Effects of starvation on the pharmacokinetics and optimal dosages of florfenicol and associated serum biochemistry in Asian seabass (Lates calcarifer).

Starvation has influence on physiology and pharmacokinetic (PK) characteristics of many drugs in land animals. However, similar PK information in fish is lacking. The current study examined the effects of starvation on fish PK, taking florfenicol (FF) in Asian seabass as an example. FF was orally administered at a single dose of 10 mg/kg into 35-day starved fish reared at 25 and 30°C and the serum FF concentration was analyzed by HPLC-FLD. At 30°C, the absorption and elimination half-lives of the starved fish were increased by 30% (from 0.44 to 0.57 h) and 55% (from 7.2 to 11.18 h), respectively. The volume of distribution, clearance, and area under the curve were changed from 1.25 to 0.71 L/kg, 0.120 to 0.044 L/kg/h, and 88 to 228 h·µg/ml, respectively. Similar starvation-induced PK changes were also observed at 25°C. The serum biochemical parameters, mainly the alanine aminotransferase, aspartate aminotransferase, and glucose levels, were significantly reduced in the starvation group. Overall, FF absorption, distribution, and elimination rates were reduced by starvation, resulting in four to five times lower optimal dosage than the non-starved fish. Drug treatment in starved fish should be treated with caution as overdosing and/or tissue residues could perceivably occur.

Effects of temperature on the pharmacokinetics, optimal dosage, tissue residue, and withdrawal time of florfenicol in asian seabass (lates calcarifer).

Drug behavior in the bodies of fish is largely influenced by the water temperature. Antimicrobial drugs are needed for the control of bacterial outbreaks in farmed fish including Asian seabass (Lates calcarifer). However, little is known about the temperature effect on appropriate drug uses in this species. The purpose of this study was to investigate the differences in pharmacokinetics (PK), optimal dosages, tissue depletion, and withdrawal time (WDT) of florfenicol (FF) in Asian seabass reared at 25 and 30 °C. In the PK study, the fish were administered with a single oral dose of 10 mg/kg FF. The optimal dosing regimen was determined by the pharmacokinetic-pharmacodynamic (PK-PD) approach. In the tissue depletion and WDT study, FF was administered at the optimal dosages once daily for 5 days and the WDT was determined by linear regression analysis based on the sum of FF and its metabolite florfenicol amine (FFA) in the muscle/skin. When the temperature was increased from 25 to 30 °C, the elimination half-life of FF was significantly decreased from 11.0 to 7.2 h. While the other PK parameters were not changed significantly, the calculated optimal dosages for the target minimum inhibitory concentration (MIC) of 2 µg/mL were 10.9 and 22.0 mg/kg/day, respectively for 25 and 30 °C. The sum of FF + FFA is a preferable marker residue for WDT determination because differential FF metabolism was observed at different temperatures. The depletion half-life of the muscle/skin was shortened from 41.1 to 32.4 h by the 5 °C temperature increase. Despite different absolute amounts of FF given between the two temperature levels, the WDTs were very similar at 6-7 days. Thus, it appears that a single temperature-independent WDT can potentially be assigned when the drug was applied at the optimal dosage.

Antiviral Properties of Pennisetum purpureum Extract against Coronaviruses and Enteroviruses.

Many severe epidemics are caused by enteroviruses (EVs) and coronaviruses (CoVs), including feline coronavirus (FCoV) in cats, epidemic diarrhea disease virus (PEDV) in pigs, infectious bronchitis virus (IBV) in chickens, and EV71 in human. Vaccines and antiviral drugs are used to prevent and treat the infection of EVs and CoVs, but the effectiveness is affected due to rapidly changing RNA viruses. Many plant extracts have been proven to have antiviral properties despite the continuous mutations of viruses. Napier grass (Pennisetum purpureum) has high phenolic content and has been used as healthy food materials, livestock feed, biofuels, and more. This study tested the antiviral properties of P. purpureum extract against FCoV, PEDV, IBV, and EV71 by in vitro cytotoxicity assay, TCID50 virus infection assay, and chicken embryo infection assay. The findings showed that P. purpureum extract has the potential of being disinfectant to limit the spread of CoVs and EVs because the extract can inhibit the infection of EV71, FCoV, and PEDV in cells, and significantly reduce the severity of symptoms caused by IBV in chicken embryos.

