrolide resistance was observed. Overall, the customized MICRONAUT-S plates are a reliable, convenient tool for AST of human mycoplasmas. Tetracycline and fluoroquinolone resistance remain limited in France. However, the prevalence of levofloxacin and moxifloxacin resistance has increased significantly in Ureaplasma spp. from 2010 to 2015 and requires monitoring. IMPORTANCE: Antimicrobial susceptibility testing of human urogenital mycoplasmas using the CLSI reference broth microdilution method is time-consuming and requires the laborious preparation of antimicrobial stock solutions. Here, we validated the use of reliable, convenient plates designed for antimicrobial susceptibility testing that allows the simultaneous determination of the MICs of eight antibiotics of interest. We then investigated the prevalence and mechanisms of resistance of each of these bacteria to tetracyclines, fluoroquinolones, and macrolides in France in 2020 and 2021. We showed that the prevalence of levofloxacin and moxifloxacin resistance has increased significantly in Ureaplasma spp. from 2010 to 2015 and requires ongoing monitoring.

Genes required for survival and proliferation of Mycoplasma bovis in association with host cells.

Mycoplasma bovis is an important emerging pathogen of cattle and bison, but our understanding of the genetic basis of its interactions with its host is limited. The aim of this study was to identify genes of M. bovis required for interaction and survival in association with host cells. One hundred transposon-induced mutants of the type strain PG45 were assessed for their capacity to survive and proliferate in Madin-Darby bovine kidney cell cultures. The growth of 19 mutants was completely abrogated, and 47 mutants had a prolonged doubling time compared to the parent strain. All these mutants had a similar growth pattern to the parent strain PG45 in the axenic media. Thirteen genes previously classified as dispensable for the axenic growth of M. bovis were found to be essential for the growth of M. bovis in association with host cells. In most of the mutants with a growth-deficient phenotype, the transposon was inserted into a gene involved in transportation or metabolism. This included genes coding for ABC transporters, proteins related to carbohydrate, nucleotide and protein metabolism, and membrane proteins essential for attachment. It is likely that these genes are essential not only in vitro but also for the survival of M. bovis in infected animals. IMPORTANCE: Mycoplasma bovis causes chronic bronchopneumonia, mastitis, arthritis, keratoconjunctivitis, and reproductive tract disease in cattle around the globe and is an emerging pathogen in bison. Control of mycoplasma infections is difficult in the absence of appropriate antimicrobial treatment or effective vaccines. A comprehensive understanding of host-pathogen interactions and virulence factors is important to implement more effective control methods against M. bovis. Recent studies of other mycoplasmas with in vitro cell culture models have identified essential virulence genes of mycoplasmas. Our study has identified genes of M. bovis required for survival in association with host cells, which will pave the way to a better understanding of host-pathogen interactions and the role of specific genes in the pathogenesis of disease caused by M. bovis.

Development and application of a cycleave dual-probe fluorescence quantitative PCR method for simultaneous detection of Mycoplasma gallisepticum ts-11 vaccine strain and non-ts-11 strains.

An attenuated vaccine against the Mycoplasma gallisepticum ts-11 strain has become an effective prevention and control method against MG infection. However, the ts-11 strain is usually difficult to distinguish from the non-ts-11 strain (including field isolates and other vaccine strains (F and 6/85)). Therefore, it is critical to establish a rapid and effective method to distinguish ts-11 strains from non-ts-11 strains. The gene sequences of the ts-11 strain (CP044225.1) and the non-ts-11 strain (including the wild-type (CP006916.3), 6/85 (CP044224.1), and F strains (NC_017503.1) were used to construct a conserved region containing a single point mutation in the potC gene in the ts-11 strain, after which a primer-probe combination method was designed. The primer-probe method was able to accurately and efficiently identify the ts-11 and non-ts-11 strains with minimum detection limits of 2.43 copies/µL and 1.65 copies/µL, respectively. Moreover, it could simultaneously distinguish the ts-11 strain from a non-ts-11 strain, and amplifications of avian influenza virus, infectious bronchitis virus, Newcastle disease virus, fowl adenovirus, infectious laryngotracheitis virus, infectious bursal disease virus, chicken anemia virus, Marek's disease virus, Mycoplasma synoviae, and Ornithobacter rhinotracheale were negative. The detection of clinical samples revealed that the established dual-probe fluorescence quantitative PCR method could be used to screen for mixed and single infections of the ts-11 strain and non-ts-11 strains effectively, with lower variation coefficients for intra- and interbatch repetition. The established cycleave dual-probe fluorescence quantitative PCR method showed good specificity, sensitivity, and repeatability and provides powerful technical support for the rapid and efficient differential diagnosis of the MG ts-11 strain from non-ts-11 strains.

