Experimental transmission of Cryptosporidium oocyst isolates from mammals, birds and reptiles to captive snakes

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1 Experimental transmission of Cryptosporidium oocyst isolates from mammals, birds and reptiles to captive snakes Thaddeus K Graczyk, Michael R Cranfield To cite this version: Thaddeus K Graczyk, Michael R Cranfield. Experimental transmission of Cryptosporidium oocyst isolates from mammals, birds and reptiles to captive snakes. Veterinary Research, BioMed Central, 1998, 29 (2), pp <hal > HAL Id: hal Submitted on 1 Jan 1998 HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

2 Original article Experimental transmission of Cryptosporidium oocyst isolates from mammals, birds and reptiles to captive snakes Thaddeus K Graczyk Michael R Cranfield Department of Molecular Microbiology and Immunology, School of Hygiene and Public Health, Johns Hopkins University, Baltimore, MD 21205, USA hmedical Department, The Baltimore Zoo, Druid Hill Park, Baltimore, MD 21217, USA Division of Comparative Medicine, School of Medicine, Johns Hopkins University, Baltimore, MD 21205, USA (Received 23 September 1997; accepted 21 November 1997) Abstract - Groups of four to five, 3-month-old rat snakes (Elnphe obsoleta) were separately gastrically inoculated with 2.0 x 106 viable oocysts of Cr vptu.sporidium muris (mice and calves), C. muri.s-like (Bactrian camels), C. wrairi (guinea pigs), C. baileyi (chickens), C. meleagridis (turkeys), CrN7)tovporidiiii!i sp. (turtles, tortoises, chameleons and lizards) and C..serpentis from clinically (fatal case) and subclinically infected snakes. None of the snakes inoculated with oocysts originating from homothennous vertebrates developed infection as determined by histology and serology, whereas all snakes challenged with reptilian oocyst isolates were infected with Cryptospnridium on weeks 6 and 10 post-inoculation (PI). On week 10 PI, the snakes displayed mild to severe, multifocal to widespread, thinning and disorganization of gastric epithelium and nine out of twelve snakes infected by oocysts originating from reptiles other than snakes displayed severe gastric hyperplasia. Three out of ten snakes infected by oocysts originating from snakes had ELISA-detectable Crvptn.s horidium-specitic antibody (Ab) titers on week 6 PI; all snakes were Cryptns pnridium-seroconverted on week 10 PI and their serum Ab titer significantly increased. The study demonstrated that Cry ptos poriclium infections in snakes maintained on the diet of rodents or birds cannot be initiated via ingestion of an infected food item; however, snakes can void ingested oocysts. Lack of host specificity among reptiles to this pathogen, demonstrated for the first time in the present study, indicates that snake-attributed C. serpentis is not distinct from Cq1.pio.%. fiv>.iéliu>n sp. infecting reptiles other than snakes, and that clinical manifestations and virulence of Cryptosporidium in snakes is modulated by the species of the host. Housing of snakes with other reptiles can enhance transmission of C!.vpto.oporidium to snakes, and therefore should be avoided. Inra/Elsevier, Paris Cryptosporidium serpentis / snake cryptosporidiosis / oocyst / captive snake * Correspondence and reprints Tel.: (1) ; fax: (1) ; tgraczyk@jhsph.edu

