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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.

In March 2022, an outbreak of Q fever (Coxiella burnetii) with non-occupational exposure was confirmed in a semi-urban area in Čavle, 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

Brucella , an extremely diverse but yet genetically highly homogenous genus of bacteria, has been a puzzle for scientists for many decades. These bacteria remain a prominent public health issue, particularly in the Balkan region. Correctly identifying and understanding the pathogen is a vital step in the epidemiology and epizootiology of any bacteria. Identification can be challenging, especially in the case of zoonotic species. This study aimed to implement fourth-generation sequencing in the typing of 11 Brucella suis strains kept in our archive and to compare this method to the classical and non-sequencing based molecular methods used to date. Classical biotyping is highly subjective and gave inconclusive results for 3 strains. Molecular methods used were multiplex PCR and RFLP methods since no one method can identify both species and biovar which is vital in the case of Brucella suis infections. Species and biovars of all the strains were successfully confirmed and in concordance with biotyping results. Oxford Nanopore long-read sequencing was used on a MinION device for next-generation sequencing (NGS). Various algorithms were implemented for genome assembly and BioNumerics 8.0 software was used for MLST identification and analysis. MLST 21 was used for biovar identification and epidemiological comparison of tested strains. The assembled genomes were 3,2 Mb in size and assembled into two chromosomes. MLST 21 analysis placed our strains into species and biovar clusters in concordance with other molecular tests used. To the extent of our knowledge, this is the first documented use of long-read sequencing in Brucella suis identification in this region. We conclude that bacteriological biotyping is outdated and host-specific identification in this genus is incorrect and that molecular characterisation is always the safer, faster and more suitable option. MinION sequencing proved to be a strong, accessible solution for species determination. Future study is required to determine how detailed genome information it can give, considering the error rate.

During regular implementation of the bovine tuberculosis-free cattle herd certification programme in the period from 2017 to 2020, the Laboratory for Bacterial Zoonoses and Molecular Diagnostics of Bacterial Diseases of Croatian veterinary institute Zagreb, Croatia tested material from 161 cattle from 27 holdings in 11 counties. The material was submitted following findings of pathoanatomical changes detected in the slaughter line suggesting tuberculosis, or after a positive reaction of cows to the tuberculin comparative methods. Species from the M. tuberculosis complex (M. bovis and M. caprae) were isolated from samples of 58 bovines (36%) from 16 holdings in eight counties. M. caprae was confirmed in 55 bovines (34%) originating from 13 holdings in seven counties, and M. bovis in three bovines (2%), each from a different holding in a different county. Saprophytic mycobacteria were isolated from four bovine samples (2.5%) from three holdings in two counties, i.e., M. gordonae (1), M. celatum (1) and two unidentified species (M. sp.). Based on the obtained results, we can conclude that the main causative agent of bovine tuberculosis in the Republic of Croatia is M. caprae, which confirmed previous findings. Control of bovine tuberculosis in the Republic of Croatia is still needed and, in the future, should be further suppressed using tuberculinisation, controls on slaughter lines, depopulation of infected herds, and etiological determination of the causative agents., U razdoblju od 2017. do 2020. godine tijekom redovitog provođenja programa certifikacije stada goveda slobodnih od tuberkuloze goveda bakteriološki je u Laboratoriju za bakterijske zoonoze i molekularnu dijagnostiku bakterijskih bolesti Hrvatskog veterinarskog instituta u Zagrebu, Hrvatska bio pretražen materijal 161 goveda iz 27 različitih uzgoja u 11 županija. Materijal je dostavljen nakon nalaza patoanatomskih promjena na liniji klanja koje upućuju na tuberkulozu ili komparativnom metodom nakon pozitivne reakcije goveda na tuberkulin. Vrste iz M. tuberculosis kompleksa (M. bovis i M. caprae) su izdvojene iz materijala 58 goveda (36 %) iz 16 uzgoja u osam županija. M. caprae je ustvrđen u 55 goveda (34 %) podrijetlom iz 13 uzgoja u sedam županija, a M. bovis u tri goveda (2 %) iz dva uzgoja u dvije županije. Saprofitske mikobakterije izdvojene su iz četiri uzorka goveda (2,5 %) dostavljenih iz tri uzgoja (21 %) u dvije županije i to M. gordonae (1), M. celatum (1) i dvije neidentificirane vrste (M. sp.). Na temelju dobivenih rezultata možemo zaključiti da je glavni uzročnik tuberkuloze goveda u Republici Hrvatskoj M. caprae, a koji je bio uzročnik i u prijašnjim istraživanjima. Kontrola tuberkuloze goveda u Republici Hrvatskoj i dalje je potrebna te bi se i u budućnosti trebala suzbijati na temelju tuberkulinizacije, kontrolama na liniji klanja te depopulacijom inficiranih stada i etiološkim dokazom vrste uzročnika.

