Thermoresistant Newcastle disease vaccine effectivelyprotects SPF, native, and commercial chickens inchallenge with virulent virus

DOI: 10.1002/ ms3.794 ORIGINAL ARTICLE The mo esis an Newcas le disease accine e ec i ely p o ec s SPF, na i e, and comme cial chickens in challenge wi h i ulen i us Mohammad Abdoshah1Mohammad Hassanzadeh2Shahin Masoudi1 Abbas Ash a i1Ali Reza Youse i1Minoo Pa o i Nas 3 1Ag icul u al Resea ch, Educa ion and Ex ension O ganiza ion (AREEO), Razi Vaccine and Se um Resea ch Ins i u e, Ka aj, I an 2Depa men o Poul y Diseases, Facul y o Ve e ina y Medicine, Uni e si y o Teh an, Teh an, I an 3Depa men o Molecula Diagnosis, Mabna Ve e ina y Lab, Ka aj, I an Co espondence Mohammad Abdoshah, Razi Vaccine and Se um Resea ch Ins i u e, Ag icul u al Resea ch, Educa ion and Ex ension O ganiza ion (AREEO), Ka aj, I an. Email: [email p o ec ed], [email p o ec ed] Funding in o ma ion Razi Vaccine and Se um Resea ch Ins i u e, G an /Awa d Numbe : 12-18-18-036-95017-950501 Abs ac Backg ound: Due o he mo e s abili y and a be e homogeneci y in immune esponse, he use o he mo esis an accines in di e en chicken ypes has been inc eased. Objec i e: This s udy aimed o e alua e he e icacy o a newly de eloped Newcas le disease accine (ND.TR.IR) o igina ing om I-2 s ain in speci ic pa hogen- ee (SPF) and na i e and b oile chickens. Me hods: Following de e mina ion o pa hogenici y indices on he candida e seed, h ee e icacy examina ions we e conduc ed. In he i s expe imen , 120 1-day- old SPF chickens we e andomly alloca ed o six g oups and ei he accina ed wi h ND.TR.IR ia eye d op a 1, 7, and 21 days o age (V1,V 7,andV 21), o conside ed as non- accina ed con ol g oups (C1,C 7,andC 21). A 20 h pos - accina ion day, se a hemag- glu ina ion inhibi ion (HI) an ibody i es agains ND i us (NDV) we e measu ed and hen he chickens we e challenged by i ulen NDV ( NDV). In he second and hi d expe imen s, he e icacy o ND.TR.IR accine was compa ed o ou ine accina ion p og am (B1 and LaSo a) in na i e and b oile chickens ha we e accina ed a 10 and 20 days o age, espec i ely. The HI an ibody i es we e measu ed on 10, 20, 30, and 40 days o age, and also challenge e icacy es wi h NDV was conduc ed on 30 days o age. Resul s: The s udied i us, as a accinal seed, complied wi h he pa hogenici y indices o a i ulen NDV and molecula iden i y o I-2 s ain. In he e icacy e alua ion i- als, he accina ed chickens had highe HI an ibody i es agains NDV compa ed wi h hei co esponding con ol chickens (p<0.05). Resul s o he challenge es s indica ed 95% and 100% p o ec ion agains NDV in na i e, SPF, and b oile - accina ed chick- ens, espec i ely. Conclusions: The p esen indings indica ed ha adminis a ion o ND.TR.IR induced app op ia e HI an ibody i es agains NDV in SPF, na i e, and b oile chickens associ- a ed wi h good p o ec ion in e icacy es . This is an open access a icle unde he e ms o he C ea i e Commons A ibu ion-NonComme cial-NoDe i s License, which pe mi s use and dis ibu ion in any medium, p o ided he o iginal wo k is p ope ly ci ed, he use is non-comme cial and no modi ica ions o adap a ions a e made. © 2022 The Au ho s. Ve e ina y Medicine and Science published by John Wiley & Sons L d. Ve Med Sci. 2022;8:1539–1546. wileyonlinelib a y.com/jou nal/ ms3 1539 1540 ABDOSHAH ET AL. KEYWORDS chicken, e icacy, Newcas le disease, he mo esis an , accine 1INTRODUCTION Newcas le disease (ND), a highly con agious and widesp ead poul y disease, is an unde lying con ibu o o economic loss in comme cial and backya d poul y locks (Lawal e al., 2016). The u al poul y a ms play a pi o al ole in ood chain supply in de eloping coun ies; how- e e , hey ha e o en been a ec ed by in ec ious diseases such as ND, occu ing by a ian pa amyxo i us-1 (Ka enbel e al., 2006; Shahid Yousa zai & Liaqua -Ahmad, 2017). Res ic ion o