Comprehensive mapping of common immunodominant epitopes in the West Nile virus nonstructural protein 1 recognized by avian antibody responses

West Nile virus (WNV) is a mosquito-borne flavivirus that primarily infects birds but occasionally infects humans and horses. Certain species of birds, including crows, house sparrows, geese, blue jays and ravens, are considered highly susceptible hosts to WNV. The nonstructural protein 1 (NS1) of WNV can elicit protective immune responses, including NS1-reactive antibodies, during infection of animals. The antigenicity of NS1 suggests that NS1-reactive antibodies could provide a basis for serological diagnostic reagents. To further define serological reagents for diagnostic use, the antigenic sites in NS1 that are targeted by host immune responses need to be identified and the potential diagnostic value of individual antigenic sites also needs to be defined. The present study describes comprehensive mapping of common immunodominant linear B-cell epitopes in the WNV NS1 using avian WNV NS1 antisera. We screened antisera from chickens, ducks and geese immunized with purified NS1 for reactivity against 35 partially overlapping peptides covering the entire WNV NS1. This study identified twelve, nine and six peptide epitopes recognized by chicken, duck and goose antibody responses, respectively. Three epitopes (NS1-3, 14 and 24) were recognized by antibodies elicited by immunization in all three avian species tested. We also found that NS1-3 and 24 were WNV-specific epitopes, whereas the NS1-14 epitope was conserved among the Japanese encephalitis virus (JEV) serocomplex viruses based on the reactivity of avian WNV NS1 antisera against polypeptides derived from the NS1 sequences of viruses of the JEV serocomplex. Further analysis showed that the three common polypeptide epitopes were not recognized by antibodies in Avian Influenza Virus (AIV), Newcastle Disease Virus (NDV), Duck Plague Virus (DPV) and Goose Parvovirus (GPV) antisera. The knowledge and reagents generated in this study have potential applications in differential diagnostic approaches and subunit vaccines development for WNV and other viruses of the JEV serocomplex.     

Seroepidemiologic Survey of Potential Pathogens in Obligate and Facultative Scavenging Avian Species in California

Throughout the world, populations of scavenger birds are declining rapidly with some populations already on the brink of extinction. Much of the current research into the factors contributing to these declines has focused on exposure to drug residues, lead, and other toxins. Despite increased monitoring of these declining populations, little is known about infectious diseases affecting scavenger bird species. To assess potential infectious disease risks to both obligate and facultative scavenger bird species, we performed a serosurvey for eleven potential pathogens in three species of scavenging birds in California: the California condor (Gymnogyps californianus), turkey vulture (Cathartes aura) and golden eagle (Aquila chrysaetos). California condors were seropositive for avian adenovirus, infectious bronchitis virus, Mycoplasma gallisepticum, avian paramyxovirus-2, West Nile virus (WNV) and Toxoplasma gondii. Golden eagles were seropositive for avian adenovirus, Chlamydophila psittaci and Toxoplasma gondii, and turkey vultures were seropositive for avian adenovirus, Chlamydophila psittaci, avian paramyxovirus-1, Toxoplasma gondii and WNV. Risk factor analyses indicated that rearing site and original release location were significantly associated with a positive serologic titer to WNV among free-flying condors. This study provides preliminary baseline data on infectious disease exposure in these populations for aiding in early disease detection and provides potentially critical information for conservation of the endangered California condor as it continues to expand its range and encounter new infectious disease threats.     

Experimental Chlamydia psittaci Infection of Japanese Quail

Japanese quail were used for the infection model of avian chlamydiosis. One-day-old Japanese quail were highly susceptible to lethal infection by a Chlamydia psittaci strain of budgerigar origin upon inoculation via the air sac route with 10^4.1 FFU of the organism, showing an acute and lethal course with chlamydial propagation. In contrast, 7-day-old quail developed resistance to the infection as shown by the lack of lethal effect with the same dose. The resistance of 7-day-old birds was abolished by immunosuppressive treatment with cyclophosphamide. Upon inoculation with a sublethal dose of 10^2.1 FFU, latent infection was established in 1-day-old birds with a minimum number of the organism. The latent infection in the birds was converted to the lethal form by treatment with cyclophosphamide along with chlamydial propagation and suppression of antibody production.     

