Cryptosporidium sp.

Li, Xunde, Pereira, Maria das Graças Cabral, Larsen, Royce, Xiao, Chengling, Phillips, Ralph, Striby, Karl, McCowan, Brenda & Atwill, Edward R., 2015, Cryptosporidium rubeyi n. sp. (Apicomplexa: Cryptosporidiidae) in multiple Spermophilus ground squirrel species, International Journal for Parasitology: Parasites and Wildlife 4 (3), pp. 343-350 : 344-345

2.4. Infectivity of Cryptosporidium sp. c-genotype oocysts

An in vivo neonatal BALB/c mouse assay ( Li et al., 2010) was used to determine if Cryptosporidium oocysts from Spermophilus squirrels were infectious for this well-studied host species. Fresh oocysts were purified as described in Section 2.3 (above) and were stored in deionized water at 4 ǫC for approximately 3 weeks before inoculation to animals. Prior to inoculating to mice, oocysts were examined with DIC microscopy and confirmed to be intact. Female BALB/c mice with neonatal pups were purchased from Harlan Laboratories (San Diego, CA, USA), housed in cages fitted with air filters and given food and water ad libitum. Oocysts were administered to neonatal mice at 5 days of age by intragastric inoculation using a 24-gauge ball ‾ point feeding needle. One hour prior to infection, the pups were removed from the dam to empty their stomachs for easier inoculation and the dam was returned to the pups after inoculation. Each litter of mice was given oocysts from only one isolate as shown in Table 2, using doses ranging from 10 2 to10 4 oocysts per mouse. C. parvum oocysts (GenBank accession no. FJ752165) purified from naturally infected California dairy calves were similarly administered to mice as a positive control, as was deionized water as a negative control. Heat inactivated (incubation at 70 ǫC for 2 h) C. parvum oocysts were also inoculated into mice to monitor passthrough of oocysts resulting from inoculation ( Li et al., 2010).

Cryptosporidium infection in mice was assessed by staining intestinal homogenates with a FITC-labeled anti- Cryptosporidium immunoglobulin M antibody (Waterborne Inc., New Orleans, LA, USA) which has been shown to be a sensitive method for detecting Cryptosporidium oocysts from intestinal homogenates of infected mice ( Hou et al., 2004). Seven days post-inoculation (PI) mice were euthanized by CO 2 asphyxiation and the entire intestine from duodenum to rectum was collected. Intestinal samples were suspended in 5 ml of deionized water in 50 ml tubes and homogenized with an IKA ® Ultra-Turrax T8 tissue homogenizer (GmbH & Co. KG, Staufen, Germany). The homogenates were washed 1× in deionized water by centrifuging at 1500 g for 10 min and the supernatant removed. The pellets were resuspended in 10 ml of deionized water and filtered through a 20 M m pore nylon net filter (Millipore, Bedford, MA, USA) fixed on a Swinnex holder (Millipore, Bedford, MA, USA). The filtrates were concentrated to 1 ml by centrifuging at 1500 g for 10 min and mixed by vortexing. Fifty M l of the final homogenates were mixed with 50 M l of anti- Cryptosporidium monoclonal antibodies (Waterborne Inc., New Orleans, LA, USA) and 2 M l of 0.5% evans blue, then incubated at room temperature for 45 min in a dark box. Three wet mount slides were prepared from each sample using 20 M l of reaction mixture per slide. Slides were mophilus ground squirrels.

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examined with a fluorescent microscope (Olympus BX 60) and a mouse was considered infected if one or more oocysts were detected in the intestinal homogenates.

