Growth of and competition between Neospora caninum and Toxoplasma gondii in vitro.

Competitive interactions between Neospora caninum and Toxoplasma gondii were studied because both species appear to have identical ecological niches in vitro. Tachyzoites of N. caninum (NC-1 isolate) and T. gondii (RH isolate) were compared in three in vitro studies: (1) rate of penetration of host cells; (2) generation time; and (3) competition between the two species when grown together in the same flask and allowed to compete for space. When tachyzoites of the two species were inoculated onto human foreskin fibroblasts, 3.24-times more N. caninum tachyzoites penetrated cells by 1 h p.i. At 3 h p.i., there were 2.87-times more N. caninum intracellular tachyzoites than T. gondii tachyzoites. The generation times for N. caninum (NC-1 isolate) and T. gondii (RH isolate) were approximately 14-15 h and 8-10 h, respectively. Before exponential growth occurred, both species displayed a lag period, which was 10-12 h for N. caninum and 8-10 h for T. gondii. To observe competition, equal numbers of tachyzoites of each species were mixed and inoculated into flasks of host cells, and the monolayers were allowed to proceed to >90% lysis before the next transfer. Competition was analysed for 31 days by labelling samples of each flask with a species-specific monoclonal antibody and determining the ratio of each species. In all trials, T. gondii outcompeted N. caninum. By 4 days p.i., 70% of the tachyzoites were T. gondii; this percentage increased to 97% by 23 days p.i. When the starting inoculum contained 75% N. caninum and 25% T. gondii tachyzoites, T. gondii was still competitively superior. When infected monolayers that were labelled with T. gondii-specific antibodies were examined, it was noted that both species can occupy and undergo endodyogeny in the same host simultaneously.

Effect of long-term insulin exposure on insulin binding in Tetrahymena pyriformis.

Previous studies have indicated that Tetrahymena pyriformis can bind the vertebrate hormone insulin. Conventional microscopic studies were conducted to determine the effect of acute and long-term (LTE) insulin exposure on insulin binding. Stock cultures included cells never exposed to insulin and cultures grown in medium containing 6 mg/ml insulin. Logarithmic cultures were exposed to porcine insulin concentrations of 0 and 6 mg/ml for 1 h (insulin treated, (IT)) after 0, 48 h, 1, 3, or 6 months of LTE insulin exposure. 24 h after the 1 h insulin treatment, the cells were fixed, exposed to porcine insulin (antigen), processed immunocytochemically using a primary antibody to porcine insulin and a secondary antibody immunocytochemistry kit, and examined for staining intensity by video image analysis. Morphological observations confirm that T. pyriformis does bind insulin whether or not the cells have had prior exposure to insulin. IT increases insulin binding (up-regulation) in previously unexposed cells (control, P < 0.01) and produces a further amplification in cells having prior acute exposure (48 h) to insulin (P < 0.01). However, LTE exposure to insulin (1, 3 and 6 months) caused a decrease in insulin binding (down-regulation) after IT (P < 0.01) such that LTE-IT cells were not different from control cells following 1, 3 or 6 months of chronic insulin exposure to insulin. Staining intensity was not different between IT cells and cells cultured with insulin throughout the six month study. Results suggest that insulin binding sites of T. pyriformis are subject to regulatory processes similar to those of metazoans.

Intracellular insulin binding in Tetrahymena pyriformis.

Insulin, a classic vertebrate hormone, produces alterations in cellular metabolism and growth in the ciliate Tetrahymena pyriformis, as well as an increase in insulin binding upon subsequent exposure, a phenomenon known as hormonal imprinting. An antibody to a peptide corresponding to the alpha-subunit of the human insulin receptor (amino acid residues 657-670) was used to investigate the location and to partially characterize immunoreactive proteins in insulin-exposed and non-insulin-exposed cells (control). Confocal microscopy revealed immunofluorescent labeling of cilia, nuclei, vesicles and an oblong structure of unknown nature. Labeling of nuclei, mitochondria and ciliary microtubules was seen with immunoelectron microscopy. Labeling was absent on the cell and ciliary membranes by immunoelectron microscopy. Polyacrylamide gel electrophoresis revealed several differences in protein composition between control and insulin-exposed ciliary membrane extracts, especially in the 30-50 kDa range. Immunoblotting revealed 2 reactive proteins in whole cell lysates but none were detected in ciliary membrane extracts or wheat germ agglutinin affinity column eluates of T. pyriformis whole cell preparations. Based on these findings it is unlikely that a cell surface structure similar to a mammalian insulin receptor exists in T. pyriformis.

