Serodiagnosis of Neospora caninum infection in cattle by enzyme-linked immunosorbent assay with recombinant truncated NcSAG1

Neospora caninum is a veterinary medically important pathogen capable of causing abortion in cattle and neuromuscular paralysis in dogs. The surface antigen 1 of N. caninum (NcSAG1) is an important candidate for the development of a diagnostic reagent for neosporosis. In order to establish an effective diagnostic method, the gene encoding truncated NcSAG1 (NcSAG1t) lacking a signal peptide and C-terminal hydrophobic regions was cloned and expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST). The purified GST-NcSAG1t was tested in an enzyme-linked immunosorbent assay (ELISA) for the detection of N. caninum antibodies in cattle. The ELISA with GST-NcSAG1t clearly differentiated between immunofluorescent antibody test (IFAT)-positive and -negative sera from cattle. In addition, the ELISA detected no cross-reactivity with sera from mice experimentally infected with the closely related parasite Toxoplasma gondii. Field serum samples collected from cattle in Brazil were examined for the diagnosis of neosporosis by using the ELISA. Of the 197 samples analyzed, 66 (33.5%) samples were positive for antibodies to N. caninum. Of the 66 ELISA-positive samples, 60 (90%) samples were confirmed as positive by Western blot analysis with whole parasite antigens. These results suggest that the recombinant NcSAG1t could be a reliable reagent for use as an antigen in ELISA for the serodiagnosis of N. caninum infection in cattle.     

用重组蛋白NcSAG1t作为ELISA诊断抗原进行土种藏系羊Neospora caninum的血清学诊断

Neospora caninum是原生动物顶器门一种原生动物性寄生虫,它最早是在一条发生瘫痪的狗上被确诊的.直到1988年,人们一直错误的把N.caninum当作Toxoplasma gondii,因为这两个寄生虫在形态学上是极为相似的.认为N.caninum是引起胎儿流产和新生犊牛死亡以及狗的神经肌肉麻痹症的主要原因.就现在掌握的资料,狗是惟一的能够排泄N.caninum卵囊的终末宿主.     

IgG avidity pattern in cattle after ingestion of Neospora caninum oocysts

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.     

Assessment of antibody avidity in aborting cattle by a somatic Neospora caninum tachyzoite antigen IgG avidity ELISA

A Neospora caninum IgG avidity ELISA was carried out on the basis of a somatic N. caninum tachyzoite antigen. The test was validated using experimentally infected calves, where a clear maturation of the IgG avidity over time could be demonstrated. At a maximum of 82 days after infection (d.p.i.), all animals showed antibody avidities ranging above 35%, and respective sera were thus defined as highly avid.Sera of 103 naturally infected seropositive cows with abortion (N. caninum association was provided by a N. caninum PCR-positivity of the fetus in 40 cases) and 139 seropositive animals without abortion history were concurrently examined. Significantly lower avidities were observed in aborting cows when compared to animals without abortion problems (P<0.01). While the avidity of sera collected before abortion remained practically constant until abortion, a significant increase of avidity could be observed in samples collected weeks to months after abortion (P<0.01).The avidities of non-aborting animals from farms with or without abortion problems did not differ significantly with time and were mainly located in the high avidity area. These data indicate that low avidities are not necessarily linked to recent N. caninum infection but can also be an indicator for increased abortion risk in cattle.     

Use of an enzyme-linked immunosorbent assay in bulk milk to estimate the prevalence of Neospora caninum on dairy farms in Prince Edward Island, Canada

