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.

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.

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.

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.

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.

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.

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.

Plasma parathyroid hormone-related peptide and bone metabolism in periparturient dairy cows.

The first weeks of lactation in dairy cows are characterised by elevated bone resorption. The connection between lactation and bone metabolism is still much discussed. In this work, changes in the concentration of plasma parathyroid hormone-related peptide (PTHrP) and markers of bone metabolism were studied in Holstein cows and heifers in the dry period and early lactation to determine the role of PTHrP in the relationship between the rate of bone remodelling and the onset of lactation in dairy cows. Blood samples were taken 14 days before calving ('D-14', n = 23) and then on day 10 ('D+10', n = 21) and day 30 after calving ('D+30', n = 23). Using enzyme immunoassay (EIA), the concentrations of PTHrP, parathyroid hormone (PTH), carboxyterminal cross-linked telopeptide of type I collagen (CTX) and oestradiol and the activity of bone specific alkaline phosphatase (BSALP) were determined. The results showed a statistically significant increase in plasma PTHrP (p < 0.005) and CTX (p < 0.0001) in cows on 'D+10' as compared to 'D-14' and CTX on 'D+30' as compared to 'D-14' (p < 0.0001). Significant negative correlations were found between the concentrations of PTHrP and oestradiol (r = -0.29, p < 0.05) and those of CTX and oestradiol (r = -0.54, p < 0.0001). In nonpregnant heifers (n = 6), the concentration of CTX and the activity of BSALP were significantly higher (p < 0.0001) than in dry cows. The observed increments of PTHrP and bone resorption after parturition reveal adaptations of bone metabolism to lactation in dairy cows.

Effects of fasting and refeeding on the antioxidant system in cockerels and pullets.

The effect of fasting and refeeding on total antioxidant status (TAS), glutathione peroxidase (GSH-Px) activity and concentration of some non-enzymatic antioxidant compounds was studied in cockerels and pullets. Blood was collected before and after 48-h fasting and 24 h after refeeding. In cockerels, fasting resulted in a significant decrease of TAS and uric acid concentration. After refeeding, the concentration of TAS remained significantly lower as compared to the control level. At the same time, blood plasma level of total lipids increased in comparison to the control and post-fasting values. In pullets, fasting resulted in a significant decrease of whole blood haemolysate GSH-Px activity and blood plasma concentrations of albumin and uric acid. Simultaneously, a significant increase in total lipids and cholesterol was obtained. In pullets, refeeding resulted in a further decrease of TAS to undetectable values, a significant decrease of blood plasma cholesterol, and a significant increase of GSH-Px in the whole blood haemolysate and in blood plasma uric acid content. The results indicate that fasting has a negative impact on the antioxidant defence system of the blood, which leads to a reduced resistance to oxidative stress in both cockerels and pullets. However, pullets seem to be more susceptible to fasting-provoked oxidative stress than cockerels.

Whole-Genome Sequence of the First Sequence Type 27 Brucella ceti Strain Isolated from European Waters.

Brucella spp. that cause marine brucellosis are becoming more important, as the disease appears to be more widespread than originally thought. Here, we report a whole and annotated genome sequence of Brucella ceti CRO350, a sequence type 27 strain isolated from a bottlenose dolphin carcass found in the Croatian part of the northern Adriatic Sea.

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.

Emerging cases of chlamydial abortion in sheep and goats in Croatia and Bosnia and Herzegovina.

In a recent lambing season (2012/2013), the seroprevalence of ovine chlamydiosis was monitored in small ruminant abortion cases in Croatia. Blood samples of 93 sheep and 69 goats were examined. In addition, 50 sheep and 61 goat samples were tested using molecular methods. Furthermore, 14 sheep blood samples, one goat blood sample and one sheep placenta sample from Bosnia and Herzegovina (BIH) were also tested as a part of inter-laboratory cooperation. Overall high seroprevalence was detected in sheep, 19.6% with the ELISA IDEXX kit and 20.5% with the ClVTEST kit. Seroprevalence in goats was 11.4%. In BIH, four sheep and one goat blood sample were seropositive for chlamydiosis. The disease causing agent, Chlamydia abortus (C. abortus) was confirmed using molecular methods in two sheep flocks in continental Croatia and in one sheep flock in BIH. In this study, C. abortus infection in sheep was identified for the first time in Croatia using species specific molecular methods. Ovine chlamydiosis is present in national sheep and goat flocks in Croatia and BIH. Thus should be subject to ongoing controls in the case of abortion. A combination of serological and molecular methods should be used for optimal laboratory diagnostics of C. abortus.

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.