Experimental modes of Caryospora bigenetica (Apicomplexa: Eimeriidae) infection in swine and the effects of temperature and salinity on parasite infectivity in porcine tissue.

Four crossbred pigs (Sus scrofa) were inoculated orally with Caryospora bigenetica oocysts derived from snake and mouse feces, and with C. bigenetica infected mouse tissue. One pig also was given i.m. injections of methylprednisolone acetate. All four pigs displayed clinical signs including erythema, edema, and lethargy. Caryocysts were observed histologically in numerous tissues including ear, tongue, jowl, shoulder, loin, intercostal, ham, hock, and feet. The four pigs each were butchered into six commercial cuts: shoulder, loin, side, ham, hock, and feet. Raw 10 g samples from each cut were bioassayed by pepsin digestion and s.c. inoculation into 12 Swiss-Webster mice (Mus musculus) and 12 cotton rats (Sigmodon hispidus). Seventeen of 24 mice and cotton rats exhibited clinical signs and C. bigenetica tissue infections. Remaining portions of the six commercial cuts were temperature or saline treated, and 10 g samples were bioassayed in 16 mice and 12 cotton rats. No clinical sign or tissue infection was observed in these animals. Our study presents evidence that swine can be infected with C. bigenetica by ingesting oocysts present in snake feces or mouse feces (following inoculation of mice with snake-derived oocysts) or by ingesting C. bigenetica infected rodent tissue, that endogenously produced C. bigenetica oocysts are not excreted in the feces of swine, and that C. bigenetica in pork can be rendered noninfective by freezing at -20 degrees C (-4 degrees F) for 21 days, frying at 84 degrees C (183 degrees F) for 17 min, microwaving at 88 degrees C (190 degrees F) for 17 min, grilling at 82 degrees C (180 degrees F) for 48 min, baking at 95 degrees C (203 degrees F) for 230 min, boiling at 100 degrees C (212 degrees F) for 60 min, or by curing at 4 degrees C (39 degrees F) for 20 days.

Experimental infections in domestic ducks with Cryptosporidium baileyi isolated from chickens.

Oral inoculation of 13 ducks (Anas platyrhynchos) with 1 x 10(6) Cryptosporidium baileyi oocysts produced patent infections but no clinical signs of disease. Intratracheal inoculation of 13 ducks with 1 x 10(6) C. baileyi oocysts produced only mild clinical signs of respiratory disease, no deaths, and gross lesions of airsacculitis in only three ducks. The distribution of developmental stages of C. baileyi in ducks was similar to that observed in experimentally infected chickens and turkeys. Results of this study indicate that ducks are more resistant to experimentally induced respiratory cryptosporidiosis caused by C. baileyi than are chickens and turkeys.

Cryptosporidium baileyi: effects of intra-abdominal and intravenous inoculation of oocysts on infectivity and site of development in broiler chickens.

Developmental stages of Cryptosporidium baileyi were observed on the epithelium of the larynx, trachea, primary and secondary bronchi, air sacs, bursa of Fabricius, and cloaca of 12 chickens inoculated intra-abdominally with oocysts. All 12 birds inoculated intra-abdominally developed clinical signs of respiratory disease and had gross lesions of airsacculitis at necropsy. Developmental stages of C. baileyi and clinical signs of disease were not observed in 12 chickens inoculated intravenously with oocysts. The response of chickens to intra-abdominal inoculation of oocysts was similar to responses recorded following intratracheal inoculation of oocysts in previous studies.

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.

Cultivation of Cryptosporidium baileyi: studies with cell cultures, avian embryos, and pathogenicity of chicken embryo-passaged oocysts.