Electromagnetic Force-Driven Needle-Free in Ovo Injection Device.

Needle-free injections are mainly used for administering human or mammalian vaccines or drugs. However, poultry vaccines, in ovo injections to embryos, subcutaneous injections to chickens, and intramuscular injections are administered using needle injections. This article presents a new needle-free in ovo injection device method that uses push-pull solenoids to eject liquid jets, mainly for embryonic eggs of chickens. Furthermore, our study investigated the suitable jet pressures for using this method and the post-injection hatching rates in 18-day-old embryonic eggs. Using this method, we could deliver the liquid to the allantoic and amniotic cavities or the muscle tissue through the egg membrane of the air chamber using a jet pressure of ~6-7 MPa or ~8 MPa. After injecting 0.25 mL of 0.9% saline into 18-day-old Lohmann breed layer embryonic eggs and specific pathogen-free (SPF) embryonic eggs at a jet pressure of ~7 MPa, we observed hatching rates of 98.3% and 85.7%, respectively. This study's electromagnetic needle-free in ovo injection device can apply vaccine or nutrient solution injection for embryo eggs and serve as a reference for future studies on needle-free in ovo injection automation systems, jet pressure control, and injection pretreatment processes.

Capsaicin exerts therapeutic effects by targeting tNOX-SIRT1 axis and augmenting ROS-dependent autophagy in melanoma cancer cells.

Although considered a sporadic type of skin cancer, malignant melanoma has regularly increased internationally and is a major cause of cancer-associated death worldwide. The treatment options for malignant melanoma are very limited. Accumulating data suggest that the natural compound, capsaicin, exhibits preferential anticancer properties to act as a nutraceutical agent. Here, we explored the underlying molecular events involved in the inhibitory effect of capsaicin on melanoma growth. The cellular thermal shift assay (CETSA), isothermal dose-response fingerprint curves (ITDRFCETSA), and CETSA-pulse proteolysis were utilized to confirm the direct binding of capsaicin with the tumor-associated NADH oxidase, tNOX (ENOX2) in melanoma cells. We also assessed the cellular impact of capsaicin-targeting of tNOX on A375 cells by flow cytometry and protein analysis. The essential role of tNOX in tumor- and melanoma-growth limiting abilities of capsaicin was evaluated in C57BL/6 mice. Our data show that capsaicin directly engaged with cellular tNOX to inhibit its enzymatic activity and enhance protein degradation capacity. The inhibition of tNOX by capsaicin was accompanied by the attenuation of SIRT1, a NAD+-dependent deacetylase. The suppression of tNOX and SIRT1 then enhanced ULK1 acetylation and induced ROS-dependent autophagy in melanoma cells. Capsaicin treatment of mice implanted with melanoma cancer cells suppressed tumor growth by down-regulating tNOX and SIRT1, which was also seen in an in vivo xenograft study with tNOX-depleted melanoma cells. Taken together, our findings suggest that tNOX expression is important for the growth of melanoma cancer cells both in vitro and in vivo, and that inhibition of the tNOX-SIRT1 axis contributes to inducting ROS-dependent autophagy in melanoma cells.

1-Nitropyrene Induced Reactive Oxygen Species-Mediated Apoptosis in Macrophages through AIF Nuclear Translocation and AMPK/Nrf-2/HO-1 Pathway Activation.

1-Nitropyrene (1-NP), one of the most abundant nitropolycyclic aromatic hydrocarbons (nitro-PAHs), is generated from the incomplete combustion of carbonaceous organic compounds. 1-NP is a specific marker of diesel exhaust and is an environmental pollutant and a probable carcinogen. Macrophages participate in immune defense against the invasive pathogens in heart, lung, and kidney infection diseases. However, no evidence has indicated that 1-NP induces apoptosis in macrophages. In the present study, 1-NP was found to induce concentration-dependent changes in various cellular functions of RAW264.7 macrophages including cell viability reduction; apoptosis generation; mitochondrial dysfunction; apoptosis-inducing factor (AIF) nuclear translocation; intracellular ROS generation; activation of the AMPK/Nrf-2/HO-1 pathway; changes in the expression of BCL-2 family proteins; and depletion of antioxidative enzymes (AOE), such as glutathione peroxidase (GPx), catalase (CAT), and superoxide dismutase (SOD) These results indicate that 1-NP induced apoptosis in macrophages through AIF nuclear translocation and ROS generation due to mitochondrial dysfunction and to the depletion of AOE from the activation of the AMPK/Nrf-2/HO-1 pathway.

