In the human lower than normal concentrations&nbsp;of IgA in saliva have been found to be associated with stress.In studies of animal welfare and prolonged stress there is a need for nominvasive methods and assays of secretory IgA would be simple to develop for many species. In the present study we studied IgA in the chicken and compared levels of IgA intears. serum and bile. Two strains of chickens were compared. The concentration of IgA in the lacrimal fluid of line C chickens was two-fold higher than that of line 151 chickens. Levels of IgA in their serum and bile where the same. Followingon from the replication of avirulent Newcastle disease virus (NDV)1n the Harderian gland of thc nietitating membiane line C chickens contained NDV — specific lacrimal IgA to a titre of 102 whereas line 151 chickens contained none at 10-1-5. Both lines conmined NDV ~specific IgA in their bile to a titre of 103. Line 151 chickens therefore have a selective deficiency in lacrimal IgA compared to biliary IgA

Hau, J

McRandle, B

O'Brien, Deirdre

Russell, P H

English

Journals from University of Tartu

Sennd .1. Lab Anim. Sei Not 3 1993 1 Volt 20Immunoglobulin A in chickens.Comparison 0ft0ta1 and immune—specific lacrimaland serum IgA levels in two lines of chickensimmunized with Newcastle Disease Virusby P. H. Rursell, B. iMcRandle, Deirdre O’Brien and J HanDepartment of Patho1ogy and Infectious Diseases,Royal Veterinary College, Royal College Street.‘ London NW1 OTU, UK.IntroductionQuantification of both total and immunespecific IgA levels in saliva in humans hasdemonstrated negative correlations betweenperception of stress and IgA concentration(Stone et al, 1987, Jemmozt & Magloirc1988).There are few markers available for the as—sessment of prolonged stress and reducedwelfare in animals and if secretory IgA levelswere depressed in animals this finding mightbe used to develop non-invasive methods forquantifying long-term stress in animals.We have initiated a series of studies an—alysing the generality of this correlation inother animal species. The intensive hus-bandry methods of Chickens make it desir-able to develop reliable non-invasive me-thods of assessing stress and low welfare inaddition to behavioral studies, and the pre-sent study was designed to explore whetherteardrops, which can be readily collectedfrom chickens without the trauma of col-1ecting blood, can be used as an alternativesource of IgA.lgA has been quantified in the serum andbile of chickens by radial immunodiffusion(Leslie at al 1976) and ELISA (Cihak el al1992). In this work we used a specific andsensitive rocket immunoelectrophoretic me—thod, which genera11y has a higher sensiti-vity than immunodiflhsion assays and arecharacterized by small intra— and interassaycotticients of variation to quantify IgAiAn advantage of this assay compared withELISA assays is that when analysing samp-les from the saliva, serum and bile, thethe environmemt of the antigen (IgA), is lesslikely to influence the results.Virus-specific antibodies to infectious bron-chitis virus (Cook. er a]. 1992) and to New-castle Disease Virus (Russell, unpublishedresults) are higher in line C White lcghornchickens than in line 151 birds. We com—pared these two lines of birds for serum. bili—ary and lacrimal IgA after vaccination withNewcastle Disease Virus to demonstratewhether virus—spccific IgA values correlatedto total IgA values.Materials and methodsBirdsWhite leghorn chickens of the ReaseheathC line with B4/4 a11e1es and the East Lans-ing 151 line with B15/15 alleles were bredfrom grandparent stock obtained fromHoughton Poultry Research Station, Eng-land. Their parents were negative for anti—bodies to Newcastle disease virus and otherinfectious agents.The chickens were housed in conventionalanimal rooms at a temperature on +/— 2 Cin a 12 hour light/IZ hour dark light cycle.The animals were kept in single or doublerabbit cages in group sizes declining withtime to two birds per cage to comply withUK Home Office guidelines. The infectedbirds were kept in isolators under negativepressure. The birds had access to water anda commercial chicken feed ad lihizumiTears were collected following applicationofa drop 01‘ glycerine which stimulated tearproduction and kept at —20 C until analysis.161?cnnd. J. Lab Anim, Sci. No. 3 . 