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.     

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.     

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.     

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.     

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.     

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 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.     

Molecular and biological characterization of Hammondia heydorni-like oocysts from a dog fed hearts from naturally infected white-tailed deer (Odocoileus Virginianus)

Neospora caninum and Hammondia heydorni are morphologically and phylogenetically related coccidians that are found in dogs. Although there is serological evidence of N. caninum infection in the white-tailed deer (Odocoileus virginianus), the parasite has not been yet isolated from the tissues of this host. In an attempt to isolate N. caninum from deer, hearts from 4 deer with antibodies to N. caninum were fed to 2 dogs. One of these dogs shed unsporulated oocysts 12-14 microm in diameter. Sporulated oocysts were not infective to Mongolian gerbils (Meriones ungulatus), and DNA isolated from these oocysts was not amplified using N. caninum-specific primers. However, positive amplification with the H. heydorni-specific first internal transcribed spacer (ITS-1) primers and common toxoplasmatiid ITS-1 primers confirmed the presence of H. heydorni DNA in the samples. The oocysts were considered to be H. heydorni on the basis of their morphology, biology, and molecular characteristics. This is the first record of a H. heydorni-like parasite in the white-tailed deer.     

The sylvatic cycle of Neospora caninum: where do we go from here?

Bovine abortions due to Neospora caninum infection are a major cattle-production problem worldwide. The parasite is readily maintained in cattle populations by vertical transmission. The domestic dog excretes oocysts in its feces and, after sporulation, these oocysts are infectious to cattle. Current control measures are aimed at culling infected cows and limiting the access of cattle to infective oocysts. The recent revelations that coyotes (Canis latrans) can excrete N. caninum oocysts in their feces and that white-tailed deer (Odocoileus virginianus) are natural intermediate hosts of the parasite demonstrate the existence of a sylvatic cycle of neosporosis in North America. This complicates parasite-prevention programs but opens many new and exciting avenues of research. Similar canid-ruminant sylvatic cycles might exist in other countries and, if so, need to be investigated.     

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.     

Protection against vertical transmission in bovine neosporosis

In this study we were interested to determine whether infection of cattle prior to pregnancy would afford any protection to the foetus if the dams were challenged with Neospora caninum at mid-gestation. The experiment comprised four groups of cattle: group 1, uninfected controls; group 2, inoculated with N. caninum tachyzoites 6 weeks prior to mating and then challenged with N. caninum at mid-gestation; group 3, naive cattle challenged with N. caninum at mid-gestation and group 4 were infected with N. caninum prior to mating and left unchallenged throughout pregnancy. Positive cell-mediated and humoral immune responses to N. caninum were recorded in groups 2 and 4 prior to pregnancy and in groups 2, 3 and 4 following challenge at mid-gestation. However there was a marked down regulation of the cell-mediated immune response in all groups around mid-gestation. There was a significant increase in rectal temperature response in animals in group 3 compared to group 2 following challenge but no other clinical symptoms of disease were recorded and all cattle proceeded to calving. At calving, pre-colostral blood samples were negative for antibodies to N. caninum in all the calves born to dams in groups 1, 2 and 4. In contrast, all the calves born to dams in group 3 had high levels of specific antibody to N. caninum indicating that they had been exposed to the parasite in utero. At post-mortem N. caninum DNA was detected in CNS, thymus and placental cotyledon samples in calves from group 3. All tissue samples from calves in the other 3 groups were negative for N. caninum DNA with the exception of one calf from group 2 where specific DNA was detected in a sample of spinal cord. These results suggest that the immune response generated in the dams in group 2 prior to pregnancy had protected against vertical transmission of the parasite following challenge at mid-gestation.     

Transplacental transmission and abortion in cows administered Neospora caninum oocysts

Neospora caninum infection is a common cause of bovine abortion. One method by which cattle can acquire infection is through ingestion of oocysts; however, this has not yet been proved to cause transplacental infection or abortion. In this study, 19 cows, pregnant between 70 and 176 days, were administered 1500 to 115,000 oocysts through an esophageal tube. Seventeen of the cows became seropositive, indicating acquisition of infection, whereas 8 negative control cows remained seronegative (P < 0.001). Offspring were examined using serology, histology, immunohistochemistry, parasite isolation, and polymerase chain reaction (PCR). Six offspring were infected and 1 of them was aborted. The aborted fetus had typical lesions and positive immunohistochemistry and PCR for N. caninum. All 6 cows with infected offspring had continuously rising antibody titers, whereas 10 of 11 infected cows with uninfected offspring had falling titers after an early apex. The risk of transplacental transmission was increased by later exposure times during gestation and by the dose of oocysts (P < 0.01 for the 2 combined variables). The lowest dose of oocysts, when administered after the 160th day of gestation, caused transplacental infection in 1 of 2 animals. This study demonstrates that infection with N. caninum oocysts can cause transplacental transmission and abortion in cattle.     

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.     

