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Mycoplasma gallisepticum (MG) is an important pathogen of the poultry industry able to cause chronic respiratory disease in chickens and infectious sinusitis in turkeys. Despite the application of biosecurity measures and the availability of vaccines for chickens, monitoring systems routinely applied for MG detection are still essential for infection control. Pathogen isolation is time-consuming and not suitable for rapid detection, albeit it is a compulsory step for genetic typing and antimicrobial susceptibility evaluation of single strains. The mgc2 gene is a species-specific molecular target adopted by most of the PCR protocols available for MG diagnosis, which are also included in the WOAH Terrestrial Manual. We describe the case of an atypical MG strain, isolated in 2019 from Italian turkeys, characterized by an mgc2 sequence not detectable by common endpoint PCR primers. Considering the potential risk of false negative results during diagnostic screenings with the endpoint protocol, the authors propose an alternative mgc2 PCR endpoint protocol, named MG600, which should be considered as a further diagnostic tool., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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.

ABSTRACT Mycoplasma iowae, a potential re-emerging avian pathogen mainly affecting turkeys, has been reported from many parts of the world. Poor hatchability, embryonic death, joint and skeletal abnormalities, poor ossification, runting-stunting, poor feathering and airsacculitis may be observed in infected flocks. The reduction of the severity of clinical signs and short-term control of M. iowae are performed by antibiotic treatment. However, M. iowae develops resistance more rapidly and is considered to be more resistant to antimicrobials than other avian pathogenic mycoplasmas. The aim of the present study was to determine the in vitro susceptibility of 101 M. iowae isolates and strains to ten clinically important antimicrobial agents, and to analyse and compare the susceptibility patterns of isolates of various origins and from a wide time-period. The examined reference strains showed high susceptibility to all antimicrobials except for spectinomycin. Low concentrations of tiamulin, florfenicol and oxytetracycline inhibited the growth of the clinical isolates. Nevertheless, slow tendency of increasing minimum inhibitory concentration (MIC) values was observed over time in the case of the above mentioned agents, while MIC values of enrofloxacin showed relatively rapid changes. Spiramycin, erythromycin, tilmicosin, tylosin, lincomycin and spectinomycin did not inhibit the bacterial growth in most of the cases. Isolates originating from captive game birds showed similar susceptibility profiles to isolates from industrial turkey hosts. The widely detected low susceptibility of M. iowae isolates to macrolides, lincomycin and spectinomycin, and the increase of MIC values of frequently used antimicrobials against this pathogen, emphasize the importance of targeted antibiotic therapy.RESEARCH HIGHLIGHTSAntimicrobial susceptibilities of 101 Mycoplasma iowae isolates were determined.Minimum inhibitory concentrations were determined by broth micro-dilution method.Tiamulin, oxytetracycline and florfenicol showed low MIC values.Isolates rapidly adapted to antimicrobial pressure.

Mycoplasma gallisepticum and Mycoplasma synoviae are bacterial pathogens that cause disease in poultry, adversely affecting their health and welfare, and are a financial burden on producers. This manuscript describes the results of the MycoPath project that is the first international antimicrobial susceptibility programme for mycoplasma pathogens isolated from poultry. Improved comparative analysis of minimal inhibitory concentration (MIC) results from participating countries was facilitated by using one laboratory determining all MICs. Chicken and turkey isolates were obtained from France, Germany, Great Britain, Hungary, Italy and Spain during 2014-2016. One isolate per farm was retained. The MIC of seven antimicrobial agents was determined using a broth microdilution method, with Friis Medium ( M. gallisepticum ) or Modified Chanock's Medium ( M. synoviae ). Of the 222 isolates recovered, 82 were M. gallisepticum and 130 were M. synoviae. M. gallisepticum MIC 50/90 values were 0.12/0.5, 2/8, 0.5/4, 0.12/>64, 0.008/0.062, 0.008/32, 0.062/4 mg/l for doxycycline, enrofloxacin, oxytetracycline, spiramycin, tiamulin, tilmicosin and tylosin, respectively. For M. synoviae , the values were 0.5/1, 8/16, 0.5/1, 0.5/8, 0.25/0.5, 0.062/2 and 0.062/16 mg/l respectively . A bimodal MIC distribution for the fluoroquinolone (enrofloxacin) and the macrolides (spiramycin, tilmicosin and tylosin) indicate that both species have sub-populations that are less susceptible in vitro to those antimicrobials. Some differences in susceptibilities were observed according to host species, Mycoplasma species, and country of origin. This study provides a baseline of novel data for future monitoring of antimicrobial resistance in poultry Mycoplasma species. Additionally, this information will facilitate the selection of the antimicrobial agents most likely to be effective, thus ensuring their minimal use with targeted and correct therapeutic treatments. Highlights First large-scale pan-European collection of representative Mg and Ms isolates.MIC values assessed in central laboratory for Mg and Ms from chickens and turkeys.Range of MIC values for 82 Mg and 130 Ms isolates to seven licenced antibiotics shown.Data can be used to help determine Mg and Ms veterinary-specific breakpoints.

