Cov txiaj ntsig ntawm Pullet Txhaj Tshuaj Tiv Thaiv Rau Kev Txhim Kho Thiab Lub Sij Hawm Ntev Ntawm Kev Tiv Thaiv Part 1
Mar 15, 2023
Abstract:
Tus kab mob avian ua pa ua rau kev poob nyiaj txiag tseem ceeb hauv kev lag luam nqaij qaib. Vim tias qhov xav tau los tiv thaiv cov tsiaj nyeg nyob ntev tiv thaiv kab mob ua pa thaum muaj hnub nyoog ntxov, kev txhaj tshuaj tiv thaiv kab mob feem ntau koom nrog kev tswj hwm ntawm ntau yam tshuaj tiv thaiv, suav nrog tus kab mob kis kab mob ntsws (IBV), kab mob Newcastle kab mob (NDV), thiab kis kab mob. laryngotracheitis virus (ILTV). Feem ntau lub sijhawm ntawm kev txhaj tshuaj tiv thaiv tsuas yog ib lub lis piam xwb, tab sis nws tseem tsis tau paub tias kev txhim kho ntawm kev tiv thaiv kab mob thiab kev tiv thaiv kev sib tw thaum cov tshuaj tiv thaiv luv luv tshwm sim hauv cov noog no, qee yam hu ua kab mob cuam tshuam. Peb lub hom phiaj yog los txiav txim seb puas muaj kev tswj hwm, nyob rau hauv cov tshuaj tiv thaiv kab mob tiv thaiv IBV, NDV, thiab ILTV cuam tshuam rau txoj kev loj hlob thiab kev tiv thaiv kab mob ntev thiab tiv thaiv kev nyuaj ntawm cov noog nyob ntev.
Raws li ib qho kev txhaj tshuaj tiv thaiv kab mob, cov kab mob tshwj xeeb-dawb dawb leghorns tau siv ntau yam tshuaj tiv thaiv kab mob tiv thaiv IBV, NDV, thiab ILTV txog 16 lub lis piam hnub nyoog (WOA), tom qab ntawd qee pawg tau tawm tsam nrog IBV, NDV, lossis ILTV ntawm 20, 24, 28, 32, thiab 36 WOA. Tsib hnub tom qab kev sib tw, kis kab mob, cov tsos mob tshwm sim, cryostasis, tracheal histopathology, thiab cov tshuaj tiv thaiv kab mob hauv cov ntshav thiab kua muag tau soj ntsuam. Peb pom tau hais tias cov hnab looj tes tau muab tshuaj tiv thaiv nyob rau hauv cov tshuaj tiv thaiv IBV, NDV, thiab ILTV tau tiv thaiv kev sib tw homologous nrog IBV, NDV, lossis ILTV tsawg kawg yog 36 lub lis piam, thiab xaus lus tias lub sijhawm ntawm kev txhaj tshuaj tiv thaiv siv hauv txoj kev tshawb no (tsawg kawg 2 lub lis piam. ) tsis cuam tshuam nrog kev tiv thaiv. Cov ntaub ntawv no tseem ceeb heev vim nws qhia tau hais tias ib qho kev txhaj tshuaj tiv thaiv kab mob sib kis uas muaj cov tshuaj tiv thaiv kab mob nyob rau hauv cov tshuaj tiv thaiv ntau yam kab mob tuaj yeem ua rau muaj kev tiv thaiv kev tiv thaiv kab mob rau txhua tus neeg sawv cev.
Hais txog kev tiv thaiv kab mob, tib neeg lub cev kuj muaj ntau yam uas tuaj yeem txhim kho kev tiv thaiv kab mob, xws li Cistanche, uas tseem muaj cov tshuaj lom neeg lom neeg, xws li peptides, terpenes, phenolic acids, thiab lwm yam, uas tuaj yeem txhawb kev ua kom lub cev tiv thaiv kab mob thiab txhim kho lub cev tiv thaiv kab mob. ua haujlwm, thiab txhim kho lub cev tiv thaiv kab mob. Cov tshuaj lom neeg lom neeg no tuaj yeem tswj hwm thiab ua kom lub cev ua haujlwm autoimmune, ntxiv dag zog rau tib neeg kev tiv thaiv.
Ntsiab lus:
Kab mob bronchitis kab mob; Tus kab mob Newcastle; kis kab mob laryngotracheitis; kev txhaj tshuaj tiv thaiv kab mob; kev tiv thaiv; kab mob ua pa; cryostasis; antibody teb.
1. Taw qhia
Kev txhaj tshuaj tiv thaiv kab mob ua pa yog kev coj ua hauv kev lag luam nqaij qaib. Ob qho tshuaj tiv thaiv kab mob nyob thiab tua yog siv rau cov nqaij qaib, thiab cov tshuaj tiv thaiv muaj sia feem ntau yog siv rau ntau yam kab mob vim tias lawv siv tau zoo thaum siv ntau thiab pheej yig [1]. Feem ntau, cov tshuaj tiv thaiv nyob ua rau muaj kev tiv thaiv kab mob hauv zos thiab hauv xov tooj ntawm tes thiab muab cov lus teb dav dav dua li cov tshuaj tiv thaiv tua, thaum cov tshuaj tiv thaiv tua feem ntau ua rau muaj kev tiv thaiv kev tiv thaiv kab mob thiab zoo li yog cov tshuaj tiv thaiv tshwj xeeb. Lub sijhawm tiv thaiv kab mob ua tiav tom qab kev tswj hwm tshuaj tiv thaiv nyob yog nyob ntawm lub hnub nyoog thiab hom noog, qib ntawm niam txiv kev tiv thaiv kab mob, kab mob tsom los ntawm cov tshuaj tiv thaiv, immunogenicity ntawm cov tshuaj tiv thaiv, txoj kev siv tshuaj tiv thaiv, tus naj npawb thiab lub sijhawm nruab nrab ntawm kev txhawb nqa, kev ua phem thiab zoo sib xws. ntawm qhov kev sib tw kis kab mob, lub caij nyoog ntawm kev txhaj tshuaj tiv thaiv thiab kev sib tw, thiab kev tiv thaiv kab mob ntawm tus tswv tsev [1–3].
