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Gregarine transmission depends upon the environmental encounter rate between viable infective oocysts and suitable hosts. Many factors determine the abundance and distribution of gregarine oocysts in the environment, but the primary factors are oocyst distribution, environmental persistence, and production rate. Prior studies have demonstrated factors affecting oocyst distribution and environmental persistence, but oocyst production rate is poorly understood. This study addresses the effects of gametocyst size on oocyst production. For each of 3 gregarine species, gametocyst size was determined, and the subsequent oocyst production of each gametocyst was quantified. Gregarine species with larger gametocysts produced more oocysts per gametocyst than species with smaller gametocysts. Likewise, within species, larger gametocysts produced more oocysts. The effect was stronger in larger gregarine species, probably as a reflection of the lower overall range of gametocyst size in the smaller gregarine species.

Several genes have recently been shown to affect the innate resistance of laboratory strains of Biomphalaria glabrata to infection with Schistosoma mansoni, including Hsp90, the expression of which following a brief exposure to elevated temperature can interfere with resistance in juvenile BS-90 snails. Because a prior study failed to see a similar effect in adult snails, juvenile BS-90 snails were exposed to 10 or 50 miracidia following a 4.5 to 6 hr incubation at 33 or 37 C. Snails were then monitored for production of secondary sporocysts or release of cercariae. In addition, snails exposed to 33 C were examined histologically between 1 and 15 days post exposure (DPE) to 30 miracidia to assess the fate of primary sporocysts. Other than elevated numbers of viable primary sporocysts in the tissues of heat shocked snails at 3 DPE, no statistically significant effect of elevated temperature was observed. This discrepancy with regard to prior studies is hypothesized to result from genetic divergence in different laboratory colonies of the same strain of snail.

Various species of Eimeria have different prepatent times and predilection sites, but their life cycles in infected poultry are similar. Practically speaking, chickens can be continuously exposed to various Eimeria species through environmental contamination. Furthermore, storage condition of the oocysts influences subsequent challenge infectivity, since coccidian oocysts contain a polysaccharide energy source known as amylopectin that is required for sporulation of oocysts and survival of the sporozoites. Here analysis of the oocyst-shedding patterns of 3 Eimeria species ( Eimeria acervulina, Eimeria maxima, and Eimeria tenella) and the effects of different oocyst storage time (64, 143, 225, and 332 days) on subsequent propagation patterns were evaluated. Based on the analysis of oocyst-shedding patterns and infectious lesions evaluated by oocyst counts and histopathology, respectively, the peak points of oocyst production and infectious lesion generation in animals infected with E. acervulina were observed to occur earlier in comparison to E. maxima- and E. tenella-infected animals. Prolonged storage of E. tenella oocysts decreased oocyst excretion (measured as oocysts per gram of feces [OPG]) and lengthened the peak period. Chickens infected with the freshest oocysts (Group A) had the highest fecal oocyst output, and animals in this group reached their peak at 7 days post-infection (dpi), which is similar to the normal pattern of oocyst output in fresh isolates. Infection with oocysts stored for longer periods showed a 1-day delay in the fecal oocyst peak count (8 dpi), and these infections also resulted in fewer OPG compared to Group A. Therefore, these results indicate that the storage period is important in affecting the peak point and pattern of oocyst shedding.

For apicomplexan parasites using an oral-fecal transmission route with significant environmental exposure, the environmental persistence and infectivity of the oocyst has a direct impact on local infection dynamics, including the ability to withstand extended periods without readily available hosts. Herein we quantify the environmental persistence and infectivity of the oocysts of 2 septate gregarine species at controlled temperature and humidity and demonstrate that they can persist over multiple generational time spans. Species of Blabericola generally complete their endogenous life cycles from oocyst to oocyst within 10 days. The median residual environmental oocyst lifetime for Blabericola oocysts in this study is 21-28 days, but a significant number of oocysts of Blabericola migrator persisted and remained infective in the environment for up to 39 days while those of Blabericola cubensis persisted and remained infective for up to 92 days. Although long-lived relative to their own generational time, the oocysts of Blabericola species infecting cockroaches are short-lived relative to gregarines infecting tenebrionid beetles. For these gregarines, oocysts can persist in the environment and remain infective for up to 787 days. Mechanistically, environmental persistence and infectivity are probably energy-limited phenomena related to the amount of stored amylopectin and the basal metabolic rate of quiescent oocysts.

