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Project description

Evaluation of the role of arthropods in the persistence and dispersal of exotic Newcastle disease (END) in southern California. (03XA006)
Program Exotic Pests and Diseases Research Program
Principal
investigators
A.C. Gerry, Entomology, UC Riverside
C.J. Cardona, Veterinary Medicine, UC Davis
Host/habitat Poultry
Pest Exotic Newcastle Disease
Disciplines Animal Sciences, Entomology
Review
panel
Agricultural Systems
Start year (duration)  2003 (Three Years)
Objectives Identify arthropods associated with poultry that are positive for Exotic Newcastle disease (END) virus and thus, may be important in the persistence and dispersal of the virus.

Characterize the persistence and site of harborage of END virus in association with arthropod vectors.

Project
Summary
Arthropods will be collected from southern California backyard and commercial poultry operations that have been recently quarantined due to the presence of exotic Newcastle disease (END) virus. Fly species such as the house fly (Musca domestica) and little house fly (Fannia canicularis) will be collected via sterile sweep net while manure-inhabiting beetles such as the lesser mealworm (Alphitobius diaperinus) will be collected using tube or pan traps. Other arthropods associated with commercial poultry operations will be collected where available in large numbers. Field collected arthropods will be sorted, identified to species, placed in pools of 50 individuals and tested by real-time RT-PCR to determine the presence and quantity of END virus RNA. Pools with positive RT-PCR results will be injected into embryonating chicken eggs for virus isolation to confirm the presence of infectious virus. House fly, little house fly, lesser mealworm, and other arthropod species from which END virus can be isolated will be colonized in the laboratory for further studies of END virus persistence. Laboratory reared arthropods will be placed into containers with a food source containing infectious END virus. Each day through day 15 post-infection for flies and day 30 post-infection for beetles, 10 arthropods will be removed from the treatment container to be tested for the presence and quantity of END virus. Half of these individual arthropods will be surface-sterilized with five percent sodium hypochlorite prior to virus isolation to determine if END virus is solely an external contaminant on the body of the arthropod. Arthropod species found to have END virus RNA present internally will be fed an infective meal, held for one to five days, and carefully dissected to separate the gut from the remainder of the arthropod body. The arthropod gut and the remainder of the body will be kept separate and will be tested by RT-PCR to determine the presence and quantity of END virus RNA. The presence of viral RNA in the insect body may indicate a disseminated infection and classification of the arthropod species as a biological vector of END virus.
Final report Sampling of fly populations was conducted at several backyard poultry operations containing birds that were infected with END virus. Flies were examined for the presence and quantity of END virus. Three species of flies were found to be infected with END in the field (Musca domestica, Fanniacanicularis, and Phaenicia cuprina. However, viral concentration associated with these flies was low.

Laboratory colonies of house fly (Musca domestica) and little house fly (Fannia canicularis) and litter beetle (Alphitobius diaperinus) were infected with END virus by forced contact with virus or choice feeding on a food/virus mixture (virus concentration similar to that found in manure from infected chicken). Insects were then held under constant laboratory conditions for up to seven days with a sub-sample of the potentially infected insects removed each day from day one to seven. Insects were either dissected to remove the gut from the remainder of the body or left intact; after which insects were pooled into groups of five insects, homogenized, and a 100 microliter aliquot was injected into embryonated chicken eggs. Flies were shown to harbor END virus for up to nine days (Fannia) or five days (Musca). Beetles were not successfully infected during these studies.

The majority of the virus was located in the gut of flies infected after choice feeding on a food/virus mixture suggesting that contamination of the external fly structures may be minimal in a field setting. Flies harbored enough virus for the first 24 to 48 hours to infect a chicken that might have consumed the fly.

Third-year
progress
Sampling of fly populations at several backyard poultry operations containing birds that were infected with END virus was conducted during the END outbreak in spring 2003. Six pools (n=5) of flies collected from these backyard poultry operations were found to harbor END virus. Positive pools included two pools of Musca domestica, one pool of Fannia canicularis, and three pools of Phaenicia cuprina. Positive flies were collected from two separate backyard flocks. Viral titers were relatively low, requiring three passages in embryonating chicken eggs to provide positive results. END is known to cause infection even when viral titers are low – however, whether these infected flies carried enough virus to cause infection to poultry following ingestion is not known.

Laboratory colonies of house fly, Musca domestica, little house fly, Fannia canicularis, and lesser mealworm beetle, Alphitobius diaperinus, have been established and maintained at UC Riverside for use in END persistence studies.

Standard Operating Procedures have been developed for handling infectious diseases at the USDA SERPL facilities. Handling and containment procedures were developed during a site visit by one of us (Gerry) conducted in January 2005 to discuss USDA requirements with our collaborators (Dr. David Swayne and Dr. Jack King, USDA SERPL). Required security clearances to work at this USDA laboratory facility have been granted for Gerry and postdoctoral researcher Dr. Seemanti Chakrabarti.

Dr. Chakrabarti is presently in Georgia, where studies on viral persistence in and on insects are currently under way at the USDA facility in Athens. Insects are being infected with END and held in isolation chambers within a quarantine facility on the grounds of the USDA research laboratory. Dr. Chakrabarti and the staff at the USDA facility have infected colony insects (house flies, little house flies, and darkling beetles), held these insects for up to 15 days post infection (p.i.) and are currently isolating virus from pools of these infected insects to determine how long virus can persist on our three representative insect species. This first infection trial will be completed for all three insect groups by May 11.

Thus far, we have found that infection of house flies and little house flies by providing a virus laden food source has been successful and that house flies were able to maintain detectable virus levels for a minimum of 72 hours. However, infection of beetles using a virus-laden substrate, while successfully accomplished previously using Newcastle Disease vaccine virus, provided spotty results with only some beetles apparently picking up virus during the 24-hour exposure period.

A second trial is scheduled for August 2006 to focus on additional questions that became evident following this first trial and to try new methods for infecting beetles.

Second-year
progress
We sampled fly populations at several backyard poultry operations containing birds that were infected with END virus. These samples are being tested at the Southeast Regional Poultry Laboratory (SERPL) in Athens, Ga., for the presence and quantity of END virus. This portion of the study is expected to be completed by late fall 2005.

Laboratory colonies of house fly (Musca domestica), little house fly (Fannia canicularis), and coastal fly (Fannia femoralis) have been established for use in END persistence studies that will begin in May and June 2005.

Standard Operating Procedures have been developed for handling infectious diseases at the USDA SERPL facilities. Handling and containment procedures were developed during a site visit by one of us (Gerry) in January 2005 to discuss USDA requirements with our collaborator (Dr. David Swayne, lab director at USDA SERPL). Required security clearances to work at this USDA laboratory facility have been applied for, and we are awaiting clearances to initiate the END persistence studies at the USDA facilities. The two researchers requiring security clearances to gain access to the USDA biosafety level 3 facilities that we will use (Gerry and postdoctoral researcher Dr. Seemanti Chakrabarti) expect to have clearances by June 2005.

First-year
progress
Sampling has been conducted of fly populations at several backyard poultry operations containing birds that were infected with END virus. These samples are currently in storage to be tested at a later date for the presence and quantity of END virus.

Laboratory colonies of house flies (Musca domestica) and little house flies (Fannia canicularis) have been established for use in END persistence studies to be conducted in 2004 and 2005.

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