Cryptosporidium, Giardia and Enterocytozoon bieneusi in cats from Bogota (Colombia) and genotyping of isolates

The prevalence of Cryptosporidium, Giardia, and Enterocytozoon bieneusi in cats from Bogota (Colombia) was determined from fecal specimens and scrapings of duodenal and ileal mucosa screened by PCR. All PCR-positive specimens were sequenced to determine the genotype(s) present. Of 46 cats, 6 (13%) were positive for Cryptosporidium, 5 (11%) were infected with C. felis and one (2%) with C. muris. Three (6.5%) cats were infected with Giardia duodenalis Assemblage F. Eight (17%) cats were infected with four genotypes of E. bieneusi: genotype D-like (9%), K (4%), Peru 10 (2%), and Peru 5 (2%). This is the first report on the presence of zoonotic species/genotypes of Cryptosporidium and E. bieneusi in cats in Colombia.     

Infectivity, pathogenicity, and genetic characteristics of mammalian gastric Cryptosporidium spp. in domestic ruminants.

Farm ruminants were infected experimentally with four mammalian gastric Cryptosporidium, namely Cryptosporidium andersoni LI03 originated from cattle and three isolates of Cryptosporidium muris from brown rat (isolate RN66), Bactrian camel (isolate CB03) and firstly characterized isolate from East African mole rat (isolate TS03). Sequence characterizations of the small-subunit rRNA gene showed that the LI03 isolate was C. andersoni and the other three isolates belonged to C. muris, although the TS03 isolate showed unique sequence variations (one single nucleotide change and four nucleotide insertions). C. andersoni LI03 was infectious for calves only, whereas lambs and kids were susceptible to C. muris CB03. C. muris TS03 and RN66 were not infectious for any farm ruminants. Infection dynamics including prepatent and patent period and infection intensity of the isolates used differed depending on the host species, but no clinical signs of cryptosporidiosis were observed in any of experimentally infected hosts. Cryptosporidium developmental stages were only detected in infected animals in the abomasum region. Histopathological changes were characterized by dilatation and epithelial metaplasia of infected gastric glands with no significant inflammatory responses in the lamina propria.     

Evaluation of fenbendazole for treatment of Giardia infection in cats concurrently infected with Cryptosporidium parvum

OBJECTIVE: To determine whether fenbendazole effectively eliminates Giardia organisms from chronically infected cats that have a concurrent Cryptosporidium parvum infection. ANIMALS: 16 clinically normal cats. PROCEDURE: Eight cats with chronic concurrent Giardia and C parvum infections received fenbendazole (50 mg/kg, PO, q 24 h) for 5 days (treatment-group cats). Feces from each cat were collected and processed 3 days weekly for 23 days after treatment. By use of an immunofluorescent assay for detection of Giardia lamblia cysts and C parvum oocysts, organism numbers were counted and scored. Fecal results from treatment-group cats were compared with those of 8 untreated cats with Giardia infection but no C parvum infection (control-group cats). RESULTS: Four of 8 treatment-group cats had consistently negative results for Giardia infection after treatment. These 4 cats had consistently positive results for C parvum oocysts prior to treatment and consistently negative results after treatment. One treatment-group cat had positive results for cysts on all fecal samples, and 3 treatment-group cats had 1 to 3 negative results and then resumed shedding large numbers of cysts; each of these cats had consistently positive results for C parvum oocysts. When compared with control-group cats, treatment-group cats shed less Giardia cysts during week 1 after treatment but not during week 2. CONCLUSIONS AND CLINICAL RELEVANCE: Administration of fenbendazole decreases Giardia cyst shedding to less than detectable numbers in some cats. In our study, persistent C parvum infection may have been associated with failure of fenbendazole to eliminate Giardia infection.     

