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    Peak viremia for West Nile virus–seronegative chicks (Gallus gallus domesticus) inoculated at various time-points PH (N = 58; age range, 1–70 days PH).

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DYNAMICS OF PASSIVE IMMUNITY TO WEST NILE VIRUS IN DOMESTIC CHICKENS (GALLUS GALLUS DOMESTICUS)

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  • 1 Department of Microbiology, Immunology and Pathology, Department of Biomedical Sciences, Colorado State University, Fort Collins, Colorado

Birds are the principle amplifying hosts for West Nile virus (WNV), and understanding the acquisition and decay of passive immunity is important to avian surveillance and diagnostics. We characterized passive transfer of WNV-neutralizing antibody from chicken (Gallus gallus domesticus) hens to eggs and chicks and the protective efficacy and decay of maternally acquired antibody over time. We also characterized age-associated changes in magnitude of viremia and examined the possibility of vertical transmission of WNV. All egg yolks and chicks from seropositive hens were maternal antibody positive. Maternal antibodies were undetectable in most chicks by 28 days post-hatch (PH), but some chicks remained protected as late as 42 days PH. By 56 days PH, chicks from immune hens had viremia profiles similar to control chicks. There were significant age-related differences in WNV-attributed morbidity and viremia levels of unprotected chicks. Vertical transmission of WNV was not detected.

INTRODUCTION

Passive transfer of maternal antibody in birds has been documented for several members of the Japanese encephalitis virus serocomplex of flaviviruses, including Murray Valley encephalitis virus, Japanese encephalitis virus, and St. Louis encephalitis virus (SLEV).16 In the case of West Nile virus (WNV; family Flaviviridae, genus Flavivirus), maternally derived neutralizing antibodies were detected in a colony of wild-caught rock pigeons (Columba livia) that bred in captivity7 and in a captive colony of Eastern screech owls (Megascops asio),8 both after natural WNV infection of adults. Maternal antibodies to WNV in pigeon squabs persisted for 19–33 days post-hatch (PH), whereas Eastern screech owlets had circulating maternal antibody when sampled between 1 and 27 days PH, although in neither case was the protective nature of these antibodies studied. Maternally derived antibodies to SLEV in house sparrow (Passer domesticus) chicks reached undetectable levels by 16 days PH, and on challenge, the responses of chicks with maternal antibody versus those without were not significantly different except for enhanced viremia levels in the former.6

Although there has been speculation as to the prevalence and effects of maternal WNV antibodies in birds,911 this phenomenon has not been examined under controlled conditions. The response of young birds to WNV infection is one key to understanding transmission because some unprotected nestling-age birds experience greater levels of WNV viremia and morbidity than older birds.1214 Details regarding the presence, prevalence, duration, and level of protection afforded by WNV maternal antibodies in birds would aid in interpreting field data and sentinel flock status, as well as in understanding the WNV transmission cycle as it continues to expand and establish itself in the New World. Transovarial transfer of maternal antibody in chickens can provide a model for other avian species, because the general mechanisms of the avian immune response are believed to apply to all bird species.15

The objectives of this study were to 1) determine the variability in transfer of passive immunity to WNV from hen to chick while quantifying and correlating antibody titers of hens, yolks, and chicks; 2) characterize the decay of passively acquired WNV antibody in chicks; 3) study the relationship between passively acquired antibody, development of viremia and clinical signs, and rate of seroconversion in chicks after WNV challenge at different time-points PH; 4) examine viremia profiles, morbidity and mortality, and seroconversion among seronegative chicks inoculated with WNV at varying ages; and 5) explore the possibility of vertical transmission of WNV from hen to egg and chick.

MATERIALS AND METHODS

Animals and animal care.

Twelve 22-week PH white leghorn laying hens were acquired from Morning Fresh Farms in Platteville, Colorado and a 30-week PH barred rock cockerel was obtained locally. On arrival, all chickens were confirmed as seronegative for WNV by plaque reduction neutralization test (PRNT). The cockerel and hens were housed individually in a biosafety level-3 room, provided Family Farm® Egg Maker® 16 crumbles (Manna Pro Corporation, St. Louis, MO) and fresh water ad libitum, and exposed to artificial lighting for 14 h/d at relatively constant temperature and humidity (~70°C and 20%, respectively).