Revolutionizing poultry hygiene: advanced electrostatic and cold fog disinfection strategies combat Mycoplasma gallisepticum in hatching eggs.

The incidence of chronic respiratory disease (CRD) due to Mycoplasma gallisepticum (MG) contamination in hatching eggs poses a serious threat to poultry health and hatchability. Implementing effective sanitization methods while safeguarding the hatching potential of embryos is crucial. This study aimed to explore novel techniques for sanitizing hatching-fertile eggs to prevent and manage MG-associated CRD. The primary objective was to assess the efficacy of acidic electrochemically stimulated water (ECS), focusing on MG disinfection. Additionally, the study investigated 2 application methods, 1) electrostatic disinfection (ED) and 2) cold fog (CF) disinfection, to evaluate their bactericidal effects against MG-contaminated eggs. Deliberately infected MG strains were used for the experimental design, which compared the disinfection efficacy of ECS with its acidic properties. The comparison involved ED, which applies an electrostatic charge to water particles, and CF disinfection, a cold mist technique. Both methods aimed to target MG without compromising egg-hatching potential. The results indicated a significant (p < 0.05) reduction in colony-forming units per milliliter (CFU/mL). However, both application methods demonstrated distinct bactericidal effects. Eggs treated with electrostatic disinfection showed a significant (p < 0.001) reduction in embryonic mortality during incubation (10%) compared to control untreated eggs (18%). Similarly, the CF method exhibited a significant (p < 0.001) decrease in embryonic mortality (13%). The ECS potential in reducing embryonic mortality within the pH range of 2.5 to 6.5 was noted. Both the ED and CF methods show promise for preventing MG-induced hatchery infection while maintaining egg-hatching potential. This study presents innovative techniques to control MG in hatching eggs, contributing to improved poultry health and reduced CRD incidence.

Mycoplasma salivarium in a Retropharyngeal abscess - nidus in a niche!

Retropharyngeal abscess (RPA) is considered one of the life threatening conditions which can present either as dysphagia or dyspnoea. Timely management for the airway obstruction along with etiology identification plays a pivotal role in saving a patient's life. Here we present a case of RPA due to a rare pathogen.

Development and Evaluation of Mycoplasma gallisepticum Challenge Model in Layer Pullets.

Manufacturers of Mycoplasma gallisepticum (MG) modified live vaccines usually recommend a single application at 8 wk of age. This makes 12-16-wk-old layer pullets suitable for challenge studies intended to evaluate these vaccines. Numerous challenge models in different poultry species and ages have been reported. However, there is not an established layer pullet challenge model for this age. The aim of this study is to develop a suitable challenge model in 12-wk-old layer pullets. MG Rlow strain was used as the challenge strain, and its ability to induce clinical signs and lesions in 12-wk-old Hy-Line W-36 layer pullets was evaluated. Three different doses (low, 7.95 × 104 color-changing units [CCU]/bird; medium, 7.95 × 106 CCU/bird; and high, 7.95 × 108 CCU/bird) via three different routes (eye drop, fine spray, and contact infection) were compared and evaluated using different parameters. At 14 days post-challenge, there were no mortalities in any of the groups throughout the study. Layer pullets directly challenged with the high dose via the fine spray route showed the clearest and most consistent results (clinical signs, positive quantitative real-time PCR [qPCR], seroconversion, air sac scoring, and histopathological changes of the tracheal mucosa). Medium and low challenge doses applied via fine spray or eye drop did not show consistent results. Rlow strain was able to spread to the contact infection birds, as confirmed by the positive qPCR results; however, none of the contact-infected birds showed any clinical signs or gross or microscopic lesions. Our results suggest that a high dose (7.95 × 108 CCU/bird) administered through a fine spray route is the model of choice in any future MG vaccine evaluation trials in 12-wk-old layer pullets.

