The current study was carried out in Fars province, in the south of Iran, where caprine coccidiosis with different Eimeria species is prevalent [5]. Kids from distinct herds referred to Veterinary Clinic of Shiraz University and that died from suspected clinical signs, mainly light green-diarrhea, were subjected to necropsy diagnosis. To obtain the oocysts, the intestinal contents were collected and checked using flotation technique by sucrose-saturated solution (specific gravity, 1.3). Samples from each Eimeria-positive feces (approximately 5 g of each positive animal) were filtered through different sieves and centrifuged at 250 g for 10 min. Filtered material was placed into a shallow layer of 2.5% (w/v) aqueous potassium dichromate (K₂Cr₂O₇) solution. The samples containing unsporulated oocysts were allowed to sporulate in petri dishes in wet chamber at approximately 26–28°C and aerated daily for about 1 month until it could be confirmed that more than 90% of oocysts were sporulated. Then, the contents of all petri dishes were mixed to form the main stock and stored at 4°C until use.

The sporulated oocysts were counted per 1.0 ml of stock solution using the hemocytometer method as described earlier [9]. The species identification of oocysts was performed based on biological and morphological characteristics (size, shape, color, shape index, presence or absence of micropyle and cap, presence or absence of residual, polar, and stieda bodies) under×400 magnification, as was done in a previous study [10]. The stock solution comprised of a proportion of 3 Eimeria species, including Eimeria caprina (57%), Eimeria ninakohlyakimovae (28%), and Eimeria arloingi (15%), as the most pathogenic species.

Twenty Iranian crossbred kids (Capra aegagrus hircus) aged 2–3 months were purchased from a non-infected herd. All animals were subjected to potentiated-sulfonamide (PANTRISUL, Pantex Holland BV, Holand, The Netherlands) antimicrobial therapy for 5 consecutive days and maintained uninfected for 14 days before starting the study and were subjected to routine parasitological tests to ensure lack of infections. The animals were kept on a ration composed of alfalfa hay and standard commercial concentrate with a ratio of 60 to 40, respectively, with total dry matter intake of 3–5% of bodyweight. Animals were divided into 2 age- and sex-matched groups and kept in completely isolated rooms under controlled conditions. Ten kids were infected orally with 5×10⁴ sporulated oocysts (E. caprina 28,500, E. ninakohlyakimovae 14,000, and E. arloingi 7,500) in 200 ml distilled water per animal. Before inoculation, the oocyst suspension was freed of potassium dichromate by multiple washings and centrifugations at 750 g for 5 min. The other 10 kids received 200 ml distilled water and remained uninfected as control. The fecal samples were examined daily for the presence of oocysts to confirm the infection of infected group and to confirm the absence of the infection in control animals. The experiment was performed under the approval of the state committee on animal ethics, Shiraz University, Shiraz, Iran (IACUC no: 4687/63). Also, the recommendations of European Council Directive (86/609/EC) of November 24, 1986, regarding the protection of animals used for experimental purposes were considered.

Blood samples were taken at days 0 (before inoculation), 3, 7, 14, 21, 28, and 35 post infection (PI). Samples were collected from the jugular vein into sterile tubes containing sodium citrate for plasma isolation (5 ml) and into sterile plain tubes for serum isolation (5 ml). Whole blood samples in sodium citrate were centrifuged at 700 g for 15 min, and the plasma portions were collected. Serum samples were prepared by centrifugation of clotted blood samples at 2,000 g for 15 min. Serum and plasma samples were distributed in 500 μl aliquots and kept at −20°C until further use. The samples with hemolysis were discarded.

Serum levels of IFN-γ, TNF-α, IL-4 and IL-6 were measured by a quantitative sandwich enzyme immunoassay using commercial goat-specific kits (Shanghai Crystal Day Biotech, Shanghai, China). The analytical sensitivities of the tests in serum have been determined as 1.95 pg/ml for IFN-γ, 3.9 pg/ml for TNF-α and IL-4, and 0.39 pg/ml for IL-6 by the manufacturer. Serum levels of AGP were determined by a commercial goat-specific kit (Shanghai Crystal Day Biotech) using ELISA and the assay sensitivity was reported to be 0.5 ng/ml. ADA was assessed by an enzymatic-calorimetric assay kit (Diazyme Laboratories, Gregg Court, California, USA). This assay is based on the enzymatic deamination of adenosine to inosine which is converted to hypoxanthine by purine nucleoside phosphorylase (PNP). Hypoxanthine is then converted to uric acid and hydrogen peroxide (H₂O₂) by xanthine oxidase (XOD). The generated quinone dye was monitored spectrophotometrically and expressed as U/L of serum.

Two-way repeated measurement ANOVA test was used to compare values within sampling time points and between study groups. Mauchly’s test of sphericity and Levene’s test of equality of error variances were performed, and data transformation was carried out in case of violations of test assumptions. P-values less than 0.05 were considered significant. All statistical analyses were carried out using SPSS software (version 22).