Trained immunity is not universal: oral heat-inactivated Mycobacterium bovis confers no protection against the non-enveloped Porcine Circovirus 2

Page 1/22 T ained immuni y is no uni e sal: o al hea - inac i a ed Mycobac e ium bo is con e s no p o ec ion agains he non-en eloped Po cine Ci co i us 2 Elisa Fe e as-Colino Ins i u o de In es igación en Recu sos cinegé icos IREC (UCLM-CSIC). Ciudad Real Jose A. Ba asona Uni e sidad Complu ense de Mad id Ma ina Sibila Uni a Mix a d'In es igació IRTA-UAB en Sani a Animal, Cen e de Rece ca en Sani a Animal (CReSA), Campus de la Uni e si a Au ònoma de Ba celona (UAB) Ma ía Maza iegos Uni e sidad Complu ense de Mad id Ri a Vaz-Rod igues Ins i u o de In es igación en Recu sos cinegé icos IREC (UCLM-CSIC). Ciudad Real Fá ima C uz Uni e sidad Complu ense de Mad id Ma inela Con e as Ins i u o de In es igación en Recu sos cinegé icos IREC (UCLM-CSIC). Ciudad Real Joseba M. Ga ido NEIKER-Ins i u o Vasco de In es igación y Desa ollo Ag a io Joaquim Segalés Uni a Mix a d'In es igació IRTA-UAB en Sani a Animal, Cen e de Rece ca en Sani a Animal (CReSA), Campus de la Uni e si a Au ònoma de Ba celona (UAB) José Fuen e Ins i u o de In es igación en Recu sos cinegé icos IREC (UCLM-CSIC). Ciudad Real Lucas Domínguez Uni e sidad Complu ense de Mad id Ch is ian Go áza  (  ch is ian.go aza @uclm.es ) Ins i u o de In es igación en Recu sos cinegé icos IREC (UCLM-CSIC). Ciudad Real Ma ia A. Risalde Uni e sidad de Có doba Page 2/22 Resea ch A icle Keywo ds: hea -inac i a ed Mycobac e ium bo is, po cine ci co i us ype 2, ained immuni y, α-Gal Pos ed Da e: May 3 d, 2023 DOI: h ps://doi.o g/10.21203/ s.3. s-2865092/ 1 License:   This wo k is licensed unde a C ea i e Commons A ibu ion 4.0 In e na ional License.  Read Full License Page 3/22 Abs ac Backg ound T ained immuni y, he enhanced esponse o inna e cells leading o an imp o ed inna e immune esponse, and an ibodies agains he glycan galac ose-α-1,3-galac ose (α-Gal), p oduced by animals unable o syn hesize α-Gal epi opes, ha e been sugges ed o p o ide he hos ce ain ad an age in in ec ions wi h en eloped i uses. Con e sely, he e idence o p o ec ion agains non-en eloped i uses a ibu ed o he e e ed mechanisms emains sca ce. Aiming o e alua e whe he a hea -inac i a ed Mycobac e ium bo is (HIMB) immunos imulan , which had p o en o p o ec agains ela ed and non- ela ed pa hogens, con e s an ad an age agains non-en eloped i uses, we pe o med an immuniza ion and challenge expe imen wi h po cine ci co i us 2 (PCV-2) in swine. Six een pigle s we e andomly assigned o he immunized g oup (n = 8), which ecei ed wo o al doses o HIMB wi h an in e al o h ee weeks, o o he con ol g oup (n = 8). All animals we e in ec ed by in anasal inocula ion wi h PCV-2 21 days la e and eu hanized a day 21 pos -challenge. Resul s No di e ences in body weigh and body empe a u e, i emia and i al bu den in a ge issues, an ibody p oduc ion and his opa hological changes in a ge issues we e obse ed be ween he immunized and he con ol g oup. O e all, o al immuniza ion wi h HIMB did no p o ec pigs agains PCV-2 in ec ion. Conclusions Ou s udy sugges s ha HIMB con e s no ad an age agains pa hogens lacking