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In Italy Neospora caninum has been reported in cattle, in buffaloes and in dogs. No data are available about the infection in sheep and goats. In this paper, the authors report the detection of protozoan cysts, identified as N. caninum by PCR, in the brain of an aborted goat foetus.
AIMS: To investigate the seroprevalence of Neospora caninum infection in breeder bulls. In addition, the level of agreement of serological methods used was evaluated. METHODS: A total of 285 bulls of different ages and breeds raised for reproductive purposes in Spain were sampled for serum antibody activity to N. caninum. Sera were tested using three validated assays: the indirect fluorescent antibody test (IFAT) and two commercially available enzyme-linked immunosorbent assays (ELISA). RESULTS: Antibodies to N. caninum were found in 13.7% of bulls sampled by at least one of the three validated serological assays. The seroprevalence of neosporosis oscillated between 11.2 and 13.3% depending on the serological technique used. No significant associations (P > 0.05) were observed between N. caninum infection and bull breed or age. The serum antibody levels were predominantly low and close to threshold levels in all positive samples. A very good agreement was found among the serological methods used. CONCLUSIONS: This is the first seroprevalence study of bovine neosporosis in breeder bulls and results showed a moderate presence of N. caninum chronic infections.
A total of 810 dairy cattle were blood sampled and used to evaluate serological screening for Neospora caninum infection in dairy cattle herds in Mashhad (Khorasan province, Iran). In 4 herds, of the 810 cattle, 123 (15.18%) were seropositive by IFA Test (VMRD Inc., Pullman, WA 99163, USA) with titer 1:200 and range of prevalence per herd from 9.92 to 19.60%. Seroprevalence was not significantly different between Holstein breed (14.88%) and Brown Swiss breed (19.60%). Comparison of N. caninum serological status with age groups (< or =2 years, 2-4 years, > or =4 years) showed no significant difference. Of the 139 aborted cattle sampled, 19.42% were seropositive for N. caninum.
To determine the prevalence of Neospora caninum antibodies and associated factors, blood sera from 623 female dairy cattle from 23 farms in the north of the state of Paraná, Brazil, were analyzed by means of the indirect immunofluorescent-antibody test (IFAT > or = 25). Serum samples from 134 dogs living on the same farms also were tested for N. caninum antibodies (IFAT > or = 50), and the presence of dogs was associated with the prevalence of N. caninum antibodies in cattle. The overall seroprevalence in cattle was 14.3%, mainly in animals over 24 months of age. Seroprevalence in Holsteins (15.1% of 558) was greater than in mixed-breed cattle (7.7% of 65). Age (> or =24 months) of cattle, feeding silage and/or concentrate produced on the farm were associated; antibodies were found in 21.6% of dogs; and the presence of dogs was associated with the prevalence of N. caninum antibodies in cattle.
A cross-sectional survey was performed to estimate the prevalences of antibodies to Toxoplasma gondii (ELISA, IFAT), Sarcocystis spp. (ELISA, using S. miescheriana as antigen) and Neospora caninum (ELISA, immunoblotting) in sows from breeding farms in southern Hesse, Germany. A total of 2041 plasma samples of sows from 94 randomly selected farms was examined. Data on farm profiles, husbandry management and sows were collected by a questionnaire and exploratively analysed. For T. gondii the ELISA results agreed well with the results obtained by IFAT (kappa=0.71). Antibodies to T. gondii were detected by ELISA in 19% of the sows. Sixty-nine percent of the farms had at least one seropositive sow, and a within-farm seroprevalence of >or=50% was observed in 14% of all farms. The prevalence of anti-T. gondii antibodies was positively correlated with the age of sows. The within-herd seroprevalence was significantly higher in farms with reproductive disorders than in those without such problems. On the farm level, the farm type 'piglet production' (versus 'pedigree breeding' or 'farrow-to-finish') was the only risk factor associated with the presence of T. gondii-seropositive sows. Antibodies to Sarcocystis spp. were found in 29% of the sows. Seventy-two percent of the farms harboured at least one seropositive sow, and a within-farm seroprevalence of >or=50% was detected in 23% of all farms. The seroprevalence increased significantly with the age of sows. On the farm level, only the farm type 'piglet production' (versus 'pedigree breeding') and the replacement of sows by purchasing (versus raising on the own farm) were identified as risk factors for seropositivity. Antibodies to N. caninum were detected in one sow using both the screening ELISA and the confirmatory immunoblotting technique. This may indicate the first natural N. caninum infection in pigs.
