Multiple Unique Cryptosporidium Isolates from Three Species of Ground Squirrels (Spermophilus beecheyi, S. beldingi, and S. lateralis) in California

Fecal collection. (i) S. beecheyi.

Fecal samples from California ground squirrels were obtained from Kern, Monterey, San Luis Obispo, Santa Barbara, Stanislaus, and Tulare counties (Fig. 1). Squirrels were captured monthly from six different regions in Kern County from February 2000 until February 2001. California ground squirrels from San Luis Obispo, Santa Barbara, Stanislaus, Monterey, and Tulare counties were captured from October 2002 to April 2004. The animals were dispatched according to the American Veterinary Medical Association's guidelines for harvesting wildlife (2), with fecal samples obtained postmortem from the lower section of the colon.

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FIG. 1.

Geographic locations of three different species of Spermophilus ground squirrels from which 49 isolates of Cryptosporidium were collected in California from 2000 to 2004.

(ii) S. beldingi.

Fecal samples from squirrels were obtained from Siskiyou, Modoc, and Tuolumne counties (Fig. 1). Squirrels from Siskiyou and Modoc counties were captured from March 2003 through March 2004, except during the winter season when animals were in hibernation, with fecal samples obtained postmortem. Belding's ground squirrels in Yosemite National Park, Tuolumne County, were captured in Tomahawk live traps, and fecal samples were collected from freshly voided samples during the summer of 2003.

(iii) S. lateralis.

Golden-mantled ground squirrels in the White Mountains of Mono County, eastern California, were captured in Tomahawk live traps, and fecal samples were collected from freshly voided samples during July and August 2003 (Fig. 1).

Ground squirrel species were determined by using stereotypical visual markings of each host species and by trapping within each species' habitat range.

Fecal samples were placed into 15-ml tubes with 5 ml of antibiotic storage solution (0.1 ml 10% Tween 20, 0.006 g penicillin G, 0.01 g streptomycin sulfate, 1.0 ml amphotericin B solution, and reagent-grade water for a total of 100 ml). Samples were stored at 4°C and transported on ice to the University of California, Davis.

Isolates.

By using a direct immunofluorescence antibody kit (Meridian Bioscience, Inc., Cincinnati, OH) and an Olympus BX60 microscope, fecal samples were screened at a magnification of ×400 for presumptive Cryptosporidium oocysts.

DNA extraction, PCR, and DNA sequencing.

Oocysts were purified by using anti-Cryptosporidium Dynabeads (Invitrogen, Lake Success, NY). DNA was extracted using five repeated freeze (−80°C) and thaw (+80°C) cycles followed by overnight incubation at 60°C in TES [N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid] buffer containing 0.8% Sarkosyl (Sigma, St. Louis, MO). DNA was precipitated in 100% cold ethanol, centrifuged, dried, and stored at 4°C in UltraPure distilled water (DNase and RNase free; Life Technologies, Carlsbad, CA). Depending on the primer pair, PCR amplification generated a DNA sequence for the entire 18S SSU rRNA gene locus, an internal sequence from the primary primer pair, or a sequence from the nested PCR according to the methodology described by Xiao et al. (15), modified by using 3 mM MgCl₂ (1). In addition, one isolate was further characterized by amplifying a section of the Cryptosporidium oocyst wall protein (COWP) gene according to the methodology described by Spano et al. (12). PCR products were purified using Qiagen spin columns and sequenced in both directions (yielding forward and reverse sequences) by using an ABI 3730 capillary electrophoresis genetic analyzer (Applied Biosystems, Foster City, CA). A C. parvum-positive control was obtained from a naturally infected dairy calf near Pixley, CA. A negative control was included by substituting RNase/DNase-free water for DNA.

Phylogenetic analyses.

