Igor Gruntar

Conversion of Borrelia garinii cystic forms to motile spirochetes in vivo

Cystic forms (also called spheroplasts or starvation forms) and their ability to reconvert into normal motile spirochetes have already been demonstrated in the Borrelia burgdorferi sensu lato complex. The aim of this study was to determine whether motile B. garinii could develop from cystic forms, not only in vitro but also in vivo, in cyst‐inoculated mice. The cysts prepared in distilled water were able to reconvert into normal motile spirochetes at any time during in vitro experiments, lasting one month, even after freeze‐thawing of the cysts. Motile spirochetes were successfully isolated from 2 out of 15 mice inoculated intraperitoneally with cystic forms, showing the infectivity of the cysts. The demonstrated capacity of the cysts to reconvert into motile spirochetes in vivo and their surprising resistance to adverse environmental conditions should lead to further studies on the role and function of these forms in Lyme disease.

Evidence of Anaplasma phagocytophilum in game animals from Slovenia

Anaplasma phagocytophilum is a tick-borne rickettsial pathogen responsible for granulocytic anaplasmosis in mammalian hosts including humans. Wild animals may play an important role in the epidemiology of this disease. The aim of this study was to estimate the prevalence of infection with A. phagocytophilum among wildlife in Slovenia. Serum samples (n = 376) from the most important game species [red deer (Cervus elaphus), roe deer (Capreolus capreolus), wild boar (Sus scrofa), chamois (Rupicapra rupicapra) and brown bear (Ursus arctos)] were examined by A. phagocytophilum-specific indirect fluorescent-antibody assay (IFA) and wild boar spleen samples (n = 160) were tested by polymerase chain reaction (PCR). A. phagocytophilum-specific antibodies were found in 72% of sera and A. phagocytophilum DNA was present in 6.2% of spleens. The data indicate that A. phagocytophilum is present and widespread in Slovenian game animals and that game species are involved in the natural life cycle of A. phagocytophilum.

High genetic similarity of ciprofloxacin-resistant Campylobacter jejuni in central Europe.

Campylobacteriosis is the leading zoonosis in the European Union with the majority of cases attributed to Campylobacter jejuni. Although the disease is usually self-limiting, some severe cases need to be treated with antibiotics, primarily macrolides and quinolones. However, the resistance to the latter is reaching alarming levels in most of the EU countries. To shed light on the expansion of antibiotic resistance in central Europe, we have investigated genetic similarity across 178 ciprofloxacin-resistant C. jejuni mostly isolated in Slovenia, Austria and Germany. We performed comparative genetic similarity analyses using allelic types of seven multilocus sequence typing housekeeping genes, and single nucleotide polymorphisms of a quinolone resistance determining region located within the DNA gyrase subunit A gene. This analysis revealed high genetic similarity of isolates from clonal complex ST-21 that carry gyrA allelic type 1 in all three of these central-European countries, suggesting these ciprofloxacin resistant isolates arose from a recent common ancestor and are spread clonally.

New Approaches on Quantification of Campylobacter jejuni in Poultry Samples: The Use of Digital PCR and Real-time PCR against the ISO Standard Plate Count Method

Campylobacteriosis is the most frequently reported bacterial food-borne illness in the European Union and contaminated broiler meat is considered the most important source of infection in humans. The aim of the present study was to evaluate real-time PCR (qPCR) and digital PCR (dPCR) for quantification of Campylobacter jejuni in 75 broiler neck-skin samples collected from a poultry slaughterhouse, and to compare them with the ISO 10272-2 standard plate count method. For qPCR standard curve, C. jejuni-negative neck-skin samples were spiked with C. jejuni suspension with a known number of bacterial cells. The observed CFU/g values by qPCR correlated greatly with the expected values and qPCR showed good performance with the reliable limit of detection (rLOD) and limit of quantification (LOQ) of three and 31 target copies per reaction, respectively. However, both rLOD (1219 CFU/g) and LOQ (12,523 CFU/g) were beyond the EFSA-proposed critical limit of 500–1,000 CFU/g of neck skin. Although C. jejuni cell counts were ≤1,000 CFU/g in only 7/75 samples by plate counting, they were ≤LOQ in 60/75 and ≤rLOD in 26/75 (≤1,000 CFU/g in 24/75) samples by qPCR. A strong and statistically significant correlation was observed between qPCR and dPCR. Both PCR-based methods correlated significantly with the plate count method; however, the correlation was moderate. Using the Bland–Altman analysis, an average agreement was noted between all three methods, although with a large standard deviation. A significant bias toward overestimation in dPCR was observed, probably due to the relatively high number of false positive calls. The linear dynamic range was comparable in both PCR-based methods; however, qPCR proved to be more suitable for routine use. In the future, the establishment of a reliable molecular quantification of C. jejuni in poultry samples showing a wide range of contamination levels down to the proposed critical limit is needed to enable time- and cost-effective surveillance throughout all stages in the food production chain. As both rLOD and LOQ were beyond this limit, a modification of the procedure is suggested to include less sample dilution prior to DNA extraction to enable PCR-based quantification of C. jejuni at the proposed microbiological criteria.

