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Almost two decades ago, in addition to a compulsory M. gallisepticum (Mg) monitoring programme of breeding stock based on European Union regulations, the Dutch poultry industry added national regulations to further reduce the Mg prevalence in Dutch commercial poultry. Currently, all commercial chicken and turkey flocks except broilers are monitored for Mg. All breeding flocks on a farm where one or more flocks tested Mg positive are culled. Mg positive layer pullets are channelled and layer pullets placed on Mg positive multi-age farms are vaccinated. The monitoring data obtained were analysed covering a period of 17 years. Moreover, 31 Dutch Mg isolates from the same period were analysed by multilocus sequence typing (MLST) and compared to available PubMLST data. The results show that in breeding stock the seroprevalence decreased from 1.6% to 0.0%, in commercial layers from 6.3% to 1.9%, and in meat turkeys from 17.6% to 2.4%. The MLST results showed the presence of closely related and identical sequence types (STs) within the different Dutch poultry types. Similar STs were found in Northern and Southern Europe only. The results show a fast decline in the Mg prevalence since 2001, although in layers the Mg prevalence has stabilized and suggests backyard poultry might pose a risk for commercial poultry. The need for Mg control across poultry sectors and in trade was confirmed by the similarity in STs found in different types of poultry and regions. These results from the Dutch poultry industry can be extrapolated to Mg control in general. [ABSTRACT FROM AUTHOR]

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Francisella tularensis subsp. holarctica isolates from Austria, Germany, Hungary, Italy, and Romania were placed into an existing phylogeographic framework. Isolates from Italy were assigned to phylogenetic group B.FTNF002-00; the other isolates, to group B.13. Most F. tularensis subsp. holarctica isolates from Europe belong to these 2 geographically segregated groups. [ABSTRACT FROM AUTHOR]

Thirteen Francisella tularensis strains were isolated from 22 seropositive brown hares ( Lepus europaeus) originating from different parts of Hungary, and further two from a patas monkey ( Erythrocebus patas) and vervet monkey ( Chlorocebus aethiops). The isolates were identified as F. tularensis ssp. holarctica on the basis of culture, morphological and biochemical characteristics. The identification was verified by polymerase chain reaction and the sequencing of the partial 16S rRNA gene. Utilization of carbon sources of the 15 F. tularensis strains was characterized with the Biolog system. The system was able to identify the strains already after 4 h of incubation, not only after the standard 24 h. After the analysis and comparison of the metabolic profiles of our strains with the Biolog database, it was concluded that not all carbon sources indicated in the database were utilized by our isolates. The Biolog software fails to distinguish the highly virulent F. tularensis ssp. tularensis and the moderately virulent F. tularensis ssp. holarctica but the Biolog microplates can be manually read to differentiate the two subspecies based on glycerol source utilization. As all the studied strains were unable to use glycerol, they could be identified as F. tularensis ssp. holarctica. The dendrogram based on the metabolic relationship of the strains shows that the isolates are very similar to each other, which correlates with the conservative genetic character of F. tularensis ssp. holarctica. [ABSTRACT FROM AUTHOR]

This article studies the pathology, histopathology and immunohistochemistry of the lesions induced by Francisella tularensis in European Brown hares. Francisella tularensis is the etiological agent of zoonotic disease tularemia. Immunohistochemistry is conducted for the demonstration of Francisella tularensis lipopolysaccharide antigen in tissue sections. The morphological characteristics of the bacteria are described.

Abstract Background Francisella tularensis, the causative agent of tularemia, displays subspecies-specific differences in virulence, geographic distribution, and genetic diversity. F. tularensis subsp. holarctica is widely distributed throughout the Northern Hemisphere. In Europe, F. tularensis subsp. holarctica isolates have largely been assigned to two phylogenetic groups that have specific geographic distributions. Most isolates from Western Europe are assigned to the B.Br.FTNF002-00 group, whereas most isolates from Eastern Europe are assigned to numerous lineages within the B.Br.013 group. The eastern geographic extent of the B.Br.013 group is currently unknown due to a lack of phylogenetic knowledge about populations at the European/Asian juncture and in Asia. In this study, we address this knowledge gap by describing the phylogenetic structure of F. tularensis subsp. holarctica isolates from the country of Georgia, and by placing these isolates into a global phylogeographic context. Results We identified a new genetic lineage of F. tularensis subsp. holarctica from Georgia that belongs to the B.Br.013 group. This new lineage is genetically and geographically distinct from lineages previously described from the B.Br.013 group from Central-Eastern Europe. Importantly, this new lineage is basal within the B.Br.013 group, indicating the Georgian lineage diverged before the diversification of the other known B.Br.013 lineages. Although two isolates from the Georgian lineage were collected nearby in the Ukrainian region of Crimea, all other global isolates assigned to this lineage were collected in Georgia. This restricted geographic distribution, as well as the high levels of genetic diversity within the lineage, is consistent with a relatively older origin and localized differentiation. Conclusions We identified a new lineage of F. tularensis subsp. holarctica from Georgia that appears to have an older origin than any other diversified lineages previously described from the B.Br.013 group. This finding suggests that additional phylogenetic studies of F. tularensis subsp. holarctica populations in Eastern Europe and Asia have the potential to yield important new insights into the evolutionary history and phylogeography of this broadly dispersed F. tularensis subspecies.

Background: Mycoplasma hyopharyngis is a commensal bacterium in the upper respiratory tract of swine. As it is recognized to be apathogenic, examinations regarding this species are scarce, compared to other swine mycoplasmas. However, in a few cases, M. hyopharyngis was detected in lesions of different organs. This report presents a case study in which M. hyopharyngis (along with other bacteria) was isolated from the joint of a pig showing lameness., Case Presentation: A Hungarian farm was repopulated with 250 gilts and 1,700 finishers after undergoing a complete depopulation and disinfection. Two days later, cases of diarrhoea and septicaemia caused by Salmonella enterica serovar typhimurium were seen in the finishers. At the same time, following the first farrowing, swollen joints were observed in 21-25 days old piglets. Joint samples were collected, and isolation of Mycoplasma sp. and other bacteria was attempted. Analysis of the joint samples revealed the presence of Staphylococcus haemolyticus, Staphylococcus hyicus, Aerococcus viridans, Trueperella pyogenes, Streptococcus agalactiae and M. hyopharyngis., Conclusions: This is the second isolation of M. hyopharyngis from joints, which highlights the necessity of a better understanding the biology of this often-overlooked species, and its role in the progress of arthritis or other lesions.

