This study was performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Ministry of Health, China. The study was approved by the Institutional Animal Care and Use Committee of Anhui Agricultural University (No. AHAU2020020). This study was carried out in compliance with the ARRIVE guidelines. In addition, all the samplings was done when the informed consent was obtained from farm owners.

The genes coding for the NcSRS2, NcSAG1 and NcGRA7 proteins of N. caninum were cloned and expressed in pET-28a expression vector. The indirect ELISA was developed using the purified recombinant protein and the results were compared with IFA and WB by evaluating 81 sera samples. Additionally, 329 serum samples from aborting dairy cattle in Ningxia were examined for N. caninum infections by using indirect ELISA.

African Green Monkey kidney cell (Vero) lines were cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 25 mM glucose and 4 mM glutamine and supplemented with 10% fetal bovine serum (FBS, Gibco, USA) as previously described (21). N. caninum tachyzoites were maintained in vitro by serial passages on confluent Vero cells. Cells and parasites were incubated at 37°C with 5% CO₂ in a humidified incubator.

A total of 329 serum samples used to evaluate the prevalence of N. caninum in Ningxia of China were collected between 2017 and 2021 from aborting dairy cattle that were the breeding adult cows suffered abortion in 11 farms located in Ningxia with approximately 450, 000 cows (Figure 1), and sera were collected within 3 days after the abortion occurred. The farms were located in 4 cities: Shizuishan (No. = 1), Wuzhong (No. = 3), Yinchuan (No. = 6), Zhongwei (No. = 1) where N. caninum was endemic (22–25). Out of the 81 sera samples used to evaluate indirect ELISA, 53 were collected from non-aborting cattle in Anhui Province, and the remaining 13 positive and 15 negative field sera samples tested by IFAT and WB were generously provided by Prof. Qun Liu of China Agricultural University. These sera were separated by centrifugation at 1500 × g for 10 min and stored at −20°C until use. A pool containing sera of 10 naturally infected cattle with immune-reactivity against N. caninum antigens in IFAT and WB was used as the positive control. Sera collected from newborn calf that did not present reproductive problems and analyzed by IFAT and WB with N. caninum negativity was used as the negative control. Fifty serum samples collected from cattle in Gansu Province that were confirmed negative for neosporosis by IFAT and WB were used to determine the cut-off value of indirect ELISA. The positive control sera, negative control sera and 50 sera negative samples were preserved in Animal Parasites and Parasitic Epidemiology Laboratory, Anhui Agricultural University, China.

All primer sequences are shown in Table 1. N. caninum NcSRS2, NcSAG1 and NcGRA7 (GenBank: AF061249, AF132217 and AF176649) genes were amplified using primers P1 and P2, P3 and P4, and P5 and P6 respectively. The expression plasmid backbone was amplified using primers P-up and P-down with plasmid pET-28a as template. The NcSRS2 and NcSAG1 genes were sequenced and ligated into the pET-28a backbone vector using the Basic Assembly Mix (TransGen Biotech; Beijing, China) according to the manufacturer's protocol, the resulting chimeric expression plasmid was named 28a-S2-G1. Then the 28a-S2-G1 carrier skeleton was amplified using primers P-up and P4-2 with plasmid 28a-S2-G1 as template, and the NcGRA7 gene was sequenced and ligated with the 28a-S2-G1 carrier skeleton, the resulting chimeric expression plasmid was named 28a-S2-G1-A7. The linker1 was added to the 5' ends of P2 and P3, and the linker2 was added to the 5' ends of P4-2 and P5, respectively.

The chimeric expression plasmid 28a-S2-G1-A7 transformed into a strain of competent E. coli cells, DH5α. The transformed colonies were selected from Luria-Bertani (LB) agar (Solarbio; Beijing, China) plates containing kanamycin (50 μg/mL) and were identified by PCR. Subsequently, the chimeric expression plasmid 28a-S2-G1-A7 was transformed into E. coli Rosseta.