Mycoplasma gallisepticum causes respiratory diseases and reproduction disorders in turkeys and chickens. The infection has considerable economic impact due to reduced meat and egg production. Because elimination programmes are not feasible in a large number of poultry farms, vaccination remains the only effective measure of disease control. Differentiating vaccine strains from field isolates is necessary in the control of vaccination programmes and diagnostics. The aim of this study was to develop a polymerase chain reaction based mismatch amplification mutation assay (MAMA) for the discrimination of K vaccine strain (K 5831, Vaxxinova Japan K.K.). After determining the whole genome sequence of the K strain, primers were designed to detect seven different vaccine-specific single nucleotide polymorphisms. After evaluating preliminary results, the MAMA-K-fruA test detecting a single guanine-adenine substitution within the fruA gene (G88A) was found to be the most applicable assay to distinguish the K vaccine strain from field isolates. The detected K strain-specific single nucleotide polymorphism showed genetic stability after serial passage in vitro, but this stability test should still be evaluated in vivo as well, investigating a large number of K strain re-isolates. The MAMA-K-fruA assay was tested on a total of 280 culture and field samples. The designed assay had 10 2 and 10 3 template copy number/µl sensitivity in melt-curve analysis based and agarose-gel based assays, respectively, and showed no cross reaction with other avian Mycoplasma species. The new MAMA provides a time- and cost-effective molecular tool for the control of vaccination programmes and for diagnostics.

Mycoplasma gallisepticum is among the most economically significant mycoplasmas causing production losses in poultry. Seven melt-curve and agarose gel-based mismatch amplification mutation assays (MAMAs) and one PCR are provided in the present study to distinguish the M. gallisepticum vaccine strains and field isolates based on mutations in the crmA , gapA , lpd, plpA , potC , glpK , and hlp2 genes. A total of 239 samples ( M. gallisepticum vaccine and type strains, pure cultures, and clinical samples) originating from 16 countries and from at least eight avian species were submitted to the presented assays for validation or in blind tests. A comparison of the data from 126 samples (including sequences available at GenBank) examined by the developed assays and a recently developed multilocus sequence typing assay showed congruent typing results. The sensitivity of the melt-MAMA assays varied between 10 1 and 10 4 M. gallisepticum template copies/reaction, while that of the agarose-MAMAs ranged from 10 3 to 10 5 template copies/reaction, and no cross-reactions occurred with other Mycoplasma species colonizing birds. The presented assays are also suitable for discriminating multiple strains in a single sample. The developed assays enable the differentiation of live vaccine strains by targeting two or three markers/vaccine strain; however, considering the high variability of the species, the combined use of all assays is recommended. The suggested combination provides a reliable tool for routine diagnostics due to the sensitivity and specificity of the assays, and they can be performed directly on clinical samples and in laboratories with basic PCR equipment., (Copyright © 2019 American Society for Microbiology.)