CD22 Is Required for Protection against West Nile Virus Infection

West Nile virus (WNV) is a RNA virus of the family Flaviviridae and the leading cause of mosquito-borne encephalitis in the United States. Humoral immunity is essential for protection against WNV infection; however, the requirements for initiating effective antibody responses against WNV infection are still unclear. CD22 (Siglec-2) is expressed on B cells and regulates B cell receptor signaling, cell survival, proliferation, and antibody production. In this study, we investigated how CD22 contributes to protection against WNV infection and found that CD22 knockout (Cd22^−/−) mice were highly susceptible to WNV infection and had increased viral loads in the serum and central nervous system (CNS) compared to wild-type (WT) mice. This was not due to a defect in humoral immunity, as Cd22^−/− mice had normal WNV-specific antibody responses. However, Cd22^−/− mice had decreased WNV-specific CD8⁺ T cell responses compared to those of WT mice. These defects were not simply due to reduced cytotoxic activity or increased cell death but, rather, were associated with decreased lymphocyte migration into the draining lymph nodes (dLNs) of infected Cd22^−/− mice. Cd22^−/− mice had reduced production of the chemokine CCL3 in the dLNs after infection, suggesting that CD22 affects chemotaxis via controlling chemokine production. CD22 was not restricted to B cells but was also expressed on a subset of splenic DCIR2⁺ dendritic cells that rapidly expand early after WNV infection. Thus, CD22 plays an essential role in controlling WNV infection by governing cell migration and CD8⁺ T cell responses.     

An avian embryo culture system for embryogenesis using an artificial vessel: possible conservation benefits in the rescue and management of endangered avian species

The development of artificial means to conserve some endangered avian species seems urgently needed. We devised an eggshell‐less embryo culture system for embryogenesis using chicken or quail embryos as a model system. As a result, 41/56 (73%) chicken embryos and 52/62 (83%) quail embryos with their own thick albumen developed as usual for 72 and 55 hr, respectively, using a Teflon membrane (Milliwrap) and an egg‐shaped plastic vessel. Furthermore, this study revealed that the most suitable vessel for culture would have the same size as the intact original eggshell. This technique opens the way for the rescue of endangered avian species even if their eggshells are abnormal or cracked. This new procedure also facilitates investigation of developmental events in many unknown birds. We discuss various aspects of the embryological techniques applicable to endangered avian species. Zoo Biol 00:000–000, 2005. © 2005 Wiley‐Liss, Inc.     

Role of CD8+ T cells in control of West Nile virus infection

Infection with West Nile virus (WNV) causes fatal encephalitis more frequently in immunocompromised humans than in those with a healthy immune system. Although a complete understanding of this increased risk remains unclear, experiments with mice have begun to define how different components of the adaptive and innate immune response function to limit infection. Previously, we demonstrated that components of humoral immunity, particularly immunoglobulin M (IgM) and IgG, have critical roles in preventing dissemination of WNV infection to the central nervous system. In this study, we addressed the function of CD8(+) T cells in controlling WNV infection. Mice that lacked CD8(+) T cells or classical class Ia major histocompatibility complex (MHC) antigens had higher central nervous system viral burdens and increased mortality rates after infection with a low-passage-number WNV isolate. In contrast, an absence of CD8(+) T cells had no effect on the qualitative or quantitative antibody response and did not alter the kinetics or magnitude of viremia. In the subset of CD8(+)-T-cell-deficient mice that survived initial WNV challenge, infectious virus was recovered from central nervous system compartments for several weeks. Primary or memory CD8(+) T cells that were generated in vivo efficiently killed target cells that displayed WNV antigens in a class I MHC-restricted manner. Collectively, our experiments suggest that, while specific antibody is responsible for terminating viremia, CD8(+) T cells have an important function in clearing infection from tissues and preventing viral persistence.     

