Vaccine 38 (2020) 5507-5515, Contonts lists available at Scioncobirect, iz \accine Vaccine journal homepage: www.elsevier.com/locate/vaccine Protection conferred by commercial NDV live attenuated and double recombinant HVT vaccines against virulent California 2018 Newcastle tar disease virus (NDV) in chickens Helena L. Ferreira®®, Alexandra M. Reilley‘, Dana Goldenberg”, Ivan R.A. Ortiz‘, Rodrigo A. Gallardo“, David L. Suarez*** Us nina Pout Research Center, Southeast Poly Resear Laboratory, 94 Clee Sain Ra Athens GA 30605, USA "Departmen of Veterinary Mec, FZEA-USP. Universo Sao Pala, Presume SP 12635000, Brac “ec Aria Hal, 39500 Wit 9st Sl Dest, KS OBD1&, USA *bepartmen of Population Heat and Rprection. schol of Veterinary Medicine Univesity of Calf, Davis, CA 95515, USA ARTICLE INFO ABSTRACT Receives in revised fot 30 May 2020 Deceted 2 Jone 2020 ‘abe ole 24 June 2020 ‘Vaccines against virulent Neweastle disease virus (NDV) are widely available and ean be protective, but, Improved vaccination protocols are needed to prevent clinical disease and reduce virus citculation, The present study evaluated the efficacy of two commercial vacines alone or in combination: a live atten fated NDV vaccine (LV) and a recombinant herpesvirus of turkeys veetor expressing the fusion pratcin of NNDV and the vis protein 2 of infectious bursa disease virus (rHVT-ND-IBD), Chickens were vaccinated with one of four vaccination protocols: live vaccine (LV) at ¥ and 11 days of age (DOA), HVT ND-IBD and —_ Vat 1 DOA, cHVT ND-IBD at 1 DOA boosted with an LV at 11 DOA, and HVT ND-IBD at 1 DOA. The vac~ Newest giscuse ‘inated birds were challenged at cfferent time points (3 or 4 weeks of age) with the California 2018 virus, nase ‘The movtaity, linia signs, mean death time (MDT) humoral response before and after vaccination and Serology Virus shedding after challenge were evaluated. All vaccination protocols were abe to prevent mortality. Vins reduce virus shedding, ad induce antibody levels before the challenge at 3 and 4 weeks-ld. Overall, the Recombinant Antibody levels before the challenge 3t 4 weeks were significantly higher inal groupe vaccinated with ve attenates the HVT ND-IBD when compared to levels in 3 week ol birds, The combination of recombinant HVT [ND-IBD with live vacine at one-day-old seems to bea better combination, due othe absence of clinical signs, higher antibody levels pre and post-hallenge, and reduced virus shedding at any time point after the challenge at 3 of 4 weeks af age withthe California 2018 virus Published by Elsevier Id. Tis isan open access article under the CC BY-NC-ND license (tp: ereative- ‘commonsorglicenses/by-ne-nd/4.0- 1. Introduction hhemagglutinin-neuraminidase (HN) (2). The F protein is one of the principal protective antigenic targets, mediates virus entry, Newcastle disease virus (NDV) belongs to the family Paramyx- cell-to-cell dissemination, and is also a major determinant for vir- oviridae, subfamily Avulavirinae, and the recently renamed genus Orthoavulavirus [1], Newcastle disease viruses (used hereafter and also commonly known as avian paramyxovirus 1, APMV-1) belong to species avian orthoavulavirus 1, This enveloped virus has a single-stranded, negative sense, non-segmented RNA genome ‘organized as follows: 3'- nucleoprotein (NP) - phosphoprotein (P) - ‘matrix protein (M) - fusion protein (F) - hemagglutini neuraminidase (HN) ~ large polymerase protein (L) - 5. The virus particle has two integral glycoproteins: the fusion (F) and the © Goresponting author at: US National Poly Resateh Center, Southeast Poultry Reseach Laboratory 934 Cll Station Ra Athens GA 30605, USA mail odes: Davi Srey gow (DA Sate) neps:do.org/10.101 6) vaccine 2020.05.04 {2264 410X/Pubished by Elser ulence [2-4] [Newcastle disease virus (NDV) often causes severe disease in young and susceptible birds depending on its pathogenicity (5). Virulent NDV isolates continue to be an ongoing threat to the poul- try industry and are reportable to the World Organization for Ani ‘mal Health (OIE) because of threat of spread between countries, Some countries on all continents, except Antarctica, reported an ‘occurrence of NDV to the World Organisation for Animat Health (OIE), including the USA 6], in the last 7 yeats. The occurrence of virulent NDV in poultry flocks of NDV-free countries can cause costly outbreaks because of control costs, effects on trade, and con- tinues to cause economic losses in endemic countries despite widespread use of vaccination [5], Vaccination against NDV in “Tis ian open aces ale under the CCBY-NCND Hens (tpereatiecommansorgiensesy ne 24/40),soe Feria eta eine 8 (2020) 5907-5515 ‘combination with biosecurity is usually done to prevent NDV infection [7] Inactivated, live attenuated, and recombinant vaccines to con- trol NDV are available in many countries [8]. Inactivated NDV vac- ‘ines can protect bisds against clinical signs and induce a strong and long-lasting, humoral response, which can be transferred to the offspring [9]. However, the