i ulen ND i uses ( NDV) ci cula ion in he backya d chickens could dec ease he isk o he i us ansmission o comme cial a ms (Adwa & Lukeso a, 2008). Vaccina ion agains ND is he mos easible and p o ec i e p e en ion app oach agains ND ou b eaks since he 1940s (Dias e al., 2001). Because o a ela i ely high cos and equi ing long imes o induce immuni y, as well as he need o be injec ed by ained people, he use o inac i a ed ND accines is limi ed in illage locks. The ou ine li e accines (B1 and LaSo a) a e cheape and easie o adminis a e, which ha e made hem mo e accep able o be adminis a ed in u al condi- ions. Ne e heless, main enance o he cold chain o li e accines is he main conce n o achie e e ec i e ND accina ion in a away a eas (Ahle s e al., 1999; Adwa & Lukeso a, 2008). In de eloping coun ies, complying wi h s anda d s o age and cold chain a e somehow di icul ha esul ed in a conside able educ ion o accine e icacy. App oxima ely, a 2-h imeline o adminis a ion o he dissol ed and p epa ed common ND accines is ega ded as a majo limi a ion on hei adminis a ion in u al locks. Al hough lyophiliza- ion and s abilizing excipien s ha e con e ed ma ginal s abili y on many accines, he lack o accines ha possess adequa e he mos a- bili y is o en a p ima y ba ie o global accina ion e o s (Rex oad e al., 2002). Unde inapp op ia e condi ions o he cold chain, adminis a ion o a he mo esis an ND accine could be he p ima y s a egy (Adwa & Lukeso a, 2008). Se e al he mo esis ance i uses d i en om I-2 o HR-V4 s ains ha e been in oduced o manu ac u e ND li e accines (Adwa & Lukeso a, 2008). I has been shown ha he e icacy o hea - esis an ND accines is kep up o 2 mon hs a 9–29◦C, and 2 weeks a 30–37◦C (Alde s, 2014). In his ega d, p e ious in es iga ions (Fen ie e al., 2014; Hassanzadeh e al., 2020; Nasse e al., 2000; Van Bo en e al., 2008) also showed a high p o ec ion agains NDV in chickens accina ed by I-2. The e a e many illage chickens and o he backya d poul y ha a e also po en ial ese oi s o h ea ening o comme cial a ms o NDV (Hassanzadeh & Bozo gme i Fa d, 2004). In his ega d, he p e- en ion o ND occu ence a illage locks is a c ucial s ep in main e- nance sus ainable ag icul u e and con ol o ND ou b eaks in he poul- y indus y. Mos o he in es iga ions on he mos able ND accines ha e ocused on hei usage in u al locks; howe e , hei e icacy on comme cial chickens is a ely s udied. The e o e, his s udy was aimed o e alua e he e icacy o a de eloping Newcas le disease I-2 accine (ND.TR.IR) agains ND in speci ic pa hogen- ee (SPF) and na i e chick- ens as well as comme cial b oile s. 2MATERIALS AND METHODS 2.1 Molecula iden i ica ion o accinal i us and bioin o ma ics analysis A iplica e molecula analysis o he F gene sequences and hei co - esponding amino acids we e ca ied ou o de e mine he iden i y o he i us. Vi us RNA was ex ac ed using a high pu e i al Nucleic Acid ki (Roche, Ge many) acco ding o he manu ac u e ’s ins uc- ions. The ex ac ed RNA was used o syn hesize cDNA using he Re e Aid RT Re e se T ansc ip ion Ki (The mo Fishe Scien i ic). An 840 bp segmen o he F gene sequence including clea age si e was ampli ied using o wa d, 3′-CATCTTCTACCAGGATCCA-5′,and e e se, 3′-CCAAGAGTTGAGTCTGTGAGTC-5′p ime s (F ozandeh- Moghadam e al., 2009). Polyme ase chain eac ion (PCR) was pe - o med using a 5 µl cDNA aliquo in 50 µ; inal olume, including 10 µl PCR bu e (10X) consis ing o 1.5 mM MgCl2,1µl 10 mM dNTPs, 1 µl o 10 pM o each p ime , 1 µl (2 U) Taq DNA polyme ase, and 31 µl double dis illed wa e . Following ini ial dena u a ion a 94◦C o 4 min, 35 cycles including dena u a ion a 94◦C o 60 s, annealing a 50◦C o 60 s, and ex ension a 72◦C o 8 s we e pe o med using a s anda d he mocycle (Eppendo -Ne hele -Hinz GmbH, 5331). The amplicon was hen elec opho esed in 1% low mel ing poin aga ose and isual- ized unde