Campylobacteriosis represents a global health challenge due to continuously increasing trends of antimicrobial resistance in

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.

Rapid-growth mycobacteria were isolated from two cases of cow mastitis with similar clinical appearance and within a narrow time frame. Mycobacteria were isolated on blood esculine agar. The isolated mycobacteria were Gram stained, Ziehl-Nielsen stained and tested for growth at 25°C, 37°C and 42°C, iron uptake, growth on Löwenstein-Jensen (LJ) agar with and without 5% NaCl, arylsulphatase (3 days), tween 80 hydrolysis, tellurite reduction, nitrate reductase and niacin synthesis. Molecular identification was performed using the Mycobacteria GenoType CM and AS tests (Hain Diagnostika, Nehren, Germany). One isolate was additionally sequenced for the hsp65, rpoB, 16S rRNA gene sequence and transcribed spacer sequence (ITS) DNA. Susceptibility testing of isolates was performed on the Sensititre Rapmycol plate (TREK Diagnostic Systems Ltd.) for trimethoprim/sulfamethoxasole, linezolid, ciprofloxacin, imipenem, moxifloxacin, cefepime, cefoxitin, amoxicillin / clavulanic acid, amikacin, ceftriaxone, doxycycline, minocycline, tigecycline, tobramycine and clarythromycine. Gram-positive acid-resistant rods were observed in stained smears. Both strains grew at 25°C, 37°C and 42°C on LJ medium, and on LJ medium containing 5 % NaCl. The conventional biochemical tests for iron uptake, arylsulphatase (3 days), Tween 80 hydrolysis, tellurite reduction and nitrate reductase were positive, while the niacin test was negative. Both isolates were identified by the GenoType Mycobacterium CM as Mycobacterium fortuitum II/ Mycobacterium mageritense, while application of the GenoType Mycobacterium AS kit identified both isolates as belonging to the species Mycobacterium smegmatis. Analysis of the isolate sequences (strain DS) for 16S ribosomal RNA confirmed a 100% identical result with Mycobacterium smegmatis strain INHR2. According to the CLSI criteria, both strains were sensitive to sulfametoxazole/trimethoprim, linezolid, doxicycline, amikacin and tobramycin. The strains differed in their sensitivity to cefoxitim, and both strains were resistant to clarithromycin. There was a strong difference between the isolates in sensitivity toward cefoxitime and tigecycline.

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.

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.

Designing an onshore wind farm is a complex planning process that requires various stages to be completed. The prime focus of this work is to assist planners and experts in finding the optimal cable layout of the onshore wind farm. The optimization algorithm is designed using mixed integer linear programming (MILP). The MILP algorithm takes into account system reliability, power transfer capacities and power quality issue. The novelty in this optimization algorithm is to simultaneously minimize cable installation cost and the cost of lost energy production and therefore maximize the reliability of the system. Additionally, the algorithm supports the optimal selection among different cable options, with different features, prices and capacities. By calculating voltage increase at the point of connection (POC), power quality issue is considered as well. The designed algorithm provides optimal results for four different wind farm layouts. Every layout is tested for three different case scenarios, where different number and type of cables are considered. The results show that more cable options contribute in lowering the total costs. Moreover, cables with higher capacity can help in improving the power quality issue. QC 20210720

