Determining potential indicators of Cryptosporidium oocysts throughout the wastewater treatment process.

Most research on wastewater treatment efficiency compliance focuses on physicochemical and microbial indicators; however, very little emphasis has been placed so far on determining suitable indicator organisms to predict the discharge level of pathogens from treatment plants. In this study, raw wastewater, activated sludge, and the resulting final effluents and biosolids in four municipal wastewater treatment plants (WWTPs A, B, C and D) were seasonally investigated for human-virulent water-borne pathogens Cryptosporidium parvum/hominis and Giardia duodenalis, and microsporidia (e.g. Encephalitozoon hellem, E. intestinalis, and Enterocytozoon bieneusi) between 2008 and 2009. A suite of potential microbial indicators for human-virulent protozoa and microsporidia was also determined. A combination of multiple fluorescent in situ hybridization and immunofluorescent antibody assays were applied to detect Cryptosporidium oocysts, Giardia cysts, and microsporidian spores. Escherichia coli, enterococci and Clostridium perfringens spores were cultivated in selective media. Positive correlations were found between the abundance of enterococci and E. coli and abundance of Cryptosporidium oocysts (r(s) > 0.47, p < 0.01) and Giardia cysts (r(s) > 0.44, p < 0.01) at WWTPs A-D. Cryptosporidium perfringens spores were positively correlated to Cryptosporidium oocysts (r(s) = 0.40, p < 0.01) and Giardia cysts (r(s) = 0.46, p < 0.01). There was a strong positive correlation between abundance of Giardia cysts and that of Cryptosporidium oocysts (r(s) > 0.89, p < 0.01). To sum up, a suite of faecal indicator bacteria can be used as indicators for the presence of Cryptosporidium oocysts and Giardia cysts in these activated-sludge systems (WWTPs A, B and C). Overall, Giardia duodenalis was noted to be the best Cryptosporidium indicator for human health in the community-based influent wastewater and throughout the treatment process.

Co-localized Crassostrea virginica and Crassostrea ariakensis oysters differ in bioaccumulation, retention and depuration of microbial indicators and human enteropathogens.

AIMS: To evaluate the bioaccumulation, retention and depuration rates of nine pathogens and surrogates when two oyster species were co-localized in tanks of seawater. METHODS AND RESULTS: Crassostrea ariakensis (n = 52) and Crassostrea virginica (n = 52) were exposed to five virus types, two protozoan and two microsporidian species for 24 h. Oysters were then placed in depuration tanks, and subsets were removed and analysed for micro-organisms at weekly intervals. The odds of C. ariakensis oysters harbouring mouse norovirus-1 (MNV-1), human norovirus (NoV) or haepatitis A virus (HAV) were significantly greater than the odds of C. virginica oysters harbouring the same viruses (MNV-1 OR = 5.05, P = 0.03; NoV OR = 6.97, P = 0.01; HAV OR = 7.40, P < 0.001). Additionally, compared to C. virginica, C. ariakensis retained significantly higher numbers of transmissive stages of all protozoan and microsporidian species (P < 0.01). Crassostrea ariakensis oysters are also capable of retaining multiple human pathogens for at least 1 month. CONCLUSIONS: Crassostrea ariakensis oysters were statistically more likely to harbour enteropathogens and microbial indicators, compared to C. virginica. Individual C. ariakensis were also statistically more likely to retain multiple viruses, protozoa and microsporidia than C. virginica, highlighting the role the species may play in the transmission of multiple diseases. SIGNIFICANCE AND IMPACT OF THE STUDY: Nonnative Crassostrea ariakensis oysters are under review for their introduction into the Chesapeake Bay. The results of this study suggest that nonnative C. ariakensis oysters may present a serious public health threat to people consuming the oysters raw from contaminated sites.

Molecular markers and sentinel organisms for environmental monitoring.

