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Waterfowl-specific mycoplasmas cause significant economic losses worldwide. However, only limited resources are available for the specific detection of three such bacteria, Mycoplasma anatis, M. anseris and M. cloacale. We developed species-specific TaqMan assays and tested their reliability across 20 strains of the respective target species as well as 84 non-target avian bacterial strains. Furthermore, we analysed 32 clinical DNA samples and compared the results with those of previously published conventional PCRs. The TaqMan assays showed 100% specificity and very high sensitivity, enabling the detection of target DNA as low as either 10 or 100 copies/μl concentration, depending on the assay. Importantly, we found that while the here developed TaqMan assays are reliable for species-specific detection of M. anatis, the previously published conventional PCR assay may give false positive results. In conclusion, the new assays are reliable, sensitive and suitable for clinical diagnostics of the target species., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Nemesházi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Mycoplasma hyorhinis is an emerging swine pathogen bacterium causing polyserositis and polyarthritis in weaners and finishers. The pathogen is distributed world-wide, generating significant economic losses. No commercially available vaccine is available in Europe. Therefore, besides improving the housing conditions for prevention, antimicrobial therapy of the diseased animals is the only option to control the infection. Our aim was to determine the minimal inhibitory concentrations (MIC) of ten antimicrobials potentially used against M. hyorhinis infection. The antibiotic susceptibility of 76 M. hyorhinis isolates from Belgium, Germany, Hungary, Italy and Poland collected between 2019 and 2021 was determined by broth micro-dilution method and mismatch amplification mutation assay (MAMA). Low concentrations of tiamulin (MIC90 0.312 μg/ml), doxycycline (MIC90 0.078 μg/ml), oxytetracycline (MIC90 0.25 μg/ml), florfenicol (MIC90 2 μg/ml) and moderate concentrations of enrofloxacin (MIC90 1.25 μg/ml) inhibited the growth of the isolates. For the tested macrolides and lincomycin, a bimodal MIC pattern was observed (MIC90 >64 μg/ml for lincomycin, tulathromycin, tylosin and tilmicosin and 5 μg/ml for tylvalosin). The results of the MAMA assay were in line with the conventional method with three exceptions. Based on our statistical analyses, significant differences in MIC values of tiamulin and doxycycline were observed between certain countries. Our results show various levels of antimicrobial susceptibility among M. hyorhinis isolates to the tested antibiotics. The data underline the importance of susceptibility monitoring on pan-European level and provides essential information for proper antibiotic choice in therapy., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: U. Klein and W. Depondt are the employees of Huvepharma® NV, the producer of various veterinary antibiotic products. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Mycoplasma synoviae infection occurs worldwide, leading to considerable economic losses in the chicken and turkey industry due to infectious synovitis, respiratory diseases and eggshell apex abnormalities. Control programs against M. synoviae infection are based on eradication, vaccination and medication with antimicrobial agents. Prudent use of antibiotics can be improved greatly by the determination of antibiotic susceptibility prior to the treatment. However, the conventional broth or agar microdilution is very labor-intensive and time-consuming method. Thus, there is an increasing need for rapid antimicrobial susceptibility tests in order to guide antibiotic therapy more effectively. The aim of this study was to develop mismatch amplification mutation assays (MAMAs) to detect resistance-associated mutations in M. synoviae. M. synoviae strains with previously determined minimal inhibitory concentrations (MICs) and whole genomes (n = 92) were used for target selection and assay specification. For the evaluation of the developed assays, 20 clinical samples and an additional 20 M. synoviae isolates derived from these specimens were also included in this study. MIC values of these 20 isolates were determined by broth microdilution method. Five MAMAs were designed to identify elevated MICs of fluoroquinolones, while three MAMAs were developed to detect decreased susceptibility to macrolides and lincomycin. The sensitivity of the MAMA tests varied between 102-104 template copy number/reaction depending on the assay. Clinical samples showed identical genotype calls with the M. synoviae isolates derived from the corresponding specimens in each case. Supporting the results of conventional in vitro sensitivity tests, our approach provides a feasible tool for diagnostics. Rapidity, robustness and cost-effectiveness are powerful advantages of the developed assays. Supporting prudent antibiotic usage instead of empirical treatment, the use of this method can reduce significantly the economic impact of M. synoviae in the poultry industry and decrease bacterial resistance-related public health concerns., Competing Interests: The authors have declared that no competing interests exist.

