Cryptosporidium Serpentis Research Articles

Molecular identification of a Cryptosporidium saurophilum from corn snake (Elaphe guttata guttata)

Fecal samples from two snakes (Elaphe guttata guttata) have been examined by immunofluorescence test (IFT) and indicated Cryptosporidium spp. infection. Subsequent polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) analysis and sequencing of small subunit (SSU) rRNA and actin genes enabled a distinct molecular characterization of the infecting organism as Cryptosporidium saurophilum. This is the first report about C. saurophilum infection in Elaphe guttata guttata. Cryptosporidium infections are common in reptiles and have been reported in at least 57 reptilian species (O’Donoghue 1995). Two main Cryptosporidium spp. are recognized in reptiles: Cryptosporidium serpentis is a gastric parasite mainly in snakes, and Cryptosporidium saurophilum is an intestinal parasite mainly in lizards (Morgan et al. 1999; Xiao et al. 2004a). Unlike other animals in which infection with Cryptosporidium spp. is usually self limiting in immunocompetent individuals, Cryptosporidium in reptiles is frequently chronic and sometimes lethal (Pasmans et al. 2007). C. serpentis infection in lizards is usually asymptomatic, whereas the infection in snakes frequently causes clinical disease (gastric hyperplasia, postprandial regurgitation and firm midbody swelling or chronic debilitating enteritis) and pathological changes (Brownstein et al. 1977; Kimbell et al. 1999; Xiao et al. 2004a; Cranfield et al. 1999). No pathological changes were found in the intestine and cloacae of adult lizards infected by C. saurophilum, but weight loss, abdominal swelling, and mortality occurred in some colonies of juvenile geckos (Eublepharis macularius) (Xiao et al. 2004b). Although C. saurophilum was originally described as a lizard parasite, it has been found in two captive snakes in Missouri (both snakes were heavily infected, and one had no clinical signs of diseases) and in three snakes (one Pituophis melanoleucuc sayi and two Pituophis ruthveni) in St. Louis Zoo (Xiao et al. 2004a, b). A group of six snakes (Elaphe obsolete, Chondrophyton viridis, Lampropeltis triangulum, Condoia asper, Pituophis melanoleucus) housed together with four lizards in the same room in Maryland also had C. saurophilum infections, but with much lower intensity than the infection of the four lizards, and all snakes were infected with multiple Cryptosporidium spp. (Xiao et al. 2004a). Studies on isolates recovered from wild and captive animals indicated that other and new Cryptosporidium spp. also exist in reptiles: a tortoise genotype identified in three tortoises, two new snake genotypes (one genotype identified in only one snake and the other genotype identified in six snakes), and another new Cryptosporidium genotype from a lizard, which was genetically distinct but was related to C. serpentis. Oocysts of the C. parvum bovine and mouse genotypes and C. muris found in some of the snakes and lizards were probably from rodents and mice ingested by these reptiles (Upton et al. 1989; Xiao et al. 2004a; Morgan et al. 1999). This possibility was supported by the fact that none of the animals with these oocysts had clinical signs and by the presence of organisms belonging to C. muris and the C. parvum mouse genotype in some of the feeder mice (Xiao et al. 2004a). In this study, we examined fecal samples from two snakes (Elaphe guttata guttata) and characterized the SSU rRNA and actin genes of Cryptosporidium-IFT positive samples by PCR-RFLP and DNA sequencing. Parasitol Res DOI 10.1007/s00436-007-0569-9

Morphologic, host specificity, and genetic characterization of a European Cryptosporidium andersoni isolate.

This study was undertaken in order to characterize a Cryptosporidium muris-like parasite isolated from cattle in Hungary and to compare this strain with other Cryptosporidium species. To date, the large-type oocysts isolated from cattle were considered as C. muris described from several mammals. The size, form, and structure of the oocysts of the Hungarian strain were identical with those described by others from cattle. An apparent difference between the morphometric data of C. muris-like parasites isolated from cattle or other mammals was noted, which is similar in magnitude to the differences between Cryptosporidium meleagridis and Cryptosporidium felis or between Cryptosporidium serpentis and Cryptosporidium baileyi. The cross-transmission experiments confirmed the findings of others, as C. muris-like oocysts isolated from cattle fail to infect other mammals. The sequence of the variable region of small subunit (SSU) rRNA gene of the strain was 100% identical with that of the U.S. Cryptosporidium andersoni and C. andersoni-like isolates from cattle. The difference between the SSU rRNA sequence of bovine strains and C. muris is similar in magnitude to the differences between C. meleagridis and Cryptosporidium parvum anthroponotic genotype or between Cryptosporidium wrairi and C. parvum zoonotic genotype. Our findings confirm that the Cryptosporidium species responsible for abomasal cryptosporidiosis and economic losses in the cattle industry should be considered a distinct species, C. andersoni Lindsay, Upton, Owens, Morgan, Mead, and Blagburn, 2000.

