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.

First isolation and genotyping of Bartonella henselae from a cat living with a patient with cat scratch disease in Southeast Europe.

BACKGROUND: The bacterial genus Bartonella is distributed worldwide and poses a public health risk. Cat-scratch disease caused by B. henselae in Croatia was first described in 1957. It is present throughout the country: a survey of serum samples from 268 Croatian patients with lymphadenopathy showed that 37.7% had IgG antibodies. Despite this prevalence, we are unaware of reports of Bartonella culturing from infected humans or cats in Croatia or elsewhere in southeast Europe. CASE PRESENTATION: Here we describe the diagnosis of a 12-year-old child with lymphadenopathy in Croatia with cat-scratch disease based on antibody detection and clinical signs, and the subsequent culturing and genotyping of B.henselae from the cat's blood. The B. henselae isolate was grown on different blood agar plates and its identity was confirmed based on polymerase chain reaction (PCR) amplification of 16S ribosomal deoxyribonucleic acid (16S rDNA) and sequencing. Multi-locus sequence typing (MLST) identified the strain genotype as sequence type 5, commonly found zoonotic B. henselae strain in cats. The child recovered after azithromycin therapy, and B. henselae in the cat was eliminated within three months after doxycycline treatment. CONCLUSIONS: This is, to our knowledge, the first report of B. henselae culturing and MLST-based genotyping from cat's blood in southeast Europe. Our ability to detect B. henselae in blood through culturing but not PCR suggests that the prevalence of infected cats with low bacteremia is very high, suggesting the need to develop faster, more sensitive detection assays.

Molecular epidemiology of Brucella melitensis strains causing outbreaks in Croatia and Bosnia and Herzegovina.

The most recent data on the incidence of brucellosis in Southeast Europe prove the persistence of this zoonosis in the area, regardless of constant efforts at controlling it as one of the most dangerous zoonoses. Forty-three Brucella melitensis strains were collected from cattle, sheep, goats and humans from Croatia as well as Bosnia and Herzegovina between 2009 and 2015. The strains were identified and genotyped in order to determine their epidemiological background. Standard biotyping methods and Bruce-ladder were used to identify the strains. Genotyping was done using multilocus variable number tandem repeats analysis (MLVA) on 16 and multilocus sequence typing analysis (MLST) on nine loci. Results were compared to each other and to internationally available data. Twenty- five novel genotypes and two sequence types were identified. All tested strains, apart from vaccine and reference strains, showed very close phylogenetic and geographic relationships. The genotyping results indicate the endemicity of brucellosis in this region. MLST showed no variation, confirming the stability of housekeeping genes. The results confirm already established routes of disease spread in this area, showing that a more detailed and vigorous control of this zoonosis is necessary.

Melitococcosis in the republic of croatia.

BACKGROUND: Melitococcosis is one of the most widespread zoonoses worldwide. In the period from 2009 to 2013, comprehensive melitococcosis testing was conducted in the Republic of Croatia. METHODS AND RESULTS: During the testing, the Rose Bengal test was applied to 344019 blood samples of sheep and goats, and positive reactions were confirmed in 1143 (0.3%) of samples. The complement fixation test (confirmatory test) was conducted on 43428 samples, with positive reactions confirmed in 768 (1.8%) of samples. The organs and tissues of 336 sheep and goats were inspected bacteriologically, and Brucella sp. was isolated in 15 (4.5%) of samples. Positive serological and bacteriological reactions were confirmed in the Karlovac, Lika-Senj and Split-Dalmatia Counties. Bacteriological and molecular techniques (Bru-up/Bru-low and Bruce-Ladder) in isolates proved the presence of Brucella melitensis biovar 3. CONCLUSION: On the basis of this study, it can be concluded that Croatia has a favourable situation concerning the infection of ruminants with B. melitensis, and that ongoing controls of the disease are necessary.

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.

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.

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.

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.

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.

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.

