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 400 μg to 10 mg. 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 10 mg 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 10 mg 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 10 mg 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.     

Adjuvant effects of interleukin-18 in DNA vaccination against infectious bursal disease virus in chickens

Interleukin-18 (IL-18) is an important cytokine with multiple functions in innate and acquired immunity. In this study, chicken IL-18 was evaluated for its adjuvant effects on the protective immunity of a DNA vaccine carrying the VP243 gene of IBDV. Groups of 14-day-old SPF chickens were given twice at 2-week intervals with 100 μg of the plasmid DNA vaccine pCAGVP243, pCAGVP243-IL-18 and the blank vector pCAGGS, respectively, and challenged with vvIBDV (HLJ0504 strain) 2 weeks later. Chickens immunized with plasmid pCAGVP243-IL-18 carrying both VP243 and IL-18 genes induced significantly higher levels of antibodies, lymphocyte proliferation responses and of the cytokines IL-4 and IFN-γ than those injected with pCAGVP243 encoding the VP243 gene alone. Furthermore, pCAGVP243-IL-18 provided higher protection (93%) against vvIBDV challenge in chickens than pCAGVP243 (60%), as evidenced by the absence of clinical signs, mortality, and bursal atrophy. These results indicated that the cytokine IL-18 could enhance the immune responses and protection efficacy of DNA vaccine against IBDV infection in chickens, highlighting the potential value of chicken IL-18 as an adjuvant in the prevention of vvIBDV infection.     

VP5-deficient mutant virus induced protection against challenge with very virulent infectious bursal disease virus of chickens

A mutant infectious bursal disease virus (IBDV) deficient in expressing VP5, rGx-F9VP2ΔVP5, was generated using reverse genetics technology. In comparison to the characteristics of rGx-F9VP2 virus in vitro, the mutant virus demonstrated lower viral titer and cytopathogenicity. To understand the role of VP5 in the pathogenicity of IBDV in vivo, animal experiments were conducted. rGx-F9VP2ΔVP5 caused reduced bursal lesion of SPF chickens compared to rGx-F9VP2. Although rGx-F9VP2ΔVP5 induced lower serum antibody than rGx-F9VP2 did, both inoculated groups were fully protected against vvIBDV challenge 4 weeks post-inoculation. In addition, immunosuppression induced by VP5-deficient virus was studied in 2-week-old SPF chickens immunized with AIV inactivated vaccine. And there was reduced immunosuppression as shown in our experimental results. The results showed that AIV hemagglutination inhibition (HI) antibodies of the rGx-F9VP2ΔVP5 inoculated group were similar to those of the mock-inoculated group, however, they were higher than those of the rGx-F9VP2 inoculated group, indicating that deficiency of VP5 decreased the immunosuppression of rGx-F9VP2ΔVP5 in chickens. All data indicated that VP5 played an important role in viral replication and pathogenesis both in vitro and in vivo. The VP5-deficient mutant virus could be a good candidate as a marked vaccine.     

Novel in-ovo chimeric recombinant Newcastle disease vaccine protects against both Newcastle disease and infectious bursal disease

Development of a safe and efficient in-ovo vaccine against Newcastle disease (NDV) and very virulent infectious bursal disease virus (vvIBDV) is of great importance. In this study, a chimeric NDV LaSota virus with the L gene of Clone-30 (rLaC30L) was used to generate a recombinant chimeric virus expressing the VP2 protein of vvIBDV (rLaC30L-VP2). The safety and efficacy of rLaC30L-VP2 in-ovo vaccination was then evaluated in 18-day-old special pathogen free (SPF) chicken embryos and commercial broiler embryos for prevention of NDV and vvIBDV. Hatchability and global survival rate of the hatched birds was not affected by in-ovo rLaC30L-VP2 vaccination. However, rLaC30L-VP2 in-ovo vaccination induced significant anti-IBDV and anti-NDV antibodies in SPF birds and commercial broilers, and 100% of vaccinated chickens were protected against a lethal NDV challenge. In-ovo rLaC30L-VP2 vaccination also provided resistance against vvIBDV challenge in a significant amount of animals. These results suggest that rLaC30L-VP2 is a safe and efficient bivalent live in-ovo vaccine against NDV and vvIBDV.     