3 Résumé - Transmission expérimentale d oocystes de Cryptosporidium isolés à partir de mammifères, oiseaux et reptiles aux serpents en captivité. Des groupes de quatre à cinq serpents ratiers (Elaphe obsoleta) de 3 mois d âge ont été inoculés séparément par voie gastrique avec 2,0 x 106 oocystes viables de Cryptosporidium muris provenant de souris et de veaux, de C. type muri,r (chameau de Bactriane), de C. wrairi (cochons dinde), de C. baileyi (poulets), C. meleagridis (dinde), de Cryptosporidium sp. (tortues terrestres et aquatiques, caméléons et lézards) et de C. serpentis provenant de serpents cliniquement (cas fatal) et subcliniquement infectés. Aucun des serpents inoculés avec des oocystes provenant de vertébrés homéothermes n a été infecté (déterminé par histologie et sérologie) ; en revanche, tous les serpents exposés aux oocystes de reptiles isolés ont été infectés par Crypto.Bporidium à la semaine 6 et 10 post-inoculation (PI). A la semaine 10 PI, les serpents ont présenté un amincissement et une désorganization de l épithélium gastrique de modérés à sévères, de multifocales à disseminés. Neuf des douze serpents infectés par des oocytes issus de reptiles autres que des serpents ont présenté une hyperplasie gastrique sévère. Trois des dix serpents infectés par des oocytes issus de serpents ont eu des titres en anticorps (Ac) détectables par Elisa spécifique de Cryptosporidium à la semaine 6 PI. Tous les serpents ont montré une séroconversion pour Cryptosporidium à la semaine 10 PI et les titres en Ac sériques ont augmenté significativement. Cette étude montre que les infections par Cryptosporidium chez les serpents ne s acquièrent pas par ingestion de souris ou d oiseaux infectés. Cependant, les serpents peuvent éliminer des oocystes ingérés. Le manque de spécificité des reptiles pour héberger ce pathogène, indique que C. serpentis attribué aux serpents n est pas différent de Cryptosporidium sp. qui infecte des reptiles autres que des serpents, et que les manifestations cliniques et la virulence de Cryptosporidiuna chez les serpents sont modulées par les différents types d hôtes. Maintenir ensemble des serpents avec d autres reptiles peut augmenter la transmission de Cryptosporidium chez les serpents ; cela doit donc être évité. Inra/Elsevier, Paris Cryptosporidium serpentis / cryptosporidiose du serpent / oocyste / serpent captif 1. INTRODUCTION Reptilian cryptosporidiosis is a common, frequently life-threatening, disease of captive snakes [6]. In snakes, Cryptosporidium serpentis infections are acquired for a life-time, as effective therapeutic anticryptosporidial compound(s) are unavailable [6, 7]. Subclinical infections are difficult to diagnosis because of the low oocyst output [14, 16], and the intermittent patterns of oocyst voiding [6, 7]. Spreading of the pathogen via mechanical contamination, e.g. cages, water bowls, utensils [5], and by direct animalto-animal contact is efficient [6]. Several Cryptosporidium epizootics were reported in ophidian collections [4]. As prevention of infection represents the most reliable means of maintaining a Cryptosporidium-free snake collection, the cross-transmission potential of Cryptosporidium attracted much attention and epizootiological interest from the ophidian center managers, researchers and practitioners concerned with reptilian cryptosporidiosis. A single Cryptosporidium oocyst isolate recovered from a lizard (Varanus exanthematicus), and multiple C. serpentis isolates from captive snakes were not cross-transmissible to neonatal mice [10, 291. Also, multiple C. serpentis oocyst isolates recovered from captive snakes did not establish infection in birds [20]. Cryptosporidium parvum, which is pathogenic to mammals, was uninfectious to fish, amphibia and reptiles [13, 17]. ]. However, no experiment was carried out in which mammal-specific C. muris (mice, bovines), and C. wrairi (guinea pig) or bird-infectious C. baileyi and C. meleagridis, have been transmitted to snakes. Cryptosporidium infections have been reported in reptiles other than snakes, such

