Partial Fusion (F) Gene Analysis of Newcastle Disease Virus Detected in Pakistan during 2021-2022

Newcastle disease (ND) virus is a leading threat to commercial and domestic poultry in Pakistan. The virus infects and constitutes irreversible impairment to the nervous system, damages the respiratory system, and marks severe gastrointestinal lesions leading to heavy mortality in short-living birds and substantial losses in layers and breeders. The continuous emergence and evolution of the virus made it inclined to evade the humoral response and indirectly the circumvention of artificial active immunization. Newcastle disease is caused by the orthoavula genus of the paramyxoviridae family and has shown high genetic diversity even in their genotypes while information regarding enzootic trends of the virus is scanty in Pakistan. A total of 40 tracheal samples of NDV were collected from different commercial broiler farms and 11 isolates of NDV were identified. In the current study, we determined the genetic diversity of the Newcastle disease virus based on the partial sequencing of the fusion protein gene available in the NCBI database. Genetic analysis showed that seven isolates belonged to class I genotype VII and four belonged to class II genotype II. Interestingly, two isolates had epidemiological connections with vaccine-like class II genotype II. Our findings, concerning the recent outbreaks of class I genotype VII and class II genotype II of NDV in vaccinated commercial flocks, suggest possible potential partial mutations in the fusion protein gene. Genetic diversity and formation of the new cleavage site in an important neutralizing protein of wild strain are linked with the potency of artificial active immunization and a major cause of vaccine failure.

MicroRNA transcriptome analysis of chicken embryo fibroblast cells infected with Newcastle disease virus variants

Variations in the pathogenicity of Newcastle disease virus(NDV),the agent causing Newcastle disease,are associated with variants of different virulence.A few studies have characterized the expression of microRNAs(miRNAs)in NDV-infected avian cells.Here,the expression of miRNAs in chicken embryo fibroblasts(CEFs)infected with Herts/33 and LaSota NDV strains(highly virulent and nonvirulent,respectively)was determined using RNA sequencing.miRNAs involved in NDV infection included 562 previously documented and 184 novel miRNAs.miRNA target genes involved transcription factors,cell apoptosis,ubiquitin-mediated proteolysis,and protein processing in the endoplasmic reticulum.Potential target genes associated with autophagy were verified by qRT-PCR.No studies have documented the miRNA profles of CEFs infected with NDVs variants.This study adds to our knowledge of the cellular miRNAs involved in NDV infection and the complex molecular mechanisms mediating virus-host interactions.The results of this study will aid the development of strategies against the chicken virus.

Development and Validation of an Indirect Whole-Virus ELISA Using a Predominant Genotype VI Velogenic NewCastle Disease Virus Isolated from Lebanese Poultry

Commercial ELISA kits are commonly used to assess the levels of chicken antibodies against NewCastle Disease Virus (NDV) and trace a field strain infection. Nevertheless, the specificity of these kits vis-à-vis endemic strains in Lebanon remains in question. This study developed an in-house indirect ELISA system to evaluate the level of chicken antibodies against a predominant velogenic NDV strain belonging to Genotype VI. A checkerboard analysis comprised a five-factorial multivariate experiment to optimize the protocol: coating antigen concentration, blocking buffer utilization, serum and conjugate dilution levels, and OD reading wavelength. The developed test was optimized and then validated through parallel testing of the sera of 20 broilers and 5 layers using standard serological assays. There was a strong correlation between the developed ELISA results and those obtained with the Hemagglutination Inhibition test (P < 0.01), and a commercial NDV ELISA kit (P < 0.05). The specificity, sensitivity, and reproducibility of the developed ELISA suggest that it can be used as the test of choice for the assessment of chicken antibody titers against locally circulating velogenic NDV strains, specifically those belonging to Genotype VI. It also offers better help in the serological detection of birds’ exposure to the said strains.