1993 . Vol. 20VirusUlster 2C and Hitehner B1 strains of NDVwere grown in the allantoic cavity of eggs toa titre of 108'6 tissue culture infectious units(iu)/0.1 ml as assayed by an indirect immu-noperoxidase monolayer assay (IPMA) withHep-Z cells (Russell 1992). 108'6 iu/0.l mlrepresent about 109-6 egg ID50/0.l m1.ExperimentsLine C and 151 birds of'the same hatch wereinfected with 109‘4 in of the Ulster-ZC orHitchner B1 strain of NDV. The virus wasapplied in 0.6 m1 allantoie fluid between theeyes, nostril and mouth of line C and 151birds of the same hatch. The birds were kil-led 10 days later when aged 65—75 days ofage and the serum tears and cystic bile werecollected for the determination of total IgAby rocket immunoelectrophoresis and foranti-NDV IgA by IPMA as previously de-scribed (Russell 1992).Rocket immunoelectrophoresisRocket immunoelectrophoresis was perfor-med as described by Hau et a]. (1980). Theantibody used was rabbit anti chicken IgA(Nordic, Tilberg, The Netherlands, Batchno. 3810) and the concentration in the gelwas 1:100 (v/v). The sample wells received 5pl samples ofehicken tears, bile and serum.ResultsLacrimal IgA which was specific to NDVwas present in all the NDV-immune line Cbirds at a geometric mean titre of 1010*0‘3“and was absent in the NDV—immune line15] birds at < 10”. Total laerimal IgAvalues were marginally higher in NDV-immune line C compared to line 151 birdsand significantly higher in the normal line Ccompared to line 151birds (Table 1).Serum IgA values did not differ significantlybetween lines or between immunized andnon-immunized birds (Table 1).If the lacrimal lgA value was divided by thecorresponding serum IgA value in order tocompensate for differences in systemic lgA,162the relative values were significantly higherboth for virus-immune line C compared toVirus—immune line 151 birds and for normalline C compared to normal line 151 birds(Table 1). Line C birds therefore containedmore lacrimal IgA than line 151 birds ir—respective ofinfection with NDV (Table l).Bile was analyzed from the NDV—immunebirds and neither its level of IgA nor its titreOfIgA specific to NDV dilTered between lineC and line 151 birds. Bile and serum IgAwere therefore the same in both lines ofbirds whereas lacrimal lgA was not (Table1).DiscussionBoth lines of White leghorn chickens had asimilar immunospecitie titre of IgA withspecificities against NDV in the bile. How-ever, line C also had a measurable titre intears, whereas line 15] had not.The differences in virus-specific lacrimalIgA between line 151 and line C birds wereassociated with similar differences in totallaerimal lgA. The lacrimal lgA concentra-tion in line 151 birds being only half that ofline C birds. This indicates that if laerimalIgA is to be used as a measure of stress, birdsof the same genotype need to be compared.The differences in lacrimal IgA were not in-dicative of difference in circulating lgA be-cause serum levels were similar in all groupsof birds and the level ofimmunospecifie IgAmolecules with specificities against NDVwas below the detection limit of the assay inall samples analysed. Serum lgA is activelysecreted into the bile (Peppard et al. 1986),and the differences in immunospccifie lgAin the tears of the two lines can not havebeen reflected in serum, albeit below the de-tection limit, baeause biliary lgA levels werenot significantly different in the two lines.The harderian gland (HG) of the nietitatingmembrane is the major source of lacrimallgA (Baba at al. 1988). The difference inNDV—speeific lacrimal IgA between line Cand line 151 birds could not be reproduciblyassociated with differences in the number ofhcand. J. Lab. Anim. Sci. No. 3 . 