Isolation of Neospora caninum genes detected during a chronic murine infection

In order to isolate genes coding for antigens of Neospora caninum which are recognised by the host immune system during a chronic murine infection, a cDNA library was immunoscreened with pooled sera from mice which survived three independent infections by N. caninum. Two new genes from N. caninum were isolated and expressed in Escherichia coli. The genes identified include one homologous to GRA1 of Toxoplasma gondii, plus another (NCP20) previously unknown in any taxon. Both genes encode small polypeptides which induced an IgG response in the mouse and were also recognised by IgG from a cow chronically infected with N. caninum. These results are consistent with the hypothesis that the polypeptides encoded by these genes are a target for the host immune system during chronic infections of N. caninum.     

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.     

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.     

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.     

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.     

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.     

Immune responses to Neospora caninum and prospects for vaccination

Developing an effective vaccine against neosporosis presents several interesting challenges. The parasite is spread efficiently from mother to foetus over several generations, and naturally infected cattle do not appear to develop adequate protective immunity. Modulation of the immune response during pregnancy favours parasite survival and multiplication. However, induction of pro-inflammatory responses that are thought to be protective against Neospora caninum would be detrimental to the pregnancy. So, is vaccination a feasible option to control the disease? This article discusses some of these issues and reports on the progress towards a vaccine for neosporosis.     

新孢子虫侵入宿主细胞的研究进展

概述了新孢子虫侵入宿主细胞的过程以及与侵入有关蛋白质的最新研究进展,指出新孢子虫侵入机制的深入探讨将为新孢子虫病的预防和快速诊断建立有效而实用的方法,并对新孢子虫疫苗以及新孢子虫新药物的研制提供新的策略。     

Factors affecting the survival of Neospora caninum bradyzoites in murine tissues.

Studies were conducted to determine factors that influence the survival of bradyzoites of Neospora caninum within tissue cysts in the brains of experimentally inoculated mice. Viable tissue cysts were detected in the brains of mice inoculated 13 mo previously with either of 2 isolates (NC-1 or NC-2) of N. caninum. Bradyzoites within tissue cysts of the NC-2 isolate survived for at least 14 days at 4 C in refrigerated brain homogenates. Bradyzoites within tissue cysts of the NC-2 isolate also survived in the intact brain of a mouse carcass refrigerated at 4 C for 7 days. Bradyzoites within tissue cysts of the NC-3 isolate were killed by freezing at -20 C for 1 day.     

Fetal infection with Neospora caninum in dairy and beef cattle in Belgium

Neospora caninum is a protozoan parasite, which causes fetal and neonatal mortality in livestock and companion animals. In 224 abortions in Belgian cattle, different diagnostic methods were used to demonstrate infection, and the presence of N. caninum. An indirect fluorescent antibody test (IFAT) was used to analyze fetal and maternal sera and immunohistochemistry (IHC) was performed when lesions consistent with neosporosis were observed in the brain, heart or liver. Twenty dairy cattle sera out of 70 (29%) and 13 beef cattle sera out of 93 (14%) were positive by IFAT. A positive titer to N. caninum was found in seven and three fetuses born to beef and dairy cows, respectively. Lesions consistent with N. caninum infection were observed in 17 fetuses. Of nine positive beef fetuses, five were confirmed by IHC while, all but one dairy fetus were confirmed using the same technique.Age had no influence on the serological status of the mother (P = 0.486) whereas husbandry system had a borderline influence (P = 0.082). However, a strong association (P = 0.004) between the level of antibodies in the dam and the occurrence of lesions in the fetus was observed and lesions were more prominent in dairy than in beef fetuses. Additionally, the distribution of intra-cerebral lesions was more extensive in dairy than in beef fetuses (P < 0.0001). Age and serological status of the fetus were found to influence the occurrence of lesions in beef fetuses (both P < 0.001) but no such significant relationships could be demonstrated in dairy fetuses. The study indicated that N. caninum must be considered as an important cause of bovine abortion in Belgium.     

