Genotyping of Giardia duodenalis from human and animal samples from Brazil using β-giardin gene: A phylogenetic analysis

Abstract

Giardia duodenalis is one of the major diarrhea agents in human and animals distributed worldwide, and present high levels of genetic diversity, showing seven genotypes: A, B, C, D, E, F, and G. Only Assemblages A and B have been detected in humans and in a wide range of other mammalians hosts, whereas the remaining Assemblages (C–G) are host-specific. Molecular characterization of cysts of human and animal origin are useful to address the co-circulate isolates between these host, and represents an objective means to evaluate zoonotic infection hypothesis. In the present work the G. duodenalis genotypes were characterized by restriction fragment length polymorphisms and DNA sequencing analysis of PCR products of the β-giardin gene. The cysts were collected in the city of Rio de Janeiro, in Brazil, from a population composed by humans (n = 366, 310 children and 56 adults), domestic animals (n = 11) from a municipal daycare center in the surroundings of a slum and neighborhood medium–high class domestic animals (n = 18). Parasitological exams were developed in human fecal samples. Parasites were found in 60% (186/310) and 66% (37/56) of the samples from children and adults, respectively. Among children's samples, 27.7% (86/310) were positive for G. duodenalis. Only 1.7% (1/56) of the adults was positive for this parasite. In general a total of 87 fecal samples (86 from children and 1 from adult) from all population studied were positive for G. duodenalis, and 62 of these were subjected to molecular analysis using a PCR that amplified a fragment of the β-giardin gene. Sixty samples were typed as genotype A1, two as genotype A2 and genotype B was not encountered. Among domestic animals samples (n = 29), eight (seven dogs and one cat) from the slum community were identified as genotype A1, and all control samples (n = 18) were negative in the molecular assay. The host-specific genotypes C, D and, F were not found. In this study we described single case of G. duodenalis infection associated with a child and her dog and both isolates characterized as genotype A1. Despite the low incidence, this data suggest the putative existence of a zoonotic cycle of G. duodenalis in the studied population.

Introduction

Giardia is a flagellate protozoan and an intestinal parasite that can infect many species in the animal kingdom including mammalian, avian and reptilian (Thompson, 2002, Appelbee et al., 2005). Of the Giardia species only Giardia duodenalis (syn. Giardia lamblia, Giardia intestinalis) is recovered from mammalians including humans and domesticated animals as dogs and cats (Adam, 2001).

G. duodenalis is the most common intestinal protozoan found worldwide. This parasitic infection has a major clinical impact on children that are 5 years older or younger. G. duodenalis are shed in feces as cysts and fecal-oral represents the main route of infection. Contaminated water, food and fomites are generally effective vehicles to transmit the infection. A great proportion of cases are asymptomatic, but symptomatic giardiasis, which may involve vomiting and recurrent intestinal symptoms, may impact in the normal growth and development of affected children (Adam, 1991, Ortega and Adam, 1997).

The prevalence of this infection varies between 2 and 5% in industrialized countries and up to 20–30% in less developed countries counting with 200 million symptomatic cases in Asia, Africa, and Latin America with an incidence of 500,000 new cases per year (CDC, 1995). However, it can be as high as 35% among children attending in daycare centers in the United States and elsewhere even in non-outbreak settings (Ish-Horowicz et al., 1989, Marshall et al., 1997, Ortega and Adam, 1997, Bilenko et al., 2004). It is estimated that 50% of infectious diarrhea disease occurring in children attending daycare affects children younger than 3 years old. G. duodenalis is one the most common recognized agents of intestinal infections in this setting along with rotavirus and Cryptosporidium spp. (Thompson, 1994, CDC, 1995, WHO, 1996, Ferson et al., 1997, Hellard and Fairley, 1997, Thompson, 2000).

The molecular typing methods of G. duodenalis cysts directly in stool samples have shown that there is a considerable diversity within this protozoan population, and that G. duodenalis should be considered as a species complex, whose members, albeit morphologically identical, can be assigned to at least seven distinct genotypes (A–G) (Monis et al., 2003, Eligio-Garcia et al., 2005). The isolates from humans and from a wide variety of mammals belong to two major genetic groups named genotypes A and B (Adam, 2001). Genotypes C and D include dog isolates, a genotype E is related to livestock animals and genotypes F and G cluster isolates from cats and rats, respectively (Ey et al., 1997, Hopkins et al., 1997, Monis et al., 1998). The β-giardin gene is an appropriate target to genotyping of G. duodenalis isolates, being able to define at least eight genotypes within genotype A and at least six genotypes within genotype B, as identified by Lalle et al., 2005a, Lalle et al., 2005b.

The control of human giardiasis may be hampered if the zoonotic cycle of transmission persists especially in domestic animals that have close contact with their owners. Dogs has a potential public health risk, since it can harbor isolates of genotypes A, B, C or D. Genotypes or Assemblages A and B are associated with human infection. Finding such genotypes in human does not provide robust evidence to associate the infection with a possible zoonotic source. For that, it is required that adequate molecular techniques be applied to characterize the genotypes that circulate among the two distinct classes of hosts.

In this study, G. duodenalis isolates from humans and domestic animals (pets) were genetically characterized at the β-giardin locus in order to investigate the prevalence of genotypes and to accrue data on occurrence, epidemiology, and potential for zoonotic transmission of Giardia infection in Brazil.