Reliability indices of a distribution system can be improved by reducing failure rate and restoration time. A resonant-earthed distribution system has a low failure rate because numerous transient faults become self-extinguishing. However, in such networks, it can be difficult and time-consuming to locate nontransient faults resulting in aggravating the restoration time. This paper analyzes how different fault location methods affect the restoration time and SAIDI. Two major fault location methods are modeled for the calculation of the reliability indices and then applied to a radial feeder of a medium-voltage distribution system. The results show that SAIDI varies depending on the applied fault location method and its accuracy. The influence of fault location methods on labour costs is also discussed.

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.

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.

Distribution system planning (DSP) is very important because it can result in reliability enhancement and large cost savings for both utilities and consumers. DSP is a complex nonlinear problem, which can be solved with different optimization methods. This paper compares two such optimization methods, conventional (mixed-integer linear programming - MILP) and meta-heuristic (genetic algorithm - GA), applied to the DSP problem: construction of feeders in distribution power system from scratch. The main objective of DSP is to minimize the total cost, where both the investment and operational outage costs are considered, while the reliability of the whole system is maximized. DSP problem is applied to an actual distribution system. Solution methods are outlined, and computational results show that even though GA gives reasonably good results in faster computation time, MILP provides a better optimal solution with simpler implementation.

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.

Unutar roda Mycobacterium (M.) identificirano je više od 170različitih vrsta mikobakterija. U odnosu prema čovjeku podijeljene su u tri skupine.M. tuberculosis kompleks u koji spada deset vrsta koje uzrokuju tuberkulozu usisavaca. Vrsta M. tuberculosis, unutar M. tuberculosis kompleksa i dalje ostaje glavniuzročnik tuberkuloze u ljudi u svijetu. Osim već do sada dugo poznatih vrsta (M.bovis, M. africanum, M. microti, M. canetti, M. caprae), u novije vrijeme otkriveni sui novi pripadnici te skupine. M. pinnipedii u tuljana, M. munghi u mungosa, M. orygisu arapske oriks antilope i M. suricattae u merkata. Isti uzročnici dokazani su u ljudikoji žive u njihovoj blizini. M. leprae je uzročnik lepre u ljudi. Bolest je dokazana uoklopnika (aramadila) (Dasypus novemcinctus), koji predstavljaju prirodni rezervoarM. leprae. Najveći broj mikobakterija (više od 160 vrsta) pripada netuberkuloznimmikobakterijama. Nalaze se u okolišu, vrlo su otporne i prilagođene rastu i razvoju,a njima su često izloženi ljudi i životinje. Neke su važne potencijalno patogene vrstei ponekad uzrokuju teške patološke procese u ljudi i životinja.

Within the genus Mycobacterium (M.) more than 170 different types ofmycobacteria have been identified. In relation to humans, they are divided into threegroups. M. tuberculosis complex, which includes ten species that cause tuberculosisin mammals. The type of M. tuberculosis within M. tuberculosis complex remainsthe major cause of tuberculosis in humans in the world. Except for a long time knownspecies (M. bovis, M. africanum, M. microti, M. canetti, M. caprae), new membersof the group have recently been discovered. M. pinnipedii in seals, M. munghi inmongoose, M. orygis in arabian oryx and M. suricattae in the meerkat. The samecauses have been proven in people living nearby. M. leprae is the cause of theleprosy in humans. The disease has been proven in nine banded armadillos (Dasypusnovemcinctus) and represents the natural reservoir of M. leprae. The largest numberof mycobacteria (more than 160 species) belongs to nontuberculous mycobacteria.They are found in the environment, they are very resistant and adapted to growthand development, and to humans and animals are often exposed to them. Someare important potentially pathogenic species and they sometimes cause severepathological processes in humans and animals.

