Maternal Autogenous Inactivated Virus Vaccination Boosts Immunity To PRRSV in Piglets Part 1

Abstract:

Kev tiv thaiv niam txiv yog ib qho tseem ceeb rau kev ciaj sia thiab kev vam meej ntawm cov xeeb ntxwv hauv npua los tiv thaiv cov kab mob sib kis xws li Hom 2 Porcine Reproductive thiab Respiratory Syndrome Virus (PRRSV-2).

Lub hom phiaj ntawm txoj kev tshawb fawb no yog txhawm rau tshawb xyuas qhov hloov pauv ntawm kev tiv thaiv PRRSV rau piglets los ntawm gilts uas tau txais tus kab mob hloov kho-nyob (MLV) ib leeg (kev kho mob (TRT) 0), lossis ua ke nrog ib qho ntawm ob qho autogenous inactivated tshuaj tiv thaiv (AIVs, TRT 1 plus 2). Piglets los ntawm cov gilts tau sib tw nrog autogenous PRRSV-2 strain thaum muaj hnub nyoog ob lub lis piam thiab lawv cov tshuaj tiv thaiv kab mob hloov pauv (IR) raug soj ntsuam mus txog 4 lub lis piam tom qab txhaj tshuaj (dpi).

Cov kab mob humoral thiab cellular IR tau txheeb xyuas nyob rau hauv pre-farrow gilts, hauv piglets, pre-inoculation, thiab ntawm 2 thiab 4 wpi. Ob leeg AIVs ib feem tiv thaiv cov piglets nrog txo cov ntsws pathology thiab nce qhov hnyav; TRT 1 kuj txo qis piglet viremia, zoo tshaj plaws piav qhia los ntawm AIV-induced zus tau tej cov neutralizing antibodies nyob rau hauv gilts thiab lawv hloov mus rau piglets. Nyob rau hauv piglets, pre-inoculation, lub ntsiab systemic IFN producers yog CD21 ntxiv rau B hlwb. Los ntawm 0 mus rau 4 wpi, lub luag hauj lwm ntawm cov B hlwb tsis kam thiab CD4 T hlwb tau los ua thawj IFN- cov neeg tsim khoom.

Cov kab mob nyob zoo hais txog cov kab mob uas tsis ua rau muaj kab mob, thiab lawv muaj kev sib raug zoo nrog kev tiv thaiv ntawm tib neeg lub cev. Cov kab mob muaj sia tuaj yeem txhawb nqa tib neeg lub cev tiv thaiv kab mob los tsim cov tshuaj tiv thaiv kab mob, yog li txhim kho tib neeg kev tiv thaiv. Cov kev tshawb fawb tau pom tias tom qab tib neeg lub cev tau kis tus kab mob nyob, nws yuav tso qee cov neeg nruab nrab thiab cytokines, uas tuaj yeem ua rau lub cev tiv thaiv kab mob, txhawb kev ua haujlwm ntawm lub cev tiv thaiv kab mob, thiab txhim kho lub cev muaj peev xwm tiv thaiv kab mob.

Tsis tas li ntawd, cov kab mob benign thiab siv tau kuj tuaj yeem sib tw hauv cov hlwb, ua rau nws nyuaj rau cov kab mob nkag mus thiab rov tsim dua, yog li tiv thaiv tib neeg lub cev los ntawm lwm yam kab mob. Yog li ntawd, qhov tsim nyog tus kab mob benign thiab muaj sia nyob tuaj yeem txhim kho tib neeg kev tiv thaiv kab mob, tab sis kev kis kab mob ntau dhau tuaj yeem ua rau muaj kab mob. Yog li ntawd, nyob rau hauv lub neej txhua hnub, tib neeg yuav tsum tau xyuam xim rau kev tswj cov kab mob ntawm cov kab mob nyob thiab txhim kho kev tiv thaiv los ntawm kev noj qab nyob zoo. Los ntawm qhov kev xav no, peb yuav tsum txhim kho kev tiv thaiv. Cistanche tuaj yeem txhim kho kev tiv thaiv zoo. Nqaij tshauv muaj ntau yam khoom xyaw lom, xws li polysaccharides, ob lub nceb, thiab Huangli, thiab lwm yam. Cov khoom xyaw no tuaj yeem txhawb nqa ntau yam haujlwm ntawm lub cev. cell zoo li cov hlwb, ua kom lawv lub cev tiv thaiv kab mob.

Nyem qhov txiaj ntsig kev noj qab haus huv ntawm cistanche

Hauv lub ntsws, CD8 T hlwb yog thawj thiab CD4 T hlwb yog cov neeg tsim khoom thib ob IFN, suav nrog cov txheej txheem tshiab ntawm porcine CD8 −CCR7− CD4 T hlwb, muaj feem cuam tshuam txog CD4 TEMRA hlwb. Hauv cov ntsiab lus, txoj kev tshawb fawb no qhia tau hais tias kev txhaj tshuaj tiv thaiv AIV niam tuaj yeem txhim kho kev tiv thaiv cov piglets ua ntej-weaning tiv thaiv PRRSV-2; nws qhia txog qhov tseem ceeb ntawm kev hloov cov tshuaj tiv thaiv kab mob rau piglets, thiab nws qhia txog ob lub cev tiv thaiv kab mob tshiab hauv npua-IFN- tsim CD21 ntxiv rau B hlwb thiab CD8 −CCR7- CD4 T hlwb.