We previously reported that alcoholic extracts of Sophora flavescens and Torilis japonica from South Korea demonstrated good efficacy in reducing replication of Toxoplasma gondii and Neospora caninum. To characterize the chemical component associated with anti-protozoal activity, specific fractions were isolated by high performance liquid chromatography (HPLC) and used for in vitro testing. These fractions were evaluated in vitro against T. gondii and N. caninum. Fractions of the herb extracts were serially diluted to final concentrations of 2.850 to 0.356 ng/ml in medium and added to wells containing replicating T. gondii and N. caninum. To determine the ability of each fraction to inhibit parasite proliferation, 3H-uracil incorporation was used to determine parasite replication. In cultures infected with T. gondii, a fraction of T. japonica (TJ2) inhibited T. gondii proliferation by 99.2, 94.4, 88.6 and 27.0% in the range from 2.850 to 0.356 ng/ml. Four fractions of S. flavescens (SF1-SF4) inhibited T. gondii proliferation by 99.6-60.6, 96.9-48.1, 92.3-68.2 and 95.4-52.9% in the range from 2.850 to 0.356 ng/ml. In cultures infected with N. caninum, a fraction of T. japonica (TJ2) inhibited N. caninum proliferation by 98.3, 95.5, 79.7 and 30.6% in the range from 2.850 to 0.356 ng/ml. Four fractions of S. flavescens (SF1-SF4) inhibited N. caninum proliferation by 97.1-25.9, 94.8-35.5, 95.9-33.7 and 95.4-49.4% in the range from 2.850 to 0.356 ng/ml. These fractions of T. japonica and S. flavescens extracts are currently undergoing in vivo evaluation in experimentally infected mice.
In order to attempt isolate the protozoan parasite Neospora caninum, an N. caninum seropositive pregnant Sahiwal Friesian cross heifer from a large-scale dairy farm in Malaysia was kept for observation until parturition at the Veterinary Research Institute, Ipoh. The heifer gave birth to a female calf that was weak, underweight and unable to rise. Precolostral serum from the calf had an N. caninum indirect fluorescent antibody test titre of 1:3200. It died 12 h after birth and necropsy was performed. Brain homogenate from the calf was inoculated into 10 BALB/c mice that were kept for 3 months after which brain tissue from the mice was inoculated onto 24 h fresh monolayer Vero cell lines. The cell cultures were examined daily until growth of intracellular protozoa was observed. DNA of the organisms from the cell cultures was analyzed by PCR and DNA sequencing. DNA fragments of the expected size were amplified from the isolate using N. caninum-specific primers, and sequence analysis of ITS1 clearly identified the isolate as N. caninum. This is the first successful isolation of N. caninum from a bovine in Malaysia, and the isolate is designated Nc-MalB1.
Seven European laboratories contributed to a multi-centre evaluation of detection techniques for Neospora caninum in bovine foetuses. Six laboratories participated in immunohistochemistry (IHC) testing. All seven laboratories participated in PCR testing, but the results from one laboratory were not included in the analysis, because of contamination problems in the preparation of the samples. A coded panel of tissue sections from 36 infected and non-infected foetuses was used to evaluate the IHC detection of parasites. A coded panel consisting of 44 homogenized foetal brain samples from natural bovine abortion cases and 32 spiked samples were used to evaluate the PCR methods. Inclusion of a duplicate dilution series of spiked samples was used to evaluate detection limits and repeatability. IHC methods had a relatively low sensitivity, but a high specificity. There was considerable variation in IHC results between participating laboratories, which may be partly explained by examination practices that depended on the experience of the operator. In addition, the use of different antibody reagents, different antibody dilutions, and different enzymatic treatments of tissues may have contributed to the observed variation. PCR methods generally had a higher sensitivity than IHC methods and also a high specificity. The agreement between the majority scores of IHC and PCR methods was low. False positive PCR results indicated contamination problems in some instances. Agreement between the PCR results of the various laboratories was better, compared with the IHC results. There appeared to be no clear relationship between the PCR format (i.e. single or nested) and diagnostic sensitivity. Consequently, an improvement of diagnostic performance of PCR might possibly be achieved by optimizing DNA extraction methods.
