Complete development of Caryospora bigenetica (Apicomplexa: Eimeriidae) in vitro

The development of Caryospora bigenetica in vitro is described by light microscopy. Sporozoites from snake-derived oocysts were purified and inoculated onto cultures of primary testicle cells of the cotton rat, cotton rat kidney cells, and human fetal lung cells. Intracellular sporozoites were observed one and two days postinoculation (DPI). Motile, extracellular first-generation merozoites were present 3 DPI, and second-generation merozoites were present 5 DPI. Mature gamonts were observed 9 DPI and developed into unsporulated oocysts by 10 DPI. Oocystes sporulated in vitro, and excystation was observed. Cells that were penetrated by in vitro-produced sporozoites formed caryocysts by 16 DPI. To test infectivity of in vitro-derived stages, merozoites were removed from cultured cells 5 DPI and inoculated intraperitoneally into a mouse; infection resulted. Sporulated oocysts removed from cell cultures 12 DPI produced facial swelling in an orally inoculated cotton rat.     

Investigation of transplacental transmission of Caryospora bigenetica (Apicomplexa: Eimeriidae) in mice.

Five pairs of female Swiss-Webster mice were caged with 5 males (2 females/1 male). Eight females were inoculated orally with 2.6 x 10(5) Caryospora bigenetica oocysts either 3 days before mating, 3 days after mating (PMD), 9 PMD, or 16 PMD. The remaining 2 females were inoculated orally with Hanks' balanced salt solution and served as controls. One female from each cage delivered naturally at full term and the second female delivered by cesarean section on postmating day 18. The number of offspring per litter ranged from 7 to 12. One female produced a litter of 3 stillborn and 5 liveborn offspring. Seven of 8 female mice exhibited swollen muzzles and footpads 8 days after inoculation. Caryospora bigenetica was identified in tissues of muzzle, tongue, footpad, uterus, and placenta at necropsy. This is the first report of C. bigenetica in uterus and placenta. Clinical signs and tissue infections were not observed in control mice or in any offspring of the 10 female mice. This study presents evidence that C. bigenetica is not transmitted transplacentally.     

Complete Development of Caryospora bigenetica (Apicomplexa: Eimeriidae) In Vitro1

The development of Caryospora bigenetica in vitro is described by light microscopy. Sporozoites from snake-derived oocysts were purified and inoculated onto cultures of primary testicle cells of the cotton rat, cotton rat kidney cells, and human fetal lung cells. Intracellular sporozoites were observed one and two days postinoculation (DPI). Motile, extracellular first-generation merozoites were present 3 DPI, and second-generation merozoites were present 5 DPI. Mature gamonts were observed 9 DPI and developed into unsporulated oocysts by 10 DPI. Oocysts sporulated in vitro, and excystation was observed. Cells that were penetrated by in vitro-produced sporozoites formed caryocysts by 16 DPI. To test infectivity of in vitro-derived stages, merozoites were removed from cultured cells 5 DPI and inoculated intraperitoneally into a mouse; infection resulted. Sporulated oocysts removed from cell cultures 12 DPI produced facial swelling in an orally inoculated cotton rat.     

Experimental Transmission of Caryospora simplex (Apicomplexa: Eimeriidae) to Palestine Vipers, Vipera xanthina palestinae (Serpentes: Viperidae)

ABSTRACT. Four littermate, laboratory-reared Palestine vipers, Vipera xanthina palestinae (#149, #150, #151, #152) (Serpentes: Viperidae) were used to determine modes by which Caryospora simplex (Apicomplexa: Eimeriidae) could be transmitted to snakes. Viper #149 was inoculated orally by stomach tube with 5.0 x 10⁴ sporulated oocysts of C. simplex obtained from the feces of an Ottoman viper, V. x. xanthina and began passing unsporulated oocysts of C. simplex 121 days post-inoculation (DPI). Viper #150 was fed five mice that had been inoculated orally £25 days previously with 5.0 x 10⁴ sporulated oocysts of C. simplex and it began passing unsporulated oocysts of C. simplex 33 days after being fed the first two of five mice. Viper #151 was inoculated orally with sporulated oocysts of C. simplex obtained from viper #150 and began passing oocysts 52 DPI. Viper #152 served as an uninoculated control and did not pass oocysts of any species of coccidian. This study demonstrates that snake/snake and mouse/snake transmission of C. simplex readily occurs.     

