Mycobacteria in aquarium fish: results of a 3-year survey indicate caution required in handling pet-shop fish.

Fish are commonly infected with non-tuberculous mycobacteria (NTM), which should be regarded as potential pathogens when handling aquarium fish and equipment. This study examined 107 aquarium fish from pet shops. Cultivation of the fish samples using different selective media was conducted for identification of NTM. Isolates were identified using the GenoType Mycobacterium common mycobacteria and additional species assays, sequencing of the 16S rRNA and rpoB genes, and real-time PCR assay for identification of Mycobacterium (M.) marinum. Among the investigated fish, 79.4% (85/107) were positive for mycobacteria, with 8.2% (7 of 85) having two mycobacterial species present. Among the positive fish, the common pathogens M. marinum, Mycobacterium fortuitum (M. fortuitum group) and Mycobacterium chelonae were identified in approx. 90% of fish and other NTM species in 10%, including Mycobacterium peregrinum/septicum, Mycobacterium gordonae, Mycobacterium arupense, Mycobacterium kansasii, Mycobacterium ulcerans and Mycobacterium setense. The well-known human pathogen M. marinum was present in 10.6% of the positive fish (9 of 85). The species of mycobacteria identified in the study are not only recognized as aquarium fish pathogens, but can also cause pathology in humans. Microbiological and clinical communities should therefore be sensitized to the role of NTM in infections associated with exposure to aquarium fish.

The evidence for clonal spreading of quinolone resistance with a particular clonal complex of Campylobacter jejuni.

Campylobacter is the most prevalent cause of bacterial gastroenteritis worldwide and it represents a significant public health risk of increasing severity due to its escalating resistance to clinically important quinolone and macrolide antibiotics. As a zoonotic pathogen Campylobacter is transmitted along the food chain and naturally cycles from environmental waters, feedstuff, animals and food to humans. We determined antibiotic resistance profiles, as well as multilocus sequence types and flaA-SVR types for 52 C. jejuni isolated in Slovenia from human, animal, raw and cured chicken meat and water samples. Twenty-eight different sequence types, arranged in ten clonal complexes, three new allele types and five new sequence types were identified, indicating the relatively high diversity in a small group of strains. The assignment of strains from different sources to the same clonal complexes indicates their transmission along the food supply chain. The most prevalent clonal complex was CC21, which was also the genetic group with 95% of quinolone-resistant strains. Based on the genetic relatedness of these quinolone-resistant strains identified by polymerase chain reaction with a mismatch amplification mutation assay and sequencing of the quinolone resistance-determining region of the gyrA gene, we conclude that the high resistance prevalence observed indicates the local clonal spread of quinolone resistance with CC21.

A survey on Tetracapsuloides bryosalmonae infections in Slovene fresh waters.

Slovenia has no history of health problems related to proliferative kidney disease (PKD) either in farmed or in wild fish. However, due to the past molecular evidence for the presence of Tetracapsuloides bryosalmonae DNA in tissues of some fish from open waters, a survey was conducted on wild salmonids that were primarily sampled for other purposes. In winter 2010-2011, specimens from a total of 244 rainbow trout, Oncorhynchus mykiss (Walbaum), and brown trout, Salmo trutta L., from 30 bodies of fresh water were examined for T. bryosalmonae using a PCR method. The sampled fish showed no clinical signs or gross pathological lesions characteristic of PKD. Nineteen (7.8%) fish from seven (23.3%) fresh waters were positive for T. bryosalmonae. The identity of PCR amplicons was confirmed by sequencing. With one exception, all the positive fish were found in waters from the regions where the average yearly temperatures and the environmental pollution are higher. This preliminary countrywide survey provided the first insight into the situation regarding T. bryosalmonae infection of wild salmonids in Slovenia.

Marteiliosis in mussels: a rare disease?