In addition to the mice infectivity assay, we also conducted a trial to measure the infectivity of the c-genotype oocysts from S. lateralis ground squirrels in two-day old Holstein calves. Newborn calves were purchased from commercial dairy farms. For each of the eight isolates of c-genotype oocysts from S. lateralis ground squirrels in Tables 1 and 2, two calves were orally inoculated, one with 100 oocysts and one with 5000 oocysts. A positive control calf was given 5000 C. parvum oocysts from dairy calves and a negative control group of two calves were not given oocysts. Fecal excretion of Cryptosporidium oocysts from calves were determined using direct immunofluorescent microscopy as described above. All animal experiments with BALB/c mice and calves were approved by the Institutional Animal Care and Use Committee (IACUC) of University of California Davis.

2.5. Multiple gene analysis of Cryptosporidium isolates from S. beecheyi

Microscopic positive fecal samples were exposed to 5 cycles of freeze (–80 ǫC) and thaw (+70 ǫC) then 0.2 g was used for DNA extraction using the QIAamp DNA Stool Mini Kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer's manual. Amplification of fragments of the 18S rRNA, actin, and HSP70 genes by nested-PCR were performed using primers and cycling conditions as previously described by Xiao et al. (2000) and Jiang et al. (2005) for the 18S rRNA gene, Sulaiman et al. (2002) for the actin gene, and Sulaiman et al. (2000) for the HSP70 gene. AmpliTaq DNA polymerase (Thermo Fisher Scientific, Grand Island, NY, USA) were used for all PCR amplifications. A positive control using DNA of C. parvum isolated from calves from a dairy near Davis, CA as template and a negative control without DNA template were included in each PCR. PCR products were verified by electrophoresis in 2% agarose gel stained with ethidium bromide. Products of the secondary PCR were purified using the QIAamp DNA Mini Kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer's manual. Purified DNA was sequenced in both directions at the University of California DNA Sequencing Facility, using an ABI 3730 Capillary Electrophoresis Genetic Analyzer (Applied Biosystems Inc., Foster City, CA, USA).

Sequences were analyzed and consensus sequences were generated using the Vector NTI Advanced 11 software (Invitrogen, Carlsbad, CA, USA). Consensus sequences were compared to Cryptosporidium sequences in the GenBank using NCBI's online BLAST tool with the default algorithm parameters to target 100 sequences (http://blast.ncbi.nlm.nih.gov/) (March 12, 2015 as last day accessed). Phylogenetic analyses were conducted using Genenious Basic 5.6.5. software (Biomatters, Auckland, New Zealand). Phylogenetic relationships were inferred by using the neighbor-joining method and the Tamura-Nei genetic distance model with bootstrapping of 1000 replicates for the three genes. Depending on the availability of sequences in the GenBank, reference sequences for constructing the phylogenetic trees were selected based on: 1) sequences representing well described Cryptosporidium species (exclude synonyms) from fish, amphibians, reptiles, birds, and mammals, 2) sequences previously used by others for species description or as reference sequences, 3) sequence length (longer sequence if full sequence not available for each species; i.e. 18S rRNA gene sequences ±700 bp, actin gene sequences ±750, and HSP70 gene sequences ±1700 bp), 4) sequences not originating from cloned PCR products due to the potential for erroneous sequence data generated from cloning PCR products ( Zhou et al., 2003; Ruecker et al., 2011), and 5) previously published c-genotypes from ground squirrels (i.e. Sbey-c, Sbld-c, and Sltl-c genotypes of the 18S rRNA gene). Names and GenBank accession numbers of selected references sequences are shown in Figs. 2 ‾ 4. The DNA sequences of 18S rRNA gene (GQ899206), actin gene (XM_003879845), and HSP70 gene (XM_003883591) of Neospora caninum were used as out-groups for constructing the phylogenetic tress. Sequences from S. beecheyi and all selected reference sequences were trimmed at both the 5 Ɩ and 3 Ɩ ends after alignment to use the same length for phylogenetic tree construction.

Figures

Fig. 2. Phylogenetic relationships of partial 18S rRNA gene sequences of Cryptosporidium sp. Sbey11c from California ground squirrels (S. beecheyi) and other Cryptosporidium spp. inferred by neighbor-joining analysis with 1000 bootstrapping replicates.