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.     

Comparison of some serological methods and coproscopic examinations for diagnosis of Giardia spp. invasion in dogs

Giardiasis was detected in 53.5% of dogs examined by FASTest Giardia Strip for use in dogs. Using the ProSpecT Giardia EZ Microplate Assay 52.2% of these results was confirmed. Cysts of Giardia spp. were found only in 6.5% of samples of feces examined by flotation or decantation techniques. The examinations confirmed problems with coproscopic diagnosis of giardiasis in dogs. They confirmed the greater usefulness of FASTest Giardia Strip for immunodiagnostic of giardiasis in carnivores.     

Evaluation of immunofluorescence microscopy and enzyme-linked immunosorbent assay in detection of Cryptosporidium and Giardia infections in asymptomatic dogs

The performance of immunofluorescence microscopy (IF) and enzyme-linked immunosorbent assay (ELISA) in canine feces was evaluated. IF and Cryptosporidium ELISA detected 10(5)oocysts/g, while the detection limit for Giardia ELISA was 10(4)cysts/g. The Cryptosporidium ELISA showed 94% specificity but only 71% sensitivity. The Giardia ELISA correlated well with IF (sensitivity 100%, specificity 96%) and was capable of detecting animal specific Giardia duodenalis genotypes. Visual interpretation appeared appropriate for assessment of ELISA results. The proportion of positive samples and possible zoonotic character of Cryptosporidium and Giardia infections in 150 asymptomatic Finnish dogs from the Helsinki area were studied. The overall proportion of dogs positive for Cryptosporidium was 5% (7/150) and that for Giardia 5% (8/150). In dogs < or =12 months old, the corresponding proportions were 17% and 19% (n=36). Sequence analyses of the 18S rDNA gene identified the isolates as Cryptosporidium canis and animal specific genotypes of G. duodenalis (assemblages C-E), indicating restricted risk of zoonotic transmission.     

Survey and molecular characterization of Cryptosporidium and Giardia spp. in owned companion animal, dogs and cats, in Japan

Compared with other countries, surveys of these parasites have been rarely performed in companion animals of Japan in spite of their significance for public health. Here, we investigated pet dogs and cats in Japan for the first time, and genetically analyzed the isolates to evaluate the risk of zoonotic infections. Seventy-seven fecal samples were collected from privately owned dogs and 55 samples from owned cats in Osaka city, Japan. Cryptosporidium oocysts were identified in 3/77 dogs (3.9%) and 7/55 cats (12.7%), and Giardia infection in 2/77 dogs (2.6%) and 1/55 cats (1.8%). Amplification of the target regions for genotyping was successful, Cryptosporidium isolates in dogs and cats were identified as C. canis and C. felis, respectively, and those of Giardia in dogs and cats were G. intestinalis Assemblages D and F. The discharge period of the oocysts varied within 3-16 weeks and that of the cysts was 12 weeks. To date, zoonotic types of both parasites have been identified in other animals in Japan, and further large-scale studies are needed to determine the distribution of zoonotic genotypes in these animals, especially those closely associated with humans.     

Comparison between natural infection by Cryptosporidium sp., Giardia sp. in dogs in two living situations in the West Zone of the municipality of Rio de Janeiro

In order to compare natural infection by Cyptosporidium sp. and Giardia sp. in clinically healthy dogs living in two situations (animal shelter and household pets), we conducted 166 faecal exams using the technique of centrifugation-flotation in a sugar-saturated solution. Of the faecal samples, 94 came from shelter dogs and 72 from house pets. Eighty-two (49.4%) contained parasite eggs, cysts or oocysts. Of these, four (2.41%) contained Cryptosporidium sp. oocysts and 52 (31.33%) contained Giardia sp. cysts. There was no statistically significant difference between the origin of the dogs and Cryptosporidium sp. infection. Infection by Cryptosporidium sp. was not associated with the living conditions and sex. With respect to Giardia sp., we observed a statistically significant difference (p < 0.001) between the shelter dogs (45%) and the household pets 12.3%. There was no association of infection by Giardia sp. with age or sex.     