inactivated vaccines are costly because they require an individual administration through subeu- taneous or intramuscular injection, Vaccinated birds also require a withdrawal period before they can be processed for human con- sumption and vaceines may cause tissue reactions which can lead to increased condemnation of carcasses (10), Live attenuated vac- cines have the potential advantage of inducing a strong humoral immune response with an early immunity onset of both cellular ‘and mucosal immunity components, [11] and are often applied by using an aerosol spray which reduces vaccination administra- tion costs, Both types of vaccines can protect against clinical signs, ‘and they also reduce the replication of the challenge virus in the host resulting in less viral shed [12 Live attenuated vaccines how- ‘ever are negatively affected by maternal antibodies and can induce ‘economically important vaccine reaction because of thei replica tion in the respiratory tract [13-15], For the past 20 years, the use of recombinant vectored vaccines has een increasing in the poultry industry largely because they can be used in ovo or subcu- taneously with one-day-old chicks in the hatchery, and unlike live attenuated vaccines, they have no respiratory vaccination reaction [16]. The fowlpox virus (FPV) expressing the F or HN genes of NDV ‘and the recombinant herpesvirus of turkeys HVT expressing the F protein of NDV (HVT ND) were proven to protect chickens from challenge with virulent NDV and have been licensed in many coun- tries. The HVT ND has been shown to provide complete clinical protection three to four weeks post-vaccination with different vir- tlent challenges [17-21]. Studies have also shown that the HVT ND can reduce virus shedding compared to control groups in birds challenged at four weeks after vaccination (18.20.22). A combina- tion of the F HVT ND witha lve attenuated vaccine, can also reduce the number of birds shedding virus [21] Finally, the HVT vector is not affected by maternal antibody and is persistent in the host which helps it overcome maternal antibody vaccine suppression which may provide a long-lasting immunity | 18,23) In 2018, the USA faced a new detection ofa virulent NDV in Los, “Angeles, California which was the frst outbreak in 15 years (prior to 2018, the most recent outbreak of vNDV was 2002-2003) [24.25], Since the first detection in May 2018, over 470 cases have been confirmed in backyard exhibition and commercial birds, mainly in California, but single cases have also been detected in two other states in the U.S. (Utah and Arizona) in backyard exhibi- tion and commercial birds (\ntips:|/wvrw.aphis.usda govlaphis) ‘ourfocus/animalhealth/animal-disease-information/avian)viru- lent-neweastlejvnd), Robust control efforts have been imple- ‘mented and approximately 72 million US dollars have been spent ‘or allocated in eradiaction efforts so far. Our group previously ‘demonstrated that the pathogenicity and transmission of the virus ‘causing the current outbreak is similar to genetically close isolates from Belize in 2008, and from the California 2002-2003 outbreak in young and adult chickens (26). The 2002-2003 California NDV-outbreak has been widely studied, and some vaccination pro- tocols were evaluated for virus control, but did not include the testing of the new generation viral vectored vaccines [27-29]. Despite widely available vaccines, control of NDY, particularly in backyard flocks, remains problematic and new vaccination proto- cols to control viral dissemination are needed, The present study ‘aimed to evaluate the efficacy of four vaccination protocols using ‘commercial vaccines ie. a live attenuated vaccine and a recombi- rant HVT expressing the fusion protein of NDV and the virus pro- tein 2(VP2) af infectious bursal disease virus (IBDV). The chickens vaccinated with these different vaccination protocols were chal- Tenged with the virus isolated in the 2018-2019 California out- break at 3 and 4 weeks post vaccination using SPF chickens and ‘were evaluated for protection from clinical diseases, development ‘of humoral immune response, and virus shedding. 2 Material and methods 2.1. Chickens In total, 185 day-old specific-pathogen-free (SPF) White Leg hhomn (WL) chicks (Callus gallus domesticus) were obtained from the Southeast Poultry Research Laboratory (SEPRL) SPF flock. Feed and water were provided! ad libitum. Birds were kept in isolators, the animal experiments were approved and performed in accor- ‘dance tothe Institutional Animal Care ancl Use Committee (IACUC) in animal biosecurity level 2 and 3 enhanced (ABSL-2E and ABSL- 3B, respectively) facilities atthe SEPRL. 