Avian coronavirus infectious bronchitis virus (IBV) yog kab mob ua pa sab sauv kab mob ntawm cov nqaij qaib thiab ua rau txo qhov hnyav nce thiab noj kom zoo, poob rau hauv qe tsim thiab qe zoo, stunted loj hlob, thiab cov kab mob thib ob ua rau airsacculitis [4]. Tus kab mob no pib rov ua dua nyob rau hauv lub ntsws sab sauv, ua raws li cov txheej txheem rov ua dua tshiab hauv lub cev xeeb tub, thiab qee hom kab mob tuaj yeem ua rau mob raum [4]. Cov noog muaj kab mob tuaj yeem pom qhov ntswg tawm, hnoos, txham, thiab tracheal rales [4]. Tus kab mob no tiv thaiv los ntawm kev txhaj tshuaj, thiab cov tshuaj tiv thaiv muaj sia feem ntau yog siv los txhawb kev tiv thaiv hauv zos thiab kev tiv thaiv. Cov tshuaj tiv thaiv nyob feem ntau yog muab rau cov noog hluas kom ua tiav kev tiv thaiv thaum ntxov, thiab cov khaubncaws sab nraud povtseg thiab cov tsiaj yug tsiaj kuj tau txhawb nqa nrog cov tshuaj tiv thaiv nyob lossis tsis ua haujlwm, uas txawv raws li lawv qhov zoo sib xws rau cov kab mob kis thoob plaws [2].
Tus kab mob Newcastle (ND) yog tshwm sim los ntawm cov kab mob virulent ntawm avian paramyxovirus type 1, uas tsis ntev los no tau muab cais ua avian avulavirus 1 (AAvV-1) [5]. Nyob ntawm tus kab mob virus, cov tsos mob ntawm tus kab mob ND tuaj yeem tsis tuaj lossis tuaj yeem cuam tshuam nrog kev nyuaj siab, ua tsis taus pa, ua pa tawm (qhov ntswg qhov ntswg, txham, hnoos), txo qis qe qe thiab qe zoo, thiab cov tsos mob ntawm lub paj hlwb (torticollis, circling, tuag tes tuag taw). ) [3]. Cov kab mob Newcastle tus kab mob (NDV) yog tus cwj pwm li lentogenic, mesogenic, thiab velogenic, raws li lawv lub sijhawm tuag hauv embryos [6]. Cov kab mob Lentogenic yog cov kab mob uas tsis tshua muaj ua rau mob me me los yog enteric kab mob, ua raws li cov kab mob mesogenic, thaum velogenic cais tawm yog cov kab mob ntau heev uas ua rau cov kab mob neurological thiab kev tuag [3]. Kev txhaj tshuaj tiv thaiv kab mob NDV sib txawv thiab tuaj yeem siv kev sib xyaw ua ke ntawm cov tshuaj tiv thaiv nyob, tsis muaj zog, thiab cov tshuaj tiv thaiv kab mob-vectored [7]. Hauv Tebchaws Meskas, cov tshuaj tiv thaiv kab mob ntau tshaj plaws muaj xws li lentogenic B1 (lossis tus kab mob clones ntawm B1 hom) thiab LaSota hom [3].
Kab mob laryngotracheitis (ILT) yog kab mob ua pa ntawm cov nqaij qaib los ntawm galled alphaherpesvirus I thiab yog ib qho tseem ceeb ntawm kev lag luam thoob ntiaj teb [8]. Kev kho mob tshwm sim ntawm ILT suav nrog kev tuag ntau ntxiv, txo qe qe, txo lub cev hnyav nce, conjunctivitis, tracheitis nrog expectoration ntawm cov hnoos qeev hauv cov neeg mob hnyav, kev nyuaj siab, mob hnyav, thiab ua rau lwm yam kab mob ua pa. Cov tshuaj tiv thaiv nyob tiv thaiv tus kab mob ILT (ILTV) tej zaum yuav yog qaib embryo keeb kwm (CEO) los yog nqaij kab lis kev cai keeb kwm (TCO), uas lawv kis tau ntau zaus hauv cov qe los yog cov nqaij mos kab lis kev cai, feem. Txawm hais tias cov tshuaj tiv thaiv recombinant rau ILT muaj kev lag luam, cov tshuaj tiv thaiv CEO yog cov tshuaj tiv thaiv ntau tshaj plaws tiv thaiv ILTV thoob ntiaj teb.
Vim tias qhov xav tau los tiv thaiv qaib tawm tsam cov kab mob sib txawv los ntawm cov hnub nyoog ntxov, kev txhaj tshuaj tiv thaiv feem ntau suav nrog ntau yam tshuaj tiv thaiv kab mob ntau yam. Cov qauv txhaj tshuaj tiv thaiv kab mob hauv cov nqaij qaib sib txawv raug tshuaj xyuas hauv Merck Veterinary Manual, uas lub sijhawm ntawm kev txhaj tshuaj feem ntau tsuas yog ib lub lis piam xwb.
