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.

Genotypes and virulence factors in Staphylococcus aureus isolated from dairy cows with subclinical mastitis in Serbia.

This study aimed to determine the prevalence of Staphylococcus aureus subclinical mastitis and to genotype the S. aureus isolates using the 16S-23S rRNA intergenic spacer (RS-PCR) method. In addition, the genes responsible for adherence, biofilm formation, host evasion, tissue necrosis, methicillin resistance, and enterotoxin production of S. aureus were investigated. The overall prevalence of S. aureus subclinical mastitis in lactating cows was 5.4% (95% confidence interval, CI=4.7-6.1%). An increased risk of S. aureus intramammary infection was observed on small family farms (odds ratio, OR=4.2, 95% CI=2.6-6.6, P < 0.001) and medium-sized farms (OR=3.5, 95% CI=2.2-5.7, P < 0.001). The RS-PCR analysis revealed 44 genotypes and genotype variants, of which 15 new genotypes and five new variants were detected within small and medium-sized farms. S. aureus isolates of new genotypes and genotype variants carried the clfA gene responsible for adherence at a lower frequency (64.8%) and enterotoxin-producing genes sea (20.4%), seb (14.8%) and sec (14.8%) at a higher frequency than the other known genotypes (P < 0.001), and were confirmed to carry the sej and sep genes. The spa gene was detected in all S. aureus isolates, whereas none harbored bap, ser, or tsst-1 genes. Methicillin-resistant strains of S. aureus (MRSA) were also detected, with a higher prevalence (19.2%) on large farms with more than 50 cows (P < 0.001). Using molecular techniques as diagnostic tools provides a better understanding of intramammary staphylococcal infections' occurrence, spread, and eradication.

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 presence of deer ked (Lipoptena cervi, Linnaeus, 1758) in Balkan chamois from the National Park Sutjeska, Bosnia and Herzegovina.

This case study describes the presence of a deer ked (Lipoptena cervi, Linnaeus, 1758) in the Balkan chamois (Rupicapra rupicapra balcanica) from the area of the National Park "Sutjeska", Bosnia and Herzegovina (B&H). This is the first finding of this ectoparasite in this animal species in B&H, which indicates that deer ked has significant adaptive abilities to different animal hosts.

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.

Molecular detection of Babesia divergens and Mycoplasma wenyonii infection in cattle from Bosnia And Herzegovina.

We report two cases of bovine babesiosis caused by Babesia divergens in a region of central Bosnia and Herzegovina. The cases were detected in June 2017 and July 2018 from two small backyard farms. Routine clinical assessments, including physical examination and haematology, revealed lethargy, fever, anaemia, leukopenia and haemoglobinuria in the affected animals. Serum alterations included an elevation of aspartate aminotransferase and a decrease of serum phosphate or hypophosphatemia. Thrombocytopenia was detected in the first clinical case. Microscopic examination of blood smears revealed intracytoplasmic protozoan parasites from the genus Babesia. Molecular screening of both animals confirmed the presence of Babesia divergens, the causative agent of bovine babesiosis. B. divergens DNA was also detected in two engorged female Ixodes ricinus ticks removed from these animals. In addition, Mycoplasma wenyonii DNA was identified by molecular screening in the animal examined in June 2017, and in I. ricinus ticks feeding on this animal. This study provides molecular confirmation of B. divergens as a cause of piroplasmosis in cattle in South-East Europe. The detection of M. wenyonii DNA ain I. ricinus also provides the first evidence of this bacterium in ticks in Europe.

A case study of vertebral fusion in a 19th-century horse from Serbia.