UV ligh using a ansillumina o (bios ep.de, GmbH). The e- a e , he 840 pb bands we e sliced and pu i ied using a high pu e PCR p oduc pu i ica ion ki (Roche) and sequenced using he PCR p ime s and Com o Read me hod (Bionee Co., Ko ea). The nucleo ide sequences we e edi ed and immed and hen we e ansla ed o F p o ein amino acid sequence o he i us. The amino acid sequences we e hen aligned and compa ed wi h comme cial ac- cinal (B1, LaSo a and i s clone, VG/GA, V4, I-2) and s anda d NDV (Roakin, He s33) s ains using BioEdi V7.0.0 so wa e. A phylogene ic analysis was also ca ied ou o iden i y e olu iona y ela ionships among he s udied i us and some i ulen and accinal NDV s ains using MEGA 6.0 so wa e wi h Neighbou -Joining algo i hm and 1000 Boo s aps (Figu e 1). 2.2 Pa hogenici y indices To measu e pa hogenici y indices, esh in ec i e allan oamnionic luid (AAF) wi h haemagglu ina ion (HA) i e >24was dilu ed in s e ile no - mal saline (W. H. Allan e al., 1978). The in ace eb al pa hogenici y 20531095, 2022, 4, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1002/ ms3.794 by Technical Uni e si y In B no, Wiley Online Lib a y on [31/07/2023]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License ABDOSHAH ET AL.1541 FIGURE 1 The phylogene ic analysis o he ND.TR.IR F-gene and some comme cial accines (B1, LaSo a and i s clone, VG/GA, V4, I-2) and s anda d i ulen Newcas le disease i us ( NDV) (Roakin, He s33) s ains da ase s, in e ed using he Neighbou -Joining me hod index (ICPI) was de e mined ia in ace eb al injec ion o 0.05 ml o he dilu ed i us in o each o 10 1-day-old SPF chickens. The chickens we e daily e alua ed o gene al and clinical signs o ND, du ing he nex 8 days and sco ed as ollow: 0 o no mal, 1 o sick, and 2 o dead chickens. The ICPI was calcula ed as he mean sco e/chicken/obse a ion o e he examina ion pe iod. The in a- enous pa hogenici y index (IVPI) was de e mined by in a enous injec ion o 0.1 ml o he dilu ed i us in o 10 6-week-old SPF chickens. Chickens we e daily obse ed o gene al and local clinical signs du ing he nex 10 days and sco ed a each obse a ion. The sco e ze o was ega ded o no mal, 1 o sick, 2 o pa alyzed, and 3 o dead chick- ens. The IVPI was calcula ed as he mean sco e pe bi d pe obse a- ion o e he 10 days (W. H. Allan e al., 1978). Mean emb yo dea h ime (MDT) was de e mined h ough inocula ion o 0.1 ml o eshly dilu ed i us om each dilu ion anged 10−6–10−9in o he allan oic ca i y o i e 9- o 10-days-old emb yona ed SPF chicken eggs a 8:00 AM, and incuba ion a 37◦C. The emaining i us dilu ions we e kep a 4◦C. Simila ly, 0.1 ml o each dilu ion was inocula ed in o ano he i e eggs a 5:00 PM and incuba ed a 37◦C. All he incuba ed eggs we e exam- ined wice each day o eco d imes o any emb yo dea hs du ing he nex 7 days. The minimum le hal dosage was de e mined as he highes i us dilu ion in which all o he inocula ed emb yos died. The MDT was calcula ed as he mean ime in hou s o he minimum le hal dose o kill emb yos (W. H. Allan e al., 1978). 2.3 Se ological and challenge e icacy es s The e icacy o he ND.TR.IR was e alua ed h ough h ee sepa a e expe imen s in SPF, comme cial b oile and backya d chickens. All he challenge es s we e unde aken by in amuscula injec ion o ≥105 LD50 o a NDV (He s33) a biosa e y le el 3 in BioFlex B40 Rigid Body Poul y Isola o (Bell Isola ion Sys ems L d, UK). 2.3.1 E icacy e alua ion in SPF chicken A o al o 120 newly ha ched SPF chickens we e andomly alloca ed o h ee accina ed(V1,V7,andV21)and h eecon ol(C 1,C 7,and C21) g oups (20 chickens/g oup). The accina ed chickens we e inocu- la ed wi h ≤106EID50 o he accinal i us (ND.TR.IR) h ough he eye d op a 1 (V1), 7 (V7), and 21 (V21) days o age. Meanwhile, he con- ols chickens ecei ed dis illed wa e by eye d op. Twen y days a e each accina ion ime, all o he accina ed and non- accina ed chick- ens we e challenged. Blood samples we e aken om he expe imen- al g oups o measu e hemagglu ina ion inhibi ion (HI) an ibody i es agains NDV immedia ely be o e he challenge es . Du ing he nex 10 days, daily mo ali y was eco ded, and he su i ed chickens we e con olled o gene al clinical signs o ND (OIE, 2012). 