Molecular discrimination of G1 and G3 genotypes of Echinococcus granulosus sensu stricto obtained from human, cattle, and sheep using the mitochondrial NADH dehydrogenase subunit 5 marker.

Cystic echinococcosis (CE) is a common zoonotic disease caused by the larval form of Echinococcus granulosus sensu lato. This study determined the genotype and haplotype differences using the NADH dehydrogenase subunit 5 gene in hydatid cyst samples. Human (n = 12), cattle (n = 28), and sheep (n = 31) hydatid cyst isolates were included. Seventy-one genomic DNA samples were successfully extracted, and a 759 bp mitochondrial NADH dehydrogenase subunit 5 gene fragment was amplified by PCR. Following the sequence analysis, E. granulosus sensu stricto isolates were identified as G1 (n = 61) and G3 (n = 10). A total of 23 haplotypes were obtained from the 71 E. granulosus s.s. G1 and G3 samples. The main haplotype was Hap01 (60.56 %), which consisted of the G1 genotype. The second largest haplotype was Hap04, which consisted entirely of the G3 genotype. Hap14 acted as a bridge between the G1 and G3 genotypes. This study identifies G1 as the dominant genotype in humans and farm animals in Turkey. High haplotype and nucleotide diversity in genotypes were observed. Additionally, this is the first report on the phylogeography and gene flow models of the E. granulosus s.s. population in Turkey using the NADH dehydrogenase subunit 5 gene, the best marker distinguishing between G1 and G3 genotypes.

An Inventory of Anthelmintic Plants across the Globe.

A wide range of novelties and significant developments in the field of veterinary science to treat helminth parasites by using natural plant products have been assessed in recent years. To the best of our knowledge, to date, there has not been such a comprehensive review of 19 years of articles on the anthelmintic potential of plants against various types of helminths in different parts of the world. Therefore, the present study reviews the available information on a large number of medicinal plants and their pharmacological effects, which may facilitate the development of an effective management strategy against helminth parasites. An electronic search in four major databases (PubMed, Scopus, Web of Science, and Google Scholar) was performed for articles published between January 2003 and April 2022. Information about plant species, local name, family, distribution, plant tissue used, and target parasite species was tabulated. All relevant studies meeting the inclusion criteria were assessed, and 118 research articles were included. In total, 259 plant species were reviewed as a potential source of anthelmintic drugs. These plants can be used as a source of natural drugs to treat helminth infections in animals, and their use would potentially reduce economic losses and improve livestock production.

Co-infection of Echinococcus equinus and Echinococcus canadensis (G6/7) in a gray wolf in Turkey: First report and genetic variability of the isolates.

Cystic echinococcosis (CE) is an important zoonotic diseases caused by larval form of Echinococcus granulosus sensu lato. The material of this study was the gray wolf (Canis lupus), which was found dead in the rural area of Bingol province of Turkey. The animal was brought to Veterinary Faculty for necropsy and many of adult Echinococcus spp. obtained. A total of 9 whole adult worms were morphologically examined under the microscope, gDNA was isolated from individual samples, a partial mt-CO1 gene fragment (875 bp) was amplified with PCR and sequenced. According to the phylogenetic analysis, six worms were characterized as E. equinus, while three were reported as E. canadensis (G6/7). It was found that the haplotypes of both species were similar to previously published haplotypes. This is the first report in which E. equinus and E. canadensis (G6/7) adult parasites were detected together in a gray wolf's intestine. The findings are important in that it draws attention to the importance of wild cycle in the spread of CE.

Multiplex PCR and Sequence Analysis to Investigate Genetic Diversity of Fasciola Isolates from Cattle and Sheep in Turkey.

Fasciolosis is a highly prevalent helminthic infection caused by Fasciola hepatica and F. gigantica. With the aim of identifying hybrid Fasciola flukes, multiplex PCR was performed to amplify the pepck gene. Furthermore, to determine Fasciola haplotypes, mitochondrial nad1 gene was amplified and sequenced, and phylogenetic analyses were performed. Adult Fasciola isolates were collected from 51 cattle and 51 sheep, genomic DNA was isolated, and one-step multiplex PCR was subsequently performed to amplify pepck. Isolates that generated a 510 bp band were identified as F. gigantica, those that generated a 241 bp band were identified as F. hepatica, and those that generated both bands were identified as hybrid (aspermic) flukes. Multiplex PCR data identified four isolates as F. gigantica and 84 as F. hepatica. Fourteen hybrid isolates (five cattle and nine sheep) were identified. On unidirectional DNA sequence analysis of nad1 PCR products, three sequences were identified as F. gigantica and 99 as F. hepatica. In addition, only 4 of 87 haplotypes detected for F. hepatica nad1 sequences were found to be previously reported, while the remaining 83 are unique to this study. To summarize, this study is the first to report the existence of hybrid Fasciola flukes and 83 unique haplotypes of F. hepatica in Turkey.