Txawm li cas los xij, muaj cov ntaub ntawv me me uas qhia tau hais tias lub sijhawm nruab nrab ntawm kev txhaj tshuaj tiv thaiv yog txaus rau cov noog los txhim kho lub cev tiv thaiv kab mob txaus tiv thaiv cov teeb meem ntawm txhua tus kab mob. Cov ntaub ntawv qhia tau hais tias kev kis kab mob sib kis tuaj yeem ua rau muaj kab mob cuam tshuam, uas ib tus kab mob thaiv cov kab mob txuas ntxiv thiab / lossis rov ua dua ntawm lwm tus kab mob hauv tus tswv [9,10], tab sis txog tam sim no nws tsis paub tias qhov tshwm sim no ua rau txo kev tiv thaiv. los ntawm serially muab tshuaj tiv thaiv nyob rau hauv cov qaib. Interestingly, nws tau raug tshaj tawm tias kev tswj hwm ib txhij ntawm cov kab mob rau qaib lossis qaib ntxhw tsis ua rau muaj kab mob cuam tshuam [11]. Hauv txoj kev tshawb no, peb tshawb xyuas seb lub sijhawm txhaj tshuaj tiv thaiv kev lag luam li cas uas muaj kev sib xyaw ua ke ntawm kev tswj hwm, nyob rau hauv cov tshuaj tiv thaiv kab mob ua pa cuam tshuam rau kev txhim kho thiab kev tiv thaiv ntev thiab tiv thaiv kev sib tw homologous.
2. Cov ntaub ntawv thiab cov txheej txheem
2.1. Kab mob
Kev lag luam IBV tshuaj tiv thaiv MILDVAC-GA-98® (Merck Animal Health, Summit, NJ, USA) tau siv rau hauv txoj kev tshawb no. Cov tshuaj tiv thaiv tau diluted raws li cov neeg tsim khoom pom zoo. Tus kab mob sib tw siv yog IBV GA98/CWL0474/98 thiab tau npaj ntawm qhov nruab nrab embryo kis kab mob koob tshuaj (EID50) ntawm 103.19. Tus kab mob titers raug xam los ntawm Reed thiab Muench txoj kev [12]. Kev lag luam tshuaj NDV tshuaj tiv thaiv B1 (NEWHATCH-C2, Merck Animal Health, Summit, NJ, USA) tau siv rau ob qho tib si txhaj tshuaj tiv thaiv thiab kev sib tw thiab tau rov tsim dua ua raws li cov chaw tsim khoom cov lus qhia.
Txij li cov kab mob mesogenic thiab velogenic ntawm NDV xav tau qib biosafety saum BSL2, uas tsis muaj nyob hauv peb lub chaw kuaj mob, peb cov qauv kev sim rau kev tiv thaiv NDV cuam tshuam nrog txo qis tus kab mob titers lossis tsis muaj tshuaj tiv thaiv kab mob tiv thaiv kab mob thib ob (kev sib tw) nrog cov tshuaj tiv thaiv kab mob. Ib qho tshuaj tiv thaiv ILTV lag luam CEO (Trachivax® Merck Animal Health, Summit, NJ, USA) thiab cov kab mob ILTV Georgia broiler strain 63140 [13] tau siv rau kev txhaj tshuaj tiv thaiv thiab kev sib tw, feem. Hom 63140 tau nthuav tawm hauv cov nqaij qaib raum tau los ntawm 3- mus rau 4-lub lim tiam cov kab mob tsis muaj kab mob (SPF) qaib [14]. Cov tshuaj tiv thaiv CEO tau npaj ua raws li cov chaw tsim khoom pom zoo. Tom qab inoculation, qhov nruab nrab cov ntaub so ntswg kab lis kev cai kis kab mob koob tshuaj (TCID50) tau lees paub los ntawm titration ntawm ob tug kab mob nyob rau hauv nqaij qaib raum hlwb raws li yav tas los piav [14].
2.2. Kev sim tsim
Cov qe tsis muaj kab mob tshwj xeeb tau txais ntawm 18 hnub ntawm incubation thiab hatched ntawm Poultry Diagnostic and Research Center, Athens, GA. Cov me nyuam qaib tau muab tso rau ntawm cov ntoo thuv tshiab hauv cov tsev colony thiab cwj mem. Cov menyuam qaib tau txhaj tshuaj tiv thaiv nrog cov tuam ntxhab pom zoo koob tshuaj hauv 100 µL ntawm txoj kev oculonasal raws li lub sijhawm hauv qab no; IBV ntawm 1 DOA, NDV ntawm 2 lub lis piam hnub nyoog (WOA), IBV ntawm 4 WOA, ILTV ntawm 8 WOA, NDV ntawm 12 WOA, thiab ILTV ntawm 16 WOA.
Tsis tas li ntawd, pawg tswj hwm tsis tau txhaj tshuaj tiv thaiv. Kev sib tw homologous tau ua nyob rau ntawm 20, 24, 28, 32, thiab 36 WOA, thiab necropsies tau ua tsib hnub tom qab kev sib tw (dpc). Ntawm qhov kev sib tw, cov noog tau txais ib qho ntawm plaub txoj kev kho mob: IBV GA98, NDV B1, ILTV 63140, lossis tsis muaj kev sib tw. Cov pab pawg kho mob rau txhua tus kab mob sib tw ib lub sij hawm muaj raws li hauv qab no: tsis txhaj tshuaj tiv thaiv, tsis nyuaj (n=9–10); txhaj tshuaj tiv thaiv, tsis nyuaj (n=9–10); txhaj tshuaj tiv thaiv, sib tw (n=17–19); tsis txhaj tshuaj tiv thaiv, sib tw (n=9–10). Txhua IBV-cov noog sib tw tau txais EID50 ntawm 1 × 103.2 rau ib tus noog hauv 100 µL intranasally. Txhua NDV-cov noog sib tw tau txais cov tshuaj tiv thaiv NDV B1 hauv 100 µL intranasally, rov tsim kho raws li cov chaw tsim khoom raws tu qauv. Txhua ILTV-kev sib tw noog tau txais 63140 kab mob kab mob ntawm qhov koob tshuaj ntawm 1 × 103.5 TCID50 rau ib tus noog hauv 100 µL faib sib npaug ntawm cov hlab muag thiab cov kab mob hauv lub cev. Rau IBV thiab NDV cov kev sib tw, noog tau pom ntawm 5 dpc rau cov paib ua pa, raws li tau piav qhia yav dhau los [15]: 0=tsis tuaj; 1=mos; 2=nruab nrab; 3=mob heev. Rau ILTV cov kev cov nyom, cov noog tau pom ntawm 3 thiab 5 dpc rau kev ua tsis taus pa, conjunctivitis, kev nyuaj siab, thiab kev tuag, raws li tau piav ua ntej [16].