Cryptosporidium serpentis Oocysts and Microsporidian Spores in Feces of Captive Snakes

Cryptosporidium serpentis Oocysts and Microsporidian Spores in Feces of Captive Snakes

Cryptosporidium serpentis oocysts and microsporidian spores in feces of captive snakes.

Fecal smears of 90 snakes, 29 lizards, and 8 turtles and tortoises were tested for Cryptosporidium spp. oocysts and microsporidian spores. Microsporidian spores measured mean = 3.7 microm in length and mean = 2.3 microm in width and were present in feces of 19 snakes and 1 lizard (16%); 13 of these snakes also shed Cryptosporidium serpentis oocysts. The oocysts were numerous in all positive samples, whereas microsporidian spores were always sparse, irrespective if whether fecal samples contained the oocysts. Retrospective examination of reptile clinical records revealed that all animals shedding microsporidian spores died naturally due to diseases, pathologic conditions, and clinical problems or were killed due to severe cryptosporidiosis. The present study indicates that microsporidian infections in reptiles have the features of an opportunistic infection.

Phylogenetic Analysis of Cryptosporidium Isolates from Captive Reptiles Using 18S rDNA Sequence Data and Random Amplified Polymorphic DNA Analysis

Sequence alignment of a polymerase chain reaction-amplified 713-base pair region of the Cryptosporidium 18S rDNA gene was carried out on 15 captive reptile isolates from different geographic locations and compared to both Cryptosporidium parvum and Cryptosporidium muris isolates. Random amplified polymorphic DNA (RAPD) analysis was also performed on a smaller number of these samples. The data generated by both techniques were significantly correlated (P < 0.002), providing additional evidence to support the clonal population structure hypothesis for Cryptosporidium. Phylogenetic analysis of both 18S sequence information and RAPD analysis grouped the majority of reptile isolates together into 1 main group attributed to Cryptosporidium serpentis, which was genetically distinct but closely related to C. muris. A second genotype exhibited by 1 reptile isolate (S6) appeared to be intermediate between C. serpentis and C. muris but grouped most closely with C. muris, as it exhibited 99.15% similarity with C. muris and only 97.13% similarity with C. serpentis. The third genotype identified in 2 reptile isolates was a previously characterized 'mouse' genotype that grouped closely with bovine and human C. parvum isolates.

Biallelic Polymorphism in the Intron Region of b-Tubulin Gene of Cryptosporidium Parasites

Nucleotide sequencing of polymerase chain reaction amplified intron region of the Cryptosporidium parvum beta-tubulin gene in 26 human and 15 animal isolates revealed distinct genetic polymorphism between the human and bovine genotypes. The separation of 2 genotypes of C. parvum is in agreement with our previous genotyping data based on the thrombospondin-related adhesion protein (TRAP-C2) gene, indicating these genotype characteristics are linked at 2 genetic loci. Characterization of Cryptosporidium muris and Cryptosporidium serpentis has further shown that non-parvum Cryptosporidium parasites have beta-tubulin intron sequences identical to bovine genotype of C. parvum. Thus, results of this study confirm the lineage of 2 genotypes of C. parvum at 2 genetic loci and suggest a need for extensive characterization of various Cryptosporidium spp.

Multiple Cryptosporidium serpentis Oocyst Isolates from Captive Snakes Are Not Transmissible to Amphibians

Viable Cryptosporidium serpentis oocysts originating from 6 captive snakes were gastrically delivered to 12 Cryptosporidium-free African clawed frogs and 9 tadpoles and 3 recently metamorphosed adults of Cryptosporidium-free wood frogs. On days 7 and 14 postinoculation, no life-cycle stage of Cryptosporidium was observed in any of the histological sections of stomach, jejunum, ileum, cloaca, and cecum. However, viable inoculum-derived C. serpentis oocysts were recovered from the water in which the amphibians were kept. Amphibians may disseminate C. serpentis oocysts in the natural habitat.