Mycoplasma anatis, M. anseris, M. cloacale and M. sp. 1220 colonise geese and ducks, and could be associated with infections of avian respiratory and nervous systems, cause mild to severe inflammation of cloaca and genital tracts, and embryo lethality. Co-occurrence of these Mycoplasma species in waterfowl is frequently detected and the identification of these mycoplasmas to the species level at a regular microbiology laboratory is difficult due to their similar morphological, cultural and biochemical properties. Moreover, species differentiation is only possible based on the sequence analysis of the product of a genus-specific PCR assay. Therefore, the aim of the current study was to develop an effective and robust method for the identification of these species in avian clinical specimens. Polymerase chain reaction (PCR) assays using species-specific primers, which target housekeeping genes in order to identify these species, were designed in the present study. The developed PCR assays can precisely identify these four mycoplasmas to the species level directly from DNA samples extracted from clinical specimens, and no cross-amplification was observed among these species and with other well-known avian mycoplasmas. The average sensitivity of the assays was 101-102 genomic equivalents per reaction. These conventional PCR assays can be run simultaneously at the same PCR cycling program, and the species can be differentiated directly (without sequence analysis) by gel electrophoresis due to the specific sizes of the amplicons. In conclusion, the presented species-specific assays were found to be suitable for routine use at regular veterinary diagnostic laboratories and promote the rapid, simple and cost-effective differentiation of these waterfowl Mycoplasma species., Competing Interests: The TOLL 96 Kft. provided support in the form of salaries for MJK. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Mycoplasma hyopneumoniae infections are responsible for significant economic losses in the swine industry. Commercially available vaccines are not able to inhibit the colonisation of the respiratory tract by M. hyopneumoniae absolutely, therefore vaccination can be completed with antibiotic treatment to moderate clinical signs and improve performances of the animals. Antibiotic susceptibility testing of M. hyopneumoniae is time-consuming and complicated; therefore, it is not accomplished routinely. The aim of this study was to determine the in vitro susceptibility to 15 different antibiotics of M. hyopneumoniae isolates originating from Hungarian slaughterhouses and to examine single-nucleotide polymorphisms (SNPs) in genes affecting susceptibility to antimicrobials. Minimum inhibitory concentration (MIC) values of the examined antibiotics against 44 M. hyopneumoniae strains were determined by microbroth dilution method. While all of the tested antibiotics were effective against the majority of the studied strains, high MIC values of fluoroquinolones (enrofloxacin 2.5 μg/ml; marbofloxacin 5 μg/ml) were observed against one strain (MycSu17) and extremely high MIC values of macrolides and lincomycin (tilmicosin, tulathromycin and lincomycin >64 μg/ml; gamithromycin 64 μg/ml; tylosin 32 μg/ml and tylvalosin 2 μg/ml) were determined against another, outlier strain (MycSu18). Amino acid changes in the genes gyrA (Gly81Ala; Ala83Val; Glu87Gly, according to Escherichia coli numbering) and parC (Ser80Phe/Tyr; Asp84Asn) correlated with decreased antibiotic susceptibility to fluoroquinolones and a SNP in the nucleotide sequence of the 23S rRNA (A2059G) was found to be associated with increased MIC values of macrolides. The correlation was more remarkable when final MIC values were evaluated. This study presented the antibiotic susceptibility profiles of M. hyopneumoniae strains circulating in the Central European region, demonstrating the high in vitro efficacy of the tested agents. The observed high MIC values correlated with the SNPs in the examined regions and support the relevance of susceptibility testing and directed antibiotic therapy., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: The SCG Diagnosztika Kft. provided support in the form of salaries for KK. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Mycoplasma synoviae is an economically significant pathogen in the poultry industry, inducing respiratory disease and infectious synovitis in chickens and turkeys, and eggshell apex abnormality in chickens. Eradication, medication and vaccination are the options for controlling M. synoviae infection. Currently there are two commercial, live, attenuated vaccines available against M. synoviae: the temperature sensitive MS-H vaccine strain and the NAD independent MS1 vaccine strain. Differentiation of vaccine strains from field isolates is essential during vaccination and eradication programs. The present study provides melt-curve and agarose gel based mismatch amplification mutation assays (MAMA) to discriminate the MS1 vaccine strain from the MS-H vaccine strain and wild-type M. synoviae isolates. The assays are based on the A/C single nucleotide polymorphism at nt11 of a HIT family protein coding gene. The melt- and agarose-MAMAs reliably distinguish the MS1 vaccine strain genotype from the MS-H vaccine strain and wild-type M. synoviae isolate genotype from 102 template number/DNA sample. No cross-reactions with other avian Mycoplasma species were observed. The assays can be performed directly on clinical samples and they can be run simultaneously with the previously described MAMAs designed for the discrimination of the MS-H vaccine strain. The developed assays are applicable in laboratories with limited facilities and promote the rapid, simple and cost effective differentiation of the MS1 vaccine strain.

Mycoplasma synoviae infection in chickens and turkeys can cause respiratory disease, infectious synovitis and eggshell apex abnormality; thus it is an economically important pathogen. Control of M. synoviae infection comprises eradication, medication or vaccination. The differentiation of the temperature sensitive (ts+) MS-H vaccine strain from field isolates is crucial during vaccination programs. Melt-curve and agarose gel based mismatch amplification mutation assays (MAMA) are provided in the present study to distinguish between the ts+ MS-H vaccine strain, its non-temperature sensitive re-isolates and wild-type M. synoviae isolates based on the single nucleotide polymorphisms at nt367 and nt629 of the obg gene. The two melt-MAMAs and the two agarose-MAMAs clearly distinguish the ts+ MS-H vaccine strain genotype from its non-temperature sensitive re-isolate genotype and wild-type M. synoviae isolate genotype, and no cross-reactions with other Mycoplasma species infecting birds occur. The sensitivity of the melt-MAMAs and agarose-MAMAs was 103 and 104 copy numbers, respectively. The assays can be performed directly on clinical samples and they can be run simultaneously at the same annealing temperature. The assays can be performed in laboratories with limited facilities, using basic real-time PCR machine or conventional thermocycler coupled with agarose gel electrophoresis. The advantages of the described assays compared with previously used methods are simplicity, sufficient sensitivity, time and cost effectiveness and specificity.

Background: Coxiella burnetii, the etiologic agent of Q fever, is a highly infectious zoonotic bacterium. Genetic information about the strains of this worldwide distributed agent circulating on the African continent is limited. The aim of the present study was the genetic characterization of C. burnetii DNA samples detected in ticks collected from Ethiopian cattle and their comparison with other genotypes found previously in other parts of the world., Methodology/principal Findings: A total of 296 tick samples were screened by real-time PCR targeting the IS1111 region of C. burnetii genome and from the 32 positive samples, 8 cases with sufficient C. burnetii DNA load (Amblyomma cohaerens, n = 6; A. variegatum, n = 2) were characterized by multispacer sequence typing (MST) and multiple-locus variable-number tandem repeat analysis (MLVA). One novel sequence type (ST), the proposed ST52, was identified by MST. The MLVA-6 discriminated the proposed ST52 into two newly identified MLVA genotypes: type 24 or AH was detected in both Amblyomma species while type 26 or AI was found only in A. cohaerens., Conclusions/significance: Both the MST and MLVA genotypes of the present work are closely related to previously described genotypes found primarily in cattle samples from different parts of the globe. This finding is congruent with the source hosts of the analyzed Ethiopian ticks, as these were also collected from cattle. The present study provides genotype information of C. burnetii from this seldom studied East-African region as well as further evidence for the presumed host-specific adaptation of this agent.