Detection of Cryptosporidium felis and Giardia duodenalis Assemblage F in a cat colony

Eighteen cats, 3-6 months of age, bred and housed in a closed colony, were transferred from that colony and placed in separate stainless steel cages in a building designed for housing animals. At daily intervals, feces were collected from the litter pans in each cage, pans and cages were cleaned, and fresh food and water were provided. Beginning 4 weeks after the transfer, oocysts of Cryptosporidium were detected in the feces of two cats by brightfield microscopy. For the following 21 days, with minor exceptions, feces from each cat were collected daily and examined by immunofluorescence microscopy and by molecular methods that included DNA extraction, 18S rDNA gene amplification, and DNA sequence analysis. Within those 22 days, every cat was found to be infected with Cryptosporidium felis and excreted oocysts for 6-18 days. Eight of these 18 cats also excreted cysts of Giardia duodenalis Assemblage F, a genotype found only in cats. Six Giardia infections were concurrent during part of the patency with C. felis infections. Neither diarrhea nor other signs of illness were observed in any of the cats during this time. Because C. felis is zoonotic these findings suggest that care should be taken by veterinary health care providers and others in close contact with cats, even when cats appear healthy and asymptomatic.     

Prevalence of Giardia sp. Cryptosporidium parvum and Cryptosporidium andersoni (syn. C. muris) [correction of Cryptosporidium parvum and Cryptosporidium muris (C. andersoni)] in 109 dairy herds in five counties of southeastern New York

A cross-sectional study was undertaken to determine the prevalence of Giardia sp. (G. duodenalis group), Cryptosporidium parvum and Cryptosporidium andersoni (C. muris) [corrected] in dairy cattle in three different age groups, and to evaluate the association of age and season with prevalence. One hundred and nine dairy farms, from a total of 212 farms, in five counties of southeastern New York volunteered to participate. On these farms, 2943 fecal samples were collected from three defined age groups. The farms were randomly assigned for sampling within the four seasons of the year. Each farm was visited once during the study period from March 1993 to June 1994 to collect fecal samples. Demographic data on the study population was collected at the time of sampling by interviewing the farm owner or manager. At collection, fecal samples were scored as diarrheic or non-diarrheic, and each condition was later related to positive or negative infection with these parasites. Fecal samples were processed using a quantitative centrifugation concentration flotation technique and enumerated using bright field and phase contrast microscopy. In this study, the overall population prevalence for Giardia sp. was 8.9%; C. parvum, 0.9%; and C. muris, 1.1%. When considering animals most at the risk of infection (those younger than 6 months of age) Giardia sp. and C. parvum was found in 20.1 and 2.4% of the animals, respectively. Giardia sp. and C. muris were found in all age groups. There was no significant seasonal pattern of infection for any of these parasites.     

Cryptosporidium muris infection in bilbies (Macrotis lagotis)

Cryptosporidiosis is an enteric disease of animals and humans that can be fatal in immunocompromised individuals. There is no known effective treatment for cryptosporidiosis. Bilbies are threatened marsupials and are bred in captivity as part of a recovery program to re-introduce this species to the southwest of Western Australia. Cryptosporidium muris infection was detected in the faeces of bilbies at a captive breeding colony. Stress associated with a high density of bilbies in enclosures may have predisposed some of the bilbies to infection with C. muris. C. muris has been described in mice and was found in the faeces of one mouse trapped in the breeding enclosures. It is likely the bilbies acquired the infection from mice by faecal contamination of food and water. The infection cleared within 2 months from some bilbies, however others remained infected for 6 months and treatment was attempted with dimetridazole. Subsequently the parasite was no longer be detectable in the faeces.     

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.     

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.     

鼠隐孢子虫卵囊及子孢子的纯化

应用不连续蔗糖密度梯度离心法，对犊牛粪便中的鼠隐孢子虫卵囊进行了提取，经过两次漂浮和一次不连续蔗糖密度梯度离心，得到纯净的卵囊，其中含有极少量的杂质，不含其他微生物。卵囊的回收率为３７％。纯化的卵囊脱囊后，经过一次不连续蔗糖密度梯度离心，得到很纯的子孢子。子孢子的回收率为４７．６％。纯化的卵囊及于孢子可用于隐孢子虫的核酸分析、生物化学、免疫学及细胞培养等方面的研究。该法具有简便、快速、大量分离卵囊的优点。     

Detection of Cryptosporidium muris type oocysts from beef cattle in a farm and from domestic and wild animals in and around the farm

Cryptosporidium muris type oocysts were detected from 21 of 516 beef cattle in a farm. Then we surveyed Cryptosporidium oocysts in 348 beef and dairy cattle, 500 pigs, 101 dogs, 38 wild animals and 11 zoo-kept animals in and around the farm. Oocysts were detected from only 2 of 25 Japanese field mice, Apodemus speciosus in the same farm. Gene analysis suggested that the oocysts were different from the C. muris type bovine isolate.     