The objective of this epidemiological study was to identify risk factors for Neospora caninum-related abortions in Ontario Holstein dairy herds. A total of 88 herds, consisting of 5080 cattle, and utilizing Dairy Herd Improvement (DHI) services, were divided into three groups. Case (n = 30) and first control (n = 31) herds were selected from 1998 and 1999 fetal abortion submissions to the Animal Health Laboratory, University of Guelph, that were histopathologically positive or negative, respectively, for N. caninum. A second control group (n = 27) was selected from multiple sources of herds sampled within the previous 4 years that had a low seroprevalence (<7%) to N. caninum. Between May and December 1999, all available cows on all farms, in parity one or greater, were blood sampled. The sera were then analyzed for antibody to N. caninum using a kinetic ELISA. A survey administered at the time of sampling recorded information on housing, animal species present, manure management, reproduction, biosecurity practices, wildlife observations, peri-parturient cow management, herd disease history and nutrition. Production and other herd parameters were obtained from DHI records. Logistic regression indicated that the following parameters were positively associated with a N. caninum abortion in a herd: the N. caninum herd seroprevalence (OR = 1.1), the total number of dogs on a farm (OR = 2.8), the frequency that dogs were observed defecating in mangers (OR = 2.8), the number of horses on a farm (OR = 3.1), the observed annual rate of retained fetal membranes (OR = 1.2) and the observed annual rate of cows returning to estrus after pregnancy confirmation (OR = 1.2). Factors negatively associated were the frequency of stray cats and wild canids observed on a farm (OR = 0.4 and OR = 0.7, respectively) and the housing of heifers on loafing packs (a housing pen divided into feed manger, scrape alley and bedded pack areas, OR = 0.1).
Sera from 1770 dogs and 94 red foxes from Austria were examined for antibodies against Neospora caninum using the indirect immunofluorescent antibody test (IFAT). 3.6% of the dogs were seropositive with titres ranging from 1:50 to 1:6400. Dogs from rural areas were significantly more often seropositive for N. caninum than those from the urban area of Vienna (5.3% versus 2.1%). There were no significant differences in sex or breed, but a slight increase in seropositivity with age was apparent, indicating postnatal infection. None of the foxes had antibodies against N. caninum. Additionally, sera from 242 dogs and 94 foxes were examined for antibodies against Toxoplasma gondii using the IFAT. Thirty-five percent foxes and 26% of the dogs were positive; 1.7% of the dogs were positive for both parasites. This is the first report of the prevalence of N. caninum infections in dogs and foxes in Austria.
The avidity (functional affinity) of specific antibodies are being used to estimate duration of bovine Neospora caninum infection. Here, we report for the first time the avidity pattern in cattle orally inoculated with N. caninum oocysts. In all, 16 pregnant cows and 7 calves were administered N. caninum oocysts. In the cows, the avidity increased during the early course of infection. In all but one, the avidity was < or = 35 during the first 6 weeks after infection and no cow had an avidity value >50 until week 9. The calves were sampled either week 6 (n = 3) or week 9 (n = 9) after infection, and by then had avidities between 2 and 17. The results are in agreement with results from previous investigations of naturally infected cattle, and calves that were experimentally infected with tachyzoites. They further validate the ability of the N. caninum iscom avidity ELISA to accurately assess the duration of infection.
In this long-term study, the changes in within-herd Neospora caninum seroprevalences and the relative importance of horizontal and vertical parasite transmission in Swedish dairy herds were investigated. The within-herd prevalences varied between 5.8% and 65.0% when the herds were first sampled. Comparing the prevalences of year 2000 with those found 2-3 years later, nine out of 14 investigated herds had a similar or lower within-herd prevalence while five herds had an increased portion of seropositive individuals. Three herds were free or almost free from infection at their last sampling. Changes in prevalence were not related to the level of prevalence that each herd had when first sampled. Antibody avidity measurements revealed that the majority of seropositive individuals in all herds were chronically infected. Dam-calf pair investigations and inspection of genealogic trees supplemented with antibody test results showed that vertical transmission was by far the dominant transmission route in all herds. Taken together, these results indicate that the prevalence of N. caninum infection in Swedish dairy cattle can be successfully reduced if actions to eliminate infection in a herd are combined with preventive measures to avoid re-infection.
