Figure. 1,2 Panel A. Results of nested Cryptosporidium parvum CP 11 gene PCR performed on pooled oyster hemolymph and gill tissues. Expected PCR product size is 344 bp. Samples analyzed were collected from Maryland Department of Natural Resources oyster harvesting sites at Mt Vernon Wharf (lanes 1-5), Wetipquin (lanes 6-8), Beacon (lane 9), and Holland Point (lane 10). Lane 11: C. parvum positive control. Lanes 12 and 13 are 1° and 2° no template controls, respectively. Panel B: Results of oyster (Crassostrea virginica) small subunit ribosomal RNA PCR performed on the same oyster tissues analyzed in Panel A, lanes 1-10. Lane 11: no template control. Expected PCR product size is 340 bp.
1A PCR product of approximately 1,325 bp was amplified first in primary PCR, by using primers 5'-TTCTAGAGCTAA
TACATGCG-3' and 5'-CCCTAATCCTTC
GAAACAGGA-3'. The PCR reaction contained 10 µl of Perkin-Elmer 10X PCR buffer, 6 mM of MgCl2, 200 µM of each dNTP, 100 nM of each primer, 2.5 U Taq polymerase, and 0.25 µl of DNA template, for a total of 100 µl reaction. Thirty-five cycles each consisted of 94oC for 45 seconds, 55oC for 45 seconds, and 72°C for 1 minute, with an initial hot start at 94°C for 3 minutes and a final extension at 72°C for 7 minutes. A secondary PCR product of 826 or 864 bp (depending on species) was then amplified from 2 µl of the primary PCR reaction, using primers 5'-GGAAGGGTTGTA
TTTATTAGATAAAG-3' and 5'-AAGGAGTAAGGA
ACAACCTCCA-3'. The PCR and cycling conditions were identical to primary PCR, except that 3 mm of MgCl2 was used in the PCR reaction. For restriction fragment analysis, 20 µl of the secondary PCR products were digested in a total of 50 µl reaction mix consisting of 20 units of Ssp I (New England BioLabs, Beverly, MA; for species diagnosis), or Vsp I (Gibco BRL, Grand Island, NY; for genotyping of C. parvum) and 5 µl of restriction buffer at 37°C for 1 hour. The digested products were fractionated on 2.0% agarose gel and stained with ethidium bromide.
2PCR products were purified first by the Wizard PCR Preps DNA purification system (Promega, Madison, WI), then sequenced by fluorescent cycle sequencing by using dye terminator chemistry on an ABI 377 Automated Sequencer (Perkin-Elmer Applied Biosystems, Foster City, CA). Sequences obtained from oyster samples were then aligned with sequences obtained from known Cryptosporidium spp. (C. parvum, C. baileyi, C. muris, C. serpentis, C. meleagridis, C. wrairi, and C. felis) and various C. parvum genotypes (human, bovine, monkey, dog, pig, mouse, ferret, marsupial, and desert monitor). Isolates corresponding to those transmissible to humans are reported as human genotype or genotype 1, and isolates corresponding to those transmissible among cattle, mice, and humans are reported as bovine genotype or genotype 2.
As an adjunct to the 18S rRNA assay, a nested PCR was also performed by using primers derived from an 11 kDa protein, extracted from C. parvum oocysts. The sequence of outer forward primer P5 is: 5' AAC ATC CAT CGA GTT TAG TA 3' and of outer reverse primer P6 is: 5'GCA AGA GCG CAT TGG TGA AT 3'; the expected PCR product size is 541 bp. The sequence of inner forward primer Cp 11/F is: 5' GTC TAG AAC CGT TAC TGT TAC TGG 3', and of outer reverse primer Cp 11/R is: 5' CAA CTC CTG GAA GCA TCT TAA CAG 3'; the expected PCR product size is 334 bp.
As a control for the quality of DNA extracted from the oyster tissues, PCR was performed by using primers derived from the small subunit ribosomal RNA sequence of the oyster, Crassostrea virginica (6). The OyF forward primer sequence is: 5' GTC TCA AAG ATT AAG CCA TGC ATG 3' (corresponding to nucleotides 34-57), and the OyR reverse sequence is: 5' TGA TTC CCC GTT ACC CGT TAC AA 3' (nucleotides 354-376). The predicted size of the PCR product is 340 bp.
For PCR, pooled oyster gill washings and hemolymph were subjected to nucleic acid extraction by using the Ambion Totally RNA® kit, followed by isopropanol precipitation of nucleic acids and a 70% ethanol wash. The pellet was resuspended in 30-50 µl of molecular biology grade water, and 5-10 µl used as template for PCR. These reactions were performed in 50-µl volumes containing 1.5 mM MgCl2, 1U taq polymerase, 1X PCR buffer, 50 pmol each primer, and 10 mM dNTP mix (Life Technologies, Gaithersburg, MD). Cycling parameters for all reactions were 2 minutes at 95°C, followed by 40 cycles of 95°C for 15 seconds, 58°C for 30 seconds, and 72°C for 30 seconds. Three microliters of primary PCR product was used as template for secondary PCR. PCR products (13-15 µl) were electrophoresed on 1% agarose gels and visualized with ethidium bromide and UV illumination.
For cycle sequencing, the amplicon was cut from a 1% agarose gel, extracted by using the QIAquick Gel Extraction kit (Qiagen, Valencia, CA) and sequenced by using dye-terminator chemistry on the ABI 373 and 377 models fluorescence automated sequencers (Perkin-Elmer Applied Biosystems, Foster City, CA). Sequences were analyzed with AutoAssembler® (Perkin-Elmer Applied Biosystems, Foster City, CA, and Genetics Computer Group, Madison, WI) software.