Seroprevalence of Neospora caninum in non-carnivorous wildlife from Spain

Serum samples from 1034 non-carnivorous wildlife from Spain were tested for antibodies to Neospora caninum by competitive screening enzyme linked immunosorbent assay (ELISA) and confirmed by an indirect fluorescent antibody test (IFAT). High agreement was observed between results in both techniques (kappa value higher than 0.9). Prevalences of N. caninum antibodies positive by both techniques were 11.8% of 237 red deer (Cervus elaphus), 7.7% of 13 barbary sheep (Ammotragus lervia), 6.1% of 33 roe deer (Capreolus capreolus) and 0.3% of 298 wild boar (Sus scrofa). In one of 53 hares (Lepus granatensis), antibodies were found in the ELISA but could not be confirmed by IFAT due to lack of sample. Antibodies to N. caninum were not found in any of 251 wild rabbits (Oryctolagus cuniculus), 79 fallow deer (Dama dama), 27 mouflon (Ovis ammon), 40 chamois (Rupicapra pyrenaica) and three Spanish ibex (Capra pyrenaica). Statistically significant differences were observed between N. caninum seroprevalence in red deer and management of hunting estates (open versus fenced) with higher prevalence in fenced estates, and among sampling sites. Seroprevalence was particularly high in some areas (MO estate in South-Central Spain or some estates of Catalonia, North-East Spain), while no contact with N. caninum was observed in others. Results indicate that in certain areas of Spain, N. caninum is present in wildlife, especially in red deer. These results have important implications in both sylvatic cycles and may influence the prevalence of infection in cattle farms in those areas. To our knowledge, this is the first report of antibodies to N. caninum in wildlife from Spain and the first report of N. caninum antibodies in barbary sheep and wild boar.     

Seroepidemiology of Neospora caninum infection in dairy cattle herds in Mashhad area, Iran

A seroepidemiology study of Neospora caninum was conducted in Mashhad area, Khorasan province, Iran on 337 cattle from 30 dairy using ELISA. All herds had seropositive cattle and antibodies were formed in 46% of the cattle. Abortion was significantly associated with seropositivity of cattle. The highest risk of abortion was observed in the 1-2-year-old cows. Our results indicate that Neospora infection is widespread in Iran.     

Nationwide seroprevalence of Neospora caninum among dairy cattle in Japan

Serum samples from 2420 clinically healthy dairy cattle, randomly selected from stored sera in 18 districts of Japan, were tested for the presence of Neospora caninum antibodies using an indirect fluorescent antibody test (titer > or =1:200). Nationwide seroprevalence is estimated at 5.7% (139/2420). Seropositive cattle were detected in all surveyed districts despite the evidence of confirmed case reports of bovine neosporosis, showing that N. caninum is widely distributed throughout Japan. Age-specific seroprevalence did not increase with cattle age, suggesting that Neospora infection is likely to be transmitted vertically rather than horizontally in Japan. Considering that N. caninum seropositive cows are thought to be more likely to abort, substantial fetal losses may be induced by N. caninum infection in Japan. Devising strategies are needed to reduce the economic impact on the Japanese dairy industry. This is the first study to investigate the nationwide seroprevalence of N. caninum in cattle in Asia.     

Experimental infection with a low virulence isolate of Neospora caninum at 70 days gestation in cattle did not result in foetopathy

