Oocyst Discharge Research Articles

Potential Development Ability of Residual Zoites, a Second-Generation Meront, Inducing Long-Term Infection by the Mouse Eimerian Parasite, Eimeria krijgsmanni.

Coccidiosis caused by eimerian parasites results in lethal watery or bloody diarrhea in hosts, and markedly impairs the growth of and feed utilization by host animals. We previously investigated detailed the life cycle of Eimeria krijgsmanni as a mouse eimerian parasite. Only second-generation meronts, as an asexual stage, were morphologically detected in the epithelium of the host cecum for at least 8 weeks after infection, even though oocyst shedding finished approximately 3 weeks after infection. The presence of zoites was of interest because infection by eimerian parasites is considered to be self-limited after their patent period. To clarify the significance of residual second-generation meronts in E. krijgsmanni infection, we performed infection experiments using immunocompetent mice under artificial immunosuppression and congenital immunodeficient mice. The number of oocysts discharged and the duration of oocyst discharge both increased in immunosuppressed mice. In immunodeficient mice, numerous oocysts were shed over a markedly longer period, and oocyst discharge did not finish until 56 days after inoculation. The present results suggest that the second-generation meronts survived in the epithelial cells of the cecum after the patent period, thereby contributing to extended infection as an asexual stage. The results obtained on E. krijgsmanni indicate that infections by Eimeria spp. are not self-limited and potentially continue for a long period of time.

In vivo Anticoccidial Efficacy of Calotropis procera Leaf Extract as an Alternative Treatment Against Eimeria papillate Infection in Mice

Background: Coccidiosis is still a serious parasite illness caused by Eimeria pps in many areas throughout the globe. As a consequence of eimeria medication resistance. Coccidiosis needs new drugs that contain natural antiparasitic chemical compounds. This study was conducted to investigate the in vivo anti-coccidia activity of Calotropis pocera leaf extract (CPLE) against infection of mice with Eimeria papillate. Methods: The first and second mouse groups were not infected, while the third, fourth, fifth and sixth were given 1x103 E. papillate sporulated oocysts. The fourth and fifth groups received 100 and 200 mg/kg CPLE orally and the sixth received toltrazuril 25 mg/kg in physiological saline. On day 5 p.i., animals were slaughtered and jejunum samples were prepared. Result: CPLE phytochemistry revealed 13 chemical compounds. 200 mg/kg was the most efficacious dose, reducing oocyst discharge in mouse faces by 68% and parasite stages in jejunal sections. Infection by E. papillata in mice causes inflammation, epithelial vacuolation, villi loss and a decrease in goblet cell density. When infected mice were given CPLE, histological damage was reduced by 69% and goblet cell number was restored to near-control levels. The infected group lost weight owing to emesis. However, the treated group CPLE gained weight.

Thyme essential oil for sheep: effect on rumen fermentation, nutrient digestibility, nitrogen metabolism, and growth

ABSTRACT Two experiments were conducted to evaluate the effect of thyme (Thymus vulgaris) essential oil (EO) doses on rumen fermentation, nutrient digestibility, and nitrogen metabolism, as well as performance and coccidia oocyst discharge. In experiment I, 20 rumen-cannulated wethers received the experimental diets containing 80% dry matter (DM) of haylage and 20% DM of concentrate. Treatments were 25mg of monensin/kg DM or doses of 1.25, 2.50, or 3.75g of thyme EO/kg DM. In experiment II, 50 ewe lambs received the same diets from experiment I, including a diet without feed additives. Wethers fed with diets containing 1.25g/kg DM of thyme EO had higher molar proportion of propionate (P= 0.03) and butyrate (P< 0.01), and lower (P= 0.04) acetate to propionate ratio than other treatments. Adding thyme EO to diets increased (P= 0.02) nitrogen retention compared to monensin. The performance of ewe lambs was not affected (P≥ 0.05) by treatments. However, lambs fed monensin had a lower (P= 0.04) number of coccidia oocyst discharge than others. Adding 1.25g/kg DM of thyme EO in high-forage diet improved ruminal fermentation. Thyme EO enhanced nitrogen metabolism, however, it did not improve performance.

