Effect of Different Levels of Maternally Derived Genotype VII Newcastle Disease Virus-Specific Hemagglutination Inhibition Antibodies on Protection against Virulent Challenge in Chicks.

Newcastle disease (ND), caused by the virulent Newcastle disease virus (NDV), is an acute, highly contagious, and economically significant avian disease worldwide. Vaccination is the most effective measure for controlling ND. In recent years, vaccines matched with the prevalent strains of genotype VII have been developed and are now commercially available. These vaccines can provide full protection for chickens against clinical disease and mortality after challenges with genotype VII viruses and significantly decrease virus shedding compared to conventional vaccines belonging to genotypes I and II. Vaccinated hens can transfer antibodies to their offspring through the egg yolk. Maternally derived antibodies can provide passive protection against diseases but can also interfere with vaccination efficacy early in life. This study was conducted on chicks hatched from hens vaccinated with a commercial genotype VII NDV-matched vaccine to investigate the correlation between hemagglutination inhibition (HI) antibody levels in chicks and hens and the decaying pattern of maternally derived HI antibodies, and to evaluate the protective efficacy of different levels of maternally derived HI antibodies against challenge with a virulent NDV strain of genotype VII based on survivability and virus shedding. The HI antibody titers in chicks at hatching were about 1.3 log2 lower than those in hens, indicating an antibody transfer rate of approximately 41.52%. The estimated half-life of these antibodies was about 3.2 days. The protective efficacy of maternally derived HI antibodies was positively correlated with the titer. These antibodies could effectively protect chicks against mortality when the titer was 7 log2 or higher, but they were unable to prevent virus shedding or infection even at a high titer of 11 log2. The obtained results will greatly assist producers in determining the immune status of chicks and formulating appropriate vaccination schedules against ND.

System to screen and purify active ingredients from herbal medicines using hydrogel-modified human umbilical vein endothelial cell membrane chromatography coupled with semi-preparative high-performance liquid chromatography-offline-high-performance liquid chromatography-mass spectrometry.

Analytical screening and validation systems based on a combination of cell membrane chromatography and two-dimensional chromatography-tandem mass spectrometry are incapable of providing prepared samples containing the active ingredients found in traditional Chinese medicine; therefore, these samples cannot be directly used in subsequent studies. In this study, a semi-preparative cell membrane chromatography column was developed using a hydrogel-modified carrier and human umbilical vein endothelial cells to optimize prepared conditions, such as hydrogel polymerization, cell fragmentation, and cell membrane volume. This increased the binding ratio of membrane protein and carrier to 15.79 mg/g. The column was systematically evaluated using multitarget tyrosine kinase inhibitors that displayed good specificity and reproducibility. Subsequently, using the column coupled with a semi-preparative high-performance liquid chromatography-offline-high-performance liquid chromatography-mass spectrometry system, 15 active ingredients were screened and purified from Indigo naturalis, and five main components were identified: l-lysine, oxyresveratrol, tryptanthrin, isorhamnetin, and indirubin. Furthermore, the pharmacological effects of the ingredients were confirmed using cell proliferation and apoptosis assays. Results revealed potent proliferation-inhibiting and apoptosis-promoting abilities on human chronic myelogenous leukemic cells and human promyelocytic leukemic cells (p < 0.001). Overall, the system presented screening and purification functions that could be used to prepare I. naturalis samples acting on the epidermal growth factor receptor and vascular endothelial cell growth factor.

Efficacy of Newcastle disease LaSota vaccine-induced hemagglutination inhibition antibodies against challenges with heterologous virulent strains of genotypes VII and IX.

