Performance and oocyst shedding in broiler chickens orally infected with Cryptosporidium baileyi and Cryptosporidium meleagridis

To study effects of experimental cryptosporidiosis, broiler chickens were infected per os with 5 x 10(5) oocysts of Cryptosporidium baileyi and Cryptosporidium meleagridis. In the first experiment, chickens were infected with oocysts of C. baileyi at the age of 7, 14, and 21 days. In the second experiment, chickens were infected with oocysts of C. baileyi, C. meleagridis, or both cryptosporidial species at the age of 7 days. Although clinical signs of infection were apparent, neither final live weight nor mortality was significanty influenced in chickens infected with a single Cryptosporidium species. In chickens infected with C. meleagridis, the growth retardation was observed in the 2-wk period after infection. The compensatory growth, however, started when the oocyst shedding had ceased. The number of oocysts in excreta specimens of chickens infected with C. meleagridis was two to three times lower than in excreta of chickens infected with C. baileyi. Chickens infected with both C. baileyi and C. meleagridis (5 x 10(5) oocysts of each) had significantly lower final live weight and worse feed efficiency than chickens of other groups. Concurrent infection did not influence individual C. baileyi or C. meleagridis oocyst shedding.     

Molecular identification of Cryptosporidium spp. in animal and human hosts from the Czech Republic

To study the diversity of Cryptosporidium spp. in various hosts, we used the variability of the small-subunit rRNA gene and the Cryptosporidium oocyst wall protein genes. Oocysts from humans, cattle, horses, dogs, field mice, chickens, reptiles, deer, goat, cat, antelope and from a sample of water reservoir were assayed. The zoonotic C. parvum bovine genotype sequence was found to be present in the most of isolates. This study shows a complex epidemiology pattern for C. parvum bovine genotype infections. The identification of cattle, horse, and deer isolates emphasizes a transmission route for C. parvum via these hosts, and identifies a potential source for human infection in the Czech Republic. Furthermore, C. andersoni from a cow, C. baileyi from a chicken, C. felis from a cat, C. meleagridis from a dog, and C. saurophilum and C. serpentis from reptiles were also identified in the isolates from the Czech Republic.     

Characterization of a factor from bovine intestine that protects against Cryptosporidium parvum infection

Cryptosporidium parvum is a protozoan parasite that causes intestinal infection in a variety of mammals. We have previously described a factor in adult rat or adult bovine intestinal mucosa that protects against C. parvum infection when fed to susceptible infant rats. This factor is absent in intestinal mucosa from bovine calves. In the present study we describe the further characterization of the active component of bovine intestinal mucosa. The ability to protect infant rats against C. parvum infection was found to be associated with the extrinsic membrane protein fraction of the intestinal mucosa. Extrinsic membrane preparations from adult cows, adult rats, and calves were separated by SDS-PAGE. A band with apparent molecular mass of 54 kDa was seen in preparations from adult rat and cow, but not calf. Protein was transferred to PVDF membrane and from this the band was excised and subjected to N-terminal sequence analysis using a gas-phase protein sequenator. A 15-amino acid consensus sequence was generated with homology to leucine aminopeptidase (LAP). Purified LAP was purchased from a commercial source and tested for ability to protect infant rats against C. parvum infection. Rats fed LAP from 7 to 11 days of age and challenged with C. parvum at 9 days were significantly less infected than controls upon necropsy at 15 days of age. These data suggest that a protein with N-terminal sequence homology to LAP may reduce susceptibility of infant rats to C. parvum infection.     

Prevalence and molecular characterization of bovine Cryptosporidium isolates in India

A survey based on PCR assay of 18S SSU rRNA gene revealed a 30.2% infection with Cryptosporidium spp., out of 457 faecal samples collected from neonatal bovine calves across three different regions of India. The PCR-RFLP pattern of the gene in all the positive cases established the species as Cryptosporidium parvum. Highest prevalence was recorded in the monsoon months (37.3%) and in the calves showing acute diarrhoea (32.3%). The calves below 15 days of age were mostly affected (45.1%). The infection was more prevalent in the northern parts (35.4%) of the country than in the eastern or southern parts. Results indicated that C. parvum was the only species of Cryptosporidium prevalent in bovine calves in three different geographical regions of India.     