The homologous and interspecies transmission of Cryptosporidium parvum and Cryptosporidium meleagridis

Experimental infections have been performed in several mammals and birds to determine the host's specificity for Cryptosporidium parvum (of human and bovine origin) and for Cryptosporidium meleagridis (isolated from broiler chicken). The C. parvum infection (of human origin) was established also in calves (Bos taurus), lambs (Ovis aries), mice (Mus musculus), and rats (Ratus norvegicus), but not in broiler chickens (Gallus domestica). The C. parvum infection (of bovine origin), was achieved in calves, lambs, dogs (Canis familiaris), cats (Felis domesticus), rabbits (Orytolagus cuniculus), mice, rats, and guinea-pigs (Cavia porcelus) but not in broiler chickens. We have demonstrated that C. meleagridis can produce infection in broiler chickens and in 5 mammalian species (calves, pigs, rabbits, rats, mice) but not in guinea-pigs.     

Comparison of direct immunofluorescence, immunoassays, and fecal flotation for detection of Cryptosporidium spp. and Giardia spp. in naturally exposed cats in 4 Northern California animal shelters

BACKGROUND: Giardia spp. and Cryptosporidium spp. are common intestinal protozoan parasites in domestic cats. Few studies have critically evaluated the performance characteristics of commercially available immunoassays for detection of these organisms in the cat. HYPOTHESIS: Human-based immunoassays are suboptimal for the detection of Giardia spp. and Cryptosporidium spp. in cats. ANIMALS: Three-hundred-and-forty-four cats with diarrheic and nondiarrheic fecal specimens at 4 northern California animal shelters. METHODS: A fecal specimen was collected from each cat in a case-controlled fashion. Fecal specimens were tested for Giardia spp. and Cryptosporidium spp. by using centrifugation flotation and 5 commercially available immunoassays (SNAP Giardia, ProSpecT Giardia Microplate Assay, ProSpecT Cryptosporidium Microplate Assay, ImmunoCard STAT! Cryptosporidium/ Giardia Rapid Assay, and Xpect Giardia/Cryptosporidium). Results were compared with a reference standard, the MeriFluor direct immunofluorescence assay. RESULTS: Overall prevalences of Giardia spp. and Cryptosporidium spp. were 9.8 and 4.7%, respectively. The ProSpecT Microplate Assay had the highest sensitivities and specificities for Giardia spp. (91.2 and 99.4%) and Cryptosporidum spp. (71.4 and 96.7%), respectively. The SNAP Giardia antigen assay was easier to use and equally sensitive (85.3%) and specific (100%) to fecal flotation. CONCLUSIONS AND CLINICAL IMPORTANCE: Caution should be exercised when using human-based immunoassays for the diagnosis of Giardia and Cryptosporidium spp. in cats. Fecal flotation remains a useful method for detection of Giardia spp., can be used to detect other parasites, and has a sensitivity of 97.8% for detection of Giardia spp. when combined with the SNAP Giardia immunoassay.     

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.     

套式PCR检测奶牛粪便中隐孢子虫

为了检测样品中的微量隐孢子虫，从含有不同数量隐孢子虫卵囊的奶牛粪便中，直接提取DNA用作初始PCR（Initial-PCR）模板，以稀释的初始PCR的产物为模板进行套式PCR（Nested-PCR），用两对人工合成寡核苷酸分别作为两PCR的引物，扩增大小分别为540pb、258pb的特异片段。PCR产物经电泳鉴定，可从阳性粪便标本DNA抽提物中扩增出目的片段，而阴性对照不能扩增目的片段；初始PCR、套式PCR的敏感小生最低可分别检测到含卵囊100、5个／g粪便。初步应用结果表明某奶牛场的奶牛自然感染率为16.4％。试验表明套式PCR的敏感性比普通PCR约高100倍，能用于奶牛隐孢子虫感染情况的调查。     

The occurrence of Cryptosporidium parvum and C. muris in wild rodents and insectivores in Spain

