Age-related presence and genetic diversity of Campylobacter spp. in young and adult yellow-legged gulls (Larus michahellis) in Croatia.

The epidemiology of Campylobacter species in wild birds is still poorly understood. This study describes the occurrence and genetic diversity of Campylobacter in adult and nestlings of yellow-legged gulls, highlighting differences between breeding locations. The gulls were captured in Croatia between 2021 and 2023. A cloacal swab was taken from each individual and tested for the presence of Campylobacter. Isolated Campylobacter species were genotyped using the multilocus sequence typing (MLST) method. A total of 1071 gulls were captured and sampled, of which 152 samples were identified as Campylobacter species, with Campylobacter jejuni (9.90%) being the most frequently isolated bacterium, followed by Campylobacter lari (3.36%) and Campylobacter coli (0.93%). Complete sequence type (ST) profiles were generated for 141 isolates: 100 C. jejuni, 33 C. lari, and 8 C. coli. A significant difference in the occurrence of positive Campylobacter species was found depending on the sampling sites, while both sampling site and age were significant for the occurrence of C. jejuni. Adults and nestlings showed high genetic diversity for C. jejuni and C. lari, and there were no significant differences between strains isolated from adults and nestlings or between sites, suggesting a high genotype flow in the studied gull population.

Isolation of Multidrug-Resistant Mycobacterium Avium Subsp. Avium from a Wild Eurasian Otter (Lutra Lutra).

Mycobacterium avium subsp. avium is pathogenic mainly to birds, although cases of mycobacteriosis caused by these bacteria have also been reported in other animals and humans. Not much is known about the effects of this pathogen on otters. The aim of this study was to report for the first time the isolation of M. avium subsp. avium in wild otter and to describe its multidrug resistance profile. A female otter injured in a car accident was found dead and subjected to postmortem examination. Apart from the trauma changes, no other macroscopic pathological changes were detected. Bacteriologic examination revealed the presence of acid-fast bacilli in the lymph nodes, which were confirmed by molecular methods as M. avium subsp. avium. Antimicrobial susceptibility testing revealed susceptibility to clarithromycin and amikacin, but resistance to linezolid, moxifloxacin, streptomycin, isoniazid, trimethoprim/sulfamethoxazole, ciprofloxacin, doxycycline, and ethionamide. This is unusual for wild species, which generally should not come into contact with antimicrobials, and may suggest that multidrug-resistant MAC strains are circulating between wild and domestic animals. These results emphasise the need for additional epidemiological studies on non-tuberculous mycobacteria in wildlife and their implications for one health.

Antimicrobial Resistance Profile, Whole-Genome Sequencing and Core Genome Multilocus Sequence Typing of B. anthracis Isolates in Croatia from 2001 to 2022.

Bacillus anthracis, the causative agent of anthrax disease, is a worldwide threat to livestock, wildlife and public health. It is also considered one of the most important pathogens of bioterrorism. Rapid and reliable diagnosis and administration of antimicrobials are essential for effective anthrax treatment. In this study, we determined the in vitro susceptibilities of 40 isolates of B. anthracis isolated in Croatia over the recent two decades to 18 antimicrobials. Whole-genome sequencing was performed, and bioinformatics tools were used to determine virulence factors and antimicrobial resistance genes. Core genome-based multilocus sequence typing was used for isolate comparison and phylogenetic analysis. All isolates were susceptible to all antimicrobials recommended for post-exposure prophylaxis or anthrax therapy. Susceptibility was found to all other tested antimicrobials that are an alternative for primary therapy. We found two beta-lactamase genes, but their expression is not sufficient to confer resistance. In all isolates used in this study, we found 21 virulence genes, 8 of which are responsible for toxin and capsule production. As far as phylogenetic analysis is concerned, the B. anthracis isolates from Croatia are categorised into two clades. The first is clade A, subclade Trans Eurasia, and the other is clade B, subclade B2.

Mycobacteriosis in a Pet Ferret (Mustela putorius furo) Caused by Mycobacterium xenopi: A Case Report on Neglected Risk of Zoonotic Transmission.