In addition to zoonotic potential, Chlamydia (C.) abortus is a very important bacterium causing serious disease in small ruminants. The main outcome of the disease is abortion in the late stages of pregnancy and the economic impact for farms is significant. During a three-year period (2015–2017), 191 vaginal swabs, 24 placentas, 210 foetal organs and 2 milk samples from small ruminant abortion cases were tested for C. abortus by real-time PCR. Positive samples were detected on eight sheep farms and two goat farms, with 8.4% of total samples testing positive samples. These samples were characterised using the MLVA method, and a single MLVA genotype (genotype [2]) was identified from sheep and goat samples, suggesting highly conserved C. abortus strains among the national flock. This study is the first description of C. abortus as a causative agent of abortion in goats in Croatia. More detailed study is required to recognize the epidemiological relevance of the abortion chlamydiosis. An open register of farms with defined health status should be established for each farm at the national level for better disease(s) control., Osim zoonotskog potencijala, vrsta Chlamydia (C.) abortus je vrlo važna bakterija koja može prouzročiti tešku bolest malih preživača. Dominantni klinički znak bolesti je pobačaj u kasnom stadiju graviditeta sa znatnim ekonomskim učinkom za farme. Tijekom trogodišnjeg razdoblja (2015.-2017.) metodom Real time (RT) PCR na C. abortus testiran je 191 vaginalni obrisak, 24 placente, 210 organa fetusa i 2 uzorka mlijeka iz slučajeva pobačaja malih preživača. Pozitivni uzorci ustvrđeni su u 8 farmi ovaca i 2 farme koza, što ukupno predstavlja 8,4 % pozitivnih uzoraka. Ovi uzorci su tipizirani metodom MLVA i identificiran je jedinstveni genotip MLVA (genotip [2]). Dobiveni rezultat ukazuje na visok stupanj podudarnosti sojeva C. abortus u nacionalnom stadu ovaca i koza. Ovo istraživanje prvi je opis vrste C. abortus kao uzročnika pobačaja u koza u Hrvatskoj. Temeljem ustvrđenog rezultata bilo bi uputno provesti daljnja, detaljnija istraživanja da bi se prepoznalo epidemiološko i ekonomsko značenje infekcija vrstom C. abortus. Radi kvalitetnijeg nadzora bolesti potrebno je uspostaviti otvoreni registar gospodarstava s definiranim zdravstvenim statusom za svaku pojedinu farmu.

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.

Security of electricity supply has become a fundamental requirement for modern societies. However, attempts to define and evaluate security of supply have differed from one another. This paper reviews relevant studies in order to give a comprehensive explanation of the security of supply concept. The paper includes theory, assessment, methodology, regulations, data and practical issues associated with the security of supply and power system reliability. Special focus is given to the methodologies used for improving the reliability and security of supply in power distribution systems.

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.