4 as lacertas, turtles, tortoises, geckos and chameleons [6]. However, virtually nothing is known about the ability to establish infection in snakes by Crypto,rporidiurrt oocyst isolates originating from non-snake reptiles. This poses an important management issue for ophidian collections and herpetological centers that possess a variety of reptiles and tend to expose them together in a geographic-or-habitat coherent display. It has been recognized that snake cryptosporidiosis has two distinguishable phases, e.g. carrier stage (= subclinical infection) and clinical stage manifested by gastric hyperplasia of the mucus secreting cells [6, 7]. Some snakes may not reach clinical stage and develop severe pathology, although they may continuously (or intermittently) void the oocysts for years [6]. It has been postulated that this phenomenon is due to various virulence of C. serpentis infecting snakes [6]; however, the subject has not been experimentally addressed. The purpose of the present study was to determine if Cryptosporidium oocyst isolates recovered from reptiles other than snakes are infectious to snakes, and if so, to describe differences in histopathology. An additional purpose of the study was to determine if Cryptosporidium infectious to rodents (C. muris, and C. wrairi), ruminants (C. muris), and birds (C. baileyi, and C. meleagridis) are able to establish infection in snakes. 2. MATERIALS AND METHODS Forty-seven, 3-month-old, rat snakes, Elaphe obsoleta, were randomly divided into two groups of five snakes, seven groups of four snakes, two groups of two snakes, and a control group of five snakes. Snakes were kept in separate cages in the same room under similar ambient conditions and management protocol [14]. Snakes were offered food [5] once a week on the same day; none were force fed. The behavior of the animals was observed daily by personnel and all snakes were examined weekly by a veterinarian. Prior to the experimental challenge, fecal specimens collected from all snakes at defecation were examined for C..serpentis oocysts by acid-fast stain (AFS) and the immunofluorescent antibody (IFA) of the MERIFGUOR TM test kit (Meridian Diagnostic, Cincinnati, OH, USA) [ 16]. Blood sample, 0.3 ml, collected as described previously [15] from all snakes was processed by the enzyme-linked immunosorbent assay (ELISA) for detection of Cryptosporidium-specific antibody (Ab) of IgY class [ 15]. The reptile oocyst isolates originated from: a) clinically infected snake, Pituophis Mf/ anc/fm CM.!, which displayed severe pathology and died due to cryptosporidiosis; b) subclinically infected snake, Lampropeltis conati, which voided oocysts for several years; c) bog turtle, Clernrnys muhlenbergi, and star tortoise Geochelone elegans; d) two mountain chameleons, Chamaeleo niontiurn and e) monitor lizard, Varanus exanthematicus (table o. The oocysts from the turtle and tortoise, and the lizard were extracted from feces [14]; the oocysts from snakes and chameleons originated from feces, and necropsy-derived gastric and intestinal contents [15]. A subclinically infected snake was euthanized as described previously [15]. Cryptosporidiurn wrairi oocysts originated from feces of experimentally infected guinea pigs, and oocysts of C. baileyi and C. meleagridis originated from feces of experimentally infected chickens and turkeys, respectively. One C. muris oocyst isolate was obtained from experimental infection of mice, another C. niuri,s isolate (IDBVS-1) originated from an experimentally infected calf. Cryptosporidiunt muris-like oocyst isolates [9] were obtained from two captive Bactrian camels, Camelus bactrianu.s. The oocysts of each isolate were purified by cesium chloride gradient centrifugation [25], counted with the aid of hematocytometer and measured [161. The viability of the oocysts in the inoculum was determined in an in vitro excystation assay [28]. The assay was performed individually for 0.5 x 10! oocysts from an individual isolate. The excystation rate [(number of oocyst shells/total number of oocysts) x 100 %] [28], and the excystation index (number of sporozoites/number of oocyst shells and intact oocysts) [24] were calculated.