An optimized,highly efficient,self-assembled,subvirus-like particle of infectious bursal disease virus (IBDV)

Infectious bursal disease virus (IBDV) causes immunosuppression in young chickens, leading to increased susceptibility to other diseases and a reduction in the immune response to other vaccines. Thus, IBDV results in great economic losses to the poultry industry. The most effective method of prevention is vaccination. However, medium-virulence vaccines can cause bursal pathological damage and immunosuppression. Here, we describe a safer, self-assembled, subvirus-like particle (sVP) vaccine without a complex purification process. The IBD-VP2 gene was cloned into Pichia pastoris, and the expressed protein self-assembled into T = 1 sVPs (∼23 nm). Immunization experiments showed that the sVP vaccine elicited high IBDV-neutralizing antibodies in each group, and all birds survived challenge with very virulent IBDV (vvIBDV). Additionally, IBDV RNA was not detected, and sterile immunity was achieved. In conclusion, the IBD-sVP is a suitable candidate for a recombinant subunit vaccine against IBDV.     

Protective efficacy of a DNA vaccine construct encoding the VP2 gene of infectious bursal disease and a truncated HSP70 of Mycobacterium tuberculosis in chickens

Infectious bursal disease (IBD) is an acute, infectious, immunosuppressive disease affecting young chicken worldwide. The etiological agent IBD virus (IBDV) is a double stranded RNA virus with outer capsid protein VP2 of IBDV is the major antigenic determinant capable of inducing neutralizing antibody. DNA vaccines encoding VP2 has been extensively studied achieving only partial protection. However, the efficacy of DNA vaccines against IBDV can be augmented by choosing a potential molecular adjuvant. The goal of the present study is to evaluate the immune response and protective efficacy of a DNA vaccine encoding the C-terminal domain of the heat shock protein 70 (cHSP70) of Mycobacterium tuberculosis gene genetically fused with the full length VP2 gene of IBDV (pCIVP2-cHSP70) in comparison to a ‘DNA prime-protein boost’ approach and a DNA vaccine encoding the VP2 gene (pCIVP2) alone. The results indicate that both pCIVP2-cHSP70 and ‘DNA prime-protein boost’ elicited humoral as well as cellular immune responses. Chickens in the pCIVP2-cHSP70 and ‘DNA prime-protein boost’ groups developed significantly higher levels of ELISA titer to IBDV antigen compared to the group immunized with pCIVP2 alone (p < 0.01). However, significantly higher levels of lymphocyte proliferative response, IL-12 and IFN-γ production were found in the pCIVP2-cHSP70 group compared to ‘DNA prime-protein boost’ group. Additionally, chickens immunized with pCIVP2-cHSP70 and ‘DNA prime-protein boost’ vaccines were completely protected against the vvIBDV whereas pCIVP2 DNA vaccine alone was able to protect only 70%. These findings suggest that the truncated C-terminal HSP70 mediated DNA vaccine genetically fused with the VP2 gene construct stimulated both humoral and cell mediated immune responses and conferred complete protection against IBDV. This novel strategy is perhaps a seminal concept in utilizing HSP70 as an adjuvant molecule to elicit an immune response against IBD affecting chickens.     

A reassortment vaccine candidate as the improved formulation to induce protection against very virulent infectious bursal disease virus

Infectious bursal disease (IBD) is a highly contagious immunosuppressive disease affecting all major poultry producing areas of the world. Infectious bursal disease virus (IBDV) is genetically prone to mutation so that vaccines have to be changed accordingly. However, the traditional method of vaccine development with blind passage could not fit the style of the emergency prevention of IBDV. In this study, for the first time, a segment-reassortment attenuated IBDV rXATB, consisting of modified segment A of a prevalent strain and segment B of an attenuated strain, was designed and rescued; rXATB was stable and could induce good humoral and cellular immune responses which resulted in excellent protection against the lethal challenge of vvIBDV without obvious immunosuppression in chicken. This study revolutionarily provides a new formulation based on reverse genetics to develop new vaccine against prevalent IBDV.     

