Molecular Detection of Theileria Annulata in Egyptian Buffaloes and Biochemical Changes Associated with Particular Oxidative Changes

This investigation is performed on 100 buffaloes native Breed in Kaliobia governorate Egypt aged from 1-3years, these animals suffered from fever (41°C) enlargement lymph node, inappetence, pale mucous membrane, increase lacrimation, emaciation and corneal opacity in progressive stages when these animals were subjected to microscopic examination the degree of parasitaema was recorded as the percentage of infected red blood cells in 1000red blood cell counted .We report the detection of Theileria annulata by PCR in blood samples obtained from buffaloes supposed to be infected. The assay employs primers specific for the gene encoding Theileria annulata 185RNA biochemical changes associated with Theileria annulata in these buffaloes particular emphasis to the oxidative stress the reduce TAC level may reflect adecrease in antioxidant capacity . Blood collected from all animals on EDTA to microscopic examination and PCR to determine type of Theileria.


Introduction
Theileria annulata, a protozoan parasite of cattle and domestic buffalo (Bubalus bubalis). It causes tropical Theileriosis, a disease which is present in northern Africa and southern Europe, extending through the middle East, India, and southern Russia into China . Dignosis of clinical T. annulata infection in bovine is usually based on the detection of macroschizonts in Giemsa-stained lymph node biopsy smears. In addition, serological tests such as the indirect immumoflurescent antibody test (IFA) can be used to detect circulating antibody either piroplasms or cultured macroschizonts as the antigen ). However, cross-reactivity with antibodies directed against other Theileria species limits the specificity of IFA . Moreover, antibodies tend to disappear in long-term carriers, whereas Theileria piroplasms persist.
Therefore, animals with negative serological test may be still infected Theileria annulata represent a major threat for Egyptian water buffaloes, causing significant economic losses as well as reduced production clinical picture that may be mistaken with other diseases. Stained thin blood film lymph node smear may be useful to detect Theileria in animals but PCR has allowed the development of sensitive diagnostic assays for Theileria annulata (Tanaka et al., 1993).
Here, we report the use of PCR for sensitive and specific amplification of T. annulata DNA from blood samples obtained from buffaloes primers were derived from the gene encoding Theileria annulata 18s RNA. Results PCR amplified DNA products were analyzed by agarose gel electrophoresis and confirmed by sequencing and blast on the NCBI website.
A significant feature of the disease is hemolytic anemia (Gill et al., 1977;Omer et al., 2002). The cause of hemolytic anemia is immune mediated hemolysis which is indicated by the presence of a hamagglutinin (Hooshmand-Rad, 1976). Although various evidence has been presented to explain the mechanism of the anemia, the exact underlying mechanism is currently unknown (Shiono et al., 2004). reported that oxidative damage to RBCs may contribute to the pathogenesis of anemia in bovine tropical theileriosis (Asri Rezaei & Dalir-Naghadeh ,2006). Oxidative stress may result from an imbalance between reactive oxygen species (ROS) and antioxidants levels (Lightboy et al. 2001). The erythrocyte has several intracellular modes of protection against oxidation, For example, it carries enzymes such as superoxide dismutase, glutathione peroxidase, and catalase capable of disarming oxidizing radicals, and it contains antioxidants such as reduced glutathione (GSH) and vitamin E (El Deeb and Younis ). Erythrocytes are particulary susceptible to an oxidative damage as a result of high polyunsaturated fatty acid content in their membranes and high concentration of oxygen and haemoglobin, the latter being a potentially powerful promoter of oxidative processes (Carrell et al., 1975) . Increasing of parasitaemia rate in cattle infected with Theileria annulata, an increase in erythrocyte osmotic fragility and a decrease in SOD activity occurred, the antioxidant status of tissues can be described by the analysis of single components in the defense systems against ROS, as well as by the determination of total antioxidant (TAC). (Cimen, 2008). Nazifi et al., (2008). The present study aimed to: 1. Detection of theileria annulata in buffaloes 2. assess the effect of theileria annulata infection on the antioxidant mechanism of RBCs.

Material and Methods
This experiment was performed on 100 buffaloes from native breed in Kaluobia Governorate ages range from 1-3years from different location all animals were clinically examined with special attention to signs related to Theileria infection samples collected from all animals Apparently Healthy and infected samples.
The blood samples we collected from Juglar vein by sterile sharp nudle with wide pore samples were collected in clean and dry test tube containing EDTA as anticoagulant for blood smear and PCR analysis.

Blood Film
Three thin blood film were prepared for every sample and left in air to dry and fixed in absolute methyle alcohol for 1-2min. staining with freshly filtered and diluted Giemsa stain for 30-45min then washed with distal water to remove excess of stain after that the slides were left to dry then but one drap afcider oil and examined under oil immersion lens according to (Coles, 1986). Examination of blood film for Theileria ¼-½ inch from end of the film and transferred from one slide to other (cross-section method) to give constant and representive examination according to (Barrent, 1965) animal be considered negative if the three slides were negative.