Dynamics of Eimeria oocyst excretion in dairy calves in the Province of Buenos Aires (Argentina), during their first 2 months of age

Clinical coccidiosis is associated with high fecal contamination and stress situations, mainly in animals under 1 year of age. Artificially fed dairy calves are one of the categories most prone to suffer from this parasitic disease. The study was carried out in a commercial dairy farm. Feces samples of heifer calves between 2 and 8 weeks old were taken monthly for oocyst counts and Eimeria spp. identification. Of the 862 feces samples analyzed, 48% presented oocysts. When grouping the results of monthly samplings of each age group, it was observed that this percentage increased in the group of calves between 20 and 40 days of life, reaching the peak average of 85% of infection prevalence in the group with between 26 and 30 days of age. The discharge of oocysts observed between 21 and 35 days of age was superior to the rest ( p < 0.05). This trend appeared every month throughout the whole year. However, during March, April, June, September and November, the curves in the group categories were higher than in the remaining months ( p < 0.05). Twelve Eimeria species were identified, being E. ellipsoidalis, E. bovis, E. zuernii and E. auburnensis those in highest numbers. E. ellipsoidalis had an important predominance in the opg composition, >75% up to 25 days of life ( p < 0.05). E. bovis reached peak values in the 26 and 30 days group ( p < 0.05), remaining without significant variations in the last stage of the artificial milk feeding period (approximately 60 days of life), when the oocyst counts were significantly low. Oocysts of E. auburnensis appeared in great proportion (46%) in the cultures later than the previous species ( p < 0.05), in calves of the age groups of between 46 and 50 days of age. Oocysts of E. zuernii showed no trend associated with age. The highest prevalence of infection and of oocyst values appeared during the periods with better environmental conditions for sporulation, survival and dispersion of oocysts (spring and autumn), coincident with the highest birth rates and an elevated number of calves in the paddock. Calves developed a process of natural “vaccination” against coccidiosis. This was demonstrated by the decrease in the quantity of animals shedding oocysts and in the number of oocysts eliminated at the end of the artificial milk feeding period.

THE LIFE CYCLE AND PATHOGENICITY OF COCCICIUM EIMERIA NOCENS (KOTLÁN, 1933) IN DOMESTIC GOSLINGS

Twenty-four 10-day-old, artificially reared, coccidia-free goslings (Anser cygnoides var. domestica) were inoculated orally with 1.0x10(5)-1.0x10(6) sporulated oocysts of Eimeria nocens, and killed at intervals from 30 to 336 hr postinoculation (PI). Parts of the visceral organs, including intestines, kidney, liver, gallbladder, and spleen from inoculated goslings, were fixed and sectioned. The life cycle of E. nocens and histologic changes during infection were examined microscopically. The results showed that at least 3 generations of meronts developed in the endogenous stage of the life cycle of E. nocens. Two types of meronts were found. The first completed maturation at 54 to 78 hr PI. These meronts were the first generation, with each forming about 12 merozoites. The second completed maturation at 102 to 240 hr PI. These meronts were the second or third generations, with each meront forming about 24 merozoites. Development of gamonts began at about 198 hr after infection. The prepatent period was 9 days and discharge of oocysts continued for 4 days. Sporulation of oocysts occurred in 60-72 hr at 25 C. Eimeria nocens invaded the posterior jejunum, ileum, caecum, rectum, and cloaca. Developmental stages were localized within the epithelial cells of villi and crypts, and in lamina propria. Marked histological changes, including desquamation and necrosis of intestinal epithelium, submucosal edema, hemorrhages, infiltration of inflammatory cells, and villous atrophy, were seen during the periods of late merogony, gamogony, and oocyst shedding. They were most pronounced in the ileum and the regions nearby. The infected goslings showed severe diarrhea, bloody feces, anorexia, emaciation, and even death, suggesting that E. nocens is highly pathogenic for goslings.