Ferrets are highly susceptible to a wide range of mycobacteria, mainly M. bovis, M. avium, and M. triplex. Therefore, ferrets pose a risk of transmission of mycobacteriosis, especially zoonotically relevant tuberculosis. The aim of this study was to describe the findings of M. xenopi mycobacteriosis in a pet ferret and emphasize its zoonotic potential. A pet ferret had a history of weight loss, apathy, hyporexia, and hair loss. Abdominal ultrasound revealed splenomegaly with two solid masses and cystic lesions of the liver. Fine-needle aspiration cytology revealed numerous acid-fast bacilli in epithelioid cells, thus leading to the suspicion of mycobacterial infection. Because of its poor general condition, the ferret was euthanized. Necropsy examination revealed generalized granulomatous lymphadenitis, pneumonia, myocarditis, splenitis, and hepatitis. Histologically, in all organs, there were multifocal to coalescing areas of inflammatory infiltration composed of epithelioid macrophages, a low number of lymphocytes, and plasma cells, without necrosis nor multinucleated giant cells. Ziehl-Neelsen staining detected the presence of numerous (multibacillary) acid-fast bacteria, which were PCR-typed as M. xenopi. This is the first study showing the antimicrobial susceptibility testing of M. xenopi in veterinary medicine, describing the resistance to doxycycline. Overall, our results could facilitate further diagnosis and provide guidelines for the treatment protocols for such infections.

Empathy and mental health in veterinary medicine.

In veterinary medicine, the relationship between empathy and mental health presents a complex and important aspect of professional well-being. Veterinarians are frequently exposed to numerous work-related stressors and are therefore more likely to experience mental health disorders and commit suicide. Due to the specific nature of the profession, veterinarians deal with negative patient outcomes, inform owners of unfavourable news, handle heavy workloads, and professional isolation. Psychological stress is a result of all these factors coming together, and it can lead to anxiety, depression, burnout, and even frequently reported suicide. Animal euthanasia has been recognised as a unique professional risk factor that can have harmful psychological effects on veterinary professionals.This paper explores the role of empathy in the mental health of veterinarians and other veterinary staff, and how this might contribute to their vulnerability to psychological stress and suicidal ideation. Empathy plays an important role in interpersonal interactions, while also influencing human-animal relationships, which adds a whole new level of complexity to the doctor-patient dynamic in this field. Veterinarians are responsible for providing compassionate care for both the animals they treat and their owners. They must manage the emotionally demanding work while preserving their mental health by balancing between providing empathetic care and sustaining their own emotional boundaries. To alleviate the negative effects of psychological stress, veterinary professionals require interventions such as peer support groups, stress management training, and mental health support programmes.

Epidemiology of Bartonella henselae infection in pet and stray cats in Croatia with risk factors analysis.

BACKGROUND: Cats are the primary reservoirs of the bacterium Bartonella henselae, the main cause of cat-scratch disease in humans. The main vector of the bacterium is the cat flea, Ctenocephalides felis. In southeastern Europe, data are lacking on the prevalence of B. henselae infection in cats, the strains of B. henselae involved and the risk factors associated with the infection. METHODS: Blood samples collected in ethylenediaminetetraacetic acid-containing tubes from 189 domestic cats (156 pet cats and 33 stray cats) from Zagreb, the capital city of Croatia, and 10 counties throughout Croatia were cultured for Bartonella spp. Following culture, bacterial isolates were genotyped at eight loci after using PCR to amplify 16S ribosomal RNA (rRNA) and the internal transcribed spacer region between the 16S and 23S rRNA sequences. Univariate and multivariate logistic regression were used to identify risk factors for B. henselae infection in cats. RESULTS: Bartonella spp. was detected in 31 cats (16.4%), and subsequent genotyping at the eight loci revealed B. henselae in all cases. Thirty complete multilocus sequence typing profiles were obtained, and the strains were identified as four sequence types that had been previously reported, namely ST5 (56.7%), ST6 (23.3%), ST1 (13.3%) and ST24 (3.3%), as well as a novel sequence type, ST33 (3.3%). The univariate analysis revealed a significantly higher risk of B. henselae infection in cats residing in coastal areas of Croatia (odds ratio [OR] 2.592, 95% confidence interval [CI] 1.150-5.838; P = 0.0191) and in cats with intestinal parasites (OR 3.207, 95% CI 1.088-9.457; P = 0.0279); a significantly lower risk was identified in cats aged > 1 year (OR 0.356, 95% CI 0.161-0.787; P = 0.0247) and in cats sampled between April and September (OR 0.325, 95% CI 0.147-0.715; P = 0.005). The multivariate analysis that controlled for age showed a positive association with the presence of intestinal parasites (OR 4.241, 95% CI 1.243-14.470; P = 0.0119) and coastal residence (OR 2.567, 95% CI 1.114-5.915; P = 0.0216) implying increased risk of infection, and a negative association with sampling between April and September (OR 0.379, 95% CI 0.169-0.848; P = 0.018) implying a decreased risk of infection. After controlling for the season, an increased risk of infection remained for the coastal region (OR 2.725, 95% CI 1.200-6.186; P = 0.012). CONCLUSIONS: Bartonella henselae is prevalent throughout Croatia and is a public health threat. Environmental and host factors can significantly affect the risk of infection, and these should be explored in more detail. The presence of intestinal parasites highlights the need to eliminate the flea vector, Ctenocephalides felis, as the most effective approach to control infections in cats and humans.