5 The inoculum of 2.0 x 10 6 oocysts of each isolate was administered to an individual snake in 0.5 ml of phosphated buffered saline (PBS) (ph 7.4) by gastric intubation [17]; 0.5 ml of PBS was administered to the control snakes. The numbers of snakes inoculated with reptilian oocyst isolates of Cryptosporidium are presented in table 1. Each of four groups of four snakes were inoculated separately with C. muris (mice), C. muris-like, C. baileyi and C. meleagridis oocysts. Two snakes were inoculated with C. ruuris (calf) oocysts and two snakes with C. wrairi oocysts. Fecal specimens collected at defecation were examined for Cryptosporidium oocysts by AFS and IFA [16]. On week 6 post-inoculation (PI), blood sample (0.3 ml) was collected from two randomly selected snakes from each group, and the sampled snakes were euthanized [ 15] and necropsied. Four weeks after the euthanasia, remaining snakes were sampled for blood and euthanized. The stomach was opened longitudinally and a stomach imprint made on a glass microscope slide processed with IFA. A longitudinal gastric tissue strip was collected, outlined spirally and fixed in buffered formalin. Spiral lining of the tissue facilitated histological examination of the entire gastric area. Tissue samples were embedded in paraffin, and 5-pm-thick sections stained with hematoxylin and eosine were examined under light microscopy for developmental stages of Cryptosporidium. The severity of infection expressed by percentage of infected gastric epithelium (infection index) was evaluated according to the standard protocol for the assessment of C. parvum infection in neonatal BALB/c mice [8]. The number of Cryptosporidium developmental stages observed in the histological sections was scored in a 1 to 5 scale range, where 5 equals the highest number of the pathogen. The mucosal surface of the remaining fragments of the gastric epithelium and the intestines collected at necropsy were opened longitudinally in PBS and gently scraped. Gastric and intestinal contents were to the cesium chlo- sieved [14], and subjected ride gradient centrifugation. The number of Cryptosporidium oocysts were determined with the aid of a hemocytometer, and the oocysts were measured [1 6). 1. Statistical analysis was carried out with Statistix 4.1 (Analytical Software, St Paul, MN, USA). The variables were examined by Runs test to determine if their distribution conformed to normal distribution, and if not, the nonparametric tests were used (Kruskal-Wallis ANOVA and Rank Sum test) to determine the significance of differences between variables. The mean values (x) were associated with their SD; statistical significance was considered to be P < RESULTS None of the snake fecal specimens contained Cryptosporidium oocysts prior to

6 - 0.41, the inoculation and all snakes were negative for Cryptosporidium-specific Ab. The overall excystation rate and the excystation index of all inoculum oocyst isolates varied within the limits of 55 to 67 x= % 6 ( 1 ± 7 %), and of 0.91 to 1.14, (x 1.07 = ± 0.09), respectively. The differences in either parameter values among oocyst isolates were not significant (Kruskal-Wallis ANOVA; F = 1.97). The snakes inoculated with C. muris, C. muri.s-like, C. wrairi, C. baileyi and C. meleagridis oocyst isolates did not develop Cryptosporidium infection as determined by histological examination of their gastric regions at 6 and 10 weeks PI. These snakes were also seronegative for Cryptosporidium-specific Ab. However, inoculumderived Cryptosporidium oocysts were detected up to 2 weeks PI in 35 % of fecal specimens. All snakes that were inoculated with Cryptosporidium oocyst isolates originating from reptiles developed infection. At 6 wk PI, the snakes displayed small areas of moderately to severely shortened and disorganized gastric epithelium infiltrated by numerous heterophils. One snake inoculated with C. serpentis oocysts originating from the subclinically infected snake (L. conati) developed mild mucus cell hyperplasia, whereas the intensity of histopathological changes of the remaining snakes were similar. At 10 weeks PI, histopathological changes had progressed considerably. All snakes displayed mild to severe, multifocal to widespread, thinning and disorganization of gastric epithelium which was infiltrated with large numbers of heterophils. Nine out of twelve snakes infected with oocysts originating from reptiles other than snakes euthanized on week 10 PI displayed gastric hyperplasia. None of the snakes showed clinical signs of infection such as midbody swelling; however, three snakes infected with C..serpenti.s from subclinically infected snake regurgitated food twice each. As determined by Rank sum test (t = 1.7, P < 0.05), the mean score of Crypto,vporidium developmental stages in snake gastric regions observed on week 6 PI (x = 2.8 ± 0.9) significantly increased on week 10 PI (x 4.1 = + 1.2). Overall, the mean scores varied from 2.8 ± 0.9 to 4.4 ± 0.9, and the only significant differences were