To My Quyen, Nguyen Khanh Thuan, Nguyen Phuc Khanh, Nguyen Thanh Lam*

1. Introduction 

Infectious bursal disease (IBD) is seen in young domestic chickens worldwide and is caused by infectious bursal disease virus (IBDV). Symptoms of the clinical disease can include depression, watery diarrhea, ruffled feathers, and dehydration. Depending on the IBDV strain and presence of maternal immunity, the disease can also present as a clinical or subclinical disease in young chicks. For both clinical and subclinical forms of the disease, all pathogenic IBDVs cause lesions in the bursa of Fabricious. The cloacal bursa can become enlarged, with a yellowish-colored transudate on the surface. Hemorrhages on the serosal and mucosal services are sometimes observed. Atrophy of the bursa, whi ch includes the loss of B-lymphocytes, occurs approximately 7-10 days after infection. Immunosuppression is directly related to this loss of B-lymphocytes, but immunosuppression and related secondary infections are typically seen in birds that recover from the disease. The severity of the immunosuppression depends on the virulence of the infecting virus and age of the host (Jackwood, 2020).
2. Etiology

2.1 Bacterial characteristics

Infectious bursal disease is caused by a birnavirus (infectious bursal disease virus; IBDV) that is most readily isolated from the bursa of Fabricius but may be isolated from other organs. It is shed in the feces and transferred from house to house by fomites. It is very stable and difficult to eradicate from premises.

Two serotypes of IBDV have been identified. The serotype 1 viruses cause disease in chickens, and, within them, antigenic variation can exist between strains. Antigenic drift is largely responsible for this antigenic variation, but antigenic differences can also occur through genome homologous recombination. Serotype 2 strains of the virus infect chickens and turkeys but have not caused clinical disease or immunosuppression in these hosts. IBDVs have been identified in other avian species, including penguins, and antibodies to IBDV have been seen in several wild avian species. The contribution of IBDV to disease in these wild birds is unknown (Jackwood, 2020).

3. Epidemiology

3.1. Susceptible hosts

IBDV is host specific, with chickens and turkeys reported to be the natural hosts. In addition, IBDV infection has been reported in ostriches, Baltic ducks, Herring gulls and sparrows. However, IBDV antibodies have been detected in pigeons, village weavers (Ploceus cucullatus) and pied cordon blues (Uraeginthus bengalus), speckled pigeons (Columba guinea), laughing doves (Streptolepia senegalensis), Antarctic penguins, and various raptors and passerines in Japan. The virus has been detected in lesser mealworm (Alphitobius spp.) fed on IBDV contaminated feed. Experimental IBDV inoculation of pheasants, partridges, guinea fowls and quails showed no signs of IBD. Furthermore, viable IBDV has been isolated from the faeces of dogs two days after oral inoculation with the virus, suggesting that dogs could be potential carriers of IBDV (Orakpoghenor et al., 2020).

3.2 Transmissions, carriers and vectors

The faecal-oral route via ingestion of contaminated feed and water constitutes the natural means by which IBDV infection occurs in chickens and turkeys. However, for experimental purposes, other mucosa routes, such as respiration, have been demonstrated. In free-living, wild birds, IBDV infection is likely to be indirect through scavenging of dead infected chickens, ingestion of contaminated water, or exposure of respiratory or conjunctival membranes to contaminated poultry dust (Gilchrist, 2005). This is enhanced by unrestricted interactions between free-living wild birds and poultry (Orakpoghenor et al., 2020). 

Infectious bursal disease is highly contagious, and the virus is persistent in the environment of a poultry house. Houses from which infected birds were removed were still infective for other birds at 54 and 122 days later. They also demonstrated that water, feed, and droppings taken from infected pens were infectious after 52 days. 

No evidence suggests that IBDV is transmitted through the egg or that a true carrier state exists in recovered birds. Resistance of the virus to heat and disinfectants is sufficient to account for virus survival in the environment between outbreaks. The lesser mealworm (Alphitobius diaperinus), taken from a house eight weeks after an outbreak, was infectious for susceptible chickens when fed as a ground suspension. In another study, the virus was isolated from several tissues of surface‐sterilized lesser mealworm adults and larvae that were fed the virus earlier. 