A pulsed-field gel electrophoresis study of the genetic diversity of Campylobacter jejuni and Campylobacter coli in poultry flocks in Slovenia.

Campylobacter jejuni and C. coli have recently become the most frequent cause of bacterial foodborne enteric infection in most industrialised countries. Consumption and handling of undercooked contaminated poultry meat was identified as an important risk factor for human campylobacteriosis. The aim of this study was to ascertain the genetic diversity of C. jejuni and C. coli strains isolated from poultry in Slovenia. A total of 68 isolates (42 C. jejuni , 26 C. coli ) from faeces (n = 48), meat (n = 15) and skin/carcasses (n = 5) of chicken (n = 60) and turkey samples (n = 5) were analysed by pulsed-field gel electrophoresis. Sma I macrorestriction discriminated between C. jejuni and C. coli isolates. C. jejuni isolates exhibited a higher degree of diversity compared to C. coli isolates. In the C. jejuni group, a number of small clusters were apparent, while C. coli strains formed less but larger clusters. Additional Kpn I digestion of selected isolates resulted in poor subtyping. Strains with identical or very similar profiles were found on different farms, either in the same or different regions and time periods. Some of the clones indicated possible cross-contamination at slaughterhouses.

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.

No evidence of Trichomonas gallinae in free-living non-fringilid passerine birds in Slovenia

Finch trichomonosis is an emerging infectious disease through European countries caused by one clonal Trichomonas gallinae strain. Initially detected in the UK in 2005, the disease spread most likely by seasonal migration of Chaffinches to continental Europe. Finch trichomonosis caused decline in population of Greenfinches and to lesser extend in Chaffinch population. Though predominant occurrence of finch trichomonosis in Greenfinches and Chaffinches the disease was infrequently simultaneously diagnosed also in other passerine species. The prevalence of T. gallinae in non-fringillids was unknown and possibility that other passerine birds could host the parasite and contribute to spreading of the disease was discussed. In the light of early reports of finch trichomonosis outbreaks from UK, preliminary testing of free-living passerines for the presence of T. gallinae started in 2006. Since we were unsuccessful to catch fringilid species in mist nets, we were looking for other migrating birds, mainly non-fringilid passerines. Crop/deep oesophageal swabs from 121 birds were taken and all were negative. After finch trichomonosis outbreak in August 2012, further observations and monitoring were carried out. In September 2013, different non-fringilid passerines were captured during autumn migration and examined for the presence on T. gallinae. Altogether, 125 birds from 26 different species and 10 families were sampled. All samples were negative on presence of T. gallinae. Based on literature data and our results, cases of T. gallinae positive birds amongst non-fringilids are very uncommon and should be rather attributed to accidental spread of infection from diseased fringilids or other primary hosts, especially columbids.

Detection of Bacillus anthracis spores in postal and environmental samples in cases of suspected bioterrorism

From October 2001 to the end of 2004, a series of postal samples containing material suspected of containing Bacillus anthracis spores were examined. Environmental samples from dwelling places of some addressees and laboratory areas, including equipment, were also examined. Classical culture methods for isolation and identification were slightly modified and conformed to environmental samples. The most common bacteria isolated from postal samples were different Bacillus spp. (B. cereus, B. licheniformis, B. subtilis, B. mycoides). In cases of isolation of nonmotile and nonhaemolytic Bacillus spp., morphologically resembling B. anthracis, identification was supplemented by molecular method and mouse bioassay. All the samples resulted negative to Bacillus anthracis but in several cases an extended procedure was necessary to confirm the negative result.

Trichomonosis outbreak in a flock of canaries (Serinus canaria f. domestica) caused by a finch epidemic strain of Trichomonas gallinae

In the present paper, an outbreak of trichomonosis in a flock of 15 breeding pairs of canaries is described. Trichomonosis was diagnosed on characteristic clinical signs, microscopic examination of crop/esophageal swabs, gross pathology and histopathology. Trichomonads were successfully grown in culture media and were characterized by multi-locus sequence typing. The three genomic loci ITS1-5.8S-ITS2, 18S rRNA and Fe-hydrogenase were analyzed. Molecular characterization confirmed the finch trichomonosis strain, identical to the strain that caused emerging disease in free-living passerine birds in Europe. Flock treatment with metronidazole (200mg/L) in drinking water for 5days was partially effective. After individual treatment with oral application of metronidazole (20mg/kg SID) for 5days no further clinical signs were observed in the flock over next 30 months.

Detection of antibodies against Borrelia afzelii spirochete in stallions from Kocevje region

In the period February 2000?August 2001, sera from horses from the Dolnja Briga farm, Koeevje, were examined for the presence of antibodies against Borrelia afzelii (B. afzelii), one of the causative agents of Lyme borreliosis. B. afzelii-specific antibodies in horse sera were detected by an indirect ELISA test. Results obtained from the sera were compared and a significant increase in the percentage of seropositive animals before and after pasturing in the endemic area has been observed.