First Report and Characterization of the mcr-1 Positive Multidrug-Resistant Escherichia coli Strain Isolated from Pigs in Croatia.

The emergence and rapid spread of the plasmid-mediated colistin-resistant mcr-1 gene introduced a serious threat to public health. In 2021, a multi-drug resistant, mcr-1 positive Escherichia coli EC1945 strain, was isolated from pig caecal content in Croatia. Antimicrobial susceptibility testing and whole genome sequencing were performed. Bioinformatics tools were used to determine the presence of resistance genes, plasmid Inc groups, serotype, sequence type, virulence factors, and plasmid reconstruction. The isolated strain showed phenotypic and genotypic resistance to nine antimicrobial classes. It was resistant to colistin, gentamicin, ampicillin, cefepime, cefotaxime, ceftazidime, sulfamethoxazole, chloramphenicol, nalidixic acid, and ciprofloxacin. Antimicrobial resistance genes included mcr-1, blaTEM-1B, blaCTX-M-1, aac(3)-IId, aph(3')-Ia, aadA5, sul2, catA1, gyrA (S83L, D87N), and parC (A56T, S80I). The mcr-1 gene was located within the conjugative IncX4 plasmid. IncI1, IncFIB, and IncFII plasmids were also detected. The isolate also harbored 14 virulence genes and was classified as ST744 and O101:H10. ST744 is a member of the ST10 group which includes commensal, extraintestinal pathogenic E. coli isolates that play a crucial role as a reservoir of genes. Further efforts are needed to identify mcr-1-carrying E. coli isolates in Croatia, especially in food-producing animals to identify such gene reservoirs.

Antimicrobial Resistance in Rapidly Growing Nontuberculous Mycobacteria among Domestic and Wild Animals Emphasizing the Zoonotic Potential.

Non-tuberculous mycobacteria (NTM) are opportunistic pathogens capable of causing infections in humans and animals. The aim of this study was to demonstrate the potential role of domestic and wild animals as a reservoir of multiple resistant, rapidly growing NTM strains representing a potential zoonotic threat to humans. A total of 87 animal isolates belonging to 11 rapidly growing species (visible colonies appear within three to seven days) were genotyped and tested for susceptibility to the 15 most commonly used antibiotics in the treatment of such infections in a human clinic. By determining the antimicrobial susceptibility, the most prevalent resistance was found to cephalosporins (>50%), followed by amoxicillin-clavulanate (31.0%), clarithromycin (23.0%), tobramycin (14.9%) and doxycycline (10.3%). Resistance to imipenem, ciprofloxacin, minocycline and linezolid was notably lower (<7.0%). All tested isolates were susceptible to amikacin and moxifloxacin. The most frequent resistance was proved in the most pathogenic species: M. fortuitum, M. neoaurum, M. vaccae and M. porcinum. Meanwhile, other species displayed a higher sensitivity rate. No significant resistance differences between domestic and wild animals were found. The established significant frequency of resistance highlights the significant zoonotic potential posed by circulating rapidly growing NTM strains, which could lead to challenges in the treatment of these infections.

Risk of zoonoses involving slow-growing non-tuberculous mycobacteria: Survey of antimicrobial resistance among strains from domestic and wild animals.

