Molecular characterization of Cryptosporidium and Giardia isolates from cattle from Portugal

Fecal samples from 291 calves and 176 adult cattle in Northern Portugal were screened for Cryptosporidium and Giardia using a formalin-ethyl acetate concentration method. Acid-fast staining techniques for Cryptosporidium oocyst identification and direct microscopic observation of fecal smears for Giardia cyst identification were performed so as immunofluorescence microscopy examination. Polymerase chain reaction methods were employed to determine the genotype of each isolate. Molecular characterization was performed using amplification and sequencing of the hsp70 and 18SrRNA genes of Cryptosporidium and beta-giardin gene and glutamate dehydrogenase for assemblage determination of Giardia duodenalis. Seventy-four out of 291 calves (25.4%) and 8 out of 176 adult bovines (4.5%) were positive for Cryptosporidium. Forty-one out of 291 calf samples (14.1%) and 1 out of 176 adults samples (0.57%) were positive for Giardia. From the Cryptosporidium positive samples we obtained 63 isolates from calves samples and 7 isolates from adult samples. Additionally, Giardia was isolated in 13 out of 41 positive samples from calves and it was also possible to isolate Giardia from the positive adult sample. Molecular characterization of the Cryptosporidium and Giardia isolates showed us that C. parvum and G. duodenalis assemblage E were the prevalent species. C. parvum may infect humans, representing a potential public health risk. On the other hand, the assemblages B and A2 of Giardia, previously described in humans, were here identified in cattle. Further studies will be needed for determine the importance of cattle as carrier of zoonotic assemblages of G. duodenalis.     

Prevalence and genotypes of Giardia duodenalis in dairy and beef cattle in farms around Charlottetown, Prince Edward Island, Canada

Prevalence of Giardia duodenalis in dairy and beef cattle on farms around Charlottetown, Prince Edward Island (Canada) was determined by analyzing feces using direct immunofluorescence antibody microscopy. Genotypes were determined by 16S-rRNA sequencing. Fecal samples (n = 892) were collected from adult cattle in dairy tie-stall, dairy free-stall, and beef herds (10 herds each), and from calves (n = 183) from 11 dairy farms. Prevalence rates were 38% and 51% in cows and calves, respectively. Giardia duodenalis was present in all dairy herds, in 9/10 beef herds and in calves from 10/11 herds examined. Prevalence rates were 40% and 41% for cows in tie- and free-stall herds, respectively, and 27% for beef cows. Zoonotic Assemblage A was found in 12.2% of calves concomitantly infected with Assemblage E. All successfully sequenced samples (114/128) from cows corresponded to Assemblage E. Giardia duodenalis is highly prevalent in cattle herds in Prince Edward Island and Assemblage A in calves is a potential public health concern.     

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.     

Survey of Cryptosporidium spp. and Giardia spp. infections in various animals at a zoo in Japan.

A total of 284 fecal samples of 89 species (43 mammalian species and 46 avian species) were examined for Cryptosporidium oocysts and Giardia cysts from 1999 to 2002. Each sample was collected at the zoo located at Osaka in Japan and examined by microscopy after performing the sucrose flotation method and by two immunofluorescent assay kits for detection of Cryptosporidium oocysts and Giardia cysts. Cryptosporidium spp. was found only in a raccoon dog (Nyctereutes procyonoides), and Giardia spp. was detected in a mandarin duck (Aix galericulata) and two ruddy shelducks (Tadorna ferruginea). In this study, the prevalences of these parasites were found to be low. However, these results suggested that the infected animals could serve as a source of contamination for surface water. This is the first report about the survey of Cryptosporidium spp. and Giardia spp. at a zoo in Japan.     

A survey of the prevalence of Giardia in dogs presented to Canadian veterinary practices

Fecal samples (1216) were collected from dogs presented to 15 veterinary practices across Canada and tested for Giardia by ELISA for group-specific antigen. If positive, the presence of Giardia was confirmed by microscopy. Eighty-eight submissions tested positive by ELISA and microscopy. The overall prevalence rate of Giardia was 7.2%.     