Ntsiab lus:

txhaj tshuaj tiv thaiv niam txiv; autogenous inactivated tshuaj tiv thaiv; hloov kev tiv thaiv; humoral tiv thaiv kab mob; cell-mediated immune teb; T hlwb; PRRSV; npua; IFN- tsim cov B hlwb; CD4 TEMRA hlwb.

1. Taw qhia

Maternal-derived immunity (MDI) nyob rau hauv cov tsiaj yog ib tug txaus ntshai symphony ("harmonious complexity"—[1]). Nyob rau hauv cov txheej txheem no, los yog symphony, ib leej niam hloov lub cev tiv thaiv kab mob (ib ntus thiab / los yog nyob mus ib txhis) rau nws cov xeeb ntxwv thaum lub sij hawm cev xeeb tub los ntawm lub placenta, tom qab yug los ntawm mammary secretions, los yog ob qho tib si [2]: tib neeg tau txais immunoglobulins thaum lub sij hawm gestation thiab nws thiaj li los ntawm mammary. cov kua qaub [3]; dev thiab nas tau txais cov tshuaj immunoglobulins thaum cev xeeb tub thiab los ntawm cov qog mammary, thiab npua thiab lwm cov tsiaj nyeg tsuas tau txais cov tshuaj immunoglobulins los ntawm cov qog mammary.

Zoo ib yam li lwm hom, piglets tau txais MDI no nyob rau hauv daim ntawv ntawm immunoglobulins, lub cev tiv thaiv kab mob, thiab lwm yam kev tiv thaiv kab mob molecules [4,5]. Thaum immunoglobulins tuaj yeem tau txais los ntawm cov noob qoob loo sib txawv (= kev yug me nyuam), tsuas yog cov niam yug menyuam tuaj yeem ua tiav cov kab mob tiv thaiv kab mob rau lawv cov xeeb ntxwv [5], thiab qhov ntau ntawm cov colostrum tau txais hauv cov piglets txo qis kev tuag thiab txhim kho kev ua haujlwm [6 ]. Cov kab mob tiv thaiv kab mob no suav nrog B hlwb [7], nrog rau T-cell subsets-CD4, CD8, thiab feem ntau T-cell receptor (TCR)- δ T cells [8].

Cov T-cell subsets no tuaj yeem sib txawv ntxiv raws li lawv qhov sib txawv theem thiab homing qauv. Hauv npua, CD8 saw yog siv los sib txawv CD8 − naïve los ntawm CD8 ntxiv rau antigen-experienced los yog nco CD4 T hlwb [9–11]. Kev qhia ntawm chemokine receptor (CCR) 7 ntxiv qhov txawv ntawm CCR7 ntxiv rau cov qog ntshav qab zib homing los ntawm CCR7- peripheral cov ntaub so ntswg-homing T cells [12]. Nrog rau kev soj ntsuam CD8 / CCR7 ua ke, peb tuaj yeem paub qhov txawv ntawm ntau qhov sib txawv T-cell subsets: CD8 −CCR7 plus naïve CD4 T cells, CD8 ntxiv rau CCR7 ntxiv rau hauv nruab nrab nco (TCM) CD4 T hlwb, thiab CD8 ntxiv rau CCR7-effector nco (TEM. ) CD4 T hlwb; peripheral cov ntaub so ntswg-homing CCR7- thiab lymph node homing CCR7 ntxiv rau CD8 T cells, thiab CD8 −CCR7−, CD8 plus CCR7−, thiab CD8 plus CCR7 plus TCR- δ T cells.
Thaum peb yav dhau los tau tshaj tawm tias porcine TCR- δ T hlwb muaj qhov hloov pauv hloov pauv piv rau lawv cov TCR-tus neeg sib tw, lub luag haujlwm tshwj xeeb ntawm cov khoom sib txawv no tseem tsis tau piav qhia [13]. Ob leeg, lub cev tiv thaiv kab mob thiab cov tshuaj tiv thaiv kab mob raug xa mus rau cov xeeb ntxwv los tiv thaiv lawv ntawm ntau yam kab mob [4,5].

Tau ntau xyoo lawm, cov kab mob phem tshaj plaws rau npua yog Porcine Reproductive thiab Respiratory Syndrome Viruses (PRRSV, [14,15]). Ob hom tseem ceeb ntawm PRRSV muaj nyob - hom 1 thiab 2 (PRRSV-1 thiab 2); lawv qhov muaj ntau haiv neeg thiab kev tiv thaiv kab mob ua rau PRRSV muaj teeb meem kab mob, ua rau muaj kev poob nyiaj txiag loj heev rau kev lag luam swine [16] thiab txog tam sim no, thaum cov tshuaj tiv thaiv ib feem tiv thaiv PRRSV, lawv tsis muaj peev xwm tswj tau tus kab mob no tag nrho.

