Enhancement of mucosal immune responses by intranasal co-delivery of Newcastle disease vaccine plus CpG oligonucleotide in SPF chickens in vivo

Immunostimulatory CpG oligodeoxynucleotides (ODN) have been tested as immunoadjuvants for various vaccines in mice and human. Findings from previous reports suggest that CpG ODN can be used to enhance magnitude and balance of an immune response while reducing undesirable side effects of commercial vaccine, when delivered by parenteral route. Recently, it has been showed that CpG ODN is a promising mucosal adjuvant in mice, but data on mucosal immune responses induced by CpG ODN in other animals, especially in chickens, are scarce. Herein, we evaluated intranasal (IN) delivery of CpG ODN with newcastle disease (ND) vaccine (NDV) to determine its potential as a mucosal adjuvant to a commercial vaccine. CpG ODN augmented systemic (IgG in serum, T cell proliferation) and mucosal (IgA in intestinal washings and feces) immune responses against antigen. CpG ODN stimulated effectively both systemic and mucosal immune responses when delivered intranasally. Results from this study indicate that stimulatory CpG ODN is a potential effective mucosal adjuvant for the NDV in SPF chickens and may be applicable to husbandry animals.     

CpG ODN对鸡新城疫LaSota活疫苗的免疫增强效应

将3种不同的未甲基化CpGODN分别与新城疫LaSota活疫苗混合后，经滴鼻和点眼免疫鸡，通过检测鸡血清中HI抗体、外周血T淋巴细胞增殖活性、诱导巨噬细胞分泌NO含量，以及刺激外周血淋巴细胞表达IFN-γ、IL-6与IL-1β mRNA量，分析各CpGODN对新城疫LaSota活疫苗免疫效果的影响。结果表明，经2次免疫后，含GTCGTT核心基序的CpG ODN1组，鸡血清平均HI抗体效价最高达8．2log2、淋巴细胞刺激指数达9．836、NO分泌量达35．833μmol／L，分别比疫苗单独免疫组高出2个滴度（P〈0．05）、4．4（P〈0．01）、27．6μmol／L（P〈0．01）；含GACGTT核心基序的CpG ODN2组增强作用不明显．与疫苗单独免疫组无差异；而CpGODN3的免疫刺激活性由于受其侧翼序列的影响，作用明显减弱甚至丧失。对细胞因子的影响，CpGODN3组IFN-γ mRNA表达量稍高于CpG ODN1组，而其余细胞因子均以CpG ODN1组表达水平最高（P〈0．05）。由此证明CpG ODN1能显著增强鸡对新城疫LaSota活疫苗的体液和细胞免疫反应，可以作为高效的免疫增强剂。     

CpG oligodeoxynucleotides augment the immune responses of piglets to swine Pasteurella multocida living vaccine in vivo

Oligodeoxynucleotides containing unmethylated CpG motifs (CpG ODN) prevent development of T-helper type 2 (Th2) immune response and reverse established allergic responses in mouse models. However, little work on immune responses in piglets has been conducted in vivo. In this report, the ability of a porcine-specific CpG ODN to act as an immunostimulant and enhance immune responses of piglets to swine Pasteurella multocida living vaccine (SPML vaccine) was determined. The titre of IgG and IgG1/IgG2 isotype to SPML vaccine in serum, the proliferation of lymphocytes, SPML-specific interferon-gamma (IFN-gamma) and IL-6, TNF-alpha, IL-4 production of PBMCs in vitro and IFN-gamma, IL-6, TNF-alpha, IL-4, IL-10 in piglets serum were examined to identify the immune responses of the piglets. Immune responses of the piglets vaccinated with SPML and CpG ODN were significantly stronger than responses of piglets vaccinated with SPML alone. All these data summarized that immunostimulatory CpG ODN could modulate the immune response towards a Th1-like response when co-administered to piglets during SPML vaccination, which suggested that the therapeutic uses envisioned for these ODNs (as vaccine adjuvants and immunoprotective agents) may be applicable to husbandry animals.     

