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.     

Udder health in dairy cattle infected with Neospora caninum

Blood samples were collected from 3449 cows on 57 representative Ontario dairy herds during the summer of 1998 and analysed for antibody to Neospora caninum using an ELISA. Forty-eight herds (2742 cattle) contained at least one N. caninum-seropositive animal. Two composite milk samples were collected from all cattle: the first on the day of blood collection and the second 68 to 365 days later. All milk samples were submitted for bacteriological culture. Ontario Dairy Herd Improvement Corporation (DHI) data were available for 3162 cattle in the 57 herds at the time of bleeding. Furthermore, complete DHI data were available for 1658 cattle that were culled between 12 and 24 months following blood collection. Using a standardised ELISA sample-to-positive (S/P) cut-off of ≥0.45, the corrected seroprevalence was 8.2% overall and 10.1% within seropositive herds. At blood collection the odds of N. caninum-seropositive cows having a high linear score (≥4.0; equivalent to a somatic cell count ≥200,000 cells/ml) was 27% less than for seronegative animals. Similarly, at the time of culling, the odds of having a high linear score was 22% less in N. caninum-seropositive cattle. Overall, linear score was lower in N. caninum-seropositive cattle at culling. After controlling for herd, parity, days in milk, and the interval between collection of milk samples, the odds of N. caninum-seropositive cattle testing positive for an environmental pathogen (i.e. environmental Streptococcus species and coliforms) on the second milk sample was 56% less than for seronegative animals. The odds were 83% less at a higher ELISA S/P cut-off of ≥0.70. Finally, the odds of N. caninum-seropositive cattle developing a new infection with a major pathogen (environmental or contagious) were 60% less than seronegative cows using the higher ELISA S/P cut-off.     

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.     

Quantification of vertical and horizontal transmission of Neospora caninum infection in Dutch dairy herds

Ninety-six of 108 randomly selected Dutch dairy herds had one or more cows with a positive serostatus for N. caninum. In these 96 herds, we have quantified the probabilities of vertical transmission (VT) and horizontal transmission (HT) of N. caninum infection by combining serostatus and pedigree data in 4091 dam-daughter pairs. The probability of animals infected by vertical transmission during pregnancy (Prob(VT)) was calculated as the proportion of seropositive daughters among daughters of seropositive dams. The probability of animals infected by horizontal transmission (Prob(HT)) was the proportion of seropositive daughters among daughters of seronegative dams. These probabilities were calculated after the frequencies of observed dam-daughter combinations were corrected for (1) imperfect test-characteristics, (2) underestimation of horizontal transmission in situations that seronegative dams were horizontally infected after the birth of their daughters and (3) overestimation of vertical transmission in situations that seronegative daughters born from seropositive dams were horizontally infected. The incidence rate for horizontal transmission was calculated based on Prob(HT) and the average age of the animals in these herds. Based on the analysis of dam-daughter serology, Prob(VT) was 61.8% (95% CI: 57.5-66.0%) and Prob(HT) was 3.3% (95% CI: 2.7-3.9%). After adjusting the observed frequencies for imperfect test-characteristics, underestimation of horizontal transmission and overestimation of vertical transmission, Prob(VT) decreased to 44.9% (95% CI: 40.0-49.9%) while Prob(HT) increased to 4.5% (95% CI: 3.9-5.2%). Prob(HT) corresponded with an incidence rate for horizontal transmission of 1.4 (95% CI: 1.2-1.7) infections per 100 cow-years at risk. When stratifying herds for the presence of farm dogs, Prob(HT) was higher (5.5% (95% CI: 4.6-6.4%)) in herds with farm dogs than in herds without farm dogs (2.3% (95% CI: 1.5-3.4%)). When stratifying for within-herd seroprevalence, Prob(HT) was higher (10.3% (95% CI: 8.6-12.2%)) in herds with high (> or =10%) within-herd seroprevalence compared with herds with low (<10%) within-herd seroprevalence (2.0% (95% CI: 1.5-2.6%)). Although there was this relation between Prob(HT) and within-herd seroprevalence (crude OR(PREV) = 5.7 (95% CI: 4.0-7.9)), in herds without farm dogs, this relationship was no longer statistical significant (OR(PREV|DOG-) = 1.9 (95% CI: 0.7-5.5)). It indicated that the association between seroprevalence and Prob(HT) depended largely on the presence of farm dogs. In addition, when looking for the presence of specific age-groups with significantly higher seroprevalence compared with the rest of the herd, there were 7 herds in which two or more horizontally-infected animals were present in specific age-groups. This was an indication of a recent point-source exposure to N. caninum. These results reiterate the current control strategies to apply strict dog-management measures as well as to minimize within-herd seroprevalence by monitoring serostatus of animals.     