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.     

Monoclonal antibody inhibition of Neospora caninum tachyzoite invasion into host cells

Monoclonal antibodies were produced against Neospora caninum tachyzoites to identify antigens which may play a role during invasion of host cells. Confocal laser microscopy showed that most antigens recognised by the mAb were located on the surface, but one mAb, 1A5, reacted to the apical end of the parasite. Some mAbs, which recognised 70, 42 and 36kDa parasite proteins, significantly inhibited the invasion of the parasite in vitro. The mAbs which recognised 42 and 36kDa parasite protein, reacted with Nc-p43 and Nc-p36 expressed by vaccinia virus and Escherichia coli, respectively. These results suggest that a 70kDa protein, Nc-p43 and Nc-p36 are involved in the invasion of the parasite into host cells.     

Comparison of the IFAT and Iscom-ELISA response in bovine foetuses with Neospora caninum infection

The study was carried out to evaluate the efficacy of foetal serology in the diagnosis of Neospora-associated bovine abortions. Fluids from 14 foetuses of cows with confirmed neosporosis (Group A), seven foetuses with confirmed bovine viral diarrhoea virus (BVD infection) (Group B) and 11 aborted foetuses without demonstrable infection (Group C) were examined. The age of the foetuses ranged from 4.5 months to 9 months. Albumin concentration (measured by Rocket Immunoelectrophoresis) was not significantly different in Group A compared with that in both Groups B and C, while that in Group B was significantly lower than in Group C. Levels of total IgG ranged from 0.01 to 1.78 (mg IgG) ml(-1) measured by single radial immunodiffusion technique. A measurable level of total IgG was found in all foetuses from Groups A and B, with no significant difference between levels in the two groups. Only one foetus in Group C had a detectable level of IgG. All foetuses in Group A had a specific IgG response (titre> or = 20) against Neospora caninum using the IFAT, while no positive responses in IFAT were found in Groups B and C. Measurement of specific IgG1 and IgG2 by Iscom-ELISA showed one and three false-negative results, respectively, in Group A. The IgG1 and IgG2 response in Group A was correlated according to the Spearman test (r = 0.66). Increasing age of the foetuses correlated significantly with the foetal IgG concentration, the specific IgG and IgG1 + IgG2. On the basis of the results obtained, it was concluded that the IFAT with a cut-off titre of 1:20, was a specific method for diagnosis of neosporosis in foetuses older than 4.5 months. The Iscom-ELISA also showed promising results as a method for screening specific antibodies against N. caninum in foetal fluid.     

Seroprevalence of Neospora caninum infection in dairy cattle of Southern China

A seroepidemiological survey of Neospora caninum infection in dairy cattle was carried out in China's southern Guangdong Province between July 2009 and March 2010. A total of 370 serum samples of dairy cattle was collected from 5 farms and examined for antibodies to N. caninum by enzyme-linked immunosorbent assay. The overall seroprevalence of N. caninum in dairy cattle was 18.9% (70/370). The seroprevalence of N. caninum in aborting cows (22.7%) was higher than that in nonaborting cows (16.3%), but the difference was not statistically significant (P > 0.05). Five-yr-old dairy cattle had the highest seroprevalence (27.8%), followed by those that were 6-yr-old (20.4%). Dairy cattle with 4 pregnancies had the highest seroprevalence (29.2%). There was no apparent association of N. caninum seropositivity with age or number of pregnancies (P > 0.05). The results of the present survey indicated that the infection with N. caninum is prevalent in dairy cattle of all ages in southern China, which may be one of the causes of bovine abortion. This is the first report of seroprevalence of N. caninum in dairy cattle in southern China.     

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.     

