Echinococcus granulosus sensu stricto G1 is the predominant genotype in human and livestock isolates from Turkey and Iran, based on mitochondrial nad5 gene differentiation

Background Different genotypes of Echinococcus granulosus sensu stricto (s.s.) isolated from livestock and humans have been identified based on cox1 and nad1 genomic fragments. The present study was performed to differentiate the G1/G3 genotypes of Echinococcus granulosus (s.s.) isolated from humans and livestock (sheep and cattle) from Azerbaijan in northwestern Iran, Fars Province in southern Iran, and Van province in Eastern Turkey, using the nad5 gene fragment as a suitable marker to distinguish these two genotypes. Methods A total of 60 pathologically confirmed human hydatid cysts and 90 hydatid cyst samples from livestock were collected from Turkey and Iran. PCR was performed on all of the samples, targeting the nad5 gene. Based on PCR product quality, host type, and the geographical area where the samples were obtained, 36 of the samples were sequenced and were used in the phylogenetic analysis. Results Out of 36 evaluated samples, 26 (72.2%) samples belonged to G1, and 10 (27.8%) samples belonged to the G3 genotype. Out of 21 samples from Turkey, 16 (76.2%) were G1 and 5 (23.8%) were G3, while out of 15 samples from Iran, 10 (66.7%) were G1 and 5 (33.3%) were the G3 genotype. None of the samples isolated from humans in Iran or from sheep in Turkey were G3. Overall, between the two countries, 18.18% of E. granulosus isolates in cattle, 41.66% of isolates in sheep, and 23.07% of human samples were identified as G3, and the others as the G1 genotype. The G3 genotype was not detected in human samples from Iran or sheep samples from Turkey. Conclusion The findings of the study revealed that the G1 genotype of E. granulosus s.s. is the predominant genotype in humans and livestock, both in Turkey and Iran. The ratio of the E. granulosus s.s. G1 to G3 genotype was 3.2 in Turkey and 2 in Iran. The study also further confirmed that the nad5 gene properly differentiated the G1/G3 isolates of E. granulosus from both humans and livestock. Graphical Abstract

Identification of Echinococcus granulosus Genotypes G1 and G3 by SNPs Genotyping Assays

Echinococcus granulosus sensu lato (s.l.) is the causative agent of cystic echinococcosis in animals and humans. Different E. granulosuss.l. genotypes exhibit great diversity in their life cycle, host selectivity and pathogenicity. For this reason, the study of genetic variation within Echinococcus species is of importance for their epidemiological implication. We employed two SNP genotyping technologies to distinguish G1 and G3 E. granulosus sensu stricto (s.s.). genotypes. The genotypes of DNA samples (n = 28) extracted from hydatid cysts of different animal species were identified by amplification and sequencing of a fragment of the mitochondrial nad5 gene. Two SYBR green and three TaqMan real time PCR assays were developed for targeting of three nad5 informative positions (SNP758, 1123, and 1380) known to be able to discriminate G1 from G3. Genotyping by SYBR Green PCR based on cycle threshold (Ct) with melting temperature (Tm) analysis and performed on SNP1123 and SNP1380 failed to identify one DNA sample. TaqMan assays for SNP758, 1123 and 1380 effectively confirmed genotype identification obtained by Sanger sequencing. Our results demonstrated that the combination of the three Taqman assays developed in this study represents a valuable and cost effective tool alternative to DNA sequencing for E. granulosus s.s. genotyping.

Specific Detection of the Wheat Blast Pathogen (Magnaporthe oryzae Triticum) by Loop-Mediated Isothermal Amplification

Wheat blast, caused by the Magnaporthe oryzae Triticum pathotype, is an economically important fungal disease of wheat. Wheat blast symptoms are similar to Fusarium head scab and can cause confusion in the field. Currently, no in-field diagnostic exists for M. oryzae Triticum. Loop-mediated isothermal amplification (LAMP) primers were designed to target the PoT2 and MoT3 loci, previously shown to be specific for M. oryzae and M. oryzae Triticum, respectively. Specificity was determined using 158 M. oryzae strains collected from infected wheat and other grasses and representing geographic and temporal variation. Negative controls included 50 Fusarium spp. isolates. Sensitivity was assessed using 10-fold serial dilutions of M. oryzae Triticum gDNA. PoT2- and MoT3-based assays showed high specificity for M. oryzae and M. oryzae Triticum, respectively, and sensitivity to approximately 5 pg of DNA per reaction. PoT2 and MoT3 assays were tested on M. oryzae Triticum-infected wheat seed and spikes and identified M. oryzae and M. oryzae Triticum, respectively, using a field DNA extraction kit and the portable Genie II system. The mitochondrial NADH-dehydrogenase (nad5) gene, an internal control for plant DNA, was multiplexed with PoT2 and MoT3 and showed results comparable with individual assays. These results show applicability for M. oryzae Triticum field surveillance, as well as identifying nonwheat species that may serve as a reservoir or source of inoculum for nearby wheat fields.