Cov choanal cleft (IBV- thiab NDV-kev sib tw thiab tswj cov noog ntawm 5 dpc) lossis trachea (ILTV-kev sib tw thiab tswj cov noog ntawm 3 thiab 5 dpc) tau swabbed rau kev kuaj kab mob, thiab swabs tau khaws cia hauv PBS ntawm −80 ◦C. Ntawm 28, 32, thiab 36 WOA, 50 µL ntawm kua muag tau sau los ntawm kev ntxiv granulated NaCl rau qhov muag. Cov ntshav tau sau los ntawm tis lossis lub plawv dhia thiab ntxiv rau lub raj microcentrifuge kom sau cov ntshav rau kev kuaj tshuaj tiv thaiv. Cov noog tau tib neeg euthanized, thiab daim tawv muag, Harderian caj pas (HG), thymus, siab, spleen, cecal tonsils, thiab bursa ntawm Fabricius tau sau thiab khaws cia ntawm −80 ◦C rau kev kuaj tus kab mob thiab hauv 10 feem pua ntxhiab buffered formalin. Cov trachea tau raug tshem tawm, ib ntu tau muab tso rau hauv 10 feem pua ntaus tsis haum cov tshuaj formalin, thiab qhov seem ntawm lub trachea tau submerged nyob rau hauv cov ntaub so ntswg kab lis kev cai xov xwm rau lub iconostasis kuaj tau piav qhia hauv qab no. Cov txheej txheem tau pom zoo los ntawm University of Georgia Institutional Animal Care and Use Committee (AUP #: A2015 05-001-R2).
2.3. Kev kuaj ciliostasis
Qhov kev sim iconostasis tau ua tiav ntawm cov tracheas khaws cia hauv xov tooj ntawm tes (Dulbecco's Modified Eagle's Medium) ntawm 37 ◦C. Rau txhua lub trachea, tsib tracheal rings ntsuas kwv yees li 1 hli tuab tau txiav thiab sawv cev rau qhov sib thooj, nruab nrab, thiab qhov nruab nrab [17,18]. Cilia kev ua haujlwm tau pom los ntawm kev siv lub tshuab ntsuas inverted (Olympus, Center Valley, PA, USA). Cov qhab nias ua raws li 0=tag nrho cov cilia ntaus; 1=75 feem pua ntawm cilia ntaus; 2=50 feem pua ntawm cilia ntaus; 3=25 feem pua ntawm cilia ntaus; 4=tsis muaj cilia ntaus raws li tau piav ua ntej [17]. Qhov qhab nia siab tshaj plaws rau txhua lub trachea yog 20. Txhua lub nplhaib tracheal tau qhab nia los ntawm peb tus neeg, thiab cov qhab nia nruab nrab ntawm txhua lub trachea raug xam. Cov qhab nia tiv thaiv iconostasis rau txhua pab pawg tau txiav txim siab los ntawm cov qauv hauv qab no: 100 − [(tag nrho ntawm cov qhab nia ntawm tus kheej rau pab pawg)/( tus naj npawb ntawm cov tib neeg hauv pab pawg × 20) × 100], thiab tus qhab nia Ntau dua lossis sib npaug rau 50 tau suav tias yog kev tiv thaiv.
2.4. Tracheal Histopathology
Ib ntu ntawm txhua lub trachea tau kho nyob rau hauv 10 feem pua ntxhiab buffered formalin, ua tiav, thiab kos rau hauv paraffin, thiab 5-µm seem raug txiav rau hematoxylin thiab eosin staining. Rau IBV qhov txhab, epithelial hyperplasia, lymphocyte infiltration, thiab epithelial deciliation tau qhab nia rau txhua lub trachea. Cov qhab nias tau txiav txim raws li hauv qab no: 1=ib txwm, 2=focal, 3=multifocal, thiab 4=diffuse, raws li tau piav yav dhau los [19]. Rau cov kab mob NDV, kev txheeb xyuas piav qhia tau ua. Rau cov kab mob ILTV, cov kab mob microscopic tau qhab nia ntawm qhov ntsuas ntawm 0-5 (ib txwm mus rau qhov hnyav heev), raws li tau piav qhia yav dhau los [20].
2.5. RNA Extraction thiab Quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR)
Rau IBV thiab NDV nrhiav pom, tus kab mob RNA rho tawm los ntawm 50 µL ntawm PBS los ntawm txhua qhov swab tau ua los ntawm 5 × MagMAX-96 Viral Isolation Kit (Thermo Fisher, Waltham, MA, USA) ntawm MagMAX™ Express{{ 3}} Deep Well Magnetic Particle Processor (Thermo Scientific, Waltham, MA, USA), raws li cov chaw tsim khoom cov lus qhia. Qhov ntau qhov rov qab hloov pauv polymerase saw cov tshuaj tiv thaiv (qRT-PCR) tau ua nrog AgPath-IDTM Ib-kauj ruam RT-PCR cov khoom siv (Thermo Fisher, Waltham, MA, USA), ua raws li cov chaw tsim khoom raws tu qauv. Txhua 25-µL cov tshuaj tiv thaiv sib xyaw muaj 12.5 µL ntawm 2 × RT-PCR tsis, 10 µM ntawm txhua tus primer, 4 µM ntawm kev sojntsuam, 1 µL ntawm 25 × RT-PCR enzyme mix, thiab 5 µL ntawm tus kab mob RNA. Cov tshuaj tiv thaiv qRT-PCR tau khiav ntawm Applied Biosystems® 7500 Fast Realtime PCR system (Life Technologies Ltd., Carlsbad, CA, USA) raws li cov hauv qab no: ib lub voj voog ntawm 50 ◦C rau 30 min thiab 95 ◦C rau 15 feeb , ua raws li 40 lub voj voog ntawm 94 ◦C rau 1 s thiab 60 ◦C rau 60 s. Cov primers thiab sojntsuam rau IBV qRT-PCR yav dhau los tau luam tawm [21], thiab muaj xws li pem hauv ntej primer IBV50GU391 (50 -GCT TTT GAG CCT AGC GTT-30 ), rov qab primer IBV50GL533 ({ {38}}GCC ATG TTG TCA CTG TCT ATT G-30 ), thiab Taqman® dual-labeled sojntsuam IBV50G sojntsuam (50 -FAM-CAC CAC CAG AAC CTG TCA CCT C-BHQ{{45} }}). Primers thiab kev sojntsuam rau NDV qRT-PCR yav dhau los tau piav qhia [22] thiab yog suav nrog rau pem hauv ntej primer NDV M ntxiv rau 4100 (5'-AGT GAT GTG CTC GGA CCT TC-3'), rov qab primer NDV M -4220 (5'-CCT GAG GAG AGG CAT TTG CTA-3'), thiab Taqman® dual-labeled sojntsuam NDV M plus 4169 (5'-FAM-TTC TCT AGC AGT GGG ACA GCC TGC -BHQ1-3').