Semen was collected from the cockerel by digital manipulation and used to inseminate hens. Fertile eggs to be hatched were labeled with the date and hen number and incubated and hatched within compartments to allow each chick to be traced back to its hen. Chicks were housed with same age cohorts, provided fresh water ad libitum on hatch, fed Family Farm Chick starter/grower medicated crumbles at ≥ 24 hours PH, and given a supplemental heat source until > 1 week PH.

The care of all animals in this study was in compliance with National Institutes of Health guidelines for the humane use of laboratory animals. Birds were killed by pentobarbital overdose delivered intravenously.

Virus strain, virus detection, and virus neutralization assays.

A NY99 strain of WNV (isolate 4132, originally from a dead crow) was used for all animal inoculations and serologic testing. Sera, egg yolk, and albumin, oral and cloacal swabs, and tissue homogenates were assayed for virus by Vero cell plaque assay as previously described.16 Briefly, Vero cell monolayers in 6-well plates were inoculated in duplicate with 0.1 mL of sample per well. After 1-hour incubation at 37°C, the cells were overlaid with 3 mL/well of 0.5% agarose in MEM medium supplemented with 2% fetal bovine serum (FBS) and antibiotics. Two days later, cells were overlaid with a second 3-mL overlay containing 0.004% neutral red dye. Viral plaques were counted on the third and fourth days of incubation. The minimum levels of WNV detection by virus isolation were as follows: ~50 plaque forming units (PFU)/mL or g for sera or tissue (except sera of chicks for vertical transmission), 25 PFU/mL for egg parts, and ~5 PFU/mL for swabs and sera of chicks examined for vertical transmission.

Sera were heat inactivated (56°C for 30 minutes) and tested for neutralizing antibody to WNV by PRNT as previously described,17 with different percentage reduction criteria used depending on the experiment (see Results). However, in most cases, 90% reduction at a dilution of 1:10 or greater was considered WNV antibody positive. Serial 2-fold dilutions were performed (starting at 1: 10) and tested in duplicate to determine endpoint titers of seropositive samples. Assays were grouped as much as possible to include all samples for a particular experiment and minimize interassay variability; the same positive control serum was used in all assays.

Passive transfer of antibody to yolk and chicks.

Eight WNV-seronegative hens were inoculated subcutaneously with ~12,000 PFU of WNV, whereas four hens served as seronegative controls throughout the maternal antibody portion of the study. Infected hens were bled daily from 1 to 6 days post-inoculation (DPI) to assess viremia. A volume of 0.2 mL whole blood was added to 0.9 mL BA-1 medium (M199-Hank’s salts, 1% bovine serum albumin, 350 mg/L sodium bicarbonate, 100 U/mL penicillin, 100 μg/mL streptomycin, 2.5 μg/mL amphotericin B in 0.05 mol/L Tris, pH 7.6), allowed to clot for 30 minutes at room temperature, centrifuged at 6,000g for 5 minutes, and frozen to −80°C as the equivalent of 10% serum until assayed for virus.

On 20 DPI, sera from all hens were tested for WNV neutralizing antibody to confirm seroconversion in infected hens and continued seronegative status in uninfected controls. Beginning on 36 DPI, hens were placed on a schedule of weekly insemination and were bled weekly (0.6 mL) for 9 weeks, during which time eggs were collected for artificial incubation. The eight seropositive hens were killed at 5 months after infection, whereas the four seronegative hens were later infected with WNV for evaluation of vertical transmission of virus.

Over the 5 weeks following the first weekly insemination, the first 2 eggs laid by each of the 12 hens were collected for yolk sampling, for a total of 10 eggs per hen. Within several hours of eggs being laid, yolk samples were collected with a sterile syringe, diluted 1:2.5 in phosphate-buffered saline, vortexed, and centrifuged at 14,000g for 10 minutes, and the resulting supernatants were stored at −80°C until testing for antibody. All eggs not used for yolk sampling were incubated until hatching and between 9 and 11 chicks from each hen (130 chicks total; 81 from seropositive hens and 49 from seronegative hens) were bled within 24 hours PH to assay for antibody. For comparison of antibody titers between a given hen and her egg yolks and chicks, the date each egg was laid was correlated to the nearest weekly hen serum sample. Because hens were bled weekly, all yolk and chick serum samples were collected within 3.5 days of serum samples from each corresponding hen.

