osed either to infectious WNV or a very closely related virus during the summer of 2004. The supplementary positive results obtained by immunoblotting and immunofluorescence microscopy also support this conclusion by demonstrating specific immune responses against the WNV envelope protein. Many of the newly hatched chicks had antibodies that neutralized WNV and to a lesser extent USUV and SINV. It is well known that maternal antibodies are concentrated in the fertile egg and that the quantity of these antibodies declines rapidly in the newly hatched chick . Our PRNT results are totally consistent with these known observations and they demonstrate that WNV, USUV and SINV (at least), or closely related viruses, must have circulated on the farm in the previous year. The decline in antibody prevalence during the first few weeks after hatching is also consistent with the idea that WNV is unlikely to have been circulating significantly during the first three or four months of the year, i.e. late winter and early spring. The detection of a significant increase in the numbers of serologically positive chickens from July onwards can probably be explained most appropriately as due to this being the time immediately after the arrival of migratory birds from Africa, Europe and the Middle East and also being the warmest time of the year when mosquitoes would be relatively active and therefore capable of transmitting arboviruses, even in England. Some chickens seroconverted even though they had been kept indoors for most of their lives. However, the ventilation system for the building in which they were housed is positive and not filtered inwards, moreover, adjoining rooms contained wild birds, inferring that the chickens could have been exposed to aerosols containing virus. In addition to virus transmission by blood transfusion and organ transplantation, there is now compelling evidence that arboviruses such as WNV may be transmitted between vertebrates using a variety of mechanisms other than direct transmission by arthropods. These include the aerosol and faecal/oral routes, transmission via direct physical contact or maternal milk, and through contaminated water. It is also clear that WNV can persist in vertebrate hosts for months if not years without inducing obvious clinical symptoms [5,14-21]. It seems likely that these properties provide WNV with the tools to circulate silently in many regions of the world and this may explain our observations of seroconversion in sentinel chickens in the UK. It is also important to emphasize that similar studies using sera from sequentially bled sentinel chickens in Italy, known to circulate WNV but with no associated disease, have been carried out and will report similar findings to those reported herein (manuscript submitted for publication).
Our observations support and extend the findings of others that although mosquitoes are important vectors in disease transmission, other modes of transmission and persistence may also be important in the transmission and circulation of WNV and other arboviruses. We now need to understand why in most cases, WNV can disperse very successfully without causing overt disease but in other situations it can cause significant epidemic outbreaks involving substantial morbidity and mortality.
Materials and methods
Three groups of chickens were hatched in early April, mid May and mid June 2004 respectively on a farm in Cambridgeshire and reared outdoors. Individual sera were collected from birds at various ages from 4 days to 20 weeks. The last samples (20 weeks) were collected at the end of October 2004, when outdoor temperatures had dropped sufficiently to reduce insect-biting activity in the UK to relatively low levels. Groups of these animals were monitored periodically for the presence, in the sera, of neutralizing antibodies to WNV, USUV and SINV. For obvious technical reasons, only very small quantities of serum were obtainable from the very young chicks, limiting the scope of their investigation. Another group of chickens was hatched and reared indoors, a