Outbreak of Campylobacteriosis Following a Dairy Farm Visit: Confirmation by Genotyping.

In April-May 2014, an outbreak of campylobacteriosis occurred after a preschool visit to a dairy farm in the South Western part of Sweden. During the visit, a meal, including unpasteurized milk, was served. A retrospective cohort study using a web-based questionnaire was performed among the participants (n = 30) of the farm visit. A total of 24 of the 30 (80%) cohort members completed the questionnaire. Eleven cases were identified, and Campylobacter jejuni was isolated from eight of them. Seven of the cases were 2- to 7-year-old children. We found the highest attack rates among those who usually drink milk (45%) and those who consumed unpasteurized milk during the farm visit (42%). No cases were unexposed (risk ratio incalculable). As result of the farm investigation, Campylobacter was isolated from cattle on the farm. Genotyping with pulsed-field gel electrophoresis and whole genome sequencing confirmed that human and cattle isolates of C. jejuni belonged to one cluster. Thus, cattle on the farm are considered the source of infection, and the most likely vehicle of transmission was contaminated unpasteurized milk. We recommend consumption of heat-treated milk only and increased awareness of the risk of consuming unpasteurized milk.

A prospective follow-up study on transmission of Campylobacter from poultry to abattoir workers.

Contact with poultry or poultry meat is a well-known risk factor for campylobacteriosis, but prospective studies on transmission of Campylobacter from chickens to humans during slaughter are scarce. In this study, we monitored transmission of Campylobacter from slaughtered chicken to originally culture-negative abattoir workers during the peak season of colonized chicken and human Campylobacter infection. Stool samples were obtained from 28 abattoir workers together with data on health status once a month between June and September 2010, with a follow-up sample collected in February 2011. Campylobacter-positive individuals and chicken flocks were identified by culture, and isolates were further characterized using molecular techniques. Campylobacter was isolated from seven asymptomatic individuals. Four of them had been newly employed and had not reported any previous Campylobacter infection. Four human isolates had matching genetic fingerprints with isolates from recently slaughtered chickens. Our results further support the role of chicken as the source of human Campylobacter infection but suggest that asymptomatic Campylobacter infection may occur even in individuals with only limited earlier exposure to Campylobacter.

Genetic diversity and host associations in Campylobacter jejuni from human cases and broilers in 2000 and 2008.

Campylobacter jejuni is an important food-borne pathogen, with a global distribution. It can colonize numerous host species, including both domestic and wild animals, but is particularly associated with birds (poultry and wild birds). For human campylobacteriosis, poultry products are deemed the most significant risk factor for acquiring infection. We conducted a genotyping and host attribution study of a large representative collection of C. jejuni isolated from humans and broilers in Sweden in the years 2000 and 2008. In total 673 broiler and human isolates from 10 different abattoirs and 6 different hospitals were genotyped with multilocus sequence typing. Source attribution analyses confirmed the strong linkage between broiler C. jejuni and domestic human cases, but also indicated a significant association to genotypes more commonly found in wild birds. Genotype distributions did not change dramatically between the two study years, suggesting a stable population of infecting bacteria.

Monitoring Campylobacter in the poultry production chain­from culture to genes and beyond.

Improved monitoring tools are important for the control of Campylobacter bacteria in poultry production. Standardized reference culture methods issued by national and international standardization organizations are time-consuming, cumbersome and not amenable to automation for screening of large numbers of samples. The ultimate goal for rapid monitoring of Campylobacter is to prevent contaminated meat from entering the food market. Currently, real-time PCR is fulfilling abovementioned criteria to a certain extent. Further development of real-time PCR, microarray PCR, miniaturized biosensors, chromatographic techniques and DNA sequencing can improve our monitoring capacity at a lower cost. Combined with innovative sampling and sample treatment, these techniques could become realistic options for on-farm and liquid-sample monitoring at slaughterhouses.

Identification of three clusters of canine intestinal spirochaetes by biochemical and 16S rDNA sequence analysis.