22. Viruses NDV obtained from the SEPRL repository was used to perform, the virus challenge: chicken/Califoria/D1806566/2018 (CA18). ‘The virus was propagated in specific pathogen free (SPF) embry- ‘onating chicken eggs (ECE), as previously described (30). Virus~ infected allantoic fluid was diluted in brain heart infusion (BHI) ‘medium (BD Bioscience, Sparks, MD) to obtain an inoculum with titers of 6 logip 50% egg infective dose (EIDsa/ml) per bird. 23. Vaccines ‘A live attenuated NDV vaccine (LV) from genotype Il (Newhatch-C2" vaccine, Merck Animal Health, DeSoto, KS, USA) was administered by spray with titers of 6.8 logo EIDso. The spray ‘vaccine was administrated using a manual pump spray. For groups ‘of 20 birds, a full dose per bird was given in a volume of 10 mL ‘administered over a 10 s period of time. The HVT recombinant, a FC126 strain, expressing the F protein from a genotype ll, clone- 30 strain of Newcastle disease virus (NDV) and the VP2 protein Of infectious bursal disease virus (IBDV), named here as HVT ND-IBD (Innovax"- ND-IBD, Merck Animal Health, DeSoto, KS, USA) was subcutaneously administrated with the HVT titer of 17540 plaque forming units (PFU)/bird. 24. Experimental design ‘The experimental design is represented in Figure Supplementary Fig. 1. In total, 147 day-old chicks were split into experimental _groups with four vaccination protocols as follows: birds vaccinated with LV vaccine at 1 and 11 days of age (LV*LV groups, n = 40) birds vaccinated with the HVT ND-IBD and the LV vaccines at 1 day of age (FHVT ND-IBD/LY groups, n ~ 38); birds vaccinated with rHIVT ND-IBD at 1 day of age and boosted with the LV vaccine at 11 days of age (sHVT ND-IBD*LV, n = 31): and birds vaccinated, with only HVT ND-ABD at 1 day of age (rHVT ND-IBD, n = 38). ‘Thirty-eight birds were kept as non-vaccinated controls. Birds receiving the same vaccination protocol were split into two groups with the groups being challenged at 3 or 4 weeks of age. Eighty- five birds, split in 11 to 20 birds per group, were challenged at 3 weeks and 100 birds (n= 20 per group) at 4 weeks-old. The chal- lenge was performed using the CAI8 isolate via the choanal cleft land the left eye of each bird at 10° 50% egg infective doses (EID) per bird. Clinical signs and mortality were monitored daily for 14 days after challenge. Oropharyngeal (OP) and cloacal (CL) swabs were collected from all birds at days 2, 4, and 7 days post1 Feria e aoc 38 (2020) 5507-5515 909 challenge (DPC) to determine virus shedding. Blood was collected before (18 DPV or 25 DPV) and after each challenge (14 DPC) to evaluate the humoral response, The mean death time was ealeu- lated by averaging the day individual died from challenged or were euthanized and reported as dead on the next day. 25. Detection of HVT in pulp feathers Feather pulp from axillary feathers were collected from all birds, vaccinated with rHVT ND-IBD vaccine and from five birds vac nated with LV and five non-vaccinated bitds at 16 days post vac nation (DPV). Feather pulps were homogenized in 500 yl of BHI supplemented with antibiotics and! DNA purified with the DNAeasy 96 blood & tissue kit (Qiagen, USA) A real-time PCR targeting (R-PCR) the SORF1 gene of the HVT was performed using a previ- ‘ously described set of primers and probe [31|. The probe was labeled with EAM fluorescence and BHQ quencher. The R-PCR reac~ tion used the TagMan™ Fast Virus 1-Step Master Mix kit (Thermo- Fisher Scientific, USA) with the following reaction volumes: 5 yl of TagMan™ Fast Virus 1-Step Master Mix (4x), 05 ul of each primer and probe, and sterile nuclease-free water to bring individual reac- tion volumes to 20 ul containing 8 jl of DNA template. After ani tial denaturation step at 95°C for 2 min, 40 cycles (95 °C for 15 sec, 60°C for 45 sec) were performed with fluorescence detection at the end of the annealing-extension step. A quantitative RT-PCR (RRT- PCR) targeting the beta-actin gene was used as an internal control [32]. The RRT-PCR reaction used the TagMan™ Fast Virus 1-Step ‘Master Mix kit (ThermoFisher Seientific, USA) with the following reaction volumes: 5 yl of TaqMan” Fast Virus 1-Step Master Mix (4x), 0.23 ul of each primer, 0.42 yl of probe, and sterile nuclease-free water to bring individual reaction volumes to 25 yl containing 2 jl of sample, Primers and probes were used ata con- centration of 20 and 6 pmol), respectively, forall reactions. After an initial denaturation step at 95 °C for 2 min, 40 cycles (95 °C for 15 sec, 60 °C for 45 sec) were performed with orescence detec- tion atthe end of the annealing-extension step. For the beta-actin detection, an initial RT step at 45 °C for 10 min was done. R-PCR reactions were carried out in an ABI750O instrument (Applied Biosystems, USA). The rHVT ND-IBD vaccine was used as a positive control for the R-PCR for HVT detection, and the limit of detection ‘was calculated at 10° PFU 26. Virus shedding OP and CL swabs were collected in 2 ml of BHI broth with a final concentration of 10 jig/ml of gentamicin, 10 units/ml. of penicillin 6, and 56 ygiml of amphotericin B, and kept frozen at ~70 °C until processed, RNA was extracted using the MagMax Al/ND RNA isola- tion kit (Ambion, Inc. Austin TX, USA) [33]. A RRT-PCR test targeting, the F gene for Newcastle disease virus (NDV) detection, which detects only the challenge virus, was performed as