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.     

Development of a rapid biological assay for determination of potency of Newcastle disease vaccine (strain I-2)

A rapid biological assay based on incubation time has been developed for determination of the potency of Newcastle disease virus strain I-2 vaccine. It is based on the observation that the interval between inoculation and the first detection of haemagglutinin (HA) depends on the titre of the vaccine inoculated. Chicken embryonated eggs were inoculated with different titres (10⁹, 10⁶ and 10³ EID₅₀/0.1 ml) of vaccine and incubated for 24 h. At hourly intervals, 5 eggs from each vaccine titre were tested for the presence of HA. The results showed that the HA activity was detected from 5, 11 and 15 h after inoculation with vaccine doses of 10⁹, 10⁶ and 10³ EID₅₀, respectively. On the basis of these results it is suggested that if there is no HA detected from 5 to 11 h after inoculation of eggs with the vaccine virus, the vaccine should not be used to vaccinate chickens as it might have an infectivity titre of less than 10⁶ EID₅₀/0.1 ml, which is equivalent to the recommended single chicken dose. It is concluded that measuring the time between inoculation of the vaccine virus and the onset of HA activity might provide an estimate of the titre of the vaccine within 24 h.     

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.     

Thermostability profile of Newcastle disease virus (strain I-2) following serial passages without heat selection

I–2 is an avirulent strain of Newcastle disease virus. During establishment of the I-2 strain master vaccine seed, a series of selection procedures was carried out at 56^°C in order to enhance heat resistance. This master seed is used to produce a working seed, which is then employed to produce the vaccine. These two passages are done without further heat selection; however, it is not known how rapidly and to what extent thermostable variants would be lost during further passage. The study was therefore conducted to determine the effect of passage on thermostability of strain I-2. The virus was serially passaged and at various passage levels samples were subjected to heat treatment at 56^°C for 120 min. The inactivation rates for infectivity and haemagglutinin (HA) titres were assayed by use of chicken embryonated eggs and HA test, respectively. Thermostability of HA and infectivity of I-2 virus were reduced after 10 and 5 passages, respectively, without heat selection at 56^°C. These results suggest that 5 more passages could be carried out between the working seed and vaccine levels without excessive loss of thermostability. This would result in increased vaccine production from a single batch of a working seed.     

Immunogenicity of a Locally Produced Newcastle Disease I-2 Thermostable Vaccine in Chickens in Uganda

A locally-produced Newcastle disease (ND) I-2 thermostable vaccine of embryo-infective dose (EID50) 10(8.5) per ml was administered to 100 laboratory chickens in four test groups, each of 25 birds. It was given by the eye-drop method, in drinking water, in drinking water freshly medicated with levamisole, or using millet grains as a vaccine carrier. A fifth control group consisting of 25 birds received the heat-sensitive La Sota vaccine (EID50 10(9) per ml) by the eye-drop method. The immunological responses were monitored by the enzyme-linked immunosorbent assay (ELISA) ND antibody technique using serum samples collected from 18 birds in each group at 3-week intervals for 3 months. The overall mean ND antibody log(10) titres and percentage positivities were 3.1, 88%; 2.9, 70%; 3.0, 83%; 3.2, 87% and 3.3, 87%, respectively. The use of water alone or medicated with levamisole for vaccine administration produced significantly lower ND antibody titres only in the first 3 weeks. The immunogenicity shown by the I-2 vaccine as a potential vaccine is discussed in relation to free-range poultry management conditions in Uganda.     

Development of a Cell Culture Method for Quantal Assay of Strain I-2 of Newcastle Disease Virus

Repeated titrations of strains of Newcastle disease virus (NDV) are more conveniently undertaken in cell cultures rather than in embryonated eggs. This is relatively easy with mesogenic and velogenic strains that are cytopathic to various cell lines, but is difficult with avirulent Australian isolates that are poorly cytopathic. Strain V4 for example has been shown to be pathogenic iin vitro only to of chicken embryo liver cells. Strain I-2 was reported to produce cytopathic effect (CPE) on chicken embryo kidney (CEK) cells. The present studies confirmed this observation and developed a quantal assay. CEK cells infected with strain I-2 developed CPE characterized by degeneration, rounding, granularity and vacuolation, and the formation of synctia. End points were readily established by microscopic examination of fixed and stained cells. In virus infectivity studies on strain I-2, where multiple titrations are required and where large numbers of samples are used, titration using CEK cell grown in microtitre plates is recommended. Such studies may not be feasible in embryonated eggs.     

