U.M. Morgan

Epidemiology of Cryptosporidium : transmission, detection and identification

There are 10 valid species of Cryptosporidium and perhaps other cryptic species hidden under the umbrella of Cryptosporidium parvum. The oocyst stage is of primary importance for the dispersal, survival, and infectivity of the parasite and is of major importance for detection and identification. Because most oocysts measure 4-6 microm, appear nearly spherical, and have obscure internal structures, there are few or no morphometric features to differentiate species and in vitro cultivation does not provide differential data as for bacteria. Consequently, we rely on a combination of data from three tools: morphometrics, molecular techniques, and host specificity. Of 152 species of mammals reported to be infected with C. parvum or an indistinguishable organism, very few oocysts have ever been examined using more than one of these tools. This paper reviews the valid species of Cryptosporidium, their hosts and morphometrics; the reported hosts for the human pathogen, C. parvum; the mechanisms of transmission; the drinking water, recreational water, and food-borne outbreaks resulting from infection with C. parvum; and the microscopic, immunological, and molecular methods used to detect and identify species and genotypes.

Differentiation between human and animal isolates of Cryptosporidium parvum using rDNA sequencing and direct PCR analysis

Sequence analysis of a polymerase chain reaction (PCR)-amplified 298-bp region of the Cryptosporidium parvum 18S rRNA gene was carried out on 10 human and 9 animal isolates. Eight of the 9 animal isolates and 3 human isolates displayed the recognition sequence TATATTT, whereas 7/10 human isolates exhibited the recognition sequence TTTTTTTTTTT. Sequence analysis of the ninth animal isolate, which was recovered from a Koala, revealed this isolate to be different from both human and animal isolates. The AT richness of the rDNA recognition sequences rendered them unsuitable for primer design and therefore a diagnostic randomly amplified polymorphic DNA fragment previously developed in our laboratory was also sequenced. Analysis of 2 human and 2 animal isolates again revealed distinct differences between animal and human isolates. On the basis of this sequence information, diagnostic primers were designed that could directly differentiate between animal and human isolates on the basis of the size of the PCR product. The ability to differentiate directly between human and animal isolates has important implications for studies of the transmission and zoonotic potential of this organism. These results also raise further doubts about the uniformity of the species C. parvum.

Cryptosporidium andersoni n. sp. (Apicomplexa: Cryptosporiidae) from Cattle, Bos taurus

Abstract A new species of Cryptosporidium is described from the feces of domestic cattle, Bos taurus. Oocysts are structurally similar to those of Cryptosporidium muris described from mice but are larger than those of Cryptosporidium parvum. Oocysts of the new species are ellipsoidal, lack sporocysts, and measure 7.4 × 5.5 μm (range, 6.0–8.1 by 5.0–6.5 μm). The length to width ratio is 1.35 (range, 1.07–1.50). The colorless oocyst wall is < 1 μm thick, lacks a micropyle, and possesses a longitudinal suture at one pole. A polar granule is absent, whereas an oocyst residuum is present. Oocysts were passed fully sporulated and are not infectious to outbred, inbred immunocompetent or immunodeficient mice, chickens or goats. Recent molecular analyses of the rDNA 18S and ITS1 regions and heat-shock protein 70 (HSP-70) genes demonstrate this species to be distinct from C. muris infecting rodents. Based on transmission studies and molecular data, we consider the large form of Cryptosporidium infecting the abomasum of cattle to be a new species and have proposed the name Cryptosporidium andersoni n. sp. for this parasite.

Molecular and phylogenetic characterisation of Cryptosporidium from birds.

Avian isolates of Cryptosporidium species from different geographic locations were sequenced at two loci, the 18S rRNA gene and the heat shock gene (HSP-70). Phylogenetic analysis of the sequence data provided support for the existence of a new avian species of Cryptosporidium infecting finches and a second species infecting a black duck. The identity of Cryptosporidium baileyi and Cryptosporidium meleagridis as valid species was confirmed. Also, C. baileyi was identified in a number of isolates from the brown quail extending the host range of this species.

CRYPTOSPORIDIUM CANIS N. SP. FROM DOMESTIC DOGS

Oocysts of Cryptosporidium, from the feces of a naturally infected dog and from an HIV-infected human, were identified as the previously reported canine genotype of Cryptosporidium parvum, hereafter referred to as Cryptosporidium canis n. sp. Also among the oocysts from the dog, a trace amount of C. parvum bovine genotype was detected. Cryptosporidium canis oocysts from both the dog and human were infectious for calves. Oocysts excreted by calf 1 (dog source) were approximately 90% C. canis and 10% C. parvum, whereas those excreted by calf 3 (human source) were 100% C. canis. Oocysts from calf 1 infected calf 2 resulting in excretion by calf 2 of oocysts ∼90% C. parvum and 10% C. canis. Oocysts of C. canis were not infectious for BALB/c neonatal mice or immunosuppressed C57 juvenile mice, although all control mice became infected with the C. parvum Beltsville isolate. Oocysts of C. canis from calf 1 and the human were structurally indistinguishable from oocysts of the C. parvum Beltsville isolate (bovine). However, C. canis oocysts differed markedly at the molecular level from all known species of Cryptosporidium based on sequence data for the 18S rDNA and the HSP 70 gene. The differences in genetics and host specificity clearly differentiate C. canis as a new species.