Avian host community structure and prevalence of West Nile virus in Chicago,Illinois

Vertebrate host diversity has been postulated to mediate prevalence of zoonotic, vector-borne diseases, such that as diversity increases, transmission dampens. This “dilution effect” is thought to be caused by distribution of infective bites to incompetent reservoir hosts. We quantified avian species richness, avian seroprevalence for antibodies to West Nile virus (WNV), and infection of WNV in Culex mosquitoes, in the Chicago metropolitan area, Illinois, USA, a region of historically high WNV activity. Results indicated high overall avian seroprevalence and variation in seroprevalence across host species; however, there was no negative correlation between avian richness and Culex infection rate or between richness and infection status in individual birds. Bird species with high seroprevalence, especially northern cardinals and mourning doves, may be important sentinels for WNV in Chicago, since they were common and widespread among all study sites. Overall, our results suggest no net effect of increasing species richness to West Nile virus transmission in Chicago. Other intrinsic and extrinsic factors, such as variation in mosquito host preference, reservoir host competence, temperature, and precipitation, may be more important than host diversity for driving interannual variation in WNV transmission. These results from a fine-scale study call into question the generality of a dilution effect for WNV at coarser spatial scales.     

An Inactivated Cell Culture Japanese Encephalitis Vaccine (JE-ADVAX) Formulated with Delta Inulin Adjuvant Provides Robust Heterologous Protection against West Nile Encephalitis via Cross-Protective Memory B Cells and Neutralizing Antibody

West Nile virus (WNV), currently the cause of a serious U.S. epidemic, is a mosquito-borne flavivirus and member of the Japanese encephalitis (JE) serocomplex. There is currently no approved human WNV vaccine, and treatment options remain limited, resulting in significant mortality and morbidity from human infection. Given the availability of approved human JE vaccines, this study asked whether the JE-ADVAX vaccine, which contains an inactivated cell culture JE virus antigen formulated with Advax delta inulin adjuvant, could provide heterologous protection against WNV infection in wild-type and β2-microglobulin-deficient (β2m^−/−) murine models. Mice immunized twice or even once with JE-ADVAX were protected against lethal WNV challenge even when mice had low or absent serum cross-neutralizing WNV titers prior to challenge. Similarly, β2m^−/− mice immunized with JE-ADVAX were protected against lethal WNV challenge in the absence of CD8⁺ T cells and prechallenge WNV antibody titers. Protection against WNV could be adoptively transferred to naive mice by memory B cells from JE-ADVAX-immunized animals. Hence, in addition to increasing serum cross-neutralizing antibody titers, JE-ADVAX induced a memory B-cell population able to provide heterologous protection against WNV challenge. Heterologous protection was reduced when JE vaccine antigen was administered alone without Advax, confirming the importance of the adjuvant to induction of cross-protective immunity. In the absence of an approved human WNV vaccine, JE-ADVAX could provide an alternative approach for control of a major human WNV epidemic.     

CD40-CD40 ligand interactions promote trafficking of CD8+ T cells into the brain and protection against West Nile virus encephalitis

Recent studies have established a protective role for T cells during primary West Nile virus (WNV) infection. Binding of CD40 by CD40 ligand (CD40L) on activated CD4+ T cells provides an important costimulatory signal for immunoglobulin class switching, antibody affinity maturation, and priming of CD8+ T-cell responses. We examined here the function of CD40-dependent interactions in limiting primary WNV infection. Compared to congenic wild-type mice, CD40(-/-) mice uniformly succumbed to WNV infection. Although CD40(-/-) mice produced low levels of WNV-specific immunoglobulin M (IgM) and IgG, viral clearance from the spleen and serum was not altered, and CD8+ T-cell priming in peripheral lymphoid tissues was normal. Unexpectedly, CD8+ T-cell trafficking to the central nervous system (CNS) was markedly impaired in CD40(-/-) mice, and this correlated with elevated WNV titers in the CNS and death. In the brains of CD40(-/-) mice, T cells were retained in the perivascular space and did not migrate into the parenchyma, the predominant site of WNV infection. In contrast, in wild-type mice, T cells trafficked to the site of infection in neurons. Beside its role in maturation of antibody responses, our experiments suggest a novel function of CD40-CD40L interactions: to facilitate T-cell migration across the blood-brain barrier to control WNV infection.     