Studies on the Inactivated Oil Emulsion Binary Vaccine against Newcastle Disease and Fowl Cholera(Ⅲ)——Safety and Immune Efficacy Test of Vaccine on Duck and Goose

[Objective] The aim of this study is to provide theoretical basis for immunizing waterfowls(duck and goose)with the inactivated oil emulsion binary vaccine against Newcastle Disease(ND)and Fowl Cholera(FC).[Method]Bacterial liquid from solid culture media inoculated avian Pasteurella multocida(APM)type A and allantoic fluid from embryonic eggs infected with Newcastle Disease Virus(NDV)attenuated strain La Sota were mixed and inactivated by formalin to prepare 5 batches of inactivated oil emulsion binary vaccine,which were then used for the safety and immune efficacy test on duck and goose.[Result]Immunized ducks and geese didn't performed any adverse reactions in the safety test of the 5 batches of vaccine;the immune efficacy test showed that ND-HI antibody titers of ducks and geese were no less than 4 log2 three weeks after inoculation,and the protection rates against NDV and APM were 100% and 66.7%-83.3%,respectively.[Conclusion]The binary vaccine against ND and FC is safe and reliable for duck and goose,and can provide them with sufficient immunity protection against ND and FC.

Study on Inactivated Vaccine in Oil Emulsion Against Newcastle Disease and Fowl Cholera IV——Field Test of Vaccine

[ Objective] The aim of this study was to evaluate the clinical effect of the inactivated vaccine in oil emulsion against Newcastle disease and Fowl cholera, and provide conditions for combined prevention and control of Newcastle disease and Fowl cholera. [ Method] The mixture of avian pasteurella multocida （type A） virulent strain 1502 and Newcastle disease virus attenuated strain La Sota was prepared into five batches of the inactivated vaccine in oil emulsion to use in the field test for assessing its safety and effects on immune protection of chicken, duck and goose. [ Result] The field safety test showed that there was no adverse reaction in the vaccinated chickens, ducks and geese. The field test of immune effect for chickens suggested that the titers of hemagglutination inhibition antibody for Nescastle disease virus （ ND-HI ） in 7 - 14 day- old chickens and 60 -90 day-old young chickens were 2 -3 log2 higher than the control group after being vaccinated for 3 weeks, which could last for more than 4 months. The protection rate against avian pasteurella multocida was over 75.0% and its immune effect could last for 6 months. The field test of immune effect for duck and goose indicated that the titers of ND-HI antibody were all higher than 4.2 log2 in vaccinated ducks and geese while lower than 2 log2 in the control group after being vaccinated for 3 weeks. The protection rate against avian pasteurella multocida in vaccinated ducks and geese was higher than 75.0% and 62.5% respectively. [ Conclusion] The binary vaccine is safe for poultry and has good immune effects.

Effects of Three Kinds of Chinese Medicine Polysaccharides on the Immune Effect of Newcastle Disease Vaccine

240 14-day-old healthy and non-immune Roman chicken were randomly divided into 8 groups, including blank control group （BC group）, immune control group （IC group）, and immunity groups of each Chinese medicine. On the day of the first immunity, 3 d before the second immunity, the day of the second immunity and 3 d after the second immunity, high-dose concentration and low-dose concen- tration of Astragalus polysaccharide （ASP）, Epimedium polysaccharide （EPP） and Isatis roots polysaccharide （IRPS） were used for the immunity groups of each Chi- nese medicine using the gavage, and 0.5 ml for each chick, and the equivalent normal saline was used for the blank control group and vaccine control group. On the 7^th, 14^th, 21^st and 28^th day after the first immunity, 10 chicken of each group were randomly got and weighed, and the antibody titer and the changes of the pro- liferation of T lymphocyte were measured. The results showed that 3 kinds of Chi- nese medicine polysaccharides all can increase the weight of chicken, improve HI antibody titer of Newcastle disease, and promote the proliferation of peripheral T lymphocyte, in which the effect of IRPS at low dosage is the best.