Among 1280 cultured and wild adult Mediterranean mussels, Mytilus galloprovincialis, collected over a 1-year surveillance period from the Slovene Adriatic Sea, 0.3% were histologically positive for the presence of Marteilia spp. The infection was concentrated in winter. Employing the molecular methods of PCR, cloning, DNA restriction and sequencing, only Marteilia refringens type M was detected in all the infected mussels. Although all life-cycle stages of M. refringens severely infected digestive glands, only sporadic disruption of epithelial cells of digestive tubules and focal destruction of digestive tubules were observed in the infected mussels. This was the first detection of M. refringens in M. galloprovincialis from the Slovene Adriatic Sea with the lowest prevalence reported to date. In addition, our results highlight the need for sequencing to complement the established PCR-RFLP analysis for correct parasite typing.

Population structure and attribution of human clinical Campylobacter jejuni isolates from central Europe to livestock and environmental sources.

Campylobacter jejuni is among the most prevalent causes of human bacterial gastroenteritis worldwide. Domesticated animals and, especially, chicken meat are considered to be the main sources of infections. However, the contribution of surface waters and wildlife in C. jejuni transmission to humans is not well understood. We have evaluated the source attribution potential of a six-gene multiplex PCR (mPCR) method coupled with STRUCTURE analysis on a set of 410 C. jejuni strains isolated from environment, livestock, food and humans in central Europe. Multiplex PCR fingerprints were analysed using Subclade prediction algorithm to classify them into six distinct mPCR clades. A subset of C. jejuni isolates (70%) was characterized by multilocus sequence typing (MLST) demonstrating 74% congruence between mPCR and MLST. The correspondence analysis of mPCR clades and sources of isolation indicated three distinct groups in the studied C. jejuni population-the first one associated with isolates from poultry, the second one with isolates from cattle, and the third one with isolates from the environment. The STRUCTURE analysis attributed 7.2% and 21.7% of human isolates to environmental sources based on MLST and mPCR fingerprints, respectively.

Mycobacterial infection of pigs in Croatia.

During a five-year period (2000 to 2004) 74,342 pigs were tested by the intradermal tuberculin test in Croatia. Of them, 248 (0.33%) pigs were positive and 91 (0.12%) were found to be suspicious in 7 out of the 13 farms included in the study. Gross pathological changes characteristic of tuberculosis were observed in tuberculin-positive and/or suspicious swine. Mycobacterium was isolated from the lymph nodes of 183 out of 234 swine (78.2%). For better epidemiological understanding, isolates were typed by conventional methods, PCR and hybridisation. The results show that most of the isolates belonged to the Mycobacterium avium complex (175 isolates, 95.7%). Other isolates belonged to M. fortuitum (6 isolates, 3.3%), M. chelonae (1 isolate, 0.5%), and M. peregrinum (1 isolate, 0.5%). Isolated strains of the M. avium complex were identified as M a. avium (37 isolates, 21.1%) and M. a. hominissuis (138 isolates, 78.9%).

Antimicrobial Susceptibility Patterns of Clostridium difficile Isolates from Family Dairy Farms.

A significant risk factor for developing Clostridium difficile infection (CDI) in humans and animals is associated with the antimicrobial use. It has often been hypothesized that farm animals could be the source for human infection with Clostridium difficile (CD). In the European Union, family-run dairy farms are the predominant farming model, which are more interlinked within the community compared to large-scale intensive dairy or beef farms. Therefore, it is important to investigate antimicrobial susceptibility patterns of CD in such environment. A total of 159 CD isolates from 20 family dairy farms were tested with a customized broth microdilution plate for their antimicrobial resistance. Seventeen antimicrobials were selected (amoxicillin, ceftriaxone, clindamycin, daptomycin, erythromycin, fusidic acid, imipenem, levofloxacin, linezolid, metronidazole, moxifloxacin, oxacillin, rifampicin, tetracycline, tigecycline, trimethoprim/sulfamethoxazole and vancomycin), which are commonly used for treatment of CDI in veterinary and human medicine, or were previously applied in CD epidemiological studies. Antimicrobials, which are used for treatment of CDI in humans (metronidazole, vancomycin, fusidic acid, tigecycline, linezolid) inhibited CD growth in vitro. Most CD isolates were resistant to erythromycin (93.1%), daptomycin (69.2%) and clindamycin (46.5%). High multiple-resistance was found in CD ribotype 012 (n = 5, 100%), some CD SLO 060 (n = 4, 25%) and one CD 033 (n = 1, 1.1%). High multiple-resistance in this study was linked with CD ribotypes and not with the origin of CD. The low prevalence of these ribotypes (6.3%; 10/159) indicates that family-run dairy farms are an unlikely source of CD with multiple-resistance to antimicrobials.