OBJECTIVES: Non-tuberculous mycobacteria are opportunistic pathogens that cause disease mainly in immunocompromised hosts. The present study assessed the prevalence of antibiotic resistance among such mycobacteria from domestic and wild animals in Croatia sampled during several years within a national surveillance program. METHODS: A total of 44 isolates belonging to nine slow-growing species were genotyped and analyzed for susceptibility to 13 antimicrobials often used to treat non-tuberculous mycobacterial infections in humans. RESULTS: Most prevalent resistance was to moxifloxacin (77.3%), doxycycline (76.9%), and rifampicin (76.9%), followed by ciprofloxacin (65.4%), trimethoprim-sulfamethoxazole (65.4%), and linezolid (61.4%). Few isolates were resistant to rifabutin (7.7%) or amikacin (6.8%). None of the isolates was resistant to clarithromycin. Nearly all isolates (86.4%) were resistant to multiple antibiotics. CONCLUSION: Our findings suggest substantial risk that human populations may experience zoonotic infections with non-tuberculous mycobacteria that will be difficult to treat using the current generation of antibiotics. Future work should clarify how resistance emerges in wild populations of non-tuberculous mycobacteria.

Genetic Diversity and Resistome Analysis of Campylobacter lari Isolated from Gulls in Croatia.

Campylobacter lari is a thermotolerant bacterium that sporadically causes gastrointestinal diseases in humans and can be found in wildlife and the environment. C. lari is an understudied species, especially in wild birds such as gulls. Gulls are potentially good carriers of pathogens due to their opportunistic behavior and tendency to gather in large flocks. During winter and their breeding period, 1753 gulls were captured, and cloacal swabs were taken to be tested for the presence of C. lari. From isolated bacteria, the DNA was sequenced, and sequence types (ST) were determined. Sixty-four swabs were positive for C. lari, and from those, forty-three different STs were determined, of which thirty-one were newly described. The whole genome was sequenced for 43 random isolates, and the same isolates were tested for antimicrobial susceptibility using the broth microdilution method to compare them to WGS-derived antimicrobial-resistant isolates. All the tested strains were susceptible to erythromycin, gentamicin, and chloramphenicol, and all were resistant to ciprofloxacin. Resistance to ciprofloxacin was attributed to a gyrA_2 T86V mutation. Genes connected to possible beta-lactam resistance (blaOXA genes) were also detected.

Coxiella burnetii (Q-fever) outbreak associated with non-occupational exposure in a semi-urban area of western Croatia in 2022.

In March 2022, an outbreak of Q fever (Coxiella burnetii) with non-occupational exposure was confirmed in a semi-urban area in Cavle, Croatia. Veterinary and human epidemiological investigations were conducted to identify the source of the outbreak and to implement appropriate control measures. Three farms were settled next to each other near the homes of the first human cases at the end of the street. The closest farm was less than 500 meters away. These farms contained 161 adult sheep and goats. Among the animal samples analysed, all 16 goats (100%) and 24/50 sheep (48%) tested positive for C. burnetii IgM/IgG antibodies, phase I and II. One out of five sheeps' vaginal swabs were C. burnetti DNA positive. Human testing revealed 20 confirmed and three probable cases (9/23 pneumonia, 2/23 hepatitis, 21/23 fever), with three hospitalizations, and one death. Twenty-seven cases were discarded following negative laboratory results. The epidemiological investigation revealed airborne transmission as the most likely route of transmission. Multiple logistic regression analyses were used to evaluate risk factors for Q fever infection. Persons who were near the farms (≤750 m) (OR 4.5; 95% CI = 1.1-18.3) and lived in the nearest street to the farms had the highest risk of contracting Q fever (OR 3.7; 95% CI = 1.1-13.6). Decreased rainfall compared to monthly averages was recorded in the months prior to the outbreak with several days of strong wind in January preceding the outbreak. This was the largest Q fever outbreak in the county in the last 16 years, which was unexpected due to its location and non-occupational exposure. To stop the outbreak, numerous intensive biosecurity measures were implemented. The outbreak highlights the importance of urban development strategies to limit the number of animal housing near residential areas while providing regular biosecurity measures to prevent infections in livestock.

Description of Mycobacterium pinniadriaticum sp. nov., isolated from a noble pen shell (Pinna nobilis) population in Croatia.