Background: The majority of vector-borne infections occur in the tropics, including Africa, but molecular eco-epidemiological studies are seldom reported from these regions. In particular, most previously published data on ticks in Ethiopia focus on species distribution, and only a few molecular studies on the occurrence of tick-borne pathogens or on ecological factors influencing these. The present study was undertaken to evaluate, if ticks collected from cattle in different Ethiopian biotopes harbour (had access to) different pathogens., Methods: In South-Western Ethiopia 1032 hard ticks were removed from cattle grazing in three kinds of tick biotopes. DNA was individually extracted from one specimen of both sexes of each tick species per cattle. These samples were molecularly analysed for the presence of tick-borne pathogens., Results: Amblyomma variegatum was significantly more abundant on mid highland, than on moist highland. Rhipicephalus decoloratus was absent from savannah lowland, where virtually only A. cohaerens was found. In the ticks Coxiella burnetii had the highest prevalence on savannah lowland. PCR positivity to Theileria spp. did not appear to depend on the biotope, but some genotypes were unique to certain tick species. Significantly more A. variegatum specimens were rickettsia-positive, than those of other tick species. The presence of rickettsiae (R. africae) appeared to be associated with mid highland in case of A. variegatum and A. cohaerens. The low level of haemoplasma positivity seemed to be equally distributed among the tick species, but was restricted to one biotope type., Conclusions: The tick biotope, in which cattle are grazed, will influence not only the tick burden of these hosts, but also the spectrum of pathogens in their ticks. Thus, the presence of pathogens with alternative (non-tick-borne) transmission routes, with transstadial or with transovarial transmission by ticks appeared to be associated with the biotope type, with the tick species, or both, respectively.

Single nucleotide polymorphisms (SNPs) are abundant in genomes of all species and biologically informative markers extensively used across broad scientific disciplines. Newly identified SNP markers are publicly available at an ever-increasing rate due to advancements in sequencing technologies. Efficient, cost-effective SNP genotyping methods to screen sample populations are in great demand in well-equipped laboratories, but also in developing world situations. Dual Probe TaqMan assays are robust but can be cost-prohibitive and require specialized equipment. The Mismatch Amplification Mutation Assay, coupled with melt analysis (Melt-MAMA), is flexible, efficient and cost-effective. However, Melt-MAMA traditionally suffers from high rates of assay design failures and knowledge gaps on assay robustness and sensitivity. In this study, we identified strategies that improved the success of Melt-MAMA. We examined the performance of 185 Melt-MAMAs across eight different pathogens using various optimization parameters. We evaluated the effects of genome size and %GC content on assay development. When used collectively, specific strategies markedly improved the rate of successful assays at the first design attempt from ~50% to ~80%. We observed that Melt-MAMA accurately genotypes across a broad DNA range (~100 ng to ~0.1 pg). Genomic size and %GC content influence the rate of successful assay design in an independent manner. Finally, we demonstrated the versatility of these assays by the creation of a duplex Melt-MAMA real-time PCR (two SNPs) and conversion to a size-based genotyping system, which uses agarose gel electrophoresis. Melt-MAMA is comparable to Dual Probe TaqMan assays in terms of design success rate and accuracy. Although sensitivity is less robust than Dual Probe TaqMan assays, Melt-MAMA is superior in terms of cost-effectiveness, speed of development and versatility. We detail the parameters most important for the successful application of Melt-MAMA, which should prove useful to the wider scientific community.