Can we improve maternal care in sows? Maternal behavioral traits important for piglet survival in loose-housed sow herds.

The primary objective of this survey was to investigate the relationship between qualitative maternal behavioral scores (nest building activities, sow communication, and sow carefulness), piglet mortality, and the number of weaned piglets on commercial farms with loose-housed lactating (Norsvin Landrace × Yorkshire) sows. Second, the impact of these scores on productivity compared with the physical condition of sows (movement disorders, body condition, and shoulder lesions) was assessed. Data on maternal care behaviors and physical condition were collected on 895 sows from 45 commercial farms. Farmers scored sows on their physical condition (movement disorders [MD], BCS, and shoulder lesions [SL]) and qualitative maternal care behaviors (nest building activities [NEST] prior to farrowing and sow communication [COM] and sow carefulness [CARE] after farrowing, while sows were standing and moving and just before lying down). There was a low positive correlation between NEST and COM ( = 0.10, = 0.026) and between NEST and CARE ( = 0.15, = 0.010) but a high positive correlation between COM and CARE ( = 0.57, < 0.001). Higher COM and CARE were associated with lower piglet mortality ( ˂ 0.001 and = 0.013, respectively), and a greater number of weaned piglets was associated with higher scores for NEST ( = 0.009), COM ( < 0.001), and CARE ( = 0.009). Maternal care behavior had a greater impact on piglet mortality and the number of weaned piglets than sow physical condition (MD, BCS, and SL). We tested 7 different models (combinations of behavioral scores) and compared their relative predictive accuracies using Akaike information criteria. The model including COM and CARE had the best predictive accuracy for piglet mortality/weaned piglets. There was between-sow variation in maternal care behaviors (COM and CARE), and both were unaffected by litter size. Because these behaviors were also easy to score for the farmers, combining COM and CARE has the greatest potential to be tested in nucleus herds for calculation of genetic variation and heritability and should be taken into account in future breeding programs for sows.

Udder characteristics of importance for teat use in purebred and crossbred pigs.

Possible side effects of the current sow selection criteria on udder characteristics and their influence on teat use and functionality have not yet been investigated. The first aim of the present study was to investigate differences in udder morphology characteristics (distance between teats in a pair, teat length, and teat diameter) in 2 different pure breeds (Norsvin Duroc [ND; = 12] and Norsvin Landrace [NL; = 12]) and 1 crossbreed (Norsvin Landrace × Yorkshire [NL×Y; = 14]) at 3 different time periods (Days 1, 21, and 35) during lactation. Second, we also investigated the association between udder morphology and teat use on d 1 and if some of these characteristics influence whether teats become nonfunctional (from d 1 to 35) during lactation. During lactation, udder morphology (teat pair distance, teat length, and diameter of functional teats) and teat use (from 6 consecutive nursings: functional teats being used/not being used and which teat row [left or right] was uppermost) were documented. The NL had shorter distance between teat pairs than ND ( = 0.030). Teat pair distance increased with sows' parity ( = 0.010) and was affected by teat position ( < 0.001). The ND had shorter teats than NL and NL×Y ( < 0.001). Teat length and diameter increased with parity ( = 0.027 and = 0.043, respectively) and were affected by teat position ( < 0.001 and < 0.001, respectively). Functional teats were less used on d 1 postpartum (21.4%) than at 2 later time periods (10.4% on Day 21 and 4.7% on Day 35; < 0.001) during lactation. On d 1, the greater the distance between teat pairs in the middle and posterior position, the higher was the proportion of not-used functional teats ( = 0.003), with a larger decline in the lower teat row ( < 0.001), where approximately half of middle and posterior teats were not used. Proportion of not-used functional teats in the lower middle position increased when the teat pair distance exceeded 16 cm, whereas in the lower posterior position, this limit was 14 cm. Furthermore, the proportion of not-used teats on d 1 influenced teats becoming nonfunctional during lactation ( < 0.001). The greater the distance between pairs, the more teats became nonfunctional irrespective of teat position ( < 0.001). The present results suggest that teat pair distance is of importance for teat use in all breeds and should be included in the breeding program to ensure colostrum intake and maintain teat functionality during lactation.