The presence of methicillin-resistant Staphylococcus aureus on large pig breeding farms in Croatia.

Methicillin-resistant Staphylococcus aureus (MRSA) have emerged worldwide and have become resistant to a variety of antibiotics. MRSA colonisation in pigs was first reported from the Netherlands in 2005, where pigs were implicated as a source of human MRSA infections (Voss et al., 2005). This paper presents the first report on the presence of MRSA on large pig breeding farms in Croatia, together with the determination of the mecA gene, the results of spa typing and susceptibility to commonly used antimicrobials. Dust samples (7-11 per farm) were collected from eight large pig farms in Croatia. Of the total 68 swabs, the mecA gene was detected in 24 isolates growing on the MRSA agar. All isolates were resistant to oxacillin, tetracycline and streptomycin, and susceptible only to vancomycin, while 92% of the strains were susceptible to ciprofloxacin. Genotyping of the MRSA strains was performed by spa typing, and revealed t011 (n = 17), t034 (n = 5) and t1451 (n = 2). The results presented here predict that MRSA is present on a large number of pig farms in Croatia.

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.

Serological, bacteriological, and molecular diagnosis of brucellosis in domestic animals in Croatia.

AIM: To present the surveillance data on Brucella melitensis, B. suis, and B. ovis infection in cattle, sheep, goats, and swine in Croatia obtained in 2008 by serological, bacteriological, and molecular methods for diagnostics of brucellosis in domestic animals. METHODS: We serologically tested 42,785 cattle serums, 22,686 sheep and goat serums, and 28520 swine serums using the Rose Bengal test, complement fixation test, and various immunosorbent assays. We also tested 10,173 ram blood samples for B. ovis infection using the complement fixation test. Bacteriological examination was conducted on 214 samples collected from 34 serologically positive animals. Different molecular methods were employed in the identification and typing of 20 isolates from the samples. RESULTS: B. melitensis biovar (bv.) 3 was confirmed with different identification methods in 2 flocks in 2 Croatian counties and B. suis bv. 2 in 3 flocks in 3 counties. B. melitensis in cows was confirmed for the first time in Croatia. Infection with B. ovis was serologically confirmed in 202 rams in 12 counties. CONCLUSIONS: In 2008, the size of the brucellosis-affected area in Croatia and the efficiency of detection and prevention of brucellosis in sheep, goats, and swine were satisfactory. Infection with B. melitensis in cattle was confirmed for the first time and possible links for infection in humans were detected. More efficient measures for suppression and control of ovine epididymitis are required and a new strategy may be necessary for complete eradication of this disease.

The first report of Brucella melitensis Rev.1 human brucellosis in Bosnia and Herzegovina.

Brucellosis is an emergent and endemic zoonotic disease in Bosnia and Herzegovina. In this report we have diagnosed the first case of human brucellosis in Bosnia and Herzegovina, using molecular and microbiological tests, caused by live attenuated Brucella melitensis Rev.1 strain. The infection was caused through unintentional exposure to vaccination of small ruminants in Bosnia and Herzegovina and without any prior accidental self-injection of vaccine suspension.

First evidence of Brucella ovis infection in Republic of Croatia.

We researched the spread of Brucella ovis (B. ovis) infection in sheep during 2002 and 2003 in Croatia. A total of 30,635 sheep blood samples were examined using the enzyme-linked immunosorbent assay (ELISA). In 2002, 1014 out of 14,404 examined sheep blood samples (7%) from six counties gave positive reactions while 2060 (14.3%) were found suspicious. In 2003, 638 out of 16,221 examined sheep blood samples in nine counties (3.9%) tested positive while 1083 (6.7%) were suspicious. In rams and sheep that were serologically positive specific pathological changes were found in 68 (43.6%) out of 156 examined rams and in 5 (3.8%) out of 133 examined sheep. B. ovis was isolated from ram tissues from three counties and identified with classical microbiological procedures and the polymerase chain reaction (PCR). This research proves that Brucella ovis is present in sheep flocks in Croatia which is also the first proof of its existence in the country.