Cov primers tau txais los ntawm Integrated DNA Technologies (Coralville, IA, USA), thiab Taqman sojntsuam tau tsim los ntawm BioSearch Technologies (Novato, CA, USA). Real-time RT-PCR Cheebtsam thiab cov thermocycler tsis tau piav qhia yav dhau los [21]. Cov ntaub ntawv raug qhia raws li qhov nruab nrab lub voj voog pib (CT) tus nqi rau tag nrho cov qauv hauv txhua pab pawg, nrog cov txiaj ntsig zoo CT raws li qhov txwv ntawm kev kuaj pom rau qhov kev sim no cuam tshuam nrog kev kuaj kab mob hauv qe [23].
Txhua qRT-PCR cov tshuaj tiv thaiv phaj suav nrog tus qauv nkhaus raws li RNA rho tawm tswj thiab tswj tau zoo. GA98 IBV cais tawm los ntawm allantoic kua tau siv los ua tus qauv rau tus qauv nkhaus. Cov kev tswj tsis zoo kuj tseem suav nrog hauv txhua lub phaj thiab muaj PCR reagents uas tsis muaj RNA.
2.6. DNA Extraction
Rau ILTV, tag nrho DNA tau muab rho tawm los ntawm tracheal swabs siv MegaZorb® DNA extraction miniprep 96-cov khoom siv zoo (Promega, Madison, WI, USA), raws li tau piav ua ntej [24].
2.7. qPCR
Kev ntsuam xyuas PCR duplex lub sij hawm uas nthuav tawm ib feem ntawm UL44 kab mob hauv ILTV thiab ib feem ntawm cov nqaij qaib alpha 2-collagen noob tau ua, raws li tau piav qhia yav dhau los [25].
2.8. Serum Infectious Bronchitis Virus (IBV)-Specific IgG Antibody Titers
IBV-specific IgG titers tau kuaj pom siv kev lag luam IgG enzyme-linked immunosorbent assay (ELISA) IBV antibody test kit (IDEXX, Westbrook, ME, USA). Luv luv, cov qauv kuaj ntshav (khov ntawm −20 ◦C) tau diluted 1:500, thiab cov txheej txheem tau ua raws li tus neeg tsim khoom raws tu qauv.
2.9. Tear-Secreted IBV-Specific IgA Antibody Titers
Tear IBV-specific IgA tau kuaj pom siv cov khoom siv coj mus muag IgG ELISA IBV antibody test (IDEXX, Westbrook, ME, USA). Luv luv, cov kua muag tau muab faib ua ob zaug hauv PBS thiab muab tso rau hauv qhov dej sib npaug thaum hmo ntuj ntawm 4 ◦C. Txhua kauj ruam ntxuav tau ua tiav siv PBS-Tween 20 (0.05 feem pua Tween 20). Cov phaj tau incubated ntawm 23 ◦C rau 2 h hauv monoclonal nas tiv thaiv qaib IgA-BIOT (1: 1000, clone A-1, Southern Biotech, Birmingham, AL, USA), ua raws li 1hr hauv Streptavidin-HRP ( 1:4000, Southern Biotech, Birmingham, AL, USA). Cov kauj ruam nrhiav tshuaj tiv thaiv zaum kawg tau ua tiav raws li cov chaw tsim khoom cov lus qhia. Endpoint titers tau txiav txim siab los ntawm kev tshaj tawm qhov qis tshaj qhov dilution ntawm qhov muag pom qhov ntom ntom (OD), sau tseg ntawm 650 nm wavelength, yog tsawg kawg peb tus qauv sib txawv saum toj no qhov txhais tau tias ntawm 12 tswj qhov dej incubated tsis muaj cov qauv tsim kua muag. Cov ntaub ntawv los ntawm lub qhov dej nrog cov xim hloov pauv vim qhov seem ntawm cov substrate lossis cov npuas huab cua tau raug cais tawm los ntawm kev tshuaj xyuas, thiab cov txiaj ntsig tau tshaj tawm raws li lub log2 ntawm qhov kawg titer.
2.10. Kev txheeb cais
Cov ntaub ntawv raug tshuaj xyuas siv Prism v.6.0 software (GraphPad Software, Inc., La Jolla, CA, USA). Rau cov ntaub ntawv kis kab mob, kev tshuaj ntsuam ib leeg ntawm qhov sib txawv (ANOVA) nrog Dunnet's posttest tau siv los sib piv cov pab pawg kho mob hauv txhua lub sijhawm sau. Tag nrho lwm cov ntaub ntawv tau txheeb xyuas siv Kruskal-Wallis test nrog Dunn's posttest los sib piv pab pawg kho mob hauv txhua lub sijhawm sau. Qhov sib txawv tseem ceeb tau txiav txim siab ntawm p < 0.05.