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.     

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.     

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.     

Characterisation of genotype VII Newcastle disease virus (NDV) isolated from NDV vaccinated chickens,and the efficacy of LaSota and recombinant genotype VII vaccines against challenge with velogenic NDV

A Newcastle disease virus (NDV) isolate designated IBS002 was isolated from a commercial broiler farm in Malaysia. The virus was characterised as a virulent strain based on the multiple basic amino acid motif of the fusion (F) cleavage site ¹¹²RRRKGF¹¹⁷ and length of the C-terminus extension of the hemagglutinin-neuraminidase (HN) gene. Furthermore, IBS002 was classified as a velogenic NDV with mean death time (MDT) of 51.2 h and intracerebral pathogenicity index (ICPI) of 1.76. A genetic distance analysis based on the full-length F and HN genes showed that both velogenic viruses used in this study, genotype VII NDV isolate IBS002 and genotype VIII NDV isolate AF2240-I, had high genetic variations with genotype II LaSota vaccine. In this study, the protection efficacy of the recombinant genotype VII NDV inactivated vaccine was also evaluated when added to an existing commercial vaccination program against challenge with velogenic NDV IBS002 and NDV AF2240-I in commercial broilers. The results indicated that both LaSota and recombinant genotype VII vaccines offered full protection against challenge with AF2240-I. However, the LaSota vaccine only conferred partial protection against IBS002. In addition, significantly reduced viral shedding was observed in the recombinant genotype VII-vaccinated chickens compared to LaSota-vaccinated chickens.     

新城疫H120、C30、C30+油剂苗疫苗免疫大法肉鸡免疫效果

鸡新城疫(ND)是一种急性、高度接触性传染病,是危害我国养鸡业最严重的烈性传染病之一。用H120、C30疫苗、C30 油剂苗对广西大法鸡进行新城疫免疫,经过一定免疫期,采血,分离血清样品,用β-微量血凝抑制试验检测其抗体效价。其结果显示：用C30 油剂苗,在同一批次、同一段时间内新城疫抗体水平最高,其免疫效果最好。     

携带新城疫病毒F基因DNA疫苗的减毒鼠伤寒沙门氏菌的构建及其免疫原性

根据GenBank中发表的新城疫病毒(NDV)融合蛋白(F)基因序列,设计1对引物,通过RT-PCR扩增出鹅源NDV分离株JS5F基因(约1700bp),测序确认后,将其克隆入真核表达载体pVAX1,获得重组真核表达质粒pVAX1-F。pVAX1-F经脂质体转染COS-7细胞,间接免疫荧光试验检测出F基因在COS-7细胞中的表达产物。将pVAX1-F转化减毒鼠伤寒沙门氏菌SL7207,构建成功携带DNA疫苗的重组沙门氏菌SL7207(pVAX1-F)。重组菌以109CFU/只的剂量2次免疫BALB/c小鼠,免疫小鼠可以检测到特异性针对NDVF蛋白的血清抗体和小肠粘膜抗体应答,SL7207(pVAX1-F)免疫组抗体水平显著高于SL7207(pVAX1)组(P<0.05)。将SL7207(pVAX1-F)以109CFU/只剂量口服免疫1日龄雏鸡,免疫保护试验结果显示,SL7207(pVAX1-F)免疫组对鸡具有良好的保护率(77.27%),与空白对照组和SL7207(pVAX1)空载体组之间存在显著性差异(P<0.05)。结果表明,该运送DNA疫苗的减毒沙门氏菌系统在体内能成功释放所携带的质粒,并能刺激机体产生免疫应答,可对NDV强毒攻击提供良好的免疫保护作用,提示该疫苗候选株对新城疫的控制有重要应用前景。     

四君子汤和补中益气汤对雏鸡免疫新城疫疫苗的影响

阐明四君子汤和补中益气汤对雏鸡免疫新城疫疫苗的影响。试验选取体质量30~35 g的1日龄海兰白雏鸡150只,饲喂全价饲料,7 d后,将其平均随机分为对照组、四君子汤组和补中益气汤组。对各组雏鸡滴鼻、点眼免疫新城疫活疫苗,免疫2 d后,对照组雏鸡每天给予纯化水,四君子汤组雏鸡每天给予四君子汤,补中益气汤组雏鸡每天给予补中益气汤,按照0.4 mL/kg的剂量连续灌服给药3 d。分别于免疫后第0天、第7天、第14天、第21天和第28天,从各试验组中随机采集10只试验鸡的血液,分离血清,用血凝抑制法测定新城疫抗体效价;翼静脉无菌采血,观察淋巴细胞转化情况,计算淋巴细胞转化率;利用碳廓清法,计算巨噬细胞吞噬率;采用全自动血球分析仪检测外周血白细胞的数量。试验结果显示,四君子汤组和补中益气汤组雏鸡的新城疫病毒抗体效价、外周血淋巴细胞转化率、巨噬细胞吞噬率均较对照组显著升高(P<0.05),白细胞总数无明显变化(P>0.05)。试验表明四君子汤和补中益气汤对雏鸡免疫新城疫疫苗均具有免疫增效作用。     

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.     

Protective efficacy of commercial inactivated Newcastle disease virus vaccines in chickens against a recent Korean epizootic strain

Despite the intensive vaccination policy that has been put in place to control Newcastle disease virus (NDV), the recent emergence of NDV genotype VII strains in Korea has led to significant economic losses in the poultry industry. We assessed the ability of inactivated, oil-emulsion vaccines derived from La Sota or Ulster 2C NDV strains to protect chickens from challenge with Kr-005/00, which is a recently isolated Korean epizootic genotype VII strain. Six-week-old SPF chickens were vaccinated once and challenged three weeks later via the eye drop/intranasal route. All vaccinated birds were fully protected from disease, regardless of the vaccine strains used. All vaccinated and challenged groups showed significant sero-conversion 14 days after challenge. However, some vaccinated birds, despite being protected from disease, shed the challenge virus from their oro-pharynx and cloaca, albeit at significantly lower titers than the unvaccinated challenged control birds. The virological, serological, and epidemiological significance of our observations with regard to NDV disease eradication is discussed.     

鸡新城疫、传染性支气管炎、禽流感(H9亚型)三联灭活苗(La Sota株+M41株+Re-9株)应用效果研究

鸡新城疫、传染性支气管炎、禽流感(H9亚型)三联灭活苗(La Sota株+M41株+Re-9株)(三联苗(Re-9株))为国内首个采用基因重组H9亚型禽流感疫苗株研制的新支流三联灭活疫苗.为研究疫苗上市后的实际应用效果,使用7日龄AA肉鸡和260日龄产蛋期蛋鸡评价三联苗(Re-9株)有效性.结果显示:肉鸡免疫后14 d...     

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     

以乳酸杆菌为载体的口蹄疫病毒VP1基因DNA疫苗猪体免疫试验

给猪口服和肌肉注射以乳酸杆菌为载体的口蹄疫病毒VP1基因DNA疫苗（L．acido SFMD-1），以正向间接血凝试验（IHA）和MTT方法分别检测了免疫后FMDV VP1抗体的动态变化和特异性T细胞增殖反应情况，并与商用FMD油佐剂疫苗、裸质粒FMDV VP1基因DNA疫苗诱导的特异性免疫抗体水平进行了比较。结果显示，口服组猪在免疫后第21d抗体效价达到了1：2^7.7，而肌肉注射组猪为1：2^3.3。加强免疫后2周，口服免疫组抗体水平下降到1：2^5.3，到第3周快速上升到1：2^8；与此相对应，肌肉途径免疫组猪抗体效价缓慢地从1：2^5.3上升到1：2^6.7。口服途径和肌肉注射途径的刺激指数（SI）分别为1．93和2．00，2种免疫途径都可以诱导特异性T细胞增殖反应。证实，该疫苗能够在猪体诱发VP1特异性T细胞和B细胞反应，以乳酸杆菌为载体是DNA免疫和预防猪口蹄疫的一种极有前景的方法。     

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.