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.     

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.     

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.     

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.     

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.     

A preliminary study of the role of ducks in the transmission of Newcastle disease virus to in-contact rural free-range chickens

The role of ducks in the transmission of Newcastle disease virus (NDV) to free-range village chicken was investigated experimentally. Newcastle disease (ND) seronegative ducklings reared in a pen were infected oronasally with velogenic NDV of intracerebral pathogenicity index (ICPI) 1.8 isolated from outbreaks in village chickens in Uganda. A first group of 3-week-old ND seronegative chicks was mixed with the ducks and they were kept together for 7 days. Both ducks and chicks were observed for ND clinical signs and any mortality, and they were bled and their sera were tested for ND antibodies by haemagglutination inhibition (HI) test. The chicks were removed, euthanized and examined for any ND lesions, while the ducks were transferred to a fresh pen and a second group of chicks was introduced and observed and treated as above. The ducks and the chicks tested positive for ND antibodies 7 days post infection and contact, respectively, but showed no clinical signs, post-mortem lesions or mortality. The mean ND antibody titre of the second group of chicks was lower than for the first group. This study has shown that although ducks can be infected with velogenic NDV, they do not show clinical signs but are able to transmit NDV to in-contact chicks. Further investigations are needed of the lack of clinical signs in the in-contact chicks and how long the ducks remain infective.     

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.     

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.     

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.     

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.     

不同新城疫弱毒苗对新城疫病毒强毒的免疫保护试验

用NDV的LaSota株、VG/GA 株、VH 株、PHY LMV 42株及Clone 30株弱毒疫苗免疫SPF鸡后 ,通过对雏鸡免疫后的血清抗体监测表明 ,4种弱毒疫苗激发雏鸡的抗新城疫病毒抗体滴度在 7log2水平以下 ,且差异不显著。通过以上 4种新城疫弱毒疫苗对新城疫病毒东台强毒株的免疫保护试验表明 :至少单独使用弱毒疫苗 ,不能对东台地区流行的新城疫病毒强毒株提供有效保护 ,保护率在 60 %～ 74%     

新城疫病毒作为疫苗载体的研究进展

在综述新城疫病毒(NDV) 的分子生物学特征、致病本质、复制机理以及利用反向遗传操作技术获得NDV的感染性克隆的基础上,着重介绍了以NDV作载体,将报告基因、功能基因包括鸡传染性法氏囊病毒VP2基因及流感病毒HA基因等插入NDV基因组的不同位置构建重组新城疫病毒,并对外源基因进行成功表达的研究进展。同时对DNV作为新的疫苗载体表达外源基因的可行性及未来应用前景进行了初探。     

新城疫病毒感染绵羊诱导免疫应答反应的实验研究

为探索新城疫病毒(NDV)作为羊病毒病疫苗载体的可行性,验证NDV感染绵羊的可能性,本研究采用NDV Clone-30株分别通过滴鼻和气管灌注两种途径接种绵羊,接种后观察临床症状,检测接种动物排毒情况,通过血凝抑制试验检测接种绵羊特异性血凝抑制抗体,ELISA试验检测接种绵羊血清特异性IgG抗体,微量中和试验检测接种绵羊血清中和抗体.研究结果表明,NDV在绵羊体内是非致病性的,并且通过两种接种途径在绵羊体内均可以产生血凝抑制抗体、NDV特异性IgG抗体和中和抗体反应.本研究结果表明NDV有可能作为宿主限制性疫苗载体用于预防无特效疫苗的羊类疾病,且与气管灌注接种途径比,滴鼻接种途径相对安全.     

鸡体内鹅源新城疫病毒抗原的免疫组织化学检测

为研究鹅新城疫（ND）的发病机理,用3株鹅新城疫病毒强毒株分别人工感染25日龄雏鸡,以单克隆抗体介导的免疫组化（IHC）法检测攻毒鸡体内NDV抗原的分布及定位,研究了鹅新城疫病毒（NDV）对鸡的组织嗜性。结果显示。在试验鸡多种组织器官中均能检测到NDV抗原,病毒抗原主要定位于淋巴细胞、网状细胞、巨噬细胞、肝细胞及各种上皮细胞的胞浆内。结果表明,鹅NDV强毒株对鸡是一种泛嗜性病毒。     

雏鸡接种新城疫霍乱毒素B亚单位佐剂苗抗体效价的检测

选取1日龄商品蛋用雏母鸡100只,随机分为对照组(接种ND疫苗)和试验组(接种NDV4．CTB苗),每组50只,免疫后1周、2周采取两组的血清和泪液,检测ND的抗体效价(HI抗体滴度)。结果显示：接种NDV4-CTB的雏鸡,血清ND抗体效价持续升高,并且泪液的ND抗体效价升高的快而持续。表明接种NDV4-CTB的雏鸡体液免疫和局部(哈氏腺)免疫都得到增强,有利于抵抗ND病毒的感染。CT-B对雏鸡具有抗免疫抑制的作用。     

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。     

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.     

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.     

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。