Quantification of the infectivity of Cryptosporidium parvum by monitoring the oocyst discharge from SCID mice

Doses of 1–10 5 oocysts of Cryptosporidium parvum HNJ-1 were inoculated into severe combined immunodeficient (SCID) mice, and the discharge of oocysts was monitored for 30 days post inoculation. None of the mice discharged any oocysts after oral inoculation of one oocyst. Only one of five SCID mice discharged oocysts after oral inoculation of 10 oocysts, and the prepatent period was 17 days. The other four mice did not discharge any oocysts. All the SCID mice discharged oocysts after oral inoculation of 10 2–10 5 oocysts. The prepatent periods were 13–17, 8–10, 8, and 10 days in SCID mice inoculated with 10 2, 10 3, 10 4, and 10 5 oocysts, respectively. A proportional correlation was observed between inoculation doses of oocysts ranging from 10 to 10 4 oocysts and the corresponding prepatent periods. The prepatent period can be used to evaluate the infectivity of C. parvum oocysts.

The periodicity of isosporan oocyst discharge in the greenfinch (Carduelis chloris)

Over a five-month period 404 faecal samples from greenfinches (Carduelis chloris L.) from Corby, Northants were examined for coccidian oocysts. Of these, 101 were positive for isosporan oocysts. By recording the time that each sample was taken the daily periodicity of oocyst discharge was shown. Only one of the 133 samples was positive from 7 am to noon, whereas 100 of 271 (37%) were positive from noon to 6 pm, the peak being between 2 pm and 5 pm when 71.5% of the samples contained oocysts.

Outbreak of cryptosporidiosis after contamination of the public water supply in Saitama Prefecture, Japan, in 1996.

An outbreak of cryptosporidiosis occurred in Ogose Town, Saitama Prefecture. Japan, in June 1996. Of 12,345 respondents to a questionnaire sent to households in the town (population; 13,809), 8,812 (71.4%) reported an acute gastrointestinal illness some time between May and July. In addition, 274 traceable visitors at local inns, golf courses, and the like during this period and 54 employees from out of town were infected. Cases of cryptosporidiosis were estimated to 9,140. Of these, 2,856 subjects were treated at outpatient clinics and 24 subjects were hospitalized (some subjects counted twice). No deaths were attributed to the outbreak. Among the visitors to Ogose who were traced, 7 persons who stayed only one day during the outbreak and drank half a glass to 2 glasses (100 to 360 ml) of tap water had cryptosporidiosis confirmed by laboratory tests. The median incubation period for the 14 persons for whom this calculation was possible was 6.4 days (range, 5 to 8 days). Of 469 pupils reporting details of their fever and diarrhea, abdominal cramps, or these combined signs and symptoms, the median maximum body temperature was 37.8 degrees C (range, 36.7 to 40.3 degrees C). The duration of illness, reported by 608 of the pupils, was 5.2 days (range, 1 to 15 days), and that reported by 187 employees was 4.8 days (range, 1 to 18 days). The longest known time for discharge of oocysts after onset was 44 days. Blood was not found in the 609 stool specimens examined. The outbreak was caused by contamination of the town's potable water by Cryptosporidium parvum oocysts. The town's water treatment plant treated river water by coagulation, sedimentation, sand filtration, and chlorination. Contamination arose because of various natural and artificial factors: one was that the monthly precipitation in May was much lower than average, causing the river water level to drop. Another factor was heavy rainfall one night in May that increased water turbidity. The amounts of the coagulant added seemed to be insufficient. There are two inns, three public lavatories, and two small-scale wastewater treatment plants upstream 400 m and 1,200 m of the intake point of the town's water treatment plant. However, there are no farms with livestock in the area. We suggest that the location of the water treatment facilities was inappropriate, and that oocysts had circulated from the potable water to humans to sewage to the river and back to the potable water.

Immunomodulatory effects of oat beta-glucan administered intragastrically or parenterally on mice infected with Eimeria vermiformis.

