Evaluation of the enzyme-linked immunosorbent assay for the rapid screening and detection of classical swine fever virus antigens in the blood of pigs.

Re . sci. ech. O . in . Epiz., 1995, 14 (3), 677-689 E alua ion o he enzyme-linked immunoso ben assay o he apid sc eening and de ec ion o classical swine e e i us an igens in he blood o pigs K. DEPNER n(1), D.J. PATON (2), C. CRUCIÈRE (3), G.M. DE MIA (4), A. MÜLLER F. KOENEN (5), R. STARK (6) and B. LIESS (1) Summa y: A wo kshop was con ened, a which se en enzyme-linked immunoso ben assays (ELISAs) we e compa ed wi h i us isola ion o he de ec ion o i aemia in se ial blood samples collec ed om six pigs a up o ou een days a e inocula ion wi h classical swine e e i us. All ELIS As we e o he double an ibody sandwich ype, using monoclonal and/o polyclonal an ibodies o de ec a a ie y o i al p o eins in leukocy es, o in an i-coagula ed blood o se um. Compa ed o i us isola ion, speci ici y o he ELISA was good: only one sample ound nega i e by i us isola ion yielded a posi i e esul in a single ELISA. Some alse-nega i e esul s occu ed wi h samples collec ed a up o eigh days a e inocula ion, bu all es s ound samples collec ed be ween nine and ou een days pos -inocula ion o be posi i e. The ELIS As equi e less-specialised acili ies and can be pe o med much mo e apidly han i us isola ion. They a e he e o e ex emely p omising ools o sc eening la ge numbe s o li e pigs. KEYWORDS: An igen - Classical swine e e - ELISA - Hog chole a - Vi aemia. INTRODUCTION Classical swine e e (CSF) is an economically impo an disease which is con olled, in gene al, by s a u o y measu es. In iew o he se ious implica ions o a diagnosis o CSF, and because clinical signs a e ne e pa hognomonic, labo a o y con i ma ion o diagnosis is e y impo an . As wi h o he i al diseases, labo a o y diagnosis is based 1) Communi y Re e ence Labo a o y o Classical Swine Fe e , Ins i u e o Vi ology, Hano e Ve e ina y School, Bün eweg 17,30559 Hano e , Ge many. 2) Cen al Ve e ina y Labo a o y (Weyb idge), Woodham Lane, Addles one, Su ey KT15 3NB, Uni ed Kingdom. 3) Cen e na ional d'é udes é é inai es e alimen ai es, Labo a oi e cen al de eche ches é é inai es, 22, ue Pie e Cu ie, BP 67, 94703 Maisons-Al o Cedex, F ance. 4) Is i u o Zoop o ila ico Spe imen ale dell'Umb ia e delle Ma che, Via G. Sal emini 1, 06100 Pe ugia, I aly. 5) Ins i u na ional de eche ches é é inai es, ue G oeselenbe g 99,1180 B ussels, Belgium. 6) Bundes o schungsans al ü Vi usk ankhei en de Tie e, Paul-Eh lich-S . 28, Pos ach 1149, 72001 Tübin gen, Ge many. P esen add ess: Ins i u ü Vi ologie, Jus us-Liebig-Uni e si ä Gießen, F ank u e S aße 107, D-35392 Giessen, Ge many. 678 on ei he de ec ion o he agen o se ology. Se ology is he ela i ely mo e simple echnique, bu is inapp op ia e o use in ecen ly-in ec ed pigs which may be shedding i us bu ha e no ye de eloped se um an ibodies. Vi us de ec ion has adi ionally been pe o med by a combina ion o luo escen an ibody es s on issue sec ions and i us isola ion in cell cul u es om ei he issues o leukocy es. Fo diagnosis in li e pigs, issues a e no eadily a ailable, and he e o e i us isola ion om leukocy es has been he me hod o choice. Al hough sensi i e and speci ic, i us isola ion is bo h ime- consuming and labo ious, and is he e o e unsui able o la ge-scale sc eening o e a long pe iod o ime. The need o a sensi i e, apid and p ac ical es o he de ec ion o CSF i us (CSFV) in blood