Twelve rabbits were used for the production of the immune sera against T. suis, T. foetus, T. vaginalis and T. gallinae antigens. Three animals were injected with each species of Tritrichomonas and Trichomonas. In the reactions between these immune sera and antigens of each of special the largest number of precipitating lines were observed between homologous systems using gel immunodiffusion. Using the Outcherlony technique the antigens of T. suis, T. foetus, T. vaginalis and T. gallinae showed 1, 2, 3 or 4 lines in the A antigenic group and 1 and 2 precipitating lines in group B. The results obtained with the immune sera of T. suis, T. foetus, T. vaginalis and T. gallinae indicated that the four trichomonads have identical antigens, or at least a closel y related antigenic structure.Doze coelhos foram usados para a produção dos imuno-soros contra antígenos de Tritriahomonas suis, T. foetus, TPiahomonas vaginalis e T. gallinae. Três animais foram injetados com cada espécie de Tritrichomonas e Trichomonas. Nas reações entre estes imuno-soros e os antígenos de cada uma das espécies, o maior número de linhas de precipitação foi observado, entre os imuno-soros e antígenos homólogos na difusão em gel. Usando-se a técnica de Outcherlony, os antígenos de T. suis, T. foetus, T. vaginalis e T. gallinae mostraram 1, 2, 3, ou 4 linhas no grupo antigênico A e 1 e 2 linhas de precipitação no grupo B. Os resultados obtidos com os imuno-soros de T. suis, T. foetus, T. vaginalis e T. gallinae indicaram que os quatro tricomomas têm antigenos idênticos, ou pelo menos com estrutura antigênica relacionada muito próxima

De Carli, Geraldo Attilio

Revista de Microbiologia

English

Lume 5.8

122 ANTIGENIC COMPARISON BETWEEN TR!TRICHOMONAS SUIS, T. FOETUS, TR!CHOMONAS VAGINALIS AND T, GALLINAE BY GEL IMMUNODIFFUSION* Geraldo Attilio de Garli Dept9 de Análises Faculdade de Farmácia UFRGS 90000 Porte Alegre RS, Brasil Twelve rabbits v;ere used for the production of the lirmune sera against T. suis, T. foetus, T. vaginalis and T. gallinae antigens. 'Jhree anirralS mre injected with each species of Tritrichomonas and Jrichomonas. In the reactions between these inno.Jne sera and antigens of each of special the largest nurober of precipitating lines v-ere observed between harologous systems using gel :irrmunodiffusion. Using the C.Utcherlony teclmique the antigens of T. suis, T. foetus, T. vaginalis and T. gallinae showed 1, 2, 3 or 4 lines in the A antigenic group and 1 and 2 precipitating lines in group B. The results obtain.ed wi:th the inm.me sera of T. suis, T. foetus, T. vaginalis and T. -gallinae indicated that the four tricharonads have identical antigens, or at least a closel y related antigenic structure. Comparação antigênica entre Tritrichomonas suis, T. foetus, Trichomonas vaginalis e T. gallinae pela imunodifusão em gel D::>ze coelhos foram usados :para a pra:iução dos i.muno-soros contra antígenos de Tritriahomonas suis, T. foetus, TPiaho-monas vagina~is e T. gallinae. Três animais foram injetados com cada espécie de lritriahomonas e Triahomonas. Nas rea-ções entre estes irnuno-soros e os antigenos de cada urra das espécies, o maior número de linhas. de precipitação foi ob-servado, entre os imun.o-soros e antigenos hanólcgos na di-fusão em gel. Usando-se a técnica de Outcherlony, os anti-genes de T. suis, T. foetus, T. vagina~is e T. gallinae rrostraram 1, 2, 3, ou 4 linhas no grupo antigênico A e 1 e 2 linhas de precipitação no grupo .B. Os resultados obtidos com os imuno-soros de T. suis, T. foetus, T. vaginalis e T. ga ~ l in a e indicaram que os quatro triccm:Jnas têm antigenos idênticos, ou pelo rrenos com estrutura antigênica relacio-nada muito próxima. *SupfOrted by a research grant of Fund.ação de Amparo à Pesquisa do Estado do Rio Grande do SUl, Brazil, project Inrnunol. 204/79. Rev. Mícrobiol., São Paulo, 14(2):122-128, Abr./Jun. 1983. 123 Introduction Studies of the antigenic ~ison between Tritrichomonas suis and T. foetv..s have l::een performed using the i.nd.irect irmrunofluorescence ( 4) , gel inmuno::liffusion (5) and :irrmunoelectrophoresis techniques (6). 