Can a super sow be a robust sow? Consequences of litter investment in purebred and crossbred sows of different parities.

The aim of this project was to study the consequences of litter investment on physical characteristics in primiparous and multiparous sows in 3 Norwegian breeds (Norsvin Duroc [ = 12], Norsvin Landrace [ = 12], and crossbreeds [Norsvin Landrace and Swedish Yorkshire { = 15}]). We predicted that the maternal sow line (Norsvin Landrace) would invest more in their litter in term of higher weight at birth, resulting in a higher litter weight of weaned piglets but with the consequence of greater loss in body condition and a higher prevalence of shoulder lesions. It was predicted that this should be more pronounced in primiparous sows than in multiparous sows. As predicted, the maternal pure line (Norsvin Landrace) had higher litter investment in terms of litter weight at birth ( = 0.003) and litter weight at weaning ( = 0.050) as well as higher total litter investment (litter weight at weaning plus weight of dead piglets [stillborn and mummified piglets and weight of piglets that died after farrowing but before weaning]; = 0.050) and suffered larger losses of body condition ( = 0.016) and had a higher prevalence of shoulder lesions ( = 0.008) during lactation than other breeds. Moreover, only in Norsvin Landrace was development of shoulder lesions related to inadequate feed consumption ( = 0.006). This has become a major welfare concern of modern pig breeding. Although primiparous and multiparous sows had similar litter sizes, primiparous sows had lower litter investment in terms of litter weight at birth ( = 0.032) and litter weight at weaning ( = 0.007) as well as total litter investment ( = 0.008). Primiparous sows suffered greater losses in body condition ( = 0.012) and developed more shoulder lesions ( = 0.026) due to lower total feed consumption ( < 0.001) during lactation than multiparous sows. Especially in the highly productive maternal line (Norsvin Landrace), development of shoulder lesions during the lactation period was more pronounced in primiparous sows than in multiparous sows ( < 0.001). The selection program has shifted the balance to greater investments in earlier life, when sows still need resources for their own growth and development. This has resulted in a larger number of weaned piglets but at a higher sow welfare cost in terms of higher losses in body condition and a higher prevalence of shoulder lesions. Our results pinpoint the importance of improving the balance between economic traits and traits that improve welfare and longevity of the sows.

Detection of mycobacteria in aquarium fish in Slovenia by culture and molecular methods.

Thirty-five aquarium fish were investigated for the presence of mycobacteria by culture and molecular methods. The following species were examined: goldfish Carassius auratus auratus, guppy Poecilia reticulata, 4 three-spot gourami Trichogaster trichopterus, dwarf gourami Colisa lalia, Siamese fighting fish Betta splendens, freshwater angelfish Pterophyllum scalare, African cichlid fish Cichlidae spp., cichlid fish Microgeophagus altispinosus, cichlid fish Pseudotropheus lombardoi, blue streak hap Labidochromis caeruleus, sterlet Acipenser ruthenus, southern platyfish Xiphophorus maculatus, and catfish Corydoras spp. Isolates of mycobacteria were obtained in 29 cases (82.9%). Two specimens were positive using Ziehl-Neelsen (ZN) staining, but the cultivation failed. Four specimens were both ZN- and culture-negative. On the basis of GenoType Mycobacterium assay (Hain Life-science) and restriction enzyme analysis of the amplified products (PCR-RFLP), 23 isolates (79.3%) were identified: 7 as Mycobacterium fortuitum, 6 as M. gordonae, 6 as M. marinum, 3 as M. chelonae, and 1 as M. peregrinum. Five isolates remained unidentified (Mycobacterium spp.). One case probably represented a mixed infection (M. marinum/M. fortuitum). Since M. marinum infections are also detected in humans, the significance of mycobacteria in aquarium fish should not be overlooked.

Prevalence of Taylorella equigenitalis infection in stallions in Slovenia: bacteriology compared with PCR examination.