Genetic Diversity and Haplotype Analysis of Cattle Hydatid Cyst Isolates Using Mitochondrial Markers in Turkey.

Echinococcus granulosus sensu lato (s.l.) causes cystic echinococcosis in ungulates and humans. The current study was designed to find the genetic diversity and haplotypic profiles of hydatid cysts from the lungs of cattle in three provinces in eastern Turkey. Individual cyst isolates (n = 60) were collected from infected cattle lungs after slaughter and then samples were stored in ethanol (70%) until further use. From each isolate, total gDNA was extracted from the cysts' germinal layers. A partial (875 bp) mt-CO1 gene was amplified by PCR and sequenced unidirectionally. The final size of the trimmed sequences was 530 bp for 60 sequences. Sequence and haplotype analyses were performed, followed by phylogenetic analyses. According to BLAST searches, all sequences were detected as E. granulosus s.s. (G1 and G3 strains). Forty-nine point mutations were identified. In addition, five conserved fragments were detected in all sequences. The haplotype analysis diagram showed E. granulosus s.s. haplotypes organized in a star-like configuration. The haplotypes were characterized by 1-17 mutations compared with the fundamental focal haplotype. Thirty-three haplotypes were determined in 60 samples of which 17 (28.3%) belonged to the main haplotype (Hap_06). The mt-CO1 sequences revealed 49 polymorphic sites, 34.5% (20/49) of which were informative according to parsimony analysis.

Molecular Characterization and Haplotype Analyses of Lung Hydatid Cyst Isolates of Cattle and First Report of Echinococcus canadensis (G6/G7) in Cattle Isolates in Turkey.

PURPOSE: Cystic echinococcosis (CE) is a zoonotic disease, caused by parasite known as Echinococcus granulosus sensu lato complex, leading to substantial economic losses in rural areas with public health problems. This study was carried out to understand the haplotypic profiles of the partial mitochondrial cytochrome c oxidase (mt-CO1) gene of cattle lung hydatid cyst samples which were obtained from two provinces in Turkey. METHODS: In this study, forty (n = 40) hydatid cyst samples from the lungs of slaughtered cattle were obtained. The germinal layers were taken separately for each individual cyst then stored in 70% ethanol. From each individual cyst sample, total genomic DNA was extracted. Amplification of the partial mt-CO1 gene (875 bp) was performed using a specific primer set by PCR, and then, the amplicons were sequenced. All sequences were analyzed individually, followed by alignment, and haplotype and phylogenetic analyses were then performed. RESULTS: By the sequence alignment process, 39 out of the 40 sequences were characterized as E. granulosus sensu stricto. However, one of them was matched with E. canadensis (G6/G7). The haplotype analyses of the E. granulosus s.s. isolates were arranged in a star-like orientation with a main haplotype, which was separated from other haplotypes by 1-10 mutation steps, and 26 haplotypes were identified. In the mt-CO1 sequences, 29 polymorphic sites were determined, and 34.5% (10/29) of them were parsimony informative. CONCLUSIONS: The findings of this study provide the first report of E. canadensis (G6/G7 genotype) among cattle in Turkey.

A Case-Study of the Molecular Diagnosis of Echinococcus multilocularis in Wild Boar with Comments on its Public Health Significance in Turkey.

Echinococcus multilocularis is a parasite species of zoonotic importance which can be fatal to humans and causes Alveolar Echinococcosis (AE). This report describes the development of a cyst from the liver of a wild boar and molecular confirmation of its identification. The cyst material was obtained from the liver of a wild boar killed by hunters. Genomic DNA was extracted from the germinal layer of the cyst material, and 875 bp mitochondrial cytochrome c oxidase subunit I (COI) gene fragment was amplified by PCR and sequenced. A BLAST search matched 100% with published Echinococcus multilocularis sequences. This study confirms the occurrence of E. multilocularis in a wild boar for the first time in Turkey.

First report of Echinococcus canadensis (G6/G7) by sequence analysis from the Khyber Pakhtunkhwa province of Pakistan.