Activity of decoquinate against Cryptosporidium parvum in cell cultures and neonatal mice

Cryptosporidium parvum is an apicomplexan parasite that is an important cause of diarrhea in neonatal calves and humans. No treatment is currently available for neonatal calves. We have recently learned from colleagues in the pharmaceutical industry that dairy practitioners are sometimes using decoquinate for the treatment of neonatal bovine cryptosporidiosis. Therefore, the present study was undertaken to determine whether the clinical observations in calves can be substantiated by laboratory investigation. Oocysts of the KSU-1 isolate of C. parvum were used to infect human ileocecal epithelial cells in vitro to measure the efficacy of treatment using an ELISA based assay. No activity was observed at 10 or 50microM decoquinate, but at 100microM an 8% inhibition of development was seen. Oocysts of the AUCp-1 isolate of C. parvum were then used to infect suckling mice. The numbers of oocysts observed in suckling mice treated with 2.5 or 5.0mg/kg decoquinate were not significantly different from untreated control suckling mice (p0.05). The results of our study suggest that decoquinate should have little efficacy for treatment of neonatal bovine cryptosporidiosis if administered once per day and that any clinical improvement observed in treated calves may be due to factors unrelated to decoquinate's effect on C. parvum.     

Genotypic characterization and phylogenetic analysis of Cryptosporidium sp. from domestic animals in Brazil

The purpose of the present study was the genetic characterization, sequencing and phylogenetic analysis of 18S rDNA sequences of Cryptosporidium isolates obtained from different animal hosts in Brazil. Fecal samples containing Cryptosporidium oocysts were obtained from chickens, ducks, quails, guinea pigs, dairy calves, dogs and cats. For amplification of 18S rDNA sequences the Secondary-PCR product of the extracted DNA from fecal suspension of each studied animal was utilized. The primary genetic characterization of Cryptosporidium sp. was performed using RFLP with the enzymes SspI and VspI. DNA samples were sequenced and subjected to phylogenetic analysis. The results showed C. baileyi infecting two ducks and one quail and C. melagridis infecting one chicken. The sequences obtained from Cryptosporidium sp. infecting guinea pigs were not identified within groups of known Cryptosporidium species. The isolates found parasitizing cats and one dog were diagnosed as C. felis and C. canis, respectively. One isolate of calf origin was identified as C. parvum. The phylogenetic analysis showed clear distribution of isolates between two Cryptosporidium sp. groups according to their gastric or intestinal parasitism. A great genetic distance was observed between C. felis and C. canis from Brazil when compared to the reference sequences obtained from GenBank. The results obtained during this study constitute the first report of rDNA sequences from C. baileyi, C. meleagridis, C. felis, C. canis and C. parvum isolated in Brazil.     

Cryptosporidium infections in birds and mammals and attempted cross-transmission studies

Infections by Cryptosporidium were detected in association with clinical disease in 11 humans (Homo sapiens), 19 calves (Bos taurus), nine common quail (Coturnix coturnix), six mallard ducks (Anas platyrhynchos), five ring-necked pheasant (Phasianus colchicus) and a single budgerigar (Melopsittacus undulatus). Infections in mammals were accompanied by transient diarrhoea and anorexia, whereas infected birds exhibited clinical signs of respiratory distress. Repeated cross-transmission studies revealed apparent strain differences or differences in the host specificity of several mammalian and avian isolates for homologous vertebrate classes only. Oocysts from humans and calves were infective to mice, pigs or lambs, but not to chickens, whereas oocysts from quail and pheasant were infective to chickens, but not to mice.     