Five rodent and two insectivore species were investigated for Cryptosporidium at seven sites in north-eastern Spain. Of the 442 animals studied, 82 Apodemus sylvaticus, 1 A. flavicollis, 5 Mus spretus, 1 Rattus rattus, 8 Clethrionomys glareolus and 13 Crocidura russula were infected with only C. parvum. Eleven A. sylvaticus and 2 C. glareolus were infected with only C. muris and 16 A. sylvaticus, 1 M. spretus and 2 C. glareolus showed mixed infections. Both cryptosporidial species were found in most study areas. No causal relationship was found between intrinsic host factors (age and sex) and the parasitic prevalence in the most captured host species (A. sylvaticus and C. russula). Extrinsic factors such as collection site of host, seasonality and covering vegetation exerted different influence on the prevalence of Cryptosporidium. Small mammals could become one of the most important sources of cryptosporidial oocysts in those areas where neither farm animals nor significant human activity are present. This is the first study to report the infection of M. spretus and C. russula by C. parvum and the first finding of C. muris in M. spretus.     

Cryptosporidium: a water-borne zoonotic parasite

Of 155 species of mammals reported to be infected with Cryptosporidium parvum or C. parvum-like organisms most animals are found in the Orders Artiodactyla, Primates, and Rodentia. Because Cryptosporidium from most of these animals have been identified by oocyst morphology alone with little or no host specificity and/or molecular data to support identification it is not known how many of the reported isolates are actually C. parvum or other species. Cryptosporidiosis is a cause of morbidity and mortality in animals and humans, resulting primarily in diarrhea, and resulting in the most severe infections in immune-compromised individuals. Of 15 named species of Cryptosporidium infectious for nonhuman vertebrate hosts C. baileyi, C. canis, C. felis, C. hominis, C. meleagridis, C. muris, and C. parvum have been reported to also infect humans. Humans are the primary hosts for C. hominis, and except for C. parvum, which is widespread amongst nonhuman hosts and is the most frequently reported zoonotic species, the remaining species have been reported primarily in immunocompromised humans. The oocyst stage can remain infective under cool, moist conditions for many months, especially where water temperatures in rivers, lakes, and ponds remain low but above freezing. Surveys of surface water, groundwater, estuaries, and seawater have dispelled the assumption that Cryptosporidium oocysts are present infrequently and in geographically isolated locations. Numerous reports of outbreaks of cryptosporidiosis related to drinking water in North America, the UK, and Japan, where detection methods are in place, indicate that water is a major vehicle for transmission of cryptosporidiosis.     

Characterization of a single dose methylprednisolone acetate immune suppression model using Cryptosporidium muris and Cryptosporidium parvum

An immunosuppressive dose of methylprednisolone acetate (MPA) was compared with a non-immunosuppressive dose using Cryptosporidium oocyst production as an indicator of immunosuppression. To be classified as immunosuppressive, the dose had to satisfy five criteria. First, the dose had to abrogate normal immune defenses allowing the propagation of an organism to which the host is normally resistant, i.e. Cryptosporidium parvum in adult mice. Second, the dose had to decrease overall circulating CD4 T-lymphocyte numbers by greater than 80%. Third, the immunosuppressive dose had to prolong the infection beyond the normal infection length, and fourth, increase the severity of an active infection. Lastly, after complete recovery from a C. muris infection, immunosuppression must suppress the naturally acquired post infection immunity and allow reinfection. In mice immunosuppression with 600 mgMPA/kg lasted approximately 14 days and satisfied all five criteria. Fecal oocyst production could be perpetuated by dosing at 10-day intervals. A 200 mgMPA/kg dose transiently lowered CD4 counts by over 80%, but failed to override the naturally acquired post infection immunity or allow infection with C. parvum. The immunosuppressed blood profile consisted of an immediate sharp rise of mature segmented neutrophils combined with a severe decrease in circulating T-lymphocyte numbers. The rise and fall of neutrophils proved to be a good indicator of the severity and duration of immunosuppression. The thymus and spleen likewise contracted and then expanded in accordance with the steroid effect. The metabolism of MPA resulted in the eventual recovery of immune function signified by the cessation of C. parvum oocyst production. The recovery blood profile was associated with circulating CD8 counts near control levels, continuing 80% depression of CD4 counts and a dropping total neutrophil count. This study shows that the 600 mg/kg MPA dose is a good model for immunosuppression, which satisfies all five criteria for immunosuppression with low morbidity and low mortality.     