Phylogenetic relationships of Cryptosporidium parasites based on the 70-kilodalton heat shock protein (HSP70) gene.

ABSTRACT We have characterized the nucleotide sequences of the 70-kDa heat shock protein (HSP70) genes of Cryptosporidium baileyi,C. felis, C. meleagridis, C. muris,C. serpentis, C. wrairi, and C. parvum from various animals. Results of the phylogenetic analysis revealed the presence of several genetically distinct species in the genus Cryptosporidium and eight distinct genotypes within the species C. parvum. Some of the latter may represent cryptic species. The phylogenetic tree constructed from these sequences is in agreement with our previous results based on the small-subunit rRNA genes of Cryptosporidium parasites. TheCryptosporidium species formed two major clades: isolates of C. muris and C. serpentis formed the first major group, while isolates of C. felis, C. meleagridis, C. wrairi, and eight genotypes ofC. parvum formed the second major group. Sequence variations were also observed between C. muris isolates from ruminants and rodents. The HSP70 gene provides another useful locus for phylogenetic analysis of the genusCryptosporidium.

Cryptosporidium systematics and implications for public health

There is controversy in the taxonomy of Cryptosporidium parasites and the public health significance of Cryptosporidium isolates from various animals. Recent advances in molecular characterization of Cryptosporidium parasites have allowed the re-examination of species structure of the genus Cryptosporidium. Non-parvum Cryptosporidium spp and new C. parvum genotypes in immunocompromised humans can now be clearly detected. In this article, Lihua Xiao and colleagues summarize the current biological and molecular evidence for different Cryptosporidium spp, and the public health importance of these species and new C. parvum genotypes.

Variation in Cryptosporidium: towards a taxonomic revision of the genus

Cryptosporidium is an important cause of enteric disease in humans and other animals. Limitations associated with conventional diagnostic methods for cryptosporidiosis based on morphological features, coupled with the difficulty of characterising parasites isolated in the laboratory, have restricted our ability to clearly identify species. The application of sensitive molecular approaches has obviated the necessity for laboratory amplification. Such studies have found considerable evidence of genetic heterogeneity among isolates of Cryptosporidium from different species of vertebrate, and there is now mounting evidence suggesting that a series of host-adapted genotypes/strains/species of the parasite exist. In this article, studies on the molecular characterisation of Cryptosporidium during the last 5 years are reviewed and put into perspective with the past and present taxonomy of the genus. The predictive value of achieving a sound taxonomy for the genus Cryptosporidium with respect to understanding its epidemiology and transmission and controlling outbreaks of the disease is also discussed.

Complete development and long-term maintenance of Cryptosporidium parvum human and cattle genotypes in cell culture

This study describes the complete development (from sporozoites to sporulated oocysts) of Cryptosporidium parvum (human and cattle genotypes) in the HCT-8 cell line. Furthermore, for the first time the complete life cycle was perpetuated in vitro for up to 25 days by subculturing. The long-term maintenance of the developmental cycle of the parasite in vitro appeared to be due to the initiation of the auto-reinfection cycle of C. parvum. This auto-reinfection is characterised by the production and excystation of new invasive sporozoites from thin-walled oocysts, with subsequent maintenance of the complete life cycle in vitro. In addition, thin-walled oocysts of the cattle genotype were infective for ARC/Swiss mice but similar oocysts of the human genotype were not. This culture system will provide a model for propagation of the complete life cycle of C. parvum in vitro.

Sequence differences in the diagnostic target region of the oocyst wall protein gene of Cryptosporidium parasites.

ABSTRACT Nucleotide sequences of the Cryptosporidium oocyst wall protein (COWP) gene were obtained from variousCryptosporidium spp. (C. wrairi, C. felis, C. meleagridis, C. baileyi,C. andersoni, C. muris, and C. serpentis) and C. parvum genotypes (human, bovine, monkey, marsupial, ferret, mouse, pig, and dog). Significant diversity was observed among species and genotypes in the primer and target regions of a popular diagnostic PCR. These results provide useful information for COWP-based molecular differentiation of Cryptosporidium spp. and genotypes.