neospora caninum ( apicomplexa : eimeriina : sarcocystidae ) is a cyst forming protozoan parasite closely related to toxoplasma gondii . the parasite is one of the most important etiological agents of abortion and neonatal mortality in cattle worldwide ( 1 - 3 ) . reported unidentified sporozoan protozoa in dogs in norway ( 4 ) . confirmed definitive hosts of n. caninum are dogs and coyotes , but other wild canids may act a role on this issue . the parasite infects a wide range of warm - blooded animals including dogs as intermediate host ( 5 - 7 ) . neospora caninum may be transmitted to intermediate hosts following ingestion of oocysts via contaminated feed or water or may be passed vertically from mother to fetus via the placenta ( 5 ) . n. caninum abortions can occur at any time of gestation but the majority of abortions are at 5 - 6 months of gestation ( 8,9 ) . there are three infection stages of the parasite : tachyzoites , bradyzoites , and sporozoites . tachyzoites and bradyzoites occur in tissues of intermediate hosts whereas sporozoites are present in oocysts that are excreted in the feces of the definitive hosts ( 10 ) . at present , serological assays including direct agglutination test ( dat ) using preserved whole tachyzoites and the elisa have been evaluated as high sensitive and specific tools for diagnosis of n. caninum ( 1,11 ) . since , there are little informative details in the literatures about validation of dot - elisa for detection of antibodies against n. caninum in naturally infected cattle for epidemiological and screening purposes , thus the presented study aimed to develop a dot - elisa for diagnosis of anti- n. caninum antibodies in naturally acquired cattle and to estimate the value of the test in comparison with commercially available elisa and dat . nc-1 strain , were grown in monolayers of vero cells cultured in rpmi 1640 medium ( sigma co. , usa ) supplemented with2% of fetal calf serum and penicillin ( 10,000 u / ml ) , streptomycin ( 100 g / ml ) and amphotericin b ( 25 g / ml ) at 37 c with 5% co2 in tissue culture flasks . tachyzoites were harvested by scraping the monolayer when 80% of the cells infected and then purified by sequential passage of the cell monolayer through a 27-gauge needle and centrifuged to remove host cell debris . the supernatant was collected , centrifuged and washed twice in phosphate - buffered saline ( pbs , ph 7.2 ) . blood samples were randomly collected from husbandry farms in fars province , south of iran . blood samples were centrifuged at 1000g and then the supernatants were frozen at 20c until the examinations have performed ( 12 ) . elisa was utilized according to the manufacturer 's instructions ( idexx , usa ) , the test samples were first diluted 1:100 with a sample diluent . then , 100 l of negative and positive controls and samples were dispensed into wells . , each well was washed with approximately 300 l of phosphate buffered wash solution four times , and then 100 l of anti - bovine conjugate was dispensed into each well . after incubation for 30 min at room temperature , the washing of each well was done . the volume of 100 l of tmb substrate solution was dispensed into each test plate well and incubated for 15 min at room temperature then 100 l of stop solution has dispensed into each well to stop the reaction . in order to the assay to be valid , the difference between the positive control mean ( pcx ) and the negative control mean ( ncx ) should be greater than or equal to 0.15 . in addition , the ncx must be less than or equal to 0.20 . the presence or absence of antibody to n. caninum s / p = sample  ncx / pcx - ncx each sample with s / p ratio below 0.50 was classified as negative for n. caninum antibodies . if the s / p ratio was greater than or equal to 0.50 , the sample was classified as positive for n. caninum antibodies . nc-1 strain , were grown in monolayers of vero cells cultured in rpmi 1640 medium ( sigma co. , usa ) supplemented with2% of fetal calf serum and penicillin ( 10,000 u / ml ) , streptomycin ( 100 g / ml ) and amphotericin b ( 25 g / ml ) at 37 c with 5% co2 in tissue culture flasks . tachyzoites were harvested by scraping the monolayer when 80% of the cells infected and then purified by sequential passage of the cell monolayer through a 27-gauge needle and centrifuged to remove host cell debris . the supernatant was collected , centrifuged and washed twice in phosphate - buffered saline ( pbs , ph 7.2 ) . blood samples were randomly collected from husbandry farms in fars province , south of iran . blood samples were centrifuged at 1000g and then the supernatants were frozen at 20c until the examinations have performed ( 12 ) . elisa was utilized according to the manufacturer 's instructions ( idexx , usa ) , the test samples were first diluted 1:100 with a sample diluent . then , 100 l of negative and positive controls and samples were dispensed into wells . after that , each well was washed with approximately 300 l of phosphate buffered wash solution four times , and then 100 l of anti - bovine conjugate was dispensed into each well . after incubation for 30 min at room temperature , the washing of each well was done . the volume of 100 l of tmb substrate solution was dispensed into each test plate well and incubated for 15 min at room temperature then 100 l of stop solution has dispensed into each well to stop the reaction . the absorbance was measured and recorded at 650 nm by the elisa reader . in order to the assay to be valid , the difference between the positive control mean ( pcx ) and the negative control mean ( ncx ) should be greater than or