observed between snakes inoculated with C..serpenti.s oocysts from the clinically infected snake (the lowest values) versus the oocysts originating from subclinically infected snake (the highest values) (Rank sum test; t 1.6, P = < 0.05) (table I). Also, snakes infected with C. serpenti.s originating from subclinically infected L. conati displayed the highest mean infection index and score (table I). Three out of ten (30 %) snakes infected with oocysts originating from snakes had ELISA-detectable relative Ab titers on week 6 PI (absorbance range; OD = F 0.36 = ± 0.04). These three snakes also had the highest scores for Crypto.sporidiurn developmental stages in the gastric region (range: 4-5). At 10 weeks PI, all snakes had Crypto.sporidium-specific Ab titer (range; OD = , x 0.45 = ± 0.06). The mean Ab titer increased significantly on week 10 PI when compared to the 6 wk PI values (Rank sum test; t 2.25, P = < 0.03). Cryptosporidium oocysts were detected in stomach imprints of eight out of ten infected snakes euthanized 6 weeks PI and in all 12 snakes euthanized on week 10 PI. The overall concentration of Crypto. Bporidium oocysts in the stools of the inoculated snakes was within the range of I 2.0 x 104to 7.5 x )0!oocysts.g-,!=5.) x 10 4 (± 4.1 x 103) oocysts-g- 1. The differences in oocyst concentration among groups of snakes inoculated with reptilian oocyst isolates were not significant (Kruskal-Wallis ANOVA; F = 1.81).

7 The overall size of Cryptosporidiurrc oocysts obtained from snakes inoculated with oocyst isolates originating from lizards, turtles, tortoises, and chameleons was 6.7 x 5.4 pm with the range of pm x pm, n = 30. The size differences among these three oocyst isolates were not significant (ANOVA, F = 2.32). Also, as determined by Rank sum test size differences between C. serpentis oocysts used to inoculate snakes and the oocysts from lizards, turtles and tortoises, and chameleons were not significant (t = 2.7). 4. DISCUSSION Snake cryptosporidiosis is an insidious disease and can devastate ophidian collections [4, 29]. The host specificity of Cryptosporidium and the ability to establish heterologous infections among vertebrate groups is important to determine the origin of snake infections and incriminate the pathogen source(s). Mammal-specific C. parvurn was noninfectious to fish, amphibians, reptiles and birds [ 13, 17, 18, 21 Multiple heterogeneous isolates of C. serpentis from captive snakes were not cross-transmissible to mice [10] and birds [20]. The present study demonstrated that C. muris (bovine and rodent isolates), C. muris-like, C. wrairi, C. baileyi and C. meleagridis are also noninfectious to snakes. We conclude that if captive snakes are maintained on the diet of birds or mammals, e.g. rodents, Crypto.sporidium infections cannot be initiated &dquo;via ingestion of infected prey&dquo; as suggested previously [12]. It is important to recognize that Cryptosporidium-infected mice or birds may sustain a source of oocysts passively transferred through snake gastrointestinal (GI) tract, and although the snake is not infected these oocysts can be detected in the stools. Such a scenario is most likely to occur if reptiles are maintained on the diet of neonatal rodents, e.g. pinkies and fuzzies, or young birds, e.g. hatchlings, as these age groups are the most susceptible to Cryptosporidium [27]. Passive transfers of oocysts through the intestine of refractory hosts, observed in the present study, have been reported in birds [18, 21 ] and reptiles, i.e. snakes and lizards [ 17]. Although minor differences in oocyst size were reported between C. muris and C. serpentis [11], it is impossible to speciate the pathogens based on oocyst morphology. Management decision toward a snake classified as intermittent shedder of oocyst, should be influenced by the fact that the oocysts detected in the snake feces may not represent infection with C. serpentis. Although Cryptosporidium has been reported in tortoises, turtles, chameleons and lizards [ 1, 2, 6, 12, 19, 23] these reports are occasional and fragmentary because the pathogen does not represent medical problems in these reptiles. Clinical signs were either not mentioned or implicated to Cryptosporidium-concurrent etiologic agents [12, 23]. As demonstrated in the present study, Cryptosporidium oocysts originating from lizard, turtles, tortoises and chameleons, induced severe infections in snakes. Transmissibility of Cryptosporidium oocyst isolates among reptiles, which is demonstrated herein for the first time, poses a serious management concern that should centers. The be addressed by ophidian pathogen that may not alert veterinarians while diagnosed in reptiles other than snakes, can be devastating for snakes. As the spread of Cryptosporidium among reptiles can be very efficient if prevention means are not undertaken or improper, the housing of snakes together with other reptiles should consider elimination of the possibility of potential Cryptosporidium transmission.