Histopathological survey of protozoa, helminths and acarids of imported and local psittacine and passerine birds in Japan.

A total of 534 psittacine and passerine birds consisting of 241 imported and 293 local birds were examined histologically. As a result, the following parasites were found: Giardia (86 cases), Knemido-coptes (26 cases), coccidia (10 cases), Ascaridia (6 cases), Cryptosporidium (5 cases), Sarcocystis (5 cases), tapeworm (4 cases), microfilaria (2 cases), Hexamita (1 case), and Spiroptera (1 case). High incidences of giardiasis and knemido-coptic infestation were detected in the local birds, but rarely in the imported birds. Giardial trophozoites were observed mainly in the duodenum of budgerigars (Melopsittacus undulatus). Knemidocoptic mites burrowed into the epidermis producing proliferative dermatitis in 25 budgerigars and 1 African Grey Parrot (Psittacus erithacus erithacus). This ectoparasite often infested the skin around the cloaca. Coccidiosis was seen only in the small intestines of the finch (Poephila gouldiae gouldiae), African Grey Parrot, Rainbow lory (Trichoglossus haematodus), Indian Ring-necked parakeet (Psittacula krameri manillensis) and peach-faced lovebird (Agapornis roseicollis). Two parrots (Amazona aestiva aestiva and Psittacus erithacus erithacus) and two budgerigars had intestinal cryptosporidiosis. Conjunctivitis associated with cryptosporidial infection was seen in a lovebird. Sarcocystis cysts containing crescent-shaped bradyzoites were found not only in the thigh and breast but also in the heart and cloacal muscles. Other organisms such as Ascaridia, tapeworm, microfilaria, Hexamita, and Spiroptera were clinically less significant. However, infections such as Giardia and Cryptosporidim might have zoonotic implications.     

Prevalence of Cryptosporidium and Giardia in roe deer (Capreolus capreolus) and wild boars (Sus scrofa) in Galicia (NW, Spain)

Faecal samples from 224 roe deer (Capreolus capreolus) and 381 wild boars (Sus scrofa) shot during the 2008-2009 hunting season (August-January) in Galicia (NW Spain) were examined to determine the presence and intensity of infection by Cryptosporidium and Giardia. Analysis of a single sample from each of the roe deer revealed that the prevalence of cryptosporidiosis and giardiosis was 1.3% and 5.3% respectively. The prevalence of Giardia infection was significantly higher in juvenile female roe deer than in adult females, but no other significant differences were found in relation to age and sex. In wild boars, the prevalence of cryptosporidiosis and giardiosis was 7.6% and 1.3% respectively. The prevalence of Cryptosporidium infection was significantly higher in juvenile male wild boars than in adult males, but no other significant differences were found in relation to age or sex. In both groups of wild animals, the number of Cryptosporidium oocysts per gram of faeces (OPG) ranged from 5 to 200 and the number of Giardia cysts per gram of faeces (CPG) was between 5 and 47; there were no significant differences between the two groups with respect to number of infections. This is the first large study of Cryptosporidium and Giardia in roe deer and wild boars in hunting areas in Spain and the results demonstrate a low, but widespread prevalence of Cryptosporidium and Giardia in these animals.     

Colloid in the anterior pituitary of helmet guinea fowl (<Emphasis Type="Italic">Numida meleagris galeata</Emphasis>): Morphometric analysis and pattern of occurrence in relation to apoptosis