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.     

奶牛新孢子虫病血清流行病学调查

本研究用酶联免疫吸附试验（ELISA）对采集自北京地区的94份奶牛血清（随机采集）和河北地区的55份奶牛血清（有流产史奶牛），进行Neospora caninum血清抗体检测。结果发现，北京地区随机采集的奶牛血清N．caninum抗体阳性率为18．1％（17／94），河北地区有流产史的奶牛N．caninum血清抗体阳性率为23．6％（13／55）。采用牛奶记录体系（DHI）对北京地区17头N．caninum血清抗体阳性牛进行了日产奶量、乳中蛋白率和乳脂率的测定，并与同群牛中134头阴性牛比较。结果表明，N．caninum血清抗体阳性牛日产奶量比阴性牛降低9．7％，乳中蛋白率和乳脂率分别降低20％和15．4％。初步证明N．caninum血清抗体阳性奶牛产奶量降低及奶品质的下降。对不同N．caninum抗体滴度阳性牛的泌乳期主要生产性能比较发现，其生产性能的变化与抗体滴度无明显相关性。     

Prevalence of Neospora caninum and bovine viral diarrhoea virus in dairy cows in Southern Vietnam

The main purpose of this study was to investigate the prevalence of Neospora caninum and bovine viral diarrhoea virus (BVDV) in some dairy herds in Southern Vietnam, and to ascertain whether there were differences in seroprevalences between herds with imported and locally bred cows. Serum samples collected on five state farms and 97 smallholder herds were analysed for the presence of antibodies to N. caninum and BVDV. All BVDV antibody-negative sera were further tested by antigen-ELISA in order to identify persistently infected individuals. The N. caninum prevalence varied between 16% and 53% in the state herds, and was higher in the four herds that had imported cows than in the herd that only had locally bred cows. Nineteen percent of the samples collected on smallholder farms, which all had only locally bred cows, had antibodies to N. caninum. The BVDV seroprevalence varied between 58% and 93% on the state farms. In smallholder herds, the prevalence of BVDV among the sampled cows was 18% and even lower on the state farms. Despite the high seroprevalence for BVDV in the state herds, no persistently BVDV infected cows were found. Given the high prevalence for Neospora and BVDV among herds with imported cows, it seems advisable to test for both infections before cattle are imported into the country.     

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.     

Neospora caninum detected in feral rodents

The role of rodents in the epidemiology of neosporosis was investigated by assaying brain tissue of feral mice (Mus musculus) and rats (Rattus norvegicus) for Neospora caninum. Both mouse and rat brain tissue were extracted for total DNA, and subjected to two different N. caninum-specific nested polymerase chain reaction (PCR) assays. A portion of brain tissue from the mice and rats were also assayed for N. caninum in gerbils or gamma-interferon gene knockout (KO) mice. Of the 105 feral mice tested, 10% were positive in the N. caninum-specific PCR assays. Of the 242 rats tested, 30% were positive in both assays. Although mice and rats had N. caninum by PCR testing, clinical signs of N. caninum infection were not observed nor were N. caninum parasites observed in gerbils or KO mice inoculated with the rodent brain tissue.     