The Impacts of Reassortant Avian Influenza H5N2 Virus NS1 Proteins on Viral Compatibility and Regulation of Immune Responses.

Avian influenza virus (AIV) can cause severe diseases in poultry worldwide. H6N1 AIV was the dominant enzootic subtype in 1985 in the chicken farms of Taiwan until the initial outbreak of a low pathogenic avian influenza (LPAI) H5N2 virus in 2003; thereafter, this and other LPAIs have been sporadically detected. In 2015, the outbreak of three novel H5Nx viruses of highly pathogenic avian influenza (HPAI) emerged and devastated Taiwanese chicken and waterfowl industries. The mechanism of variation in pathogenicity among these viruses is unclear; but, in light of the many biological functions of viral non-structural protein 1 (NS1), including interferon (IFN) antagonist and host range determinant, we hypothesized that NS genetic diversity contributes to AIV pathogenesis. To determine the impact of NS1 variants on viral infection dynamics, we established a reverse genetics system with the genetic backbone of the enzootic Taiwanese H6N1 for generation of reassortant AIVs carrying exogenous NS segments of three different Taiwanese H5N2 strains. We observed distinct cellular distributions of NS1 among the reassortant viruses. Moreover, exchange of the NS segment significantly influenced growth kinetics and induction of cytokines [IFN-α, IFN-ß, and tumor necrosis factor alpha (TNF-α)] in an NS1- and host-specific manner. The impact of NS1 variants on viral replication appears related to their synergic effects on viral RNA-dependent RNA polymerase activity and IFN response. With these approaches, we revealed that NS1 is a key factor responsible for the diverse characteristics of AIVs in Taiwan.

Preparation of Chicken Anemia Virus (CAV) Virus-Like Particles and Chicken Interleukin-12 for Vaccine Development Using a Baculovirus Expression System.

Chicken infectious anemia (CIA) is a poultry disease that causes huge economic losses in the poultry industry worldwide. Commercially available CIA vaccines are derived from wild-type chicken anemia viruses (CAVs) by serial passage in cells or chicken embryos. However, these vaccinal viruses are not completely attenuated; therefore, they can be transmitted vertically and horizontally, and may induce clinical symptoms in young birds. In this study, we sought to eliminate these issues by developing a subunit vaccine exploiting the CAV structural proteins, engineering recombinant baculovirus-infected Spodoptera frugiperda (Sf9) cells that contained both the viral protein 1 (VP1) and VP2 of CAV. Moreover, we produced single-chain chicken interleukin-12 (chIL-12) in the same system, to serve as an adjuvant. The recombinant VP1 was recognized by chicken anti-CAV polyclonal antibodies in Western blotting and immunofluorescence assays, and the bioactivity of the recombinant chIL-12 was confirmed by stimulating interferon-γ (IFN-γ) secretion in chicken splenocytes. Furthermore, the ability of the recombinant VP1 to generate self-assembling virus-like particles (VLPs) was confirmed by transmission electron microscopy. Specific pathogen-free (SPF) chickens inoculated with VLPs and co-administered the recombinant chIL-12 induced high CAV-specific antibodies and cell-mediated immunity. Taken together, the VLPs produced by the baculovirus expression system have the potential to be a safe and effective CIA vaccine. Finally, we demonstrated the utility of recombinant chIL-12 as an adjuvant for poultry vaccine development.

Immune response evoked by tumor-associated NADH oxidase (tNOX) confers potential inhibitory effect on lung carcinoma in a mouse model.