Nota de investigación- Desarrollo y evaluación del modelo de desafío para Mycoplasma gallisepticum en pollitas de postura. Los fabricantes de vacunas vivas modificadas contra Mycoplasma gallisepticum (MG) suelen recomendar una sola aplicación a las ocho semanas de edad. Esto hace que las pollitas de postura de 12 a 16 semanas de edad sean adecuadas para estudios de desafío destinados a evaluar estas vacunas. Se han reportado numerosos modelos de desafío en diferentes especies y edades de aves de corral. Sin embargo, no existe un modelo de desafío establecido para pollitas de postura de esta edad. El objetivo de este estudio fue desarrollar un modelo de desafío adecuado en pollitas ponedoras de 12 semanas de edad. Se utilizó la cepa Rlow de Mycoplasma gallisepticum como cepa de desafío y se evaluó su capacidad para inducir signos clínicos y lesiones en pollitas ponedoras Hy-Line W-36 de 12 semanas de edad. Tres dosis diferentes (baja, 7.95 × 104 unidades de cambio de color [CCU]/ave; media, 7.95 × 106 CCU/ave; y alta, 7.95 × 108 CCU/ave) a través de tres rutas diferentes (gota en el ojo, aerosol con gota fina e infección por contacto) se compararon y evaluaron utilizando diferentes parámetros. A los 14 días posteriores al desafío, no hubo mortalidades en ninguno de los grupos durante todo el estudio. Las pollitas de postura expuestas directamente a la dosis alta a través de la ruta de aerosol con gota fina mostraron los resultados más claros y consistentes (signos clínicos, PCR cuantitativa en tiempo real [qPCR] positiva, seroconversión, puntuación de lesiones en los sacos aéreos y cambios histopatológicos de la mucosa traqueal). Las dosis de desafío medias y bajas aplicadas mediante aerosol con gota fina o gota en el ojo no mostraron resultados consistentes. La cepa Rlow pudo propagarse a las aves infectadas por contacto, como lo confirmaron los resultados positivos de qPCR; sin embargo, ninguna de las aves infectadas por contacto mostró signos clínicos o lesiones macroscópicas o microscópicas. Estos resultados sugieren que una dosis alta (7.95 × 108 CCU/ave) administrada a través de una ruta de aerosol con gota fina es el modelo de elección en cualquier ensayo futuro de evaluación de vacunas para M. gallisepticum en pollitas de postura de 12 semanas de edad.

Functional surface expression of immunoglobulin cleavage systems in a candidate Mycoplasma vaccine chassis.