Screening of Anti-Newcastle Disease Virus Ingredients of Phyllanthus urinaria

[ Objective ] The aim of the study was to screen the antiviral ingredients of Chinese herbal medicine Phyllanthus urinaria in order to devel- op new drugs for the treatment of viral diseases of poultry. [ Method ] The whole grass of phyllanthus was respectively decocted in 75% ethanol （E） and pure water （PF）, and then the active ingredients were separately extracted in petroleum ether （PE）, ethyl acetate （EA）, and n-butyl alcohol （BU）. The extracts were inoculated on chicken embryo fibroblast （CEF） to observe their inhibitory effect on cytopathic effect （CPE） of Newcastle disease virus （NDV）, inoculated into chicken embryos to observe the changes of the hemagglutination titer of NDV, and inoculated in chickens to determine the mortality and evaluate their effect of immune protection. [ Result] The E -PE and E -BU extracts, especially E -BU extract, inhibi- ted CPE of NDV on CEF and significantly inhibited the proliferation of NDV in chicken embryo （P〈0.05） ; the survival rate of E-PE and E-BU group was extremely significantly higher than that of ribavirin group and the normal saline control （ P 〈0.01 ）, with E - PE group more higher. [ Condusion] The water extract of phyllanthus is less effective; E -PE extract and E -BU extract should effectively inhibit the CPE and proliferation in chicken embryo of NDV and also have better immune protection against NDV.

Protective Efficacy of Newcastle Disease Vaccines against Prevalent Strains in Recent Years

[Objective] To investigate the protective efficacy of currently available Newcastle disease vaccines against currently prevalent strains and thus provide guidance for the application of vaccines and the prevention and control of Newcastle disease. E Method] The 6-week-old SPF chickens were respectively inoculated with five live attenuated Newcastle disease vaccines （A, B, C, D, and E） at the dose of 1-fold or 0.01-fold usage via intranasal, intraocular or oral route. After 14 d post immunization, the titers of HI antibody were detected. And all the chickens were chal- lenged by Newcastle disease virus （NDV） FEo standard strain or the isolated wild strains, NDV-2007-HB and NDV-2008-YB. The clinical symptoms of chickens were continuously observed, and the morbidity and mortality were determined. [ Resalt] After 14 d post immunization, antibodies were induced at a protective level in chickens immunized with the five vaccines. As shown by the animal experiment, the five vaccines at the dose of 1-fold or 0.01-fold usage protected all vaccinated chickens from the death caused by NDV strain, and more than 90% of vaccinated chick- ens from the death caused by NDV-2007-HB strain, while the vaccines, A, B, and C, at the dose of 0.01 -fold usage protected more than 90% of vaccinated chickens from the death caused by NDV-2008-YB strain. E Conclusion] Under laboratory conditions, the currently available Newcastle disease vaccines have better protective efficacy against the two currently prevalent NDV strains and prevent the occurrence of Newcastle disease.

Assessing the quality of studies in meta-analyses: Advantages and limitations of the Newcastle Ottawa Scale

One of the most important points in the meta-analyses is certainly represented by the assessment of the quality of the studies included in such research. The meta-analyses are considered the highest level of evidence in science. Also for this reason, the quality of the studies included should be accurately evaluated by standardized tools. The overall results of the metaanalysis depend indeed also on a rigorous evaluation of the studies quality. Among all the possible tools for this complex evaluation, the Newcastle Ottawa Scale(NOS) is one of the most used worldwide, above all for observational studies. In this review, we will discuss the strengths and limitation of the NOS, also on the basis of the branch of science in which it has been applied.