previously described [26] on an AB! 7500 (Applied Biosystems) A standard curve for virus quantification was established with 10-fold serial dilutions in nuclease-free water of the titrated CA18 virus, and results were reported as ElDso/ml equivalents. The calculated [RT-PCR lower limit of detection was 10%” EIDzo/ml. 27, Hemagglutination inhibition (HI) test ‘The HI test was used to quantify antibody responses after vaccination and challenge using the homologous antigen to the challenge as previously described [34]. Serum was collected from all birds before the challenge (18 of 25 DPV) and from surviving. birds atthe end ofthe experiment (14 DPC). Titers were calculated as the highest reciprocal serum dilution providing complete ‘hemagglutination inhibition, Serum titers of 3 Logs or above were considered as positive. 28. Indirect ELISA for detection of antibodies specific to NDV “The ID screen Newcastle disease indirect ELISA (IDvet, Grables, France) for recombinant vaccines was also used to assess the antibody titers against NDV before and after the challenge using serum diluted at 1/500. The test, as well as the antibody titer calculation, were performed according to the manufacturers recommendations. 29, Statistical analysis Data were analyzed using Prism (v.7.03) software, and outliers, were identified using the ROUT test (GraphPad Software Inc, La Jolla, CA, USA). Survival curves were tested using the Log-rank (Mantel-Cox) test with a significance level of P< 0.0001. For virus shedding and humoral response, the D'Agostino-Pearson normality test was performed to estimate if the values in each group come {rom a Gaussian distribution, Based on the normality distribution, ‘non-paramettic ANOVA test was used for multiple comparisons of ‘mortality rates and viral titers in OP or CL swab samples from the lifferent viruses on the same day. Hi titers and ELISA were also tested using the non-parametric ANOVA test comparing titers from serum collected before or after challenge. Statistical significance ‘was set at a P value of <005. The means * standard errors with ‘no common letters differ significantly. The Pearson correlation ‘was tested to compare the ELISA titers before the challenge and the HVT shedding from birds vaccinated with the rHVT ND-IBD alone or in combination with the live vaccine. 3. Results 3.1, Replication of HVT in feather pulp and antibodies against NDV ‘The HVT vaccine was detected by R-PCR in 88 out of 105 tested birds, eepresenting 84% detection in vaccinated bitds (Table 1). The birds from rHVT ND-IBD/LV group challenged at 3 weeks of age had 100% detection while those challenged at 4 week-old had a lower tate of detection (65.5%). The rate of detection in the HVT ND-IBD*LV was 91%, and 70% in the birds challenged at 3 and 4 week-old, respectively. The rate of detection in the groups that received only the rHVT ND-IBD vaccine was over 94%. All tested samples were positive for the housekeeping gene (beta-actin) with Jow Ct values (mean of 23.6), including those that were negative by the R-PCR for HVT. 3.2, Challenge at 3 week-old 32.1. Mortality, MDT, and clinical signs 'Non-vaccinated birds inoculated with the CATS virus had 100% ‘mortality, showing typical clinical signs of Newcastle disease, suci as moderate to severe lethargy, rufled feathers, and conjunctivitis at 2 to 4 DPC. The MDT for this group was 4.1 DPC (Fig. 1). As for the vaccinated groups, no mortality nor clinical signs were ‘observed in most vaccinated birds, with the only exception of the THVT ND-IBD*LV group that had two birds that displayed mild neurological symptoms. One bird showed a mild head tlt at 9 DPC with recovery two days later, and a second bird displayed a head tilt at 11 DPC, which remained until the end of the experi ‘ment (14 DPC),Feria eta eine 8 (2020) 5907-5515 Detect of rT in the feather pulp from bids vaccinate with HVT ND-BDslane rin combination with he Live vaccine by R-FCR at 16 DPV Chaenze ‘Group name E pstves values ‘306% 3 week ol HVT ND-BOIL 100, Bis 4 4 weekcld 65 29 23 3 cecal 3 soa 39 4 weekcld 70 289 a9 ce ot 235 33 ota All groups a 25 33 Challenge at 3 week-old two birds (one in the LV*LV group and another bird in the rHVT ND-IBD*LV group that displayed clinical signs after challenge). A fen ‘The mean ELISA titers in sera were: 4082, 7421, 4646, and 5926 in LV*LV, HVT NDABD/LY, rHVT ND-IBD+LV, HVT ND-IBD groups, 8 revT NO BD/IV respectively. ELISA titers from vaccinated groups were statistically rHVTNDIBOUY" rev No-80 A control MoT: 4.1 0) Time (orc) Challenge at 4 week-old 7 THVT ND-BO/W ee a THVT NDABDHIV Waiver Ne NoweD 4, control MoT: 4.10°¢) ‘ime (ore) Fg. Survival rates after challenge at 3 or 4 week-ld Corto birds ad the same MDI ater challenge at (A) 308). woekol. Tw birds challenged st woeko}d in the HHVT ND-IBD-LV proup displayed tit head 3t 9 and 11 DPC. he fst one recovered a0 11 DPC. "2 birds inthe LVSLY grou challenged at 4 week ld ‘splayed lethargy at 3 DOC. The suecumed to death a and 10 DPC 3.2.2, Humoral responses before