2.1. Samples

From April to August 2021, 145 broiler farms were randomly selected, taking samples from five dead chickens per farm at slaughterhouses with ages ranging from 24-day-old to 38-day-old that originated from each farm in nine provinces. The number of farms for surveillance at slaughterhouses was determined based on the proportions of the number of chickens slaughtered per abattoir in 2019 [11]. The dead chicken carcasses were necropsied the same day or the following day after refrigeration to avoid misinterpretation of expected postmortem changes.

During the necropsy, samples for bacteriological culture, virus detection, and histopathological examination were collected. The surface of the livers was seared with a hot spatula, and samples were obtained using a sterile swab for bacterial examination. Swab samples were subsequently cultured on blood agar and MacConkey agar plates. After incubation overnight at 37°C, a single colony was selected, and species identity was determined using the Vitek 2 system (bioMérieux, France) according to the manufacturer’s instructions. Tracheas, livers, cecal tonsils, bursas, and kidneys were obtained from five carcasses per farm and were pooled by organs.

The collected organs for the virus detection were homogenized in 10% phosphate-buffered saline and stored at −70°C until processing. Sections of the liver organs with lesions, including tracheas, liver, kidneys, and bursa, were collected and fixed in 5% buffered formalin. The samples were embedded in paraffin blocks, and paraffin wax sections were cut (5 µm), dewaxed, stained with hematoxylin and eosin, and examined using light microscopy.

2.2. Detection of FAdV, IBDV, and IBV

DNA/RNA was extracted from homogenized samples (liver and cecal tonsil for FAdV, trachea, kidney, and cecal tonsil for IBV and bursa for IBDV) using a Maelstrom 4000 DNA/RNA auto-extraction machine (TAN bead, Taiwan). PCR and RT-PCR assays were conducted using the specific primer pairs for the hexon gene of the FAdV, VP2 gene of the IBDV, and 3^′untranslated region of the IBV as described previously [12–14] (Table S1).

2.3. Isolation of IBV

Samples that were positive for IBV using RT-PCR were propagated in 10 days old specific pathogen-free embryonated chicken eggs at 37°C for 72 h. Allantoic fluids were harvested and centrifuged at 2851 g for 10 min. The supernatants were collected. RT-PCR amplifying the S1 region was used to confirm the genotype of IBV, as described previously [15].

2.4. Sequencing and Phylogenetic Analysis

Direct sequencing of the PCR product was performed using an ABI 3730xl DNA analyzer. The sequences of the isolated viruses were aligned and edited with CLC Main Workbench 7.7.2 (CLC bio, Aarhus, Denmark). The partial hexon gene of FAdV sequences, the partial VP2 gene of IBDVs, and the partial S1 gene of IBVs used in this study were deposited in the GenBank database under accession numbers (Table S2). Nucleotide (nt) and amino acid (aa) sequences were aligned and trimmed using the CLC Main workbench. The phylogenetic tree of FAdV was generated by a maximum likelihood method with 1000 bootstrap replications, and phylogenetic trees of IBDV and IBV were generated by a neighbor-joining method with 1000 bootstrap replications using MEGA 11 software [16], with related species published in the DDBJ/EMBL/GenBank database. The phylogenetic tree of IBDV was displayed with iTOL (Interactive Tree Of Life) [17]. Sequences showing similarities were found using a GenBank nucleotide BLAST search, and nt and aa identity were calculated using BioEdit software (version 7.2.5).

2.5. Broiler Production Index (PI)

2.6. Statistical Analysis

The PI between coinfected farms and single-infected or noninfected farms was analyzed using Student’s t-test of Microsoft Excel software. The probability value (p) < 0.05 was considered statistically significant.

3.1. Detection of FAdV

FAdV was confirmed in 64 of 145 farms, which account for 44.1% of tested farms. According to the phylogenetic analysis based on hexon gene, including the L1 loop region, FAdV-11/D was detected the most in 23 farms, FAdV-5/B was positive in 19 farms, FAdV-8b/E in 12 farms, and FAdV-8a/E and FAdV-1/A in three farms, respectively. Two species of concurrent infections were detected in three farms (two farms for FAdV-5/B and 11/D and one for FAdV-11/D and 8b/E). One farm was infected with FAdV-2/D, but FAdV-4 /C was not detected (Table 1 and Figure 1a). By region, FAdV was detected at a moderate level nationwide, with a detection rate of 30.0%–55.6% in Korea. Chungnam and Jeonbuk had relatively low detection rates compared to other regions, at 30.0% and 35.9%, respectively, and no cases were detected in Jeju (Figure 2).

