Evasion of maternal antibody protection by an IBDV Argentine variant.

Infectious bursal disease (IBD) is a viral disease that affects the ability of chickens to produce humoral immune responses. One way to prevent the disease is the passage of maternally derived antibodies (MDA) from dams to offsprings via the yolk. Despite sanitary measures, which include immunization with genogroup 1 (G1) vaccines, infections with IBDV genogroup 4 (G4) in young animals have been detected. The aim of this study was to determine whether a local IBDV isolate belonging to G4 could evade the immunity generated by MDAs. Twelve-day-old animals positive for MDA, were inoculated with G1 or G4 isolates or phosphate buffered saline (PBS) as a control. After 1 wk, the animals were sacrificed and the following parameters were evaluated: bursa-body (BB) ratio, viral load, and histologic damage in the bursa of Fabricius. Results showed that G4-infected animals had significant differences in the BB ratio compared to the PBS group. In addition, viral load was significantly higher in the G4 group than in the G1 group. Histologic damage in the bursa of Fabricius was detected only in G4-infected MDA chickens. Our results suggest that infection with G4 local isolate can circumvent the immunity generated by MDA and, furthermore, that G4 isolate does not differ in its pathogenicity from G1 isolate, which underlines the need to include variant strains in vaccine formulations to reduce potential losses caused by these viruses.

Study of coinfection with local strains of infectious bursal disease virus and infectious bronchitis virus in specific pathogen-free chickens.

Immunosuppressive diseases cause great losses in the poultry industry, increasing the susceptibility to infections by other pathogens and promoting a suboptimal response to vaccination. Among them, infectious bursal disease virus (IBDV) arises as one of the most important around the world. IBDV infects immature B lymphocytes, affecting the immune status of birds and facilitating infections by other pathogens such as avian infectious bronchitis virus (IBV). Although it has been reported that the interaction between these viruses increases IBV clinical signs, there are no actual studies about the interaction between regional circulating isolates that validate this statement. In this context, the objective of our work was to evaluate the effect of the interaction between local isolates of IBDV (belonging to genogroup 4) and IBV (lineage GI-16) in chickens. Thus, specific pathogen-free chickens were orally inoculated with IBDV genogroup (G) 4 or with PBS at 5 d of age. At 14-days postinoculation (dpi) the animals were intratracheally inoculated with a GI-16 IBV or with PBS. At multiple time points, groups of birds were euthanized and different parameters such as histological damage, viral load, lymphocyte populations and specific antibodies were evaluated. The success of IBDV infection was confirmed by the severity of bursal atrophy, viral detection, and presence of anti-IBDV antibodies. In IBV-infected animals, the presence of viral genome was detected in both kidney and bursa. The coinfected animals showed higher degree of lymphocyte infiltration in kidney, higher rate of animals with IBV viral genome in bursa at 28 dpi, and a clear decrease in antibody response against IBV at 28, 35, and 40 dpi. The results indicate that the infection with the local isolate of IBDV affects the immune status of the chickens, causing major severe damage, in response to IBV infection, which could consequently severely affect the local poultry industry.

Comparative Study of the Immune Response Induced by an Argentinian Classical Strain of IBDV in Two Chicken Breeds.

