First isolation of Neospora caninum from the feces of a naturally infected dog.

Neospora caninum is a major cause of abortion in cattle worldwide. Cattle become infected with N. caninum by ingesting oocysts from the environment or transplacentally from dam to fetus. Experimentally, dogs can act as definitive hosts, but dogs excrete few oocysts after ingesting tissue cysts. A natural definitive host was unknown until now. In the present study, N. caninum was isolated from the feces of a dog. Gerbils (Meriones unguiculatus) fed feces from the dog developed antibodies to N. caninum in the Neospora caninum agglutination test, and tissue cysts were found in their brains. Neospora caninum was isolated in cell culture and in gamma-interferon gene knockout mice inoculated with brain homogenates of infected gerbils. The DNA obtained from fecal oocysts of the dog, from the brains of gerbils fed dog feces, and from organisms isolated in cell cultures inoculated with gerbil brains was confirmed as N. caninum. The identification of N. caninum oocyst by bioassay and polymerase chain reaction demonstrates that the dog is a natural definitive host for N. caninum.     

Improved production of Neospora caninum oocysts,cyclical oral transmission between dogs and cattle,and in vitro isolation from oocysts

Scarce information is available about Neospora caninum oocysts and sporozoites, in part because only small numbers of oocysts have typically been produced by experimentally infected dogs. We hypothesized that I reason for low experimental production of oocysts is that dogs have been fed tissues from experimentally infected mice instead of tissues from cattle (which are natural intermediate hosts of N. caninum). In this study, 9 dogs were fed tissues from N. caninum-infected calves, and oocyst production was compared with 6 dogs that were fed infected mouse carcasses. The number of oocysts produced by dogs that ingested infected calf tissues (mean = 160,700) was significantly greater (P = 0.03) than the number of oocysts shed by dogs that ingested infected mice (mean = 5,400). The second goal of our experiment was to demonstrate cyclical oral transmission of N. caninum between dogs and cattle. As few as 300 oocysts were used to successfully infect calves, and tissues from these calves induced patent infections in 2 of 3 dogs; oocysts from I of these dogs were administered to another calf, and tissues from this calf subsequently induced a third dog to shed oocysts. Oocysts were confirmed to be N. caninum using a species-specific polymerase chain reaction technique. In addition, sporulated oocysts were used to recover N. caninum in vitro after digestion in an acid-pepsin solution and inoculation of cell monolayers.     

Ingestion of Neospora caninum tissue cysts by Mustela species

Dogs are a definitive host of Neospora caninum, a protozoal parasite that causes abortion in cattle. Mustelids were tested to determine if they could also be definitive hosts. The procedures used were the same as those previously used to test dogs. Ermine (Mustela erminea), weasels (Mustela frenata) and ferrets (Mustela putorius) were fed N. caninum-infected mice. Neospora caninum oocysts were not observed. Mustelid faeces were fed to mice. The mice did not seroconvert and N. caninum was not detected in murine brains using tissue culture and PCR. The hypothesis that Mustela spp. are definitive hosts of N. caninum is not supported.     

A structural study of the Neospora caninum oocyst

Oocysts of Neospora caninum were collected from the faeces of a dog fed mouse brains containing tissue cysts of the NC-beef strain of N. caninum. Sporulated oocysts were spherical to subspherical, and were 11.7x11.3 microm. The length/width ratio was 1.04. No micropyle or oocyst residuum was present. Polar granules were not present, although occasionally tiny refractile granules were seen among sporocysts. Sporocysts were ellipsoidal, did not contain a Stieda body, and were 8.4x6.1 microm. The length/width ratio for sporocysts was 1.37. A spherical or subspherical sporocyst residuum was present, and was composed of a cluster of small, compact granules of 4.3x3.9 microm, or was represented by many dispersed granules of similar size. Sporozoites were elongate and 7.0-8.0x2.0-3.0 microm in situ. No refractile bodies were present and the nucleus was centrally or slightly posteriorly located. The features of the oocyst of N. caninum are similar to those of Hammondia heydorni oocysts from dog faeces and Toxoplasma gondii and Hammondia hammondi oocysts from cat faeces.     