不同纯化浓缩方法制备的新城疫疫苗对SPF鸡免疫效果的研究

为研究利用不同纯化浓缩方法制备的新城疫疫苗对SPF鸡免疫效果的影响,试验取经过纯化浓缩的新城疫抗原制备的疫苗、经离心未经浓缩的抗原制备成的疫苗和经浓缩未离心的抗原制备成的疫苗分别免疫30日龄SPF鸡,免疫后分别在第7d、14 d、21 d、28 d、35 d、45 d、70 d、90 d采集血清检测新城疫抗体水平。结果表明,经浓缩纯化后抗原制备的疫苗与其他两组抗原制备的疫苗相比,物理外观性状基本相同,浓缩纯化后抗原制备的疫苗其单位抗原含量更高、杂质更少,抗体水平更高,统计数据显示差异显著。因此研究得出,浓缩倍数(即抗原含量)越高,抗体水平越高,持续的时间也越长。     

Effect of lipopolysaccharide on intranasal administration of liposomal Newcastle disease virus vaccine to SPF chickens

In order to potentiate the low immunogenicity of the inactivated Newcastle disease virus immunized into chickens by mucosal route, liposomes as a drug delivery system and LPS (lipopolysaccharide) as an immuno-stimulator were evaluated. Here, we report a new nasal delivery system of inactivated Newcastle disease virus (NDV) vaccine. The intranasal vaccine was based on different lipids to form MLV (multi-lamellar vehicles) liposomes. The liposomes had combined carrier and adjuvant activities, which induced strong systemic (serum) and local (lung and nasal) humoral responses in SPF (specific-pathogen-free) chickens, and provided protective immunity. PC-Lip (phosphatidylcholine-liposome) elicited significant mucosal secretary immunoglobulin A (s-IgA) levels (p < 0.05) in tracheal lavage fluid and serum IgG levels (p < 0.05). In response to virulent viral challenge, birds treated with PBS (phosphate buffered saline) as control group died, whereas 80% of chickens which received PC-Lip, PC-Lip-LPS, PS-Lip (phosphatidylserine-liposome), and PS-Lip-LPS survived. HAI titers were 1:2560 in the PS-Lip-LPS group and 1:1280 in the PC-Lip, PC-Lip-LPS, and PS-Lip groups after two vaccinations. The results suggest that PC-Lip or PS-Lip might thus be suitable as a potential adjuvant for mucosal vaccination against NDV in chickens.     

Calcium phosphate coupled Newcastle disease vaccine elicits humoral and cell mediated immune responses in chickens

Calcium phosphate (CaP) particles were coupled with inactivated Newcastle disease virus (NDV) vaccine. The surface morphology of CaP particles coupled to NDV was found to be spherical, smooth and with a tendency to agglomerate. The mean (± SE) size of CaP particles was found 557.44 ± 18.62 nm. The mean percent encapsulation efficiency of CaP particles coupled to NDV assessed based on total protein content and haemagglutination (HA) activity in eluate was found to be 10.72 ± 0.89 and 12.50 ± 2.09, respectively. The humoral and cell mediated immune responses induced by CaP coupled NDV vaccine were assessed in comparison to a commercial live vaccine (RDV ‘F’). CaP coupled NDV vaccine elicited prolonged haemagglutination inhibition (HI) and enzyme linked immunosorbent assay (ELISA) titres in the serum even at fourth and fifth week post-vaccination (PV), unlike RDV ‘F’ inoculated chickens whose titres declined to insignificant levels by this time. CaP coupled NDV vaccine could stimulate HI antibodies in tracheal washings and tears from second and first week PV, respectively. IgA ELISA antibodies were also seen in tracheal washings of these birds from third week PV and in tears from second week PV. CaP coupled NDV vaccine elicited cell mediated immune responses (CMI) from two to four weeks PV. The stimulation indices obtained after stimulation with specific antigen was not significantly different between CaP coupled antigen and live NDV virus except on first week PV. However, CaP coupled antigen did not cause suppression of lympo proliferation as indicated by statistically similar responses to mitogen, concanavalin A between the two groups. Overall, CaP coupled NDV vaccine elicited stronger and prolonged immune responses in comparison to the commercial live vaccine. No increase in the serum calcium and phosphorous levels were seen in CaP coupled NDV vaccine inoculated chickens.     

Effect of difference doses of Newcastle disease vaccine immunization on growth performance,plasma variables and immune response of broilers

Background

As a member of the Paramyxoviridae group, Newcastle disease virus (NDV) is the key causative agent of Newcastle disease (ND) that attacks chickens, turkeys and other avian birds. Surviving birds showed lower feed utilization, growth performance or egg production, which results in severe economic losses. The purpose of this study was to determine the effect of different doses of NDV immunization on growth performance, plasma variables and immune response of broiler chickens.