First demonstration of protective immunity against foetopathy in cattle with latent Neospora caninum infection

The parasite Neospora caninum is an important cause of abortion in cattle world-wide. Chronically infected dams transmit the parasite transplacentally and infected foetuses may be aborted or born chronically infected but clinically normal. Chronically infected cows repeatedly transmit the parasite to foetuses in several pregnancies and some may abort more than once suggesting that the immune response in these cattle is compromised during pregnancy. To investigate the nature of the immune response in chronically infected cattle, five naturally, chronically infected cows were challenged with N. caninum tachyzoites at 10 weeks of gestation. No foetopathy occurred and all five delivered live calves at full-term. In four naive pregnant cows challenged at the same time, all four foetuses died within 3-5 weeks of challenge. Of the five live calves born to the chronically infected challenged cows, three were transplacentally infected with N. caninum. The kinetics of the maternal anti-N. caninum antibody responses during gestation suggested that these transplacental infections were not the result of the superimposed challenge, but the result of the recrudescence of the maternal chronic infection-which occurred concurrently in non-challenged, chronically infected pregnant controls. These data provide the first experimental evidence that protective immunity occurs in neosporosis. They also suggest that whilst immunity to a pre-existing infection will protect against an exogenous challenge, this protective immunity will not prevent transplacental infection. This implies that a subtle form of concomitant immunity exists in chronically infected cattle and has important implications for vaccine development.     

Seroprevalence of Neospora caninum infection on dairy cattle in farms from southern Romania

Neospora caninum, a coccidian parasite closely related to Toxoplasma gondii, is one of the major causes of abortion in cattle worldwide. Conventional serological techniques, such as the indirect fluorescent antibody test and enzyme-linked immunosorbent assay (ELISA), are routinely used in adult animals and aborted fetuses for the detection of anti- N. caninum antibodies. In Romania, infection with N. caninum in cattle has been reported recently, but only in limited areas from the north and central parts of the country. Therefore, the aim of this study was to obtain additional seroepidemiological data on infection with N. caninum on dairy farms from the south of Romania. A total of 258 blood samples was analyzed from 230 dairy cows and 28 calves from 9 dairy farms in southern Romania; the presence of specific IgG antibodies against N. caninum was determined using an indirect ELISA test. The average seroprevalence was 40.3%, but the within-herd prevalence ranged between 11.5 and 80.0%; the seroprevalence in dairy cows was 41.7%, while in calves it was 28.6%. Of the positive samples, 74.0% (77/104) had a high positive reaction (S/P ratio more than 1.0), while 26.0% (27/104) had a low positive reaction (S/P ratio between 0.5 and 1.0). This study indicates that N. caninum infection is widespread in the south of Romania, which could explain the causes of abortions registered in some herds in the studied area. However, a serological screening across the country is planned in order to assess the actual national prevalence of N. caninum infection, followed by implementation of a prevention and control program.     

Use of an immunodominant P17 antigenic fraction of Neospora caninum in detection of antibody response in cattle

A Neospora caninum 17 kDa protein fraction (p17) has been described as an immunodominant antigen (IDA) under reducing and non-reducing conditions. The aim of the present study was to investigate the diagnostic utility of p17 in cattle. In order to achieve this, p17 was purified by electroelution from whole N. caninum tachyzoite soluble extract and a p17-based Western blot (WB-p17) was developed. The p17 recognition was measured by densitometry and expressed as OD values to check the validity of the WB-p17. A total of 131 sera including sequential samples from naturally- and experimentally-infected calves and breeding cattle were analysed by WB-p17 and compared with IFAT using whole formalin-fixed tachyzoites as a reference test. The results obtained highlight the feasibility of using the N. caninum p17 in a diagnostic test in cattle. Firstly, the assay based on the p-17 antigen discriminated between known positive and negative sera from different cattle populations, breeding cattle and calves. Secondly, the p17 antigen detected fluctuations in the antibody levels and seroconversion in naturally- and experimentally-infected cattle. Significant differences in p-17 antigen recognition were observed between naturally infected aborting and non-aborting cattle, as well as significant antibody fluctuations over time in experimentally infected cattle, which varied between groups. Furthermore, the results obtained with WB-p17 are in accordance with the results obtained with the IFAT as high agreement values were obtained when all bovine subpopulations were included (kappa = 0.86).     

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.     

Canine and bovine Neospora caninum control sera examined for cross-reactivity using Neospora caninum and Neospora hughesi indirect fluorescent antibody tests

Neospora caninum is a well known protozoan parasite of domestic and wild animals. Neospora hughesi is a closely related protozoan with an unknown life cycle, host range, and infection prevalence. Many serologic surveys of N. caninum have been performed without consideration of potential cross-reactions with N. hughesi, which could confound results. The aim of this study was to investigate whether postexposure sera from animals experimentally infected with N. caninum exhibit significant reactivity differences when tested using N. caninum and N. hughesi Immunofluorescent Antibody Tests (IFAT). Pre- and postinfection serum samples from 10 dogs, 20 calves, and 17 cows were tested by dual IFATs. All pre-exposure samples for N. caninum tested seronegative for both organisms. All postexposure samples that were seropositive for N. caninum were also positive for N. hughesi, although N. hughesi antibody titers were usually 1 dilution lower (P < 0.02). Serologic surveys for N. caninum may be confounded by cross-reacting titers with N. hughesi, but true positive N. caninum antibody titers are greater than, or equal to, cross-reacting N. hughesi antibody titers.     

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.