The Mycoplasma Immunoglobulin Binding/Protease (MIB-MIP) system is a candidate 'virulence factor present in multiple pathogenic species of the Mollicutes, including the fast-growing species Mycoplasma feriruminatoris. The MIB-MIP system cleaves the heavy chain of host immunoglobulins, hence affecting antigen-antibody interactions and potentially facilitating immune evasion. In this work, using -omics technologies and 5'RACE, we show that the four copies of the M. feriruminatoris MIB-MIP system have different expression levels and are transcribed as operons controlled by four different promoters. Individual MIB-MIP gene pairs of M. feriruminatoris and other Mollicutes were introduced in an engineered M. feriruminatoris strain devoid of MIB-MIP genes and were tested for their functionality using newly developed oriC-based plasmids. The two proteins are functionally expressed at the surface of M. feriruminatoris, which confirms the possibility to display large membrane-associated proteins in this bacterium. However, functional expression of heterologous MIB-MIP systems introduced in this engineered strain from phylogenetically distant porcine Mollicutes like Mesomycoplasma hyorhinis or Mesomycoplasma hyopneumoniae could not be achieved. Finally, since M. feriruminatoris is a candidate for biomedical applications such as drug delivery, we confirmed its safety in vivo in domestic goats, which are the closest livestock relatives to its native host the Alpine ibex.

Molecular prevalence and phylogenetic analysis of hemotropic Mycoplasma species in cats in different regions of Iran.

BACKGROUND: Hemotropic Mycoplasma species (hemoplasmas) cause hemolytic anemia in cats worldwide and are recognized as emerging zoonotic pathogens. There is no comprehensive study on the prevalence and species diversity of hemoplasmas in domestic cat populations in different regions in Iran. Thus, the aims of the present study were to provide data on the prevalence and molecular characterization of hemotropic Mycoplasma species in apparently healthy cats from six Iranian provinces with different climates. In addition, potential risk factors associated with hemoplasmosis in cats were assessed. RESULTS: Mycoplasma spp. DNA was detected in the blood of 56 / 361 cats (15.5%) using genus-specific PCR. Further examinations with species-specific PCR and Sanger sequencing showed that 38 cats (10.5%) tested positive for Candidatus Mycoplasma haemominutum (CMhm), 8 cats (2.2%) tested positive for Mycoplasma haemofelis (Mhf), and 2 cats (0.6%) tested positive for Candidatus Mycoplasma turicensis (CMt). Co-infection with CMhm, and Mhf was observed in 7 cats (1.9%). One cat (0.3%) showed mixed infection with CMhm, Mhf, and CMt. There were statistically significant relationships between Mycoplasma positivity and being female, living in shelter (cattery), and being over 3 years old (P < 0.05). No significant association was observed for the cat breed and sampling localities. CONCLUSIONS: Current study findings revealed that hemoplasma infections are common among Iran cat populations. Considering the impact of such emerging zoonotic pathogens on the One Health, routine screenings, increasing public awareness, effective control, and prophylactic strategies for minimizing infection in cats and subsequently in human are strongly recommended.

Analysis of the Spectrum of Common Pathogens and the Resistance of Mycoplasma in Sialidase-Positive Bacterial Vaginosis.

BACKGROUND: The objective of this study is to understand the characteristics of the common spectrum of pathogen and the resistance of Mycoplasma in Sialidase-positive bacterial vaginosis. METHODS: The vaginal secretion specimens collected from August 2018 to October 2018 for the analysis of bacterial vaginosis (BV) were subjected to various techniques. These included routine leukorrhea examination, bacterial vaginosis sialidase testing, routine culture for common pathogens, mass spectrometry identification, and Mycoplasma resistance testing. RESULTS: A total of 238 patients with BV were identified. The cleanliness grading was mostly clean (+) and clean (2+), accounting for 38.24% and 30.67%, respectively. The bacterial vaginosis test for vaginal secretions showed leukocyte esterase positivity in 220 cases, resulting in a positivity rate of 92.44%. The spectrum of routine culture was analyzed and divided into four groups: A, B, C, and D. Group A consisted of Candidal vaginitis (13.45%); group B consisted of Gardnerella vaginalis vaginitis (32.77%); group C consisted of gram-negative bacillus vaginitis (46.22%); and group D consisted of Streptococcus agalactiae vaginitis (7.56%). The identification and antimicrobial susceptibility testing results for Mycoplasma showed a high detection rate of BV, with a positivity rate of 86.13%. There was a high sensitivity to tetracyclines for Ureaplasma urealyticum and Mycoplasma hominis, but a high resistance to macrolides and quinolones. CONCLUSIONS: Bacterial vaginosis existed in various complex forms, including Candida, Gardnerella vaginalis, Gram-negative bacillus, and Streptococcus agalactiae types. Moreover, there was an increasing trend of multi-drug resistance in Mycoplasma hominis. Therefore, it is crucial to pay attention to this condition and make accurate judgments based on the etiological characteristics and common antimicrobial susceptibility tests. This will enable the implementation of effective therapeutic interventions.