A cross-sectional serological survey was conducted on cattle pasturing in an area of the southern Italian Apennines to evaluate the seroprevalence to Neospora caninum, and to investigate the climatic, environmental, farm management, and individual animal factors that influence the distribution of this protozoan. Blood samples were collected from 864 pastured cattle raised on 81 farms. Serum samples were tested for antibodies to N. caninum using an ELISA assay (HerdCheck), IDEXX). A geographical information system (GIS) for the study area was constructed using the following remote sensing (RS) and landscape feature data: autumn-winter, spring and summer normalized difference vegetation index (NDVI), land cover, elevation, slope, aspect, mean rainfall and minimum, mean, and maximum temperature in spring, summer, autumn and winter. Data on each of these features were then extracted for "buffer zones" consisting of the area included in a circle of 3 km diameter centered on the 81 geo-referenced centroids of the main cattle pastures. Climatic and environmental data obtained from RS and GIS and individual animal characteristics and farm management data obtained from a questionnaire were analyzed in relation to N. caninum seropositivity and antibody titres both by linear and logistic regression models. Out of the 81 farms sampled, 63 (77.8%) had at least one tested animal positive for N. caninum. Out of the 864 bovine sera samples, 266 (30.8%) were found to have antibodies to N. caninum. The results of the logistic regression model show that significantly high seroprevalence to N. caninum was found in heifers/steers and adults, in cattle raised on farms having a large number of dogs, and in cattle raised in buffer zones having high minimum temperatures in the spring, and a narrow extension of summer NDVI. In addition, positive linear correlations were found between N. caninum antibody titres and the number of dogs on farm, and the minimum temperature in spring. All the above determined risk factors for N. caninum seroprevalence indicate that horizontal infection resulting from the ingestion of oocysts shed by dogs is the most probable route of N. caninum infection in pastured cattle of the southern Italian Apennines.
AIM: To determine the contribution of Neospora caninum to abortions on a dairy farm in NSW (Australia), determine the mode of transmission and develop and trial a control option for infection. METHODS: Two whole herd bleeds were conducted 12 months apart and the association between serological status and abortion events were calculated for a number of bovine abortifacients. Family trees were constructed for N. caninum seropositive cattle in the herd. Some N. caninum seropositive cows were culled from the herd and no female offspring was retained from seropositive cows. RESULTS: At the first whole-herd bleed in December 2002 a seroprevalence of 10.2% for N. caninum infection was detected. Cows with N. caninum infection were 13 times more likely to abort than uninfected ones. Seventy-five percent of seropositive animals in the herd were related, suggesting a high degree of congenital infection/transmission. Only 15% of infections were likely to be postnatally acquired. Selective culling of seropositive cows and not breeding from them reduced the number of seropositive animals. Only one newly sero-converted cow was detected at the second whole-herd bleed 12 months later. CONCLUSIONS: Seroepidemiological approaches were able to establish a high degree of association between N. caninum infection and low-level abortion in the dairy herd. Vertical transmission of infection was the predominant mode of infection and hence control efforts aimed at selectively culling seropositive animals from the herd were highly successful in reducing the level of infection.
Neospora caninum is a coccidian parasite that causes disease in captive and domesticated animals and has been found in wild animals such as cervids. Sera from 150 cervids of the genus Mazama, were collected from 31 captive herds and 16 zoos from different Brazilian regions and analyzed by indirect fluorescent antibody test for anti-N. caninum antibodies. Positive reactions were found in 42% (63) of the samples and the titers varied from 50 to 51,200. Of the 86 cervids from the captive herds, 38 (44.2%) had anti N. caninum antibodies and of the 64 samples from the zoo, 25 (39.1%) were positive. No significant difference (p>0.05) was found for the occurrence values observed between the animals from captive herds and zoos as well as within the values documented for each one of the species analyzed. Therefore, the results indicate that the agent is prevalent from cervids in captivity in Brazil.