Cystic echinococcosis (CE) is a zoonotic parasitic disease that can result in human and animal health problems globally. Although the disease is known to be endemic in Asia and the Middle East, there are few epidemiological studies on CE in Pakistan. The purpose of the present study was to identify the Echinococcus granulosus sensu lato species and genotypes contributing to human CE cases in the Khyber Pakhtunkhwa (KPK) province of Pakistan. A total of fifty-six formalin fixed paraffin embedded (FFPE) CE cyst samples of human origin were collected from the Pathology Department, Rehman Medical Institute (RMI), KPK for the years 2012-2017. Cyst samples came from the liver (26/56; 46.4%), lungs (3/56; 5.3%), spleen (3/56; 5.3%), pelvis (1/56; 1.8%), breast (1/56; 1.8%), and thigh (1/56; 1.8%). The organ location for 21 of the cysts was not recorded. World Health Organization-Informal Working Group on Echinococcosis (WHO-IWGE) ultrasound-based cyst staging was available for 17 of the 26 (65.4%) hepatic cysts. Five of these cysts (29.4%) were CE3 (transitional), nine (52.9%) were CE4 (inactive), and three (17.6%) were CE5 (inactive). Most of the cysts were obtained from CE patients that were ethnically Afghan Pashtuns (44/56; 78.6%), while 12.5% (7/56) were from patients that were Pakistani Pashtuns. The majority (41/56; 73.2%) of patients reported having close interactions with dogs. Using 12SrRNA primers, 33 cyst samples were identified as being caused by E. granulosus sensu stricto (s.s.). Mitochondrially encoded cytochrome C oxidase 1 (mt-CO1) was evaluated for the remaining 23 samples. PCR product was obtained from six of these 23 samples. Of these six samples, one was identified as Echinococcus canadensis (G6/7). Haplotype analysis showed high haplotype and low nucleotide diversity for the mt-CO1 gene. There were 26 polymorphic sites for the mt-CO1 sequence, of which 65.3% (17/26) were parsimony informative. The E. canadensis mt-CO1 haplotype network consisted of 11 haplotypes, with a main central haplotype. In conclusion, it appears that E. granulosus s.s. and E. canadensis (G6/7) are circulating in the northwestern region of Pakistan. Further molecular epidemiological studies are needed to explore the local genetic diversity of the parasite.

Molecular survey on cattle and sheep hydatidosis and first detection of Echinococcus canadensis (G6/G7) in sheep in Turkey.

Larval stage of genus Echinococcus is the causing agent for the zoonotic infection which is life threatening known as Echinococcosis. The purpose of this study was the identification, molecular analysis and characterization of Echinococcus spp. in sheep and cattle. The sampling was done from slaughterhouse of Elazig, Turkey. A total of 85 isolates (sheep, n = 19 and cattle, n = 66) have been collected after slaughtering. Following the gDNA isolation and PCR products of mt-CO1 gene (446 bp) of all the samples were sequenced. Out of 85 isolates, 84 were recognized as Echinococcus granulosus sensu stricto and one sheep isolate was found as Echinococcus canadensis (G6/G7 ) which is identified for the first time in Turkey. However, single nucleotide polymorphism has been observed not only in samples of different animals but also in samples collected from the same cattle. Six liver and three lung hydatid cysts have been detected in cattle. Although no nucleotide differences have been observed in the liver samples, there was single nucleotide polymorphism (C→T) in 40th nucleotide of two lung cysts. As a result of haplotype analysis, 16 haplotypes of E. granulosus s.s. were detected in 66 cattle isolates whereas 7 haplotypes of E. granulosus s.s. were identified in 19 sheep samples.

Reduce Disease Burden of Human Schistosomiasis in Asia Through Biological Control.

Schistosomiasis is a chronic parasitic disease caused by a trematode blood fluke of the genus Schistosoma that belongs to the Schistosomatidae family. It is a neglected disease in different regions of Asia. In this review, 218 articles (between 2000 and 2017) related to the topic were collected from PubMed and Google scholar and reviewed. After thoroughly reading collected articles, due to irrelevant topic requirements, 94 articles were excluded. Articles that have data associated with Asian regions are considered. In Asia, the disease is prevalent in China, Philippines, Indonesia, Yemen, Nepal and Laos, etc. While in Pakistan, India and Bangladesh, the disease is not endemic and very few cases were reported. The disease was eliminated from Japan and Iran. The current review highlights the geographical distribution among Asian countries, transmission patterns, diagnosis, control strategies based on the use of anthelmintic plants and management practices implemented in Asia for the control of schistosomiasis. However, new implementations to treat schistosomiasis in humans should be proved to eliminate the disease finally in the future. This review emphasizes the biological control of schistosomiasis for the eradication of the disease from Asia in the near future.

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.

Occurrence of Liver Hydatid Cysts in a Donkey and Molecular Characterization of Echinococcus equinus.

Larval tapeworms of Echinococcus granulosus have been viewed as the etiological agent for the zoonotic disease cystic echinococcosis, but the species is a complex readily divided into several species and genotypes. Cystic echinococcosis is an important public health issue. Here, the case of liver hydatid cyst in a donkey and molecular characterization of the cyst is presented. The fluid-filled hydatid cyst materials were obtained from the liver of a necropsied donkey. Genomic DNA was extracted and PCR amplification of mitochondrial 12S rRNA gene as well as partial sequencing of mitochondrial cytochrome c oxidase subunit 1 (COI) gene were performed. All cysts were fertile. Traditional polymerase chain reaction (PCR) amplification of 12S rRNA and COI yielded bands (254 and 446 base pairs, respectively) for all 3 cyst samples. However, partial COI gene sequences were identical to those reported for Echinococcus equinus (formerly E. granulosus genotype G4). Thus E. equinus is still transmitting among the equids in Turkey but the mitochondrial 12S rRNA gene primers may not be sufficient for the molecular characterization of members of the E. granulosus species/genotype complex. Molecular diagnosis must be confirmed by partial COI sequence analysis.

