Maja Zdelar-Tuk

Characterisation of Brucella suis isolates from Southeast Europe by multi-locus variable-number tandem repeat analysis.

Porcine brucellosis is a common bacterial zoonosis which can cause significant financial losses. Its diverse and often complicated factors have hampered efforts to control disease spread. The aim of the study was to assess the epidemiological situation of porcine brucellosis primarily in Croatia and its relationship to genotypes present in other, mostly European countries. One hundred and seven Brucella suis strains isolated from swine, hares, cattle, humans, wild hares, a wild boar and a mare originating mainly from Croatia (112), but also a few from Slovenia, Bosnia and Herzegovina, Serbia and Macedonia (15) were tested using classical microbiological testing, Bruce-ladder, RFLP, Multiplex-suis and genotyped using multi-locus variable-number tandem repeat analysis (MLVA). We determined 43 Brucella suis genotypes. Strains were grouped according to phylogenetic and geographic relationships, revealing both regional specificity and uniqueness and suggesting possible sources and modes of spread among animals. Our study also confirmed problems with Bruce19 locus that may hinder comparisons of new types with those in the international database. Forty-one novel genotypes were identified and deposited into the international database. Our study supports the idea of wild animals as a source of disease in domestic animals and also gives evidence to hypothesis of cross-border animal trafficking between former Yugoslavian countries. It also highlights the need to expand such research across more of southeast Europe, especially to countries with poorer social and economical situation in order to prevent a realistic outbreak and for better understanding of the biology of this pathogen.

Identification of Coxiella burnetii genotypes in Croatia using multi-locus VNTR analysis.

Although Q fever affects humans and animals in Croatia, we are unaware of genotyping studies of Croatian strains of the causative pathogen Coxiella burnetii, which would greatly assist monitoring and control efforts. Here 3261 human and animal samples were screened for C. burnetii DNA by conventional PCR, and 335 (10.3%) were positive. Of these positive samples, 82 were genotyped at 17 loci using the relatively new method of multi-locus variable number tandem repeat analysis (MLVA). We identified 13 C. burnetii genotypes not previously reported anywhere in the world. Two of these 13 genotypes are typical of the continental part of Croatia and share more similarity with genotypes outside Croatia than with genotypes within the country. The remaining 11 novel genotypes are typical of the coastal part of Croatia and show more similarity to one another than to genotypes outside the country. Our findings shed new light on the phylogeny of C. burnetii strains and may help establish MLVA as a standard technique for Coxiella genotyping.

Evidence of Brucella strain ST27 in bottlenose dolphin (Tursiops truncatus) in Europe

Marine mammal brucellosis has been known for more than 20 years, but recent work suggests it is more widespread than originally thought. Brucella (B.) pinnipedialis has been isolated from pinnipeds, while B. ceti strains have been associated with cetaceans. Here we report a Brucella strain isolated from multiple lymph nodes of one bottlenose dolphin (Tursiops truncatus) during routine examination of dolphin carcasses found in the Croatian part of the northern Adriatic Sea during the summer of 2015. Classical bacteriological biotyping, PCR-based techniques (single, multiplex, PCR-RFLP) and 16S rRNA DNA sequencing were used to identify Brucella spp. Multiple-locus variable number tandem repeat analysis of 16 loci and multilocus sequence typing of 9 loci were used for genotyping and species determination. The combination of bacteriological, molecular and genotyping techniques identified our strain as ST27, previously identified as a human pathogen. This report provides, to our knowledge, the first evidence of ST27 in the Adriatic Sea in particular and in European waters in general. The zoonotic nature of the strain and its presence in the Adriatic, which is inhabited by bottlenose dolphins, suggest that the strain may pose a significant threat to human health.

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.

