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.

Development of molecular biological tools for the rapid determination of antibiotic susceptibility of Mycoplasma hyopneumoniae isolates.

Mycoplasma hyopneumoniae is the etiologic agent of porcine enzootic pneumonia, a contagious respiratory disease, causing significant economic losses worldwide. Antibiotic treatment is commonly utilised in the pig industry to control M. hyopneumoniae infection. Since the conventional antibiotic susceptibility test is time-consuming, taking up to weeks' period, antibiotics are usually empirically chosen. Certain single nucleotide polymorphisms in the parC (C239A/T, G250A) and gyrA (G242C, C247 T, A260 G) genes show correlation with decreased fluoroquinolone susceptibility by the change of the target site. Furthermore, the nucleotide alteration A2059 G in the 23S rRNA sequence correlates with significantly decreased macrolide and lincosamide susceptibility of M. hyopneumoniae. Mismatch amplification mutation assays (MAMA) and high resolution melt (HRM) analysis, capable to detect the mentioned resistance markers, were developed in the present study, in order to provide susceptibility data in a considerably shorter time than the conventional methods. The results of the MAMA and HRM assays were congruent with the results of the conventional antibiotic susceptibility method of the tested M. hyopneumoniae field isolates. The sensitivity of the MAMAs was 103-104 copy numbers, while that of the HRM assay was 105-106 copy numbers. To the best of our knowledge this was the first time that MAMA and HRM assays were developed for the rapid detection of decreased fluoroquinolone, macrolide or lincosamide susceptibility in M. hyopneumoniae strains.

Genotyping Mycoplasma hyorhinis by multi-locus sequence typing and multiple-locus variable-number tandem-repeat analysis.

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.

Antibiotic susceptibility profiles of Mycoplasma hyorhinis strains isolated from swine in Hungary.

Mycoplasma hyorhinis is a common pathogen of swine causing mainly polyserositis and arthritis, but it has also been implicated as a cause of pneumonia. The economic losses due to M. hyorhinis infection could be reduced by antibiotic treatment. The aim of this study was to determine minimal inhibitory concentrations (MIC) of antibiotics potentially used to combat M. hyorhinis in swine production. Thirty-eight Hungarian M. hyorhinis strains isolated between 2014 and 2017 were examined by microbroth dilution tests for fifteen antimicrobial agents. Low MIC values of tetracyclines (MIC50 0.078 µg/ml for doxycycline, ≤0.25 µg/ml for oxytetracycline) and pleuromutilins (MIC50 0.156 µg/ml for tiamulin, ≤0.039 µg/ml for valnemulin) were detected against all strains. Fluoroquinolones (MIC50 0.625 µg/ml), gentamicin (MIC50 1 µg/ml) and florfenicol (MIC50 2 µg/ml) inhibited the growth of Hungarian isolates at moderate MIC values. Most of the strains were inhibited by spectinomycin with low or moderate MIC values (MIC50 4 µg/ml) except one strain (>64 µg/ml). Numerous isolates showed decreased susceptibility to macrolides and lincomycin (MIC90 >64 for tylosin, tilmicosin, tulathromycin, gamithromycin, lincomycin, 8 µg/ml for tylvalosin). This study serves as evidence for the increasing resistance to macrolides and lincomycin in mycoplasmas, and also reports the occurrence of strains with extremely high MIC values to spectinomycin thus emphasizes the importance of the prudent use of antibiotics. Based on our results, tetracyclines and pleuromutilins are the most active compounds in vitro against the Hungarian M. hyorhinis strains.

Combination therapy of rabies-infected mice with inhibitors of pro-inflammatory host response, antiviral compounds and human rabies immunoglobulin.