The Nc-Spain 1H isolate of Neospora caninum, which was newly obtained from the brain of a congenitally asymptomatic infected calf, demonstrated a reduced in vitro tachyzoite yield and viability rate, as well as low virulence in mouse models. The objective of the present study was to determine the ability of this isolate to induce foetal death in a pregnant bovine model. For this purpose, 13 naïve pregnant heifers were divided into three groups and were experimentally challenged with either 10⁷ tachyzoites of Nc-1 (group 1, n = 5), Nc-Spain 1H (group 2, n = 5) isolates or phosphate-buffered saline (group 3, n = 3) intravenously at 70 days of gestation. After inoculation, pregnancy was monitored and dams were sacrificed when foetal death was detected. The remaining animals were slaughtered at 45 days post-infection. Maternal and foetal samples were collected for examination by histology and parasite DNA detection. Parasitaemia, specific anti-N. caninum IgG and interferon γ responses were also studied. At 3–4 weeks after infection, foetal death was detected in 3 out of 5 Nc-1-infected dams. However, no evidence of foetal death was observed in either Nc-Spain 1H-infected or control groups during the period studied. The most severe histopathological lesions were observed in the placenta and foetal organs from Nc-1-infected cattle that exhibited foetal death. It was in these animals that N. caninum DNA was more frequently detected. Parasitaemia was observed in all Nc-1-infected dams and in only 3 out of 5 Nc-Spain 1H-infected animals. The magnitude of the immune response was significantly higher in the Nc-1-inoculated group than in the group inoculated with the Nc-Spain 1H isolate. These data reveal the reduced virulence of the Nc-Spain 1H isolate in cattle.     

Prevalence of antibodies to Neospora caninum in local and imported cattle breeds in the Kars province of Turkey

This study investigates the seroprevalence of Neospora caninum in cattle in the province of Kars in north-eastern Turkey. A total of 301 serum samples, 228 from local breeds of cows with a history of recent abortion and the remaining 73 collected at random from Simmental cows imported from Germany or their offspring, were tested for anti-Neospora antibodies by ELISA. All the serum samples from local breeds were negative for N. caninum antibodies, apart from one which tested inconclusive. In contrast six of the samples (8.2% with 95%CI: 2-14.5%) from Simmental cows tested positive. There was a significant difference (P<0.001) in the seroprevalence of N. caninum between local and Simmental breeds, even if the inconclusive test was considered positive, and the odds ratio of its occurrence in the Simmental breed was 20.3. In conclusion, it is unlikely that N. caninum contributes to abortion in local cattle breeds and our results also suggest that N. caninum was introduced to the region by the importation of Simmental cattle and that the vertical transmission of the parasite in cattle is important in the region.     

Comparison of serological methods for the diagnosis of Neospora caninum infection in cattle

The aims of this study were to evaluate the performance and agreement of various commercial and in-house Neospora caninum antibody assays used in dairy cattle in North America, and to investigate reproducibility of two assays performed in different laboratories. From 1998 to 2005, three enzyme linked immunosorbent assays (ELISAs, a competitive ELISA-VMRD Inc., an indirect ELISA-Biovet Inc., and another indirect ELISA-Herdchek IDEXX Corp.), two indirect fluorescent antibody tests (IFATs, VMRD Inc., and in-house USDA) and one N. caninum agglutination test (NAT, in-house USDA) were utilized to test 397 randomly selected dairy cattle serum samples from 34 herds in eastern Canada for antibodies to N. caninum. The manufacturers' recommended cut-off values were used to evaluate test performance and agreement between tests. One IFAT (VMRD Inc.) performed well (sensitivity and specificity: 0.97 and 0.97, respectively) using reference sera (n = 452), therefore, results from this IFAT on the 397 samples could subsequently be used as the reference standard to calculate test characteristics for the other assays. Only 11% of the 397 sera were found to be N. caninum-positive with the IFAT. Prevalence-adjusted bias-adjusted kappa (PABAK) ranged from 0.06 to 0.99. Positive agreement was moderate to very good (P(pos) = 0.25-0.96). Negative agreement was very good for all assays (P(neg) > 0.94) except NAT (P(neg) = 0.66). Sensitivity was > or =0.89 for all assays except the NAT, which had a significantly lower sensitivity (0.66). Specificity was high (>0.94) for all assays except for one indirect ELISA (specificity = 0.52). This indirect ELISA did not perform satisfactorily when used in 1998, but an improved version of the ELISA performed as one of the best assays in 2004. Reproducibility of the competitive ELISA was excellent, but the reproducibility of the indirect ELISA that was improved was low (concordance correlation coefficient = 0.90 and 0.36, respectively). The performance characteristics observed for most assays in this study make them useful for screening antibodies to N. caninum in cattle.     