α-Gal, mainly non- en eloped i uses such as PCV-2, in α-Gal-p oducing hos s, such as he swine. Backg ound The e m “ ained immuni y” was p oposed byNe ea e al. (2011)[1] o e e o an adap a i e esponse elici ed by he inna e immune sys em owa ds a subsequen encoun e no only wi h he same pa hogen bu also wi h non- ela ed ones. T ained immuni y o “inna e immune memo y” occu s in a T and B cell- independen and non-specic manne and, hus, he inna e immuni y may p o ide c oss-p o ec ion agains a wide ange o pa hogens e en in he absence o an adap a i e esponse[2]. T ained immuni y in ol es epigene ic modica ions and me abolic changes unc ioning in e dependen ly wi hin inna e cells, mainly na u al kille (NK) cells and monocy es/mac ophages[3–5]. The Bacille Calme e-Gué in (BCG) accine, a li e a enua ed Mycobac e ium bo is s ain, beyond being he only comme cialized accine o p e en human ube culosis, has eme ged as one o he mos widely explo ed induce s o ained immuni y[6]. Indeed, nume ous in i o and in i o expe iences ha e demons a ed ha BCG boos s p oinamma o y cy okines p oduc ion in NK cells and Page 4/22 monocy es/mac ophages upon es imula ion wi h non- ela ed mic oo ganisms, hus leading o non- specic and adap a i e-independen p o ec ion in he hos [6]. Since 2020, wo ldwide labelled as he yea o he pandemic o co ona i us disease 19 (COVID- 19) caused by se e e acu e espi a o y synd ome co ona i us 2 (SARS- CoV- 2), he scien ic in e es ega ding he ole o “ ained immuni y” in i al in ec ions has isen (o e 60 en ies pe yea in 2020, 2021 and 2022 e sus 20 o less in p e ious yea s in he Scopus da abase). Ne e heless, he p o ec ion agains i al in ec ion a ibu ed o ained immuni y is s ill unclea (Table 1). P elimina y expe iences o immuniza ion wi h BCG o β-glucans e idenced p o ec ion agains di e se i uses. Fu he assessmen o he e ec o bo h immunos imulan s in i al in ec ions con med he educ ion in i al bu den, mo ali y, and mo bidi y, as well as a shi in he cy okine p ole (Table 1). Howe e , ce ain expe imen al s udies epo ed ailu e o he he e ologous p o ec ion expec ed om ained immuni y induce s. Fo ins ance, no p o ec i e e ec o p io BCG accina ion was obse ed ega ding inuenza in ec ion[7], and no p o ec i e e ec o β-glucans in zeb ash in ec ed wi h sp ing i emia ca p i us[8]no in u bo s in ec ed wi h i al hemo hagic sep icemia i us[9]. In addi ion, BCG does no educe lesions, mo bidi y, mo ali y o i al bu den in he SARS-CoV-2 in ec ion model in mice[10]o p ima es[11]. Mos o he li e a u e e e s o a benecial e ec o ained immuni y agains i uses ha a e en eloped, while e idence o p o ec ion agains non-en eloped i uses is limi ed ( o non-en eloped i uses see:[12–18]). P ima es, including humans, as well as sh, amphibians, ep iles, and bi ds, e ol ed wi h he inabili y o syn hesize he glycan galac ose-α-1,3-galac ose (α-Gal). The e o e, hey na u ally p oduce an ibodies in esponse o his modica ion p esen in glycop o eins, glycolipids and o he biomolecules p esen in o he species’ cells, including gas oin es inal mic obio a, and i uses[19,20]. Thus, an ibodies agains α-Gal ha e been sugges ed o con e non-specic p o ec ion agains en eloped i uses con