8 The first complete study on Cry h- tosporidium in snakes was commenced in 1977 [3], and in 1980 the name C. serpentis was assigned to snakes [26]. Since then, C..serpentis has been numerously reported from snakes, and Cryptosporidium sp. (or spp.) from other reptiles; this convention is also used in the present based on the study. It has been suggested, minor differences in size of oocysts originating from a variety of wild and captive reptiles, that multiple species of Cryptos poridium may exist [30!. Unfortunately, transmission experiment(s) did not follow this suggestion [30]. Although four Crypto.sporidium species (C. parvum, C. MtM!.y, C. felis and C. wrairi) infect mammals, and two species were reported from birds (C. baile yi and C. me leagridis), the sizes of oocysts of these species are similar and overlap. However, numerous experimental cross-transmission studies have establiahed that species of Cryptosporidium are specific to mammals, birds and reptiles (see [22] for review). Some species, such as C. wrairi or C.,f elis, are even more specific infecting only guinea pigs or cats, respectively. The current state-of-the-art knowledge on the distinction of Cry h- to,sporidium species is based on the recognition of specificity of a particular oocyst isolate to the host species or to the host group. In the present study, size differences among oocyst isolates originating from lizard, turtle and tortoise, and chameleons were not significant, and all isolates were perfectly transmissible to snakes, which posed a serious doubt that they were distinct from C..s<:v /: <?M<M. Also, it is important to point out that without the knowledge on the origin of an isolate, the oocysts recovered from a snake infected with lizard, turtle and tortoise, and chameleon oocysts would be classified as C. pentis. ser The results of the present study, however, do not eliminate a possibility of the existence of Crypto.sporidium species that would infect certain species (or groups) of reptiles and would be non-transmissible to other reptilian hosts. The assemblage of host species in the present study was not exhaustive and includes only captive reptiles. Snake cryptosporidiosis is an insidious disease owing to its different clinical manifestations, which are thought to be attributed to snake species [6] although the interplay between species of the host and pathogen virulence has been never experimentally addressed. To simplify interpretation of results on virulence of oocyst isolates to snakes, only similar age, one species snakes were used as oocyst recipients in the present study. Cryptosporidillll1 oocysts recovered from lizards, turtles and tortoises, and chameleons caused similar histopathological changes in snakes. The severity of pathology and the number of Cr yptosporidillll1 developmental stages in the snake gastric region were similar indicating that these three oocyst isolates were equally virulent to E. ob.soleta. Striking results, however, were obtained when C. serpentis oocysts derived from subclinical and clinical infections were transferred to healthy E. ob.soleta. Cnptos poridill l1 serpelltis, which caused severe and fatal infection of P. mela ll o!eucus, only induced mild histopathological changes in E. oh.soleta, whereas the most severe histopathological changes in the same snake species from sub- ati. The lack of differences in virulence of the oocyst isolates from reptiles other than snakes in the and the considerable were induced by C. serpelltis clinically infected L. wll same snake species, differences in the clinical outcome of C. serpelltis infection in three snake species suggests that the clinical manifestations and virulence of Cryptosporidiul/1 in snakes is modulated by the species host. of the