has become all oo e iden du ing ecen CSF epidemics in Belgium and Ge many, when a la ge numbe o animals had o be es ed. One way o o e come hese p oblems has been h ough he ecen de elopmen o enzyme-linked immunoso ben assay (ELISA) echniques o de ec ing CSF i al an igens (14). Sensi i e and speci ic an igen-cap u e ELISA sys ems a e al eady success ully used in he ou ine diagnosis o he ela ed pes i i us diseases o ca le and sheep, namely bo ine i us dia hoea (BVD) and Bo de disease (BD), espec i ely. The p esen epo summa ises he indings o a wo kshop on an igen de ec ion p ocedu es o CSFV, held unde he auspices o he Commission o he Eu opean Union (EU) a he Communi y Re e ence Labo a o y (CRL) o CSF, Ins i u e o Vi ology, Hano e Ve e ina y School, om 19 o 21 Ma ch 1995. Rep esen a i es o he CRL and o he Na ional Swine Fe e Labo a o ies o Belgium, F ance, Ge many, I aly and he Uni ed Kingdom a ended he wo kshop o e alua e a ious ELISA echniques o de ec ing CSF i al an igen in blood. Emphasis was pu on e alua ing he abili y o he es s o de ec i aemia in pigs in he ea ly phase o in ec ion be o e classical signs o CSF we e p esen . MATERIALS AND METHODS Ma e ials a ailable o an igen de ec ion Each wo kshop pa icipan was in i ed o es 55 po cine blood samples (ei he blood wi h e hylenediamine e aace ic acid [EDTA], o se um), using his/he own ELISA sys em o a comme cial ELISA. Twen y o he samples o igina ed om pigs which we e ee om CSFV, and 35 samples we e aken om six weaned pigs a a ious ime in e als a e hey had been inocula ed wi h CSFV. One blood sample om a cal pe sis en ly in ec ed wi h BVD i us was also included. P inciples o e alua ed ELISAs Se en di e en ELISAs we e e alua ed. Some had al eady been ex ensi ely es ed o he de ec ion o CSFV, whils o he s we e p o o ypes which had no been ully alida ed. All used a double an ibody sandwich p inciple, wi h an an igen-cap u e an ibody coa ed o ELISA pla e mic o i e wells and a de ec o an ibody e ealing bound an igen. The ype and speci ici y o he an ibodies used a e compa ed in Table I, oge he wi h o he es cha ac e is ics. The nega i e samples p o ided we e used o he calcula ion o he cu -o alues o each ELISA. Values highe han he means ob ained wi h nega i e samples plus h ee imes he s anda d de ia ion we e sco ed posi i e. Values equi alen o he mean o nega i es plus be ween wo and h ee imes he s anda d de ia ion we e in e p e ed as inconclusi e. The esul s o he 'Se elisa 679 HCV-Ag' es we e calcula ed acco ding o he ins uc ions o he manu ac u e . The sensi i i y o each ELISA was calcula ed by di iding he numbe o posi i e and doub ul posi i e ELISA esul s by he numbe o posi i e i us isola ions. Se elisa HCV-Ag (Rhône-Mé ieux) This comme cial ELISA used a mix u e o h ee monoclonal an ibodies (MAbs) which ecognise epi opes on he pl20/80 p o ein (1). These an ibodies we e coa ed o he solid phase and cap u ed an igen om an i-coagula ed blood o se um. An an i-pl20/80 abbi an ise um was used o de ec bound an igen in combina ion wi h a goa an i- abbi ho se adish pe oxidase (HRPO)-conjuga e. The an ise um had been p epa ed by immunisa ion o a abbi wi h a ecombinan pl20/80 p o ein. This es was o iginally es ablished o use wi h EDTA blood bu , in he p esen exe cise, se a o unsepa a ed EDTA blood we e dispensed in o he ELISA pla e mic o i e wells oge he wi h a lysis bu e . This es is panpes i i us-speci ic and can be pe o med in i e hou s. The ELISA was in oduced