Background: Mycoplasma synoviae (MS) is a major poultry pathogen which causes severe economic losses in all the productive sectors. The prevalence of MS in European countries has increased in the last few years, leading to greater attention to the available methods to prevent its spread. The main strategy currently applied for its containment is the development and maintenance of MS-free breeder flocks. A live MS vaccine (MS-H) obtained by mutagenizing an Australian field strain has recently been introduced in Italy. The aim of the present study was to evaluate the vaccine behaviour in broiler breeder groups at different production stages and the effectiveness of the available laboratory tests in discriminating the MS-H from a field strain., Results: The vaccine diffused extensively through the population, shown by the wide serological response (over 80% of positive samples in RSA and 85% in ELISA), the high serological titres, the positivity of all the tracheal samples collected during the production phase by MS PCR and the positivity by cultivation from tracheal swabs at the end-point (55 weeks after vaccination). In contrast, only one swab from a sternal bursa was positive in MS PCR, while all the joint and oviduct samples were negative. There was no evidence of vertical transmission. Different genotyping techniques were used to achieve a clear classification of the MS positive samples. The vlhA and the obg gene analysis showed that most of the strains were homologous with the vaccine, but some ambiguous samples were further investigated with the multi locus sequence typing (MLST) scheme which confirmed the homology., Conclusions: The development of a multi-technique approach to monitor vaccinated avian flocks, based both on serological and biomolecular methods, is advised as well as the use of effective genotyping techniques to analyse the MS strains circulating in high densely populated poultry areas.

Control of one of the most important avian mycoplasmas, Mycoplasma synoviae, and tracing the spread of the infection can be challenging as the pathogen is transmissible by both horizontal and vertical routes, and it can be disseminated through long distances via the hatching eggs, day-old chicks or pullets during intensive international trade. Genetic information provided by molecular typing methods support control programmes and epizootiologic studies. The aims of the present study were to develop a multi-locus variable number of tandem-repeats analysis (MLVA) method for the typing of M. synoviae isolates and to evaluate the currently used molecular typing methods which are applicable directly on clinical samples. Tandem repeat (TR) regions were selected from the whole genome sequence of the M. synoviae type strain (WVU1853) to characterise the genetic diversity of 86 M. synoviae strains originating from 15 countries. The strains were also submitted to multi-locus sequence typing (MLST) assays, vlhA gene sequence analysis and to assays designed to differentiate live vaccine strains from field strains. The developed MLVA involves the examination of seven TR regions and provides similar genetic resolution as the tested MLST assays by identifying 35 genotypes among the tested strains. Differentiation of the live vaccine strains from field strains was also successful with the developed assay. The provided MLVA method proved to be a highly discriminative, rapid and cost-effective alternative typing technique for the genetic characterisation of M. synoviae and it is also suitable for the complementation of live vaccine strain differentiating assays in ambiguous cases., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Mycoplasmas of humans and animals are usually associated with respiratory, autoimmune, genital and joint diseases. Human mycoplasmas have also been known to affect the brain. Severe central nervous system (CNS) diseases, such as encephalitis, have been linked to Mycoplasma pneumoniae and ureaplasma infections. Less well known is the sheep and goat pathogen, Mycoplasma agalactiae, which has been found in large quantities in the brain where it may be responsible for non-purulent encephalitis as well as ataxia in young animals. Experimental intra-mammary infections of sheep with this mycoplasma have resulted in histopathological changes in the CNS. The cattle pathogen, M. bovis, has been reported occasionally in the brains of calves and adult cattle showing a range of histopathological lesions including abscesses and fibrinous meningitis. Two avian pathogens, M. gallisepticum and M. synoviae have been isolated from the brains of poultry showing meningeal vasculitis and encephalitis. There have been no reported detections of two other avian pathogens, M. meleagridis or M. iowae in the CNS. Over the last few decades, mycoplasmas have been isolated from the brains of sea mammals dying in large numbers in the North Sea although it was concluded that their role may be secondary to underlying viral disease. Finally, evidence has been advanced that certain Spiroplasma species may have a role in the development of the transmissible spongiform encephalopathies (TSE). Invasion of the brain by mycoplasmas may be as a result of direct entry following damage to the inner ear as seen with M. bovis or across the blood brain barrier by mechanisms as yet uncertain., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Mycoplasma synoviae (Ms) is considered to be an economically important poultry pathogen. Although the full economic costs of infection in layer chickens are still under debate, the prevalence of Ms is known to be high in some countries and earlier reports have shown a correlation between infection and Eggshell Apex Abnormality (EAA). This work is a continuation of an earlier study of a clinical case of EAA on a layer hen farm where the presence of two different strains of Ms, based on the sequence of the 5' end of the vlhA gene, was demonstrated. Both strains could be detected in the trachea but only one (designated strain PASC8) appeared able to colonize the oviduct, while the other (designated TRACH) was not found in the oviduct and has not been related to EAA. The PASC8 partial vlhA gene sequence differs from that of the TRACH in having a 39 nucleotide deletion in the proline rich region and three point mutations in the RIII region. Based on this information an experimental infection was performed in SPF chickens using groups infected with either the PASC8 or the TRACH strain and a non-infected control group. Both Ms strains were detected in the trachea of infected birds, but only the PASC8 strain was found in the oviduct. Furthermore, EAA developed only in the group infected with PASC8 strain. Compared to the control group, both strains produced an adverse impact on egg production: a decrease in the numbers laid and in their average weight (P<0.05) This work demonstrates a difference in oviduct tropism between two Ms strains and a possible relationship to the production of EAA in experimental conditions., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: The role of wild birds in the transmission and spread of mycoplasmas is not clear. Up to now different Mycoplasma species have been isolated from wild birds many of which are not considered pathogens sensu stricto for domestic flocks. This report describes the first isolation of Mycoplasma synoviae in a captive lesser flamingo (Phoeniconaias minor) held in a zoo in Italy and the laboratory investigations performed to elucidate its origin. Results showed that the strain was similar to the MS-H vaccine strain using the vlhA methods although no vaccination with this product was used in the zoo., Case Presentation: This paper describes investigations into a case in which 10 of 12 adult lesser flamingos (Phoeniconaias minor) died after having recently been moved from the Netherlands to a new zoo in Northern Italy. While most of the birds appeared to have died from the stress of movement and poor adaptation to their new environment, Mycoplasma synoviae, an important poultry pathogen in the layer and meat industry, was isolated for the first time from the trachea of one animal presenting catarrhal tracheitis and fibrinous airsacculitis. Genetic analysis of the conserved region of the vlhA was not able to differentiate the flamingo strain from the MS-H vaccine strain. However differences in the sequences of the obg gene of the flamingo and vaccine strain were detected. A test for temperature-sensitivity (ts) gave a ts (-) phenotype for the flamingo strain, in contrast to the ts (+) status of the MS-H strain. Based on this information and knowing that the flamingos were not vaccinated against M. synoviae, it is highly likely that the flamingo was infected with a genetically similar wild strain by contact with infected birds., Conclusions: This case provides evidence for the potential role of international trade of ornamental birds as a possible route of introduction of new mycoplasma strains between countries, and moreover highlight that vlhA gene sequencing was not sufficient to discriminate the wild strain isolated from the flamingo from the MS-H vaccine strain.

