Introduction

Cryptosporidium spp. are widespread protozoa considered to be important causes of gastrointestinal disease in humans and vertebrates.

Cryptosporidium infections have been reported in a wide spectrum of mammals belonging to several orders – Primata, Artiodactyla, Perissodactyla, Carnivora, Lagomorpha, Rodentia, Marsupialia, Monotrema and Proboscidea – and in numerous reptilian species belonging to orders Testudines and Squamata (Graczyk et al. 1997; Majewska et al. 1997; Mtambo et al. 1997; Muriuki et al. 1997; Gómez et al. 2000; Graczyk and Cranfield 2000). Recent molecular studies have shown that there is an extensive genetic diversity in Cryptosporidium parasites infecting mammals, reptiles and birds and in addition to the 14 Cryptosporidium accepted species – C. andersoni, C. hominis, C. parvum, C. canis, C. felis, C. wrairi, C. suis, C. muris, C. saurophilum, C. serpentis, C. baileyi, C. meleagridis, C. galli and C. molnari – many host-adapted genotypes have been described such as the deer, mouse, pig, bear, cervine, mongoose, marsupial, snake and lizard genotypes (Morgan et al. 1999a; Ryan et al. 2004; Xiao and Ryan 2004; Xiao et al. 2004a, 2004b).

Studies carried out previously in artiodactyl ruminants at the Lisbon Zoo, showed a 4% prevalence of cryptosporidiosis (Delgado et al. 2003) and molecular characterization has identified the isolates as C. parvum (Alves et al. 2001, 2003). In the present study, we extended the range of animals from the Lisbon Zoo examined for Cryptosporidium to several other mammalian orders and to reptiles. We also characterized part of the small subunit (SSU) rRNA gene of Cryptosporidium-positive samples by PCR amplification and sequence analysis.

Materials and methods

Between April 2002 and February 2003, 274 stool samples were collected from 100 mammals (Table 1) and 28 reptiles (Table 2) kept at the Lisbon Zoo.

Table 1 List of mammals studied
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Table 2 List of reptiles studied
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With the exception of two baby lions, all mammals were adults, and had no Cryptosporidium-related symptoms. The reptiles studied, adults and young animals, were all asymptomatic, with the exception of one Indian star tortoise, which was part of a large group of confiscated tortoises in the black market in Singapore, that was severely sick due to crowded facilities, bad nutrition status, extreme temperature changes, and transport-induced stress. This tortoise arrived at the Lisbon Zoo 3 days prior stool collection and died 2 weeks later.

Direct and concentrated faecal smears were prepared and Cryptosporidium oocysts identified by light microscopy at a ×400 magnification after modified Ziehl-Neelsen staining. In samples that were positive by microscopy, oocysts’ DNA was extracted by a KOH/QIAamp DNA stool mini kit protocol (QIAGEN GmbH) (Alves et al. 2003). The species and genotype of the isolates identified were determined by nested PCR of a fragment of the small subunit (SSU) rRNA gene (Xiao et al. 2001) and sequencing of the secondary PCR products in both directions on an ABI Prism 3100 analyser (Applied Biosystems). The nucleotide sequences obtained were aligned with additional Cryptosporidium SSU rRNA sequences obtained from GenBank using the ClustalX program. A neighbour-joining tree was constructed from the aligned sequences with the TreeconW program using a sequence of Eimeria tenella (AF026388) as an outgroup. Genetic distance was calculated based on the Kimura 2-parameter model. Bootstrap analyses were conducted using 1,000 pseudo replicates.

Results

Oocysts were found in faeces of two artiodactyl bovids, a black wildebeest (Connochaetes gnou) and a Prairie bison (Bison bison bison), and in one Testudinidae, the Indian star tortoise (Geochelone elegans) that came from Singapore. In both mammals, the level of oocyst shedding was low and none had apparent symptoms of cryptosporidiosis. Subsequent stool collection was conducted on these two animals, but no oocysts were found. The level of oocyst shedding in the Indian star tortoise was low.