The positive transformants were cultured in LB medium (Solarbio; Beijing, China) containing 50 μg/mL kanamycin with vigorous shaking at 37 °C until the 600 nm optical density (OD) of bacteria cultures reached approximately 0.4–0.6, were induced chemically by using 1 mM isopropyl-β-D-thiogalactoside (IPTG) for 5 h. The expression of chimeric proteins was analyzed by 12% (v/v) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the gels were stained with coomassie brilliant blue. Induced cells were washed in phosphate-buffered saline (PBS) two times, then lysed by sonication in an ice-water bath. The lysed cells were centrifuged at 10,000 g for 15 min. The supernatant and the precipitate were then subjected to SDS-PAGE analysis. The chimeric protein was mainly expressed as insoluble form. The inclusion body was dissolved in 8 M urea solution and purified by affinity chromatography on a HisTrap™ Sepharose nickel column (Beyotime Biotechnology; Shanghai, China). And the concentration was determined using a BCA kit (Beyotime Biotechnology; Shanghai, China) as previously described (20).

Western blotting to detect the cross-reactivity with T. gondii of chimeric protein rSRS2-SAG1-GRA7 was performed as described previously (26). Purified chimeric protein was subjected to 12% (v/v) SDS-PAGE, and the gel was prepared for WB as follows. Chimeric proteins separated in the gel were electrically transferred to a PVDF membrane and the membranes were blocked 1 h at room temperature with phosphate-buffered saline which contained 5% (w/v) skimmed milk powder. The membrane was then incubated with mouse serum positive for neosporosis and mouse serum positive for toxoplasmosis for 1 h at room temperature on a plate shaker. The mouse serum was performed as described previously (26). Following this incubation, the membrane was washed three times with PBS buffer and reacted with HRP-goat anti-mouse IgG (1:1000 dilution in PBS) at room temperature for 1 h. After three washes, the reactive bands were visualized using enhanced chemiluminescence reagents (Beyotime Biotechnology; Shanghai, China).

Detection of specific N. caninum antibodies was carried out by WB as described previously (27). Tachyzoites of NC-1 isolate were used for antigen preparation under non-reducing conditions. Bovine serum samples were diluted 1:100. A HRP-conjugated anti-cattle total IgG (1:1000 dilution in PBS) (Solarbio; Beijing, China) was used. The WB process and reactive bands visualization were performed as described above.

The antigen was produced by culturing tachyzoites of N. caninum Nc1 strain as described previously (28). 1 × 10⁵ N. caninum tachyzoites were spotted on slides in 12-well plates and allowed to air-dry overnight. All slides were fixed with formaldehyde and stored at −20°C until used. The IFAT procedures were performed as described previously with some modifications (29). Each serum sample was diluted at 1:50 using sterile saline solution buffered by phosphate (PBS), pH of 7.0, for screening. 20 μL of each sera samples were placed on slides and the slides were incubated at 37°C for 30 min in a box containing moisture content 100%. The slides were washed three times in a sterile carbonated buffer, pH of 9.0 with 5 min for each washing. Fluorescein isothiocyanate (FITC) conjugated goat anti-bovine IgG (Solarbio; Beijing, China) diluted at 1:100 was added to each slide before the slides were incubated again at 37°C for 30 min in a box containing moisture content 100%. The slides were washed in PBS, as was described earlier, and mounted in buffered glycerin (pH of 8.0). Observation of complete peripheral fluorescence of the tachyzoites was considered a positive result (28).

Polystyrene 96-well microplates (NEST; Wuxi, China) were coated with chimeric protein rSRS2-SAG1-GRA7 diluted in carbonate buffer (pH 9.6) at a final concentration of 0.5 μg/ml, and plates incubated for 1 h at 37 °C. After five washes with PBST (PBS containing 0.05% Tween 20), the plates were blocked by PBST plus 5 % skimmed milk powder (SMP) for 2 h at 37 °C. Then, the plates were washed five times with PBST, and 100 μL of sera sample diluted at 1:100 in serum dilutions (10 mM PBS, 0.1% Tween 20, 0.2% Fish gelatin and 0.3% BSA) (Baiditai; Jining, China) was added in duplicate. After 1 h of incubation at 37 °C and five washes with PBST, 100 μL of HRP-conjugated anti-cattle total IgG (Solarbio; Beijing, China) diluted at 1:3000 in PBST, were added to the plates for 1 h at 37 °C. The reaction was developed with 100 μL of TMB coloring solution, at 37 °C in the dark for 20 min, TMB coloring solution (Solarbio; Beijing, China) becomes blue color at HRP-conjugated anti-cattle. The reaction was terminated by 50 μL of stop solution. Subsequently, the OD value was measured at a wavelength of 450 nm using a microplate reader (Bio-Rad; Hercules, USA). ELISA was performed in duplicate for all samples, with three positive and three negative controls on each plate. The OD value of the blank was automatically subtracted from each sample value.