The mechanism responsible for acquired decreased susceptibility to macrolides (14-membered erythromycin [Ery], 16-membered tylosin [Ty] and tilmicosin [Tm]) and to lincosamides (lincomycin [Ln]) was investigated in Mycoplasma synoviae, a pathogen that causes respiratory infections and synovitis in chicken and turkey. Sequence analysis of domains II and V of the two 23S rRNA alleles and ribosomal proteins L4 and L22 was performed on 49 M. synoviae isolates, M. synoviae type strain WVU1853, and reference strain FMT showing minimal inhibitory concentrations (MICs) to Ty (≤ 0.015 to 2 μg/ml), Tm (0.03 to ≥ 8 μg/ml), and Ln (0.125 to 8 μg/ml); MICs to Ery ranged from 32 to ≥ 128 μg/ml. Our results showed that the nucleotide substitution G748A (Escherichia coli numbering) in domain II of one or both 23S rRNA alleles may account for a slight increase in MICs to Ty and Tm (up to 0.5 and 2 μg/ml, respectively). No correlation between the presence of G748A and decreased susceptibility to Ln was found. However, the presence of the point mutations A2058G or A2059G in domain V of one or both alleles of the 23S rRNAs was correlated with a more significant decrease in susceptibility to Ty (1-2 μg/ml), Tm (≥ 8 μg/ml), and Ln (≥ 8 μg/ml). All M. synoviae isolates tested had a G2057A transition in the 23S rRNAs consistent with previously described intrinsic resistance to Ery. Mutations G64E (one isolate) and Q90K/H (two isolates) were identified in the L4 and L22 proteins, respectively, but their impact on decreased susceptibility to macrolides and lincomycin was not clear.

