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Jounwl of WUiUtfe 31(4), 1995, EXPERIMENTAL INFECTION OF WADING BIRDS WITH EASTERN EQUINE ENCEPHALITIS VIRUS Robert G. McLean, Wayne J. Crans,’ Donald F. Caccamise,2 James McNelly," Larry J. Kirk,’ Carl J. Mitchell,’ and Charles H. Calisher3 Division of Vector-Some infectious Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Fort Collins, Colorado 80522, USA Mosquito Research and Control, Cook College, Rutgers University, New Brunswtok, New Jersey 08903, -USA Artriropod-borne and Infectious Diseases Laboratory, Colorado State University, Fort Collins, Colorado 8(1523, USA To study the susceptibility of wading birds to eastern equine encephalitis (EEE) virus and to determine their potential reservoir amplifying hosts, fledgling glossy ibises (Ptegadis and captured in New Jersey (USA) and shipped falcinellus) snowy egrets (Egretta thula) to Colorado (USA) where they experimentally inoculated with EEE virus. All 16 snowy egrets and 14 (93%) of 15 of the glossy ibises inoculated became viremic with moderate liters, and all survivors developed neutralizing antibody. Six ibises and two egrets died during the first week after inoculation, and EEE virus isolated from the tissues of three birds. Our experimental results support field evidence about the relative involvement of glossy ibises and snowy egrets in the epizootiology of EEE virus in New Jersey. Key words: Arbovirus, eastern equine encephalitis virus, wading birds, experimental infection, reservoir competence, glossy ibis, Plegadis falcinellus, snowy egret, Egretta thula. INTRODUCTION Eastern equine encephalitis (EEE) virus (Alphavirus, Family Togaviridae) from Canada to Argentina (Acha and Szyfres, 1987) and produces clinical disin humans; equines and other domestic animal species; exotic game bird species, including recently introduced (Dromaius novaehollandiae, Tully et al., 1992); and number of native wild bird species, most notably the whooping (Crus americana) (Dein et al., 1986). Within the United States, EEE virus first isolated in the mid-Atlantic coastal gion during epizootic in equines in 1933, and it probably responsible for epizootics in equines early 1831 (Morris, in the Atlantic 1989). The virus and Gulf coastal states and in isolated, foci in inland states east of the Mississippi River, such Michigan and New York (USA). Human and equine ported nearly every year within the zootic areas; 141 human ported during the 29-yr period of 1964 to 1992 (Calisher, 1994). In the U.S., EEE virus is maintained in enzootic foci in freshwater swamp habitats in wild bird-mosquito transmission cycles months in northern during the latitudes (Dalrymple et al., 1972; Morris et al., 1980; McLean et al., 1985b) and throughout the year in southern latitudes (Stamm et al., 1962; Bigler et al., 1976). Many species of wild birds, mostly passerines, have been incriminated natural maintenance and amplifying hosts and Culiseta melanura has been implicated the principal mosquito vector in these zootic foci (Morris, 1989). Well-established EEE foci have been identified in cedar swamp habitats in southern New Jersey (USA), where the viis maintained in the usual wild birdCs. melanura cycle (Crans et al., 1994). The virus generally remains confined to these swamp habitats; however, it sionally escapes from these foci, probably during the peak of seasonal virus transmission, carried by either infected viremic birds. Two quitos species of wading birds, the glossy ibis {Plegadis falcinellus) and the snowy egret (Egretta thula), roost in wooded swamps and feed in open salt marshes of southern New Jersey. Both species regularly posed to EEE virus there, although glossy MCLEAN ET ibises have significantly higher antibody prevalence than do snowy egrets (W. J. Crans, unpubl.)