Examination of macroscopic and microscopic lesions in IBDV-infected organs and molecular characterization of IBDV VP1 gene fragments obtained from commercial broiler farms in Indonesia

Background and Aim: Infectious bursal disease (IBD) is an infectious immunosuppressive disease that affects young chickens. Instead of strict biosecurity practices, vaccination is used to control IBD. However, the disease has not been effectively managed. Variations in the observed clinical symptoms lead to confounding diagnoses. The study aimed to obtain pathological lesion data from chickens suspected of IBD virus (IBDV) infection by gross pathology, confirm IBDV infection through molecular diagnostics, and genotype the VP1 gene fragments of circulating IBDV in the field. Materials and Methods: The bursa of Fabricius, thymus, spleen, proventricular–ventricular junction, thigh muscles, and kidneys samples were collected from chickens suspected of IBDV infection from four commercial broiler farms in Central Java and The Yogyakarta Special Region Province between 2021 and 2022. The collected samples were examined histopathologically. Infectious bursal disease virus RNA was extracted from the bursa of Fabricius and VP1 gene was identified by reverse-transcriptase polimerase chain reaction (RT-PCR). The RT-PCR positive sample were sequenced and analyzed in Mega X for homology search and phylogenetic tree analysis. Results: Macroscopic pathological lesions in the bursa of Fabricius were demonstrated by enlarged edema and thickened plica, presence of gelatinous exudate, hemorrhage, atrophy, and caseous exudate in the lumen. Moreover, the thymus had atrophy and small gray foci were observed in the spleen. Petechiae or hemorrhage was detected on the thigh muscle, and the kidney was dull and pale. Hemorrhage in the proventricular–ventricular junction was distinct. The histopathological examination of the bursa of Fabricius showed follicular vacuolization, edema, heterophilic infiltration, follicular atrophy, congestion, and hemorrhage. The thymus and spleen showed the presence of multifocal necrosis. Hemorrhage was observed in thigh muscle and mucosal part of proventricular–ventricular junction. Vacuolization was seen in renal tubules (nephrosis). Reverse transcriptase-PCR of 26 bursa of Fabricius samples from chickens suspected of IBDV infection showed four negative and 22 positive samples. Phylogenetic analysis of the VP1 gene fragment has indicated very virulent IBD (vvIBD) and belonged to B2 genotype. Conclusion: Infectious bursal diseases virus infection in broiler chicken generated macroscopic and microscopic primary lesions in the bursa of Fabricius and thigh muscle. Other organs such as the spleen, thymus, proventricular–ventricular junction, and kidney, were also involved. Molecular analysis of the VP1 gene confirmed the causative agent and grouped the virus into vvIBD and B2 genotype. All samples were collected from vaccinated birds therefore, the efficacy of available vaccine is required for urgent evaluation. Since most studies only focused on VP1, further exploration on VP2 gene is suggested notably for new-generation vaccines. Monitoring clinical signs’ transformation over time could assist field diagnostics.