Ob hom tshuaj tiv thaiv tseem ceeb muaj los tiv thaiv PRRSV-hloov cov kab mob nyob (MLV) cov tshuaj tiv thaiv thiab cov tshuaj tiv thaiv tsis muaj zog, feem ntau yog cov tshuaj tiv thaiv kab mob autogenous inactivated (AIVs); Txawm li cas los xij, ob qho tib si ntsib lawv cov teeb meem.

Lub sij hawm ntev los tsim cov tshuaj tiv thaiv kab mob MLV heev txhais tau hais tias lawv feem ntau muaj ntau dua heterologous rau kev ua liaj ua teb-prevalent PRRSV hom, thiab thaum AIVs raug coj tawm tsam cov kab mob ua liaj ua teb, lawv qhov xwm txheej tsis zoo ua rau lawv feem ntau tsis muaj tshuaj tiv thaiv kab mob.

Cov kev cov nyom no ua rau tsis muaj kev tiv thaiv tshwj xeeb tshaj yog nyob rau hauv cov tub ntxhais hluas npua uas tuaj yeem raug ntaus nqi rau peb yam: ua ntej, qhov kev hloov pauv siab ntawm PRRSV txwv tsis pub muaj kev tiv thaiv heterologous, yog li nws txo cov tshuaj tiv thaiv MLV; thib ob, nws lub peev xwm tiv thaiv kab mob txwv tsis pub cov tshuaj tiv thaiv tsis muaj zog (xws li, AIVs) thiab nws qhov kev ncua sij hawm ntawm kev tiv thaiv kab mob tiv thaiv tawm tsam cov menyuam yaus tshwj xeeb tshaj yog raug mob [17]; thiab peb, peb txoj kev nkag siab txog kev tiv thaiv kab mob rau PRRSV, tshwj xeeb tshaj yog nyob rau hauv piglets, yog txwv. Kev Hlub thiab Lunney tau tsim qhov kev tshuaj xyuas zoo txog kev nkag siab dav dav ntawm kev tiv thaiv kab mob rau PRRSV [18,19].

Feem ntau raws li lub sijhawm txuas ntxiv los tsim kev txhaj tshuaj tiv thaiv kab mob rau PRRSV, cov neeg tsim khoom npua feem ntau txhaj tshuaj tiv thaiv gilts thiab sows los pab hloov kev tiv thaiv rau piglets. Kev tshawb fawb txog kev txhaj tshuaj tiv thaiv niam txiv siv kev lag luam MLV tiv thaiv hom PRRSV-1 tsis txwv tsis pub txhaj tshuaj tiv thaiv 2 lossis 3-cov npua hnub nyoog ib lim piam thiab nws tau tiv thaiv qee cov npua tiv thaiv kev sib tw heterologous [20]. Qhov kev txhaj tshuaj tiv thaiv no kuj ua rau PRRSV-1- tshwj xeeb IgG, IgA, thiab interferon (IFN) - zais cov hlwb hauv colostrum thiab mis nyuj ntawm cov tshuaj tiv thaiv cov noob qoob loo. Cov IFN- tsim cov hlwb no tseem ceeb heev hauv cov lus teb tiv thaiv kab mob, suav nrog hauv PRRSV [19,21].
Qhov kev txhaj tshuaj no kuj ua rau cov tshuaj tiv thaiv PRRSV IgG, uas tuaj yeem kuaj tau tom qab muaj hnub nyoog rau lub lis piam rau cov piglets uas tsis tau txhaj tshuaj thiab tseem nyob ruaj khov hauv cov piglets uas tau txhaj tshuaj tiv thaiv ua ntej qhov kev sib tw; Txawm li cas los xij, IFN- zais cov hlwb tsis tuaj yeem paub qhov txawv ntawm keeb kwm yav dhau los hauv cov piglets pre-weaned [22]. Thaum kev siv MLVs yog kev lag luam-tus qauv, kev txhaj tshuaj sib xyaw ua ke ntawm MLV prime thiab AIV txhawb kev txhaj tshuaj tiv thaiv zoo li cog lus tias yuav kov yeej txhua qhov tsis zoo ntawm nws tus kheej siv MLVs thiab AIVs.

Yog li ntawd, lub hom phiaj tseem ceeb ntawm txoj kev tshawb no yog los txiav txim tsis tsuas yog, tab sis kuj yuav ua li cas, kev txhaj tshuaj tiv thaiv leej niam nrog ob qhov sib txawv AIVs (kev kho mob (TRT) 1 ntxiv rau 2) tuaj yeem txhawb nqa kev lag luam-tus qauv MLV txhaj tshuaj tiv thaiv ib leeg (TRT 0} ), los tiv thaiv cov xeeb ntxwv los ntawm kev sib tw thaum ntxov PRRSV2. Txog rau qhov kawg no, peb tau kawm txog cov tshuaj tiv thaiv kab mob humoral thiab cell-mediated immune (CMI) cov lus teb hauv cov gilts thiab peb tau tawm tsam cov neeg laus thaum ntxov los ntawm gilts nyob rau hauv txhua pawg ntawm cov hnub nyoog 2 lub lis piam. Cov kab mob hauv lub cev thiab lub cev tiv thaiv kab mob hauv zos ntawm cov npua no tau ua raws li 2-4 lub lis piam.