This study evaluated the use of bulk milk as a diagnostic tool for estimation of herd-level Neospora caninum exposure in Atlantic Canada; it was used to estimate the prevalence of dairy farms with a within-herd N. caninum-seroprevalence > or = 15% in Prince Edward Island (PEI). The variation over time of N. caninum antibodies in bulk milk is also reported. Skimmed bulk milk and individual serum samples were analyzed for N. caninum antibodies by using an enzyme-linked immunosorbent assay (ELISA). Bulk milk samples were collected in May 2004 (n = 235), May 2005 (n = 189), and June 2005 (n = 235). The prevalence of dairy farms with a within-herd seroprevalence > or = 15% on PEI was 6.4% in May 2004. In May and June 2005, respectively, 10.1% and 10.2% of farms had a > or = 15% within-herd seroprevalence. In 11 farms that were considered positive based on bulk milk samples, blood samples were collected from all adult cows in September 2005, in conjunction with a 4th bulk milk sample on the same day. The correlation coefficient between serology and bulk milk ELISA was 0.87. The results of this study demonstrate that the prevalence of N. caninum in dairy farms can be estimated by using a bulk milk ELISA.     

Congenital Neospora caninum infection in dairy cattle and associated calfhood mortality.

A prospective cohort study was undertaken on two central California dairies, A and B, to estimate prevalence of congenital infection with Neospora caninum, to characterize temporal variation in prevalence, to determine if occurrence of congenital infection was associated with specific dam and calf attributes, and to estimate the effect of congenital infection on calfhood mortality. Of the 405 calves enrolled over a period of 2 1/2 y on dairy A and dairy B, 30.6% (85/278) and 53.5% (68/127), respectively, were seropositive precolostrally to N. caninum, as determined by an ELISA test. Adult cow seroprevalence at calving was 36.0% (82/228) for dairy A, and 57.9% (33/57) for dairy B. No evidence was found for a significant increasing or decreasing trend in adult and precolostral seroprevalence through the study period (P > or = 0.26). For both herds combined, 81% of seropositive cows (93/115) and 5% of seronegative cows (8/170) had congenitally infected calves. Seroprevalence did not increase with cow age on either dairy (P > or = 0.47). The probability of a calf being congenitally infected was not associated with dam age, dam lactation number, dam history of abortion, calf gender, or length of gestation (P > or = 0.11). High dam ELISA values at calving were significantly associated (P < or = 0.001) with an increased probability of congenital infection in her calf. Results of survival analyses of female calves available for follow-up indicated a consistently greater survivorship to 90 d in congenitally infected calves than in noninfected calves on both dairies, which was significant for dairy A (P = 0.07, n = 186) but not for dairy B (P = 0.69, n = 72), thus indicating that congenital infection does not necessarily have a detrimental effect on calf health. The findings of a similar magnitude in congenital infection rate and adult cow prevalence, the lack of increasing seroprevalence with cow age, the lack of an effect of dam age on precolostral seropositivity, and the constant seroprevalences during the study period, suggest that, in the two dairies studied, congenital transmission constituted a substantial amount of infection and was likely the major mode of transmission of N. caninum.     

Use of an enzyme-linked immunosorbent assay to estimate prevalence of paratuberculosis in cattle of Florida

A survey was conducted from 1986 through 1987, for which an ELISA was used to obtain information on the prevalence of Mycobacterium paratuberculosis infection in cattle of Florida. Results revealed prevalence of 8.6% in beef cattle and 17.1% in dairy cattle. In beef and dairy cattle, prevalence increased with increasing herd size. It was concluded that ELISA-detectable circulating antibodies to M paratuberculosis are widespread in cattle of Florida.     

Evaluation of an enzyme-linked immunosorbent assay for the serological diagnosis of Neospora caninum infection in sheep and determination of the apparent prevalence of infection in New Zealand

Neospora caninum has recently been shown to be a cause of abortions of sheep in New Zealand. A commercially available enzyme-linked immunosorbent assay (ELISA) was validated for use in sheep with sera from experimentally infected sheep. A cut-off threshold was established that demonstrated sero-conversion between 7 and 14 days post-infection. Higher inocula led to earlier sero-conversion. This ELISA was applied to 640 sera collected from rams across New Zealand and 0.625% (+/-0.61%) (4/640) were shown to be serologically positive. The four positive sera were also demonstrated to be positive by indirect fluorescent antibody test (IFAT). The ELISA evaluated here lends itself more readily to large-scale investigations than IFAT. The low background of N. caninum infection in the New Zealand sheep population suggests that N. caninum abortions could be more easily diagnosed by serological means than in populations with higher background sero-prevalence.     