aining α-Gal epi opes in i s glycop o eins[19,21,22]. An i-α-Gal an ibodies bound o i uses may ac i a e he complemen sys em o i us lysis o up ake by dend i ic cells and mac ophages and be p esen ed o T cells o igge adap a i e esponses[19]. T ained immuni y has been also p oposed o be associa ed wi h α-Gal[22,23]. Acco dingly, se e al expe imen al s udies ha e demons a ed ha an i-α-Gal an ibodies con ibu e o neu aliza ion o en eloped i uses[24,25]; hus, ecacy o en eloped i us accines can be inc eased by exp ession o α-Gal epi opes in he i al su ace[26–29]. Al hough i has been mo e ex ensi ely explo ed in oden s[30–32]and humans[14,15,33], he no el phenomenon o aining he inna e immuni y a ouses g owing in e es in domes ic animals ( e iewed inAngulo and Angulo, 2022). In pa icula , pigle s ha had been adminis e ed Saccha omyces ce e isae β-glucan o ally p io o being challenged wi h he en eloped swine inuenza i us displayed milde clinical signs and lung lesions, as well as highe in e e on (IFN)-γ and ni ic oxide (NO) le els[35]. Likewise, p iming wi h β-glucan impai ed cell in asion and eplica ion o he en eloped A ican Swine Fe e i us and boos ed IFNα and in e leukin (IL)-6 le els in po cine al eola mac ophages[36]. Recen ly, By ne e al. (2020)[37]p o ided he  s e idence o ained immuni y induced by BCG in pigs, as hey obse ed ha s imula ing po cine monocy es wi h BCG in i o enhanced IL-1β and umo nec osis ac o (TNF)-α gene exp ession and p o ein p oduc ion upon LPS e-s imula ion. In line wi h his nding, pigs Page 5/22 ha had ecei ed an immunos imulan composed o hea -inac i a ed M. bo is (HIMB) ia he o al ou e p io o being challenged wi h Salmonella en e ica se o a Chole aesuis displayed inc eased p oinamma o y cy okines p oduc ion and an ioxidan ac i i y, oge he wi h educed pulmona y lesions, compa ed wi h non-immunized pigs[38]. Mo eo e , HIMB also elici s pa ial immuni y agains p o ozoal[39]and a h opod ( icks;[40]in ec ions in di e en animal models, al hough i s c oss- p o ec ion agains i uses has no been e alua ed o da e. Po cine ci co i us 2 (PCV-2), a small non-en eloped i us, is conside ed one o he mos ele an pa hogens ha a ec swine wo ldwide, mainly due o he subs an ial economic losses i causes o he pig indus y[41]. PCV-2 in ec ion has been associa ed wi h a ious disease synd omes in bo h domes ic pigs and wild boa , such as PCV-2-sys emic disease (PCV-2-SD, o me ly known as pos weaning mul isys emic was ing synd ome), PCV-2- ep oduc i e disease, PCV-2-subclinical in ec ion and po cine de ma i is and neph opa hy synd ome (PDNS). Impo an ly, PCV-2-SD a ec ed pigs su e om immunosupp ession[41–44]. Cu en ly, all comme cialized PCV-2 accines a e only indica ed o he pa en e al ou e[41], which is a limi a ion o a ge wild boa and ee- ange pigs [45,46]. Consequen ly, al hough se e al a emp s o de elop o al accines composed o ecombinan bac e ia[47,48]o yeas s[49–52]exp essing PCV-2 Cap p o ein ha e e ealed p omising esul s, he e is s ill a long way o go be o e achie ing an o ally deli e ed p epa a ion ha is easible o p o ec swine agains PCV-2. Thus, he objec i e o he p esen s udy was o e alua e he e ec o he o al HIMB immunos imulan in