2.3.2 E icacy e alua ion in na i e chicken A o al o 320 1-day-old na i e chickens we e ea ed du ing 50 days o he expe imen al pe iod. Ma e nally de i ed an ibody (MDA) agains NDV was de e mined on 20 andomly selec ed chickens on 1 and 10 days o age (10 chickens/sampling imes). The sampled chickens we e emo ed om he ial, and he emaining chickens we e an- domly allo ed in o h ee expe imen al g oups (100 chickens/g oup) including B1+LaSo a g oup: inocula ion o comme cial B1 and LaSo a accines, a 10 and 20 days o age, espec i ely; ND.TR.IR g oup: inocu- la ion o ND.TR.IR accine a 10 and 20 days o age and Con ol g oup: no accine adminis a ion. Vaccine inocula ion a 10 and 20 days o age was conduc ed h ough eye d op and d inking wa e , espec i ely. To measu e HI an ibody i es agains NDV, blood samples we e collec ed (10 chickens/g oup) a 20, 30, and 40 days o age. On day 30 o age, 20 chickens pe g oup we e ans e ed o isola o s and challenged. [Co ec ion added on 8 Ap il 2022, a e i s online publica ion: “10 chickens” was co ec ed o “20 chickens” in he p eceding sen ence] Du ing he nex 10 days, daily mo ali y was eco ded, and he su i ed chickens we e checked o gene al clinical signs o ND. 2.3.3 E icacy e alua ion in b oile chicken To e alua e he e icacy o ND.TR.IR in b oile chickens, a o al o 470 1-day-old Ross 308 b oile chickens (20 chickens o de e mining MDA, and 450 expe imen al chickens [n=150 chickens /g oup]) we e ea ed unde s anda d en i onmen al and nu i ional condi ions acco ding o Ross B oile Managemen Handbook. Excep o he numbe o 20531095, 2022, 4, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1002/ ms3.794 by Technical Uni e si y In B no, Wiley Online Lib a y on [31/07/2023]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License 1542 ABDOSHAH ET AL. FIGURE 2 Compa ison o he amino acid sequence deduced om he F-gene nucleo ide sequence o ND.TR.IR i us wi h se e al comme cial accinal (B1, LaSo a and i s clone, VG/GA, V4, I-2) i uses and i ulen Newcas le disease i us ( NDV) (Roakin, He s33) s ains da ase s. The ND.TR.IR amino acid sequence is accessible unde AHH29550.1. No e: he mo i s loca ed in he box ep esen ing ND.TR.IR and o he NDVs F-p o ein clea age si e challenged chickens which was 10 pe g oup, all o he p ocedu es we e as same as Sec ion 2.3.2 (OIE, 2012). 2.4 Da a analysis Da a ob ained om se a HI an ibody i es we e s a is ically analyzed using a gene alized linea model (GLM) p ocedu e o SAS, 9.4 (SAS, 2004). Be o e he analysis, he no mal dis ibu ion o he da a was es ed by he Shapi o–Wilk es and UNIVARIATE p ocedu e. Mo al- i y a e, as bina y dis ibu ed da a, was analyzed by GENMOD p oce- du e using a logi odds a io link unc ion. The ma hema ical model was as ollows: Yij =µ+Ti+eij whe e Yij is obse a ions, Tiis a ea men e ec , and eij is esidual andom e o . Resul s we e epo ed as mean ±s anda d e o (SE). Tukey’s es was used o mul iple compa isons, and s a is ical di e ences we e decla ed a p<0.05. 