Seroprevalence of <Emphasis Type="Italic">Neospora caninum</Emphasis> antibodies in beef cattle in three southern states of Mexico

The aims of the present study were to examine the seroprevalence of neosporosis in beef herds from three southern states of Mexico and determine the association with several risk factors. A cross-sectional serological survey for Neospora caninum was carried out by sampling a total of 596 animals from 31 herds in Chiapas, Veracruz, and Yucatan States and tested using an ELISA assay (IDEXX) to detect anti-N. caninum antibodies. The overall prevalence was 11.6%, (95% CI: 0.93–0.14), however, the prevalence for Chiapas was 15% (30/200) (95% CI: 0.11–0.21), in Yucatan 11.3% (21/186) (95% CI: 0.07–0.17) and in Veracruz 8.6% (18/210) (95% CI: 0.05–0.13). Of the 596 serum samples taken, 578 were from females and 18 were bulls., Only one bull sample was found seropositive from one herd in Veracruz State. The overall herd-prevalence was 70.9% (22/31) (95% CI: 0.53–0.84), and by State was: Chiapas 90% (9/10) (95% CI: 0.60–0.98); Yucatan 72.7% (8/12) (95% CI: 0.39–0.86); and Veracruz 50% (5/10) (95% CI: 0.24–0.76), respectively. The highest age-prevalences were found in animals 1 yr old (19.4% [95% CI: 0.01–0.35]) and 4 yrs old (19.6% [95% CI: 0.12–0.29]), and the lowest in animals 3 –yrs of age (6.2%,[95% CI: 0.02–0.20]). No association was found among all the variables tested in this study since most of the ranches have similar management conditions. In conclusion, N. caninum infection is common among beef herds in the Mexican tropics.     

新孢子虫SAG1-SRS2融合基因真核表达质粒的构建及表达

根据犬新孢子虫NcSAG1-NcSRS2融合基因序列,设计了1对含有Kozak序列、终止密码子、BamHⅠ和XhoⅠ酶切位点的引物,以含有NcSAG1-NcSRS2融合基因的质粒pGEX-tNcP43-P36为模板,经PCR扩增获得NcSAG1-NcSRS2融合基因片段,用BamHⅠ和XhoⅠ双酶切该片段,回收得到含有以上2个酶切位点黏端的NcSAG1-NcSRS2融合基因,将此基因片段克隆至相同酶切回收后的pcDNA3.1（＋）真核表达载体中,获得重组质粒pcNCSAG1-SRS2。经PCR鉴定、限制性内切酶酶切分析和克隆片段序列测定、比较,证实了重组质粒的正确性。将构建好的真核表达质粒转染到COS-7细胞中进行瞬时表达,经免疫荧光检测,证实了该载体能在细胞内进行蛋白表达。     

Maternal and foetal immune responses of cattle following an experimental challenge with Neospora caninum at day 70 of gestation

The immune responses of pregnant cattle and their foetuses were examined following inoculation on day 70 of gestation either intravenously (iv) (group 1) or subcutaneously (sc) (group 2) with live NC1 strain tachyzoites or with Vero cells (control) (group 3). Peripheral blood mononuclear cell (PBMC) responses to Neospora antigen and foetal viability were assessed throughout the experiment. Two animals from each group were sacrificed at 14, 28, 42 and 56 days post inoculation (pi). At post mortem, maternal lymph nodes, spleen and PBMC and when possible foetal spleen, thymus and PBMC samples were collected for analysis. Inoculation with NC1 (iv and sc) lead to foetal deaths in all group 1 dams (6/6) and in 3/6 group 2 dams from day 28pi; statistically significant (p ≤ 0.05) increases in cell-mediated immune (CMI) responses including antigen-specific cell proliferation and IFN-γ production as well as increased levels of IL-4, IL-10 and IL-12 were observed in challenged dams compared to the group 3 animals. Lymph node samples from the group 2 animals carrying live foetuses showed greater levels of cellular proliferation as well as significantly (p ≤ 0.05) higher levels of IFN-γ compared to the dams in group 2 carrying dead foetuses. Foetal spleen, thymus and PBMC samples demonstrated cellular proliferation as well as IFN-γ, IL-4, IL-10 and IL-12 production following mitogenic stimulation with Con A from day 14pi (day 84 gestation) onwards. This study shows that the generation of robust peripheral and local maternal CMI responses (lymphoproliferation, IFN-γ) may inhibit the vertical transmission of the parasite.     