Recent studies demonstrated that inhibitors of pro-inflammatory molecular cascades triggered by rabies infection in the central nervous system (CNS) can enhance survival in mouse model and that certain antiviral compounds interfere with rabies virus replication in vitro. In this study different combinations of therapeutics were tested to evaluate their effect on survival in rabies-infected mice, as well as on viral load in the CNS. C57Bl/6 mice were infected with Silver-haired bat rabies virus (SHBRV)-18 at virus dose approaching LD50 and LD100. In one experimental group daily treatments were initiated 4 h before-, in other groups 48 or 96 h after challenge. In the first experiment therapeutic combination contained inhibitors of tumour necrosis factor-α (infliximab), caspase-1 (Ac-YVAD-cmk), and a multikinase inhibitor (sorafenib). In the treated groups there was a notable but not significant increase of survival compared to the virus infected, non-treated mice. The addition of human rabies immunoglobulins (HRIG) to the combination in the second experiment almost completely prevented mortality in the pre-exposure treatment group along with a significant reduction of viral titres in the CNS. Post-exposure treatments also greatly improved survival rates. As part of the combination with immunomodulatory compounds, HRIG had a higher impact on survival than alone. In the third experiment the combination was further supplemented with type-I interferons, ribavirin and favipiravir (T-705). As a blood-brain barrier opener, mannitol was also administered. This treatment was unable to prevent lethal consequences of SHBRV-18 infection; furthermore, it caused toxicity in treated mice, presumably due to interaction among the components. In all experiments, viral loads in the CNS were similar in mice that succumbed to rabies regardless of treatment. According to the findings, inhibitors of detrimental host response to rabies combined with antibodies can be considered among the possible therapeutic and post-exposure options in human rabies cases.

Genotyping Mycoplasma hyopneumoniae isolates based on multi-locus sequence typing, multiple-locus variable-number tandem repeat analysis and analysing gene p146.

Mycoplasma hyopneumoniae is a swine pathogen bacterium, causing significant economic losses worldwide. Epidemiological investigations based on molecular typing methods support the prevention and eradication strategies for the control of M. hyopneumoniae, through tracing the spreading of the pathogen. The present study describes the genotyping of 44 M. hyopneumoniae strains isolated from Hungarian, Czech and Slovakian porcine lung samples by multi-locus sequence typing (MLST), multiple-locus variable-number tandem repeat analysis (MLVA) and analysing gene p146, and the evaluation of the used methods. The resolution of the three-gene (adk, rpoB, tpiA) and the seven-gene (efp, metG, pgiB, recA, adk, rpoB, tpiA) based MLST systems was identical with 27 sequence types. MLVA utilising loci P97-RR1 and Locus1 extended with the serine repeat numbers of gene p146 showed the highest resolution power among the studied methods differentiating 40 genotypes. The independent analysis of gene p146 revealed 31 different types among the isolates. High variability of M. hyopneumoniae strains was detected by the used typing methods. The results confirmed that utilization of the minimal MLST is suitable for phylogenetic analyses of M. hyopneumoniae strains. The MLVA method extended with the evaluation of serine repeat numbers of gene p146 is adequate for the resolution of genetic relationships within MLST groups. Examination of the p146 gene is suitable to complement both MLST and MLVA methods in order to refine closer genetic relationships.

Antibiotic susceptibility testing of Mycoplasma hyopneumoniae field isolates from Central Europe for fifteen antibiotics by microbroth dilution method.

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

Genotyping Mycoplasma synoviae: Development of a multi-locus variable number of tandem-repeats analysis and comparison with current molecular typing methods.

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

Genotyping of Brucella melitensis strains from dromedary camels (Camelus dromedarius) from the United Arab Emirates with multiple-locus variable-number tandem repeat analysis.

Camel brucellosis is a widespread zoonotic disease in camel-rearing countries caused by Brucella melitensis and Brucella abortus. The aim of this study was the first genetic analysis of B. melitensis strains isolated from dromedary camels (Camelus dromedarius) using multiple-locus variable-number tandem repeat analysis (MLVA). MLVA 16 and its MLVA 8 and MLVA11 subsets were used to determine the genotypes of 15 B. melitensis isolates from dromedary camels (11 strains) and other host species (4 strains) from the United Arab Emirates and the results were then compared to B. melitensis MLVA genotypes from other parts of the world. Five, including two novel genotypes were identified with MLVA 8. MLVA 16 further discriminated these five genotypes to ten variants. The eleven camel isolates clustered into four main genetic groups within the East-Mediterranean and African clades and this clustering correlated with the geographic origin of the hosts (United Arab Emirates, Kingdom of Saudi Arabia and Sudan) and the date of their isolation. The camel strains were also genetically related to strains isolated from wild and domestic ruminants from their close habitat or from other parts of the world. Although limited number of strains were analysed, based on our data imported animals from foreign countries, local small ruminants and wildlife species are hypothesized to be the main sources of camel brucellosis in the United Arab Emirates. MLVA was successfully applied to determine the epidemiological links between the different camel B. melitensis infections in the United Arab Emirates and it can be a beneficial tool in future disease control programs.

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.