The aim of the present study was to monitor the dynamics and to measure the safety and efficacy of a live, attenuated, thermosensitive Mycoplasma anserisalpingitidis vaccine candidate, namely MA271, in geese breeder flocks under field conditions. Two rearing flocks were vaccinated with MA271 at 4 weeks of age and boosted at 24 weeks of age by cloaca inoculation (1 ml) and eye-dropping (60 µl). The geese then were transported to multi-aged breeding farms. Two breeding flocks served as controls. Colonization of the cloaca by MA271 showed 75% maximum prevalence between 4 and 6 weeks after the first vaccination. Then the prevalence decreased to 25% until the cooler, humid fall months which coincided with the booster vaccination. Boosting raised cloacal colonization to 100%. No clinical signs were observed in the vaccinated birds. After transportation to five multi-aged breeding farms, the wild-type strain appeared as well as MA271 in three flocks. In one flock, the wild-type strain completely displaced MA271, while in one flock only MA271 was detected. Only wild-type strains were detected in the control flocks; however, due to an HPAI outbreak, both flocks were exterminated before the end of the study. Based on the available data, the median percentage of infertile eggs was 3.7-5.1% in the MA271 vaccinated flocks, and 7.7% in the non-vaccinated flock. In conclusion, MA271 can colonize the cloaca of geese under field conditions. MA271 proved to be safe and presumably protects against M. anserisalpingitidis- induced reproduction losses.

Mycoplasma iowae is a worldwide spread and economically important avian pathogen that mostly infects turkeys. Currently, multi-locus sequence typing (MLST) serves as the gold standard method for strain identification in M. iowae. However, additional robust genotyping methods are required to effectively monitor M. iowae infections and conduct epidemiological investigations. The first aim of this study was to develop genotyping assays with high resolution, that specifically target M. iowae, namely a multiple-locus variable number of tandem-repeats analysis (MLVA) and a core genome multi-locus sequence typing (cgMLST) schema. The second aim was the determination of relationships among a diverse selection of M. iowae strains and clinical isolates with a previous and the newly developed assays. The MLVA was designed based on the analyses of tandem-repeat (TR) regions in the six serotype reference strains (I, J, K, N, Q and R). The cgMLST schema was developed based on the coding sequences (CDSs) common in 95% of the examined 99 isolates. The samples were submitted for a previously published MLST assay for comparison with the developed methods. Out of 94 TR regions identified, 17 alleles were selected for further evaluation by PCR. Finally, seven alleles were chosen to establish the MLVA assay. Additionally, whole genome sequence analyses identified a total of 676 CDSs shared by 95% of the isolates, all of which were included into the developed cgMLST schema. The MLVA discriminated 19 distinct genotypes (GT), while with the cgMLST assay 79 sequence types (ST) could be determined with Simpson's diversity indices of 0.810 (MLVA) and 0.989 (cgMLST). The applied assays consistently identified the same main clusters among the diverse selection of isolates, thereby demonstrating their suitability for various genetic analyses and their ability to yield congruent results., 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 The Authors. Published by Elsevier B.V. All rights reserved.)

Organizations like the Clinical and Laboratory Standards Institute (CLSI) or the European Committee of Antimicrobial Susceptibility Testing (EUCAST) provide standardized methodologies for antimicrobial susceptibility testing of a wide range of nonfastidious and fastidious bacteria, but so far not for Mycoplasma spp. of animal origin. Recently, a proposed method for the standardized broth microdilution testing of Mycoplasma hyorhinis using commercial Sensititre microtiter plates was presented. In this study, we evaluated this broth microdilution method with 37 field isolates and tested their susceptibility toward the following antimicrobial agents: doxycycline, enrofloxacin, erythromycin, florfenicol, gentamicin, marbofloxacin, tetracycline, tiamulin, tilmicosin, tulathromycin, and tylosin. The isolates originated from different countries, isolation sites, and years. The broth microdilution method was carried out using a modified Friis broth as the culture and test medium. For macrolides and lincosamides, a bimodal distribution with elevated MIC values could be observed for almost half of the tested field isolates, deducing reduced susceptibility toward these substances. With a recently published protocol, we were able to test a variety of field isolates, and consistent data could be obtained. Using this method, monitoring studies of Mycoplasma hyorhinis isolates can be carried out in a comparable manner, and the observed susceptibility profiles can be screened for possible changes in MIC values in the future.

Background: Among hard ticks (Acari: Ixodidae), the genus Ixodes comprises the highest number of species, which in turn are most numerous in the Afrotropical zoogeographic region. In South Africa extensive morphological studies have been performed on Ixodes species but only few reports included molecular analyses., Methods: In this study, 58 Ixodes spp. ticks, collected from ten mammalian and eight avian host species in South Africa, were molecularly and phylogenetically analyzed. In addition, a newly collected sample of the Palearctic Ixodes trianguliceps was included in the analyses., Results: Among the ticks from South Africa, 11 species were identified morphologically. The majority of ticks from mammals represented the Ixodes pilosus group with two species (n = 20), followed by ticks resembling Ixodes rubicundus (n = 18) and Ixodes alluaudi (n = 3). In addition, single specimens of Ixodes rhabdomysae, Ixodes ugandanus, Ixodes nairobiensis and Ixodes simplex were also found. Considering bird-infesting ticks, Ixodes theilerae (n = 7), Ixodes uriae (n = 4) and ticks most similar to Ixodes daveyi (provisionally named I. cf. daveyi, n = 2) were identified. Molecular analyses confirmed two species in the I. pilosus group and a new species (I. cf. rubicundus) closely related to I. rubicundus sensu stricto. Phylogenetic trees based on concatenated mitochondrial or mitochondrial and nuclear gene sequences indicated that the subgenus Afrixodes forms a monophyletic clade with bird-associated exophilic ticks (subgenus Trichotoixodes). Ixodes trianguliceps clustered separately whereas I. alluaudi with their morphologically assigned subgenus, Exopalpiger., Conclusions: Phylogenetic analyses shed new lights on the relationships of Ixodes subgenera when including multiple sequences from subgenus Afrixodes and African as well as Palearctic species of subgenera Trichotoixodes and Exopalpiger. Subgenera Afrixodes and bird-associated Trichotoixodes share common ancestry, suggesting that the latter might have also originated in Africa. Regarding the subgenus Exopalpiger, I. alluaudi is properly assigned as it clusters among different Australian Ixodes, whereas I. trianguliceps should be excluded., (© 2023. The Author(s).)