9 As demonstrated in the present study, a 10-week period was sufficient for development of severe histopathological changes in the snake gastric region due to Cryptosporidium infection. Serological testing indicates that three of ten infected snakes were able to mount a specific, ELISA-detectable, Ab response 6 weeks after contracting the pathogen, and all snakes were seropositive on week 10 postinfection (PI). Detectable Crypto.rporidium-seroconversion in all infected animals and increased Ab response on week 10 PI versus week 6 PI, indicate that serodiagnosis provides a reliable means to detect snake infection. A certain time is necessary for a snake to mount an Ab response against Cryptosporidium, and it is therefore recommended to serologically test a snake several times before determining his Cryptosporidium-infection status. The fact that none of the control snakes housed in the same room with infected animals contracted the pathogen, as determined by histology and serology, indicates that strict hygiene [6] is a reliable means to prevent transmission of reptilian Cryptosporidium. ACKNOWLEDGEMENTS Technical assistance of B. Hainie, L. Campbell and S. Davis is greatly acknowledged. We thank R.J. Montali (National Zoological Society, Washington, DC), J. Guy (North Carolina State University, Raleigh, NC), B.C. Anderson (University of Idaho, Caldwel, ID), B.L. Blagburn (Auburn University, Auburn, AL) and C. Chrisp (University of Michigan, Ann Harbor, MI) for providing us with the oocysts from lizards and camels, turkeys, mice and calf, chickens, and guinea pigs, respectively. We acknowledge A.P. Wisnieski (Reptile Department, The Baltimore Zoo, Baltimore, MD) for providing the snakes. The study was supported by the Maryland Zoological Society, and the AKC Fund of New York. REFERENCES [1 ] Barnard S.M., Upton S.J., A Veterinary Guide to the Parasites of Reptiles, vol. I, Protozoa, Krieger Publishing Company, Malabar, Florida, 1994, p [2] Bourdeau P., Diseases of turtles: disease of the skin and digestive tract, Point Vet 20 (1988) [3] Brownstein D.G., Strandberg J.D., Montali R.J., Bush M., Fortner J., Cryptosporidium in snakes with hypertrophic gastritis, Vet. Pathol. 14 (1977) [4] Carmel B.P., Groves V., Chronic cryptosporidiosis in Australian elaphid snakes: control of an outbreak in a captive colony, Aust. Vet. J. 70 (1993) [5J Cranfield M.R., Graczyk T.K., Experimental infection of elaphid snakes with Cryptosporidium serpentis (Apicomplexa: Cryptosporidiidae), J. Parasitol 80 (1994) [61 Cranfield M.R., Graczyk T.K., Cryptosporidiosis, in: Mader W.B. (Ed.), Manual of Reptile Medicine and Surgery, W.B. Saunders Company, The Curtis Center, Philadelphia, Pennsylvania, 1996, pp [7] Cranfield M.R., Graczyk T.K., Cryptosporidia in reptiles, in: Anderson N., Meloni D., Bonagura J.D. (Eds.), Current Veterinary Therapy, W.B. Saunders Company, The Curtis Center, Philadelphia, Pennsylvania, 1998, in press. [8] Fayer R., Effect of sodium hypochlorite exposure on infectivity of Cryptosporidium parvum oocysts for neonatal BALB/c mice, Appl. Environ. Microbiol. 61 (1995) [9] Fayer R., Lindsay P., Anderson B.C., Bush M., Chronic cryptosporidiosis in a bactrian camel (Camelus bactrianus), J. Zoo. Wildl. Med. 22 (1991) [ l OJ Fayer R., Graczyk T.K., Cranfield M.R., Multiple heterogenous isolates of Cryptosporidium serpentis from captive snakes are not transmissible to neonatal BALB/c mice (Mus mtisculus), J. Parasitol. 80 (1995) [1 I] Fayer R., Speer C.A., Dubey J.P., General biology of Cryptosporidium, in: Fayer R. (Ed.), Cryptnsporidium and Cryptosporidiosis, CRC Press, Boca Raton, Florida, 1997, pp [ I 2] Funk R.S., Implications of cryptosporidiosis in emerald tree boas (Corallus caninus), in: Rosenberg M.J. (Ed.), l lth International Herpetological Symposium on Captive Propagation and Husbandry, Chicago, Illinois, Zoological Consortium, Inc., Thurmont, Maryland, 1987, pp [131 Gillespie D., Cryptosporidiosis in reptiles, in: Kirk R.W. (Ed.), Current Veterinary Ther-

