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.     

Coprodiagnosis of Hammondia heydorni in dogs by PCR based amplification of ITS 1 rRNA: differentiation from morphologically indistinguishable oocysts of Neospora caninum

Hammondia heydorni is thought to be a non-pathogenic coccidian parasite of dogs that is closely related to Neospora caninum, an important parasite of cattle and dogs. Oocysts of these two species are morphologically indistinguishable from each other. A population of 2240 dogs in the Czech Republic was screened for the presence of H. heydorni/N. caninum oocysts and five (0.22%), represented by five of 3135 faecal samples (0.16%), were positive. The internal transcribed spacer 1 region of the rRNA gene (ITS1) from two isolates were cloned and the DNA sequences were identical with those of the ITS1 of H. heydorni. Based on the rRNA sequences available for H. heydorni and related coccidia, the primer pair JS4-JS5 was designed to amplify the 3' end of the small subunit (SSU) rRNA gene and ITS1 of H. heydorni. When tested on DNA extracted from a variety of parasites, the primers amplified a specific 267 bp fragment in our isolates only. The presence of DNA equivalent to 10 oocysts was sufficient for the amplification of the ITS1. We present a PCR-based diagnostic method as the only fast and reliable method for the diagnosis of H. heydorni in dogs.     

Studies on serological cross-reaction of Neospora caninum with Toxoplasma gondii and Hammondia heydorni

The enzyme-linked immunosorbent assay (ELISA) was used to examine cross-reactivity of Neospora caninum with Toxoplasma gondii and Hammondia heydorni. Anti-T. gondii mouse and cat sera cross-reacted with N. caninum soluble antigen (NLA), but not with the recombinant surface antigen (NcSRS2). Anti-H. heydorni dog sera showed no cross-reactivity with either the NLA antigen or the NcSRS2. Lack of cross-reactivity between anti-H. heydorni sera and N. caninum antigens, and the cross-reactivity of anti-T. gondii sera with the NLA suggest that N. caninum has common antigens to T. gondii except for NcSRS2 based on serology. In light of several studies suggesting a closer relationship between N. caninum and H. heydorni than with T gondii, examination of serological cross-reactivity with N. caninum may be necessary to further classify the parasites in addition to molecular and morphological studies and clarification of the life cycle.     

Hammondia heydorni oocysts in the faeces of a greyhound in New Zealand

AIMS: To identify oocysts found in faecal material of a greyhound. METHODS: Polymerase chain reaction (PCR) and DNA sequencing were used to study genomic DNA isolated from oocysts purified from faeces of a greyhound. RESULTS: Database searches with the DNA sequences obtained showed they were derived from Hammondia heydorni. A species-specific PCR was developed to detect H. heydorni DNA. CONCLUSIONS: Light microscopy in conjunction with PCR and DNA sequencing definitively identified the presence of H. heydorni oocysts in faeces of a greyhound. CLINICAL RELEVANCE: This study confirms the presence of H. heydorni in New Zealand and indicates the need to correctly identify similar oocysts from dogs, rather than assume they are Neospora caninum.     

Dog shedding oocysts of Neospora caninum: PCR diagnosis and molecular phylogenetic approach

Results of molecular determination of a dog isolate of Neospora caninum in the Czech Republic are presented. Colorless bisporocystic oocysts measuring 10-13 micro m x 10-11 micro m were recovered from feces and used for DNA isolation. A diagnostic PCR procedure using previously described molecular methods was performed to determine the species. The N. caninum species-specific primers based on the Nc 5 region produced a positive result, while primers specific for Hammondia heydorni rDNA internal transcribed spacer 1 (ITS1) was negative. Sequencing and phylogenetic comparison of ITS1 rDNA and the D2 domain of the large subunit rDNA (D2 LSU) determined our isolate to be N. caninum. Phylogenetic analysis of closely related genera Toxoplasma, Neospora and Hammondia based on ITS1 and D2 LSU robustly distinguished three clades: (i). Toxoplasma gondii + Hammondia hammondi, (ii). N. caninum + Neospora hughesi, and (iii). H. heydorni. Based on phylogenetic relationships we propose three acceptable suggestions to solve the problem of taxonomy of these genera.     

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.     