Newcastle disease (ND), caused by virulent Newcastle disease virus (NDV), still remains one of the most important avian diseases affecting the poultry industry worldwide, despite intensive vaccination programs have been implemented in many countries. All NDV isolates characterized to date are of one serotype and classified into classes I and II, with class II being further divided into twenty-one genotypes. Antigenic and genetic diversity is observed among the different genotypes. Current commercially available vaccines belonging to genotypes I and II are genetically divergent from strains that caused ND outbreaks worldwide in the last two decades. Reports of vaccination failures on their insufficient ability to inhibit infection or virus shedding have created renewed interest in developing vaccines homologous to virulent NDV circulating in the field. In this study, after vaccination with the most widely used LaSota vaccine (genotype II), chickens with different hemagglutination inhibition (HI) antibody levels were challenged with heterologous virulent NDV strains of genotypes VII and IX to evaluate how antibody levels relate to clinical protection and infection or virus shedding. Under the experimental condition, LaSota vaccine could fully protect birds from morbidity and mortality, but higher antibody levels were required to inhibit virus shedding. The number of birds shedding virus generally tended to decrease as the HI antibody titers increase in vaccinated birds. When the HI antibody titers reached ≥ 13 log2 and ≥ 10 log2, the virus shedding from JSC0804 strain (genotype VII) and F48E8 strain (genotype IX) could be completely inhibited, respectively, but it may be difficult to ensure that all individuals reach and maintain those levels in chicken flocks vaccinated according to routine procedure. Furthermore, the virus shedding in vaccinated birds was correlated with the amino acid similarity between the vaccine and challenge strains; more similarity, less virus shedding. The results obtained highlight that stringent biosecurity measures combined with vaccination are crucial for chicken farms to maintain a virulent NDV-free status.

Antileukemic effects of indigo naturalis constituents by "target constituent knock-out" coupled with semipreparative liquid chromatography and quadrupole time-of-flight mass spectrometry.

A novel approach is presented to identify constituents with antileukemic properties in extracts of Indigo naturalis (Qingdai in Chinese). Target compounds (A+ , BC+ , and ABC+ ) that knocked out specific constituents displayed antileukemic effects in a total extract of I. naturalis and negative constituents (A- , BC- , and ABC- ) that knocked out target compounds were separated, identified and knocked out by semipreparative liquid chromatography (semipreparative HPLC) and quadrupole time-of-flight mass spectrometer. Quantitative methods were used to evaluate the content of each knocked-out constituent in the total extract (D). Subsequently, interactions between the antileukemic effects of knocked-out constituents and D were screened and evaluated at the cellular level. Negative constituents including A- (65.47% ± 1.20%), BC- (54.61% ± 2.43%) and ABC- (67.49% ± 3.28%) displayed a greater inhibitory effect than D (47.16% ± 0.072%), which was not knocked out after 24 h of incubation, whereas the target compounds had not superior. Target compounds may have caused an antagonistic effect on the corresponding negative constituents. After 48 h, inhibition of proliferation by D (75.48% ± 3.78%) increased compared with that by negative constituents, whereas the antagonistic effect of target components on negative constituents was diminished. This result may reflect competitive antagonism. Comparing the reactions after 24 and 48 h, the inhibitory ratio of ABC- (79.29% ± 1.22%) in these knocked-out constituents and D was always the highest. With different concentrations tested after 48 h, ABC- significantly increased the rate of apoptosis on K562 cells (P < 0.01), indicating that in addition to indirubin, tryptanthrin and isorhamnetin, other antileukemic constituents may be present. Our study presents an approach that is a truer reflection of the antileukemic effects of knocked-out constituents in I. naturalis supported by reference to pharmacodynamic actions and the quality of I. naturalis. The approach may be useful for the analysis of other herbal extracts found in traditional Chinese medicine.

The endogenous development and pathogenicity of Eimeria anseris (Kotlan, 1932) in domestic goslings.

Twenty-one, 25-day-old, artificially reared, coccidia-free goslings (Anser cygnoides var. domestica) were inoculated orally with 0.5 × 104, 1 × 104, or 100 × 104 sporulated oocysts of Eimeria anseris and sacrificed at intervals from 24 to 216 h post-inoculation (HPI). Nine uninfected goslings served as negative controls. Parts of the visceral organs from goslings, including the intestines, kidneys, and liver, were fixed, sectioned, and observed microscopically. The results revealed that two generations of meronts occurred in the life cycle of E. anseris. The first generation of meronts developed at 24-96 HPI and the second generation at 90-128 HPI. Each meront contained 4-10 merozoites. Development of gamonts began at 128 HPI and mature oocysts appeared at 168 HPI. Developmental stages presented mainly in the epithelial cells of crypts and lamina propria in the posterior parts of the jejunum and ileum. Parasites localized mostly in the cytoplasm and occasionally in the nuclei of host cells. Histological lesions were pronounced in the jejunum and ileum. Desquamation and necrosis of the epithelium of intestine and crypts, infiltration of inflammatory cells, and hemorrhage and mucosal edema were associated with aggregates of endogenous stages. The infected goslings mainly showed severe diarrhea, depression, anorexia, and emaciation, suggesting that E. anseris is highly pathogenic in goslings.