It has been suggested that canine intestinal spirochaetes consist of Brachyspira pilosicoli and a group of strains that has been provisionally designated 'Brachyspira canis'. The purpose of the present study was to compare 22 spirochaete isolates that were obtained from intestinal specimens of dogs in Sweden (n = 12), Norway (n = 4), the United States (n = 3), Australia (n = 2) and Germany (n = 1) with type and reference strains, as well as field isolates, of Brachyspira species by five biochemical tests and determination of almost-complete 16S rDNA sequences. In an evolutionary tree derived from 16S rDNA sequences, the canine isolates grouped into three clusters. One cluster included the type strain of porcine B. pilosicoli, whereas a second larger cluster, which was monophyletic, contained a canine strain that was identified previously as 'B. canis'. The third cluster consisted of three canine isolates of Scandinavian origin, which grouped together with the type strain of the species Brachyspira alvinipulli (pathogenic to chicken). These three genotypes, which were identified on the basis of 16S rDNA sequences, corresponded to four phenotypic groups based on biochemical testing. Two biochemical tests, hippurate hydrolysis and alpha-galactosidase production, were sufficient for rapid identification of each canine cluster.

Species identification by genotyping and determination of antibiotic resistance in Campylobacter jejuni and Campylobacter coli from humans and chickens in Sweden.

Campylobacter is today the most common cause of human bacterial enteritis in Sweden, as well as in most other industrialized countries. Common sources of infection are undercooked chicken meat, unpasteurized milk and contaminated drinking water. One aim with our present study was to identify the species Campylobacter jejuni and Campylobacter coli strains from humans and chickens using a polymerase chain reaction/restriction enzyme analysis (PCR/REA) method, as well as traditional hippurate hydrolysis test. Another aim was to investigate the antibiotic resistance pattern of the human domestic C. jejuni/C. coli isolates from infected patients and isolates from healthy Swedish chicken, as well as isolates from humans infected abroad. If discrimination between C. jejuni and C. coli was based on testing for hippurate hydrolysis, 95% of the human domestic strains and 88% of the chicken strains were identified as C. jejuni. Based on genotyping by PCR/REA, 100% of the human domestic strains and 98% of the chicken strains were attributed to C. jejuni. The E-test and disc diffusion methods were used for phenotypic antibiotic resistance studies. The two methods gave similar results. Most Swedish C. jejuni/C. coli isolates both from humans and chickens were sensitive to doxycycline and erythromycin, which are antibiotics used to treat human infection. Only 7% of the human domestic strains and 2% of the chicken strains were resistant to the quinolones tested. As a comparison, more than 94% of strains isolated from travelers to Asia and southern Europe showed antibiotic resistance to one or more drugs.

Multilocus sequence typing of Campylobacter jejuni from broilers.

Campylobacter jejuni isolates from a national Swedish Campylobacter monitoring in broilers were characterized by multilocus sequencing typing (MLST) in order to study the genetic diversity of this bacterial population. Isolates were initially characterized by pulsed-field gel electrophoresis (PFGE). One hundred were chosen for MLST genotyping. PFGE identified 69 distinct types compared to 44 different sequence types (STs) identified with MLST. Eighteen STs had not been described previously, while the remaining 26 STs were assigned to previously known clonal complexes. The majority of isolates were of genotypes noted in broilers and in humans in earlier studies. However, three clonal complexes, ST-206 complex, ST-677 complex and ST-1034 complex, previously associated with wild bird and environmental samples, were among the genotypes found. This study shows that most of the Swedish broiler isolates were of genotypes noted as common in broilers. However, it also highlights the potential influence of environmental sources on the broiler C. jejuni genotypes.

Within-flock variations of Campylobacter loads in caeca and on carcasses from broilers.

The proportions of broiler caeca and carcass rinse samples positive for Campylobacter spp. within broiler flocks were determined. Twenty intact caeca per flock from 27 flocks were analyzed individually. Rinse samples were obtained from 5 to 25 carcasses from 27 other flocks. In total, 540 caecum samples and 445 carcass rinse samples were analyzed. The proportion of positive caeca within flocks ranged from 10 to 100%, and the proportion of positive carcasses ranged from 85 to 100%. The highest and lowest numbers of Campylobacter spp. found in positive caecum samples were 8.6 and 1.7log cfu/g caecal contents, respectively. The number of Campylobacter spp. in the caeca from individual broilers within a flock varied by up to 6.3log cfu/g caecal contents. The highest number found on one carcass was 4.2log cfu/ml carcass rinse sample, and the within-flock variation in Campylobacter spp. was up to 3.2log cfu/ml rinse sample. There was thus great variation in the load of Campylobacter spp. in individual caecum and carcass samples obtained from each positive broiler flock. This large variation in the numbers of Campylobacter spp. carried by individual birds should be considered when only one or a few samples are collected from a flock and the results are used for risk assessments.

Identification of nine sequence types of the 16S rRNA genes of Campylobacter jejuni subsp. jejuni isolated from broilers.