The water buffalo (Bubalus bubalis) is an important natural host for Neospora caninum. Serologic responses to N. caninum were studied in experimentally and naturally infected water buffaloes in Brazil. Antibodies were assayed by the indirect fluorescent antibody test (IFAT) using a cut off value of 1:25. Six buffaloes were each inoculated subcutaneously with 5 x 10(6) live culture-derived tachyzoites of the cattle Illinois strain of N. caninum, and two calves were kept as uninoculated controls. Post-inoculation (p.i.) blood samples were collected weekly for 8 weeks and then monthly until 1 year p.i. All inoculated buffaloes developed IFAT titers of 1:100 or more between 7 and 11 days p.i. and the titers remained elevated until 7 weeks p.i. Antibody titers peaked to 1:1600 in 1, 1:800 in 3 and 1:400 in 2, usually by 3 weeks p.i. Antibody titers declined to 1:25 or 1:50 in all the six buffaloes by 12 months p.i. IFAT titers to N. caninum remained at an undetectable level (< 1:25) in both control uninoculated buffaloes. To follow the dynamics of N. caninum antibodies, sera from 29 buffaloes and their calves were collected for 1 year and assayed for N. caninum antibodies; 23 of 29 calves were seropositive (IFAT of 1:100 or more) at 1-2 day of age. Of these 23 calves, 17 remained seropositive during the study, while six became seronegative at four (two calves), six (one calf) seven (two calves) and eight (one calf) months of age. These findings suggest a high rate of neonatal transmission of N. caninum in buffaloes.
To examine the relationship between occurrence of vertical transmission and type 1/type 2 immune responses induced by Neospora caninum infection in BALB/c mice, pregnant (group 1 p) and non-pregnant mice (group 1 np) were inoculated with 2 x 10(6) of the N. caninum parasites and then we examined the vertical transmission rate and production of IFN-gamma and IL-4. We also studied chronically infected mice, which were bred at 4 weeks or more after infection (group 2), and mice inoculated during pregnancy and re-bred at 4 weeks or more after delivery (group 3). In groups 1p, 2 and 3, vertical transmission was observed in 27.4, 41.4, and 50% of the offspring, respectively. The serum IFN-gamma level increased on days 1 and 5 post-inoculation (p.i.) in groups 1 p and 1 np, while no increase level was observed in groups 2 and 3 during pregnancy or after delivery. When the mice in groups 2 and 3 were re-inoculated, all mice showed a transient increase in serum IFN-gamma on day 1 post-re-inoculation. The serum IL-4 level in both of groups 1p and np increased in a similar manner following infection. In group 3, the serum IL-4 level was somewhat higher than that in group 2 after re-inoculation. The anti-N. caninum antibody IgG1 titer in group 3 increased on day 10 post-re-inoculation. These results suggest that the mice infected during pregnancy may acquire a weaker immune response to the parasite than mice infected when they are not pregnant, and that mice infected during pregnancy may show an enhanced type 2 immune response in the recrudescence of the infection.
Serum samples from 145 dairy and 65 beef cattle with reproductive disorders and 54 normally calving dairy cattle (controls) in Japan were tested for presence of Neospora caninum antibodies by use of an indirect fluorescent antibody test (IFAT, titer 1:200). Overall, seroprevalence of N. caninum was significantly higher (P < 0.001) in dairy cattle (20.0%, 29/145) than in beef cattle (1.5%, 1/65). In cattle which aborted, seroprevalence of N. caninum was significantly higher (P = 0.041) in dairy cattle (26.1%, 23/88, compared with controls (3.7%, 2/54)) than in beef cattle (5.0%, 1/20), indicating that neosporosis might be a more common problem in dairy cattle than in beef cattle in Japan. Seropositive cattle were 9.2 times more likely to abort compared to seronegative cows. Abortions associated with N. caninum seropositivity in this study were most frequently observed in the second trimester, and the mean gestational age of the fetuses aborted from seropositive dams was 5.7 months. In conclusions, N. caninum seems to be causing serious economic losses in the dairy industry in Japan. This is the first report on an objective comparison of seroprevalence of dairy and beef cattle with reproductive disorders in Asia.