Introduction: Mycoplasma hyorhinis is an emerging swine pathogen with high prevalence worldwide. The main lesions caused are arthritis and polyserositis, and the clinical manifestation of the disease may result in significant economic losses due to decreased weight gain and enhanced medical costs. We aimed to compare two challenge routes to induce M. hyorhinis infection using the same clinical isolate., Methods: Five-week-old, Choice hybrid pigs were inoculated on 2 consecutive days by intravenous route (Group IV-IV) or by intravenous and intraperitoneal routes (Group IV-IP). Mock-infected animals were used as control (control group). After the challenge, the clinical signs were recorded for 28 days, after which the animals were euthanized. Gross pathological and histopathological examinations, PCR detection, isolation, and genotyping of the re-isolated Mycoplasma sp. and culture of bacteria other than Mycoplasma sp. were carried out. The ELISA test was used to detect anti- M. hyorhinis immunoglobulins in the sera of all animals., Results: Pericarditis and polyarthritis were observed in both challenge groups; however, the serositis was more severe in Group IV-IV. Statistically significant differences were detected between the challenged groups and the control group regarding the average daily weight gain, pathological scores, and ELISA titers. Additionally, histopathological scores in Group IV-IV differed significantly from the scores in the control group. All re-isolated strains were the same or a close genetic variant of the original challenge strain., Discussion: Our results indicate that both challenge routes are suitable for modeling the disease. However, due to the evoked more severe pathological lesions and the application being similar to the hypothesized natural route of infection in Group IV-IV, the two-dose intravenous challenge is recommended by the authors to induce serositis and arthritis associated with M. hyorhinis infection., Competing Interests: JF was employed by Euvet Bt. ZK and MG were employed by MolliScience Kft. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Földi, Nagy, Belecz, Szeredi, Földi, Kollár, Tenk, Kreizinger and Gyuranecz.)

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.)

Introduction: Mycoplasma anserisalpingitidis is one of the most important waterfowl-pathogenic mycoplasmas. Due to inadequate antibiotic treatment, many strains with high minimal inhibitory concentration (MIC) values for multiple drugs have been isolated lately. Decreased antibiotic susceptibility in several Mycoplasma species are known to be associated with mutations in topoisomerase and ribosomal genes, but other strategies such as active efflux pump mechanisms were also described. The scope of this study was the phenotypic and genetic characterization of the active efflux mechanism in M. anserisalpingitidis ., Methods: We measured the MIC values in the presence and absence of different efflux pump inhibitors (EPIs), such as carbonyl cyanide m-chlorophenylhydrazine (CCCP), orthovanadate (OV), and reserpine (RSP). Moreover, bioinformatic tools were utilized to detect putative regulatory sequences of membrane transport proteins coding genes, while comparative genome analysis was performed to reveal potential markers of antibiotic resistance., Results: Out of the three examined EPIs, CCCP decreased the MICs at least two-fold below the original MICs (in 23 cases out of 36 strains). In the presence of OV or RSP, MIC value differences could be seen only if modified dilution series (10% decrease steps were used instead of two-fold dilutions) were applied (in 24/36 cases with OV and 9/36 with RSP). During comparative genome analysis, non-synonymous single nucleotide polymorphisms (nsSNPs) were identified in genes encoding ABC membrane transport proteins, which were displayed in higher percentages in M. anserisalpingitidis strains with increased MICs. In terms of other genes, a nsSNP was identified in DNA gyrase subunit A ( gyrA ) gene which can be related to decreased susceptibility to enrofloxacin. The present study is the first to highlight the importance of efflux pump mechanisms in M. anserisalpingitidis ., Discussion: Considering the observed effects of the EPI CCCP against this bacterium, it can be assumed, that the use of EPIs would increase the efficiency of targeted antibiotic therapy in the future control of this pathogen. However, further research is required to obtain a more comprehensive understanding of efflux pump mechanism in this bacterium., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Nagy, Kovács, Wehmann, Bekő, Földi, Bányai, Kreizinger and Gyuranecz.)

Mycoplasma anserisalpingitidis is a waterfowl colonizing mycoplasma, mainly found in geese. In this study, we compared the whole genomes of five atypical M. anserisalpingitidis strains originating from China, Vietnam and Hungary, with the rest of the collection. Common methods used in the description of species are genomic analyses like the analysis of 16 S - intergenic transcribed spacer (ITS) - 23 S rRNA, of housekeeping genes, of the average nucleotide identity (ANI) and average amino acid identity (AAI) and phenotypic analyses like testing the growth inhibition and the growth parameters of the strains. The atypical strains showed notable genomic differences in all of the genetic analyses: on average ANI and AAI 95% (M. anserisalpingitidis ANI Minimum: 92.45, Maximum: 95.10; AAI Minimum: 93.34, Maximum: 96.37). The atypical strains formed a separate branch among the M. anserisalpingitidis strains in all phylogenetic studies. The small genome size and possibly higher mutation rate of the M. anserisalpingitidis species likely contributed to the observed genetic difference. Based on genetic analyses, the studied strains clearly represent a new genotype of M. anserisalpingitidis. The atypical strains showed slower growth in the medium containing fructose and three of the atypical strains showed diminished growth in the inhibition test. However, no definitive geno-phenotype associations were found regarding the fructose metabolism pathway in the atypical strains. The atypical strains are potentially at an early stage of speciation., 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 The Authors. Published by Elsevier B.V. All rights reserved.)