- Therefore, these birds could to facilitate the movement of EEE virus from enzootic foci if they susceptible to the virus and able to infect mosquitoes. Our objective to determine the laboratory susceptibility and viremic response of the glossy ibis to infection with EEE virus, to confirm and extend previous the susceptibility of the vestigations snowy egret to EEE virus (Kissling et aL, 1954), and to determine the competence and potential of these species vertebrate reservoir hosts. MATERIALS AND METHODS Both glossy ibises and snowy egrets captured by hand fledglings at their in coastal breeding colonies at Stone Harbor in southern New Jersey (3902’N, 7446’W). This of the known history of EEE virus activity state has and is virtually free of mosquitoes (W. ]. Crans, unpubl.). For shipment, the birds placed in large animal shipping containers and immediately transported by overnight air shipto Denver, Colorado (USA), where they collected and directly transported by hicle to Fort Collins, Colorado. Because ibises have earlier breeding season, they captured and shipped first (24 June 1992); two sepshipments of egrets (15 and 22 July 1992) followed. The birds examined and marked with numbered leg bands. A blood sample taken from the brachial vein with ml syringe and 22-gauge needle; the birds then placed in wire cages the floor of biocontainment level 2 (Subcommittee Arbovirus Laboratory Safety, 1980) at the Centers for Disease Control and Prevention laboratory in Fort Collins- Steam-sterilized sand placed the floor of each cage; water and food containers placed inside the cages top of the sand. A roosting bar attached within each cage. Birds fed their natural food; glossy ibises fed horseshoe oral? {Limulus polyfirst phemus) eggs which collected in and shipped from New Jersey, but they gradually switched to commercial, canned cat food containing fish shrimp products provided twice each day. Snowy egrets maintained live flathead minnows {Pim-ephales promelas} obtained from local commercial and placed in fresh water containers twice each day. EEC WAOING 503 Each species divided into three separate groups of five birds each, with the exception of group of egret’s which comprised six birds. The birds in each group inoculated subcutaneously with 0.1 ml of particular dosages of EEE virus (strain NJO/60, World Health Organization Center for Reference and Research, Centers for Disease Control & Prevention, Fort Collins, Colorado); 2.9 login plaque-forming units (PFU), 3.9 Iog,o PFU, and 5,0 logio PFU of virus, respectively. There uninoculated trol birds because of the multiple experimental group’; used and because preinoculation blood samples from alt of the birds tested for virus and specific antibody. The birds in each treatment group kept together in separate cages. Blood samples (0.5 ml) taken described, daily for 7 days and then days 14, 21, and 28 postinoculation (PI); samples drawn from of the snowy egrets at 36 days PI, The blood samples added to equal amount of diluent consist-ing of M199 dium (Gibco, Life Technologies, Inc., Grand Island, New York), antibiotics, 1% bovine albumin (Intergen Company, Purchase, New York), and 20% heat-inactivated fetal bovine (Hyclone Laboratories Inc., Logan, Utah, USA,). The samples kept cool wet ice; the removed after centrifugation at 860 G for 15 min and stored in labeled vials 70 C until tested. Birds observed twice daily for signs of illness. Dead birds, which ally discovered in the morning, placed immediately into sealed bag and kept at 70 C until thawed for the collection of tissue ples, At the conclusion of the experiment, euthanized with COa gas, viving birds For the determination of viremia, 0.1 ml of each diluted specimen placed onto monolayer of Vero cells (American Type Culture Collection,,. Rockville, Maryland, USA), grown in six-well plastic plates; the sample allowed to absorb for hr at 37 C (McLean aL, 1985a). An overlay containing nutrient dium, 1% Noble agar (Difco Laboratories Inc., Detroit, Michigan), and 1:25,000 of neutral red added to the cultures, which then (Gibco) incubated at 37 C in 5% CO.; until plaques counted, for 10 days. When specimens contained plaques too to count, 