Marja-Liisa Hänninen

Campylobacter spp., Giardia spp., Cryptosporidium spp., Noroviruses, and Indicator Organisms in Surface Water in Southwestern Finland, 2000-2001

ABSTRACT A total of 139 surface water samples from seven lakes and 15 rivers in southwestern Finland were analyzed during five consecutive seasons from autumn 2000 to autumn 2001 for the presence of various enteropathogens (Campylobacter spp., Giardia spp., Cryptosporidium spp., and noroviruses) and fecal indicators (thermotolerant coliforms, Escherichia coli, Clostridium perfringens, and F-RNA bacteriophages) and for physicochemical parameters (turbidity and temperature); this was the first such systematic study. Altogether, 41.0% (57 of 139) of the samples were positive for at least one of the pathogens; 17.3% were positive for Campylobacter spp. (45.8% of the positive samples contained Campylobacter jejuni, 25.0% contained Campylobacter lari, 4.2% contained Campylobacter coli, and 25.0% contained Campylobacter isolates that were not identified), 13.7% were positive for Giardia spp., 10.1% were positive for Cryptosporidium spp., and 9.4% were positive for noroviruses (23.0% of the positive samples contained genogroup I and 77.0% contained genogroup II). The samples were positive for enteropathogens significantly (P < 0.05) less frequently during the winter season than during the other sampling seasons. No significant differences in the prevalence of enteropathogens were found when rivers and lakes were compared. The presence of thermotolerant coliforms, E. coli, and C. perfringens had significant bivariate nonparametric Spearmans rank order correlation coefficients (P < 0.001) with samples that were positive for one or more of the pathogens analyzed. The absence of these indicators in a logistic regression model was found to have significant predictive value (odds ratios, 1.15 × 108, 7.57, and 2.74, respectively; P < 0.05) for a sample that was negative for the pathogens analyzed. There were no significant correlations between counts or count levels for thermotolerant coliforms or E. coli or the presence of F-RNA phages and pathogens in the samples analyzed.

Detection and Typing of Campylobacter jejuni and Campylobacter coli and Analysis of Indicator Organisms in Three Waterborne Outbreaks in Finland

ABSTRACT Waterborne outbreaks associated with contamination of drinking water by Campylobacter jejuni are rather common in the Nordic countries Sweden, Norway, and Finland, where in sparsely populated districts groundwater is commonly used without disinfection. Campylobacters, Escherichia coli, or other coliforms have rarely been detected in potential sources. We studied three waterborne outbreaks in Finland caused by C. jejuni and used sample volumes of 4,000 to 20,000 ml for analysis of campylobacters and sample volumes of 1 to 5,000 ml for analysis of coliforms and E. coli, depending on the sampling site. Multiple samples obtained from possible sources (water distribution systems and environmental water sources) and the use of large sample volumes (several liters) increased the chance of detecting the pathogen C. jejuni in water. Filtration of a large volume (1,000 to 2,000 ml) also increased the rate of detection of coliforms and E. coli. To confirm the association between drinking water contamination and illness, a combination of Penner serotyping and pulsed-field gel electrophoresis (digestion with SmaI and KpnI) was found to be useful. This combination reliably verified similarity or dissimilarity of C. jejuni isolates from patient samples, from drinking water, and from other environmental sources, thus confirming the likely reservoir of an outbreak.

Swimming and Campylobacter infections.

A matched case-control study was conducted to study risk factors for domestically acquired sporadic Campylobacter infections in Finland. Swimming in natural sources of water was a novel risk factor. Eating undercooked meat and drinking dug-well water were also independent risk factors for Campylobacter infection.

Longitudinal Study of Finnish Campylobacter jejuni and C. coli Isolates from Humans, Using Multilocus Sequence Typing, Including Comparison with Epidemiological Data and Isolates from Poultry and Cattle

ABSTRACT We describe a study on the application of multilocus sequence typing for the analysis of Campylobacter jejuni and C. coli isolates from human domestically acquired infections in the Helsinki-Uusimaa area of Finland in 1996, 2002, and 2003. In addition, isolates from poultry meat and fecal samples of cattle from the seasonal peak (July to September) in 2003 were included in the study. In total, 361 Finnish C. jejuni and C. coli strains were typed. Sequence type 45 (ST-45) (45%), ST-21 (21%), and ST-677 (11%) clonal complexes were the most prevalent. The ST-45 and ST-677 complexes were overrepresented in comparison with previous studies. The longitudinal study revealed an association between C. coli (ST-828 complex) infection and elderly patients (≥60 years). Analysis of exposure factors, determined by a previous case-control study conducted during the seasonal peak in 2002, revealed that the ST-48 complex was significantly (P < 0.05) associated with the tasting or eating of raw minced meat. New and unassigned STs were associated with swimming in natural bodies of water, whereas the ST-677 complex was related to drinking nonchlorinated water from a small water plant or water from natural sources. The ST-45 complex was associated with contact with pet cats and dogs. In 2003, ST-45 occurrence was significantly associated with poultry whereas ST-50 was associated with isolates from humans. In contrast, ST-53, ST-58, ST-61, and ST-883 were significantly associated with isolates from cattle. Further studies are needed to reveal the significance of the observed associations.