A fragment of the SSU rRNA gene was successfully amplified and sequenced for these three isolates. BLASTn GenBank searches performed on the sequences obtained from the Prairie bison and the Indian star tortoise, showed 100% similarity with the sequences of Cryptosporidium mouse genotype (AF112571) and Cryptosporidium tortoise genotype (AY120914), respectively (Xiao et al. 1999, 2004b).

The black wildebeest isolate revealed a unique sequence, different from all other animal Cryptosporidium sequences, and thus constituted a new Cryptosporidium genotype in animals. The SSU rRNA partial nucleotide sequence of the black wildebeest Cryptosporidium isolate was deposited in the GenBank database under accession number AY883022. The new genotype from black wildebeest had the highest sequence homology to the W5 genotype (AF262332) previously found in a storm water sample collected in the Eastern Catskill Mountains, NY, with an A to G mutation, a TA insertion and a T deletion (Xiao et al. 2000). A neighbour-joining analysis showed that this genotype from wildebeest formed a cluster with several intestinal Cryptosporidium species (C. parvum, C. hominis, C. wrairi, C. suis, C. canis, C. felis, C. meleagridis and C. saurophilum) and genotypes (rabbit, horse, ferret, mouse, skunk, cervine, fox, squirrel, deer mouse, bear, opossum I and II, marsupial I and II, and muskrat I and II), with 87% of bootstrap support (Fig. 1).

Fig. 1
figure 1

Phylogenetic relationships of Cryptosporidium parasites inferred by neighbour-joining analysis of the SSU rRNA gene based on genetic distances calculated by Kimura 2-parameter model. The tree was rooted with an SSU rRNA sequence from Eimeria tenella (AF026388)

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Discussion

The present finding of Cryptosporidium mouse genotype in a Prairie bison constitutes the first report of this genotype in a ruminant. Gómez et al. (2000) also identified Cryptosporidium oocysts in a Prairie bison at the Barcelona Zoo, but the molecular identification was not performed. The mouse genotype is very common in mice and small rodents (Morgan et al. 1999b; Bajer et al. 2003). Whether the bison was infected with mouse genotype parasites or merely passing oocysts through the gastrointestinal tract after ingestion of food/water in facilities contaminated with rodents’ faeces remains to be determined. Phylogenetically, the mouse genotype has the closest relatedness to C parvum, which infects mainly ruminants (Xiao et al. 2004a). Thus, it is possible that the bison was actually infected by the mouse genotype. However, the absence of clinical signs, the low level of oocyst shedding and its detection only once in only one individual animal cannot exclude the possibility of the mere passage of oocysts in the animal. The turtle genotype found in the Indian star tortoise has already been found in three Indian star tortoises from the Saint Louis Zoo and thus should represent a true parasite of these reptiles (Xiao et al. 2004b).

Cryptosporidium infection is apparently common in wildebeests, as an earlier study identified Cryptosporidium spp. in 27% of black wildebeests from the Mikumi National Park, Morogoro, Tanzania (Mtambo et al. 1997). Because no molecular characterization of the isolates was done, the identity of Cryptosporidium spp. in these animals was not clear. Cryptosporidium was also previously found in faeces of a blue wildebeest (Connochaetes taurinus taurinus) at the Barcelona Zoo (Gómez et al. 2000). Molecular characterization of the parasite identified it as C parvum (Morgan et al. 1999b). The black wildebeest in this study was apparently passing oocysts of a new Cryptosporidium genotype. Due to its similarity with the W5 genotype from storm water, this new genotype may represent a new Cryptosporidium parasite that the black wildebeest acquired from other animals in the Zoo, rather than a native parasite from Africa. Thus, like many species or groups of vertebrates (Xiao et al. 2004a), wildebeests may be infected with at least two Cryptosporidium parasites, C. parvum and this new Cryptosporidium genotype.