We determined WNV viremia profiles for 25 species of birds representing 17 families and 10 orders (). Four (a Budgerigar, a Monk Parakeet, and two Japanese Quail) of 87 birds did not develop a detectable viremia (threshold of detection 50 PFU/mL serum) (). Four birds sustained detectable viremias of 7 days (a Ring-billed Gull, a House Finch, and two Fish Crows). Fish Crows were bled daily after 7 dpi for an additional 4 days to investigate whether viremias may endure >7 days (). They did not, although one moribund Fish Crow became viremic at 11 dpi, shortly before dying. Generally, viremias averaged greater in magnitude and duration in passerine and charadriiform birds than in other orders. Psittacine and gallinaceous birds had the lowest titered and shortest duration viremias.
Comparative West Nile virus viremia profiles for 10 orders of birds.
Mean West Nile virus viremias (shown as log10 PFU/mL serum, with ranges) for each of 7 days postinoculation by mosquito bite, and mean duration of detectable viremia (days, with ranges)a,b
Daily viremia determinations for nine Fish Crows infected with West Nile virus by mosquito bitea
Illness and Death
Of the 87 mosquito-exposed birds, we observed obvious signs of illness in 28 birds, including members of certain passerine species (in particular, the corvids) and the Ring-billed Gull. Signs of illness included generalized lethargy, ruffled feathers, unusual posture (Blue Jay), inability to hold head upright (Ring-billed Gull), and ataxia (Ring-billed Gull). In most cases, clinical signs were followed by death within 24 h. Moribund birds were euthanized, although ill birds were rarely found moribund because death occurred rapidly. External hemorrhage, either from the mouth or from the cloaca, was noted in a small number of American Crows that died. Although our sample sizes and controls were insufficient to generate accurate estimates of mortality rates, our observations can be used to generate preliminary estimates ().
Illness observed in eight species of birds exposed to West Nile virus (WNV) by mosquito bitea
We evaluated oral susceptibility to WNV infection for 15 species of birds representing 11 families and seven orders (). We confirmed susceptibility to orally acquired WNV infection in Great Horned Owl, American Crow, Common Grackle, House Finch, and House Sparrow. The owl that ingested infected mice developed viremia and seroconverted. American Crows also became infected after consuming a WNV-infected House Sparrow carcass (83% susceptibility, n=6); three Black-billed Magpies and a Fish Crow did not become infected after consuming infected House Sparrows or infected mice. American Crows and House Sparrows became infected after ingesting an aqueous solution containing 107.4 PFU (100% susceptibility; n=6 and n=3, respectively). Grackles became infected after ingesting an aqueous solution containing1,000 PFU (100% susceptibility; n=4) but were resistant to a dose of 100 PFU (n=2). One of two House Finches that ate an infected mosquito, representing a dose of about 107 PFU, became viremic. Three each of Mourning Doves and Budgerigars did not become infected after ingesting an infected mosquito; three each of Japanese Quail and Monk Parakeet and two Bobwhite, did not become infected after ingesting an aqueous suspension containing about 3,400 PFU. Viremias generated from oral infection were similar to those from mosquito bite–derived infection, although the onset of detectable viremia was consistently delayed by at least a day (), except for the one House Finch and the Great Horned Owl. Viremia profiles of these birds were similar to their mosquito-exposed counterparts, with no delay in the onset of viremia.
Figure 2 West Nile virus viremia profiles in American Crows that were mosquito-exposed (n=8), orally exposed by ingestion of sparrow carcasses (n=5), or contact-exposed (n=4). A fifth contact-exposed crow developed an ephemeral low-titered viremia (102.2/mL serum) (more ...)
We monitored for direct transmission between mosquito-exposed birds and their cage mates among 18 species of bird representing 12 families and seven orders (). Transmission to cage mates was detected only in Ring-billed Gulls, Blue Jays, Black-billed Magpies, and American Crows (). The viremia profile of contact-exposed American Crows was similar to that of mosquito-exposed and orally exposed crows (). In contact-exposed American Crows and Black-billed Magpies, onset of viremia occurred subsequent to death of their mosquito-exposed cage mates, suggesting that infection occurred near the time of death of the mosquito-exposed birds. The two contact-exposed Blue Jays both became infected while their mosquito-exposed cage mates were still viremic and apparently healthy. The one contact-exposed Ring-billed Gull that became infected did not develop viremia within 7 days of the inoculation of its two mosquito-exposed companions but was viremic at 14 dpi.