Longitudinal Study of Escherichia coli O157 in a Cattle Finishing Unit

ABSTRACT In a longitudinal study in a Finnish cattle finishing unit we investigated excretion and sources of Escherichia coli O157 in bulls from postweaning until slaughter. Three groups of 31 to 42 calves were sampled in a calf transporter before they entered the farm and four to seven times at approximately monthly intervals at the farm. All calves sampled in the livestock transporter were negative for E. coli O157 on arrival, whereas positive animals were detected 1 day later. During the fattening period the E. coli O157 infection rate varied between 0 and 38.5%. The animals were also found to be shedding during the cold months. E. coli O157 was isolated from samples taken from water cups, floors, and feed passages. E. coli O157 was detected in 9.7 to 38.9% of the fecal samples taken at slaughter, while only two rumen samples and one carcass surface sample were found to be positive. E. coli O157 was isolated from barn surface samples more often when the enrichment time was 6 h than when the enrichment time was 24 h (P < 0.0001). Fecal samples taken at the abattoir had lower counts (≤0.4 MPN/g) than fecal samples at the farm (P < 0.05). E. coli O157 was isolated more often from 10-g fecal samples than from 1-g fecal samples (P < 0.0001). Most farm isolates belonged to one pulsed-field gel electrophoresis (PFGE) genotype (79.6%), and the rest belonged to closely related PFGE genotypes. In conclusion, this study indicated that the finishing unit rather than introduction of new cattle was the source of E. coli O157 at the farm and that E. coli O157 seemed to persist well on barn surfaces.

Aeromonas species in fish, fish-eggs, shrimp and freshwater

Aeromonas spp. are common contaminants of fish and seafood. They also are ubiquitous in the water environment. Aeromonas spp. were identified in 27 (93%) of 29 fish, in 17 (100%) fish-egg, in two (16%) of 12 shrimp samples and in 23 (100%) freshwater samples. In total, 117 Aeromonas strains were isolated from 69 positive samples, several samples having had two or three Aeromonas species. Included in this were also 26 mesophilic Aeromonas strains isolated in association with the study on fish diseases. The distribution of the species into 13 known hybridization groups (HGs) were studied by phenotypic and molecular methods. Ribopattern analysis of SmaI digested DNA was used for the identification of HGs. The predominant HG in fish, fish-eggs and freshwater samples was A. hydrophila HG 3 because 63% (22/37), 28% (16/57) or 80% (16/20) of the strains, respectively, were in HG 3. A. hydrophila HG 2 was also common in fresh fish samples but was not identified in fish-egg samples. HG 7 was common in fish samples studied for fish diseases and in freshwater samples. Strains which were not allotted to any HGs were common (19 of 143 strains). A. hydrophila HG 1, A. caviae HG 4, A. veronii subspecies sobria or subspecies veronii HG 8/10 known to be associated with human diarrhea were uncommon in all samples. The three strains isolated from frozen shrimp during two suspected food-borne outbreaks were A. hydrophila HG 2 and HG 3.

Occurrence and species level diagnostics of Campylobacter spp., enteric Helicobacter spp. and Anaerobiospirillum spp. in healthy and diarrheic dogs and cats

In order to study the occurrence and co-infection of different species of Campylobacter, enteric Helicobacter and Anaerobiospirillum in dogs and cats and define a possible association between these microrganisms and gastrointestinal disorders, 190 dogs and 84 cats, either healthy or with diarrhea, were sampled between 2002 and 2003. Thirty-three C. upsaliensis, 17 C. jejuni, 2 C. helveticus, 1 C. lari isolates from dogs and 14 C. helveticus, 7 C. jejuni, 6 C. upsaliensis isolates from cats were identified using species-specific PCR and phenotypic tests. Whole cell protein profile analysis, phenotypic tests, PCR-RFLP of gyrB and a phylogenetic study of partial groEL and 16S rRNA sequences were used to identify 37 H. bilis, 22 H. canis and 14 H. cinaedi in dogs and 12 H. canis, 5 H. bilis and 2 H. cinaedi in cats. Whole cell protein profile analysis, phenotypic tests and species-specific PCR of 16S rRNA were used to identify 14 A. succiniciproducens, 12 A. thomasii isolates and one unidentified Anaerobiospirillum sp. isolate in dogs and 3 A. thomasii isolates in cats. Fifty-two animals (19%) were positive for the isolation of more than one genus. No significant statistical correlation was found between any isolates of Campylobacter, Helicobacter or Anaerobiospirillum spp. or the various co-infection rates, and the presence of diarrhea in either dogs or cats. Campylobacter isolates were also tested for antibiotic resistance using the agar dilution method.

