All animal procedures were reviewed and approved by the Animal Care Committee of South China Agricultural University (approval ID: SYXK-2024-0136). All procedures complied with the Guidelines for the Care and Use of Laboratory Animals issued by the Ministry of Science and Technology of China. To minimize distress, birds were euthanized following deep isoflurane anesthesia and subsequent intravenous pentobarbitone administration, according to established broiler anesthesia protocols (17).

A systematic epidemiological investigation of IC was conducted from September 2024 to October 2025 across nine provinces in China (Guangdong, Guangxi, Jiangsu, Zhejiang, Sichuan, Hubei, Hebei, Henan, and Anhui). Swabs from the infraorbital sinuses and/or nasal exudates were collected from chickens exhibiting characteristic clinical signs of IC, including facial swelling and nasal discharge. Samples were transported under cold-chain conditions and refrigerated at 4 °C until processing. A total of 200 samples were obtained, and their geographic distribution is detailed in Table 1. The number of samples obtained from each province was determined by the availability of clinically suspected IC cases and voluntary diagnostic submissions during the study period, rather than by a predefined or evenly stratified sampling design. Consequently, sample sizes varied substantially among provinces.

Following surface disinfection of the head, infraorbital sinuses were aseptically incised. Exudates were collected using sterile inoculating loops and streaked onto tryptic soy agar (TSA) supplemented with 10% heat-inactivated chicken serum and 0.02% β-nicotinamide adenine dinucleotide (β-NAD). Plates were incubated at 37 °C in a 10% CO₂ atmosphere for 24 h. Presumptive colonies exhibiting smooth, dew-drop-like morphology were subcultured in tryptic soy broth (TSB) containing 10% chicken serum and 0.01% NAD, and incubated at 37 °C for 6 h with shaking (180 rpm). For purification, 3–5 single colonies were streaked to obtain pure cultures. Colony and cellular morphology were examined visually and via Gram staining and microscopy, respectively. Species-level identification was confirmed by PCR amplification of an ~1,600 bp fragment from the hmtp210 gene using established primers (18). Isolates yielding the expected amplicon were identified as A. paragallinarum. A previously characterized A. paragallinarum reference strain (CVCC4341) was included as a positive control in each PCR run to verify assay specificity and performance, and its identity had been confirmed by sequencing. Due to limitations related to sample quality, transport conditions, and biosafety requirements, bacterial isolation was attempted only for a subset of PCR-positive samples with sufficient volume and appropriate storage conditions.

Bacterial genomic DNA was extracted exclusively from purified A. paragallinarum colonies obtained as described in this study. DNA extraction was conducted using the FastPure Microbiome DNA Isolation Kit (Cat. no. DC502; Vazyme Biotech, Nanjing, China) according to the manufacturer’s instructions (Version 24.1). Briefly, approximately 400 μL of bacterial culture was transferred to a lysis tube and centrifuged at 10,000 rpm for 3 min to pellet the cells. After supernatant removal, the pellet was resuspended and incubated at 70 °C for 5 min to ensure efficient cell lysis. Following centrifugation (10,000 rpm, 1 min), the lysate was mixed with absolute ethanol and loaded onto a silica-based DNA binding column. The column was washed sequentially with Buffer WP and Buffer WB, dried by high-speed centrifugation, and genomic DNA was eluted in 50 μL of elution buffer. DNA concentration and purity were assessed using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA), and extracted DNA was stored at −20 °C until further use.

