Coprological Survey of Helminths in Reindeer (Rangifer tarandus) in 50 Selected Zoos and Menageries in Russia

1. Introduction

Helminths of reindeer (Rangifer tarandus) have already been studied quite thoroughly, and the knowledge on them was summarized in thematic books [1,2,3] and chapters [4,5,6,7,8]. Ongoing studies expand our views on many aspects of reindeer helminths [9,10,11,12,13], and sometimes even add new species to the checklist of parasitic worms of Rangifer [14,15].

Reindeer exist in domestic and wild forms totaling in more than 4 mln animals worldwide [16]. Their popularity as zoo cervids in the 21st century is unprecedentedly growing. According to Species360 ZIMS database, there are more than 500 Rangifer spp. distributed in more than 150 zoos around the world [17]. Some research on reindeer helminths in captive facilities were published [3,16,18,19,20], but they are still too rare and fragmentary.

Meanwhile zoo conditions are unique for reindeer, because even domestic reindeer mostly are not kept in farms and captive facilities of the kind like cattle. In the zoo, reindeer are usually surrounded by many different animals they would never encounter naturally. It might pose a threat of picking up some new helminths. Numerous petting zoos raise another issue: the safety of such tactile communications for human visitors.

Therefore, the aim of our study was to conduct a large-scale survey of reindeer helminths in Russian zoos. Fecal examination being non-invasive method (an essential trait for zoo animals) offers wide diagnostic capabilities, scoping gastrointestinal, lung, brain, and muscle parasitic worms. This study was done to find out, what kind of helminths inhabit zoo reindeer across Russia, how widespread they are, how typical they are, and how dangerous they are both for reindeer and humans.

2. Materials and Methods

2.1. Helminths Recovery

Our study collected fecal samples from 233 reindeer kept in 50 selected zoos and menageries located in Russia (Table 1). Those include: all the state zoos in which collections reindeer are present (17); other state organizations having live reindeer for exhibition (Children's ecological station, Natural-ethnographic complex, and Arctic Tourism Center); private organizations-members of the Union of Zoos and Aquariums of Russia (5 private zoos and an urban farm); and 25 private menageries. The latter constitute of “clubs”, “parks: (“recreation-“, “ethnic-“, “fauna-“, and “safari park”), “reserve” (self-proclaimed), “mini zoo”, “ranch”, “farm”, “farmstead”, “pets' corner” and no name menageries. Many of these organizations participated in our research anonymously; therefore, for uniformity, we do not provide the actual names of all the 50 of them. However, their location combined with the type of ownership makes the data quite transparent. For brevity, further on all of these organizations would be referred to as zoos.

Qualitative and quantitative fecal analyses, light microscopic study, and measurements of obtained eggs and larvae of helminths were performed as described by Loginova et al. [21].

2.2. DNA Analysis

For trematodes, the QIAamp DNA Accessory Set, Micro kit (Quiagen, Netherlands) was used to extract DNA from embryonated eggs as described by Loginova et al. [22]. For nematodes, DNA was extracted either via digestion of first larval stages (L1) in presence of Proteinase K [23] as described by Loginova et al. [24] or by using the QIAamp DNA Accessory Set, Micro kit (Quiagen, Netherlands). We targeted the region of the internal transcribed spacer (ITS rDNA). For trematodes, we used BD1, BD2 primers [25] and corresponding protocols [26]. For nematodes we used either NC1, NC2 primers and protocols as described by Gasser et al. [27] or 18S, 26S primers [28] and protocols as described by Loginova et al. [24]. Visual check of PCR, cleaning of the PCR products, sequencing, and analysis of obtained chromatograms were performed as described by Loginova et al. [21].

3. Results

Flukes, tapeworms and round worms were found at the stage of egg or larva in feces of 106 reindeer (45%) kept in 31 zoos (62%). Identification of helminths was based on their morphology, morphometric data, and (where possible) DNA analysis (Table 2).

Appearance of eggs and larvae of helminths obtained from zoo reindeer feces is shown in Figure 1.

Prevalence rates and distribution of helminths obtained from zoo reindeer feces are summarized in Table 3. For brevity, only helminth-positive zoos are included.

Prevalence rates for total 233 reindeer examined are shown in Figure 2.