Experimental transmission of Caryospora simplex (Apicomplexa: Eimeriidae) to Palestine vipers, Vipera xanthina palestinae (Serpentes: Viperidae)

Four littermate, laboratory-reared Palestine vipers, Vipera xanthina palestinae (#149, #150, #151, #152) (Serpentes: Viperidae) were used to determine modes by which Caryospora simplex (Apicomplexa: Eimeriidae) could be transmitted to snakes. Viper #149 was inoculated orally by stomach tube with 5.0 X 10(4) sporulated oocysts of C. simplex obtained from the feces of an Ottoman viper, V. x. xanthina and began passing unsporulated oocysts of C. simplex 121 days post-inoculation (DPI). Viper #150 was fed five mice that had been inoculated orally greater than or equal to 25 days previously with 5.0 X 10(4) sporulated oocysts of C. simplex and it began passing unsporulated oocysts of C. simplex 33 days after being fed the first two of five mice. Viper #151 was inoculated orally with sporulated oocysts of C. simplex obtained from viper #150 and began passing oocysts 52 DPI. Viper #152 served as an uninoculated control and did not pass oocysts of any species of coccidian. This study demonstrates that snake/snake and mouse/snake transmission of C. simplex readily occurs.     

Serial transmission of Caryospora bigenetica Wacha and Christiansen, 1982 (Apicomplexa: Eimeriidae) between different species of rodents

Transmission of Caryospora bigenetica by cannibalism between cotton rats, between cotton rats and mice, and between mice was demonstrated. All experimental animals developed swollen muzzles and scrota 8 days after ingestion of infected tissues. Infections were confirmed by light microscopy of fresh tissue smears. Tissues of cannibalized animals contained caryocysts that, after ingestion by the next host, released sporozoites that underwent merogony, gamogony, sporogony, and caryocyst formation in dermal tissues. This study demonstrates that C. bigenetica can be transmitted by predation between species of rodents and that, in the recipient host, asexual and sexual reproduction occur before caryocysts appear.     

Fatal Caryospora bigenetica (Apicomplexa: Eimeriidae) infections in cotton rats, Sigmodon hispidus

Four groups of cotton rats, Sigmodon hispidus, were shown to be suitable secondary hosts for the viperid coccidium, Caryospora bigenetica, following oral inoculation of a mixture of oocysts and sporocysts. Swelling of the face, ears, and scrota and hemorrhagic ears were the predominant clinical signs and some cotton rats died in 3 of 4 experiments. Developmental stages of C. bigenetica were found in connective tissue components of the ear, nose, cheeks, anal skin, scrotum, and penile sheath of all cotton rats in which these tissues were examined. Additionally, developmental stages of C. bigenetica were found in connective tissue components of the following tissues examined from some cotton rats: tongue, lung, testicle, epididymis, rectum, base of the tail, footpad, and bone marrow. The present study shows that C. bigenetica can be pathogenic for cotton rats and demonstrates many new anatomic sites for developmental stages of this parasite in the secondary host.     

Ultrastructure of in vivo-produced caryocysts containing the coccidian Caryospora bigenetica (Apicomplexa: Eimeriidae)

A cotton rat was inoculated orally with oocysts of Caryospora bigenetica from the feces of a rattlesnake. Sixteen days later the rat was euthanized, and portions of the scrotum, foot pad and muzzle were processed for histological sections and transmission electron microscopy. Sporozoites within caryocysts had typical coccidian features such as an anterior and posterior refractile body, centrally located nucleus, micronemes, rhoptries, a conoid, a micropore near the anterior refractile body, a posterior pore, amylopectin granules, lipid bodies, a Golgi-like body, a mitochondrion and subpellicular microtubules. The infected host cell was spherical and surrounded by a fibrous wall-like covering, 0.35-1.00 microns thick. This outer covering, when viewed in stained histological sections, was periodic acid-Schiff (PAS)-positive.     

Ultrastructure of In Vivo-Produced Caryocysts Containing the Coccidian Caryospora bigenetica (Apicomplexa: Eimerüdae)

A cotton rat was inoculated orally with oocysts of Caryospora bigenetica from the feces of a rattlesnake. Sixteen days later the rat was euthanized, and portions of the scrotum, foot pad and muzzle were processed for histological sections and transmission electron microscopy. Sporozoites within caryocysts had typical coccidian features such as an anterior and posterior refractile body, centrally located nucleus, micronemes, rhoptries, a conoid, a micropore near the anterior refractile body, a posterior pore, amylopectin granules, lipid bodies, a Golgi-like body, a mitochondrion and subpellicular microtubules. The infected host cell was spherical and surrounded by a fibrous wall-like covering, 0.35–1.00 μm thick. This outer covering, when viewed in stained histological sections, was periodic acid-Schiff (PAS)-positive.     

