Toxoplasma gondii is an obligate intracellular protozoan parasite which has significant medical and veterinary impact in all around the world. The precise detection of Toxoplasmosis as a zoonotic disease in food producing animals like water buffaloes has important public health significance. Tracking of specific antigens or antibodies for Toxoplasma gondii is important in its diagnosis. Recombinant proteins will improve sensitivity and specificity and reduce problems of standardization and reproducibility of diagnostic kits. The aim of this study was development of enzyme-linked immunosorbent assay (ELISA (using a combination of recombinant TgGRA7 and TgSUB1 antigens for diagnosis of toxoplasmosis in water buffalo. For this purpose at first using bioinformatics softwares the highly antigenic regions of these proteins were analyzed and selected. Then selected DNA fragments of these proteins were amplified, and cloned into pET28a expression vector. The fusion recombinant 6xHis-tagged proteins were expressed in E. coli BL21 (DE3) and then purified by Ni-NTA affinity chromatography under denaturation condition. Identity of the purified GRA7 (27 kDa) and SUB1 (31 kDa) proteins were confirmed by SDS-PAGE and Western blot using Anti-HisG-HRP Antibody. The TgGRA7 and TgSUB1 antigens and also a combination of them were evaluated as potential diagnostic markers for toxoplasmosis infection in 223 sera obtained from water buffaloes. Results of the three ELISAs were statistically analyzed and compared with a commercial indirect ELISA kit (ID.vet, France). The statistical analysis of the results are indicating that in differentiation of positive and negative sera there are no significant difference between these tests (p >0.05). Moreover, almost perfect agreements were observed between the ELISA tests and commercial kit (k= 0.812, 0.919 and 0.874, respectively; p <0.001). In statistical analysis of receiver operating characteristic (ROC), the cut-off values of GRA7-ELISA, SUB1-ELISA and Combination-ELISA (0.357, 0.2445 and 0.4115, respectively) were selected somehow with highest relative sensitivity and specificity compared with the results obtained by commercial kit. At these cut-off values the three ELISA tests showed 98% relative sensitivity compared to the commercial ELISA kit. The relative specificity of the ELISA tests was 88.3%, 95.3% and 92.4%, respectively. The results of this study demonstrate that TgGRA7 and TgSUB1 are promising serodiagnostic markers for T. gondii infection in water buffaloes. However, it seems that TgSUB1 is better than TgGRA7 in order to use as antigen in ELISA. The developed ELISAs have high sensitivity and specificity but should be tested in a larger collection of samples to evaluate their reliability and reproducibility for serodiagnosis of toxoplasmosis in water buffaloes.
 

Equine piroplasmosis (EP) is a tick-borne disease caused by two hemoprotozoan parasites, Theileria equi and Babesia caballi, which leads to fever, anemia and icteric mucous membranes in horses. This disease is one of challenges for international movement of horses and is monitored by World Organization for animal health (OIE). Economic loss associated with EP are huge and include the cost of treatment, especially in acutely infected horses, abortions, loss of performance, death and restrictions in exportation or participation of infected horses in equestrian sports. One of considerable aspects of EP is mechanism of anemia. Better cognition of this mechanism can be helpful in choosing of more proper therapeutic and prophylactic methods. In this study, indicators of oxidative and anti-oxidative status were investigated to evaluate possible role of oxidative stress in generation of anemia in EP caused T. equi in horses. Horse population investigated in this study was Iran Equestrian Federation registered purebred Arabian horses of Ahvaz area. Samples required were obtained by two methods: 1- From horses with clinical signs associated with EP (14 samples) and 2- From clinically healthy horses from four stables in Ahvaz (45 samples). Hematological and parasitological examinations were carried out in clinical pathology laboratory (College of Veterinary Medicine of Shahid Chamran University). PCR was also performed to evaluate T. equi and B. caballi infections in samples. Samples would be discarded If sequence related to B. caballi was found and/or this parasite or other parasites were detected during microscopic examination of blood smears. The horses were divided in three groups, Control, Infected and Diseased, regarding their clinical signs and PCR results. Oxidative stress was evaluated by measuring protein carbonyl (PCO), malondialdehyde (MDA), glutathione (GSH), total antioxidant activity (TAA), catalase and iron, copper and zinc concentrations. Statistical analysis of hematological and biochemical results in defined groups was performed using 16th version of SPSS software and One-way ANOVA or Kruskal-Wallis tests. Cohen's kappa statistics and McNemar´s test were used to test for agreement and significant differences between diagnostic methods, microscopic examination and PCR, in detection of infections. In the present study, Only T. equi was detected in samples. This is the first PCR confirmation of T. equi infection in Ahvaz area. Examination of stained blood smears revealed T. equi presence in 28 samples while PCR identified 35 of horses were infected, which this difference was statistically significant (P<0.05). Hematological results showed that EP will cause anemia, increase in numbers of total white blood cells, Neutrophils, Monocytes and in Neutrophil/Lymphocyte ratio and decrease in Platelet numbers in diseased horses, While in infected group except to mild anemia, No obvious difference with control group is seen. There is also significant differences between diseased and infected groups with control group in PCO, MDA, TAA, catalase, red blood cells GSH content and in copper and zinc concentrations (P<0.05). Results of this research shows that in addition to hematological alterations in EP, Oxidative stress indices will change which may be indicative of oxidative damage to red blood cells. Hence with regard to roles of oxidative stress and damage in generation of anemia, it is necessary to choose proper therapeutic methods for improvement of condition and/or elimination of infection in horses infected with piroplasmosis.