3RESULTS 3.1 Vi us iden i y Phylogene ic analysis was based on usion (F) p o ein gene o ND.TR.IR i us, and some comme cial accines and s anda d NDV s ains a e ep esen ed in Figu e 1. The sequence analysis demons a ed a com- ple e iden i y (100%) be ween ND.TR.IR and I-2 s ain, esul ing in dis- ibu ion a he same clus e . The F gene nucleo ide sequence o he s udied i us is a ailable a NCBI gene bank as ND.TR.IR unde he accession numbe KF805771.1. Amino acid sequence compa ison o he s udied i us wi h some comme cial accinal i us and s anda d NDV s ains is shown in Figu e 2. The s udied i us showed a comple e consis ency wi h I-2 s ain, while i had conside able F-p o ein amino acid sequence di e - ences compa ed o o he NDV s ains. The p o eoly ic clea age si e o ND.TR.IR is cha ac e ized by h ee basic amino acids a he ca boxy e minus o F2 and leucine a he amino e minus o F1 (112R-K-Q- G-R*L117) associa ed wi h he a i ulen NDVs. [Co ec ion added on 8 Ap il 2022, a e i s online publica ion: “ i e basic amino acids” was co ec ed o “ h ee basic amino acids” in he p eceding sen ence] TABLE 1 In i o pa hogenici y indices o ND.TR.IR and speci ica ions o accinal Newcas le disease i us (NDV) s ains Pa hogenici y indices S anda d accep able alue*Resul ICPI <0.5 0.0 IVPI 0.0 0.0 MDT (h) >90 138 Abb e ia ions: ICPI, in ace eb al pa hogenici y index; IVPI, in a enous pa hogenici y index; MDT, mean dea h ime. *Values ep esen ing speci ica ions o accinal NDV s ains. 3.2 Pa hogenici y indices Resul s o con en ional in i o pa hogenici y es s and speci ica ions o accinal NDV s ains a e p esen ed in Table 1. Bo h ICPI and IVPI alues measu ed o ND.TR.IR we e ze o. The mean dea h ime (MDT) was 138 h. 3.3 Se ological and challenge e icacy esul s An ibody i es agains NDV in SPF chickens ha we e inocu- la ed wi h ND.TR.IR a e shown in Figu e 3. Vaccina ion inc eased mean HI an ibody i es in V1, V7, and V21 g oups compa ed o hei co esponding con ol g oups (p<0.05). I is wo h no - ing ha accina ion in elde chickens caused highe an ibody i es han ha o younge s (p<0.05). Resul s o he challenge es showed ha ND.TR.IR adminis a ion caused 100% p o ec ion in he accina ed chickens compa ed o hei co esponding con ol chickens. Mean HI an ibody i es agains NDV in na i e chickens immunized ia B1 and Laso a o ND.TR.IR accines a e ep esen ed in Figu e 4. The B1+LaSo a and ND.TR.IR g oups had no signi ican di e ence du - ing he expe imen al pe iod; howe e , hey had highe HI an ibody i es compa ed o he con ol chickens (p<0.05). The MDA i e was dec eased om i s (7.2 ±0.24) o en h (5.6 ±0.26) days o he expe - imen . The dec easing end in MDAwas con inued in he con ol g oup and eached 2.7 ±0.24, 2.0 ±0.21 and <1 a 20, 30, and 40 days o age, 20531095, 2022, 4, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1002/ ms3.794 by Technical Uni e si y In B no, Wiley Online Lib a y on [31/07/2023]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License ABDOSHAH ET AL.1543 FIGURE 3 Hemagglu ina ion inhibi ion (HI) an ibody i es agains Newcas le disease i us in speci ic pa hogen ee chickens accina ed by he I-2 s ain (ND.TR.IR) a di e en ages. No e: Chickens we e accina ed wi h a dose o ND.TR.IR (V1, V7, and V21) o ecei ed dis illed wa e (C1,C 7,andC 21) h ough eye d op a 1, 7, and 21 days o age. An ibody i es we e measu ed 21 days a e each adminis a ion ime. Values wi h di e en supe sc ip s (a and b) indica e a signi ican di e ence (p<0.05). E o ba =s anda d e o o he mean espec i ely. Mean HI an ibody i es in he B1+LaSo a and ND.TR.IR g oups showed a g adually dec easing end be ween 10 and 20 days o age; howe e , ollowing he boos e accina ion a day 20 o age, he an ibody le els emained a a cons an le el ( anged be ween 4.1 ± 0.36 and 4.4 ±0.42). Resul s o he challenge es showed 95% p o ec- ion in B1+LaSo a and ND.TR.IR g oups which was highe han 35% o he con ol g oup (p<0.05; Table 2). Mean HI an ibody i es agains NDV in b oile s inocula ed wi h di e en ND accines a e ep esen ed in Figu e 5. The an ibody le - els we e no di e en be ween B1+LaSo a and ND.TR.IR g oups o e he expe imen al pe iod (p<0.05), bu hey had signi ican ly highe HI an ibody i es compa ed o he con ol chickens (p<0.05). A se e e dec easing end in MDA i es was no ed om i s (5.8 ±0.18) o en h (1.7 ±0.22) days o he expe imen . Howe e , ollowing he i s ND accina ion, he an ibody le els we e inc eased in B1+LaSo a (4.30 ±0.45) and ND.TR.IR (4.90 ±0.27) g oups compa ed o he con- ol chickens on day 20 o age (p<0.05). The ea e , he an ibodies i e o B1+LaSo a and ND.TR.IR g oups showed a ela i ely cons an end and each 4.8 ±0.41 and 4.7 ±0.42 on day 30, and 4.3 ±0.49 and 4.2 ±0.39 on day 40 o age, espec i ely. Resul s o he challenge es in his expe imen showed ull p o ec ion (100%) in bo h B1+LaSo a and ND.TR.IR accina ed chickens, while all he un accina ed challenged chickens (con ol) died (Table 2). 