Experimental Caryospora bigenetica (Apicomplexa: Eimeriidae) infections in swine (Sus scrofa).

Two Hampshire-Landrace crossbred pigs were found to contain developmental stages of Caryospora bigenetica following oral inoculation with 1 x 10(8) oocysts. One pig was given intramuscular injections of methylprednisolone acetate. Both pigs displayed clinical signs of dermal coccidiosis from 3 to 10 days after inoculation, including swollen jowls and hocks, bilateral ocular discharges, generalized erythema, and lethargy. Meronts and gamonts were observed histologically in numerous tissues including jowl, ear, footpad, tongue, and lung at 10 days postinoculation. The present study is the first report of C. bigenetica infections in swine.     

Extraintestinal development of Caryospora simplex (Apicomplexa: Eimeriidae) in experimentally infected mice, Mus musculus

Developmental stages of Caryospora simplex were found in connective tissue of the cheek, tongue, and nose of Swiss-Webster and C57 BL/6 mice (Mus musculus) from 8 through 70 days after oral inoculation with 50,000 or 250,000 oocysts, or 60,000 free sporocysts of the same species obtained from an Ottoman viper, Vipera xanthina xanthina. The earliest developmental stages were seen on day 8 post-inoculation (PI) and consisted of two types of meronts and gamonts (undifferentiated sexual stages). Gamonts, microgametocytes, macrogametes, and unsporulated oocysts were found on days 10 and 12 PI. Fully sporulated, thin-walled oocysts containing eight sporozoites surrounded by a thin sporocyst membrane were first seen 12 days PI. Monozoic cysts (caryocysts) were first seen 12 days PI and appeared fully viable throughout the duration of the study, 70 days PI. Four mice injected intra-peritoneally with 150,000 free sporozoites and killed 12 days PI contained unsporulated and sporulated oocysts in connective tissues of the cheek, tongue, and nose, suggesting that sporozoites may be carried to the site of infection via the lymphatic/circulatory system. Four cotton rats, Sigmodon hispidus, inoculated orally with 250,000 oocysts all had unsporulated and sporulated oocysts of C. simplex in connective tissue of the cheek, tongue, and nose when killed on day 12 PI, indicating extraintestinal development in the secondary host is not species specific. This is the first report of a heteroxenous coccidium with both asexual and sexual development in the primary (predator) and secondary (prey) hosts.     

Extraintestinal Development of Caryospora simplex (Apicomplexa: Eimeriidae) in Experimentally Infected Mice,Mus musculus1

Developmental stages of Caryospora simplex were found in connective tissue of the cheek, tongue, and nose of Swiss-Webster and C57 BL/6 mice (Mus musculus) from 8 through 70 days after oral inoculation with 50,000 or 250,000 oocysts, or 60,000 free sporocysts of the same species obtained from an Ottoman viper, Vipera xanthina xanthina. The earliest developmental stages were seen on day 8 post-inoculation (PI) and consisted of two types of meronts and gamonts (undifferentiated sexual stages). Gamonts, microgametocytes, macrogametes, and unsporulated oocysts were found on days 10 and 12 PI. Fully sporulated, thin-walled oocysts containing eight sporozoites surrounded by a thin sporocyst membrane were first seen 12 days PI. Monozoic cysts (caryocysts) were first seen 12 days PI and appeared fully viable throughout the duration of the study, 70 days PI. Four mice injected intra-peritoneally with 150,000 free sporozoites and killed 12 days PI contained unsporulated and sporulated oocysts in connective tissues of the cheek, tongue, and nose, suggesting that sporozoites may be carried to the site of infection via the lymphatic/circulatory system. Four cotton rats, Sigmodon hispidus, inoculated orally with 250,000 oocysts all had unsporulated and sporulated oocysts of C. simplex in connective tissue of the cheek, tongue, and nose when killed on day 12 PI, indicating extraintestinal development in the secondary host is not species specific. This is the first report of a heteroxenous coccidium with both asexual and sexual development in the primary (predator) and secondary (prey) hosts.     

