Cryptosporidium parvum is not transmissible to fish, amphibia, or reptiles

A recent report suggested that an isolate of Cryptosporidium parvum had established infections in fish, amphibians, and reptiles and raises concern that animals other than mammals might be a potential source of waterborne Cryptosporidium oocysts. To test this possibility, viable C. parvum oocysts, infectious for neonatal BALB/c mice, were delivered by gastric intubation to bluegill sunfish, poison-dart frogs, African clawed frogs, bearded dragon lizards, and corn snakes. Histological sections of the stomach, jejunum, ileum, and cloaca prepared from tissues collected on days 7 and 14 postinoculation (PI) were negative for Cryptosporidium developmental stages. However, inoculum-derived oocysts were detectable by fluorescein-labeled monoclonal antibody in feces of inoculated animals from day 1 to day 12 PI in fish and frogs, and up to day 14 PI in lizards. Snakes did not defecate for 14 days PI. Impression smears taken at necropsy on days 7 and 14 PI revealed C. parvum oocysts in the lumen of the cloaca of 2 fish and 1 lizard on day 7 PI only. Because tissue stages of the pathogen were not found, it appears that C. parvum was not heterologously transmitted to lower vertebrates. Under certain circumstances, however, such as after the ingestion of C. parvum-infected prey, lower vertebrates may disseminate C. parvum oocysts in the environment. Cryptosporidium parvum-associated cryptosporidiosis is a worldwide recognized zoonotic disease (Miron et al., 1991; Thompson, 1994; Colley, 1995) that causes acute, self-limiting diarrhea in immunocompetent humans (Heijbel et al., 1987) and severe chronic diarrhea in immunocompromised patients (Goodgame et al., 1993). Transmission of the infectious oocyst stage via water contamination has been documented (Hayes et al., 1989; Gradus et al., 1994), and the majority of human cryptosporidiosis outbreaks are classified as waterborne (Moore et al., 1994). Although C. parvum infects a wide spectrum of mammalian species (Heuschele et al., 1986; Fayer et al., 1990) and is readily transmissible to humans, data on cross-transmission to lower vertebrate groups are scant. Bovine-derived C. parvum oocysts infected the respiratory tract but not the digestive tract of chickens (Lindsay et al., 1987), whereas, fish (Lebistes reticulatus) fed on bovine-originated C. parvum oocysts remained uninfected (Gillespie, 1987). The host specificity of Cryptosporidium oocysts originating from lower vertebrates (fish, amphibians, and reptiles), and their ability to establish infections among these heterologous groups (or in mammals) remains unresolved (Cranfield and Graczyk, 1995). Avian isolates of Cryptosporidium were not cross-transmissible to mammals (O'Donoghue et al., 1987), and Cryptosporidium oocysts from a lizard (Varanus exanthematicus) and Cryptosporidium serpentis from a snake (Elaphe obsoleta obsoleta) failed to establish infections in mammals (Upton, 1990). It has been demonstrated that multiple heterogeneous isolates of C. serpentis from 7 snake species were not cross-transmissible to neonatal BALB/c mice (Fayer et al., 1995). Based on these results, it has been concluded that snake cryptosporidial infections cannot be acquired via ingestion of Cryptosporidium-infected mice (Fayer et al., 1995). The foregoing reports have led to the assumption that C. parvum cannot establish infection in lower vertebrates. Consequently, fish, amphibians, and reptiles have not received epidemiological and epizootiological attention as a reservoir for this water-transmitted human pathogen. Surprisingly, it was recently reported (Arcay et al., 1995) that C. parvum oocysts recovered from human stool were infectious for fish (Hypostomus plecostomus and Hoplosternum thoracatum), amphibia (Bufo marinus and Hyla crepitans), and reptiles (Caiman crocodillus, Iguana iguana, Cnemidophorus lemniscatus, Boa constrictor, and Geochelone carbonaria). Histological sections from each of these animals revealed a wide range of cryptosporidial developmental stages, including mature and immature oocysts, sporozoites, trophozoites, schizonts, gamonts, and zygotes (Arcay et al., 1995). Oocysts recovered from the C. parvum-inoculated animals were infectious to laboratory mice (Arcay et al., 1995). The conclusions reached in that study (Arcay et al., 1995) stand in contrast to previous concepts of host specificity of C. parvum and suggest that either the host range for C. parvum is greater than commonly known and accepted, or a new isolate of C. parvum, able to cross-infect all vertebrate groups, had been recovered. If aquatic lower vertebrates can serve as sources of this waterborne pathogen of humans and domestic animals, the results of that report (Arcay et al., 1995) have serious epidemiological and epizootiological implications. In addition, the study mentioned above (Arcay et al., 1995) constituted the first report on the respiratory cryptosporidiosis in amphibians and reptiles documenting pathological changes due to Cryptosporidium in the respiratory system of those animals. Considering the potential epidemiological importance of these results, the present study was undertaken to substantiate host species specificity and cross-transmission potential of C. parvum by challenging fish, amphibians, and reptiles with this human pathogen. MATERIALS AND METHODS Cryptosporidium parvum (AUCP-1 strain) oocysts were obtained from the feces of experimentally infected Holstein calves (Fayer and Ellis, 1993), purified by cesium chloride (CsC1) gradient centrifugation (Kilani and Sekla, 1987), and stored for 2 wk at 4 C in potassium dichromate (K2Cr207). To remove K2Cr20,, the oocysts were washed 5 times with phosphate-buffered saline (PBS) (pH 7.4), followed by centrifugation (750 g, 5 min) at 4 C. The oocysts were numerically evaluated (Fayer and Ellis, 1993) with a hemacytometer, and morphologically assessed Received 1 February 1996; revised 17 April 1996; accepted 17 April 1996. * Medical Department, The Baltimore Zoo, Druid Hill Park, Baltimore, Maryland 21217. t Immunity and Disease Prevention Laboratory, Livestock and Poultry Sciences Institute, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, Maryland 20705. $ Division of Comparative Medicine, Department of Pathology, School of Medicine, Johns Hopkins University, Baltimore, Maryland 21205.