Fas ligand interactions contribute to CD8+ T-cell-mediated control of West Nile virus infection in the central nervous system

West Nile virus (WNV) is a neurotropic flavivirus that causes encephalitis, most frequently in elderly and immunocompromised humans. Previous studies demonstrated that CD8+ T cells utilize perforin-dependent cytolytic mechanisms to limit WNV infection. Nonetheless, the phenotype of perforin-deficient CD8+ T cells was not as severe as that of an absence of CD8+ T cells, suggesting additional effector control mechanisms. In this study, we evaluated the contribution of Fas-Fas ligand (FasL) interactions to CD8+ T-cell-mediated control of WNV infection. Notably, the cell death receptor Fas was strongly upregulated on neurons in culture and in vivo after WNV infection. gld mice that were functionally deficient in FasL expression showed increased susceptibility to lethal WNV infection. Although antigen-specific priming of CD8+ T cells in peripheral lymphoid tissues was normal in gld mice, increased central nervous system (CNS) viral burdens and delayed clearance were observed. Moreover, the adoptive transfer of WNV-primed wild-type but not gld CD8+ T cells to recipient CD8(-/-) or gld mice efficiently limited infection in the CNS and enhanced survival rates. Overall, our data suggest that CD8+ T cells also utilize FasL effector mechanisms to contain WNV infection in Fas-expressing neurons in the CNS.     

Naturally Induced Humoral Immunity to West Nile Virus Infection in Raptors

West Nile virus (WNV) infection can be fatal to many bird species, including numerous raptors, though population- and ecosystem-level impacts following introduction of the virus to North America have been difficult to document. Raptors occupy a diverse array of habitats worldwide and are important to ecosystems for their role as opportunistic predators. We documented initial (primary) WNV infection and then regularly measured WNV-specific neutralizing antibody titers in 16 resident raptors of seven species, plus one turkey vulture. Most individuals were initially infected and seroconverted between July and September of 2003, though three birds remained seronegative until summer 2006. Many of these birds became clinically ill upon primary infection, with clinical signs ranging from loss of appetite to moderate neurological disease. Naturally induced WNV neutralizing antibody titers remained essentially unchanged in some birds, while eight individuals experienced secondary rises in titer presumably due to additional exposures at 1, 2, or 3 years following primary infection. No birds experienced clinical signs surrounding or following the time of secondary exposure, and therefore antibodies were considered protective. Results of this study have implications for transmission dynamics of WNV and health of raptor populations, as well as the interpretation of serologic data from free-ranging and captive birds. Antibodies in raptors surviving WNV may persist for multiple years and protect against potential adverse effects of subsequent exposures.     

Exposure of Eurasian magpies and turtle doves to West Nile virus during a major human outbreak, Greece, 2011

A major number of West Nile virus (WNV) infections in humans occurred in 2010 in northern Greece, with 262 laboratory confirmed cases. In 2011, fewer cases were reported, but the pattern was more dispersed throughout the Greek mainland. Isolated strains were similar to lineage 2 strains detected in previous years in Austria and Hungary from birds of prey. We conducted a serological surveillance study on hunter-harvested wild birds, to determine possible exposure of avian species during the current outbreak. Serum samples from a total of 113 Eurasian magpies and 85 turtle doves (abundant resident and migratory avian species, respectively, with potential roles in WNV epidemiology) were tested. These birds were hunter-harvested during 2011 from various prefectures both affected and not affected by the WNV outbreak in Greece. Sera were tested for the presence of WNV IgG antibodies by indirect immunofluorescence assay (IFA). Verification of positive results by a micro-virus neutralization test (VNT) was also performed. A total of 23 out of 113 (20.4%) Eurasian magpies and 6/85 (7.1%) turtle doves were found positive. Results showed association of human cases with wild birds’ exposure to the virus; no avian sera were found positive in prefectures not affected by the WNV outbreak. In contrast, positive avian sera were found in every prefecture that human WNV cases occurred in 2011. High seroprevalence in Eurasian magpies suggests high activity of WNV in the areas. Findings of past exposure of migratory birds like turtle doves to WNV upon their arrival in resting areas in Greece suggest various avian species with similar migration traits as target species for viral isolation studies, as they can be considered candidates for the introduction of WNV lineage 2 in Greece from Central Europe.     