Maleus je kontagiozna bolest konja, magaraca i njihovih križanaca, a mogu oboljeti i mesojedi, uključujući i čovjeka. Obično je kronična, a očituje se tvorbom specifičnih čvorića u koži, potkožju, plućima i drugdje u organizmu. U Hrvatskoj je bolest zadnji puta zabilježena 1961. godine. U Arhivi sojeva Laboratorija za bakterijske zoonoze, Hrvatskog veterinarskog instituta pronađen je liofilizat oznake B. mallei - Karlovac (5.2.1957.). Zbog iznimno rijetkog uzorka namjera je bila ponovno kultivirati soj te ga identificirati daljnjim standardnim i molekularnim testovima potvrditi radi li se zaista o vrsti Burkholderia (B.) mallei. Liofilizirani izolat B. mallei nismo uspjeli kultivirati opisanim bakteriološkim postupcima. Izvršili smo izolaciju DNK iz uzorka otopljenog liofilizata. Koncentracija izolirane DNK izmjerena je na DS-11 spektrofotometru (DeNovix, SAD). Umnožavanjem za vrstu B. mallei specifičnog fliP-IS407A odsječka veličine 989-bp za soj „B.mallei Karlovac 5.2.1957.“ 221. utvrđeno je da se radi o vrsti B. mallei. Prema trenutno dostupnim podatcima molekularne genotipizacije primjenom high resolution melting PCR metode na 15 filogenetski značajnih polimorfizama jedne baze vrste B. mallei, hrvatski soj izdvojen 1957. godine nalazi se u grupi filogenetski srodnih sojeva iz Italije, Mađarske, Turske i Irana iz istog vremenskog razdoblja. Maleus je klasična, reemergentna bakterijska zoonoza i bolest koja ne poznaje granice kako po klinički nespecifičnoj slici i kroničnim oblicima bolesti bez vidljivih simptoma tako i po činjenici da se slučajevi bolesti u konja dijagnosticiraju i danas u zemljama s višegodišnjim statusom bez bolesti. Kontrola konja u međunarodnom prometu je i dalje neophodna., Glanders is a contagious disease of horses, donkeys and their crossbreeds, though it is also known to infect carnivores, including humans. The disease is usually chronic and is manifested by the formation of specific nodes in skin, subcutaneous tissue, lungs and elsewhere. In Croatia, the last record of the disease was in 1961. In the archive of strains in the Laboratory for Bacterial Zoonoses at the Croatian Veterinary Institute, a lyophilisate marked B. mallei – Karlovac (5.2.1957) was found. As these specimens are exceptionally rare, the intention was to re-cultivate the strain and identify it with further standard and molecular tests, to confirm whether it was really Burkholderia (B.) mallei. The lyophilized isolate could not be cultivated using the described bacteriological procedures. DNA extraction from the sample of dissolved lyophilizate was performed and a concentration of extracted DNA was measured on the DS-11 Spectrophotometer (DeNovix, USA). By amplifying B. mallei specific, 989- bp long, flip-IS407A region, it was possible to determine that the strain “B. mallei Karlovac 5.2.1957” belongs to the B. mallei species. According to the currently available data on molecular genotyping using high-resolution melting PCR method on 15 phylogenetically significant single nucleotide polymorphisms of B. mallei, the Croatian strain isolated in 1957 falls within the group of phylogenetically related strains from Italy, Hungary, Turkey and Iran from the same time period. Glanders is a classical, re-emerging bacterial zoonosis and a disease that knows no boundaries, both in the clinically unspecific and chronic forms of illness without visible symptoms, and in the fact that the cases of the disease in horses are still diagnosed today in countries with long- term disease-free status. The control of horses in international traffic is still necessary.

Patogene mikobakterije imaju svoje domaćine, a većina njih i prirodne rezervoare u prirodi. Tuberkuloza prouzročena vrstom M. tuberculosis i dalje ostaje glavni uzročnik tuberkuloze u svijetu. M. bovis ima najrašireniji raspon prirodnih rezervoara od svih poznatih patogena. Različite vrste divljih životinja predstavljaju trajan rezervoar infekcije vrstom M. bovis za domaće životinje i ljude. U novije vrijeme otkrivene su i nove vrste mikobakterija (M. caprae, M. pinnipedii, M. mungi, M. orygis i M. suricattae) koje po svojim karakteristikama pripadaju u kompleks M. tuberculosis. Sve navedene vrste mikobakterija imaju svoje rezervoare u prirodi i kao zoonoze predstavljaju stalnu prijetnju za infekciju ljudi. M. leprae prouzroči lepru u ljudi. Dugo vremena se mislilo da je samo čovjek domaćin i rezervoar uzročnika. Danas je poznato da su prirodni rezervoari lepre devetopojasni pasanci (Dasypus novemcinctus) u SAD i Južnoj Americi, crvene vjeverice (Sciurus vulgaris) u Velikoj Britaniji i različite vrste majmuna u Africi i Aziji., Pathogenic micobacteria have their hosts and most also have natural reservoirs. Tuberculosis caused by M. tuberculosis remains the main cause of tuberculosis in the world. M. bovis has the widest natural reservoir range of all known pathogens. Different wildlife species represent a permanent reservoir of M. bovis infection for domestic animals and humans. New species of mycobacteria have recently been discovered (M. caprae, M. pinnipedii, M. mungi, M. orygis and M. suricattae) which by their characteristics belong to the M. tuberculosis complex. Each of these mycobacteria species have their own reservoirs in nature, and as zoonoses, present a constant threat to humans. M. leprae causes leprosy in humans. It was previously thought that only men were the host and reservoir of the causative agent. Today, it is well-known that natural reservoirs are nine banded armadillo (Dasypus novemcinctus) in the United States and South America, red squirrels (Sciurus vulgaris) in the United Kingdom and various types of monkeys in Africa and Asia.