3. Cov txiaj ntsig
3.1. Kab mob bronchitis kab mob
Ntawm 5 hnub tom qab qhov kev sib tw nrog IBV GA98, cov tshuaj tiv thaiv / cov noog tau sib tw tau txo qis RNA loads piv rau cov kev tswj zoo ntawm txhua lub sij hawm sau thiab hauv txhua cov ntaub so ntswg, tshwj tsis yog cov cecal tonsil ntawm 24 WOA (Table 1). Hauv kev txhaj tshuaj tiv thaiv, tsis muaj IBV RNA tau kuaj pom ntawm txhua lub sijhawm sau tshwj tsis yog hauv cecal tonsils ntawm 20 thiab 24 WOA thiab choanal cleft ntawm 20 WOA. IBV loads nyob rau hauv cov kev tswj tsis zoo nyob rau hauv tag nrho cov ntaub so ntswg yog qis dua qhov kev txwv ntawm kev tshawb nrhiav siv CT tus nqi ntau dua los yog sib npaug rau 36.17 raws li tau tshaj tawm yav dhau los [23], ntawm txhua lub sijhawm sau tshwj tsis yog 24 WOA HG tswj tsis zoo.
Cov tsos mob ntawm tus kab mob IBV ntsuas ntawm 5 hnub tom qab kev sib tw tau txo qis hauv cov tshuaj tiv thaiv / cov noog uas muaj kev sib tw thaum piv rau kev tswj tau zoo hauv txhua lub lis piam tshwj tsis yog 28 WOA, tab sis cov txiaj ntsig ntawm cov qhab nia kho mob tau qis dua ntawm cov tshuaj tiv thaiv / cov noog sib tw (Table 2) . Cov tsos mob tshwm sim tsis muaj nyob hauv cov noog uas tsis muaj kev sib tw thiab cov tshuaj tiv thaiv kab mob, thiab cov cim qhia hauv cov noog uas tau txhaj tshuaj tiv thaiv / sib tw tsis sib txawv ntawm cov cim hauv cov kev tswj tsis muaj kev sib tw.
Table 1. Qhov nruab nrab ntau qhov rov qab hloov pauv polymerase saw cov tshuaj tiv thaiv (qRT-PCR) lub voj voog pib (CT) qhov tseem ceeb rau tus kab mob bronchitis (IBV) RNA sau los ntawm choanal cleft, Harderian caj pas, conjunctiva, thiab cecal tonsil 5 hnub tom qab sib tw nrog IBV GA98 ntawm 20, 24, 28, 32, thiab 36 lub lis piam. Cov ntawv (a–d) qhia qhov sib txawv tseem ceeb ntawm cov tshuaj tiv thaiv thiab cov pab pawg sib tw rau txhua lub lim tiam (p <0.05).
Table 2. Cov tsos mob thiab cov kab mob microscopic raug ntsuas 5 hnub tom qab qhov kev sib tw IBV GA98. Cov ntawv (a–b) qhia qhov sib txawv tseem ceeb ntawm cov tshuaj tiv thaiv thiab cov pab pawg sib tw rau txhua lub lim tiam (p < 0.05).
Histopathological kuaj ntawm tracheas los ntawm tag nrho cov pab pawg muaj xws li nyob rau hauv ib txwm txwv mus rau focal mus rau multifocal tsawg heev mus rau nruab nrab lymphocytic tracheitis; Txawm li cas los xij, qhov nruab nrab lymphocytic infiltration tau pom ntau dua hauv kev tswj hwm zoo. Nyob rau hauv txhua lub lis piam, qhov kev faib ua feem ntawm cov tshuaj tiv thaiv / nyuaj noog nrog deciliation los yog mob tracheal necrosis raug txo qis piv rau cov kev tswj zoo thiab tsis txawv ntawm qhov tsis zoo thiab kev txhaj tshuaj tiv thaiv (Table 2).
Ciliostasis, defined as the cessation of tracheal ciliary movement, was measured at 5 DPC, and the number of birds positive for cryostasis and cryostasis protection scores was calculated for each group (Figure 1). At all collection times, vaccinated/challenged birds were protected from cryostasis (scores were >50), thiab kev tswj tau zoo tsis muaj kev tiv thaiv (cov qhab nia yog<50). The non-challenged negative controls and vaccinated controls were protected (scores were >50) ntawm txhua lub sijhawm sau.
Figure 1. Ciliostasis protection scores among tracheas were collected 5 days post-challenge with IBV GA98 for different groups of birds challenged at 20, 24, 28, 32, and 36 weeks of age (WOA). Groups with a protection score >50 (kab kab rov tav) tiv thaiv, thiab pab pawg nrog cov qhab nia tiv thaiv<50 are not protected. Fractions above each bar represent the proportion of birds positive for cryostasis for the respective group. −/− = non-vaccinated/non-challenged, +/− = vaccinated/non-challenged, +/+ = vaccinated/challenged, −/+ = non-vaccinated/challenged.
IBV-specific IgG titers tau ntsuas hauv cov ntshav sau ntawm 5 DPC. Txhua lub sijhawm tshwj tsis yog ntawm 32 WOA, txhaj tshuaj tiv thaiv noog los ntawm ob pawg uas tsis muaj kev sib tw thiab cov neeg sib tw tau nthuav tawm cov titers siab dua piv rau cov noog uas tsis tau txhaj tshuaj los ntawm ob pawg uas tsis muaj kev sib tw thiab kev sib tw (Daim duab 2). Ntawm 32 WOA, titers hauv cov noog uas tau txhaj tshuaj tiv thaiv, tsis hais txog kev sib tw, tau nce siab dua piv rau kev tswj hwm zoo. Titers hauv cov tshuaj tiv thaiv / cov noog sib tw tsis sib txawv ntawm cov titers hauv kev txhaj tshuaj tiv thaiv kom txog rau thaum 36 WOA, thaum txhaj tshuaj tiv thaiv / sib tw noog muaj ntau dua titers. Piv nrog rau cov kev tswj tsis zoo, cov tshuaj tiv thaiv / cov noog sib tw tau muaj ntau dua titers txhua lub sijhawm.