Multilocus sequence types of Finnish bovine Campylobacter jejuni isolates and their attribution to human infections

BackgroundCampylobacter jejuni is the most common bacterial cause of human gastroenteritis worldwide. Due to the sporadic nature of infection, sources often remain unknown. Multilocus sequence typing (MLST) has been successfully applied to population genetics of Campylobacter jejuni and mathematical modelling can be applied to the sequence data. Here, we analysed the population structure of a total of 250 Finnish C. jejuni isolates from bovines, poultry meat and humans collected in 2003 using a combination of Bayesian clustering (BAPS software) and phylogenetic analysis.ResultsIn the first phase we analysed sequence types (STs) of 102 Finnish bovine C. jejuni isolates by MLST and found a high diversity totalling 50 STs of which nearly half were novel. In the second phase we included MLST data from domestic human isolates as well as poultry C. jejuni isolates from the same time period. Between the human and bovine isolates we found an overlap of 72.2%, while 69% of the human isolates were overlapping with the chicken isolates. In the BAPS analysis 44.3% of the human isolates were found in bovine-associated BAPS clusters and 45.4% of the human isolates were found in the poultry-associated BAPS cluster. BAPS reflected the phylogeny of our data very well.ConclusionsThese findings suggest that bovines and poultry were equally important as reservoirs for human C. jejuni infections in Finland in 2003. Our results differ from those obtained in other countries where poultry has been identified as the most important source for human infections. The low prevalence of C. jejuni in poultry flocks in Finland could explain the lower attribution of human infection to poultry. Of the human isolates 10.3% were found in clusters not associated with any host which warrants further investigation, with particular focus on waterborne transmission routes and companion animals.

Prevalence of Campylobacter spp. in cattle in finland and antimicrobial susceptibilities of bovine Campylobacter jejuni strains

ABSTRACT The study investigated the prevalence of Campylobacter spp. in Finnish cattle at slaughter and carcass contamination after slaughter. During the period January to December 2003, bovine rectal fecal samples (n = 952) and carcass surface samples (n = 948) from 12 out of 15 Finnish slaughterhouses were examined. In total, campylobacters were detected in 31.1% of fecal samples and in 3.5% of carcass surface samples. Campylobacter jejuni was isolated from 19.5%, Campylobacter coli from 2.2%, and presumptive Campylobacter hyointestinalis from 10.8% of fecal samples. Campylobacters were detected in 4.4% and 37.4% of the fecal samples examined both by direct culture and by enrichment (n = 730), respectively, suggesting a low level of campylobacters in the intestinal content. A slightly increasing trend was observed in the overall prevalence of campylobacters towards the end of summer and autumn. Seventeen different serotypes were detected among the fecal C. jejuni isolates using a set of 25 commercial antisera for serotyping heat-stable antigens (Penner) of C. jejuni by passive hemagglutination. The predominant serotypes, Pen2 and Pen4-complex, were isolated from 52% of the fecal samples. Subtyping by pulsed-field gel electrophoresis (SmaI) yielded 56 and 20 subtypes out of 330 fecal and 70 carcass C. jejuni isolates, respectively. MICs of ampicillin, enrofloxacin, erythromycin, gentamicin, nalidixic acid, and oxytetracycline for 187 C. jejuni isolates were determined using a commercial broth microdilution method. Sixteen (9%) of the isolates were resistant to at least one of the antimicrobials tested. Resistance to nalidixic acid was most commonly detected (6%). No multiresistance was observed.

Genomic relatedness within five common Finnish Campylobacter jejuni pulsed-field gel electrophoresis genotypes studied by amplified fragment length polymorphism analysis, ribotyping and serotyping

ABSTRACT Thirty-five Finnish Campylobacter jejuni strains with five SmaI/SacII pulsed-field gel electrophoresis (PFGE) genotypes selected among human and chicken isolates from 1997 and 1998 were used for comparison of their PFGE patterns, amplified fragment length polymorphism (AFLP) patterns,HaeIII ribotypes, and heat-stable (HS) serotypes. The discriminatory power of PFGE, AFLP, and ribotyping withHaeIII were shown to be at the same level for this selected set of strains, and these methods assigned the strains into the same groups. The PFGE and AFLP patterns within a genotype were highly similar, indicating genetic relatedness. The same HS serotypes were distributed among different genotypes, and different serotypes were identified within one genotype. HS serotype 12 was only associated with the combined genotype G1 (PFGE-AFLP-ribotype). These studies using polyphasic genotyping methods suggested that common Finnish C. jejuni genotypes form genetic lineages which colonize both humans and chickens.