The hybrid chicken Negra INTA, which originated at the National Institute of Agricultural Technology (INTA), is the product of the cross between Barred Plymouth Rock females and Rhode Island Red males, and it is used as a laying hen for egg consumption. It has been characterized by productive parameters, but the characterization from an immunological perspective has not been done yet. Infectious bursal disease virus (IBDV) causes a highly contagious viral disease that affects the bursa of Fabricius. Although most chickens are regularly vaccinated against IBDV, this virus still generates negative impacts on production with significant economic losses. The aim of the present work was to compare the immune responses of the Negra INTA hybrid and the White Leghorn layer line to the infection with a field isolate of IBDV. Four-week-old chickens were infected with a single dose of IBDV and at 3, 5, 7, and 30 days postinfection (dpi), bursae were removed, and different parameters were evaluated. Results showed that the reduction of the bursa body (BB) ratio and the histopathological damage were maximum on day 7 postinfection (pi). The viral load was greater in the hybrid Negra INTA at 5 dpi. The humoral immune response between both breeds was similar, although more animals from the commercial line showed higher titers of neutralizing antibodies. Flow cytometry analysis revealed that Bu+ bursal lymphocytes reached a minimum at 7 dpi. Meanwhile, T cell infiltration measured by the percentage of CD3+, CD4+, and CD8+ cells in the bursa was at its maximum at 5 dpi. To our knowledge, this work describes for the first time the pathogenesis and the immune response caused by an Argentinian IBDV isolate in two different chicken lines.

Oral Immunization With Plant-Based Vaccine Induces a Protective Response Against Infectious Bursal Disease.

Infectious bursal disease virus (IBDV) is the etiological agent of an immunosuppressive and highly contagious disease that affects young birds causing important economic losses in the poultry industry worldwide. We have previously developed a plant-based vaccine candidate for infectious bursal disease (IBD) that is able to protect against infection with IBDV when administered through intramuscular (im) route. Given that oral vaccination is non-invasive and stimulates the immunity of the mucosal gastrointestinal surface, the initial site of contact and entry of IBDV, the aim of this work was to study if our immunogen was also able to elicit a protective immune response when orally administered. We demonstrated that 85% of the animals that received two oral doses of the vaccine formulation and all animals that were orally boosted after an im prime scheme developed virus neutralizing antibodies and were protected against IBDV infection, evidenced by the bursa/body weight (BB) ratio, absence of T-cell infiltration, and low viral load in bursa. Although mild to moderate bursal damage was observed in some of these animals, these lesions were not as severe as the ones observed in challenged control groups, which also presented signs of acute inflammation, bursal atrophy, T-cell infiltration, and absence of viral clearance. These results show that two immunizations with our recombinant immunogen are able to induce a specific and protective immune response in chicken against IBDV when orally administered in a prime/boost scheme or when the oral boost follows an im prime scheme. In conclusion, our oral plant-based vaccine candidate could represent a viable alternative to conventional vaccines and is of great interest to the poultry industry.

Development of diagnostic tools for IBDV detection using plants as bioreactors.

Infectious bursal disease virus (IBDV) is the etiological agent of an immunosuppressive and highly contagious disease that affects young birds, thus causing important economic losses in the poultry industry. Multimeric particles with different architectures based on the capsid protein VP2 have been widely produced for different purposes. We hereby show the production and easy recovery of IBDV subviral particles (SVP) from transiently transformed Nicotiana benthamiana. The SVP, which were observed by electronic microscopy, proved to be antigenically and immunogenically similar to the virion. Indeed, anti-IBDV antibodies from samples of infected birds recognized these SVP and, when injected intramuscularly, these subviral particles also evoked a humoral immune response in chickens. We developed an in-house ELISA using SVP as coating reagent that demonstrated to be highly accurate and in good agreement with a commercial ELISA. This study demonstrates that the recombinant antigen generated and the technology used to produce it are suitable for developing a diagnostic tool against Infectious bursal disease.

Plant-based vaccines for potential human application: a review.

The worldwide need to produce safe and affordable vaccines with a minimum requirement of manufacture and processing, together with the advancements achieved in biotechnology, have promoted the development of efficient alternatives to traditional ones. One of the available options is the use of transgenic plants, not only as a protein production system but as an antigen transportation system as well, being capable of delivering antigens to the mucosal immune targets, becoming what is known as edible vaccines. The versatility of the plant production system allows for instance, to express and to accumulate foreign antigens in edible plant tissues. Thus, the hypothesis for the choice of plant-based vaccines is that once a plant-based vaccine is eaten, the susceptible host mounts a mucosal immune response against the antigen that is expressed in the plant, becoming protected against the pathogen from which the antigen was selected. This idea is still under study. Here, we described the basis of the system, the promising future and the possible drawbacks.