Infectious bursal disease virus was isolated from mosquitoes (Aedes vexans) that were trapped in an area where chickens were being raised in southern Ontario. The isolate was nonpathogenic for chickens. The IBDV antibodies were detected by the agar‐gel precipitin (AGP) test in 6 of 23 tissue samples from rats found dead on four poultry farms that had histories of IBDV infection. There has been no further evidence to support that either mosquitoes or rats act as vectors or reservoirs of the virus. 

A dog fed chickens that had died of acute IBD shed viable vvIBDV in its feces for up to two days after ingestion (Eterradossi and Saif, 2020).

4. Pathogenesis

Under natural conditions, the most common mode of infection appears to be via the oral route. From the gut, the virus is transported to other tissues by phagocytic cells, most likely resident macrophages. Although viral antigen has been detected in liver and kidney within the first few hours of infection, extensive viral replication takes place primarily in the bursa of Fabricius.

In vivo and in vitro studies have shown that the target cell is an IgM-bearing B lymphocyte. Within hours of exposure, virus-containing cells appear in the bursa and the virus spreads rapidly through the bursal follicles. Virus replication leads to extensive lymphoid cell destruction in the medullary and the cortical regions of the follicles. The cellular destructive process may be accentuated by apoptosis of virus-free bystander cells. The acute lytic phase of the virus is associated with a reduction in circulating IgM+ cells, although there is no detectable reduction in circulating immunoglobulins (Igs). 

T cells are resistant to infection with IBDV. Although the thymus undergoes marked atrophy and extensive apoptosis of thymocytes during the acute phase of virus infection, there is no evidence that the virus actually replicates in thymic cells. Gross and microscopic lesions in the thymus are quickly overcome and the thymus returns to its normal state within a few days of virus infection. 

Clinical signs associated with acute disease include anorexia, depression, ruffled feathers, diarrhea, prostration and death. The incidence of mortality is highly variable ranging from 100% to negligible. Lesions include bursal atrophy, dehydration and darkened discoloration of pectoral muscles, often hemorrhages may be present in the thigh and pectoral muscles and the bursa. The birds that survive the acute phase of the disease clear the virus and recover from clinical disease. IBDV-induced inhibition of B and T cell functions is also overcome. General aspects of IBDV induced pathogenesis and immunosuppression are outlined in Fig. 2 (Sharma et al., 2000). 

Target cells. All compartments of the bird’s immune system will be affected during infection with IBDV. IBDV targets the chicken’s immune cells in a very comprehensive and complex manner by destroying B lymphocytes, attracting T cells and activating macrophages (Fig. 3). 

The target organ for IBDV is the Bursa of Fabricius (BF) at its maximum development, which is a specific source for mature B lymphocytes in avian species. Bursectomy can prevent illness in chicks infected with virulent virus. The severity of the disease is directly related to the number of susceptible cells present in the BF; therefore, the highest age susceptibility is between 3 and 6 weeks, when the BF is at its maximum rate of development. This age susceptibility is extended in the case of vvIBDV infection. Depletion of lymphoid B cells in the Bursa of Fabricius after IBDV infection is due to both necrosis and apoptosis. Actively dividing, surface immunoglobulin M-bearing B cells are lysed by IBDV infection. Apoptosis, characterized by nuclear fragmentation and cellular breakdown into apoptotic vesicles also plays an important role in IBDV pathogenesis (Ingrao et al., 2013)

