Epidemiological and Molecular Characterization of Echinococcus granulosus Isolated from Small Ruminants in Kashmir Valley, India

Background: Cystic Echinococcosis (CE) is an emergent or re-emergent zoonosis and remains a public health and economic problem all over the world. Methods: The present study was carried on the prevalence and genotypes of Echinococcus present in small ruminants in Kashmir valley. A total of 2100, sheep (2052) and goats (48), slaughtered or spontaneously dead, from various areas of Kashmir valley were screened for the presence of hydatidosis. In case of goat none of the cases were found positive for hydatidosis, whereas, all the positive cases (85) were recorded in sheep only. The overall prevalence of hydatidosis was 4.04%. The prevalence was higher in female sheep (5.46%) compared to males (2.83%). Season-wise highest prevalence was in summer (4.55%), followed by autumn (4.1%), spring (3.89%) and winter (2.5%).The liver was observed to be the most frequently infected organ with relative prevalence of 61.17% followed by lungs (38.82%).The rDNA-ITS1 fragment of positive samples was amplified with BD1 / 4S primers. Results: The length of amplified fragment for all isolated samples was 1000bps. The products obtained on PCR were digested with four restriction enzymes (Rsa 1, Alu 1, Msp 1 and Taq1). Rsa 1, Alu 1, Msp 1 yielded identical fragments, 300 and 700 bp in sheep. TaqI restriction enzyme had no effect on PCR product and after digestion; intact 1000bps fragment was seen. Conclusion: Phylogenetic analysis of ITS1 gene revealed that the common sheep strain (G1) is the predominant genotype in sheep in Kashmir valley.

Employing NADH Dehydrogenase Subunit 1 in the Determination of Echinococcus granulosus Strain in Sheep, Cattle and Human in Thi-Qar Province, Iraq

Echinococcosis is a zoonotic disease caused by the larval stage of the tapeworm Echinococcus granulosus. This disease is an important public health and a significant economic issue in Iraq, where the lungs and livers are the popular places of infection. The aim of the current study focused on using the molecular techniques in the detection of an E. granulosus strain that causes cystic echinococcosis to human, sheep and cattle in Thi-Qar province, Iraq. In the current study, thirty isolates of E. granulosus were collected from 10 human hydatid cysts through surgery done at Al-Hussein Imam Teaching Hospital in Thi-Qar province and 10 sheep with 10 cattle hydatid cysts were obtained from the slaughterhouse in Thi-Qar province, Southern of Iraq to identify strains of E. granulosus which infect human and other intermediate hosts (sheep and cattle). The molecular study was carried out on the isolates and a specific primer set for the mitochondrial dehydrogenase NADH subunit 1 (NAD1) gene was used. This primer set was amplified 400 bp of the NAD1 gene in all selected isolates. The PCR products for the twelve selected isolates of E. granulosus (4 isolates per intermediate host) were sequenced and the results for these twelve isolates showed that all sequenced isolates, except one isolate Eg_5, belonged to the sheep strain G1 and a slight genetic diversity was observed with the reference sequences of the strain G1. The exception was in the isolate Eg_5 isolated from a cattle liver, which was similar to the buffalo strain G3. This study concludes that the common E. granulosus strain in Thi-Qar province is G1.

Comparative Genomics of Mycobacterium avium Subspecies Paratuberculosis Sheep Strains

Mycobacterium avium subspecies paratuberculosis (MAP) is the aetiological agent of Johne's disease (JD), a chronic enteritis that causes major losses to the global livestock industry. Further, it has been associated with human Crohn's disease. Several strains of MAP have been identified, the two major groups being sheep strain MAP, which includes the Type I and Type III sub-lineages, and the cattle strain or Type II MAP lineage, of which bison strains are a sub-grouping. Major genotypic, phenotypic and pathogenic variations have been identified in prior comparisons, but the research has predominately focused on cattle strains of MAP. In countries where the sheep industries are more prevalent, however, such as Australia and New Zealand, ovine JD is a substantial burden. An information gap exists regarding the genomic differences between sheep strain sub-lineages and the relevance of Type I and Type III MAP in terms of epidemiology and/or pathogenicity. We therefore investigated sheep MAP isolates from Australia and New Zealand using whole genome sequencing. For additional context, sheep MAP genome datasets were downloaded from the Sequence Read Archive and GenBank. The final dataset contained 18 Type III and 16 Type I isolates and the K10 cattle strain MAP reference genome. Using a pan-genome approach, an updated global phylogeny for sheep MAP from de novo assemblies was produced. When rooted with the K10 cattle reference strain, two distinct clades representing the lineages were apparent. The Australian and New Zealand isolates formed a distinct sub-clade within the type I lineage, while the European type I isolates formed another less closely related group. Within the type III lineage, isolates appeared more genetically diverse and were from a greater number of continents. Querying of the pan-genome and verification using BLAST analysis revealed lineage-specific variations (n = 13) including genes responsible for metabolism and stress responses. The genetic differences identified may represent important epidemiological and virulence traits specific to sheep MAP. This knowledge will potentially contribute to improved vaccine development and control measures for these strains.

