Prevalence of Antibodies to Neospora caninum and Toxoplasma gondii in Swedish Dogs

Neospora caninum is a newly described coccidian parasite which has been found in various species such as the dog, cattle, horse, sheep and goat. Morphologically it resembles Toxoplasma gondii with which it is related (Holmdahl et al. 1994), and with which it has earlier been confused. The life cycle of N caninum is only partially known. Tachyzoites and tissue cysts are the only known stages of the parasite, and transplacental transmission is the only known route of infection. Subclini-cally infected dams can transmit the parasite to their fetuses and successive offspring from the same mother might be born infected (Dubey et al. 1990b). Clinical neosporosis is mostly seen in pups or young dogs, and the majority or all pups in a litter are often affected. The disease is characterized by ascending paralysis of the legs, with the hind legs more severely affected than the front legs, paralysis of the jaw, difficulty in swallowing and muscle flaccidity and atrophy (Dubey 1992, Dubey & Lindsay 1993). Fatal infections with N caninum in dogs have been reported from many countries, e.g. Norway (Bjerkäs & Presthus 1988), USA (Dubey et al. 1988), Sweden (Uggla et al. 1989a,b) and the United Kingdom (Dubey et al. 1990a). Serological surveys for antibodies to N. caninum in dogs from Kansas, USA and England have shown a prevalence of 2 and 13%, respectively (Lindsay et al. 1990, Trees et al. 1993).     

Neospora caninum in wildlife

Neosporosis, which is caused by the coccidian parasite Neospora caninum, is recognized as a major disease of domestic animals that causes high abortion rates in cattle and fatal neurological disease in dogs. A life cycle of N. caninum in wild animals (i.e. sylvatic) has long been suspected because neosporosis has been detected in several wildlife species. Recently, the transmission of N. caninum has been confirmed in coyotes and white-tailed deer. The newly confirmed wild hosts and other wild animals are probably involved in the sylvatic cycle of the parasite. Control measures for neosporosis could now become more complicated, given the participation of wildlife in the life cycle of N. caninum.     

Dogs shed Neospora caninum oocysts after ingestion of naturally infected bovine placenta but not after ingestion of colostrum spiked with Neospora caninum tachyzoites

An experiment was carried out to determine whether bovine colostrum or placenta could be a source of infection of Neospora caninum for dogs. For this purpose, two dogs were fed bovine colostrum to which culture-derived N. caninum tachyzoites were added and two other dogs were fed placental cotyledonary tissue from N. caninum seropositive cows. One dog served as a negative control during the start of the experiment but this control dog was fed cotyledonary tissue later on. None of the dogs did produce serum antibodies to N. caninum. All three dogs that were fed cotyledonary tissue did shed N. caninum oocysts, but no oocyst shedding was seen in the two dogs that were fed colostrum with N. caninum tachyzoites. Oocyst excretion did not resume in two dogs after repeated feeding of N. caninum infected placenta. The identity of the oocysts was confirmed by a bioassay in gerbils. It is concluded that ingestion of bovine placenta by dogs is an effective mode of transmission of N. caninum from cattle to dogs.     

Infection of primary canine duodenal epithelial cell cultures with Neospora caninum

According to current knowledge, sexual development of the apicomplexan parasite Neospora caninum takes place in the canine intestine. However, to date there is no information on the interaction between the parasite and the canine intestinal epithelium, and, next to the clinical and in vivo research tools, an in vitro model comprised of canine intestinal cells infected with N. caninum would be very helpful for investigations at the cellular level. Following the isolation of cells of neonatal canine duodenum and growth of cell cultures to monolayers for 5-6 days, canine intestinal epithelial cells were exposed to cell culture-derived N. caninum tachyzoites and bradyzoites. The host cells remained viable during in vitro culture for an average of 2 wk. During this time span, N. caninum was found to readily adhere to any surface area of these cells, but infection took mostly place at sites where microvilli-like structures were missing, e.g., at the cell periphery, with tachyzoites exhibiting at least 3-4 times increased invasive capacities compared to bradyzoites. Once intracellular, parasites resided within a parasitophorous vacuole, moved toward the vicinity of the nucleus and the more distal portion of the epithelial cells, and proliferated to form vacuoles of not more than 2-4 parasites, which were surrounded by numerous mitochondria. Immunofluorescence staining and TEM of infected cells showed that the expression of cytokeratins and the structural integrity of desmosomes and tight junctions were not notably altered during infection. Furthermore, no changes could be detected in the alkaline phosphatase activities in cell culture supernatants of infected and noninfected cells. Canine duodenal epithelial cell cultures represent a useful tool for future studies on the characteristics of the intestinal phases of N. caninum infection.     