the challenge Al birds in the control group were negative by the HI test before ‘challenge (Fig. 2)- Only a few birds that had atleast one inoculation ‘of the LV had lov positive titers by HI test with no significant dif ference (P > 0.05) detected among them. In the LV*LV and rHVT IND-IBDLV groups, the mean of all HI titers was below 3 logs Only 22% and 18% ofthe sera from the birds were positive (above 3 logs). respectively, These positive sera included the two birds that dis- played clinical signs in the HVT ND-IBD+LV group. The rHVT ND-IBD/LV group had the highest positive rates with 70% of tested birds having positive titers with a mean of 3:9 logo. All birds vacci- nated only with HVT ND-IBD were negative by Hl and had sigai icantly different titers (P < 0.0001) to other vaccinated groups. ‘The ELISA test was also used to detect specific fusion antibodies against NDV before the challenge (Fig. 2). The non-vaccinated birds had ELISA titers below 993, the cut-off for positive samples. On the ‘ther hand, all vaccinated birds had ELISA titers above 993, except higher (P = 0.0029) when compared to the control group. ‘The cortelation of HVT detection in feathers and ELISA titers was evaluated Figure Supplementary Fig, 2 comparing birds vacci- nated only with tHVT ND-IBD or with the LV. No clear correlation ‘could be observed when analyzing the birds challenged at 3 week- ‘old with Pearson values of ~0.2292 and 0.2240 for birds vacci- nated with HVT ND-IBD only and all rliVT ND-IBD groups, respectively, 3.23. NDV shedding Al birds in the control group shed challenge virus by oral and cloacal routes with titers above § logyo EIDsa(ml after challenge (Fig. 3). Vaccinated birds shed significantly less virus (at least 1 Jogo less) compared to the control group at 2 DPC. except the birds in the rHVT ND-IBD*LV group by the oropharyngeal swabs. The peak of shedding in all vaccinated birds was detected at 4 DPC with titers varying from 4.2 to 5.4 logio£1Dsaiml and 3.3 t0 4.0 logio- E1Dso/ml by oral and cloacal routes, respectively, but more pro- nounced in the oral route as more tested birds shed the virus through this route, The THVT ND-IBD/LY group had the lowest amounts of virus shedding among the vaccinated groups by both ‘OP and CL swabs at any time point, except at 2 DPC by the cloacal route. The birds in the rHVT ND-IBD/LV group shed significantly (P< 0.05) less virus from the oropharynx than other birds in the LV¢LV group at 7 DPC, in the HVT ND-IBDSLY group at all time points, and in the rHVT ND-IBD group at 4 DPC. Overall, the rHVT ND-IBD#LV group shed the highest virus titers at alltime points in vaccinated birds, 3.24. Humoral response after the challenge ‘Alter the challenge, the mean of HI titers from all surviving birds in the vaccinated groups varied from 9.0 to 102 logs (Fig. 20). The birds in the LVsLV group had the highest HI titers, ‘which were significantly different from HI titers in the cHVT ND- IBDILY group (P = 0.0042), Similar to the HI test, all remaining birds in the vaccinated ‘groups had high titers measured by the ELISA test ranging from 21,885 to 26,891 (Fig. 2D). The ELISA titers in the LV+LV group ‘were significantly different (P = 0.0002) compared to other vacci- nated groups with the highest mean titer (26,891), and no signif- ‘cat difference was detected among the tH1VT ND-IBD groups. ‘The ELISA titers before and after challenge at 3 weeks of age were compared Figure Supplementaty Fig. 3 (Supplemental Fig. 34). The LV*LV group also had the highest increase of ELISA titer (560%) when comparing to the titers before the challenge. ‘The THVT ND-IBDLV group had the second highest increase of ELISA titer (3742), followed by the tHVT ND-IBD (269%) and rHVT ND-IBD/LV group (199%.1 Feria e aoc 38 (2020) 5507-5515 ss A Hitest B FUIsA A A a ae eer sao eerie crows owe asa c Hitest o A A » , ow anos oo . : 3° ry i i 4 ? =| 5 sos E = 10000) : é OTe ae Sener cela er ee o Ta aaa era ewe crows cious @ control ME Wvetv | A HVT NDIBD/LV |W FHVTNDIBDHLY = @ HVT ND-IBD ig. 2. Serology in ids challonge 2 3 weokold. Humoral response (Aan) before and (Cand D) ater challenge a -week- ol was measured by Hand ELISA ets. No cormmon eters difer significantly (®< 005) NS: no survivors 33, Challenge at 4 week-old 3.3.1. Mortality, MDT, clinical signs Consistent with birds challenged at 3-week-old, non-vaccinated birds in the control group challenged with the CA18 virus had 100% ‘mortality (Fig. 1B). The non-vaccinated birds also displayed mod ate to severe lethargy, rifled feathers, and conjunctivitis at 2 to 4 DPC. The MDT for the control group challenged at 4 week-old was the same as recorded for birds challenged at 3 weeks-old (4.1 DPC). 