1he results obtained illd:icated the existence of antigens that are· cormnn to b:>th species or perhaps associated with a generic antigeníc identity. In the present investigation the antigenic relation between T. suis, T. foetus, T. vaginalis and T. gallinae was studied through gel irmum.cxliffusion with the purpose of obtaining new infonration on the antigenic afinity of the four species. M3.terial and Mothods Protozoan species - Tritriehomonas suis - This strain has been described previously {3). O.li:tures of the protozoa w=re rnaintained in axenic cultures in m:::xlified Diarrúnd rredium (3) at 35 ,5CC and transferred every 48 hours. Tritrichomonas foetus - 'Ihe hi.story of this specie was given in a previous rep;Jrt (4). At first the sarrple was spread in Fe:inberg & Whittmgton rredium and transferred every 72 hours (8). After that the axemc culture was mairltamed in rrodified Diarrond rredium at 35,5CC and transferred every 48 hours. Trichomonas vaginalis - The sample TV 1 was isolated from the vaginal secretion frcm a w:xran, patient in a gynecolcqical clinic in Porto Alegre, RS. 'Ihis strain has been kept in axeffic culture in rrodified Diarrond rredium since 1973 (2). 1he cult.ures v;ere .incubated at 3S,SOC and transferred every 48 hours. Triehomonas gallinae - 'nle rreth.cxls of isolation, cultivation and preservation of the 'TI:; 1 isolated by De Carli, Pansera & Guerrero fran l..lJ?P3r digestive tract of a darestic pigeon ~e described previously (7) • 'lhe culture of the parasites v;ere maintained in m:xlified Diarrond :rrediurn at 35 , SOC and transferred every 48 hours. Preparation of antigens for immunization - Antigens of each species were extracted from each organisrn as previousl y descri.bed. ( 4) . 'lhe strains of T. vaginal is and T. ga ll i na e which had the sarre treat:rrent during the preparation o f the antigens, were included. All disrupted antigens ..ere stored at -20CC. Coneentration of total nitrogen and protein of the antigens - The conoentration of total nitrogen was determmed by technique of Kjeldhal rrodified by Taveira & Bethelem (21) and total prctein was detenn:ined by the rrethod of Foling-Ciccoalteau as descr:ibed by IDNry (16) . 1he results of these analyses are given in Table 1 . Immunization of rabbits and immune sera -Twelve rabbits \<.'ere use:l for the prcrluction of the :immune sera against each of the four species. 'Ihe rrethcd of preparation was as previousl y descr:ibed ( 4) . 1he :immune sera and the rabbit I10nnal rea. (NRS) , prepared for the illdirect :i!mo.mofluorescence study ( 4) ~e used i..11 the present investigation. Gel immunodifusion teohnique - The technigue of :inmunodifusion described by Outcher lony ( 18) was used for the identification o f Tritriehomonas and Triehomonas antigens. Glass slides (75 x 51Jrrun) were used for this technique. 1he gel layer was prepared by addi tion o f 5rnl o f liquid purified agar (BBL) at 1% over the glass slides. Scdiurn azide Table 1 - Protein and total ni-trogen of the antigens used in the gel diffusion precipitin reactions Protein Total N l\ntigens "'J/ml "'J/ml T. suis TS 1,8 0,37 T. foetus TF 16,0 2,61 T. vaginaZis 'IV 5,0 0,8 T. gaZZinae 1G 6,0 0,96 124 (Difco) VJa.S used as a preservative of the gel in a final concentration of O, 1%. A seven wells pa.ttern cutter used being the inte:r:nal diarrrreter of the wells of Smn with a distance of Snm l::etween them, or five v..ells pattexn cutter was used being the internal d.iamneter of the wall of Smn with clistance of 1 Omrn between them. The external wells of the r::erforated gel ~re used for the .imnune sera and the internal well was used for the antigens. For all reactions, the v.~E:!lls were loadeà v1ith 20pl of antigens aP.d 50yl of undíluted antiserum. T'ne antigen anti.l::ody reactions were processed. in humid charnl:::er at roam temperature (220C) • The readings were made 48 hours after the beginning of the diffusion. Obliquous lighting against a dark field was used for reacling the reactions. 