he po cine model o in ec ion wi h he non-en eloped i us PCV-2. Resul s Clinical signs No clinical sings we e obse ed h oughou he expe imen . No signican e ec o he immuniza ion was obse ed on ei he body empe a u e (Figu e 2A) o weigh gain (Figu e 2B). PCV-2 load Vi emia and i al load we e es ima ed weekly by de ec ing PCV-2 DNA load in blood and he p og ession was simila in bo h g oups: s eadily inc eased a e in ec ion ( i emia: HIMB = 3/8, con ol = 3/8; i al load: HIMB = 2.97 x 104copies/ml, con ol = 3.38 x 104copies/ml), eaching a peak 14 dpi ( i emia: HIMB = 8/8, con ol = 8/8; i al load: HIMB = 5.75 x 104copies/ml, con ol = 5.59 x 104copies/ml), and hen dec eased un il he end o he expe imen ( i emia: HIMB = 3/8, con ol = 5/8; i al load: HIMB = 2.20 x 104copies/ml, con ol = 2.40 x 104copies/ml) (Figu e 3A). No di e ences in pe cen age o i emic animals and mean i al loads be ween g oups we e de ec ed a any imepoin . PCV-2 an igen in a ge issues was e alua ed a e nec opsy, de ec ing low amoun o an igen in bo h g oups (mean o al sco e: HIMB = 0.0892; con ol = 0.0892). Al hough he immunized g oup showed an appa en ly highe p opo ion o animals wi h one o mo e PCV-2-immunolabeled issues (HIMB: 4/8 (50%), con ol: 3/8 (37.5%)), he di e ences be ween g oups we e no signican ( P = 0.72). Page 6/22 Humo al esponse Humo al esponse was measu ed h ough he p oduc ion o an i-PCV-2 an ibodies. As expec ed, an ibody i e s s a ed aising upon in ec ion, eaching maximum le els wen y-one days a e in ec ion (Figu e 3B). No di e ences in an ibody S/P a ios be ween g oups we e de ec ed. No an i-P22 an ibodies we e de ec ed. Pa hological ndings          To assess p o ec ion agains PCV-2, mac oscopic and mic oscopic lesions we e examined a he end o he expe imen . All indi iduals showed a good gene al condi ion a nec opsy. Among he mac oscopic lesions, he main ndings we e mode a e conges ion and lymphadenomegaly in se e al LNs, especially in he mandibula , acheob onquial, medias inal, mesen e ic and inguinal ones (Figu e 4A). The p opo ion o animals wi h lymphadenomegaly in he pulmona y LNs ( acheob onquial and medias inal) was signican ly highe in he HIMB g oup (7/8, 87.5%) compa ed wi h he con ol g oup (1/8, 12.5%) (P<0.04). Mic oscopically, mandibula and pulmona y LNs p esen ed mild lymphocy e deple ion and low numbe o his iocy es and mul inuclea ed gian cells inl a e (Figu e 4B, and Figu es 5A, B), occasionally associa ed o low amoun o PCV-2 an igen (Figu e 4B inse and Figu es 5A, B). Sca ce p esence o PCV-2 an igen also was obse ed in he onsil o some animals, along wi h ligh inl a ing o his iocy es and mul inuclea ed gian cells, and lymphocy e deple ion (Figu e 5C). Mo eo e , mild o mode a e in e s i ial pneumonia was equen ly obse ed in bo h g oups, composed by inl a e o mononuclea cells in he pulmona y pa enchymal (Figu es 4C, D and Figu e 5D). In addi ion, one indi idual om each g oup showed mild in e s i ial neph i is (da a no shown). The appea ance and se e i y o mic oscopic lesions was simila be ween g oups and no signican di e ences we e de ec ed (Figu e 5). α-Gal con en in PCV-2 The p esence o α-Gal in PCV-2 was assessed by a di ec ELISA o he