Introduction
Infectious bursal disease (IBD) or Gumboro, is an immunosuppressive disease that affects young chickens. The virus is an RNA virus of the genus Avibirnavirus of the Birnaviridae family. The virus has an icosahedral symmetrical shape, is 55-65 nm in diameter, and is non-clustered. The viral genome is dsRNA with two segments: A and B. Segment A (3,261 bp) encodes VP5 and a precursor polyprotein (VP2-VP3-VP4). This protein is autocatalytically cleaved by the viral protease VP4 into the main structural proteins of the virion VP2 and VP3. Segment A also encodes a non-structural protein VP5. [1,2]. VP2 also plays a crucial role in virulence, cell tropism, pathogenicity phenotype, and protective immunity [3]. Segment B (2,827 bp) encodes VP1, an enzyme with Available at www.veterinaryworld.org/Vol.16/May-2023/19.pdf RNA-dependent-RNA polymerase activity [1], which is crucial for the replication and genetic evolution of IBD virus (IBDV) [4]. Immature B-lymphocytes in the follicles of the bursa of Fabricius are targets of IBDV since they contain immunoglobulin M specific for viral infection [5,6]. Actively proliferating cells are the primary target of IBDV. The affinity of IBDV is greater toward immature B-lymphocytes [7]. In B-lymphocytes, Gumboro virus inhibits proliferation and promotes apoptosis [8]. Allan et al. [9] reported that defects in B-lymphocytes lead to impaired response to vaccination. Chickens infected with Gumboro virus at an early stage may suffer from reduced antibody response, increasing susceptibility to other diseases. The clinical manifestations of and mortality caused by IBD vary depending on virulence, infection dose, age, chicken type, and presence of passive immunity [10]. The very virulent IBD (vvIBD) type causes severe lesions to immune-related organs, such as the thymus, spleen, bursa of Fabricius, as well as the liver, kidneys, heart, proventriculus, gizzard, and ceca tonsil [11]. Lesions in the pectoral and thigh muscles, including dark and dehydrated muscles, and hemorrhage have been observed [1]. A very virulent infectious bursal disease virus (vvIBDV) can cause extensive thymus cell necrosis. Further reports of IBD cases exhibited edema, lymphoid cell follicle draining, also follicle connective tissue fibrosis, and proliferation of endothelial reticular cells [12,13]. Chicken mortality due to IBDV infection was 59.09% in broilers and 25.08% in the commercial layer. In vvIBDV, mortality rate may reach 35%-75% [14].
Infectious bursal disease virus is classified into pathogenic serotype I and non-pathogenic serotype II. Based on pathogenicity, IBDV belonging to serotype I is classified into mild, intermediate, intermediate plus, classical, variant, and very virulent strains [15]. Due to the increasingly complex development of IBDV, classification based on antigenicity and pathogenicity has become more complicated. Classification based on gene sequence data is more consistent and reliable [16]. To add, current report on IBD genotyping mostly focus on VP2 only [17]. Seven genogroups have been reported thus far. A1 is a classic IBDV, A2 is an antigenic variant virus, A3 is a vvIBDV, and A4 is a distinct (d) IBDV, characterized by 222S, 272T, 289P, 290I, and 296F. A5 is a variant virus or a classic recombinant IBDV, A6 is an Italian variant, and A7 is an Australian variant [16,18]. The identified IBDV strain enters the A8 genogroup [19], and IBDV serotype 2 is classified in the all genotype [19]. Initially, the evolutionary analysis of IBDV focused on the role of VP2 in virulence, antigenicity, and tropism. The function of segment B, specifically VP1, attract great attention. Several studies have shown the role of VP1 in the evolution and emergence of vvIBDV [4]. Therefore, a molecular epidemiological investigation should include VP1 in genogroup analysis [19]. A previous study [19] has proposed genotyping based on segment B of VP1 gene. Serotype I is grouped into cluster B1 (including non-very virulent strain), B2 (vvIBDV), B3 (IBDV originated in other poultry or wild-bird reservoirs), and B4 (European lineage IBDV). Serotype II is classified into the BII genogroup [19].
In Indonesia, subclinical IBD cases in 1991 caused low mortality. The first outbreak of IBD reported an acute increase and expansion of IBD cases, with high mortality reaching 25% in broilers and 60% in laying hens [14]. Based on VP2 gene, IBDV Indonesia isolates are majority grouped in vvIBDV, although classical viruses were also detected [20,21]. Infectious bursal disease outbreaks have been reported in many areas of Indonesia, although vaccination and stringent biosecurity have long been used in farms. Diagnosis in the field is arduous to perform because classical signs are not perpetually present. In addition, genetic characterization of latest IBDV Indonesia VP1 gene is lacking. This gene is important in virus encapsidation, virulence, and replication. Genetic data would assist control strategies development and support field diagnostics.
Thus, this study aimed to obtain pathological lesion data and VP1 gene characterization of the latest cases of IBD in commercial broiler farms in Indonesia. This is the first genotype analysis of IBDV VP1 gene from Indonesia.

Ethical approval
The samples were obtained from IBDV case in commercial broiler farms. There were no challenge or live animal experiment involved. Thus, ethical clearance is not required. The method has been reviewed and obtained approval from Faculty of Veterinary Medicine, Universitas Gadjah Mada, Indonesia (approval number 4100/UN1/FKH/TU/2022).

