SYTTY
HAZARD OF ANIMAL SHIGATOXIC ESCHERICHIA COLI FOR HUMAN HEALTH
Project leader: Sinikka Pelkonen, National Veterinary and Food
Research Institute, Kuopio Regional Laboratory, P.O. Box 92, FIN-70701
Kuopio, Finland, tel. +358-17-201 458, +358-9-3931826, e-mail: Sinikka.Pelkonen@eela.fi
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| TIIVISTELMÄ SUOMEKSI |
Researchers:
National Veterinary and Food Research Institute (EELA), Kuopio Department:
Sirpa Heinikainen, tel.+357-17-201 493, Sirpa.Heinikainen@eela.fi
Tarja Pohjanvirta, tel. +358-17-201 493, Tarja.Pohjanvirta@eela.fi
Hideki Kobayashi, visiting scientist from the National Institute
of Animal Health, Japan
Virpi Seppänen
National Public Health Institute (KTL), Helsinki:
Anja Siitonen tel. +358-9-4744 8245, Anja.Siitonen@ktl.fi
Marjut Saari tel. +358-9-47441, Marjut.Saari@ktl.fi
Matti Sarvas, tel. +358-9-4744 8241, Matti.Sarvas@ktl.fi
Nina Klinger tel. +358-9-47441, Nina.Klinger@ktl.fi
Pertti Koski tel. +358-9- 47441, Pertti.Koski@ktl.fi
Kuopio Regional Institute of Occupational Health:
Markku Seuri tel. +358-17-201 223, Markku.Seuri@occuphealth.fi
Financing SYTTY organisation: The Ministry of Agriculture and
Forestry, Makera
Funding from SYTTY / Total funding of project (€): 141277
/ 340057
Person-months of work funded by SYTTY / Total person-months of work:
58 / 96,5
KEY WORDS: shiga toxin-producing Escherichia coli (STEC), EHEC,
foodborne disease
EXTENDED ABSTRACT
1 Introduction
Certain shiga toxin-producing strains of Escherichia coli (STEC) may cause diarrhoea, haemorrhagic colitis and, in children in particular, haemolytic uraemic syndrome (HUS). Such STECs are also called enterohaemorrhagic E. coli (EHEC). Ruminants are regarded as the major source of STECs, and the disease is transmitted from animals to humans via contaminated foodstuffs and water. The aim of this project was to analyse what kind of hazard animal STECs, other than serotype O157:H7, pose for human health in Finland. The project included studies on prevalence of STECs in animals, such as healthy and diarrhoeic calves, cattle at slaughter, gulls and reindeer, and epidemiology of STECs at a farm level. The isolates from animals were compared by their virulence factor genes, genotypes and serotypes with non-O157 STEC isolates from human cases. Exposure of cattle farmers to STECs is at present analysed by bacteriology of stool samples and by measurement of anti-shiga toxin antibodies.
2 Materials and Methods
Materials: Stool samples were obtained from 568 calves aged under 14 weeks (371 diarrhoeic, 197 clinically healthy; altogether from 272 farms in different parts of the country), from 200 cattle at slaughter, from calves on three 3-site production units at the age of 2 to 12 wk, from 178 healthy reindeer (Rangifer tarandus) at slaughter representing all reindeer ranging areas of northern Finland, from 923 elk and 37 deer during hunting from all parts of the country, from 23 broiler chicken flocks before slaughter (altogether 199 samples from two major chicken rearing companies), from 30 urban pigeons from the city of Kuopio, and from 86 healthy gulls (Larus ridibundus, n=54, and L. argentatus, n=32) from eastern Finland. Two cattle farms were sampled bimonthly for one year (1 dairy farm with 42-60 animals sampled and 1 beef farm with 21 heifers sampled). Faecal and serum samples have been obtained from 104 farmers from cattle farms.
Methods: The prevalence of the stx and eae genes in stool samples was analysed by PCR from mixed cultures on SMAC plates cultured from faecal samples enriched overnight at 37oC in mTSB with novobiocin. Different enrichment broths, times and temperatures were tested. From PCR-positive mixed cultures E. coli strains were isolated by single colony PCR or colony hybridisation with stx or eae DNA probes. The lower limit of isolation was 1 STEC colony per 300 E. coli colonies. The strains were characterised by PCR for various virulence factor genes, such as stx, bfp, and ehlyA and the LEE genes espB, espD, espA and tir, and further intimin gene types ?and ? Intimin genes not conforming to any previously described type were sequenced. Isolates with stx2 gene were subtyped by PCR-RFLP. Selected isolates were analysed by pulsed field gel elctrophoresis and ribotyping, and selected ribotypes serotyped or sent for serotyping to the WHO E. coli centre in Copenhagen.