10-fold dilutions of the specimens made, and the dilutions retested in Vero cell cultures to determine endpoints. About 1-cm3 portions of brain, liver, spleen, and heart tissue aseptically removed from thawed carcasses, washed three times with sterile, phosphate-buffered saline, and macerated in 2 ml of diluent described with mortar and pestle. The mixture G for 10 min, centrifuged at 310 and 10-fold dilutions made from each JOURNAL DISEASES, VOL. 31, 4, OCTOBER 1985 isolated from bloods taken viruses from glossy ibises snowy egrets before had the start of the experiment, and detectable SN antibody against EEE virus. All 16 snowy egrets and 14 (93%) of 15 glossy ibises became viremic following inoculation with EEE virus. No significant Days after Inoculation viremic responses of glossy FIGURE 1. Daily equine ibises and snowy egrets inoculated with encephalitis virus (NJO\60 strain). Virus (PFU)/ml of expressed logic plaque-forming blood in Vero cell culture. Bars represent virus titers and lines represent the percentage viremic. Bars with represent glossy diagonal lines and lines with ibises; bars with dots and lines with square boxes represent snowy egrets. pernatant. An 0.1-ml aliquot of each dilution inoculated onto monolayers of Vero cells for isolation of virus and for determination of described for virus titers specimens. heat-inactivated at 56 Serum specimens C for 30 min and tested for neutralizing (SN) antibody against EEE (NJO/60 strain) virus by the constant virus, serum-dilution neutralization test in Vero cell culture grown in six-well plastic plates (McLean et al., 1998). Equal volumes of and virus, diluted contain 100 to 200 mixed and incubated overnight at 4 PFU, added to C. Then 0.1 ml of the mixture Vero cell cultures and allowed to absorb for hr at 37 C. The inoculated cultures laid and incubated described until plaques counted. A reduction in PFU by 80% compared with positive and negative considered positive. trol specimens Serum specimens positive for SN antibody diluted two-fold and retested to confirm the sults and determine antibody titers. The data analyzed with the Chi-square and Student’s <-tests (Sokal and Rohlf, 1981). With single exception, all birds vived their capture and shipment to Colorado and readily adapted to the laboraglossy ibis failed to eat tory situation; in captivity and died of malnutrition. No found in the viremic differences sponses of either species to the three different dosages of EEE virus inoculated; combined for therefore, the results further analyses. The (+/- SE) peak viremia titer (3.83 +/- 0.42 logic PFU/mI for egrets and 3.43 +/- 0.35 for ibises), overall viremia titer (3.2 +/- 0.23 and 3.0 +/- 0.21, respectively), and duration of viremia (2.2 +/-0.16 days and 1.9 +/- 0.29 all slightly but not days, respectively) significantly higher in the snowy egrets variable responses (Table 1). There for both glossy ibises and snowy egrets (Fig. 1); most birds had low viremia titers of 2 days’ duration, while others had mod4 days erate to high viremia titers of 3 duration. Two ibises had viremia patterns that quite different from each other (Fig. 2), and both died by day 6 PI; EEE isolated from the tissues of only virus of these birds (Bird 1). One snowy unusually high viremia titer egret had (109’3 PFU/ml) of day’s duration; it died the next day. Six ibises and two egrets died during the apparently well egret died experiment; day 3 PI. Generally, during handling sick birds first stopped eating and became had drooping wings and lethargic; ataxia, and all died to 3 days after these noted. One behavioral changes first snowy egret that did not die had bloody day discharge from its mouth, starting iso5 PI and lasting for 4 days. Virus of the six glossy lated from tissues from ibises and from both snowy egrets that died (Table 2), Eastern equine encephalitis viisolated from heart tissue of these three birds and only from the heart of the egret that died during handling. Virus also isolated from the spleen of