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).     

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.     

Viability and infectivity of Cryptosporidium parvum oocysts are retained upon intestinal passage through a refractory avian host.

Six Cryptosporidium-free Peking ducks (Anas platyrhynchos) were each orally inoculated with 2.0 x 10(6) Cryptosporidium parvum oocysts infectious to neonatal BALB/c mice. Histological examination of the stomachs jejunums, ilea, ceca, cloacae, larynges, tracheae, and lungs of the ducks euthanized on day 7 postinoculation (p.i.) revealed no life-cycle stages of C. parvum. However, inoculum-derived oocysts extracted from duck feces established severe infection in eight neonatal BALB/c mice (inoculum dose, 2.5 x 10(5) per mouse). On the basis of acid-fast stained direct wet smears, 73% of the oocysts in duck feces were intact (27% were oocyst shells), and their morphological features conformed to those of viable and infectious oocysts of the original inoculum. The fluorescence scores of the inoculated oocysts, obtained by use of the MERIFLUOR test, were identical to those obtained for the feces-recovered oocysts (the majority were 3+ to 4+). The dynamics of oocyst shedding showed that the birds released a significantly higher number of intact oocysts than the oocyst shells (P < 0.01). The number of intact oocysts shed (87%) during the first 2 days p.i. was significantly higher than the number shed during the remaining 5 days p.i. (P < 0.01) and significantly decreased from day 1 to day 2 p.i. (P < 0.01). The number of oocyst shells shed during 7 days p.i. did not vary significantly (P > 0.05). The retention of infectivity of C. parvum oocysts after intestinal passage through an aquatic bird has serious epidemiological and epizootiological implications. Waterfowl may serve as mechanical vectors for the waterborne oocysts and may enhance contamination of surface waters with C. parvum. As the concentration of Cryptosporidium oocysts in source waters is attributable to watershed management practices, the watershed protection program should consider waterfowl as a potential factor enhancing contamination of the source water with C. parvum.     

Azithromycin: antimalarial profile against blood- and sporozoite-induced infections in mice and monkeys

The spectrum of antimalarial activity of the new macrolide antibiotic azithromycin was evaluated against blood- and sporozoite-induced infections with a chloroquine-resistant strain of Plasmodium yoelii nigeriensis (N-67) in Swiss mice and with simian parasite Plasmodium cynomolgi B in rhesus monkeys. Against experimental rodent malaria, a 70 mg/kg/day dose showed curative blood-schizontocidal activity in a four-dose regimen administered orally from day 0 to day 3 or from day 2 to day 5 to mice harboring established infection. The curative response was also obtained with a 40 mg/kg/day dose administered in an extended seven-dose (days 0-6) regimen. Azithromycin was also effective in the causal prophylactic test, since a 50 mg/kg dose from day -1 to day +2 protected mice against P. y. nigeriensis (N-67) sporozoite challenge. In comparison, erythromycin did not show either of the above activities up to a 405 mg/kg/day dose in identical regimens. Comparison of the ED(90) values showed that azithromycin was 31-fold more effective than erythromycin as a blood schizontocide. In the simian model, trophozoite-induced infections of P. cynomolgi B were cured with 25 mg/kg/day azithromycin administered for 7 days. In the causal prophylactic test, the prepatent period was significantly extended in monkeys challenged with P. cynomolgi B sporozoites, presumably because of the growth inhibition of preerythrocytic schizonts in hepatocytes. Azithromycin did not exhibit any hypnozoitocidal (dormant exoerythrocytic stages) activity at 25 mg/kg/day in a seven-dose regimen.     

Development of patent infection in immunosuppressed C57Bl/6 mice with a single Cryptosporidium meleagridis oocyst

The ability of Cryptosporidium meleagridis to produce patent infection was studied in adult C57BL/6 mice that were immunosuppressed with dexamethasone phosphate provided in the drinking water at a dosage of 16 microg/ml. Four days after the onset of immunosuppression, mice were orally challenged with 1, 3, 10, or 1,000 C. meleagridis TU1867 oocysts per mouse. The mice were monitored daily for 18 days postinoculation for oocyst shedding. Five of 10 mice given a single oocyst, 4 of 5 mice given 3 oocysts, and all 9 mice given either 10 or 1,000 oocysts became infected and began shedding oocysts 5-7 days after challenge and continued to shed oocysts until the end of the experiment on day 18 postchallenge. Approximately 10(7) oocysts per mouse per day were excreted, regardless of the challenge dose. Neither the noninfected, immunosuppressed nor the inoculated, nonimmunosuppressed control mice shed oocysts. The excreted oocysts were confirmed to be those of C. meleagridis by polymerase chain reaction-restriction fragment length polymorphism analysis. We show that C. meleagridis, originally classified as an avian pathogen but recently found in humans with cryptosporidiosis, can produce patent infection in mice infected with a single oocyst. Moreover, we demonstrate that the immunosuppressed C57BL/6 adult mouse is an ideal host for the propagation of clonal populations of C. meleagridis isolates for laboratory studies.     