BACKGROUND: Campylobacter is the most commonly reported bacterial cause of enteritis in humans in the EU Member States and other industrialized countries. One significant source of infection is broilers and consumption of undercooked broiler meat. Campylobacter jejuni is the Campylobacter sp. predominantly found in infected humans and colonized broilers. Sequence analysis of the 16S rRNA gene is very useful for identification of bacteria to genus and species level. The objectives in this study were to determine the degree of intraspecific variation in the 16S rRNA genes of C. jejuni and C. coli and to determine whether the 16S rRNA sequence types correlated with genotypes generated by PFGE analysis of SmaI restricted genomic DNA of the strains. METHODS: The 16S rRNA genes of 45 strains of C. jejuni and two C. coli strains isolated from broilers were sequenced and compared with 16S rRNA sequences retrieved from the Ribosomal Database Project or GenBank. The strains were also genotyped by PFGE after digestion with SmaI. RESULTS: Sequence analyses of the 16S rRNA genes revealed nine sequence types of the Campylobacter strains and the similarities between the different sequence types were in the range 99.6-99.9%. The number of nucleotide substitutions varied between one and six among the nine 16S rRNA sequence types. One of the nine 16S rRNA sequence profiles was common to 12 of the strains from our study and two of these were identified as Campylobacter coli by PCR/REA. The other 10 strains were identified as Campylobacter jejuni. Five of the nine sequence types were also found among the Campylobacter sequences deposited in GenBank. The three 16S rRNA genes in the analysed strains were identical within each individual strain for all 47 strains. CONCLUSION: C. jejuni and C. coli seem to lack polymorphisms in their 16S rRNA gene, but phylogenetic analysis based on 16S rRNA sequences was not always sufficient for differentiation between C. jejuni and C. coli. The strains were grouped in two major clusters according to 16S rRNA, one cluster with only C. jejuni and the other with both C. jejuni and C. coli. Genotyping of the 47 strains by PFGE after digestion with SmaI resulted in 22 subtypes. A potential correlation was found between the SmaI profiles and the 16S rRNA sequences, as a certain SmaI type only appeared in one of the two major phylogenetic groups.

Granulocytic ehrlichiosis in Swedish dogs and horses.

Granulocytic ehrlichiosis is a frequently diagnosed tick-borne disease in Swedish dogs and horses. The infection is caused by a granulocytic Ehrlichia species belonging to the Ehrlichia phagocytophila genogroup. In the acute stage, the disease is mainly characterized as a febrile illness and diagnosis can be confirmed by the demonstration of ehrlichial inclusions in blood granulocytes. Seropositivity in many healthy dogs and horses indicate that the infection also can be transient without clinical signs. The infection can persist in experimentally inoculated animals for months, but to what extent this persistance also occurs in naturally infected animals and is associated with clinical signs, is not clarified yet.

Serological survey of Toxoplasma gondii infection in pigs slaughtered in Sweden.

The prevalence of Toxoplasma gondii infection in Swedish pigs was investigated by analysis of 807 meat juice samples collected in 1999 from 10 abattoirs in different parts of the country. When analysed using ELISA, 42 (5.2%) of the samples were found to be positive. The seroprevalence was 3.3% in fattening pigs (n = 695) and 17.3% (n = 110) in adult swine. Alternative interpretations of the results, considering estimates of the true prevalence based on the sensitivity and specificity of the test method, are discussed. It is concluded that the risk of contracting T. gondii infection as a result of eating undercooked pork from Swedish pigs, especially adult animals, is not negligible.

Isolation and identification of thermophilic Campylobacter species in faecal samples from Swedish dogs.

To investigate the role of Swedish dogs as potential reservoirs of thermophilic Campylobacter species, faecal samples were analysed from 91 dogs in 2001. The majority of dogs (n = 84) were healthy family dogs. Campylobacter spp. were isolated from 51 of the 91 dogs (56%). A significant difference in isolation rates was observed between younger and older dogs: 76% of the younger dogs (5-12 months) were positive, compared with 39% of dogs > or = 13 months (p < 0.01). Two different selective media, Preston and CAT, were used for isolation of Campylobacter species. 104 Campylobacter isolates were identified to species level using polymerase chain reaction and restriction enzyme analysis techniques. Campylobacter upsaliensis predominated and was isolated from 39 dogs, C. jejuni from 10, C. coli from 2, C. helveticus from 2 and C. lari from 1 dog. Four dogs had mixed flora with 2 different Campylobacter species. These data clearly show that younger dogs in particular frequently shed thermophilic Campylobacter spp, which could be of impact for public health. To establish the zoonotic potential of canine Campylobacter isolates, both human and canine isolates have to be further characterized and compared.