Development of Caryospora simplex (Apicomplexa: Eimeriidae) from sporozoites to oocysts in human embryonic lung cell culture

Leighton tubes containing monolayers of human embryonic lung cells were inoculated with 70,000 or 30,000 sporozoites of the viperid coccidium Caryospora simplex and examined at 1, 2, 4, 6, 8, 10, 12, 14, 16, and 18 days post-inoculation (PI). By day 1 PI, sporozoites had penetrated cells and were within parasitophorous vacuoles. Most sporozoites became spherical and then underwent karyokinesis several times between days 2 and 6 PI. Mature Type I meronts were found on days 6-16 PI and contained 8 to 22 short, stout merozoites. Mature Type II meronts were present on days 10-18 PI and contained 8 to 22 long, slender merozoites. Developing gamonts (undifferentiated sexual stages) were observed on days 14 and 16 PI. Mature micro- and macrogametes and thin-walled unsporulated oocysts were present on days 16 and 18 PI. Attempts to sporulate oocysts in tissue culture medium or in a 2.5% (w/v) aqueous solution of K2Cr2O7 at 25 degrees C and 37 degrees C were unsuccessful; only a few oocysts developed to the contracted sporont stage. Four Swiss-Webster mice injected intraperitoneally with merozoites obtained from Leighton tubes on day 10 PI did not acquire infections. This is the second coccidium reported to complete its entire development, from sporozoite to oocyst, in cell culture.     

Effects of route of inoculation on the site of development of Caryospora bigenetica (Apicomplexa: Eimeriidae)

The sites of infection by Caryospora bigenetica in Swiss-Webster mice (Mus musculus) were demonstrated after 7 routes of inoculation: oral, intraperitoneal, intravenous, intramuscular, subcutaneous, dermal, and intraocular. All mice exhibited clinical signs of dermal coccidiosis 9 days after inoculation regardless of the inoculation route. Signs included swelling of the facial tissue, footpads, and scrota (male mice). Developmental stages of the parasite were found in the muzzle, tongue, footpad, lumbar subcutaneous tissue, biceps femoris muscle, conjunctiva, and eye; the latter 3 sites represent new sites of development. The site of development of the parasite in the host tissue was independent of experimental inoculation route.     

Effect of disinfection of drinking water with ozone or chlorine dioxide on survival of Cryptosporidium parvum oocysts.

Demineralized water was seeded with controlled numbers of oocysts of Cryptosporidium parvum purified from fresh calf feces and subjected to different treatments with ozone or chlorine dioxide. The disinfectants were neutralized by sodium thiosulfate, and neonatal mice were inoculated intragastrically and sacrificed 7 days later for enumeration of oocyst production. Preliminary trials indicated that a minimum infection level of 1,000 oocysts (0.1-ml inoculum) per mouse was necessary to induce 100% infection. Treatment of water containing 10(4) oocysts per ml with 1.11 mg of ozone per liter (concentration at time zero [C0]) for 6 min totally eliminated the infectivity of the oocysts for neonatal mice. A level of 2.27 mg of ozone per liter (C0) was necessary to inactivate water containing 5 x 10(5) oocysts per ml within 8 min. Also, 0.4 mg of chlorine dioxide per liter (C0) significantly reduced infectivity within 15 min of contact, although some oocysts remained viable.     

Effect of disinfection of drinking water with ozone or chlorine dioxide on survival of Cryptosporidium parvum oocysts

Demineralized water was seeded with controlled numbers of oocysts of Cryptosporidium parvum purified from fresh calf feces and subjected to different treatments with ozone or chlorine dioxide. The disinfectants were neutralized by sodium thiosulfate, and neonatal mice were inoculated intragastrically and sacrificed 7 days later for enumeration of oocyst production. Preliminary trials indicated that a minimum infection level of 1,000 oocysts (0.1-ml inoculum) per mouse was necessary to induce 100% infection. Treatment of water containing 10(4) oocysts per ml with 1.11 mg of ozone per liter (concentration at time zero [C0]) for 6 min totally eliminated the infectivity of the oocysts for neonatal mice. A level of 2.27 mg of ozone per liter (C0) was necessary to inactivate water containing 5 x 10(5) oocysts per ml within 8 min. Also, 0.4 mg of chlorine dioxide per liter (C0) significantly reduced infectivity within 15 min of contact, although some oocysts remained viable.     

Biology and pathogenicity of Eimeria spinosa Henry, 1931 in experimentally infected pigs.