Study period and location
The study was conducted from December 2021 to October 2022. The samples were collected from commercial broiler farms in the Special Region of Yogyakarta (DIY) and Central Java. The histopathology slides were processed at the Wates Disease Investigation Center, Yogyakarta. The RT-PCR test was carried out at the Department of Microbiology, Faculty of Veterinary Medicine, Universitas Gadjah Mada University.

Samples
Samples were obtained from chickens suspected of IBDV by gross pathology in four commercial broiler farms: Sragen, Wonogiri, Batang District of Central Java Province, and Sleman District of Yogyakarta Special Region Province. A total of five chickens from each farm with indicative clinical symptoms of IBD were selected. The clinical signs were recorded, and necropsies were performed to observe lesions on the bursa of Fabricius, thymus, spleen, proventricular-ventricular junction, thigh muscles, and kidneys. The collected organs were preserved in 10% formalin prior to histology slides processing. A part of the bursa of Fabricius was stored at −20°C for molecular analysis.
Histology slides were processed at the Wates Disease Investigation Centre, Yogyakarta Special Region Province, according to the standard protocols of the institution. Histopathology examination was conducted at the Department of Pathology, Faculty of Veterinary Medicine, UGM.

RNA extraction, reverse transcriptase polymerase chain reaction (RT-PCR), and sequencing
All samples were tested for the VP1 gene by RT-PCR. The genetic material of IBDV was extracted from the bursa of Fabricius using Geneaid Viral Nucleic Acid Extraction Kit II (Geneaid, Taiwan) according to the manufacturer. The VP1 gene was amplified using specific primers: Forward 5'-cta cgg gag tgg gac cta ca-3' and reverse 5'-acc acg tgt tgg agt gaa ca-3', which yielded a 749-bp amplicon [22]. The polymerase chain reaction was performed in 50-μL reaction volume, using MyTaq One-Step RT-PCR Kit (Bioline Reagents Ltd., United Kingdom). Reverse transcriptase-PCR was performed under the following cycling conditions: transcription at 50°C for 30 min; pre-denaturation at 95°C for 5 min; 40 cycles of denaturation at 94°C for 45 s, annealing at 62°C for 45 s, extension at 72°C for 45 s; and final extension at 72°C for 5 min. The PCR product was visualized by electrophoresis on 1.2% agarose in 1× Tris-borate-EDTA buffer. Amplified samples were sequenced at First BASE (Apical Scientific, Selangor, Malaysia) and analyzed using Mega X (https://www.megasoftware.net/) [23]. The sequences reading were compared with VP1 gene sequences of IBDV available in GenBank [24].
Macroscopic lesions of involved organs in IBD cases were collected from commercial broiler farms are shown in Table-

Histopathological analysis
Obtained histopathological changes involving organs are grouped according to lesion type (Table-3 (Table-4). The phylogram (Figure-3) formed two subclusters within the B2 genotype. Furthermore, 19 out of 22 samples fell into a separate cluster from vvIBD in the B2 genotype, whereas three samples clustered with the vvIBD reference virus genogroup B2 from China, and Japan.