The Mycoplasma synoviae live attenuated vaccine strain MS-H (Vaxsafe MS; Bioproperties Pty., Ltd., Australia) is commonly used around the world to prevent chronic infections caused by M. synoviae in birds and to minimize economic losses in the poultry industry. MS-H is a temperature-sensitive strain that is generated via the chemical mutagenesis of a virulent M. synoviae isolate, 86079/7NS. 32 single nucleotide polymorphisms have been found in the genome of MS-H compared to that of 86079/7NS, including 25 in predicted coding sequences (CDSs). There is limited information on the stability of these mutations in MS-H in vitro during the propagation of the vaccine manufacturing process or in vivo after the vaccination of chickens. Here, we performed a comparative analysis of MS-H genomes after in vitro and in vivo passages under different circumstances. Studying the dynamics of the MS-H population can provide insights into the factors that potentially affect the health of vaccinated birds. The genomes of 11 in vitro laboratory passages and 138 MS-H bird reisolates contained a total of 254 sequence variations. Of these, 39 variations associated with CDSs were detected in more than one genome (range = 2 to 62, median = 2.5), suggesting that these sequences are particularly prone to mutations. From the 25 CDSs containing previously characterized variations between MS-H and 86079/7NS, 7 were identified in the MS-H reisolates and progenies examined here. In conclusion, the MS-H genome contains individual regions that are prone to mutations that enable the restoration of the genotype or the phenotype of wild-type 86079/7NS in those regions. However, accumulated mutations in these regions are rare. IMPORTANCE Preventative measures, such as vaccination, are commonly used for the control of mycoplasmal infections in poultry. A live attenuated vaccine strain (Vaxsafe MS; MS-H; Bioproperties Pty. Ltd., Australia) is used for the prevention of disease caused by M. synoviae in many countries. However, information on the stability of previously characterized mutations in the MS-H genome is limited. In this study, we performed a comparative analysis of the whole-genome sequences of MS-H seeds used for vaccine manufacturing, commercial batches of the vaccine, cultures minimally passaged under small-scale laboratory and large-scale manufacturing conditions, MS-H reisolated from specific-pathogen-free (SPF) chickens that were vaccinated under controlled conditions, and MS-H reisolated from vaccinated commercial poultry flocks around the world. This study provides a comprehensive assessment of genome stability in MS-H after in vitro and in vivo passages under different circumstances and suggests that most of the mutations in the attenuated MS-H vaccine strain are stable.

Mycoplasma anserisalpingitidis is economically the most important pathogenic Mycoplasma species of waterfowl in Europe and Asia. The lack of commercially available vaccines against M. anserisalpingitidis had prompted this study with the aim to produce temperature-sensitive (ts + ) clones as candidates for an attenuated live vaccine. The production of ts + clones was performed by N-methyl-N'-nitro-N-nitrosoguanidine (NTG)-induced mutagenesis of Hungarian M. anserisalpingitidis field isolates. The clones were administered via eye-drop and intracloacally to 33-day-old geese. Colonization ability was examined by PCR and isolation from the trachea and cloaca, while the serological response of the birds was tested by ELISA. Pathological and histopathological examinations were performed in the eighth week after inoculation. Whole-genome sequence (WGS) analysis of the selected clone and its parent strain was also performed. NTG-treatment provided three ts + mutants (MA177/1/11, MA177/1/12, MA271). MA271 was detected at the highest rate from cloacal (86.25%) and tracheal (30%) samples, while MA177/1/12 and MA271 elicited remarkable serological responses with 90% of the birds showing seroconversion. Re-isolates of MA271 remained ts + throughout the experiment. Based on these properties, clone MA271 was found to be the most promising vaccine candidate. WGS analysis revealed 59 mutations in the genome of MA271 when compared to its parent strain, affecting both polypeptides involved in different cellular processes and proteins previously linked to bacterial fitness and virulence. Although further studies are needed to prove that MA271 is in all aspects a suitable vaccine strain, it is expected that this ts + clone will contribute to the control of M. anserisalpingitidis infection. RESEARCH HIGHLIGHTS Three M. anserisalpingitidis ts + vaccine candidates were produced by NTG-mutagenesis.Clone MA271 was able to colonize geese and induce a serological response.MA271 re-isolates remained ts + during the 8-week-long experiment.WGS analysis revealed 59 mutations in the genome of MA271.

Polyctenidae bugs are rarely studied, hematophagous, and highly specialized ectoparasites of bats. There are only 32 described species worldwide, including six species in the Afrotropical region. Knowledge on these parasites is limited, and most studies are restricted to the New World polyctenid species. Here we report additional records of Adroctenes horvathi from Kenya and South Africa, as well as Hypoctenes faini from Rwanda. We present an updated list of published polyctenid records in the Afrotropical region indicating their host specificity and their geographical distribution. We report global infection patterns and sex ratio of polyctenids based on previously published data, including Old and New World species. Lastly, we demonstrate the first molecular phylogeny of Polyctenidae, showing their phylogenetic relationship with the closely related family Cimicidae., Competing Interests: The authors declare no competing interests., (© 2022 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

Several Mycoplasma species can form biofilm, facilitating their survival in the environment, and shielding them from therapeutic agents. The aim of this study was to examine the biofilm-forming ability and its potential effects on environmental survival and antibiotic resistance in Mycoplasma anserisalpingitidis, the clinically and economically most important waterfowl Mycoplasma species. The biofilm-forming ability of 32 M. anserisalpingitidis strains was examined by crystal violet assay. Biofilms and planktonic cultures of the selected strains were exposed to a temperature of 50 °C (20 and 30 min), to desiccation at room temperature (16 and 24 h), or to various concentrations of eight different antibiotics. Crystal violet staining revealed great diversity in the biofilm-forming ability of the 32 tested M. anserisalpingitidis strains, with positive staining in more than half of them. Biofilms were found to be more resistant to heat and desiccation than planktonic cultures, while no correlation was shown between biofilm formation and antibiotic susceptibility. Our results indicate that M. anserisalpingitidis biofilms may contribute to the persistence of the organisms in the environment, which should be taken into account for proper management. Antibiotic susceptibility was not affected by biofilm formation; however, it is important to note that correlations were examined only in vitro.

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.

