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.

Phenotypic and genetic insights into efflux pump mechanism in Mycoplasma anserisalpingitidis.

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.

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.

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.

Diversity of tick species and associated pathogens on peri-urban wild boars - First report of the zoonotic Babesia cf. crassa from Hungary.

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.

Identification and detection of mutations potentially associated with decreased susceptibility to macrolides and lincomycin in Mycoplasma anserisalpingitidis isolates.

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.

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.

Novel prophage-like sequences in Mycoplasma anserisalpingitidis.

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.

Multilocus sequence typing of the goose pathogen Mycoplasma anserisalpingitidis.

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.

Antimicrobial susceptibility of pathogenic mycoplasmas in chickens in Asia.

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 MIC90 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.

Development of molecular assays for the rapid and cost-effective determination of fluoroquinolone, macrolide and lincosamide susceptibility of Mycoplasma synoviae isolates.

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.

Mutations potentially associated with decreased susceptibility to fluoroquinolones, macrolides and lincomycin in Mycoplasma synoviae.

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.

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.

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.

Genotyping Mycoplasma gallisepticum by multilocus sequence typing.

Mycoplasma gallisepticum causes chronic respiratory disease and reproductive disorders in many bird species, resulting in considerable economic losses to the poultry industry. Maintenance of M. gallisepticum-free flocks is the most adequate method to control infection. To this end, monitoring systems and vaccination programs with live vaccine strains are applied worldwide. There is strong demand for efficient epidemiological investigation tools to distinguish M. gallisepticum strains in order to control disease. Up to now, multilocus sequence typing (MLST) has been regarded as gold standard for genotyping bacteria due to its good reproducibility and high discriminatory power. The aim of this study was to develop an MLST assay which can determine phylogenetic distances between M. gallisepticum strains. After analysing more than 30 housekeeping genes, six loci (atpG, dnaA, fusA, rpoB, ruvB, uvrA) were selected for the MLST assay due to their genomic location and high diversity. Examination of 130 M. gallisepticum strains with this MLST method yielded 57 unique sequence types (STs) with a 0.96 Simpson's index of diversity. Considering the large number of STs and high diversity index, this MLST method was found to be appropriate to discriminate M. gallisepticum strains. In addition, the developed method was shown to be suitable for epidemiological investigations, as it confirmed linkage between related strains from outbreaks in different farms. Besides, MLST also suggested high impact of extensive international trade on the spread of different M. gallisepticum strains. Furthermore this method can be used for differentiation among vaccine and field strains.

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.

P2X7 Receptors Drive Poly(I:C) Induced Autism-like Behavior in Mice.

Maternal immune activation (MIA) is a principal environmental risk factor contributing to autism spectrum disorder (ASD), which compromises fetal brain development at critical periods of pregnancy and might be causally linked to ASD symptoms. We report that endogenous activation of the purinergic ion channel P2X7 (P2rx7) is necessary and sufficient to transduce MIA to autistic phenotype in male offspring. MIA induced by poly(I:C) injections to P2rx7 WT mouse dams elicited an autism-like phenotype in their offspring, and these alterations were not observed in P2rx7-deficient mice, or following maternal treatment with a specific P2rx7 antagonist, JNJ47965567. Genetic deletion and pharmacological inhibition of maternal P2rx7s also counteracted the induction of IL-6 in the maternal plasma and fetal brain, and disrupted brain development, whereas postnatal P2rx7 inhibition alleviated behavioral and morphological alterations in the offspring. Administration of ATP to P2rx7 WT dams also evoked autistic phenotype, but not in KO dams, implying that P2rx7 activation by ATP is sufficient to induce autism-like features in offspring. Our results point to maternal and offspring P2rx7s as potential therapeutic targets for the early prevention and treatment of ASD.SIGNIFICANCE STATEMENT Autism spectrum disorder (ASD) is a neurodevelopmental psychiatric disorder caused by genetic and environmental factors. Recent studies highlighted the importance of perinatal risks, in particular, maternal immune activation (MIA), showing strong association with the later emergence of ASD in the affected children. MIA could be mimicked in animal models via injection of a nonpathogenic agent poly(I:C) during pregnancy. This is the first report showing the key role of a ligand gated ion channel, the purinergic P2X7 receptor in MIA-induced autism-like behavioral and biochemical features. We show that genetic or pharmacological inhibition of both maternal and offspring P2X7 receptors could reverse the compromised brain development and autistic phenotype pointing to new possibilities for prevention and treatment of ASD.

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.

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.

Analyses of separate and concatenated cox1 and 18S rRNA gene sequences indicate that the bat piroplasm Babesia vesperuginis is phylogenetically close to Cytauxzoon felis and the 'prototheilerid' Babesia conradae.

Babesia vesperuginis is the only piroplasm known to infect bats. Unlike most members of the genus Babesia, it is probably transmitted by a soft tick species (i.e. Argas vespertilionis). Recently, two studies have been conducted to clarify the phylogenetic status of this species, and both agreed on placing it into a basal position among Babesia sensu stricto (s.s.). However, several important groups of piroplasms were not included in the already reported phylogenetic trees of B. vesperuginis isolates. Therefore, the aim of the present study was to amplify an approx. 950-bp fragment of the cytochrome c oxidase subunit 1 (cox1) gene of B. vesperuginis from A. vespertilionis specimens, and to compare its sequences with those from other piroplasmid groups in a broader phylogenetic context. Sequence comparisons focusing on either 18S rRNA or cox1 genes, as well as phylogenetic analyses involving separate and concatenated 18S rRNA and cox1 sequences indicate that B. vesperuginis is more closely related to the phylogenetic group of Theileriidae than to Babesia s.s. In particular, B. vesperuginis clustered closest to Cytauxzoon felis and the 'prototheilerid' B. conradae. The results of this study highlight that B. vesperuginis is a unique and taxonomically important species, which should be included in future studies aimed at resolving the comprehensive phylogeny of Piroplasmida.