Decay of passively acquired antibody in chicks.

A group of maternal antibody positive chicks (N = 33) was used to characterize loss of maternally acquired antibody over time. These birds were housed with same age cohorts and bled weekly for up to 10 weeks PH. All of these chicks were bled weekly through 4 weeks PH, and at 6, 8, and 10 weeks PH, subsets (consisting of between 6 and 13 maternal antibody positive chicks) were challenged with WNV (see below), so that the final 10-week group consisted of six chicks that were maternal antibody on hatch (derived from immune hens). In-contact seronegative chicks were included among each of the aforementioned age groups and were bled following the same schedule. Sera were evaluated by PRNT to determine antibody titers.

Relationship between passively acquired antibody and protection from WNV challenge.

At various times PH, groups of chicks that hatched from immune hens were inoculated subcutaneously with ~1,000 PFU of WNV. Each of these challenge groups also contained chicks from seronegative hens to serve as susceptible controls and to characterize age-associated differences in WNV viremia and morbidity (see below). All chicks were bled immediately before WNV inoculation to assess serologic status at the time of challenge, and daily from 1 to 7 DPI to determine magnitude and duration of viremia by Vero cell plaque assay. Ages of challenge groups consisting of maternal antibody positive chicks (derived from immune hens) were from 1 day to 10 weeks PH. After challenge, birds were killed on 10 DPI, at which time a final serum sample was collected and assayed to determine neutralizing antibody titers by PRNT. In addition, for all seronegative chicks (derived from non-immune hens) that were inoculated at < 14 days PH, sera obtained on 5 and 7 DPI were screened for antibody to assess the timing of initial antibody detection after WNV infection in naïve chicks.

Clinical response and magnitude of viremia as a function of chick age.

Groups of seronegative chicks originating from non-immune hens were challenged at various time-points PH to evaluate age-based changes in magnitude of viremia and clinical response to WNV challenge. Ages of these infection groups ranged from 1 day to 10 weeks PH. These birds were challenged in concert with the chicks from seropositive hens and therefore used to evaluate and compare the protective effect of maternally acquired antibody (see previous section).

Vertical transmission of WNV from viremic hens to eggs and chicks.

Eggs were collected from six hens after inoculation of hens with ~12,000 PFU of WNV and also from two uninfected control hens. Hens were bled daily from 1 to 6 DPI to evaluate viremia, and eggs from each hen were collected daily from 1 to 8 DPI. Eggs from two of the infected and one uninfected control hen were sampled immediately after laying to test egg parts for virus. For these eggs, yolk and albumin were diluted 1:5 with BA-1 with 20% FBS and stored at −80°C until testing for virus by Vero cell plaque assay.

Eggs from the remaining four inoculated hens (and one seronegative control hen) were collected from 1 to 8 DPI and incubated until hatch. Within 12 hours PH of each chick, blood was collected by jugular venipuncture, after which chicks were immediately killed. After death, oral and cloacal swabs and tissue samples were collected. Tissue samples included heart, brain, spleen, kidney, liver, lung, intestine, muscle, eye, and yolk sac. Cotton-tipped applicators were used to swab the oropharyngeal and cloacal cavities and were placed in 0.5 mL BA-1 with 20% FBS. Tissues were placed in 1 mL BA-1 with 20% FBS as a 10% tissue suspension with a single steel 4.5 mm BB added to each tissue sample, which was homogenized in a mixer mill (Retsch, Haan, Germany) for 5 minutes at 25 cycles/s, clarified by centrifugation for 4 minutes at 16,000g, and frozen at −80°C until testing.

RESULTS

Passive transfer of antibody to yolk and chicks.

All hens and the cockerel were WNV-seronegative before experimental infection. Of the 12 hens inoculated with WNV, 10 developed viremia. In two of these hens, viremia was detected by 1 DPI, and viremia was detected as late as 5 DPI in one hen. The average duration of detectable viremia was 2 days, with peak levels ranging from 102.0 to 103.7 PFU/mL serum, except for the two hens that failed to reach detectable viremia levels. None of the hens showed any clinical signs during the study. On 20 DPI, eight experimentally infected hens had endpoint 90% neutralization titers (PRNT90 titers) ranging from 80 to 1,280, and these levels remained relatively constant (within 2-fold difference) until 100 DPI, when hen antibody levels were last measured.