The hmtp210 gene was amplified using specific primers: HMTp210F (5’-ATGAATAAAGTTTTTAAAATTAAATATTCTGTTATTAAACAAGAAAT-3′) and HMTp210R (5’-CTAAAAGGTAAATCCAACACTCATTGCTGCCC-3′), which generate an approximately 6100 bp amplicon (19). PCR reactions (50 μL) consisted of 25 μL PrimeSTAR HS Premix (Takara Bio), 10 pM of each primer, and 2 μL of template DNA. Reactions were carried out on a T100 Thermal Cycler (Bio-Rad Laboratories, Hercules, CA, United States) under the following cycling conditions: an initial denaturation at 98 °C for 3 min; 32 cycles of denaturation at 98 °C for 15 s, annealing at 55 °C for 10 s, and extension at 72 °C for 35 s; followed by a final extension at 72 °C for 5 min. PCR products were held at 4 °C until further analysis. Amplified PCR products were separated by 1% agarose gel electrophoresis. Target bands were excised and purified using a gel extraction kit (Solarbio). Purified amplicons were A-tailed using a DNA A-Tailing Kit (Takara Bio), ligated into the pMD19-T vector (Takara Bio), and transformed into Escherichia coli DH5α competent cells. For each isolate, three independent positive clones were selected for bidirectional sequencing by Sangon Biotech (Shanghai, China). Sequences were assembled and analyzed using the SeqMan program of DNASTAR Lasergene 7.1 software. The hmtp210 sequences from the 22 obtained isolates were aligned with those from 15 reference strains (Table 2) using the ClustalW algorithm in MEGA11. A phylogenetic tree was constructed via the neighbor-joining method with 1,000 bootstrap replicates to assess genetic relationships and molecular serotype classification.

The serotype A isolate Apg-159-GX and the serotype C isolate Apg-108-GX used in this study were clinical isolates obtained in the present investigation and were selected from the 22 confirmed Avibacterium paragallinarum isolates for challenge experiments. To assess the pathogenicity and transmissibility, an experimental infection study was performed using 50-day-old specific pathogen-free (SPF) chickens. Birds were randomly assigned into three groups: Group 1 (n = 15) was inoculated with serotype A isolate Apg-159-GX, Group 2 (n = 15) with serotype C isolate Apg-108-GX, and Group 3 (n = 15) served as the unchallenged control. For Groups 1 and 2, ten birds per group were directly challenged via inoculation into the left infraorbital sinus with 0.2 mL of a bacterial suspension containing 1 × 10⁴ CFU/mL. The remaining five birds per group were housed as in-contact sentinels to assess horizontal transmission. Control group birds received 0.2 mL of phosphate-buffered saline (PBS) via the same route. The challenge dose was selected based on previously published experimental infection models of infectious coryza, in which an inoculum of approximately 10⁴ CFU was demonstrated to reliably induce typical clinical signs without causing excessive mortality (20).

All chickens were monitored daily for 8 days post-challenge (dpc). Clinical signs were evaluated using a standardized clinical scoring system adapted from a previously published method for infectious coryza, with minor modifications to better reflect the severity of infraorbital sinus involvement observed under the present experimental conditions (8). Briefly, clinical signs were scored as follows: 0 = no signs; 1 = mild facial swelling/serous exudate; 2 = moderate swelling with mucous nasal discharge; 3 = severe swelling with copious exudate and/or transient blindness. At 8 dpc, all surviving birds were humanely euthanized. Necropsy was performed, with a specific focus on the infraorbital sinuses to confirm the presence and severity of caseous exudate characteristic of infectious coryza.

Statistical analyses were conducted using GraphPad Prism 9 (GraphPad Software Inc., San Diego, United States). Differences in clinical scores between groups across time points were analyzed using two-way analysis of variance (ANOVA). A p-value < 0.05 was considered statistically significant.

A total of 200 clinical samples collected from nine major poultry-producing provinces were analyzed to determine the geographic distribution, seasonal patterns, and serotype composition of A. paragallinarum. A. paragallinarum detection varied substantially among provinces (Figure 1A; Table 1). The highest positivity rates were observed in Anhui (100.0%, 4/4), followed by Guangxi (75.8%, 47/62) and Guangdong (73.3%, 11/15). Moderate detection rates were recorded in Jiangsu (65.8%, 52/79), Henan (66.7%, 2/3), and Hebei (63.6%, 7/11), while a relatively low rate was found in Zhejiang (30.8%, 4/13). All samples from Sichuan (0/7) and Hubei (0/6) tested negative, revealing a pronounced regional heterogeneity with circulation concentrated primarily in southern and eastern China. These provincial detection rates should be interpreted with caution, particularly for regions with limited sample numbers, as the present study was not designed to estimate province-level prevalence.

