Can preening contribute to influenza A virus infection in wild waterbirds?

Abstract

Wild aquatic birds in the Orders Anseriformes and Charadriiformes are the main reservoir hosts perpetuating the genetic pool of all influenza A viruses, including pandemic viruses. High viral loads in feces of infected birds permit a fecal-oral route of transmission. Numerous studies have reported the isolation of avian influenza viruses (AIVs) from surface water at aquatic bird habitats. These isolations indicate aquatic environments have an important role in the transmission of AIV among wild aquatic birds. However, the progressive dilution of infectious feces in water could decrease the likelihood of virus/host interactions. To evaluate whether alternate mechanisms facilitate AIV transmission in aquatic bird populations, we investigated whether the preen oil gland secretions by which all aquatic birds make their feathers waterproof could support a natural mechanism that concentrates AIVs from water onto birds' bodies, thus, representing a possible source of infection by preening activity. We consistently detected both viral RNA and infectious AIVs on swabs of preened feathers of 345 wild mallards by using reverse transcription-polymerase chain reaction (RT-PCR) and virus-isolation (VI) assays. Additionally, in two laboratory experiments using a quantitative real-time (qR) RT-PCR assay, we demonstrated that feather samples (n = 5) and cotton swabs (n = 24) experimentally impregnated with preen oil, when soaked in AIV-contaminated waters, attracted and concentrated AIVs on their surfaces. The data presented herein provide information that expands our understanding of AIV ecology in the wild bird reservoir system.

Figure 1. Field Studies: The novel sampling approach and virological results from feather and cloacal swabs taken from wild mallards.

(A) Feather swabs were obtained by rubbing feather surface around the waterline level of birds. (B) Molecular results showing percentages of RT-PCR–positive ducks (n = 345) for matrix (M) gene of influenza A virus: 64/345 (18.6%) mallards were positive from feathers only (F), 9/345 (2.6%) from cloaca only (C), and 29/345 (8.4%) from both feathers and cloaca (F and C). (C) Influenza A virus isolation (VI) results: 13 ducks (3.8%) were VI-positive from feathers only (F), 12 (3.5%) from cloaca only (C), and 3 (0.9%) from both feathers and cloaca (F and C). Denominator for prevalence calculation is the same as in A (n = 345) because, after the initial molecular screening, we assumed that PCR-negative ducks were VI-negative ducks as well.

Figure 2. Interactions between preened feather tufts and AIV–contaminated salt water.

(A) Feather tufts surrounding the oil gland papilla were taken from slaughtered mallards (n = 5) and preened by squeezing the uropygial gland. (B) Preened feather tufts were soaked in AIV-contaminated salt water for 24 h before qRRT-PCR assays to detect the M gene. (C) The mean ±2 SEMs of log₁₀ of M gene copies per reaction from each mallard (UPG+1 to UPG+5) is shown; the contaminated salt water was used for comparison.

Figure 3. AIV capture from AIV-contaminated fresh water and concentration on preen oil impregnated swabs.

(A) Swabs impregnation with preen oil (UPG+). (B) Swabs soaked in AIV-contaminated water. (C) Bar graph representing mean ±2 SEMs of log₁₀ of vRNA M gene copies per reaction calculated at each interval time from 3 preened (UPG+) swabs and 3 unpreened (UPG–) swabs that were soaked in AIV-contaminated fresh water; the contaminated fresh water samples were used for comparison. (D) Pair comparison of qRRT-PCR results. Bold type indicates significant differences calculated by 2-tailed Student's t-test. (E–G) Relationship between log₁₀ of vRNA M gene copies detected in (E) UPG+ swabs with respect to swab-soaking hours, (F) water with respect to water-collection hours, (G) UPG– swabs with respect to swab soaking hours. UPG+ and UPG–: Swabs impregnated with preen oil and non-impregnated Swabs, respectively.