All egg yolks (N = 80) and 1 day PH chick sera (N = 81) originating from seropositive hens tested positive for WNV antibody. Alternatively, all yolks (N = 40) and 1 day PH chick sera (N = 49) originating from seronegative hens tested negative for WNV antibody. All 10 egg yolk samples from each of eight hens yielded antibody titers equal to or within 2-fold difference to titers of corresponding hen sera and were therefore not considered significantly different. However, chick hatch-day antibody titers exhibited a greater range compared with their corresponding hens’ serum antibody titer. Chick serum antibody titers were usually at least 4-fold (and up to 32-fold) below those of their hens (Table 1).

Decay of passively acquired antibody in chicks.

Thirty-one of 33 chicks (93.9%) had PRNT90 titers of < 10 by 28 days PH and were considered negative for WNV maternal antibody; PRNT90 titers of all chicks were < 10 by 35 days PH (Table 2). With a less stringent criteria of PRNT50, 8/33 (24.2%) of chicks had levels < 10 at 28 days PH (PRNT50 range 10–80), but by 35 days PH, all 33 chicks had PRNT50 titers of < 10. Chicks that still had detectable PRNT90 titers at 28 days PH originated from the same two hens, both of which had the highest PRNT90 titers of all hens (PRNT90 = 1,280). However, PRNT90 titers of three other chicks from hens with this high level titer had dropped to < 10 by 28 DPI. In-contact seronegative chicks (N = 14) bled weekly remained seronegative for the duration of the study.

Relationship between passively acquired antibody and protection from WNV challenge.

None of the six maternal antibody positive chicks challenged with WNV on 1 day PH showed signs of morbidity, whereas all four of their seronegative counterparts succumbed to infection by ~5 DPI. Morbidity was not observed in any chick inoculated at > 1 day PH regardless of maternal antibody status. However, all WNV-inoculated chicks that were seronegative on hatch (derived from seronegative hens) became detectably viremic between 1 and 5 DPI, whereas all maternal antibody positive chicks inoculated at ≤ 28 days PH failed to become detectably viremic. At 42 days PH, none of seven chicks that hatched from immune hens had detectable serum antibody (< 1:10 PRNT50), but three of these seven chicks failed to become viremic after challenge. Viremia levels of the remaining four chicks were later in onset and of lower magnitude than those of their seronegative counterparts. All chicks from immune hens inoculated on 56 and 70 days PH became viremic, with profiles similar to those of chicks from seronegative hens (Table 3).

The serologic responses of chicks that were maternal antibody positive on hatch (derived from WNV immune hens) also differed according to age PH of WNV challenge. After challenge, some chicks of immune hens that were ≤ 28 days PH when challenged had declining (2- to 8-fold) antibody levels evident on 10 DPI compared with inoculation day titers; these low antibody titers detected on 10 DPI were presumed to be declining levels of maternal antibody. Two of the chicks derived from immune hens that were seronegative when challenged on 28 days PH subsequently seroconverted by 10 DPI, whereas the remaining 11 chicks did not show evidence of seroconversion by 10 DPI. The range of hatch day PRNT90 titers of the 11 chicks that failed to seroconvert (20–320) encompassed titers of the chicks that did seroconvert after challenge (40, 160). Of the chicks derived from immune hens that were challenged on 42, 56, and 70 days PH, six of seven, six of seven, and six of six seroconverted by 10 DPI, respectively (Table 3).

For the evaluation of seroconversion in chicks derived from seronegative hens, a detectable antibody response was defined as ≥ 90% neutralization at a 1:20 dilution, because these blood samples were diluted immediately on collection as the equivalent of 10% serum so that the lowest dilution possible for neutralization assays (in which serum is added to an equal volume of virus solution) was 1:20. Few seronegative chicks (3/36; 8.3%) inoculated at < 14 days PH had formed a detectable antibody response by 5 DPI, whereas 5/32 (15.6%) met this criteria by 7 DPI. However, by 10 DPI the majority of these chicks (21/32; 65.6%) had detectable PRNT90 titers, ranging from 10 to 160. The four seronegative chicks that died after inoculation on 1 day PH had not mounted a detectable antibody response by the time of death. All seronegative chicks inoculated at ≥ 14 days PH had detectable PRNT90 titers (range, 10–320) on 10 DPI.