Genetic,antigenic and pathogenic characterization of four infectious bursal disease virus isolates from China suggests continued evolution of very virulent viruses

Infectious bursal disease virus (IBDV) causes an economically significant disease of young chickens worldwide. The emergence of very virulent IBDV (vvIBDV) strains has brought more challenges for effective prevention and control of this disease. The aim of the present study was to characterize four IBDV isolates from various regions of China between late 1990s and recent years and to compare them with previously isolated European IBDV strains. In this study, one Chinese vvIBDV strain isolated in 1999 and three strains isolated between 2005 and 2011 were analyzed at the genetic, antigenic and pathogenic levels. Strain SH99 was closely related and clustered in the same genetic lineage as the typical vvIBDV based on the genomic sequences of segments A and B. However, the three more recent Chinese vvIBDV (HLJ0504, HeB10 and HuN11) showed several genetic changes in both segments and clustered in a distinct lineage from the typical vvIBDV and the previously known Chinese vvIBDV. Based on the binding to a panel of neutralizing monoclonal antibodies in antigen capture enzyme-linked immunosorbent assays, all Chinese vvIBDVs exhibited similar antigenicity with the European typical vvIBDV strains. Nonetheless, the pathogenicity caused by the recent Chinese vvIBDV was higher than that induced by the European typical vvIBDV. This study calls for a sustained surveillance of IBD situation in China in order to support a better prevention and control of the disease.     

Phylogenetic analysis of the polyprotein coding region of an infectious South African bursal disease virus (IBDV) strain

Infectious bursal disease virus (IBDV) causes Gumboro disease, which is highly contagious and immunosuppressive in young chickens. A virulent form of IBDV reached South Africa in 1989 and to date there has been little molecular information available for this strain. In this study, the polyprotein coding region of the South African strain SA-KZN95 was sequenced and analysed along with 52 representative sequences of other serotype I and II strains. We explored the relative impact of recombination on phylogenetic reconstruction using a multidimensional scaling approach. Phylogenetic analyses consistently placed the South African isolate within the very virulent IBDV clade. Selection analyses were also conducted to identify evolutionarily relevant amino acid residues. Previously, 19 residues in the polyprotein were shown to be potentially diagnostic for the different IBDV pathotypes. This study identified an additional two unique residues in the polyprotein which may be used as genetic signatures in future viral identifications. Better strain identification would aid in the development and application of vaccines.     

Plant-based vaccine candidate against Infectious bursal disease: An alternative to inactivated vaccines for breeder hens

Infectious bursal disease (IBD) is an acute, highly contagious immunosuppressive disease that affects young birds causing important economic losses in the poultry industry worldwide. Strict hygiene management together with effective vaccination programs are the most important strategies to prevent Infectious bursal disease virus entry in poultry production facilities. Hyperimmunisation of dams with inactivated vaccines just before the laying period provides passive immunity to the progeny that protects them during the critical first few weeks after hatching before vaccination with live attenuated virus takes place. In the present study, a safe and economic plant-based vaccine candidate against IBD intended for breeder hens was evaluated. We demonstrated that the recombinant immunogen is effective as booster for previously primed hens since it increases specific antibodies against VP2 that are transmitted to the offspring with titres and decay rate similar to those achieved by inactivated vaccine. Moreover, these maternally derived antibodies have virus neutralising activity and are able to confer protection against challenge in progeny, as evidenced by absence of bursal damage and low viral titres in this organ. Taking into account the disadvantages of inactivated vaccines as well as the benefits of plants as expression systems, such as time and cost efficiency, lower risk of contamination from animal pathogens and nearly unlimited scalability, a plant-based subunit IBD vaccine represents a viable alternative in the veterinary field.     

Recombinant infectious laryngotracheitis virus expressing Newcastle disease virus F protein protects chickens against infectious laryngotracheitis virus and Newcastle disease virus challenge