Sporozoites of Cryptosporidium baileyi did not undergo development in primary cell cultures from either avian or mammalian hosts, or in mammalian cell lines. Oocysts of C. baileyi produced infections resulting in complete development to sporulated oocysts in chicken embryos and embryos of 8 other avian species examined. Inoculation of 4 X 10(5) oocysts was not pathogenic for avian embryos as evidenced by the lack of gross lesions or death. Oocysts obtained after C. baileyi had been passaged 10 times (first experiment) or 20 times (second experiment) in chicken embryos still caused clinical respiratory disease and gross airsacculitis when inoculated intratracheally into 2-day-old broiler chickens. Oocysts that had been passaged 10 times in chicken embryos were similarly pathogenic for 4-day-old turkeys after intratracheal inoculation.

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.

Host specificity of Cryptosporidium sp. isolated from chickens.

The host specificity of Cryptosporidium sp. infecting chickens was evaluated by oral inoculation of oocysts into 6 different species of neonatal rodents, adult nude mice (athymic), neonatal conventional and gnotobiotic pigs, turkeys, muscovy ducks and bobwhite quail. Examinations of tissue sections, ileal mucosal smears, fecal flotations and stained feces failed to reveal any infections in the mammalian species examined. Oocysts were observed in the feces, and developmental stages were observed in tissue sections, of turkeys and muscovy ducks but not bobwhite quail. This study indicates that Cryptosporidium sp. infections in avian species are probably not a zoonotic threat to humans.

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.

Immunohistochemical diagnosis of Toxoplasma gondii: potential for cross-reactivity with Neospora caninum.

A monoclonal antibody (MAB 3F5) was compared with a commercial polyclonal antibody for specificity for Toxoplasma gondii and cross-reactivity with Neospora caninum in paraffin-embedded tissue sections. Both antibody preparations reliably recognized T. gondii tachyzoites (RH isolate) in animal tissues but did not always label tissue cysts (ME-49 isolate). The commercial antibody strongly cross-reacted with N. caninum tachyzoites, but MAB 3F5 did not when the immunoperoxidase, immunofluorescence, or immunogold procedures were employed, nor did it cross-react with other apicomplexans, e.g., Isospora suis, Eimeria bovis. Sarcocystis cruzi, Hammondia hammondi, or Caryospora bigenetica. Immumoelectron microscopy revealed that MAB 3F5 bound to dense granules in extracellular T. gondii tachyzoites. In western blots of T. gondii tachyzoites, a major band at 38 kDa and a minor band at 32 kDa were labeled by MAB 3F5, but no labeling of the proteins of N. caninum tachyzoites or uninfected host cells occurred at the ultrastructural level or in immunoblots.

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.

Molecular phylogeny of the other tissue coccidia: Lankesterella and Caryospora.

Nearly complete sequences were obtained from the 18S rDNA genes of Eimeria falciformis (the type species of the genus), Caryospora bigenetica, and Lankesterella minima. Two clones of the rDNA gene from C. higenetica varied slightly in primary structure. Parsimony-based and maximum likelihood phylogenetic reconstructions with a number of other apicomplexan taxa support 2 major clades within the Eucoccidiorida, i.e., the isosporoid coccidia (consisting of Toxoplasma, Neospora, Isospora [in part], and Sarcocystis spp.) and a second clade containing Lankesterella and Caryospora spp., as well as the eimeriid coccidia (Cyclospora, Isospora [in part], and Eimeria spp.). Our observations suggest that Caryospora spp. may not belong in the family Eimeriidae but rather may be allied with the family Lankesterellidae with which they share molecular and life history similarities. This may be a third lineage of coccidian parasites that has independently evolved a unique heteroxenous transmission strategy.

Caryospora-associated dermatitis in dogs.

Numerous developmental stages of Caryospora sp. were seen in skin and lymph node of a 2-mo-old-dog with severe pyogranulomatous dermatitis. Schizonts were up to 20 microns long and contained up to 25 merozoites. Gamonts, unsporulated oocytes, sporulated oocysts, and caryocysts were seen in macrophages, connective tissue cells, and in unidentifiable host cells. Caryocysts were up to 18 microns long, and had thin walls, and contained a host cell nucleus and 1-3 sporozoites. All developmental stages of the coccidium reacted positively with anti-Caryospora bigenetica serum when examined in an immunoperoxidase test.

Doublet cells in Tetrahymena as indicators of culture media composition.

