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Species and Strain-specific Typing of Cryptosporidium Parasites in Clinical and Environmental Samples

Abstract

Cryptosporidiosis has recently attracted attention as an emerging waterborne and foodborne disease as well as an opportunistic infection in HIV infected individuals. The lack of genetic information, however, has resulted in confusion in the taxonomy of Cryptosporidium parasites and in the development of molecular tools for the identification and typing of oocysts in environmental samples. Phylogenetic analysis of the small subunit ribosomal RNA (SSU rRNA) gene has shown that the genus Cryptosporidium is comprised of several distinct species. Our data show the presence of at least four species: C. parvum, C. muris, C. baileyi and C. serpentis (C. meleagridis, C. nasorum and C. felis were not studied). Within each species, there is some sequence variation. Thus, various genotypes (genotype 1, genotype 2, guinea pig genotype, monkey genotype and koala genotype, etc.) of C. parvum differ from each other in six regions of the SSU rRNA gene. Information on polymorphism in Cryptosporidium parasites has been used in the development of species and strain-specific diagnostic tools. Use of these tools in the characterization of oocysts various samples indicates that C. parvum genotype 1 is the strain responsible for most human Cryptosporidium infections. In contrast, genotype 2 is probably the major source for environmental contamination of environment, and has been found in most oysters examined from Chesapeake Bay that serve as biologic monitors of surface water. Parasites of Cryptosporidium species other than C. parvum have not been detected in HIV+ individuals, indicating that the disease in humans is caused only by C. parvum.

Cryptosporidium; phylogeny; genotype; ribosomal RNA


Species and Strain-specific Typing of Cryptosporidium Parasites in Clinical and Environmental Samples

Vol. 93: 687-692

Lihua Xiao/+, Irshad Sulaiman, Ronald Fayer*, Altaf A Lal

Division of Parasitic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, US Department of Health and Human Services, Atlanta, GA 30341 *Parasite Immunobiology Laboratory, Agriculture Research Service, U.S. Department of Agriculture, Beltsville, MD 20705, USA

Cryptosporidiosis has recently attracted attention as an emerging waterborne and foodborne disease as well as an opportunistic infection in HIV infected individuals. The lack of genetic information, however, has resulted in confusion in the taxonomy of Cryptosporidium parasites and in the development of molecular tools for the identification and typing of oocysts in environmental samples. Phylogenetic analysis of the small subunit ribosomal RNA (SSU rRNA) gene has shown that the genus Cryptosporidium is comprised of several distinct species. Our data show the presence of at least four species: C. parvum, C. muris, C. baileyi and C. serpentis (C. meleagridis, C. nasorum and C. felis were not studied). Within each species, there is some sequence variation. Thus, various genotypes (genotype 1, genotype 2, guinea pig genotype, monkey genotype and koala genotype, etc.) of C. parvum differ from each other in six regions of the SSU rRNA gene. Information on polymorphism in Cryptosporidium parasites has been used in the development of species and strain-specific diagnostic tools. Use of these tools in the characterization of oocysts various samples indicates that C. parvum genotype 1 is the strain responsible for most human Cryptosporidium infections. In contrast, genotype 2 is probably the major source for environmental contamination of environment, and has been found in most oysters examined from Chesapeake Bay that serve as biologic monitors of surface water. Parasites of Cryptosporidium species other than C. parvum have not been detected in HIV+ individuals, indicating that the disease in humans is caused only by C. parvum.

Key words: Cryptosporidium - phylogeny - genotype - ribosomal RNA

Cryptosporidiosis is a coccidian infection of humans, domestic animals and other vertebrates. In young farm animals, especially preweaned dairy calves, it causes a severe enteritis resulting in significant morbidity, mortality and economic loss. In humans, it results in an acute but self-limiting infection of the digestive system in immunocompetent individuals, and chronic, life-threatening disease in immunocompromised patients. Several transmission routes, including person-to-person, contamination of water or food, and zoonotic infection, are possible. The specific source of Cryptosporidium oocysts involved in infection or contamination is frequently unknown, largely due to a lack of detailed epidemiologic investigation and strain-typing tools. The latter results from the lack of molecular characterization and acceptance of the taxonomy of Cryptosporidium species and genotypes.