4DISCUSSION The welling o adminis a ion he mo esis an accines, especially in illage condi ion, is inc easing du ing las decades. The I-2 is he main ND s ain used o p oducing he mo esis an ND accines o u al chickens. Howe e , i s po en ial use in eme gency accina ion and no e idence o e e s o i ulence has also made i a candida e ac- cine o con ol ND in comme cial locks (Bisschop e al., 2021). In an a emp o p oduce a he mo esis an ND accine, in his s udy, an I- 2 o igina ed accinal seed was success ully cha ac e ized and i s e i- cacy agains ND was in es iga ed in SPF, na i e, and b oile chickens. Resul s o he p esen s udy e ealed a comple e iden i y be ween genomic sequences o he s udied i us and I-2 s ain showing he po en ial o he s udied i us as a accinal seed. Acco ding o W. H. Allan e al. (1978), an a i ulen NDV complies wi h he IVPI, ICPI, and MDT indices when hei co esponding alues a e ze o, less han 0.5, and longe han 90 h, espec i ely. Consis en wi h he molecula FIGURE 4 E ec o adminis a ion a he mo esis an Newcas le disease (ND) accine (ND.TR.IR) e sus common accina ion p og amme (B1 and LaSo a) on an ibody i e agains ND in na i e chickens. No e: The chickens ecei ed a dose o ND.TR.IR o B1 and LaSo a ND accines a days 10 and 20 o age ia eye d op and d inking wa e , espec i ely. Values wi h di e en supe sc ip s (a and b) wi hin each sampling ime indica e a signi ican di e ence (p<0.05). E o ba =s anda d e o o he mean 20531095, 2022, 4, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1002/ ms3.794 by Technical Uni e si y In B no, Wiley Online Lib a y on [31/07/2023]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License 1544 ABDOSHAH ET AL. TABLE 2 P o ec ion le el o speci ic pa hogen- ee (SPF), na i e, and b oile chickens accina ed wi h common accina ion p og am (B1 and LaSo a) o a he mo esis an Newcas le disease (ND) accine (ND.TR.IR) in he challenge wi h i ulen ND i us Expe imen al g oups V1 V7 V21 Con ol (C1,C 7, and C21) SPF1100% (10/10)a100% (10/10)a100% (10/10)a0% (0/10)b,2 Expe imen al g oups – B1+LaSo a ND.TR.IR Con ol B oile 3– 100% (10/10)a100% (10/10)a0% (0/10)b Na i e chicken3–95% (19/20)a95% (19/20)a35% (7/20)b 1Chickens in V1, V7, and V21 g oups we e accina ed wi h a dose o ND.TR.IR o ecei ed dis illed wa e (C1,C 7,andC 21) h ough eye d op a 1, 7, and 21 days o age, espec i ely. 2Resul s o con ol g oups in C1, C7, and C21 we e he same. 3The chickens ecei ed B1 and LaSo a o ND.TR.IR accines a days 10 and 20 o age ia eye d op and d inking wa e , espec i ely. a,bWi hin each ow, alues wi h di e en supe sc ip s a e signi ican ly di e en (p<0.05). FIGURE 5 E ec o adminis a ion a he mo esis an Newcas le disease (ND) accine (ND.TR.IR) e sus common accina ion p og am (B1 and LaSo a) on an ibody i e agains ND in b oile chickens. No e: The chickens ecei ed a dose o ND.TR.IR o B1 and LaSo a ND accines a days 10 and 20 o age ia eye d op and d inking wa e , espec i ely. Values wi h di e en supe sc ip s (a and b) wi hin each sampling ime indica e a signi ican di e ence (p<0.05). E o ba =s anda d e o o he mean iden i ica ion esul s, he ob ained alues o he pa hogenici y indices app o ed ha he ND.TR.IR is a candida e accinal seed. In he p esen s udy, h ee expe imen s we e conduc ed o e alu- a e he e icacy o ND.TR.IR. Acco ding o he OIE (2012), he e icacy e