Stomatogenesis in ciliates is a complex and carefully orchestrated event. The exo- mutant SB255 of Tetrahymena thermophila has defects in mucocyst formation and docking and can also have one or two mouths. Three common culture media (proteose peptone, Medium 357, and yeast extract) were analyzed for total C, N, and inorganic elements and then tested for their effect on the number of mouths present in SB255. Cultures of SB255 grown in Medium 357 consisted of a mixed population of cells with either two mouths (doublet) or one mouth. Cultures from the same original stock grown in Medium 357 (SBm) and in 1% proteose peptone (SBpp) had different percentages of doublet cells in 1-, 2-, 3-, and 4-d-old cultures. When transferred to and grown in 1% yeast medium, both SBpp and SBm cultures had increased percentages of doublets over a 4-d culture period. When grown in 0.1, 0.5, or 1% yeast medium for 2 d, both SBpp and SBm cultures had more doublets in 1% than in either 0.1 or 0.5% yeast medium. Cultures of SBm grown in Medium 357 or 1% yeast medium for 2 d had a 10-fold increase in doublet cells compared to the inoculum. After 2 d in 1% proteose peptone, SBm cultures had percentages of doublet cells almost equal to that of the inoculum. Immunofluorescence and scanning electron microscopy (SEM) were used to examine cellular morphology of the doublet cells. These findings suggest that enriched media promote the growth of doublet cells. Furthermore, these doublets could prove to be a useful model system for the study of biological roles of trace elements.

Effect of broiler chicken age on susceptibility to experimentally induced Cryptosporidium baileyi infection.

Clinical signs of respiratory tract disease were observed in chickens that were inoculated intratracheally with 1 x 10(6) oocysts of Cryptosporidium baileyi at 2 or 14 days of age (10 chickens/group), but not in chickens inoculated at 28 or 42 days of age (10 chickens/group). Orally inoculated chickens in all age groups (10 chickens/group) did not develop clinical signs of disease. Orally and intratracheally inoculated chickens in all age groups were infected, as determined by the finding of cryptosporidia in tissue sections of the trachea, bursa of Fabricius, and cloaca, and by the recovery of oocysts from their feces. Chickens inoculated at 2 and 14 days of age excreted oocysts for a longer period and had greater numbers of cryptosporidia in their tissues, compared with chickens inoculated at 28 and 42 days of age.

Experimental Cryptosporidium infections in chickens: oocyst structure and tissue specificity.

Oocysts of an avian isolate of Cryptosporidium were used to inoculate 21 chicks orally and 7 chicks intratracheally to determine the tissue specificity of this organism. Oocysts were passed in the feces 4 to 5 days after inoculation. Oocysts (6.8 by 5.0 microns) were fully sporulated and they were passed for at least 17 days by infected chicks. The mode of inoculation did not influence the distribution of cryptosporidia within the digestive tract. Cryptosporidia were found in the cloaca (100%), bursa of Fabricius (95.7%), terminal portion of the colon (26.1%), and cecum (4.3%) of chicks that were positive for developmental stages. Of 21 chicks inoculated orally, 4 had cryptosporidia in their trachea, whereas 6 of 7 chicks inoculated intratracheally had cryptosporidia in the trachea, bronchi, and air sacs. Cryptosporidium was found in the ducts of the salivary glands and nasal turbinates of chicks inoculated intratracheally that had clinical signs of respiratory tract disease. None of the chicks died or had intestinal disease.

Chemoprophylaxis of cryptosporidiosis in chickens, using halofuginone, salinomycin, lasalocid, or monensin.

Chemoprophylaxis of Cryptosporidium baileyi infections was attempted by feeding 4 groups of chicks diets containing 3 mg of halofuginone/kg of feed, 60 mg of salinomycin/kg, 75 mg of lasalocid/kg, or 110 mg of monensin/kg. Rations were fed 5 days before oral or intratracheal inoculation with oocysts and were continued for 20 days. None of the drugs prevented C baileyi infections. Clinical signs of respiratory tract disease and gross lesions of airsacculitis were observed in intratracheally inoculated birds in all treatment groups and nonmedicated controls. Orally inoculated birds did not develop clinical signs of infection. Pathogenic bacteria were not isolated from the respiratory tract systems of any chicks. Halofuginone delayed the establishment of infections of the bursa of Fabricius and cloaca, but not of the trachea.