A high level of apoptosis can be evidenced in peripheral blood lymphocytes of chickens infected with serotype 1 IBDV. Very virulent strains causing increased pathology and earlier mortality also induce also a higher level of chIFN- γ mRNA in bursal tissue. The viral proteins VP2 and especially VP5 have been suspected to play a crucial role in IBDV replication by inducing cell death. It was shown in vitro that IBDV infection activates effector caspase 3 and the initiation caspase 9 as well as nuclear factor кB (NFкB), likely through the accumulation of oxygen reactive species, resulting in apoptosis late in the infective cycle. More recently, VP5 was confirmed to be a major apoptosis inducer by interacting with the voltage-dependent anion channel 2 (VDAC2) in the mitochondrion. Additionally, the RNA-binding VP3 polypeptide likely ensures the continuity of the IBDV replication cycle by inhibiting PKR-mediated apoptosis. On the other hand, apoptosis has also been observed in viral antigen-negative bursal cells, reinforcing the role of immunological mediators in the process. The interaction between IBDV and the host cell has become clearer over the years. It was first pointed out that the virus prerequisites a certain stage of cell differentiation for its replication. The old hypothesis was that this fact may be due to special receptors or to a potential synthesis apparatus being present in such cells. It was then demonstrated that IBDV could be mainly controlled by the presence of a virus receptor composed of a N-glycosylated protein on the surface of IgM-bearing cells. A decrease in the IgM B-cell population relative to IgA and IgG B-cell following IBDV infection was observed and, afterwards, two distincts IgM B-cell subpopulations were identified. More recently, it was also suggested that the IBDV might use the a4b1 integrin as a specific binding receptor in avian cells. Although membrane perforation was suggested as the means of penetration mediated by IBDV, the cellular mechanism being hijacked to facilitate its entry is still largely unknown. Recent result suggests that the intact IBDV particle is transported to the V-ATPase positive vesicles for uncoating and implicates an essential role of clathrin independent endocytosis during the viral entry. Cells of the monocyte-macrophage lineage can also be infected in a persistent and productive manner and play a crucial role in the dissemination of the virus as well as in the onset of the disease. In bursal macrophages, viral RNA was detected by RT-PCR and viral proteins by immunochemistry between 1 and 7 dpi. Confocal microscopic examination revealed cells that were positive for both KUL01 (macrophage surface marker) and R63 (IBDV-VP2 marker), thus confirming the presence of the virus in macrophages. As a consequence, the macrophage functions, notably the phagocytic activity, are modified by the infection with IBDV and cytokine gene expression is upregulated, therefore influencing normal immune responsiveness of the affected birds. Finally, although they are not susceptible to infection, T cells and IFN-γ play an important indirect role in the pathogenesis of IBD (Fig. 4). Indeed, there is an influx and infiltration of CD4+ and CD8+ cells into the BF between 1 and 10 dpi,most probably enhancing cellular damage (Ingrao et al., 2013).  

5. Clinical signs and pathology

5.1 Clinical signs 

There were no obvious clinical signs in the collected turkey poults, but some of them showed dullness, dehydration, ruffled feathers (Fig. 5a) and whitish diarrhoea with soiled vent in few birds (Fig. 5b), with no mortality. The necropsy findings were observed in few birds as atrophied bursae without haemorrhages (Fig. 5c) and mild nephritis (Fig. 5d). Interestingly, some turkey poults showed petechial haemorrhage on thigh muscles (Fig. 5e and f) which is very characteristic in the IBDV infection. The observed clinical signs of suspected infected chickens with IBDV in the vicinity of collected turkey poults were depression, ruffled feathers, anorexia, whitish diarrhoea and mortality that ranged from 20 to 30%, and the lesions included swollen bursa with a gelatinous exudate, haemorrhagic thigh muscle and nephritis (Orakpoghenor et al., 2020).

a. Turkey poult with dullness and ruffled feathers. b. Turkey poult with whitish diarrhoea and soiled vent. c. Bursae of 5-week-old turkey poults, (1) normal-sized bursa of healthy turkey poult and (2) atrophied bursa of turkey poult suspected to be infected with IBDV. d. Lesion of mild nephritis (arrow) and whitish diarrhoea inside rectum (arrow). e, f. Petechial haemorrhages on thigh muscles of turkey poult suspected to be naturally infected with IBDV.

The clinical signs also showed dwarfing with subcutaneous oedema and subcutaneous haemorrhages in inoculated dead embryos (Fig. 6a). Oedema, greenish coloration and necrosis in liver were indicated (Fig. 6b), and CAMs thickened in the three egg passages. Some inoculated embryos (1/5 or 2/5) died between the 4th and 5th day post-inoculation, while the negative control showed no embryo mortality.