Methods

A total of 480 one-day-old Arbor Acres broilers were randomly administrated with 0, 4, 6 or 8 doses of NDV at 12 d and 28 d, respectively. Each group consisted of ten replicates with 12 birds each. Growth performance and organ weight were recorded. Plasma concentration of glucose, total protein, cholesterol, triglycerides and nonesterified fatty acid was determined using commercial kits. The concentration of plasma corticosterone and insulin was measured using commercially available radio immune assay kits. Serum antibody titer and peripheral blood lymphocyte proliferation were also recorded.

Results

The results showed that NDV decreased body weight gain (BWG), and increased Feed:Gain ratio at 1–21 d at all doses (P < 0.05). Plasma insulin concentration was lower in all immunization groups after the first immunization at 12 d (P < 0.01). The rest of the plasma indexes were not affected by NDV immunization, including glucose, total protein, cholesterol, triglycerides, nonesterified fatty acid, heterophil/lymphocyte ratio, as well as the proliferation of peripheral blood lymphocyte (P > 0.05). Compared with the control group, NDV treatment elevated NDV antibody titer at 10 d after the first inoculation (P < 0.05), and at d 5, 9 and 13 after the second inoculation (P < 0.05). Repeated NDV inoculation had no deleterious impacts on body composition at 42 d, and nutrient accretion rates at 8–42 d (P > 0.05).

Conclusions

In conclusion, NDV challenge decreased BWG and feed efficiency in earlier stage of growth. However, NDV treatment at 6 doses down-regulated the Feed:Gain ratio by 6.36 % throughout the whole growing period. These data suggest that appropriate lower doses of NDV inoculation increase feed efficiency of broiler chickens.

Electronic supplementary material

The online version of this article (doi:10.1186/s40104-015-0019-y) contains supplementary material, which is available to authorized users.     

Reduced mucosal injury of SPF chickens by mast cell stabilization after infection with very virulent infectious bursal disease virus

Recent studies here have demonstrated that increased mast cell populations and tryptase activity contribute to lesion formation in regions of immune organs in special-pathogen-free chickens after infection with very virulent infectious bursal disease virus (vvIBDV). Mast cells and their mediators have been implicated in acute inflammatory injury after vvIBDV infection, but their precise role in this process remains elusive. In this study, the role of mast cells in the vvIBDV infection process was examined using ketotifen, a mast cell membrane stabilizer. On days 1, 2, and 3 postinfection, the bursa of Fabricius (BFs) were collected to quantify mast cells, tryptase and histamine contents by cytochemistry, immunohistochemistry and fluorospectrophotometry analyses, respectively. The results showed that the mast cell populations, tryptase expression, and histamine released increased significantly in the BFs (p < 0.01) of infected birds compared to controls, and acute inflammatory responses were observed in the former. In contrast, in infected chickens pretreated with ketotifen, mast cells, tryptase, and histamine were markedly decreased (p < 0.01) and probably as a result, the BFs remitted significantly. The overall results suggest that mast cells are positively involved in BF injury induced by vvIBDV infection. Inhibition of mast cell degranulation and concurrent mediator release may represent a novel strategy to modulate this process. This study, thus, advances the understanding of the acute inflammatory injury mechanisms triggered by vvIBDV infection and the contribution of mast cell activity in this process.     

切除结膜结合淋巴组织鸡对新城疫疫苗点眼免疫反应 Ⅱ.组织学与免疫组织化学研究

60只 1日龄健康雏鸡随机分为 A、B、C三组。A组于 1日龄手术切除下眼睑结膜。A、B两组于 12日龄时结扎鼻泪管 ,用新城疫克隆 30苗点眼。C组为对照组。 2 3日龄分别采取其 CAL T和哈德氏腺 ,比较观察其组织细胞。结果 ,1日龄手术切除下眼睑结膜后造成鸡 CAL T缺失 ,点眼免疫后 ,哈氏腺的免疫细胞也较正常免疫鸡少 ,而正常接种 ND克隆 30 (B组 )后 ,CAL T发生早 ,生长快 ,淋巴细胞数量多 ,故本实验证实 CAL T对哈氏腺免疫细胞的数量与成分有一定的调控作用。 CAL T缺失鸡是研究黏膜免疫系统比较理想的模型     

