Designing and developing of high-resolution melting technique for separating different types of Toxoplasma gondii by analysis of B1 and ROP8 gene regions.

BACKGROUND: Determination of Toxoplasma gondii genotypes plays an important role in the health management and epidemiology of toxoplasmosis. We developed HRM analysis to differentiate genotypes of T. gondii using the B1 and ROP8 genes, through comparing the sensitivity and specificity of both genes and methods used for the detection of T. gondii. METHODS: A total of 96 DNA samples of muscle tissue of livestock and poultry brain tissue with three standard strains RH (type I), PRU (type II) and VEG (type III) were prepared and analyzed. Three methods of nested PCR, PCR-PCR and nested-qPCR-HRM were used. Specific new primers were designed and synthesized for developing HRM. Thirty positive samples obtained from nested-qPCR-HRM were sequenced (18 B1 and 12 ROP8). RESULTS: Overall, 87 infected samples were identified using both genes. Through the B1 gene, we could separate type I (Tm = 84.8 °C) from II/III types (Tm = 84.6 °C). Also, the ROP8 gene could separate type II (Tm = 84.5 °C) from I/III types (Tm = 84.12 °C). Highest sensitivity (100%) and specificity (78.72%) were observed by nested-qPCR-HRM assays of the B1 and ROP8 genes than by other methods, respectively. Thus, the B1 gene can be used to most accurately detect T. gondii, while the ROP8 gene was more appropriate for T. gondii genotyping. PCR-sequencing results were consistent with HRM results in most selected samples. CONCLUSION: HRM analysis is a powerful diagnostic tool for rapid detection and determination of main clonal lineages, and even unusual T. gondii genotypes.

Phylogenetic Analysis and Genetics Polymorphisms Evaluation of ROP8 and B1 Genes of Toxoplasma gondii in Livestock and Poultry Hosts of Yazd, Qom and Golestan Provinces of Iran.

BACKGROUND: A high correlation is observed between specific clonal lineages and host types in toxoplasmosis. The main objectives of this study were comparing polymorphism and evolutionary analysis of the B1 and ROP8 genes, as well as the evaluation of phylogenic and Toxoplasma gondii isolates obtained from different hosts and regions. METHODS: Overall 96 brain/diaphragm tissue samples of livestock and poultry from three provinces of Iran (cows: 9 from Yazd, 9 from Qom; sheep: 19 from Yazd, 7 from Qom; goats: 7 from Yazd, 4 from Qom; one camel from Yazd and 37 chickens, 2 roosters and one duck from Golestan) were tested during 2018-19. A nested PCR and PCR-PCR methods were developed with the B1 and ROP8 genes. Evaluation of genetic proximity, genetic diversity and evolutionary analysis were done using MEGA-X and DnaSP5 software. Thirty samples of both genes were sequenced (18 B1 and 12 ROP8 genes), and submitted to the GenBank (MN275903-MN275932). RESULTS: Tajima's D index analyses showed that both genes were in the negative direction of evolution. The B1 gene was more sensitive than the ROP8 gene. The ROP8 gene showed better and more acceptable results in terms of the relationship between the host and the genotyping of the samples. CONCLUSION: The B1 gene was only an attractive target for rapid detection of T. gondii parasites, whereas the ROP8 gene due to a high level of polymorphism was able to isolate the three clonal lineages (type I, II and III), intertypes and even atypical strains from different isolates of T. gondii.

Toxoplasma gondii in Aborted Fetuses of Sheep in Ardebil Area, North-West of Iran.