Incidence of coccidia in dogs in Brno and the surrounding area

In the course of two years, 663 dogs from Brno and its vicinity were subjected to clinical and parasitological examination with a special reference to the occurrence of coccidia. A saturated sugar solution with the specific weight of 1150 was used as the flotation medium. Coccidia were found in 2.17% of the dogs examined. The oocysts of the groups Isospora ohioensis, I. burowsi and I. neorivolta were identified most frequently (2.11% of the dogs). Four dogs (0.60%), all alike, eliminated the oocysts of Hammondia heydorni and sporocysts of Sarcocystis species, and two dogs oocysts of Hammondia heydorni and sporocysts of Sarcocystis species, and two dogs (0.30%) excreted oocysts of I. canis. Out of the animals infected, 58% were younger than six months. Clinical signs were observed only at a medium to strong invasion with oocysts of the species I. ohioensis, I. burowsi and I. neorivolta. The digestive tract was affected most severely--the animals suffered from bloodless diarrhoeas lasting two to twelve days, from occasional vomiting and systemic dehydration. Invasion by I. canis, Hammondia heydorni and Sarcoystis sp. did not cause any clinical changes.     

Camel (Camelus dromedarius) and sheep (Ovis aries) meat as a source of dog infection with some coccidian parasites.

Experimental infection of dogs with camel (Camelus dromedarius) meat resulted in infection of the dogs with Isospora canis, Hammondia heydorni and Sarcocystis cameli. The dogs fed sheep (Ovis aries) meat passed oocysts of Isospora canis, Isospora ohioensis and sporocyts of Sarcocystis spp. Extraintestinal stages were detected in the intestinal lymph node of a rabbit killed 4 days following inoculation with Isospora ohioensis oocysts. Dogs fed the rabbit (killed 4 days after inoculation with I. ohioensis) passed I. ohioensis oocysts in their faeces 8 days post-infection.     

Failure of dogs to shed oocysts after being fed bovine fetuses naturally infected by Neospora caninum

Neospora caninum is a protozoan that causes abortion in cattle. The dog has recently been identified as a definitive host for N. caninum. To verify if bovine fetuses can infect dogs, nine 2-4-month-old dogs were fed bovine fetuses naturally infected by N. caninum. None of the dogs excreted oocysts, seroconverted, had clinical signs or lesions compatible with N. caninum infection. Additional studies will be necessary to determine the natural mode of infection of dogs by N. caninum.     

Occurrence of Toxoplasma gondii and Hammondia hammondi oocysts in the faeces of cats from Germany and other European countries

Faecal samples of 24,106 cats from Germany and other European countries were examined microscopically in a veterinary laboratory in Germany between October 2004 and November 2006 to estimate the prevalence of animals shedding Toxoplasma gondii or Hammondia hammondi oocysts. Oocysts of 9-15 microm size with a morphology similar to that of H. hammondi and T. gondii were found in 74 samples (0.31%). A total of 54 samples were further characterised to achieve a species diagnosis and to determine the genotype of T. gondii isolates by PCR and PCR-RFLP. From these samples, 48 isolates were obtained: 26 (0.11%) were finally identified as T. gondii and 22 (0.09%) as H. hammondi. T. gondii-positive samples came from Germany, Austria, France and Switzerland while H. hammondi was detected in samples from Germany, Austria and Italy. In two samples (one T. gondii and one H. hammondi), PCR indicated the presence of Hammondia heydorni DNA. No Neospora caninum DNA was detected in any of the feline faecal samples. Twenty-two of the 26 T. gondii isolates could be genotyped. A PCR-RFLP analysis for the SAG2, SAG3, GRA6 and BTUB genes revealed T. gondii genotype II in all cases. Morphologically, H. hammondi oocysts exhibited a statistically significantly smaller Length-Width-Ratio than T. gondii oocysts.     

Experimental infection of dogs (Canis familiaris) with sporulated oocysts of Neospora caninum