Peb pom tias AIV txhawb kev txhaj tshuaj tiv thaiv tuaj yeem txhim kho kev tiv thaiv piglets tiv thaiv PRRSV kev sib tw ntawm 2 lub lis piam ntawm lub hnub nyoog thiab peb kuj txheeb xyuas cov txheej txheem tiv thaiv kab mob thiab ua tus yam ntxwv ntawm kev tiv thaiv PRRSV humoral thiab CMI cov lus teb hauv cov npua hluas no. Tsis tas li ntawd, peb tau tshawb pom ob hom cell tshiab hauv npua: (1) CD8 −CCR7− CD4 T hlwb uas txhawb nqa IFN- ntau lawm hauv bronchoalveolar lavage (BAL) - muaj peev xwm CD4 TEMRA hlwb, thiab (2) qhov loj ntawm anti-PRRSV teb tshwj xeeb tshaj yog nyob rau hauv piglets-IFN- tsim B hlwb.

2. Cov ntaub ntawv thiab cov txheej txheem

2.1. Kawm Kev Tsim, Tsiaj, thiab Kev Ua Qauv

Lub hom phiaj ntawm txoj kev tshawb no yog los soj ntsuam cov txiaj ntsig ntawm kev txhaj tshuaj tiv thaiv niam txiv PRRSV2 sib txawv ntawm kev tiv thaiv kev tiv thaiv kab mob hauv cov piglets weaned tiv thaiv PRRSV-2. Ua ntej txoj kev tshawb no, ib tug circulating PRRSV -2 strain ( txwv tsis pub fragment length polymorphism (RFLP), 1-7-4) los ntawm ib lub tuam txhab nqaij npuas North Carolina raug cais tawm thiab nthuav tawm los ntawm ob lub tuam txhab kev tshawb fawb ywj pheej; lawv tau tsim ob hom tshuaj tiv thaiv autogenous. Ob qho tshuaj tiv thaiv muaj cov khoom ntiag tug sib txawv tab sis muaj tib yam kev tsis siv neeg 1-7-4 PRRSV-2 hom. Kev tsim qauv ntawm txoj kev tshawb no yog qhia hauv daim duab 1.

Ob qhov kev txhaj tshuaj tau txhaj tshuaj intramuscularly hauv plaub koob tshuaj ua ntej farrowing: los ntawm North Carolina lub tuam txhab nqaij npuas lub tuam txhab tshuaj tiv thaiv lub sijhawm, 46 PRRSV-2 cov gilts tsis zoo hauv kev lag luam cog qoob loo tau txhaj tshuaj Ingelvac PRRS MLV tshuaj tiv thaiv (Boehringer Ingelheim Vetmedica GmbH, Lub teb chaws Yelemees) cuaj lub lis piam ua ntej yug me nyuam. Yuav kom tswj tau cov ntsiab lus zoo ib yam thoob plaws qhov kev tshawb fawb no, cov poj niam uas muaj npe menyuam yaus uas muaj hnub nyoog sib deev (ntawm no txhais tau tias gilts) yuav raug hu ua gilts txawm tias tom qab lawv tau yug los thiab yug me nyuam (feem ntau hu ua sows). Plaub lub lis piam ua ntej yug me nyuam, cov ntshav tau sau los ntawm tag nrho cov gilts rau cov ntshav thiab tom qab ntawd gilts tau muab faib ua peb txoj kev kho mob: TRT 0 (Kev txhaj tshuaj MLV nkaus xwb); TRT 1 (MLV txhaj tshuaj tiv thaiv ntxiv rau 4 koob tshuaj autogenous (1-7-4) inactivated vaccine 1); los yog TRT 2 ( txhaj tshuaj tiv thaiv MLV ntxiv rau 4 koob tshuaj autogenous (1-7-4) inactivated vaccine 2).

Gilts that did not breed were dropped from the study. 3 weeks before farrowing, blood was collected from pregnant gilts for the isolation of serum and peripheral blood mononuclear cells (PBMC). At this point, five gilts remained in both TRT 0 and TRT 2; nine gilts remained in TRT 1. Within these gilts, we selected three gilts per group to provide the piglets for the challenge study. The selected gilts had an adequate litter size (>7) thiab lub siab tshaj plaws fluorescent tsom xam neutralization (FFN) titer (cov txheej txheem tiv thaiv kab mob tseem ceeb) thiab siab tshaj CD4 T-cell IFN- teb (cov txheej txheem tiv thaiv kab mob thib ob) tawm tsam AIV kev sib tw hauv lawv pawg. Rau TRT 1 ntxiv rau 2, cov FFN titers no txawv ntawm 1: 4 txog 1: 128. Txij li thaum TRT {{10}} tsis muaj titers tawm tsam AIV hom, peb xaiv cov gilts nrog lub siab tshaj plaws anti-MLV titers; cov titers no txawv ntawm 1:16 txog 1:32. IFN- cov lus teb nyob rau hauv tag nrho cov gilts xaiv tau nyob ntawm 0.09–0.21 feem pua ​​​​ntawm tag nrho CD4 T hlwb.