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.

Mucosal immunity induced by orally administered transgenic plants.

Oral immunization is an efficient means to induce protection at the portal entrance for many pathogens. Therefore, the design of efficient edible vaccines through transgenic plants represents a challenging alternative to the traditional injectable ones. We have previously reported the construction of transgenic potato plants expressing the genes coding for the immunogenic proteins of Newcastle Disease Virus (NDV) and their immunogenicity in mice. All mice receiving transgenic plant extracts in incomplete Freund's adjuvant produced specific antibodies. Animals fed with transgenic leaves also showed a specific response against NDV. The aim of the present study was to continue the evaluation of the mucosal immune response. Adult Balb/c mice were fed with potato leaves for a month and on day 36 mucosal samples were collected. ELISAs performed on intestinal washes showed that transformed plants elicited the synthesis of NDV-specific IgG and IgA antibodies. In addition, anti-NDV IgA antibodies were detected in supernatants of cultured small intestine fragments of mice fed with the recombinant immunogens, suggesting the presence of NDV-specific IgA secreting plasma cells in the intestinal tissue. Moreover, we detected specific anti-NDV antibodies in intestinal fluids after oral immunization with F and HN transgenic plants. Also, indirect immunofluorescence on intestinal tissue was performed. The present results suggest that these immunogens, F and HN glycoproteins of NDV, when orally administered, would enhance the number of IgA(+) B cells, and the cytotoxic cellular immune response via CD8(+) T cells, found in the gut lamina propria that is in accordance with our first findings.

Comparison of homologous and heterologous prime-boost immunizations combining MVA-vectored and plant-derived VP2 as a strategy against IBDV.

Different immunogens such as subunit, DNA or live viral-vectored vaccines against Infectious Bursal Disease virus (IBDV) have been evaluated in the last years. However, the heterologous prime-boost approach using recombinant modified vaccinia Ankara virus (rMVA), which has shown promising results in both mammals and chickens, has not been tried against this pathogen yet. IBD is a highly contagious and immunosuppressive disease of poultry that affects mainly young chicks. It is caused by IBDV, a double-stranded RNA virus carrying its main antigenic epitopes on the capsid protein VP2. Our objective was to evaluate the immune response elicited by two heterologous prime-boost schemes combining an rMVA carrying the VP2 mature gene (rVP2) and a recombinant VP2 protein produced in Nicotiana benthamiana (pVP2), and to compare them with the performance of the homologous pVP2-pVP2 scheme usually used in our laboratory. The SPF chickens immunized with the three evaluated schemes elicited significantly higher anti-VP2 antibody titers (p<0.001) and seroneutralizing titers (p<0.05) and had less T-cell infiltration (p<0.001), histological damage (p<0.001) and IBDV particles (p<0.001) in their bursae of Fabricius when compared with control groups. No significant differences were found between both heterologous schemes and the homologous one. However, the rVP2-pVP2 scheme showed significantly higher anti-VP2 antibody titers than pVP2-rVP2 and a similar tendency was found in the seroneutralization assay. Conversely, pVP2-rVP2 had the best performance when evaluated through bursal parameters despite having a less potent humoral immune response. These findings suggest that the order in which rVP2 and pVP2 are combined can influence the immune response obtained. Besides, the lack of a strong humoral immune response did not lessen the ability to protect from IBDV challenge. Therefore, further research is needed to evaluate the mechanisms by which these immunogens are working in order to define the combination that performs better against IBDV.

Immunomodulatory effect of baculovirus in chickens: How it modifies the immune response against infectious bursal disease virus.