Plasmodium parasites (Plasmodiidae) cause malaria in many species of terrestrial vertebrates and are transmitted mainly by mosquitoes (Culicidae). Avian malaria is often caused by Plasmodium relictum , a cosmopolitan hemosporidian infection. Numerous genetic lineages of P. relictum have been described. However, it remains unclear if these lineages can be transmitted by Culex pipiens pipiens form molestus, which is widespread but has been insufficiently investigated as a possible vector of avian malaria. The aim of this study was to test experimentally if 2 common P. relictum lineages complete sporogony in the experimentally infected insects. The mosquitoes were cultivated under laboratory conditions. Unfed females were allowed to take blood meals on domestic canaries experimentally infected with the lineages pSGS1 and pGRW11 of P. relictum . These lineages are widespread and cause malaria in many species of birds. Infected female flies were examined for sporogonic development of each parasite lineage. Both exposed groups were maintained under the same laboratory conditions. Mosquitoes were dissected at intervals, and the midguts and salivary glands were prepared in order to detect sporogonic stages. Numerous ookinetes, oocysts, and sporozoites of both parasite lineages were observed. Polymerase chain reaction confirmed the presence of corresponding parasite lineages in the experimentally infected insects. Sporogonic stages of both lineages were morphologically similar and developed synchronously in this mosquito; however, the lineage pGRW11 developed a significantly greater number of oocysts than did the lineage pSGS1. This study shows that Culex p. pipiens f. molestus is susceptible to 2 widespread lineages of P. relictum and worth more attention in avian malaria epidemiology. We recommend C. p. pipiens f. molestus for experimental research of avian malaria parasites, principally because it willingly takes blood meals on birds and because it is easy to establish and maintain new colonies of this insect under laboratory conditions using wild-sampled eggs, larvae, or imago.

Abstract :  Oral administration of Toxoplasma gondii oocysts to cats (i.e., monoxenous transmission) typically induces patent infections in fewer than half of test subjects. In the present study, oral administration of T. gondii oocysts to 5 kittens induced a patent infection in 2 of them, but only 1 kitten shed enough oocysts to enable further study. Those monoxenously-produced oocysts were administered to another kitten, which produced a second generation of monoxenous oocysts, and then those were used to induce a third generation of monoxenous oocysts. These results provide a rationale to develop a strain of T. gondii that has efficient direct transmission. The isolate of T. gondii that was able to be passaged in this manner has been designated the Dubey strain and cultured tachyzoites have been donated to a repository.

Cats serve as definitive hosts for zoonotic Toxoplasma gondii , a protozoan that threatens human reproductive health, but they also excrete sporocysts of related protozoan that pose no known human health risk. Here we provide the first definitive evidence for natural infection with the enzootic parasite Sarcocystis muris, one such enzootic parasite. Sporulated Sarcocystis sp. sporocysts were found in rectal contents of an adult feral cat ( Felis catus ) in Giza, Egypt. After these sporocysts were orally inoculated into 2 Swiss Webster mice, sarcocysts were found to have developed in skeletal muscles 114 days later. As observed through transmission electron microscopy, the cyst wall corresponded to Type 1, and the parasitophorous vacuolar membrane had tiny outpocketing of blebs (<200 nm thick) that were not invaginated into the interior of the cyst; these structures were identical to the sarcocyst wall described for a Costa Rican isolate of S. muris that has served as an experimental model for nearly 4 decades. Two parasite-free cats fed sarcocyst-infected muscles developed patent infections; fully sporulated sporocysts (10-11 × 7.0 μm) were found in the lamina propria of small intestines of cats killed 6 and 7 days postinoculation (PI). Interferon gamma gene knockout (KO) mice were orally inoculated with sporocysts from experimentally infected cats, and their tissues were examined histologically; sarcocysts were found in 5 KO mice killed 87, 115, 196, 196, 196 days PI, but no stages were seen in 5 KO mice 10, 14, 14, 18, and 39 days PI. Bradyzoites were released from intramuscular sarcocysts of a KO mouse killed 115 days PI and orally inoculated into 5 KO mice. No stage of Sarcocystis was found in any organ (including intestinal lamina propria) of KO mice killed 4, 8, 81, 190, and 190 days PI, confirming that the definitive host is required to complete the life cycle even in the case of immunodeficient mice. This is the first confirmation of S. muris infection in a naturally infected cat anywhere.

Outbreaks of avian coccidiosis may occur when susceptible chickens are raised on litter containing viable Eimeria oocysts. The purpose of this study was to compare the relative sensitivities of Eimeria acervulina, Eimeria maxima, and Eimeria tenella oocysts to dessication. Sporulated E. acervulina, E. maxima, or E. tenella oocysts were incorporated into gelatin beads and incubated at 32 C for 0, 1, 2, or 3 days. In vitro oocyst excystation rates were measured for each combination of Eimeria species and incubation time. Day-old broiler chicks were allowed to ingest the oocysts-containing beads, and total oocyst production was measured from days 5-8 post-inoculation. Although no effect on excystation was observed, E. maxima oocysts displayed greater resistance to drying compared to E. acervulina and E. tenella oocysts. Eimeria acervulina oocyst production decreased 100-fold after 1-2 days incubation. Eimeria tenella oocysts were slightly more resistant to drying in that a 100-fold decrease in oocyst production was delayed until 2 days. For both E. acervulina and E. tenella , very few oocysts were observed after 3 days incubation. Eimeria maxima oocyst production remained high at all time points. Subsequent studies revealed E. maxima oocyst production was ablated only after 5 days incubation. These findings may explain in part the observed prevalence of E. maxima in litter from commercial poultry operations.

