Experimental assessment of houseflies as vectors in avian influenza subtype H5N1 transmission in chickens.

In this study, laboratory-reared houseflies were experimentally exposed to the high pathogenicity avian influenza virus (HPAI) subtype H5N1 virus to evaluate the houseflies as vectors in HPAI-H5N1 virus transmission in chickens. One hundred and fifty houseflies (Musca domestica L.) were equally allocated into three groups. Groups 2 and 3 were exposed to the HPAI-H5N1 virus by allowing the flies to consume food containing the virus for 15 min, while the flies in group 1 were allowed to consume H5N1-free food and would serve as a negative control group. Group 2 flies were euthanatized immediately after H5N1 exposure, while group 3 were held at room temperature for 24 hr and euthanatized. The houseflies in the transmission of the HPAI-H5N1 virus were examined by challenging three groups of housefly homogenates into layer chickens via the oral drop. Morbidity and mortality were observed for 14 days, and virus shedding monitored via oropharyngeal swabs (OS) and cloacal swabs (CS), which were collected daily and determined by real-time reverse transcription-PCR and virus titration. Experimental challenge showed that all the chickens of groups 2 and 3 died within 7 days of inoculation. The OS had higher concentrations of virus than CS. Moreover, the chickens of group 2 had higher concentrations of virus shedding than the chickens of group 3. Immunohistochemistry detected the nucleoprotein of the type A influenza virus in all tissue samples collected, including the trachea, duodenum, pancreas, and brain. In summary, this study demonstrates that houseflies could serve as vectors in HPAI-H5N1 virus transmission in chickens under experimental conditions.

The potential of house flies to act as a vector of avian influenza subtype H5N1 under experimental conditions.

The objective of the present study was to determine the potential for house flies (Musca domestica L.) (Diptera: Muscidae) to harbour the avian influenza (AI) H5N1 virus. Laboratory-reared flies were experimentally fed with a mixture containing the AI virus. Exposed flies were washed with brain-heart infusion broth and followed by 70% alcohol before preparation of whole fly homogenate. The homogenate was inoculated into six 10-day-old embryonated chicken eggs (ECEs). Allantoic fluids were collected to determine the virus using the haemagglutination (HA) test, reverse transcription-polymerase chain reaction (RT-PCR) or quantitative real-time RT-PCR (RRT-PCR). In the first experiment, ECEs that were inoculated with the 50 AI virus exposed fly homogenates died within 48 h and HA and RT-PCR were positive for AI virus. In the second experiment, ECEs that were inoculated with only one fly died with positive HA test and RT-PCR. In the last experiment, a group of exposed flies was collected at 0, 6, 12, 24, 36, 48, 72 and 96 h post-exposure. Fly homogenates of each time point were tested by virus titration in ECEs and RRT-PCR. Virus titres declined in relation to exposure time. Furthermore, RRT-PCR results were positive at any time point. The present study shows that the flies may harbour the AI virus and could act as a mechanical vector of the AI virus.