Colloidal accumulations in the pars distalis of helmet guinea fowls at various ages from 1 to 450 days were examined by Periodic acid-Schiff reaction, immunohistochemistry and electron microscopy. Round, ovoid and elongated colloids were observed. Colloids (69.5 ± 2.997) with 0.169 ± 0.014 μm mean diameter were already present in a 1-day-old bird. Numerous colloids were encountered in 450 days old birds (2931.333 ± 29.847) with 2.263 ±  0.078 μm mean diameter of round colloids. A significant difference in the mean colloidal number and diameter between young and adult birds was observed. In young birds (aged 1–30 days) both Periodic acid-Schiff reaction positive colloids and S-100 positive folliculostellate (FS) cells were found to appear first on or near the posterolateral region. In adult birds, FS cells were found to completely surround the colloids. We examined the biochemical components of colloids and the relationship with apoptosis by immunohistochemistry. Results showed that the colloids are composed of clusterin protein. Apoptotic cells detected by single stranded DNA (ssDNA) were abundant and localized preferentially near colloids. To define clearly the type of cells undergoing apoptosis in the anterior pituitary, we performed electron microscopy. Numerous endocrine cells at different stages of apoptosis were found engulfed by FS cells that were in close association with the colloidal accumulations. The occurrence of extremely large number of colloids in relation to apoptotic profiles in anterior pituitary of helmet guinea fowl is discussed.     

Presence and molecular characterisation of Giardia and Cryptosporidium in alpacas (Vicugna pacos) from Peru

The presence of Giardia and Cryptosporidium was investigated in 274 faecal samples of alpacas (Vicugna pacos) from 12 herds from Peru by immunofluorescence microscopy and PCR amplification and sequencing of fragments of the ssu-rRNA and β-giardin genes from Giardia spp., as well as the ssu-rRNA gene from Cryptosporidium spp. A total of 137 samples (50.0%) were positive for Giardia spp., and 12 samples (4.4%) for Cryptosporidium spp. In ten samples (3.6%), co-infection by both pathogens was found. Herd prevalence was found to be 91.7% (11/12 herds) for Giardia and 58.3% (7/12 herds) for Cryptosporidium. Regarding the age of the animals, although Giardia was detected in animals as young as 1 week, the prevalence increased with age, reaching 80% by 8 weeks. Similarly, the highest percentage of Cryptosporidium detection (20%) was also found in the 8 week-old group. By PCR, 92 of the 274 analysed samples were positive for Giardia. Sequencing of the amplicons showed the existence of Giardia duodenalis assemblage A in 67 samples; G. duodenalis assemblage E in 24 samples; and inconsistent results between the two molecular markers used in a further sample. Cryptosporidium was only detected by PCR in 3 of the 274 samples; Cryptosporidium parvum was identified in two samples and Cryptosporidium ubiquitum in one sample. This study is the first performing molecular characterisation of both parasites in Peruvian alpacas, and the first report of C. ubiquitum in this host. The identification of G. duodenalis assemblage A, C. parvum and C. ubiquitum, suggests that zoonotic transmission of these enteropathogens between alpacas and humans is possible.     

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.     

Identification of genotypes of Giardia intestinalis isolates from a human and calf in Japan

Giardia intestinalis is recognized as a significant pathogen in humans and animals, causing diarrhea. Recent molecular studies indicate that G. intestinalis is composed of genetically distinct multiple genotypes. Therefore, it is valuable to distinguish among genotypes in the epidemiology of Giardia infection in humans and animals. Although G. intestinalis has been found in humans and animals in Japan, the genotype of isolates remains unclear except for several isolates from dogs, because identification has been performed only by conventional microscopy. We report herein the genotypes of G. intestinalis isolates distinguished by a phylogenetic analysis. G. intestinalis isolates originated from a patient and a calf were found to have Assemblage B and E, respectively.     

A cross-sectional study of Leishmania spp. infection in clinically healthy dogs with polymerase chain reaction and serology in Greece