Myeloid differentiation factor 88 is required for resistance to Neospora caninum infection

Neospora caninum is an intracellular parasite that causes major economic impact on cattle raising farms, and infects a wide range of warm-blooded hosts worldwide. Innate immune mechanisms that lead to protection against this parasite are still unknown. In order to investigate whether myeloid differentiation factor 88 (MyD88) is required for resistance against N. caninum, genetically deficient mice (MyD88^−/−) and wild type littermates were infected with live tachyzoites and the resistance to infection was evaluated. We found that sub-lethal tachyzoite doses induced acute mortality of MyD88^−/− mice, which succumbed to infection due to uncontrolled parasite replication. Higher parasitism in MyD88^−/− mice was associated with the lack of IL-12 production by dendritic cells, delayed IFN-γ responses by NKT, CD4⁺ and CD8⁺ T lymphocytes, and production of high levels of IL-10. MyD88^−/− mice replenished with IL-12 and IFN-γ abolished susceptibility as the animals survived throughout the experimental period. We conclude that protective IFN-γ-mediated immunity to N. caninum is dependent on initial MyD88 signaling, in a mechanism triggered by production of IL-12 by dendritic cells. Further knowledge on Toll-like receptor recognition of N. caninum antigens is encouraged, since it could generate new prophylactic and therapeutic tools to control parasite burden.     

Seroepidemiology of Neospora caninum and Toxoplasma gondii in cattle and water buffaloes (Bubalus bubalis) in the People's Republic of China

A seroepidemiological survey of Neospora caninum and Toxoplasma gondii in cattle and water buffaloes was carried out in the People's Republic of China. Serum samples were obtained from dairy (n=262, 9 herds in 9 provinces) and beef cattle (n=10, 1 herd) and water buffaloes (n=40) in China. All sera were tested for antibodies to N. caninum and T. gondii by an enzyme-linked immunosorbent assay (ELISA) and an indirect agglutination test (IAT), respectively. The overall seroprevalence of N. caninum in dairy cattle was 17.2% (45/262), and the herds seroprevalence of N. caninum was 88.9% (8/9), and antibodies to T. gondii were present in 6 cows (2.3%). None of the cows had antibodies against both T. gondii and N. caninum. Antibodies to T. gondii or N. caninum were not found in beef cattle or water buffaloes. The seroprevalence of N. caninum in aborting cows (20.2%) was higher than that in non-aborting cows (16.6%) with an odds ratio of 1.26 (95% CI, 0.54-2.95), but the difference was not statistically significant (P>0.05). There was no apparent association of N. caninum seropositivity with age or number of pregnancies. This is the first report on the seroprevalence of N. caninum in cattle and water buffaloes in China.     

Influence of Neospora caninum intra-specific variability in the outcome of infection in a pregnant BALB/c mouse model

Previous assays in pregnant animals have demonstrated the effect of different host factors and timing of infection on the outcome of neosporosis during pregnancy. However, the influence of Neospora caninum isolate itself has been poorly investigated. Here, we compared the effects on clinical outcome and vertical transmission observed in a pregnant mouse model following infection with 10 different N. caninum isolates. The isolates in our study included the Nc-Liv isolate and nine N. caninum isolates obtained from calves. Female BALB/c mice were inoculated with 2 × 10⁶ tachyzoites at day 7 of pregnancy. Morbidity and mortality, in both dams and offspring during the course of infection, and transmission to progeny at day 30 postpartum were evaluated. The serum IgG1 and IgG2a production in dams were also examined. All dams showed elevated IgG1 and IgG2a responses, confirming N. caninum infection, although signs of disease were only exhibited in dams infected with 4 of the 10 isolates (Nc-Spain 4H, Nc-Spain 5H, Nc-Spain 7 and Nc-Liv). In neonates, clinical signs were observed in all N. caninum-infected groups, and neonatal mortality rates varied from greater than 95% with the isolates mentioned above to less than 32.5% with the other isolates. Vertical transmission rates, as assessed by parasite PCR-detection in neonate brains, also varied from 50% to 100% according to the isolate implicated. These results confirm the wide pathogenic and transmission variability of N. caninum. The intra-specific variability observed herein could help us explain the differences in the outcome of the infection in the natural host.     