Oocyst excretion in dogs fed mouse brains containing tissue cysts of a cloned line of Neospora caninum

Neospora caninum is an apicomplexan parasite that causes neonatal neuromuscular disease in dogs and abortions in cattle. Bovine neosporosis is a major production problem worldwide. The parasite is transmitted to cattle via oocysts excreted by dogs or by transplacental transmission. Dogs are the only proven definitive host for N. caninum. One of 3 dogs fed mouse brains containing tissue cysts of a wild-type N. caninum strain CK0160SC3B (CKO) excreted oocysts in its feces. Two of 3 dogs fed mouse brains containing tissue cysts from a cloned line of the CKO strain excreted N. caninum oocysts in their feces. The results indicate that a single N. caninum tachyzoite contains all the genetic information needed to produce the asexual and sexual cycles in the canine intestine.     

The antigenic composition of Neospora caninum

Neospora caninum is an apicomplexan parasite which causes neosporosis, namely stillbirth and abortion in cattle, and neuromuscular disease in dogs. Although N. caninum is phylogenetically and biologically closely related to Toxoplasma gondii, it is antigenically clearly distinct. In analogy to T. gondii, three stages have been identified. These are: (i) asexually proliferating tachyzoites; (ii) tissue cysts harbouring slowly dividing bradyzoites; and (iii) oocysts containing sporozoites. The sexually produced stage of this parasite has only recently been identified, and has been shown to be shed with the faeces from dogs orally infected with N. caninum tissue cysts. Thus dogs are definitive hosts of N. caninum. Tachyzoites can be cultivated in vitro using similar techniques as previously described for T. gondii. Methods for generating tissue cysts containing N. caninum bradyzoites in mice, and purification of these cysts, have been developed. A number of studies have been undertaken to identify and characterise at the molecular level specific antigenic components of N. caninum in order to improve serological diagnosis and to enhance the current view on the many open questions concerning the cell biology of this parasite and its interactions with the host on the immunological and cellular level. The aim of this paper is to provide an overview on the approaches used for detection of antigens in N. caninum. The studies discussed here have had a great impact in the elucidation of the immunological and pathogenetic events during infection, as well as the development of potential new immunotherapeutic tools for future vaccination against N. caninum infection.     

Protracted shedding of oocysts of Neospora caninum by a naturally infected foxhound

Feces from 15 dogs at 2 different foxhound kennels in the U.K. were examined microscopically for the presence of oocysts of Neospora caninum. One sample containing approximately 400 candidate oocysts per gram was positive in a polymerase chain reaction (PCR) using N. caninum-specific primers. In a sample taken 4 mo later from the same hound. N. caninum oocysts were again detected visually and by PCR. This is the third reported case of a dog naturally excreting oocysts of N. caninum and suggests that oocyst excretion can occur over a relatively long period of time in some circumstances or that reshedding may occur.     

Vaccination with gamma-irradiated Neospora caninum tachyzoites protects mice against acute challenge with N. caninum

Neospora caninum, an apicomplexan parasite, is a leading cause of bovine abortions worldwide. The efficacy of gamma-irradiated N. caninum strain NC-1 tachyzoites as a vaccine for neosporosis was assessed in C57BL6 mice. A dose of 528 Gy of gamma irradiation was sufficient to arrest replication but not host cell penetration by tachyzoites. Female C57BL6 mice were vaccinated with two intraperitoneal inoculations of 1 x 10(6) irradiated tachyzoites at 4-wk intervals. When stimulated with N. caninum tachyzoite lysates, splenocytes of vaccinated mice, cultured 5 and 10 wk after vaccination, secreted significant (P<0.05) levels of interferon gamma, interleukin (IL)-10, and small amounts of IL-4. Antibody isotype-specific ELISA of sera from vaccinated mice exhibited both IgG1 and IgG2a isotypes of antibodies. Vaccinated mice were challenged intraperitoneally with 2 x 10(7)N. caninum tachyzoites. All vaccinated mice remained healthy and showed no obvious signs of neosporosis up to the 25th day post-challenge when the study was terminated. All unvaccinated control mice died within 1 wk of infection. Gamma-irradiated N. caninum tachyzoites can serve as an effective, attenuated vaccine for N. caninum.     