High Prevalence of West Nile Virus in Domestic Birds and Detection in 2 New Mosquito Species in Madagascar

West Nile virus is an arthropod-borne zoonosis transmitted by a large number of mosquito species, and birds play a key role as reservoir of the virus. Its distribution is largely widespread over Africa, Asia, the Americas and Europe. Since 1978, it has frequently been reported in Madagascar. Studies described a high seroprevalence level of the virus in humans in different areas of the island and a human fatal case of WNV infection was reported in 2011. Despite these reports, the epidemiology of WNV in Madagascar, in particular, viral circulation remains unclear. To explore the transmission of WNV in two rural human populations of Madagascar, we investigated local mosquitoes and poultry for evidence of current infections, and determined seroprevalence of candidate sentinel species among the local poultry. These 2 areas are close to lakes where domestic birds, migratory wild birds and humans coexist. Serological analysis revealed WNV antibodies in domestic birds (duck, chicken, goose, turkey and guinea fowl) sampled in both districts (Antsalova 29.4% and Mitsinjo 16.7%). West Nile virus nucleic acid was detected in one chicken and in 8 pools of mosquitoes including 2 mosquito species (Aedeomyia madagascarica and Anopheles pauliani) that have not been previously described as candidate vectors for WNV. Molecular analysis of WNV isolates showed that all viruses detected were part of the lineage 2 that is mainly distributed in Africa, and were most closely matched by the previous Malagasy strains isolated in 1988. Our study showed that WNV circulates in Madagascar amongst domestic birds and mosquitoes, and highlights the utility of poultry as a surveillance tool to detect WNV transmission in a peri-domestic setting.     

CD4+ T-cell responses are required for clearance of West Nile virus from the central nervous system

Although studies have established that innate and adaptive immune responses are important in controlling West Nile virus (WNV) infection, the function of CD4(+) T lymphocytes in modulating viral pathogenesis is less well characterized. Using a mouse model, we examined the role of CD4(+) T cells in coordinating protection against WNV infection. A genetic or acquired deficiency of CD4(+) T cells resulted in a protracted WNV infection in the central nervous system (CNS) that culminated in uniform lethality by 50 days after infection. Mice surviving past day 10 had high-level persistent WNV infection in the CNS compared to wild-type mice, even 45 days following infection. The absence of CD4(+) T-cell help did not affect the kinetics of WNV infection in the spleen and serum, suggesting a role for CD4-independent clearance mechanisms in peripheral tissues. WNV-specific immunoglobulin M (IgM) levels were similar to those of wild-type mice in CD4-deficient mice early during infection but dropped approximately 20-fold at day 15 postinfection, whereas IgG levels in CD4-deficient mice were approximately 100- to 1,000-fold lower than in wild-type mice throughout the course of infection. WNV-specific CD8(+) T-cell activation and trafficking to the CNS were unaffected by the absence of CD4(+) T cells at day 9 postinfection but were markedly compromised at day 15. Our experiments suggest that the dominant protective role of CD4(+) T cells during primary WNV infection is to provide help for antibody responses and sustain WNV-specific CD8(+) T-cell responses in the CNS that enable viral clearance.     

Experimental infections of birds with Turlock virus

Two experiments were conducted with five gallinaceous and one passerine bird species to determine their responses to Turlock (TUR) virus inoculations. Inoculation of TUR strain 847-32 into bobwhites, chukars, ring-necked pheasants, chickens, and Japanese quail did not product detectable viremias. The first four species, however, did respond with neutralizing antibody. Inoculation of chickens with strain 69V-1095 resulted in a viremia which lasted 5 days and had a peak mean titer of 2.0 log 10 PFU per 0.2 ml of blood on the third day after infection. The observation that viremic birds exhibited no noticeable virus-associated morbidity or mortality suggested that TUR virus does not have a detrimental effect on free-ranging populations of the avian hosts studied during this investigation.     