Oocysts of Cryptosporidium from snakes are not infectious to ducklings but retain viability after intestinal passage through a refractory host

Six 2-week-old Cryptosporidium-free Peking ducklings ( Anas platyrhynchos) each received 2.0×10 6 viable Cryptosporidium serpentis oocysts from 6 naturally infected captive snakes. Histological sections of digestive (stomach, jejunum, ileum, cloaca, and cecum) and respiratory tract tissues (larynx, trachea, and lungs) did not contain life-cycle stages of Cryptosporidium in any of the inoculated ducklings. Because ducklings were refractory to infection, C. serpentis transmission via a diet of Peking ducklings is improbable. Viable (per in vitro excystation assay) inoculum-derived oocysts were detected in duckling feces up to 7 days post-inoculation (PI); the number of intact oocysts excreted during the first 2 days PI was significantly higher than for the remaining 5 days PI ( P<0.01). The dynamics of oocyst shedding showed that overall the birds released a significantly higher number of intact oocysts than oocyst shells ( P<0.01). Retention of the viability of C. serpentis oocysts following intestinal passage through a refractory avian species may have epizootiological implications. Under certain circumstances such as after the ingestion of C. serpentis-infected prey, herpetivorous birds may disseminate C. serpentis oocysts in the environment.

Therapeutic efficacy of hyperimmune bovine colostrum treatment against clinical and subclinical Cryptosporidium serpentis infections in captive snakes

Therapy based on the protective passive immunity of Hyperimmune Bovine Colostrum (HBC) (raised against Cryptosporidium parvum in dairy cows immunized during gestation) was tested for heterologous efficacy in subclinical and clinical infections of 12 captive snakes with C. serpentis. Six gastric HBC treatments of 1% snake weight at 1-week intervals each, have histologically cleared C. serpentis in three subclinically infected snakes, and regressed gastric histopathological changes in one of these snakes. In all snakes, each subsequent HBC treatment significantly decreased the number of oocysts recovered in gastric lavage eluants ( P<0.03). The treatments induced oocyst-negative gastric eluants and stools in all snakes, and improved clinical signs of infection. Clinically infected snakes displayed severe histopathological changes in the gastric region; however, the numbers of developmental stages of C. serpentis were moderate. Considering the severity of pathology, much lower than expected pathogen numbers were observed, and it is believed that clinically infected snakes did not have enough time to repair tissue damage that had occurred over the years of infection. As the HBC treatment was safe and highly efficacious, it is recommended to gastrically administer the HBC therapeutically to snakes that are clinically or subclinically infected with C. serpentis. Hyperimmune bovine colostrum can also be used in snake supportive therapy or prophylaxis.

Diagnosis of subclinical cryptosporidiosis in captive snakes based on stomach lavage and cloacal sampling

The applicability of stomach lavage and cloacal swab techniques for diagnosis of subclinical cryptosporidiosis were tested in eight captive snakes subclinically infected with Cryptosporidium serpentis. Two feeding regimes were employed. The snakes were first fed 7 days prior to stomach and cloaca sampling, and then 3 days prior to sampling, and the oocysts were detected by fluorescein labeled monoclonal antibody (mAb) and by acid-fast stained (AFS) direct wet smear (DWS). The overall sensitivity of AFS DWS was 95% for stomach samples and 57% for cloacal samples, with false-negativity of 5% and 43%, respectively. A significant relationship ( P<0.01) was found between stomach and cloacal samples when mAb were used for oocyst detection. Stomach sampling was diagnostically superior to cloacal sampling for identifying snake subclinical cryptosporidiosis. Based on gastric aspirates, cryptosporidial infection was diagnosed in all eight animals, and only in two or four snakes when cloacal swab material was processed by AFS or by mAb, respectively. Feeding snakes 3 days prior to sampling facilitated diagnosis based on stomach samples; however, it did not improve diagnosis when cloacal samples were used. The fraction of oocyst-positive stomach samples was significantly higher ( P<0.05) for snakes fed 3 days prior to gastric lavage when compared with the fraction of positive samples of snakes fed 7 days prior to lavage. If subclinical cryptosporidiosis is suspected in a non-eating snake patient, force-feeding and stomach lavage, 3 days after the meal, is recommended.