In this study, we isolated and identified an infectious laryngotracheitis virus (ILTV) that was naturally avirulent in specific pathogen-free (SPF) chickens, with the aim of developing a more efficacious vaccine against ILTV and Newcastle disease virus (NDV). We constructed a US9-deleted ILTV mutant based on this avirulent ILTV, and then constructed a recombinant ILTV (designated ILTV-ΔUS9-F) expressing the fusion protein (F) of the genotype VII NDV based on the US9-deleted ILTV mutant. Expression of the F protein in ILTV-ΔUS9-F-infected cells was confirmed by indirect immunofluorescence assay and western blotting. The inserted F gene was stably expressed in ILTV-ΔUS9-F. The growth kinetics of ILTV-ΔUS9-F were comparable to those of the wild-type ILTV strain. Vaccination of SPF chickens with ILTV-ΔUS9-F produced no clinical signs but did induce low levels of NDV-specific enzyme-linked immunosorbent assay and neutralizing antibodies. A single vaccination with 10⁴ plaque-forming units (PFU) of ILTV-ΔUS9-F provided good protection against both genotype VII and IX NDVs based on clinical signs, similar to the protection provided by the commercial live La Sota vaccine. Notably, ILTV-ΔUS9-F limited the replication and shedding of genotype VII NDV from oropharyngeal swabs more efficiently than the La Sota vaccine. In addition, vaccination with lower doses (10³ and 10² PFU) of ILTV-ΔUS9-F also provided sufficient clinical protection. These results indicated that ILTV-ΔUS9-F may be a bivalent vaccine candidate against both ILTV and NDV.     

Porcine lactoferrin administration enhances peripheral lymphocyte proliferation and assists infectious bursal disease vaccination in native chickens

The purpose of this study was to investigate the effects of dietary supplementation with recombinant porcine lactoferrin (rPLF) produced by yeast culture on peripheral lymphocyte proliferation and serum antibody titers in chickens vaccinated against the infectious bursal disease (IBD) virus. Treatment groups were fed with rPLF powder in their diet (2.0%, w/w), and the IBD vaccine was administrated at 1 and 3 weeks of age. At 8, 12, and 16 weeks after vaccination, serum IBD antibody titers were measured via the micro-method and T cell proliferation rates were evaluated. In gene expression analyses, rPLF-treated chicken peripheral T lymphocytes were stimulated with concanavalin A (ConA) for 24 h. The mRNA expression levels of interleukin-2 (IL-2), interferon-γ (IFN-γ), interleukin-4 (IL-4), and interleukin-12 (IL-12) were determined using a semi-quantitative RT-PCR assay. The results revealed that the rPLF additive led to significant increases in serum IgG and IBD-specific antibody titers (P < 0.05). The rPLF administration significantly increased chicken intestinal villous lengths and also enhanced the expression of IFN-γ and IL-12 in chicken T lymphocytes. These data suggest that rPLF enhances cell-mediated immunity and augment the ability of IBD vaccination to benefit chicken industry in disease resistance.     

Recombinant Newcastle disease virus expressing the infectious bronchitis virus S1 gene protects chickens against Newcastle disease virus and infectious bronchitis virus challenge

The recombinant LaSota strain expressing a chimeric IBV S1 gene (rLaSota-S1) was constructed with the S1 gene of the LX4 type IBV ck/CH/LDL/091022. The expression of the S1 protein was detected by an indirect immunofluorescence assay and Western blotting. The rLaSota-S1 strain was slightly attenuated, and its growth dynamics were similar to that of the parental LaSota strain. Vaccination of specific pathogen-free chickens with the rLaSota-S1 strain induced NDV hemagglutination inhibition antibodies, and it protected chickens from challenge with virulent NDV. In addition, vaccination with the rLaSota-S1 strain induced IBV-specific IgG antibodies and cellular immunity; however, a single vaccination provided partial protection with reduced virus shedding. Better protection efficiency was observed after a booster vaccination, which resulted in higher antibody titers, significantly fewer disease symptoms, and reduced virus replication and shedding. Our results suggest that the rLaSota-S1 strain is a bivalent vaccine candidate against both NDV and IBV.     

Development and large-scale use of recombinant VP2 vaccine for the prevention of infectious bursal disease of chickens

Infectious bursal disease virus (IBDV) is the causative agent of Gumboro disease, an infectious disease of global economic importance in poultry. One of the most effective types of inactivated IBDV vaccine is produced by infecting young chickens with a virulent strain, sacrificing them and extracting the virus from the bursa of Fabricius. The goal of this study was to produce an effective subunit vaccine against IBDV thereby providing an effective means of combating the disease. In areas in which the bursa-derived vaccine is in use, this subunit vaccine would eliminate the use of live birds for the production of inactivated vaccines. The gene for viral protein 2 (VP2) of IBDV was cloned into a Pichia pastoris expression system. This efficient system allowed us to meet the need for inexpensive vaccines required by the poultry industry. Following expression and scale-up, the protein was used to vaccinate chickens, against either Gumboro disease alone or in combination with inactivated Newcastle disease virus (NDV). Full protection was conferred against IBDV following vaccination with the subunit recombinant vaccine. No untoward influence on the response to the NDV vaccine was recorded. Over 250 million birds have already been vaccinated with this vaccine. The advantages of a subunit vaccine over an inactivated one are discussed. This approach will enable rapid adjustment to new virulent strains if and when they appear.     