10% mortality was observed only in LV*LV group, as two birds dis- played mild lethargy, ruffled feathers, and conjunctivitis (one bird) at 3 DPC, which progressed to death at 4 and 10 DPC. The MDT for the LVsLV group was 7 DPC. No mortality nor clinical signs were ‘observed in birds from HVT ND-IBD/LV, rHVT ND-IBD+LV, and THVT ND-IBD groups. The mortality, MDT, and clinical signs from birds in the control sroup challenged at 4 weeks were not significantly different when ‘compared tothe challenge at 3 weeks of age. Two birds in the HVT ND-BD*LV group displayed clinical signs after the challenge at 3 weeks, but none of the birds in the same vaccinated group showed any clinical sign after the challenge at 4 weeks of age. On the other hand, birds in the LV*LV group were healthy after the challenge at 3 week-old, but two birds died when the same vaccinated group was challenged one week after 3.32. Humoral response before the challenge ‘The control group and the rHVT ND-IBD group had negative HI titets (Fig. 4). The groups that received at least one inoculation of the LV vaccine (LV+LV, rHVT ND-IBD/LV, and sHVT ND-IBD+LV) hhad mean Hi titers above 3.2 logs. In the LV*LV, rHIVT ND-IBD/ LV, and rHiVT ND-IBD+LV groups, 40%, 50% and 35% of tested birds, respectively, had titers above 3 loge by the HI test. Al vaccinated birds, except one bird in the LVLV group that, died, were positive with high titers (Fig. 4) by ELISA before the challenge. The mean of ELISA titers varied from 6739 to 13,462 among the vaccinated groups, The LV*LV group had significantiy lower ELISA titers than the FHIVT ND-IBD/LV and HVT ND-IBD groups (P < 0.05 = 0.0008 and 0.0126, respectively). but not than the tHVT ND-IBD+LV group (P > 005 « 0.52). {s for the correlation of HVT detection in feathers and ELISA titers (Supplemental Fig. 2), the Pearson correlation had even lower values when all ELISA titers were higher at 4 week-old. Pearson values were 0.06335 for birds vaccinated only with HVT ND- IBD vaccine and 0.07396 winen testing all rHVT ND-IBD groups. ELISA titers from vaccinated birds before challenge at 4 weeks of age were higher compared to the vaccinated birds before chal- lenge at 3 week-old. After one week, the titers were up to 125% and 106% higher by the ELISA test in the rHVT ND-IBD+LV and THVT ND-IBD groups, respectively (Supplemental Fig. 3B). How- ever, only significant differences (P < 0.05) occurred when compar- ing rHVT ND-IBD groups, No significant difference was observed in the HVT ND-IBDsLV group, probably due to the high standard deviation. 3.3.3, NDV shedding Comparable to the birds challenged at 3 weeks of age. virus, shedding was detected in birds from all groups, mainly by the ‘orophayngeal route after challenge at 4 weeks of age (Fig. 5). TheFeria eta eine 8 (2020) 5907-5515 2ore sore ore a, re Tt. 20)6 ao te soo ge Zz . ol : g i a ei: ace ie Ez? 1. E Bd ow [f ar" i 2 2 7s ‘7 DPC 3 ” a a 3 gota i 3 . i Bowe @ contro! A HVT ND-IBD/LV VV orHVTNDABD+LV — @ = FHVT ND-IBD Fig. 5. Shedding at 2.4. and 7 DPC in oral and cloacal ruts in binds challenged with CANS virus at 3 weekold. No common eters difer significantly (# < 005). NS: no virus shedding through the cloacal route was limited in all vacel- nated groups at alltime points, All birds in the control group shed significantly (P< 0.05) higher virus titers (at least 1.1 logioE1Dolml higher) than vaccinated bieds at 2 and 4 DPC by the oral route. The birds in the LV+LV group shed less virus than birds in the rHVT ND- IBD*LV or sHIVT ND-IBD groups, but more than the birds in the THVT ND-IBD/LY group at 2 and 4 DPC by the oral route only. At these same time points and route, birds in the HVT ND-IBD/LV ‘group shed significantly less virus with titers around 4 logig E1Dsp/mi than the other birds vaccinated with the FHVT ND-IBD vaccine (FHVT ND-BD+LV or rHVT ND-IBD) (1. logigElDso/ml higher. At 7 DPC, 35% to 65% of the vaccinated birds shed low amounts of virus (up to 3.1 logioE1Dsq/ml) by the oropharyngeal route with no significant differences. As for the shedding through the cloacal route, birds in the LV*LV group shed significantly higher titers, when comparing the HVT ND-IBD/LY and rHVT ND-IBD groups, but only at 4DPC. ‘Overall, birds in the LV*LV group shed slightly higher virus titers when challenged one week later, primarily through the eloa- ‘al route. As for the other vaccinated groups, no significant difer~ ‘ence was detected, except for birds in the rHVT ND-IBD*LV group that shed significantly less virus by the cloacal route at 7 DPC at 4 week-old than at 3 week-old challenge. 3.34. Humoral response after the challenge ‘All surviving vaccinated birds had high HL titers at 14 DPC when challenged at 4 weeks of age (Fig. 4 (1). The birds in the LV¥LV ‘group had significantly (P < 005) higher titers than birds vaccl- nated with rHVT ND-IBD vaccine alone or in any combination with LV. The mean ofthe Hi titers was 10.4 log, in the LV+LV group and below 9.4 log, for the other vaccinated groups. No difference in the Hitters was found among the same vaccinated groups after chal- lenge with the 3 or 4 week-old birds. Similar to the challenged birds at 3-week-old, al birds in the vaccinated groups challenged at 4 weeks of age also had high titers by ELISA test (Fig. 4). The mean ELISA titers varied from 14,866 to 23,337 in the vaccinated