Intensity of invasion was mostly low in terms of National Standard of the Russian Federation (GOST R 54627-2011) Ruminant animals—Methods of Laboratory Helminthological Diagnostics [29]. Nematode eggs / gram of feces ranged from 1 to 56 in all reindeer samples. Nematode larvae / gram varied from 1 to 98. Cestode eggs / gram ranged from 1 to 17. Trematode eggs / gram varied from 1 to 8. Exceptions were: fecal sample of reindeer from the zoo #9 that showed 537 E.rangiferi larvae / gram; fecal sample of reindeer from the zoo #30 that showed 56 Paramphistomum sp. eggs / gram. Those numbers are estimated as medium intensity (GOST R 54627-2011).

4. Discussion

Our study revealed trematodes (Fasciola hepatica and Paramphistomum sp.), cestodes (Moniezia expansa and Moniezia sp.), and nematodes (so called small strongylids, Nematodirus spp., Dictyocaulus sp., Elaphostrongylus rangiferi, Trichuris sp., and Capillaria sp.) in zoo reindeer via fecal examination. All of these helminths were previously reported for reindeer [3,8].

In many samples, only one kind of helminths was found. However, we have also discovered combinations of two or three kinds of helminths (Table 4).

Common liver fluke F. hepatica inhabits bile ducts of its host. Previously it was reported for reindeer only in Eurasia, and one of the cases also involved zoo reindeer [3]. It is a rare finding for reindeer, and the latter is considered as an accidental host. Infestation of reindeer with F. hepatica usually is associated with other ruminants in contact and captive facilities [3]. In two private zoos where F. hepatica was found in reindeer, there were sheep, goats and sika deer (Cervus nippon) also infected.

Rumen fluke Paramphistomum sp. is widespread in Russian reindeer. When numerous, they cause atrophy of the rumen papillae and may lead to death [3,8]. Reindeer from the private zoo #30 with medium level of invasion with rumen flukes died within 6 month afterwards.

Tapeworms found were present with M. expansa (easily recognizable by its triangular eggs) and Moniezia sp. These helminths inhabit small intestine and may be quite pathogenic, especially for calves [3,8]. However, in our study it was found only in adults. Reindeer is considered an accidental host for M. expansa, and its infestation is also associated with sympatric livestock and captive facilities [3].

Supposedly, small strongylids found in zoo reindeer are of two different genera (Figure 1D,I) which may belong to fam. Chabertiidae, Cooperiidae, Haemonchidae or Trichostrongylidae. Coproculture is needed to obtain larvae and clarify the diagnosis. In any case, small strongylids inhabit gastrointestinal tract, and their impact depends on many circumstances [8,10].

Eggs of Nematodirus obtained from the reindeer fecal sample from the zoo #40 certainly belong to two different species. Apart from their differences in size and shape, their development rates were also different. Figure 1H shows Nematodirus sp. with 8 blastomeres, whereas Figure 1C shows Nematodirus sp. with gastrulated embryo. These two types of eggs were found in the same fecal sample. In total, 4 eggs (two per type) were obtained from that sample. We made an attempt to get third larval stages (L3s) for DNA analysis, but did not succeed. However, light micrographs (taken daily during three weeks after the finding of these eggs) show their embryonic development, and are available as Supplementary Materials. Helminths of Nematodirus sp. inhabit small intestine. In reindeer, they are often accompanied by Nematodirella longissimespiculata [3], but in this study the latter was not found.

Lung worm Dictyocaulus sp. is the most concerning finding, because its presence may be life threatening. In the only zoo (#30) where it was found, there was also a sika deer infected with Dictyocaulus sp. It is hard to tell, which animal was the source of invasion, or was it a coincidental infestation.

Brain worm E. rangiferi was the most widespread helminth in our research. Its pathogenicity is tricky, because it may as well cause lethal epizootics or have no manifestation of its presence [3,8,30]. Due to localization of adult worms in the nervous system or between the muscles, there is no effective treatment. On the other hand, authors observed few reindeer that were severely infected in 2018 and eventually outlived their brain worms. At least, no E. rangiferi larvae have been found in their fecal samples since 2023. Probably, it would be impossible in the wild, but in human care reindeer can live much longer and, apparently, get rid of some of their parasites naturally [Loginova, unpublished].

Whipworms Trichuris sp. inhabit large intestine and can cause hemorrhagic diarrhoea [8]. They are more expected in captive animals. There are reindeer specific species and those common to ruminants that can also infect reindeer [3]. Egg morphology is not sufficient for the identification up to the species level.

Round worm Capillaria sp. inhabits intestine and is often associated with young animals [3,8]. Still the impact of this parasite is not known. Infection with Capillaria sp. was previously reported for zoo reindeer as well [3].