Introduction: Shortly before the mass mortality event of the noble pen shell (Pinna nobilis) population in the south-eastern Adriatic coast, two rapidly growing Mycobacterium strains CVI_P3T (DSM 114013 T, ATCC TSD-295 T) and CVI_P4 were obtained from the organs of individual mollusks during the regular health status monitoring. Methods: The strains were identified as members of the genus Mycobacterium using basic phenotypic characteristics, genus-specific PCR assays targeting the hsp65 and 16S rRNA genes and the commercial hybridization kit GenoType Mycobacterium CM (Hain Lifescience, Germany). MALDI-TOF mass spectrometry did not provide reliable identification using the Bruker Biotyper Database. Results and discussion: Genome-wide phylogeny and average nucleotide identity (ANI) values confirmed that the studied strains are clearly differentiated from their closest phylogenetic relative Mycobacterium aromaticivorans and other validly published Mycobacterium species (ANI ≤ 85.0%). The type strain CVI_P3T was further characterized by a polyphasic approach using both phenotypic and genotypic methods. Based on the phenotypic, chemotaxonomic and phylogenetic results, we conclude that strains CVI_P3T and CVI_P4 represent a novel species, for which the name Mycobacterium pinniadriaticum sp. nov. is proposed.

Prevalence of Virulence Genes Among Escherichia Coli Strains Isolated from Food and Carcass Swabs of Different Animal Origins in Croatia.

Introduction: Escherichia coli is present in the normal intestinal flora but some strains can cause intestinal and extraintestinal diseases, and research on its presence in food of animal origin is in the interests of public health. This study was designed to characterise E. coli strains according to their origin, their carriage of virulence genes specific for certain pathogroups, and phylogenetic group affiliation. Material and Methods: The study was carried out on 100 E. coli strains isolated from food samples of various animal origin as well as pig and cattle carcass swabs. Isolation of the strains was performed using two methods. One method included colony count and the other an overnight enrichment of the samples. Isolation was followed by DNA extraction and detection of virulence genes and phylogenetic group with conventional and multiplex PCRs. Results: In this study, the most prevalent gene was EAST1 (20%) and strains which carried it were identified as enteroadherent E. coli. Other pathogroups were represented in lower incidences. Phylogenetic group analysis revealed the prevalence of the A and B1 groups, with B1 mainly present in game and cattle strains, while the majority of pig and poultry strains were assigned to group A. Conclusion: This study provides an overview of the presence of potentially pathogenic strains and E. coli phylogenetic groups in Croatia, for which the data are limited. Further microbiological and molecular research is required to examine the epidemiological situation in the country.

Resistome Analysis of Campylobacter jejuni Strains Isolated from Human Stool and Primary Sterile Samples in Croatia.

Campylobacteriosis represents a global health challenge due to continuously increasing trends of antimicrobial resistance in Campylobacter jejuni. C. jejuni can sometimes cause life-threatening and severe systematic infections (bacteremia, meningitis, and other extraintestinal infections) with very few antibiotics left as treatment options. Bearing in mind that C. jejuni is the predominant species in humans, in this paper, we present a study of the C. jejuni differences in antimicrobial resistance and genotype distribution between strains isolated from stool and primary sterile sites. We compared the genomic data obtained through whole genome sequencing (WGS) and phenotypic susceptibility data of C. jejuni strains. Once antimicrobial susceptibility testing of C. jejuni strains was carried out by the broth microdilution method for six of interest, results were compared to the identified genotypic determinants derived from WGS. The high rate of resistance to fluoroquinolones presented in this study is in accordance with national surveillance data. The proportion of strains with acquired resistance was 71% for ciprofloxacin and 20% for tetracycline. When invasive isolates were analysed separately, 40% exhibited MIC values of ciprofloxacin higher than the ECOFFs, suggesting a lower flouroquinolone resistance rate in invasive isolates. All isolates demonstrated wilde-type phenotype for chloramphenicol, erythromycin, gentamicin, and ertapenem. A special focus and review in this study was performed on a group of C.jejuni strains found in primary sterile samples. Apart from demonstrating a lower resistance rate, these isolates seem genetically more uniform, showing epidemiologically more homogenous patterns, which cluster to several clonal complexes, with CC49 being the most represented clonal complex.