Prevalence of selected zoonotic and vector-borne agents in dogs and cats in Costa Rica

To estimate the prevalence of enteric parasites and selected vector-borne agents of dogs and cats in San Isidro de El General, Costa Rica, fecal and serum samples were collected from animals voluntarily undergoing sterilization. Each fecal sample was examined for parasites by microscopic examination after fecal flotation and for Giardia and Cryptosporidium using an immunofluorescence assay (IFA). Giardia and Cryptosporidium IFA positive samples were genotyped after PCR amplification of specific DNA if possible. The seroprevalence rates for the vector-borne agents (Dirofilaria immitis, Borrelia burgdorferi, Ehrlichia canis, and Anaplasma phagocytophilum) were estimated based on results from a commercially available ELISA. Enteric parasites were detected in samples from 75% of the dogs; Ancylostoma caninum, Trichuris vulpis, Giardia, and Toxocara canis were detected. Of the cats, 67.5% harbored Giardia spp., Cryptosporidium spp., Ancylostoma tubaeforme, or Toxocara cati. Both Cryptosporidium spp. isolates that could be sequenced were Cryptosporidium parvum (one dog isolate and one cat isolate). Of the Giardia spp. isolates that were successfully sequenced, the 2 cat isolates were assemblage A and the 2 dog isolates were assemblage D. D. immitis antigen and E. canis antibodies were identified in 2.3% and 3.5% of the serum samples, respectively. The prevalence of enteric zoonotic parasites in San Isidro de El General in Costa Rica is high in companion animals and this information should be used to mitigate public health risks.     

Survey on giardiosis in shelter dog populations

Faecal samples from 183 dogs living in three different shelters in the Rome metropolitan area were randomly collected and examined for the prevalence of giardiosis. Giardia infections were detected by a commercially available ELISA test (ProspecT Giardia Microplate Assay). Overall prevalence was 55.2%. Prevalence rates in single shelters were 74.3, 35.5, and 20.9%, respectively. Using multivariate analysis, no association was found between Giardia-positivity and shelter or sex, breed, or diarrhoea. Giardia-positive dogs were more likely to be younger than 5-year-old (odds ratio [OR] = 2.87; 95% confidence interval [95% CI]: 1.07-7.77; p = 0.038), living together (OR = 2.58; 95% CI: 1.12-5.93; p = 0.026), and fed commercial wet food, both alone and combined with dry food (OR = 5.67; 95% CI: 1.59-20.24; p = 0.008). Correlation between Giardia infection and type of food has not been previously reported in dogs. Possible use of the ELISA test for detection of Giardia infection in dogs and zoonotic implications are discussed.     

Detection of zoonotic intestinal parasites in public parks of Spain. Potential epidemiological role of microsporidia

Several studies have demonstrated that the soil of public parks presents an important source of infection which has a significant impact on public health. Children are the main group affected by accidentally ingestion of contaminated soil. This study was performed in order to identify the presence of zoonotic parasites in dog and cat faecal and soil samples from public parks of Madrid, Spain. Six hundred twenty-five and seventy-nine soil and faecal samples (presumably from dogs and cats) respectively were collected from 67 parks. Intestinal parasites were identified in 27 parks (40.3%), which were contamined with Giardia sp. (19.4%), microsporidia (19.4%), Toxocara spp. (16.4%), Cryptosporidium sp. (6%), Entamoeba histolytica (3%) and Ancylostomidae (3%). Combinations of two or more intestinal parasites were found in 11 parks, and it was common to find Giardia and microsporidia together in samples. Intestinal parasites were detected in 18% (112/625) of soil samples. The most frequent parasite species found in the examined soil samples were Toxocara spp. (16.4%), followed by Giardia sp. (4.5%) and Strongyloides sp. larvae (3%). The zoonotic parasites found in the 79 faecal samples were Giardia sp. (17.7%), Cryptosporidium sp. (9%), E. histolytica (2.5%), Trichuris vulpis (1.3%), Toxascaris leonina (1.3%) and microsporidia spores (28%). Microsporidia characterization by amplification of DNA confirmed 10 samples as positive, eight for E. bieneusi and two for E. hellem by PCR. The role of those parasites in the environment are discussed.     