Lactobacillus salivarius ameliorates Mycoplasma gallisepticum-induced inflammation via the JAK/STAT signaling pathway involving respiratory microbiota and metabolites.

Mycoplasma gallisepticum (MG) can cause chronic respiratory disease (CRD) in chickens, which has a significant negative economic impact on the global poultry sector. Respiratory flora is the guardian of respiratory health, and its disorder is closely related to respiratory immunity and respiratory diseases. As a common probiotic in the chicken respiratory tract, Lactobacillus salivarius (L. salivarius) has potential antioxidant, growth performance enhancing, and anti-immunosuppressive properties. However, the specific mechanism through which L. salivarius protects against MG infection has not yet been thoroughly examined. This study intends to investigate whether L. salivarius could reduce MG-induced tracheal inflammation by modulating the respiratory microbiota and metabolites. The results indicated that L. salivarius reduced MG colonization significantly and alleviated the anomalous morphological changes by using the MG-infection model. L. salivarius also reduced the level of Th1 cell cytokines, increased the level of Th2 cell cytokines, and ameliorated immune imbalance during MG infection. In addition, L. salivarius improved the mucosal barrier, heightened immune function, and suppressed the Janus kinase/Signal transducer, and activator of transcription (JAK/STAT) signaling pathway. Notably, MG infection changed the composition of the respiratory microbiota and metabolites, and L. salivarius therapy partially reversed the aberrant respiratory microbiota and metabolite composition. Our results highlighted that these findings demonstrated that L. salivarius played a role in MG-mediated inflammatory damage and demonstrated that L. salivarius, by altering the respiratory microbiota and metabolites, could successfully prevent MG-induced inflammatory injury in chicken trachea.

A novel high sensitive, specificity duplex enzyme-activated differentiating probes PCR method for the SNP detection and differentiation of MS-H vaccine strains from wild-type Mycoplasma synoviae strains.

Mycoplasma synoviae (MS) is a contagious pathogen that poses a significant threat to the poultry industry. Detection plays an important role in the prevention and control of MS, particularly in differentiating between wild-type MS and live attenuated vaccine strains for vaccination selection and culling of animals with wild-type only. The live attenuated ts+ vaccine strain MS-H is recognized as the most effective and widely used vaccine. In this study, we have developed a method called double enzyme-activated differentiation probes PCR (DEA-probes PCR) for the differentiation of MS-H vaccine strain from wild-type strain by targeting the single nucleotide polymorphism (SNP) of the 367th nucleotide in the Obg gene sequence. We developed 2 modified probes with the ribonucleotide insert. When the probe perfectly complements with the target, the ribonuclease H2 (RNase H2) will cleave the ribonucleotide, resulting in the generation of fluorescent signal. With a detection limit of 5.8 copies/µL, the DEA-probes PCR method demonstrates 100% specificity in distinguishing wild-type MS from MS-H strains in 1 h. The method demonstrated great performance in real application of 100 superior palate cleft swab samples from chickens in poultry farms. Twenty-eight samples were detected as MS positive, consistent with the results of the Chinese industry standard method. Additionally, our method was able to distinguish 19 wild-type MS strains from 9 MS-H vaccine strains. The DEA-probes PCR method is rapid, specific and sensitive for SNP detection, overcoming the misidentification in MS detection and differentiation. It can be also applied to the differentiation of infected from vaccinated animals (DIVA) for other pathogens.