The prevalence of gastrointestinal parasites in stray dogs, and dogs with owners was investigated by fecal examinations from 271 dogs employing sedimentation, simple flotation and centrifugation-flotation methods. The centrifugation-flotation method, when compared to simple flotation or sedimentation methods was generally more accurate in the diagnosis of all intestinal parasites, but statistical differences were detected only in relation to Giardia spp. and Cystoisospora spp. (synonym Isospora spp.). The following parasites, with their respective prevalence, were diagnosed in the fecal samples: Ancylostoma spp. (23.6%); Toxocara canis (5.5%); Trichuris vulpis (4.8%); Spirocerca lupi (1.9%); Dipylidium caninum (0.7%); Giardia spp. (12.2%); Hammondia heydorni (2.6%); Cystoisospora spp. (8.5%); and Sarcocystis spp. (2.2%). The prevalence of most parasites was similar for dogs of mixed-breed and for dogs of a defined-breed, except for Cystoisospora spp. and T. canis which showed a significantly higher prevalence in mixed-breed dogs. The prevalence of Ancylostoma spp. (17.1%) was significantly lower in stray dogs than in those with an owner (31.9%) and the prevalence of Giardia spp. and Cystoisospora spp. was higher in stray dogs (P < 0.05). No effect of season on the occurrence of the different parasite genera could be observed, except for Ancylostoma spp., for which an increase in the percentage of dogs shedding eggs was observed at the beginning of Summer with a peak occurrence during April and May (Autumn). The prevalence of Ancylostoma spp., T. canis, T. vulpis, Giardia spp. and Cystoisospora spp. was higher in adult males than in adult females, but significant differences between the two groups occurred only with Giardia spp. Young animals were found to more frequently shed Nematode eggs in feces than adult animals.     

Metronidazole for the treatment of feline giardiasis

There are several drugs available for the treatment of giardiasis in cats, including metronidazole. The purpose of this study was to determine whether metronidazole benzoate administered at a dose of 25 mg/kg, orally, twice a day for 7 days lessens or eliminates Giardia cyst shedding in cats with chronic infection. Twenty-six, adult, laboratory-reared cats were used in this study. Sixteen cats had been inoculated orally with cysts of a human Giardia sp. isolate and had completed a Giardia vaccine study in one animal holding room. The other ten cats were infected with the same Giardia sp. presumably by contamination from the adjacent room where the Giardia vaccine study cats were located. From each cat, a fecal sample was collected within 1 week of the start of treatment and then every 2 to 4 days for 15 days after treatment was completed. Fecal samples were analyzed for the presence of Giardia cysts using a commercially available direct immunofluorescence test (IFA). Clinical signs of drug toxicity were not detected during the study.     

Identification of genotypes of Giardia intestinalis isolates from dogs in Japan by direct sequencing of the PCR amplified glutamate dehydrogenase gene

Giardia has been detected in domestic dogs in Japan, but the genotype of isolates has remained unclear because identification has relied on conventional microscopy. Here we tried to identify the genotypes of four isolates from dogs in Japan by direct sequencing of the PCR amplified Giardia glutamate dehydrogenase (GDH) gene. The primer pair GDHF3 and GDHB5, targeting the GDH gene, was designed to prime a region of the GDH gene sequence conserved in the strains found to have the dog-specific genotype. The specific PCR product (approximately 220 bp), amplified with this primer pair, was only observed when Giardia DNA was used as the template. The sequences of the diagnostic fragments were identical among the isolates from dogs, and were differed by 15 bp or 1 bp from the strains, which were found to be the dog-specific genotypes, Assemblage C or D respectively. To verify the identity of the amplified DNA, a phylogenetic analysis was performed. Consequently, the sequence of the isolates from dogs clearly clustered with the strain found to be Assemblage D with neighbor-joining analyses. Therefore, all the isolates from dogs examined were identified as the dog-specific genotype, Assemblage D. In the present study, we revealed the genotype of Giardia isolates in Japan, and showed that direct sequencing of the PCR product amplified with the primer pair GDHF3 and GDHB5 was a useful tool for distinguishing between the zoonotic and dog-specific genotypes.     

Prevalence of Microsporidia, Cryptosporidium spp., and Giardia spp. in beavers (Castor canadensis) in Massachusetts.

