Introduction

Cryptosporidium is among the most common parasites of domestic and wild animals and humans. Interest in Cryptosporidium has heightened in the veterinary field, not only because of the potential for zoonotic transmission, but also because of difficulties controlling economic losses in production animals (Ramirez et al. 2004). A large body of work has been published on Cryptosporidium and cryptosporidiosis in domestic and captive animals (Kváč et al. 2014a). Although cryptosporidiosis was initially considered to occur rarely in horses and was associated with immunodeficiency (Snyder et al. 1978), more recent studies have shown that horses are frequently infected with Cryptosporidium (Olson et al. 1997; Xiao and Herd 1994). Natural equine cryptosporidiosis has been reported in many countries worldwide, including America (Cole et al. 1998; de Souza et al. 2009; Xiao and Herd 1994), Canada (Gajadhar et al. 1985; Olson et al. 1997), New Zealand (Grinberg et al. 2003, 2009), Africa (Laatamna et al. 2013) and a number of European countries (Majewska et al. 2004; Ryan et al. 2003; Sturdee et al. 2003; Veronesi et al. 2010). Horses appear susceptible to at least three Cryptosporidium spp.: Cryptosporidium horse genotype, Cryptosporidium parvum and Cryptosporidium erinacei (previously known as hedgehog genotype) (Grinberg et al. 2003; Laatamna et al. 2013; Ryan et al. 2003). All Cryptosporidium spp. detected in horses to date are also infectious for humans (Kváč et al. 2014b; Robinson et al. 2008; Xiao et al. 2009). The aim of this study was to determine the diversity of Cryptosporidium spp. in horses under various conditions in the Czech Republic and Poland and to determine any associations between infection occurrence and age, sex, housing systems and consistency of faeces.

Material and methods

During 2011 and 2012, faecal specimens were collected from 352 horses of different ages (5 days to 32 years) on 23 horse farms throughout the Czech Republic (CR; n = 20) and Poland (P; n = 3). The farms were screened without previous knowledge of parasitological status. The management systems in operation on the farms, which were exclusively horse farms were as follows: (i) year-round grazing with shelters (CR farm #1–4), (ii) year-round grazing with overnight housing in stables during winter (CR #5–7), (iii) year-round housing in stables on concrete floors (CR #8–10), (iv) year-round housing in the stables on the deep straw bedding (CR #11), (v) daytime grazing and overnight housing in stables on deep straw bedding (CR #12–16) and (vi) daytime grazing and overnight housing in stables on concrete floors (CR #17–20; P #21–23). Each sample was individually placed in a plastic dish without fixation, stored in at 4 °C and analysed within 48 h for the presence of Cryptosporidium using the aniline-carbol-methyl violet staining method (Miláček and Vítovec 1985). The faecal consistency (loose if it took the form of the container and solid if it maintained its original shape) was noted at the time of sampling. Repeated analyses of the same animals were excluded from the survey to avoid estimating cumulative prevalence.

Total DNA was extracted from 200 mg of faecal samples from each specimen by bead disruption for 60 s at 5.5 m/s using 0.5 mm glass beads in a FastPrep®24 Instrument (MP Biomedicals, CA, USA) using QIAamp® DNA Stool Mini Kit (QIAGEN, Hilden, Germany).