Clinical response and magnitude of viremia as a function of chick age.

Age and magnitude of viremia level were significantly negatively correlated (Spearman rank order correlation coefficient, rs = −0.9542; N = 58; one-tailed P < 0.0001; Figure 1). The mean peak viremia levels of unprotected chicks inoculated on 1, 7, and 21 days PH were 107.3, 105.2, and 103.8 PFU/mL serum, respectively; the mean peak viremia among the 12 hens (≥ 6 months of age when inoculated) was 102.9 PFU/mL serum. All four of the chicks inoculated at 1 day PH were evidently succumbing to WNV infection, and on 5 DPI, three of these chicks were killed and one died. These chicks developed detectable viremia of 5-day duration before death, unlike almost all other chicks, in which detectable viremia lasted from 1 to 4 days (in one 2-day PH unprotected chick, viremia was also detectable for 5 days). Clinical signs in these birds included intermittent somnolence and reluctance to move. No morbidity was observed in chicks inoculated at > 1 day PH.

Vertical transmission to eggs and chicks.

Fourteen eggs (2 of which were laid during detectable viremia of hens) and 21 chicks (5 of which hatched from eggs laid during detectable viremia of hens) were collected from 1–8 DPI hens. During the time of egg collection, hens were viremic from 2 to 3 DPI, with peak viremia levels ranging from 10<1.7 to 102.9 PFU/mL serum. Virus was not detected in any egg parts, chick tissues, sera, or swabs.

DISCUSSION

Adult chickens have not been reported to experience morbidity caused by WNV infection, although chicken carcasses have tested positive for WNV.18 In addition, WNV infection of chickens elicits significant antibody titers,19 artificial insemination is relatively easy to perform, and hens provide a constant supply of eggs under defined conditions, making domestic chickens an excellent candidate for evaluation of passive transfer of WNV immunity from hen to chick. Immunoglobulins (Ig) are transferred from hen to egg and embryo through various routes. IgA and IgM in oviduct secretions are transferred to the egg as it passes down the oviduct, whereas IgG is transferred from hen serum by yolk into the embryo’s circulation. Immunoglobulins are also passed to the chick by swallowed amniotic fluid.20 To our knowledge, maternal transfer of antibody to WNV in birds has not been explored experimentally, although other viral agents have been associated with the passive transfer of maternal antibodies to neonatal birds in an experimental setting.1,2124

A strong correlation between chicken hen serum and yolk IgG levels has been previously shown.25,26 In this study, the correlation between hen, yolk, and chick WNV antibody titers suggests that levels of antibody circulating in hen sera are fully transferred to their eggs but that levels begin to decline before hatch (Table 1). Subsequent to hatch, WNV maternal antibody underwent relatively rapid decay in chicks, as most had undetectable antibody levels by 28 days PH (Table 2). These results are consistent with the negative correlation observed between maternal antibody levels in sera of Eastern screech owl chicks and the time of sampling PH.8 Additionally, maternal Ig levels in eggs of black-headed gulls (Larus ridibundus L.) decreased in eggs laid later within a clutch.27 While we did not find evidence of the latter in chickens, hens in our study were commercial quality, continuous egg-layers, which may have affected the pattern of passive transfer. In addition, siblings from eggs laid over a 100-day span did not exhibit a decline in 1 day PH antibody titers during this time period. Our results suggest that, as hen WNV antibody levels remain constant, the passive transfer of antibody to their eggs behaves similarly, and whereas sibling chicks exhibit a range of hatch-day antibody titers, these levels do not seem to decline over time.

In our study, maternal antibody seemed to decay relatively rapidly and was undetectable in most chicks by 28 days PH. However, undetectable levels of maternal antibody remained protective against the development of viremia in some chicks for up to 42 days PH (Table 3). Furthermore, most chicks that were inoculated at ≤ 28 days PH failed to seroconvert on WNV challenge, suggesting that maternal antibody persisted and therefore chicks failed to recognize or respond immunologically to infection. However, nearly all chicks derived from immune hens that were inoculated at 42, 56, and 70 days PH seroconverted after WNV challenge (Table 3), indicating that, at these later times PH, there was a lack of protective, circulating maternal antibody. We did not observe that antibody levels of hens or subsequent maternal antibody levels of chicks at time of hatch or on challenge had an effect on rate of decay or level of protectiveness at later time-points, although these data were relatively limited and not quantified.

