Institute for Veterinary Medical Research

Background: Vancomycin is a critically important antimicrobial in human medicine, and vancomycin-non-susceptible enterococci represent a One Health concern when animal reservoirs contribute to the wider resistance ecology. We aimed to characterize vancomycin non-susceptibility among poultry-derived Enterococcus spp. from Hungary, using a combined phenotypic-genomic approach. Methods: Following a phenotypic pre-screen with antimicrobials authorized for poultry, 218 isolates with elevated minimum inhibitory concentrations (MICs) were selected for extended broth microdilution testing including vancomycin. Vancomycin susceptibility was interpreted using Clinical and Laboratory Standards Institute (CLSI) clinical breakpoints and European Committee on Antimicrobial Susceptibility Testing (EUCAST) epidemiological cut-off values (ECOFFs). Whole-genome sequencing was performed on a targeted multidrug resistant (MDR) subset (n = 42), enriched for elevated or borderline vancomycin MICs and stratified by region and host species (chicken, turkey), and resistance determinants were annotated against the Comprehensive Antibiotic Resistance Database (CARD) using stringent similarity/coverage thresholds. Results: Among the 218 pre-screened isolates (126 from chickens; 92 from turkeys), 196 (89.9%) met MDR criteria. For vancomycin, 15.6% of isolates were resistant and 9.2% intermediate by CLSI, while EUCAST ECOFF classification placed 34.9% in the non-wild-type group. The vancomycin MIC distribution was right shifted, with high-end MICs observed. In the sequenced subset, vancomycin-associated determinants consistent with the vanC pathway (including regulatory and auxiliary components) were detected in five isolates. Beyond vancomycin-related determinants, the WGS subset harbored common resistance genes consistent with the observed multidrug-resistant phenotypes. Conclusions: Vancomycin non-susceptibility was detected among pre-screened poultry-derived Enterococcus isolates in Hungary, and genomic analysis revealed vanC-associated and other peptide antibiotic resistance signatures. These findings support targeted One Health surveillance integrating MIC distributions with genomic resistance determinants in food animal reservoirs.

The rapid evolution of coronaviruses (CoVs) requires researchers to develop specific yet broad-spectrum detection methods to monitor their constant genomic changes. The goal of the present study was to establish a current pan-coronavirus RT-PCR system capable of detecting a wide variety of CoVs and useful for the investigation of virus diversity and host spectrum. For optimization, one-step and two-step nested RT-PCRs with three RT enzymes were examined, amplifying a ~600 bp long product of the RNA-dependent RNA polymerase. As templates, the in vitro transcribed RNA of ten pathogenic CoVs (SARS-CoV, SARS-CoV-2, NL-63, OC43, feline CoV, porcine epidemic diarrhea virus or PEDV, transmissible gastroenteritis virus or TGEV, canine CoV, bat CoV, and infectious bronchitis virus) were applied instead of the often-used DNA standards. A limit of detection of 5-50 copies/reaction was achieved with a random hexamer-primed two-step RT-PCR and a touchdown cycling profile, representing a lower detection limit and higher specificity compared to previously published primer sets. Swine origin pooled samples (n = 121), collected from apparently healthy herds in Hungary, were tested with the novel RT-PCR system. Sequences of porcine respiratory CoV/TGEV and porcine hemagglutinating encephalomyelitis virus were identified in 24 oral fluid and nasal swab pools, demonstrating the circulation of these viruses in this country, as well as the suitability of the new PCR for their detection. The results highlighted the importance of adequate RT enzyme selection and the use of RNase inhibitors in sample preparation and conservation.

IntroductionAntimicrobial resistance (AMR) poses an escalating global health threat. Potentiated sulphonamides are widely used in both veterinary and human medicine. This study aimed to investigate the in vitro adaptation of Escherichia coli strains to increasing concentrations of potentiated sulphonamides, focusing on co-selection and the genetic mechanisms of resistance.MethodsThe MEGA-plate evolutionary model was used to expose E. coli ATCC 25922 to increased concentrations (0 × to 1000×) of a potentiated sulphonamide. Clones isolated from different concentration zones were analyzed for phenotypic resistance via minimum inhibitory concentration (MIC) testing and genotypically through next-generation sequencing.ResultsIn strains adapted to 1000 × potentiated sulphonamide, MIC values significantly increased for most tested antibiotics. Mutations were identified in key folate pathway genes (folP, folA), as well as in efflux pump regulator genes (emrR, marR, acrR, mdtM). These genetic changes indicated activation of multiple multidrug efflux systems, including acrAB-tolC, emrAB-tolC, and mdtEF-tolC. Mutations were also detected in genes associated with SOS response regulation (recN, recQ, uvrB), suggesting stress-induced genetic adaptation. In vitro microevolutionary adaptation to potentiated sulphonamide exposure induced broad genetic changes in E. coli, potentially driving cross-resistance through co-selection. The MEGA-plate method proved to be a robust tool for tracking resistance development and dissecting complex resistance mechanisms.DiscussionThese findings underscore the need for cautious use of combination antimicrobials, as they may elicit pleiotropic resistance responses beyond their intended targets.