by J.C. Thibaul (Rhône-Mé ieux) and pe o med by F. Koenen (Ins i u na ional de eche ches é é inai es, Belgium), C. C uciè e and M. Gonzague (Cen e na ional d'é udes é é inai es e alimen ai es, F ance). CVL-1 and CVL-2 (Cen al Ve e ina y Labo a o y, Uni ed Kingdom) The CVL-1 ELISA is based on cap u ing an igen om leukocy es using an an i-pl20/80 MAb. The bound an igen was de ec ed wi h a second an i-pl20/80 MAb which is bio in-conjuga ed. Bo h MAbs we e aised agains BVDV, bu he es is panpes i i us-speci ic and can be pe o med in h ee hou s. The p inciple o he CVL-2 ELISA is he same as o CVL-1, excep ha he cap u e MAb and he bio inyla ed MAb a e CSFV-speci ic, eac ing wi h sepa a e epi opes on gp55. The es is CSFV-speci ic, bu does no ecognise all s ains o CSFV. Leukocy es we e p epa ed by exp essing 5 ml o EDTA blood h ough a sy inge-end il e . The apped leukocy es we e lysed and elu ed simul aneously by passage h ough he il e o 0.5 ml o 2% oc yl-ß-D glucopy anoside in phospha e-bu e ed saline (5). Elua e was added a 0.05 ml pe well o he ELISA pla e as he es sample. Bo h ELISAs we e in oduced and pe o med by DJ. Pa on. Pe ugia ELISA (Is i u o Zoop o ila ico, Pe ugia, I aly) In his ELISA, an igen om lysed bu y coa s was cap u ed using a s ongly eac i e swine polyclonal pes i i us an ise um. Bound an igen was de ec ed wi h an HRPO conjuga e p oduced om he same an ise um. An igen was p epa ed om leukocy es pipe ed om he bu y coa ac ion o cen i uged EDTA blood samples by ea men wi h 1 % Nonide P-40. This es is panpes i i us-speci ic and can be comple ed wi hin i e hou s ollowing sepa a ion o he bu y coa . The ELISA was in oduced and pe o med by G.M. De Mia. CSFV E0-SADA (Tübingen, Ge many) The CSFV E0-SADA (SADA: se um an igen de ec ion assay) is based on cap u ing an igen om se um using a s ongly eac i e abbi -monospeci ic polyclonal an ise um agains he E0 (gp44/48) p o ein o CSFV. The bound an igen was de ec ed wi h a bio in- conjuga ed e sion o he same an ise um. The es is panpes i i us-speci ic and can be pe o med in ou hou s. This ELISA was in oduced and pe o med by R. S a k. HAN-1 and HAN-2 (Communi y Re e ence Labo a o y, Ge many) These wo ELISA ki s a e based on cap u ing an igen om an i-coagula ed blood. The HAN-1 es uses a panpes i i us-speci ic MAb eac i e wi h pl20/80, while he 680 TABLE I Cha ac e is ics o a ious an igen enzyme-linked immunoso ben assays (ELISAs) ELISA Cap u e an ibody Speci ici y o cap u e Re ealing an ibody Speci ici y o e ealing Pes i i us speci ici y Sample es ed Time aken Se elisa MAb (x 3) pl20/80 PAb ( abbi ) pl20/80 Pan Blood/se um 5h CVL-1 MAb pl20/80 MAb pl20/80 Pan Leukocy es 3h CVL-2 MAb gp55 MAb gp55 CSFV Leukocy es 3h Pe ugia PAb (pig) CSFV PAb (pig) CSFV Pan Leukocy es 5h EO-SADA PAb ( abbi ) gp44/48 (E0) PAb ( abbi ) gp44/48 (E0) Pan Se um 4h HAN-1 MAb pl20/80 PAb (pig) CSFV Pan Blood 4h HAN-2 MAb (x2) pl20/80+ gp55 PAb (pig) CSFV Pan Blood 4h CVL: Cen al Ve e ina y Labo a o y (Weyb idge, Uni ed Kingdom) SAD A: se um an igen de ec ion assay HAN: Hano e Ve e ina y School, Ge many MAb: monoclonal an ibody PAb: polyclonal an ibody CSFV: classical swine e e i us Pan: panpes i i us-speci ic (i.e. ecognises CSFV, bo ine i us dia hoea i us and Bo de disease i us) 681 HAN-2 es employs a mix u e o wo MAbs speci ic o pl20/80 and gp55, espec i ely. The bound an igen was de ec ed wi h a panpes i i us-speci ic polyclonal HRPO- conjuga ed an ise um. This an ise um