Mycoplasma bovis is a major bovine pathogen associated with bovine respiratory disease complex and is responsible for substantial economic losses worldwide. M. bovis is also associated with other clinical presentations in cattle, including mastitis, otitis, arthritis, and reproductive disorders. To gain a better understanding of the genetic diversity of this pathogen, a multilocus sequence typing (MLST) scheme was developed and applied to the characterization of 137 M. bovis isolates from diverse geographical origins, obtained from healthy or clinically infected cattle. After in silico analysis, a final set of 7 housekeeping genes was selected (dnaA, metS, recA, tufA, atpA, rpoD, and tkt). MLST analysis demonstrated the presence of 35 different sequence types (STs) distributed in two main clonal complexes (CCs), defined at the double-locus variant level, namely, CC1, which included most of the British and German isolates, and CC2, which was a more heterogeneous and geographically distant group of isolates, including European, Asian, and Australian samples. Index of association analysis confirmed the clonal nature of the investigated M. bovis population, based on MLST data. This scheme has demonstrated high discriminatory power, with the analysis showing the presence of genetically distant and divergent clusters of isolates predominantly associated with geographical origins., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Subclinical mastitis in dairy cows is a big economic loss for farmers. The monitoring of subclinical mastitis is usually performed through Somatic Cell Count (SCC) in farm but there is the need of new diagnostic systems able to quickly identify cows affected by subclinical infections of the udder. The aim of this study was to evaluate the potential application of thermographic imaging compared to SCC and bacteriological culture for infection detection in cow affected by subclinical mastitis and possibly to discriminate between different pathogens. In this study we evaluated the udder health status of 98 Holstein Friesian dairy cows with high SCC in 4 farms. From each cow a sample of milk was collected from all the functional quarters and submitted to bacteriological culture, SCC and Mycoplasma spp. culture. A thermographic image was taken from each functional udder quarter and nipple. Pearson's correlations and Analysis of Variance were performed in order to evaluate the different diagnostic techniques. The most frequent pathogen isolated was Staphylococcus aureus followed by Coagulase Negative Staphylococci (CNS), Streptococcus uberis, Streptococcus agalactiae and others. The Somatic Cell Score (SCS) was able to discriminate (p<0.05) cows positive for a pathogen from cows negative at the bacteriological culture except for cows with infection caused by CNS. Infrared thermography was correlated to SCS (p<0.05) but was not able to discriminate between positive and negative cows. Thermographic imaging seems to be promising in evaluating the inflammation status of cows affected by subclinical mastitis but seems to have a poor diagnostic value.

Mycoplasma iowae, an occasional pathogen of turkeys, was isolated for the first time from captive grey partridges (Perdix perdix). Clinical signs including respiratory and intestinal disorder were seen in birds of all ages but mainly in those kept housed during rearing. Mortality rates averaged over 20% during the year. Treatment with antibiotics and antiparasitic drugs produced only a transient improvement in condition. The gross pathology findings included poor body growth, lack of development of the breast muscles, abnormalities in the keel development, and bone fragility. Some birds showed infraorbital sinusitis with serous or fibrinous exudates and catarrhal tracheitis, while others presented serofibrinous airsacculitis and splenomegaly. Laboratory investigations revealed pure cultures of M. iowae in the gut as well as sinus and air sacs. While other organisms such as coccidia, Trichomonas, Escherichia coli, Clostridium perfringens, and Aspergillus spp. were detected, the similarity of the disease with that seen in turkeys infected with M. iowae strongly suggests that this mycoplasma may be the primary pathogen here. The presence of M. iowae in game birds commonly released into the wild could have serious implications particularly in areas where industrial poultry farms are concentrated.

Eggshell abnormalities were seen in the apex of eggs in two of three flocks of multi-age, Hy-Line layer chickens housed on a farm in Northern Italy. Approximately 1.3% to 1.8% of eggs in one flock were affected, amounting to 300-400 eggs per day; the abnormalities resulted in a great deal of breakage and spoilage of healthy eggs. The mean weight of eggs was also reduced. Egg abnormalities in a second flock were less severe. Mycoplasma synoviae was detected in birds from both of the affected flocks by serologic, cultural, and molecular techniques, but not in a third, adjacent flock where no eggshell abnormalities were seen. Treatment with tylosin, administered in the drinking water for 5 days, resulted in an immediate improvement of eggshell quality and egg weight. There was no evidence of infectious bronchitis virus in the flocks.