Neospora caninum is widely distributed in the world and this parasite is one of the major causes of abortion in cattle. Dogs and coyotes are definitive hosts of N. caninum and several species of domestic and wild animals are intermediate hosts. Dogs can become infected by the ingestion of tissues containing cysts and then excrete oocysts. It is not yet known whether sporulated oocysts are able to induce a patent infection in dogs, i.e. a shedding of N. caninum oocysts in feces. The objective of this study was to experimentally examine the infection of dogs by sporulated oocysts. The oocysts used in the experiment were obtained by feeding dogs with brain of buffaloes (Bubalus bubalis) positive for anti-N. caninum antibodies by indirect fluorescent antibody test (IFAT ≥200). Oocysts shed by these dogs were confirmed to be N. caninum by molecular methods and by bioassay in gerbils, and sporulated N. caninum oocysts were used for the oral infection of four dogs. The dogs were 8 weeks old and negative for antibodies to N. caninum and Toxoplasma gondii. Dogs 1 and 4 received an inoculum of 10,000 sporulated oocysts each; dog 2 an inoculum of 5000 sporulated oocysts and dog 3 received 1000 sporulated oocysts of N. caninum. The total feces excreted by these dogs were collected and examined daily for a period of 30 days. No oocysts were found in their feces. The dogs were monitored monthly for a 6-month period to observe a possible seroconversion and when this occurred the animals were eliminated from the experiment. Dogs 1 and 4 seroconverted 1 month after the infection with titer, in the IFAT, of 1600 and 800, respectively; the other two dogs presented no seroconvertion during the 6-month period. Dogs 1 and 2 were euthanized 180 days after infection and were examined for the detection of N. caninum in tissues (brain, muscle, lymph node, liver, lung, heart and bone marrow) by immunohistochemistry and PCR with negative results in both techniques. Bioassay in gerbils with brain of these dogs was also performed and again the results were negative. In conclusion, dogs infected with sporulated oocysts of N. caninum were not able to shed oocysts in feces. However, a higher dose of infection stimulated the production of antibodies against N. caninum in the dogs.     

Effects of host maturity and prior exposure history on the production of Neospora caninum oocysts by dogs

To investigate whether dogs shed Neospora caninum oocysts more than once, five dogs with a previous history of shedding oocysts were fed infected bovine tissues. Two of three dogs shed oocysts when they were re-exposed 18-20 months after the first challenge; two other dogs re-exposed earlier, only 8 months after the primary exposure, did not produce oocysts. These results suggest that dogs may become refractory to shedding N. caninum oocysts for a period approximately between 8 and 18 months after a primary infection; however, this possibility requires statistical validation by testing of more dogs. The development of a high antibody titer did not ensure that a dog would completely resist shedding oocysts after consuming an infected meal. Oocyst production was also compared between puppies and adult dogs with primary infections. Twelve puppies (three from the present study and nine from a previous study) shed significantly more oocysts (mean: 166,400) compared with five adult dogs following primary exposure (mean: 2900), indicating that a dog's age can influence N. caninum oocyst production (P=0.02).     

The role of the dog in the epidemiology of neosporosis in cattle

Neospora caninum is an intracellular protozoan parasite that was discovered in a dog in 1988. Since then, N. caninum has been demonstrated in a variety of animal species and it has been recognized as an important cause of abortion in cattle. An infection with N. caninum can be maintained in cattle herds for several generations by transplacental transmission from cow to calf. Recently, it was demonstrated that dogs can act as definitive hosts of N. caninum and therefore may be a source of infection for other species by shedding oocysts. Further evidence of a role of the dog in spreading the infection to cattle has been derived from epidemiological studies. The present state of knowledge is reviewed in this paper.     

Studies on the role of the red fox (Vulpes vulpes) as a potential definitive host of Neospora caninum

Neospora (N.) caninum is a protozoan parasite which is regarded as a major cause of abortion in cattle. Dogs and coyotes are definitive hosts of N. caninum which may shed environmentally resistant stages, oocysts, in their feces. Epidemiological studies in Germany showed that the presence of dogs increased the risk of a bovine herd to be N. caninum-positive in a bulk-milk ELISA test. However, there were also N. caninum-positive herds where dogs were not kept together with cattle.This leads to the question whether canids other than dogs, e.g., foxes, might be involved in the horizontal transmission of N. caninum. Therefore, the aim of our examinations in wild animals was to find out whether there are indications for a sylvatic cycle with foxes as definitive hosts and deer, roe deer and wild mice samples contained structures which resembled those of coccidian oocysts. In 13 of these 65 samples coccidian DNA was detected using a 18S rRNA gene based polymerase chain reaction (PCR).The examination of the 65 samples in a N. caninum-specific PCR revealed no positive result. Hammondia (H.) heydorni-DNA was detected in two samples. In addition, brain samples from 528 foxes, 224 wild mice, 16 deer and roe deer as well as from 1 wild boar were examined for the presence of N. caninum DNA by real time PCR. All samples tested negative by PCR. In conclusion, our study yielded no evidence indicating that the examined animals were part of a sylvatic cycle for N. caninum.     