Tumor-associated NADH oxidase (tNOX, ENOX2), which belongs to a family of growth-related NADH oxidases, was originally identified as a plasma membrane protein of rat hepatoma and is inhibited or downregulated by several anti-cancer drugs. The objective of this study was to evaluate the anti-tumor effects of tNOX used as an immunogen against Lewis lung cancer. Human tNOX was expressed in Escherichia coli, purified by His-Tag affinity chromatography, and emulsified with the adjuvant, ISA 201 VG. Immunological analyses of the generated tNOX vaccine were performed in mice. The results of ELISA and ELISpot were significantly higher in tNOX vaccine group compared to the control group. In vivo, we examined the anti-tumor effects of mice that received the tNOX vaccine via the intraperitoneal or subcutaneous routes. Mice were vaccinated three times at 2-week intervals, challenged at 2 weeks after the final vaccination, and terminated at 34 days post-challenge. Antibody titers, tumor volume and histopathological scores were used to evaluate the anti-tumor effects of the tNOX vaccine. Our results revealed that tNOX-vaccinated mice had significantly higher antibody titers than negative control (NC) and challenge control (CC) mice. When compared to the corresponding CC groups, the intraperitoneal and subcutaneous vaccination with tNOX showed a significantly smaller tumor mass volume (P < 0.05) and a significantly lower histological lesion score (P < 0.05), respectively. Our results demonstrate that the use of a xenogeneic tNOX as an immunogen in mice activates immune responses and anti-tumor effects against Lewis lung cancer.

Prevalence and Genotyping of Chlamydia psittaci from Domestic Waterfowl, Companion Birds, and Wild Birds in Taiwan.

Chlamydia psittaci, the causative agent of avian chlamydiosis, an important zoonotic disease, infects a wide range of birds. Infected birds, whether symptomatic or asymptomatic, intermittently shed the agent through respiratory and intestinal routes. Therefore, it is essential to investigate the epizootiology of C. psittaci in poultry, pet birds, and wild birds. In this study, cloacal or fecal swabs collected from domestic waterfowl, psittacine birds, Columbidae, and wild birds were used to determine the prevalence of C. psittaci in Taiwan between 2014 and 2017. The C. psittaci infection rate was as high as 34.2% among domestic waterfowl farms. The waterfowl isolates clustered into two groups based on ompA phylogeny: one group (G1-like) clustered with the Polish G1 strains; the other group (waterfowl-TW) clustered near, but independently from, the classical ABE genotype cluster. Separately, 3.1% of parrot samples tested positive for C. psittaci belonging to genotype A. C. psittaci isolates of genotype B were detected in 10.1% of racing pigeons and other Columbidae. Wild bird samples from a wildlife refuge had a 2.2% prevalence rate; among these, two atypical C. psittaci genotypes were detected in samples from a Malayan night heron (Gorsachius melanolophus) and a Taiwan barbet (Megalaima nuchalis). Taken together, our results revealed that the risk of C. psittaci transmission from domestic waterfowl and Columbidae birds to humans could be underestimated, given the high prevalence rates in these birds. Furthermore, the free-range rearing system of waterfowl in Taiwan may promote C. psittaci transmission between poultry and wild birds. Pet birds and racing pigeons, which are in close contact with people, are also possible sources for cross-species transmission. Further studies are necessary to elucidate the virulence, biological and genetic characteristics, and modes of transmission of Taiwanese C. psittaci isolates to facilitate the prevention and control of C. psittaci infection.

Identification and characterization of infectious bursal disease virus subviral particles by capillary zone electrophoresis: potential application for vaccine production and quality control.

The infectious bursal disease (IBD) causes immunosuppression in chicken of all ages and high mortality in young chicken, posing serious threat to poultry industry worldwide. One promising strategy for preventing this highly contagious disease is using recombinant subunit vaccine, employing VP2 subviral particles (SVP) as epitomic antigen. Analytical techniques of viral-like particles such as SDS-PAGE, western blot, or high-performance size-exclusion chromatography have been widely applied, but mostly unsatisfactory. In the present study, a simple, fast and cost-effective capillary zone electrophoresis (CZE) method with UV-detection was developed to analyze purified IBDV-SVP (expressed by Escherichia coli system) using commercial monoclonal antibody (mAb) against VP2. To find satisfying CZE conditions, injection mode, separation voltage, and separation buffer were explored. Through the modified CZE, mAb and SVP could be well separated and shown distinct peaks in the electropherogram. Furthermore, to determine the stoichiometry, the area of the mAb peak versus SVP/mAb binding ratio was plotted and indicated that 2 or 3 receptor molecules were bound per SVP. The purity and integrity of SVP and the interactions between SVP and mAb could be analyzed by the developed simple CZE-UV method in less than half hour. This CZE-UV method proved to be a valuable and useful tool in detection, characterization, and quantification of IBDV-SVP and the mAb, offering potential applications of in-process quality control of vaccine production, surveillance of serum antibody produced against IBDV infection, or vaccine immunization.