Application of Newcastle disease virus in the treatment of colorectal cancer

Colorectal cancer (CRC) is one of the main reasons of tumor-related deaths worldwide.At present,the main treatment is surgery,but the results are unsatisfactory,and the prognosis is poor.The majority of patients die due to liver or lung metastasis or recurrence.In recent years,great progress has been made in the field of tumor gene therapy,providing a new treatment for combating CRC.As oncolytic viruses selectively replicate almost exclusively in the cytoplasm of tumor cells and do not require integration into the host genome,they are safer,more effective and more attractive as oncolytic agents.Newcastle disease virus (NDV) is a natural RNA oncolytic virus.After NDV selectively infects tumor cells,the immune response induced by NDV’s envelope protein and intracellular factors can effectively kill the tumor without affecting normal cells.Reverse genetic techniques make NDV a vector for gene therapy.Arming the virus by inserting various exogenous genes or using NDV in combination with immunotherapy can also improve the anti-CRC capacity of NDV,and good results have been achieved in animal models and clinical treatment trials.This article reviews the molecular biological characteristics and oncolytic mechanism of NDV and discusses in vitro and in vivo experiments on NDV anti-CRC capacity and clinical treatment.In conclusion,NDV is an excellent candidate for cancer treatment,but more preclinical studies and clinical trials are needed to ensure its safety and efficacy.

Newcastle disease virus-based MERS-CoV candidate vaccine elicits high-level and lasting neutralizing antibodies in Bactrian camels

Middle East respiratory syndrome coronavirus （MERS-CoV）, a member of the Coronavifidae family, is the causative pathogen for MERS that is characterized by high fever, pneumonia, acute respiratory distress syndrome （ARDS）, as well as extrapul- monary manifestations. Currently, there are no approved treatment regimens or vaccines for MERS. Here~ we generated recombinant nonvirulent Newcastle disease virus （NDV） LaSota strain expressing MERS-CoV S protein （designated as rLa- MERS-S）, and evaluated its immunogenicity in mice and Bactrian camels. The results revealed that rLa-MERS-S showed similar growth properties to those of LaSota in embryonated chicken eggs, while animal immunization studies showed that rLa-MERS-S induced MERS-CoV neutralizing antibodies in mice and camels. Our findings suggest that recombinant rLa- MERS-S may be a potential MERS-CoV veterinary vaccine candidate for camels and other animals affected by MERS.

Activity of T Cells Stimulated by Hemagglutinin-neuraminidase of Newcastle Disease Virus in vivo

To investigate the stimulated activity of T cells and the anti-tumor properties of hemagglutinin-neuraminidase（HN） of Newcastle disease virus（NDV） strain Changchun（NDVcc）, the expression of HN gene in hepatoma cells（human HepG-2 and mouse H22 cells） infected with the recombinant adenovirus（Ad-HN） was identified by Western blot analysis and flow cytometry. Sialidase activity of NDVcc HN expressed by Ad-HN was assayed by the periodate-resorcinol method. The in vivo anti-tumor effects of NDVcc HN were evaluated in the H22 solid tumor model. Regional lymph nodes of the mouse model treated with Ad-HN were removed to harvest T lymphocytes and evaluating the specific cytotoxicity of cytotoxic T lymphocyte（CTL） and natural killer（NK） cells by an L-lactate dehydrogenase（LDH） assay, in the mean time, the secretion of cytokines was analyzed by enzyme linked immunosorbent assays（ELISA）. The results show that NDVcc HN was effectively expressed by Ad-HN in HepG-2 and H22 cells. The sialidase activity assay showed that Ad-HN significantly reduced sialic acid level of the hepatoma cells compared with the cells infected the empty adenovirus vector（Ad-mock）. When treated with Ad-HN, the growth of subcutaneous H22 primary tumors in C57BL/6 mice was suppressed, and the mean mice survival increased. In addition, the treatment of Ad-HN elicited strong NK and CTL responses, and high levels of Th1 cytokines, such as IL-2 and IFN-γ. In conclusion, NDVcc HN effectively elicits T cell-mediate anti-tumor cytotoxicity via sialidase activity and may be a novel strategy for cancer immunotherapy.