Because of outbreaks of cryptosporidiosis in humans, some Cryptosporidium spp. have become a public health concern. Commercial swine operations can be a source of this protozoan parasite. Although the species distribution of Cryptosporidium is likely dominated by Cryptosporidium suis , a fraction may be comprised of other Cryptosporidium species infectious to humans such as Cryptosporidium parvum . To better understand the survival dynamics of Cryptosporidium spp., oocysts associated with swine operations, 2 experiments were performed to determine die-off rates of C. parvum oocysts in a swine waste lagoon (2009 and 2010) and its spray field (2010 and 2011). Sentinel chambers containing a lagoon effluent suspension of C. parvum oocysts were submerged in the lagoon, and triplicate chambers were removed over time; oocysts were extracted and assayed for viability. For comparative purposes, inactivation rates of Ascaris suum eggs contained in sentinel chambers were also determined. For 2 spray field experiments, air-dried and sieved surface soil was placed in sentinel chambers, hydrated, and inoculated with a lagoon effluent suspension of C. parvum oocysts. Sentinel chambers and control oocysts in PBS contained in microcentrifuge tubes were buried 1.5 cm below the soil surface in 3 blocks. Triplicate chambers and controls were removed over time; oocysts were extracted and assayed for viability. Based on the first order decay equation, days to reach 99% die-off (T(99)) were determined. T(99)-values determined for the 2 lagoon experiments were 13.1 and 20.1 wk, respectively. A T(99)-value for C. parvum in the spray field was significantly longer at 38.0 wk than the control oocysts in PBS at 29.0 wk. The waste lagoon and spray field system of manure management at this large-scale farrowing operation appeared to reduce the load of C. parvum oocysts before they can be hydrologically transported off the operation and reduces their likelihood of contaminating surface waters and threatening public health.

Toxoplasma gondii infections are common in humans and other animals, but clinical disease is relatively rare. It is unknown whether the severity of toxoplasmosis in immunocompetent hosts is due to the parasite strain, host variability, or to other factors. Recently, attention has been focused on the genetic variability among T. gondii isolates from apparently healthy and sick hosts. Whether T. gondii genetic makeup plays a part in the pathogenesis of clinical feline toxoplasmosis is uncertain because little is known of genetic typing of strains associated with clinical feline toxoplasmosis. A 6-mo-old domestic male cat was hospitalized because of lethargy, anorexia, fever, and diarrhea. Numerous (6 million in 1 sample) T. gondii oocysts were found in feces of the cat and antibodies to T. gondii (titer 1:800) were found in its serum by the modified agglutination test. The cat was medicated orally with Clindamycin for 10 days; it became asymptomatic after 10 days and was discharged from the hospital. Viable T. gondii (designated TgCatUs9) was isolated from feces (oocysts) by bioassays in mice. Genetic typing using the DNA extracted from the brains of infected mice and 10 PCR-restriction fragment length polymorphism (RFLP) markers revealed Type II allele at the SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, and PK1 loci and Type I at the L358 and Apico loci; therefore, this isolate belongs to the ToxoDB PCR-RFLP genotype no. 4, which is grouped into the Type 12 lineage that is dominant in wildlife from North America. To our knowledge, this is the first T. gondii isolate characterized genetically from a sick cat in the USA.

Toxoplasma gondii oocysts are environmentally resistant and can survive outdoors for many months in dry and cold climates. In the present study, sporulation and survival of T. gondii oocysts was studied in different types of cat litters commercially available in the United States. Oocysts sporulated within 2-3 days in all types of cat litters and occasionally remained viable for 14 days. Results indicate that cat litter should be changed daily to prevent sporulation and infectivity to people.

Between April 2008 and March 2009, we analyzed the pattern of coccidian oocysts present in the feces of the European bison (Bison bonasus L., 1758) and found 4 species (Eimeria bovis , E. canadensis, E. ellipsoidalis, E. zuernii) previously reported from this host and 3 species (Eimeria alabamensis, E. cylindrica, E. pellita) that are new host and locality records. All the species occurred in bison females, and only 4 occurred in males; E. bovis was the most prevalent in both sexes. The overall prevalence of Eimeria spp. invasion reached 34.7% in cows and 13.9% in bulls. The highest prevalence was noted in early spring, with a peak in April, and the lowest in late autumn and winter. The oocyst count per gram of feces (OPG) varied from 50 to 1,350; no symptoms of clinical coccidiosis were observed. We found a significant influence of winter aggregations of bison on shedding of coccidian oocysts. The prevalence and OPG values were higher in bison congregating in large numbers around winter-feeding sites in comparison to other sites. We suggest that the coming together of cows during the growing season impacts the gender-related differences in prevalence and the number of coccidian species involved. This observation probably results from an increased production of oocysts by sub-clinically infected individuals in high-density bison populations.