Serotyping identified clear geographic differences in serotype distribution (Table 1). Serotype A was the most widespread, detected in Jiangsu (29 positive samples), Guangxi (24 positive samples), Guangdong (8 positive samples), Hebei (7 positive samples), Anhui (4 positive samples), and Henan (2 positive samples). In contrast, serotype B was identified in only one sample from Guangxi and one from Guangdong. Serotype C exhibited regional clustering, predominating in Jiangsu (23 positive samples) and Guangxi (22 positive samples), with lower occurrence in Zhejiang (4 positive samples) and Guangdong (2 positive samples). Serotype C was absent from Sichuan, Hubei, Hebei, Anhui, Henan. These results indicate co-circulation of serotypes A and C in key provinces (e.g., Jiangsu, Guangxi, Guangdong), a more restricted distribution of serotype A in northern/central regions, and an apparent absence of serotype B during the study period.

Seasonal dynamics revealed that a clear seasonal pattern was observed (Figure 1B). The positive rate increased from 33.3% in September 2024 to a peak of 87.0% in December 2024. A sustained high-incidence phase occurred from January to March 2025 (100.0%), followed by a sharp decline to 0% in April 2025. Positivity remained low throughout May–September. A distinct resurgence was observed in October 2025 (54.2%), establishing a stable, annual epidemic cycle with peak activity during late autumn and winter.

A. paragallinarum was isolated from infraorbital sinus exudates collected from clinically suspected IC cases. From the 200 field samples, 22 isolates with phenotypic characteristics consistent with A. paragallinarum were recovered. Although a substantially larger number of samples tested positive by PCR, successful bacterial isolation was limited to these 22 isolates. On TSA supplemented with 10% chicken serum and NAD, colonies appeared as grey-white, round, smooth, and dew-drop-like (Figure 2A). Microscopic examination after Gram staining confirmed Gram-negative coccobacilli (Figure 2B). All isolates exhibited the characteristic satellite growth around Staphylococcus aureus streaks, confirming their NAD dependency (Figure 2C). To ensure clonal purity, isolates were subcultured, and 3–5 well-isolated colonies from each were subjected to species-specific PCR targeting the hmtp210 gene. All selected colonies yielded the expected ~1,600 bp amplicon (Figure 2D), confirming their identity as A. paragallinarum and verifying the successful isolation of genetically representative strains.

A phylogenetic tree was constructed based on the hmtp210 gene sequences from the 22 isolates and 15 reference strains representing serotypes A, B, and C. The GenBank accession numbers of the clinical isolates and reference strains are listed separately in Table 2. The resulting phylogeny (Figure 3) revealed that all isolates clustered into three distinct, well-supported clades corresponding to serotypes A, B and C, demonstrating strong concordance between molecular genotyping and conventional serotyping.

The majority of isolates (15 out of 22) formed a monophyletic clade with 7 reference serotype A strains, indicating a genetically homogeneous lineage circulating in the sampled regions. Two isolates clustered closely with a reference serotype B strain Spross. The remaining 5 isolates clustered with 2 reference serotype C strains (TW07, TW13), reflecting their close evolutionary relationship. These results confirm the predominance of serotype A among circulating strains in the sampled regions during the study period.

Among the 22 confirmed A. paragallinarum isolates, Apg-159-GX (serotype A) and Apg-108-GX (serotype C) were selected for experimental challenge. Following direct infraorbital sinus inoculation of SPF chickens, both representative strains—Apg-159-GX (serotype A) and Apg-108-GX (serotype C)—induced typical infectious coryza. Clinical signs emerged at 2 dpc, peaked at 4 dpc, and largely resolved by 8 dpc. As shown in Figure 4, directly inoculated birds from both serotype groups exhibited significantly higher clinical scores than controls at 2 and 4 dpc (p < 0.05). At 4 dpc, clinical scores in the Apg-159-GX direct-challenge group were also significantly higher than in its contact-exposed group (p < 0.05), whereas no significant difference was observed between the Apg-108-GX challenge and contact groups (p > 0.05). By 8 dpc, clinical scores did not differ significantly among any groups (p > 0.05). No significant differences in mean clinical scores were observed between the two serotypes under direct-challenge conditions, indicating comparable overall clinical virulence in this model.

Gross pathology. Gross lesion assessment corroborated the clinical findings (Figure 5). Control birds showed no abnormalities (Figures 5A,B). Directly challenged birds from both serotype groups developed classic IC lesions, including periocular swelling, conjunctivitis, and accumulation of yellow caseous exudate in the infraorbital sinus (Figures 5C–F). Contact-exposed birds displayed similar but milder lesions (Figures 5G–J), consistent with a lower infectious dose during natural transmission.