Antimicrobial resistance profiles of human Brucella melitensis isolates in three different microdilution broths: the first multicentre study in Bosnia and Herzegovina.

OBJECTIVES: Brucellosis is a ubiquitous emergent bacterial zoonotic disease causing significant human morbidity in Bosnia and Herzegovina. So far, a high rate of resistant Brucella has been found worldwide. This study prospectively analysed the rates of resistance among human Brucella melitensis strains isolated in Bosnia and Herzegovina. METHODS: This study included 108 B. melitensis isolates from 209 patients diagnosed at five medical centres in Bosnia and Herzegovina. The resistance profiles of the B. melitensis isolates for the 13 most commonly used antimicrobials were studied in standard Brucella broth (BB) and cation-adjusted Mueller-Hinton broth (CAMHB) supplemented with 4% lysed horse blood or 5% defibrinated sheep blood. RESULTS: Of the 209 patients, B. melitensis blood cultures were positive for 111 (53.1%). Among the 108 isolates investigated, 91 (84.3%) were resistant to trimethoprim-sulfamethoxazole on BB, but not on either CAMHB. Nearly all isolates (>90%) were resistant to azithromycin on BB and both CAMHBs. CONCLUSION: We observed a high rate of B. melitensis resistance to azithromycin. The high rate of resistance to trimethoprim-sulfamethoxazole that we observed was related to BB, so an alternative broth should be used, such as the enriched CAMHBs in this study, for evaluating resistance to trimethoprim-sulfamethoxazole. Whole-genome sequencing studies are needed to understand the development of antimicrobial resistance in B. melitensis strains isolated from humans.

A Suggested Diagnostic Approach for Sporadic Anthrax in Cattle to Protect Public Health.

The repeated occurrence of anthrax in grazing animals should be a reminder of a widespread presence of Bacillus anthracis spores in the environment. Its rapid diagnosis is critical to protect public health. Here, we report a case of anthrax in cattle that was investigated using conventional and molecular methods. In 2015, six cows suddenly died within three days and the number of dead animals increased to a total of 12 within two weeks. At necropsy, anthrax was suspected. Therefore, spleen tissue samples were collected (from 6/12 animals) and laboratory tests (microscopy, cultivation, and real-time PCR) performed. The results of tissue staining for microscopy and cultivation were in congruence, while B. anthracis real-time PCR outperformed both. Spleen tissues from all six animals were real-time PCR-positive, while B. anthracis was successfully cultivated and detected by microscopy from the spleen of only three animals. Additionally, the ear tissue from another (1/12) cow tested positive by real-time PCR, supporting the suitability of ear clippings for molecular confirmation of B. anthracis. Genotyping of the isolates using multiple-locus variable-number tandem repeat analysis (MLVA) revealed a common source of infection as all three typed isolates had an indistinguishable MLVA genotype, which has not been observed previously in Europe. The results indicate that molecular testing should be selected as the first-line tool for confirming anthrax outbreaks in animals to ensure timely protection of public health.

Multi Locus Sequence Typing and spa Typing of Staphylococcus Aureus Isolated from the Milk of Cows with Subclinical Mastitis in Croatia.

BACKGROUND: The bacterial species S. aureus is the most common causative agent of mastitis in cows in most countries with a dairy industry. The prevalence of infection caused by S. aureus ranges from 2% to more than 50%, and it causes 10-12% of all cases of clinical mastitis. AIM: The objective was to analyze 237 strains of S. aureus isolated from the milk of cows with subclinical mastitis regarding the spa, mecA, mecC and pvl genes and to perform spa and multi-locus sequence typing (MLST). METHODS: Sequencing amplified gene sequences was conducted at Macrogen Europe. Ridom StaphType and BioNumerics software was used to analyze obtained sequences of spa and seven housekeeping genes. RESULTS: The spa fragment was present in 204 (86.1%) of strains, while mecA and mecC gene were detected in 10 strains, and the pvl gene was not detected. Spa typing successfully analyzed 153 tested isolates (64.3%), confirming 53 spa types, four of which were new types. The most frequent spa type was t2678 (14%). MLST typed 198 (83.5%) tested strains and defined 32 different allele profiles, of which three were new. The most frequent allele profile was ST133 (20.7%). Six groups (G) and 15 singletons were defined. CONCLUSION: Taking the number of confirmed spa types and sequence types (STs) into account, it can be concluded that the strains of S. aureus isolated from the milk of cows with subclinical mastitis form a heterogenous group. To check the possible zoonotic potential of isolates it would be necessary to test the persons and other livestock on the farms.