Wild boars show increasing numbers and population densities throughout Europe, including Hungary. While their presence is appreciated as game animals, they are also responsible for significant agricultural damage, habitat degradation and water quality issues. In addition, wild boars may harbor ticks and can act as reservoirs of tick-borne pathogens, thus posing a risk of transmission towards humans and domestic animals. This latter aspect of their veterinary-medical and epidemiological significance has become especially important in recent years, because increasing numbers of wild boars are reported to enter urban areas. Despite of this, reports on tick infestations of wild boars are scarce in Europe. For this study, 333 ixodid ticks were collected from 51 wild boars at 32 peri-urban locations in 14 counties of Hungary, during 2005-2008 (older samples) and 2019-2020 (new samples). Five species of ticks were identified: Dermacentor reticulatus (n = 165), Ixodes ricinus (n = 90) and Haemaphysalis concinna (n = 29) in both sample groups, while H. inermis (n = 29) and D. marginatus (n = 20) were only found among the old samples. The seasonality of collected ticks corresponded to their known activities. After DNA extraction, ticks were screened for three groups of tick-borne pathogens. All samples were negative for brucellae, recently reported to be carried and transmitted transovarially by D. marginatus. Four D. reticulatus contained Babesia canis DNA, while in one H. concinna nymph the recently discovered zoonotic B. cf. crassa (reported in Slovenia within 80 km of our sampling site) was detected. In addition, Anaplasma phagocytophilum was identified in D. reticulatus (n = 1), H. concinna (n = 3) and in its known vector, I. ricinus (n = 15). Phylogenetically, three out of four A. phagocytophilum genotypes clustered with zoonotic ones. In conclusion, despite of the high prevalence of Brucella suis in wild boars in Hungary, no evidence was found in support of the epidemiological role of ticks in transmitting brucellae. On the other hand, wild boars might introduce B. canis-carrier D. reticulatus into urban areas, unlike birds (which are not known to carry this tick species in the country). Most importantly, tick-infested wild boars can contribute to the spread of a novel zoonotic Babesia sp. and of the zoonotic variants of A. phagocytophilum., (Copyright © 2022. Published by Elsevier GmbH.)

Mycoplasma anserisalpingitidis infection is associated with the inflammation of the genital tract and cloaca, embryo lethality and decreased egg production in geese, leading to serious economic losses. This bacterium has so far been described in Europe and Asia. There is no commercially available vaccine against M. anserisalpingitidis, thus treatment of waterfowl mycoplasmosis relies mainly on antimicrobial therapy. However, M. anserisalpingitidis isolates with decreased susceptibility to macrolides and lincomycin have been reported before. The minimal inhibitory concentration (MIC) values of tilmicosin, tylosin, tylvalosin and lincomycin were determined against 82 M. anserisalpingitidis isolates originating from Hungary, Poland, China and Vietnam. Whole-genome sequence analyses revealed two mutations in the 23S rRNA coding regions and one mutation in the 50S ribosomal protein L22 coding gene possibly correlating with decreased susceptibility to the examined antibiotics. Mismatch amplification mutation assays coupled with melt analysis (melt-MAMAs) were designed to detect the nucleotide substitutions. This study is the first to describe resistance-related mutations in the goose pathogen M. anserisalpingitidis. The developed molecular assays support targeted antibiotic usage, hence their use may help to reduce the development and spread of antibiotic resistance., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

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., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The objective of this study was to clarify whether the most common species of Mycoplasma can be detected in the reproductive organs and the cloaca, as well as in the semen of asymptomatic native Hungarian male geese. As it is necessary for the semen of that breed to be preserved pathogen-free in an in vitro gene-conservation programme, the presence of and sources of infection, as well as prevention of the survival of pathogens following semen cryopreservation, are key issues. Ten asymptomatic, 2-year-old ganders were tested. For the detection of mycoplasmas, samples were taken from both fresh and frozen/thawed semen, cloaca, phallus lymph, testes and vas deferens; that is five samples from each of the 10 ganders. The semen was statically frozen using dimethyl-formamide as a cryoprotectant and stored in liquid nitrogen at -196°C. Species-specific PCR systems targeting M. anserisalpingitidis , M. anseris and M. cloacale were used for screening and identification. Results of this study have shown, for the first time, that (1) among the three Mycoplasma species examined, all were detectable in the indigenous Hungarian ganders, with no clinical signs; (2) the pathogens could be detected in the cloaca, in both fresh and cryopreserved semen samples, but remained undetected within the inner reproductive organs; and (3) as pathogens were able to survive the freezing/storing/thawing procedures, the possibility of vertical transmission of the pathogens during artificial inseminations does exist, which causes problems in the in vitro gene-conservation programmes for this breed.

The family Cimicidae includes obligate hematophagous ectoparasites (bed bugs and their relatives) with high veterinary/medical importance. The evolutionary relationships of Cimicidae and their hosts have recently been reported in a phylogenetic context, but in the relevant study, one of the six subfamilies, the bat-specific Latrocimicinae, was not represented. In this study the only known species of Latrocimicinae, i.e., Latrocimex spectans, was analyzed with molecular and phylogenetic methods based on four (two nuclear and two mitochondrial) genetic markers. The completed subfamily-level phylogeny of Cimicidae showed that Latrocimicinae is most closely related to Haematosiphoninae (ectoparasites of birds and humans), with which it shares systematically important morphologic characters, but not hosts. Moreover, in the phylogenetic analyses, cimicid bugs that are known to infest phylogenetically distant bat hosts clustered together (e.g., Leptocimex and Stricticimex within Cacodminae), while cimicid subfamilies (Latrocimicinae, Primicimicinae) that are known to infest bat hosts from closely related superfamilies clustered distantly. In conclusion, adding Latrocimicinae significantly contributed to the resolution of the phylogeny of Cimicidae. The close phylogenetic relationship between Latrocimicinae and Haematosiphoninae is consistent with long-known morphologic data. At the same time, phylogenetic relationships of genera within subfamilies are inconsistent with the phylogeny of relevant hosts., (© 2021. The Author(s).)