The effect of age on the pathogenesis of WNV infection in birds is poorly studied, although some information has been obtained from domestic geese.14 Differential effects of age with other flavivirus infections have also been observed for Murray Valley and Japanese encephalitis viruses in domestic chickens,28,29 and SLEV in mourning doves (Zenaida macroura) and house finches (Caropodacus mexicanus).30 Experimental WNV infections in chickens showed that adults reach relatively low peak viremia levels (102.4–105.0 PFU/mL serum) with no clinical signs of illness,19 whereas 1- to 3-day-old chicks undergo higher peak viremia levels (106.5–107.5 PFU/mL serum) and exhibit significant morbidity.13,31,32 In our study, magnitude of viremia in unprotected chicks had a strong negative correlation with age (Figure 1), and no chicks inoculated at > 14 days PH reached viremia levels considered efficiently infectious to mosquitoes (> 105 PFU/mL).33 Furthermore, no chicks infected at > 1 day PH showed signs of illness.

The phenomenon of passive transfer of maternal WNV antibody has important implications on transmission dynamics and the differential survival of young birds. First, existing data indicate that antibody produced in response to infection may persist7 and remain protective for ≥ 1 year in some species of birds (N. M. Nemeth and others, unpublished data), and our data suggest that all offspring of seropositive female birds will benefit from protective maternal antibody. If exposed to WNV in the wild, maternal antibody positive chicks at ≤ 42 days PH would potentially be partially or fully protected from the effects of infection and therefore less likely to play role in transmission. Chicks that resist WNV infection because of the presence of maternal antibody are susceptible at a later age after maternal antibody wanes, but at this time, they are less likely to experience morbidity and higher viremia levels, possibly contributing to decreased rates of mosquito WNV transmission34 and increased rates of survival for some bird species. However, unprotected chicks infected early in life have a lesser chance of survival because they are more susceptible to higher viremia levels and the ill effects of WNV on their health. The potentially devastating effects of WNV on younger, naïve chicks, as well as the occurrence and persistence of maternal antibody, are important considerations in management and conservation of endangered avian species within endemic areas. Understanding these dynamics is especially important because the occurrence of neonatal and juvenile birds corresponds temporally to the WNV transmission season in many regions of the United States.35

The interpretation of serologic results from young birds can be confused by the presence of maternal antibody, whether from a diagnostic or surveillance perspective. From a diagnostic standpoint, a maternal antibody positive chick might lead to mistaken consideration of WNV as a possible differential diagnosis. Furthermore, a chick derived from an immune hen that tests positive for WNV antibody at a young age represents a false positive from a surveillance standpoint, because it does not represent a recent WNV exposure. In turn, if a natural exposure were to occur in a bird protected by maternal antibody, this chick would likely fail to seroconvert in response to this exposure, and after waning of maternal antibody, would be considered a false negative. We concluded that antibody formation in response to WNV infection in the majority of young, naïve chicken chicks likely occurs between 7 and 10 DPI, so that there is a reasonable likelihood that WNV antibody detected in chicks at < 7 days PH is maternally derived. Further confusion may arise if a maternal antibody positive chick is retested at a later date, at which time, barring a subsequent natural exposure event, it will test seronegative. In addition, within the time frame that chicks from immune hens no longer have detectable antibody but remain partially protected from WNV infection (~28–42 days PH), a negative PRNT result would be misleading because these chicks may not be susceptible to viremia and morbidity after a natural exposure event.

An additional application regarding passive transfer of WNV antibody involves vaccination of chicks, whether within a zoological collection, involved in wildlife rehabilitation or education programs, or part of endangered species programs. Based on results from this study, we recommend that initial vaccination of chicks from mothers that are likely seropositive be delayed until after 8 weeks PH to circumvent potential interference of maternal antibody with vaccination success. However, if vaccination at a younger age is deemed necessary, a booster vaccination at ~8 weeks PH would be advisable. More research is needed in examining the efficacy of currently available WNV vaccines in birds and future vaccines that might show promise.

