To assess the genetic diversity in Cryptosporidium parvum, we have sequenced the small subunit (SSU) rRNA gene of seven Cryptosporidium spp., various isolates of C. parvum from eight hosts, and a Cryptosporidiumisolate from a desert monitor. Phylogenetic analysis of the SSU rRNA sequences confirmed the multispecies nature of the genusCryptosporidium, with at least four distinct species (C. parvum, C. baileyi, C. muris, and C. serpentis). Other species previously defined by biologic characteristics, including C. wrairi, C. meleagridis, and C. felis, and the desert monitor isolate, clustered together or within C. parvum. Extensive genetic diversities were present among C. parvum isolates from humans, calves, pigs, dogs, mice, ferrets, marsupials, and a monkey. In general, specific genotypes were associated with specific host species. A PCR-restriction fragment length polymorphism technique previously developed by us could differentiate mostCryptosporidium spp. and C. parvum genotypes, but sequence analysis of the PCR product was needed to differentiateC. wrairi and C. meleagridis from some of theC. parvum genotypes. These results indicate a need for revision in the taxonomy and assessment of the zoonotic potential of some animal C. parvum isolates.
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.
A sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of Echinococcus multilocularis coproantigens (EM-ELISA) was developed with polyclonal rabbit (solid phase) and chicken egg (catching) antibodies that were directed against E. multilocularis coproantigens and somatic worm antigens, respectively. In experimentally infected dogs and cats, coproantigens were first detectable 6-17 days postinfection (PI) in samples of 8 dogs (worm burdens at necropsy: 6,330-43,200) and from 11 days PI onward in samples of 5 cats infected with 20-6,833 worms. After anthelmintic treatment of 4 dogs and 5 cats at day 20 PI, coproantigen excretion disappeared within 3-5 days. The sensitivity of the ELISA was 83.6% in 55 foxes infected with 4-60,000 E. multilocularis, but reached 93.3% in the 45 foxes harboring more than 20 worms. The EM-ELISA was used in surveys of "normal" dog and cat populations in Switzerland. Among 660 dogs and 263 cats, 5 dogs and 2 cats exhibited a positive reaction. In 2 of these dogs (0.30%) and 1 cat (0.38%), intestinal E. multilocularis infections were confirmed by necropsy, polymerase chain reaction PCR, or both. The specificities of the ELISA in these groups were found to be 99.5% and 99.6%, respectively, if positive ELISA results that could not be confirmed by other methods were classified as "false positive" reactions. An accurate determination of the prevalence of E. multilocularis in populations of final hosts is an essential requirement for establishing epidemiological baseline data and for estimating the potential infection risk for humans (Eckert, 1998). Currently, the most reliable technique for the diagnosis of E. multilocularis infection in foxes and other definitive hosts is parasitological examination of the small intestine at necropsy. Until recently, methods for an accurate and sensitive identification or exclusion of the infection in living animals were not available. The standard purgation technique with arecoline hydrobromide routinely used for screening dog populations for Echinosoccus granulosus is not applicable to foxes and cats. In dogs, this technique is hampered by its relatively low sensitivity (65.2% after 1 dose, 78.3% after 2 doses), and it is inefficient in up to 32% of the dogs that do not purge (Schantz, 1997). Moreover, the technique is biohazardous, labor intensive, and costly.Recent developments in serum antibody, fecal antigen, and DNA detection for the diagnosis of intestinal infections with E. granulosus or E. multilocularis have provided alternatives to current techniques (reviewed by Craig et al., 1996; Deplazes and Eckert, 1996). In particular, the detection of parasite coproantigens by sandwich enzyme-linked immunsorbent assay (ELISA) has become a general focus of interest in the diagnosis In a previous publication, Deplazes et al. (1992) reported for the first time the detection of E. multilocularis coproantigens in fecal samples of foxes and dogs by a sandwich ELISA. However, the sensitivity of this test system designed for the detection...
Seven-to 8-day-old Arc/Swiss mice were infected with 100,000-120,000 Cryptosporidium parvum oocysts. At 8 days postinfection (PI) the jejunum, ileum, cecum, colon, and rectum were removed. Using a simple extraction procedure and purification by Ficoll gradient centrifugation, we rountinely obtained between 3-6 million and up to 15 million purified oocysts per mouse. For in vitro cultivation, purified oocysts were pretreated in a low pH (2.5-3) 0.5% trypsin solution for 20 min, resuspended in supplemented RPMI-1640 containing glucose 0.1 g (5.55 mM), sodium bicarbonate 0.3 g, bovine bile 0.02 g, folic acid 25 tg, 4-aminobenzoic acid 100 Mg, calcium pantothenate 50 Mg, ascorbic acid 875 gg, penicillin G 10,000 U and streptomycin 0.01 g per 100 ml, and 1% fetal bovine serum (pH 7.4 before filtration), and used to inoculate confluent monolayers of the human adenocarcinoma cell line HCT-8. Incubation was in a candle jar at 37 C. We tested numerous supplements to RPMI-1640, different pHs, and atmospheric conditions and found the parameters described above produced the greatest parasite numbers in vitro. We obtained significantly superior growth of C. parvum grown in HCT-8 cells using the conditions described above than in culture conditions described previously. . The serious nature of cryptosporidiosis in high risk groups is compounded by the lack of curative treatment strategies. In this respect, a major limiting factor has been the lack of a cultivation system for biochemical studies and for the systematic assessment of potential anticryptosporidial chemotherapeutic agents.An important prerequisite for the establishment of a suitable cultivation system for C. parvum is a reliable and easily obtainable source of oocysts, a simple oocyst purification procedure, and identification of optimal conditions for growth. Although many studies have reported the in vitro development of C. parvum in various cell lines, most of these studies have found that, despite some development of C. parvum, the percentage of cells infected is low. The work of Upton, Tilley, and Brillhart (1994a, 1994b, 1995) and Upton, Tilley, Nesterenko, and Brillhart (1994) has identified a number of important growth additives, culture conditions, and a cell line that has been shown to greatly enhance growth of C. parvum in vitro. We have examined a number of these variables and have optimized conditions in our laboratory that produce superior growth of C. parvum in vitro. In addition, we report a simple method for obtaining large quantities of purified oocysts from mice suitable for in vitro cultivation and for molecular and biochemical studies. MATERIALS AND METHODS Cryptosporidium isolatesThe C. parvum isolate used for the majority of in vivo and in vitro manipulations in this study was originally obtained from a calf in Millicent, South Australia in 1993 and has subsequently been passaged through mice in our laboratory. This C. parvum isolate was designated Cl. Cryptosporidium parvum isolates from humans were obtained from individuals in We...
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