CRYPTOSPORIDIUM SPECIES

CRYPTOSPORIDIUM PARVUM GENOTYPES

CRYPTOSPORIDIUM GENOTYPES IN CLINICAL SAMPLES

CRYPTOSPORIDIUM PARASITES IN ENVIRONMENTAL SAMPLES

CRYPTOSPORIDIUM SPECIES

Since the discovery of Cryptosporidium muris and C. parvum in rodents, over 20 Cryptosporidium species have been described in various animal hosts (O'Donoghue 1995). Species were named based on the historical the belief that Cryptosporidium spp. are coccidian parasites, and therefore share the strict host specificity demonstarted by many other coccidian parasites. Studies conducted in late 1970s and early 1980s, however, indicated that some isolates of Cryptosporidium were infectious for several animal species. Thus, one group of investigators suggested that all Cryptosoridium parasites were the same species, C. muris (Tzipori et al. 1980). Others demonstrated that host specificity was present among isolates from different classes of vertebrates (O'Donoghue 1995). Based on these observations, Levine (1984, 1986) classified the parasites from mammals, birds, reptiles and fish as C. muris, C. meleagridis, C. serpentis, and C. nasorum, respectively. Subsequent studies demonstrated that C. parvum from mammals and C. baileyi from birds were biologically and morphologically different from C. muris and C. meleagridis (Upton & Current 1985, Current et al. 1986). Thus, C. parvum, C. muris, C. baileyi, C. meleagridis, C. serpentis and C. nasorum were considered valid Cryptosporidium species (O'Donoghue 1995). More recently, based on published reports of host specificity, Fayer et al. (1997) added C. felis from cats and C. wrairi from guinea pigs to the list of valid species, whereas Tzipori and Griffiths (1998)suggested that current evidence does not support the concept that there is more than one species of Cryptosporidium parasites.

The lack of genetic information and the presence of erroneous sequences in a few published studies have added to the present state of taxonomic confusion. Cai et al. (1992) compared the small subunit (SSU) ribosomal RNA (rRNA) gene, and showed a greater than 99% identity between one C. parvum and one C. muris isolate. Alignment of sequences (accession numbers X64430 to 64343) from that study with sequences from us and others indicates that all four sequences from Cai et al. (1992) are the C. muris type, suggesting that their C. parvum sample was contaminated with C. muris oocysts. Furthermore, numerous sequencing errors were found in these sequences, including in regions that are highly conserved in apicomplexans (Xiao et al. unpublished results). Minor sequence errors (one insertion and 12 deletions of nucleotides) were found in the SSU rRNA sequence (L25642) of another published study (Kilani & Wenman 1994). These sequences and five other sequences deposited in the GenBank were used recently by Tzipori and Griffiths (1998) in a phylogenetic analysis of Cryptosporidium parasites. Based on this analysis, they concluded that the observed inter-species and intra-species variation did not favor the designation of separate Cryptosporidium species, and therefore all Cryptosporidium oocysts, including those from lower vertebrates, should be considered hazardous to humans.

We have recently sequenced the SSU rRNA genes from various isolates of C. parvum, C. muris, C. baileyi and C. serpentis, and used these sequences in a phylogenetic analysis (Xiao et al. unpub. data). Results of the analysis have shown that Cryptosporidium parasites are a multi-species complex containing at least four species: C. parvum, C. baileyi, C. muris and C. serpentis (C. felis, C. nasorum and C. meleagridis were not studied). The evolutionary distance between the Cryptosporidium guinea pig isolate and C. parvum is too small to warrant a separate species designation.