OBJECTIVE: To evaluate the etiology of skeletal changes noted in equid vertebrae from a 19th- century context near Belgrade, Serbia. MATERIALS: A vertebral column consisting of 15 fused thoracic vertebrae (T2-T16), with right ribs fused to T4 and T5 and small remnants of ribs articulating with T4, T5 and T6 on the left side were accidentally recovered during industrial exploitation of sand. METHODS: The specimen was subjected to morphological analysis and collagen fingerprinting by mass spectrometry for species identification. In order to determine the absolute temporal context, radiocarbon dating was employed. Pathological changes were analyzed macroscopically and then underwent X-ray and (CT) imaging. RESULTS: Species identification indicates that the vertebral column belonged to a domestic horse (Equus caballus) living in the early 19th century. Pathological changes included exuberant bone proliferation, fusion of small articulations, enthesopathy formation, complete fusion between the vertebral bodies, and ossification of the anterior longitudinal ligament. CONCLUSIONS: Pathological changes represent signs of an advanced stage of vertebral fusion consistent with diffuse idiopathic skeletal hyperostosis (DISH). SIGNIFICANCE: This case study provides a clear distinction between diffuse idiopathic skeletal hyperostosis (DISH) and other vertebral column diseases in equids. It also presents a new and significant contribution to the nascent discipline of animal paleopathology in present-day Serbia. LIMITATIONS: Given that only 15 thoracic vertebrae were discovered, the impact of this disease on other parts of the horse skeleton remains unknown, as does the archaeological context of the remains. SUGGESTIONS FOR FURTHER RESEARCH: Research into the frequency of DISH in equids, as well as the historical context of equine husbandry in Serbia will allow greater insight into the causes and effects of this pathological condition.

Severe Verminous Pneumonia Caused by Natural Mixed Infection with Aelurostrongylus abstrusus and Angiostrongylus chabaudi in a European Wildcat from Western Balkan Area.

AIMS: Aelurostrongylus abstrusus (Railliet, 1898) and Angiostrongylus chabaudi (Biocca, 1957) are important cardiopulmonary metastrongyloids in felids. This case report describes, for the first time, a natural and patent mixed infection caused by A. abstrusus and A. chabaudi in a European wildcat (Felis silvestris silvestris) from Bosnia and Herzegovina. Most important, this is a rare report of fatal parasitism in wildlife, involving a severe verminous bronchopneumonia and gastrointestinal nematode and cestode infection with Toxocara cati, Taenia taeniaeformis, Aonchotheca putorii, and Ancylostoma spp. RESULTS: Emphasis is set to detailed description of granulomatous, interstitial verminous bronchopneumonia, morphological description of A. abstrusus and A. chabaudi, and molecular confirmation of diagnosis by triplex PCR. CONCLUSION: The data provided in this study contribute to the knowledge on the epizootiology and pathological effect of these neglected metastrongyloids in European wildcat.

Clinical Case of Respiratory Eucoleosis in a Dog from Bosnia and Herzegovina.

AIMS: This case report describes a clinical case of patent and natural infection with trichuroid Eucoleus aerophilus in a 5-month-old, mixed-breed female dog in Bosnia and Herzegovina. METHODS: A detailed history was taken from the owner. Physical exam and thoracic radiography were performed. Faecal sample was processed by the passive flotation tube test with saturated zinc sulphate solution. After the final diagnosis, a dose of 0.4 mg /kg ivermectin was applied subcutaneously to the diseased dog. RESULTS: The history revealed intermittent, non-productive cough after physical exercise. During the physical exam, mild signs of general distress, moderate cachexia with bad body condition, increased heart and respiratory rates, mild dyspnoea and increased bronchovesicular respirations were observed. Thoracic radiography showed mixed bronchial-alveolar lung pattern compatible with chronic bronchopneumonia. Trichuroid eggs of Eucoleus aerophilus were detected in faecal samples from the affected dog. Ivermectin administration at a dose of 0.4 mg/kg was sufficient to eliminate parasitic infection with Eucoleus aerophilus. CONCLUSION: This is the first report of respiratory eucoleosis in a dog from Bosnia and Herzegovina.

The first reported case of advanced aelurostrongylosis in Eurasian badger (Meles meles, L. 1758) in Bosnia and Herzegovina: histopathological and parasitological findings.

This paper represents the first description of advanced aelurostrongylosis in the Eurasian badger (Меles meles, L. 1758) from Bosnia and Herzegovina, which is an autochthonous species of the country. An adult female badger was found dead on a road; the cause of death was trauma but the emphasis in the paper is on severe verminous pneumonia caused by metastrongylids from genus Aelurostrongylus spp. This parasitological and histopathological finding confirms the presence of Aelurostrongylus in mustelids in Bosnia and Herzegovina and the Balkans.