A preliminary analysis was conducted using Vector NTI Advance software (Invitrogen Corporation, Carlsbad, CA) followed by a BLAST search with each Cryptosporidium 18S rRNA sequence to compare it against existing sequences deposited in GenBank (www.ncbi.nlm.nih.gov ). Phylogenetic and molecular evolutionary analyses were conducted using MEGA version 4 (www.megasoftware.net ) (14). All sequences were shortened at the 5′ and 3′ ends to match the number of base pairs from the Cryptosporidium sp. muskrat genotype IV and C. bovis domain sequences, respectively. Phylogenetic relationships were inferred by neighbor-joining analysis of these partial sequences based on distance measures calculated by the Kimura two-parameter method (14). By using the same analytical approach, the entire 18S rRNA sequences of various Cryptosporidium isolates from each of the ground squirrel species were compared to available complete 18S rRNA sequences of existing species and genotypes of Cryptosporidium. Bootstrap analyses were conducted with 1,000 replicates. The sequences corresponding to the following GenBank accession numbers were downloaded and used in the above-described phylogenetic analysis: AF442484, EF641018, EF641019, AY120906, AY120904, EF641020, AF115377, GQ121021, AF112574, AF093493, AF115378, EU250845, AF093492, AY204228, FJ262726, AF112573, AF108862, AY741305, AF093495, AF093502, AF093498, AB089285, and AF112576. The DNA sequence (accession no. GSU41086) of Gymnodinium simplex was used as an out-group.

Based on direct immunofluorescence microscopy, the overall prevalences of fecal shedding of Cryptosporidium oocysts by squirrels from the trap locations shown in Fig. 1 were 20% for California ground squirrels (S. beecheyi), 22% for Belding's ground squirrels (S. lateralis), and 14% for golden-mantled ground squirrels (S. beldingi). Partial or complete 18S rRNA gene sequences were determined for 49 different Cryptosporidium isolates obtained from these infected ground-dwelling squirrels in locations varying from low-elevation coastal rangeland and valley farmland to alpine grass meadow and bristlecone pine forest (Fig. 1; Table 1). Based on a GenBank BLAST search for each Cryptosporidium isolate, none of the 49 isolates had an 18S rRNA gene with 100% homology to any entry in GenBank as of 21 January 2010, except for the isolates with sequences assigned accession numbers AY462231 to AY462233, which correspond to sequences in a previously published article reporting novel Cryptosporidium genotypes in California ground squirrels (S. beecheyi) (1). Furthermore, this collection of 49 Cryptosporidium isolates segregated into four different groups according to contiguous 18S rRNA gene sequences. For identification purposes, we labeled these DNA sequence groups by an abbreviation of the squirrel's scientific name, followed by the year in which the isolate was sequenced and ultimately by a letter that identified one of these four unique Cryptosporidium DNA sequences (a, b, c, and d). For example, Cryptosporidium sp. Sbey05c designates Cryptosporidium DNA sequence group C isolated in 2005 from a California ground squirrel (S. beecheyi). Table 1 shows the percentage of similarity between each ground squirrel Cryptosporidium isolate, listed by ground squirrel species, and the Cryptosporidium isolate(s) in GenBank that had the maximal similarity score.

Isolates of Cryptosporidium sequence group A (Cryptosporidium sp. Sbey03a and Cryptosporidium sp. Sbld05a) were identified in two different squirrel species, California and Belding's ground squirrels, from different geographic regions in three California counties, with prevalences of 4% (1 in 23) and 17% (3 in 18) in the respective squirrel species. The prevalence of sequence group A among all 49 Cryptosporidium isolates was 8.2% (4 in 49). A BLAST search with 785 bp from the Cryptosporidium sp. Sbey03a sequence revealed a maximum of 97% homology to the chipmunk genotype III and Cryptosporidium sp. vole genotype sequences, whereas the partial and entire 18S rRNA gene locus sequences (831 and 1,744 bp) from Cryptosporidium sp. Sbld05a revealed maximums of 97 to 98% homology to sequences from C. hominis and C. meleagridis. Additionally, an 828-bp internal sequence of the COWP gene (accession no. EU847640) from a Cryptosporidium sp. Sbld05a isolate showed a maximum of 92% homology to sequences from C. suis, C. bovis, and C. hominis.

Isolates of Cryptosporidium sequence group B (Cryptosporidium sp. Sbey03b and Cryptosporidium sp. Sbey05b) accounted for 13% of isolates (3 of 23) from California ground squirrels (S. beecheyi). A BLAST search with the partial sequence showed a maximum of 97% homology to sequences from the Cryptosporidium sp. lemur genotype (accession no. AF442484), the Cryptosporidium sp. cervine genotype (accession no. EU827398), and C. suis (accession no. AF115377).

