Campylobacter spp., Enterococcus spp., Escherichia coli, Salmonella spp., Yersinia spp., and Cryptosporidium oocysts in semi-domesticated reindeer (Rangifer tarandus tarandus) in Northern Finland and Norway

The specific aim of this study was to assess the faecal shedding of zoonotic enteropathogens by semi-domesticated reindeer (Rangifer tarandus tarandus) to deduce the potential risk to human health through modern reindeer herding. In total, 2,243 faecal samples of reindeer from northern regions of Finland and Norway were examined for potentially enteropathogenic bacteria (Campylobacter species, Enterococcus species, Escherichia coli, Salmonella species and Yersinia species) and parasites (Cryptosporidium species) in accordance with standard procedures. Escherichia coli were isolated in 94.7%, Enterococcus species in 92.9%, Yersinia species in 4.8% of the samples and Campylobacter species in one sample only (0.04%). Analysis for virulence factors in E. coli and Yersinia species revealed no pathogenic strains. Neither Salmonella species nor Cryptosporidium oocysts were detected. The public health risk due to reindeer husbandry concerning zoonotic diseases included in this study has to be considered as very low at present but a putative epidemiological threat may arise when herding conditions are changed with respect to intensification and crowding.


Introduction
Zoonotic organisms such as viruses, bacteria or parasites can possess the potential to cause severe diseases both in humans and animals. Free-ranging animals with sporadic or indirect contact to domestic livestock and humans may serve as reservoirs or sentinels for diseases. Transmission of these pathogens can occur directly from a reservoir to the susceptible animal or human being, for example through direct contact with free-ranging animals including cervids [1]. Indirect transmission occurs via vectors, i.e. mosquitos, by contamination of the environment through faecal shedding [2] or by consumption of venison [3]. The epidemiological situation in free-ranging, semi-domesticated animals like reindeer (Rangifer tarandus tarandus), however, is difficult to assess, in contrast to domestic animals. Conclusions derived from studies which focus on domestic animal species in extensive husbandry systems can only be used with caution. But it can be assumed that pathogens are transmitted easily through close animal contact and lead to high animal losses as it is known from intensive husbandry systems. This is of special importance as the crowding of reindeer for winter feeding is becoming more and more common, particularly in southern parts of Northern Finland.
With regard to the occurrence of possibly zoonotic pathogens in reindeer, data is rare. In view of the zoonotic enteropathogens examined in this study, Campylobacter species, Enterococcus species, Escherichia coli, Salmonella species and Yersinia species are among the most important bacteria which cause severe enteric diseases. In detail, various Campylobacter species (C. coli, C. jejuni subspecies jejuni, C. hyointestinalis, C. lari and C. upsaliensis) were isolated from humans with gastro-enteritis [4,5]. Campylobacter hyointestinalis was detected in healthy Finnish reindeer [6]. In a recent study about Campylobacter species and other bacteria in wild cervids in Norway, no Campylobacter species were detected in wild reindeer [7]. Enterococcus species are known as pyogenic organisms and can cause hospital acquired infections, but are primarily natural inhabitants of the gastrointestinal tract of humans and animals. Enterococcus species in reindeer have not yet been described. The virulence and pathogenesis of the examined bacteria depends on different factors. In E. coli, for instance, the ability to cause severe disease in humans and animals is associated with the occurrence of several virulence factors such as shigatoxins. Therefore, the presence of shigatoxin genes can indicate the virulence of certain strains, also known as shigatoxin-producing E. coli (STEC). In 50 faecal samples from wild Norwegian reindeer, 42 were positive for E. coli, but no shigatoxin-producing strains were identified [7]. Like STEC, Campylobacter species, Salmonella species and Yersinia species are a problem in meat production with a high infection risk for humans who consume contaminated products. Salmonella species have been found to be associated with mortality in reindeer in Finland  No data is available on the occurrence of zoonotic protozoa in Northern European reindeer such as Cryptosporidium species as a causative agent for heavy diarrhoea in humans and animals.