Inoculation of northern bobwhite quail ( Colinus virginianus ) with low doses of Eimeria lettyae oocysts stimulates a protective immune response, suggesting immunization may be an option for controlling coccidiosis. However, the oocyst production of inoculated birds could be considerable, leading to subsequent outbreaks. To determine the oocyst production following inoculation with E. lettyae, we orally infected 12-wk-old bobwhites with 100, 1,000, or 10,000 sporulated oocysts. Fecal materials were collected on days 5-9 post-inoculation, and total oocyst production was counted in McMaster chambers. Oocyst production/bird was 49.75, 89.5, and 436 × 10(6) for 100, 1,000, or 10,000 oocysts administered, respectively. Estimated oocysts produced/oocyst administered was 49.75, 8.95, and 4.36 × 10(4) for 100, 1,000, or 10,000 oocysts administered, respectively. These findings not only illustrate the crowding effect of larger oocyst inocula but also illustrate the fecundity of E. lettyae at low doses. This suggests that successful immunization of bobwhites against coccidiosis with live vaccines might require attenuated strains with reduced reproductive potential.

The aim of the present study was to evaluate the viability of Neospora caninum sporulated oocysts after various chemical and physical treatments. Bioassays in gerbils and molecular techniques (PCR-RFLP) were used for identification of the oocysts shed by experimentally infected dogs. Sporulated oocysts were purified and divided into 11 treatment groups as follows: absolute ethanol for 1 hr; 20 C for 6 hr; 4 C for 6 hr; 60 C for 1 min; 100 C for 1 min; 10% formaldehyde for 1 hr; 10% ammonia for 1 hr; 2% iodine for 1 hr; 10% sodium hypochlorite for 1 hr; 70% ethanol for 1 hr; and one group was left untreated and kept as a positive control. All chemical treatments were performed at room temperature (37 C). A total of 33 gerbils, or 3 gerbils per treatment, were used for bioassays. After treatment, the oocysts were divided into aliquots of 1,000 oocysts and orally administered to gerbils. After 63 days, the gerbils were anesthetized and killed with 0.2 ml of T61; blood and tissue samples were collected for serological (IFAT and western blotting), molecular (real-time PCR), histopathology, and immunohistochemical tests. Treatments were considered effective only if all 5 detection techniques tested negative. High temperatures at 100 C for 1 min and 10% sodium hypochlorite for 1 hr were the only treatments that met this condition, effectively inactivating all oocysts.

Metam sodium (MS, sodium N-methyldithiocarbamate) is a widely used soil pesticide. Fumigation or chemical sterilization of poultry litter containing infectious oocysts could be an effective strategy to block the transmission of avian coccidia. In the current study, the effect of MS on the viability and infectivity of ocysts was investigated. The development of isolated, unsporulated oocysts of both Eimeria tenella and Eimeria maxima was inhibited, in a dose-related manner (IC(50) 8 to 14 microg/ml), by exposure to aqueous MS. Most treated oocysts failed to develop beyond early stages of sporulation. To determine the effect of MS on infectivity, isolated oocysts of E. tenella , Eimeria acervulina , and E. maxima were exposed for 24 hr to aqueous concentrations of MS ranging from 0 to 1,000 microg/ml. Treated oocysts were inoculated into chickens, and parameters of coccidiosis infection were compared to chickens inoculated with equal numbers of untreated oocysts. In a dose-related manner, MS significantly reduced the infectivity of oocysts with maximum effect observed at a dose of 300 microg/ml. When a mixture of oocysts containing 3 coccidian species was exposed to 300 microg/ml MS, from 0 to 24 hr, infectivity of oocysts was significantly reduced after a minimum of 12 hr of exposure. Treatment of aqueous slurries of litter samples obtained from commercial poultry houses, with 300 microg/ml MS for 24 hr, prevented the sporulation of eimerian oocysts in the litter samples relative to untreated control samples. The results indicate that MS could be used to reduce coccidial contamination of poultry litter.

Avian coccidiosis, a major parasitic disease of poultry, is caused by Eimeria spp. infection. It inflicts severe economic losses on the poultry industry worldwide. To further understand the molecular basis of sporulation and invasion of Eimeria spp., suppression subtractive hybridization and microarray approaches were combined to identify novel and important genes involved in the development and invasion of the early stages of Eimeria tenella . Three subtractive cDNA libraries were constructed for 3 stages of E. tenella including unsporulated oocysts, sporulated oocysts, and sporozoites. A subset of clones was selected from the 3 subtractive libraries to construct cDNA microarrays. Microarray analysis was used to assay expression changes of these clones. A total of 657 valid expressed sequence tags (ESTs) was obtained, including 119 unique sequences, 31 from sporulated oocysts and 88 from sporozoites. Homology searches of the public sequence databases showed that, among the 119 ESTs, 32 genes encoded proteins homologous with previously reported proteins including microneme proteins and surface antigen proteins of E. tenella , small heat shock proteins, rhoptry proteins of Toxoplasma gondii , and calcium-dependent protein kinase of Plasmodium spp. Thus, the remaining 87 ESTs have not previously been reported. Further characterization of these differentially expressed genes will be useful in understanding those responsible for sporulation, invasion, growth, and development of E. tenella .