IBV-specific IgA titers tau ntsuas hauv cov kua muag ntawm 5 DPC ntawm 28, 32, thiab 36 WOA. Ntawm 28 WOA, titers hauv cov tshuaj tiv thaiv / sib tw noog tau ntau dua piv rau titers hauv kev tswj tsis zoo (Daim duab 3). Ntawm 32 WOA, cov tshuaj tiv thaiv / cov noog sib tw tau pom cov titers qis dua piv rau kev tswj tau zoo. Tsis tas li ntawd, cov noog uas tau txhaj tshuaj tiv thaiv / sib tw muaj cov titers ntau dua li cov tshuaj tiv thaiv / tsis muaj kev sib tw ntawm 28 WOA. Tsis pom muaj lwm qhov sib txawv tseem ceeb. Virus 2019.
Daim duab 2. IBV tshwj xeeb IgG titers hauv cov ntshav tau sau 5 hnub tom qab kev sib tw nrog IBV GA98 rau ntau pawg noog sib tw ntawm 20, 24, 28, 32, thiab 36 WOA. Cov ntawv (a–c) qhia qhov sib txawv tseem ceeb ntawm cov tshuaj tiv thaiv thiab cov pab pawg sib tw rau txhua lub lim tiam (p <0.05). −/−=non-vaccinated/non-challenged, plus /−=vaccinated/non-challenged, plus / plus=vaccinated/challenged, −/ ntxiv=tsis txhaj tshuaj tiv thaiv / nyuaj.
Daim duab 3. Tear IBV-specific IgA titer hauv kua muag sau 5 hnub tom qab kev sib tw nrog IBV GA98 rau ntau pawg noog sib tw ntawm 20, 24, 28, 32, thiab 36 WOA. Cov ntawv (a–c) qhia qhov sib txawv tseem ceeb ntawm cov tshuaj tiv thaiv thiab cov pab pawg sib tw rau txhua lub lim tiam (p <0.05). −/−=non-vaccinated/non-challenged, plus /−=vaccinated/non-challenged, plus / plus=vaccinated/challenged, −/ ntxiv=tsis txhaj tshuaj tiv thaiv / nyuaj.
3.2. Newcastle Disease Virus (NDV)
Ntawm 5 dpc nrog NDV B1, txhaj tshuaj tiv thaiv / sib tw noog txawm tias muaj qhov tsis paub meej RNA loads lossis qis qis dua piv rau cov tswj tau zoo ntawm txhua lub sijhawm sau thiab hauv txhua cov ntaub so ntswg kuaj (Table 3). Kev tswj tsis tau qhov tsis zoo thiab kev txhaj tshuaj tiv thaiv tsis zoo rau tus kab mob RNA siv CT tus nqi ntau dua lossis sib npaug rau 35.0 raws li tau tshaj tawm yav dhau los [22].
Table 3. Qhov nruab nrab qRT-PCR CT qhov tseem ceeb rau NDV RNA sau los ntawm choanal cleft, Harderian caj pas, thiab conjunctiva 5 hnub tom qab sib tw nrog NDV B1 tshuaj tiv thaiv. Cov ntawv (a–c) qhia qhov sib txawv tseem ceeb ntawm cov tshuaj tiv thaiv thiab cov pab pawg sib tw rau txhua lub lim tiam (p < 0.05).
Cov tsos mob ntawm kev ntsuas ntawm 5 dpc yog qhov tseem ceeb ntawm 20 WOA hauv kev tswj hwm zoo, tom qab ntawd cov tsos mob ntawm kev tswj xyuas zoo tsis txawv ntawm lwm pawg kho mob (Table 4). Tsis muaj qhov sib txawv tseem ceeb hauv cov tsos mob tshwm sim ntawm kev txhaj tshuaj tiv thaiv / sib tw noog thiab tsis muaj kev sib tw tswj txhua lub sijhawm. Nyob rau hauv txhua lub lis piam, histopathological kev ntsuam xyuas ntawm lub tracheas yog nyob rau hauv ib txwm txwv los yog qhia focal rau multifocal tsawg heev rau me me lymphocytic tracheitis, thiab tsis muaj ib pab pawg neeg sib txawv.
Tracheas kuj raug soj ntsuam rau cryostasis, thiab tsis muaj pab pawg pom tias cryostasis.
NDV-specific serum IgG titers nyob rau hauv cov tshuaj tiv thaiv / sib tw noog thiab kev txhaj tshuaj tiv thaiv tau ntau dua li cov titers hauv ob qho tib si zoo thiab tsis zoo ntawm txhua lub sijhawm sau (Daim duab 4). Tsis muaj qhov sib txawv tseem ceeb hauv titers ntawm cov tshuaj tiv thaiv / cov noog sib tw thiab kev txhaj tshuaj tiv thaiv txhua lub sijhawm. Ib yam li ntawd, tsis muaj qhov sib txawv tseem ceeb hauv titers tau kuaj pom ntawm qhov tsis muaj tshuaj tiv thaiv / kev sib tw thiab kev tswj tsis zoo, tshwj tsis yog ntawm 28 WOA nyob rau hauv uas titers los ntawm cov tsis txhaj tshuaj tiv thaiv / kev sib tw tswj tau ntau dua.
Table 4. Cov tsos mob tshwm sim tau ntsuas 5 hnub tom qab qhov kev sib tw NDV B1. Cov ntawv (a–b) qhia qhov sib txawv tseem ceeb ntawm cov tshuaj tiv thaiv thiab cov pab pawg sib tw rau txhua lub lim tiam (p < 0.05).