groups. The LV+LV group had titers signif icantly higher (P< 0.0001) than the rHVT ND-IBD*LV and VT ND- IBD groups, but not compared to the HVT ND-IBDJLV group that also had ELISA titers above 21,000. The ELISA titers in the HVT ND-IBD/LY group were significantly higher (P < 0.05 = 0.003) than the titers in the HVT ND-IBDYLV vaccinated group at 14 DPC, “The ELISA titers in surviving birds after challenge at 4 weeks of age also increased when comparing the titers before challenge (Supplemental Fig. 3A); however, not as pronounced as observed in birds challenged at 3 week-old, The LVLV group also had the highest increase of ELISA titer (245%) when compared to the titer before challenge at 4 weeks of age. The rHVT ND-IBD/LV, HVT ND-IBDMLV, and riVT ND-IBD groups had an increase of ELISA titers around 55% to 70%. Vaccinated birds challenged at 4 weeks of age had a smaller Increase of ELISA titers compared to the vaccinated birds chal- lenged at 3 weeks of age after the challenge. The ELISA titers post-challenge from birds in the group HVT ND-IBD+LY chal- Tenged at 4 weeks were significantly (P< 0.04) lower (18%) than birds challenged 3 week-old (Supplemental Fig. 3). 4. Discussion Virulent Newcastle disease virus continues to be a problem for the poultry industry despite the widespread availability of vaccines for over 50 years. NDV remains as a single Serotype, as antibodies against NDV isolates can provide protection against other NDV1 Feria e aoc 38 (2020) 5507-5515 seta A Hi test B ELISA 20000 = Z 3 5 ood = 3 10000 ¢ c Ht test D A 2 = | 20000 a4 z & iz 200004 re i = B 10000 4 5 ss Le = Gros @ controt ME WHY A rHVTNDABD/LV J rHVTNDABDHVY = @ = FHVT ND-IBD Fig. 4 Serology in bins challenge at 4 weck-old, Humoral response (Aan 8) before and (C and D) after challenge at 4-eekold measured by Hl and ELISA tests. No oimon letters dif significantly (®< 005}. Nino survivors isolates [5,7]. However, NDV, in part because itis a RNA virus, has considerable genetic diversity. Molecular epidemiology studies have identifed 2 different classes of NDV, and in class Tl, at least 20 unique genotypes have been identified with at least 10% diff fences between genotypes [35,36]. These genetic and antigenic di ferences, although still providing cross-protection between genotypes, have been proposed as one reason why vaccines have rot been more protective for the poultry industry and is supported by better clinical protection when using antigenically matched DV vaccines to the challenge virus 12,28,29,37]. The vaccine industry nas more recently developed a new class of vaccines using the herpesvirus of turkeys or avian fowlpax viruses to act as vital vectors that can express NDV antigens, from lentogenic viruses ‘belonging to genotypes | or I, that are protective from NDV chal- lenge [75]. The evaluation of vaccination strategies using, all avai able vaccines should provide the best prediction of protection ‘when evaluating a new virus lke the California 2018 NDV, a geno- type V NDV. Here, we compared different vaccination regimens using a live NDV vaccine, a recombinant HVT expressing F NDV and VP2 IBDY proteins combined or administered alone in chickens at day of age andjor at 11 days of age to control the NDV infection with the California NDV from the 2018-2019 outbreak. An R-PCR test specific for the detection of rHVT and a commercial EUSA test confirmed the replication of the HVT virus in the vaccinated birds before challenge. Although the tHVT virus ‘was not detected in all tested feather pulp samples, all vaccinated birds had detectable NDV antibody titers before challenge, con- firming the vaccine replication. Nevertheless, differences in the virus replication in feather samples could lead to. the non- devection of the virus in some birds. Variation in HVT detection ‘was also found 14 days after vaccination in another study [33]- Live and recombinant HVT vaccines expressing the fusion pro- tein of NDV usually provide good protection in SPF chickens ‘against clinical signs after NDV challenge and virus shedding has ‘been used as a more discriminating indicator of vaccine efficiency [20.21,28,37). In our study, all vaccination protocols were able to ‘not only prevent mortality, but also to reduce virus shedding com- pared to unvaccinated controls, and induce high antibody levels as measured by ELISA before the challenge at 3 weeks-old, Overall, the rHVT ND-IBD with the live vaccine at one-day-old seems to bbe better combination, due to the absence of clinical signs, higher antibody levels pre and post-challenge, and reduced virus shed- dling at any time point. Even so, the only significant difference ‘between the rHVT ND-IBD and the rHVT ND-IBD/LV was observed at the peak of virus shedding in groups challenged at 3 week-old by the oral route, Besides, the FHVT ND-IBD*LV group displayed clin- ical signs and a higher virus shedding when challenged at 3-weeks ‘of age. The boost with the live vaccine did not seem to offer addi- tional protection in bitds challenged at 3 