New Brucella variant isolated from Croatian cattle.

BACKGROUND: A novel Brucella strain closely related to Brucella (B.) melitensis biovar (bv) 3 was found in Croatian cattle during testing within a brucellosis eradication programme. CASE PRESENTATION: Standardised serological, brucellin skin test, bacteriological and molecular diagnostic screening for Brucella infection led to positive detection in one dairy cattle herd. Three isolates from that herd were identified to species level using the Bruce ladder method. Initially, two strains were typed as B. melitensis and one as B. abortus, but multiplex PCR based on IS711 and the Suis ladder showed that all of them to belong to B. melitensis, and the combination of whole-genome and multi-locus sequencing as well as Multi-Locus Variable numbers of tandem repeats Analysis (MLVA) highlighted a strong proximity within the phylogenetic branch of B. melitensis strains previously isolated from Croatia, Albania, Kosovo and Bosnia and Herzegovina. Two isolates were determined to be B. melitensis bv. 3, while the third showed a unique phylogenetic profile, growth profile on dyes and bacteriophage typing results. This isolate contained the 609-bp omp31 sequence, but not the 723-bp omp31 sequence present in the two isolates of B. melitensis bv. 3. CONCLUSIONS: Identification of a novel Brucella variant in this geographic region is predictable given the historic endemicity of brucellosis. The emergence of a new variant may reflect a combination of high prevalence among domestic ruminants and humans as well as weak eradication strategies. The zoonotic potential, reservoirs and transmission pathways of this and other Brucella variants should be explored.

Occurrence of Campylobacter jejuni in Gulls Feeding on Zagreb Rubbish Tip, Croatia; Their Diversity and Antimicrobial Susceptibility in Perspective with Human and Broiler Isolates.

Campylobacteriosis is the most common gastrointestinal bacterial disease in the European Union (EU). Wild birds are one of the natural reservoirs of these pathogens. In this study we tested cloacal swabs of 643 gulls captured on rubbish tip in Zagreb, Croatia for the presence of Campylobacter spp. and found 168 Campylobacter positive samples. We used multilocus sequence typing (MLST) to genotype 62 random C. jejuni isolates from gulls, 24 isolates from broiler caeca, 27 isolates from broiler neck skins and 23 human isolates. Altogether, we identified 44 different STs, from which 19 were newly described. Most of the new STs (14) originate from gulls. Although humans and broilers share the majority of STs and isolates from gulls are separated from these, there was one ST present in all three hosts: 45. Additionally antimicrobial susceptibility to six antimicrobials was performed on 123 C. jejuni strains isolated from broiler caeca (n = 22), neck skins of broilers (n = 20), gulls cloacal swabs (n = 50) and human faeces (n = 31) by the broth microdilution method. Results show lower resistance of gull isolates to NAL and CIP, while resistance to TET was as high as in human and broiler isolates.

First reports of indigenous lethal infection with Anaplasma marginale, Anaplasma bovis and Theileria orientalis in Croatian cattle.

Anaplasmosis and theileriosis are important diseases with great economic impact that affect the cattle industry worldwide. In this study, we describe the first molecularly confirmed clinical cases of anaplasmosis caused by Anaplasma marginale and of a concurrent infection with A. bovis and Theileria orientalis in Croatian cattle. Clinical signs of A. marginale-infected cows included fever, lethargy, dark urine, as well as icteric and reddish mucous membranes. Postmortem examination revealed icterus, urinary bladder filled with dark urine and splenomegaly. A marginale was observed within erythrocytes on Giemsa-stained spleen imprints. Three affected cows were successfully treated with oxytetracycline, after which no new deaths occurred in the herd. Three cows in a different herd died suddenly and were found to be concurrently infected with A. bovis and T. orientalis. Postmortem examination revealed generalized icterus and urinary bladder filled with dark urine. These cases of A. marginale, A. bovis and T. orientalis infection show that bovine anaplasmosis and theileriosis are present within Croatian cattle and should be included in differential diagnostic protocols.