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

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

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

Abstract 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

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

Establishment of a Mycoplasma hyorhinis challenge model in 5-week-old piglets

IntroductionMycoplasma hyorhinis is an emerging swine pathogen with high prevalence worldwide. The main lesions caused are arthritis and polyserositis, and the clinical manifestation of the disease may result in significant economic losses due to decreased weight gain and enhanced medical costs. We aimed to compare two challenge routes to induce M. hyorhinis infection using the same clinical isolate.MethodsFive-week-old, Choice hybrid pigs were inoculated on 2 consecutive days by intravenous route (Group IV-IV) or by intravenous and intraperitoneal routes (Group IV-IP). Mock-infected animals were used as control (control group). After the challenge, the clinical signs were recorded for 28 days, after which the animals were euthanized. Gross pathological and histopathological examinations, PCR detection, isolation, and genotyping of the re-isolated Mycoplasma sp. and culture of bacteria other than Mycoplasma sp. were carried out. The ELISA test was used to detect anti-M. hyorhinis immunoglobulins in the sera of all animals.ResultsPericarditis and polyarthritis were observed in both challenge groups; however, the serositis was more severe in Group IV-IV. Statistically significant differences were detected between the challenged groups and the control group regarding the average daily weight gain, pathological scores, and ELISA titers. Additionally, histopathological scores in Group IV-IV differed significantly from the scores in the control group. All re-isolated strains were the same or a close genetic variant of the original challenge strain.DiscussionOur results indicate that both challenge routes are suitable for modeling the disease. However, due to the evoked more severe pathological lesions and the application being similar to the hypothesized natural route of infection in Group IV-IV, the two-dose intravenous challenge is recommended by the authors to induce serositis and arthritis associated with M. hyorhinis infection

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

Abstract 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. Keywords: Antibiotic, China, Mycoplasma, Swan goose, Phallus inflammation, Venereal disease, Whole genom

Neplodnost u mliječnih krava – mogući bakterijski ili virusni uzroci

In this research uterine swab and biopsy samples were collected from 40 infertile dairy cows kept at five dairy cattle farms in Hungary. Samples were tested for bacteria including Coxiella burnetii chlamydiae, Mycoplasma and Ureaplasma, and for the viruses Bovine herpesvirus 1 (BoHV-1) and Bovine viral diarrhoea virus (BVDV). Chlamydiaceae DNA was detected by real-time PCR in 22/40 (55%) samples. Coxiella burnetii DNA was detected in 3/40 (7.5%) cases by real-time PCR. Mycoplasma and Ureaplasma DNA was found in 2/40 (5%) and 4/40 (10%) cows, respectively. BVD and BoHV-1 DNA was not detected in any samples. Escherichia coli as a recognised uterine pathogen was found in two cases. The following potential uterine pathogens were found: Bacillus licheniformis (one case), non- haemolytic streptococci (five cases), Histophilus somni (two cases) and Candida krusei (two cases). Blood samples were collected at same time as swab samples from all 40 cows, and their examination for C. burnetii antibodies by ELISA revealed seropositivity in 26/40 cows (65%). Histological examination of the uterine biopsy samples showed the presence of mild lympho-histiocytic infiltration in the mucosain 22 cases (59%). Moderatelympho-histiocytic infiltration of the endometrium was evident in 13 cases (35%), while in two cases (6%) severe inflammatory cell infiltration of the endometrium with lympho-histiocytes and neutrophil granulocytes was found. Although no statistical correlation could be demonstrated between the severity of histological lesions of the endometrium and the uterine pathogenicity of the bacteria (P = 0.8555), endometritis of a certain severity grade and/or a recognised or potential uterine pathogen were found in all samples. The latter may play a role in the development of infertility either collectively or independently.U ovom su istraživanju prikupljeni brisevi maternice i biopsijski uzorci 40 neplodnih mliječnih krava s pet mliječnih farmi u Mađarskoj. Uzorci su testirani na bakterije, uključujući; Coxiella burnetii, klamidiju, mikoplazmu i ureaplazmu te na viruse uključujući goveđi herpesvirus 1 (BoHV- 1) i virus virsnog proljeva goveda (BVDV). DNK Chlamydiaceae otkriven je PCR testom u stvarnom vremenu u 22/40 (55 %) uzoraka. DNK bakterije Coxiella burnetii otkriven je u 3/40 (7,5 %) slučajeva PCR testom u stvarnom vremenu. DNK mikoplazme i ureaplazme pronađen je u 2/40 (5 %), odnosno 4/40 (10 %) krava. DNK virusa BVD i BoHV-1 niti u jednom uzorku nije otkriven. Escherichia coli kao priznati maternični patogen pronađen je u dva slučaja. Pronađeni su sljedeći potencijalni maternični patogeni: Bacillus licheniformis (jedan slučaj), nehemolitički streptokoki (pet slučajeva), Histophilus somni (dva slučaja) i Candida krusei (dva slučaja). Uzorci krvi su istovremeno prikupljeni kad i brisevi od svih 40 pokusnih krava. Njihova pretraga na protututijela C. burnetii ELISA metodom otkrila je seropozitivnost u 26/40 krava (65 %). Histološka pretraga uzoraka biopsije maternice pokazala je prisutnost blage limfohistiocitotske infiltracije u sluznici u 22 slučaja (59 %). Umjerena limfohistiocitotska infiltracija endometrija bila je prisutna u 13 slučajeva (35 %), dok je u dva slučaja (6 %) otkrivena ozbiljna upalna stanična infiltracija endometrija s limfohistiocitima i neutrofilnim granulocitima. Premda nije bilo moguće dokazati statističku korelaciju između ozbiljnosti histoloških lezija endometrija i maternične patogenosti bakterija (P = 0,8555), endometrioza određenog stupnja ozbiljnost i/ ili priznati ili potencijalni maternični patogeni pronađeni su u svim uzorcima. Ovi posljednji mogu prouzročiti razvooj neplodnosti bilo skupno ili neovisno

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