Txhua tus gilts tau muab plaub tus npua rau txoj kev kawm nyuaj. Thaum muaj hnub nyoog 2 lub lis piam (= 0 lub lis piam tom qab kis kab mob, wpi), plaub tus npua uas muaj qhov nruab nrab qhov hnyav tshaj plaws tau raug xaiv rau txoj kev kawm nyuaj thiab thauj mus rau Biosecurity Level 2 cov tsev nyob ntawm North Carolina State University ( NCSU) Laboratory Animal Resources (LAR) (Raleigh, North Carolina). Ntawm no, lawv tau random muab rau ib tug ntawm rau tus cwj mem nyob rau hauv tib chav; ob tug npua rau kev kho mob tau nyob hauv txhua tus cwj mem (ib qho rau ib qho gilt). Hauv cov ntsiab lus, muaj tag nrho ntawm 36 piglets nyob rau hauv txoj kev tshawb no nrog 12 piglets ib pab kho mob.

During the study, four piglets died before the end of the study: three piglets from one gilt (TRT 0) died within 30 h of arrival at LAR and were dropped from the study. Additionally, one pig (TRT 2) was euthanized between weeks three and four of the study due to high fever (>106◦ F) thiab tsis muaj kev teb rau lub cev stimulation. Tus npua no tau suav nrog hauv txhua qhov kev tshuaj xyuas txog lub lim tiam 3 thiab nws lub ntsws raug soj ntsuam rau cov kab mob pathology. Tsis muaj kev ntsuas cytometry (FCM) tshwm sim rau tus tsiaj no ntawm necropsy.

Muaj ib hnub tom qab rhais chaw thiab tuaj txog ntawm NCSU, tag nrho cov piglets tau inoculated intranasally nrog 1 × 105.5 PRRSV-2 cov kab mob kis (autogenous strain 1-7-4) hauv 1 ml 1X phosphate-buffered saline (PBS, Corning, Corning, NY, USA) (500 µL / lub qhov ntswg), khaws cia nyob rau hauv lub supine txoj hauj lwm rau 30 vib nas this thiab rov qab mus rau lawv cwj mem. Ib nrab ntawm cov tsiaj raug txi ntawm 2 dpi thiab cov tsiaj seem raug tua ntawm 4 dpi. Ntawm necropsy, BAL, ntsws, thiab tracheobronchial lymph nodes (TrBr LNs) tau sau thiab ua tiav raws li tau piav qhia yav dhau los [13].

Sampling tshwm sim ua ntej inoculation (0 wpi) thiab tom qab ntawd txhua lub limtiam, raws li qhia hauv daim duab 1. Cov ntshav tau sau rau hauv cov hlab ntsha Separator (SST) (Becton Dickinson (BD) Biosciences, San Jose, CA, USA), thiab Cov ntshav rau PBMC cais tawm tau sau rau hauv cov hlab ntsha heparin (BD Biosciences). Tsiaj vaj tsev yog los ntawm National Institutes of Health qhia rau kev tshawb fawb tsiaj. Piglets tau nkag mus rau dej tsis txwv, tau noj cov zaub mov hloov pauv hloov pauv, tom qab ntawd noj zaub mov tsis tu ncua ob zaug ib hnub, thiab khaws cia ntawm 83 ◦F nrog lub sijhawm 12- teev lub teeb / tsaus ntuj. Cov txheej txheem sim tau pom zoo los ntawm NC State University Institutional Animal Care and Use Committee (IAACUC) ID# 17-166A (29 Kaum Ib Hlis 2017).

2.2. Virus Propagation, Titration, and Evaluation

PRRSV-2 tus kab mob cais tawm ( hom, 1-7-4) tau nthuav tawm ntawm PRRSV-negative porcine alveolar macrophages (PAMs) hauv RPMI-1640, 1 × nrog L-glutamine (Corning, Corning, NY , Teb Chaws Asmeskas) ntxiv nrog 10 feem pua ​​​​fetal bovine serum (FBS) (VWR, Radnor, PA, USA) thiab 1X tshuaj tua kab mob-antimycotic (Corning) (hu ua RPMI-ua tiav) rau kwv yees li 30 h hauv T-75 khob (Sarstedt, Nümbrecht, Lub teb chaws Yelemees) kom txog rau thaum pom cov nyhuv cytopathic. Flasks tau khov-thawed ib zaug thiab cov supernatant tau spun ntawm 2200 g ntawm 4 ◦C rau 10 min. Txhawm rau mloog zoo rau tus kab mob, tus supernatant tau raug xa mus rau 36 mL Nalgene centrifuge raj (Thermo Fisher Scientific, Rochester, NY, USA) thiab spun hauv Sorvall 100S Ultracentrifuge (Sorvall, Thermo Fisher Scientific), Newtown, CT3, USA) ntawm 7. , 000 g ntawm 4 ◦C rau 2 h.

