Short communication
Development of new microsatellites for the hookworm Ancylostoma caninum and analysis of genetic diversity in Brazilian populations

https://doi.org/10.1016/j.meegid.2017.03.008Get rights and content

Highlights

  • Development of new microsatellites markers to Ancylostoma caninum

  • Significant deviation from Hardy-Weinberg equilibrium in hookworm populations

  • Low to moderate population differentiation in hookworm populations

Abstract

Considering the great efforts towards formulating a vaccine against hookworms, and the concerns about the spread of drug resistance through hookworm populations, it is justified to study the molecular diversity and population genetic structure of these nematodes. This work had the aim to develop microsatellite markers to investigate the genetic structure and the molecular diversity of Brazilian populations of Ancylostoma caninum. Seven microsatellites markers were successfully used to characterize five Brazilian populations. These findings may contribute to a better comprehension of the ecology, patterns of transmission, drug resistances and development of immunotherapeutic strategies in hookworms.

Introduction

The hookworm Ancylostoma caninum is considered one of the primary causes of death in newborn dogs (Miller, 1966). Besides its importance in animal health, this parasite has zoonotic potential, with several reports of cutaneous larva migrans (Bowman et al., 2010). Furthermore, it is considered a causative agent of eosinophilic enteritis in humans (Prociv and Croese, 1990).

Recurrent drug resistance has been reported to the drug of choice, benzimidazole, and genetic polymorphism related to drug resistance has been described in A. caninum populations (Furtado et al., 2014). Furthermore, despite all efforts to control hookworms, no effective vaccine has been discovered (Hawdon, 2014). Knowledge of population genetic structure in this species may therefore inform us about the likelihood that drug resistance alleles selected for in one population will rapidly spread to other populations via gene flow. Similarly, knowledge of genetic structure can inform us about the likelihood that vaccines developed against one strain of the parasite will work on all populations (Chow et al., 2008).

Although genetic diversity of hookworm populations has been studied using mitochondrial markers (Hawdon et al., 2001, Hu et al., 2004, Miranda et al., 2008) and microsatellites markers have been described for A. caninum (Schwenkenbecher and Kaplan, 2007), no study used microsatellites to characterize the genetic diversity in this worm. This study identified and characterized seven microsatellite loci in the genome of A. caninum, and used them to evaluate the genetic diversity of Brazilian populations of this parasite.

Section snippets

Population studied and DNA extraction

Populations of A. caninum were obtained from 5 different geographical regions from Brazil. The localities and number of worms per population are shown in Table S1. DNA extraction was performed as described in a previous study from our group (Miranda et al., 2008).

Identification and selection of DNA microsatellite loci of A. caninum

Approximately 104.000 A. caninum sequences were retrieved from the GenBank (www.ncbi.nlm.nih.gov), and submitted to the Tandem Repeats Finder software (http://tandem.bu.edu/trf.html) (Benson, 1999).

Thirty eight microsatellite loci

Results and discussion

The thirty eight primer pairs were tested by nested PCR reaction under different annealing temperatures (data not shown). Most of the evaluated loci produced no amplicon. Seven loci were successfully amplified and genotyped. Table S1 shows number of alleles found in each loci and the number of individuals sampled per population. Not all samples amplified at all loci, most probably due to the presence of polymorphisms at the primer annealing sites. Indeed some sequence difference is expected

Acknowledgements

This work received financial support from CNPq (Process number 470968/2014-1) and FAPEMIG (Process number APQ-02417-16).

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