'l1le precipitating lines were stained by Ponceau fixing stain. All of the reactions were photographed as previou.sly described (22) . All of the reactions v;ere photographed 48 hours after t.he incubati.on using the technigue described by Williams & Chase (22). Resul ts and Discussion Nega tive resul ts \Vere found in the reactions that invol ved normal rabbi t senim (NRS) and four protozoan antigens studied. i\'b precipitating lines were fomed when the four antisera reacted with the sterile culture mediurn, sterile carponents of these rredia, horse sterile carp::.nents of these rredia, house sterile and i..P..activated horse serum diluted and not diluted. l\b precipitin lines were forned in the slides containing sterile distilled water, sterile physiological saline solution. Negative reactions am:mg baseline bleecling serurn and T-Pi tY'iehomonas and Tf'·ichomonas antigens proved. that the rabbits utilizes in this study had not a previous contact · with these antigens. In each series o f experirrents the specific inmune serum reacted. against the hc:m:llogous and heterologous antigens. The imnune reactio.."lS bet:v.reen the irrrm.me sera of OOth systems \<iere represented by two groups of precipitation lines or bands. According to G:>ldrnan & Honigberg (11) these group were arbitrarily designated group A and group B based on their position. Group A of precipitation lines always developed closer to the serum t,o;ells, and w:;re concave to their side. Group B li..r1es, were located closer to the antigen wells and concave to its side. 'lhe precipitationg lines obtained in the heterologous systems were classified according to Wilscn & Pringle (23); Outcherlony (18) and cr<::Mle (9, 1 0). The tests perfoJ::IIEd with each antiserurn show" reactions carparing the irrmune sera and NRS with the heterologous and hcm::üogous antigens as follows; one-well reactions wi th the hom::ücgous antigens and three-well reactions with the heterolcgous antigens. Anti-Tritriehomonas suis serwn - When T. suis (TS) antigen reacted against its ha:rolcgous antise..rum it shc:1.-.ed the strongest reaction. A great nl.JITll::er of lines from groups A and B wrere forrred in the reaction arrong TS antigen and their hc::mJlcgous antiserurn. l'b reactions cccured bet:v>een TS antigen and T. vaginalis antiserurn {tv} (Fig. 1 and 2) . When the antigen TS reacted against harologous and heterologous sera it shov.:ed tv.:o, three and four lines in group A and one and two li.nes in group B (Fig. 1, 2 and 3). When the TS antigen reacted against T. j'oetus (tf) and T. gaUinae (tg) irru:nune sera shcmed lines with total identity (Fig. 1 and 3). Most of these lines from both hc::nnlogous and heterologous eysterns s.howed total identi ty (Fig. 1 and 3) and partial identity (Fig. 6). Kb conclusion can be described abcut any lin.es of group B antigenic eysterns. The results of these experirrents 'i-Vith anti-T. suis serum indicated that OOth T. foetus and T. gallinae have antigen 'Which are identical or at least very closely structurally related or identical antigen in the A and B group. Anti-Tf'itriehomonas foetus serum - This serum gave the largest number of lines with its horrúlc:çfQus antigen. When the T. foetus {'IF} antigen reacted against the ha:rologous and heterologous antisera one to three lines in group A anel two lin.es in group B were forrred (Fig. 5 and 7) . ~'Íh.en 'IF antigen reacteà. against the irmn.rne sera of T. suis (ts) and T. gaUinae (tg) only lines with partial identity \<iere developed (Fig. 4 and 6). i•bst of these lines fran bcth harologous and heterologous 125 system ~ total identity (Fig. 4) and partial identity (Fig. 6). N::> reaction cccured betlooen TF antigen and T. vaginalis (tv) antiserum (Fig. 5). It is evident fran the results that either T. foet:us, T. suis and T. gallinae have identical antigens, or are very closely relatei in the group A. The T. foetus aparently appear to share one o f the group B antigen of T. ga U inae (Fig. 5) • Figures 1, 2, 3- Precipitatíon lines of reaction between T. suis antigen (TS) and antí-T. vaginalis antisera '(tv), anti-T. gallinae antisera (tg), anti-T. foetus antisera (tf and wells 4, 