had been p epa ed by immunisa ion o a pig wi h CSFV. An igens we e p epa ed by ea men o EDTA/dex an blood samples wi h 1 % T i on X-100® de e gen . Bo h es s a e panpes i i us-speci ic and can be pe o med in ou hou s. The ELISAs we e in oduced by A. Mülle and K. Depne , and we e pe o med by U. Beh (Ins i u e o Vi ology, Hano e Ve e ina y School, Ge many). Expe imen al in ec ion o pigs wi h CSFV Six weaned pigs weighing app oxima ely 15 kg and ee om neu alising an ibodies agains pes i i uses (BVDV s ain NADL [Na ional Animal Disease Labo a o y] and CSFV s ain Al o /187) we e used o he p o ision o i aemic pigs. Each animal was inocula ed in anasally wi h 5 ml o pig kidney (PK)15 cell-cul u e suspension con aining 1,000 TCID50 (50% issue cul u e in ec i e dose) o he ecen Ge man CSFV isola e Diepholz 1/Han94. Pai s o pigs we e inocula ed wel e days (nos 1 and 2), eigh days (nos 3 and 4) and i e days (nos 5 and 6) be o e he wo kshop commenced. The s age o in ec ion o he pigs du ing he h ee days o he wo kshop was be ween 5 and 14 days pos -inocula ion (d.p.i.). Clinical signs and body empe a u es we e eco ded daily, and blood samples (EDTA blood and se um) we e collec ed h ee imes weekly p io o he wo kshop and daily du ing he wo kshop. Blood samples aken mo e han wo days in ad ance we e s o ed a -20°C p io o use. Vi us isola ion and se ology Re-isola ion o CSFV om bu y coa s and se um was a emp ed using PK15 cell cul u es, as desc ibed elsewhe e (3). Se um samples we e es ed o he p esence o neu alising an ibodies agains he homologous CSFV isola e Diepholz 1/Han94, he CSFV s ain Al o /187 and he BVDV s ain NADL, using a di ec neu alising pe oxidase-linked assay (NPLA) in PK15 and e al cal kidney cells, espec i ely (9). RESULTS Clinical cou se o he disease All animals became eb ile be ween 6 d.p.i. and 8 d.p.i. (Fig. 1). No clinical signs ypical o CSF (e.g. skin haemo hages, ne ous signs) we e isible du ing he wo kshop pe iod. Pig 1 died due o a up u e o he ao a c anialis a 11 d.p.i., be o e he wo kshop s a ed. The emaining i e pigs we e eu hanised one day a e he wo kshop. Vi us isola ion and se ology Resul s o i us isola ion om bu y coa s and se um a e gi en in Figu e 1 and Table II, espec i ely, and show ha i us was de ec ed soone in bu y coa s in i e o six animals. Vi us was no eco e ed om he se um o pig 5. CSFV was i s e-isola ed om leukocy es a be ween 5 d.p.i. and 7 d.p.i. I is no ewo hy ha CSFV could be e- isola ed om he blood o pigs 3,4 and 6 du ing he incuba ion pe iod, one o h ee days be o e he onse o e e . 682 H body empe a u e abo e 40°C KS in anasal inocula ion o pigs wi h CSFV 0 i us isola ion O no i us isola ion FIG.1 Vi us isola ion om bu y coa and body empe a u es o six pigs in anasally inocula ed wi h classical swine e e i us (CSFV) isola e Diepholz 1/Han94 None o he pigs de eloped neu alising an ibodies agains BVDV o CSFV du ing he es pe iod. ELISA esul s A single 3 d.p.i. sample om pig 4 was posi i e wi h he HAN-1 es . O he wise, all samples ound o be nega i e by i us isola ion also es ed nega i e by ELISA. Pigs 1-4 we e de ec ed as posi i e by all ELISAs (Table III). Samples aken om pig 1 a 11 d.p.i. and om pig 2 a 11,12,13 and 14 d.p.i. we e posi i e in all es s. Samples aken om pigs 3 and 4 a 10 d.p.i. we e no es ed wi h all ELISAs, bu we e posi i e in hose used. Fo pigs 5 and 6, blood was a ailable du ing he wo kshop pe iod up o 7 d.p.i. Pig 5 es ed nega i e by all ELISAs h oughou