weeks-old, probably because of the early challenge only 10 days after the boost vaccination, In the group challenged at 4 weeks of age, the LV+LV combina- tion had the lowest efficacy with two out of 20 birds dying, lower antibody levels at time of challenge, and slightly higher virus shed- ding. One of the two birds that succumbed to death in the LV+LV group did not have positive titers by ELISA and HI test and had high2oPc ec Vrortrfana af) ahr a) OP swabs Feria eta eine 8 (2020) 5907-5515 ppc ar 70°C ar a) 2oPc me or £ HAgege } Cloacal swabs ppc bias Bm oiww © control A rHVT NDIBD/LV © rivt ND-BD V tiv nsepstv Fig 5. Sheng at 2.4, and 7 DAC in oral and coacal routes in birds challenged with CAIS virus at 4 weekold. No common eters difer significantly (P< 005) NS: no virus shedding, suggesting no vaccine response. Although these differences were not significantly different, the boost at 11 days of age using the live vaccine seemed not t0 have a clear benefit ‘even when it was used in combination with the HVT ND-IBD vac ‘ine. The live vaccine used in this study, a B1-Iike vaccine, is not as immunogenic as the LaSota vaccine, but itis more attenuated and ‘causes less of a post-vaccination reaction in young, birds (39). A ‘more immunogenic vaccine might be a better candidate as a boos- ter vaccine to overcome the initial vaccine immunity and stimulate ‘a stronger boost response (13.40) Birds vaccinated with only the eH1VT ND-IBD vaccine had unde- tectable HI titers before the challenges at 3 or 4 weeks of age, which isin agreement with previous studies [18,20]. Birds in the HVT ND-IBD groups vaccinated with the LV vaccine at one or 11 days of age had low Hi titers before the challenge using the CCAS virus, the homologous antigen to the challenge. However, specific antibody levels as measured by the ELISA test were detected in almost all vaccinated birds. The antibody levels were significantly higher in all HVT ND-IBD groups at 4 week-old when ‘compared to levels one week before. The turkey herpesvirus is con- sidered to persistently infect chickens and is thought to provide an ‘ongoing antigen presentation that maintains or even increases the ‘antibody response ver time [4], A positive effect was observed using the combination ofthe HVT ND-IBD virus with the live vac- cine at day one, as birds in this group had the highest antibody levels before challenge and the lowest increase of antibody levels measured by ELISA after the challenge at 4 week-old, This ‘decreased antibody response after challenge is thought to be from, the decreased replication of the challenge virus, which provides less antigen to stimulate an immune response, because of the improved protection. The high antibody levels specific to NDV in this group were able to decrease the replication ofthe virus chal- lenge at 2 and 4 DPC in the oral route compared to the group that received only the rHVT ND-IBD vaccine. Indeed, the levels of anti- bodies are inversely correlated to the virus shedding [37]. An important caveat in this study is that specific pathogen free chick- ‘ens were used in this study. The presence of maternal antibody to NDV is known to negatively affect the immune response {18,23}, but it is unclear if NDV maternal antibody would also affect the response to the NDV part ofthe HVT NDV vaccination, ‘Our results indicate that the live NDV vaccine and the F NDV protein were able to reduce the virus shedding even after a chal- lenge using a heterologous NDV strain, such as California 2018 virus from genotype V. Moreover, the protection using a recombi- nant HVT expressing the NDV fusion protein was protective against the California 2018 challenge on its own, but some improvement was seen when combined with a live vaccine at day one against challenge using a high dose as early as 3-week post- ‘vaccination, This combination could induce higher antibody levels, which were able to reduce the load of the virus shedding, not only in the cloacal but also in the oral route. Declaration of Competing Interest ‘The authors declare that they have no known competing finan- ial interests or personal relationships that could have appeared to influence the work reported in this paper. ‘Acknowledgements ‘The authors gratefully acknowledge the technical assistance provided by Suzanne DeBlois, Timothy L Olivier, Ricky Zoller, as well as animal care assistance provided by Keith Crawford and Roger Brock. ‘The mention of trade names or commercial products in this publication is solely for the purpose of providing specific1 Feria e aoc 38 (2020) 5507-5515 sis information and does not imply recommendation or endorsement by the US. Department of Agriculture. The USDA is an equal oppor- ‘unity provider and emplayer. Funding ‘This work was supported by the US. Department of Agriculture, [ARS CRIS Project 6612-32000-072-00D and a trust agreement with Merck Sharp & Dohme Corp., a subsidiary of Merck & Co, Inc, Kenilworth, NJ, USA (#58-6040-9-007) Appendix A. 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