in ec i e i al inoculum, wi h an unin ec ed PK15 cell cul u e as con ol. As expec ed o non-en eloped i uses such as PCV-2, α-Gal con en was lowe in pu ied i uses (0.20 ng/μg) han in cul u ed cells om an α-Gal p oducing animal such as he pig (0.43 ng/μg). Discussion S imula ing he immune sys em wi h ained immuni y induce s, such as mycobac e ial o ungal de i ed compounds, cons i u es a s a egy o elici non-specic p o ec ion agains a wide ange o pa hogens, including ce ain i uses [5]. Howe e , o al immuniza ion wi h HIMB, an immunos imulan ha had p e iously demons a ed p o ec i e e ec a ibu ed o ained immuni y in he e ologous in ec ions [38, 39, 61] ailed o exe p o ec i e e ec s in he PCV-2 in ec ion model. A pa amoun conce n abou PCV-2-SD in he pig indus y is he de imen in he a e age daily weigh gain [62]. In he p esen s udy, o al HIMB immuniza ion did no inuence weigh gain o body empe a u e in Page 7/22 pigs in ec ed wi h PCV-2. Al hough [63] did no obse e a benecial e ec on g ow h pe o mance a e immuniza ion, mos eld ials showed be e weigh gain in pigs accina ed wi h a comme cial accine [64, 65]. O he hallma ks o PCV-2-SD in pigs a e lymphocy e deple ion oge he wi h g anuloma ous inamma ion o lymphoid issues and in e s i ial pneumonia [66]. In ag eemen wi h p e ious s udies [57, 67, 68], in e s i ial pneumonia and enla ged LNs we e ou p edominan ndings, wi h occasional p esence o lymphocy e deple ion and his iocy ic inl a e wi h mul inuclea ed gian cells, pigs o ally immunized wi h HIMB no showing lowe lesion sco es he compa ed o he con ol animals. The capaci y o comme cialized accines o alle ia ing pa hology in na u ally PCV-2 in ec ed pigs has been widely demons a ed in he eld [69, 70], and he benecial e ec has also been e iden unde expe imen al condi ions. Fo ins ance, Seo e al. (2014) [71] obse ed lowe lymphoid deple ion and g anuloma ous eplacemen in pigs accina ed wi h ei he Fos e a PCV/Su axyn PCV-2 one dose (Zoe is), Ci co ac (Me ial), Ci coex (Boeh inge Ingelheim Ve medica) and Po cilis PCV (Me ck, Sha p and Dohme Animal Heal h) and in ec ed wi h PCV-2 compa ed wi h non- accina ed pigs. O al immuniza ion wi h HIMB did no educe PCV-2 load in pig, illus a ed by i emia and i al load in issues, and did no s imula e a specic humo al esponse. Con e sely, he ecacy o comme cialized specic accines o educe i emia and i al bu den in issues in pigs in ec ed wi h PCV-2, associa ed o a s ong an ibody esponse, has been con med unde expe imen al and eld condi ions [63–66, 71, 72]. The p esen s udy challenges he p e ailing pa adigm o ained immuni y being uni e sal and sugges s ha i migh no be ex endable o all pa hogens, o ins ance, non-en eloped i uses such as PCV-2. Simila ly, he lack o e ec o β-glucan o BCG in expe imen al o na u al in ec ions wi h en eloped i uses had been p e iously epo ed [7–11, 73, 74] Howe e , o he bes o ou knowledge, only a ew s udies ha e explo ed he p o ec i e ecacy o he compounds o in e es agains non-en eloped i uses [12–18]. Xue e al. (2017) [16] assessed he e ec o As agalus spp. polysaccha ides on PCV-2 in ec ion in an in i o model using po cine PK15 cells and in an in i o model using mice, and concluded ha