Detection of undeclared animal by-products in commercial canine canned foods: Comparative analyses by ELISA and PCR-RFLP coupled with slab gel electrophoresis or capillary gel electrophoresis.

BACKGROUND: The potential presence of undeclared animal by-products in pet foods is not subject to routine examination. Previously published methods for species-based identification of animal by-products have not been used routinely owing to inconsistent results. The present study evaluated the utility of several approaches for accurate identification of animal by-products in 11 commercial brands of canine canned foods. RESULTS: Canine canned foods from several countries were analysed by ELISA, PCR-RFLP coupled with slab-gel electrophoresis (SGE) and capillary gel electrophoresis (CGE) to test for evidence of by-products derived from cattle, chicken, sheep or pig. While CGE-based analysis detected all (24) animal-derived by-products that were reported for the 11 test samples, SGE and ELISA detected only 22/24 (92%) and 14/24 (58%) of labelled by-products, respectively. In addition, undeclared animal by-products were found using all three analytical approaches with CGE detecting more positives (19) than SGE (17) or ELISA (5). CONCLUSION: Significant disparities were evident between the labelled contents and the detected content of animal by-products. CGE-based testing for PCR products appears to provide greater sensitivity and accuracy than either SGE or ELISA-based methods. As testing of commercial products becomes more reliable and mainstream, manufacturers will need to develop more thorough and accurate labelling protocols.

Loss of the Capsule Increases the Adherence Activity but Decreases the Virulence of Avibacterium paragallinarum.

Avibacterium paragallinarum is the causative agent of infectious coryza, an important respiratory disease of chickens. The capsule is an important virulence determinant of many pathogenic bacteria, but the function of the capsule in Av. paragallinarum is not well defined. In this study, acapsular mutants of Av. paragallinarum were constructed by inactivation of the hctA gene using the TargeTron gene knockout system. The acapsular mutants were found to have greater hemagglutination activity than did the wild-type strain. Further, acapsular mutants exhibited an increased ability to adhere to DF-1 cells and to form biofilms on abiotic surfaces. Virulence assays showed that acapsular mutants were less virulent than the wild-type strain. Taken together, these results indicated that loss of capsule increases hemagglutination and adhesion activities but decreases the virulence of Av. paragallinarum. These results could be valuable to further elucidate the function of the capsule and the mechanism of pathogenicity of Av. paragallinarum.

Eliciting specific humoral immunity from a plasmid DNA encoding infectious bursal disease virus polyprotein gene fused with avian influenza virus hemagglutinin gene.

DNA vaccine coding for infectious bursal disease virus (IBDV) polyprotein gene and that for avian influenza virus (AIV) hemagglutinin (HA) gene have been shown to induce immunity and provide protection against the respective disease. The present study was carried out to determine whether an IBDV polyprotein gene-based DNA fused with AIV HA gene could trigger immune response to both IBDV and AIV. After transfection, VP2 and HA were detected in the cytoplasm and at cell membrane, respectively, by immunofluorescent antibody double staining method, suggesting the fusion strategy did not affect the location of protein expression. VP4 cleavage between VP2 and HA was confirmed by Western blot, indicating the fusion strategy did not affect VP4 function in transfected cells. After vaccination in chickens, the DNA construct VP24-HA/pcDNA induced ELISA and virus neutralizing antibodies against VP2 and hemagglutination inhibition antibody against the HA subtype. The results indicated that a single plasmid construct carrying IBDV VP243 gene-based DNA fused with AIV HA gene can elicit specific antibody responses to both IBDV and AIV by DNA vaccination.