the ibis and from the brain, liver, and spleen of the AL-EXPERIMENTAL TABLE 1. Response of three species of (strain NJO/60). birds equine encephalitis inoculation with (PI^U/ml)- (+/-SE) Range Number tested Species Number (+/-SE) of peak overall duration antibody- (days) positive Glossy ibis 15 3.4 +/-0.35 2.0-6.5 3.0 0.21 1.9 100 3.8 0.42 2.0-9.3 3.2 0.23 2.2 100 3.3 0.27 2.5-4.2 2.7 0.18 2.1 100 4.3 0.21 3.4 0.32 2.9 100 Snowy egret Whistling ducks’" 16 16 Snowy 6 egret- 6 login plaque-forming Aguirre al. (1992). expressed Results Results from Kissling al, (1954), expressed 3.3-4.8 cell culture (PFU) login LDso ml of ml of blood. blood 24-hr intervals. passerine species, viously observed in second egret. High liters of virus presimilar to viremia titers present in the tissues of this; egret (Table but the bird with the very high viously in snowy egrets and other wading 2), which the day bird species by Kissling et al. (1954) and viremia titer (1093 PFU/ml.) in whistling ducks (Dendrocygna autumbefore its death. Serum neutralizing antibody began to naUs) by Aguirre et al. (1992) (Table 1). Both species developed moderate SN appear by the end of the first: week PI, and all surviving birds of both species had de- antibody response (100% positive by 3 wk tectable antibody by the third week PI PI, Fig. 3), in contrast to the rapid response (Fig. 3). Antibody developed faster in of snowy egrets infected during previous antibody positive study (Kissling et al., 1954) in which all glossy ibises (78% antibody positive by wk PI. The 50% of snowy egrets at 2 wk PI), but significantly higher (T antibody liters 3.68, P 0.001) in snowy egrets up to 4 wk PI (1:116 1:76). Both glossy ibises and snowy egrets highly susceptible to inoculation with EEE the lowest dose of virus used virus; above the 50% infectious in the study dose (IDso) tor these two species. Both species developed similar viremic responses, not uniform although these responses among individual birds. As used in this study, artificially induced infections in ter birds has been shown to yield results similar to those produced by transmission from infected Aedes aegypti mosquitoes (Kissling et al., 1954). The viremia titers of the glossy ibises and snowy egrets in this considerably lower than prestudy OFWILDLIFE DISEASES, 506 TABLE 2. Virus isolation 31, 4, OCTOBER 1995 from tissues of glossy ibises and snowy egrets that died following inoculation with (strain NJO/60). equine encephalitis Species Glossy ibis umber positm!/ number tested 1/6 tested Brain <1.0 Heart 4.3 <1.0 Liver Snowy egret 2/2 Spleen 2.7 Brain 6.7 6.6 Heart Liver 8.3 Spleen Blood (pre vious day) 2.0 9.3 Brain <1.0 Heart Liver Spleen ’xpressed <1.0 <1.0 login plaque-forming snowy egrets in this study had significantly higher liters of antibody during the first few weeks, particularly at 3 wk PI, than the glossy ibises. However, the ibises produced equal liters after 3 wk PI. Apparently, antibody titers had not reached their peak then since the titers continued to in- FIGURE 3. Neutralizing antibody response of glossy ibises and snowy egrets inoculated with equine encephalitis virus (NJO\60 strain). Bars repthe deterreciprocal antibody titer the plaque-reduction neutralization mined in Vero cell culture and lines represent the percentage positive. Bars with diagonal lines and lines with represent glossy ibises; bars with dots and lines with square boxes represent snowy egrets. egrets that group of additional week. In associated study, colony-reared Aedes albopictus mosquitoes allowed selected EEEto feed, and fed readily inoculated snowy egrets used in this study (Mitchell et al., 1993). These mosquitoes became infected with EEE virus after viremic egrets; 60% became feeding infected after ingesting 1031 PFU of virus in the bloodmeal from of the egrets. The IDgo for Ae. albopictus calculated to be 1028 PFU, and the minimum thresh^10 PFU per bloodold of infection meal. The infection threshold and IDso observed for Ae. albopictus (Mitchell et al., similar to those parameters de1993) termined previously for Ae. sollicitans (Sudia et al., 1956). Aedes sollicitans is suspected to be important epizootic and epidemic vector for EEE in southern New Jersey (Crans et al., 1986), and, if given the opportunity, this vector will feed ciconiform birds (Crans et al., 1990). Mortality higher for the glossy ibises than for egrets; however, since histopathologic examination performed and since there separate control group, death caused by EEE virus could only be assumed in the egret that had disseminated, high-titered infection, includin the kept for ET AL-EXPERIMENTAL system. ing infection of the central Death from EEE infection has been documented in number of exotic avian species (Morris, 1989; Tully et al., 1992) and drocygna autumnalis). WADING BIRDS 507 Journal of Wildlife Dis- 28;521-525. BICLER, W. J., E. G. LASSING, E. E, BUFF, E. C. G. L. HOFF. 1976. PRATHER, E. C. BECK, Endemic equine encephalomyelitis in Florida; A twenty-year analysis, 1955-1974. The American Journal of Tropical Medicine and Hygiene 25: 884-890. CALISHER, C. H. 1994. Medically important arboviruses of the United States and Canada. Clinical Microbiology Reviews 7: 89-116. CHAMBERLAIN, R. W., R. K. SIKES, D. B. NELSON, the North W. D. SUDIA. 1954. Studies American arthropod-borne encephalitides. VI. Quantitative determinations of virus-vector lationships, The American Journal of Hygiene 60: 278-285. A. CRANS, W. J., J. MCNELLY, T. L. SCHULZE, equine J. MAIN. 1986. Isolation of cephalitis virus from Aedes sollicitans during epizootic southern New Jersey. Journal of the 68American Mosquito Control Association (Kissling et al., 1954) and Gruidae (Dein et al., 1986) species. The extreme differences in the viremic birds, particularly the sponses of snowy egret with the very high titers of disseminated virus, and death in birds could have resulted from synergistic factors not tested in this study, such debilitating avian retroviruses. From these studies, conclude that glossy ibises and snowy egrets susceptible hosts for the transmission of EE^E vifrom the enzootic vector Cs. melanura in freshwater swamp habitats and could for the virus subsequently 72. epizootic vector Ae. sollicitans in salt marsh D. A. SPRENGERL. J. MCCUISTON, habitats of New Jersey. However, the glossy 1990. The blood-feeding habits of Aedes solliibis appears to be equine (Walker) in relation important bridgof New Jersey I. cephalitis virus coastal ing host for the escape of EEE virus from Host selection determined in by precipitin enzootic swamp habitats in southern New tests wild-caught specimens. Bulletin of the Jersey because of its early and long feeding Society for Vector Ecology 15: 144-148. period in the salt marsh during the D. F. CACCAMISE, J. MCNELLY. 1994. relaEastern equine encephalomyelitis season, its relatively high natural the avian community of coastal cedar tion exposure to EEE virus (W. J. Crans, 31: of Medical 711swamp. Entomology Journal publ.), and its susceptibility and potential 728. reservoir competence for the virus. In DALRYMPLE, J. M., 0. P. YOUNG, B. F. ELDRIDGE, trast, both species appear to be inefficient P. K. RUSSELL. 1972. Ecology of arboviamplifying hosts within the swamp habiMaryland freshwater swamp. III. Vertebrate hosts. American Journal of Epidemiology tats because they had only moderate vi96:129-140. remia titers and Cs. melanura has much F- J., J. W. CARPENTER, G. C. CLARK. R. J. higher infection threshold (Chamberlain DEIN,MONTALI, D. E. C. L. CRABBS, T. F. TSAI, et al., 1954) than Ae. sollicitans. DOCHERTY. 1986- Mortality of captive whoopin native Icteridae ing ACKNOWLEDGMENTS We thank Ronald Shriner, David Duty, and John Liddell for assisting with the transport and of the birds, and to Gordon Smith and George Wiggett for their technical assistance. LITERATURE CITED B. SZYFRES. 1987. Zoonose.s and ACHA, P. N-, and communicable diseases imals, 2nd ed. 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