Transmission of Neospora caninum between wild and domestic animals

To determine whether deer can transmit Neospora caninum, brains of naturally infected white-tailed deer (Odocoileus virginianus) were fed to 4 dogs; 2 of these dogs shed oocysts. Oocysts from 1 of the dogs were tested by polymerase chain reaction and found to be positive for N. caninum and negative for Hammondia heydorni. The internal transcribed spacer 1 sequence of the new strain (designated NC-deer1) was identical to N. caninum from domestic animals, indicating that N. caninum is transmitted between wild and domestic animals, often enough to prevent divergent evolution of isolated populations of the parasite. NC-deerl oocysts were administered to a calf that developed a high antibody titer, providing evidence that N. caninum from wildlife can infect cattle. In addition, N. caninum antibody seroprevalence was detected in 64/164 (39%) free-ranging gray wolves (Canis lupus), 12/113 (11%) coyotes (Canis latrans), 50/193 (26%) white-tailed deer, and 8/61 (13%) moose (Alces alces). These data are consistent with a sylvatic transmission cycle of N. caninum between cervids and canids. We speculate that hunting by humans favors the transmission of N. caninum from deer to canids, because deer carcasses are usually eviscerated in the field. Infection of canids in turn increases the risk of transmitting the parasite to domestic livestock.     

In vitro development of Neospora caninum (Protozoa: Apicomplexa) from dogs

The development of Neospora caninum isolated from naturally infected dogs was examined in mammalian cell cultures. Tachyzoites developed by endodyogeny when inoculated onto bovine monocyte or bovine cardiopulmonary artery endothelial cell cultures. Tachyzoites were 5.0 by 2.0 microns and had a posteriorly located nucleus. Cytopathogenic effects of parasite development consisted of the formation of holes in the cell monolayer associated with the rupture of infected host cells. Serial passage of tachyzoites was achieved by subinoculation of tachyzoites onto non-infected bovine monocyte cell cultures. It appears that N. caninum can be continuously grown in cell cultures.     

Seroepidemiological evidence for a relationship between Neospora caninum infections in dogs and cattle

Dogs from dairy farms with a known prevalence of Neospora caninum antibodies in the cattle were examined for the presence of N. caninum antibodies using an ELISA. Data of farm dogs were compared with those of dogs examined at a university clinic, which originated mainly in urban areas. Of the 152 farm dogs, 36 (23.6%) were seropositive to N. caninum, which was significantly higher than the proportion of seropositives in the clinic dog population (19 of 344, 5.5%). Seroprevalence was significantly higher (P = 0.01) in female dogs than in male dogs. Seroprevalence in dogs increased with age, indicating postnatal infection. Seropositivity to N. caninum in farm dogs was strongly correlated with a high prevalence of N. caninum antibodies in the cattle. At farms where no dogs were present, the seroprevalence to N. caninum in the cattle was significantly lower (P = 0.0002) than in farms where dogs were present. These findings suggest that there is a relationship between N. caninum infection of farm dogs and cattle. Since dogs have been shown to be definitive hosts of N. caninum, cattle may be infected by exposure to canine oocysts. Further research is needed to find out whether and how dogs may acquire the infection from cattle.     

Transplacental Neospora caninum infection in cats

Transplacental transmission of Neospora caninum was studied in 2 pregnant cats (queens). Queen 1 was inoculated subcutaneously with 2 x 10(6) cell culture-derived N. caninum tachyzoites on day 47 of gestation. She gave birth to a full-term kitten on the 17th day after inoculation. The kitten died the second day after birth due to generalized N. caninum infection. The mother cat was killed on the third day after parturition and was found to have a macerated kitten in the uterus. Severe placentitis, metritis, hepatitis, and nephritis due to N. caninum were seen in tissues from the queen. Queen 2 was fed N. caninum tissue cysts and mated 111 days later. She gave birth to 3 healthy full-term kittens. The kittens were necropsied at 2, 22, and 30 days of age. Neospora caninum was recovered from the organs and was seen in histologic sections in 1 of the 3 kittens. Results indicate that N. caninum can be transplacentally transmitted in cats during acute and chronic stages of infection. Neospora caninum-specific IgG antibodies were demonstrated in the sera of inoculated cats and nursing kittens.     

Biologic, morphologic, and molecular characterisation of Neospora caninum isolates from littermate dogs

Isolation and biologic and molecular attributes of Neospora caninum from three littermate dogs are described. Tissue cysts were confined to the brain and striated muscles. N. caninum was isolated (isolates NC-6, NC-7, and NC-8) in rodents and cell culture that had been inoculated with brain tissue from the dogs. Schizont-like stages reactive with N. caninum antibodies were seen in cell cultures seeded with bradyzoites released from Percoll-isolated N. caninum tissue cysts from the brain of one dog. Tissue cysts were infective orally to mice and gerbils, but not to cats and dogs. The isolates were also identified as N. caninum by PCR and sequence analysis.     

