Multilocus sequence analysis (MLSA) of a Nocardia cyriacigeorgica strain causing severe bovine mastitis in Bosnia and Herzegovina.

Nocardia cyriacigeorgica is a well-known agent of human nocardiosis and is considered an emerging pathogen, however, its identification to the species level is complex for many clinical laboratories. Available data on the clinical significance of N. cyriacigeorgica in veterinary medicine are sparse and mainly concern isolated reports of pyogranulomatous lesions in domestic animals. We report a case of severe bovine mastitis caused by N. cyriacigeorgica that did not respond to conventional antimicrobial therapy in a small holding in Bosnia and Herzegovina. After isolation of the pathogen, further identification by routine microbiological methods was not possible. Susceptibility to antimicrobials was tested using the disc diffusion method according to published recommendations. The sample was also tested by MALDI-ToF MS with inconclusive results. In addition, 16S rRNA sequence analysis, verified by multilocus sequence analysis (MLSA) using the gyrB, 16S rRNA, secA1, and hsp65 sequences, confirmed the species N. cyriacigeorgica. To our knowledge, this is the first report of isolation of N. cyriacigeorgica from a clinical case of bovine mastitis in a European dairy farm and the first MLSA method approach to distinguish a Nocardia spp. strain isolated from animals.

Development of multiplex PCR based NGS protocol for whole genome sequencing of West Nile virus lineage 2 directly from biological samples using Oxford Nanopore platform.

West Nile virus (WNV) can affect humans, birds, horses and another mammals, causing asymptomatic infection, mild febrile disease, neurological and systematic disease and death. In order to gain insight into the prevalence of WNV, a monitoring program has been established in the Republic of Serbia. Whole genome sequencing is essential for the molecular epizootiological analysis of virus entry and transmission routes, especially in high-risk regions. This paper describes the development of a multiplex PCR based NGS protocol for whole genome sequencing of WNV lineage 2 directly from biological samples using Oxford Nanopore (ONT) platform. The results obtained using this platform, confirmed by Sanger sequencing, indicate that this protocol can be applied to obtain whole sequences of the WNV genome, even when the virus concentration in the sample is medium, Ct value is approximately 30. The use of this protocol does not require prior virus isolation on cell culture nor the depletion of host nucleic acids.

The presence of malignant ovine babesiosis in Bosnia and Herzegovina indicates a possible emerging risk for Balkan region.

Malignant ovine babesiosis or ovine babesiosis caused by Babesia ovis is an important hemoprotozoan tick-borne disease of sheep. After 70 years, a study was conducted in Bosnia and Herzegovina, in the geographical areas of Podrinje and Eastern Herzegovina, aiming to molecularly identify and determine the presence of B. ovis infection in the sheep population. From 2019-2021, a clinical and molecular study was conducted in 53 flocks, and 192 sheep were examined, divided into two groups: clinically suspected and asymptomatic sheep. The presence of B. ovis was confirmed by PCR in blood samples of 75 % and 11.21 % of clinically affected and asymptomatic sheep, respectively. The majority of clinical cases of malignant ovine babesiosis were confirmed in the Rudo epidemiological unit (78.78 %) within the Podrinje region indicating typical seasonal pattern of disease occurrence and endemic focus. Rhipicephalus bursa was only tick species identified in Podrinje and Eastern Herzegovina. Acute form of disease was observed with dominant clinical signs of depression, fever, loss of appetite and respiratory distress. Fatal outcome was recorded in 12.28 % of cases. This study describes the molecular detection of B. ovis in sheep in Bosnia and Herzegovina for the first time. To the authors knowledge, this survey represents a report of the highest number of clinical cases of malignant ovine babesiosis in Europe. Since the disease has been recorded in the south-eastern border of Bosnia and Herzegovina, there is a high risk of disease transmission to a wider area of the Balkan region.

The detection and phylogenetic analysis of equine herpesviruses 1, 4 and 5 identified in nasal swab samples of asymptomatic horses from Serbia and Bosnia and Herzegovina.

Nasal swabs originating from 112 apparently clinically healthy and unvaccinated horses of different age, breed and from diverse rearing conditions from Serbia and Bosnia and Herzegovina were examined for the presence of equine herpesviruses 1, 4 and 5 using multiplex nested PCR (Mn­PCR) and virus isolation. The detected viruses were subsequently characterised by gB gene nucleotide sequencing and their phylogenetic analysis was performed. The infections with EHV­1, EHV­4, and EHV­5 in the examined horse populations are apparently chronic, subclinical and persistent, whilst the shedding of EHV­1 and EHV­5 was confirmed by their successful isolation. A connection was established between the finding of EHVs and rearing conditions since horses kept together in stables were positive for at least one EHV in contrast to animals held free grazing or individually. EHV­5 was found most often in younger horses, however descending in frequency in animals up to 10 years of age. The phylogenetic analysis showed that the identified EHV strains group mostly with Turkish and German strains of respective viruses. A certain degree of genetic heterogeneity was determined regarding the identified EHV­5 strains in contrast to EHV­1 and EHV­4.