Toxoplasma gondii is now recognized as an important pathogen in costal marine mammals. Oocysts from cat feces are believed to be washed into seawater and serve as a source of infection via transport hosts. Experimentally, it has been demonstrated that T. gondii oocysts can sporulate in seawater and remain infectious for mice for up to 6 mo. The present study examined the long-term survival of T. gondii in seawater (15 ppt NaCl) kept at 4 C or at room temperature. Oocysts kept at 4 C for 24 mo were orally infectious for mice, while those kept at room temperature for 24 mo were not.

Oocysts obtained from a guanaco and an alpaca with natural infections were identified as Eimeria macusaniensis and evaluated for host specificity and infectivity over time. In 3 separate trials conducted over 4 yr, 4 adult llamas were fed 500-5,000 sporulated oocysts obtained from guanaco feces stored under laboratory conditions for 41-84 mo. Infections with prepatent periods of 36-41 days and patent periods of 38-55 days developed in 4/4 llamas. In a fourth trial, 3 adult llamas and 1 alpaca were each fed 1,000 sporulated E. macusaniensis oocysts obtained from alpaca feces stored in the laboratory for 3 mo. Infections with prepatent periods of 33-34 days and patent periods of 14-20 days developed in 3/3 llamas. Infection in the alpaca had a prepatent period of 58 days and a patent period of 1 day. Clinical signs associated with infection, if any, were minimal and included increased fecal mucus and occasional soft feces. These results provide evidence that E. macusaniensis is a single species transmissible amongst alpacas, llamas, and guanacos and that oocysts of this species can remain infective for many years.

Oocysts are the environmentally resistant life stage of Toxoplasma gondii. Humans can become infected by accidentally ingesting the oocysts in water or from contaminated produce. Severe disease can occur in immunocompromised individuals, and nonimmune pregnant women can infect their offspring. Chronic infection is associated with decreased mental functions, vision and hearing problems, and some mental disorders such as schizophrenia. High pressure processing (HPP) is a commercial method used to treat food to eliminate pathogens. Treatment of produce to eliminate viable T. gondii oocysts would provide a means to protect consumers. The present study was done to better define the effects of HPP on oocysts placed on raspberries. Raspberries were chosen because they are a known source of a related human intestinal parasite, Cyclospora cayetanensis. Raspberries were inoculated with 5 x 10(4) oocysts of the VEG strain of T. gondii for 20 hr prior to HPP. Individual raspberries were exposed to 500 MPa, 400 MPa, 340 MPa, 300 MPa, 270 MPa, 250 MPa, 200 MPA, 100 MPa, or no MPa treatment for 60 sec in a commercial HPP unit (1 MPa = 10 atm = 147 psi). Treatment of raspberries with 340 MPa for 60 sec was needed to render oocysts spot inoculated on the raspberries noninfectious for mice. Treatment of raspberries with 200 MPa or less for 60 sec was not effective in rendering oocysts noninfectious for mice.

To estimate isotopic changes caused by trematode parasites within a host, we investigated changes in the carbon and nitrogen isotope ratios of the freshwater snail Lymnaea stagnalis infected by trematode larvae. We measured carbon and nitrogen stable isotopes within the foot, gonad, and hepatopancreas of both infected and uninfected snails. There was no significant difference in the delta13C and delta15N values of foot and gonad between infected and uninfected snails; thus, trematode parasite infections may not cause changes in snail diets. However, in the hepatopancreas, delta15N values were significantly higher in infected than in uninfected snails. The 15N enrichment in the hepatopancreas of infected snails is caused by the higher 15N ratio in parasite tissues. Using an isotope-mixing model, we roughly estimated that the parasites in the hepatopancreas represented from 0.8 to 3.4% of the total snail biomass, including the shell.

Sarcocystis neurona is the most important cause of equine protozoal myeloencephalitis (EPM), a neurologic disease of the horse. In the present work, the kinetics of S. neurona invasion is determined in the equine model. Six ponies were orally inoculated with 250 x 10(6) S. neurona sporocysts via nasogastric intubation and killed on days 1, 2, 3, 5, 7, and 9 postinoculation (PI). At necropsy, tissue samples were examined for S. neurona infection. The parasite was isolated from the mesenteric lymph nodes at 1, 2, and 7 days PI; the liver at 2, 5, and 7 days PI; and the lungs at 5, 7, and 9 days PI by bioassays in interferon gamma gene knock out mice (KO) and from cell culture. Microscopic lesions consistent with an EPM infection were observed in brain and spinal cord of ponies killed 7 and 9 days PI. Results suggest that S. neurona disseminates quickly in tissue of naive ponies.