Development of a tailored vaccine against challenge with very virulent infectious bursal disease virus of chickens using reverse genetics

Due to the problems associated with traditional methods for infectious bursal disease virus (IBDV) vaccine development and the pressure of evolution and variation of very virulent strains, it is urgent to develop IBDV vaccine rapidly with novel approaches. Using reverse genetics, the aim of this study was to generate a tailored vaccine strain (rGtHLJVP2) with its VP2 gene similar to very virulent IBDV (vvIBDV) to prevent the prevalence of IBDV. Characteristics of rGtHLJVP2 were evaluated in both cell culture and SPF chickens. rGtHLJVP2 replicated well as its parental strain Gt in vitro and in vivo. Immunization of SPF chickens with rGtHLJVP2 resulted in comparable antibody titers against IBDV as that of the medium virulent live vaccine B87, which was significant higher than that of attenuated vaccine Gt. Challenge studies with 10⁴ ELD₅₀ of prevalent homogeneous or heterogenous vvIBDV revealed complete (100%) protection in the groups immunized with rGtHLJVP2. No significant clinical and pathological lesions were observed in chickens immunized with rGtHLJVP2. Our data demonstrated that rGtHLJVP2 could be used as a novel vaccine candidate for prevention against vvIBDV.     

Modified live infectious bursal disease virus (IBDV) vaccine delays infection of neonatal broiler chickens with variant IBDV compared to turkey herpesvirus (HVT)-IBDV vectored vaccine

Chickens are commonly processed around 35–45 days of age in broiler chicken industry hence; diseases that occur at a young age are of paramount economic importance. Early age infection with infectious bursal disease virus (IBDV) results in long-lasting immunosuppression and profound economic losses. To our knowledge, this is the first study comparing the protection efficacy of modified live (MdLV) IBDV and herpesvirus turkey (HVT)-IBDV vaccines against early age variant IBDV (varIBDV) infection in chicks. Experiments were carried out in IBDV maternal antibody (MtAb) positive chicks (n = 330), divided into 6 groups (n = 50–60/group), namely Group 1 (saline), Group 2 (saline + varIBDV), Group 3 (HVT-IBDV), Group 4 (HVT-IBDV + varIBDV), Group 5 (MdLV) and Group 6 (MdLV + varIBDV). HVT-IBDV vaccination was given via the in ovo route to 18-day-old embryonated eggs. MdLV was administered via the subcutaneous route in day-old broilers. Group 2, Group 4 and Group 6 were orally challenged with varIBDV (SK-09, 3 × 10³ EID₅₀) at day 6 post-hatch. IBDV seroconversion, bursal weight to body weight ratio (BBW) and bursal histopathology were assessed at 19 and 35 days of age. Histopathological examination at day 19 revealed that varIBDV-SK09 challenge caused severe bursal atrophy and lower BBW in HVT-IBDV but not in MdLV vaccinated chicks. However by day 35, all challenged groups showed bursal atrophy and seroconversion. Interestingly, RT-qPCR analysis after varIBDV-SK09 challenge demonstrated an early (9 days of age) and significantly high viral load (∼5744 folds) in HVT-IBDV vaccinated group vs unvaccinated challenged group (∼2.25 folds). Furthermore, flow cytometry analysis revealed inhibition of cytotoxic CD8⁺ T-cell response (CD44-downregulation) and decreased splenic lymphocytes counts in chicks after HVT-IBDV vaccination. Overall, our data suggest that MdLV delays varIBDV pathogenesis, whereas, HVT-IBDV vaccine is potentially immunosuppressive, which may increase the risk of early age varIBDV infection in broilers.     