Financial analysis of various strategies for the control of Neospora caninum in dairy cattle in Switzerland

The present study was conducted to estimate the direct losses due to Neospora caninum in Swiss dairy cattle and to assess the costs and benefits of different potential control strategies. A Monte Carlo simulation spreadsheet module was developed to estimate the direct costs caused by N. caninum, with and without control strategies, and to estimate the costs of these control strategies in a financial analysis. The control strategies considered were “testing and culling of seropositive female cattle”, “discontinued breeding with offspring from seropositive cows”, “chemotherapeutical treatment of female offspring” and “vaccination of all female cattle”. Each parameter in the module that was considered to be uncertain, was described using probability distributions. The simulations were run with 20,000 iterations over a time period of 25 years.

The median annual losses due to N. caninum in the Swiss dairy cow population were estimated to be € 9.7 million. All control strategies that required yearly serological testing of all cattle in the population produced high costs and thus were not financially profitable. Among the other control strategies, two showed benefit–cost ratios (BCR) >1 and positive net present values (NPV): “Discontinued breeding with offspring from seropositive cows” (BCR = 1.29, NPV = € 25 million) and “chemotherapeutical treatment of all female offspring” (BCR = 2.95, NPV = € 59 million). In economic terms, the best control strategy currently available would therefore be “discontinued breeding with offspring from seropositive cows”.     

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.     

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.     

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.     

Seroprevalence of antibody to NcSAG1 antigen of Neospora caninum in cattle from Western Java,Indonesia

Neospora caninum can cause fetal abortion and neonatal mortality incattle, and is a cause of economic concern worldwide. This study aimed to determine theprevalence of Neospora caninum-specific antibodies in cattle from WesternJava, Indonesia. Serum samples from 991 cattle from 21 locations were tested forantibodies to N. caninum by using an enzyme-linked immunosorbent assay(ELISA) on the basis of recombinant NcSAG1. The overall seroprevalence was 16.6%, rangingfrom 0 to 87.5% in the sampled locations. The results of this study indicate latentinfection rates of sampled animals were different in each location. Further studies arenecessary to elucidate the relationship between N. caninum infection andabortion in cattle, and to identify risk factors for infection in high-prevalenceenvironments.     

Serological survey of Neospora caninum in dogs in the Czech Republic and a long-term study of dynamics of antibodies

The main aim of the present study was to establish the prevalence of antibodies against Neospora caninum dogs from the Czech Republic and to examine the dynamics of antibody titers during a long-term period. For this purpose, sera of 858 dogs were examined for the presence of anti-N. caninum antibodies using an indirect immunofluorescent antibody test (IFAT). Four groups of dogs of various origins were included in the survey: the first group (A, n=470) comprised dogs purchased by the Czech Army from the civilian sector throughout the Czech Republic, with 22 (4.7%) N. caninum-positive dogs, second group (B, n=115) represented police dogs with no seropositive animal, third group (C, n=195) were pet dog sera collected for veterinary clinic with 5 (2.6%) anti-N. caninum sera and the fourth group (D, n=78) of canine shelter dogs with the seroprevalence of 19.2%. The differences in seroprevalence were significant (P< or =0.01) between groups B and A, and between D and A. None of the serologically positive animals had clinical signs of neurological disorders. Coprological examination did not reveal any dog shedding N. caninum oocysts. The seropositivity rates for N. caninum were analyzed in relation to other data, such as age, breed and gender. Increased prevalence rates of anti-N. caninum antibodies were found in the older age strata of the dog population sample tested in the present study. We found significantly higher (P=0.02) prevalence in 3-3.5-year-old dogs (11.1% of 36), as compared to 1-1.5-years-old dogs (2% of 98). A longitudinal study of antibody dynamics was carried out in 19 initially seropositive dogs over a period of 4 years. The second and third examinations revealed that antibody titers decreased in majority of positive dogs (10, 52.6%), of which in seven cases (36.8%) the titers fell to levels that are currently considered as being seronegative (titer <1:50), or even became undetectable (titer <1:25).     

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.     

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.