Molecular methods are useful for both to monitor anthropogenic viral, bacterial, and protozoan enteropathogens, and to track pathogen specific markers in a complex environment in order to reveal sources of these pathogens. Molecular genetic markers for fecal viruses, bacteria, and protozoans hold promise for monitoring environmental pollution and water quality. The demand for microbiologically safe waters grows exponentially due to the global demographic rise of the human population. Economically important shellfish, such as oysters, which are harvested commercially and preferentially consumed raw can be of public health importance if contaminated with human waterborne pathogens. However, feral molluscan shellfish which do not have an apparent economic value serve as indicators in monitoring aquatic environments for pollution with human waterborne pathogens and for sanitary assessment of water quality. Current technology allows for multiplexed species-specific identification, genotyping, enumeration, viability assessment, and source-tracking of human enteropathogens which considerably enhances the pathogen source-tracking efforts.

The role of non-biting flies in the epidemiology of human infectious diseases.

The feeding and reproductive habits of non-biting synanthropic flies make them important mechanical vectors of human pathogens. Synanthropic flies are major epidemiologic factors responsible for the spread of acute gastroenteritis and trachoma among infants and young children in (predominantly) developing countries. House flies are involved in mechanical transmission of nosocomial infections with multiple antibiotic-resistant bacteria in hospital environments.

A model for the prediction of relative titres of avian malaria and Aspergillus spp. IgG in Jackass penguin (Spheniscus demersus) females based on maternal IgG in egg-yolk.

A model for the prediction of IgG titres in females of the Jackass penguin (Spheniscus demersus) was developed, based on IgG which was maternally transmitted to the yolk of unembryonated eggs produced by these females. However, prediction of the female titre based on the titre of embryonated eggs may be inadequate. Blood samples from 10 S. demersus females and their corresponding embryonated (n = 10) and unembryonated (n = 49) eggs were analysed by indirect ELISA for avian malaria (Plasmodium relictum, P. elongatum) IgG and Aspergillus spp. IgG. There was no correlation between humoral responses to avian malaria and Aspergillus spp. in females or in their eggs. Avian malaria and Aspergillus spp. titres were significantly higher (P < 0.05) in the eggs than in the corresponding females and were significantly correlated (P < 0.01) with the blood titres, r = 0.84, r = 0.89, respectively. No correlation was found between titres of embryo yolk-sac (embryonated eggs) and the blood of their female parent; however, the embryo blood and the corresponding female titres were significantly (P < 0.05) correlated (avian malaria, r = 0.74; Aspergillus spp., r = 0.69). Blood and the corresponding egg-yolk (unembryonated eggs) IgG titres regressed significantly (P < 0.01). Female IgG titre (y) is related to (unembryonated) egg-yolk IgG titre by the significant (P < 0.05) regression y = -0.61 + 1.46X for avian malaria, and y = -0.02 + 0.85X for Aspergillus spp., with +/- 95% prediction limits of +/- 0.15 and +/- 0.12, respectively. This model provides access to serological information on the remote free-ranging Jackass penguins and captive Jackass-penguin colonies without the necessity of stressful blood collection.

Effects of environmental change on emerging parasitic diseases.

Ecological disturbances exert an influence on the emergence and proliferation of malaria and zoonotic parasitic diseases, including, Leishmaniasis, cryptosporidiosis, giardiasis, trypanosomiasis, schistosomiasis, filariasis, onchocerciasis, and loiasis. Each environmental change, whether occurring as a natural phenomenon or through human intervention, changes the ecological balance and context within which disease hosts or vectors and parasites breed, develop, and transmit disease. Each species occupies a particular ecological niche and vector species sub-populations are distinct behaviourally and genetically as they adapt to man-made environments. Most zoonotic parasites display three distinct life cycles: sylvatic, zoonotic, and anthroponotic. In adapting to changed environmental conditions, including reduced non-human population and increased human population, some vectors display conversion from a primarily zoophyllic to primarily anthrophyllic orientation. Deforestation and ensuing changes in landuse, human settlement, commercial development, road construction, water control systems (dams, canals, irrigation systems, reservoirs), and climate, singly, and in combination have been accompanied by global increases in morbidity and mortality from emergent parasitic disease. The replacement of forests with crop farming, ranching, and raising small animals can create supportive habitats for parasites and their host vectors. When the land use of deforested areas changes, the pattern of human settlement is altered and habitat fragmentation may provide opportunities for exchange and transmission of parasites to the heretofore uninfected humans. Construction of water control projects can lead to shifts in such vector populations as snails and mosquitoes and their parasites. Construction of roads in previously inaccessible forested areas can lead to erosion, and stagnant ponds by blocking the flow of streams when the water rises during the rainy season. The combined effects of environmentally detrimental changes in local land use and alterations in global climate disrupt the natural ecosystem and can increase the risk of transmission of parasitic diseases to the human population.