5.2 Pathology

a. Bursa displays depletion of lymphoid tissue with numerous plasma cells (arrow). b. Kidney displays degenerative changes and necrosis of renal tubular epithelium (arrow). c. Liver is showing coagulative necrosis of hepatocytes and lymphocytic infiltrate into hepatic tissue (arrow). d. Spleen displays plugged splenic arteriole with heterophilic (arrow) and recruitment into splenic tissue. e. Cecal tonsils displays marked lymphoid depletion (arrow). f. Thymus displays coagulative necrosis of lymphoid tissue in thymic cortex (arrow) and congestion with expansion at thymic medulla.

For histopathological examination, the bursa of Fabricius displayed marked depletion of lymphoid tissue with numerous plasma cells (Fig. 7a). The kidney displayed degenerative changes and necrosis of renal tubular epithelium (Fig. 7b). The liver showed coagulative necrosis of hepatocytes and lymphocytic infiltrate into hepatic tissue (Fig. 7c). The spleen displayed plugged splenic arterioles with heterophilic and recruitment into splenic tissue (Fig. 5d). Caecal tonsils showed marked lymphoid depletion (Fig. 7e). Moreover, the thymus displayed coagulative necrosis of lymphoid tissue in the thymic cortex and congestion with expansion at the thymic medulla (Fig. 7f). Using monoclonal antibody, viral antigen of IBD was detected in the bursa inside lymphocyte of lymphoid follicles. Viral antigen was detected in the renal lining tubular epithelium, and in the cytoplasm of hepatic cells. Moreover, it was detected in the lymphocyte of red and white pulps and lymphocytes of caecal tonsils and thymus. All sections were tested negative for avian influenza virus and Newcastle disease virus antigens for their exclusion.

6. Diagnosis

Diagnosis can be accomplished by clinical evaluation of the cloacal bursa for macroscopic and microscopic lesions followed by molecular detection of the viral VP2 gene using RT-PCR.

Sequence analysis of the VP2 gene is used to identify the IBDV genotype.

Virus isolation in chicken embryos or chicken embryo fibroblast cell cultures is possible but often not necessary.

Initial diagnosis of infectious bursal disease is accomplished by the observation of gross lesions in the cloacal bursa. This is followed by microscopic analysis of the bursa for lymphocyte depletion in the follicles. Molecular diagnostic assays are most often used to identify IBDV in diagnostic samples. The reverse-transcriptase-PCR assay is used to identify the viral genome in bursa tissue. Sequence alignments and phylogenetic analysis of the VP2 coding region has been used to further characterize the viruses into genogroups. Samples for molecular diagnostic testing are typically collected after maternal antibodies have waned.

IBDV may be isolated in 8- to 11-day-old, antibody-free chicken embryos with inocula from birds in the early stages of disease. The chorioallantoic membrane is more sensitive to inoculation than is the allantoic sac. Some strains of IBDV may also be isolated in cell cultures that include chicken embryo fibroblasts, cells from the cloacal bursa, and established avian and mammalian cell lines. Cell culture–adapted strains of IBDV produce a cytopathic effect and may be used for quantitative titration of the virus and virus-neutralization assays.

Serology can be used to detect the presence of antibodies to IBDV in convalescent chicks. Commercially available ELISA kits are most often used to quantitate IBDV antibodies. The presence of IBDV antibodies in chicks is not always an indication of infection because most young chicks have maternal antibodies (Jackwood, 2020).

7. Treatment 

No practical therapeutic or supportive treatment has been found to change the course of IBDV infection (Parkhurst, 1964). Experimental immunotherapy where passively transferred antibody is injected intraperitoneally after challenge greatly reduced birds showing clinical signs, but this approach has not been tested in the field (Malik et al., 2006). There are no reports in the literature concerning the use of some of the newer antiviral compounds and interferon inducers for the treatment of IBD. Ketotifen was reported experimentally to prevent the development of bursal damage, and reduce clinical signs and mortality induced by vvIBDV challenge when administered one hour before IBDV inoculation, but it is not licensed for food animal veterinary use (Wang et al., 2009).