Evaluation of three enzyme-linked immunosorbent assays for detection of antibodies to Neospora caninum in bulk milk

Three ELISAs for the detection of antibodies against Neospora caninum in bulk milk were evaluated in 162 Dutch dairy herds. The first ELISA was the Dutch Animal Health Service (AHS) in-house ELISA, developed from the routine in-house serum ELISA. The other two ELISAs were commercial milk ELISAs from IDEXX and LSI. Blood samples of all lactating cows in 162 dairy herds were tested using the AHS in-house serum ELISA. Based on previous studies in the Netherlands a within-herd N. caninum seroprevalence of 15% was associated with increased risk for reproductive losses. This percentage was therefore used as positive seroprevalence cut-off value. Repeatability of the ELISAs was evaluated by testing on three different days. The AHS in-house ELISA lacked specificity, probably due to use of a different batch of antigen on the second and third test-day. Cut-off values were determined using misclassification costs term calculations. At cut-off values 0.6 for the IDEXX and 0.2 for the LSI, a herd sensitivity of 61% (95% CI: 49--73%) and 47% (95% CI: 35--60%) was estimated. Herd specificity at these cut-off values was 92% (95% CI: 87--98%) for the IDEXX and 94% (95% CI: 90--99%) for the LSI ELISA. The positive and negative predictive values were 84% (95% CI: 68--100%) and 86% (95% CI: 79--94%) for the IDEXX ELISA, and 85% (95% CI: 67--100%) and 82% (95% CI: 74--90%) for the LSI ELISA. The agreement between all possible combinations of test-days was expressed by kappa values. These were found to be slightly higher for the IDEXX than for the LSI ELISA. It is concluded that both commercial ELISAs performed satisfactorily to detect a within-herd seroprevalence of N. caninum in lactating cows of at least 15%.     

Natural postnatal Neospora caninum infection in cattle can persist and lead to endogenous transplacental infection

A serological follow-up study of 3.5 years duration was done of a dairy herd that had experienced a mass seroconversion to Neospora caninum following a point source exposure shortly before the 17th of January 2000. A total of 913 blood samples of 244 animals at seven sampling dates were used to investigate the seroprevalence dynamics in the herd. Most postnatally infected cattle remained seropositive during the period of investigation but 11 animals became seronegative after 6-27 months indicating transient infection. Six animals seroconverted later than the main group of 45 animals and 5 animals became seronegative after at least two seropositive records possibly due to a low infection dose or difference in the haplotypes of the infected animals. In total 58% (14/24) of the offspring of postnatally infected dams was seropositive. Nine of 16 (56%) daughters originating from inseminations after the postnatal infection of their dams were seropositive indicating endogenous transplacental infection.     

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

Seroprevalence of Neospora caninum in 879 beef and dairy cattle in different locations of Paraguay was determined by an ELISA. In the survey, 262 (29.8%) cattle were positive to N. caninum, and animals with anti-Neospora antibody titre were observed in all the locations tested in the country. Serum samples taken from a herd that exhibited persistent abortion had the highest percentage of animals being positive to the parasite (17/30, 56.7%). In the same herd, abortion was significantly more likely in animals with high anti-Neospora antibody titre. Immunoblot analysis demonstrated that the banding pattern from positive Paraguayan cattle was similar to that seen with the positive control sample. In conclusion, N. caninum infection is present among Paraguayan beef and dairy cattle, and it may be an important cause of bovine abortion in Paraguay.     