Removal of Toxoplasma gondii Oocysts from Sea Water by Eastern Oysters (Crassostrea virginica)

SUMMARY. Toxoplasma gondii infections have been reported in a number of marine mammals. Presently it is not known how these animals acquire T. gondii from their aquatic environment. The eastern oyster, Crassostrea virginica , has been shown to remove Cryptosporidium oocysts from seawater and a similar phenomenon may be occurring with T. gondii oocysts and marine invertebrates. The present study was done to determine if eastern oysters could remove and retain T. gondii oocysts from seawater. Oocysts of the VEG strain of T. gondii (1 × 10⁶ oocysts) were placed in seawater (32 ppt NaCl) containing live eastern oysters. The infected seawater was removed one day postinoculation (PI) and replaced with fresh seawater. Selected oysters were removed at 1, 3 and 6 days PL Hemolymph, gill washes, and oyster tissue were collected separately at each observation time. The oyster tissue was homogenized and all 3 samples fed separately to mice. Toxoplasma gondii positive mice were observed at each time period. The results indicate that T. gondii oocysts can be removed from seawater by eastern oysters and retain their infectivity. Contaminated raw oysters may serve as a source of T. gondii infection for marine mammals and humans.     

Plasmodium yoelii: activity of azithromycin in combination with pyrimethamine or sulfadoxine against blood and sporozoite induced infections in Swiss mice

The efficacy of pyrimethamine or sulfadoxine administered in combination with azithromycin was examined in a rodent malaria model. Outbred Swiss mice infected with blood stage parasites were treated from day 0 to day 3 and efficacy of different regimens was monitored in terms of the curative response and the delay time to reach 2% parasitaemia (2% DT). Administration of azithromycin alone at 60 mg/kg/day produced curative response while lower doses showed marginally delayed 2% DT. A marked potentiation in activities of pyrimethamine (100-fold) or sulfadoxine (10-fold) was observed when administered at non-curative doses of 0.1 mg/kg/day in combination with azithromycin (30 mg/kg/day) against blood stage parasites. A combination of 10 mg/kg/day azithromycin with 0.3 mg/kg/day sulfadoxine was also curative. Likewise in the causal prophylactic test, a combination regimen comprising 1/16th and 1/3rd the individual curative doses of pyrimethamine and azithromycin, respectively, prevented the development of patent infection after Plasmodium yoelii sporozoite challenge. Our results suggest that a combination of azithromycin with the second line treatment regimen of fansidar may enhance the therapeutic efficacy of the latter and also provide better prophylaxis against Plasmodium falciparum malaria.     

A comparison of the effects of two dinitroanilines against Cryptosporidium parvum in vitro and in vivo in neonatal mice and rats

The effects of two dinitroanilines, oryzalin and trifluralin, were compared against Cryptosporidium parvum, in vitro using HCT-8 cells and in vivo using neonatal Swiss ARC mice and Wistar neonatal rats. In vitro, oryzalin and trifluralin exhibited IC(50) values (concentration necessary to cause a 50% inhibition) of 750 and 800 nM, respectively. A viability assay showed that neither compound produced a cytotoxic effect on the host cells at concentrations as high as 1 microM. The in vivo component of this study consisted of inoculation of neonatal mice and neonatal rats with 10(5) viable oocysts of C. parvum per animal and the subsequent treatment of this infection with trifluralin and oryzalin administered via gastric intubation. At doses of 100 mg kg(-1) body weight administered twice daily for 3 consecutive days, trifluralin had no statistically significant effect on the number of oocysts recovered from the gut of either rats or mice compared with controls, whereas at the same concentration, oryzalin caused 90 and 79% inhibition of oocysts recovered from mice and rats, respectively.     