We obtained the entire 18S rRNA gene sequence for each of the three ground squirrel species infected with Cryptosporidium sequence group C (Cryptosporidium sp. Sbey05c, Cryptosporidium sp. Sbld05c, and Cryptosporidium sp. Sltl05c). This group revealed maximum homology of 97% to C. suis and C. meleagridis 18S rRNA gene sequences with accession numbers AF115377 and AF111271, respectively (Table 1). The Cryptosporidium group C sequence was found in the majority of Cryptosporidium isolates from these wildlife species, about 84% of total sequenced isolates (41 of 49). By squirrel species, the Cryptosporidium group C sequence was found in 83% of sequenced isolates (19 of 23) from California ground squirrels (S. beecheyi), 78% of sequenced isolates (14 of 18) from Belding's ground squirrels (S. beldingi), and 100% of sequenced isolates (8 of 8) from golden-mantled ground squirrels (S. lateralis). Given the higher prevalence of sequence group C than of the other groups in all three naturally infected ground squirrel species from throughout California, we consider this sequence group to represent the primary Cryptosporidium sp. for this group of Spermophilus ground-dwelling squirrels.

A single isolate obtained from a Belding's ground squirrel captured in Modoc County in northern California was classified as group D (Cryptosporidium sp. Sbld05d). This Cryptosporidium sequence group was found in just 5.5% of isolates (1 of 18) from this host species, with an overall prevalence of 2% (1 in 49) among our bank of 49 Spermophilus isolates. In contrast to A, B, and C sequence groups, sequence group D showed 99% similarity to C. parvum isolated from a human host (Table 1).

Phylogenetic analysis of partial 18S rRNA sequences was conducted to determine the relationships of the DNA sequences from these 49 isolates of Cryptosporidium sp. from S. beecheyi, S. lateralis, and S. beldingi to sequences of Cryptosporidium species and genotypes that were retrieved from GenBank (Fig. 2). This analysis placed Cryptosporidium isolates from groups A and B (Cryptosporidium sp. Sbey03b, Cryptosporidium sp. Sbey05b, Cryptosporidium sp. Sbey03a, and Cryptosporidium sp. Sbld05a) into a joint clade that exhibited minimal distance from or DNA dissimilarity to a Cryptosporidium sp. chipmunk genotype. In contrast, Cryptosporidium isolates from the more common group C (Cryptosporidium sp. Sbey03c, Cryptosporidium sp. Sbey05c, Cryptosporidium sp. Sltl05c, and Cryptosporidium sp. Sbld05c) were placed into a completely separate clade from Spermophilus sequence groups A and B and shared internal nodes with various genotypes of Cryptosporidium, such as Cryptosporidium sp. muskrat genotype I, Cryptosporidium sp. opossum genotype II, and Cryptosporidium sp. deer mouse genotype IV. In other words, Spermophilus sequence group C exhibited more DNA sequence similarity to other genotypes of Cryptosporidium isolated from non-Spermophilus mammals than to Spermophilus sequence groups A and B, which were more similar to each other. Lastly, Spermophilus sequence group D (Cryptosporidium sp. Sbld05d) was most similar to a C. parvum human isolate genotype when the 836-bp sequence was analyzed by a BLAST search in GenBank and to a Cryptosporidium sp. rabbit genotype when the sequence was trimmed to 771 bp to construct a phylogenetic tree (Fig. 2). Sequence group D (Cryptosporidium sp. Sbld05d) showed 99% similarity to C. parvum isolated from a human host (Table 1). This isolate was placed into a separate clade apart from the three other ground squirrel sequence groups, A, B, and C (Fig. 2).

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FIG. 2.

Phylogenetic relationships of partial (∼750-bp) 18S SSU rRNA sequences from Cryptosporidium species, genotypes, and novel Cryptosporidium isolates from three species of Spermophilus ground squirrels, S. beecheyi, S. lateralis, and S. beldingi, inferred by neighbor-joining analyses with 1,000 bootstrap replicates.

Phylogenetic analysis of the entire 18S rRNA gene sequence may provide a more accurate representation of the similarity among the various species and genotypes of Cryptosporidium, but fewer isolates are available in GenBank for such an analysis. We were able to sequence the entire 18S rRNA gene for several isolates from Spermophilus sequence groups A and C. Similar to the phylogenetic analysis using a partial sequence of the 18S rRNA gene, this analysis placed Spermophilus sequence group C into a separate and distinct clade from group A, tentatively suggesting that sequence groups A and C are not the same genotype or species of Cryptosporidium. Furthermore, Spermophilus sequence group C did not directly share a terminal node of the constructed phylogenetic tree with any known species or genotype of Cryptosporidium deposited in GenBank (Fig. 3), indicative that the sequence is unique. This common sequence group was obtained from numerous Cryptosporidium isolates from each of the three Spermophilus squirrel species throughout California (Table 1).

