Encephalomyelitis Infection in the Snowshoe Hare

Transcription

1 INFECTION AND IMMUNITY, My 1975, P Americn Society for Microbiology Environmentl Modifiction of Western Equine Encephlomyelitis Infection in the Snowshoe Hre (Lepus mericnus) Vol. 11, No. 5 Printed in U.SA. A. L. KIORPES* AND T. M. YUILL Deprtment of Veterinry Science, College of Agriculture nd Life Sciences, Univerity of Wisconsin-Mdison, Mdison, Wisconsin Received for publiction 18 December 1974 The snowshoe hre (Lepus mericnus) could be infected with western equine encephlomyelitis (WEE) virus nd produce viremi. Furthermore, viremi in hres exposed to vrible climtic conditions differed significntly from viremis seen in the control nimls held t constnt tempertures. The viremi durtion nd titer were incresed in nimls subjected to fluctuting temperture nd humidity. The time of onset of viremi ws ccelerted. Antibody response lso incresed in nimls exposed to vrying tempertures when compred with controls held t constnt temperture nd humidity. Snowshoe hres were studied t two distinct sesonl periods: winter, before reproductive ctivity; nd summer, during reproductive midseson. Winter nimls experienced greter viremi thn did summer hres when exposed to fluctuting tempertures, suggesting sesonlity in the hre's susceptibility to host modifiction by environmentl influences. These findings implicte the snowshoe hre s possible mmmlin mplifying host for WEE virus in the borel forest. Westem equine encephlomyelitis (WEE) virus is n gent of significnt public helth importnce, the epizootiology of which is not yet completely understood. Throughout most of its rnge, the virus is believed to be mintined in sylvn cycle between mosquitoes nd birds (15). In the borel forest, however, there is no known vector for WEE virus nd the role tht smll mmmls ply in the ecology of this virus is unstudied. It ws previously believed tht smll mmmls were not importnt in the epizootiology of WEE (11, 15). However, during the course of long-term popultion study in the borel forest ner Rochester, Albert, Cnd, neutrlizing ntibody to WEE virus hs been detected on severl occsions in the snowshoe hre (7). On two occsions, epizootics of WEE swept through tht hre popultion severl months prior to epidemics in humn beings nd horses in the trnsitionl spen prklnd re 100 miles south of Rochester (8, 22). Serologicl studies indicte tht snowshoe hres in Albert hve been infected in the field (23). Demonstrtion of the presence of neutrlizing ntibodies, however, does not prove tht this species responds to infection by the development of viremi dequte for the infection of rthropod vectors. Vertebrte host response to virl infection hs been studied with both domestic nd cptive wild nimls by rtificilly infecting them in the lbortory nd monitoring viremi nd ntibody production. These re trditionl procedures for estblishing epidemiologicl reltionships between host nd prsite. It hs been ssumed tht such reltionships re not significntly ltered by the lbortory milieu, nd tht the course of infection under experimentl nd nturl conditions is essentilly the sme. Consequently, reltively little considertion hs been given to the role the environment plys in modifying the response of vertebrte host to replicting virus. In these studies, the effect of environmentl modifiction of nturlly occurring host-virus reltionship ws investigted. This investigtion employed the snowshoe 986 hre to determine whether it could develop viremi gret enough to serve s source of infectious blood mels for susceptible rthropods. Since chnges in viremi levels would lter the bility of n infected niml to serve s potentil mplifying host for the cquisition of virus for trnsmission by mosquito vectors, we lso exmined the effect temperture-humidity fluctutions, cting s environmentl stressors, hd on the levels of circulting virus nd ntibody in WEE virus-infected snowshoe hres. This communiction reports the results of our experiments. MATERIALS AND METHODS Experimentl nimls. Snowshoe hres from the re of Rochester, Albert (540 north ltitude), were trpped, tgged, shipped to Mdison, nd held in