Gastrointestinal parasites of domestic cats in Perth, Western Australia

A study was conducted to determine the prevalence of gastrointestinal parasites in a sample of domestic cats in Perth and the knowledge of their owners about the control and potential for zoonotic transmission of these parasites. Faecal samples (418), collected from cats originating from five sources, were examined by microscopy and questionnaires administered to cat owners. Forty randomly selected samples were also screened using PCR in order to detect cysts of Giardia and oocysts of Cryptosporidium that may have been present in a faecal sample at very low levels. The overall prevalence of gastrointestinal parasites in domestic cats by microscopy was 8.6%. Pet shop kittens had the highest parasite prevalence (34.3%), followed by cats and kittens from breeding establishments (15.8%), refuge cats and kittens (8.3%), privately owned cats (2.3%), and boarding cats and kittens (1.6%). Surprisingly, 80% of the 40 cats tested by PCR were positive for Giardia duodenalis and 10% for Cryptosporidium. None of these cats were positive on microscopy. After adjusting for other factors with multiple logistic regression, kittens less than 6 months of age, and cats living in households with more than one cat or with a dog were significantly more likely to be parasitised. In the logistic regression model, the presence of parasitism was also significantly influenced by the number of anthelmintic doses administered in the 12 month period prior to the study. The majority (64.5%) of cat owners were aware that feline parasites could be transmitted to humans, however less than half (42.8%) were aware of the modes of transmission to humans.     

Cryptosporidium and Giardia in wild otters (Lutra lutra)

A total of 437 faecal samples from wild otter (Lutra lutra) were collected from 161 sites in Galicia (northwest Spain) and were analysed by a direct immunofluorescence antibody test (IFAT). Cryptosporidium sp. oocysts and Giardia sp. cysts were detected in 17 (3.9%) and 30 (6.8%) samples, respectively. The results demonstrate that otters may contribute to the contamination of watercourses, although further studies are required to establish which species or genotypes of Cryptosporidium and Giardia infect these animals and also the significance in terms of public health.     

Detection of Cryptosporidium oocysts in soil samples by enzyme-linked immunoassay

An ELISA protocol was adapted for detection of Cryptosporidium parvum oocysts in soil samples and the limit of detection of the test was determined. A modified indirect antigen capture ELISA protocol was developed using monoclonal antibodies against the oocyst outer wall. The accuracy of the ELISA was compared to spiked soil samples and measured in terms of sensitivity and specificity of the test. The performance of the ELISA was evaluated in field soil samples and measured using the kappa-statistics. Similarly, the performance of the ELISA was compared to the concentration flotation method, to a modified concentration flotation method and to a commercial ELISA (ProSpecT) in field fecal and soil samples.The limit of detection of the test was selected to be 10,000 oocysts/g. At this limit of detection, the ELISA had a sensitivity of 95% and specificity of 100%. The agreement between the ELISA and the modified flotation-concentration method in detecting Cryptosporidium oocysts in soil samples was 32% (kappa=0.32). The ELISA had the same relative sensitivity (82%) in comparison to both the flotation and ProSpecT in determining Cryptosporidium-infection status of an animal. The kappa-statistics was 0.26 for both tests. The developed ELISA proved to be a valuable diagnostic test for detecting oocysts in soil samples and has a potential application in determining the infection status of animals.     

Prevalence of Giardia and Cryptosporidium species in dog park attending dogs compared to non-dog park attending dogs in one region of Colorado

Dog parks are very popular in urban areas, but there are no current studies attempting to correlate visits to dog parks and risk of colonization by enteric parasites. The purpose of this study was to determine whether dog park visitation is associated with an increased prevalence of enteric parasites or an increase in prevalence of gastrointestinal signs in dogs in northern Colorado. Feces from dogs owned by veterinary students or Veterinary Teaching Hospital staff members were submitted with a completed survey form detailing dog park attendance rates, fecal character scores, and other clinical information. Feces were examined microscopically for parasites after sugar centrifugation, for Giardia spp. cysts and Cryptosporidium spp. oocysts by a commercially available immunofluorescence assay (FA) and the FA positive samples were genotyped after PCR amplification. The Giardia assemblages were determined using the glutamate dehydrogenase (GDH) β-giardin and triose phosphate isomerase (TPI) genes and the Cryptosporidium species were determined using the heat shock protein-70 gene. A total of 129 fecal samples were assayed; 66 were from dog park attending dogs and 63 were from non-dog park-attending dogs. The overall parasite prevalence rate was 7.0% (9 of 129 samples). Dog park attending dogs were more likely to be positive for Giardia or Cryptosporidium than non-dog park-attending dogs (p=0.0279), but there was no association of gastrointestinal signs with dog park attendance or with fecal flotation or FA results. The five Giardia isolates were assemblage C and/or D and the one Cryptosporidium isolate was Ctenocephalides canis.     