10 apy, W.B. Saunders Company, Philadelphia, Pennsylvania, 1987, pp [141 Graczyk T.K., Cranfield M.R., Assessment of the conventional detection of fecal Cryptosporidium serpentis oocysts of subclinically infected captive snakes, Vet. Res. 27 (1996) [15J Graczyk T.K., Cranfield M.R., Detection of Cryptosporidium-specific immunoglobulins in captive snakes by a polyclonal antibody in the indirect ELISA, Vet. Res. 2 (1997) [16] Graczyk T.K., Cranfield M.R., Fayer R., A comparative assessment of direct fluorescence antibody, modified acid fast stain, and sucrose flotation techniques for detection of Cryptosporidium serpentis oocysts in snake fecal specimens, J. Zoo. Wildl. Med. 26 (1995) 396!02. [ 17! Graczyk T.K., Fayer R., Cranfield M.R., Cryptosporidium parvum is not transmissible to fish, amphibians, and reptiles, J. Parasitol. 82 ( 1996) [181 Graczyk T.K., Cranfield M.R., Fayer R., Anderson M.S., Viability and infectivity of are retained Cryptosporidium parvum oocysts upon intestinal passage through a refractory avian host, Appl. Environ. Microbiol. 62 ( 1996) [ 19] Graczyk T.K., Cranfield M.R., Fayer R., Evaluation of commercial enzyme immunoassay (EIA) and immunofluorescent antibody (IFA) tests kits for detection of Cryptosporidium oocysts other than Crypto.vporidium parvum, Am. J. Trop. Med. Hyg. 53 (1996) Graczyk T.K., Cranfield M.R., Fayer R., Oocysts of Cryptosporidiurn from snakes are not infectious to ducklings but retain viability after intestinal passage through a refractory host, Vet. Parasitol. 69 (1998) in press. [211] Graczyk T.K., Cranfield M.R., Fayer R., Trout J., Goodale J.H., Infectivity of Cryptosporidium parvum oocysts is retained upon intestinal passage through a migratory waterfowl species (Canada goose, Branfa canadensis), Trop. Med. Int. Hlth. 2 (1997) [22] Graczyk T.K., Fayer R., Cranfield M.R., Zoonotic transmission of Cryptosporidium parvum: implications for waterborne cryptosporidiosis, Parasitol. Today 13 (1997) [23J] Heuschele W.P., Osterhuis J., Janssen D., Robinson P.T., Ensley P.K., Meier J.E., Olson T., Anderson M.P., Benirschke K., Cryptosporidial infections in captive wild animals, J. Wildl. Dis. 22 (1986) [24] Holton J., Nye P., McDonald V., Efficacy of selected disinfectants against Mycobacteria and cryptosporidia, J. Hosp. Infect. 27 (1994) [25 ] Kilani R.T., Sekla L., Purification of Cryptn.sporidium oocysts and sporozoites by cesium chloride and percoll gradients, Am. J. Trop. Med. Hyg. 36 ( 1987) [26 ] Levine N.D., Some corrections of coccidian (Apicomplexa: Protozoa) nomenclature, J. Parasitol. 66 (1980) [27] O Donoghue P.J., Crvptosporidium and cryptosporidiosis in man and animals, Int. J. Parasitol. 25 (1995) [28] Roberston L.J., Campbell A.T., Smith H.V., Survival of Cryptosporidium parvum oocysts under various environmental pressures, Appl. Environ. Microbiol. 58 (1992) [29J] Upton S.J., Cryptosporidium spp. in lower vertebrates, in: Dubey J.P., Speer C.A., Fayer R. (Eds.), Cryptosporidiosis in Man and Animals, CRC Press, Boca Raton, Florida, 1990, pp [30] Upton S.J., McAllister C.T., Freed P.S., Barnard S.M., Cryptosporidium spp. in wild and captive reptiles, J. Wildl. Dis. 25 (1989)