Kinga M. Sulyok

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

BackgroundMycoplasma 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.ResultsThirty 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.ConclusionsMLST 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.

Genetic relatedness of Brucella suis biovar 2 isolates from hares, wild boars and domestic pigs.

Porcine brucellosis generally manifests as disorders in reproductive organs potentially leading to serious losses in the swine industry. Brucella suis biovar 2 is endemic in European wild boar (Sus scrofa) and hare (Lepus europeus, Lepus capensis) populations, thus these species may play a significant role in disease spread and serve as potential sources of infection for domestic pigs. The aim of this study was an epidemiologic analysis of porcine brucellosis in Hungary and a comparative analysis of B. suis bv. 2 strains from Europe using multiple-locus variable-number tandem repeat analysis (MLVA). MLVA-16 and its MLVA-11 subset were used to determine the genotypes of 68 B. suis bv. 2 isolates from Hungary and results were then compared to European MLVA genotypes. The analyses indicated relatively high genetic diversity of B. suis bv. 2 in Hungary. Strains isolated from hares and wild boars from Hungary showed substantial genetic divergence, suggesting separate lineages in each host and no instances of cross species infections. The closest relatives of strains from Hungarian wild boars and domestic pigs were mainly in the isolates from German and Croatian boars and pigs. The assessment of the European MLVA genotypes of wild boar isolates generally showed clustering based on geographic origin. The hare strains were relatively closely related to one another and did not cluster based on geographic origin. The limited relationships between geographic origin and genotype in isolates from hares might be the result of cross-border live animal translocation. The results could also suggest that certain B. suis strains are more adapted to hares. Across Europe, isolates from domestic pigs were closely related to isolates originating from both hares and wild boars, supporting the idea that wild animals are a source of brucellosis in domestic pigs.

Q fever epidemic in Hungary, April to July 2013

We investigated a Q fever outbreak with human patients showing high fever, respiratory tract symptoms, headache and retrosternal pain in southern Hungary in the spring and summer of 2013. Seventy human cases were confirmed by analysing their serum and blood samples with micro-immunofluorescence test and real-time PCR. The source of infection was a merino sheep flock of 450 ewes, in which 44.6% (25/56) seropositivity was detected by enzyme-linked immunosorbent assay. Coxiella burnetii DNA was detected by real-time PCR in the milk of four of 20 individuals and in two thirds (41/65) of the manure samples. The multispacer sequence typing examination of C. burnetii DNA revealed sequence type 18 in one human sample and two manure samples from the sheep flock. The multilocus variable-number tandem repeat analysis pattern of the sheep and human strains were also almost identical, 4/5-9-3-3-0-5 (Ms23-Ms24-Ms27-Ms28-Ms33-Ms34). It is hypothesised that dried manure and maternal fluid contaminated with C. burnetii was dispersed by the wind from the sheep farm towards the local inhabitants. The manure was eliminated in June and the farm was disinfected in July. The outbreak ended at the end of July 2013.

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

BackgroundInformation 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.ResultsThree 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.ConclusionsEven 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.

Antibiotic susceptibility profiles of Mycoplasma bovis strains isolated from cattle in Hungary, Central Europe

BackgroundMycoplasma bovis is a worldwide pathogen, causative agent of pneumonia, mastitis, arthritis, and a variety of other symptoms in cattle. The economic losses due to mycoplasma pneumonia could be reduced by antibiotic treatment. The aim of the present study was to determine the in vitro susceptibility of M. bovis strains isolated from cattle in Hungary to eleven antibiotics.ResultsMinimal inhibitory concentration (MIC) values of 35 M. bovis strains collected from different parts of Hungary between 2010 and 2013 were determined by the microbroth dilution method. Strains with high MIC values were found in the case of all applied antibiotics. The most effective antibiotics tested in vitro were fluoroquinolones (MIC90 danofloxacin 0.312 μg/ml, enrofloxacin 0.312 μg/ml, marbofloxacin 0.625 μg/ml). Our results confirm the observations of increasing MIC values to antibiotics commonly used in the therapy of mycoplasma infections, primarily to tetracyclines; tetracycline (MIC90 16 μg/ml) and oxytetracycline (MIC90≥64 μg/ml) and macrolides; tylosin (MIC90≥128 μg/ml) and tilmicosin (MIC90≥128 μg/ml). The growth of many M. bovis strains was not inhibited by gentamicin (MIC90 8 μg/ml), spectinomycin (MIC90≥256 μg/ml), florfenicol (MIC90 8 μg/ml) or lincomycin (MIC90≥64 μg/ml).ConclusionsOur results emphasize the necessity of periodic testing for antibiotic susceptibility in this geographic region. Based on our in vitro examinations, fluoroquinolones could be the most effective drugs for the therapy of M. bovis infections in Hungary. However, current antimicrobial use policies have to be taken into account to avoid further antibiotic resistance development and to reserve fluoroquinolones for the treatment of severe infections which have responded poorly to other classes of antimicrobials.

Influence of the Biotope on the Tick Infestation of Cattle and on the Tick-Borne Pathogen Repertoire of Cattle Ticks in Ethiopia

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

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

ABSTRACT 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.

Rapid, Simple and Cost-Effective Molecular Method to Differentiate the Temperature Sensitive (ts+) MS-H Vaccine Strain and Wild-Type Mycoplasma synoviae Isolates.

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

Occurrence of Coxiella burnetii and Chlamydiales species in abortions of domestic ruminants and in wild ruminants in Hungary, Central Europe

Coxiella burnetii and certain members of the Chlamydiales order are zoonotic, intracellular, Gram-negative bacteria, with abortigenic potential in ruminants. These pathogens have a broad host range and worldwide geographical distribution. The current study aimed to reveal the importance of C. burnetii and Chlamydiales spp. in abortions in domestic ruminants and their occurrence in wild ruminants with real-time polymerase chain reaction (qPCR) assays, histology, and immunohistochemical staining (IHC). From the 111 abortion cases of domestic ruminants examined, C. burnetii was detected in 33 placenta samples (cattle, n = 22; sheep, n = 10; goat, n = 1), and members of the Chlamydiales order were detected in 32 placenta samples (cattle, n = 14; sheep, n = 16; goat, n = 2) using qPCR. Coinfection with both C. burnetii and Chlamydiales spp. were identified in 12 cases (cattle, n = 3; sheep, n = 8; goat, n = 1) out of the qPCR-positive samples. The presence of the relevant antigen was confirmed by IHC in 20 cases (C. burnetii, n = 2, in sheep; Chlamydiaceae, n = 17, in sheep [n = 15] and goat [n = 2]; and both pathogens in 1 sheep). Coxiella burnetii was identified in 2.2% (2/91) of the wild ruminants, but the samples were negative by IHC. Uncultured Chlamydiales spp. were detected in 4.4% (4/91) of the placenta samples by qPCR. In conclusion, Q fever is widespread among domestic ruminants in Hungary, and, in several cases, C. burnetii was implicated as the primary cause of abortions. Waddlia chondrophila, Parachlamydia spp., and uncultured Chlamydiales spp. were present only sporadically in samples from cattle and wild ruminants.

Identification of novel Coxiella burnetii genotypes from Ethiopian ticks

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