Oral infection of calves with Neospora caninum oocysts from dogs: humoral and cellular immune responses

Neospora caninum has been identified as a major cause of abortion in cattle in a number of countries throughout the world. Until the recent demonstration that dogs can serve as a definitive host of this parasite, it was not possible to study the infection in cattle orally exposed to oocysts. The aim of this study was to investigate the potential of N. caninum oocysts to infect calves, and to define initial immune responses that arise after oral infection. Seven calves were fed approximately 10(4)-10(5) N. caninum oocysts, three calves served as uninfected controls. Before infection, all calves were serologically negative for anti-Neospora antibodies and the calves were non-reactive to Neospora antigen in an in vitro lymphocyte proliferation assay. Peripheral blood lymphocytes from inoculated calves were able to mount in vitro proliferative responses to crude N. caninum antigen extract as early as 1 week p.i. Within 2 and 4 weeks p.i., Neospora-specific IgG1 and IgG2 antibodies were detected by IFAT and ELISA in serum from infected calves but not from sham-infected calves. The continued presence of reactive cells in the blood, spleen and mesenteric, inguinal, bronchial lymph nodes was seen as late as 2.5 months p.i., and parasite DNA was detected in the brain and spinal cord of the infected animals by PCR, indicating that the cattle were infected by oral inoculation of N. caninum oocysts collected from dogs, and that the animals were systematically sensitised by parasite antigen.     

Experimental infection of sheep with Neospora caninum oocysts

The purpose of the present study was to investigate the potential of Neospora caninum oocysts to infect sheep and determine whether N. caninum DNA could be detected by polymerase chain reaction (PCR) assay in blood and brain of sheep after oocyst inoculation. Six ewes were inoculated per os with 10(4) N. caninum oocysts, whereas 2 ewes served as uninoculated controls. All sheep were bled weekly for 7 wk after inoculation. Blood was analyzed for the presence of N. caninum DNA by 2 different PCR assays, as well as for the presence of antibodies to recombinant and native N. caninum antigens. Neospora caninum DNA was detected in 2 sheep as early as 7 days after oocyst inoculation (DAOI). All 6 sheep were PCR positive by 32 days and remained positive until the end of the study at 49 DAOI. Aside from 1 ewe, all sheep inoculated with N. caninum oocysts contained detectable N. caninum DNA in the brain tissue collected at 49 DAOI. Unlike with PCR, no lesion or parasite was detected by immunohistochemistry. Antibodies were detected by enzyme-linked immunosorbent assay, Neospora agglutination test, or immunoblotting to either native or recombinant N. caninum antigens in sheep inoculated with oocysts.     

Fatal Neospora caninum infection in kittens

Three 3-day-old kittens were inoculated subcutaneously and orally with Neospora caninum tachyzoites. A littermate and the queen were not inoculated with N. caninum and served as controls. Kitten 1 died between 14 and 16 days postinoculation (DPI) and was eaten by the mother. Kitten 2 died 17 DPI and kitten 3 was euthanized 29 DPI in a moribund condition. The control littermate and the dam remained healthy. Granulomatous skeletal myositis and nonsuppurative encephalomyelitis were the main lesions and were associated with numerous N. caninum tachyzoites in kittens 2 and 3. Cysts were found in kitten 3. Oocysts were not found in any cats. Neither lesions nor parasites were found in control cats.     

Protective efficacy of vaccination with Neospora caninum multiple recombinant antigens against experimental Neospora caninum infection

Protective efficacy of vaccination with Neospora caninum multiple recombinant antigens against N. caninum infection was evaluated in vitro and in vivo. Two major immunodominant surface antigens (NcSAG1 and NcSRS2) and two dense granule proteins (NcDG1 and NcDG2) of N. caninum tachyzoites were expressed in E. coli, respectively. An in vitro neutralization assay using polyclonal antisera raised against each recombinant antigen showed inhibitory effects on the invasion of N. caninum tachyzoites into host cells. Separate groups of gerbils were immunized with the purified recombinant proteins singly or in combinations and animals were then challenged with N. caninum. Following these experimental challenges, the protective efficacy of each vaccination was determined by assessing animal survival rate. All experimental groups showed protective effects of different degrees against experimental infection. The highest protection efficacy was observed for combined vaccination with NcSRS2 and NcDG1. Our results indicate that combined vaccination with the N. caninum recombinant antigens, NcSRS2 and NcDG1, induces the highest protective effect against N. caninum infection in vitro and in vivo.     