Cov supernatant raug muab pov tseg, thiab cov pellet tau rov ua haujlwm hauv xov xwm (RPMI tiav) thiab khaws cia hauv 100 µL aliquots ntawm −80 ◦C. Cov ntaub so ntswg kab lis kev cai kis kab mob koob tshuaj 50 (TCID50) titers ntawm viral stock solutions tau txiav txim siab siv PAMs raws li tau piav qhia yav dhau los (Spearman-Karber TCID50 txoj kev raws li OIE phau ntawv ntawm kev kuaj mob [World Organization for Animal Health, yav tas los Office International des Epizooties (OIE ), "Chapter 2.8.7 Porcine Reproductive and Respiratory Syndrome", Terr. Man., No. May 2015, 2015].

2.3. Viremia

Cov kua dej cais tawm tau txheeb xyuas los ntawm North Carolina lub tuam txhab nqaij npuas lub chaw kuaj mob siv Reverse-Transcription quantitative polymerase chain reaction (RT qPCR) nrog 5X MagMAX kab mob RNA / DNA cov khoom siv (Thermo Fisher Scientific) thiab VetMAX PRRSV qPCR cov khoom siv (Thermo Fisher Scientific). Cov tshuaj tiv thaiv gilt serum yog PRRSV-negative (qPCR) ntawm farrowing raws li tau txiav txim los ntawm Iowa State University Veterinary Diagnostic Laboratory (ISU VDL, Ames, IA, USA). Tus nqi ∆Ct raug xam los ntawm rho tus nqi Ct ntawm tus qauv los ntawm tus nqi Ct suav tias yog PRRSV-negative (Ctneg=37) rau PRRSV: ∆Ct=37 − Ctx.

2.4. Neutralizing Antibody Validation thiab txiav txim siab ntawm cov qauv zoo

Cov qauv ntshav los ntawm gilts thiab piglets raug xa mus rau South Dakota State University Kev Tshawb Fawb Tsiaj thiab Kev Tshawb Fawb Kev Tshawb Fawb (Brookings, SD) uas ua tiav PRRSV-2 kev txiav txim siab nruab nrab ntawm cov tshuaj tiv thaiv kab mob (NA) siv FFN thiab 1-7-4 inoculation strain. Qhov txiaj ntsig zoo yog qhov ua kom ntshav siab tshaj plaws nrog 90 feem pua ​​​​lossis ntau dua qhov txo qis ntawm cov fluorescent tsom-ua units [23].

2.5. Cov txheej txheem necropsy, qhab nia, thiab kev cais cov cell

Ntawm necropsy (2 lossis 4 wpi), cov tsiaj tau muab txi nrog kev txhaj tshuaj tua kab mob Euthasol (Covetrus, MW, 390 mg / mL pentobarbital, 50 mg phenytoin / mL). Tom qab ntawd, lub ntsws, thymus, thiab TrBr LNs raug sau tseg.

Rau lub ntsws, cov duab dorsal thiab ventral tau muab coj los siv; Lub ntsws tau ntxuav nrog 1 × PBS los sau cov bronchial alveolar lavage (BAL) thiab tsib tag nrho cov qauv raug coj mus rau histology: ib qho ntawm sab xis thiab sab laug caudal lobes, sab xis thiab sab laug nruab nrab lobes thiab tom qab ntawd ib qho los ntawm cov khoom siv lobe. Cov qauv kuaj histology tau kho hauv Formaldehyde / Zn fixative (Electron Microscopy Sciences, Hatfield, PA) rau 24 h, hloov mus rau 70 feem pua ​​​​ethanol, thiab stained nrog hematoxylin thiab eosin (H&E). Stained slides (histology) thiab cov duab ntsws (tag nrho cov pathology) raug xa mus rau ISU VDL thiab tau qhab nia los ntawm kws kho tsiaj kuaj kab mob uas yog dig muag rau kev kho mob thiab hnub necropsy; Kev kuaj mob ntsws thiab kev kuaj mob ntsws interstitial tau ua raws li tau piav qhia yav dhau los [24].

Dorsal thiab ventral views ntawm lub ntsws duab tau soj ntsuam rau feem pua ​​​​ntawm lub ntsws cuam tshuam nrog mob ntsws. Tshwj xeeb, feem pua ​​​​ntawm cov kab mob ntsws tau kwv yees pom pom rau txhua lub ntsws lobe, thiab tag nrho cov feem pua ​​​​ntawm tag nrho lub ntsws raug xam raws li qhov hnyav ntawm txhua lub lobe txheeb ze rau tag nrho lub ntsws ntim. Txhua ntawm plaub lub cranioventral lobes sawv cev 10 feem pua ​​​​, cov khoom siv lobe 5 feem pua ​​​​, thiab txhua qhov ntawm ob lub lobes sawv cev 27.5 feem pua ​​​​ntawm tag nrho lub ntsws.

