Application of a species-specific PCR-RFLP to identify Ancylostoma eggs directly from canine faeces
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.
Section snippets
Study area and design
The study area, design have already been described in detail elsewhere (Traub et al., 2002).
Molecular methods
One hundred and one dog feacal samples, regardless of microscopic results, were subjected to molecular screening using PCR-RFLP in order to assess the sensitivity of the molecular technique in comparison to conventional microscopy techniques.
Hookworm controls
Ancylostoma caninum adult worms and eggs were obtained from an 8-week old puppy that presented with hemorrhagic diarrhoea at a local veterinary hospital in Perth.
Phylogenetic analysis of unidentified hookworm sequence
Clear and readable sequences were obtained for the PCR products RTGHF1-RTGHR1 of D6, D9, D10, D12 and the A. ceylanicum and A. caninum controls. PCR derived sequences of D10, D12 and the adult A. caninum control were found to be 100% homologous to previously published sequences of A. caninum (GenBank accession numbers Y19181, Z70739, AJ001592) and with each other. The PCR derived sequence for the adult and eggs of the A. ceylanicum controls were found to be 100% homologous to previously
Discussion
The focus of this study was to develop a tool to rapidly and efficiently identify the zoonotic species of canine hookworms A. caninum, A. ceylanicum and A. braziliense directly from eggs in faeces, in order to carry out diagnostic and epidemiological screening with ease and accuracy. The PCR-RFLP technique described herein using highly specific and sensitive primers capable of selectively detecting and differentiating the species of Ancylostoma directly from feacal samples was successfully
Conclusion
In conclusion, development of this highly species-specific and sensitive PCR-RFLP technique to detect and differentiate canine Ancylostoma spp directly from eggs in faeces has allowed epidemiological screening 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.
Acknowledgements
We thank Williamson Magor & Co. for permission to conduct research at the tea estates owned by them, and the managerial and medical staff at the Phulbari and Addabarie Tea Estates for their help and cooperation during sample collection. Bayer Healthcare AG, Animal Health Division, Leverkusen, Germany, for providing financial support for this project.
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