Genetic Diversity of Echinococcus granulosus Isolated from Humans: A Comparative Study in Two Cystic Echinococcosis Endemic Areas, Turkey and Iran

Cystic echinococcosis (CE) is one of the most important zoonotic parasitic diseases caused by the larval stage of Echinococcus granulosus. Based on molecular studies and DNA sequencing, E. granulosus has been classified into 10 different genotypes (G1 to G10). Two neighboring countries, Turkey and Iran, are considered the two main foci of CE in the Middle East. The current study is aimed at examining the genotype diversity of E. granulosus isolated from human clinical samples in Turkey and Iran. Surgically removed human hydatid cysts were collected from East Azerbaijan and Fars provinces in Iran and Van province in Turkey. After extracting DNA, performing PCR, targeting the cox1 gene, the PCR products were purified from the gel and were sequenced from both directions. The sequences were aligned and compared, using BioEdit and also the BLAST program of GenBank. The maximum likelihood tree was constructed based on the Tamura-Nei model, using the MEGAX software. Phylogenetic analysis showed that the human isolated samples were classified into two major clades: G1 (from Iran and Turkey) and G3 (5 samples from northwestern Iran and one sample from Turkey). The mean and degree of genetic divergence (K2P) between the two major clades, G1 and G3, were 0.2% and 0.7±0.4%, respectively. The findings of the current study revealed that the sheep strain (G1) and the less important strain G3 have major roles in the transmission cycle of CE in two neighboring countries, Iran and Turkey. Therefore, it is necessary to interpose the life cycle of this parasite and reduce the disease burden in livestock and humans by adopting common regional preventive and control policies.

Genotyping of Echinococcus granulosus Isolates from Human in Khorasan Province, North-Eastern Iran

Background: Human hydatidosis is endemic in northeastern Iran. The present study aimed to investigate molecular diversity of Echinococcus granulosus isolates collected from human surgically. Methods: Sixty human hydatid cysts (58 lung cysts and 2 liver cysts) were collected through surgery from Ghaem and Emam Reza hospitals in Mashhad University of Medical Sciences during 2015-2016. Cysts were characterized using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the internal transcribed spacer 1 (ITS1) gene and sequencing fragments of the genes coding for mitochondrial cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase subunit I (nad1). Results: Overall, 55 out of 60 Echinococcus granulosus cysts (91.6%) were determined as the G1 strain, 4 cases (6.6%) were determined as the G6 strain and 1 sample was not identified. Conclusion: Although sheep strain (G1) is dominated in human patients in Great Khorasan, the prevention of camel-dog cycle should pay attention in this region.

Retrospective study on Cystic Echinococcosis in cattle of Italy

Introduction: Cystic Echinococcosis (CE) is one of the most widespread zoonosis of veterinary and medical importance still constituting a sanitary, economic and socio-cultural problem in Italy. Methodology: The aim of this study was to update epidemiological data on cattle CE in Italy. Data on CE positivity of 5,336 cattle were acquired from abattoir registers between January 2009 and July 2010. Morphobiological characterization of hydatids was performed by direct examination of liver and lungs of 1,664 animals butchered in the same slaughterhouses in 2010. Strain typing of parasites was carried out through the amplification and sequencing of nd1 and cox1 mitochondrial genes. Results: Overall CE prevalence was of 8.1% (430/5,336). Parasitological examination of hydatids showed an overall prevalence of 8.6% with a fertility rate of 0.7% (12/1,664). Regarding localization, hydatids were found in 8% of the livers and in 7.6% of the lungs, respectively. Among positive animals, higher prevalence was observed in the liver (93%) compared to lungs (88.1%) (p > 0.05). Conclusion: The economic loss due to organs condemnation related to CE in cattle amounted to almost € 24,000 per year in the examined abattoir during 2010. Sequence analysis showed the presence of G1 (sheep strain) or Echinococcus granulosus sensu strictu in all examined samples. The G1 confirmed, once more, its possible development into several intermediate hosts such as cattle, especially in areas like southern Italy and Sardinia where the lifecycle of the parasite is still to date carried on by sheep and dogs.

