The testing of dead birds is a reliable surveillance tool for monitoring the enzootic and epizootic activity of West Nile virus (WNV). Fresh specimens, i.e., birds dead less than 24 h, are preferred for WNV antigen testing by VecTest®, RAMP® test, or viral RNA testing by real-time RT-PCR (rRT-PCR) and badly decomposed or scavenged carcasses are thought to be of limited diagnostic value. Signs that a bird has been dead longer than 24 to 48 h are the presence of maggots, extremely light weight, missing eyes, skin discoloration, skin or feathers that rub off easily, strong odor, or a soft, mushy carcass (California Department of Public Health 2008).
Six dead American crows, Corvus brachyrhynchos (Passeriformes: Corvidae), in advanced stages of decomposition and heavily infested with fly maggots collected in 2008 (Figure 1) were used to validate the feasibility of testing WNV antigen and viral RNA in decomposed carcasses and their infested fly maggots. The dead crows studied were coded 08–5267, 08–5465, 08–5637, 08–5764, 08–5996, and 08–5997 according to the reporting and filing system of the California Department of Public Health. Oral swabs were obtained from five out of the six dead birds and tested with VecTest® (Microgenics Corporation) and RAMP® test (Response Biomedical Corporation) for WNV antigen, and by rRT-PCR for viral RNA (Lanciotti et al. 2000, Pei et al. 2001). Of the five oral swabs, three tested WNV antigen positive by the VecTest® (08–5367, 08–5996 and 08–5997), four tested WNV antigen positive by the RAMP® test (08–5267, 08–5764, 08–5996, and 08–5997) with RAMP values all greater than 640 units, and all five tested WNV RNA positive by rRT-PCR. An oral swab was not obtained from the sixth crow (08–5637) because it was in such an advanced decomposition state that its head and neck regions were nearly dry (Table 1). Therefore, neither antigen test by either VecTest® or RAMP® test, nor RNA test by rRT-PCR was performed on this specimen.
|Dead Bird State No.||Species||VecTest||Ramp (units*)||rRT-PCR|
|08–5267||American crow||Pos||Pos (> 640)||Pos|
|08–5465||American crow||Neg||Neg (26.7)||Pos|
|08–5764||American crow||Neg||Pos (> 640)||Pos|
|08–5996||American crow||Pos||Pos (> 640)||Pos|
|08–5997||American crow||Pos||Pos (> 640)||Pos|
Some maggots were removed from the dead crows and reared to adulthood for identification. They were identified as Phoridae and Calliphoridae. As shown in Table 2, up to five 2nd to 3rd instar maggots recovered from each of the six dead birds were pooled in 1 ml of RAMP® buffer. Each maggot pool was macerated thoroughly and centrifuged, and supernatant was used for an immediate WNV antigen test by RAMP® test. The remaining supernatants of the six maggot samples were stored at –80° C for later assay by rRT-PCR for WNV RNA. Two additional samples (sample codes F08–009 and F08–015), with ten maggots each, were collected from dead birds 08–5996 and 08–5997 (without RAMP® buffer), and were stored at –80° C, also for an rRT-PCR test.
|Dead Bird State No.||With RAMP® Buffer||# of Maggots||Maggot Sample Code||RAMP® Results (units)*||rRT-PCR Results|
|08–5267||Yes||5||F08–001||Pos (> 640)||Pos|
|08–5997||Yes||5||F08–014||Pos (> 640)||Pos|
The six maggot pools in RAMP® buffer that were tested by the RAMP® test had values ranging from 36.8 to greater than 640. When processing the remaining supernatants along with the two additional maggot pools without RAMP® buffer from all six dead crows in a rRT-PCR test (Table 2), all tested positive for the WNV RNA, including the maggots (sample code F08–003) collected from dead bird 08–5637, whose oral-pharyngeal cavity was too dry to get an oral swab.
Some maggots collected from three dead crows, 08–5637, 08–5996, and 08–5997 were transferred to a culture medium (Chakrabarti et al. 2008) and allowed to develop further to pupae and adults. From these, larval, pupal, or adult flies were sampled at different time intervals (from one to ten days) and pooled up to five individuals per pool (without RAMP® buffer) and stored at –80º C for an rRT-PCR test. A total of 11 samples (one maggot and two pupal samples from 08–5637, two maggot and one each of pupal and adult samples from 08–5996, and two maggot and one each of pupal and adult samples from 08–5997) were assayed and the results are shown in Table 3. Only one of the 11 samples with ten maggots (F08–010), which had been on the culture medium for only one day, tested positive for viral RNA by rRT-PCR; the remaining ten samples all tested WNV RNA negative (Table 3).
|Dead Bird State No.||Fly Sample Description||# of Flies||Fly Sample Code||rRT-PCR Results|
|08–5637||Maggots in RAMP buffer (3 d on medium)||5||F08–004||Neg|
|Pupae in RAMP buffer (3 d on medium)||5||F08–005||Neg|
|Pupae without RAMP buffer (3 d on medium)||12||F08–006||Neg|
|08–5996||Maggots without RAMP buffer (1 d on medium)||10||F08–010||Pos|
|Maggots without RAMP buffer (5 d on medium)||4||F08–011||Neg|
|Pupae without RAMP buffer (5 d on medium)||12||F08–012||Neg|
|Adults without RAMP buffer (8 d on medium)||10||F08–013||Neg|
|08–5997||Maggots without RAMP buffer (1 d on medium)||10||F08–016||Neg|
|Maggots without RAMP buffer (5 d on medium)||7||F08–017||Neg|
|Pupae without RAMP buffer (8 d on medium)||10||F08–018||Neg|
|Adults without RAMP buffer (10 d on medium)||10||F08–019||Neg|
Dead birds in stages of advanced decomposition generally are not recommended for submission for viral antigen tests, such as the VecTest® or RAMP® test, nor for viral RNA detection. However, our preliminary results indicate that WNV antigen and RNA may continue to persist in dead crow carcasses even in an advanced decomposition state. Furthermore, fly maggots freshly removed from the carcasses remained positive by the RAMP® test and rRT-PCR assay, which provides an alternate source of sample for testing in case oral swab sample collection is not practical. It appears that the WNV acquired through ingestion of, and physical contact with, infected bird tissues by fly larvae do not replicate in fly larvae. Once the larvae have left the infected dead host for more than 24 h and/or proceeded to molt to the next developmental stage, the viral components became undetectable. The evidence argues for possibly extending the post-mortem interval of WNV infection testing in dead crows for at least three days depending on weather conditions and fly species which infest the carcasses.