Blood samples were collected from a total of 150 cows with a history of abortion in different period of pregnancy from different dairy farms. Serum samples were stored at -20℃ until use. The N. caninum NC-1 isolate, was obtained from Razi Vaccine and Serum Research Institute, Shiraz, Iran, which was cultured in Vero cell line with fresh DMEM medium (Sigma Co., USA) and 2% fetal calf serum. DMEM medium was supplemented with an antibiotic/antimycotic solution, containing Penicillin (10,000 U/ml), Streptomycin (100 µg/ml), and Amphotericin B (25 µg/ml) (Invitrogen, USA). The cells were incubated with 5% CO₂ and observed daily. Tachyzoites were harvested when more than 80% of the Vero cells were infected. Then the suspended parasites were pelleted by centrifugation with 1,800 g for 15 min and passed through a 27-gauge needle to rupture the host cells. Finally, the tachyzoites were separated from the host cell debris by centrifugation in a 40% Percoll density gradient. Cell-free tachyzoites were pelleted (800 g for 20 min), washed three times in PBS, resuspended in sterile distilled water, and sonicated for six 30-s pulses (MSE Soniprep 150, SANYO, Japan). Cell debris and intact cells were removed by centrifugation (1,000×g for 20 min at 4℃) [14,15]. Disperse dye, BLUE SP (DNBS), was provided from DA-YU Chemical Co. (Taiwan ROC). Rabbit anti-bovine IgG was purchased from Sigma. A nonenzymatic detection reagent was prepared by conjugation of anti-bovine IgG antibody to colloidal dye particles as described previously [16]. Dye particle suspensions were prepared using a washing/centrifugation procedure. The concentration of dye in the aqueous solution used for antibody conjugation was monitored at its optimum wavelength (λ max) and expressed as the multiples of dye concentration corresponding to an A (absorbance at λ max)=1. To determine optimum concentration; various concentrations were tested. Ten µg of the antibody per one milliliter dye was mixed and shaken in a 37℃ incubator for 1 hr. The antibody and colloidal dye conjugate was stored at 4℃ until use. To optimize the DDIA, dye-antibody conjugates (various ratios of dye and rabbit anti-bovine antibody) were used to detect the antibody presence in the bovine's sera in order to select the most effective combinations of dye and secondary antibody. To analyze the sensitivity and specificity of the assay, parallel test was applied to compare the results of DDIA and ELISA kit (iscom ELISA kit, Svanova Biotech AB, Sweden). The procedures of antibody detection using DDIA are described as follow. The nitrocellulose membrane was divided into 3 segments for suspected serum, positive and negative control 2 µl of N.caninum soluble antigen was dotted on suspected and 2 µl of uninfected Bovine serum was dotted on positive control segment of nitrocellulose membrane. Dots were allowed to dry on the NC strips. The non specific binding sites on the NC surface were blocked with 2% BSA for 1 hr and then washed 3 times with tween 20 solution in PBS (0.5 ml Tween-20 per 1 L PBS). The strips were placed in serum samples which were diluted 1:50 with PBS for 1 hr. The strips were then washed 3 times with PBS tween 20 and were put in dye/antibody reagent for 45 min. The blue color dot on suspected segments was considered as positive.

To determine the optimal conditions, various dye concentrations were used to link the dye and antibodies in the conjugation procedure. The optimum concentration was A (λ max) =15. The procedure of dye-antibody conjugation should be carefully optimized to achieve high sensitivity and specificity in the assay. According to our results the pH values around 7 the temperature at 37℃ and the dyeing incubation time of 30 min were the most suitable conditions for dyed-antibody preparation. Out of 150 field serum samples collected from cattle, 39 (26%) were positive in DDIA and ELISA tests and 91(66.6%) were negative in both tests. The kappa statistic and apparent correlation rate were used to measure the strength of agreement between the results of the tests by the ELISA and DDIA. A kappa statistic value of >0.75 represents excellent agreement between the results [17]. In the method we used in our study good agreement (Kappa=0.697) between the prevalence estimated by the DDIA and ELISA tests was found (Table 1).

Due to the fact that N. caninum is closely related to Toxoplasma gondii and the potential cross-reaction might occur the DDIA was evaluated by using 5 bovine confirmed T. gondii seropositive samples previously diagnosed by ELISA and IFA tests. The result of DDIA showed no cross reaction with bovine Toxoplasma positive sera.

N. caninum is associated with abortion and reproductive loss in cows worldwide. Hence researchers have developed a number of diagnostic tests to investigate the epidemiology of infection and disease. The different type of ELISA techniques are mostly used world-wide for the diagnosis of Neospora infection in cattle [18,19]. Despite the high specificity and sensitivity of microplate ELISA tests, the use of these methods requires an ELISA reader which is an expensive instrument which is not available in many underdeveloped areas of the world. Moreover, ELISA procedure is time consuming, requires technical skills and experience to run. In contrast, the DDIA requires only non-expensive commercially available materials and reagents with long half-lives and the test can be assessed visually [20]. Serological investigation for several infection agents was recently accomplished using a dipstick colloidal dye immunoassay [21-26]. The dipstick dye is a variation of labeled immunoassay that uses nitrocellulose membrane as a support and colloidal dye as the label for the rapid reaction of Ag and Ab. In this study, a DDIA strip test for the detection of N. caninum infection in cattle was developed. We successfully applied the blue color colloidal dye particles to label rabbit anti-bovine IgG to detect IgG antibodies against N. caninum. Comparing the developed DDIA with commercial ELISA kit, there was no significant difference between the two tests, but the DDIA was faster and much easier to perform than the commercial ELISA kit and also more suitable for use in the field. The dye antibody suspension, with dye concentration at A (λmax)=15 giving the highest color intensity without background was chosen. It is suggested that at a lower dye concentration, only little amount of immuno-protein were dyed which resulted in a lower color intensity on the strip. On the other hand, at a higher dye concentration the epitopes of the antibody might be shielded by dye molecules which cause antibody inactivation. Also, in the higher dye concentrations, the aggregation of dyed-antibody may occur, which could result in background of NC membrane during the assay process [28]. It is also observed that higher color intensities were obtained when dyeing was conducted with a longer incubation time. The DDIA which utilize rabbit anti bovine IgG-colloidal dye conjugates as a visualizing agent may also be used for detection of specific antibodies in other infectious diseases with specific detectable antigens. The DDIA was also associated with other advantages such as easy conjugate preparation and test performance. Where costs are an issue, and in the absence of more elaborated diagnostic tests in many developing countries, simple tests such as DDIA could still deliver reliable information that could monitor individuals and/or herds infected with N. caninum. To our best knowledge this is the first report of DDIA for the detection of N. caninum.