Performance characteristics and optimisation of cut-off values of two enzyme-linked immunosorbent assays for the detection of antibodies to Neospora caninum in the serum of cattle

AIM: To determine the performance characteristics of two enzyme-linked immunosorbent assays (ELISAs) manufactured by Institut Pourquier (IP) for the detection of antibodies against Neospora caninum in bovine sera. METHODS: Sera from 526 cattle were assayed in two ELISAs (IP) for the detection of anti-N. caninum antibodies. Results from a further ELISA (IDEXX) were used to provide the "gold standard"N. caninum infection status of the cattle and the ELISA results assessed by two-graph receiver operating characteristic (TG-ROC) analysis. RESULTS: TG-ROC analysis suggested changes to one of the IP ELISA protocols, arriving at a cut-off threshold that was different to the one recommended by the manufacturer. With that change, both of the ELISAs performed with high sensitivity and specificity (in excess of 98%) for bovine sera. CONCLUSIONS: The analysis of the two IP ELISAs when used on individual bovine sera demonstrated high sensitivity and specificity. TG-ROC analyses optimised the cut-off point suggested by the manufacturer for one of these commercial diagnostic assays and found agreement with the manufacturer's cut-off regarding the other assay. This will help with the accurate identification of infected animals and thereby contributing to the control of neosporosis.     

Use of the enzyme-linked immunosorbent assay in the diagnosis of Dictyocaulus filaria infection

The enzyme-linked immunosorbent assay (ELISA) was used to detect and quantify antibodies to whole worm extract antigens of Dictyocaulus filaria. The results provided evidence that the test was highly sensitive and could be used as a tool for epidemiological studies. No cross reactions were observed with sera from lambs infected with the hookworm Gaigeria pachyscelis.     

Induced transplacental transmission of Neospora caninum in cattle.

Three Jersey cows were inoculated SC and IM with 26 million Neospora caninum tachyzoites at 129 (cow 1), 126 (cow 2), and 81 (cow 3) days after mating. Cows remained clinically normal for at least 1 month after inoculation of N caninum. Cow 1 was euthanatized 32 days after inoculation because of gangrenous mastitis. Cow 1 had a live fetus with no gross lesions; however, microscopic lesions were seen in the fetus and consisted of severe nonsuppurative necrotizing encephalitis of the cerebral white matter. Neospora caninum was identified in lesions by staining with anti-N caninum serum in an immunohistochemical test, by bioassays in mice, and by inoculation of bovine monocyte cultures with fetal tissue homogenate. Neither N caninum nor lesions were associated with infection with the protozoon identified in tissues of cow 1. Cows 2 and 3 aborted small autolysed fetuses 101 and 74 days, respectively, after inoculation with N caninum; the fetuses and attached placenta were unsuitable for laboratory investigations. Cows 2 and 3 remained clinically normal 4 months after abortion. Results of this study indicated that N caninum can be transmitted transplacentally in cattle.     

Agreement between three serological tests for Neospora caninum in beef cattle.

During 1999, serum samples were collected from beef cows on pastures in western Canada. Some of the herds had a history of confirmed abortions associated with Neospora caninum infection. All these samples were initially analyzed using a single application of 1 common commercial enzyme-linked immunosorbent assay (ELISA) for antibodies to N. caninum. From these initial results, 239 positive and 250 negative samples were randomly selected for further testing. This group of samples was retested using the 3 commercially available ELISA tests for N. caninum as per the manufacturer's recommendations. The agreement between 2 of the ELISAs was good (K = 0.76); agreement of these 2 tests with the third test was much lower (K = 0.46 and 0.60). Quantitative agreement between tests measured by intraclass correlation coefficients was also acceptable between the first 2 tests but was almost zero when the first 2 tests were compared with the third. This information is necessary to understand the differences in seroprevalence reported in different regions from laboratories using different methods.     

