Sandra S. CloudA
Brian S. LadmanA
Mariano SalemB
John K. RosenbergerA
A Department of Animal and Food Sciences, College of Agriculture and Natural Resources, University of Delaware
B Lasher Laboratory, Georgetown, Delaware, U.S.A.
Introduction and the "High Mortality" concept for Avian Influenza surveillance
Virus isolation attempts conducted during Delmarva's 2004 avian influenza (AI) outbreak yielded the recovery of low pathogenicity (path) H7N2 from only three premises, the Viva and Hobo Farms in Delaware and the Minh Ma Farm in Maryland. However, unprecedented AI surveillance efforts encompassing the entire Delmarva commercial poultry production region resulted in 361 isolations of avian viruses from a total of 922 attempts. The vast majority of the attempts (94%) were from commercial flocks, although backyard poultry and dead wild birds in the region were also sampled.
As part of the AI surveillance program, the Emergency Disease Task Force implemented a "High Mortality" sampling program for flocks three weeks and older with an established daily rate of mortality. At the outset of the outbreak (February 6-March 11), the criterion for sampling was a mortality rate of greater than or equal to four mortalities per thousand in the flock per day. From March 12- April 14, the threshold for sampling was dropped to three mortalities per thousand in the flock per day.
Methods for sampling and virus isolation
Sampling personnel received specific training to maximize virus recovery as well as to prevent accidental viral cross-contamination of samples and potential transmission of disease agents to other flocks.
Tracheal swabbings were collected from ten "fresh" mortalities (within the last day) or recently euthanized broilers with clinical signs. Swabbings were taken using culturettes (Fisherbrand™ transport swab with liquid Stuart's media). The culturettes were placed in a cooler with frozen cold packs and transported to the Lasher Laboratory.
At the Lasher Laboratory, swabbings were pooled into two pools of five birds per tube. Each tube contained 2 ml of sterile 0.85% saline. The last processed swab of each pool of five was placed into 2 ml tryptose phosphate broth (TPB) containing antibiotics and vortexed. Using the saline samples, AIV was identified by real time RT-PCR (matrix and HA-gene-specific primers and probes) (NVSL Protocol AVPRO1510.01) and antigen-capture (Directigen™ Flu A) testing.
The USDA National Veterinary Service Laboratory, in Ames, Iowa, provided confirmation of the AI virus isolations. The TPB samples were transported to Newark for performing virus isolation attempts.
Virus isolation attempts for respiratory viruses were conducted in 9-11-day-old specific-pathogen-free (SPF) chicken embryos inoculated via the chorioallantoic sac route. Prior to inoculation, the TPB swab media was filtered to remove bacteria and fungi. The inoculated embryos were candled daily and incubated for seven days. Up to three embryo passages were made before a sample was considered negative for virus isolation. The allantoic fluids from all living and dead eggs were checked for hemagglutination (HA) of chicken erythrocytes. HA positive fluids were tested with Newcastle disease virus specific antibody by hemagglutination-inhibition (HI). Fluids that were NDV HI positive were re-checked for AIV by real time RT-PCR to verify that they were indeed AIV negative.
HA negative virus samples were identified as infectious bronchitis virus (IBV) by RT-PCR (Kingham et al., 2000) or avian adenovirus PCR (Meulemans et al., 2001). Viruses listed as unknown (UNK) are still under evaluation. These may include reoviruses, infectious bursal disease virus, or infectious laryngotracheitis virus.
References
Kingham, B. F., C. L. Keeler, Jr., W. A. Nix, B. S. Ladman, and J. Gelb, Jr. Identification of avian infectious bronchitis virus by direct automated cycle sequencing of the S-1 gene. Avian Diseases. 44:325-335. 2000.
Meulemans, G., M. Boschmans, T. P. van den Berg, and M. Decaesstecker. Polymerase chain reaction combined with restriction enzyme analysis for detection and differentiation of fowl adenoviruses. Avian Pathology 30:655-660. 2001.
From Proceedings of the 39th National Meeting on Poultry Health and Processing, Ocean City, Maryland.
