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.

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.

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.

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.

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.

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.

Intraperitoneal administration of Shiga toxin 2 induced neuronal alterations and reduced the expression levels of aquaporin 1 and aquaporin 4 in rat brain.

Shiga toxin-producing Escherichia coli produces watery and hemorrhagic diarrhea, and hemolytic uremic syndrome (HUS) characterized by thrombocytopenia, microangiopathic hemolytic anemia, and acute renal failure. Central nervous system (CNS) complications are observed in around 30% of infant population with HUS. Common signs of severe CNS involvement leading to death include seizures, alteration of consciousness, hemiparesis, visual disturbances, and brain stem symptoms. The purpose of the present work was to study the effects of Shiga toxin 2 (Stx2) in the brain of rats intraperitoneally (i.p.) injected with a supernatant from recombinant E. coli expressing Stx2 (sStx2). Neurological alterations such as postural and motor abnormalities including lethargy, abnormal walking, and paralysis of hind legs, were observed in this experimental model of HUS in rats. Neuronal damage, as well as significant decrease in aquaporin 1 (AQP1) and aquaporin 4 (AQP4) expression levels were observed in the brain of rats, 2 days after sStx2 injection, compared to controls. Downregulation of aquaporin protein levels, and neuronal alterations, observed in brain of rats injected with sStx2, may be involved in edema formation and in neurological manifestations characteristic of HUS.