Characterization and Sequencing of a Genotype VIId Newcastle Disease Virus Isolated from Laying Ducks in Jiangsu, China.

We report here the complete genome sequence and biological characterization of a virulent Newcastle disease virus (NDV) strain, NDV/duck/Jiangsu/JSD0812/2008, isolated from laying ducks in Jiangsu Province, China. The genome is 15,192 nucleotides in length and is classified in subgenotype VIId of genotype VII, class II.

Development of a loop-mediated isothermal amplification targeting a gene within the pyruvate dehydrogenase complex, the pdhA gene, for rapid detection of Mycoplasma gallisepticum.

Mycoplasma gallisepticum infections impose a significant economic burden on the poultry industry. In the current study, a loop-mediated isothermal amplification (LAMP) assay was developed and optimized to detect M. gallisepticum based on a gene within the pyruvate dehydrogenase complex, the pdhA gene, which codes for the major subunit (E1α) in the complex. The reaction conditions were optimized, and the specificity was confirmed by successful amplification of several M. gallisepticum strains, while no amplification was detected with 20 other major bacterial and viral pathogens of poultry. Additionally, the LAMP assay achieved 10-fold higher sensitivity than an existing polymerase chain reaction (PCR) method. The LAMP assay was applied to swab samples collected from poultry farms and compared with PCR. The positive detection rate was 20.2% (37/183) by LAMP and 13.1% (24/183) by PCR. The LAMP assay could provide a cost-effective, quick, and sensitive method for the detection of M. gallisepticum.

Experimental infection of duck origin virulent Newcastle disease virus strain in ducks.

BACKGROUND: Newcastle disease (ND) caused by virulent Newcastle disease virus (NDV) is an acute, highly contagious and fatal viral disease affecting most species of birds. Ducks are generally considered to be natural reservoirs or carriers of NDV while being resistant to NDV strains, even those most virulent for chickens; however, natural ND cases in ducks have been gradually increasing in recent years. In the present study, ducks of different breeds and ages were experimentally infected with duck origin virulent NDV strain duck/Jiangsu/JSD0812/2008 (JSD0812) by various routes to investigate the pathogenicity of NDV in ducks. RESULTS: Six breeds (mallard, Gaoyou, Shaoxing, Jinding, Shanma, and Pekin ducks) were infected intramuscularly (IM) with JSD0812 strain at the dose of 5 × 108 ELD50. Susceptibility to NDV infection among breeds varied, per morbidity and mortality. Mallard ducks were the most susceptible, and Pekin ducks the most resistant. Fifteen-, 30-, 45-, 60-, and 110-day-old Gaoyou ducks were infected with JSD0812 strain at the dose of 5 × 108 ELD50 either IM or intranasally (IN) and intraocularly (IO), and their disease development, viral shedding, and virus tissue distribution were determined. The susceptibility of ducks to NDV infection decreased with age. Most deaths occurred in 15- and 30-day-old ducklings infected IM. Ducks infected IN and IO sometimes exhibited clinical signs, but seldom died. Clinical signs were primarily neurologic. Infected ducks could excrete infectious virus from the pharynx and/or cloaca for a short period, which varied with bird age or inoculation route; the longest period was about 7 days. The rate of virus isolation in tissues from infected ducks was generally low, even in those from dead birds, and it appeared to be unrelated to bird age and infection route. CONCLUSIONS: The results confirmed that some of the naturally occurring NDV virulent strains can cause the disease in ducks, and that ducks play an important role in the epidemiology of ND. The prevention of NDV spread in ducks should receive more attention and research in terms of preventing the occurrence and prevalence of ND.

Complete genome sequence of a novel infectious bronchitis virus strain circulating in China with a distinct S gene.

An avian infectious bronchitis virus (IBV) was isolated and identified from a commercial layer flock vaccinated with live attenuated H120 vaccine in China, designed as ck/CH/IBTZ/2012. To determine the origination and evolution of this isolated strain, we have carried out a complete genome sequencing of this strain. The genome of the ck/CH/IBTZ/2012 strain is 27,691 nucleotides in length and includes more than 10 open reading frames. Sequence comparison and phylogenetic analysis based on the full-length genomic sequences showed that ck/CH/IBTZ/2012 is mostly related to the LX4-like strains. However, sequence analysis based on the spike protein (S) gene sequences revealed that ck/CH/IBTZ/2012 possesses a distinct S gene setting it apart from the Massachusetts-type strains and LX4-type strains. The cleavage site within the spike protein (S) of ck/CH/IBTZ/2012 is HRRKR, which is different from the majority of the IBVs in China for their cleavage sits are HRRRR. Recombination analysis showed that ck/CH/IBTZ/2012 is a chimeric virus with a LX4-like backbone except S gene which might be from an unknown strain. Based on the data presented in this paper, it can be concluded that genetic changes due to adaptive evolution and recombination both contributed to the origin of strain ck/CH/IBTZ/2012, which belongs to a new genotype.