CRYPTOSPORIDIUM PARVUM GENOTYPES

Results of various studies indicate that there is variation within the species C. parvum. Two dimensional gel electrophoresis has revealed minor differences between human and bovine C. parvum isolates (Mead et al. 1990), which has been confirmed by immunoblot (Nichols et al. 1991, Nina et al. 1992), isozyme (Ogunkolade et al. 1993, Awad-El-Kariem et al. 1995), and restriction fragment length polymorphism (RFLP) analysis (Ortega et al. 1991). More recently, random amplified polymorphic DNA (RAPD) markers have revealed two distinct groups of human C. parvum isolates, one containing most human isolates and the other containing some human isolates and all animal isolates (Morgan et al. 1995), indicating the possibility of zoonotic infection. Similar results have been obtained by sequence data or PCR-RFLP analysis of a repetitive sequence (Bonnin et al. 1996), bifunctional dihydrofolate reductase thymidylate synthase (DHFR) (Vasquez et al 1996), rRNA repeats (Carraway et al. 1996), polythreonine motifs (Carraway et al. 1997), oocyst wall protein (COWP) gene (Spano et al. 1997), and thrombospondin anonymous protein-2 (TRAP-C2) gene (Peng et al. 1997 Sulaiman et al. unpub. data). It remains unclear, however, whether the same two genotypes are present in all these polymorphic loci. Results of our multi-locus analysis suggest that indeed the same genotypes are linked across all polymorphic genes (SSU rRNA, TRAP-C1, TRAP-C2, CP15, and ß-tubulin intron) examined.

Our phylogenetic analyses of the SSU rRNA gene have revealed diversities in C. parvum than not previously observed (Table I). Human C. parvum isolates differ from bovine isolates in four regions of the SSU rRNA gene. Likewise, the Cryptosporidium isolate from guinea pigs (C. wrairi) also differs from the bovine isolates in four regions, two of which are the same polymorphic regions between the human and bovine genotypes, thus representing a third genotype of C. parvum. Partial sequences obtained from a monkey by us and from a koala by Morgan et al.(1997) indicate the presence of two additional genotypes. The difference between the human and bovine genotypes in nucleotides 689-699 has also been observed recently by Morgan et al. (1997). We, however, have observed that some human isolates have the sequence TTTTTT instead of TTTTTTTTTTT. Based on a partial SSU rRNA gene sequence, another group also identified a new C. parvum genotype (Carraway et al. 1994, 1996). The new genotype sequence(ICP), however, is identical to the C. muris bovine isolate and possibly resulted from sample contamination.

CRYPTOSPORIDIUM GENOTYPES IN CLINICAL SAMPLES

Results of the molecular characterization have been used by us in the development of molecular diagnostic tools. A PCR-RFLP technique based on the polymorphism in the TRAP-C2 gene was developed and used in the analysis of human clinical samples from various outbreak and non-outbreak cases (Sulaiman et al., unpub. data). Results of our studies and those by others indicate that anthroponotic organisms account for the majority of the cases and person-to-person transmission is likely to be an important transmission route of cryptosporidiosis in non-outbreak cases. This is evident from the large number of genotype 1 parasites in sporadic cases and HIV patients (Sulaiman et al., unpub. data). This is in agreement with some recent observations by others (Table II). Even in outbreak cases, the majority of cryptosporidiosis outbreaks are caused by anthroponotic (genotype 1) parasites (such as the waterborne outbreaks in Milwaukee in 1993, Nevada in 1994, and Florida in 1995; the Atlanta day care outbreak in 1995, and the Washington outbreak in 1997). It appears that genotype 2 parasites largely cause human infection through contamination of water or food or direct contact with infected animals, especially in rural areas. Examples are the Maine apple cider outbreak in 1993, the British Columbia waterborne outbreak in 1996, and the Pennsylvania multi-family outbreak in 1997. Even in AIDS patients, there is some similarity in the epidemiology of cryptosporidiosis, since most HIV+ individuals from both New Orleans and Guatemala were infected with genotype 1 parasites (Sulaiman et al., unpub. data). The reason for the high percentage of genotype 2 in AIDS patients (6/13 patients) in France (Bonnin et al. 1996) is not clear. Taken together, there are two distinct population of C. parvum parasites, one cycling only in humans and one cycling predominantly in animals. The latter can sometimes cause human infections. We have so far found neither non-parvum Cryptosporidium parasites nor genotypes of C. parvum other than genotypes 1 and 2 in HIV+ individuals.