Diagnosis of Fasciola sp. infections in cattle by enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay (ELISA) was evaluated for diagnosis of experimental or naturally occurring Fasciola sp. infections in cattle. The positive rate for the ELISA in calves inoculated with Fasciola metacercariae were 21.1% by 2 weeks postinoculation (PI), 94.6% by 4 weeks PI and 100% by 6-21 weeks PI. The positive rate for the immunodiffusion test (Ouchterlony test) reached 91.7% by 2 weeks PI, however, it dropped to 77.8% by 10 weeks PI. The positive rate for the fecal egg examination was 0% by 10 weeks PI, 77.8% by 12 weeks PI and 100% by 14-21 weeks PI. The practical application of ELISA was tested by using 165 cows raised under field condition. All the 24 cows that were positive both in the fecal egg examination and the Ouchterlony test were ELISA positive. Of the 6 cows that were egg positive and Ouchterlony negative, 5 showed ELISA positive reactions. Of the 27 cows that were egg negative and Ouchterlony positive, 24 were ELISA positive. Of the 108 cows that were egg negative and Ouchterlony negative, 90 were ELISA negative. However, the other 18 cows had ELISA positive reactions. Our results suggested that the ELISA using crude adult antigen was superior to the Ouchterlony test and fecal egg examination for diagnosis of experimental or naturally occurring Fasciola sp. infections in cattle.     

Herd evaluation of Fasciola hepatica infection levels in Louisiana cattle by an enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay (ELISA) was evaluated as a method of determining relative immunoconversion rates in calves and how immunoconversion rates and strength of optical density values correlate with prevalence of Fasciola hepatica fecal egg shedding. Ten to 55 calves and cows were examined from each of 10 separate beef cattle herds in central and southern Louisiana. Infection prevalence rates for calves averaged 8% higher when ELISA optical density values were used than those when fecal egg count data were used. Of 55 calves in 8 herds that were ELISA positive, 39 were shedding F hepatica eggs; of 53 calves that were shedding eggs, 14 were ELISA negative. Significant correlation of calf fecal and ELISA prevalence was observed for 8 herds by linear regression analysis. A chi 2 analysis showed that calf ELISA and fecal egg shedding data were not independent. Results indicate that positive ELISA reactions for as few as 10 to 15 calves from the fall calf crop of a given herd are sufficiently accurate to be used to assess F hepatica herd infection rates, the likelihood of liver condemnations at feedlot destinations, and variation between individual farms in fascioliasis infection risk. The test was less valuable as a diagnostic test when used in adult animals previously exposed to F hepatica or on an individual animal basis.     

Vertical transmission of Neospora caninum in BALB/c mice in both acute and chronic infection

To examine the frequency of congenital infection by Neospora caninum, BALB/c mice were inoculated intraperitoneally with tachyzoites of N. caninum either during pregnancy (Group 1) or 4 weeks or more before pregnancy (Group 2). Further, the mice inoculated during pregnancy were bred at 4 weeks or more after delivery to form Group 3. Congenital transmission was observed in 76% of the neonates of the mice in Group 1 and in 50% of the neonates of the mice in Group 2. Interestingly, congenital transmission was observed in 86% of the neonates from Group 3. These results suggest that chronically-infected BALB/c mice efficiently transmit N. caninum infection to their offspring.     

Natural transmission routes of Neospora caninum between farm dogs and cattle

Twelve dairy herds with evidence of post-natal infection with Neospora caninum were compared with 21 control herds with no evidence of post-natal infection. On the former farms, dogs consumed placenta or licked uterine discharge in 75 and 67% of the farms, respectively, while on control farms these activities occurred in 38 and 24% of the farms, respectively. On all control farms and all but three post-natally infected farms the dogs were fed colostrum or milk. Defecation of dogs on the feeding alley was observed in 92% of the post-natally infected farms and in 24% of the control farms. The same trend was observed for defecation of dogs in grass silage, in 75% of the post-natally infected farms and in 19% of the control farms; and in corn silage, in 50% of the post-natally infected farms and in 10% of the control farms. Consumption of placenta, material of aborted foetuses or uterine discharge in combination with defecation on the feeding alley, storage of grass or corn silage was observed in 19% of the control farms and in 75% of the post-natally infected farms. This study supports the hypothesis that farm dogs may become infected by foetal fluids or placental material of infected cattle, and may subsequently cause a post-natal infection of cattle in the herd by shedding oocysts.     

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.