Group A rotaviruses (RVAs) are a major cause of acute dehydrating diarrhea in infants and young animals worldwide. In rabbits, RVAs are associated with enteric disease, likely in combination with other pathogens. We report the identification and characterization of a lapine RVA strain in an Italian rabbit breeding farm. Increased mortality rates associated with enteric symptoms were reported in the facility in post-weaning rabbits around 40 days of age. By quantitative RT-PCR, an RVA strain was identified in the intestinal contents of deceased rabbits. A PCR-based enrichment protocol coupled with Nanopore sequencing allowed the reconstruction of the nearly complete genome of a rabbit RVA strain, Rabbit-wt/ITA/36-9/2022/G3P[14], with a genotype constellation (G3-P[14]-I2-R2-C2-M3-A9-N2-T6-E5-H3) conserved among lapine RVAs. Each of the 11 gene segments displayed high nucleotide identity and phylogenetic clustering with lapine rotavirus strains, as well as two Belgian human G3P[14] strains, which had been shown to have a zoonotic (lapine) origin. However, the NSP2 gene of strain 36-9 clustered closer with a group of rare human G3P[9] strains, suggesting a common path during their evolution. Gathering sequence data on animal RVAs is pivotal to reconstructing the history of homologous and heterologous RVAs in various mammals, including humans.

Heartworm disease-caused by Dirofilaria immitis-is an emerging mosquito-borne zoonotic disease with potentially fatal outcomes in dogs. Due to factors such as climate change, animal importation, and animal transport, the prevalence of the infection has increased in countries in Central and Eastern Europe, where the parasite was not detected before. The first autochthonous case of D. immitis in dogs in Hungary was reported in 2009. This study assessed the prevalence of D. immitis infection and the associated risk factors in domestic dogs in Hungary. A total of 1071 blood samples were collected between March 2023 and February 2024 and were tested using Knott's test, ELISA, and real-time PCR method. The countrywide prevalence of D. immitis was 17.0%, suggesting a continuous increase in the parasite's spread compared to the previously reported 11.3% prevalence, with age and spending time outdoors being the main risk factors. These results highlight the ongoing need for preventive measures against heartworm disease.

ASFV-G-ΔI177L is a modified-live African swine fever virus (ASFV) strain that has been incorporated into a commercially available vaccine. Its safety in pregnant sows and genetic stability in an in vivo passaging experiment were investigated. Upon inoculation of two pregnant sows with ASFV-G-ΔI177L, one developed moderate ASF-related clinical signs. In terms of reproductive performance, 43% of the offspring was born dead and the live-born piglets developed ASF-specific clinical signs, became viremic, and only 17% survived until the end of study. During passaging in pigs, ASFV-G-ΔI177L reverted to virulence with severe ASF-specific clinical signs at passages 3 and 4, associated with increased viremia. Whole genome sequencing identified C257L mutations as a potential driver of increased replication fitness and virulence. The data show that ASFV-G-ΔI177L is not genetically stable and, therefore not safe for use in ASF vaccines and suggest that ASF vaccine candidates should be tested for safety in pregnant animals.

Here we provide a comprehensive update on the diversity and genetic relatedness of adenoviruses occurring in rodents. Extensive PCR screenings revealed the presence of adenoviral DNA in samples originating from representatives of 17 rodent species from four different suborders of Rodentia. Distinct sequences of 28 different adenoviruses were obtained from the positive samples. Out of these, 20 were from hitherto unknown, putative novel adenoviruses, whereas 6 were variants of previously published murine adenoviruses. Additionally, two known viruses, guinea pig adenovirus 1 and squirrel adenovirus 1 were also detected. By PCR and primer walking, we determined the sequence of a considerable part of the genomic DNA of squirrel adenovirus 1, detected in red squirrel (Sciurus vulgaris) samples from Germany previously. We annotated the almost complete genome sequence of a novel mastadenovirus found by data mining in the bulk data of the Ord's kangaroo rat (Dipodomys ordii) genome project. We revisited the sequence of the gene of E1B 19K protein of mouse adenovirus 3. In contrast to the prototype strain, where a truncated version of this gene has been found, in our sample of mouse adenovirus 3, it seemed to be intact. Based on phylogeny reconstructions, all rodent adenoviruses clustered in the genus Mastadenovirus. Interestingly, however, there wasn't a common monophyletic clade encompassing every adenovirus of rodent origin. Instead, three major lineages were observed. Because two lineages contained viral sequences deduced from samples of three suborders, and one consisted almost exclusively of adenoviruses from the family Muridae, we hypothesize there has been a long-term coevolution with the rodent hosts, as a result of possible ancient host-switch events. Several putative viruses appeared in distinct branches further away from the three clades. Thus, the evolutionary past of the adenoviruses of rodents remains to be studied further.

Cochimibdella mexicana n. gen. n. sp. is described based on morphological data and comparative analysis of DNA sequences, including two mitochondrial (cytochrome oxidase subunit I and 12s rRNA) and nuclear (18S rRNA and 28S rRNA) loci. Leech specimens were found in the mantle cavity and external surface of the octopus Octopus bimaculatus from Bahía de los Ángeles, Baja California, México. In vivo observations of recently hatched leeches in the laboratory confirm a broad prey range, including fishes, the most common hosts of the family species. The new taxon differs from species of the family Piscicolidae in possession of weakly developed tubercles in a3 of the urosome. In addition, molecular analysis places Cochimibdella mexicana n. gen. n. sp. sister to Austrobdella californiana and Myzobdella lugubris. This work amends a previous record of the elasmobranch-feeding leech Pontobdella (=Stibarobdella) moorei as a parasite of Octopus bimaculatus from the type locality of the new taxon.