1993 . V0120Table I. The level ot’lgA in line C and line 151birds and in NDV—immune line C and line 151 birds. Exp. Line Age of Strain of Total IgA in arbitary units Log 10 titre of IgA()1 birds in NDV in Tears/ to NDVbbirds days inoculuma Tears Serum Bile Serum Tears Serum Bile1 C 65 Ulster 17 23 650 0.74 2.5 <1.5 3.0C 65 Ulster 17 17 850 1.00 2.0 <1.5 3.0C 65 Ulster 20 20 1300 1.00 2.0 <1.5 3.0C 72 Hitchncr B1 10 26 850 0.39 1.5 <1.5 3.0C 72 Hitchner B1 20 20 450 1.00 2.0 <1.5 3.0C 72 Hitehner B1 37 20 750 1.90 2.0 < 1.5 3.020.2d 21.0 775 0.990 2.0 < 1.5 3.0151 67 Ulster 1 23 750 0.04 <1.5 <1.5 3.0151 67 Ulster 5 20 300 0.25 <1.5 <1.5 4.5151 67 Ulster 10 20 750 0.50 <1.5 <1.5 3.0151 75 Hitehner B1 5 32 1150 0.16 <1.5 <1.5 2.0151 75 Hitchner B1 26 42 2050 0.62 <1.5 <1.5 2.0151 75 Hitchner B1 15 29 1300 0.52 <1.5 <1.5 2.010.3d 21.7 1050 035° <1.5 <1.5 2.82 C 70 None 26 20 1.30 <1.5 <1.5C 70 None 41 15 2,73 <1.5 <1.5C 70 None 26 20 1.30 <1.5 <1.531.0c 18.3 1.77“ <1.5 <1.53 151 70 None 5 15 0.33 <1.5 <1.5151 70 None 8 15 0.53 <1.5 <1.5151 70 None 8 20 0.40 <1.5 <1.5151 70 None 5 20 0.25 <1.5 <1.5151 70 None 15 15 1.00 <1.5 <1.519.23 19 050° <1.5 <1.5 2' NDV, Newcastle the disease virus. NDV was inoculated 10 days earlier by the oeulotopieal route.b IgA, measured by an indirect immunoperoxidase monolayer assay.3 Significance difference between line C and line 151 groups p < 0.02 by unpaired students ’t‘ test.d Marginal difference between line C and line 151 group by unpaired students ’t’ test 0.05 < p < 0.1.antibody forming cells secreting IgA t0 NDVin the HG (Russell, unpublished results) ands0 differences in the transport of IgA into thelacrimal fluid or its dilution by the lacrimalfluid may also contribute to the differencesbetween line C and line 151. Dilution by thelacrimal fluid is less likely because Virus-speeifie IgG titres in the tears and serumwere the same from the line C and 15] birds(Russell, unpublished results).Cihak et ul. (1992) found IgA level to be43-fold higher in the bile compared to theserum and we also found an identical43-fold difference for virus immune birds(Table 1). Biliary IgA would therefore be themost accurate measure of central IgA pro-duction in the chicken if birds were to besacrified. Emptying of the gall bladder oc‘curs during feeding and so bile collectionmay have to be standardised to 12 hoursafter starvation as utilised in a study of theontogeny of IgA production (Leslie et a1.1976).Cihak et a1. (1992) showed that the deple-tion of T cells With the V131 receptor fromchickens led to a diminution of IgA but notIgG when biliary IgA decreased by 8,000fold and serum IgA decreased by 15 fold.Stress is generally regarded to depress thecellular immune system more than the hu-163Seanil..1.1_flb Anim. Sc1. No. 3. 1993 . Vol 20moral immune system (.Manser 1992) and Tcells may therefore be one link betweenstress level and IgA level.In future experiments laerimal and serumIgA will be compared in inbred birds stres—sed by housing conditions or inoculated withcorticosteroids in place of stress to assesswhich eouree of IgA is a better indicator ofstress.AcknowledgementsThe present study was supported by SmithKlineBeecham, Welwyn, UK, The Leverhulme Foun-dation and the AFRC and Welcome Trust.SummaryIn the human lower than normal concentrationsof IgA in saliva have been found to be associatedwith stress.In studies of animal welfare and prolonged stressthere is a need for nominvasive methods and as-says of secretory IgA would be simple to developfor many species. In the present study we studiedIgA in the chicken and compared levels of IgA intears. serum and bile. Two strains of chickenswere compared. The concentration of IgA in thelacrimal fluid of line C chickens was two-foldhigher than that of line 151 chickens. Levels ofIgA in their serum and bile where the same. Fol-lowing nn trom the replication of avirulent New-castle disease virus (NDV)1n the Harderian