Prevalence and age-related variation of Cryptosporidium species and genotypes in dairy calves

Fifteen dairy farms in seven states on the east coast of the US were each visited on two consecutive years to determinate the prevalence of Cryptosporidium species in pre-weaned (5 days to 2 months) and post-weaned calves (3-11 months), respectively. After each of 971 fecal specimens collected directly from each calf was sieved and subjected to density gradient centrifugation to remove debris and concentrate oocysts, specimens were examined by immunofluorescence microscopy, and polymerase chain reaction (PCR). For all PCR-positive specimens the 18S rRNA gene of Cryptosporidium was sequenced. Cryptosporidium was identified from all farms. Types of housing appeared to have no influence with regard to prevalence of infection. Of 971 calves, 345 were infected with Cryptosporidium (35.5%), but more pre-weaned calves (253 of 503; 50.3%) than post-weaned calves (92 of 468; 19.7%) were found to be infected. A total of 278 PCR-positive specimens characterized by gene sequencing revealed Cryptosporidium parvum, Cryptosporidium andersoni, and two unnamed Cryptosporidium genotypes Bovine B (AY120911) and deer-like genotype (AY120910). The prevalence of these Cryptosporidium species and genotypes appeared to be age related between pre- and post-weaned calves. C. parvum, the only zoonotic species/genotype, constituted 85% of the Cryptosporidium infections in pre-weaned calves but only 1% of the Cryptosporidium infections in post-weaned calves. These findings clearly demonstrate that earlier reports on the presence and prevalence of C. parvum in post-weaned cattle that were based solely on oocyst morphology must be reassessed using molecular methods to validate species and genotype. This finding also indicates that persons handling or otherwise exposed to calves under 2 months of age are at greater risk of zoonotic infection from Cryptosporidium than the risk of infection from exposure to older calves.     

Molecular characterization of Cryptosporidium spp. in native breeds of cattle in Kaduna State, Nigeria

Despite numerous molecular epidemiologic studies of cryptosporidiosis in dairy cattle in industrialized countries, there are very few studies on the diversity and public health significance of Cryptosporidium species in native cattle in developing countries. In this study, a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis of the small-subunit (SSU) rRNA gene was used to detect and identify Cryptosporidium spp. in 194 fecal specimens from 2 to 365 days old calves in 20 White Fulani and Sokoto Gudali herds in Nigeria. Thirty one (16.0%) of the specimens were positive for Cryptosporidium. Restriction digestion of the PCR products showed the presence of Cryptosporidium bovis (7.2%), Cryptosporidium ryanae (4.1%), Cryptosporidium andersoni (2.5%), and concurrent occurrence of C. bovis and C. ryanae (1.5%), and C. bovis and C. andersoni (0.5%). There were no significant differences (p>0.05) in Cryptosporidium infection rates by sex, herd location, management system, breed of calves, or fecal consistency. However, calves 180 days or younger had a higher infection rate of Cryptosporidium than older calves (p=0.034). Likewise, younger calves also had higher occurrence of C. bovis and C. ryanae (p=0.022). The absence of zoonotic Cryptosporidium parvum in the calves studied suggests that native breeds of cattle may not be important in the transmission of human cryptosporidiosis in Kaduna State, Nigeria.     

Age-related and housing-dependence of Cryptosporidium infection of calves from dairy and beef herds in South Bohemia, Czech Republic

Data of the prevalence, age-related and housing-dependence of naturally acquired cryptosporidiosis on 11 dairy and 11 beef farms in South Bohemia (Czech Republic) were collected. The farms were visited over four consecutive years (from 2002 to 2005). The prevalence of Cryptosporidium in pre-weaned (animals until second month of age) and post-weaned (animals from the third month of age) calves was determined. A total of 7001 faecal samples were collected, concentrated by Sheather's floatation method and stained by aniline-carbol-methyl violet. All samples were examined by light microscopy. Cryptosporidium parvum and C. andersoni oocysts were differentiated on morphological criteria. Of the 7021 specimens, 1814 (25.8%) were positive for Cryptosporidium oocysts; 561 samples (8%) for C. parvum and 1253 (17.8%) for C. andersoni. Pre-weaned dairy calves had higher infection levels of C. parvum than pre-weaned beef calves. The prevalence of C. parvum ranged from 1.4 to 56.5% on dairy farms. Only three cases of C. parvum oocysts shedding in pre-weaned calves on beef farms were found. Only one case of C. andersoni infection in pre-weaned calves was detected and no infections of C. parvum in post-weaned calves were found. The prevalence of C. andersoni reached 35.5% on dairy farms and 61.7% on beef farms. Calves that were on pasture all year long, had a lower probability of C. andersoni infection than those calves kept in a cowshed during the winter season.     