Several reports have shown that baculoviruses (BVs) have strong adjuvant properties on the mammalian immune system. Recent studies of our group demonstrated the ability of BV to stimulate the innate immunity in chickens. In this investigation, we aimed to assess the potential antiviral effect of BV given both, before and after infectious bursal disease virus (IBDV). In the first case, specific pathogen free chickens were intravenously inoculated with 5 × 10(7) pfu of Autographa californica nuclear polyhedrosis virus and 3 h later were orally administered 2.5 × 10(5) egg infectious doses 50 of IBDV. In the second case, chickens received IBDV 3 h before BV inoculation. Five days later, chickens were bled and euthanized. RNA from the bursa was analyzed for cytokine production. Also, bursae were used for virus recovery, and processed for lymphocyte isolation. The results showed that the administration of BV 3 h after the inoculation with IBDV produced important changes in the effect that IBDV causes in the bursa. BV reduced the infiltration of T lymphocytes, decreased the expression pattern of IL-6 and IFN-γ and inhibited IBDV replication. The results herein presented demonstrate that this Lepidopteran virus shows antiviral activity in chickens under experimental conditions. Investigations under field conditions have to be done to probe this strategy as a valuable sanitary tool for the treatment and prevention of chicken diseases.

High-level expression of recombinant 3AB1 non-structural protein from FMDV in insect larvae.

For its potential usefulness in diagnosis, the non-structural protein 3AB1 from foot-and-mouth disease virus was expressed as a soluble protein by using Autographa californica nuclear polyhedrosis virus as a vector. The 3AB1 coding sequence was introduced into AcNPV genome via pBAcPAK3AB1 transfer vector to originate Ac3AB1 recombinant baculovirus of phenotype occ-. Rachiplusia nu larvae were injected with supernatants of Sf9 cells infected with Ac3AB1 and 5 days post-infection total protein extracts were obtained. An intense band of approximately 21.5 kDa was observed when total larvae extracts were SDS-PAGE resolved and the recombinant protein detected by an FMDV-infected guinea pig serum. ELISA tests and Western blot experiments were carried out using sera both from FMDV-infected cattle and from vaccinated animals. The recombinant protein was only recognized by sera from infected animals, suggesting that this method of production in insect larvae could be applied to an efficient mass production of proteins of diagnostic interest.

Molecular characterization and phylogenetic analysis of Newcastle disease virus isolates from healthy wild birds.

Wild waterfowl is considered a natural reservoir of potentially infectious agents and a source of pathogenic viruses like avian paramyxoviruses type 1 (APMV 1). In 1997, commercial poultry in Argentina had reached the status of being free from virulent Newcastle disease virus (NDV) infections. Vaccination and biosecurity measures are actively performed to maintain this preferential sanitary condition. However, the risk of reintroduction of pathogenic viruses is always present. In this context, we conducted a study to describe the status of wild healthy birds in a geographic region relevant for the poultry industry. The presence of anti-NDV antibodies was determined in different species in all areas sampled suggesting previous contact with NDV. Seven ND viruses were isolated and characterized as apathogenic strains by biological and molecular methods. The phylogenetic analysis revealed that the majority of the Argentinian isolates form a subgroup related to viruses of genotype II. The results presented here highlight the importance of maintaining strict biosecurity measures and vaccination programs in poultry industries in order to preserve the virulent NDV-free status for commercial flocks in the country.

Infectious Bursal Disease Virus non-structural protein VP5 is not a transmembrane protein.

Infectious Bursal Disease Virus (IBDV) causes a highly relevant poultry disease that affects young chickens causing, among other effects, immunosuppression. IBDV is a bi-segmented double stranded RNA virus. The smaller ORF of larger RNA segment encodes VP5, a 17-kDa non-structural protein. Although it is an important protein for viral replication cycle, the definition of its specific role and subcellular localization remains unclear. In the present work we demonstrate, using imaging techniques, that VP5 is not a type II transmembrane protein but an intracellular membrane-associated protein. This finding might provide evidences of VP5 interaction with cellular proteins and its functions.

Heteroduplex mobility assay for detection of new avian influenza virus variants.