Protection of chickens from Newcastle disease with a recombinant baculovirus subunit vaccine expressing the fusion and hemagglutinin-neuraminidase proteins

Recombinant baculoviruses containing the fusion (F) and hemagglutinin-neuraminidase (HN) glycoprotein gene of the viscerotropic velogenic (vv) Newcastle disease virus (NDV) isolate, Kr-005/00, and a lentogenic La Sota strain of the NDV were constructed in an attempt to develop an effective subunit vaccine to the recent epizootic vvNDV. The level of protection was determined by evaluating the clinical signs, mortality, and virus shedding from the oropharynx and cloaca of chickens after a challenge with vvNDV Kr-005/00. The recombinant ND F (rND F) and recombinant HN (rND HN) glycoproteins derived from the velogenic strain provided good protection against the clinical signs and mortality, showing a 0.00 PI value and 100% protection after a booster immunization. On the other hand, the combined rND F + HN glycoprotein derived from the velogenic strain induced complete protection (0.00 PI value and 100% protection) and significantly reduced the amount of virus shedding even after a single immunization. The rND F and rND HN glycoproteins derived from the velogenic strain had a slightly, but not significantly, greater protective effect than the lentogenic strain. These results suggest that the combined rND F + HN glycoprotein derived from vvNDV can be an ideal subunit marker vaccine candidate in chickens in a future ND eradication program.     

Oral vaccination of chickens against Newcastle disease with I-2 vaccine coated on oiled rice

Antibody response produced by Newcastle disease virus (NDV, strain I-2) when given orally through oiled rice to chickens was determined. Serum samples were collected before and at a weekly interval for 28 days after vaccination and tested for haemagglutination inhibition (HI) antibody to NDV. The results showed 7 days after vaccination HI antibody titre log₂ was 3.8. Moreover, 14 and 28 days after vaccination HI antibody titre log₂ reached 6.5 and 8.0, respectively. All unvaccinated chickens were negative to NDV antibody throughout the study. Significant finding from the present study is that 7 days after vaccination chickens had produced protective antibody against NDV; this is in contrast to previous studies. Therefore, I-2 vaccine coated on the oiled rice is efficacious as it protects chickens from challenge with NDV. Wambura, P. N., 2008. Oral vaccination of chickens against Newcastle disease with I-2 vaccine coated on oiled rice. Tropical Animal Health and Production.     

Effect of Newcastle disease and infectious bronchitis live vaccines on the immune system and production parameters of experimentally infected broiler chickens with H9N2 avian influenza

H9N2 Avian influenza (AI) is an infectious disease which considered to have low pathogenic virulence, but in the case of coinfection with other pathogens it has the potential to become a major threat to the poultry industry. Infectious bronchitis (IB) and Newcastle diseases (ND) are other common problems to the poultry industry, which there are an extensive vaccination program against these viral pathogens. To investigate the effects of administration of infectious bronchitis and Newcastle disease live vaccines (IBLVs and NDLVs) in the presence of H9N2 AI infection on the immune system and some production parameters, 180 one-day-old broiler chicks were randomly allocated into six groups with different vaccination programs including H120 IBLV, 4/91 IBLV, B1 NDLV and LaSota NDLV. At the age of 20 days, all birds of the experimental groups except the negative control group, were inoculated intra-nasally (at dose of 10⁶ EID₅₀) with H9N2 AIV. After the inoculation, gross and microscopic lesions of the immune organs, serological changes and some production parameters were examined. The findings of this study showed that coinfection of H9N2 AI with NDLVs exacerbated the gross and microscopic injuries in the immune organs; especially the bursa of Fabricius. LaSota + AIV group had the most severe lesion in the bursa of Fabricius, spleen and thymus. Furthermore, the birds of LaSota + AIV group consumed the least amount of feed and water and their final body weight were significantly (P ≤ 0.05) lower in comparison with the other groups. Interestingly, in the context of this experiment both 4/91 and H120 IB live vaccines enhanced the HI antibody titers against H9N2 AIV, but the 4/91 showed the most significant (P ≤ 0.05) increase compared to the other experimental groups.     