The effect of heating against Cryptosporidium oocysts

The effect of heat treatment was examined against oocysts of Cryptosporidium parvum, Cryptosporidium muris and chicken Cryptosporidium sp. isolated in Japan. The oocysts of these species were exposed at 50, 55, 60 and 70 degrees C for 5, 15, 30 and 60 sec in water bath, respectively. To determine the infectivity of heated oocysts, the nice and chickens were inoculated with the treated oocysts and the oocyst output in the feces after inoculation was examined. In C. parvum and chicken Cryptosporidium sp., the oocysts were not detected from mice or chickens which were received oocysts heated at 55 degrees C for 30 sec, 60 degrees C for 15 sec and 70 degrees C for 5 sec. In C. muris, the oocysts were not detected from mice which were received oocysts heated at 55 degrees C for 15 sec, 60 degrees C for 15 sec and 70 degrees C for 5 sec. Consequently, it was clarified that the infectivity of Cryptosporidium oocysts to mice and chickens was lost by heating at 55 degrees C for 30 sec, 60 degrees C for 15 sec and 70 degrees C for 5 sec.     

Long-term study of Cryptosporidium prevalence on a lowland farm in the United Kingdom

A longitudinal sample survey testing for Cryptosporidium in livestock and small wild mammals conducted over 6 years (1992-1997) on a lowland farm in Warwickshire, England, has shown the parasite to be endemic and persistently present in all mammalian categories. Faecal samples were taken throughout the year and oocysts concentrated by a formal ether sedimentation method for detection by immunofluorescence staining using a commercially available genus specific monoclonal antibody. Cryptosporidium parvum was identified by morphology and measurement of modified Ziehl-Neelsen stained oocysts. C. muris was rarely found in wild mammals and C. andersoni oocysts were never detected in livestock. Faecal samples from 3721 individuals gave cumulative 6-year prevalences for C. parvum as follows: bull beef, 3.6%; dairy cows, 3.5%; ewes, 6.4%; horses, 8.9%; calves (home bred), 52%; calves (bought-in) 23.2%; lambs, 12.9%; small wild mammals (rodents) living in and around farm buildings, 32.8%; small wild mammals (mainly rodents) living in areas of pasture, 29.9%. Animal categories with the highest prevalences also shed the highest average oocyst numbers per gram of faeces (ranging from 1.4 x 10(3) for bull beef to 1.1 x 10(5) for calves). Analysis of annual and seasonal data for each animal category revealed that patterns of infection were variable and sporadic; this means that short-term sampling was never likely to provide a true or representative picture. Seasonally combined data for adult livestock, young livestock and small wild mammals showed all three categories tended to have the highest Cryptosporidium prevalences in the autumn. Calves were separated from their dams within 24h of birth and yet showed high prevalence of infection in most years despite the low prevalence for the dairy herd. It is possible the coincidence of high autumn prevalence in mice with the main period for the rearing of calves contributed to the infection of the latter. The farming estate was used to teach students of agriculture and took pride in good land management and husbandry practices that produced well fed and healthy livestock. The data from this estate may represent, therefore, the baseline, the lowest possible levels to be expected, for Cryptosporidium infection and oocyst production on a lowland farm in the United Kingdom.     

Prevalence and molecular characterization of Cryptosporidium and Giardia species and genotypes in sheep in Maryland

In the United Kingdom and Australia sheep have been implicated as sources of Cryptosporidium and Giardia that infect humans, but no such studies have been conducted in North America. Therefore, a study was undertaken to investigate the prevalence of these parasites in sheep on a farm in Maryland. Feces were collected from 32 pregnant ewes 1, 2, and 3 days after parturition and from each of their lambs 7, 14, and 21 days after birth. The presence of Cryptosporidium oocysts and Giardia cysts was determined by both immunofluorescence microscopy and PCR/gene sequence analysis. PCR was consistently more sensitive than microscopy. The prevalence, by PCR, of Cryptosporidium in ewes and lambs was 25 and 77.4%, respectively. Three species/genotypes of Cryptosporidium were identified: C. parvum, a novel C. bovis-like genotype, and Cryptosporidium cervine genotype. Cryptosporidium parvum and the cervine genotype have been reported worldwide in human infections. The novel C. bovis-like genotype is reported here for the first time. The prevalence of Giardia in ewes and lambs was 12 and 4%, respectively. Most infections were Assemblage E which is not zoonotic; however, one ewe was infected with zoonotic Assemblage A. The identification of only two lambs infected with C. parvum and one ewe infected with G. duodenalis Assemblage A suggests a low prevalence of these zoonoses. However, the high prevalence of the zoonotic cervine genotype indicates that sheep should be considered a potential environmental source of this human pathogen.     

