Crab-eating fox (Cerdocyon thous), a South American canid, as a definitive host for Hammondia heydorni

Hammondia heydorni is a cyst forming coccidia closely related to other apicomplexans, such as Toxoplasma gondii, Neospora caninum and Hammondia hammondi with a two-host life cycle. Dogs and other canids as red foxes (Vulpes vulpes) and coyotes (Canis latrans) may serve as definitive hosts for H. heydorni. Sporulated oocysts are infective for cattle, sheep and goats, which may serve as intermediate hosts. Herein, we describe the ability of crab-eating fox (Cerdocyon thous), a wild carnivore that is commonly found from northern Argentina to northern South America, to serve as definitive host of H. heydorni. The whole masseter muscle and brain from two 2-year-old bovines were collected, minced and pooled together for the fox infection. The bovine pooled tissues were equally administered to four foxes, in two consecutive days. Two foxes shed subspherical unsporulated oocysts measuring 10-15 mu m, after 8 and 9 days post-infection, respectively. One of the foxes eliminated oocysts for 5 days, while the other fox shed oocysts for 9 days. A DNA sample of oocysts detected at each day of oocyst elimination was tested by two PCRs, one of them carried out employing primers directed to the common toxoplasmatiid 18S and 5.8S ribosomal RNA coding genes (PCR-ITS1) and the other based on heat-shock protein 70 kDa coding gene (PCR-HSP70). These samples were also submitted to a N. caninum specific nested-PCR protocol based on a N. caninum specific gene (Nc5-nPCR). All of them were positive by PCR-ITS1 and PCR-HSP70 but negative by Nc5-nPCR. The PCR-ITS1 and PCR-HSP70 nucleotide sequences amplified from the oocysts shed by the foxes revealed 100% identity with homologous sequences of H. heydorni. In conclusion, it is clear that H. heydorni also uses the crab-eating fox as a definitive host. The crab-eating fox is usually reported to live in close contact with livestock in several regions of Brazil. Therefore, it is reasonable to infer that such carnivores may play an important role in the sylvatic and domestic cycles of H. heydorni infection. (C) 2009 Elsevier B.V. All rights reserved.

Identification of Hammondia heydorni oocysts by a heminested-PCR (hnPCR-AP10) based on the H-heydorni RAPD fragment AP10

Toxoplasma gondii, Hammondia hammondi, Neospora caninum, Neospora hughesi and Hammondia heydorni are members of the Toxoplasmatinae sub-family. They are closely related coccidians with similarly sized oocysts. Molecular diagnostic techniques, especially those based on polymerase chain reaction (PCR), can be successfully applied for the differentiation of Hammondia-like oocysts. In this paper, we describe a rapid and simple method for the identification of H. heydorni oocysts among other members of the Toxoplasmatinae sub-family, using a heminested-PCR (hnPCR-AP10) based on a H. heydorni RAPD fragment available in molecular database. DNA of oocysts of H. heydorni yielded a specific fragment of 289-290 bp in the heminested-PCR assay. No product was yielded when the primers were used for the amplification of DNA extracted from T. gondii, N. caninum, N. hughesi and H. hammondi, thus allowing the differentiation of H. heydorni among other members of the Toxoplasmatinae sub-family. The hnPCR-AP10 was capable of detecting H. heydorni genetic sequences from suspensions with at least 10 oocysts. In conclusion, the hnPCR-AP10 proved to be a reliable method to be used in the identification of H. heydorni oocysts from feces of dogs. (C) 2010 Elsevier B.V. All rights reserved.

Shedding of Neospora caninum oocysts by dogs fed different tissues from naturally infected cattle

Neospora caninum is one of the most important causes of abortion in dairy cattle worldwide. The distribution of N. caninum in tissues of adult cattle is unknown and the parasite has not been demonstrated histologically in tissues of cows. In the present study the distribution of N. caninum in different tissues of adult cattle was evaluated by bioassays in dogs. Seventeen dogs (2-3 month-old) were fed different tissues of 4 naturally exposed adult cattle (indirect fluorescent antibody test N. caninum titer >= 400): 5 were fed with masseter: 5 with heart, 3 with liver, 4 with brain, and 3 pups were used as non-infected control. Two dogs fed masseter, 2 fed heart, 1 fed liver, and 3 fed brain shed oocysts, and all dogs presented no seroconvertion to N. caninum during the observation period of 4 weeks. The oocysts were confirmed as N. caninum based on the detection of N. caninum-specific DNA by PCR and sequencing. The results indicate that dogs can be infected by N. caninum with different tissues of infected cattle. (C) 2011 Elsevier B.V. All rights reserved.