Dynamics of West Nile Virus Persistence in House Sparrows (Passer domesticus)

West Nile Virus (WNV) is now endemic throughout North America, with annual recurrence dependent upon successful overwintering when cold temperatures drive mosquito vectors into inactivity and halt transmission. To investigate whether avian hosts may serve as an overwintering mechanism, groups of eight to ten House Sparrows were experimentally infected with a WN02 genotype of WNV and then held until necropsy at 3, 5, 7, 9, 12, 15, or 18 weeks post-infection (pi) when they were assessed for the presence of persistent infection. Blood was collected from all remaining birds every two weeks pi, and sera tested for WNV RNA and WNV neutralizing antibodies. West Nile virus RNA was present in the sera of some birds up to 7 weeks pi and all birds retained neutralizing antibodies throughout the experiment. The detection of persistently infected birds decreased with time, from 100% (n = 13) positive at 3 weeks post-infection (pi) to 12.5% (n = 8) at 18 weeks pi. Infectious virus was isolated from the spleens of birds necropsied at 3, 5, 7 and 12 weeks pi. The current study confirmed previous reports of infectious WNV persistence in avian hosts, and further characterized the temporal nature of these infections. Although these persistent infections supported the hypothesis that infected birds may serve as an overwintering mechanism, mosquito-infectious recrudescent viremias have yet to be demonstrated thereby providing proof of principle.     

A recombinant influenza A virus expressing domain III of West Nile virus induces protective immune responses against influenza and West Nile virus

West Nile virus (WNV) continues to circulate in the USA and forms a threat to the rest of the Western hemisphere. Since methods for the treatment of WNV infections are not available, there is a need for the development of safe and effective vaccines. Here, we describe the construction of a recombinant influenza virus expressing domain III of the WNV glycoprotein E (Flu-NA-DIII) and its evaluation as a WNV vaccine candidate in a mouse model. FLU-NA-DIII-vaccinated mice were protected from severe body weight loss and mortality caused by WNV infection, whereas control mice succumbed to the infection. In addition, it was shown that one subcutaneous immunization with 10(5) TCID(50) Flu-NA-DIII provided 100% protection against challenge. Adoptive transfer experiments demonstrated that protection was mediated by antibodies and CD4+T cells. Furthermore, mice vaccinated with FLU-NA-DIII developed protective influenza virus-specific antibody titers. It was concluded that this vector system might be an attractive platform for the development of bivalent WNV-influenza vaccines.     

CD8+ T cells require perforin to clear West Nile virus from infected neurons

Injury to neurons after West Nile virus (WNV) infection is believed to occur because of viral and host immune-mediated effects. Previously, we demonstrated that CD8+ T cells are required for the resolution of WNV infection in the central nervous system (CNS). CD8+ T cells can control infection by producing antiviral cytokines (e.g., gamma interferon or tumor necrosis factor alpha) or by triggering death of infected cells through perforin- or Fas ligand-dependent pathways. Here, we directly evaluated the role of perforin in controlling infection of a lineage I New York isolate of WNV in mice. A genetic deficiency of perforin molecules resulted in higher viral burden in the CNS and increased mortality after WNV infection. In the few perforin-deficient mice that survived initial challenge, viral persistence was observed in the CNS for several weeks. CD8+ T cells required perforin to control WNV infection as adoptive transfer of WNV-primed wild-type but not perforin-deficient CD8+ T cells greatly reduced infection in the brain and spinal cord and enhanced survival of CD8-deficient mice. Analogous results were obtained when wild-type or perforin-deficient CD8+ T cells were added to congenic primary cortical neuron cultures. Taken together, our data suggest that despite the risk of immunopathogenesis, CD8+ T cells use a perforin-dependent mechanism to clear WNV from infected neurons.     