DNA Extraction from Blood
The DNA was extracted from each sample by chloroform-isoamyl extraction method (All buffers used according to . Blood samples typically were obtained as 1 ml of whole blood stored in EDTA vacutainer tubes. To each 1 ml sample, add 0.8 ml 1X SSC buffer, and mix. Centrifuge for 1 minute at 12,000 rpm in a microcentrifuge. Remove 1 ml of the supernatant and discard into disinfectant. Add 1 ml of 1X SSC buffer, vortex, and centrifuge as above for 1 minute, and remove all of the supernatant. Add 375 ul of 0.2M NaOAc to each pellet and vortex briefly. Then add 25 ul of 10% SDS and 5 ul of proteinase K (20 mg/ml H2O) (Sigma P-0390), vortex briefly and incubate for 1 hour at 55°C. Add 120 ul phenol/chloroform/isoamyl alcohol and vortex for 30 seconds. Centrifuge the sample for 2 minutes at 12,000 rpm in a microcentrifuge tube. Carefully remove the aqueous layer to a new 1.5 ml microcentrifuge tube, add 1 ml of cold 100% ethanol, mix, and incubate for 15 minutes at -20°C. Centrifuge for 2 minutes at 12,000 rpm in a microcentrifuge. Decant the supernatant and drain. Add 180 ul 10:1 TE buffer, vortex, and incubate at 55°C for 10 minutes. Add 20 ul 2 M sodium acetate and mix. Add 500 ul of cold 100% ethanol, mix, and centrifuge for 1 minute at 12,000 rpm in a microcentrifuge. Decant the supernatant and rinse the pellet with 1 ml of 80% ethanol. Centrifuge for 1 minute at 12,000 rpm in a microcentrifuge. Decant the supernatant, and dry the pellet in a Speedy-Vac for 10 minutes (or until dry). Resuspend the pellet by adding 200 ul of 10:1 TE buffer. Incubate overnight at 37C, vortexing periodically to dissolve the genomic DNA. Store the samples at -20°C.

PCR
Specific PCR has been used to detect the gene encoding 18sRNA for T. annulata within DNA extracts of the suspected animals for infection. Forward and reverse primers were designated using NCBI website and the contribution of genebank, the primers sequences were: Forward: CGGAAGGGCACCACCAGGC; Reverse GCATCAGTGTAGCGCGCGTG The PCR master mix was comprised of PCR buffer (300 mM Tris, 75 mM ammonium sulfate, pH 9.0), 2.5 mM MgCl, 400 µM dNTPs, 20 pmol of each primer, and 2 U µl-1 taq DNA polymerase. The PCR cycling parameters were one cycle of 94°C for 5 min, 35 cycles of 94°C for 30 s, 52°C for 30 s and 72°C for 1 min, with a final extension step of 72°C for 10 min. PCR products were analyzed by electrophoresis on 1.5% agarose gel and documented with documentation system.

Reduced Glutathione (R.GSH)
The activity of R.GSH was carried out using commercially available test kits, according to the method described by Beutler et al., (1963).

Total Antioxidant Capacity (TAC)
The activity of TAC was carried out using commercially available test kits, according to the method described by Koracevic et al., (2001).      The oxidative stress appears when the production of free radicals and reactive metabolites of oxygen exceeds their safe disposal by antioxidant mechanisms this investigation is agreement with (El Deeb and Younis ).

Results
With the availability of sequenced parasite genes and PCR, it is possible to detect parasites within samples of blood (Tanaka et al., 1993). In this study, we developed a PCR assay for improved detection of T. annulata in blood samples from carrier buffalloes. Previously, we amplified a 341-bp fragment by using a primer pair from all blood samples taken from animals suspected to be infected.
Using 18sRNA primer, the expected 341-bp product was amplified from blood samples and the specificity of this fragment was confirmed by DNA product sequencing. Successful amplification of T. annulata from Kalubia region indicates that the targeted gene sequence is conserved within T. annulata.
Exploitation of both the highly conserved and hypervariable sequences genes like 18sRNA permits designing of a platform primers capable of early detection and specific pathogen identification in a single rapid detection platform. PCR analyses, permitting identification of definitive pathogen characterization of the species. Diagnostic accuracy of our assay was evaluated against conventional light microscopy-based methods. This assay may be a useful early diagnostic for T. annulata.
In the present study, a significant decrease in erythrocyte TAC and GSH levels were detected in buffalloes infected with Theileria annulata in contrast to the healthy control animals. In buffalloes infected with T. annulata, the antioxidant levels of RBCs decreased during the progression of anaemia this results are agreement with (Murat Guzel et.al. 2007). The findings in this study may suggest the alteration in antioxidative and oxidative balance due to oxidative stress and ROS generation in the course of theileriosis. This alteration may be due to a decrease in the levels of enzymatic and non-enzymatic antioxidants, which are the component of antioxidant-defence system this record as (Rezai and Dalir-Naghadeh, 2006).

Conclusions
The diagnosis of theileria spp must be confirmed by PCR because there is a great morphological similarity among theileria spp. The decreased levels of erythrocytic GSH and TAC in cattle infected with Theileria annulata may indicate oxidative damage to RBCs.