REASONS FOR PERFORMING STUDY: The prevalence of Taylorella equigenitalis infection in Slovenia is unknown and methods used to refine identification in these stallions are required. HYPOTHESIS: In diagnosis of T. equigenitalis, polymerase chain reaction (PCR) would have advantages over culture methods, especially in cases where small numbers of causal agent or intensive contamination of genital swabs are involved. METHODS: Culture method and PCR were used to examine a total of 980 genital swabs from the urethra and fossa urethralis of 245 stallions for the presence of the contagious equine metritis organism. RESULTS: Among 245 examined stallions, 225 (91.8%) were negative to T. equigenitalis by both methods. From the swabs of 17 stallions (6.9%) T. equigenitalis was isolated at first and/or second sampling. Swabs of 3 (13%) stallions were PCR positive but the isolation of T. equigenitalis failed. The rate of T. equigenitalis detection was higher with PCR than with the classic bacteriological examination. CONCLUSIONS AND POTENTIAL RELEVANCE: PCR protocol used in this study provided a specific, sensitive, and simple tool for rapid detection of T. equigenitalis. PCR is especially valuable in cases of intensive bacterial and fungal contamination of swabs where the isolation of T. equigenitalis usually fails.

Isolation and characterisation of Mycobacterium avium and Rhodococcus equi from granulomatous lesions of swine lymph nodes in Slovenia.

Granulomatous lesions in bovine and especially swine lymph nodes are still frequently observed during routine veterinary meat inspections even though Mycobacterium bovis infections are no longer detected in domestic animals in Slovenia. Different lymph nodes of pigs (n = 260) were investigated using classical bacteriological and molecular methods. Mycobacterium avium alone was isolated in 47.3% of pigs and in mixed infection with Rhodococcus equi in 3.9% of pigs. R. equi alone was isolated in 27.3% and in mixed infection with mycobacteria other than M. avium in 1.5% of pigs. A total of 133 M. avium isolates were typed using the IS1245, IS901 and FR300 PCR. Almost two thirds (60.9%) of isolates belonged to M. avium hominissuis (IS901-, IS1245+ genotype), 33.8% of isolates belonged to M. avium avium (IS901+, IS1245+ genotype) and 5.3% of isolates remained non-typed. Fifty out of 85 R. equi isolates were tested for the virulence-associated antigens (VapA and VapB). Nearly two thirds (60.0%) were positive for VapB while all the other isolates were VapA- and VapB-negative.

Wild boars (Sus scrofa) as reservoirs of Brucella suis biovar 2 in Croatia.

This work presents the results of findings for brucellosis in wild boars and domestic swine in two regions of Croatia. In the region of Djakovo the blood samples of 211 wild boars were analysed and in 29.4% of the samples serologically positive reactions were established. In the same region the blood samples of 1080 domestic swine on pastures were also analysed and positive serological reactions were established in 12.3%. In the regions around Lonjsko Polje the blood samples of 53 wild boars were analysed and in 22.6% of them positive serological reactions were established. On several locations around Lonjsko Polje the blood samples of 901 domestic swine were serologically analysed and 13.5% of the swine were found to be seropositive. Bacteriological analyses of submitted materials from 24 wild boars resulted in isolation of Brucella from seven (29.2%) samples, and from 43 samples originating from domestic swine that had aborted and had been serologically positive, Brucella were isolated from 25 (58.1%) swine, as well as from 10 (62.5%) out of 16 aborted piglets. In all the isolates Brucella suis biovar 2 was identified. Wild boars are carriers and reservoirs of Brucella suis biovar 2 in Croatia.

Cutaneous listeriosis in a veterinarian with the evidence of zoonotic transmission--a case report.

A case of Listeria monocytogenes skin infection in a man is presented. A 54-year-old male veterinary practitioner developed pustular changes on the skin of arms and hands after assisting with the delivery of a stillborn calf. Listeria monocytogenes was isolated from the skin lesions on the arms and from the bovine placenta. Listeria monocytogenes isolates were serotyped and genotyped with pulsed-field gel electrophoresis (PFGE) to confirm the suspected transmission of the pathogen from animal to human. All isolates were of serotype 4b with identical pulsotype. To the best of our knowledge, this is the first case of cutaneous listeriosis in which the evidence for zoonotic transmission of L. monocytogenes is supported by genotyping methods.