2 VOL. 11, 1975 ENVIRONMENTAL MODIFICATION OF WEE INFECTION qurntine fcility t Chrmny Frm. Hres were housed individully in cges (84 by 38 by 61 cm), fed specilly mde rbbit rtion (University of Wisconsin feedmills), nd wtered d libitum. Animls were bled for serologicl studies from the centrl er rtery using 5-ml plstic syringes equipped with 23-guge needles. Serum ws septiclly collected nd stored in 1-drm vils t -25 C. The BHK-21 microneutrliztion test (13) ws employed for ntibody ssy. Cell cultures nd medi. BHK-21 cells (clone 13) dpted to fetl bovine serum, originlly obtined from the Deprtment of Virology, U.S. Army Medicl Component, SEATO, Bngkok, Thilnd, were grown in 32-oz (bout liters) glss prescription bottles in 30 ml of medium. Growth medium for cell cultures consisted of medium 199 in Hnks blnced slt solution without sodium bicrbonte (GIBCO, Grnd Islnd, N.Y.), 5% fetl bovine serum heted t 56 C for 30 min, nd ntibiotics giving finl concentrtion of 200 U of potssium penicillin G per ml nd 200 Ag of streptomycin sulfte per ml, djusted to ph of 7.2. Outgrowth medium for use in the microneutrliztion test ws similr to the bove except tht 10% fetl bovine serum ws used nd the finl ph ws djusted to 7.5. All ph djustments were ccomplished using either 0.1 N HCl or 7.5% NHCO3. Virus. WEE virus employed in experimentl inocultion ws lyophilized stock of the Fleming isolte from ftl humn cse in Cliforni. Seed virus ws supplied by the Center for Disese Control with recorded history of ten pssges in mice nd two pssges in eggs. The wet-stock virus used in the microneutrliztion test ws of the sme origin with history of 11 mouse pssges nd ws stored s 10% solution of growth medium contining 10% fetl bovine serum in mechnicl freezer t -65 C. Host modifiction experiments. Two experiments were performed t two distinct periods of the yer. Snowshoe hres inoculted in Februry 1973 (winter study) were reproductively inctive. Those inoculted in June 1973 (summer study) were reproductively ctive. Prior to ech experiment, hres were segregted into two groups, experimentl nd control. Animls were evenly distributed mong groups by ge nd sex. All experimentl group nimls were exposed to fluctuting temperture (rnge, -10 to 25 C) nd humidity (rnge, 70 to 80% reltive humidity). Control nimls for these studies were held t 10 C nd 50% reltive humidity. The fluctuting-temperture progrm ws bsed on ctul wether dt gthered t the sttion in Athbsc, Albert, ner the Rochester reserch site. All host modifiction experiments were performed in the University of Wisconsin Biotron, controlledenvironment fcility. Experimentl nd control snowshoe hres were individully cged in two seprte but identicl rooms (2.6 by 3.7 m), where temperture, humidity, nd photoperiod were controlled within nrrow limits by centrl computer. Prior to inocultion, ll nimls were cclimted for 6 dys to cyclicl dily high (15.5 C) nd then low (4.5 C) temperture t constnt 50% reltive humidity. Within 12 h fter the termintion of the cclimtion sequence nd the beginning of the experimentl environmentl temperture regimen, snowshoe hres were inoculted subcutneously with ml suspension of virus described below for ech experiment. All nimls were bled from the centrl er rtery for 6 dys t 24-h intervls for titrtion of ntibody nd circulting virus. For viremi titrtions, 1 ml of fresh whole blood ws plced in 1-drm vil, cpped, seled with Prfilm (Americn Cn Co., Neenh, Wis.), nd quick frozen in n lcohol/dry-ice bth. The remining blood ws llowed to clot for serum extrction. After the initil 6-dy period, blood ws tken for serum smples only on the following schedule: every other dy for week 2, every third dy for week 3, once in week 4, nd once in the following month. Viremi ws quntified by intrcerebrl inocultion in 3- to 4-dy-old suckling mice, using the Reed nd Muench end-point determintion method (14). Unless otherwise stted, ll viremi titers were expressed s log10 of the virus dilution. Antibody titrtion ws performed on seril twofold dilutions of ser mde from the initil 1:2 dilution nd tested ginst 100 men tissue culture lethl doses of virus. End points for serum titrtion were red microscopiclly s the highest serum dilution which prevented cytopthic effect. Sttisticl nlysis. Differences in viremi mplitude nd durtion nd ntibody response were tested for significnce by the simple, unpired, one-tiled Student's t test. Although nimls were evenly distributed s to ge nd sex, this ws not piring. The one-tiled test of significnce ws used since onedirectionl devition could be resonbly expected from the literture. Differences in the time of onset of viremi between