Immunological and DNA-based identification of the screwworm, Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae).

摘要

The screwworm, Cochliomyia hominivorax (Coquerel), is an obligate parasite of warm-blooded animals and one of the most important arthropod pests of animals in the Western Hemisphere. Immature stages and males of screwworms are difficult to identify morphologically from several closely-related blow fly species. The enzyme-linked immunosorbent assay (ELISA) and random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) methods were used to identify the screwworm from other myiasigenic flies.; A monoclonal antibody-based enzyme-linked immunosorbent assay (MAb-ELISA) specific for the screwworm was developed which rapidly and accurately distinguished all stages of the screwworm from non-screwworm species. Both microtiter plate assays and nitrocellulose blots were evaluated and results showed that they can positively identify screwworm samples. A simplified, field-usable dot-ELISA procedure, utilizing nitrocellulose strips and microcentrifuge tubes, was designed that allows a visual on-the-spot diagnosis of screwworm specimens in about two hours. Blind test results showed that dot-ELISA accurately and consistently discriminates all life stages of screwworm better than the conventional microplate ELISA. The accuracy of the dot-ELISA test depends on sample preservation methods and storage conditions of assay reagents: a 100% sensitivity, specificity and overall accuracy were achieved in dot-ELISA blind tests using frozen samples and freshly-prepared assay reagents. This rapid, simple, and inexpensive method can be used as a field identification kit for population monitoring, quarantine, and eradication efforts against the screwworm.; RAPD-PCR was used for inter- and intraspecific discrimination of screwworm populations. Nine out of 40 decameric primers screened were found suitable for distinguishing all life stages (interspecific) of screwworms based on clear and reproducible RAPD profiles against other wound-inhabiting flies including C. macellaria (Fabr.), Phormia regina (Meigen), Phaenicia sericata (Meigen), Calliphora vicina Robineau-Desvoidy, Chrysomya rufifacies (Macquart), Sarcophaga sp., and Musca domestica L. Unidentified first instars collected from Nicaragua in 1998 were tested by this method, and results agreed with morphological identification that the samples were not screwworms. DNA polymorphisms produced by three other primers (intraspecific) were observed from screwworm populations originating from Mexico, Costa Rica, Panama, Jamaica, and Brazil such that it may be possible to determine the geographic origin of the sample by RAPD-PCR.