Differentiating human from animal isolates of Cryptosporidium parvum.

We analyzed 92 Cryptosporidium parvum isolates from humans and animals by a polymerase chain reaction/restriction fragment length polymorphism method based on the thrombospondin-related anonymous protein 2 gene sequence. Used as a molecular marker, this method can differentiate between the two genotypes of C. parvum and elucidate the transmission of infection to humans.


Dispatches
Cryptosporidium parasites cause infection in humans and other vertebrates, including mammals, birds, reptiles, and fish. More than 20 species of Cryptosporidium have been reported, of which six are considered valid species on the basis of oocyst morphologic features and site of infection (1,2). Cryptosporidium parvum, the species that infects humans and most mammals, has a monoxenous life cycle in which all stages of asexual and sexual development occur within one host. The parasite generates large numbers of viable oocysts in feces. Cross-infection studies in various mammalian systems have indicated zoonotic transmission to humans (1,3). C. parvum has caused waterborne outbreaks of cryptosporidiosis and (in AIDS patients) life-threatening diarrhea for which no effective treatment exists (4). A waterborne outbreak of cryptosporidiosis in Milwaukee, Wisconsin, in 1993 affected more than 400,000 people (5).
In a previous article, we identified several mutations in the gene thrombospondin-related anonymous protein 2 of C. parvum (TRAP-C2) that differentiate between anthroponotic and zoonotic infection in humans (14). Our objective in the present study was to develop a simple, rapid protocol that can be used as a diagnostic tool to differentiate between the two genotypes of C. parvum and elucidate the transmission of infection in humans. We analyzed 92 C. parvum isolates from humans, calves, deer, dogs, and monkeys and found that this new PCR/RFLP method based on the TRAP-C2 gene sequence can be used as a molecular marker to differentiate between the two genotypes of C. parvum.

Isolates
We summarized data for 92 isolates of C. parvum, 50 from human and 42 from animal sources (Tables 1, 2). Twenty-one of the 50 human isolates were from AIDS patients; the rest were primarily from cryptosporidiosis outbreak case-patients. Seven of the human isolates came from a previous TRAP-C2 sequencing study (14), but because of the lack of DNA, other isolates we used in the previous study were not used in this study. Fecal samples were stored at 4° C in 2.5% potassium dichromate before oocysts were isolated. Oocysts were purified from fecal samples by first using the discontinuous density sucrose gradient centrifugation and then the Percoll gradient centrifugation (15,16).

Extraction of Genomic DNA and PCR Amplification
We followed the protocol of Kim et al. in isolating the total genomic DNA from the purified oocyst (17). A 369 base pair (bp) fragment of the TRAP-C2 gene of C. parvum was amplified by using a forward (cua cua cua cua CAT ATT CCC TGT CCC TTG AG) and a reverse (cau cau cau cau TGG ACA ACC CAA ATG CAG

DNA Sequencing and Analysis
PCR products were purified by the Wizard PCR Preps DNA purification system (Promega, Madison, WI) and cloned by the CLONEAMP pAMP1 System for Rapid Cloning of Amplification Products (GIBCO BRL, Frederick, MD) according to the manufacturers protocol. DNA sequencing of recombinant clones that had the correct size insert was carried out on an ABI 377 Automated Sequencer by the dRhodomine Terminator Cycle Sequencing Kit (Perkin Elmer-Applied Biosystems).

RFLP
To develop an RFLP technique for differentiating between the two genotypes of C. parvum, the TRAP-C2 sequences were aligned and mapped for restriction enzyme sites by the Genetics Computer Group program (18). Enzymes with predicted exclusive cutting in each genotype were used in RFLP development and analysis. For RFLP analysis, 10 µl of amplification products was digested in a 30-µl reaction mix consisting of 10 units of BfaI (New England BioLabs), and 3 µl of respective restriction buffer for 1 hr, under conditions recommended by the supplier. The digested products were fractionated on 2.0% agarose gel and visualized by ethidium bromide staining.