BACKGROUND: One of the severe complications of toxoplasmosis is the induction of abortion in sheep, goats, and human beings. The rate of abortions related to toxoplasmosis in sheep varies between 10% and 23% in the United States and European countries. In Iran, Toxoplasma infection were diagnosed in aborted ovine fetuses between 13.5% and 69% in the brain samples based on PCR. The purpose of this study was to investigate the prevalence of abortions related to T. gondii among the aborted fetuses of sheep in Ardabil area, North-West of Iran. METHODS: The brains of 75 aborted sheep fetuses in Ardebil area were investigated with nested PCR method using fragments of the GRA6 gene during lambing seasons in 2014-2015. Meanwhile, thoracic and abdominal fluids of aborted fetuses and 200 serum samples collected from the herds affected by abortion were examined for the existence of anti-T. gondii IgG. RESULTS: For 48 out of the 75 fetal brain samples (64%), the results of nested PCR test were found to be positive. Furthermore, antibodies against Toxoplasma was observed in 136 (68%) collected samples of sheep and 21 samples (28%) of fetal fluids. CONCLUSION: There is the significant role of T. gondii in the abortions of sheep in Ardabil area. Meanwhile, this condition can also be dangerous for human beings because of their consumption of sheep meat.

Identification of Antigenic and Immunogenic Proteins of Toxoplasma gondii in Human and Sheep by Immunoproteomics.

BACKGROUND: Toxoplasmosis is a parasitic disease caused by the intracellular protozoan parasite, Toxoplasma gondii, which can infect humans and warm-blooded animals. This infection can lead to still birth and abortion among some susceptible hosts especially sheep and human in pregnancy. Development of a vaccine against T. gondii infection is very important-especially for use in immunocompromised patients, pregnant women, and sheep. Different antigens of T. gondii can be potential candidates for immunization. The aims of this study were to identify the immunodominant and antigenic proteins of T. gondii in sheep and man. METHODS: Tachyzoites' proteins were separated by two-dimensional polyacrylamide gel electrophoresis (2-DE), and subjected to western blot analysis probed with T. gondii positive sera of sheep and human (Biotechnology Department of Pasteur Institute of Tehran, Iran, from April 2016 to March 2017). Finally, the immunoreactive proteins were identified by mass spectrometry (MALDI-TOF/MS and MS/MS) technique. RESULTS: Five immunoreactive and antigenic proteins were recognized by Toxoplasma positive sera of human and sheep. These identified proteins were Enolase 2, rhoptry protein 4 (ROP4), dense granular protein 14 (GRA14), rhoptry protein 15 (ROP15) and rhoptry protein 9 (ROP9). CONCLUSION: The identified immunodominant proteins have potential to be used as diagnostic antigens and as diagnostic markers of Toxoplasma infection in sheep and human.

Detection of Theileria orientalis in Iran by semi-nested PCR.

In order to identify and differentiate Theileria orientalis in cattle which may be infected with Theileria annulata simultaneously, a semi-nested PCR was performed. Thus, 160 blood samples were collected from apparently healthy native cattle in Golestan province of northern Iran, during 2009 to 2011. The Tbs-S/Tbs-A primer set derived from the 18S rRNA encoding gene was used for first PCR amplification, and the amplified sequence weight by this primer set for Theileria sp. was 426-430 bp. Then, DNA solution from purified PCR product was used for the semi-nested PCR analysis. The first PCR product amplified using T. orientalis primer set (To-S/Tbs-A) derived from the 18SrRNA encoding gene, and this specific primer weight was 235 bp. Also, the first PCR product amplified using T. annulata primer set (Ta-S/Tbs-A) derived from the 18SrRNA encoding gene and this specific primer weight was 193 bp. Having extracted DNA of each sample, using Tbs-S/Tbs-A primer set for PCR and analyzing the PCR products on the 2% agarose gel electrophoresis, 13 out of 160 blood samples (8.12%) were positive for Theileria sp. Meanwhile, performing semi-nested PCR with T. orientalis-specific primers, 9 out of 13 blood samples (5.62%) were positive and performing semi-nested PCR with T. annulata-specific primers, 12 out of 13 blood samples (7.5%) were also positive. This molecular assay approves the presence of T. orientalis in the native cattle of northern parts of Iran for the first time. In addition, this procedure will detect the concurrent infection of T. orientalis and T. annulata in the cattle too.