Infection of immunosuppressed C57BL/6N adult mice with a single oocyst of Cryptosporidium parvum

The present study was designed to determine the minimum number of Cryptosporidium parvum oocysts capable of producing patent infections in immunosuppressed C57BL/6N adult mice. Sixty-four female mice were divided into 6 groups of 8 mice each, except group 1 that contained 24 mice. Mice in groups 1-3 were immunosuppressed with dexamethasone and inoculated with 1, 5, and 10 oocysts per mouse, respectively. The accuracy of the inoculum size was microscopically confirmed. Mice in groups 4-6 served as controls: they received either only oocyst inoculation (group 4), or immunosuppression (group 5), or no treatments (group 6). Fecal oocyst shedding was monitored daily for each mouse using an indirect immunofluorescent assay. Parasite colonization in the terminal ileum of each mouse was evaluated histologically. Four of 24 mice in group 1 developed patent infections, with a prepatent period of approximately 6 days. All mice in groups 2 and 3 developed patent infections, with prepatent periods ranging from 4 to 7 days. Mice in groups 4-6 remained uninfected. Parasite colonization was observed in the terminal ilea of all mice in groups 1-3 that shed fecal oocysts. The present study experimentally demonstrates that a single viable oocyst can induce patent C. parvum infections in immunosuppressed C57BL/6N adult mice and indicates that this mouse model could be used for the parasite genotype or isolate cloning.     

Infection kinetics and developmental biology of Cryptosporidium muris (strain MCR) in Korean native kids and Corriedale lambs

A total of nine Korean native kids and two Corriedale lambs, 1-20 days old, were each inoculated per os with a single dose of 2 × 10⁷ oocysts of Cryptosporidium muris (strain MCR) originated from mice to elucidate the kinetics and developmental stages of the coccidium in small ruminants. Irrespective of host''s age, the prepatent period for both animals ranged from 19 to 35 days (28.1 days, on the average) and the patent period 16-85 days (47.8 days), and the total oocyst outputs showed enormous differences. Infection with greater numbers of oocyst outputs was not ordinarily established by transmission experiments. Oocysts discharged from the kids retained their infectivity by the mouse titration method. The immunogenicity of the coccidium and oocyst reproduction were proven by challenge infection and administration of prednisolone acetate, respectively. All the developmental stages of the coccidium in parasitophorous vacuoles were found by transmission electron microscopy in the pits of the gastric glands of a kid inoculated with oocysts and then necropsied on day 44 postinoculation. It indicated the full course of the host-parasite relationship in kids and lambs as well as mice.     

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.     

Digestive and metabolic utilization of lauric,myristic and stearic acid in cows,and associated effects on milk fat quality

A study was carried out to examine the effect of dietary supplementation of oregano essential oil on performance of broiler chickens experimentally infected with Eimeria tenella at 14 days of age. A total of 120 day-old Cobb-500 chicks separated into 4 equal groups with three replicates each, were used in this study. Two groups, one infected with 5·10⁴ sporulated oocysts of E. tenella and the other not, were given a basal diet and served as controls. The other two groups also infected with E. tenella were administered diets supplemented with oregano essential oil at a level of 300 mg/kg, or with the anticoccidial lasalocid at 75 mg/kg. Following this infection, survival rate, bloody diarrhoea and oocysts excretion as well as lesion score were determined. Throughout the experimental period of 42 days, body weight gain and feed intake were recorded weekly, and feed conversion ratios were calculated. Two weeks after the infection with E. tenella supplementation with dietary oregano oil resulted in body weight gains and feed conversion ratios not differing from the non-infected group, but higher than those of the infected control group and lower than those of the lasalocid group. These parameters correspond with the extent of bloody diarrhoea, survival rate, lesion score and oocyst numbers and indicated that oregano essential oil exerted an anticoccidial effect against E. tenella, which was, however, lower than that exhibited by lasalocid.     

Seasonal shedding of multiple Cryptosporidium genotypes in California ground squirrels (Spermophilus beecheyi)

Twelve percent of 853 California ground squirrels (Spermophilus beecheyi) from six different geographic locations in Kern County, Calif., were found to be shedding on average 44,482 oocysts g of feces(-1). The mean annual environmental loading rate of Cryptosporidium oocysts was 57,882 oocysts squirrel(-1) day(-1), with seasonal patterns of fecal shedding ranging from <10,000 oocysts squirrel(-1) day(-1) in fall, winter, and spring to levels of 2 x 10(5) oocysts squirrel(-1) day(-1) in summer. Juveniles were about twice as likely as adult squirrels to be infected and shed higher concentrations of oocysts than adults did, with particularly high levels of infection and shedding being found among juvenile male squirrels. Based on DNA sequencing of a portion of the 18S small-subunit rRNA gene, there existed three genotypes of Cryptosporidium species in these populations of squirrels (Sbey03a, Sbey03b, and Sbey03c; accession numbers AY462231 to AY462233, respectively). These unique DNA sequences were most closely related (96 to 97% homology) to porcine C. parvum (AF115377) and C. wrairi (AF115378). Inoculating BALB/c neonatal mice with up to 10,000 Sbey03b or Sbey03c fresh oocysts from different infected hosts did not produce detectable levels of infection, suggesting that this common genotype shed by California ground squirrels is not infectious for mice and may constitute a new species of Cryptosporidium.     

