Molecular characterization of cattle and sheep isolates of Echinococcus granulosus from Elazig province in Turkey and expression analysis of the non-coding RNAs, egr-miR-7, egr-miR-71 and egr-miR-96.

Cystic Echinococcosis (CE) is a common zoonotic disease seen in human and animals worldwide, caused by the larval form of Echinococcus granulosus. In this study, E. granulosus s.l. species and haplotypes were determined in hydatid cysts isolated from cattle and sheep, and the expression levels of egr-miR-7, egr-miR-71 and egr-miR-96 miRNAs were compared in different cyst structures. A total of 82 (cattle, n = 41; sheep, n = 41) hydatid cyst isolates (germinal membranes and/or protoscoleces) were collected from a slaughterhouse in Elazig province of Turkey. After mt-CO1 gene sequences were made, 81 out of 82 hydatid cyst isolates were determined as E. granulosus s.s. (G1 and G3), while an isolate of cattle origin was determined as Echinococcus canadensis (G6/7). A total of 26 nucleotide polymorphisms and 29 haplotype groups were identified in the samples. miRNA expressions in germinal membranes of sterile cysts and germinal membrane and protoscoleces of fertile cysts were investigated by qRT-PCR and Real Time PCR analyses. It was determined that miRNAs were expressed at high levels in 79.31% of the 29 haplotype groups and at low levels in the remaining 10.34%. In 10 fertile samples of sheep origin, egr-miR-7, egr-miR-71 and egr-miR-96 miRNAs were found to be 44, 168, and 351-fold higher in expression, respectively, in the germinal membrane compared to the protoscoleces. Especially egr-miR-96 may have the potential to be used as biomarkers in the diagnosis of active CE.

Identification of antigen B (AgB) Gene polymorphism in cattle and sheep isolates of Echinococcus granulosus and investigation of effects on serological diagnosis.

Cystic Echinococcosis (CE) is a worldwide common helminth disease caused by the larval form of Echinococcus granulosus. The aim of this study is to determine the genetic differences between distinct isolates of E. granulosus obtained from cattle and sheep and determine the polymorphism of the AgB1 gene by DNA sequence analysis, as well as investigating its relationship with serological response using ELISA and Western Blot tests. For this aim, germinal membranes of hydatid cysts of 30 cattle and 30 sheep from the provinces of Elazig and Erzincan in Turkey and serum samples of these animals were collected. Following isolation of the total genomic DNA, the 12S rRNA gene of all isolates was amplified by PCR for genetic characterization, and the mt-CO1 gene region was examined by DNA sequence analysis. The gDNAs were then amplified by PCR using AgB1-specific primers, and genetic variation was investigated by DNA sequence analysis. At the final stage, all serum samples were analyzed by ELISA and Western Blot tests using a partially purified hydatid cyst fluid antigen. As a result, 114 (95%) of the 120 isolates were determined to be E. granulosus sensu stricto by using 12S rRNA-PCR. Subsequently, the DNA sequence analysis of the remaining 6 samples of the mt-CO1 gene revealed that all samples were E. granulosus sensu stricto. According to the DNA sequence analysis that followed, nucleotide changes in the AgB1 gene were observed in 13 (10.8%) of 120 samples. With this study, 9 (69.2%) out of 13 hydatid cysts in which polymorphism was detected by DNA sequence analysis in their AgB1 gene were found to be positive with ELISA, and 6 (46.1%) were found positive by WB. While 80 (74.7%) of 107 non-polymorphic samples in the AgB1 gene were found to be positive with ELISA, and 75 (70.9%) were positive with WB. As a result, variation in different ratios was determined in the AgB1 gene of E. granulosus sensu stricto, and it was determined that this had a partial effect on serological response.

Molecular characterization and detection of variants of Taenia multiceps in sheep in Turkey.

Taenia multiceps is a cestode (family Taeniidae) that in its adult stage lives in the small intestine of dogs and other canids. The metacestode, known as Coenurus cerebralis, is usually found in the central nervous system including brain and spinal card in sheep and other ruminants. The presence of cysts typically leads to neurological symptoms that in the majority of cases result in the death of the animal. Coenurosis could cause high losses in sheep farms because the disease commonly affects young animals. A total of 20 C. cerebralis isolates collected from naturally infected sheep in Mardin province of Turkey were characterized through the polymerase chain reaction and sequencing of a fragment of cytochrome c oxidase subunit 1 (CO1) gene. The results showed that the CO1 gene sequences were highly conserved in C. cerebralis isolates. Phylogenetic analysis based on partial CO1 gene sequences revealed that C. cerebralis isolates were composed of three different variants.

Epidemiological survey and molecular characterization of Echinococcus granulosus in cattle in an endemic area of eastern Turkey.

Cystic echinococcosis (CE) is a zoonotic disease affecting mainly various species of livestock and humans. A survey of cystic echinococcosis in cattle was conducted from December 2008 to April 2009 in an endemic area of eastern Turkey. A total of 1758 cattle were examined and hydatid cysts were found in 33.9% of the cattle (595/1758). Most of the cattle (75.8%) had hydatid cysts only in the lungs, 10.9% only in the liver, 12.4% in the both liver and lungs, 0.7% in the spleen and 0.2% in the heart. 220 of these cysts were examined by PCR of 12S rRNA gene and sequencing of mt-CO1 gene. 147 of 220 cattle isolates showed the same band pattern with 12S rRNA analyses and were identified as G1-G3 complex (Echinococcus granulosus sensu stricto) and also 28 of these were confirmed by mitochondrial CO1 sequencing as G1 genotype. The other 73 samples that did not amplified with 12S rRNA gene specific primers were analyzed for mt-CO1 gene and only 7 samples yielded 446bp product and after the CO1 sequencing these were identified as G3 genotype. This study confirms the predominance of the sheep strain (G1 genotype) in Turkey and this is the most comprehensive genetic survey of cattle CE in Turkey.

Molecular genetic characterization of different isolates of Echinococcus granulosus in east and southeast regions of Turkey.

We used PCR-RFLP analysis of ribosomal ITS1 fragment using four different restriction enzymes and DNA sequencing of mitochondrial CO1 gene to investigate the genetic characteristics of isolates of Echinococcus granulosus obtained from different hosts (179 sheep, 19 cattle, 7 goat, 1 camel, 1 dog and 1 human) and regions (Elazig, Malatya, Erzurum, Van, Diyarbakir and Sanliurfa) of Turkey. The report represents the most comprehensive genotypic investigation of E. granulosus isolates undertaken in Turkey, with Turkish samples of cattle, goat, camel and dog origin being characterized for the first time. We show that the predominant genotype involved in E. granulosus transmission in Turkey is the common sheep strain (G1 genotype) infecting humans, cattle, sheep, goats, camels as well as the dog definitive host. Nevertheless, we urge that coordinators of local control programs in Turkey should take into consideration the potential occurrence and risk of additional strains of E. granulosus infecting humans and animal hosts, and plan accordingly.

[Determination of Hypoderma bovis and Przhevalskiana silenus somatic antigens using SDS-PAGE.]. / Hypoderma bovis ve Przhevalskiana silenus somatik antijenlerinin SDS-PAGE ile tanimlanmasi

The aim of this study was to examine larval antigens obtained from the third instar larvae of Hypoderma bovis and Przhevalskiana silenus using the sodium dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE) method. SDS-PAGE separation was performed using 12% separation gel and 5% stacking gel. At the end of the SDS-PAGE examination, 11 and 19 different polypeptide bands were detected between 6-66 kDa and 6-100 kDa in the third instar larvae antigen of H. bovis and P. silenus, respectively.