Effects of colostral antibody on susceptibility of calves to Cryptosporidium parvum infection

Effects of colostral antibody on susceptibility of calves to Cryptosporidium parvum infection were examined. Six calves were fed pooled colostrum that contained C parvum antibody, 6 times daily (at 4-hour intervals) for 7 days and then milk replacer for 7 days. Colostrum was obtained from healthy cows or cows inoculated parenterally with C parvum oocysts before parturition. Antibody content was determined in serum and colostrum whey, using an ELISA for anticryptosporidia immunoglobulin. Six calves were fed colostrum from healthy cows 1 time, and then milk replacer 6 times daily for 14 days. On day 1, all calves were challenge exposed with C parvum, PO, and were monitored daily for diarrhea and oocyst shedding. Bovine colostrum containing specific antibody to C parvum, at ELISA titers up to 10,240, was not effective in protecting calves against challenge exposure to C parvum.     

Cryptosporidium ryanae n. sp. (Apicomplexa: Cryptosporidiidae) in cattle (Bos taurus)

A new species, Cryptosporidium ryanae, is described from cattle. Oocysts of C. ryanae, previously identified as the Cryptosporidium deer-like genotype and recorded as such in GenBank (AY587166, EU203216, DQ182597, AY741309, and DQ871345), are similar to those of Cryptosporidium parvum and Cryptosporidium bovis but smaller. This genotype has been reported to be prevalent in cattle worldwide. Oocysts obtained from a calf for the present study are the smallest Cryptosporidium oocysts reported in mammals, measuring 2.94-4.41micromx2.94-3.68microm (mean=3.16micromx3.73microm) with a length/width shape index of 1.18 (n=40). The pre-patent period for two Cryptosporidium-na?ve calves fed C. ryanae oocysts was 11 days and the patent period was 15-17 days. Oocysts were not infectious for BALB/c mice or lambs. Fragments of the SSU-rDNA, HSP-70, and actin genes amplified by PCR were purified and PCR products were sequenced. Multi-locus analysis of the three unlinked loci demonstrated the new species to be distinct from all other species and also demonstrated a lack of recombination, providing further evidence of species status. Based on morphological, molecular and biological data, this geographically widespread parasite found only in Bos taurus calves is recognized as a new species and is named C. ryanae.     

Prevalence of Cryptosporidium parvum infection and pattern of oocyst shedding in calves in Japan

Cryptosporidium parvum infection and the pattern of oocyst shedding were observed in calves. A total of 480 fecal samples were collected from 30 calves (age, < or =30 days) over a period of 10 months from June 1998 to March 1999. A sucrose centrifugal flotation technique revealed 28/30 (93%) calves were passing Cryptosporidium oocysts. Oocyst shedding was first detected on the sixth day after birth, with 8% of the calves testing positive. This rate increased day by day and reached approximately 80% by day 15. Oocyst shedding varied from 1 to 13 days, with a mean of 7 days. Calves infected with C. parvum had a significantly higher rate of diarrhea (33%) than non-infected calves (8%) (P<0.05), suggesting C. parvum infection as the likely cause. The mean number of oocysts excreted by calves < or =30 days old was approximately 6x10(7) per gram of feces. These results indicated that one calf would excrete some 6x10(11) oocysts in the first month after birth, taking both the quantity of feces in a day and the period of excretion into consideration. Accordingly, it is clear that calves are important in the spread of cryptosporidiosis to calves and humans.     

Malabsorption of vitamin A in preruminating calves infected with Cryptosporidium parvum.