The extracted DNA was kept frozen at −20 °C until used for genotyping. Nested PCR protocols amplifying a fragment of the small-subunit (SSU) rRNA, the 60-kDa glycoprotein (gp60) and four minisatellite markers, including the MS1 (encoding a hypothetical protein), MS2 (encoding a 90-kDa heat shock protein), MS3 (encoding a hypothetical protein) and MS16 (encoding a leucine-rich repeat family protein) genes of Cryptosporidium were performed in duplicate as previously described by Alves et al. (2003); Jiang et al. (2005); and Feng et al. (2011). Negative (PCR water) and positive controls (samples containing DNA of Cryptosporidium suis for SSU, Cryptosporidium hominis for gp60, Cryptosporidium andersoni for MS genes) were included with each PCR amplification. PCR products were visualized following electrophoresis on a 1 % agarose gel containing 0.2 g/ml ethidium bromide. All sequences were confirmed by sequencing amplicons from two independent DNA extractions. PCR products were sequenced in both directions on an ABI 3730XL sequence analyser (Applied Biosystems, Foster City, CA). Sequences were assembled, manually edited and aligned using the ChromasPro 1.7.4 (Technelysium, Pty, Ltd.), BioEdit v7.0.5.3 (Hall 1999 and MAFFT version 7 online server with automatic selection of alignment mode (http://mafft.cbrc.jp/alignment/server/), and were compared with sequences in GenBank. Phylogenetic trees were inferred by the neighbour-joining method, with pairwise deletions, from distances estimated using the Kimura 2-parameter distance model (MEGA5) (Tamura et al. 2011). Bootstrap support for branching was based on 1000 pseudoreplicates. Phylograms were edited for style using CorelDrawX5. Sequences have been deposited in GenBank under the accession numbers KJ469983, KJ469985-KJ469989 and KP176787-KP176793.

Relationships between Cryptosporidium spp. presence and the age or sex of the animal or farm management practices were determined using a classical chi-squared test of independence without Yates’ continuity correction. Statistical analyses were performed using R (version 2.15.0), a software environment for statistical computing.

Results

Cryptosporidium spp. was detected in 12/352 (3.4 %) samples tested by PCR and none of the samples was tested by microscopy. Positive samples were from 4/23 (17.4 %) of farms. Statistical analyses did not show any association between sex or age of the animal and Cryptosporidium prevalence (data not shown). Cryptosporidium was detected only on farms operating under management systems combining pasture grazing and housing in stables. None of horses showed signs of diarrhoea at the time of sampling or during the 2 weeks prior to sampling. Analysis of partial sequences of the SSU gene showed the presence of C. parvum (n = 1), Cryptosporidium horse genotype (n = 1) and C. muris (n = 9) (Fig. 1a). Neighbour-joining trees constructed using on gp60 sequences obtained in this study and sequences published in GenBank revealed the presence of C. parvum (n = 1), C. tyzzeri (n = 1) and Cryptosporidium horse genotype (n = 1) belonging to family IIa, IXb and VIa, respectively. Based on the established gp60 nomenclature (Sulaiman et al. 2005), the Cryptosporidium subtypes were named IIaA15G2R1 (C. parvum), IXbA22R9 (C. tyzzeri) and VIaA15G4 (horse genotype) (Fig. 1b). Cryptosporidium parvum and horse genotype were identified in two young stallions on the same farm (#15). At the four minisatellite loci (MS1, MS2, MS3 and MS16), six, five, zero and seven C. muris-positive isolates were sequenced, respectively (Table 1). A total of four out nine C. muris isolates were subtyped at three loci.

Fig. 1
figure 1

Phylogenetic relationships between Cryptosporidium spp. found in present study (highlighted) and other Cryptosporidium spp. as inferred by a neighbour-joining analysis of a SSU (820 base positions in the final dataset), b gp60 (692 base positions in the final dataset), c MS1 (encoding a hypothetical protein; 436 base positions in the final dataset), d MS2 (encoding a 90-kDa heat-shock protein; 406 base positions in the final dataset) and e MS16 (encoding a leucine-rich repeat family protein; 549 base positions in the final dataset) genes. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates). Numbers at the nodes represent bootstrap values for the nodes gaining more than 50 % support. A scale bar is included in each tree. Interrupted branch has been shortened fivefold. CZ Czech Republic, PL Poland

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Table 1 Specimens detected in the study and their species/subtype identity at the SSU, gp60 and four minisatellite MS loci
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Discussion