不同禽流感病毒含量的新-流二联灭活疫苗免疫SPF鸡后抗体水平的监测

将未浓缩的新城疫抗原分别与未浓缩的、浓缩3倍、浓缩6倍的禽流感抗原混合,并制备成三组鸡新城疫、禽流感（H9N2 HP株）二联灭活疫苗（简称新-流二联灭活疫苗）,分别免疫21日龄SPF鸡,每羽0.3 mL,同时设置未免疫的空白对照组,免疫组与对照组均在免疫前及免疫后7、14、21、28、35 d进行采血,检测新城疫和禽流感抗体。结果发现,各免疫组在免后不同日龄的新城疫抗体基本一致,禽流感病毒抗原浓缩倍数越高（即禽流感病毒含量越高）的新-流二联灭活疫苗,免后14、21 d的抗体也越高;从免后21 d开始,各免疫组的禽流感抗体水平差异逐渐减小,免疫后禽流感抗体水平的高低可以反映该疫苗的免疫效果。试验结果表明,该疫苗可以通过浓缩提高抗原病毒含量的方法来提高免后早期抗体水平,取得良好的早期免疫效果。     

Improved immunogenicity of Newcastle disease virus inactivated vaccine following DNA vaccination using Newcastle disease virus hemagglutinin-neuraminidase and fusion protein genes

The present study describes the development of DNA vaccines using the hemagglutinin-neuraminidase (HN) and fusion (F) genes from AF2240 Newcastle disease virus strain, namely pIRES/HN, pIRES/F and pIRES-F/HN. Transient expression analysis of the constructs in Vero cells revealed the successful expression of gene inserts in vitro. Moreover, in vivo experiments showed that single vaccination with the constructed plasmid DNA (pDNA) followed by a boost with inactivated vaccine induced a significant difference in enzyme-linked immunosorbent assay antibody levels (p < 0.05) elicited by either pIRES/F, pIRES/F+ pIRES/HN or pIRES-F/HN at one week after the booster in specific pathogen free chickens when compared with the inactivated vaccine alone. Taken together, these results indicated that recombinant pDNA could be used to increase the efficacy of the inactivated vaccine immunization procedure.     

Vaccination of chickens using raw rice coated with novel trehalose nano-organogels containing Newcastle disease (strain I-2) vaccine

The formulation and evaluation of trehalose nano-organogels for storage and oral delivery of Newcastle disease (ND) strain I-2 vaccine to chickens were carried out in this study. Trehalose sugar was blended with vegetable oil to form nano-organogels where trehalose also acted as a stabilizer against thermal inactivation of I-2 ND virus. Results from infectivity titration assay indicated that the titre of 10^7.5 EID₅₀/0.1 mL was maintained after 12 weeks of storage of nano-organogel I-2 vaccine at ambient room temperature. Serology results showed that 33% chickens which were vaccinated with nano-organogel I-2 vaccine after 14 days had HI antibody titres of ≥ 3.0 log₂ with GMT of 2.3. Moreover, results showed 100% of chickens vaccinated with nano-organogel I-2 vaccine had the mean antibody titres of 3.4 and 3.7 log₂ at 21 and 28 days after vaccination, respectively. All vaccinated chickens (100%) survived the challenge of virulent ND virus whereas all unvaccinated chickens succumbed to challenge and died of signs consistent with ND. The findings from this study showed that the nano-organogel I-2 vaccine was stable at room temperature, safe and produced protective antibody response in vaccinated chickens. Moreover the nano-organogel I-2 vaccine was used for oral administration and hence is suitable for mass vaccination. However, optimization of the formulation of trehalose nano-organogel vaccine is required in order to achieve its application potentials.     

疫尅灵与新城疫活疫苗（LaSota株）诱导雏鸡黏膜免疫的对比试验

新城疫病毒是副黏病毒,对鸡的黏膜有特殊的亲嗜性,极易经呼吸道和消化道感染,利用弱毒疫苗的局部免疫作用封锁新城疫强毒的入侵门户,是防制新城疫的重要措施。本试验利用疫尅灵与新城疫活疫苗（LaSota株）分别免疫SPF鸡雏,于免疫后不同时间检测免疫鸡雏泪液中的IgA,发现疫尅灵免疫组均高于LaSota株免疫组和对照组,说明疫尅灵在新城疫免疫上具高的诱导黏膜免疫能力。     

Effect of transgenic expression of porcine interleukin-6 gene and CpG sequences on immune responses of newborn piglets inoculated with Pseudorabies attenuated vaccine

Porcine interleukin-6 gene and CpG sequences were used as immunoadjuvants to enhance the immune responses of newborn piglets to Pseudorabies attenuated vaccine (PAV). The titer of specific antibodies to PAV, the proliferation of lymphocytes and induced IL-2 activities were all examined to identify the immune response of the piglets. The results showed that the immune responses with CpG ODN and porcine interleukin-6 gene were significantly stronger than routine immunities. The data suggests that porcine IL-6 and CpG motifs could be employed as effective immunoadjuvants to raise the humoral and cellular responses of newborn piglets to Pseudorabies attenuated vaccine.     