Discussion
The clinical symptoms observed in this study are consistent with those reported elsewhere. Chickens infected with IBDV exhibit general weakness, whitish diarrhea, dirty cloaca, and tremors [25][26][27][28]. These clinical symptoms are distinct from those of similar diseases, such as inclusion bodies hepatitis (IBH), reported by Wibowo et al. [29], or experimental study post-challenge [30]. Furthermore, chickens with coccidiosis exhibit general weakness, sudden death, and depression without thigh muscle hemorrhage and bursal edema [31]. The mortality rate in this study is between 3.82% to 20.41%. Zannah et al. [26] reported the mortality rate of IBDV infection was 4.65%. However, higher mortality rates have been claimed to 40% [15], 51% [25], and 77.73%-98.56% [32].  The bursa of Fabricius is the primary organ affected by IBDV infection and generally presents with inflammation, edema, hyperemia, hemorrhage, and atrophy [33]. Bursal atrophy may occur 3-5 d after infection [34]. In sub-chronic or chronic IBD, bursal weight was reported to be significantly   decreased [35]. This is due to atrophy following recovery from infection. Atrophy was also detected in other lymphoid organs, such as the thymus [36]. Bursal atrophy was also found in Marek's disease and chicken anemia virus infection [31]. In addition to the major immune organ bursa of Fabricius, other organs, such as the thymus, spleen, proventricular-ventricular junction, kidney, and thigh muscles, are targets of IBDV infection [37]. Akter et al. [38] consistently reported hemorrhage in the proventricular-ventricular junction and thigh muscle, as well as swollen kidneys.
Wibowo et al. [29] reported swelling and hemorrhaging of the bursa of Fabricius, swollen kidneys with uric acid deposits, and hemorrhaging in IBH. A pale and swollen liver with multifocal necrosis is always present in IBH. Hydro pericarditis is frequently seen  in cases of IBH [29]. Liver lesions are distinct characteristic that differentiates between IBH and IBD [29].
In an experimental mix infection model of IBD and IBH, liver necrosis and bursa of Fabricius atrophy were seen after 5 d post infection [39]. Histopathological lesions from bursal samples ( Table-2) exhibited follicular vacuolization (23.08%), edema (3.85%), heterophile infiltration (3.85%), follicle atrophy (61.54%), congestion (15.38%), and hemorrhage (15.38%). Bursal hemorrhage was less observed than atrophy, which may be related to sample collection time at the post-peak period of the outbreak [40][41][42]. Lymphocyte defects and vacuolization of the bursa of Fabricius in this study are consistent with Kulsum et al [43]. Furthermore, histopathological studies have revealed severe lymphoid depletion and heterophile and macrophage infiltration in the interfollicular space of the bursa of Fabricius [38,43]. Widespread acute lymphoid necrosis, follicular hemorrhage, and stromal edema in the bursa of Fabricius were identified as an acute form of IBD [44]. Challenge experimental study of IBDV field isolate has been reported to generate a liver lesion. The liver of 25-day-old chickens inoculated with the field isolate of IBDV exhibited congestion on day 1 post-inoculation, followed by localized necrosis, hemorrhage, and fatty degeneration [45]. However, liver lesion is uncommon in IBD field cases (interviews report with poultry practitioner). The IBD lesion could be differentiated with IBH which mostly exhibits swollen and multifocal necrosis liver [46]. Other differential diagnoses of IBD are avian coccidiosis, chicken anemia virus infection, mycotoxicosis, and Marek's disease [31]. Multifocal follicle necrosis was observed in the thymus and spleen by Zahid et al. [47]. A previous study has reported renal tubule hypertrophy in the kidney on days 3-4 post-inoculation of IBD. The proximal segment of the tubules had empty areas (vacuoles) that extended to the lumen of the distal tubule [47]. The renal tubules exhibited necrosis and the glomerulus showed atrophy and an increased size of capsular (vacuolization) [48].
The molecular analysis could only confirm 22 samples by RT-PCR, possibly the viral load was too low because samples were collected after peak infection [49]. This evidence is supported by macroscopic and microscopic analyses indicated chronic infection of the bursa of Fabricius. Infectious bursal disease virus load concentrations peaked 3-4 days after infection; however, chronic lesions in the bursa of Fabricius occurred 7-10 days after infection [49].
Based on sequence analysis, some variations were detected in amino acid residues D242E, T287A, L360M, and E393D of all isolates in this study. Mutations in residues D242E, T287A, L360M, and E393D were reported to alter pathogenicity from classic to very virulent strain [50,51,52,53].
Our study has limitations. The genetic properties of identified vvIBD strains must be further explored. Moreover, this study should include a larger area of samples and include cases from layer farms.

Conclusion
Broiler chickens infected with IBDV showed specific macroscopic and microscopic lesions in the bursa of Fabricius and thigh muscle. Moreover, other organs such as the spleen, thymus, proventricular-ventricular junction, and kidney were affected. Molecular analysis of the VP1 gene fragment has grouped the virus into vvIBD of B2 genotype. This study has provided evidence of the latest IBDV genotype circulating in broilers in Indonesia. Most studies on IBDV have focused on the VP1 gene; however, some studies have suggested using VP2 for generating more effective IBDV vaccines. Periodic monitoring of the latest isolate for the probability of genome mutation and clinical signs update of IBD field cases. Because all samples were obtained from vaccinated chickens, our findings provide a new outlook regarding vaccine efficacy.