Highly pathogenic avian influenza (HPAI) in poultry causes high morbidity and mortality, and it is a List A disease of the Office International des Epizooties. An outbreak of HPAI in commercial poultry not only causes direct disease losses but often results in trade restrictions for the affected country. Because HPAI viruses can mutate from H5 and H7 low pathogenic avian influenza viruses, it is necessary to monitor and control even the low pathogenic form of the virus. We report a practical approach for screening large numbers of isolates that uses amplification by reverse transcriptase-polymerase chain reaction of a segment of the hemagglutinin (HA) gene (536-560 bp) of H7 avian influenza viruses followed by the heteroduplex mobility assay (HMA). The HMA test compares the amplified polymerase chain reaction product from unknown samples with reference isolates, which allows the identification of new variants. The HMA test results were compared with sequence analysis of the isolates used in the study. On the basis of the HMA, we could identify several new variant viruses present in the live bird markets in the northeastern United States. New strains gave a distinct pattern of bands in the gels in accordance with the different heteroduplexes formed when their HA region amplification products were incubated together with the same amplification product of a reference strain. These differences correlate with phylogenetic analysis from sequence data.

Immune response elicited by the oral administration of an intermediate strain of IBDV in chickens.

The immune response elicited by the oral inoculation of an intermediate strain of infectious bursal disease virus was studied in chickens. A strong over expression of IL-6, IL-8, IFNα and IFNγ was observed in bursa at 3 days post inoculation together with an increase in splenic NO2 release. An influx of T-lymphocytes was also detected.

Enhanced IRES activity by the 3'UTR element determines the virulence of FMDV isolates.

A reverse genetics approach was used to identify viral genetic determinants of the differential virulence displayed by two field foot-and-mouth disease virus (FMDV) strains (A/Arg/00 and A/Arg/01) isolated in Argentina during the 2000-2001 epidemics. A molecular clone of A/Arg/01 strain and viral chimeras containing the S-fragment or the internal ribosome entry site (IRES) of A/Arg/00 in the A/Arg/01 backbone were constructed and characterized. The IRES appeared as a determining factor of the lower level of A/Arg/00 replication in cell culture. High-throughput RNA probing revealed structural differences between both IRESs. Translation experiments using either synthetic viral RNAs (in vitro) or bicistronic plasmids (in vivo) showed that these IRESs' activities differ when the viral 3' untranslated region (UTR) is present, suggesting that their function is differentially modulated by this region. This work provides experimental evidence supporting the role of the IRES-3'UTR modulation in determining the level of FMDV replication in field strains.

Transient expression of VP2 in Nicotiana benthamiana and its use as a plant-based vaccine against infectious bursal disease virus.

Infectious Bursal Disease Virus (IBDV) is the etiological agent of an immunosuppressive and highly contagious disease that affects young birds. This disease causes important economic losses in the poultry industry worldwide. The VP2 protein has been used for the development of subunit vaccines in a variety of heterologous platforms. In this context, the aim of this study was to investigate VP2 expression and immunogenicity using an experimental plant-based vaccine against IBDV. We determined that the agroinfiltration of N. benthamiana leaves allowed the production of VP2 with no apparent change on its conformational epitopes. Chickens intramuscularly immunized in a dose/boost scheme with crude concentrated extracts developed a specific humoral response with viral neutralizing ability. Given these results, it seems plausible for a plant-based vaccine to have a niche in the veterinary field. Thus, plants can be an adequate system of choice to produce immunogens against IBDV.

Modulation of innate immunity in chickens induced by in vivo administration of baculovirus.