Intestinal Cryptosporidium sp. infection in the Egyptian tortoise, Testudo kleinmanni.

An adult Egyptian tortoise (Testudo kleinmanni) presented with clinical signs of enteritis and died 5 weeks after initiation of antibiotic therapy. Histological examination of the small intestine revealed heavy infection with Cryptosporidium sp.; over 80% of epithelial cells harboured the pathogen. No Cryptosporidium developmental stages were present in the stomach or the lungs. The intestinal lamina propria and mucosa were infiltrated by heterophils, lymphocytes and macrophages. The present study constitutes the first report of Cryptosporidium sp. infection in T. kleinmanni, and the first histological documentation of intestinal cryptosporidiosis in Chelonia.

Recovery of avian schistosome cercariae from water using penetration stimulant matrix with an unsaturated fatty acid.

Avian schistosome cercariae that emerge from aquatic snails can penetrate human skin causing cercarial dermatitis resulting in serious skin disease in sensitized and immunocompromised people. A trap developed for Schistosoma mansoni cercariae was tested for recovery of avian schistosome cercariae. A matrix with an unsaturated fatty acid, linoleic acid stimulates attachment and penetration of Trichobilharzia spp. cercariae, and the immobilized larvae can be subsequently visualized. The number of trapped cercariae exceeded by 3 to 7 times the number of larvae expected on the surface of the trap, based on their random distribution in the water. Recognition, attachment, and penetration of Trichobilharzia spp. cercariae led to injection of more secretory products into the stimulant matrix than by Schistosoma mansoni cercariae. This method can assist in the identification of waters infected with avian schistosome cercariae so that human exposure to these parasitic larvae can be minimized.

Echinostomiasis: a common but forgotten food-borne disease.

Human echinostomiasis, endemic to southeast Asia and the Far East, is a food-borne, intestinal, zoonotic parasitosis attributed to at least 16 species of digenean trematodes transmitted by snails. Two separate life cycles of echinostomes, human and sylvatic, efficiently operate in endemic areas. Clinical symptoms of echinostomiasis include abdominal pain, violent watery diarrhea, and anorexia. The disease occurs focally and transmission is linked to fresh or brackish water habitats. Infections are associated with common sociocultural practices of eating raw or insufficiently cooked mollusks, fish, crustaceans, and amphibians, promiscuous defecation, and the use of night soil (human excrement collected from latrines) for fertilization of fish ponds. The prevalence of infection ranges from 44% in the Philippines to 5% in mainland China, and from 50% in northern Thailand to 9% in Korea. Although the patterns of other food-borne trematodiases have changed in Asia following changes in habits, cultural practices, health education, industrialization, and environmental alteration, human echinostomiasis remains a health problem. The disease is most prevalent in remote rural places among low-wage earners and in women of child bearing age. Echinostomiasis is aggravated by socioeconomic factors such as poverty, malnutrition, an explosively growing free-food market, a lack of supervised food inspection, poor or insufficient sanitation, other helminthiases, and declining economic conditions. Furthermore, World Health Organization control programs implemented for other food-borne helminthiases and sustained in endemic areas are not fully successful for echinostomiasis because these parasites display extremely broad specificity for the second intermediate host and are capable of completing the life cycle without involvement of the human host.

Evaluation of commercial enzyme immunoassay (EIA) and immunofluorescent antibody (FA) test kits for detection of Cryptosporidium oocysts of species other than Cryptosporidium parvum.