he obse a ion pe iod, excep o a doub ul posi i e esul wi h CVL-2 a 7 d.p.i. Pig 6 es ed posi i e a 6 d.p.i. only by EO-SADA. A 7 d.p.i., his animal es ed posi i e by CVL-2 and doub ully posi i e by HAN-1 and HAN-2. No single ELISA was able o de ec ea ly i aemia as consis en ly as i us isola ion om leukocy es. The sensi i i y o he ELISAs is compa ed in Table IV. Compa ed o i us isola ion, alse-nega i e esul s we e ob ained wi h blood samples aken du ing he i s 8 d.p.i. By 8 d.p.i., howe e , he sensi i i y o he ELISAs inc eased o up o 100%. The blood sample om he cal which was pe sis en ly-in ec ed wi h BVDV sco ed posi i e in all ELISAs, excep in he CSFV-speci ic CVL-2 ELISA. 683 TABLE II Re-isola ion o classical swine e e i us* om se um o pigs a a ious imes pos -inocula ion (shaded a ea indica es wo kshop pe iod) dpi-Pigi Pig 2 Pig 3 Pig 4 Pig 5 Pig 6 4 -- NT NT NT NT 5 NT NT - - - - 6 NT NT NT NT - 0.7 7 0.7 - 2.0 2.0 - 1.5 8 NT NT 2.7 3.0 NT NT 9 2.7 2.5 2.7 3.3 1.7 2.7 10 NT NT 3.3 3.0 NT NT 11 3.0 2.5 NT NT NT NT 12 NT 3.3 NT NT NT NT 13 NT 2.0 2.7 NT NT NT 14 NT 2.0 NT 2.3 3.3 3.3 * log10 i e o i us as 50% issue cul u e in ec i e dose pe 0.1 ml o se um d.p.i.: no. o days pos -inocula ion NT: no es ed - no i us isola ed This s udy concen a ed on e alua ion o i aemia in he ea ly pos -inocula ion pe iod, as his is he ime when clinical signs may be inappa en o non-speci ic and when se ological es s a e gene ally inapp op ia e. The Diepholz 1/Han94 isola e was chosen o inocula ion o he pigs, as i is a ecen ield isola e. I has been shown o cause 90% mo ali y in inocula ed weane s, al hough dea hs did no occu un il 17-25 d.p.i. (4). In he p esen s udy, he pigs we e killed a 15 d.p.i. and, al hough hey became py e ic om 6 d.p.i., hey did no de elop ma ked signs o illness o become mo ibund. As p e iously epo ed, i us isola ion om leukocy es was mo e sensi i e han isola ion om se um and, using he o me echnique, h ee o six pigs became i aemic be o e he onse o e e . Vi aemia pe sis ed in all six pigs o as long as samples we e aken and up o 14 d.p.i. In iew o he ange o i ulence among CSFV isola es, o he challenge s ains would be expec ed o p oduce i aemia and clinical signs o a iable du a ion and in ensi y. As mos o he ELISAs u ilised had no been ully alida ed, posi i e/nega i e cu -o le els we e es ablished by e e ence o he esul s ob ained wi h he p e-challenge samples. Mo e ex ensi e alida ion, as had been conduc ed in he case o he Se elisa, migh ha e equi ed di e en cu -o poin s o be used. In ac , i he same me hod was used o calcula ing he cu -o o he Se elisa as o he o he es s, only a ma ginal quali a i e di e ence in he Se elisa eadings esul ed, wi h one sample becoming posi i e ins ead o doub ul posi i e and one sample becoming doub ul posi i e ins ead ° nega i e. Compa ed o i us isola ion om leukocy es, he e alua ed an igen-cap u e ELISAs we e speci ic, bu less sensi i e o he de ec ion o e y ea ly i aemia, o en equi ing an ex a day o wo o ha e elapsed be o e a posi i e esul was ob ained om indi idual pigs. By 9 d.p.i., howe e , all samples we e consis en ly posi i e wi h all assays. I should be bo ne in mind ha he ela i ely low sensi i i ies o some an igen-cap u e ELISAs shown in Table IV e lec he many weakly posi i e samples 684 TABLE III De ec ion o i al an igens in selec ed samples collec ed om pigs a a ious in e als pos -inocula ion Assay Pig1 Pig 2 Pig 3 Pig 4 Assay 4 d.p.i. 7 d.p.i. 9 