glandof thc nietitating membiane line C chickens con—tained NDV — specific 1ac1ima1 IgA to a titre of102 whereas line 151 chickens contained none at10-1-5. Both lines conmined NDV~specific IgA intheir bile to a titre of 103. Line 151 chickens there-fore have a selective deficiency in lacrimal IgAcompared to biliary IgA.Sammc'ndragStudier af studenter har vist, at stress giver anled»ning til lavere end normals niveauer af IgA i spyt.l studier af dyrevelfzerd og vedvarende stress hosdyr er der et behov for non-invasive metoder 0gassays til maling af sekretorisk IgA er thrholdsvisenkle at udvikle for mange arter. 1 natrvarendestudie undersagte vi IgA 1 kyllinger og sammenlig-nede niveauet i tarer, serum 0g galde. To stammeraf kyllinger blev sammenlignet. Koncentrationenaf IgA 1 tareva3sken at" linie C kyllinger var togauge hejere end i linie 151 Niveauet a1 IgA 1deres serum 0g galde var det samme. Efterimmu-nisering med Newcastle disease Virus (NDV) fand-tes signifikaut hejerc immunspeeifik IgA titer itarevzesken hos linie C sammenlignet med linie151, Begge linier havde samme koneentration igaldevzeske.164theenveta/K PelkonmIhmisessa’ on havaittu etta stressiin Iiittyy syljessaalentunut IgA: n maar'a. Elainten hyvinvoinninJapitkuaikaisen strcssin tutkimuksessa olisi kfiyttoaei-invasiivisille mcnctclmille ja olisi helppoa ke-1111155 monille lajeillc sopiva sekretorisen IgAznmaariyusmenetelma. Tessa tutkimuksessa selvitet-tiin kanan IgA:11 maaria ja verrattiin lgAzn tasojakyynelnesteessa, seerumissa Ja sapcssa. Tutkimuk-sessa verrattiin kahta eri kanakantaa. C-liniassaIgNzn vakevyys kyynelnesteessa oli kaksi kertaakorkcampi kuin linjassa 151. Seerumin ja sapenIgA-tasot olivat samoja.Harde1ian rauhasen avi-rulentin Neweastlen virus- (NDW-infektion ja'l-keen C-1injen kanojen kyynelneesteesséi NDV-spcsiflsen IgA: n tiitte1i 102 mutta 151-linJankanoissa se 011 alle 1015 Molemmissa linjoissaNDV-specihsen IgA:11 tiitteri sapessa oli 103.1%-nollcn linjan 151 kanoilla 011 kyynelnesteesséi sap-peen verrattuna selektiivinen IgApuutos.Rcfi’rencwBalm T. K Maxwmoto, 5 Nislu'da. T Kajikawa &M Mitsui: Harderian gland dependency of im-munoglobulin A production in lacrimal fluidof chicken. Immunology 1988, 65, 67—71.(‘ilmk G, G Hq/frnann-Fezer, HWL Ziegler—Hci-bmrk H Stein B Kaspers. CH (hen, MDCooper & U Losch: T cells expressing the VplT-eell receptor are 1equired for IgA produc-tion in the chicken. Proc.Nat1. AcademicSci., USA, 1991. 88, 10951—10955.Cook JKA, 0131,1111 K NR da Silva Martins, MMEllis & MB IIuggins: The secretory antibodyresponse of inbred lines of chicken to avianinfectious bronchitis virus infection. AvianPathology 1992, 21, 681—692.H1114 J, P Svendxen, B Teisner & G ThomsenPedersen: Immunochemical characterizationand serum concentrations of pregnaney—asso—ciated serum proteins in mice. J. Reprod. Fen,1980, 58, 389—392.Jemmott 111 JB & K Maglozre: Academic Stress,Social Support, and Secretory ImmunogIobu-lin A. Journal of Personality and SocialPsychology 1988, 55, 803—810.Leslie GA. RP Slrmkus & LN Martin: SecretoryImmunological System of Fowl. InternationalArchives Allergy and Applied Immunology1976, 51, 175—185..Manxer CE: The assessment of stress in labora-torg animals. Published by RSPCA 1992, p.11 .Peppard J V, SM HobbS, LE Jackson, ME Rose& AP/1 Mackelt: Biochemical characterizationof chicken secretory component. EuropeanJournal ofhnmunology 1986, 16, 225—229.Stone AA, DS Cox, H Vuldimarsdottir, L Jan~dorf & JM Neale: Journal of Personality andSocial Psychology 1987, 52. 988—993.

Immunoglobulin A in chickens. Comparison 0ft0ta1 and immune—specific lacrimal and serum IgA levels in two lines of chickens immunized with Newcastle Disease Virus

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Immunoglobulin A in chickens. Comparison 0ft0ta1 and immune—specific lacrimal and serum IgA levels in two lines of chickens immunized with Newcastle Disease Virus

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