Syphacia (Syphacia) maxomyos sp. n. (Nematoda: Oxyuridae) from Maxomys spp. (Rodentia: Muridae) from Sulawesi and Sumatra,Indonesia

The present report describes Syphacia (Syphacia) maxomyos sp. n. (Nematoda: Oxyuridae) from two species of spiny rats, Maxomys musschenbroekii from Sulawesi and M. whiteheadi from Sumatra. It is characterized by a cephalic plate extending laterally with dorsoventral constriction and stumpy eggs with an operculum rim reaching pole. It is readily distinguishable by the former feature from all of hitherto known representatives of this genus in Indonesia, but it resembles parasites in Murini and Hydromyni rodents in continental Asia and Sahul. This is the first Syphacia species distributed in both the Sunda Shelf and Sulawesi with the exception of Syphacia muris, a cosmopolitan pinworm found in rodents of the of genus Rattus. It is surmised that S. maxomyos is specific to Maxomys and that it was introduced to Sulawesi by dispersal of some Maxomys from the Sunda Shelf.     

Studies on Neospora caninum DNA detection in the oocytes and embryos collected from infected cows

Neosporosis is a major cause of abortion in cattle over the world. One of the methods of preventing vertical transmission within the herd is to avoid breeding replacement heifers from infected dams. Another procedure suggested and recommended by the International Embryo Transfer Society (IETS) is embryo transfer (ET) from infected dams into uninfected recipients. Oocytes and embryos taken from seropositive cows were examined for the presence of Neospora caninum DNA. A modified PCR protocol using Np21 and Np6 primers was applied to detect parasite DNA in the samples. The expected 328 bp product was not obtained in oocytes and/or embryos collected from seropositive dams. The results confirmed that transfer of the embryos from seropositive donors into seronegative recipients is an appropriate method to eliminate vertical transmission of neosporosis in a herd. The present study demonstrated that oocytes and embryos are not exposed to N. caninum in the uterine cavity of seropositive dams.     

Neospora caninum associated with epidemic abortions in dairy cattle: The first clinical neosporosis report in Turkey

Neospora caninum, a protozoan parasite, has been considered as one of the most important etiological agents responsible for abortion in dairy cattle throughout the world since it was first identified in dogs in 1988. In this report, characteristics of neosporosis, detected in a dairy cow ranch having epidemic abortions as high as 18.4%, were described. Blood samples were collected from 25 infertile or aborted dairy cattle, 6 calves born in 2006 and 40 heifers that were born in 2005 and raised in the same ranch. Necropsy was conducted in a 20-day-old Simmental calf that exhibited neurological signs including incoordination, head shaking, hyperextension in forelimbs and hindlimbs and tremor. The seroprevalance in aborted or infertile dairy cattle, heifers, and calves was 60%, 40%, and 33.3%, respectively. The mothers of seropositive two calves including clinically affected calf and its dam were N. caninum seropositive. In immunoperoxidase examinations, N. caninum antigen immunopositivity was observed in the degenerative and necrotic neurons in the brain, cerebellum as well as neurons in dorsal root ganglia of the cervical and thoracic regions of the spinal cord. In the heart, myocytes and Purkinje cells exhibited granular and linear patterns of immunoreactivity. Striated myofibers around the eyeball also showed immunolocalization for N. caninum antigen. Ultrastructurally, tachyzoites with typical apical complex, rhoptries and double-layered parasitic membrane were detected in the brain and heart sections. In conclusion, this report described clinical neosporosis for the first time in Turkey with tissue localization of the causative agents. This scientific communication also discusses the possible impact of cattle neosporosis by clinical, serologic and pathologic evidences collected from the survey of calves born in two successive generations in a ranch.     