The immunostimulatory effect of intragastrically or parenterally administered beta-(1-->3; 1-->4) glucan, extracted from oats (ObetaG), on disease resistance to Eimeria vermiformis was studied in C57BL/6 mice. Multiple administrations of ObetaG by intragastric or subcutaneous routes reduced fecal oocyst shedding compared to the non-treated control group. The administration of ObetaG by subcutaneous route resulted in higher levels of total serum immunoglobulins and antigen (sporozoite and merozoite)-specific immunoglobulins as compared with the non-treated group. To evaluate the effect of a single subcutaneous dose, groups of mice were treated with ObetaG 2 days before E. vermiformis infection, at the time of infection and at 2 or 6 days after infection. From day 11 post-infection the oocyst discharge was significantly diminished (P<0.05-0.01) in the ObetaG-treated groups, except in those treated 6 days after infection, as compared to the non-treated control group. The proliferative responses to E. vermiformis sporozoite antigen of lymphocytes isolated from the spleen were significantly increased (P<0.05) when ObetaG was administered 2 days before or at the time of E. vermiformis infection. Lymphocyte proliferative responses to merozoite antigen were not influenced by treatment. In conclusion, ObetaG appeared to up-regulate immune mechanisms and provide enhanced resistance against eimerian coccidiosis in mice.

Effects of toltrazuril on oocyst discharge and growth in kids with naturally-acquired coccidial infections

Effect of toltrazuril on Eimeria spp. oocyst output and on animal growth in 4–6 months old French Alpine kids was evaluated in two field trials. A single administration of 20 mg/kg BW of toltrazuril reduced total oocyst excretion and Eimeria ninakohlyakimovae oocyst excretion for 7 d. At 30 and 40 mg/kg BW of toltrazuril the reduction of oocyst excretion was observed for 14 and 21 days, respectively. No differences in weight gains were seen between control and treated groups (Trial 1). Compared with a sulfadimidine-diaveridine medication (67 mg/kg BW/d for 5 d), a single administration of toltrazuril at 40 mg/kg BW resulted in a persistent decrease of oocyst discharge during at least one week in Trial 2. Sulfadimidine-diaveridine plus toltrazuril treated animals gained more weight than did controls. At the three different doses used, toltrazuril was safe in kids.

Experimental Cryptosporidium parvum infection in a Korean native calf isolated from a Korean mouse

This study was performed to investigate experimental transmission of Cryptosporidium parvum in a calf. A 25-day-old Korean native calf was inoculated per os with 1 x 10(6) C. parvum oocysts isolated from a Korean mouse. The calf commenced oocyst discharge in feces on post-inoculation day 4, and continued until the day 11. The number of discharged oocysts peaked (4.9 x 10(5)) on post-inoculation day 6. However, the calf did not show signs of diarrhea. The present results indicate that C. parvum is cross-transmissible between the calf and the mouse.

Isolation and identification of Cryptosporidium from various animals in Korea. III. Identification of Cryptosporidium baileyi from Korean chicken

Each of SPF chicken (Hi-Line strain, 2-day-old males) was inoculated with 2.5 or 5 x 10(4) oocysts by stomach tube. The oocyst was the medium type of Cryptosporidium previously isolated from Korean chicken origin, and passed in 2-day-old SPF chicken. The patterns of oocyst discharge were monitored daily, and in order to observe the ultrastructure of the developmental stages, the bursa of Fabricius of the chicken was examined by transmission electron microscopy (TEM) on the 12th day postinoculation. The prepatent period for 8 chicken was 5.9 days postinoculation on the average, and the patent period was 12.9 days. The number of oocysts discharged per day for the chicken was reached peak on day 12 postinoculation on the average. A large number of oocysts was found in fecal samples obtained from inoculated chicken on days 8-14 postinoculation. The ultrastructural feature of almost every developmental stage of the medium type from chicken was very similar to that of Cryptosporidium previously isolated from mammalia including human and birds except for the attachment site of C. muris to the mucus cell from mammalia, but dimension of the oocysts from fecal samples of the medium type was different from those of C. meleagridis and mammalia origin. The above results reveal that the medium type of Cryptosporidium of Korean chicken origin is identified as Cryptosporidium baileyi.