West Nile virus cage mate transmission trialsa
Development of Neutralizing Antibodies
Most mosquito-exposed birds that survived WNV infection were euthanized at 14 dpi (House Finches were held until 21 dpi, and Rock Doves were held for 64 dpi). We evaluated final serum samples for neutralizing antibodies. Only two birds, both Budgerigars, did not produce at least 70% neutralization activity in the final serum sample (tested at a 1:10 dilution). One of these also did not develop detectable viremia. The other had a detectable viremia only at 24 h postinoculation (log10 titer 2.8 PFU/mL serum), which may have represented residual virus from the injection rather than viral multiplication. The neutralizing antibody response of Rock Doves was tracked weekly for 9 weeks postinoculation (). Between weeks 2–9 postinoculation, reciprocal 90%-neutralization titers ranged from 10 to 640 and tended to rise early, then fall, and then rise again between weeks 3–7 postinoculation.
West Nile virus-neutralizing antibody response of six mosquito-exposed Rock Doves (pigeons). Rock Dove 175 reached a titer of 1:640 at 4 weeks postinoculation and then died of other causes.
Cloacal and in some cases oral (nasopharyngeal) swabs were collected from 24 species of birds that were exposed to mosquitoes for subsequent virus isolation attempts. By swabbing the cloaca, we documented that most birds shed WNV in feces (17 [71%] of 24 species; 46 [59%] of 78 individual birds). Some passerine birds shed large quantities of WNV through the cloaca (). Cloacal shedding was generally first detected after several days of viremia and persisted longest in Fish Crows (>9 days), with peak cloacal swab titers occurring at 4–5 dpi. Although the highest cloacal swab titers were detected in American Crows and Blue Jays, these did not persist beyond 4 days because the birds died. By swabbing the oral cavity, we documented that most birds shed WNV in oral exudates (11 [85%] of 13 species; 29 [69%] of 42 individuals birds); the highest titers were observed in Great Horned Owl, American Crow, and American Kestrel (). Shedding per os persisted longest in the American Kestrel (up to 10 days). Shedding (either per cloaca or per os) was observed in representatives of 8 of the 10 orders, with the exceptions of Psittaciformes (n=6 individual birds) and Piciformes species (n=1). Although environmental sampling was not undertaken rigorously, we did detect infectious WNV in a water dish that had been contaminated with Blue Jay feces and from bloody oral effusion collected underneath a dead American Crow.
West Nile virus shedding in living birds, as determined by daily cloacal swabbing of four species of birds exposed by mosquito bitea,b
West Nile virus shedding in living birds, as determined by plaque assay of oral swabs collected daily from 14 species of birds exposed by mosquito bitea,b
Viral Load and Viral Persistence in Organs
Some birds that died acutely were necropsied to determine viral load in different organs (). Almost all organs evaluated were infected, although certain organs harbored consistently more virus. Among the four species of corvids evaluated, titers were higher in American Crows and Blue Jays than in Fish Crows and Black-billed Magpies. Titers were lowest in Ring-billed Gulls, but most tissues were still infected.
Viral load, determined by Vero plaque assay, in organs harvested from fatal cases of West Nile virus infection in experimentally infected birdsa
All surviving birds were necropsied after euthanization to determine whether infectious WNV could be detected in any of 11 organs, including brain, eye, kidney, heart, spleen, liver, lung, intestines, gonads, esophagus, and skin. This analysis determined that 18 of 41 birds sampled at 14 dpi sustained virus infections in one or more organs for up to 13 days beyond the period of viremia and, in two cases, in birds with no detectable viremia ().
Viral load, determined by Vero plaque assay in organs harvested from surviving birds 14 days after West Nile virus (WNV) infection by mosquito bitea,b
We analyzed viremia data from mosquito-exposed birds to determine values for susceptibility, mean infectiousness, and duration of infectious viremia; from these, we calculated competence indices (). Species with high mean peak viremias and long duration of viremia generally also had high competence index values.
West Nile virus reservoir competence index values derived for 25 species of birds