Prevalence of Cryptosporidium, Giardia and Isospora species infections in pet cats with clinical signs of gastrointestinal disease

This study reports the prevalence of Cryptosporidium, Giardia and Isospora species in cats showing signs of gastrointestinal disease. Records from a United Kingdom commercial diagnostic laboratory between December 2003 and December 2005 were reviewed. Of 1355 cats, Cryptosporidium species oocysts were found in 13 cats (1%), Giardia species trophozoites in 74 (6%), and Isospora felis oocysts in 46 (3%). In a second group of 48 cats, prevalence of Giardia species was 15% using an immunoassay for detection of antigen compared to 4% detected with microscopy. Prevalence of Giardia (9%) and Isospora (9%) species was higher in cats less than 6 months old. Gender and breed did not affect prevalence. There was a trend for Cryptosporidium and Isospora species infections to be detected in late autumn and early winter. Regional differences in prevalence were not detected. None of these organisms show a characteristic pattern of clinical signs. This study demonstrates that enteric protozoal infection is common in domestic cats showing signs of alimentary disease.     

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.     

Screening for Echinococcus granulosus in dogs: Comparison between arecoline purgation, coproELISA and coproPCR with necropsy in pre-patent infections

Echinococcus granulosus is an important zoonotic infection of dogs. The purpose of the present study assessed the performance of two laboratory diagnostic methods with arecoline purgation and necropsy in infected dogs. In total 65 dogs were successfully experimentally infected with protoscoleces of E. granulosus from ovine infection. At 14-34 days post-infection groups of dogs were purged with arecoline hydrobromide and then necropsied. Faecal samples were tested at weekly intervals by coproantigen ELISA and coproPCR. The necropsy infection rate with E. granulosus was 89.2 per cent. Only 43 per cent of dogs were successfully purged after one arecoline dose; this percentage increased to 76.9% for two doses of arecoline purgation. E. granulosus coproantigen was detected by coproELISA in 82.8% of faeces. The positive and negative predictive values for coproantigen ELISA were 96 and 44.4% respectively. E. granulosus DNA was detected in pre-patent faecal samples by coproPCR in 25.9% of dogs. These results indicate that coproELISA is more sensitive than arecoline purgation for the detection of pre-patent E. granulosus infection in dogs. CoproPCR detected E. granulosus DNA in dog faeces by 21 days post-infection before egg production.     

Prevalence of Giardia duodenalis and Cryptosporidium spp. in dogs from different living conditions in Uberlândia, Brazil

Infection rates with Giardia duodenalis and Cryptosporidium spp. were compared among dogs living under different conditions. Stool samples (n = 433) collected from dogs of different ages, gender, living conditions and origin were analyzed using three techniques, i.e., centrifugal flotation in zinc sulfate solution, centrifugal flotation in sucrose solution, and methylene blue gram safranin staining. Eighty-nine of the samples were from stay dogs living in shelters run by animal protection societies, 199 were from kennels and 122 from households. A total of 119 (29.0%) had G. duodenalis cysts and six (1.4%) were positive for Cryptosporidium spp. oocysts. Dogs from kennels were most frequently affected by G. duodenalis (49.7%) while those from shelters showed a higher prevalence of Cryptosporidium spp. (2.2%). A significant difference (p < or = 0.05) was observed between immature dogs and adults only with respect to Giardia infection. There was no significant difference between the gender with regard to the presence of either protozoan.     

P-58 Three cases of pathological dermal deposits associated with internal diseases in dogs (xanthomatosis, calcinosis and amyloidosis)