alua ion o a li e ND accine mus be conduc ed on 10 o mo e SPF o o he ully suscep ible chickens, a he minimum ecommended age using he minimum ecommended dose. Meanwhile, o ob ain mo e conclusi e esul s, in he i s expe imen , he e icacy o he ND.TR.IR accine was examined in di e en ages o chickens. Ou esul s showed ha he inocula ion o 1-day-old SPF chickens wi h ND.TR.IR was asso- cia ed wi h se a an ibody i es g ea e han 23, a 21 days pos - accina ion. The highe an ibody i es which we e obse ed in he chickens inocula ed a 7 and 21 days o age is explained by he de el- oped immune sys em in he elde chickens (Ambali e al., 2017;Al- Ga ib e al., 2003). The app op ia e se ological immune esponse in chickens ecei ed ND.TR.IR was suppo ed by he esul o he chal- lenge e icacy es , whe e he accina ed chickens we e ully p o ec ed agains NDV in ec ion. I has been documen ed ha chickens wi h HI an ibody i e s ≥23would be p o ec ed ollowing he challenge e i- cacy es (W. Allan & Gough, 1974; Hossain e al., 2010; Hassanzadeh e al., 2020; Van Bo en e al., 2008). Howe e , he e a e pieces o e i- dence showing ha he chickens wi h lowe i e also may su i e a e he challenge (Mee s & Sp adb ow, 2006). In addi ion o he ci cula ing an ibody, cellula and mucosal immuni y a e also in ol ed in acqui ing a good p o ec ion le el in a challenge wi h he i ulen i us; howe e , because o de e mina ion o ci cula ing an ibody is mo e easible and easie , i is conside ed as he p ima y index o e alua ion p o ec ion agains diseases. In he second expe imen , he e icacy o ND.TR.IR on na i e chickens was e alua ed and compa ed wi h a ou ine accina ion 20531095, 2022, 4, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1002/ ms3.794 by Technical Uni e si y In B no, Wiley Online Lib a y on [31/07/2023]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License ABDOSHAH ET AL.1545 p og am. Resul s showed an app op ia e HI an ibody aise and p o- ec ion agains NDV in all o he accina ed chickens. In ag eemen wi h 95% p o ec ion eco ded in his expe imen , Ilonggo e al. (2008) epo ed ha he mos able I-2 ND accine p o ided 100% and 89% p o ec ion agains ND in-housed and unhoused chickens, espec i ely. Mo eo e , in es iga ions on u al locks ha e e ealed ha I-2 o igi- na ed accines we e associa ed wi h app oxima ely 80% co e age and long- e m immuni y (Adwa and Lukeso a, 2008). His o ically, he - mos able ND accines ha e been de eloped a he Queensland Uni- e si y and used in se e al de eloping coun ies o con ol ND in u al locks (Mee s & Sp adb ow, 2006); howe e , due o mo e s abili y in ambien empe a u e and la e al ansmission, he he mos able ND accines a e usually associa ed wi h a lowe a ia ion in an ibody i es and cause be e p o ec ion agains ND (Alde s & Sp adb ow, 2001; Illango e al., 2008; Shahid Mahmood e al., 2014). These ad an ages ha e made he he mos able ND accine as a good op ion o he use in comme cial b oile a ms. Resul s o he hi d expe imen e icacy es showed ha immune esponse de i ed om ND.TR.IR o ou ine ND accines (B1 and LaSo a) was no di e en in he comme cial b oile s. Despi e he negli- gible di e ence in se a an ibody i es, bo h immuniza ion p og ammes we e associa ed wi h ull p o ec ion agains NDV. These indings we e in line wi h he esul s o he second expe imen in na i e chickens. Following accina ion, he immunized chickens acqui e humo al and cellula as well as mucosal immuni y. Howe e , by he in amuscula ou e o challenge, he NDV is bypassed h ough mucosal immuni y ha is one o he i s de ensi e ba ie s agains pa hogens. The e- o e, esis ance o challenge wi h he in amuscula ou e is ega ded as he gold s anda d o e alua ing he e icacy o i al accines (Mee s & Sp adb ow, 2006). I has been widely accep ed ha he d immu- ni y p o ides some p o ec ion o subop imal o un accina ed chick- ens (Hassanzadeh & Bozo gme i Fa