Mycoplasma anserisalpingitidis is a bacterial waterfowl pathogen. In these days of growing antibiotic resistance, it is necessary to search for alternative methods of defense against Mycoplasma impacts in flocks. In order to identify prophage-like sequences, three established bioinformatics tools (PHASTER, PhiSpy, Prophage Hunter) were used in this study for the in silico screening of 82 M. anserisalpingitidis whole genomes. The VIBRANT software was used as a novel approach to further investigate the possibility of prophages in the sequences. The commonly used softwares found prophage-like sequences in the strains, but the results were inconclusive. The VIBRANT search resulted in multiple hits, and many of them were over 10,000 base pairs (bp). These putative prophages are comparable in size to the few described mycoplasma phages. The translated coding DNA sequences of the putative prophages were checked with protein BLAST. The functions of the proteins found by the BLASTP search are common among bacteriophages. The BLASTN search of the sequences found that many of these were more similar to the M. anatis NCTC 10156 strain, rather than the available M. anserisalpingitidis strains. The initial screening pointed at the presence of novel bacteriophages in the M. anserisalpingitidis and M. anatis strains. The VIBRANT search results were very similar to each other and none of these sequences were part of the core genome of M. anserisalpingitidis, with a few exceptions. The VIBRANT analysis explored presumably intact, novel prophages., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

The control of Mycoplasma hyorhinis infection relies mainly on antimicrobial therapy. However, the antibiotic susceptibility testing of the bacteria is usually not performed before applying the treatment, and thus therapeutic failures are not uncommon. In the case of M. hyorhinis, several antibiotic-resistance-related single nucleotide polymorphisms (SNPs) are known but assays for their detection have not been described yet. The aims of the present study were to investigate macrolide- and lincomycin-resistance-related SNPs in Hungarian M. hyorhinis isolates and to develop mismatch amplification mutation assays (MAMA) to detect the identified resistance markers. Minimal inhibitory concentrations (MIC) of different drugs and whole genome sequences of 37 M. hyorhinis isolates were used to find the resistance-related mutations. One MAMA assay was designed to detect the mutation of the 23S rRNA gene at nucleotide position 2058 (Escherichia coli numbering). For further evaluation, the assay was challenged with 17 additional isolates with available MIC data and 15 DNA samples from clinical specimens. The genotypes of the samples were in line with the MIC test results. The developed assay supports the practice of targeted antibiotic usage; hence it may indirectly reduce some bacterial resistance-related public health concerns.

Q fever is a disease of high zoonotic potential, but interest in its causative agent is rather low although it causes some public health problems in Hungary. The prevalence of Q fever is highly variable by country. The main reservoirs of the disease are the same domestic ruminant species everywhere, but the epidemiological profile depends on the features of the specific reservoir. The aim of this large-scale study was to demonstrate the importance of Q fever in different species as a possible source for human infection in most regions of Hungary. A total of 851 serum samples from 44 dairy farms, 16 sheep flocks, 4 goat farms and 3 zoos located in different parts of Hungary were tested. The presence of antibodies to Coxiella burnetii was surveyed in dairy cattle (n = 547), goats (n = 71), sheep (n = 200) and zoo animals (n = 33). The animal species tested in Hungary showed different seroprevalence values of C. burnetii infection. Seropositivity by the enzyme-linked immunosorbent assay was found in 258 out of 547 (47.2%) cows and in 69 out of 271 (25.5%) small ruminants, among them in 47 out of 200 (23.5%) sheep and in 22 out of 71 (31.0%) goats. Antibodies to C. burnetii were not detected in zoo animals. Seropositivity was demonstrated in 44 out of 44 (100%) dairy cattle farms, with at least one serum sample found to be positive on each farm. The seropositivity rate of small ruminant farms was 55.0% (11 positive out of 20 tested), with 9 out of 16 (56.3%) sheep flocks and 2 out of 4 (50.0%) goat herds showing seropositivity.

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 anserisalpingitidis infection is associated with the inflammation of the genital tract and cloaca, embryo lethality, and decreased egg production in geese, leading to serious economic losses. M. anserisalpingitidis has been detected mainly in Central and Eastern Europe, especially in Hungary, but the pathogen was identified recently in China, predicting it's worldwide occurrence. In this study, a novel multilocus sequence typing (MLST) scheme was developed to analyse phylogenetic relationships between M. anserisalpingitidis field isolates and clinical specimens originating from different geographical locations. Five loci (atpG, fusA, pgiB, plsY, and uvrA) were selected for the final MLST study. The examined 89 M. anserisalpingitidis samples yielded 76 unique sequence types with a 0.994 Simpson's index of diversity. The samples were originated from Hungary, Poland, Ukraine, China, and Vietnam. Phylogenetic analysis revealed the existence of three distinct clades (A-C) and six subclades within clade C. Generally, samples originating from the same geographical locations or livestock integration clustered together. Isolates in clade A showed the closest relationships to the M. anatis outgroup due to sequence similarity of the plsY locus. The highest genetic distance was observed in 5C among the subclades of clade C, containing the Asian and some Hungarian field isolates. The developed MLST assay revealed high diversity of the investigated M. anserisalpingitidis samples. The method proved to be a valuable and cost-effective tool for sequence typing of this waterfowl Mycoplasma species, enabling the better understanding of its phylogeny and providing a robust assay for future molecular epidemiological investigations., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Mycoplasma hyopneumoniae is the causative agent of porcine enzootic pneumonia, a chronic respiratory disease, causing significant economic losses. Results from the 2015-2016 MycoPath pan-European antimicrobial susceptibility monitoring survey of M. hyopneumoniae are presented. In total, 147 M. hyopneumoniae porcine isolates from Belgium, France, Germany, Great Britain, Hungary, Italy, and Spain were tested. One isolate per farm was retained from pigs that had not been recently treated with antimicrobial agents. The minimal inhibitory concentration (MIC) of 13 antimicrobial agents was determined in a central laboratory using a broth microdilution method, with Friis Medium, incubated at 35 ± 1 °C for 5-12 days. M. hyopneumoniae NCTC 10110 was used as Quality Control. MIC 50 /MIC 90 (mg/L) values were: enrofloxacin 0.06/1; marbofloxacin 0.06/2; spiramycin 0.06/0.25; tulathromycin ≤0.001/0.004; gamithromycin 0.06/0.5; tylosin 0.016/0.06; tilmicosin 0.06/0.5; florfenicol 0.5/1; doxycycline 0.25/1; oxytetracycline 0.25/2; lincomycin 0.06/0.25; tiamulin 0.016/0.06 and valnemulin ≤0.001/0.004. Compared with the data from 2010 to 2012 MycoPath study (50 isolates), MIC 50/90 results were similar and the majority were within ± two dilution steps, except for the MIC 50 of oxytetracycline which is more than two dilution steps higher in the present study. Between-country comparisons show some differences in the MIC values for the fluoroquinolones, tulathromycin and tylosin, but the limited sample size per country precludes performing meaningful country comparisons for several countries. Standardized laboratory methods and interpretive criteria for MIC testing of veterinary mycoplasmas are clearly needed; there are currently no clinical breakpoints available to facilitate data interpretation and correlation of MICs with in vivo efficacy., (Copyright © 2020 Elsevier B.V. All rights reserved.)