Intestinal parasites have been anecdotally associated with pruritus in dogs. A relationship was proposed when the elimination of a specific parasite resolved the pruritus, and re-infection with a parasite caused the pruritus to recur. Ascarids, coccidia, hookworms, tapeworms and whipworms have all been incriminated. Recent reports in the human and veterinary literature have suggested that there may be a relationship between Giardia infection and the development of pruritus and other allergic signs. In order to determine if there is an association between infection with Giardia and pruritus in dogs, faecal samples were collected from 71 pruritic dogs and 101 clinically normal dogs. Three faecal samples from each animal were analysed using a zinc-sulphate concentration technique for the presence of Giardia cysts, and one randomly selected faecal sample from each dog was analysed with an ELISA for Giardia -specific antigen. Ten of 101 dogs (9.9%) in the clinically normal group tested positive with the ELISA, and Giardia cysts were detected in the faeces of seven of these antigen-positive dogs. No Giardia cysts were found in the dogs that tested negative with the ELISA. None of the dogs in the pruritic group tested positive for Giardia by either method. The results indicated a negative association between Giardia infection and pruritic skin disease ( P  = 0.006). Cephalexin was commonly used in the pruritic group, but not in the control group, and may therefore be an explanatory variable.
Funding: Ontario Veterinary College Pet Trust.     

Occurrence of Giardia and Cryptosporidium in Norwegian red foxes (Vulpes vulpes)

Faecal samples from 269 Norwegian wild red foxes (Vulpes vulpes) shot during the hunting season (October-April) in 2002-2004 were examined for the presence of Giardia and Cryptosporidium. Cryptosporidium oocysts were detected in samples from 6 (2.2%) of the foxes, and Giardia cysts in 13 (4.8%) of the foxes. The prevalence of Giardia infection was significantly higher in juvenile male foxes than in adult male foxes, but no other significant differences between age and sex were found. No significant differences in prevalence related to geographical origin of animals were found. Insufficient nucleated Cryptosporidium oocysts were isolated for successful PCR, but genotyping of Giardia duodenalis isolates from seven foxes demonstrated a high degree of heterogeneity amongst them, with all isolates belonging to the zoonotic Assemblages A and B.     

Cryptosporidium spp. and other zoonotic enteric parasites in a sample of domestic dogs and cats in the Niagara region of Ontario

To determine the prevalence of Cryptosporidium spp. and other zoonotic enteric parasites in a sample of domestic dogs and cats in the Niagara region, Ontario, 5 of 26 clinics invited by mail survey reported their parasitological findings over 24 months. Stool samples collected by 1 clinic over 68 days were investigated for parasites by using several techniques (fecal concentration, acid-fast staining, and a Cryptosporidium immunoassay). The 5 clinics that provided data indicated Toxocara spp. as the most frequent finding. Parasitological study of 111 stool samples showed a high overall positivity rate in samples from both dogs (40%) and cats (36.6%). Cryptosporidium spp. antigen was detected in 7.4% and 7.3%, Toxocara spp. in 14.2% and 12.2%, and Giardia spp. 7.1% and 2.4% of dog and cat samples, respectively. The high prevalence of zoonotic parasites in the Niagara region is important, and increased awareness of their potential threat to human health is necessary. Additionally, further research into the zoonotic capacity of Cryptosporidium spp. and Giardia spp. is necessary.     

Pathogenic bacteria and parasites in wildlife and surface water

In the period October 2003 to August 2005, 897 faecal samples were collected from wild animals and examined for Salmonella spp., Campylobacter spp., and Shiga toxin producing Escherichia coli (STEC) O157, the prevalence of which was found to be 0.1%, 13.8%, and 0.5 %, respectively. Campylobacter spp. were isolated mainly from faecal samples collected from corvidae (59.8%), and meadow birds and waterfowl (22.4%). A subset of these samples was also examined for Cryptosporidium and Giardia oocysts and cysts. None of the 247 samples examined contained C. parvum oocysts, and only 1 sample (roe faeces) contained G. lamblia assemblage A cysts. In the period September to November 2006, samples of running or still surface water were collected at 10 sites on 5 days, to investigate the presence of Salmonella spp., Campylobacter spp., and STEC O157. Twenty (40.8%) of the surface water samples were positive for one or more bacterial pathogens. Seven (14.3%) samples were positiveforSalmonella spp., 14 (28.6%) samples were positive for Campylobacter spp., and 1 (2.0%) sample was positivefor E. coli O157. Samples collected at only 2 of the 10 sites were negative for the pathogens tested; samples collected at the other 8 sites were positive for the pathogens at least once. To gain a better picture of the potential human health risk, this study should be followed up with a more quantitative study of the occurrence of human pathogens in wildlife, taking into account the different natural habitats and behaviour of the different animal populations and a possible seasonal effect. Furthermore, the contamination of surface water with human pathogens should be investigated more extensively.