groups were tested for significnce in contingency tble (2 by 2) (18). Significnce of this test ws computed with one-tiled z vlues t the 0.05 level. Antibody nlysis ws bsed on geometric men log2 titers. RESULTS Effect of temperture-humidity fluctution on viremi. (i) Winter study. A totl of 14 snowshoe hres ws inoculted with 7.0 x 10' men suckling mouse lethl doses of WEE virus. All seven experimentl hres nd six of seven controls developed demonstrble viremi. The men viremi titers of experimentl hres on dys 1, 2, nd 3 (2.54, 4.65, 3.17) were significntly higher (P < 0.05) thn those of controls (1.40, 2.93, 2.53) (Tble 1). Control hre 64 ws not included in these clcultions becuse it developed no demonstrble viremi during the 5-dy period of observtion. Dy 4 viremis were found not to be significntly different becuse of the smll number of nimls hving persistent viremi. The differences in viremi durtion nd time of onset between the two groups were lso tested for significnce. Durtion ws clculted s the totl dys of viremi in ech group divided by the number of nimls in tht group which developed demonstrble viremi during the 5-dy observtion period. The difference, 3.57

3 988 KIORPES AND YUILL INFECT. IMMUN. dys (experimentl) compred with 2.51 dys (control), ws significnt t P < Time of viremi onset ws compred in contingency tble (2 by 2) s described. All of the experimentl hres developed detectble viremi on dy 1, wheres only hlf (three of six) of the controls did so. This difference ws significnt t P < (ii) Summer study. All six experimentl nd six control nimls inoculted with 3 x 105 men suckling mouse lethl doses of WEE virus responded with viremi. Men viremi titers for experimentl nd control hres for dys 1, 2, nd 3 post-inocultion were 1.8, 3.2, 2.7 nd 1.0, 3.1, 1.5. These differences were not significnt t P < 0.05 (Tble 2). Viremi durtion nd onset were compred s previously described. Viremi lsted 3.2 dys in experimentl hres nd 2.2 dys in controls. This difference of 1.0 dys ws highly significnt t P < On dy 1 post-inocultion, four of five experimentl hres developed detectble viremi, wheres only two of five controls did. These differences were not significnt t P < Effect of temperture-humidity fluctution TABLE 1. Men viremi titer of snowshoe hres inoculted with 7 x 104 men suckling mouse lethl doses of WEE virus t fluctuting low men or constnt low tempertures (winter study) Dys post- Temperture group Level of inocultion Low fluctuting Low constnt significnceb ± ± ± NS Titer expressed s the log10 per milliliter + the stndrd error. bclculted s one-tiled Student's t test. NS, Not significnt (P < 0.05). TABLE 2. Men viremi titer of snowshoe hres inoculted with 3 x 105 men suckling mouse lethl doses of WEE virus t fluctuting low men or constnt low tempertures (summer study) Temperture group Dys postinocultion Low fluctuting Low constnt Level of significnce NS NS NS NS Titer expressed s the log,0 per milliliter ± stndrd error. bclculted s one-tiled Student's t test. NS, Not significnt (P > 0.05). on ntibody. (i) Winter study. All experimentl nd six of seven control hres responded with t lest fourfold increse in ntibody titer ginst WEE virus. Above this level, responses were highly vried nd were reflected in lrge stndrd error (Tble 3). Comprison of log2 geometric men titers showed experimentl hres hd significntly higher ntibody titers thn did controls. (ii) Summer study. All surviving experimentl hres (three of five) nd four of five controls developed fourfold increses in ntibody titer. Antibody to WEE virus ppered on dy 4 nd rose slightly on dys 5 nd 6. On dy 6, this study ws terminted nd ll surviving hres were removed to the niml holding fcilities t Chrmny Frm. On dy 14, these nimls were bled. Comprison of log2 geometric titers showed no difference between experimentl nd control hres (Tble 4). DISCUSSION Our investigtion showed tht snowshoe hres could be infected with WEE virus, developed trnsient viremis, nd produced neutrlizing ntibodies. This is the first report of WEE virus infection with viremi in the snowshoe hre. In ddition, the results indicte tht the responses of the snowshoe hres to WEE virus infection re not sttic with respect to either seson or environmentl conditions. When exposed to fluctuting low men tempertures, these nimls exhibited chnges in viremi nd ntibody compred with controls held t constnt