The haemagglutinin of Avibacterium paragallinarum is a trimeric autotransporter adhesin that confers haemagglutination, cell adherence and biofilm formation activities.

The haemagglutinin (HA) protein plays a key role in the immunogenicity and pathogenicity of Avibacterium paragallinarum. A 210-kDa protein (HMTp210) was previously reported to be the HA of Av. paragallinarum, but the biological function of HMTp210 is not well defined. In this study, mutant strains that lacked HMTp210 were constructed using the TargeTron(®) gene knockout system. Haemagglutination and haemagglutination-inhibition (HI) assays showed that the HMTp210-deficient mutants exhibited no HA activity and failed to elicit HI antibodies in immunized chickens. Additionally, HMTp210-deficient mutants exhibited reduced ability to adhere to HeLa cells and to form biofilms on abiotic surfaces. Virulence assays showed that HMTp210-deficient mutants are less virulent than their isogenic wild-type strains. HMTp210 bears significant similarity to proteins of the trimeric autotransporter adhesin (TAA) family, and recombinant HMTp210 expressed in E. coli formed a trimeric structure. Taken together, these results indicated that HMTp210 is a trimeric autotransporter adhesin that confers haemagglutination, cell adherence and biofilm formation activities. These results should prove valuable to further elucidate the biological function of HA and the mechanism of pathogenicity of Av. paragallinarum.

Tail loss compromises immunity in the many-lined skink, Eutropis multifasciata.

Tail autotomy incurs energetic costs, and thus, a trade-off in resource allocation may lead to compromised immunity in lizards. We tested the hypothesis that tailless lizards will favor constitutive innate immunity responses over an energetically costly inflammatory response. The influence of fasting and colorful ornamentation was also investigated. We experimentally induced tail autotomy in the lizard Eutropis multifasciata and found that inflammation was suppressed by tail loss, but not further affected by fasting; the suppressive effect of colorful ornamentation was manifested only in males, but not in females. Constitutive innate immunity was not affected by any of these factors. As expected, only costly inflammation was compromised, and a less expensive constitutive innate immunity might be favored as a competent first-line defense during energetically demanding periods. After considering conventional trade-offs among tail regeneration and reproduction, further extending these studies to incorporate disease risk and how this influences escape responses to predators and future reproduction would make worthwhile studies.

Capsaicin-mediated tNOX (ENOX2) up-regulation enhances cell proliferation and migration in vitro and in vivo.

Cancer chemoprevention is employed to block or reverse the progression of malignancies. To date, several thousands of agents have been found to possess chemopreventative activity, one of which is capsaicin, a component of chili peppers that exhibits antigrowth activity against various cancer cell lines. However, the role of capsaicin in tumorigenesis remains controversial because both cancer prevention and promotion have been proposed. Here, we made the unexpected discovery that treatment with low concentrations of capsaicin up-regulates tNOX (tumor-associated NADH oxidase) expression in HCT116 human colon carcinoma cells in association with enhanced cell proliferation and migration, as evidenced by down-regulation of epithelial markers and up-regulation of mesenchymal markers. Importantly, tNOX-knockdown in HCT116 cells by RNA interference reversed capsaicin-induced cell proliferation and migration in vitro and decreased tumor growth in vivo. Collectively, these findings provide a basis for explaining the tumor-promoting effect of capsaicin and might imply that caution should be taken when using capsaicin as a chemopreventive agent.

Infectious bursal disease DNA vaccination conferring protection by delayed appearance and rapid clearance of invading viruses.

The present study was undertaken to determine the kinetics of viral load and immune response in protection against infectious bursal disease virus (IBDV) by DNA vaccination. Chickens were DNA-vaccinated and challenged with IBDV one week after the third vaccination. Tissues were collected at 12 hours postinfection (HPI), 1 day postinfection (DPI), 3, 5, 7 and 10 DPI. The vaccinated chickens had less viral RNA, with delayed appearance and shorter duration in the bursa of Fabricius, spleen, and cecal tonsil than the challenged control chickens. Their ELISA and neutralizing antibody titers were decreased at 12 HPI and significantly lower (P < 0.05) than those in the challenged control chickens at later time points. Their spleen IFNγ expression was up-regulated compared to that in the DNA-vaccinated chickens without IBDV challenge. These results indicate that DNA vaccination confers protection against IBDV challenge by delayed appearance and rapid clearance of the invading viruses.