Findings Sequence Analysis of Human and Bovine Isolates
Two genotypes of C. parvum exist in humans, as shown by the primary sequence of the TRAP-C2 gene (14). Nucleotide sequences differed at five positions between most human and bovine isolates. To confirm and extend this observation, we sequenced additional human and bovine isolates, as well as isolates from dogs, deer, and monkeys. We obtained 42 additional sequences of the TRAP-C2 gene from animal sources and 27 additional sequences of the TRAP-C2 gene from human sources; results of DNA sequencing confirmed that C. parvum is highly conserved at the TRAP-C2 locus. All animal isolates, including those from nonbovine animals, showed bovine genotype characteristics ( Table 2). Differences between the two genotypes are shown in Table 3. Of the additional 23 human isolates showing human genotype pattern, four isolates (HGMO7, HGMO9, HGMO10, and HNO18) showed C at the fifth place, whereas the rest showed T.

PCR-RFLP Method To Discriminate between Human and Bovine Genotype Isolates
To avoid expensive and lengthy DNA sequencing when determining the genotype of C. parvum isolates, we developed a simpler, quicker methodPCR amplification of the TRAP-C2 gene followed by RFLP. Restriction Dispatches enzyme mapping on the aligned sequences of both genotypes showed five human-genotype specific (HaeI, HaeIII, NruI, PacI, and ThaI) and six bovine-genotypespecific (BfaI, BsetEI, Eco571, HphI, MaeIII, and Tsp45I) restriction enzymes. All human-genotype and bovinegenotypespecific restriction enzymes except HaeI and ThaI were tested for the TRAPC-2 PCR-amplified products of genomic DNA of C. parvum. After restriction and gel electrophoresis, the resulting bands were the size predicted by the mapping analysis ( Figure). Digestion of PCR products with these enzymes resulted in a distinct band pattern for the human genotype and bovine genotype isolates. In all cases, the DNA sequencing and PCR-RFLP mapping data matched.

Using PCR-RFLP in Outbreak Investigations
We validated the PCR-RFLP technique by using isolates from outbreaks and sporadic cases of human cryptosporidiosis. Human genotype characteristics were evident in all samples from HIV-infected patients from Guatemala and most patients with sporadic clinical cases, as well as samples from the following outbreaks: Milwaukee (1993), Florida (1995), Atlanta (1995), Canada (1995), Nevada (1994), and Washington (1998). Of the 17 samples from HIV-infected patients in New Orleans, two demonstrated bovine-genotype pattern, while the rest were similar to human genotype. However, bovinegenotype characteristics were evident in the human isolates from outbreaks in British Columbia, Canada (1996), Minnesota (1997), and Pennsylvania (1997).

Conclusions
We examined a large number of C. parvum isolates (92) from human and animal sources from patients in outbreak and nonoutbreak settings to determine the two transmission routes of the parasite in humans. Molecular markers were generated by restriction digestion of PCR-amplified TRAP-C2 products with one of the 12 enzymes to differentiate the two genotypes of C. parvum. The results based on TRAP-C2 gene PCR-RFLP showed that this method could also be used in future cryptosporidiosis outbreak investigations.
Results of our characterization of outbreak and nonoutbreak cases of human cryptosporidiosis indicate that anthroponotic organisms account for most cases. We find a large number of human genotype parasites in sporadic cases and in HIVinfected patients. Most cryptosporidiosis outbreaks examined are caused by anthroponotic (human genotype) parasites. Our results suggest similar epidemiologic features of cryptosporidiosis in HIV-infected persons from New Orleans and Guatemala because both were infected with human genotype parasites.
The results of this study confirm the polymorphic nature of C. parvum. As we showed in a previous study, two alleles of the TRAP-C2 gene exist, each representing a distinct genotype of C. parvum with different transmission cycles in humans. The simple PCR-RFLP technique we developed can effectively differentiate between these two genotypes and transmission cycles and can be used as a tool in outbreak investigations of cryptosporidiosis. Information generated from these investigations will be useful not only in Figure. Human-and bovine-specific restriction enzymes showed distinct banding pattern for genotypes of Cryptosporidium parvum isolates. The different lanes represent the TRAP-C2 PCRamplified products belonging to AGA43, AMD36, AOH6, HM3, and HM5 isolates of C. parvum, respectively, after digestion with HaeIII (Lanes 1-5, human-specific marker) and BstE II (Lanes 7-11, bovine-specific marker) restriction enzymes and agarose gel electrophoresis. Lane 6 is the 100 bp marker. Samples AGA43, AMD36 and AOH6 are bovine (bovine genotype) and samples HM3 and HM5 are human (human genotype). Human-or bovinespecific restriction enzyme markers can cut only the TRAP-C2 amplified product of the respective genotype of C. parvum isolates.
Dispatches identifying the sources of contamination but also in controlling the disease.