Anthrax of domestic animals in Vrbas Banate: from traditional beliefs to the first scientific views on the ancient disease.

From 1929 to 1941, the Vrbas Banate was one of nine provinces of the Kingdom Yugoslavia, and according to historical data, the poorest one, without well-organized and sustainable agriculture production. Naturalistic production and poor animal health control in the Vrbas Banate were the most important risk factors for infectious disease spreading. Anthrax was very prevalent infectious disease in domestic animals and humans in that period, but some data on this disease remain scarce. In this paper epidemiology and clinical investigation of anthrax in the Vrbas Banate are reviewed. Apart from many aggravating factors that influenced the control of anthrax, the veterinary service of Banate contributed to the development of animal husbandry, animal health and public health in that period.

Spavin in red deer: A case study from the Early Neolithic Blagotin, Serbia.

Pathological modifications are rarely observed in the remains of wild animals from archaeological sites. We present one such specific, pathological change - a case of spavin in a red deer specimen from the Early Neolithic site of Blagotin, in central Serbia. The left tarsal joint presented proliferative new bone formation, which was analyzed macroscopically, then subjected to X-ray and computed tomography (CT) imaging. We assume that the initial degenerative changes in this red deer tarsal joint were probably caused by ageing, although the environment may have likely contributed to the progression of the disorder. Spavin usually results in stiffness of the joints and in lameness, perhaps contributing in the animal's capture by Neolithic hunters. This case is important in that it demonstrates that spavin is not necessarily a consequence of riding or traction work.

The first report of Trichinella pseudospiralis presence in domestic swine and T. britovi in wild boar in Bosnia and Herzegovina.

The Balkans is endemic for nematodes of the genus Trichinella in both domestic and wild animals. The high prevalence of these zoonotic pathogens in animals linked with the food habits to consume raw meat and meat derived products resulted in a very high prevalence of trichinellosis in humans living in this European region. In spite of numerous epidemiological investigations carried out in this region, very few information is available on the Trichinella species circulating in Bosnia and Herzegovina. Trichinella spp. larvae were isolated from a domestic pig reared in a backyard and from a hunted wild boar whose meat had been the source of trichinellosis in one case. Both Trichinella pseudospiralis and T. spiralis have been identified in the domestic pig, whereas, T. britovi was detected in the wild boar. While, T. spiralis is the Trichinella species most frequently detected in domestic pigs, T. pseudospiralis has been previously documented in domestic pigs only three times in Russia, Slovakia and Croatia. The detection of T. britovi in the wild boar confirms that this nematode is the most frequent species circulating among wildlife of Europe.

[Use of phytotherapy as a form of ethnoveterinary medicine in the area of Stara planina mountain in Serbia].

Ethnoveterinary medicine (EVM) considers implementation of folk knowledge and skills in animal health care, production and breeding. Also it means understanding of diagnostic, preventive, therapeutic skills and treatments in order to improve public health. EVM has a wide importance in the organic farming and environmental protection. Our studied geographic area is important because it is inhabited with Shops or Torlaks, a special ethnographic group of the South Slavs. Torlaks have developed special animal treatment and methods of animal breeding, particularly in sheep. The aim of this study was to examine the knowledge of ethno veterinary medicine in villagers from the eastern part of Stara planina in the Dimitrovgrad municipality, District of Pirot, to point out the specificity and importance of knowledge in relation to the geographic places and determine the applicability of knowledge in veterinary medicine and organic production. The research included 50 inhabitants from area of villages: Gornji Krivodol, Boljev Dol, Kamenica and Senokos. All villages are in the territory of the municipality of Dimitrovgrad, District of Pirot, Republic of Serbia. Performed methods were: non-structural, semi-structured and field interviews. According to the collected data, 64 different recipes and methods were noted, of which 43 include medicinal plants recipes, and other data include different methods of diagnosis, treatment, prevention and animal nutrition.