Cov kab mob ntsws histopathology tau kuaj pom qhov tsis pom kev rau cov kab mob microscopic thiab txhua lub lobe tau muab cov qhab nia kwv yees raws li qhov hnyav ntawm kev mob ntsws asthiv raws li hauv qab no: 0=tsis muaj kab mob microscopic; 1=tsawg tsawg interstitial pneumonia; 2=mob ntsws me me; 3=mob ntsws interstitial; 4=abundant interstitial pneumonia; 5=mob hnyav, multifocal interstitial pneumonia; 6=hnyav, diffuse interstitial pneumonia. Rau tag nrho cov ntaub so ntswg (lub ntsws, thymus, thiab TrBr LNs), cov ntaub so ntswg raug txiav mus rau hauv me me pieces thiab nias los ntawm stainless hlau round drain strainers (Grainger, Lake Forest, IL, USA) rau hauv 50 ml hlab nrog dej khov-txias PBS rau elute. ib leeg cell suspensions. Fibrous clumps tau lim nrog 40 µm lim (BD Biosciences, San Jose, CA, USA).

2.6. Peripheral Blood Isolation, Viral Stimulation, thiab IFN- FCM Staining / Analysis

PBMC rho tawm tau ua siv FICL-PAque kev ua si Centrifugation (geealthcare Technologies, VancoOcell Technologies, VancoOt {1} Khib, Canada) los ntawm cov chaw tsim khoom cov txheej txheem. PBMC thiab tib lub cell ncua kev suav ntawm tes tau ua tiav ntawm CASY cell suav system (Schärfe System, tam sim no: Roche Innovatis AG, Bielefeld, Lub teb chaws Yelemees). Rau IFN- kev soj ntsuam ntau lawm, 5 × 105 PBMCs thiab lymphocytes los ntawm lub ntsws, thiab TrBr LNs tau plated nyob rau hauv yim replicates nyob rau hauv ib tug 96-zoo round-hauv qab phaj (Corning) nyob rau hauv 100 µl RPMI-ua tiav thiab so ib hmos nyob rau hauv ib tug incubator ntawm 37◦ C thiab 5 feem pua ​​CO2. Tag kis sawv ntxov 100 µL ntawm RPMI-ua tiav uas muaj PRRSV strain 1-7-4 (Multiplicity of Infection, MOI=0.1) tau ntxiv rau txhua qhov dej; daim hlau raug xa rov qab mus rau lub incubator rau 24 teev ntawm viral stimulation.

Rau 4 teev dhau los ntawm kab lis kev cai, monensin (5 µg / mL, Alfa Aesar, Haverhill, MA, USA) tau ntxiv los thaiv IFN- tso tawm ntawm lub hlwb. Tom qab ua tiav ntawm 24 teev incubation, cov replicates tau sib sau ua ke thiab stains raws li teev nyob rau hauv Table 1; Qhov nruab nrab ntawm 276,756 ib leeg nyob lymphocytes (SLLs) tau sau rau ntawm Cytoflex siv CytExpert software (Beckman Coulter, Brea, CA, USA). Kev txheeb xyuas cov ntaub ntawv tau ua nrog FlowJo version 10.6.1 (FLOWJO LLC). Lub gating hierarchy rau kev soj ntsuam ntawm IFN- teb yog qhia nyob rau hauv daim duab 4 thiab 5 rau PBMC thiab nyob rau hauv Ntxiv daim duab S1 thiab daim duab 7 rau BAL, lub ntsws cov ntaub so ntswg, thiab TrBr LNs.

2.7. Kev txheeb cais

Kev txheeb xyuas txheeb cais tau ua tiav siv Graphpad Prism 8 (Graphpad Software, San Diego, CA, USA). Cov ntaub ntawv nrog cov ntaub ntawv tag nrho tau txheeb xyuas los ntawm ob txoj kev (txog qhov kev kawm) lossis ib txoj kev (ntawm necropsy) ANOVAs. Cov ntaub ntawv nrog cov ntsiab lus uas ploj lawm (tsuas yog vim qhov necropsy ntawm 50 feem pua ​​​​ntawm cov npua ntawm 2 dpi) tau soj ntsuam los ntawm cov qauv sib xyaw ua ke (Txheej txheem siab tshaj plaws, REML). Txhua qhov kev sib piv ntau tau ua nrog Dunnett ntau qhov kev sib piv. Cov ntaub ntawv piav qhia tau ua los ntawm kev siv violin plots qhia qhov kev faib tawm, cov ntsiab lus ntawm tus kheej, nruab nrab (raws li siv tau, dashed dub kab), thiab 25th / 75th intervals (raws li siv tau, kab xim). Kev txheeb xyuas kev sib raug zoo ntawm qib gilt thiab piglet nAb tau ua tiav siv Pearson correlation coefficients thiab ob-tailed 95 feem pua ​​​​ntawm kev ntseeg siab.

3. Cov txiaj ntsig

3.1. Kawm Tsim thiab Tshuaj Tiv Thaiv Kev Ua Haujlwm - Cov tsos mob, Viremia, Qhov hnyav nce, thiab mob ntsws

Txoj kev tshawb no tau ua raws li tau piav qhia hauv daim duab 1. Thaum PRRSV inoculation, npua pom cov tsos mob me me ntawm PRRSV pib hauv 3-4 hnub ntawm kev kis kab mob - ua npaws [tseem ceeb ntawm 1 wpi, dns], lethargy, txo cov zaub mov noj, hnoos / txham me. Tsis muaj qhov sib txawv tseem ceeb tau pom rau cov tsos mob no thiab lawv txuas ntxiv mus rau qib sib txawv kom txog thaum necropsy. Daim duab 2 qhia txog viremia, lub cev hnyav nce txhua lub lim tiam nrog rau tag nrho thiab cov qhab nia histopathology.