Endogenous and exogenous transplacental infection in Neospora caninum and Toxoplasma gondii

It is clear from researching the vertical transmission of Neospora caninum in cattle that the terms 'vertical', 'congenital' and, indeed, 'transplacental' are inadequate for describing two extremely different situations that have fundamentally different immunological, epidemiological and control implications. A similar situation pertains to Toxoplasma gondii in different hosts. We advocate the use of the terms 'endogenous transplacental infection (TPI)' to define foetal infection from a recrudescent maternal infection acquired before pregnancy (and probably prenatally) and 'exogenous TPI' to define foetal infection that occurs as a result of an infection of the dam during pregnancy.     

Seroprevalence of Neospora caninum infection in dairy and beef cattle in Spain

In recent years, neosporosis has been identified as a major cause of abortion in dairy and beef cattle. Although the disease has been described worldwide, there is a Jack of information concerning the prevalence of this infection in different cattle production systems. The aim of this study was to investigate the seroprevalence of Neospora caninum infection in a representative area of beef and dairy cattle production in Spain. A cross-sectional study was undertaken in which herds constituted the initial sampling unit and two strata (dairy and beef herds) were considered. Using a 95% level of confidence and setting 5% (beef) and 5.4% (dairy) error limits, 216 beef and 143 dairy herds were randomly selected and sampled. Nine animals (> 1 year old) were randomly sampled in each herd to detect the presence of the infection. A herd was considered infected when at least one animal was seropositive. In total, serum samples from 1121 dairy and 1712 beef animals were collected and tested for specific anti-N. caninum IgG using an ELISA. Specific antibodies were detected in 55.1% (119/216) beef and 83.2% (119/143) dairy herds. Individual prevalences obtained were 17.9% (306/1712) for beef and 35.9% (402/1121) for dairy animals. Presence of N. caninum infection was higher in dairy than in beef herds and the association between infection and the cattle production system (dairy or beef) was statistically significant [(chi2)Y= 29.21, P < 0.001, OR = 4.04 (2.35-6.99)]. Herd size of dairy cattle did not appear to be associated with N. caninum infection. On the contrary, infection was associated with herd size in beef cattle (chi2 = 12.79, P < 0.01). Finally, no association was found between replacement or pasture management and infection in beef herds.     

Neospora caninum infection does not affect early pregnancy in dairy cattle

This study was designed to evaluate the relationship between Neospora caninum infection prior to pregnancy, as determined through maternal serology, and the subsequent occurrence of abortion in dairy cattle. Special emphasis was placed on pregnancy losses in the first trimester of pregnancy. Neospora caninum antibodies were analyzed by commercial ELISA in 2773 pregnant animals (2022 parous cows and 751 heifers) from six herds. The mean seroprevalence of antibodies to N. caninum in the herds was 15.1% (n = 419). From gestation Day 34 to the 90th day of pregnancy, there were 183 abortions (6.6% of all pregnancies) (23 in Neospora positive animals). After 90 days of pregnancy, the number of abortions was 146 (5.3%); 126 occurring during the second and 20 during the third trimester of pregnancy (105 in Neospora positive animals). Multiple logistic regression analyses were performed on data from each animal using abortion before or after 90 days of pregnancy as the dependent variable, and Neospora positivity, herd, pregnancy season, and parity (parous or nonparous) as independent factors. No significant effects of Neospora positivity and herd were found on the abortion rate before 90 days of pregnancy. Based on the odds ratio, the abortion rate was 4 times higher (P < 0.0001) in animals that became pregnant in the warm than in the cool period, and 3.7 times higher (P < 0.0001) in parous than in nonparous animals. Neospora positivity was the only variable included in the logistic regression model for abortions occurring after 90 days of pregnancy. Seropositivity in an animal increased the probability of abortion by an odds ratio of 18.9 (P < 0.0001; 95% confidence interval 12.9-27.8). Season, parity, and herd showed no effect. The results of the present study suggest that chronic N. caninum infection prior to pregnancy appears not to affect the early fetal period, but does have a significant abortive effect after 90 days of gestation.