MIRU-VNTR typing of Mycobacterium avium in animals and humans: heterogeneity of Mycobacterium avium subsp. hominissuis versus homogeneity of Mycobacterium avium subsp. avium strains.

Epidemiological studies on Mycobacterium avium are requisite for revealing infection sources and disease transmission. They are based upon genotyping methods like RFLP and MIRU-VNTR. In our study, MIRU-VNTR typing was applied to 121 previously RFLP typed M. avium field isolates to compare the discriminatory power of both methods. The applicability of MIRU-VNTR typing was studied for isolates from a limited geographic area, namely 41 M. avium subsp. avium and 80 M. avium subsp. hominissuis isolates. Among the former, exhibiting 12 IS901 RFLP types, five MIRU-VNTR types were found with discriminatory index (DI) of 0.716. Among the latter, exhibiting 56 IS1245 RFLP types, 18 MIRU-VNTR types were found with DI of 0.866. Concomitant use of both methods increased DI to 0.981 and 0.995, respectively. MIRU-VNTR typing employing the selected markers provided discernible discrimination among M. avium subsp. hominissuis isolates, but more discriminative markers are needed for M. avium subsp. avium isolates.

Outbreak of tuberculosis caused by Mycobacterium caprae in a zoological garden.

In the autumn of 2004, tuberculosis caused by Mycobacterium caprae occurred in a zoo in Slovenia. A dromedary camel (Camelus dromedarius) was killed after a history of progressive emaciation. Necropsy findings indicated disseminated tuberculosis, which was confirmed by cultivation of M. caprae. Consequently, a tuberculin skin test was performed in all epidemiologically linked animals and another dromedary camel and six bison (Bison bison) were positive and killed. Mycobacterium caprae was isolated from two bison while M. scrofulaceum and Mycobacterium spp. were found in two other bison, respectively. The second dromedary camel was found to be negative for mycobacteria under both microscopic and culture tests. The isolates were investigated with commercial identification kits, IS6110 PCR, IS6110 restriction fragment length polymorphism analysis, spoligotyping and mycobacterial interspersed repetitive units typing. Genotyping results revealed that the dromedary camel and the two bison were infected by the same M. caprae.

Haemolytic Rhodococcus equi Isolated from a Swine Lymph Node with Granulomatous Lesions.

Rhodococcus equi is generally thought to be non-haemolytic although some earlier investigations reported minor haemolytic activity. A case of a haemolytic R. equi isolate from a swine lymph node with granulomatous lesions is described. This is a new contribution to knowledge of the cultural properties of R. equi.

Microbiological features of Staphylococcus schleiferi subsp. coagulans, isolated from dogs and possible misidentification with other canine coagulase-positive staphylococci.

Staphylococcus schleiferi subsp. coagulans has only rarely been isolated and identified from the external auditory meatus of dogs suffering from external otitis. Its morphological and basic biochemical characteristics are of relatively little value for identification, as it phenotypically resembles another coagulase-positive staphylococci (CPS) and, consequently, may be easily misidentified as S. intermedius or even as S. aureus. In the present work, differentiation of S. schleiferi ssp. coagulans was therefore based on specific biochemical and genetic methods. All the strains were evaluated with the following commercial methods: Api Staph System (bioMérieux, Marcy l'Etoil, France), BBL Crystal Identification Systems (Gram-Positive ID Kit and Rapid Gram-Positive ID Kit; Becton Dickinson), and GEN-PROBE AccuProbe, Staphylococcus aureus identification test (bioMérieux). Gram-Positive ID System/GP database includes the broadest range of staphylococcal species and correctly identifies the majority of strains important in veterinary medicine. Therefore, it is an acceptable alternative to conventional methods for identification of canine staphylococcal isolates. Reliable differentiation of S. aureus from S. schleiferi ssp. coagulans and S. intermedius was feasible with AccuProbe for S. aureus, which gave positive results only for S. aureus; all other CPS tested were negative.