Comparison of immune responses of brown-headed cowbird and related blackbirds to west Nile and other mosquito-borne encephalitis viruses

The rapid geographic spread of West Nile virus (family Flaviviridae, genus Flavivirus, WNV) across the United States has stimulated interest in comparative host infection studies to delineate competent avian hosts critical for viral amplification. We compared the host competence of four taxonomically related blackbird species (Icteridae) after experimental infection with WNV and with two endemic, mosquito-borne encephalitis viruses, western equine encephalomyelitis virus (family Togaviridae, genus Alphavirus, WEEV), and St. Louis encephalitis virus (family Flaviviridae, genus Flavivirus, SLEV). We predicted differences in disease resistance among the blackbird species based on differences in life history, because they differ in geographic range and life history traits that include mating and breeding systems. Differences were observed among the response of these hosts to all three viruses. Red-winged Blackbirds were more susceptible to SLEV than Brewer's Blackbirds, whereas Brewer's Blackbirds were more susceptible to WEEV than Red-winged Blackbirds. In response to WNV infection, cowbirds showed the lowest mean viremias, cleared their infections faster, and showed lower antibody levels than concurrently infected species. Brown-headed Cowbirds also exhibited significantly lower viremia responses after infection with SLEV and WEEV as well as coinfection with WEEV and WNV than concurrently infected icterids. We concluded that cowbirds may be more resistant to infection to both native and introduced viruses because they experience heightened exposure to a variety of pathogens of parenting birds during the course of their parasitic life style.     

Neuroendocrine and behavioral implications of endocrine disrupting chemicals in quail

Studies in our laboratory have focused on endocrine, neuroendocrine, and behavioral components of reproduction in the Japanese quail. These studies considered various stages in the life cycle, including embryonic development, sexual maturation, adult reproductive function, and aging. A major focus of our research has been the role of neuroendocrine systems that appear to synchronize both endocrine and behavioral responses. These studies provide the basis for our more recent research on the impact of endocrine disrupting chemicals (EDCs) on reproductive function in the Japanese quail. These endocrine active chemicals include pesticides, herbicides, industrial products, and plant phytoestrogens. Many of these chemicals appear to mimic vertebrate steroids, often by interacting with steroid receptors. However, most EDCs have relatively weak biological activity compared to native steroid hormones. Therefore, it becomes important to understand the mode and mechanism of action of classes of these chemicals and sensitive stages in the life history of various species. Precocial birds, such as the Japanese quail, are likely to be sensitive to EDC effects during embryonic development, because sexual differentiation occurs during this period. Accordingly, adult quail may be less impacted by EDC exposure. Because there are a great many data available on normal development and reproductive function in this species, the Japanese quail provides an excellent model for examining the effects of EDCs. Thus, we have begun studies using a Japanese quail model system to study the effects of EDCs on reproductive endocrine and behavioral responses. In this review, we have two goals: first, to provide a summary of reproductive development and sexual differentiation in intact Japanese quail embryos, including ontogenetic patterns in steroid hormones in the embryonic and maturing quail. Second, we discuss some recent data from experiments in our laboratory in which EDCs have been tested in Japanese quail. The Japanese quail provides an excellent avian model for testing EDCs because this species has well-characterized reproductive endocrine and behavioral responses. Considerable research has been conducted in quail in which the effects of embryonic steroid exposure have been studied relative to reproductive behavior. Moreover, developmental processes have been studied extensively and include investigations of the reproductive axis, thyroid system, and stress and immune responses. We have conducted a number of studies, which have considered long-term neuroendocrine consequences as well as behavioral responses to steroids. Some of these studies have specifically tested the effects of embryonic steroid exposure on later reproductive function in a multigenerational context. A multigenerational exposure provides a basis for understanding potential exposure scenarios in the field. In addition, potential routes of exposure to EDCs for avian species are being considered, as well as differential effects due to stage of the life cycle at exposure to an EDC. The studies in our laboratory have used both diet and egg injection as modes of exposure for Japanese quail. In this way, birds were exposed to a specific dose of an EDC at a selected stage in development by injection. Alternatively, dietary exposure appears to be a primary route of exposure; therefore experimental exposure through the diet mimics potential field situations. Thus, experiments should consider a number of aspects of exposure when attempting to replicate field exposures to EDCs.