Specific detection of the Old World screwworm fly, Chrysomya bezziana, in bulk fly trap catches using real-time PCR.

The Old World screwworm fly (OWS), Chrysomya bezziana Villeneuve (Diptera: Calliphoridae), is a myiasis-causing blowfly of major concern for both animals and humans. Surveillance traps are used in several countries for early detection of incursions and to monitor control strategies. Examination of surveillance trap catches is time-consuming and is complicated by the presence of morphologically similar flies that are difficult to differentiate from Ch. bezziana, especially when the condition of specimens is poor. A molecular-based method to confirm or refute the presence of Ch. bezziana in trap catches would greatly simplify monitoring programmes. A species-specific real-time polymerase chain reaction (PCR) assay was designed to target the ribosomal DNA internal transcribed spacer 1 (rDNA ITS1) of Ch. bezziana. The assay uses both species-specific primers and an OWS-specific Taqman MGB probe. Specificity was confirmed against morphologically similar and related Chrysomya and Cochliomyia species. An optimal extraction protocol was developed to process trap catches of up to 1000 flies and the assay is sensitive enough to detect one Ch. bezziana in a sample of 1000 non-target species. Blind testing of 29 trap catches from Australia and Malaysia detected Ch. bezziana with 100% accuracy. The probability of detecting OWS in a trap catch of 50 000 flies when the OWS population prevalence is low (one in 1000 flies) is 63.6% for one extraction. For three extractions (3000 flies), the probability of detection increases to 95.5%. The real-time PCR assay, used in conjunction with morphology, will greatly increase screening capabilities in surveillance areas where OWS prevalence is low.

Cattle herd inspections and fly trapping for the detection of the Old World screw-worm fly (Chrysomya bezziana)

ObjectivesTo compare the sensitivity of inspections of cattle herds and adult fly trapping for detection of the Old World screw-worm fly (OWS). ProceduresThe incidence of myiases on animals and the number of OWS trapped with LuciTrap (R)/Bezzilure were measured concurrently on cattle farms on Sumba Island (Indonesia) and in peninsular Malaysia (two separate periods for the latter). The numbers of animal inspections and traps required to achieve OWS detection at the prevalent fly densities were calculated. ResultsOn Sumba Island, with low-density OWS populations, the sensitivity of herd inspections and of trapping for OWS detection was 0.30 and 0.85, respectively. For 95% confidence of detecting OWS, either 45 inspections of 74 animals or trapping with 5 sets of 4 LuciTraps for 14 days are required. In Malaysia, at higher OWS density, herd inspections of 600 animals (twice weekly, period 1) or 1600 animals (weekly, period 2) always detected myiases (sensitivity = 1), while trapping had sensitivities of 0.89 and 0.64 during periods 1 and 2, respectively. For OWS detection with 95% confidence, fewer than 600 and 1600 animals or 2 and 6 LuciTraps are required in periods 1 and 2, respectively. ConclusionsInspections of cattle herds and trapping with LuciTrap and Bezzilure can detect OWS populations. As a preliminary guide for OWS detection in Australia, the numbers of animals and traps derived from the Sumba Island trial should be used because the prevailing conditions better match those of northern Australia.

Composition of chemical attractants affects trap catches of the Australian sheep blowfly, lucilia cuprina, and other blowflies

Numbers of Lucilia cuprina (Australian sheep blowfly), Chrysomya spp., and Calliphora spp. blowflies caught on sticky traps baited with various synthetic attractants or a standard liver/sodium sulfide attractant in western Queensland were recorded. Numbers of each genus collected were influenced by the composition of the chemical attractants. Attractant mixtures based on 2-mercaptoethanol, indole, butanoic/pentanoic acid, and a sodium sulfide solution gave 5- to 20-fold higher L. cuprina catches than the liver standard. These blends attracted similar numbers of Chrysomya spp. (0.85–2.7× ) and fewer Calliphora spp. (0.02–0.2× ) compared to the liver standard. These synthetic attractants were more effective and selective for L. cuprina than the standard liver/sodium sulfide attractant, and they can be packaged in controlled-release dispensers to generate constant, prolonged release of the attractant.