Characterization of a Cryptosporidium muris infection and reinfection in CF-1 mice

To establish control values for circulating cells and immune associated organs over the course of a self-limiting Cryptosporidium muris infection and rechallenge infection, mice were sacrificed at intervals starting before oral inoculation and ending after oocyst shedding had ceased. These values were used in other experiments to evaluate changes in these parameters induced by a single dose glucocorticoid immunosuppression model and in other immunosuppression studies. Flow cytometry counts of circulating T-lymphocytes and neutrophils, differential leukocyte counts, leukocyte morphology, spleen and thymus changes, and oocyst shedding were evaluated. Immediately after C. muris oocyst inoculation and up to the start of oocyst production (day 0 to day 7), the circulating blood profile showed a 50% drop in all leukocytes, including both large and small lymphocytes and CD3, CD4 and CD8 T-lymphocytes. There was an initial slight rise in circulating mature neutrophils after oocyst inoculation but numbers promptly dropped below normal and remaineded below normal. In the differential cell counts, monocytes with a fat, oval morphology increased by 60% at 24 h and remained high through oocyst shedding and beyond (day 8 through day 36). During oocyst shedding and continuing past the end of shedding, T-lymphocytes increased 100%. Monocytes with a flat, angular morphology increased in a similar manner. Immediately after oocyst inoculation the spleen contracted by 29%, but became 92% larger than its pre-inoculation size by day 14 when heavy oocyst shedding began. It remained enlarged through the end of oocyst shedding (day 29) and beyond (day 36). Spleen volume decreased and increased similar to changes in T-cell numbers. Throughout the C. muris infection the thymus remained largely unchanged. The transit of an oocyst bolus was followed from the stomach through the gut to the colon. No oocysts could be found in the stomach, caecum or feces of mice one half hour after oocyst inoculation. Likewise, an oral bolus of India ink passed from the stomach entirely into the colon after 3 h; therefore, no oocysts from the inoculum passed completely through the intestine and out into the feces. Recovered mice rechallenged with C. muris showed increased B-lymphocyte numbers; however, T-lymphocyte numbers remained level. The large lymphocytes increased after rechallenge, peaking on day 3, then decreased through day 10. B-cell numbers followed a pattern similar to the large lymphocytes. On day 10 of infection monocytes with a fat oval morphology rose sharply while B-cells fell in number. In both the initial infection and the rechallenge there was no unique blood profile which could definitely indicate a protozoal disease or identify a specific point during the course of the disease. There was no increase in the number of either small or large lymphocytes prior to increases in fat or flat monocytes.     

Cross-reactivities with Cryptosporidium spp. by chicken monoclonal antibodies that recognize avian Eimeria spp

In a previous study, we have developed several chicken monoclonal antibodies (mAbs) against Eimeria acervulina (EA) in order to identify potential ligand molecules of Eimeria. One of these mAbs, 6D-12-G10, was found to recognize a conoid antigen of EA sporozoites and significantly inhibited the sporozoite invasions of host T lymphocytes in vitro. Furthermore, some of these chicken mAbs showed cross-reactivities with several different avian Eimeria spp. and the mAb 6D-12-G10 also demonstrated cross-reactivities with the tachyzoites of Neospora caninum and Toxoplasma gondii. Cryptosporidium spp. are coccidian parasites closely related to Eimeria spp., and especially C. parvum is an important cause of diarrhea in human and mammals. In the present study, to assess that the epitopes recognized by these chicken mAbs could exist on Cryptosporidium parasites, we examined the cross-reactivity of these mAbs with Cryptosporidium spp. using an indirect immunofluorescent assay (IFA) and Western blotting analyses. In IFA by chicken mAbs, the mAb 6D-12-G10 only showed a immunofluorescence staining at the apical end of sporozoites of C. parvum and C. muris, and merozoites of C. parvum. Western blotting analyses revealed that the mAb 6D-12-G10 reacted with the 48-kDa molecular weight band of C. parvum and C. muris oocyst antigens, 5D-11 reacted the 155 kDa of C. muris. Furthermore, these epitopes appeared to be periodate insensitive. These results indicate that the target antigen recognized by these chicken mAbs might have a shared epitope, which is present on the apical complex of apicomplexan parasites.