Differential diagnosis of oocysts of Hammondia-like organisms of dogs and cats by PCR-RFLP analysis of 70-kilodalton heat shock protein (HSP70) gene

Organisms of the genera Toxoplasma, Hammondia and Neospora, the Hammondia-like organisms, are closely related coccidian with similarly sized oocysts. Therefore, a diagnosis based on microscopy of oocysts in feces is not a method of choice for species identification of these important parasites. In this paper, we present a polymerase chain reaction coupled with restriction fragment length polymorphism (PCR-RFLP) method to differentially diagnose oocysts of Toxoplasma gondii from oocyst of Hammondia hammondi. Another PCR-RFLP was designed to differentiate oocysts of Hammondia heydorni from oocysts of Neospora spp. Both PCR-RFLP are based on nucleotide sequences of the Hsp70 coding gene. In conclusion, we presented two alternative molecular diagnostic assays that can be successfully applied for the differentiation of oocysts of Hammondia-like organisms shed by felids and canids.

Prevalence of intestinal parasites in dogs from São Paulo State, Brazil

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. (C) 2002 Elsevier B.V. B.V. All rights reserved.

A SURVEY OF NEOSPORA CANINUM AND TOXOPLASMA GONDII INFECTION IN URBAN RODENTS FROM BRAZIL

Toxoplasma gondii is a protozoan parasite that infects humans and other warm-blooded animals; it uses feral and domestic cats as the definitive hosts. Neospora caninum is a protozoan parasite of animals whose life cycle is very similar to T. gondii but uses canids as definitive hosts. Small rodents play an important role in the life cycle of T. gondii, and a few findings indicated that they may be natural intermediate hosts for N. caninum. The present study was aimed at identifying infections by T. gondii and N. caninum in urban rodents. Infections by T. gondii were quantified using isolation of the parasite by bioassay in mice; molecular methods were also used for both parasites. Overall, 217 rodents were captured. Brain and heart tissues of all rodents were bioassayed in mice for the detection of T. gondii infection. Brain and heart tissues of 121 rodents had the DNA extracted for molecular analysis. Toxoplasma gondii was isolated by bioassay from a single rodent. From the 121 rodents tested for the presence of T. gondii DNA, 2 animals were positive. In contrast, DNA of N. caninum was not detected in any of the samples. In conclusion, the surveys of N. caninum and T. gondii infection in Rattus rattus, Rattus norvegicus, and Mus musculus captured in urban areas of Sao Paulo reveal a striking low frequency of occurrence of these infections.

Incidence of Neospora caninum and other intestinal protozoan parasites in populations of Swiss dogs

The protozoan parasite Neospora caninum is one of the most important abortifacient organisms in cattle worldwide. The dog is known to act as definitive host although its potential role as infection source for bovines still remains unelucidated. The aim of the present study was to compile initial epidemiological data on the prevalence and incidence of N. caninum in Swiss dogs acting as definitive hosts. Thus, 249 Swiss dogs were investigated coproscopically in monthly intervals over a period of 1 year. A total of 3289 fecal samples was tested by the flotation technique. Among these, 202 were shown to contain Sarcocystis sp. (6.1%), 149 Cystoisospora sp. (=Isospora sp.; 4.5%) and 25 Hammondia/Neospora-like oocysts (HNlO) (0.7%). All but one sample containing HNlO were from different dogs; one dog shed HNlO at two subsequent time points. Calculation of the yearly incidence for HNlO resulted in the surprisingly high value of 9.2%. Farm dogs exhibited a higher incidence for HNlO than urban family dogs. Thirteen out of the 25 HNlO-samples showed sporulation after 5 days incubation at room temperature. HNlO were further differentiated by species-specific PCR. However, all HNlO-samples were negative for N. caninum, Hammondia heydorni and Toxoplasma gondii. One reason may be the low oocyst density found in most fecal samples, which did not permit us to carry out PCR under optimal conditions. Three out of the 25 HNlO-cases contained enough oocysts to allow further enrichment and purification by the flotation technique. Subsequently, twenty to fifty sporulated HNlO-oocysts were orally administered to Meriones unguiculatus. All gerbils were seronegative for N. caninum at 5 weeks p.i. A N. caninum-seroprevalence of 7.8% was determined by ELISA upon 1132 serum samples collected from dogs randomly selected by veterinarians among their clinical patients.