Vertical transmission of WNV has been reported in mice36 but has not been described in birds.37 While various modes of WNV transmission (e.g., mosquito, oral) have been documented in birds,38,39 the potential for transmission of WNV from mother to egg and chick remains unexplored. While we had small sample sizes of eggs and chicks, our data suggest a lack of vertical WNV transmission in chickens. However, this does not rule out the possibility in other avian species, especially those that reach relatively high peak viremia levels but survive infection, such as the American kestrel (Falco sparverius), American robin (Turdus migratorius), common grackle (Quiscalus quiscula), fish crow (Corvus ossifragus), great horned owl (Bubo virginianus), and house sparrow.39,40 The hens in our study had very low viremia levels of short duration, thereby decreasing the potential for virus transmission to eggs or chicks. More data are needed from a wider representation of avian species to better assess the potential for vertical WNV transmission in birds, although achievement of fertile eggs laid within the viremic phase of captive, experimentally manipulated adult female birds represents a challenge. Additionally, sampling of eggs during various phases of embryonic development before hatch may be necessary to fully explore viral transmission from mother to chick, because embryos of susceptible species may not survive to hatching if infected in ovo.

In summary, despite potential variances in WNV maternal antibody transfer among avian species, the results from this study will aid in the interpretation of wild bird WNV sero-surveys, as well as epidemiologic data involving the distribution of WNV antibodies of birds of varying age groups. In addition, these results should be considered in concert with avian management and conservation schemes, especially those involving endangered species propagation within WNV endemic areas. While chicks with maternal antibody are protected for only a limited period of time after hatching, this period likely includes much of the nestling stage for many altricial and semialtricial bird species, at which time these chicks are relatively immobile, sparsely feathered, and seemingly more vulnerable to mosquito feeding. This also corresponds to the time in a bird’s life when they are likely most susceptible to high-level viremia and morbidity if infected with WNV. The dynamics of maternal antibody decay and subsequent immunologic naïveté of previously maternal antibody positive birds are additional factors that affect WNV transmission and population health of birds. The role of maternally derived WNV antibody, age-related differences in viremia and morbidity, and the possibility of vertical WNV transmission in free-ranging avian species must be explored to better understand their effects on WNV transmission and implications on the health of free-ranging avian populations.

Table 1

Range of antibody titers measured from West Nile virus–seropositive chicken (Gallus gallus domesticus) hens with corresponding maternal antibody titer ranges of their egg yolks and chicks at < 1 day PH

HenHen PRNT90 range*Yolk PRNT90 range†Chick PRNT90 range‡
* Hens were bled over a 10-week period.
† Ten yolks per hen were sampled over a 10-week period.
‡ Between 9 and 11 chicks per hen were sampled at < 1 day PH over a 5-week period.
1640–1280320–1,28020–160
2160–320160–32020–160
3640–1,280640–2,560160–640
4320–640160–32020–160
580–16080–16020–160
6320–640320–64040–320
780–32080–32020–160
8640320–64040–160
Table 2

Decay of maternally derived West Nile virus antibodies in chicken (Gallus gallus domesticus) chicks (N = 33) as measured by end-point 90% neutralization titers (PRNT90) from 1–4 weeks PH

Chick ID1 week PH2 weeks PH3 weeks PH4 weeks PH
1802010< 10
2804010< 10
54010< 10< 10
84020< 10< 10
98020< 10< 10
113208020< 10
19320804010
212010< 10< 10
238020< 10< 10
24402010< 10
25804010< 10
264020< 10< 10
292010< 10< 10
331608020< 10
344020< 10< 10
35802010< 10
37802010< 10
381608010< 10
42804010< 10
45802010< 10
461604010< 10
47804010< 10
50402010< 10
514020< 10< 10
532010< 10< 10
54802010< 10
59160802010
601604020< 10
628020< 10< 10
638020< 10< 10
644010< 10< 10
6610< 10< 10< 10
70802010< 10
Table 3

Serologic responses of West Nile virus maternal antibody positive and seronegative chicken (Gallus gallus domesticus) chicks of ≥ 21 days PH when inoculated*