Fatal congenital Neospora caninum infection in a lamb

Neospora caninum tissue cysts were found in the brain and spinal cord of a 1-wk-old lamb that was unable to stand after birth. The lamb was originally diagnosed as having toxoplasmosis, but ultrastructural and immunohistochemical techniques used in the present study permitted a definitive diagnosis of Neospora caninum tissue cysts in the brain and spinal cord of this lamb. This is the first report of N. caninum in sheep.     

Neospora caninum: comparative gene expression profiling of Neospora caninum wild type and a temperature sensitive clone

To understand the genetic basis of virulence, gene expression profiles of a temperature-sensitive clone (NCts-8, relatively avirulent) and its wild type (NC-1) of Neospora caninum were characterized and compared using a high-density microarray with approximately 63,000 distinct oligonucleotides. This microarray consists of 5692 unique N. caninum sequences, including 1980 Tentative Consensus sequences and 3712 singleton ESTs from the TIGR N. caninum Gene Index (NCGI, release 5.0). Each sequence was represented by 11 distinct 60mer oligonucleotides synthesized in situ on the microarray. The results showed that 111 genes were significantly repressed and no up-regulated genes were identified in the NCts-8 clone. The level of 10 randomly selected genes from the repressed genes was confirmed using real-time RT-PCR. Of the 111 repressed genes, 58 were hypothetical protein products and 53 were annotated genes. Over 70% of the repressed genes identified in this study are clustered on five chromosomes (I, VII, VIII, X and XII). These results suggest that the down-regulated genes may be in part responsible for the reduced pathogenesis of NCts-8; further characterization of the regulated genes may aid in understanding of molecular basis of virulence and development of countermeasures against neosporosis.     

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.     

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.     

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.     

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.     

Immunological characterization of Neospora caninum cyclophilin

Neospora caninum is an intracellular parasite that poses a unique ability to infect a variety of cell types by causing host cell migration. Although previous studies demonstrated that parasite-derived proteins could trigger host cell migration, the related molecules have yet to be determined. Our study aimed to investigate the relationship between Neospora-derived molecules and host cell migration using recombinant protein of N. caninum cyclophilin (NcCyp). Indirect fluorescent antibody test revealed that NcCyp was expressed in the tachyzoite cytosol. Furthermore, NcCyp release from extracellular parasites was detected by sandwich enzyme-linked immunosorbent assay in a time-dependent manner. Recombinant NcCyp caused the cysteine-cysteine chemokine receptor 5-dependent migration of murine and bovine cells. Furthermore, immunohistochemistry indicated that NcCyp was consistently detected in tachyzoites distributed within or around the brain lesions. In conclusion, N. caninum-derived cyclophilin appears to contribute to host cell migration, thereby maintaining parasite/host interactions.     

Serological diagnosis of Neospora caninum infection

Since the first isolation of the apicomplexan parasite Neospora caninum, a range of serological assays have been developed for use in dogs, cattle and a variety of other potential host species. The tests include the indirect fluorescent antibody test, the direct agglutination test and different enzyme-linked immunosorbent assays. This article reviews the principles and properties of the available tests which are discussed in relation to different applications.     

Neospora caninum (Apicomplexa) in a stillborn goat.

Tissue cysts of Neospora caninum were found in sections of brain from a stillborn pygmy goat. The tissue cysts had 1-2-microns-thick cyst walls and stained with anti-N. caninum serum in an immunohistochemical test. Glial nodules, mononuclear perivascular cuffing, and foci of inflammation were associated with N. caninum tissue cysts throughout the brain. This report indicates that N. caninum can be transmitted transplacentally in goats.     

Neospora caninum in birds: A review

Neospora caninum is an obligate intracellular protozoan parasite that infects domestic and wild animals. Canids are considered to be definitive hosts since they may shed oocysts into the environment through their feces. The disease is recognized as one of the major causes of bovine abortion worldwide, leading to important economic losses in the dairy and beef cattle industries. Previous studies have reported N. caninum infection in different species of birds; infection in birds has been associated with increased seroprevalence and reproductive problems in dairy cattle. Although the role of birds in the epidemiological cycle of neosporosis is unknown, birds are exposed to infection because they feed on the ground and could thus contribute to parasite dissemination. This review is focused on the current state of knowledge of neosporosis in birds.     

Review of Neospora caninum and neosporosis in animals

Neospora caninum is a coccidian parasite of animals. It is a major pathogen for cattle and dogs and it occasionally causes clinical infections in horses, goats, sheep, and deer. Domestic dogs are the only known definitive hosts for N. caninum. It is one of the most efficiently transmitted parasite of cattle and up to 90% of cattle in some herds are infected. Transplacental transmission is considered the major route of transmission of N. caninum in cattle. Neospora caninum is a major cause of abortion in cattle in many countries. To elicit protective immunity against abortion in cows that already harbor a latent infection is a major problem. This paper reviews information on biology, diagnosis, epidemiology and control of neosporosis in animals.     