More than 1000 humans have acquired the febrile disease tularemia in Spain since the first notification of human cases in 1997. We here aimed to study the recent molecular evolution of the causative bacterium Francisella tularensis during disease establishment in Spain. Single-nucleotide polymorphisms (SNPs) and variable-number tandem repeats (VNTRs) were analyzed in whole-genome sequences (WGS) of F. tularensis . Short-read WGS data for 20 F. tularensis strains from humans infected in the periods 2014-2015 and 2018-2020 in Spain were generated. These data were combined with WGS data of 25 Spanish strains from 1998 to 2008 and two reference strains. Capillary electrophoresis data of VNTR genetic regions were generated and compared with the WGS data for the 11 strains from 2014 to 2015. Evolutionary relationships among strains were analyzed by phylogenetic methods. We identified 117 informative SNPs in a 1,577,289-nucleotide WGS alignment of 47 F. tularensis genomes. Forty-five strains from Spain formed a star-like SNP phylogeny with six branches emerging from a basal common node. The most recently evolved genomes formed four additional star-like structures that were derived from four branches of the basal common node. VNTR copy number variation was detected in two out of 10 VNTR regions examined. Genetic clustering of strains by VNTRs agreed with the clustering by SNPs. The SNP data provided higher resolution among strains than the VNTRs data in all but one cases. There was an excellent correlation between VNTR marker sizing by capillary electrophoresis and prediction from WGS data. The genetic data strongly support that tularemia, indeed, emerged recently in Spain. Distinct genetic patterns of local F. tularensis population expansions imply that the pathogen has colonized a previously disease-free geographical area. We also found that genome-wide SNPs provide higher genetic resolution among F. tularensis genomes than the use of VNTRs, and that VNTR copy numbers can be accurately predicted using short-read WGS data.

Mycoplasma gallisepticum (MG) infects many avian species and leads to significant economic losses in the poultry industry. Transmission of this pathogen occurs both horizontally and vertically, and strategies to avoid the spread of MG rely on vaccination and the application of biosecurity measures to maintain breeder groups as pathogen-free. Two live attenuated MG vaccine strains are licensed in Italy: 6/85 and ts-11. After their introduction, the implementation of adequate genotyping tools became necessary to distinguish between field and vaccine strains and to guarantee proper infection monitoring activity. In this study, 40 Italian MG isolates collected between 2010-2019 from both vaccinated and unvaccinated farms were genotyped using gene-targeted sequencing (GTS) of the cythadesin gene mgc2 and multilocus sequence typing (MLST) based on six housekeeping genes. The discriminatory power of GTS typing ensures 6/85-like strain identification, but the technique does not allow the identification ts-11 strains; conversely, MLST differentiates both vaccine strains, describing more detailed interrelation structures. Our study describes MG genetic scenario within a mixed farming context. In conclusion, the use of adequate typing methods is essential to understand the evolutionary dynamics of MG strains in a particular area and to conduct epidemiological investigations in the avian population.

The arctic fox ( Vulpes lagopus ) is the only native terrestrial mammal in Iceland. While red foxes ( V. vulpes ) are known to be epidemiologically important carriers of several vector-borne pathogens in Europe, arctic foxes have never been evaluated in a similar context on this continent. This has become especially relevant in the last decade, considering the establishing populations of the tick species Ixodes ricinus in Iceland. In this study, liver DNA extracts of 60 arctic foxes, hunted between 2011-2012, were molecularly screened for vector-borne protozoan parasites ( Trypanosomatidae , Babesia , Theileria , Hepatozoon ) and bacteria ( Anaplasma , Ehrlichia , Rickettsia , Borrelia , hemotropic Mycoplasma ). One sample was real-time qPCR positive for Anaplasma phagocytophilum , though this positivity could not be confirmed with sequencing. Samples were negative for all other tested vector-borne pathogens. Results of this study indicate that, except for A. phagocytophilum , Icelandic arctic foxes were apparently "not yet infected" with vector-borne pathogens in 2011-2012, or their infections were "below the detection limit" of applied methods. Taking into account the broad range of target microorganisms analyzed here, as well as the warming climate and increasing presence of the vector I. ricinus in Iceland, our results will be very useful as baseline data for comparison in future monitoring of the emergence of ticks and tick-borne diseases in this country.

Mycoplasma synoviae (n = 26) and M. gallisepticum (n = 11) isolates were gained from 164 clinical samples collected from China, India, Indonesia, Malaysia, Philippines, Republic of Korea and Thailand. Most isolates were from commercial chicken production systems. A method of filtering (0.45 μm) samples immediately after collection was convenient allowing over a week for transit to the laboratory. Minimum inhibitory concentrations (MICs) were characterized by a broth microdilution method to enrofloxacin, difloxacin, oxytetracycline, chlortetracycline, doxycycline, tylosin, tilmicosin, tylvalosin, tiamulin, florfenicol, lincomycin, spectinomycin and lincomycin and spectinomycin combination (1:2). Increased MICs to various antimicrobials were seen in different isolates but appeared largely unrelated to the antimicrobial treatment histories. Overall, the results were similar to other MIC surveys around the world. Generally, low MICs to tetracyclines, tiamulin and tylvalosin were observed. Increased tilmicosin MICs were observed in both M. synoviae and M. gallisepticum isolates (≥64 μg/ml MIC 90 values) and this was seen in all isolates with high tylosin MICs. Increases in lincomycin MICs were mostly associated with increases in tilmicosin MICs. The results also suggested that antimicrobial use after mycoplasma vaccination may interfere with vaccine strain persistence and efficacy (field strains were more commonly observed in flocks that had treatments after vaccination) and this area warrants more investigation. The study shows that isolation and MIC determination can be done from remote locations and suggests that this may provide information that will allow more effective use of antimicrobials or other methods of control of avian mycoplasma in chickens (e.g. live vaccines) and therefore more responsible use of antimicrobials from a one health perspective., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