d, 2004; Kapczynski e al., 2013) i mo e han 85% o he chickens acqui e HI an ibody i es g ea e han 23a e wo ND accina ions (Van Bo en e al., 2008). The cu - en esul s we e co obo a ed by he p e ious s udies which epo ed ha an ibody i e ≥23is he possible p o ec i e le el in chickens ac- cina ed agains ND (W. Allan & Gough, 1974; Bensink & Sp adb ow, 1999; Wambu a, 2011). Meanwhile, Kapczynski and King (2005)sug- ges ed ha a comme cial b oile housing, only chickens wi h HI i es g ea e han 24su i ed in NDV challenge and 66% o chickens wi h lowe an ibody i es emained as unp o ec ed. Mo e commonly, HI i es ≥25a e ypically sugges ed as a p o ec i e le el (W. H. Allan e al., 1978; Kapczynski e al., 2013). In his s udy, 100%, 80%, and 60% o he ND.TR.IR-inocula ed b oile chickens had HI an ibody i es ≥23,2 4,and2 5on day 30 o age, espec i ely (da a we e no shown). Howe e , he ob ained an ibody i es we e associa ed wi h 100% p o- ec ion agains NDV, unde a con olled examina ion. As he inci- dence o ND in chicken locks usually occu ed a 4–5 weeks o age (Hassanzadeh & Bozo gme i Fa d, 2004), he highe p opo ion o chickens wi h p o ec i e i es would dec ease he mo bidi y and mo - ali y a e in he ea ly ND challenge. I is wo h no ing ha he a e age MDA i e was highe in il- lage chickens compa ed o b oile s. P obably, lowe biosecu i y mea- su es in na i e locks han he s ic condi ions obse ed in b oile b eede s cause mo e expose wi h NDV, esul ing in a highe MDA i e in hei o sp ing. In addi ion, he mo e apid dec easing end in MDA i es o b oile s han ha o na i e chickens is explained by he highe me abolic a e associa ed wi h gene ic selec ion o mo e g ow h a e and weigh gain. In acco dance, Gha aibeh and Mahmoud (2013) epo ed ha a ia ions in he MDA hal -li e may s em om ac- o s ela ed o di e ences in chicken lines and ypes. 5CONCLUSION In his s udy, a ecei ed I-2 o igina ed ND i us success ully complied wi h he equi ed speci ica ions o a accinal seed. Also, adminis a ion o he p oduced ND.TR.IR accine p ope ly aised HI an ibody i es associa ed wi h a high p o ec ion le el agains NDV in SPF, na i e, and b oile chickens. These indings sugges ha no only ND.TR.IR migh be use ul o p e en ND ou b eaks in u al locks bu also i could be ad ised o be adminis a ed in comme cial b oile s a ms. Howe e , u he s udies a e wa an ed. ACKNOWLEDGEMENT The au ho s would like o hank he Razi Vaccine and Se um Resea ch Ins i u e o inancial suppo o his p ojec unde g an numbe 12- 18-18-036-95017-950501. CONFLICT OF INTEREST No po en ial con lic o in e es is epo ed by he au ho s. ETHICS STATEMENT The expe imen was conduc ed unde app o al gi en om Razi Vac- cine and Se um Resea ch Ins i u e (RVSRI) E hics Commi ee, Ka aj, I an. AUTHOR CONTRIBUTIONS Concep ualiza ion, me hodology, supe ision, and p ojec adminis a ion: Mohammad Abdoshah and Mohammad Hassanzadeh. Visualiza ion, in es iga ion, w i ing—o iginal d a , and w i ing— e iew and edi ing: Shahin Masoudi and Abbas Ash a i. Fo mal analysis, isualiza ion, w i ing—o iginal d a , and w i ing— e iew and edi ing: Ali Reza Youse i. Me hodology and w i ing—o iginal d a : Minoo Pa o i Nas . DATA AVAILABILITY STATEMENT The da a ha suppo he indings o his s udy a e a ailable on eques om he co esponding au ho . PEER REVIEW The pee e iew his o y o his a icle is a ailable a h ps://publons. com/publon/10.1002/ ms3.794. 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The mo esis an Newcas le disease accine e ec i ely p o ec s SPF, na i e, and comme cial chickens in challenge wi h i ulen i us. Ve e ina y Medicine and Science,8, 1539–1546. h ps://doi.o g/10.1002/ ms3.794 20531095, 2022, 4, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1002/ ms3.794 by Technical Uni e si y In B no, Wiley Online Lib a y on [31/07/2023]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License