Suppression of populations of Australian sheep blowfly, Lucilia cuprina (Wiedemann) (Diptera: Calliphoridae), with a novel blowfly trap

The Australian sheep blowfly, Lucilia cuprina initiates more than 85% of fly strikes on sheep in Australia with an estimated average annual cost of A$280 million to the Australian wool industry. LuciTrap® is a commercially available, selective trap for L. cuprina consisting of a plastic bucket with multiple fly entry cones and a synthetic attractant. The impact of LuciTrap on populations of L. cuprina on sheep properties in five Australian states was evaluated by comparing L. cuprina populations on paired properties with and without LuciTraps over seasons when significant fly populations could be expected. Twenty-four comparisons (trials) were conducted over four years. During times of ‘higher fly density’ (when the 48 h geometric mean of trap catches on the control property was greater than five L. cuprina), the overall geometric mean trap catches for control and trapped properties differed significantly (P<0.001) with mean trap catches of 19.4 and 7.74 L. cuprina respectively. The selectivity of the LuciTrap was confirmed with 59% of all trapped flies being L. cuprina. Chrysomya spp. and Calliphora spp. constituted 9.3% and 1.1% of the catches with a variety of other flies (mainly Sarcophagidae and Muscidae) providing the remainder (31%). L. sericata was only trapped in Tasmania and made up 7.7% of the Lucilia spp. catch in this State. Seventy-two percent of the trapped L. cuprina were female. The deployment of LuciTrap on sheep properties at one trap per 100 sheep from the beginning of the anticipated fly season suppressed the populations of L. cuprina by 60% compared to matched control properties. The LuciTrap is a selective and easy to use fly trap and constitutes an effective, non-insecticidal tool for use in integrated management programs for L. cuprina.

An improved real-time PCR assay for the detection of Old World screwworm flies

The Old World screwworm (OWS) fly, Chrysomya bezziana, is a serious pest of livestock, wildlife and humans in tropical Africa, parts of the Middle East, the Indian subcontinent, south-east Asia and Papua New Guinea. Although to date Australia remains free of OWS flies, an incursion would have serious economic and animal welfare implications. For these reasons Australia has an OWS fly preparedness plan including OWS fly surveillance with fly traps. The recent development of an improved OWS fly trap and synthetic attractant and a specific and sensitive real-time PCR molecular assay for the detection of OWS flies in trap catches has improved Australia's OWS fly surveillance capabilities. Because all Australian trap samples gave negative results in the PCR assay, it was deemed necessary to include a positive control mechanism to ensure that fly DNA was being successfully extracted and amplified and to guard against false negative results. A new non-competitive internal amplification control (IAC) has been developed that can be used in conjunction with the OWS fly PCR assay in a multiplexed single-tube reaction. The multiplexed assay provides an indicator of the performance of DNA extraction and amplification without greatly increasing labour or reagent costs. The fly IAC targets a region of the ribosomal 16S mitochondrial DNA which is conserved across at least six genera of commonly trapped flies. Compared to the OWS fly assay alone, the multiplexed OWS fly and fly IAC assay displayed no loss in sensitivity or specificity for OWS fly detection. The multiplexed OWS fly and fly IAC assay provides greater confidence for trap catch samples returning negative OWS fly results. © 2014 International Atomic Energy Agency.

Chemical containment and eradication of screw− worm incursions in Australia

Screwworms are obligate, invasive parasites of warm-blooded animals. The female flies lay batches of eggs at the edge of wounds or other lesions. These eggs hatch to larvae or screw-worms which feed on affected animals for 6-7 days, burrowing deeply into subcutaneous tissues and causing severe trauma to animals, production loss and potentially death. Susceptible sites include wounds resulting from management practices such as castration, de-horning and ear tagging and lesions caused by the activities of other parasites such as buffalo flies and ticks. The navels of the new born and the vulval region of their mothers following parturition are highly susceptible and body orifices such as nose and ears are also frequent targets for ovipositing screwworm flies. The Old World screw-worm, Chrysomya bezziana (OWS) is considered one of the most serious exotic insect pest threatening Australia's livestock industries and is endemic in a number of our closest neighbouring countries. New World screwworm (NWS), Cochliomyia hominivorax, endemic to South America, has also entered Australia on at least 2 occasions. Many tropical and subtropical areas of Australia are suitable for the establishment of OWS and the potential range is expected to increase with climate change. The Australian screwworm preparedness strategy indicates a program of containment with chemical treatments followed by eradication of OWS using sterile male release and parasiticides. However, there is no longer an operational OWS sterile insect screw-worm facility anywhere in the world and establishing a large scale production facility would most optimistically take at least 2 years. In the interim, containment would be almost totally dependent on the availability of effective chemical controls. A review of chemical formulations available for potential use against OWS in Australia found that currently only one chemical, ivermectin administered by subcutaneous injection (s.c.) is registered for use against OWS and that many of the chemicals previously shown to be effective against OWS were no longer registered for animal use in Australia.18 From this review a number of Australian-registered chemicals were recommended as a priority for testing against OWS. The Australian Pesticides and Veterinary Medicines Authority (APVMA) can issue an emergency use permit for use of pesticides if they are registered in Australia for other animal uses and shown to be effective against OWS. This project tested the therapeutic and prophylactic efficacy of chemicals with potential for use in the treatment and control of OWS.