temperture. To our knowledge, this is the first report of environmentl modifiction of the response of non-hibernting wild mmmlin host fter infection by togvirus. We did not investigte the possible mechnisms underlying the observed viremi nd ntibody chnges. Perhps the most likely explntion is the concept of stress response of mmmls (16) nd the effects tht stress nd corticoids hve been shown to hve s immunosuppressnts (1, 5, 6, 17, 20) nd on nonimmune (nonspecific) defense systems (10), including reduction of interferon production (3, 4, 9). Antibody titers of winter study experimentl hres were observed to be significntly higher thn those of controls. There re three possible interprettions of this observtion: (i) fluctuting temperture stress induced enhncement of the immune response in experimentl hres; (ii) exposure to constnt temperture cused depression of the immune response in control hres; (iii) the different temperture regimen hd little effect on the immune response, nd differences observed in ntibody titers reflected

4 VOL. 11, 1975 ENVIRONMENTAL MODIFICATION OF WEE INFECTION TABLE 3. Comprison of men ntibody titers of snowshoe hres inoculted with 7 x 104 men suckling mouse lethl doses of WEE virus t fluctuting nd constnt low men tempertures (winter study) Dys post Temperture group" Lvel of inocultion Low fluctuting Low constnt cnceb NS ± ± ± ± NS ± ± i ± i ± Reciprocl of twofold dilution end point (4 stndrd error) in microneutrliztion test. "Clculted using Student's one-tiled t test nd geometric men titers. NS, Not significnt (P > 0.05). TABLE 4. Comprison of men ntibody titers of snowshoe hres inoculted with 3.0 x 10" men suckling mouse lethl doses of WEE virus t fluctuting low nd constnt tempertures (summer study). Dys post- Temperture group Level of inocultion Low fluctuting Low constnt significnce" NS NS NS Reciprocl of twofold dilution end point (+ stndrd error) in microneutrliztion test. bclculted using Student's one-tiled t test nd geometric men titers. NS, Not significnt (P > 0.05). 989 the levels of viremi which were in turn indictive of ntigenic mss. --Although stress-induced enhncement of ntibody production hs been postulted, such phenomenon hs been lrgely discredited. Neither ppernce nor titer of ntibody elicited in stressed or control mice infected with vesiculr stomtitis virus differed (21). Our observtion of incresed viremi under conditions of fluctuting tempertures does not pper to be consistent with the hypothesis tht these sme conditions enhnce the immune response. Nor does the hypothesis tht constnt tempertures suppress the immune response seem likely. Exposures to constnt cold temperture did not lter the immune response of mice (2) or rbbits (12). The most likely interprettion of the ntibody dt presented here is tht fluctuting temperture stress hs little effect on the ntibody-producing system of snowshoe hres infected with WEE virus. In our study, the significntly greter ntibody titers in experimentl hres closely prlleled the significntly greter viremi in this group compred to controls. In summer study hres, no difference between experimentl nd control group ntibody titers ws ssocited with no difference between group viremi titers. A comprison of winter nd summer study experimentl group viremi dt suggests tht nimls were more susceptible to stress plus infection erlier in the yer thn lter. This difference in host modifiction by environmentl influences cnnot be explined by seson lone, since winter nd summer studies showed similr viremi response under constnt tempertures. Similr sesonl phenomen hve been observed in lbortory hmsters (19). These nimls showed greter susceptibility to stress plus infection with poliomyelitis virus in Februry thn in either My or September. These differences ppered to be correlted with drenl corticl ctivity, lthough further investigtion in this re is needed. In this study, the more nturl condition of vrible climtic conditions must be considered more stressful thn the rtificil lbortory or control sitution of sttic temperture nd humidity. One impliction of these findings is tht in lbortory studies designed to test host-pthogen reltionships mong wild species, erroneous conclusions my be reched if the results of such studies re extrpolted to the field condition without considering environmentl effects. Tht the snowshoe hre my be host of WEE virus in the wild is