Results of this study are consistent with the reported low worldwide prevalence of Cryptosporidium infection in horses (e.g. de Souza et al. 2009; Majewska et al. 2004; Olson et al. 1997; Veronesi et al. 2010; Xiao and Herd 1994). Unlike pigs or cattle, we found no association between management system or sex and Cryptosporidium infection (Maddox-Hyttel et al. 2006; Mohammed et al. 1999). However, this may be due to the very low prevalence of Cryptosporidium in horse populations. Most studies, including the present one, have found no association between Cryptosporidium infection and clinical signs in horses and foals, suggesting that cryptosporidiosis is frequently subclinical in healthy horses (e.g. McKenzie and Diffay 2000; Majewska et al. 2004; Veronesi et al. 2010; Xiao and Herd 1994). However, Grinberg et al. (2009), Perrucci et al. (2011) and Díaz et al. (2012) reported an association between infection by C. parvum and diarrhoea primarily in foals. The C. parvum subtypes, IIa A15G2R1 and IIaA18G3R1, associated with clinical cryptosporidiosis in foals (Díaz et al. 2012; Grinberg et al. 2008) also cause diarrhoea in humans and livestock (Glaberman et al. 2002; Trotz-Williams et al. 2006; Wielinga et al. 2008). Absence of clinical disease in horses infected with IIa A15G2R1 in this study could be due to the age of infected horses (older than 3 years). In addition, the failure to detect C. parvum oocysts by microscopy suggests that the infection intensity was low. It is possible that asymptomatically infected adult horses are sources of C. parvum causing cryptosporidiosis in foals.

The Cryptosporidium horse genotype detected was identical to an isolate found in a foal in Italy (VIaA15G4; Caffara et al. 2013) and belonged to the same subtype family as a horse genotype previously found in Prezewalski’s and domestic horses (Burton et al. 2010; Ryan et al. 2003). In contrast, two human isolates from England (Robinson et al. 2008) and New Mexico (Xiao et al. 2009) belonged to the Cryptosporidium horse genotype VIb family, which has never been found in horses. Consistent with previous studies, we found a low frequency of Cryptosporidium horse genotype occurrence (e.g. Grinberg et al. 2003, 2008, 2009; Veronesi et al. 2010). Unexpectedly, we also found rodent-specific C. muris and C. tyzzeri in horses in this study. Both rodent-specific Cryptosporidium species have been found previously in non-specific hosts, including humans and domestic animals such as pigs, cattle and camels (Kváč et al. 2014a). It is not known whether the presence of C. muris and C. tyzzeri DNA in horse faeces was due to an active infection or mechanical passage of oocysts through the digestive tract. Previous studies suggested that the presence of C. muris and C. tyzzeri in faecal samples of snakes, lizards, raptors and pigs was due to the ingestion of mice or contamination from the environment. The association of these rodent species with horses kept in stables with straw bedding supports the hypothesis of passive transport. However, the C. tyzzeri subtype (IXbA22R9) was previously found only in Mus musculus domesticus, and all positive horses were bred within an area where mice (Mus musculus musculus) were infected with IXa family only (Kváč et al. 2013). The presence of C. tyzzeri IXb in horses kept in an area of IXa distribution could be explained by an ongoing infection in incoming horses, as the farm served as breeding centre for young horses, which could be more susceptible to infection.

MLST subtyping provided a data on the occurrence of C. muris subtypes on the monitored farms. The C. muris subtypes obtained from horses in the Czech Republic and Poland included variants of MS1-M1, previously found in a camel, Tawny frogmouth, laboratory mouse and human; MS2-M4, found in an ostrich, Siberian chipmunk, hamster, mara, laboratory mouse and human; and MS16-M1, found in a siberian chipmunk, hamster, mara, cat, domestic and laboratory mouse (Feng et al. 2011; Wang et al. 2012). While isolates originating from horses kept in Poland had the same subtypes at MS1, MS2 and MS16 loci as isolates from Czech Republic, novel variant at the MS2 locus, named M7, were found in horses in the Czech Republic. Although it is likely that various subtypes of C. muris could cause infections in various hosts, the susceptibility of horses to C. muris and C. tyzzeri remains unclear. Understanding of the epidemiology of Cryptosporidium infections in horses is gradually improving with an increasing number of studies supported by molecular analyses, but there remains much to discover.