Application of a species-specific PCR-RFLP to identify Ancylostoma eggs directly from canine faeces

Abstract

Apart from their veterinary importance, the hookworms Ancylostoma caninum, Ancylostoma braziliense and Ancylostoma caninum are also capable of causing zoonotic disease in humans. A highly sensitive and species-specific PCR-RFLP technique was utilised to detect and differentiate the various canine Ancylostoma spp directly from eggs in faeces. This technique was utilised to screen 101 canine faecal samples from parasite endemic tea growing communities in Assam, India, as part as an ongoing epidemiological investigation into canine parasitic zoonoses. The prevalence of hookworms in dogs was found to be 98% using a combination of PCR and conventional microscopy. Overall, 36% of dogs were found positive for single hookworm infections with A. caninum, 24% positive for single infections with A. braziliense and 38% had mixed infections with both A. caninum and A. braziliense. No dogs were found positive for A. ceylanicum in the community under study. The high prevalence of A. caninum and A. braziliense in dogs in this community may account for the high incidence of cutaneous larva migrans (CLM) observed among the human population residing at the tea estates. The PCR-RFLP technique described herein allows epidemiological screening of canine hookworms to be conducted rapidly, with ease and accuracy, and has the potential to be applied to a number of different clinical, pharmacological and epidemiological situations.

Introduction

Ancylostoma caninum, Ancylostoma braziliense and Ancylostoma ceylanicum are ubiquitous hookworms found in dogs, cats and other mammals in tropical climates, where conditions are conducive for hookworm survival. The principal veterinary importance of these hookworms arises from their ability to suck blood in their primary host. From a morbidity standpoint, the most important difference between the three species is that A. caninum causes far greater blood loss (∼1–2 ml/day per worm) (Georgi et al., 1969) than A. ceylanicum (∼0.033 ml/day per worm) (Areekul et al., 1975) or A. braziliense (∼0.001 ml/day per worm) (Miller, 1966) as demonstrated by clinical studies using radioactive chromium labeling of erythrocytes. Uncinaria stenocephala is another genus of hookworm found in dogs, however, its distribution is limited to cooler regions such as those found in Europe (Smith et al., 2003, Gorski et al., 1996) and Australia (Beveridge, 2002). In puppies and immunosuppressed hosts even light to moderate infections with A. caninum can result in moderate to severe anaemia, hypoproteinemia and bloody diarrhoea and may result in per acute or acute fatalities (Georgi and Georgi, 1989). In adult animals, a more chronic and sub clinical disease manifests (Reinemeyer, 1995). The principal public health importance of these hookworms however, is their ability to cause zoonotic diseases in man.

Cutaneous larva migrans (CLM) or “creeping eruptions” is a hypersensitivity reaction caused by migrating nematode larvae, of which Ancylostoma braziliense is the most frequently implicated aetiological agent in humans (Chaudhry and Longworth, 1989, Malgor et al., 1996). CLM is commonly reported in travelers returning from the tropics (Bouchaud et al., 2001, Jelinek et al., 1994, Tremblay et al., 2000) and in areas endemic for canine hookworm infection (Malgor et al., 1996, Taranto et al., 2000).

Recent studies in Australia have implicated A. caninum to be the leading cause of human eosinophilic enteritis (EE) (Prociv and Croese, 1990). An outbreak totaling 150 cases of EE between 1988 and 1992 was reported and the dog hookworm A. caninum was convincingly implicated as the causal agent in a number of cases via biopsy, laparotomy or colonoscopy (Croese et al., 1994b, Loukas et al., 1992). Prior to this, sporadic accounts of A. caninum occurring in the intestine of humans were also reported from the Philippines, South America and Israel (Croese et al., 1994a). A. caninum implicated EE syndromes have also been recognised in the US (Khoshoo et al., 1995, Khoshoo et al., 1994) and Egypt (Bahgat et al., 1999). The widespread nature of A. caninum and obscure clinical presentation makes it plausible that the condition may be more widespread than reported but misdiagnosed.

A. ceylanicum has been shown to produce natural and experimental patent infection in humans. Natural infections with A. ceylanicum have been reported in Dutch servicemen returning from West New Guinea, who suffered heavy infections with concurrent anaemia (Anten and Zuidema, 1964), and mostly light infections have been reported from humans in the Philippines (Velasquez and Cabrera, 1968), Taiwan (Yoshida et al., 1968) Thailand (Areekul et al., 1970) and India (Chowdhury and Schad, 1972). Recently, A. ceylanicum infections in dogs and cats have been shown to be endemic, especially in the South East Asian region (Choo et al., 2000, Setasuban et al., 1976, Yoshida et al., 1968, Yoshida et al., 1973) and Australia (Beveridge, 2002). However, there have been no reports of natural human infections for at least 30 years. Since morphological differentiation of Ancylostoma eggs in faeces is impossible, it is probable that recent studies have not aimed to identify the species of hookworm and assumed the species found in human surveys to be A. duodenale. Current methods of identification of hookworm species in dogs requires coprological or post-mortem examinations of the adult worms, based on morphological differences between the species of Ancylostoma (Soulsby, 1982, Yoshida et al., 1971). This is very time consuming, labor intensive, requires skilled personnel and also risks the possibility of overlooking mixed infections, especially if the burden of one species of hookworm is low compared to the other species. Molecular based techniques for rapid and specific diagnosis of developmental stages of human hookworms have been developed (Hawdon, 1996, Monti et al., 1998, Verweij et al., 2001, Zhan et al., 2001). Similar use of highly sensitive and species-specific molecular tools to identify hookworms of dogs will not only aid veterinary diagnosis, but also allow epidemiological screening to be conducted with ease in order to assess the public health risks these parasites pose in a community.

This study was carried out as part of an ongoing epidemiological investigation aimed at studying the prevalence and risk factors associated with canine gastrointestinal parasitic zoonoses in tea growing communities in northeast India (Traub et al., 2002). Ninety-three percent of dogs were found to harbor hookworm eggs by conventional parasitological techniques such as feacal floatation and microscopy. The high prevalence of hookworms among dogs coupled with widespread feacal contamination was hypothesised to account for the high incidence of “creeping eruptions” observed among the human population in this community. In this paper we describe the application of a highly sensitive and species-specific PCR-RFLP tool to screen 101 dog feacal samples for Ancylostoma spp. The internal transcribed spacer (ITS)-1, 5.8S and ITS-2 regions were utilised as genetic markers to determine the prevalence of various Ancylostoma spp of dogs directly from eggs in faeces. Ancylostoma caninum (Sahasrabudhe et al., 1969) and A. ceylanicum (Chowdhury and Schad, 1972) have been previously identified in dogs in India, however there have been no reports of the presence of A. braziliense.