Occurrence of Neospora caninum antibodies in sera from dogs of the city of São Paulo,Brazil

Neospora caninum is an important cause of abortion in dairy cattle worldwide. Dogs are important in the epidemiology of this parasite because they are the only hosts known to excrete N. caninum oocysts. In order to understand the prevalence of N. caninum in dogs, sera from 500 owned dogs and from over 600 feral street dogs from the city of S?o Paulo, Brazil were assayed for antibodies to N. caninum. Sera were examined by the Neospora agglutination test (NAT) using mouse-derived tachyzoites. Antibodies (> or =1:25) to N. caninum were found in nearly 10% (49/500) of owned dogs and in 25% (151/611) of stray dogs. NAT titers for owned dogs were 1:25 in 28 (5.6%) dogs, 1:50 in 20 (4%) dogs, and > or =1:500 in 1 (0.2%) dog. NAT titers for stray dogs were 1:25 in 79 (12.9%) dogs, 1:50 in 68 (11.1%) dogs, and > or =1:500 in 4 (0.6%) dogs. These data indicate that feral dogs may be important in the epidemiology of N. caninum infection.     

Prevalence of antibodies to Neospora caninum in dogs from Amazon,Brazil

Neospora caninum is an important cause of abortion in dairy cattle worldwide. Dogs are important in the epidemiology of this parasite because they are the only hosts known to excrete N. caninum oocysts. Antibodies to N. caninum were assayed in serum samples from 157 dogs from Monte Negro, Rond?nia, Amazon, Brazil using the indirect fluorescent antibody test. Antibodies to N. caninum were found in 13 (8.3%) of dogs in titers of 1:50 in 1, 1:100 in 2, 1:200 in 5, 1:800 in 1, 1:1600 in 2, and 1:3200 in 2 dogs. These data indicate that N. caninum infection is prevalent even in remote areas of the Amazon.     

Isolation and molecular detection of Neospora caninum from naturally infected sheep from Brazil

Neospora caninum was isolated from a naturally infected sheep from Brazil by bioassay in dogs. Approximately 70g of brain from each of two 4-month-old sheep with indirect fluorescent antibodies (>or=1:50) to N. caninum was offered to a different IFAT negative dog (Sheep n. 302, IFAT 1:400-Dog 1 and Sheep n. 342, IFAT 1:50-Dog 2). Parasite DNA was detected in both sheep brains using a PCR targeting the Nc-5 gene of N. caninum. Shedding of Neospora-like oocysts was noticed only in Dog 1, from 10 days post-inoculation (PI) to 25 days PI (a total of approximately 27,600 oocysts). Seventy days after infection, Dog 1 was euthanized and brain/cerebellum and medulla were collected and submitted to molecular methods, as were the oocysts, to confirm the identity of the isolate. Serum samples collected weekly from both dogs from the infection to the end of the experimental period had no antibodies anti-N. caninum by IFAT (<1:50). Oocysts, brain/cerebellum and medulla specimens of Dog 1 proved positive by a PCR assay targeting the Nc-5 gene of N. caninum. In addition, the oocysts have the DNA amplified by a PCR based on primers directed to the common toxoplasmatiid ITS1 sequence. The PCR products of ITS1 were sequenced, confirming again the isolate as N. caninum. Oocysts were also orally inoculated in two Swiss white mice two Mongolian gerbils (Meriones ungulatus) and two large vesper mice (Calomys callosus) (10(3)oocysts/animal). The rodents were sacrificed 2 months PI, and fresh preparations of brains showed Neospora thick-walled cysts in gerbil brains, but molecular detection using the Nc-5 PCR assay revealed DNA parasite in gerbil and also C. callosus brains. This is the first report of isolation and sequencing of N. caninum from a Brazilian sheep and the first report of molecular detection of N. caninum from C. callosus.     

Confirmation that the dog is a definitive host for Neospora caninum.

Two mixed-breed littermate dogs were fed mouse brains containing tissue cysts of the NC-beef isolate of Neospora caninum. Both dogs excreted N. caninum oocysts in their feces. Dog 1 which was given methylprednisolone acetate (MPA) prior to ingesting tissue cysts, excreted oocysts on days 5 to 10 inclusive and on day 17 after ingesting tissue cysts. Dog 1 had a serum antibody titer of 1:200 in the indirect fluorescent antibody test (IFAT) 35 days after it was fed tissue cysts. Dog 2, which was not treated with MPA, excreted oocysts on Day 6 and Day 9 after ingesting tissue cysts. Antibodies to N. caninum were not found in a 1:25 dilution of serum on any examination period for Dog 2 during the study. Neospora caninum was not found in the tissues of either dog by histological or immunohistochemical means following necropsy 42 days after being fed tissue cysts. The identity of the oocysts excreted in the feces of the dogs was confirmed by mouse inoculation studies.     

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.