Genetic characterization of Hookworm in soil and community dogs in upper Northern Thailand

Abstract

Ancylostoma ceylanicum is a zoonotic helminth that is frequently found in domestic dogs and cats throughout Asia and Southeast Asia but largely neglected in many countries, including Thailand. In this study, we estimated the prevalence of A. ceylanicum in community dogs and soil environments in four provinces of Thailand, including Chiang Mai, Chiang Rai, Lampang, and Phayao provinces. In a total of 299 dog fecal samples and 212 soil samples from 53 temples, the prevalence of hookworm infection by microscopic examination were 26.4% (79/299) and 10.4% (22/212) in dog and soil samples, respectively. A PCR with restriction fragment length polymorphism assay targeting the internal transcribed spacer region of nuclear ribosomal DNA was used to identify the hookworm species. In the present study, A. ceylanicum was the predominant hookworm species infecting dogs, and the proportion of A. ceylanicum and A. caninum infections were 96.6% and 3.5%, respectively. In addition, we confirmed the presence of A. ceylanicum larvae in the soil. The genetic characterization and evolutionary analyses of A. ceylanicum among Thai and Asian populations were conducted using the mitochondrial cytochrome c oxidase subunit 1 gene, consecutively. There were 9 haplotypes which identified from Thai A. ceylanicum, and the haplotype diversity and the nucleotide diversity were 0.4436 and 0.0036, respectively. Phylogenetic analysis revealed that A. ceylanicum from this study clustered in the same clade of A. ceylanicum from other reference countries. The median-joining network demonstrated six unique haplotypes in Thailand; nevertheless, three haplotypes were communally found in Malaysia, Cambodia, and China. The highest nucleotide diversity of Chinese A. ceylanicum populations advocated that Chinese A. ceylanicum could be the ancestor of the populations. Pairwise fixation indices implied that Thai population was closely related to Malaysian A. ceylanicum, suggesting gene flow between these populations. At last, A. ceylanicum genotypes in dogs and soil samples in this study were similar, and the temples with hookworm-positive dogs were correlated with the presence of hookworm-contaminated soil, as these levels showed about 4-fold increase compared with temples with hookworm-negative dogs (OR=4.38, 95% CI: 1.55-12.37). Hence, increased awareness and public concern in the communities with regards to the responsibility of temples and municipal offices to provide proper deworming programs to community dogs should be intensively endorsed to reduce the risk of transmission of this zoonotic disease. Moreover, parasitic examination and treatment should be strongly applied before dogs are imported and exported worldwide.