Days PH when inoculatedHen WNV immune statusPRNT90† on inoculation dayPeak viremia (log PFU‡/mL serum)Viremia duration (DPI)§PRNT90 on 10 DPI
* Age groups of < 21 days PH when inoculated with WNV included: 1 day PH (N = 4 seronegative, 6 maternal antibody positive), 2 days PH (N = 5 seronegative, 7 maternal antibody positive), 4 days PH (N = 4 seronegative, 4 maternal antibody positive), 5 days PH (N = 4 seronegative, 1 maternal antibody positive), 7 days PH (N = 6 seronegative, 9 maternal antibody positive), 14 days PH (N = 6 seronegative, 6 maternal antibody positive). Additional seronegative chicks in the age-associated viremia and morbidity analysis included 6 days PH (N = 7) and 9 days PH (N = 6).
† Endpoint 90% neutralization antibody titer (PRNT90).
‡ Plaque forming units (PFU) of West Nile virus.
§ Days post-inoculation (DPI); duration indicates which days post-infection on which viremia was detected.
¶ Seronegative (SN) indicates chicks that hatched from seronegative hens and were seronegative upon hatch.
|| This chick showed some evidence of seroconversion, with PRNT80 = 10 on 10 DPI.
21Immune10< 1.7< 10
Immune10< 1.7< 10
Immune40< 1.710
Immune10< 1.7< 10
Immune< 10< 1.7< 10
Immune10< 1.7< 10
Immune20< 1.7< 10
Non-immuneSN¶4.11–210
Non-immuneSN3.61–220
Non-immuneSN3.41–210
28Immune< 10< 1.740
Immune< 10< 1.7< 10
Immune< 10< 1.710
Immune< 10< 1.7< 10
Immune< 10< 1.7< 10
Immune10< 1.7< 10
Immune10< 1.7< 10
Immune< 10< 1.7< 10
Immune< 10< 1.7< 10
Immune< 10< 1.7< 10
Immune< 10< 1.7< 10
Immune< 10< 1.7< 10
Immune< 10< 1.7< 10
Non-immuneSN3.01–310
Non-immuneSN4.11–310
Non-immuneSN4.62–3160
Non-immuneSN4.42–310
Non-immuneSN4.22–340
42Immune< 5< 1.720
Immune< 52.0320
Immune< 52.72–380
Immune< 5< 1.740
Immune< 52.62–3160
Immune< 5< 1.740
Immune< 5< 1.7< 10
Non-immuneSN3.91–2320
Non-immuneSN3.21–340
Non-immuneSN2.81–2160
56Immune< 52.32–310
Immune< 52.23–480
Immune< 52.52–340
Immune< 52.72–340
Immune< 52.52–3< 10||
Immune< 52.72–310
Immune< 52.2340
Non-immuneSN2.61–340
Non-immuneSN3.6280
Non-immuneSN3.42–380
70Immune< 53.02–420
Immune< 53.61–340
Immune< 52.52–320
Immune< 53.72–340
Immune< 53.61–380
Immune< 52.52–310
Non-immuneSN3.52–340
Non-immuneSN3.61–240
Figure 1.
Figure 1.

Peak viremia for West Nile virus–seronegative chicks (Gallus gallus domesticus) inoculated at various time-points PH (N = 58; age range, 1–70 days PH).

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 76, 2; 10.4269/ajtmh.2007.76.310

*

Address correspondence to Richard Bowen, Department of Biomedical Sciences, 3801 West Rampart Road, Foothills Campus, Colorado State University, Fort Collins, CO 80523-1683. E-mail: rbowen@colostate.edu

Authors’ addresses: Nicole M. Nemeth, Department of Microbiology, Immunology and Pathology, 3801 West Rampart Road, Foothills Campus, Colorado State University, Fort Collins, CO 80523-1683, Telephone: 970-491-8165, Fax: 970-491-3557, E-mail: nnemeth@colostate.edu. Richard A. Bowen, Department of Biomedical Sciences, 3801 West Rampart Road, Foothills Campus, Colorado State University, Fort Collins, CO 80523-1683, Telephone: 970-491-5768, Fax: 970-491-3557, E-mail: rbowen@colostate.edu.

Acknowledgments: The authors thank Derek Yancy and the personnel at Morning Fresh Farms for donating the hens in this study and sharing their expertise and Kate Huyvaert and Paul Doherty for donating the cockerel for this study. James Graham generously donated his time and expertise in semen collection and artificial insemination and allowed us use of his egg incubator, and Paul Gordy, Paul Oesterle, and Angela Bosco-Lauth provided laboratory and husbandry support.

Financial support: This research was funded by NIH Contract N01-AI25489.

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