d.p.i. 4 d.p.L 7 d.p.i. 9 d.p.i. 5 d.p.i. 7 d.p.i. 8 d.p.i. 3 d.p.i. 5 d.p.i. 7 d.p.i. Vi us isola ion (a> - + + - + + + + + - + + Vi us isola ion (b> - + + -- + - + + -- + Se elisa w - - + - +/-+ - + + -- + Se elisa <<•> - - +/-- - • - - - +/- -- - CVL-1 NT - + - - + -- NT -- + CVL-2 - + - + + - + + -- + Pe ugia - - + --+/- + + + -- + EO-SADA NT + + - + + - - + - + + HAN-1 NT - +/- - + + - + + + + + HAN-2 NT - +/-NT + + - - + - + + a) om bu y coa s b) om se um c) using blood wi h e hylenediamine e aace ic acid d) using se um d.p.i.: no. o days pos -inocula ion - nega i e +/- doub ul posi i e + posi i e NT: no es ed 685 TABLE IV P opo ion o samples posi i e a di e en days pos -inocula ion O e all 1-4 d.p.i. S d.p.i. 6 d.p.i. 7 d.p.i. 8 d.p.i. 9-14 d.p.i. sensi i i y* Vi us isola ion (a) 0/6 3/4 2/2 6/6 2/2 11/11 100% (24/24) Vi us isola ion (b) 0/10 0/4 1/2 4/6 2/2 17/17 75% (18/24) Se elisa (c) 0/10 0/4 0/2 3/6 2/2 10/10 65 % (15/23) Se elisa (d) 0/10 0/4 0/2 0/6 1/2 10/11 45% (11/24) CVL-1 0/9 0/4 0/2 1/6 1/1 10/10 60% (12/20) CVL-2 0/10 0/4 0/2 6/6 2/2 10/10 78% (18/23) Pe ugia 0/8 1/4 0/2 2/4 2/2 8/8 68% (13/19) EO-SADA 0/7 1/4 1/2 3/4 2/2 8/8 78% (15/19) HAN-1 1/9 1/4 0/2 4/6 2/2 11/11 75% (18/24) HAN-2 0/6 1/4 0/2 3/6 2/2 10/10 66% (16/24) d.p.i.: no. o days pos -inocula ion * no. o samples posi i e o doub ul posi i e as a p opo ion o no. posi i e by i us isola ion om bu y coa samples, excluding esul s whe e i us isola ion om bu y coa s was no pe o med a) om bu y coa s b) om se um c) using blood wi h e hylenediamine e aace ic acid d) using se um examined, and g ea e sensi i i y could p obably be achie ed wi h ield samples om na u ally-occu ing cases. A p e ious compa ison be ween i us isola ion and an an igen-cap u e ELISA, using a hype -immune BVDV an ise um as cap u e an ibody and an i-pl20/80 MAbs as immunological de ec o s, also concluded ha ELISA was highly speci ic bu sligh ly less sensi i e (14). A challenge inocula ion wi h a low i ulen CSFV s ain (Bake A) was ound o p oduce a i aemia which was no de ec ed a all by ELISA. Mos p e ious publica ions on an igen-cap u e ELISAs o pes i i uses ha e epo ed on he de ec ion o i us in issues, leukocy es sepa a ed om pe iphe al blood (5, 6, 7, 8, 11, 12, 13, 14) o unsepa a ed, an i-coagula ed blood (1, 2, 15). Leukocy e sepa a ion has been achie ed by cen i uga ion, lash lysis o il a ion. In he case o ELISAs o he de ec ion o ca le pe sis en ly in ec ed wi h BVDV, mos es s ha e u ilised an i-pl20/80 MAbs and do no eliably de ec hese p o eins in se um (D.J. Pa on, unpublished indings). J.C. Thibaul (pe sonal communica ion), howe e , epo s ha a pl20/80-speci ic es can iden i y pes i i us an igen in se a collec ed om pe sis en ly-in ec ed ca le, albei less eliably han i leukocy es a e included. P elimina y indica ions a e ha simila esul s can be achie ed o he de ec ion o CSFV in pig se a (2,10). A es o BVDV i aemia has been epo ed using bo h an i-gp48 and an i-pl20/80 MAbs (12) and, in his case, he au ho s s a ed ha unsepa a ed an i-coagula ed blood did con ain de ec able le els o gp48. In he p esen s udy, he di e en an igen-cap u e ELISAs u ilised a a ie y o me hods o blood sample p epa a ion. Se um can be p epa ed eadily om clo ed blood, and i would be highly ad an ageous o be able o pe o m bo h se ology and Sensi i i y o di e en an igen enzyme-linked immunoso ben assays (ELISAs) in es ing o classical swine e e a a ious imes pos -inocula ion