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 PRAA IAŒEV, POVZROAŒENA Z BAKTERIJO Brucella suis BIOVAR 2 NA HRVAA KEM Brucelozo pri praAiiAih smo spremljali od leta 2011 do leta 2015 v 13 hrvaAikih obAinah. S seroloAiko 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 % A¾ivali. S seroloAikimi testi smo preverili prisotnost protiteles proti bruceli tudi pri 641 plemenskih svinjah, ki so zvrgle ali imele teA¾ave z zabrejitvijo. Pozitivna reakcija je bila ugotovljena pri 34 svinjah, kar predstavlja 5,3 % vseh testiranih A¾ivali. Notranje organe 68 svinj iz Aiestih obAin 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 domaAih 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 kaA¾ejo, da je popolno izkoreninjenje bruceloze teA¾avno, Aie posebej v prostih rejah praAiiAev, kjer obstajajo moA¾nosti stika z divjimi praAiiAi. Zato bi bilo v prihodnje potrebno razmisliti o dodatnih naAinih nadzora nad to nevarno boleznijo praAiiAev. KljuAne besede: Brucella suis biovar 2; praAiiAi; zvrg; pojavnost; HrvaAika

The bacteria Anaplasma platys, Anaplasma phagocytophilum and Ehrlichia canis are tick-borne agents that cause canine vector-borne disease. The prevalence of these pathogens in South Eastern Europe is unknown with the exception of an isolated case of A. platys detected in a dog imported into Germany from Croatia. To gain a better insight into their presence and prevalence, PCR-based screening for these bacterial pathogens was performed on domesticated dogs from different regions of Croatia. Blood samples from 1080 apparently healthy dogs from coastal and continental parts of Croatia as well as tissue samples collected from 63 deceased dogs with a history of anaemia and thrombocytopenia were collected for molecular screening by an Anaplasmataceae-specific 16S rRNA conventional PCR. Positive samples were confirmed using a second Anaplasmataceae-specific PCR assay with the PCR product sequenced for the purpose of bacterial species identification. All sequenced isolates were georeferenced and a kernel intensity estimator was used to identify clusters of greater case intensity. 42/1080 (3.8% ; CI 2.7–5.0) of the healthy dogs were PCR positive for bacteria in the Anaplasmataceae. Sequencing of the 16S rRNA gene amplified from these positive samples revealed the presence of A. platys in 2.5% (CI 1.6–3.4%, 27 dogs), A. phagocytophilum in 0.3% (CI 0–0.6%, 3 dogs) and a Wolbachia endosymbiont in 1.1% (CI 0.4–1.6%, 12 dogs) of dogs screened in this study. Necropsied dogs were free from infection. Notably, no evidence of E. canis infection was found in any animal. This survey represents a rare molecular study of Anaplasmataceae in dogs in South Eastern Europe, confirming the presence of A. platys and A. phagocytophilum but not E. canis. The absence of E. canis was surprising given it has been described in all other Mediterranean countries surveyed and raises questions over the regional vector capacity of the Rhipicephalus sanguineus tick.