equal to 0.15 . in addition , the ncx must be less than or equal to 0.20 . the presence or absence of antibody to n. caninum s / p = sample  ncx / pcx - ncx each sample with s / p ratio below 0.50 was classified as negative for n. caninum antibodies . if the s / p ratio was greater than or equal to 0.50 , the sample was classified as positive for n. caninum antibodies . igg antibodies were also assayed by direct agglutination test based upon the direct agglutination of fixed tachyzoites of n. caninum , incorporating 2-mercaptoethanol to prevent non - specific igm agglutination ( 13 ) . gondii antibodies using the modified agglutination test ( mat ) with sera pre - treated with 2 mercaptoethanol ( 14 - 16 ) . to set up the suitable concentration of antigen and the best dilutions of sera and conjugate , checkerboard procedure using positive and negative controls was performed . positive and negative controls had been prepared in another performed work by authors of this manuscript . these sera had been confirmed by two commercial elisa kits ( idexx and idvet ) and also indirect fluorescent antibody test ( ifat ) . antigens were dotted onto strips of nitrocellulose membranes ( cut into 0.7 cm 5 cm ) on square zones and after air drying , fixed on pbs for 30 minutes . the dismounted free binding sites of membranes were blocked with skim milk in 0.05% pbs - tween ( ph=7.4 ) for 60 minutes at 37 c . sera were diluted in pbs - t and incubated with the strips for 60 minutes at 37 c with constant shaking and then washed four times with 0.05% pbs - tween . after that , rabbit anti - bovine igg peroxidase conjugate ( sigma ) was added in incubation circumstances same as previous stage and also washed as described . the strips were soaked in 5 ml of a chromogen substrate consisted of 6 mg of 4-chloro--naphthol diluted in 2 ml of absolute methanol , 5 l of 100-vol hydrogen peroxide and 10 ml of tbs for 10 minutes and at last the reaction has stopped by washing with distilled water . kappa statistical test ( k ) was utilized to assess the agreement between dot- elisa and two other mentioned procedures . < 0.2 = slight agreement ; 0.20.4 = fair ; 0.40.6 = moderate ; 0.6 0.8 = substantial ; > 0.8 = almost perfect ( 17 ) . mc nemars analyzing was used for detection the agreement of dot- elisa with elisa as well as the dat to reveal the positive results . revealed in table 2 , sensitivity and specificity of dot- elisa in comparison with elisa and dat have also calculated . sensitivity was calculated as the percentage of positive sera in the dot- elisa that had also been positive by the elisa . specificity was also defined as the percentage of negative sera in elisa that were also negative in the dot- elisa . for measuring the sensitivity and specificity of dot- elisa in comparison with dat , these calculations were also performed according to dat results ( 18 ) . to set up the suitable concentration of antigen and the best dilutions of sera and conjugate , checkerboard procedure using positive and negative controls was performed . positive and negative controls had been prepared in another performed work by authors of this manuscript . these sera had been confirmed by two commercial elisa kits ( idexx and idvet ) and also indirect fluorescent antibody test ( ifat ) . antigens were dotted onto strips of nitrocellulose membranes ( cut into 0.7 cm 5 cm ) on square zones and after air drying , fixed on pbs for 30 minutes . the dismounted free binding sites of membranes were blocked with skim milk in 0.05% pbs - tween ( ph=7.4 ) for 60 minutes at 37 c . sera were diluted in pbs - t and incubated with the strips for 60 minutes at 37 c with constant shaking and then washed four times with 0.05% pbs - tween . after that , rabbit anti - bovine igg peroxidase conjugate ( sigma ) was added in incubation circumstances same as previous stage and also washed as described . the strips were soaked in 5 ml of a chromogen substrate consisted of 6 mg of 4-chloro--naphthol diluted in 2 ml of absolute methanol , 5 l of 100-vol hydrogen peroxide and 10 ml of tbs for 10 minutes and at last the reaction has stopped by washing with distilled water . kappa statistical test ( k ) was utilized to assess the agreement between dot- elisa and two other mentioned procedures . strength of agreements was evaluated as follows : < 0.2 = slight agreement ; 0.20.4 = fair ; 0.40.6 = moderate ; 0.6 0.8 = substantial ; > 0.8 = almost perfect ( 17 ) . mc nemars analyzing was used for detection the agreement of dot- elisa with elisa as well as the dat to reveal the positive results . revealed in table 2 , sensitivity and specificity of dot- elisa in comparison with elisa and dat have also calculated . sensitivity was calculated as the percentage of positive sera in the dot- elisa that had also been positive by the elisa . specificity was also defined as the percentage of negative sera in elisa that were also negative in the dot- elisa . for measuring the sensitivity and specificity of dot- elisa in comparison with dat using positive and negative serum samples , 2500 whole tachyzoites per microliter , the dilution of 1/20 for sera and 1/1000 for conjugate were determined as the best amounts on dot - elisa . based on these settings , out of 178 examined sera 54.4% were positive for n. caninum antibodies whereas 53.3% and 56.1% were positive utilizing elisa and dat respectively ( table 1 ) . only two samples reacted with t. gondii using the dat . according to the kappa statistical analysis , furthermore , mc nemars analyzing revealed that dot - elisa has acceptable potential to discriminate the positive results . relative sensitivity