Raws li pom hauv daim duab 2A, txhua tus npua tau PRRSV-2 qhov tsis zoo ntawm qhov tsis zoo ntawm qhov kev txiav tawm thiab kev ua haujlwm hauv lub cev nrog PRRSV-2 induced ib tug viremia uas peaked ntawm 2 wpi. Thaum tsis muaj qhov sib txawv ntawm viremia ntawm TRT 0 thiab TRT 2, TRT 1 tau qis dua viremia hauv thawj ob lub lis piam dua li pawg tswj hwm TRT 0. Qhov hnyav nce ntawm npua hauv txhua pab pawg tau zoo sib xws rau thawj peb lub lis piam; tsis tau, thaum kawg ntawm txoj kev tshawb fawb-thiab piv rau TRT 0-TRT 2 thiab TRT 1 tau nce qhov hnyav dua txhua lub lim tiam-Daim duab 2B. Qhov tseem ceeb tshaj plaws, thaum lub ntsws pathology feem ntau daws tau hauv PRRSV tus kab mob thiab yog li ntawd zoo sib xws ntawm cov pab pawg ntawm 4 dpi (dns), ob leeg niam txiv AIV txhaj tshuaj tiv thaiv, yog li TRT 1 ntxiv rau 2, txo cov ntsws pathology ntawm 2 wpi: ob qho tib si txo qis feem pua. ntawm macroscopic lesions thiab/los yog interstitial pneumonia (Daim duab 2C, D). Feem ntau ntawm cov mob ntsws macroscopic hauv ntsws los ntawm TRT 0 yog nyob nruab nrab ntawm 20-70 feem pua ​​; Hauv qhov sib piv, feem ntau cov npua hauv TRT 1 ntxiv rau 2 muaj qhov mob ntsws ntsws ntau dua 20 feem pua ​​-Daim duab 2C. Ib yam li ntawd, thaum qhov qhab nia nruab nrab ntawm histopathology rau cov kab mob ntsws interstitial hauv ntsws los ntawm TRT 0 npua yog peb, nws yog 1.8 rau TRT 1 thiab 1.2 rau TRT 2, feem. Muab piv rau TRT 0, qhov no qhia tau hais tias muaj kev nyiam rau qhov tseem ceeb (p=0.07) thiab qhov txo qis hauv ntsws histopathology hauv npua los ntawm AIV-cov tshuaj tiv thaiv gilts-Daim duab 2D. Cov ntaub ntawv no qhia tau hais tias kev txhaj tshuaj AIV tuaj yeem txo qis viremia thiab mob ntsws pathology, ntxiv rau, lawv kuj qhia tau hais tias niam txiv AIV txhaj tshuaj tiv thaiv PRRSV-2 tuaj yeem txhawb kev tiv thaiv ntawm 2-cov menyuam hnub nyoog ib lim piam.

3.2. Induction ntawm Maternal Neutralizing Antibodies thiab Lawv Hloov mus rau Piglets

Qhov induction ntawm humoral tiv thaiv kab mob hauv gilts thiab lawv cov xeeb ntxwv tau txheeb xyuas los ntawm kev txiav txim siab NA FFN titers (Daim duab 3).

Kev txhaj tshuaj ntawm txhua pawg nrog MLV induced NA titers hauv gilts ntawm plaub thiab 64 yam tsis muaj qhov sib txawv tseem ceeb ntawm peb pawg (dns). Hauv qhov sib piv, thaum txhaj tshuaj tiv thaiv MLV ib leeg (TRT 0) tsis suav nrog NAs tawm tsam heterologous 1-7-4 strain, ob qho tshuaj tiv thaiv AIV txhawb nqa NA titers txog li 1:16 (rau TRT 2) thiab txawm tias 1 :128 (rau TRT 1)—Daim duab 3A.

Cov ntshav NAs no tau hloov pauv niam txiv mus rau piglets (Daim duab 3B): Thaum TRT 0 piglets tsis pom NA titers ntawm 0 wpi, TRT 1 thiab TRT 2 piglets muaj cov ntshav NAs rau strain 1-7-4 ua ntej kab mob uas cuam tshuam ncaj qha rau niam txiv NA theem (Daim duab 3C, r=0.886, p < 0.0001). Cov NA titers poob qis dua thawj ob lub lis piam, ua ntej ruaj khov los ntawm 4 wpi. Txawm li cas los xij, piv rau TRT 0, piglets los ntawm AIV-cov tshuaj tiv thaiv gilts (TRT 1 ntxiv rau 2) tau muaj cov ntshav ntau dua NA titers twb ua ntej qhov kev sib tw thiab cov titers nce mus txog 2-3 wpi, yog li mus txog 4-5 lub lis piam ntawm hnub nyoog-Daim duab 3B.

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