First isolation of Neospora caninum from an aborted bovine fetus in Spain

Neospora caninum was isolated from the brain of a 6-mo-old aborted bovine fetus from Galicia, Spain. The fetal brain homogenate was inoculated intraperitoneally into cortisonized mice. The peritoneal exudate from the infected mice, along with mouse sarcoma cells (Tg180), was inoculated into a second group of mice, and parasites were harvested from the peritoneal exudate. The parasites were adapted to in vitro growth in Vero monolayers. The tachyzoites from the peritoneal exudate reacted positively with anti-N. caninum antibodies and not with anti-Toxoplasma gondii antibodies on indirect fluorescent antibody test. The tachyzoites were lethal to interferon gamma gene knock out (KO) mice and could be identified immunohistochemically in the tissues. The identity of the parasite was also confirmed by polymerase chain reaction amplification of N. caninum-specific fragments. The sequences of the amplified gene 5 fragments (GenBank AY494944) were found to be identical to that of an Austrian isolate of N. caninum but not to that of NC-1. This is the first isolation of viable N. caninum from Spain.     

Growth of and competition between Neospora caninum and Toxoplasma gondii in vitro

Competitive interactions between Neospora caninum and Toxoplasma gondii were studied because both species appear to have identical ecological niches in vitro. Tachyzoites of N. caninum (NC-1 isolate) and T. gondii (RH isolate) were compared in three in vitro studies: (1) rate of penetration of host cells; (2) generation time; and (3) competition between the two species when grown together in the same flask and allowed to compete for space. When tachyzoites of the two species were inoculated onto human foreskin fibroblasts, 3.24-times more N. caninum tachyzoites penetrated cells by 1 h p.i. At 3 h p.i., there were 2.87-times more N. caninum intracellular tachyzoites than T. gondii tachyzoites. The generation times for N. caninum (NC-1 isolate) and T. gondii (RH isolate) were approximately 14-15 h and 8-10 h, respectively. Before exponential growth occurred, both species displayed a lag period, which was 10-12 h for N. caninum and 8-10 h for T. gondii. To observe competition, equal numbers of tachyzoites of each species were mixed and inoculated into flasks of host cells, and the monolayers were allowed to proceed to >90% lysis before the next transfer. Competition was analysed for 31 days by labelling samples of each flask with a species-specific monoclonal antibody and determining the ratio of each species. In all trials, T. gondii outcompeted N. caninum. By 4 days p.i., 70% of the tachyzoites were T. gondii; this percentage increased to 97% by 23 days p.i. When the starting inoculum contained 75% N. caninum and 25% T. gondii tachyzoites, T. gondii was still competitively superior. When infected monolayers that were labelled with T. gondii-specific antibodies were examined, it was noted that both species can occupy and undergo endodyogeny in the same host simultaneously.     

Characterization of the Oregon isolate of Neospora hughesi from a horse

Neospora hughesi was isolated in cell cultures inoculated with homogenate of spinal cord from a horse in Oregon. Tachyzoites of this Oregon isolate of N. hughesi were maintained continuously by cell culture passage and tachyzoites were infective to immunosuppressed mice. Gamma interferon gene knockout (KO) mice injected with tachyzoites developed fatal myocarditis and numerous tachyzoites were seen in lesions. Gerbils (Meriones unguiculatus) inoculated with tachyzoites developed antibodies (> or = 1:500) as indicated by the Neospora caninum agglutination test but did not develop clinical signs, and Neospora organisms were not demonstrable in their tissues. Tissue cysts were not found in gerbils, nude mice, KO mice, immunosuppressed outbred Swiss Webster mice, or BALB/c mice injected with the Oregon isolate of N. hughesi. Ultrastructurally, tachyzoites of the Oregon isolate from the myocardium of infected KO mice and from cell culture were similar to N. caninum tachyzoites. Western blot analysis using NcSAG1 and NcSRS2 polyclonal and monoclonal antibodies and characterization of the internal transcribed spacer 1 sequences from the equine isolates and different isolates of N. caninum from dogs and cattle indicated that the Oregon isolate of N. hughesi is distinct from N. caninum isolates from cattle and dogs.