Determination the causative strain for hydatid cyst in Iraqi cattle by using ND1 gene

Hydatid Cysts were obtained from 15 cows from liver, lung, spleen, heart, and peritoneal cavity, between December 2014 and October 2015. Hydatid cysts (protoscoleces) were used for deoxyribonucleic acid extraction by using mechanical grinder. The purification of mtDNA was done by (promega kit, USA). The mitochondrial NADH dehydrogenase subunit 1 (ND1) genes was used as targets for polymerase chain reaction amplification, all hydatid cysts yielded amplification products. Polymerase chain reaction product for NADH1 800 basic pair. The polymerase chain reaction products were purified and partial sequences were generated. The sequences obtained were found to align with corresponding region for ND1 gene in the Gene Bank nucleotide database confirming to genotype of sheep strain (G1) in Iraq, Phylogenetic analysis of partial sequence data from ND1 genes for obtained Phylogenetic tree. G1 genotype was the most common taxon and the actual source of infection of Iraqi's cattle. All of 15 strains were G1 genotype (sheep strain) based on the partial sequences of NADH dehydrogenase 1 (ND1).

DETERMINATION OF THE INFECTIVE STRAIN OF HYDATID CYST IN IRAQI CATTLE BY USING CO1 GENE

Hydatid Cysts were obtained from liver, lungs, spleen, heart, and peritoneal cavity of 15 cows, from different Iraqi regions between December 2014 and October 2015. Hydatid cysts (protoscoleces) were used for mitochondrial DNA extraction by using mechanical grinder, and the purification of mtDNA was done by (promega kit, USA). "The mitochondrial cytochrome c oxidase subunit 1 (CO1) gene" was used as target for "polymerase chain reaction (PCR)" which successfully amplified the targeted this gene with 450 bp. The PCR products were purified and partial sequences were determine. The obtained sequences were aligned with the corresponding region of co1 gene in the Gene Bank nucleotide database to confirm the infection with hydatid cyst sheep strain (G1) in Iraq. The amplified CO1 targeted region was analyzed to obtain the phylogenetic tree. G1 genotype was the most common strain and the actual source of infection of Iraqi's cattle. All of 15 samples were G1 strain (sheep strain) according to the partial sequences of (CO1) genes.

Molecular classification of Echinococcus granulosus strains from livestock animals in Libya

This study demonstrates the distribution of various E. granulosus strains in sheep, cattle and camel. The common sheep strain G1 is mainly found in sheep and cattle, but also parasitized camels. In contrast, the camel strain G6 is found mainly in camels and rarely in sheep and cattle. However, the study also revealed the possible presence of cryptic species that are closely related to both genotypes in livestock of Libya as evident by high mutations in several specimens. Based on the occurrence of overlapping hosts of E. granulosus in Libya, more research on the transmission cycles and genotypes of E. granulosus in Libya is required. In addition, it is suggested that surveys on potential intermediate hosts, including in humans with dogs as the major final host in Libya using higher resolution molecular tools such as microsatellite markers is recommended.

Identification of Echinococcus granulosus strains using polymerase chain reaction–restriction fragment length polymorphism amongst livestock in Moroto district, Uganda

A descriptive study was conducted to identify the different strains of Echinococcus granulosus occurring in livestock in Moroto district, Uganda. Echinococcus cysts from 104 domestic animals, including cattle, sheep, goats and camels, were taken and examined by microscopy, polymerase chain reaction with restriction fragment length polymorphism and Sanger DNA sequencing. Echinococcus granulosus genotypes or strains were identified through use of Bioinformatics tools: BioEdit, BLAST and MEGA6. The major finding of this study was the existence of a limited number of E. granulosus genotypes from cattle, goats, sheep and camels. The most predominant genotype was G1 (96.05%), corresponding to the common sheep strain. To a limited extent (3.95%), the study revealed the existence of Echinococcus canadensis G6/7 in three (n = 3) of the E. granulosus–positive samples. No other strains of E. granulosus were identified. It was concluded that the common sheep strain of Echinococcus sensu stricto and G6/7 of E. canadensis were responsible for echinococcal disease in Moroto district, Uganda.