Isolation and identification of Cryptosporidium from various animals in Korea. II. Identification of Cryptosporidium muris from mice

Each of SPF mice(Scl: ICR strain, 3-week-old males) was inoculated with 5 x 10(4) oocysts of Cryptosporidium by stomach tube. The oocysts were large type one which was previously isolated from Korean mice, and passaged in 3-week-old SPF mice. The patterns of oocyst discharge were monitored daily, and in order to observe the ultrastructure of developmental stages the stomach of the mice was examined by transmission electron microscopy (TEM) at 4 weeks post-inoculation. The prepatent period for 6 mice was 5.6 days post-inoculation on the average, and the patent period was 63.2 days. The number of oocysts discharged per day from the mice reached peak on day 36.6 post-inoculation on the average. A large number of oocysts were found in fecal samples obtained from inoculated mice on days 30-50 post-inoculation. C. muris was larger than C. parvum at almost every developmental stages, the size difference being 1.4 times in oocysts, 2.4 times in sporozoites, 1.6 times in merozoites, and 1.5 times in microgametes. The ultrastructural features of the attachment site of C. muris to the mucus cells were remarkably different from those of C. parvum and its closely related species. The anterior projection of the protozoa (C. muris), the outer aspect of which was surrounded by a thick filamentous process of the host cell, has not been reported at any developmental stages of C. parvum or its closely related species. The size of the oocysts of strain RN 66 was larger than that of Korean mice origin. The above results reveal that the large type Cryptosporidium of Korean mice origin is identified as Cryptosporidium muris and this type was named as C. muris (strain MCR).

Prepatent and Patent Periods, and Production and Sporulation of Oocysts of Eimeria subspherica Isolated in Japan.

Eight calves were inoculated with 10(6) or 10(7) sporulated oocysts of Eimeria subspherica, Christensen, 1941. All the calves, except one, were successfully infected. Prepatent period was 10 to 11 days with a mean of 10.6 days and patent period was 1 to 5 days with a mean of 2.9 days. Peak oocyst discharge was found on days 11 and 12 after inoculation. The number of oocysts passed in the feces was very small and varied among calves, and no relationship was detected between the inoculum dose and the number of oocysts passed in the feces. No clinical symptoms were associated with the infection in any calves. Infectivity, reproductive potential and pathogenicity of E. subspherica were suggested to be low in the experimentally infected calves, even though relatively large numbers of oocysts were given. The sporulation process of E. subspherica oocysts was observed under a condition of 29 degrees C with constant aeration. Completely sporulated oocysts increased in percentage following a sigmoid curve, and the expected 50% and 95% sporulation times were determined as 161 and 194 hours, respectively, by regression analysis.

Efficacy of narasin against hepatic and intestinal coccidiosis in rabbits.

The anticoccidial effect of narasin against Eimeria flavescens, E. intestinalis, E. magna, E. perforans and E. stiedai was tested in experimentally infected rabbits. The drug was highly effective in reducing oocyst output and in preventing clinical signs. Maximum activity as measured by oocyst discharge was achieved with feed levels from 12 to 24 p.p.m.; there was no influence on oocyst sporulation. Optimal weight gain and food consumption were obtained with levels between 8 and 12 p.p.m.; at higher levels there was an adverse effect on growth, food intake, relative liver and heart weights and on serum potassium and glucose.

Effects of Acquired Resistance on Infection with Eimeria falciformis var. pragensis in Mice

Mice immunized with infections of 500, 5,000, or 20,000 oocysts of E. falciformis var. pragensis were reinfected with 20,000 and 100,000 oocysts at 20 and 38 days, respectively, after the initial infection. After the first challenge infection, none of the immunized mice showed clinical signs of coccidiosis; a few mice passed very low numbers of oocysts, and oocyst discharge seemed to correlate negatively with immunizing dose. None of the mice immunized twice passed oocysts after challenge. Mice immunized with three infections were completely immune to challenge for 4 months. The effect of the immune response on the life cycle of the coccidium was determined by histological examination of the intestines of immune and nonimmune mice infected with the parasite. In both the immune and nonimmune groups, sporozoites penetrated absorptive epithelial cells and migrated to crypt epithelial cells during the first 6 to 24 h postinfection. At 48 to 72 h postinfection, the sporozoites developed into mature first-generation schizonts in the nonimmune mice, whereas the developing first-generation schizonts degenerated within the crypt epithelial cells of the immune mice. In nonimmune mice, third-generation merozoites, inoculated intracecally, developed into mature fourth-generation schizonts, whereas in immune mice the developing fourth-generation schizonts degenerated before maturing. The possibility that a cellmediated immune mechanism is responsible for the arrest in schizogony is discussed.