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.     

Genotype and animal infectivity of a human isolate of Cryptosporidium parvum in the Republic of Korea

Cryptosporidium parvum oocysts were isolated from a child suffering from acute gastroenteritis and successfully passaged in a calf and mice (designated hereafter SNU-H1) in the Republic of Korea; its molecular genotype has been analyzed. The GAG microsatellite region was amplified by a polymerase chain reaction (PCR), with a 238 base pair product, which is commonly displayed in C. parvum. The isolate was shown to be a mixture of the genotypes 1 (anthroponotic) and 2 (zoonotic). To study its infectivity in animals, 2 calves and 3 strains of mice were infected with the SNU-H1; in these animals, the propagation of both genotypes was successful. In immunosuppressed (ImSP) BALB/c and C57BL/6 mice the number of oocysts decreased after day 10 post-infection (PI); but in ImSP ICR mice, they remained constant until day 27 PI. The results show that both the C. parvum genotypes 1 and 2 can be propagated in calves and ImSP mice.     

Anti-cryptosporidial drug activity screened with an immunosuppressed rat model

Cryptosporidium parvum, a coccidian parasite can cause mild or severe self-limiting diarrhea in immunocompetent humans, but chronic and life-threatening in immunocompromised individuals. An immunosuppressed rat model with persistent cryptosporidiosis was used to investigate the anti-cryptosporidial activity of drugs. Using curative procedures, no activity was found with 6 antibiotics assayed, including spiramycin (31-100%). Mepacrine at a dose of 100 mg/kg had mild activity (19%), while lasalocid (10 mg/kg) and sulfadimethoxine (60 mg/kg) exhibited a 0.1% and 13%, activity respectively. This experimental immuno-suppressed rat model appears suitable for screening candidate drugs either for preventive or curative treatment of cryptosporidiosis.     

Quantitative Flow Cytometric Evaluation of Maximal Cryptosporidium parvum Oocyst Infectivity in a Neonate Mouse Model

The importance of waterborne transmission of Cryptosporidium parvum to humans has been highlighted by recent outbreaks of cryptosporidiosis. The first step in a survey of contaminated water currently consists of counting C. parvum oocysts. Data suggest that an accurate risk evaluation should include a determination of viability and infectivity of counted oocysts in water. In this study, oocyst infectivity was addressed by using a suckling mouse model. Four-day-old NMRI (Naval Medical Research Institute) mice were inoculated per os with 1 to 1,000 oocysts in saline. Seven days later, the number of oocysts present in the entire small intestine was counted by flow cytometry using a fluorescent, oocyst-specific monoclonal antibody. The number of intestinal oocysts was directly related to the number of inoculated oocysts. For each dose group, infectivity of oocysts, expressed as the percentage of infected animals, was 100% for challenge doses between 25 and 1,000 oocysts and about 70% for doses ranging from 1 to 10 oocysts/animal. Immunofluorescent flow cytometry was useful in enhancing the detection sensitivity in the highly susceptible NMRI suckling mouse model and so was determined to be suitable for the evaluation of maximal infectivity risk.     

Evaluation of water treatment plant UV reactor efficiency against Cryptosporidium parvum oocyst infectivity in immunocompetent suckling mice

Aim: To assess the efficiency of a medium‐pressure UV reactor under full‐scale water treatment plant (WTP) conditions on the infectivity of Cryptosporidium parvum oocysts in an Naval Medical Research Institute (NMRI) suckling mice infectivity model. Methods and Results: Six/seven‐day‐old mice were administered orally 2–10 × 10⁴Cryptosporidium parvum oocysts. Compared with nonirradiated oocysts, 40 mJ cm^?2 UV irradiation of ingested oocysts resulted 7 days later in a 3·4–4·0 log₁₀ reduction in the counts of small intestine oocysts, using a fluorescent flow cytometry assay. Conclusion: Present data extend to industrial conditions previous observations of the efficiency of UV irradiation against Cryptosporidium parvum oocyst in vivo development. Significance and Impact of the study: Present results suggest that in WTP conditions, a medium‐pressure UV reactor is efficient in reducing the infectivity of Cryptosporidium parvum oocysts, one of the most resistant micro‐organisms present in environmental waters.