黄芪、白术水煎剂对鸡新城疫疫苗免疫效果的影响

探讨鸡口服黄芪、白术水煎剂对鸡新城疫疫苗免疫效果的影响。给7日龄健康梅岭土鸡连续7 d饮用口服黄芪或白术水煎剂,12.5 g/kg.d,后分2次间隔14 d滴鼻接种鸡新城疫Ⅳ系弱毒苗(LaSota株)。分别在首免前、及免疫后1、2、3、4周采血,分离血清,用血凝抑制试验检测鸡新城疫抗体效价(HI)。结果显示,口服中药组的鸡血清HI效价高于未服中药对照组,黄芪水煎剂组于二免后1周,白术水煎剂组于二免后1、2周,HI效价显著高于未服中药对照组。结果表明,在疫苗免疫之前给鸡口服白术水煎剂或黄芪水煎剂可以显著增强免疫效果。     

Regulating effects of porcine interleukin-6 gene and CpG motifs on immune responses to porcine trivalent vaccines in mice

In order to develop novel immunoadjuvants to boost immune response of conventional vaccines, experiments were conducted to investigate the regulating effects of porcine interleukin-6 gene and CpG motifs as the molecular adjuvants on immune responses of mice that were co-inoculated with trivalent vaccines against Swine fever, the Pasteurellosis and Erysipelas suis. Synthetic oligodeoxynuleotides containing CpG motifs were ligated into pUC18, forming recombinant pUC18-CpG plasmid. Eukaryotic plasmid expressing porcine interleukin-6 (VPIL-6) were also constructed as molecular adjuvants in an attempt to enhance levels of immune responses of mice co-administered with the trivalent vaccines in this paper. The cellular and humoral immune responses of mice were systematically analysed, and the experimental results were observed that the number of white blood cells, monocytes, granuloytes and lymphocytes significantly increased, respectively, in the mice immunized with VPIL-6, compared with those of the control; the IgG content and titre of specific antibodies to the trivalent vaccine mounted remarkably in the sera from the VPIL-6 vaccinated mice; the proliferation of lymphocytes and induced IL-2 activities were significantly increased in the vaccinated groups. The above-mentioned immune responses of mice co-inoculated with pUC18-CpG plasmid were significantly stronger than those of co-inoculated with pUC18 plasmid, suggesting that the immunostimulatory effect of oligodeoxynuleotides CpG is closely connected with the number of CpG motifs. These results suggest that the porcine IL-6 gene and CpG motifs could be employed as effective immunoadjuvants to elevate immunity to conventional vaccines.     

Protective antibody response produced by the chickens vaccinated with green coloured thermostable Newcastle disease virus

The efficacy of green-coloured (GC) I-2 Newcastle disease vaccine was determined in the present study. I-2 vaccine was mixed with a green coloured dye and stored at 4°C for 6 months while assayed for the virus infectivity at a monthly interval. Chickens were vaccinated with the GC vaccine by eye drop. Serum samples were collected from all birds before and after vaccination at weekly interval for 4 weeks and tested for haemagglutination-inhibition (HI) antibody against Newcastle disease virus (NDV). These chickens were challenged with NDV virulent strain four weeks after vaccination. The results showed that there was no difference between the infectivity titres of GC and uncoloured vaccines. However, chickens vaccinated with GC vaccine produced higher HI antibody titres than chickens vaccinated with uncoloured vaccine. Results from the challenge trial showed that all vaccinated chickens survived whereas all unvaccinated chickens died. The findings from this study have shown that the GC vaccine is safe and produced protective antibodies against NDV in vaccinated chickens. Wambura, P. N., 2008. Protective antibody response produced by the chickens vaccinated with green coloured thermostable Newcastle disease virus. Tropical Animal Health and Production.