CRYPTOSPORIDIUM PARASITES IN ENVIRONMENTAL SAMPLES

One difficulty facing the investigation of waterborne outbreaks of cryptosporidiosis is the lack of a sensitive, specific diagnostic tool. Most of the current PCR diagnostic and genotyping tools are designed for analysis of clinical samples. Because they cannot differentiate Cryptosporidium species and have low sensitivities, they have limitations in the analysis of water samples. Two PCR-RFLP techniques based on the SSU rRNA gene have claimed to differentiate C. parvum from other Cryptosporidium parasites (Awad-El-Kariem et al. 1994, Leng et al. 1996). One technique (Leng et al. 1996) used conserved sequences for primers and therefore amplify the SSU rRNA gene of all eukaryotic organism. The other technique (Awad-El-Kariem et al. 1994) used erroneous sequence by Cai et al. (1992) as primers, reducing the efficiency of amplification and making interpretation of the data difficult or misleading. Nor have the present genotyping techniques been subjected to cross-species testing, making interpretation of results from environmental samples that could contain non-parvum Cryptosporidium virtually impossible. Our experience with these techniques indicate the PCR-RFLP method by Bonnin et al. (1996) has extremely low sensitivity, and the PCR of RAPD fragment by Morgan et al. (1997) fails to differentiate the two genotypes. The PCR-RFLP methods by Spano et al. (1997) and Sulaiman et al. (unpub. data) also amplify non-parvum Cryptosporidium parasites.

Based on sequence information on the SSU rRNA gene, we have developed a PCR-RFLP technique for both species identification and genotyping of Cryptosporidium parasites. Because the technique employs nested PCR and targets the multi-copied rRNA gene, it is sensitive for use in environmental samples. We have used this technique in the analysis of Cryptosporidium oocysts recovered from the gill washings and hemolymph of oysters (Crassostrea virginica) collected from the Chesapeake Bay. We are interested in oysters because they are filter feeders that concentrate and accumulate Cryptosporidium oocysts they have removed from surface waters. The use of oysters enables investigators to avoid the temporal problems and poor recovery rate often associated with filtering hundreds of liters of water for to determine the presence or absence of Cryptosporidium oocysts. Before applying our technique Cryptosporidium oocysts were identified in oysters, but the species of most of the oocysts was unconfirmed (Fayer et al. 1998).

Preliminary analysis of 65 pooled oyster samples using the SSU rRNA-based PCR-RFLP technique has shown the presence of Cryptosporidium oocysts in 26 samples. Twenty four of these positive samples were C. parvum, and each of the others was C. baileyi and C. serpentis. The majority of Cryptosporidium oocysts were of genotype 2 (22 samples), indicating animals were probably the source of most Cryptosporidium oocyst contamination in water in the Chesapeake Bay area. Even though this is a highly populated area, only two samples had genotype 1 sequences . These results demonstrate that oysters can serve as a biologic monitor for Cryptosporidium oocyst contamination in waters. Because raw oysters are often consumed by humans, Cryptosporidium oocysts in oysters also pose a potential health concern. Other filter-feeders such as freshwater clams and marine mussels have also been shown to accumulate Cryptosporidium oocysts (Graczyk et al. 1998, Chalmers et al. 1997). They may serve as similar biologic monitors for Cryptosporidium oocyst contamination in waters.

CONCLUSIONS

Although the traditional classification of species based on vertebrate classes is largely accurate, it has greatly underestimated the diversity present among various Cryptosporidium isolates. This has presented problems in the identification of parasites in environmental samples. Molecular techniques are now available to identify species of Cryptosporidium and to differentiate known genotypes of C. parvum, and should be very useful in the investigation of clinical outbreaks of cryptosporidiosis. The performance of these techniques in the analysis of environmental samples, however, has yet to be thoroughly demonstrated. Because of the nature of environmental samples, Cryptosporidium isolates from various hosts must be more extensively characterized before enough data have been acquired and interpreted to instill full confidence in the method.In the interim, oysters and other filter feeders can serve as biologic indicators for contamination of Cryptosporidium oocysts in waters.

ACKNOWLEDGMENTS

To Joself Limor for technical assistance.

REFERENCES

This work was supported in part by inter-agency agreements (#DW75937730-01-0 and DW7593784-01-0) from CDC and EPA, and Emerging Infectious Diseases and Opportunistic Infectious Diseases funds from CDC, USA.

+Corresponding author. Fax: +770-488-4454.

Received 15 June 1998

Accepted 30 July 1998

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Publication Dates

  • Publication in this collection
    14 Oct 1998
  • Date of issue
    Sept 1998

History

  • Accepted
    30 July 1998
  • Received
    15 June 1998
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