Activation of the PKR/eIF2α signaling cascade inhibits replication of Newcastle disease virus.

BACKGROUND: Newcastle Disease virus (NDV) causes severe and economically significant disease in almost all birds. However, factors that affect NDV replication in host cells are poorly understood. NDV generates long double-stranded RNA (dsRNA) molecules during transcription of single-stranded genomic RNA. Protein kinase R (PKR) is activated by dsRNA. The aim of this study was to elucidate the role of PKR in NDV infection. RESULTS: NDV infection led to the activation of dsRNA-dependent PKR and phosphorylation of its substrate, translation initiation factor eIF2α, in a dose-dependent manner by either the lentogenic strain LaSota or a velogenic strain Herts/33. PKR activation coincided with the accumulation of dsRNA induced by NDV infection. PKR knockdown remarkably decreased eIF2α phosphorylation as well as IFN-ß mRNA levels, leading to the augmentation of extracellular virus titer. Furthermore, siRNA knockdown or phosphorylation of eIF2α or okadaic acid treatment significantly impaired NDV replication, indicating the critical role of the PKR/eIF2α signaling cascade in NDV infection. CONCLUSION: PKR is activated by dsRNA generated by NDV infection and inhibits NDV replication by eIF2α phosphorylation. This study provides insight into NDV-host interactions for the development of candidate antiviral strategies.

Infectivity and pathogenicity of Newcastle disease virus strains of different avian origin and different virulence for mallard ducklings.

Experimental infections of Newcastle disease virus (NDV) strains of different avian origin and different virulence in mallard (Anas platyrhynchos) ducklings were undertaken to evaluate infectivity and pathogenicity of NDV for ducks and the potential role of ducks in the epidemiology of Newcastle disease (ND). Ducklings were experimentally infected with seven NDV strains, and their clinical sign, weight gain, antibody response, virus shedding, and virus distribution in tissues were investigated. The duck origin virulent strain duck/Jiangsu/JSD0812/2008 (JSD0812) and the Chinese standard virulent strain F48E8 were highly pathogenic for ducklings. They caused high morbidity and mortality, and they distributed extensively in various tissues of infected ducklings. Other strains, including pigeon origin virulent strain pigeon/Jiangsu/JSP0204/2002 (JSP0204), chicken origin virulent strain chicken/Jiangsu/JSC0804/2008 (JSC0804), goose origin virulent goose/Jiangsu/JSG0210/2002 (JSG0210), and vaccine strains Mukteswar and LaSota had no pathogenicity to ducklings. They produced neither clinical signs of the disease nor adverse effect on growth of infected ducklings, and they persisted in duck bodies for only a short period. Virus shedding was detectable in all infected ducklings, but its period and route varied with the virulence of NDV strains. The results suggest that NDV with high pathogenicity in ducks may arise from the evolution within its corresponding host, further confirming that the ducks play an important role in the epidemiology of ND.

[Expression of green fluorescent protein using an infectious cDNA clone of infectious bronchitis virus].

An infectious cDNA clone of H120 vaccine strain of infectious bronchitis virus (IBV) was constructed to demonstrate its potential as a gene transfer vector. Primers were designed according to the published genome sequence of H120 strain, and ten cDNA fragments covering the entire genome of H120 strain was amplified by RT-PCR. All the PCR products were ligated into pMD19-T vector and sequenced, and the ORF5a open reading frame in the pMDTM9 plasmid was replaced by an enhanced green fluorescent protein (EGFP) gene. Recombinant plasmids were digested by the restriction enzyme Bsa I, and all the cDNA fragments were recovered. By using appropriate ligation strategy, the genomic cDNA of H120 strain were reconstituted. Then genome RNA was synthesized in vitro by T7 RNA polymerase and transfected into BHK-21 cells. Recombinant virus expressing the green fluorescent protein was rescued and identified by RT-PCR and sequencing. The characteristics of recombinant virus were evaluated by passage in embryonated chicken eggs. This study showed that the 5a ORF is a good candidate for an insertion site of recombinant genes for the development of IBV infectious cDNA clone as a gene transfer vector.