Molecular characterization and haplotypes of sheep and goat isolates of Cysticercus tenuicollis in Turkey.

Taenia hydatigena, is a common parasite, lived mostly in dogs and wild carnivores in its mature stage, and the larvae, Cysticercus tenuicollis, is found on ruminants and pigs. The aim of the current study was to determine the genetic diversity in 20 isolates of the sheep and goats. After the isolation of total genomic DNA from C. tenuicollis isolates, genetic characterization of the mitochondrial NADH dehydrogenase subunit 1 gene region was amplified using specific JB11-JB12 primers in PCR and the PCR products were sequenced and haplotype and genetic diversity analyses were utilized. As a result, multiple nucleotide changes were determined in the sequence analyses of the isolates leading to detection of 16 and 15 different haplotypes in sheep and goat samples, respectively. These findings are important in terms of showing the diversity of nucleotide variation in C. tenuicollis in Turkey.

A Retrospective Analysis on the Cystic Echinococcosis Cases Occured in Northeastern Punjab Province, Pakistan.

A retrospective study was performed to report the case occurrence of cystic echinococcosis (CE) in three hospitals of north-eastern region in Punjab Province, Pakistan. We reviewed retrospectively the clinical records of patients in 4 hospitals which were diagnosed with CE during 2012-2017. A total of 198 cases, 82 (41.4%) male and 116 (58.6%) females were detected as CE. The most Highest incidence was revealed in.... 21-30 years-old group (24.2%) followed by 41-50 (22.7%), 31-40 (16.2%), 11-20 (13.6%), 51-60 (8.1%), below 10 (5.5%), over 71 (5.1%) and 61-70 year-old group (4.5%). CE was detected in various organs of infected individuals. However, most of CE cases were detected in the liver (47.4%) and lung (18.6%). The present study indicated that CE is more or less prevalent in surveyed areas and one of the most important public health problems in Punjab Province, Pakistan.

Molecular Characterization of Hypoderma SPP. in Domestic Ruminants from Turkey and Pakistan.

The aim of this study was to determine the morphological and molecular characterization of Hypoderma spp. in cattle and yak from provinces in Turkey and Pakistan. In total, 78 Hypoderma larvae were collected from slaughtered animals in Turkey and Pakistan from October 2015 to January 2016. Thirty-eight of these 78 Hypoderma larvae were morphologically classified as third instar larvae (L3s) of Hypoderma bovis, 37 were classified as Hypoderma lineatum, and 3 were classified as suspected or unidentified. The restriction enzyme TaqI was used to differentiate the Hypoderma spp. by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP). According to the sequences and the PCR-RFLP results, all larval samples from cattle from Turkey were classified as H. bovis, except for 1 sample classified as H. lineatum. All Hypoderma larvae from Pakistan were classified as H. lineatum from cattle and as Hypoderma sinense from yak. This study provides the first molecular characterization of H. lineatum (cattle) and H. sinense (yak) in Pakistan based on PCR-RFLP and sequencing results.

First Molecular Characterization of Hypoderma actaeon in Cattle and Red Deer (Cervus elaphus) in Portugal.

Hypoderma spp. larvae cause subcutaneous myiasis in several animal species. The objective of the present investigation was to identify and characterize morphologically and molecularly the larvae of Hypoderma spp. collected from cattle (Bos taurus taurus) and red deer (Cervus elaphus) in the district of Castelo Branco, Portugal. For this purpose, a total of 8 larvae were collected from cattle (n=2) and red deer (n=6). After morphological identification of Hypoderma spp. larvae, molecular characterization was based on PCR-RFLP and mitochondrial CO1 gene sequence analysis. All larvae were morphologically characterized as the third instar larvae (L3) of H. actaeon. Two restriction enzymes were used for molecular identification of the larvae. TaqI restriction enzyme was not able to cut H. actaeon. However, MboII restriction enzyme differentiated Hypoderma species showing 210 and 450 bp bands in H. actaeon. Furthermore, according to the alignment of the mt-CO1 gene sequences of Hypoderma species and to PCR-RFLP findings, all the identified Hypoderma larvae were confirmed as H. actaeon. This is the first report of identification of Hypoderma spp. (Diptera; Oestridae) from cattle and red deer in Portugal, based on morphological and molecular analyses.