5 and 6) and anti-T. suis antisera (ts wells 1, 2 and 3) Anti-Trichomonas vaginalis serum - Reactions beb\e.en T. vaginalis (tv) antiserum and its horrologous antigen resulted in the fonnation of one and t:w;:) lines on the group A (Fig. 8). lfuen T. vaginalis (tv) antigen reacted against T. gaUinae (tg) antiserum, there was one line with total identity in group A (Fig. 9). N::> reactions occured betlooen W antigen and T. suis (ts) and T. foetus (tf) antiserum (Fig. 8 and 9). vlhen W antigen reactel againet harol09ous and heterol09QUS sera, these "Were one and tvK> lines in A group alone, and not lines in B group. The results indicatel a very close antigenic relation of superficial antigens bet~ooen the t= species of Trichomonas. Figures 4, 5, 6 - Precipitation lines of reactions between T. foetus antigen (TF) and anti-T. vaginalis antisera (tv), anti-T. gaZZinae antisera (tg), anti-T. foetus antisera (tf and wells 4, 5 and 6) and anti-T. suis antisera (ts wells 1, 2 and 3) 126 Anti-Trichomonas ga~linae serum - The strongest reactions was observed l:e~ T. gallinae (TS) antigen and its harologous antisennn (Fig. 10). When the 1G antigen reacted against harologous and heterologous sera it ~ ooe and two lines in the A group (Fig. 1 O) • fue reactions reh>:en 1G antigen against T. vaginalis (tv) serum shcmed ~ lines in group A with partial identity (Fig. 11) • fue precipitin lines observed in reaction involving TG antigen and T. suis (ts) and T. foetus (tf) inmlJne sera ~ ooe line on the A group with total identity. fue results of experirtents with T. gaUinae (TI;) antigen against T. suis (ts) , T. foetv.s (tf) and T. vaginaUs (tv) antiserurn ~ evidence thet this tricham:>nads have identical antigens or very closely related antigenic structure. COnclusions fue results of previous study on indirect :im:nunofluorescence (4), gel :inrnunodiffusion (5) and :im:nunoelectrophoresis ( 6) suggested the close antigenic relationships l:etween T. suis and T. foetus. Recent light and electronic abservations pmvided a similar !lDrpbology in the two Figure 7 - Precipitation lines of reactions between T. foetus antigen (TF) and anti-T. vaginalis antisera (tv), anti-T. gallinae antisera (tg), anti-T. foetus antisera (tf and wells 4, 5 and 6) and anti-T. suis antisera (ts and wells 1, 2 and 3) Tritrichomonas and. two Trichomonas species (7, 13, 15, 17, 19). The close antigenic relationships reh>:en these two genera could reveal the existence of cmss reacting antigens but did not give infoJ:lllation cancerning the nurnber of camon antigens res:p::>nsible for tbese r~ons (12, 14, 20). 'Ihey indicated, also that T. suis, T. foetus anel T. gallinae v.ere antigenically rrore closely related to each other.. It has l:x=en proved through gel imrnmo:liffusion that T. vaginalis, T. gallinae and T. foe tus share structurally identical or closely related antigens, oonfil:ming the results suggested previously through stndies with tricham:>nads of genera Tritrichomonas. Figures 8, 9 - Precipitation lines of reactions between T. vaginalis antigen (TV) and anti-T~ vaginalis antisera (tv), anti-T. gallinae antisera (tg), anti-T. foetus antisera (tf)~ and anti-T. suis antísera (ts) Figure 10 - Precipítation lines of reactions between T. gallinae antigen (TG) and anti-T .. vaginalis antisera (tv), anti-T. gallinae antisera (tg), anti-T. foetus antisera (tf)~ and anti-T. suis antisera (ts) Figure 11- Precípítatíon lines of reactions between T. gallinae antígen (TG) and antí-T.vaginalis antisera (tv), anti-T. gallinae antisera (tg), anti-T. foetus antisera (tf), and anti-T. suis antisera (ts) References Abbreviations TS - T. suis antigen; TF - T. foetus antigen; TV T. vaginalis antigen; TG - T. gallinae antigen. ts - T. suis antisera; tf - T. foetus antisera; tv T. vaginalis antisera; tg - T. gallinae antisera. 1,2 and 3- antisera of T. suis; 4, 5 and 6 antisera of T. foetus. Ackn<Mledgements 127 The author indebet to Dr. Jorge Gurerrero, Manager Pre-clinical Research, Pitrnan-f:Ioore, Inc., New Jersey, USA, for his kind revision of the manuscript, and thankas are due to Professor Paulo Jaconi Saraiva, Mr. Eni Pereira Nunes and Miss Silvia Guterres. 