Complete Nucleotide Sequence of a Newly Avirulent Newcastle Disease Virus Hubei 92(HB92) Strain

A new avirulent, heat-resistance HB92 strain of newcastle Disease Virus (NDV) was acquired from Australia V4 strain. Its complete nucleotides sequence was first determined. The entire genome of NDV HB92 consists of 15 186 nucleotides (GenBank accession no. AY225110). It was demonstrated by sequence analysis that nucleotides homology of HB92 strain with virulent strain ZJ1 were 83.9%, and the homology compared with avirulent vaccine strain La Sota and B1 were 94.0% and 93.5%, respectively. These results might be contributive to the study of the molecular mechanism of evolution of the NDV strain HB92 and to develop the engineered recombinant vaccine. Key words newcastle disease virus - genomic sequence - sequence analysis CLC number S 852. 65 Foundation item: Supported by Hubei Natural Science Foundation (2002AB144)Biography: Pan Zhi-shu(1961-), male, Ph. D, Associate professor, research direction: molecular biology and pathogenesis of eucaryotic viruses.

Production and application of recombinant haemagglutinin neuraminidase of Newcastle disease virus

Objective:To discuss the possibility of expressing the haemagglutinin-neuraminidase（HN） protein in prokaryotic system such as Escherichia coli（E.coli） cells by cloning the full length HN gene.Methods:The full length HN gene of Newcastle Disease Virus（NDV） of size 1 734 bp was preciously isolated by RT-PCR.The sequence was assessed and submitted to Nucleic Acid Databank（NCBI） and the gene ID was EU215390.1 after cloning and sequencing.Now the assessed HN gene was subcloned into pET 32 a+ expression vector for production the HN protein in E.coli, BL21（DE3） PLYSS cells following standard protocols.The crude lysate protein from the induced positive clone was size assessed by sodium dodecyl sulfale-polyacrylamide gel electrophoresis （SDS-PAGE） and their haemagglutination（HA） property against chicken RBC was assessed by standard micro HA test.Results:The molecular size of the full HN gene of NDV as assessed by cloning and digesting the positive clone to release the insert was 1.7 kb.The expressed protein in both crude and pure form was assessed to be 63 kDa and 81 kDa,respectively.The HA activity of the crude protein of the positive clone was 1 in 40.Conclusions:This finding indicates that the fusion protein retains the biological activity of native protein in the crude form and therefore could be used as a diagnostic reagent for antibody detection and for routine assessment of immune status in commercial layer forms.

Antiviral Effects of Compound Traditional Chinese Medicine on Newcastle Disease Virus

[Objective] To investigate the mechanism of compound traditional Chinese medicine （TCM） on Newcastle disease virus （NDV） and to provide a scientific basis for the reasonable usage of antiviral drugs in clinic. [Method] The compound TCM was composed of Hedyotis diffusa, Lonicera japonica Thunb, Radix astragali and Glycyrrhiza uralensis. Different dilutions of fluid extract were prepared. Its antiviral effects on NDV were observed through three inoculation ways, first, inoculation with the medicine and NDV mixture which had been incubated at 37 ℃; second, incubating chicken embryo fibroblasts （CEF） with the medicine followed by inoculation with NDV; third, inoculation with N DV followed by incubating CEF with the medicine. The A,= was determined by M]-r [ 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide） ~ method. Therapeutic indexes were used to evaluate the antiviral effects. [ Result] The minimum effective concentration of the compound TCM which acted through the three ways was 1.0 × 2^-10 1.0 × 2^-8 and 1.0 × 2^-7 g/ml, respectively. The antiviral effects of the compound TCM were the best through inoculation with the incubated medicine and NDV mixture, followed by the second method and the third method. [ Conclusion] The compound TCM can not only kill NDV directly in vitro but also inhibit viral propagation.

Characterization of Pigeon Paramyxoviruses (Newcastle disease virus) Isolated in Kazakhstan in 2005

Isolates of Newcastle disease virus (NDV) from deceased wild and domestic pigeons in Kazakhstan were obtained from the Almaty region during 2005 and were genotypically analyzed by using reverse transcription polymerase chain reaction (RT-PCR) with primers specific to the viral fusion (F) protein gene. Part of the amplified F protein DNA product (nucleotide sequence 47-422) and the deduced amino acid sequences were compared phylogenetically with those from strains previously reported in other geographic regions. Phylogenetic analysis indicated that the Kazakhstanian pigeon paramyxovirus type 1 (PPMV-1) isolates belong to genotype VI or 4bii. To our knowledge, this is the first reported VI isolates that possess the sequences of 112 GKRQKR116* F117 within the F0 protein. The information is fundamental to improving the efficiency of control strategies and vaccine development for NDV.