Disseminated tuberculosis in hyrax (Procavia capensis) caused by Mycobacterium africanum.

Tuberculosis due to Mycobacterium africanum was diagnosed in an adult female hyrax (Procavia capensis). Pathologic examination revealed disseminated tuberculous lesions. The same pathologic changes were also found in a male hyrax that died a year later. Both animals were imported from the United Arab Emirates and were held in captivity at the Zagreb Zoo in Croatia. The source of infection remains unknown. The acid-fast bacteria isolated from the lungs of the female hyrax were identifyed by polymerase chain reaction as Mycobacterium tuberculosis complex and Geno Type MTBC test confirmed the strain to be M. africanum I.

Epidemiological, Clinical and Molecular Characterization of Human Brucellosis in Bosnia and Herzegovina - An Ongoing Brucellosis Outbreak.

OBJECTIVE: The aim of this study was to evaluate an ongoing outbreak of brucellosis in southern region of Bosnia and Herzegovina (BIH) on the epidemiological, clinical and molecular level. PATIENTS AND METHODS: This study included 19 patients affected by brucellosis between 2015 and 2017, in Trebisevo (BIH). Out of 19 patients, 16 were admitted to and treated at the Department of Infectious diseases of the University Clinical Hospital Mostar, while three patients were treated in ambulatory care setting. Epidemiological, clinical and microbiological parameters were investigated. The Rose Bengal test (RBT) positive sera were serologically confirmed by complement fixation test (CFT). We also analyzed blood cultures, and isolates were additionally serotyped. Molecular analyses were performed with Bruce-ladder multiplex polymerase chain reaction (PCR) and multiple locus variable number of tandem repeat analysis of 16 loci (MLVA-16) assay. RESULTS: Fifteen out of 19 patients had been professionally exposed to the bacterium, while four patients acquired brucellosis without prior contact with infected animals. In seven out of eight (87.5%) patients with localized form of brucellosis, we detected significantly higher values of C-reactive protein (CRP) or erythrocyte sedimentation rate (P<0.001). B. melitensis was isolated from 13/16 (81.3%) blood culture samples, and additionally serotyped as biovar 3. Using MLVA16 assay, 11 isolates were genotyped. We observed complete genotype matches among 8/11 B. melitensis isolates, while 3/11 isolates differed in Bruce04 locus. CONCLUSION: Overall, our study confirms the usefulness of MLVA-16 method in the epidemiological and molecular research of brucellosis during epidemic that, most likely, originated from the same source.

New trends in human and animal leptospirosis in Croatia, 2009-2014.

Leptospirosis is a geographically widespread and globally underestimated zoonosis that affects humans and variety of animals. To identify trends and possible risk factors, joined medical and veterinary teams investigated epidemiology and epizootiology of leptospirosis in Croatia. Retrospective analysis of data obtained from referent diagnostic laboratories included a total of 1917 human and 123964 animal sera tested in the period from 2009 to 2014. We found high human leptospirosis average incidence rate of 1.53/100000 with clear predominance of male patients older than 40 years (sex ratio M/F:3.2; median age 51±15.1years). Statistical analysis revealed seasonal and annual variations of incidence in humans that were primarily associated with favourable weather conditions (temperature 10-19, 9°C and precipitation above 100mm/m2). Majority of infections in humans were caused by serogroups Sejroe, Australis and Icterohaemorrhagiae. Notable variations in seroprevalence and changing trends in prevailing serogroups were recorded in most of the domestic animals and during the entire period of investigation. All of the observed findings underline leptospirosis as a significant human and veterinary public health threat and emphasize the importance of continuous multidisciplinary surveillance. We also argue that only input from both professions improves our overall knowledge on leptospirosis and leads to better and more efficient prevention and control strategies.