As agalus spp. polysaccha ide supp essed PCV-2 in ec ion by inhibi ing endoplasmic e iculum s ess. S ill, he la e esul s should be ad essed ca e ully and u he esea ch is needed o ex apola e hem o PCV-2 in ec ion in i s na u al hos , he pig. Ul ima ely, we con med ha , as expec ed om a non-en eloped i us [19, 21], PCV-2 does no con ain α- Gal. Unlike o en eloped i uses (because he ca bohyd a e chains o he en elope glycop o eins a e adqui ed om he hos golgi appa a us du ing i al eplica ion), ac i a ion o he inna e componen s, such as he complemen sys em and an igen p esen ing cells, media ed by na u al an i-α-Gal an ibodies [19, 21] would no con ibu e in he hos immuni y agains non-en eloped i uses including PCV-2. Fu he mo e, non-specic immune an ibody-media ed and non-an ibody-media ed mechanisms in esponse o α-Gal may no be ac i a ed in α-Gal-posi i e hos s. Conclusions Page 8/22 In conclusion, ou esul s demons a e ha o al immuniza ion wi h HIMB does no p o ec swine agains challenge wi h PCV-2, a non-en eloped DNA i us. Conside ing he absence o α-Gal in PCV-2, ou s udy sugges s ha HIMB con e s no ad an age agains pa hogens lacking α-Gal in hos s p oducing his molecule. Me hods Animals and expe imen al design Six een 21 days-old Land ace x La ge Whi e hyb id emale pigle s wi h homogeneous weigh s and es ed as PCV-2 PCR nega i e wi h low an i-PCV2 an ibody i e s we e ob ained om a comme cial pig p oduc ion a m. Animals we e housed in class III biocon ainmen animal acili ies (BSL-3) loca ed a VISAVET Heal h Su eillance Cen e (Mad id, Spain) o acclima iza ion one week be o e he expe imen . All animals ecei ed con inuous access o wa e , non-medica ed pig eed and e e ina y moni o iza ion. Indi iduals we e andomly assigned o ei he he immunized g oup (n=8), which ecei ed wo o al doses o HIMB wi h an in e al o h ee weeks (-42 and -21 days p e-in ec ion), o he con ol g oup (n=8), which ecei ed PBS ins ead o immunos imulan . Twen y-one days a e he second immuniza ion dose (day 0), all animals om bo h g oups we e in ec ed by in anasal inocula ion wi h 105 TCID50 o PCV-2 pe animal (Figu e 1). All pigs we e con med as PCV-2 PCR nega i e and PCV-2 ELISA nega i e be o e challenge. Animals we e handled on 0, 7, 14 and 21 days pos -in ec ion (dpi) o blood sampling, ec al empe a u e and weigh measu emen s. All pigle s we e eu hanized 21 dpi by cap i e bol a e seda ion wi h xylazine(Xilagesic 2%, Labo a o ies Calie , Ba celona, Spain)and we e subjec ed o nec opsy o assess PCV-2 g oss lesions in pala ine onsil, as well as mandibula , acheob onchial and medias inal lymph nodes (LNs), lung (c anial and caudal lobes) and kidney. Typical PCV-2 g oss lesions such as lymphadenomegaly, in e s i ial pneumonia and neph i is we e e alua ed byexpe ienced pa hologis s.Lesion se e i y was g aded wi h a lesion sco e (0 =absen , 1 = mild, 2 = mode a e o 3 = se e e). Samples om pala ine onsil, lung (c anial and caudal lobes), as well as mandibula , acheob onchial and medias inal LNs we e xed by imme sion in 10% neu al bu e ed o malin du ing 24 hou s a oom empe a u e (RT),dehyd a ed in a g aded se ies o e hanol, imme sed in xylol, and embedded in pa an wax using an