Faecal samples
In total, 2,243 faeces samples from healthy reindeer, adults and calves, of both genders were taken over eleven month (June 2001 -April 2002) from eight Finnish and Norwegian free-ranging and corralled reindeer herds, considering parameters such as the degree of intensity of herding, location and season. The origin of samples and further information are given in Table 1. Samples were taken off the ground or per rectum from slaughter animals, sent to the laboratory directly after collection and kept frozen (-4°C) until being processed further within one week.

Examination for Campylobacter species
The examination was carried out by inoculating 1 g faecal material into 9 ml Preston broth (Oxoid). After 24 hours incubation in a microaerophilic atmosphere (5% oxygen, 10% carbon dioxide, 3% hydrogen and 82% nitrogen) at 37°C, a loopful of the enriched suspension was plated on Preston agar (Oxoid, Wesel, Germany) and incubated for 48 hours under the above-mentioned conditions. Campylobacter-like colonies were analysed by Gram-staining, catalase and oxidase tests, and further biochemical reactions (ApiCampy, bioMérieux, Marcy-l'Etoile, France). For all bacteria species, positive controls (ATCC, Manassas, USA) were used to approve the sensitivity of the culture methods.

Examination for Enterococcus species
For the selective enrichment of Enterococcus species, 1 g faecal material was diluted in 9 ml glucose-azide broth (Merck, Darmstadt, Germany) and incubated for 48 hours at 37°C. A loopful of broth was then spread both on kanamycin-aesculin-azide agar (Merck) and Slanetz and Bartley agar (Oxoid). After 48 hours at 37°C suspicious colonies were Gram-stained and their biochemical reactions were analysed further by catalase and oxidase tests.

Examination for Escherichia coli
Escherichia coli was isolated by adding 1 g faeces to 9 ml Gram-negative broth (Becton & Dickinson, Franklin Lakes, USA). After 24 hours of incubation at 37°C a loopful of broth was then plated onto Endo-c agar (Merck) and incubated under the above-mentioned conditions for 24 h. Typical metallic shiny colonies were subcultured on blood agar (Oxoid), incubated for 24 hours at 37°C and tested for their biochemical reactions applying API 20E (bioMérieux). PCR was used to detect the occurrence of shigatoxin1 and 2 genes (stx1, stx2), the intimin gene (eae) and EHEC-haemolysin gene (hly EHEC ) as indicators for the pathogenicity of the isolated strains. Primers were developed with help of the European Molecular Biological Library database and the oligo 6.0 software (Molecular Biology Insight, Cascade, USA) and produced commercially (Invitrogen, Paisley, UK).

Examination for Salmonella species
For the selective enrichment of Salmonella species 1 g faeces was inoculated into 14 ml of tetrathionate broth (Merck) and incubated for 24 hours at 37°C as described by Baird [11]. One ml of this enriched broth was brought into tetrathionate broth the next day and incubated for another 24 hours at 37°C. This enrichment step was repeated one more time. On the fourth day, one loopful of the cultured medium was plated both on Salmonella-Shigella agar (Difco) and Leifson agar (Merck). After 24 hours of incubation at 37°C presumptive Salmonella spe-cies colonies were Gram-stained and tested by API 20E (bioMérieux).

Examination for Yersinia species
Cultural examination of Yersinia species was performed by adding 1 g faeces into 9 ml of Gram-negative broth and incubating for 48 hours at 21°C. One loopful of broth was then plated on Yersinia-selective agar (Difco) and incubated for another 48 hours at 21°C. Colonies with the typical bull's-eye appearance were subcultured on blood agar and Gram-stained and biochemical tests were subsequently carried out by use of API 20E (bioMérieux) and Micronaut (Merlin, Bornheim-Hersel, Germany). To detect various Yersinia-genes, crucial for the pathogenicity of the detected strains, PCR was performed using primers to detect the genes encoding 16SrRNA, yadA and v-antigen.