Protection of chickens,with or without maternal antibodies,against IBDV infection by a recombinant IBDV-VP2 protein

The use of avian herpesviruses (Marek's disease virus, MDV) as vectors to express the capsid protein of infectious bursal disease virus (IBDV) was well established, and its protection against IBDV challenge has been evaluated previously. However, there is little data about rMDV1 expressing the VP2 protein of IBDV protecting SPF and commercial chickens against virulent IBDV (vIBDV) challenge. In this study, we constructed a stable rMDV1 expressing the VP2 protein of IBDV by inserting the coding sequence within the US10 gene of MDVl by homologous recombination and designated this as rMDVl-US10L, and evaluated effectiveness of the recombinant VP2 protein with SPF chickens and commercial chickens with maternal antibodies in vIBDV challenge. The results can be summarized as follows: (1) We constructed a rMDV1 expressing IBDV-VP2 under the control of the MDV1 glycoprotein B (gB) promoter [rMDV1-US10L]. (2) rMDV-VP2 protein was readily expressed and induced 53% protection against a vIBDV challenge in SPF chickens with 10³ PFU/chicken, whereas 10⁴ PFU induced 73% protection. (3) Vaccination of commercial chickens having maternal antibodies to rMDV1-VP2 induced 87% protection in vIBDV challenge, which was similar to results using the live vaccine, BJ87 IBDV strain, in commercial chickens. These results demonstrate that the VP2 antigen expressed in the MDV vector was an effective and stable vaccine in correlation with the vaccine efficacy against lethal IBDV challenge, and can provide a better protective effect that is likely to persist for the life of the chickens.     

Infectious laryngotracheitis virus (ILTV) vaccine intake evaluation by detection of virus amplification in feather pulps of vaccinated chickens

Infectious laryngotracheitis (ILT) is a respiratory disease of poultry caused by an alphaherpesvirus, ILTV. The live vaccine is applied worldwide by drinking water or by the respiratory route, and by the vent application in Israel. No system of direct evaluation of the efficacy of vaccination exists today, except of antibody elicitation, which is an indirect indication of vaccination intake and might happen due to environment exposure. We suggest for the first time an assay for evaluating the accuracy of the vaccination process by spotting the spread of the live vaccine systemically, namely by virus detection in the feather shafts of the vaccinated birds. The feathers are particularly beneficial as they are easy to collect, non-lethal for the bird, therefore advantageous for monitoring purposes. Moreover, the continuous survey of the vaccine virus unveiled the different kinetics of viremia by the different vaccination routes; while after the vent vaccination the systemic viremia peaks during the first week afterwards, after two consecutive vaccine administration by drinking water with 6 day interval, the vireamia peaks only after the second administration. A robust amplification was needed because the vaccine ILTV was present in the bird in minute quantities compared to the wild-type virus. For the vaccine virus identification in feather shafts a nested real-time PCR for the TK ILTV gene was developed. The sensitivity of detection of the nested rtPCR was greater by 1000 compared to conventional nested PCR and 10 times that real-time PCR.     

Superinfection and recombination of infectious laryngotracheitis virus vaccines in the natural host

Infectious laryngotracheitis virus (ILTV, Gallid alphaherpesvirus 1) causes severe respiratory disease in chickens and has a major impact on the poultry industry worldwide. Live attenuated vaccines are widely available and are administered early in the life of commercial birds, often followed by one or more rounds of revaccination, generating conditions that can favour recombination between vaccines. Better understanding of the factors that contribute to the generation of recombinant ILTVs will inform the safer use of live attenuated herpesvirus vaccines. This study aimed to examine the parameters of infection that allow superinfection and may enable the generation of recombinant progeny in the natural host. In this study, 120 specific-pathogen free (SPF) chickens in 8 groups were inoculated with two genetically distinct live-attenuated ILTV vaccine strains with 1–4 days interval between the first and second vaccinations. After inoculation, viral genomes were detected in tracheal swabs in all groups, with lowest copies detected in swabs collected from the groups where the interval between inoculations was 4 days. Superinfection of the host was defined as the detection of the virus that was inoculated last, and this was detected in tracheal swabs from all groups. Virus could be isolated from swabs at a limited number of timepoints, and these further illustrated superinfection of the birds as recombinant viruses were detected among the progeny. This study has demonstrated superinfection at host level and shows recombination events occur under a very broad range of infection conditions. The occurrence of superinfection after unsynchronised infection with multiple viruses, and subsequent genomic recombination, highlight the importance of using only one type of vaccine per flock as the most effective way to limit recombination.