au oma ic p ocesso  o u he his opa hology and immunohis ochemical s udies. Hea -inac i a ed Mycobac e ium bo is (HIMB) immunos imulan  The o al immunos imulan consis ed o 2 mL o s e ile PBS con aining app oxima ely 107 hea - inac i a ed colony o ming uni s (CFU)/mL o a eld isola e (S ain 1403; spoligo ype SB0339) o iginally ob ained om a na u ally in ec ed wild boa . HIMB was p epa ed a Neike -Tecnicalia (De io, Spain) ollowing he p o ocol desc ibed by Ga ido e al. (2011)[53], excep o an ex ended inac i a ion s ep a 83 °C o 45 minu es (min). Bac e ial concen a ion was de e mined p io o inac i a ion by measu ing Tu bidi y in a VITEK® DensiCHEK® (BioMe ieux) and by pla ing a se ially dilu ed aliquo on o aga - Page 9/22 solidied Middleb ook 7H9 wi h glyce ol (0.2% / ) and OADC (10% / ) (Bec on Dickinson, F anklin Lakes, NJ, USA). Po cine ci co i us 2 PCV-2 geno ype b (Sp-10-7-54-13 s ain a 14 hpassage) was used as he inoculum[54]. This s ain was isola ed om he lymphoid issues o a eld case o PCV-2-SD in 2006 in Spain. Fo his s udy, he PCV-2 s ain was p opaga ed in PCV- ee PK15 cells in minimal essen ial medium (MEM)[55] o a i e o 105 TCID50/mL. Humo al esponse Blood was ex ac ed h ough punc u e o he sinus oph halmicus a in e als o one week upon in ec ion. Blood samples we e cen i uged a 400 g o 10 min o ex ac se a which was u he s o ed a – 20 °C. Se a we e es ed in duplica e by means o Ingezim CIRCO IgG ELISA (R.11.PCV.K1; Eu ons Technologies, SA; Mad id, Spain) o de ec an ibodies agains PCV-2 VP2 p o ein, ollowing manu ac u e ’s ins uc ions. Se um samples we e also analyzed by ELISA o de ec an ibodies agains Mycobac e ium ube culosis complex using P22 as an igen in an indi ec in-house ELISA p e iously desc ibed by Thomas e al. (2019)[56]. The es ima ed sensi i i y and specici y o his ELISA in swine was 84.1% and 98.4%, espec i ely. De ec ion o po cine ci co i us 2 in ec ion by eal ime quan i a i e PCR (qPCR) DNA was ex ac ed om 200 μL o se um by using he MagMAX™ Pa hogen RNA/DNA Ki (The mo Fische Scien ic Bal ics. Vilnius, Li huania) ollowing he manu ac u e ’s ins uc ions. The DNA ob ained was suspended in 90 μL o elu ion solu ion. The comme cial QPCR ki Ve MAX™ Po cine PCV2 Quan Ki (Applied Biosys ems, Lisseu, F ance) was used o de ec and quan i y PCV-2 DNA in se um samples. PCV- 2 qPCR esul s we e exp essed as PCV-2 copies/mL o se um. A cu o alue o 104 PCV-2 DNA copies/mL was conside ed he quan ica ion limi o he PCV-2 qPCR. Vi emia ep esen ed he p opo ion o posi i e animals (quan iable and non-quan iable) pe g oup, and i al load ep esen ed he mean o he quan iable indi iduals pe g oup. De ec ion o po cine ci co i us 2 in ec ion by immunohis ochemis y Immunohis ochemis y was pe o med o de ec PCV-2 as p e iously desc ibed in Chianini e al. (2003)[57]. B iey, issue sec ions we e depa anized wi h xylene and ehyd a ed h ough g aded alcohols. Endogenous pe oxidase ac i i y was blocked by incuba ion wi h hyd ogen pe oxide 3% in dis illed wa e o 30 min. A e wa ds, an igen e ie al was pe o med using 0.1% P o ease XIV (Sigma, e . P5147) in PBS o 8 min a 37ºC. A monoclonal PCV-2 an ibody (Ingenasa, M.11.PCV.I36A9) able o ecognize he Cap p o