A commercial enzyme immunoassay (EIA) (ProSpect Rapid Assay), a direct immunofluorescence antibody (IFA) test for stool testing (MERIFLUOR Cryptosporidium/Giardia), and an indirect IFA test for environmental testing (Hydrofluor-Combo Cryptosporidium/Giardia) were evaluated for detection of low public health risk Cryptosporidium oocyst isolates, and for C. parvum oocyst isolates from human and bovine feces that represent a high public health risk. There was no cross-reactivity of EIA with ova of eight medically important helminths, three Eimeria species oocysts, Sarcocystis cruzi sporocysts, and two Candida sp. isolates. All nine snake oocyst isolates (C. serpentis), two of seven lizard oocyst isolates, one turtle oocyst isolate, two avian oocyst isolates (turkey, C. meleagridis), one C. wrairi oocyst isolate from guinea pigs, one C. muris oocyst isolate from hyrax, one heifer C. muris isolate, and two C. muris-like oocyst isolates from a camel were positive by both IFA tests; six of these 19 oocyst isolates were EIA-positive. There was no difference in the sensitivity and specificity between direct and indirect IFA tests. The sensitivity of the EIA and both IFA tests to the C. parvum oocysts was 100%. The EIA showed less cross-reactivity with the non-C. parvum oocysts (24%) than direct or indirect IFA (76%), and was less sensitive to those isolates (20%) than both IFA tests (63%). A simulated sampling model for high and low public health risk Cryptosporidium oocysts showed that the low risk oocyst isolates may constitute up to 35% of all positive environmental samples by direct or indirect IFA determination, and up to 12% of all EIA positive samples. This study indicates a superiority of direct and indirect IFA and EIA for screening of human-or-bovine-origin fecal specimens, whereas testing of environmental samples may lead to misidentification of medically important isolates. The results demonstrated that the EIA kit can more accurately identify environmental samples containing oocytes pathogenic for humans than both IFA tests. The specificity of commercially available diagnostic kits to C. parvum should be critically examined for cross-species identification before they are recommended or adopted for use in testing environmental samples.

Recovery of waterborne oocysts of Cyclospora cayetanensis by Asian freshwater clams (Corbicula fluminea).

Asian freshwater clams (Corbicula fluminea) were exposed for 24 hr in 38 liters of water contaminated with 1.0 x 10(5) Cyclospora cayetanensis oocysts (2.6 x 10(3) oocysts/L). The hemolyph and gill smears of 30 clams were examined by acid-fast stain on days 1, 3, 5, 7, 10, 13, and 18 postexposure (PE). Since no oocysts were detected in the water 24 hr after contamination by the membrane filter-dissolution method, the oocyst retention rate was 4.6 X 10(2) oocysts/clam. The prevalence of oocyst-positive clams significantly decreased (P < 0.01) from 93% to 47% during 13 days PE. None of the clams contained oocysts on day 18 PE; no oocysts were detected in the clam feces. The numbers of oocysts recovered from six clam size classes varied and significantly decreased with smaller clam size (P < 0.01). The lowest prevalence values of oocyst-positive clams, 45% and 34%, were observed in the two lowest size classes: 12.1-14.0 mm and 14.1-16.0 mm, respectively. The prevalence values in the remaining four classes ranged from 84% to 100%. The sampling program demonstrated that the population of 180 clams examined during the study up to 13 day PE could be assessed for C. cayetanensis positivity by random testing of a minimum of 75 clams (42%). When the two lowest clam size classes are eliminated, the population of 114 clams could be assessed by sampling a minimum of 32 clams (28%). The results demonstrate that Corbicula fluminea can recover waterborne oocysts of C. cayetanensis, and could be used as biological indicators of contamination of water with C. cayetanensis oocysts.

House flies (Musca domestica) as transport hosts of Cryptosporidium parvum.