Thus, via fecal examination we managed to find liver and rumen flukes, GINs, lung worms, brain worms, whipworms, and capillarids. However, this method is not suitable for those helminths that inhabit ligaments (like Onchocerca), abdominal cavity (like Setaria), muscles, lungs, liver and brain (like larval stages of Taeniidae). Therefore, despite the large scale of our study, the helminth status of zoo reindeer is still incomplete.

Another bias of this work concerns different seasons of fecal sampling (which, probably, affected reproductive activity of helminths) and differences in age and sex of studied animals [3,8]. These circumstances should be kept in mind while comparing obtained results between zoos.

As we said earlier, popularity of reindeer as zoo animals in 21 century is outstanding. Only one zoo (#17) has 100 years of its history of keeping reindeer in captivity. It is not surprising that fecal samples from their reindeer were helminth negative not only in this study, but years prior to it. Many other zoos got their first reindeer only in 2023. Majority of the private zoos purchase domestic reindeer from agricultural husbandries. The latter are all private and run by families of indigenous peoples of the North. Zoos are able to buy only those reindeer that indigenous people are willing to sell. Reindeer herders want to keep the best individuals in the herd, and reindeer for sale may be less resistant to helminths.

Domestic reindeer in contrast to wild reindeer can be pure white (leucism, not albinism) throughout the year [31,32]. White reindeer attract most people [33], and numerous private zoos tend to have exactly the white reindeer. However, reindeer herders noticed that white reindeer are not good survivors [33,34]. It might add another disadvantage to such zoo reindeer in terms of helminth resistance, and requires special research.

Apart from husbandry “heritage”, how can zoo reindeer become infected with helminths? In particular, through the birds. Helminths eggs were reported to survive through sparrow digestive system and remain invasive [35]. Thus, birds can be vectors for helminths within the zoo and out of its boundaries.

It is noteworthy that even in helminth positive zoos, about 30% (0 to 83%) reindeer fecal samples were helminth free, probably, due to the different immune status of reindeer.

Besides helminths, we found mites in reindeer fecal samples (Figure A1). They were either ingested during autogruming and passed digestive tract or contaminated fecal samples afterwards. Having found a mite egg in the sample from the zoo #30, we contacted that zoo and shared our considerations. It turned out that the zoo indeed had problems with mange caused by mites (Figure A1E). One adult mite found in a sample from the zoo #40 was identified as Chorioptes sp. (Figure A1D). Chorioptes mites were reported for reindeer in Canada, Norway, and Finland [8]. Another adult mite found in reindeer fecal sample (zoo #12) met morphological criteria for Demodex sp. To authors’ knowledge, Demodex has never been reported for reindeer [3,8,36]. This finding also requires additional research which may result in the description of the new species.

Tactile contact between humans and animals in petting zoos may pose a risk of helminth infestation. For instance, red fox (Vulpes vulpes) and Arctic fox (V. lagopus) are recognized definitive hosts for Echinococcus spp. The latter produces numerous eggs which are shed with feces. Those eggs are very light and flying, and easily contaminate animal’s fur. Stroking such sick fox or even breathing closely to it may result in ingestion of Echinococcus eggs which are instantly invasive [37].

Among helminths found in zoo reindeer in our research, there are few genera, reported for humans. Those are Fasciola, GINs (Trichostrongylus, Oesophagostomum), Trichuris, and Capillaria [37,38,39,40]. However, infected reindeer pose no direct threat for human visitors or zoo employees for the following reasons. Trematode F. hepatica is indeed the species, that can infect both cervids and humans, but its life cycle requires intermediate host (fresh water mollusk) [3]. Eggs shed by reindeer are not the infective stage for any vertebrate host. Moreover, eggs are excreted without miracidium, and it takes time for it to develop (that is possible only in water). Species of Trichostrongylus, Oesophagostomum, Trichuris, and Capillaria that can infect humans are not the same species that infect reindeer [3,8,37,40]. Besides, all these nematodes shed eggs that are not embryonated by the moment of their excretion with feces. Larvae development also requires few days or weeks. Thus, if reindeer dung is regularly removed, even reindeer infected with helminths are safe for humans.

5. Conclusions

In our study F. hepatica, Paramphistomum sp., Moniezia spp. (including M. expansa), gastrointestinal strongylids (including Nematodirus spp.), Dictyocaulus sp., E. rangiferi, Trichuris sp., and Capillaria sp. were found in 106 Russian zoo reindeer out of 233 ones via fecal examination. All these helminths were previously reported for reindeer and pose no direct danger for humans. Intensity of invasions was mostly low. Fecal examination might be considered as an indirect method for mange diagnostics, as Chorioptes and Demodex mites were found in reindeer fecal samples. The latter may represent a novel species of mite specific for reindeer.