Serum retinol, retinyl palmitate, and total vitamin A concentrations, and jejunoileal morphology were examined in neonatal calves infected with Cryptosporidium parvum. Group-1 calves served as noninfected controls and, after an adjustment period, were given 50 ml of saline solution i.v. every 12 hours for 6 days. Group-2 calves were inoculated with 10(7) C parvum oocysts and, after the onset of diarrhea, were given 50 ml of saline solution i.v. every 12 hours for 6 days. Group-3 calves were inoculated with 10(7) C parvum oocysts and, after the onset of diarrhea, were treated with difluoromethylornithine (DFMO, 200 mg/kg of body weight i.v., q 12 h) for 6 days. Group-4 calves were naturally infected with C parvum. Jejunoileal biopsy specimens were excised from calves of groups 1-3 at 3 and again at 15 to 16 days of age. During the course of diarrhea and 3 days after saline or DFMO administration, water-miscible retinyl palmitate was administered orally (2,750 micrograms/kg) to each calf in each group. Cryptosporidium parvum infection was associated with significant (P < or = 0.05) reduction in postadministration serum retinol, retinyl palmitate, and total vitamin A concentrations in calves of groups 2, 3, and 4. Cryptosporidium parvum infection caused significant (P < or = 0.05) reduction in villus height. Decreased villus height, villus blunting and fusion, and attenuation of the intestinal mucosa were associated with reduced absorption of vitamin A, as indicated by lower peak postadministration retinyl palmitate concentration in C parvum-infected calves. Intravenous administration of DFMO to group-3 calves did not improve retinol absorption.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isoenzyme activities of different strains of Cryptosporidium parvum

Different isoenzyme activities have been assayed in three strains of Cryptosporidium parvum, C1 (C. parvum from infected calves, UK), C2 (C. parvum from infected calves, Egypt) and C3 (C. parvum from infected goats, Egypt). The electrophoretic variations of five enzymes; lactate dehydrogenase (LDH), glucose phosphate isomerase (GPI), hexokinase (HK), malate dehydrogenase (MDH) and glutamate dehydrogenase (GLDH) were compared among the three different isolates using native polyacrylamide gel-electrophoresis. LDH showed an identical pattern in the three isolates. GPI showed two different bands in C3 and C1, with both bands present in C2. HK activity showed a weak band in C1 but no reaction was detected with C2 and C3. Malate dehydrogenase (MDH) showed no reaction in C1, but similar bands in C2 and C3. Glutamate dehydrogenase (GLDH) showed two different patterns, C2 and C3 had one pattern and C1 showed additional zones of reaction. Rat liver homogenate was run at the same time as the parasite extracts as a positive control. This investigation suggests that GPI, HK and GLDH could be used to characterise different Cryptosporidium isolates.     

Giardia and Cryptosporidium in dairy calves in British Columbia.

A study was undertaken to determine the prevalence of Giardia infections in dairy calves and to compare Giardia and Cryptosporidium infections in calves of different ages. Fresh fecal samples were collected from 386 male and female Holstein calves (newborn to 24 wk) in 20 dairies located in the lower Fraser river valley area of British Columbia. Giardia intestinalis, Cryptosporidium parvum, and Cryptosporidium muris were enumerated in each sample after concentration by sucrose gradient centrifugation and immunofluorescent staining. Giardia was identified at all farm locations. The overall prevalence of Giardia in calves was 73% with a geometric mean cyst count of 1180 cysts per gram of feces (CI, 41 to 5014). Cryptosporidium parvum and C. muris were identified in 80% and 40% of the farms, respectively. The prevalence of C. parvum was 59%, and the geometric mean for oocysts was 457 oocysts per gram of feces (CI, 18 to 160). The prevalence of C. muris was only 2% and the mean oocyst counts were 54 oocysts per gram of feces. Giardiasis was not age dependent, and approximately 80% of the calves from 2 to 24 wk were infected. In contrast, C. parvum infections were predominant in calves 2 to 4 wk, while C. muris was demonstrated in calves older than 4 wk. Fourty-seven percent of calves with diarrhea had high numbers of Giardia cysts in their feces. Giardia infections are highly prevalent in dairy calves and should be considered in animals with diarrhea or failure to thrive.