consistent with two epidemiologicl studies mde during the lst decde. Between the period April to June 1963, nd gin in 1965, epizootics of WEE swept through the hre popultion in the borel forest round Rochester, Albert (23). Within these 3-month periods, neutrlizing ntibody prevlence in cptured hres rose from less thn 10% to greter thn 90% for WEE virus. After June, the prevlence declined nd then leveled off. On both occsions, the epizootics in hres were followed 2 to 3 months lter by epidemics of WEE in humn beings nd epizootics in horses in the trnsitionl spen prklnd to the south. The present report supports the erlier suggestion by Yuill nd Hnson (22) tht the snowshoe hre my hve cted s virus mplifier on both occsions. ACKNOWLEDGMENTS This study ws supported by Public Helth Service grnt AI from the Ntionl Institute of Allergy nd Infectious

5 990 KIORPES AND YUILL INFECT. IMMUN. Diseses. Prtil support for the senior uthor ws provided by the Institute for Environmentl Studies nd College of Agriculturl nd Life Sciences, University of Wisconsin, Mdison. We wish to thnk Pm Lthm, Sue Brton, nd Judy Christofferson for their superb technicl ssistnce, nd Don Bosmn nd Dvid Bermn for their dvice nd criticism. The id of Lloyd B. Keith nd students of the Deprtment of Wildlife Ecology in trpping hres is recognized. LITERATURE CITED 1. Brown, G. L., M. V. Rothluf, R. H. Heinzerling, nd T. S. Myers Antibody formtion in rbbits dpted nd nondpted to high ltitude. Infect. Immun. 3: Chffee, R. R. J., nd C. M. Mrtin The immune response in cold cclimted mice. Proc. Soc. Exp. Biol. Med. 111: Chng, S-S, nd A. F. Rsmussen, Jr Stressinduced suppression of interferon products in virus infected mice. Nture (London) 205: DeMeyer, E., nd J. DeMeyer Two-sided effect of steroids on interferon in tissue culture. Nture (London) 197: Elliot, E. V., nd N. R. St. C. Sinclir Effect of cortisone cette on 19S nd 7S hemolysin ntibody. A time course study. Immunology 15: Gisler, R. H., nd L. Schenkel-Hulliger Hormonl regultion of the immune response. II. Influence of pituitry nd drenl ctivity on immune responsiveness in vitro. Cell. Immunol. 2: Hoff, G. L., T. M. Yuill, J. 0. Iversen, nd R. P. Hnson Selected microbil gents in snowshoe hres nd other vertebrtes of Albert. J. Wildl. Dis. 6: Iversen, J. O., G. L. Sewright, nd R. P. Hnson Serologic survey for rboviruses in Centrl Albert. Cn J. Public Helth 62: Jensen, M. M Possible mechnisms of impired interferon production in stressed mice. Proc. Soc. Exp. Biol. Med. 142: Kss, E. H., nd M. Finlnd Adrenocorticl hormones in infection nd immunity. Annu. Rev. Microbiol. 7: Miles, J. A. R Epidemiology of rthropod-borne encephlitides. Bull. W. H : Northey, W. T Studies on the interreltionships of cold environment, immunity nd resistnce to infection. I. Qulittive nd quntittive studies on the immune response. J. Immunol. 94: Pntuwtn, S., W. H. Thompson, D. M. Wtts, nd R. P. Hnson Experimentl infection of chipmunks nd squirrels with LCrosse nd Trivitttus viruses nd biologicl trnsmission of LCrosse virus by Aedes triseritus. Am. J. Trop. Med. Hyg. 21: Reed, L. J., nd H. Muench A simple method of estimting fifty percent endpoints. Am. J. Hyg. 27: Reeves, W. C., nd W. McD. Hmmon Epidemiology of the rthropod-borne encephlitides in Kern County, Cliforni, Univ. Clif. Berkeley Publ. Public Helth. 4: Selye, H The physiology nd pthology of exposure to stress. Act Inc. Med. Publ., Montrel. 17. Solomon, G. F Stress nd ntibody response in rts. Int. Arch. Allergy 35: Steele, R. G. D., nd J. Torrie Principles nd procedures of sttistics, p McGrw-Hill, New York. 19. Teodoru, C. V., nd G. Schwrtzmn Endocrine fctors in pthogenesis of experimentl poliomyelitis in hmsters. Role of inocultory nd environmentl stress. Proc. Soc. Exp. Biol. Med. 91: Vessey, S. H Effect of grouping on levels of circulting ntibodies. Proc. Soc. Exp. Biol. Med. 115: Ymd, A., M. M. Jensen, nd A. F. Rsmussen, Jr Stress nd susceptibility to virl infection. III. Antibody response nd virl retention during voidnce-lerning stress. Proc. Soc. Exp. Biol. Med. 116: Yuill, T. M., nd R. P. Hnson Serologic evidence of Cliforni encephlitis virus nd western equine encephlomyelitis in snowshoe hres. Zoo. Res. 3: Yuill, T. M., J. 0. Iversen, nd R. P. Hnson Evidence for rbovirus infections in popultion of snowshoe hres. A possible mortlity fctor. Bull. Wildl. Dis. 5: