Evaluating the dynamics and efficacy of a live, attenuated Mycoplasma anserisalpingitidis vaccine candidate under farm conditions.

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.

In vitro susceptibility of Mycoplasma iowae isolates to antimicrobial agents.

ABSTRACTMycoplasma 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.

Development and evaluation of temperature-sensitive Mycoplasma anserisalpingitidis clones as vaccine candidates.

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.

Biofilm formation and its impact on environmental survival and antibiotic resistance of Mycoplasma anserisalpingitidis strains.

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 species in the male reproductive organs and the fresh and frozen semen of the Hungarian native goose.

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.

Development of a molecular biological assay for the detection of markers related to decreased susceptibility to macrolides and lincomycin in Mycoplasma hyorhinis.

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.

The core genome multi-locus sequence typing of Mycoplasma anserisalpingitidis.

BACKGROUND: Mycoplasma anserisalpingitidis is a waterfowl pathogen that mainly infects geese, can cause significant economic losses and is present worldwide. With the advance of whole genome sequencing technologies, new methods are available for the researchers; one emerging methodology is the core genome Multi-Locus Sequence Typing (cgMLST). The core genome contains a high percentage of the coding DNA sequence (CDS) set of the studied strains. The cgMLST schemas are powerful genotyping tools allowing for the investigation of potential epidemics, and precise and reliable classification of the strains. Although whole genome sequences of M. anserisalpingitidis strains are available, to date, no cgMLST schema has been published for this species. RESULTS: In this study, Illumina short reads of 81 M. anserisalpingitidis strains were used, including samples from Hungary, Poland, Sweden, and China. Draft genomes were assembled with the SPAdes software and analysed with the online available chewBBACA program. User made modifications in the program enabled analysis of mycoplasmas and provided similar results as the conventional SeqSphere+ software. The threshold of the presence of CDS in the strains was set to 93% due to the quality of the draft genomes, resulting in the most accurate and robust schema. Three hundred thirty-one CDSs constituted our cgMLST schema (representing 42,77% of the whole CDS set of M. anserisalpingitidis ATCC BAA-2147), and a Neighbor joining tree was created using the allelic profiles. The correlation was observed between the strains' cgMLST profile and geographical origin; however, strains from the same integration but different locations also showed close relationship. Strains isolated from different tissue samples of the same animal revealed highly similar cgMLST profiles. CONCLUSIONS: The Neighbor joining tree from the cgMLST schema closely resembled the real-life spatial and temporal relationships of the strains. The incongruences between background data and the cgMLST profile in the strains from the same integration can be because of the higher probability of contacts between the flocks. This schema can help with the epidemiological investigation and can be used as a basis for further studies.

Development of mismatch amplification mutation assay for the rapid differentiation of Mycoplasma gallisepticum K vaccine strain from field isolates.

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 102 and 103 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.

Isolation of Mycoplasma anserisalpingitidis from swan goose (Anser cygnoides) in China.

BACKGROUND: Mycoplasma anserisalpingitidis causes significant economic losses in the domestic goose (Anser anser) industry in Europe. As 95% of the global goose production is in China where the primary species is the swan goose (Anser cygnoides), it is crucial to know whether the agent is present in this region of the world. RESULTS: Purulent cloaca and purulent or necrotic phallus inflammation were observed in affected animals which represented 1-2% of a swan goose breeding flock (75,000 animals) near Guanghzou, China, in September 2019. From twelve sampled animals the cloaca swabs of five birds (three male, two female) were demonstrated to be M. anserisalpingitidis positive by PCR and the agent was successfully isolated from the samples of three female geese. Based on whole genome sequence analysis, the examined isolate showed high genetic similarity (84.67%) with the European isolates. The antibiotic susceptibility profiles of two swan goose isolates, determined by microbroth dilution method against 12 antibiotics and an antibiotic combination were also similar to the European domestic goose ones with tylvalosin and tiamulin being the most effective drugs. CONCLUSIONS: To the best of our knowledge this is the first description of M. anserisalpingitidis infection in swan goose, thus the study highlights the importance of mycoplasmosis in the goose industry on a global scale.

Development of Molecular Methods for Rapid Differentiation of Mycoplasma gallisepticum Vaccine Strains from Field Isolates.

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 101 and 104M. gallisepticum template copies/reaction, while that of the agarose-MAMAs ranged from 103 to 105 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.

Mutations Associated with Decreased Susceptibility to Seven Antimicrobial Families in Field and Laboratory-Derived Mycoplasma bovis Strains.

The molecular mechanisms of resistance to fluoroquinolones, tetracyclines, an aminocyclitol, macrolides, a lincosamide, a phenicol, and pleuromutilins were investigated in Mycoplasma bovis For the identification of mutations responsible for the high MICs of certain antibiotics, whole-genome sequencing of 35 M. bovis field isolates and 36 laboratory-derived antibiotic-resistant mutants was performed. In vitro resistant mutants were selected by serial passages of M. bovis in broth medium containing subinhibitory concentrations of the antibiotics. Mutations associated with high fluoroquinolones MICs were found at positions 244 to 260 and at positions 232 to 250 (according to Escherichia coli numbering) of the quinolone resistance-determining regions of the gyrA and parC genes, respectively. Alterations related to elevated tetracycline MICs were described at positions 962 to 967, 1058, 1195, 1196, and 1199 of genes encoding the 16S rRNA and forming the primary tetracycline binding site. Single transversion at position 1192 of the rrs1 gene resulted in a spectinomycin MIC of 256 µg/ml. Mutations responsible for high macrolide, lincomycin, florfenicol, and pleuromutilin antibiotic MICs were identified in genes encoding 23S rRNA. Understanding antibiotic resistance mechanisms is an important tool for future developments of genetic-based diagnostic assays for the rapid detection of resistant M. bovis strains.

Antibiotic susceptibility profiles of Mycoplasma synoviae strains originating from Central and Eastern Europe.

BACKGROUND: Mycoplasma synoviae causes infectious synovitis and respiratory diseases in chickens and turkeys and may lead to egg shell apex abnormalities in chickens; hence possesses high economic impact on the poultry industry. Control of the disease consists of eradication, vaccination or medication. The aim of the present study was to determine the in vitro susceptibility to 14 different antibiotics and an antibiotic combination of M. synoviae strains originating from Hungary and other countries of Central and Eastern Europe. RESULTS: Minimal inhibitory concentration (MIC) values of a total of 41 M. synoviae strains were determined by the microbroth dilution method. The strains were collected between 2002 and 2016 and originated from Hungary (n = 26), Austria (n = 3), the Czech Republic (n = 3), Slovenia (n = 3), Ukraine (n = 3), Russia (n = 2) and Serbia (n = 1). Tetracyclines (with MIC50 values of 0.078 µg/ml, ≤0.25 µg/ml and 0.5 µg/ml for doxycycline, oxytetracycline and chlortetracycline, respectively), macrolides (with MIC50 values of ≤0.25 µg/ml for tylvalosin, tylosin and tilmicosin), pleuromutilins (with MIC50 values of 0.078 µg/ml and ≤0.039 µg/ml for tiamulin and valnemulin) and the combination of lincomycin and spectinomycin (MIC50 1 µg/ml (0.333/0.667 µg/ml)) were found to be the most effective antibiotic agents against M. synoviae in vitro. High MIC values were detected in numerous strains for fluoroquinolones (with MIC50 values of 1.25 µg/ml and 2.5 µg/ml for enrofloxacin and difloxacin), neomycin (MIC50 32 µg/ml), spectinomycin (MIC50 2 µg/ml), lincomycin (MIC50 0.5 µg/ml) and florfenicol (MIC50 4 µg/ml). Nevertheless, strains with elevated MIC values were detected for most of the applied antibiotics. CONCLUSIONS: In the medical control of M. synoviae infections the preliminary in vitro antibiotic susceptibility testing and the careful evaluation of the data are crucial. Based on the in vitro examinations doxycycline, oxytetracycline, tylvalosin, tylosin and pleuromutilins could be recommended for the therapy of M. synoviae infections in the region.

Comparison of virulence of Francisella tularensis ssp. holarctica genotypes B.12 and B.FTNF002-00.

BACKGROUND: Two main genetic groups (B.12 and B.FTNF002-00) of Francisella tularensis ssp. holarctica are endemic in Europe. The B.FTNF002-00 group proved to be dominant in Western European countries, while strains of the B.12 group were isolated mainly in Northern, Central and Eastern Europe. The clinical course of tularemia in the European brown hare (Lepus europaeus) also shows distinct patterns according to the geographical area. Acute course of the disease is observed in hares in Western European countries, while signs of sub-acute or chronic infection are more frequently detected in the eastern part of the continent. The aim of the present study was to examine whether there is any difference in the virulence of the strains belonging to the B.FTNF002-00 and B.12 genetic clades. RESULTS: Experimental infection of Fischer 344 rats was performed by intra-peritoneal injection of three dilutions of a Hungarian (B.12 genotype) and an Italian (B.FTNF002-00 genotype) F. tularensis ssp. holarctica strain. Moderate difference was observed in the virulence of the two genotypes. Significant differences were observed in total weight loss values and scores of clinical signs between the two genotypes with more rats succumbing to tularemia in groups infected with the B.FTNF002-00 genotype. CONCLUSIONS: Results of the experimental infection are consistent with previous clinical observations and pathological studies suggesting that F. tularensis ssp. holarctica genotype B.FTNF002-00 has higher pathogenic potential than the B.12 genotype.

Antibiotic susceptibility profiles of Mycoplasma sp. 1220 strains isolated from geese in Hungary.

BACKGROUND: Mycoplasma sp. 1220 can induce inflammation primarily in the genital and respiratory tracts of waterfowl, leading to serious economic losses. Adequate housing and appropriate antibiotic treatment are promoted in the control of the disease. The aim of the present study was to determine the in vitro susceptibility to thirteen different antibiotics and an antibiotic combination of thirty-eight M. sp. 1220 strains isolated from geese and a duck in several parts of Hungary, Central Europe between 2011 and 2015. RESULTS: High MIC50 values were observed in the cases of tilmicosin (>64 µg/ml), oxytetracycline (64 µg/ml), norfloxacin (>10 µg/ml) and difloxacin (10 µg/ml). The examined strains yielded the same MIC50 values with spectinomycin, tylosin and florfenicol (8 µg/ml), while enrofloxacin (MIC50 5 µg/ml), doxycycline (MIC50 5 µg/ml), lincomycin (MIC50 4 µg/ml) and lincomycin-spectinomycin (1:2) combination (MIC50 4 µg/ml) inhibited the growth of the bacteria with lower concentrations. Tylvalosin (MIC50 0.5 µg/ml) and two pleuromutilins (tiamulin MIC50 0.625 µg/ml; valnemulin MIC50 ≤ 0.039 µg/ml) were found to be the most effective drugs against M. sp. 1220. However, strains with elevated MIC values were detected for all applied antibiotics. CONCLUSIONS: Valnemulin, tiamulin and tylvalosin were found to be the most effective antibiotics in the study. Increasing resistance was observed in the cases of several antibiotics. The results highlight the importance of testing Mycoplasma species for antibiotic susceptibility before therapy.

Antimicrobial susceptibility of Bacillus anthracis strains from Hungary.

The susceptibility of 29 Bacillus anthracis strains, collected in Hungary between 1933 and 2014, was tested to 10 antibiotics with commercially available minimum inhibitory concentration (MIC) test strips. All strains were susceptible to amoxicillin, ciprofloxacin, clindamycin, doxycycline, gentamicin, penicillin, rifampicin, and vancomycin. Intermediate susceptibility to erythromycin and cefotaxime was detected in 17.2% (5/29) and 58.6% (17/29) of the strains, respectively. Correlations were not observed between the isolation date, location, host species, genotype, and antibiotic susceptibility profile of strains.

First isolation and characterization of Brucella microti from wild boar.

BACKGROUND: Brucella microti was first isolated from common vole (Microtus arvalis) in the Czech Republic in Central Europe in 2007. As B. microti is the only Brucella species known to live in soil, its distribution, ecology, zoonotic potential, and genomic organization is of particular interest. The present paper is the first to report the isolation of B. microti from a wild boar (Sus scrofa), which is also the first isolation of this bacterial species in Hungary. RESULTS: The B. microti isolate was cultured, after enrichment in Brucella-selective broth, from the submandibular lymph node of a female wild boar that was taken by hunters in Hungary near the Austrian border in September 2014. Histological and immunohistological examinations of the lymph node sections with B. abortus-, B. suis- and B. canis-specific sera gave negative results. The isolate did not require CO2 for growth, was oxidase, catalase, and urease positive, H2S negative, grew well in the presence of 20 µg/ml basic fuchsin and thionin, and had brownish pigmentation after three days of incubation. It gave strong positive agglutination with anti-A and anti-M but had a negative reaction with anti-R monospecific sera. The API 20 NE test identified it as Ochrobactrum anthropi with 99.9% identity, and it showed B. microti-specific banding pattern in the Bruce- and Suis-ladder multiplex PCR systems. Whole genome re-sequencing identified 30 SNPs in orthologous loci when compared to the B. microti reference genome available in GenBank, and the MLVA analysis yielded a unique profile. CONCLUSIONS: Given that the female wild boar did not develop any clinical disease, we hypothesize that this host species only harboured the bacterium, serving as a possible reservoir capable of maintaining and spreading this pathogen. The infectious source could have been either a rodent, a carcass that had been eaten or infection occurred via the boar rooting in soil. The low number of discovered SNPs suggests an unexpectedly high level of genetic homogeneity in this Brucella species.

Complement sensitivity and factor H binding of European Francisella tularensis ssp. holarctica strains in selected animal species.

Francisella tularensis is a Gram-negative bacterium, the causative agent of the zoonotic disease tularaemia. The bacterium has developed several extracellular and intracellular strategies to evade the hosts' innate and adaptive immune responses. The aims of the study were to examine complement sensitivity of wild and attenuated F. tularensis ssp. holarctica strains in animal hosts of distinct sensitivity to the bacterium, to compare the complement-evading ability of wild strains of different phylogeographic background, and to examine the role of factor H in the host-pathogen interactions. Complement sensitivity assays were carried out on various F. tularensis ssp. holarctica wild strains and on the attenuated live vaccine strain (LVS) with sera of the highly sensitive house mouse (Mus musculus), the moderately sensitive European brown hare (Lepus europaeus) and the relatively resistant cattle (Bos taurus). Specific binding of complement regulator factor H to bacterial membrane proteins was examined by Western blot assays. All wild strains interacted with the hosts' complement system and showed no significant differences in their survivability. The attenuated LVS was resistant to serum killing in mouse, but was lysed in the sera of hare and cattle. Direct binding of factor H to F. tularensis membrane proteins was not detected.

Worldwide phylogenetic relationship of avian poxviruses.