DNA-mediated vaccination conferring protection against infectious bursal disease in broiler chickens in the presence of maternal antibody.

The objective of the present study was to determine if a DNA vaccine carrying large segment gene of infectious bursal disease virus (IBDV) could confer protection against infectious bursal disease (IBD) in broiler chickens in the presence of maternal antibody. Broiler chickens with maternal antibody titers to IBDV were intramuscularly injected with a DNA plasmid coding for VP2, VP3, and VP4 genes of IBDV strain variant E (VE) (P/VP243/E) at 1-day, 1-week, and/or 2 weeks old. The dose of P/VP243/E used ranging from 400microg to 10mg. Broiler chickens at 3 weeks old were orally challenged with IBDV strain (VE) and observed for 10 days. Only broiler chickens vaccinated with 7.5 or 10mg of P/VP243/E 3 times had 90 or 100% protection against challenge by IBDV strain VE and protected broiler chickens had significantly higher (P<0.05) bursa weight/body weight (B/B) ratios, significantly lower (P<0.05) bursal lesion scores, and the absence of IBDV antigens in bursae determined by immunofluorescent antibody assay (IFA). Antibody titers to IBDV as determined by enzyme-linked immunosorbent assay (ELISA) or virus neutralization (VN) assay in chickens of each group in each trial were gradually decreased prior to challenge. There was no significant difference (P>0.05) in ELISA or VN titers to IBDV among all groups of broiler chickens or among the groups of broiler chickens vaccinated with various dose of P/VP243/E before challenge. Broiler chickens in the groups receiving 7.5 or 10mg of P/VP243/E had significantly lower (P<0.05) ELISA or VN titers to IBDV than those in the challenge control (CC) groups or the other groups vaccinated with various dose of P/VP243/E after challenge. Broiler chickens in the groups vaccinated with 10mg of P/VP243/E 3 times had significantly higher (P<0.05) stimulation indices for IBDV-stimulated lymphocyte proliferation response than those in the vector control (VC) or CC group at 14, 21, 24, or 31 days after first DNA vaccination. The results indicated that DNA vaccination with DNA encoding large segment gene of IBDV confers protection against challenge by IBDV in broiler chickens with maternal antibody to IBDV.

Priming with DNA vaccine and boosting with killed vaccine conferring protection of chickens against infectious bursal disease.

The present study, including seven trials, was conducted to determine if priming with DNA carrying a large segment gene of the IBDV and boosting with killed IBD vaccine could adequately confer protection of specific pathogen free (SPF) chickens against IBD. One-day-old chickens were intramuscularly injected with DNA plasmid coding for the large segment gene of the IBDV strain variant E (VE) (P/VP243/E) followed by an intramuscular injection of killed IBD vaccine containing both standard and variant IBDV at 1 or 2 weeks of age. Chickens were orally challenged with IBDV strain VE or standard challenge strain (STC) at 3 weeks of age. Chickens primed with 50, 100, 200, or 400mug of P/VP243/E at 1 day of age and boosted with 0.5ml of killed IBD vaccine at 1 or 2 weeks of age had 80-100% protection against challenge by IBDV strain VE or 71-100% protection against challenge by IBDV strain STC. Protected chickens had higher (P<0.05) B/B ratios and lower (P<0.05) bursal lesion scores than chickens in the challenge control (CC) groups and groups primed with saline or vector plasmid and boosted with killed IBD vaccine. No IBDV antigen was detected by immunofluorescent antibody assay (IFA) in bursae of chickens protected by the DNA vaccine prime and killed vaccine boost vaccination. Prior to challenge, chickens (21 days of age) in the groups primed with P/VP243/E and boosted with killed IBD vaccine had higher (P<0.05) ELISA and VN titers to IBDV and lymphoproliferation stimulation indices. These results indicate that a prime-boost approach by priming with DNA vaccine encoding the large segment gene of the IBDV and boosting with killed IBD vaccine can adequately protect SPF chickens against challenge by homologous or heterologous IBDV.