A line of Eimeria necatrix was selected by repeated passages of oocysts that were collected after peak oocyst production from feces or cecal contents of previously infected chickens. When compared with the parent strain, the new line of E. necatrix after 16 successive passages had the following characteristics: (1) the peak of oocyst production was delayed by 2 days; (2) the sizes of 9 endogenous development stages became larger; and (3) the reproductive capacity and the immunogenicity were both enhanced. This new line of E. necatrix may be used for the development of new coccidiosis vaccines.

Effects of temperature on the sporulation of the parasite Cyclospora cayetanensis were studied in 2 food substrates, dairy and basil. Unsporulated Cyclospora oocysts were subjected to freezing and heating conditions for time periods ranging from 15 min to 1 wk. Oocysts were then removed from the food substrates and placed in 2.5% potassium dichromate for 2 wk to allow viable unsporulated oocysts to differentiate and fully sporulate, and to determine the percentage sporulation as an indicator of viability. Sporulation occurred when oocysts resuspended in dairy substrates were stored within 24 hr at -15 C. When oocysts were placed in water or basil, sporulation occurred after incubation for up to 2 days at -20 C, and up to 4 days at 37 C. Few oocysts sporulated when incubated for 1 hr at 50 C. Sporulation was not observed in basil leaves or water at -70 C, 70 C, and 100 C. Sporulation was not affected when incubated at 4 C and 23 C for up to 1 wk, which was the duration of the experiment in both of the tested substrates.

The influence of lectins on Cryptosporidium parvum oocyst agglutination and on attachment to both fixed Madin Darby Canine Kidney (MDCK) cells and fixed HCT-8 (human colorectal epithelial) cells was examined. Oocyst cell wall characteristics were examined by transmission electron microscopy. Lectin-free oocysts were shown to adhere equally to both MDCK cells and HCT-8 cells. In MDCK cells, the addition of 1-25 microg/ml Codium fragile lectin, 10 microg/ml Maclura pomifera lectin, 10 microg/ml Helix pomatia lectin, and 10-200 microg/ml Artocarpus integrifolia lectin significantly increased attachment to at least 1 of the cell cultures as compared to oocysts incubated without any lectin. The lectin-enhanced attachment was reversed by co-incubation of lectin treated-oocysts with 250 mM of each specific sugar (for a given lectin). In agglutination assays, concentrations as low as 0.5 microg/ml of C. fragile, M. pomifera, and A. integrifolia lectin agglutinated oocysts within 60 min. Finally, in TEM samples, colloidal gold conjugated-lectins from A. integrifolia, C. fragile, H. pomatia, and M. pomifera attached to oocysts, and this could be competitively inhibited by a lectin-specific sugar. This suggests that C. parvum oocysts are highly reactive to N-acetyl galactosamine-binding lectins and that the presence of N-acetyl-galactosamine containing molecules on oocysts can potentially help in oocyst attachment to host cells.

High pressure processing (HPP) has been shown to be an effective non-thermal method of eliminating non-spore forming bacteria from a variety of food products. The shelf-life of the products is extended and the sensory features of the food are not or only minimally effected by HPP The present study examined the effects of HPP using a commercial scale unit on the viability of Toxoplasma gondii oocysts. Oocysts were exposed from 100 to 550 MPa for 1 min in the HPP unit and then HPP treated oocysts were orally fed to groups of mice. Oocysts treated with 550 MPa or less did not develop structural alterations when viewed with light microscopy. Oocysts treated with 550 MPa, 480 MPa, 400 Mpa, or 340 MPa were rendered noninfectious for mice. Mice fed oocysts treated with no or 100 to 270 MPa became infected and most developed acute toxoplasmosis and were killed or died 7 to 10 days after infection. These results suggest that HPP technology may be useful in the removal of T. gondii oocysts from food products.

Hepatozoon polytopis, described from Coluber constrictor priapus from Palm Beach County, Florida, has short, usually broad gamonts 12.8 x 4.6 microm (10.0-15.0 x 3.5-6.0), with LW 58.5 microm2 (42-84) and L/W 2.84 (1.8-3.7). Nuclei commonly extend into first quarter of gamont (45%), are always present in second quarter, and seldom in third quarter (11%), with dimensions 4.5 x 3.4 (3.0-6.0 x 2.5-4.5) and LW 15.1 (10.0-24.0). Spherical to ovoid oocysts, 122.1 x 104.9 (62-240 x 57-190), with L/W 1.17 (1.0-1.9), contain 31.3 (3-103) sporocysts. Spherical to ovoid sporocysts, 38.0 x 33.9 (28-73 x 25-58), with LW 1,325.1 (756-4,168) and L/W 1.12 (1.0-1.4), contain 42.9 (22-64) sporozoites. Thamnophis sauritus sackenii from Palm Beach County is infected also by H. polytopis, as indicated from similar gamont dimensions and verified by isolation of an identical haplotype of the 18S ribosomal RNA gene from both host species.