Baculoviruses stimulate cytokine production in mammalian cells. They induce a strong innate immune response in animals and have adjuvant properties. The purpose of this work was to study the in vivo effect of baculovirus on chicken innate immune response. SPF chickens were inoculated intravenously with Autographa californica nuclear polyhedrosis virus (BV). Three hours later, chickens were bled, euthanized and their spleen, duodenum and cecal tonsils were excised in order to take samples for RNA extraction and real time PCR, and to isolate lymphocytes, which were stained and analyzed by flow cytometry. The results obtained showed that baculovirus inoculation up-regulates the expression of IFN-γ, IL-6 and LITAF in spleen cells. This result (IFN-γ) correlated with that obtained by ELISA which showed a very strong increase of IFN-γ in chicken plasma. Flow cytometry analysis revealed that BV inoculation induced in spleen an increase in the percentage of monocyte/macrophage population together with an increase in CD3(+)CD4(+) T lymphocytes. On the other hand, BV inoculation decreased the percentage of CD3(+)CD4(+) T lymphocytes and increased the percentage of NK cells in cecal tonsils. However, intraepithelial lymphocytes of the gut did not show differences between BV and control treated animals. Even though further studies in order to understand the mechanisms by which BVs affect the avian immune response are needed, results obtained in the present work demonstrate the ability of BVs to stimulate the innate immunity in chickens, modifying the expression pattern of related genes and the profile of the immune cells involved.

Expression of a secreted version of the hemagglutinin-neuraminidase glycoprotein of Newcastle disease virus: its evaluation as a diagnostic reagent.

The hemagglutinin-neuraminidase (HN) glycoprotein of Newcastle disease virus (NDV) constitutes, together with the fusion glycoprotein, the main surface antigen of this avian pathogen, which causes a highly contagious disease, relevant economically worldwide. The purpose of this work was to obtain the HN glycoprotein as a soluble antigen in culture supernatants of recombinant baculovirus-infected Spodoptera frugiperda (Sf9) cells and to evaluate its application to the development of a recombinant enzyme-linked immunosorbent assay (rELISA) for the analysis of chicken sera. A transfer vector for baculovirus containing the sequence of a melittin signal peptide was constructed and the sequence coding for HN protein without its own signal peptide was cloned. The recombinant protein was secreted and recovered easily from the culture medium of Sf9-infected cells. The recombinant protein was evaluated as antigen for ELISA coating the plates with the recovered HN using 79 positive and 142 negative samples. The Cohen kappa value resulted 0.91, indicating excellent agreement between the rELISA and the hemagglutinin inhibition tests. The rELISA was also compared with a commercial ELISA, finding high levels of agreement between both assays. The present results show that the cloning strategy developed yielded the HN protein free in the cell culture supernatant and that the recombinant protein retained its reactivity with anti-NDV HN antibodies in chicken sera.

Activation of the immune response against Infectious Bursal Disease Virus after intramuscular inoculation of an intermediate strain.

Infectious bursal disesase is a highly contagious, wide spread immunosuppressive chicken disease caused by the Infectious Bursal Disease Virus (IBDV). IBDV is a two segmented double-strand RNA virus, member of the Birnaviridae family. In order to study the interaction between IBDV and the immune system, chickens were exposed to an intermediate IBDV strain by intramuscular route, and using Real Time PCR the expression of a panel of avian cytokines and chemokines in duodenum, spleen and bursa of Fabricius was analyzed. Also, splenic nitrite (NO2) production and the frequencies of different mononuclear cell populations were evaluated by Griess reaction and flow cytometry, respectively. Intramuscular (i.m.) IBDV inoculation promoted an over expression of proinflammatory cytokines IL-6, IL-15 and gIFN in spleen, which correlated with an increase of gIFN plasma concentration measured by ELISA, together with an increment of NO2 concentration in splenocyte supernatants at 1dpi. Results obtained in the present work showed that IBDV of intermediate virulence, given i.m., induced similar effects to those previously described for highly virulent IBDV in early innate immune responses. Considering that the i.m. route is the route of choice for the delivery of new generation vaccines, and that the use of recombinant antigens also requires the addition of adjuvants for proper immune stimulation, results presented here could contribute to identify suitable cytokines to be used or to be stimulated when utilizing subunit vaccines, for the improvement of prevention tools for avian health.