1. Abraham, R. & Honigberg, B.M .. - Structure of Trichomonas gallinae (Ri volta) . .J. Parasitol., 5:608-619, 1964. 2. ~ Carli, G .. A .. & M:lchado, B. Epidemiologia e diagnóstico la:l:;oratorial de Irichomonas vaginalis. Rev .. Microbiol .. (S .. Paulo), 4:33-36, 1973. 3. De Carli, G .. A. & Guerrero-Ramirez, J.. T'.f'ichomonas suis: isolamento, rrorfoloqia e incidência na cavidade nasal de porcos dcmésticos SUs Sere f a.. Rev .. Fac. Med. Vet. Zootec. Univ. S. Paulo, 12:269-276, 1975. 4. IE Carli, G.A. & Guerrero, J. -Comparação antigênica entre ·rntrichomonas suis e T. foetus. I - Irmmofluorescência indireta (IFI). Rev. t-licrobioL (S.Paulo}, 6:55-58, 1975. 5. :ce Carli, G.A. & Guerrero, J. - Antigenic a:::uparison between Tr-itrichomonas suis and T. foetus. II -Gel imm.mcxliffusion (ID). Rev. Lat-amer. Microbiol., 18:167-171, 1976. 6. De carli, G.A. & Guerrero, J .. - Cc:inparação antigênica entre Tritrichomonas suis e T. foetus. III Imunoeletroforese (IEF). Rev. Microbiol. (S.Paulo), 8:107-109, 1977. 7. :ce Carli, G.A.;. Pansera, M.C.G. & Guerrero 1 J.- Trichomonas gaUinae (Rivolta, 1878) Stabler, 1938, no trato digestivo de p:::rnl::os domésticos, ColVJnba livia, no Rio Grande do Sul: prirreiro registro. Acta Biol. Ieopoldesía, 1:85-95, 1979. 8. :ce carli, G.A. & D2 Carli, M. um rreio silq?les para o cultivo de Tritrichomonas foetus in vitro. Acta Biol. Leopoldesia, 2:89-90, 1980. 9. cro.vle, A.J. - Interpretation of irmn.mcx:liffusion test. Ann. R.ev. .Hicrobiol., 14:161-176, 1960. 128 10. Crc:w'le, A.J. - Irmruno::liffusion. 3.ed. New York, Academic Press, 333p., 1961. 11. Goldmann, M. & Honigberg, B.H. - IrrmunolCY::JiC analysis by gel diffusion techniques of effects of prolonged cultivation on Trichomonas gallinae. J. Protozool., 15:350-352, 1968. 12. Honigberg, H.F.; friedrran, H. & StepkCMSki, S. - Effect of diferent llnmunization procedures on agglutination and precipitation reactions of ~iehomonas gallinae. J. Parasitol., 57:363-369, 1971. 13. Honigberg, B.!--1. & King, V.M. - Structure of T:t-iehow;-r:.as vaginaUs Donné. J. Parasitol., 50:345-364, 1964. 14. Honígberg, B.M. & Goldman, H. - Intnunologic analysis by quantitative fluorescent antil:x::d.y :rrethcà.s of the effects of prolonged. cul ti vation on Jriahomonas gallinae. J. Protozool., 15:176-184, 1968. 15. Kirby, H. - 'Ihe structure of the corrmron intestinal tricharonad o f rnan. J. Parasitol., 31:163-175, 1945. 16. U;Mry, O.H. - Pratein neasurerrent with the folin phenol reagent. J. Biol. Chem., 193:265-275, 1951. 17. Nielsen, Ivl.H. - 'Ihe ultrastructure of Trichomonas vaginalis Donné before and after transfer from vaginal secretion to Diarronds :rredium. Acta Path. l'ücrobioL Scand. Sect. B, 83:581-589, 1975. 18. ü.ltcherlony, O. - Innn.modiffusion and. írmn.melectrophoresis. In: - Weir, M. Handl:x:lok of e><perilrental ilmnmology. Oxford, Black>.ell, cap. 19, p. 655-706, 1967. 19. Srnith, B.F. & Stewart, B.T. -Fine structure of Trichomonas vaginalis. Exp. Parasitol., 19:52-63, 1966. 20. Stepkowski, S. & Honigberg, B.i'l. - Antigenic analysis of virulent and avirulent strains o f Triehomonas ga Z Z i na e ~ gel diffusion rrethc:ds. J. Protozool. , 19:306-315, 1972. 21. 'Iaveira, N. & Bethelm, M.L. - DJsearrento de proteínas. In: brana.tolÓjica: métodos de análises de alimentos. Rio de Janeiro, Fannácia 1 Univ. Fed. do Brasil, fase. 1, p. 75-80 1 1975. QuÍmica Fac. de 22. Williams, C.A. & Cllase, M.W. - Photography of precipitates in gels. In: Mothods ilmnmology and irrmJnochemistry. New York, Academic Press, v. 3 cap. 14, p. 321-339, 1971. 23. Wilson, H.W. & Pringle, B.H. - Interpretation of OUtcherlany precipitin test. J. ImnRL~l., 75:460-469, 1955. 

Comparação antigênica entre tritrichomonas suis, t. foetus, trichomonas vaginalis e t. gallinae pela imunodifusão em gel

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Comparação antigênica entre tritrichomonas suis, t. foetus, trichomonas vaginalis e t. gallinae pela imunodifusão em gel

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