A single-species isolate of E. spinosa from a diarrheic weaned pig was used to determine the endogenous development and pathogenicity of this swine coccidium. Seven out of 14 inoculated pigs developed endogenous stages or passed oocysts of E. spinosa in their feces. Immunosuppressive treatment with cyclophosphamide had no effect on the susceptibility to infection with E. spinosa in young pigs. The endogenous stages developed within the apical cytoplasm of the enterocytes lining the distal part of the villi in the posterior jejunum. The asexual development comprised three generations of meronts, which were seen at 5, 7 and 9 days post-infection (DPI). Meronts of the first generation measured 6-8 microns and produced 10-14 merozoites 4-6 microns in length. The second generation of meronts measured 6-8 microns and contained 10-20 merozoites 4-6 microns in length. Third generation mature meronts (8-10 microns) on DPI 9 contained 12-20 merozoites measuring 5-7 microns, which were more crescent-shaped and less blunt than the merozoites at 5 and 7 DPI. Merogony continued after formation of the gametes and the first fully developed macrogametes (10-14 microns), microgametes (9-12 microns), and oocysts were also seen at 9 DPI. The prepatent period was 8 or 9 days, but the patent period was not determined. In the present study E. spinosa infection did not produce overt clinical signs. Pathological changes consisted of an inflammatory infiltration in the lamina propria of the posterior jejunum, Peyer's patches activation and sporadic erosions scattered at the villous tips. No villous atrophy in association with a large number of endogenous stages was observed.     

Isospora spilogales n. sp. and I. sengeri n. sp. (Protozoa: Eimeriidae) from the Spotted Skunk, Spilogale putorius ambarvalis

SYNOPSIS. Two new species of Isospora are described from the feces of an eastern spotted skunk Spilogale putorius ambarvalis imported into Montana from Florida. The oocysts of I. spilogales n. sp. are 29 to 38 by 22 to 28 μ and contain 2 sporocysts measuring 17 to 22 by 13 to 16 μ; a few Caryospora -like oocysts containing a single sporocyst were considered abnormal I. spilogales oocysts. The oocysts of I. sengeri n. sp. are 16 to 23 by 12 to 18 μ and contain 2 sporocysts measuring 10 to 14 by 8 to 12 μ.     

Development of Caryospora simplex (Apicomplexa: Eimeriidae) from Sporozoites to Oocysts in Human Embryonic Lung Cell Culture1

Leighton tubes containing monolayers of human embryonic lung cells were inoculated with 70,000 or 30,000 sporozoites of the viperid coccidium Caryospora simplex and examined at 1, 2, 4, 6, 8, 10, 12, 14, 16, and 18 days post-inoculation (PI). By day 1 PI, sporozoites had penetrated cells and were within parasitophorous vacuoles. Most sporozoites became spherical and then underwent karyokinesis several times between days 2 and 6 PI. Mature Type I meronts were found on days 6–16 PI and contained 8 to 22 short, stout merozoites. Mature Type II meronts were present on days 10–18 PI and contained 8 to 22 long, slender merozoites. Developing gamonts (undifferentiated sexual stages) were observed on days 14 and 16 PI. Mature micro- and macrogametes and thin-walled unsporulated oocysts were present on days 16 and 18 PI. Attempts to sporulate oocysts in tissue culture medium or in a 2.5% (w/v) aqueous solution of K₂Cr₂O₇ at 25/°C and 37°C were unsuccessful; only a few oocysts developed to the contracted sporont stage. Four Swiss-Webster mice injected intraperitoneally with merozoites obtained from Leighton tubes on day 10 PI did not acquire infections. This is the second coccidium reported to complete its entire development, from sporozoite to oocyst, in cell culture.     

Biology and pathogenicity of Eimeria neodebliecki Vetterling, 1965 in experimentally infected pigs

The endogenous development and pathogenicity of Eimeria neodebliecki Vetterling, 1965 are described in weaned pigs inoculated with 250 000 oocysts. The endogenous stages developed within the apical cytoplasm of the enterocytes of the middle and posterior jejunum. The asexual development comprised two generations of meronts. Immature and mature meronts were found in groups up to five per host cell. The first fully developed macrogametes and mature microgamonts were seen at 9 days post-infection (DPI). The prepatent period was 10 days, and the patent period lasted 6–8 days. Sporulation of oocysts was completed within 12 days at 25°C, and 16 days at 20°C. E. neodebliecki infection produced clinical signs of coccidiosis in weaned pigs which developed frothy or mucoid diarrhea from 9 to 12 DPI. Pathological changes were situated in the second half of the small intestine, with the predilection for the posterior jejunum. At 9 and 10 DPI, macroscopically, ranged from catarrhal to focal, pseudomembranous inflammatory lesions. Histopathological and SEM examinations revealed moderate villous atrophy with focal epithelial erosions and fibrinonecrotic material at the villous tips. E. neodebliecki is pathogenic for pigs and can be associated with clinical manifestation of diarrhea, stunted growth and poor condition in pigs.