ein was used a 1/1000 dilu ion in 0.1 M T is-bu e ed saline and incuba ed o e nigh a 4-8ºC. Dako EnVision Sys em-HRP Labelled Polyme An i-Mouse (Dako, e . K4001) was Page 16/22 4. Beach NM, Meng XJ. Ecacy and u u e p ospec s o comme cially a ailable and expe imen al accines agains po cine ci co i us ype 2 (PCV2). Vi us Res [In e ne ]. Else ie B.V.; 2012;164:33–42. A ailable om: h p://dx.doi.o g/10.1016/j. i us es.2011.09.041 47. Xu XG, Zhao HN, Zhang Q, Ding L, Li ZC, Li W, e al. O al accina ion wi h a enua ed Salmonella en e ica se o a Typhimu ium exp essing Cap p o ein o PCV2 and i s immunogenici y in mouse and swine models. Ve Mic obiol [In e ne ]. Else ie B.V.; 2012;157:294–303. A ailable om: h p://dx.doi.o g/10.1016/j. e mic.2012.01.008 4. Wang L, Zhao D, Sun B, Yu M, Wang Y, Ru Y, e al. 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Figu es Page 19/22 Figu e 1 Expe imen al design. Six een 21 days-old Land ace x La ge Whi e hyb id emale pigle s we e andomly assigned o he immunized g oup (n=8), which ecei ed wo o al doses o HIMB wi h an in e al o 3 weeks, o he con ol g oup (n=8), which ecei ed PBS ins ead o he immunos imulan . Twen y-one days a e he second immuniza ion dose, all he animals o bo h g oups we e in ec ed by in anasal inocula ion wi h 105 TCID50 o PCV-2. Twen y-one days a e in ec ion, all animals we e eu hanized and nec opsied. Page 20/22 Figu e 2 Tempe a u e and body weigh inc emen s. Mean ± SE o body empe a u e (A) and body weigh (B) in immunized and con ol g oups o animals in ec ed wi h PCV-2. Figu e 3 Vi al bu den and an ibody S/P a io o PCV-2. A) PCV-2 DNA copies/mL o se um by indi iduals om immunized and con ol g oups. A cu o alue o 104 PCV-2 DNA copies/mL (black dashed line) was conside ed he quan ica ion limi o he PCV-2 qPCR. B) Mean ± SE o an i-PCV2 an ibody S/P a io by g oup a each ime poin a e PCV2 in ec ion in immunized and con ol g oups. An S/P a io o 0.29 (black dashed line) o g ea e was conside ed se oposi i e o an i-PCV-2 an ibodies. Page 21/22 Figu e 4 PCV-2 lesions a 21 days pos -in ec ion. PCV-2-in ec ed pigs om bo h g oups showed simila lesions, p esen ing a mode a e conges ion and lymphadenomegaly, e.g. medias inal lymph node (LN) (A). Mild inl a ing o his iocy es and mul inuclea ed gian cells was obse ed in he mandibula and pulmona y LNs o some animals om bo h g oups (B), associa ed o he p esence o PCV-2 an igen (B, inse ). Bo h, immunized and con ol g oups, also p esen ed mild o mode a e in e s i ial pneumonia (C), mainly cha ac e ized by mononuclea leukocy e inl a es in he pulmona y pa enchyma (D). Hema oxylin and eosin s ain. Page 22/22 Figu e 5 Sco e o his opa hological ndings and an igen de ec ion associa ed o PCV-2 in ec ion. Mean o he sco e o each mic oscopic lesion and PCV-2 an igen de ec ion in mandibula (A) and pulmona y ( acheob onchial and medias inal) lymph nodes (B), onsil (C) and lung (D). Lesions we e g aded by sco e as absen o 0% (0), mild o 1% o 30% o he pa enchyma a ec ed (1), mode a e o 30% o 70% a ec ed (2), and se e e o 70% o 100% a ec ed (3). PCV-2 an igen de ec ion by immunohis ochemis y was g aded by sco e as low (1), mode a e (2) and in ense (3) amoun o an igen.