Cryptosporidium parvum is Not Transmissible to Fish, Amphibia, or Reptiles

<i>Cryptosporidium parvum</i> is Not Transmissible to Fish, Amphibia, or Reptiles

Therapeutic efficacy of halofuginone and spiramycin treatment against Cryptosporidium serpentis (Apicomplexa: Cryptosporidiidae) infections in captive snakes.

The therapeutic effect of halofuginone hydrobromide (Steronol) and spiramycin solubilized (Spirasol) applied to clinical Cryptosporidium serpentis infections in captive snakes was investigated. Pathological changes induced by C. serpentis were typical for snake cryptosporidiosis. Spiramycin induced no significant change in the pattern of shedding of fecal Cryptosporidium oocysts; biopsies and necropsies revealed cryptosporidiosis in gastric mucosa of all spiramycin-treated animals. In all, 8 of 21 (38%) halofuginone-treated snakes stopped shedding C. serpentis oocysts; examination of gastric tissue of 6 of these 8 animals revealed cryptosporidiosis in 2 snakes. Hepatotoxic and nephrotoxic pathological changes induced by halofuginone included focal or multifocal, severe, acute liver necrosis; severe liver hemosiderosis; and bilateral, severe, acute diffuse cortical and tubular necrosis and iron deposition. Postprandial regurgitation associated with midbody swelling, observed in 4 halofuginone-treated and 2 spiramycin-treated snakes at 15 and 21 weeks after drug withdrawal, did not coincide with oocyst-positive feces. Neither halofuginone nor spiramycin treatment produced a satisfactory therapeutic outcome when applied against clinical C. serpentis infections in snakes.

Multiple Heterogenous Isolates of Cryptosporidium serpentis from Captive Snakes Are Not Transmissible to Neonatal BALB/c Mice (Mus musculus)

Oral inoculations of 9 litter-groups of 3 5-day-old suckling BALB/c mouse pups (Mus musculus) with 6.7 x 10(3) to 1.2 x 10(5) per pup of viable, Cryptosporidium serpentis oocysts from snakes resulted in no transmission. Mice showed normal development; the litter-group weight gain was not altered significantly (P > 0.05) relative to the total number of C. serpentis oocysts inoculated or to the initial group weight (P > 0.05). Histological sections of stomach, duodenum, jejunum, ileum, cecum, and colon 4 days postinoculation did not contain life-cycle stages of Cryptosporidium in any inoculated mice. Because these neonatal, C. parvum-susceptible BALB/c mice were resistant to infection it is unlikely that C. serpentis transmission to the snakes "via infected prey" results when captive snakes are maintained on a diet of BALB/c mice.

Experimental Infection of Elaphid Snakes with Cryptosporidium serpentis (Apicomplexa: Cryptosporidiidae)

The shedding pattern of fecal Cryptosporidium serpentis oocysts, histopathologic changes in the gastric region, and the effect of spiramycin treatment were investigated in 6 experimentally infected, captive black rat (Elaphe obsoleta obsoleta), 4 yellow rat (Elaphe obsoleta quadrivittata), and 2 corn snakes (Elaphe guttata guttata). Feces were monitored for up to 2 years postinfection (PI). No significant (P > 0.07) differences were observed between expected and observed numbers of PI oocyst-positive feces. Two of 5 control animals acquired natural infections of C. serpentis over the period of study. No morphological differences were observed between oocysts from experimental and natural infections. Clinical signs included postprandial regurgitation in 5 of 13 (38%) snakes, not coinciding with the shedding of fecal oocysts. Midbody swelling and self-cure were not observed. Spiramycin treatment of 4 of 12 experimentally infected animals resulted in negative fecal examinations in 2 snakes and reduced the percentage of oocyst-positive feces in 2 other snakes from 75.5% to 24.5% and from 83.9% to 33.6%. Biopsies and necropsies revealed stages of Cryptosporidium in the gastric mucosa of all spiramycin-treated animals. The gastric mucosa was thickened and edematous, with focal necrosis, mucosal petechiae, and brush hemorrhages. Fibroplasia of lamina propria associated with chronic mucosal inflammation were common. Examination of direct fecal smears was found not to be a reliable technique for diagnosis of cryptosporidial infections in snakes.