The effect of Eimeria neischulzi infection on leukocyte levels in the rat.

Rats inoculated with 10,000 sporulated oocysts of Eimeria neischulzi had significantly higher total leukocyte counts on postinoculation days (PI) 1, 5, 6 and 7 when compared to control rats. Relative and absolute neutrophil counts increased concomitantly with a decrease in the relative lymphocyte levels in E. nieschulzi-infected rats on PI day 7. Absolute and relative neutrophil counts in infected rats on PI days 7 and 8 were closely correlated with the host's total oocyst discharge. The E. nieschulzi infection had no significant effect on the relative or absolute levels of monocytes or eosinophils. The described changes in leukocyte levels were not paralleled by a significant change in the erythrocyte count.

The Immunizing Potential of Sporulated Oocysts of Eimeria nieschulzi Exposed to Heat and CO-60 Gamma-Radiation

Sporulated oocysts of Eimeria nieschulzi Dieben 1924, a rat coccidium, were exposed to radiation, heat, or both in an effort to attenuate the parasite. Moderate levels of each treatment or combination thereof attenuated the parasite, reduced pathogenesis (as judged by oocyst discharge during primary infection), and produced immunity to challenge when the oocysts were subsequently inoculated into rats. Thus, heat- and/or radiation-treated E. nieschulzi oocysts fed to rats could reduce pathogenesis during a primary infection and yet give good homologous protection.

Life cycles of two isopora species in the canary, Serinus canarius Linnaeus.

The endogenous stages of Isospora serini Arogão and Isospora canaria Box are described from experimentally infected canaries, Serinus canarius Linnaeus. Unlike other Coccidia, the first part of the I. serini life cycle takes place in mononuclear phagocytes. Five asexual generations are described from this cell type; 2 additional asexual generations and the sexual stages take place in the intestinal epithelium. Isospora canaria, on the other hand, has a conventional coccidian life cycle in that all of the endogenous stages are in the epithelium of the small intestine, with 3 asexual generations and the sexual generation described in the duodenal epithelium. The 2 species differ in their position relative to the nucleus of the intestinal epithelial cell. Isospora serini is usually on the lumenal side of the nucleus while I. canaria is below the nucleus, toward the basement membrane. The prepatent period is 4-5 days for I. canaria and 9-10 days for I. serini. Patency lasts for 11-13 days in I. canaria infections, but duration of oocyst output is more chronic in I. serini infections, persisting for as long as 231 days. Both species have a diurnal periodicity of oocyst discharge which occurs in late afternoon and evening.

The prepatent and patent periods of Eimeria alabamensis and further description of the exogenous stages

Eimeria alabamensis infections were established in calves 5 to 6 weeks of age by adminestering 10 million, 80 million, or 100 million sporulated oocysts. The prepatent period was 6 to 8 days (mean 6.6). Oocyst discharges usually lasted for 2 to 3 days although a few calves passed oocysts throughout the rest of the 3-week observation period. Calves with oocyst discharge exceeding 1 million oocysts per g of feces had a moderate diarrhea at the time of peak oocyst discharge. No other clinical signs were observed in any of the infected calves. Reinoculations with 100 million sporulated oocysts given 3 weeks after the initial inoculations of 10 million or 80 million oocysts resulted in infections characterized by greatly reduced oocyst discharges. Sporulated oocysts of E. alabamensis were 16 to 24 μ by 12 to 16 μ and were usually ovoid. The oocyst wals consisted of two layers. Sporocysts were elongate-ellipsoid, had a distinct Stieda body, and were 10 to 12 μ by 4 to 6 μ. Completely sporulated oocysts were first observed after 5 days at 25 °C, and most were sporulated after 8 days. Oocysts did not sporulate at 4 °C, 33 °C, and 37 °C.