SWINE BRUCELLOSIS CAUSED BY Brucella suis BIOVAR 2 IN CROATIA

Brucellosis in swine was surveyed from 2011 to 2015 in 13 counties in Croatia. A total of 3230 breeding males were tested serologically, and positive reactions were confirmed in 42 (1.3%) males from 17 farms. A total of 641 sows with abortion or reproductive problems were tested, and positive reactions were confirmed in 34 (5.3%). Organs from 68 swine were tested for bacteria, and Brucella spp. was isolated from 47 (69.1%). B. suis was identified in 45 isolates from domestic swine and 2 isolates from wild boar in six counties in Croatia, and all isolates were found to be B. suis biovar 2 based on Bru-up/Bru-low, Bruce-ladder, Suis-ladder and RFLP-based PCR assays. These results indicate that brucellosis is difficult to eradicate in free-range and semi-free-range swine farming, particularly in areas where contact with wild boar is possible. Further disease control measures are required. Key words: Brucella suis biovar 2; swine; abortion; prevalence; Croatia BRUCELOZA PRASICEV, POVZROCENA Z BAKTERIJO Brucella suis BIOVAR 2 NA HRVASKEM Brucelozo pri prasicih smo spremljali od leta 2011 do leta 2015 v 13 hrvaskih obcinah. S serolosko analizo smo preverili prisotnost bruceloze pri 3230 samcih v razplodu. Pozitivne reakcije smo ugotovili pri skupno 42 merjascih s 17 farm, kar predstavlja 1,3 % živali. S seroloskimi testi smo preverili prisotnost protiteles proti bruceli tudi pri 641 plemenskih svinjah, ki so zvrgle ali imele težave z zabrejitvijo. Pozitivna reakcija je bila ugotovljena pri 34 svinjah, kar predstavlja 5,3 % vseh testiranih živali. Notranje organe 68 svinj iz sestih obcin smo uporabili za osamitev bakterij Brucella spp. Bakterije smo ugotovili pri 47 vzorcih (69,1 %). Bakterijo Brucella suis smo odkrili v vseh 47 vzorcih, izmed katerih jih je bilo 45 od domacih plemenskih svinj, dva vzorca pa sta bila od divjih svinj. Vse izolirane bakterije so pripadale sevu B. suis biovar 2, kot so pokazale dodatne analize z uporabo metod Bruup/Bru-low, Bruce-ladder, Suis-ladder in RFLP. Ti rezultati kažejo, da je popolno izkoreninjenje bruceloze težavno, se posebej v prostih rejah prasicev, kjer obstajajo možnosti stika z divjimi prasici. Zato bi bilo v prihodnje potrebno razmisliti o dodatnih nacinih nadzora nad to nevarno boleznijo prasicev. Kljucne besede: Brucella suis biovar 2; prasici; zvrg; pojavnost; Hrvaska

Bruceloza u morskih sisavaca, s posebnim osvrtom na Republiku Hrvatsku

After almost two decades of research and evidence of brucellosis in marine mammals, it has become widely known that this disease is prevalent in marine mammals on a global scale. Positive animals have been found in the northern Atlantic, Mediterranean Sea and in the Arctic, including the Barents Sea. Infected or exposed animals have been detected along the Atlantic and Pacific coasts of North America, off the coasts of Peru, Australia, New Zealand, Hawaii, the Solomon Islands, and in the Antarctic Ocean. Brucellosis most commonly occurs in the harbour porpoise (Phocoena phocoena), followed by the striped dolphin (Stenella coeruleoalba), Atlantic white-sided dolphin (Lagenorhynchus acutus), bottlenose dolphin (Tursiops truncatus), shortbeaked common dolphin (Delphinus delphis) and minke whale (Balaenoptera acutorostrata). A positive serological reaction has been confirmed in 35 species of whales and 14 species of seals, two subspecies of sea otters, one species of freshwater otter and the polar bear. To date, brucellosis has been found in marine mammals in the Mediterranean Sea in Italy and Spain. The first research on brucellosis in dolphins in the Republic of Croatia began in 2015. Brucella sp. was isolated from the lymph node of a bottlenose dolphin found dead in the Poreč region. This was the first evidence of brucellosis in dolphins in the Adriatic Sea. The isolated sample was identified as Brucella ceti strain ST27, making this the first record of this strain in Europe, which may present a significant threat to human health.