PLASMID VACCINE ENCODING HN GENE FROM NEWCASTLE DISEASE VIRUS HAS MARKED ANTITUMORAL EFFECT IN VITRO

Objective: To explore the antitumor effects of hemaagglutinin-neuraminase gene (HN gene) from Newcastle disease virus. Methods: Plasmid vaccine of pIRHN was constructed and transfected into HeLa cells. The expression of HN was analyzed by Western blot analysis, and the mode of cell death was detected by fluorescence microscope, gel electrophoresis and TUNEL assay and the expression of p53 and bcl-2 was also analyzed in transfected Hela cells. The effect of pIRHN on sialic acid contents in the Hela cell was examined. Results: pIRHN nucleic acid vaccines could be expressed in eukaryotic cell. pIRHN could induce apoptosis after HeLa cells were transfected. The effect of antitumor responses of pIRHN was correlated with the contents of sialic acid in tumor cells, and there was no prominent evidence for the relatedness of the antitumor effect with the expression of p53 and bcl-2. Conclusion: pIRHN may become a new antitumor biological agent.

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 Paromyxoviridoe 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 1-2 ] 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, NDVtreatment 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.

Comparisons of the Pathogenicity between Pigeon-derived and Chickenderived Genotype Ⅵ Newcastle Disease Virus(NDV) Strains in Pigeons

[ Objective]This study aimed to compare differences in the pathogenicity between genotype VI Newcastle disease virus （NDV） strains from pigeons and chickens in pigeons. [ Method] Two-month-old pigeons were artificially inoculated with ZJ3 strain from chickens and WX-10-07-Pi strain from pigeons. After inoc- ulation, the clinical symptoms, pathological anatomical changes, tracheal and cloacal detoxification, and histological lesions of experimental pigeons were observed. [ Result] Both ZJ3 strain and WX-10-07-Pi strain could infect pigeons with the incidence rate of 100%, but the mortality rate was 0. The cloacal detoxification time of pigeons in WX-10-07-Pi infection group was longer, and the virus detection rate was higher; in addition, the virus could be detected in various tissues and organs of inoculated pigeons. [ Conclusion] Different genotypes of NDV are pathogenic to pigeons, but the pathogenicity is related to the features of NDV strains. Genotype VIb NDV from pigeons can be carried and discharged for a long term in pigeons, which can spread in pigeon groups more easily.

Preparation and Examination of Inactivated Emulsion Vaccine against Newcastle Disease, Infectious Bronchitis and H9 Subtype Avian Influenza

[ Objective] To prepare inactivated emulsion vaccine against Newcastle disease, infectious bronchitis and H9 subtype avian influenza. [ Method] Antigen fluid of Newcastle disease virus （NDV） La Sota strain, infectious bronchitis virus （IBV） M41 strain and HgN2 subtype avian in- fluenza virus （AIV） WD strain was prepared by propagation in chicken embryos, respectively. The antigen fluid was concentrated with FILTRON Cassette ultra-filtration system and inactivated by formalin. The antigen fluid of NDV, IBV and AIV was mixed at a volume ratio of 1：1：1. Then the mixture was emulsified by Span-80 and Tween-80 and added medical white oil as adjuvant. The sterility and physical characteristics of the prepared ND-IB-AI combined vaccine were detected. [ Result] The three batches of ND-IB-AI combined vaccine were germ-free, milky white, with water-in- oil pattern and with viscosity of 6.3 -6.8 s. The water and oil were not separated after rest at 37 ~C for 21 d or centrifugation. [ Conclusion] The three batches of ND-IB-AI combined vaccine were germ-free and reached the standard for physical characteristics of vaccines.