Detection and Isolation of Exotic Newcastle Disease Virus from Field-Collected Flies

Chakrabarti, Seemanti; King, Daniel J.; Afonso, Claudio; Swayne, David; Cardona, Carol J.; Kuney, Douglas R.; Gerry, Alec C.

Flies were kept frozen on dry ice during identiÞcation in a biological safety cabinet, and then they were pooled into groups of Þve or fewer ßies of the same species from the same home. Fly pools were stored at Ϫ80ЊC until shipment on dry ice to the USDA Southeast Poultry Research Laboratory (SEPRL), in Athens, GA, where they were again placed at Ϫ80ЊC until tested for the presence of ENDV. Pools were homogenized in 1.5 ml of brain-heart infusion (BHI) broth with antibiotics (200 g of gentamicin/ml, 2000 U of penicillin/ml, and 4 g of amphotericin B/ml, Sigma-Aldrich, St. Louis, MO) by using a tissue grinder with sterile plastic pestles in microfuge tubes and centrifuged at 16,000 ϫ g for 10 min. Virus isolation was performed by inoculating 100 l of the supernatant into the allantois of each of three 9-or 10-d-old embryonated chicken eggs (ECE). Eggs were incubated at 37ЊC in a standard humidiÞed incubator. The embryonated chicken eggs were obtained from the SEPRL speciÞc-pathogen-free White Leghorn ßock. Eggs were candled to determine embryo death each 24 h through 7 d postinoculation. Embryos that died within the Þrst 24 h were discarded. Embryos that died between 24 h and 7 d as well as all survivors at 7 d were chilled at 4ЊC. Amnio-allantoic ßuid (AAF) harvested from chilled eggs was tested for hemagglutination (HA) activity to detect ENDV. Virus presence in HApositive samples was conÞrmed by hemagglutinationinhibition (HI) with Newcastle disease virus (family Paramyxoviridae, genus Avulavirus, NDV)-speciÞc antiserum (King 1996a) . Amnio-allantoic ßuids from HA negative dead embryos and embryos alive at 7 d postinoculation were subjected to a second serial pas-sage by inoculation of 100 l of the AAF into each of three additional embryonated chicken eggs. Eggs were candled, and killed embryos were handled as before. If by day 7 postinoculation there was no HA activity in the AAF of the second passage dead or surviving embryos, the specimen was regarded as negative for ENDV.