Daim duab 4. NDV-specific IgG titers hauv cov ntshav tau sau 5 hnub tom qab sib tw nrog NDV B1 tshuaj tiv thaiv rau ntau pawg noog sib tw ntawm 20, 24, 28, 32, thiab 36 WOA. Cov ntawv (a–c) qhia qhov sib txawv tseem ceeb ntawm cov tshuaj tiv thaiv thiab cov pab pawg sib tw rau txhua lub lim tiam (p <0.05). −/−=non-vaccinated/non-challenged, plus /−=vaccinated/non-challenged, plus / plus=vaccinated/challenged, −/ ntxiv=tsis txhaj tshuaj tiv thaiv / nyuaj.
3.3. Infectious Laryngotracheitis Virus (ILTV)
Ntawm 5 dpc nrog ILTV hom 63140, txhaj tshuaj tiv thaiv / sib tw noog tau qis dua cov kab mob DNA ntau dua li kev tswj tau zoo ntawm txhua lub sijhawm sau thiab hauv txhua cov ntaub so ntswg (Table 5). DNA loads nyob rau hauv lub trachea thiab HG yog undetectable los yog tsawg thiab tsis txawv heev ntawm DNA loads nyob rau hauv uas tsis yog-kawg tsis zoo thiab txhaj tshuaj tiv thaiv, uas yog tsis zoo siv tus nqi CT ntawm ntau dua los yog sib npaug rau 38.0 raws li yav dhau los qhia [26]. Hauv cov kab mob sib kis, DNA loads hauv cov tshuaj tiv thaiv / cov noog sib tw tau ntau dua li DNA loads los ntawm ob qho tib si tsis muaj kev sib tw tsis zoo thiab kev txhaj tshuaj tiv thaiv, tshwj tsis yog ntawm 28 WOA uas tsis muaj qhov sib txawv tseem ceeb ntawm kev txhaj tshuaj tiv thaiv / sib tw noog thiab kev txhaj tshuaj tiv thaiv.
Table 5. Qhov nruab nrab qPCR CT qhov tseem ceeb rau ILTV DNA sau los ntawm trachea, Harderian caj pas, thiab conjunctiva 5 hnub tom qab sib tw nrog cov kab mob ILTV strain 63140. Cov ntawv (a–c) qhia qhov sib txawv tseem ceeb ntawm cov tshuaj tiv thaiv thiab cov pab pawg sib tw rau txhua lub lim tiam (p <0.05).
Cov tsos mob thiab cov kab mob DNA tau kuaj pom nyob rau hauv cov noog sib tw ntawm 3 DPC, tab sis cov cim qhia hauv kev tswj tau zoo tau dhau los ua hnyav dua los ntawm 5 dpc raws li cov kab mob kis tau nce ntxiv (Table 6). Cov qhab nia ntawm kev kuaj mob ntsuas ntawm 5 dpc tau txo qis hauv cov tshuaj tiv thaiv / cov noog sib tw piv rau cov kev tswj tau zoo, ntawm txhua qhov sau cov ntsiab lus tshwj tsis yog 36 WOA thaum cov qhab nia kuaj mob tau raug txo qis. Txhua qhov kev tswj tsis zoo thiab kev txhaj tshuaj tiv thaiv tsis muaj cov tsos mob tshwm sim, thiab cov tsos mob tshwm sim hauv cov tshuaj tiv thaiv / cov noog sib tw tsis sib txawv heev piv rau cov kev tswj hwm no, tshwj tsis yog ntawm 36 WOA thaum cov qhab nia kuaj mob tau siab dua.
Table 6. Cov tsos mob tshwm sim tau ntsuas 5 hnub tom qab ILTV 63140 kev sib tw. Cov ntawv (a–b) qhia qhov sib txawv tseem ceeb ntawm cov tshuaj tiv thaiv thiab cov pab pawg sib tw rau txhua lub lim tiam (p <0.05).
Kev kuaj mob tracheal histological yog nyob rau hauv ib txwm txwv lossis qhia tawm focal kom nthuav tawm tsawg heev rau me me lymphocytic tracheitis hauv tracheas los ntawm txhua pawg. Rau cov kev tswj zoo, 3/9 thiab 1/10 hauv lub lis piam 20 thiab 28, feem, muaj mob focal necrotizing tracheitis nrog syncytia thiab intranuclear inclusions (Daim duab 5).
Daim duab 5. Tracheal microscopic lesions zoo ib yam nrog cov kab mob laryngotracheitis kis tau thaum muaj hnub nyoog 20 lub lis piam. (A) Ntau syncytia nrog intranuclear inclusions ( xub xub). (B) Lwm trachea los ntawm tib pab pawg tau pom syncytia nrog intranuclear inclusions ( xub xub).
Tsis muaj iconostasis tau pom nyob rau hauv tracheas ntawm cov noog los ntawm ib qho ntawm cov pab pawg.
ILTV-specific IgG titers hauv cov ntshav tau sau 5 hnub tom qab kev sib tw tau nce siab dua hauv cov noog uas tau txhaj tshuaj los ntawm ob pawg sib tw thiab tsis muaj kev sib tw, piv rau cov kev tswj zoo thiab tsis zoo (Daim duab 6).
Daim duab 6. ILTV tshwj xeeb IgG titers hauv cov ntshav tau sau 5 hnub tom qab kev sib tw nrog ILTV hom 63140 rau ntau pawg noog sib tw ntawm 20, 24, 28, 32, thiab 36 WOA. Cov ntawv (a–b) qhia qhov sib txawv tseem ceeb ntawm cov tshuaj tiv thaiv thiab cov pab pawg sib tw rau txhua lub lim tiam (p <0.05). −/−=non-vaccinated/non-challenged, plus /−=vaccinated/non-challenged, plus / plus=vaccinated/challenged, −/ ntxiv=tsis txhaj tshuaj tiv thaiv / nyuaj.
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