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 hyorhinis is a swine pathogen bacterium, which causes significant economic losses. The infection spreads through direct contact between the animals. Powerful genotyping methods like PCR based multi-locus sequence typing (MLST) and multiple-locus variable-number tandem-repeat analysis (MLVA) are necessary to monitor the infections and to conduct epidemiological investigations; hence supporting the control of the disease. The aims of the present study were to examine M. hyorhinis isolates originating mainly from Hungary with MLST and MLVA developed in the study, and to compare the results of the two typing methods. To characterize 39 M. hyorhinis isolates and the type strain (NCTC 10,130), six house-keeping genes were selected for MLST and six tandem-repeat regions were chosen for MLVA. We were able to differentiate 31 sequence types and 37 genotypes within the 40 analyzed isolates by the MLST and the MLVA, respectively. With the combination of the two newly developed assays all examined isolates were distinguished with the exception of the ones originating from the same animal. The developed MLST assay provided a robust and high resolution phylogenetic tree, while the MLVA system is suitable for the differentiation of closely related isolates from the same farm, hence the assay is appropriate for epidemiologic studies., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

The aim of the present study was to assess the prevalence of Coxiella burnetii in dairy herds in Central and Eastern European countries based on ELISA and PCR tests. A total of 370 bulk tank milk samples were collected in 2019 originating from Croatia (n = 13), Czech Republic (n = 138), Hungary (n = 126), Serbia (n = 24), Slovakia (n = 53) and Slovenia (n = 16). Prevalence of C. burnetii differed according to the country of origin with Croatia showing 100.00%, the Czech Republic 98.55%, Hungary 97.61%, Serbia 70.83%, Slovakia 90.56% and Slovenia showing 62.50% average percentages of the positive herds. C. burnetii specific ELISA showed 100.00% positivity in all examined countries if herds consisted of equal or above 250 milking cows. The growing number of farms managing large number of animals, where cattle density is high correlates with the increasing prevalence of C. burnetii in the region., (Copyright © 2020. Published by Elsevier Ltd.)

Q fever is one of the commonest infectious diseases worldwide. A Coxiella burnetii prevalence of 97.6% has been found by ELISA and PCR tests of the bulk tank milk in dairy cattle farms of Hungary. The herd- and individual-level seroprevalence rates of C. burnetii in the examined dairy cows and farms have dramatically increased over the past ten years. Three high-producing industrial dairy farms were studied which had previously been found ELISA and PCR positive for C. burnetii by bulk tank milk testing. Coxiella burnetii was detected in 52% of the 321 cows tested by ELISA. Pregnancy loss was detected in 18% of the cows between days 29-35 and days 60-70 of gestation. The study found a higher seropositivity rate (80.5%) in the cows that had lost their pregnancy and a seropositivity of 94.4% in the first-bred cows that had lost their pregnancy at an early stage. The ELISA-positive pregnant and aborted cows were further investigated by the complement fixation test (CFT). In dairy herds an average of 66.6% individual seropositivity was detected by the CFT (Phase II) in previously ELISA-positive animals that had lost their pregnancy and 64.5% in the pregnant animals. A higher (Phase I) seropositivity rate (50.0%) was found in the cows with pregnancy loss than in the pregnant animals (38.5%). The high prevalence of C. burnetii in dairy farms is a major risk factor related to pregnancy loss.

Mycoplasma synoviae is one of the economically most significant avian Mycoplasma species. It can cause great financial losses to the poultry industry by inducing respiratory diseases, infectious synovitis, or eggshell apex abnormalities. There are different approaches to control M. synoviae infection. Although antimicrobial therapy cannot replace long-term solutions, like eradication and vaccination, this strategy can be effective in the short term, as adequate antibiotic treatment can relieve economic losses through the attenuation of clinical signs and reduction of transmission. Using broth microdilution method, minimal inhibitory concentration (MIC) values to fourteen antibiotics related to eight antimicrobial groups were determined in 96 M. synoviae strains. Whole genome sequencing and sequence analysis revealed mutations potentially associated with decreased susceptibility to fluoroquinolones, macrolides and lincomycin. Molecular markers responsible for the high MICs to fluoroquinolones were found in the gyrA, gyrB, parC and parE genes. Besides, single nucleotide polymorphisms identified in genes encoding the 23S rRNA were found to be responsible for high MICs to the 50S inhibitor macrolides and lincomycin, while amino acid change in the 50S ribosomal protein L22 could be associated with decreased susceptibility to macrolides. The revealed mutations can contribute to the extension of knowledge about the genetic background of antibiotic resistance in M. synoviae. Moreover, the explored potentially resistance-related mutations may serve as targets for molecular biological assays providing data of antibiotic susceptibility prior to the laborious and time-consuming isolation of M. synoviae strains., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

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.

Increasing amount of data attest that (in the context of vector-borne infections) birds are not only important as hosts of blood-sucking arthropod vectors, but also as reservoirs of vector-borne pathogens. From 2015 to 2019 cadavers of 100 birds (from 45 species, nine orders) were collected in Hungary, and their organs were screened for DNA from a broad range of vector-borne bacteria with PCR and sequencing. Molecular analyses revealed the presence of Anaplasmataceae, and sequencing identified bacteria closely related to Neorickettsia helminthoeca and Ehrlichia chaffeensis in a Eurasian teal (Anas crecca) and a song thrush (Turdus philomelos), respectively. All samples were PCR negative for rickettsiae, borreliae, Francisella and Coxiella spp., as well as for piroplasms. To our knowledge, this is the first report of a Neorickettsia and an Ehrlichia sp., which belong to the phylogenetic groups of N. helminthoeca and E. chaffeensis, respectively, from Europe. The potential presence of these two vector-borne bacteria needs to be taken into account during future studies on the eco-epidemiology of Anaplasmataceae in Europe.