Hepatozoon priapus n. sp. from Coluber constrictor priapus has robust gamonts with broadly rounded ends, 18.0 x 4.2 microm (17.0-20.0 x 3.5-6.0), with LW 76.4 microm2 (59-105) and L/W 4.31 (2.9-5.4). The nucleus is always present in second quarter of gamont, seldom extend into first quarter but often into third quarter, 6.0 x 3.0 (5.0-7.0 x 2.5-4.0), with LW 17.9 (13.7-21.0). Erythrocyte cytoplasm is always thin, appearing dehemoglobinized, with infected cells always distorted. Infected erythrocytes are much longer and wider than uninfected cells, with longer nuclei. Oocysts are spherical to ovoid, 92.5 x 86.0 (55-123 x 47-115) and L/W 1.08 (1.0-1.3), contain 14.0 (6-31) sporocysts. Sporocysts, which are also spherical to ovoid, 26.3 x 23.3 (19-50 x 16-38), LW 641.2 (320-1,500) and L/W 1.13 (1.0-2.2), contain 12.6 (5-18) sporozoites. Hepatozoon confusus n. sp., also from C. constrictor priapus, has slender gamonts with rounded ends, 15.6 x 4.1 (14.0-17.0 x 3.5-5.0), with LW 64.3 (52-80) and L/W 3.82 (2.8-4.4). The nucleus is always present in second quarter of gamont, commonly extending into first and third quarters, 5.0 x 2.7 (2.5-4.4 x 4.0-6.0), with LW 13.5 (11.0-16.5). Erythrocyte cytoplasm is sometimes thin, appearing partially dehemoglobinized, with infected cells usually distorted. Infected erythrocytes are longer than uninfected cells but similar in width, with erythrocyte nuclei longer. Oocysts are spherical to ovoid, 115.5 x 108.9 (52-278 x 50-278), with L/W 1.06 (1.0-1.2), and contain 25.0 (7-111) sporocysts. Sporocysts are spherical to ovoid, 27.6 x 25.2 (21-38 x 20-33), LW 701.3 (420-1,125) and L/W 1.09 (1.0-1.4), containing 20.2 (12-32) sporozoites.

Cryptosporidium parvum has been associated with outbreaks of human illness by consumption of contaminated water, fresh fruits, and vegetables. Free-living nematodes may play a role in pathogen transmission in the environment. Caenorhabditis elegans is a free-living soil nematode that has been extensively studied and serves as a good model to study possible transmission of C. parvum oocysts that may come into contact with produce before harvest. The objective of this study was to determine whether C. elegans could serve as a potential mechanical vector for transport of infectious C. parvum and Cyclospora cayetanensis in agricultural settings and whether C. elegans could ingest, excrete, and protect oocysts from desiccation. Seventy to 85% of worms ingested between 0 and 500 oocysts after 1 and 2 hr incubation with oocysts. Most of the nematodes ingested between 101 and 200 oocysts after 2 hr. Intact oocysts and empty shells were excreted by nematodes. Infectivity was determined by the neonatal assay with different treatments of worms (intact or homogenized) or oocysts or both. Adult C. elegans containing C. parvum kept in water were infective for mice. In conclusion, C. elegans adults can ingest and excrete C. parvum oocysts. Caenorhabditis elegans containing C. parvum oocysts can infect mice but does not seem to protect oocysts from extreme desiccation at 23 C incubation of a day or longer. Cyclospora oocysts were not ingested by C. elegans. The role of free-living nematodes in produce contamination needs to be further examined.

This study was undertaken to observe the effects of hydrogen peroxide on Cryptosporidium parvum oocysts with respect to protease activity in comparison to known protease inhibitors. In assessing the possible mechanisms of action of hydrogen peroxide, treatment effectiveness was analyzed using 3 assays and the potential roles of proteases and cations were considered. Treatment of C. parvum oocysts with hydrogen peroxide inhibited protease activity up to 50% compared with untreated controls. Treatment of oocysts with chemicals that affect sulfhydryls, including N-ethylmaleimide and dithiolthreitol, inhibited protease activity by >90%. Treatment of oocysts with these chemicals, along with the protease inhibitors, phenylmethylsulfonyl fluoride (PMSF), ethylenediamine-tetraacetic acid, and cystatin, inhibited protease activity as well as in vitro excystation and infection in a cell culture assay. Several mechanisms may result in the successful inhibition of infection and excystation by hydrogen peroxide treatment, including: oxidation of oocyst wall proteins or lipids, chelating of cations necessary for infection, or hydroxyl radical-induced DNA damage to sporozoites, or both.