and specificity of dot - elisa were respectively 92.63 and 89.16% in comparison with elisa and 93.4% and 90.8% compared with the dat ( table 2 ) . the main aim of most diagnostic procedures is diagnosis of the infections and screening or epidemiological studies . for instance , neospora - associated abortions are mainly identified by examination of the aborted fetus or fetal fluids by immunohistochemistry or pcr ( 19 - 21 ) . however , in many situations when the aborted fetuses are not available for screening and or epidemiologic studies , serological investigations in herd populations are methods of choice . many serologic procedures including elisa , indirect fluorescent antibody test ( ifat ) or dat and etc . are just carrying out in laboratory sites but some procedures such as dot - elisa are performable in both laboratory and field ( 22 ) . caninum antibodies in cattle or other hosts ( 23 - 27 ) but there is a little information about dot - elisa for this purpose . dot- elisa has a good potential to diagnose the neosporosis in aborted cattle with acceptable sensitivity and remarkable specificity ( 28 ) . in the presented study , we aimed to assess the dot - elisa in comparison with traditional elisa and the dat which does not require species - specific conjugates and is utilized as routine procedure in seroepidemiology of neosporosis in various hosts ( 29 ) . in our study , the dat was slightly better than elisa and dot - elisa to detect positive results but , the differences between three performed procedures to identify the positive reaction against n. caninum was not statistically significant and dot - elisa revealed a good and acceptable competence to distinguish the positive results in comparison with elisa and dat . detected high sensitivity and specificity of dot - elisa in our study to identify the naturally infected cattle with n. caninum clarified that this test could be considered as a useful tool . nine sera which revealed negative reactions by elisa had positive results utilizing dot - elisa maybe due to cross - reactive antibodies . from those nine elisa negative sera , only 1 had dat positive result to n. caninum and no reaction to t. gondii were seen using the mat . these results also , replicating a good sensitivity of dot - elisa to detect the anti- n. caninum antibodies and to discriminate them from t. gondii as a highly cross reacting agent with this parasite . dat has 100% sensitivity and 97% specificity in comparison with the elisa ( 30 ) . therefore , it seems that this situation is due to false - positive reactions in testing sera with dot - elisa . reported 94% sensitivity and 73% specificity for dot - elisa for detection of anti - n . caninum antibodies in dogs in comparison with ifat at a cut - off point of 1:50 ( 31 ) . one of the preferences of the elisa and dat is that a huge number of animal sera can be examined simultaneously and in a same condition and maybe it interprets some false positive results of dot - elisa . on the other hands , elisa is often expensive tool and requires an elisa reader and also huge numbers of tachyzoites are needed for the dat . the accuracy of the elisa as well as the dat in this condition is critically important because the evaluation of dot - elisa was dependent upon the accuracy of these methods . in our study , overall prevalence of anti - n. caninum antibodies was 54.4% , 53.3% and 56.1% using dot - elisa , elisa and dat respectively . these findings indicate that the variations in different screening studies are affected with either applying different serologic tests or detecting variable cut - off points . thus , there are serious difficulties for comparing the prevalence among different studies . in iran , prevalence of neosporosis in cattle reported 38.8% in tehran ( 32 ) , 21% in ahvaz ( 33 ) , 10.5% in tabriz ( 34 ) , 46% in mashhad ( 35 ) and 12.6% in kerman ( 36 ) . therefore , we can conclude that this disease can cause serious problems in our district especially when occur simultaneously with other infectious diseases . for instance , 9.4% of dogs in meshkin - shahr infected with n. caninum had afflicted with leishmania infantum infection ( 37 ) . we suggest that the dot - elisa test may be superior to elisa when we aim to screen the cattle on herd populations and slaughterhouses or when the laboratory equipment is not available . this procedure is easy , inexpensive and do not need high experienced person to read the results . also , since the dat needs enough time and elucidating the results of this test is difficult , the dot - elisa could be considered as alternative .
backgroundneospora caninum is a protozoan parasite from phylum apicomplexa and an important agent causing abortion in cattle which produce notable economic loss all around the world.methodsdot-elisa was set up performing checker board procedure and then 178 sera of cattle examined with commercial indirect elisa and direct agglutination test ( dat ) were also evaluated by dot - elisa afterwards.resultskappa statistical analysis revealed that dot - elisa has good agreements with elisa as well as the dat and also , mc nemar s analyzing showed that this procedure has acceptable ability to discriminate positive results . 
 relative sensitivity and specificity of dot - elisa were respectively 92.63% and 89.16% and 93.4% and 90.8% in comparison with elisa and dat.conclusionsince the dot - elisa is easy , inexpensive and not needed high experience to interpret the results , it is superior to elisa and dat when we aim to screen the cattle on the farm and slaughterhouses or when the laboratory equipment is not available .