The in vitro development of Neospora caninum bradyzoites

Neospora caninum is a recently identified apicomplexan protozoan parasite that is closely related to Toxoplasma gondii. Neospora caninum is of significant economic importance as it causes neurological disease and abortion in numerous animals. Antibodies to BAG1/hsp30 (also known as BAG5), a T. gondii bradyzoite-specific protein, have been demonstrated to react with N. caninum tissue cysts in vivo. Bradyzoite differentiation of N. caninum in vitro was investigated using culture conditions previously utilised for T. gondii in vitro bradyzoite development. Utilising the NC-Liverpool isolate of N. caninum, cyst-like structures developed within 3-4 days of culture of this parasite in human fibroblasts. In addition, an antigen reacting with mAb 74.1.8 (anti-BAG1) and rabbit anti-recombinant BAGI was demonstrable by immunofluorescence, fluorescence-activated cell sorter, and immunoblot analyses. Expression of this antigen was increased by stress conditions, similar to that which has been described for T. gondii bradyzoite induction. Cyst-wall formation in vitro, as assayed by lectin binding, did not occur as readily for N. caninum as it does for T. gondii.     

Abortion risk in progeny of cows after a Neospora caninum epidemic

A study was done of the descendants of cows from 4 dairy herds in which there had been N. caninum abortion outbreaks. Precolostral antibodies to N. caninum were demonstrated in 34 of 50 (68%) F1 calves and in 14 of 17 (82%) F2 calves from cows that aborted during the outbreaks. In 214 F1 progeny, N. caninum seroprevalence was nearly 50%, and there was a significant association between serostatus of the offspring and serostatus of dams. These observations indicated that congenital infection was an important mode of transmission after abortion outbreaks in these herds. A total of 52 abortions was recorded in 293 pregnancies of F1 progeny cows (1 to 3 pregnancies per animal). It was found that seropositive F1 cows had a three-fold increased abortion risk compared with seronegative F1 cows. In 2 of 10 abortions in seronegative cows evidence for N. caninum infection was found, suggesting that a low level of postnatal infection may also have occurred. It is concluded that N. caninum-infected calves should not be used as replacement stock, to decrease the future risk of abortion in dairy herds.     

Seroprevalence of Neospora caninum antibodies in dogs in India

Neospora caninum is one of most important causes of abortion in cattle worldwide, and dogs are an important risk factor for N. caninum infection in cattle. Antibodies to N. caninum were determined in 184 (126 rural, 58 urban) dogs from the Punjab State, India, using commercial monoclonal antibody-based competitive ELISA and found in 16.8% of the animals. The prevalence of N. caninum antibodies was significantly higher in rural dogs (21.4%, 27 of 126) than city dogs (6.9%, 4 of 58). To our knowledge this is the first report of N. caninum infection in canines from India.     

Fecal and Molecular Survey of Neospora caninum in Farm and Household Dogs in Mashhad Area, Khorasan Province, Iran

Neospora caninum is an important cause of abortion in dairy cattle worldwide. Dog is the definitive host for N. caninum and can infect dairy cattle. The aim of this study is to determine the prevalence of Neospora oocysts in feces of dogs from dairy farms. A total of 174 fecal samples was collected from 89 farm dogs and 85 household dogs during 2006 and 2008. Fecal samples of dogs were microscopically examined for detecting Hammondia Neospora-like oocysts (HNLO) by Mini Parasep®SF fecal parasite concentrator. HNLO were microscopically detected in 4 fecal samples (2.2%). The fecal samples with HNLO were examined by N. caninum-specific PCR. Two of the samples were positive for N. caninum. The 2 positive fecal samples were selected for inoculation to calves. Two inoculated calves were seronegative by ELISA for 4 months post-infection. This is the first report of finding N. caninum DNA in feces of farm dogs in Mashhad area, Iran.     

Characterization of temperature-sensitive strains of Neospora caninum in mice

Temperature-sensitive (ts) strains of the Neospora caninum tachyzoites were selected by chemical mutagenesis and selection for growth at 32 C. Three ts strains and the parental, N. caninum wild-type strain, NC-1, were examined in the present study for their ability to cause disease in inbred BALB/c mice, outbred ICR mice, and chemically immunosuppressed ICR mice. In BALB/c mice, all 3 strains failed to induce clinical disease, whereas infection with the NC-1 strain caused central nervous system disease and death in some mice. No disease was observed in ICR mice inoculated with the 3 ts strains or the NC-1 strain. All immunosuppressed ICR mice inoculated with the NC-1 strain died, whereas no immunosuppressed mice inoculated with the NCts-4 strain and only 1 of 5 mice inoculated with the NCts-8 and NCts-12 strains died. The NCts-4 and NCts-12 strains reverted to a wild-type phenotype when grown at 37 C. Vaccination of BALB/c mice with live, but not frozen NCts-8 strain tachyzoites induced significant (P < 0.05) protection following NC-1 strain challenge.