Feces from 62 beavers (Castor canadensis) in Massachusetts were examined by fluorescence microscopy (IFA) and polymerase chain reaction (PCR) for Microsporidia species, Cryptosporidium spp., and Giardia spp. between January 2002 and December 2004. PCR-positive specimens were further examined by gene sequencing. Protist parasites were detected in 6.4% of the beavers. All were subadults and kits. Microsporidia species were not detected. Giardia spp. was detected by IFA from four beavers; Cryptosporidium spp. was also detected by IFA from two of these beavers. However, gene sequence data for the ssrRNA gene from these two Cryptosporidium spp.-positive beavers were inconclusive in identifying the species. Nucleotide sequences of the TPI, ssrRNA, and beta-giardin genes for Giardia spp. (deposited in GenBank) indicated that the four beavers were excreting Giardia duodenalis Assemblage B, the zoonotic genotype representing a potential source of waterborne Giardia spp. cysts.     

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.     

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.     

Deaths in yellow-eyed penguins (Megadyptes antipodes) on the Otago Peninsula during the summer of 1990

About 150 rare, adult, yellow-eyed penguins died over a short period during the summer of 1989-1990 on the Otago Peninsula. These were from a total mainland population estimated at 240 breeding pairs. Penguin chicks and non-breeding birds were not affected, but there were indications of shortages in feed supply for birds that bred successfully. Thirteen dead penguins were examined. In ten birds, the cause of death was not established. Although it was commonly found that adult birds had little or no food in their gut, none were considered to have starved to death. No consistent pathological lesions were found, nor were any viruses, Chlamydia or significant bacteria recovered from selected tissues. Toxicology tests ruled out poisoning by copper, zinc, iron, lead, arsenic, selenium, cadmium, mercury, organophosphates, organochlorines, polychlorinated biphenyls, hexachlorobenzene or the toxins of Clostridium perfringens, Clostridium botulinum and dinoflagellates. The problem did not recur during the following summer.     

Treatment of giardiasis in adult Greyhounds, using ipronidazole-medicated water

Two of 3 adult Greyhounds with clinical signs of giardiasis were treated by the addition of ipronidazole hydrochloride to their water supply (126 mg/L) for 7 days. Within 15 hours, the dogs had improved clinically, and large numbers of degenerating Giardia cysts were passed. After 54 hours, cyst shedding had ceased. The nontreated control dog continued to shed Giardia cysts. During an additional 7 days, the dogs were treated with ipronidazole-medicated water (378 mg/L) and remained clinically normal.     

Prevalence and genotypic characterisation of Giardia in dairy calves from Western Australia and Western Canada

In this study, the prevalence of Giardia duodenalis infections was determined in Western Canadian and Western Australian dairy calves. Faecal samples were collected from Holstein calves located on a commercial dairy near Lethbridge, Alta., Canada (N=28) and from calves located on two commercial dairies located near Perth, WA, Australia (N=36). Faecal samples were examined for the presence of Giardia cysts using sucrose gradient centrifugation, followed by immunofluoresence microscopy. DNA was then extracted from Giardia isolates obtained from positive samples. A PCR based method was employed to amplify and sequence a 292bp region of the 16S-rRNA gene. Genetic sequences obtained from Giardia isolates were compared to each other and to previously sequenced isolates. Following a single faecal sample, 58% of Western Australian calves and 57% of Western Canadian calves were positive for Giardia. Geometric mean cyst counts/g of faeces were 839 for Western Australian calves and 3475 for Western Canadian calves, but these values did not differ significantly. Genetic sequences were obtained from 10 calves from Western Canada, while six sequences were obtained from Western Australian calves. Of the Western Canadian isolates, eight aligned with the proposed 'Hoofed livestock' genotype. Of the five isolates obtained from Western Australian calves, four sequences were identical to the 'Hoofed livestock' genotype. Two isolates from the Western Canadian calves and one isolate from the Western Australian calves had the identical genetic sequence to the Genotype (Assemblage) A sequence, a common human genotype. The results of this study demonstrate, for the first time, that Giardia infections occur in Western Australian calves. Also, calves from different geographical locations appear to be primarily infected with a Giardia genotype unique to hoofed livestock. However, calves can shed Giardia cysts potentially infective for humans. Thus, Giardia infections should be considered important to Australian dairy producers, and infections in calves may pose a risk to public health regardless of geographical location.