With the benefit of increasing the natural immunity against IBDV diseases, ANTI-GUM is provided following its indication that helps strengthen the immune system of infected poultry. 

Indication: 

Increases the natural immune, helps relieve stress and against infectious disease or secondary infection after Gumboro, and improves recovery during convalescence Additionally, it also supplies electrolytes for the animal with diarrhea.

Dosage: 

Dissolve in water for 5-7 days

• Increases natural immune, helps relieve stress: 100g/ 150 liters of water.
• During the period of disease: 100g/ 50-100 liters of water.
• Note: medicated water for use within a day.

8. Prevention and control 

The IBDV is contagious and contact with infected birds and contaminated fomites could result in the spread of infection. The virus is environmentally stable and resistant to many chemical and physical agents. The spread between flocks can be restricted through implementation of strict biosecurity measures. However, with the integrated nature of commercial poultry operations, litter reuse and the possibility of interaction with free-living wild birds, the control of IBD faces difficulties. The use of therapeutic treatment has been reported to have no effect on the course of infection. The prevention of IBD outbreaks in the field has been achieved globally through vaccination. Commercially available vaccines against IBDV are live attenuated and inactivated vaccines, whereas, in some countries, the use of recombinant and subunit vaccines has been licensed (Orakpoghenor et al., 2020). 

Contact with infected birds and contaminated fomites readily causes spread of the infection. The relative stability of this virus to many physical and chemical agents increases the likelihood that it will be carried over from one flock to a succeeding flock. The sanitary precautions that are applied to prevent the spread of most poultry infections must be rigorously used in the case of IBD; this includes control of personal and material movements. In their study of the epidemiological factors associated with the spread of vvIBDV in Denmark, it was demonstrated that the highest risk for farms was when another case of IBD occurred during a short period of time or within a short range, a finding that emphasizes the role of local factors in the spread of IBDV. The possible involvement of other vectors (e.g., the lesser mealworm, mosquitos, dogs, and rats) has already been discussed; they could certainly pose extra problems for the control of this infection (Eterradossi and Saif, 2020). 

9. References

Delmas, B., Attoui, H., Ghosh, S., Malik, Y.S., Mundt, E., Vakharia, V.N., 2019. ICTV virus taxonomy profile: Birnaviridae. Journal of General Virology 100, 5-6.

Eterradossi, N., Saif, Y.M., 2020. Infectious bursal disease, Diseases of Poultry, pp. 257-283.

Gilchrist, P., 2005. Involvement of free-flying wild birds in the spread of the viruses of avian influenza, Newcastle disease and infectious bursal disease from poultry products to commercial poultry. World's poultry science journal 61, 198-214.

Jackwood, D.J., 2020. Infectious Bursal Disease in Poultry. MSD MANUAL. Veterinary Manual.

Malik, M.W., Ayub, N., Qureshi, I.Z., 2006. Passive immunization using purified IgYs against infectious bursal disease of chickens in Pakistan. Journal of Veterinary Science 7, 43-46.

Ingrao, F., Rauw, F., Lambrecht, B., van den Berg, T., 2013. Infectious bursal disease: a complex host–pathogen interaction. Developmental & Comparative Immunology 41, 429-438.

Orakpoghenor, O., Oladele, S.B., Abdu, P.A., 2020. Infectious bursal disease: transmission, pathogenesis, pathology and control - An overview. World's Poultry Science Journal 76, 292-303.

Parkhurst, R.T., 1964. On-the-farm studies of Gumboro disease in broilers. Avian Diseases 8, 584-596.

Sharma, J.M., Kim, I.J., Rautenschlein, S., Yeh, H.Y., 2000. Infectious bursal disease virus of chickens: pathogenesis and immunosuppression. Developmental and Comparative Immunology 24, 223-235.

Wang, D., Liu, Y., She, R., Xu, J., Liu, L., Xiong, J., Yang, Y., Sun, Q., Peng, K., 2009. Reduced mucosal injury of SPF chickens by mast cell stabilization after infection with very virulent infectious bursal disease virus. Veterinary Immunology and Immunopathology 131, 229-237.