Host/parasite relationship in the in vitro infection of rat gliocytes by Neospora caninum: evaluation of cell respiration

Neospora caninum is a protozoon that causes abortion in cattle and neuromuscular lesions in dogs, with the formation of cysts mainly in the central nervous system. Since N. caninum is an intracellular parasite with tropism for the cells of nervous system, this study evaluated the respiratory metabolism of glial cells infected by this* parasite. Glial cultures obtained from the cerebral cortex of newborn rats were kept in DMEM enriched with 10% fetal bovine serum, 1 mM pyruvic acid and 2 mM of L-glutamine. They were infected at a ratio of approximately 1:1 (cell/parasite). Oxygen consumption was evaluated by polarography in the non infected and N. caninum infected groups, 24 and 72 h following infection. Glial cell respiration after 24 and 72 h was 307.2 +/- 34.7 and 308.9 +/- 64.1 microL of oxygen per mug of total protein per minute, and 566.2 +/- 54.6 and 579 +/- 117.5 microL O2/microg of total protein/minute in the control and infected groups, respectively. These results show that N. caninum does not interfere with glial respiration in vitro.     

GC/MS analysis of high-performance liquid chromatography fractions from Sophora flavescens and Torilis japonica extracts and their in vitro anti-neosporal effects on Neospora caninum

We analyzed alcoholic extracts of herbs possessing anti-neosporal activity against Neospora (N.) caninum. To identify the chemical components of Sophora (S.) flavescens and Torilis (T.) japonica associated with anti-neosporal activity, specific fractions were isolated by high-performance liquid chromatography (HPLC). In vitro activity of the fractions against N. caninum was then assessed. Gas chromatography/mass spectrometry (GC/MS) was used to identify and quantify specific anti-neosporal molecules in the herbal extracts. Almost all HPLC fractions of S. flavescens and T. japonica had higher levels of anti-neosporal activity compared to the not treated control. Active constituents of the extracts were sophoridane, furosardonin A, and tetraisopropylidene-cyclobutane in S. flavescens; 5,17-β-dihydroxy-de-A-estra-5,7,9,14-tetraene, furanodiene, and 9,12-octadecadienoic acid (Z,Z)-(CAS,1) in T. japonica.     

Adjuvant and immunostimulatory effects of a D-galactose-binding lectin from Synadenium carinatum latex (ScLL) in the mouse model of vaccination against neosporosis

Vaccination is an important control measure for neosporosis that is caused by a coccidian parasite, Neospora caninum, leading to abortion and reproductive disorders in cattle and serious economic impacts worldwide. A D-galactose-binding lectin from Synadenium carinatum latex (ScLL) was recently described by our group with potential immunostimulatory and adjuvant effects in the leishmaniasis model. In this study, we evaluated the adjuvant effect of ScLL in immunization of mice against neosporosis. First, we investigated in vitro cytokine production by dendritic cells stimulated with Neospora lysate antigen (NLA), ScLL or both. Each treatment induced TNF-α, IL-6, IL-10 and IL-12 production in a dose-dependent manner, with synergistic effect of NLA plus ScLL. Next, four groups of C57BL/6 mice were immunized with NLA + ScLL, NLA, ScLL or PBS. The kinetics of antibody response showed a predominance of IgG and IgG1 for NLA + ScLL group, whereas IgG2a response was similar between NLA + ScLL and NLA groups. Ex vivo cytokine production by mouse spleen cells showed the highest IFN-γ/IL-10 ratio in the presence of NLA stimulation for mice immunized with NLA + ScLL and the lowest for those immunized with ScLL alone. After parasite challenge, mice immunized with NLA + ScLL or ScLL alone presented higher survival rates (70-80%) and lower brain parasite burden as compared to PBS group, but with no significant changes in morbidity and inflammation scores. In conclusion, ScLL combined with NLA was able to change the cytokine profile induced by the antigen or lectin alone for a Th1-biased immune response, resulting in high protection of mice challenged with the parasite, but with low degree of inflammation. Both features may be important to prevent congenital neosporosis, since protection and low inflammatory response are necessary events to guide towards a successful pregnancy.