3.2. Diagnosis of IBH

Grossly, swollen livers and petechial hemorrhages on the liver were present in a few cases infected with FAdV-8b/E and FAdV-11/D strains, but it was not a distinct lesion.

Intranuclear inclusion bodies (INIB), including necrosis of hepatocytes and inflammatory cell infiltration, which are typical findings of IBH, were observed in FAdV-8b/E and FAdV-11/D infections in the histopathology. We confirmed IBH based on PCR assay for FAdV detection, sequencing, and microscopy. As a result, IBH was positive in 13.8% of the 145 farms tested. Of the 12 cases infected with FAdV-8b/E, six cases were confirmed as IBH, and of the 23 cases infected with FAdV-11/D, 12 cases were IBH. Not all cases infected with FAdV-11/D or FAdV-8b/E showed IBH (Table 1). Among cases diagnosed with IBH, microscopic lesions by FAdV-8b/E infections were evident, but in lesions caused by FAdV-11/D, INIB were rarely identified, and necrosis and infiltration inflammatory cells in the hepatocytes were weaker than lesions by FAdV-8b/E. Cases of other species of FAdV were not observed IBH (Figure 3).

3.3. Detection of Other Diseases

Of the 145 farms, 91 (62.8%) were confirmed to have an infection with field IBDV using RT-PCR, sequencing, and microscopic lesions. According to the phylogenetic analysis based on hypervariable regions of the VP2 gene, 75 (51.7%) farms were infected with the G2d variant, 12 (8.3%) farms were positive with the G2b variant, two farms were positive G3 virulent virus, and two farms were infected with two variant viruses together (G2b and G2d) (Figure 1b and Table 2). Histopathologically, G2 IBDV-infected bursas showed some epithelial loss, moderate lymphocyte depletion, and lymphoid follicle diffusion. G3 IBDV-infected bursas showed more severe findings. In total, 43 (29.7%) IBV-positive farms were confirmed using RT-PCR, sequencing and microscopic lesions. Three genotypes based on S1 partial gene were identified, and the predominant genotype of IBV was GI-19 (Figure 1c and Table 2). Histopathologically, IBV-infected tracheas showed loss of epithelium and lymphocyte infiltration, and GI-19 IBV-infected kidney showed damage to the renal tubular epithelial cell and lymphocyte infiltration. In total, 46 (31.7%) farms out of 145 were confirmed colibacillosis using bacterial culture and microscopic lesions.

3.4. Association of Productivity of Broiler and Coinfection of Other Pathogens

PI index was calculated on 95 of the farms tested. Among these, 17 farms had less (<300) productivity, and 39 farms each had good (<350) and excellent (>350) productivity. PI value criteria were referenced from a previous study [19]. Positive rates for each of the four diseases, IBH, G2 IBDV infection, IB, and colibacillosis, were lowest on farms with PI above 350, but there was no significant difference between farms with good productivity or less (< 350 and <300 of PI). The positive rates for two or more diseases were highest at 70.6% in farms with PI index of less than 300 and lowest at 46.2% in farms with PI of more than 350. This showed that coinfections with two or more diseases were significantly related to the productivity of the broiler (Table 3).

To determine whether the factor related to PI was the number of coinfection diseases or the impact of any viral disease infection, the PIs of six groups (three or more diseases coinfection, including IBH / G2 IBDV infection/IB, two diseases coinfection, including IBH / G2 IBDV Infection/IB) were analyzed. And those were compared with PI of single infected or uninfected farms. In three or more diseases coinfected farms, PI of groups, including IBH, G2 IBDV infection, and IB, showed significantly lower PI (p < 0.05), but there was no significant difference in PI whether or not any viral disease was included in farms with two diseases coinfection (Table 4). It means that the PI decreases significantly when infected with three or more diseases, including any one of the three diseases (IBH, IB, G2 IBDV infection).