Chemiluminescent detect of E. coli O157:H7 using immunological method based on magnetic nanoparticles.

The system of chemiluminescent magnetic enzyme-linked immunoassay was developed. E. coli O157:H7 was sandwiched between rabbits anti-E. coli O157:H7 polyclonal antibody-coated magnetic nanoparticles (immunomagnetic nanoparticles or IMNPs) and mouse anti-E. coli O157:H7 monoclonal antibody. Commercial alkaline phosphatase conjugated horse anti-mouse immunoglobulin (ALP-Ab) was used to bind with the monoclonal antibody, finally the chemiluminescent signals were detected by adding 3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl-1,2-dioxetane (AMPPD) which was the substrate reagent of ALP. Different solvents of AMPPD were compared to get an optimal chemiluminescent signal. The effects of sodium borohydride and glycine on blocking the aldehyde groups of IMNPs were compared either, and the specificity and sensitivity of this system for detecting E. coli O157:H7 were researched. The results indicated that Tris buffer was the best solvent of AMPPD, sodium borohydride was better than glycine in blocking IMNPs, and this method was of good specificity when using E. coli Top 10F' and Vibrio cholera as negative controls. The detection limit was 10(3) cells mL(-1) when the antigen solution was 1 mL, and the procedure duration was about 3 h.

Protective efficacy of commercial Newcastle disease vaccines against challenge of goose origin virulent Newcastle disease virus in geese.

Since 1997, severe outbreaks of Newcastle disease (ND) in geese in many regions throughout China have resulted in high morbidity and mortality, and great economic loss to farmers; however, no licensed, specific vaccine is yet available for this disease in China. In this study, goslings were immunized with different combinations and dosages of several commercial ND vaccines including La Sota vaccine, Mukteswar vaccine, recombinant live vaccine against avian influenza (AI) and ND (rL-H5 strain), and inactivated ND oil-emulsion vaccine (La Sota strain). The protective effects were evaluated based upon the level of antibody response and the degree of protection against the goose-origin virulent NDV strain. The result showed that two doses (i.e., one more than that for chicken) of La Sota vaccine priming, followed by 2-5 doses of Mukteswar vaccine boosting 2-3 weeks later, not only induced higher HI antibody levels, but also conferred longer-lasting protection. This immunization procedure can be recommended for prevention of ND in geese.

The life cycle and pathogenicity of coccicium Eimeria nocens (Kotlán, 1933) in domestic goslings.

Twenty-four 10-day-old, artificially reared, coccidia-free goslings (Anser cygnoides var. domestica) were inoculated orally with 1.0x10(5)-1.0x10(6) sporulated oocysts of Eimeria nocens, and killed at intervals from 30 to 336 hr postinoculation (PI). Parts of the visceral organs, including intestines, kidney, liver, gallbladder, and spleen from inoculated goslings, were fixed and sectioned. The life cycle of E. nocens and histologic changes during infection were examined microscopically. The results showed that at least 3 generations of meronts developed in the endogenous stage of the life cycle of E. nocens. Two types of meronts were found. The first completed maturation at 54 to 78 hr PI. These meronts were the first generation, with each forming about 12 merozoites. The second completed maturation at 102 to 240 hr PI. These meronts were the second or third generations, with each meront forming about 24 merozoites. Development of gamonts began at about 198 hr after infection. The prepatent period was 9 days and discharge of oocysts continued for 4 days. Sporulation of oocysts occurred in 60-72 hr at 25 C. Eimeria nocens invaded the posterior jejunum, ileum, caecum, rectum, and cloaca. Developmental stages were localized within the epithelial cells of villi and crypts, and in lamina propria. Marked histological changes, including desquamation and necrosis of intestinal epithelium, submucosal edema, hemorrhages, infiltration of inflammatory cells, and villous atrophy, were seen during the periods of late merogony, gamogony, and oocyst shedding. They were most pronounced in the ileum and the regions nearby. The infected goslings showed severe diarrhea, bloody feces, anorexia, emaciation, and even death, suggesting that E. nocens is highly pathogenic for goslings.