Poxvirus infections have been found in 230 species of wild and domestic birds worldwide in both terrestrial and marine environments. This ubiquity raises the question of how infection has been transmitted and globally dispersed. We present a comprehensive global phylogeny of 111 novel poxvirus isolates in addition to all available sequences from GenBank. Phylogenetic analysis of the Avipoxvirus genus has traditionally relied on one gene region (4b core protein). In this study we expanded the analyses to include a second locus (DNA polymerase gene), allowing for a more robust phylogenetic framework, finer genetic resolution within specific groups, and the detection of potential recombination. Our phylogenetic results reveal several major features of avipoxvirus evolution and ecology and propose an updated avipoxvirus taxonomy, including three novel subclades. The characterization of poxviruses from 57 species of birds in this study extends the current knowledge of their host range and provides the first evidence of the phylogenetic effect of genetic recombination of avipoxviruses. The repeated occurrence of avian family or order-specific grouping within certain clades (e.g., starling poxvirus, falcon poxvirus, raptor poxvirus, etc.) indicates a marked role of host adaptation, while the sharing of poxvirus species within prey-predator systems emphasizes the capacity for cross-species infection and limited host adaptation. Our study provides a broad and comprehensive phylogenetic analysis of the Avipoxvirus genus, an ecologically and environmentally important viral group, to formulate a genome sequencing strategy that will clarify avipoxvirus taxonomy.

Genotyping of Coxiella burnetii from domestic ruminants and human in Hungary: indication of various genotypes.

BACKGROUND: Information about the genotypic characteristic of Coxiella burnetii from Hungary is lacking. The aim of this study is to describe the genetic diversity of C. burnetii in Hungary and compare genotypes with those found elsewhere. A total of 12 samples: (cattle, n = 6, sheep, n = 5 and human, n = 1) collected from across Hungary were studied by a 10-loci multispacer sequence typing (MST) and 6-loci multiple-locus variable-number of tandem repeat analysis (MLVA). Phylogenetic relationships among MST genotypes show how these Hungarian samples are related to others collected around the world. RESULTS: Three MST genotypes were identified: sequence type (ST) 20 has also been identified in ruminants from other European countries and the USA, ST28 was previously identified in Kazakhstan, and the proposed ST37 is novel. All MST genotypes yielded different MLVA genotypes and three different MLVA genotypes were identified within ST20 samples alone. Two novel MLVA types 0-9-5-5-6-2 (AG) and 0-8-4-5-6-2 (AF) (Ms23-Ms24-Ms27-Ms28-Ms33-Ms34) were defined in the ovine materials correlated with ST28 and ST37. Samples from different parts of the phylogenetic tree were associated with different hosts, suggesting host-specific adaptations. CONCLUSIONS: Even with the limited number of samples analysed, this study revealed high genetic diversity among C. burnetii in Hungary. Understanding the background genetic diversity will be essential in identifying and controlling outbreaks.

Phylogeny of Mycoplasma bovis isolates from Hungary based on multi locus sequence typing and multiple-locus variable-number tandem repeat analysis.

BACKGROUND: Mycoplasma bovis is an important pathogen causing pneumonia, mastitis and arthritis in cattle worldwide. As this agent is primarily transmitted by direct contact and spread through animal movements, efficient genotyping systems are essential for the monitoring of the disease and for epidemiological investigations. The aim of this study was to compare and evaluate the multi locus sequence typing (MLST) and the multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) through the genetic characterization of M. bovis isolates from Hungary. RESULTS: Thirty one Hungarian M. bovis isolates grouped into two clades by MLST. Two strains had the same sequence type (ST) as reference strain PG45, while the other twenty nine Hungarian isolates formed a novel clade comprising five subclades. Isolates originating from the same herds had the same STs except for one case. The same isolates formed two main clades and several subclades and branches by MLVA. One clade contained the reference strain PG45 and three isolates, while the other main clade comprised the rest of the strains. Within-herd strain divergence was also detected by MLVA. Little congruence was found between the results of the two typing systems. CONCLUSIONS: MLST is generally considered an intermediate scale typing method and it was found to be discriminatory among the Hungarian M. bovis isolates. MLVA proved to be an appropriate fine scale typing tool for M. bovis as this method was able to distinguish closely related strains isolated from the same farm. We recommend the combined use of the two methods for the genotyping of M. bovis isolates. Strains have to be characterized first by MLST followed by the fine scale typing of identical STs with MLVA.