Refuse and promiscuous-landing synanthropic filth flies, such as house flies (Musca domestica), are recognized as transport hosts for a variety of protozoan and metazoan parasites in addition to viral and bacterial pathogens of public health importance. Exposure of adult M. domestica to 20 ml of bovine diarrheal feces containing Cryptosporidium parvum oocysts (2.0 x 10(5) oocysts/ml) resulted in intense deposition of the oocysts through fly feces on the surfaces visited by the flies (mean = 108 oocysts/cm2). Cryptosporidium parvum oocysts were detected by immunofluorescent antibodies on the exoskeleton of adult flies and in their digestive tracts. An average of 267, 131, 32, 19, and 14 oocysts per adult fly were eluted from its exoskeleton on days 3, 5, 7, 9, and 11 after they emerged, respectively. Approximately 320 C. parvum oocysts per pupa were eluted from the external surface of the pupae derived from maggots that breed in a substrate contaminated with the bovine feces; the oocysts were numerous on maggots (approximately 150 oocysts/maggot). Adult and larval stages of house flies breeding or having access to C. parvum-contaminated substrate will mechanically carry the oocysts in their digestive tracts and on their external surfaces.

Giardia duodenalis cysts of genotype A recovered from clams in the Chesapeake Bay subestuary, Rhode River.

Filter-feeding molluscan shellfish can concentrate zoonotic and anthroponotic waterborne pathogens. Cysts of Giardia sp. were detected by immunofluorescent antibodies in tissues of the clams Macoma balthica and M. mitchelli from Rhode River, a Chesapeake Bay (Maryland) subestuary. Molecular tests identified the cysts as Giardia duodenalis Genotype A, the most common genotype recovered from humans. Macoma clams are burrowers in mud or sandy-mud substrata and preferentially feed on the surface sediment layer. Waterborne Giardia cysts settle rapidly to the bottom in slow-moving waters and contaminate the sediment. Macoma clams do not have economic value, but can serve as biologic indicators of sediment contamination with Giardia sp. cysts of public health importance. These clams can be used for sanitary assessment of water quality.

Mechanical transport and transmission of Cryptosporidium parvum oocysts by wild filth flies.

Over the course of six months wild filth flies were collected from traps left for 7-10 days in a barn with or without a calf shedding Cryptosporidium parvum Genotype 2 oocysts in diarrheic feces. The oocysts of C. parvum transported on the flies' exoskeletons and eluted from their droplets left on visited surfaces were infectious for mice. The mean number of oocysts carried by a fly varied from 4 to 131, and the total oocyst number per collection varied from 56 to approximately 4.56 x 10(3). Fly abundance and intensity of mechanical transmission of infectious C. parvum oocysts were positively correlated, and both increased significantly when an infected calf was in the barn. Molecular data showed that the oocysts shed by infected calves were carried by flies for at least 3 weeks. Filth flies can acquire infectious C. parvum oocysts from unsanitary sites, deposit them on visited surfaces, and therefore may be involved in human or animal cryptosporidiosis.

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.

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 x 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.

Prevalence of Cryptosporidium, Giardia and Eimeria infections in post-weaned and adult cattle on three Maryland farms.

The prevalence of Cryptosporidium, Giardia and Eimeria, in healthy, asymptomatic, post-weaned and mature cattle was investigated on three Maryland farms. One farm, a dairy research facility, had 150 multiparous Holstein milking cows; 24 were examined and Cryptosporidium andersoni was detected in three (12.5%) but neither Giardia nor Eimeria was detected. The second farm, a commercial dairy, had 57 multiparous Holstein milking cows and an equal number of heifers. Of 19 cows examined, C. parvum, Giardia duodenalis, and Eimeria bovis and/or E. ellipsoidalis were detected in two (10.5%), two (10.5%) and one (5.26%) cow, respectively. Of 23 heifers examined, C. parvum, Giardia, and E. bovis and E. ellipsoidalis, was detected in two (8.7%), four (17.4%), and five (21.7%), heifers, respectively. The third farm, a beef cattle breeding and genetics research facility, had 180 7- to 9-month old purebred black Angus. Of 118 examined for C. parvum and Giardia, 34 (28.8%) and 44 (37.3%) were positive, respectively, of 97 examined for E. bovis and/or E. ellipsoidalis 32 (33.0%) were positive. These findings, based on a method with a minimum detection level of 100 oocysts of C. parvum/g of feces, which underestimates the number of infected cattle, clearly demonstrate the presence of low level, asymptomatic infections in post-weaned and adult cattle in the United States and indicate the potential role of such cattle as reservoirs of infectious parasites.

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.

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.

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.