3 Results and Discussion
The prevalence of STEC in cattle at slaughter was roughly 30%, in calves up to 93% (Table 1). The detection limit was 104 cfu/g. The farm epidemiology of STEC varied: on one farm a particular strain predominated in different animals over longer time, whereas on an other farm animals harboured several STEC strains at a time. Prevalence was the highest in the end of pasture period and the lowest before summer, and calves harboured more STEC bacteria than adult animals. Herd prevalence can be regarded to be up to 100%, because not all animals do excrete or carry the organisms at a certain time point and the detection limit was high.
Table 1. Prevalence of stx positive faecal samples among different animal groups, detection by stx PCR from mixed culture.
| Animal group | stx+¤/no. samples | % |
| calf, diarrhoea | 98/371 | 26# |
| calf, healthy | 65/197 | 33# |
| calf, 3-site production& | 125/257 | 34-93 |
| cattle at slaughter | 57/200 | 29 |
| reindeer | 1/178 | 0.6 |
| elk, deer | 97/960 | 10 |
| gull | 0/86 | 0 |
| urban pigeon | 0/30 | 0 |
| broiler chicken | 0/199$ | 0 |
The number of STEC bacteria in calves was surprisingly high: about one third of the PCR-positive calves had STEC as major type of E. coli, one third had few colonies per 300 E. coli, and one third had less than 1 colony per 300 E. coli. In calves, there were STECs as often present in samples from diarrhoeic and healthy calves, but a certain type of STEC (O26:H11; ribotype V1; virotype stx1, eae, ehly, intimin gene ?) was associated with diarrhoea. The O26 serogroup was also the most common STEC type among calves collected from different farms to 3-site production units. This serogroup is one of the most important EHEC serogroups, too.
The proportion of EHEC serotypes other than O157:H7 among human EHEC infections in Finland has varied from 40 to 71% in the last few years. Twenty different O serogroups have been detected from human EHEC cases in Finland, of these eight were detected from cattle. In addition, the serogroup O113 was recovered from calves. This serogroup belongs worldwide to the most important EHEC O groups, but has not yet caused human disease in Finland. Identical STEC types, according to serotype, virotype and genotype, were detected both from calves and humans. Roughly 11% of the farms with calves harboured such STEC types that, based on O serogroup and virulence factors, may be hazardous to humans.
The high prevalence of STECs in Finnish cattle indicates that farmers and slaughterhouse employees who are in a daily contact with cattle are exposed to STECs. The ongoing part of the project which analyses this exposure by bacteriological and immunological methods has, however, found only one human faecal sample positive for STEC by PCR among 104 samples that have been obtained from farmers. Sampling from slaughter house workers could not be organised in this study.
STECs could not be isolated from birds, and only one out of 178 reindeer samples was positive for STECs (Table 1). Thus not all ruminants, such as reindeer, carry STEC at a high frequency. In contrast, E. coli isolates containing the intimin gene, eae, were present in 70% of the reindeer samples, 26% of the elk and deer samples, 57% of the broiler flocks, 41% of the gull samples and 7% of the pigeon samples. The prevalence of eae positive E. coli at such a high frequency in healthy birds and ruminants is interesting because the eae gene of the LEE locus is regarded as the hallmark of enteropathogenic E. coli (EPEC). Similar subtypes of eae gene and O serogroups occur in the eae positive strains from humans and animals, but the isolates from birds clearly differ from those from humans.
4 Conclusions
Shiga toxin-producing E. coli are highly prevalent in Finnish cattle and elk populations, but very rare in reindeer and birds. Detailed analysis of virulence genes can identify animal STEC types that have occurred in human infections and may thus be considered hazardous to human health. However, the prevalence of human EHEC infection is extremely low with regard to the high prevalence of STEC in animals. This indicates that not enough is known about the factors that predispose humans to EHEC infection or make bacteria pathogenic.
Combination of the virotype result with the serotype identifies certain types of STECs from cattle that are similar to the isolates from human EHEC infections. Similarity can further be analysed by genotyping, keeping in mind the limitation that some serogroups are genetically homogenous and others heterogenous. The high prevalence of STEC in animals makes it cumbersome and often impossible to trace back a human infection to an animal level.
The study indicates that control of EHEC infection has to be focused
to milk and meat hygiene whereas there are no means of eradicating or even
reducing the level of STEC in animals. Thus the study has provided data
and methodology to the risk assessment of food-borne enterohaemorrhagic
E. coli infection and to the control and surveillance measures in the food
production chain.