The study was performed in Mashhad area as the center city of Khorasan Razavi province from April 2016 to August 2017. The city is located at 36.20º North latitude and 59.35º East longitude, in the valley of the Kashafrud River near Turkmenistan, between the two mountain ranges of Binalood and Hezar Masjed Mountains. The city benefits from the proximity of the mountains, having cool winters, pleasant springs, and mild summers. (Fig.1)
One hundred seventy five stray cats were trapped from different areas of the Mashhad in with the help of local municipality. Furthermore, Thirty-one samples were from killed stray cats during driving accidents. The trapped cats and carcasses were transferred to the diagnostic laboratory of the parasitology department for laboratory examination. They were sedated by premedication with intramuscular (IM) Ketamine hydrochloride (6mg/kg). The approximate age of cat was determined by teeth examination. All of the adult teeth are in place by 6 months of age, and the growth is no longer useful in determining a cat's age. In older cats, the amount of staining, or tartar, on a cat's teeth is also an indicator of age. Then, the data related to their age, the sex were recorded and collected feces from each cat. The killed cats were necropsied and feces and the brain were collected. Collected fecal and brain samples were kept in a refrigerator at 4 ˚C for further examination. The trapped cats were released after sampling with help of the Mashhad municipality.
Feces (1g) of each animal were emulsified in sucrose solution (specific gravity 1.203), filtered through gauze, and centrifuged in a 15 mL tube at 400g for 10 min. A drop of the float from the meniscus was examined microscopically at × 400 magnifications for the presence of T. gondii oocysts . The size of oocysts was measured by a calibrated ocular micrometer (Zeiss Company, Germany).
Oocysts of fecal samples were repeatedly washed in PBS and homogenized by grinding with 0.5mm glass beads for 30min. DNA of homogenized oocysts, fecal and brain samples was extracted by commercial kit ( Molecular and Biological Transmission Systems (MBST), Tehran, Iran) as per manufacturer’s recommendations. T. gondii B1 gene PCR amplification was carried out using a nested-PCR as previously described by Burg et al., 1989. Amplification in 25 μL reaction volumes (Accupower PCR premix kit, Bioneer®, South Korea) in first reaction contained: 250 μM of each dNTP, 10mM Tris-HCl pH 9.0, 30mM KCl and 2mM MgCl2, 1U Taq DNA polymerase and 10 pmol of each PCR primer (Denazist, Mashhad, Iran). Then 1 μL of DNA template (250-500 ng) was added to each reaction and the remaining 25 μL reaction volume was filled with sterile distilled water.
After 3 minutes of initial denaturation at 94°C, 38 cycles of amplification (each cycle: 1 minute at 94°C, 1 minute at 50°C, and 1 minute at 72°C) and a final extension step for 7 minutes at 72°C were performed in an automated thermocycler (MJ Mini Thermal Cycler, Bio-Rad Co , USA). The PCR products were visualized by electrophoresis on a 1.5% agarose gel. One μL of the diluted (1:10) each reaction is then used in second reaction in the same mixture and cycling condition, except for the annealing temperature which was 52 °C; the number of cycles was 30. The presence of specific bands of 193 bp in primary PCR and 96 bp in nested -PCR on agarose gel was considered a positive sample .Distilled water was used as a negative control and T. gondii strains (RH) was used as positive controls.
Isolation of T.gondii
The tissue homogenates were prepared from the brain tissue of the cats as the method that described by Dubey . Briefly, 100 grams of brain samples were homogenized in 0.5 L of normal saline (0.85%) with penicillin 100 IU/mL and streptomycin 1mg/mL by the electrical mixture. The tissue homogenate was strained through 2 layers of gauze to remove coarse material. The homogenate was kept at room temperature for three hours and centrifuged at 1.500 g for 5 min. The homogenate (0.5 mL per each mouse) was inoculate subcutaneously to Swiss Webster mice (Razi Vaccine& serum research institute, Mashhad, Iran).None inoculated mice were shown clinical signs. Six weeks after inoculation, blood samples were collected from the tail of mice. Serum samples were separated and analyzed for the presence of antibodies against T. gondii by ELISA test (ID.vet Innovative Diagnostics, Grabels, France). Seropositive mice were killed at 42 days post-infection by chloroform-inhalation. Then, mouse brain was homogenized with an equal volume of sterile normal saline by passing through a 16 g needle ten times by mean a syringe. One drop of given suspension placed on a slide and spread out covered with a slip and microscopically examined. At least five slides should be examined. The isolation of T.gondii was successful if Toxoplasma cysts were found in the mouse brain.
PCR-RFLP with SAG1, SAG2, SAG3, BTUB and GRA6 markers were performed to determine the T. gondii genotype in fecal and brain samples according to described methods [8-11]. Briefly, the PCR reaction was performed in a 25 µl reaction mixture containing containing 1 µl of extracted DNA, 75 mM Tris-HCl (pH 8.5), 20 mM (NH4)2SO4, 1.5 mM MgCl2, 0.1% Tween 20, 0.2 mM dNTPs, 0.025 U/µl amplicon Taq DNA polymerase, inert red dye, a stabilizer and 10 pmol of each primer described in Table.1 After 5 minutes of initial denaturation at 95°C, 35 cycles of amplification followed by 30 sec at 94˚C, 1 min at 60˚C, 2 min at 72˚C, and a final extension of 72˚C for 10 min ( MJ Mini Thermal Cycler, Bio-Rad Co , USA).
After that, 1.5 U of enzymes endonuclease with 2 U buffers was added to 15-mL of each PCR product and incubated as the manufacturer’s protocol. The digested products were electrophoresed to separate restriction fragments in 1.6% agarose gel. Finally the agarose gel was stained with ethidium bromide and visualized under UV. Extracted DNA of RH strain was used as a positive control.
The purified PCR products of B1 with primers were sent to DNA sequencing in the Bioneer Inc. (Bioneer Company, Seoul, Company).The assembling and editing of nucleotide sequences were used by CLc bio software.
The relationship between infection rate and variables such as age and gender was analyzed by the chi-square test. A significant association was identified when a p-value of less than 0.05 was observed . The agreement between the different tests was showed as k-value. The agreement as poor if k-values between 0.2 and 0.4, moderate if k-values between 0.4 and 0.6, substantial if 0.6 and 0.8 and good if it exceeds 0.8 and 1.3 .