Examination for Cryptosporidium oocysts
For the detection of Cryptosporidium oocysts, immunomagnetic separation was applied using Dynabeads anti-Cryptosporidium (Dynal Biotech, Oslo, Norway). Twenty µl of the immuno-concentrate were used for a direct immuno-fluorescence test (medac, Wedel, Germany) [12].Cryptosporidium parvum oocysts from a calf (Iowa isolate, Waterborne, USA) served as the positive control. Using a fluorescence microscope at x400-x1000 magnification Cryptosporidium oocysts appear as 6-10 µm in size, round or oval in shape with bright green fluorescence.
For statistical analyses, the data was evaluated with the Statistica 5.0 software (StatSoft Inc., Tulsa, USA), following the instructions of Trampisch & Windeler [13]. For all analyses, differences were considered significant at P ≤ 0.05.

Results
In 2,224 (99.2%) out of the total number of 2,243 faecal samples, one or more of the examined bacteria species were isolated.
No significant differences were found for Enterococcus species and E. coli with regard to the degree of intensity of reindeer herding, the season or the geographic origin, whereas the prevalence of Yersinia species differed significantly (p ≤ 0,001): prevalence for Yersinia species in freeranging reindeer in summer and autumn were significantly higher than in fenced reindeer during winter, as shown in Figure 1. A direct comparison of fenced (n = 100) and free-living (n = 147) reindeer during different seasons was possible in Näkkälä, but with prevalences of 100,00% (fenced) and 65,99% (free-living) for Enterococcus species, 85,00% and 91,63% for E. coli and 1,00% and 0 % for Yersinia species no significant differences could be detected.   . The seasonal pattern of Yersinia species with an increase in the summer period can be attributed to the constant accidental incorporation of environmental strains which in this extent is not given in winter with limited survival conditions for bacteria.

Discussion
Campylobacter hyointestinalis was isolated from one sample only. As the cultivation of Campylobacter species is difficult, the actual prevalence might be higher. Campylobacter hyointestinalis has hitherto been associated only sporadically with human gastrointestinal disorders [5,24]. Even though the prevalence of Campylobacter species in this study was very low, it shows that reindeer can be carriers. This is supported by a study from Hänninen et al. [ All bacteria analysed in this study may be found in Northern Europe in the environment in aquatic, terrestrial and animal reservoirs [28] and have been isolated before from the intestinal tract of healthy or diseased ruminants world-wide [29,30]. Even though most of the isolated bacteria strains do not have the potential to cause severe human or animal health problems, certain strains might be a risk, especially for immuno-supressed, old or very young persons and animals. Therefore, one has to regard the epidemiological impact of transmission of these infectious agents from the environment to reindeer and man and vice versa, depending on a number of local factors. To recapitulate, the excretion risk of pathogens by reindeer has to be considered in the context of the extreme climatic conditions in the research area. Permafrost soils of the tundra and taiga are a domain of psychrophilic and psychrotolerant organisms [31], but as enteropathogens, living in intestines of warm-blooded animals at 37°C, are not adapted to these extreme northern environmental conditions, fast destruction is probable.
In conclusion, the enteropathogens examined were either not detected at all (Salmonella species and Cryptosporidium species), in very small numbers (Campylobacter species) or if detected, their virulence and pathogenicity was very low (E. coli and Yersinia species). The potential human and animal health risk from reindeer excreting various important enteropathogenic bacteria and Cryptosporidium species should be regarded very low at present. No differences could be found in the flora between fenced and free-living animals. However, especially if reindeer are crowded, e.g. for winter feeding, an increased prevalence of enteric pathogens excreted by reindeer and eventually an increased risk to the consumer has to be considered as is already known from other intensive animals husbandry systems worldwide.

Conclusion
With respect to the investigated pathogens, the analysis of faecal samples from Norwegian and Finnish reindeer indi-cates that the animals do not represent an important source for zoonotic diseases at the moment. Enterococcus species and E. coli belong to the normal intestinal flora of reindeer. Climate conditions in the northern regions are a limiting factor to the survival of enteropathogens in the environment and might be a reason for the low prevalences of the other pathogens examined.