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FIG. 3.

Phylogenetic relationships of the entire 18S SSU rRNA sequences (∼1,750 bp) from Cryptosporidium species, genotypes, and novel Cryptosporidium isolates from three species of Spermophilus ground squirrels, S. beecheyi, S. lateralis, and S. beldingi, inferred by neighbor-joining analyses with 1,000 bootstrap replicates.

In order to conduct phylogenetic analyses of multiple Cryptosporidium isolates, one has to restrict the length of the analyzed DNA sequence so that all sequences have comparable domains. This trimming or shortening of the 5′ and/or 3′ end can result in the placement of isolates into the wrong clades and thus the wrong phylogenetic tree nodes, thereby altering the topology of the constructed phylogenetic tree for Cryptosporidium and reducing the probability of obtaining the correct tree topology (9). This trimming of DNA sequences for phylogenetic tree analysis can also induce discrepancies between analyses like the one shown in Fig. 1 and analyses that compare one isolate at a time to sequences of similar length in GenBank, as in Table 1. For example, it is common to use 700 to 800 bp of sequence from the 18S rRNA gene for phylogenetic analyses involving Cryptosporidium (10, 11, 15). When these smaller DNA sequences are used in our analysis, isolates of Cryptosporidium sp. deer mouse and C. meleagridis are identified as having maximal percent similarity (96 to 97%) to Cryptosporidium sp. Slt05C (Table 1). When the entire 18S rRNA gene sequence of 1,756 bp is used instead, isolates of C. meleagridis and C. suis show maximal percent similarity (97%) to this golden-mantled ground squirrel isolate. Similarly, with respect to a Cryptosporidium sp. Sbld05c isolate from a Belding's ground squirrel, use of 784-, 821-, and 1,755-bp DNA sequences resulted in different species or genotypes of Cryptosporidium (Cryptosporidium sp. cervine genotype, Cryptosporidium sp. lemur genotype, and C. meleagridis, respectively) showing maximal percent similarity to this isolate, indicating the dependency of phylogenetic analysis of Cryptosporidium isolates on DNA sequence length.

These findings support an earlier assertion regarding the uniqueness of these Cryptosporidium isolates shed in the feces of ground squirrels from the Spermophilus squirrel genus (1) relative to the many Cryptosporidium strains that have been submitted to GenBank. Groups A and C were isolated from California ground squirrels (Cryptosporidium sp. Sbey03a, Cryptosporidium sp. Sbey03c, and Cryptosporidium sp. Sbey05c) and Belding's ground squirrels (Cryptosporidium sp. Sbld05a and Cryptosporidium sp. Sbld05c) trapped in a widespread geographic region spanning 500 miles in length (north to south) and 150 miles in width (east to west). This 75,000-square-mile area encompasses a variety of habitats and ecosystems, including central to northern California agricultural systems, the area east to the White Mountains, the Sierra Nevada range and its western foothills, and the region down to the central coastal valleys of San Luis Obispo and Santa Barbara County. The golden-mantled ground squirrels in this study were from the White Mountains, which are a remote and isolated mountain range that is separated from the other study locations by hundreds of miles, including the highest elevations of the Sierra Nevada Mountains. Interestingly, this remote population of high-elevation ground squirrels shed only the common C sequence group (Cryptosporidium sp. Sltl05c) of Cryptosporidium.

In conclusion, given the unique 18S rRNA and COWP gene sequences of these 49 different Cryptosporidium isolates compared with those of any known species or genotype of Cryptosporidium, the placement of sequence groups A and C into different topological locations in the phylogenetic tree, and the inability of genotypes B and C to infect BALB/c neonatal mice (1), it is likely that Spermophilus-derived Cryptosporidium isolates represent one or more new species of this protozoan parasite. Further work is in progress to verify or refute this assertion of the existence of one or more new species of Cryptosporidium in ground-dwelling squirrels in California.

Nucleotide sequence accession numbers.

The new 18S rRNA nucleotide sequences for Cryptosporidium isolates from S. beecheyi, S. lateralis, and S. beldingi were deposited in GenBank under accession numbers AY462231 to AY462233 and DQ295012 to DQ295017.