Fifty serum samples of cattle were collected to determine the cut-off value of this assay. These serum samples were confirmed negative for neosporosis by IFAT and WB described, and then were used to define the cut-off value in the rSRS2-SAG1-GRA7 indirect ELISA. The OD450 value of these cattle serum negative for neosporosis obtained in this rSRS2-SAG1-GRA7 indirect ELISA were defined to calculate the cut-off value. The mean OD450 value + 3 × standard deviations was defined as the cut-off value. OD450 value of samples greater than or equal to the cut-off value were considered positive serum for neosporosis.

A total of 81 serum samples of cattle tested using this ELISA method, the results obtained from the sera samples were subjected to receiver operating characteristic (ROC) analysis using the MedCalc statistical software (version 19.5.6). As a comparison, IFAT and WB were applied to test these samples and act as reference methods to distinguish positive or negative samples. The results of the three methods were compared, and the sensitivity and specificity of detection were calculated to evaluate the accuracy of the indirect ELISA. All other statistical data were analyzed using GraphPad Prism 5 (version 5.01; GraphPad Software, San Diego, CA, USA). The results were expressed as mean ± SD and evaluated by non-parametric tests. Values of P < 0.05 were considered statistically significant.

The genes for NcSRS2, NcSAG1 and NcGRA7 were amplified by PCR (Figure 2A) and cloned into a pET-28 system, and the inserted genes were sequenced to ensure the correctness of the reading frame. As shown by SDS-PAGE (Figure 2B), the chimeric protein rSRS2-SAG1-GRA7 was expressed as soluble and insoluble forms, up to 90% of all proteins are insoluble, and the molecular mass of the chimeric protein was estimated at approximately 53 kDa, which was in agreement with that deduced from its amino acid sequence (Figure 2C). The reactivity of the purified chimeric protein was detected by WB. The result revealed that the chimeric protein was specifically bound by mouse serum positive for neosporosis, but unbound by mouse serum positive for toxoplasmosis (Figure 2D).

This developed rSRS2-SAG1-GRA7 indirect ELISA was applied to 81 serum samples with varied N. caninum antibody status. In these samples, 30 of them were tested N. caninum-positive by IFAT and WB, 51 of them were negative. The rSRS2-SAG1-GRA7 indirect ELISA detected 28 N. caninum-positive samples, 53 N. caninum-negative samples. And there are 26 samples that have been detected by these three methods as positive samples, 49 samples as negative samples. Based on ROC analysis (Figure 3A), an ELISA OD value of 0.438 was chosen as the threshold to distinguish between positive and negative samples, yielding a specificity of 96.1%, a sensitivity of 86.7%, and a kappa-value of 0.84 (Table 2). Figure 3B shows the frequency distribution of the positive and negative samples tested by IFAT and WB. In summary, the overall coincidence rate of the rSRS2-SAG1-GRA7 indirect ELISA to IFAT and WB was 92.6% (true positives + true negatives/total samples).

Sero-epidemiological data of for N. caninum infections in cattle from Ningxia are presented in Table 3. The 329 sera samples were collected from 11 farms with an average herd of more than 500 cows in 4 cities and examined by the ELISA technique. The herd prevalence was 100% and the overall seroprevalence was around 41.64%. The N. caninum antibody positive rate in each farm ranged from 19.05 to 57.89%. As shown in Table 4, the seroprevalence varied in different age groups, ranging from 28.26 to 44.04%, with the highest prevalence of 44.04% in >4 years dairy cattle, followed by 2–4 years dairy cattle (43.33%), indicating that the seroprevalence increased with age.