Sarcocystis neurona has become recognized as the major causative agent of equine protozoal myeloencephalitis (EPM) in the Americas. At least 3 pathogenic species of Sarcocystis, including S. neurona, can be isolated from opossums. Methods are needed to ascertain whether these isolates are viable and capable of causing infections. In this study, the nuclear stain propidium iodide (PI) was used to differentiate between live (viable) and heat-killed (nonviable) S. neurona sporocysts. PI was excluded by live sporocysts but penetrated compromised sporocyst membrane and stained sporozoite nuclei of dead sporocysts. After live and dead sporocysts were mixed at various ratios, the number of unstained sporocysts detected after the staining procedure correlated significantly (r2 = 0.9978) with the expected numbers of live sporocysts. Sporocyst mixtures were also assayed for in vitro excystation and development in tissue cultures. The correlation between the percentage of plaques formed in tissue cultures and the percentage of expected infectious (live) sporocysts in each mixture was r2 = 0.6712. By analysis of variance, no statistically significant difference was measured between the percentage of viable sporocysts and the percentage of infectious sporocysts (P = 0.3902) in each mixture. In addition, there was evidence of a relation between PI impermeability of sporocysts and animal infectivity. These results suggest that the PI dye-exclusion technique can be a useful tool in identifying viability and potential infectivity of S. neurona sporocysts and in differentiating between viable and nonviable sporocysts.

This is the first report of blue autofluorescence as a useful characteristic in the microscopic detection of Toxoplasma gondii, Hammondia hammondi, Hammondia heydorni, Neospora caninum, Besnoitia darlingi, and Sarcocystis neurona oocysts or sporocysts. This autofluorescence is of sufficient intensity and duration to allow identification of these oocysts from complex microscopic sample backgrounds. As with the autofluorescence of related coccidia, the oocysts glow pale blue when illuminated with an ultraviolet (UV) light source and viewed with the correct UV excitation and emission filter set.

Changes in proteins during sporulation of Eimeria tenella oocysts were investigated. Unsporulated E. tenella oocysts collected from cecal tissue at 7 days postinoculation were sporulated in aerated media at 28 C for 0-48 hr. Gel analysis of soluble protein extracts prepared from oocysts from their respective time points indicated the presence of 2 prominent bands with relative molecular weight (Mr) in the range of 30 kDa and making up 20% of the total protein. These 2 bands, designated as major oocyst proteins (MOPs), were absent or barely detectable by 21 hr of sporulation. MOP bands were weakly reactive with glycoprotein stain but showed no mobility shift on deglycosylation. By gel analysis it was shown that the purified MOPs consisted of 2 bands of Mr 28.7 and 30.1 kDa. However, by matrix-assisted laser deabsorption-time of flight analysis it was shown that masses were about 17% lower. Internal sequence analysis of the 28.7-kDa protein generated 2 peptides of 17 and 14 amino acids in length, consistent with a recently described protein coded by the gam56 gene and expressed in E. maxima gametocytes. Rabbit antibodies made against MOPs were localized to outer portions of sporocysts before excystment and to the apical end of in vitro-derived sporozoites. These same antibodies were found to react with bands of Mr 101 and 65 kDa by Western blot but did not recognize MOPs in soluble or insoluble sporozoite extracts. The data suggest that the MOPs are derived from part of a gametocyte protein similar to that coded by gam56 and are processed during sporulation into sporocyst and sporozoite proteins. Alternatively, the binding of anti-MOP to 101- and 65-kDa proteins may result from alternatively spliced genes as the development of parasite proceeds.

Scarce information is available about Neospora caninum oocysts and sporozoites, in part because only small numbers of oocysts have typically been produced by experimentally infected dogs. We hypothesized that I reason for low experimental production of oocysts is that dogs have been fed tissues from experimentally infected mice instead of tissues from cattle (which are natural intermediate hosts of N. caninum). In this study, 9 dogs were fed tissues from N. caninum-infected calves, and oocyst production was compared with 6 dogs that were fed infected mouse carcasses. The number of oocysts produced by dogs that ingested infected calf tissues (mean = 160,700) was significantly greater (P = 0.03) than the number of oocysts shed by dogs that ingested infected mice (mean = 5,400). The second goal of our experiment was to demonstrate cyclical oral transmission of N. caninum between dogs and cattle. As few as 300 oocysts were used to successfully infect calves, and tissues from these calves induced patent infections in 2 of 3 dogs; oocysts from I of these dogs were administered to another calf, and tissues from this calf subsequently induced a third dog to shed oocysts. Oocysts were confirmed to be N. caninum using a species-specific polymerase chain reaction technique. In addition, sporulated oocysts were used to recover N. caninum in vitro after digestion in an acid-pepsin solution and inoculation of cell monolayers.