Detection of Fasciola gigantica infection in snails by polymerase chain reaction
Introduction
Fasciolosis is an economically important helminth disease of livestock having a significant impact on growth, development and productivity in domestic ruminants (Dargie, 1987, Fabiyi, 1987). Two species, Fasciola hepatica and Fasciola gigantica are responsible for causing the disease in ruminants worldwide. In India, fasciolosis in domestic ruminants is mainly caused by F. gigantica, however, the prevalence of F. hepatica has been reported from Kashmir valley, a temperate agroclimatic region in India (Dhar et al., 1988, Sharma et al., 1989). Lymnaea auricularia, an aquatic snail is the intermediate host of F. gigantica in India. The development of a reliable and sensitive method for parasite diagnosis and characterisation is necessary for proper understanding of the epidemiology. Such understanding is an important requirement in designing rational Fasciola control programs. Recently, a number of sensitive and specific techniques have been applied in the investigations of Fasciola epidemiology, however, all these methods have their limitations when applied to studies on Fasciola population dynamics.
As the conventional techniques used for the detection of intramolluscan stages of Fasciola species, suffer on account of sensitivity and specificity, the development of a highly sensitive and specific method for detecting infected snails would greatly aid in the epizootiological studies on F. gigantica or F. hepatica, by enabling the generation of accurate data on infection rates in field populations of snails. In this context, development of DNA probe assay with its high level of sensitivity and specificity for Fasciola infected snails (Kaplan et al., 1995, Kaplan et al., 1997, Kramer and Schnieder, 1998) has been a forward step in this direction. It was the first field use of a nucleic acid based technique for determining the prevalence of F. hepatica infection in its snail intermediate host. The development of this probe by these authors was based on the identification of a 124 bp non-coding repetitive and highly abundant DNA sequence, representing about 15% of the total F. hepatica genome (Kaplan et al., 1995). Earlier also, DNA probes using highly repetitive sequences have been developed for identification of several different parasite species (Shah et al., 1987, Sirisinha et al., 1991).
Taking this background research information into consideration, polymerase chain reaction (PCR) assay was designed to detect in situ intramolluscan stages of F. gigantica. This PCR assay was designed because of high attributes of simplicity, sensitivity and specificity of this technique.
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Isolation of DNA from snails and F. gigantica parasites
The protocol for isolation of genomic DNA described by Kaplan et al., 1995, Kaplan et al., 1997 was followed with certain modifications. F. gigantica infected L. auricularia were thoroughly washed in PBS and individually crushed in 400 μl lysis buffer (8% Triton X 100, 250 mM sucrose, 50 mM TE pH 8.0) and frozen in liquid nitrogen. Snail lysate were thawed, 20 μl of freshly prepared proteinase K (10 mg ml−1) was added and samples were incubated in a water bath at 68 °C for 2 h. Following incubation,
Polymerase chain reaction
Fasciola specific primers amplified a 124 bp fragment in PCR when the genomic DNA isolated from F. gigantica infected snail was used as template. In addition to the 124 bp amplicon, a ladder of DNA fragments (ranging in size from 248, 372, 496 and 620 bp) representing amplification of 124 bp repetitive sequences was observed (Fig. 1). Certain amplifications would reveal only the 124 bp fragment (Fig. 2). Genomic DNA of the parasite was used as a positive control, which also gave an amplification of
Discussion
For detecting F. gigantica or F. hepatica infection in snails, conventionally the snails are screened for cercarial shedding or teased for the microscopic examination of the developmental stages of the parasite. This process is tedious, expensive and lacks specificity, as earlier intramolluscan developmental stages of the parasite cannot be specifically identified. Therefore, there is a role of PCR for the specific and early detection of infection in snails. PCR is a highly sensitive and
Acknowledgements
The author is grateful to the Indian Council of Agricultural Research for financial support in the form of a Senior Research Fellowship. Research facilities arranged through NATP (MM) by Principal Investigator and Mission leader are thankfully acknowledged.
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