The animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Hebei Normal University of Science and Technology, Qinhuangdao, China, and were conducted in accordance with the Regulations for the Administration of Laboratory Animals issued by the Hebei Provincial Department of Science and Technology (HPDST 2020-17) and internationally accepted guidelines for the care and use of laboratory animals. All procedures involving Kunming mice (SPF (Beijing) Biotechnology Co., Ltd., Beijing, China) were performed under specific-pathogen-free conditions by trained personnel. Animals were monitored throughout the study, and humane endpoints were applied to minimize pain and distress. Mice exhibiting severe clinical signs, including marked respiratory distress, inability to access food or water, or prostration, were humanely euthanized under isoflurane anesthesia followed by cervical dislocation.

Clinical samples from diseased cattle were collected as part of routine veterinary diagnostic investigations with the permission of farm owners and did not involve additional invasive procedures beyond standard veterinary practice. Sample collection was performed by licensed veterinarians in accordance with institutional animal welfare and biosafety requirements.

From March 2023 to April 2025, clinical samples were collected from diseased cattle in seven prefecture-level cities of Hebei Province (Qinhuangdao, Chengde, Tangshan, Shijiazhuang, Hengshui, Xingtai, and Handan).

This cross-sectional study involved isolation, molecular characterization, antimicrobial susceptibility testing, and in vivo pathogenicity assessment of P. multocida isolates.

A total of 162 clinical samples were collected from diseased cattle in seven prefecture-level cities in Hebei Province, including Qinhuangdao, Chengde, Tangshan, Shijiazhuang, Hengshui, Xingtai, and Handan. The samples comprised nasal swabs from cattle with apparent respiratory symptoms and lung tissues from cattle that died of pneumonia or septicemia. All samples were transported under refrigeration and subjected to bacterial isolation immediately upon arrival. During isolation and purification, bacterial isolates were cultured on sheep blood agar plates (Wenzhou Kangtai Biotechnology Co., Ltd., Wenzhou, China). Purified P. multocida isolates were cultured on Brain Heart Infusion (BHI) agar or in BHI (Qingdao Hope Biotechnology Co., Ltd., Qingdao, China) broth at 37 °C, and the cultures were preserved in 25% (v/v) glycerol at −80 °C for long-term storage.

Clinical samples from dairy and beef cattle were streaked onto blood agar plates for bacterial isolation. Dominant grayish-white, drop-like colonies were selected and purified. Purified isolates were subjected to Gram staining (Beijing Boao Kuoda Biotechnology Co., Ltd., Beijing, China) and identified by polymerase chain reaction (PCR) amplification of the kmt gene, following methods described by Townsend et al. [22]. Primer (Sangon Biotech Co., Ltd., Beijing, China) sequences are listed in Table 1. Bacterial genomic DNA was prepared by boiling. Briefly, 1 mL of logarithmic-phase P. multocida culture was centrifuged at 12,000 r/min for 3 min, and the supernatant was discarded. The pellet was resuspended in an equal volume of sterile saline, heated in a dry bath at 100 °C for 10 min, and centrifuged again; the supernatant was used as the DNA template. The PCR reaction mixture (20 μL) contained 10 μL of 2× Taq PCR MasterMix (CW Biotech Co., Ltd., Taizhou, China), 1 μL each of forward and reverse primers for the kmt gene, 2 μL of DNA template, and 6 μL of double-distilled water (ddH₂O), with a negative control included. The PCR amplification program was as follows: initial denaturation at 94 °C for 5 min; 30 cycles of denaturation at 94 °C for 30 s, annealing at 30 s (annealing temperatures are listed in Table 1), and extension at 72 °C for 1 min; and a final extension at 72 °C for 10 min. PCR products were detected by electrophoresis on 1.5% agarose gels. The isolates were identified as P. multocida through a combination of microbiological morphology and molecular biology methods.

Primers for capsular and LPS typing of P. multocida were synthesized according to published methods. Capsular multiplex PCR [15] and LPS multiplex PCR [9] were used to determine capsular types and LPS genotypes, respectively. Primer (Sangon Biotech Co., Ltd.) sequences are listed in Table 1. The capsular multiplex PCR reaction was carried out in a 20 μL mixture containing 10 μL of 2× Taq PCR MasterMix (CW Biotech Co., Ltd.), 0.4 μL each of five primer pairs (forward and reverse), 2 μL of DNA template, and 4 μL of ddH₂O. The LPS multiplex PCR reaction was carried out in a 25 μL mixture containing 10 μL of 2× Taq PCR MasterMix, 0.5 μL each of eight primer pairs (forward and reverse), 2 μL of DNA template, and 5 μL of ddH₂O. The PCR amplification program was identical to that described above, with annealing temperatures listed in Table 1. A negative control was included in each assay. Ambiguous results were confirmed by single-pair PCR.

According to the sequences provided in the PubMLST database, seven housekeeping genes (adk, est, pmi, zwf, mdh, gdh, and pgi) used in the RIRDC MLST scheme for P. multocida were selected. Primer (Sangon Biotech Co., Ltd.) sequences are shown in Table 1. Each housekeeping gene was amplified by PCR [23]. The PCR reaction mixture (50 μL) contained 25 μL of 2× Taq PCR MasterMix (CW Biotech Co., Ltd.), 1 μL each of forward and reverse primers for each individual housekeeping gene, 2 μL of DNA template, and 21 μL of ddH₂O, with a negative control included. The PCR amplification program was identical to that described above, with annealing temperatures listed in Table 1. PCR products with clear specific bands were sent to Sangon Biotech (Shanghai) Co., Ltd. for bidirectional sequencing and assembly. The assembled sequences of each housekeeping gene were submitted to the P. multocida RIRDC MLST database for comparison to obtain the corresponding allele numbers, and the RIRDC MLST type of each isolate was subsequently determined based on the combination of the seven allele numbers.

Based on published primer sequences, 15 virulence genes in nine categories of P. multocida [17, 24] were detected, including outer membrane protein genes (ompA and oma87), fimbrial gene (fimA), filamentous hemagglutinin gene (pfhA), neuraminidase genes (nanB and nanH), iron acquisition-related genes (exbB, exbD, tonB, and tbpA), hemoglobin-binding protein genes (hgbA and hgbB), hyaluronidase gene (PmHAS), dermonecrotic toxin gene (toxA), and adhesin gene (tadD). Primer (Sangon Biotech Co., Ltd.) sequences are listed in Table 2. The PCR reaction mixture (20 μL) contained 10 μL of 2× Taq PCR MasterMix (CW Biotech Co., Ltd.), 1 μL each of forward and reverse primers, 2 μL of DNA template, and 6 μL of ddH₂O, with a negative control included. The PCR amplification program was identical to that described above, with annealing temperatures listed in Table 2.

Based on published primer sequences, 12 resistance genes in eight antimicrobial classes [25, 26] were detected, including resistance genes of aminoglycosides (strA and strB), resistance genes of macrolides (ermA and mphE), resistance genes of tetracyclines (tetA), resistance genes of quinolones (qnrA and qnrB), beta-lactams (blaTEM and blaOXA), resistance genes of lincosamides (lnuA), resistance genes of phenicols (floR), and resistance genes of sulfonamides (sul2). The PCR reaction mixture and amplification program were identical to those used for virulence gene detection. Primer (Sangon Biotech Co., Ltd.) sequences and annealing temperatures are listed in Table 3.

The susceptibility of all isolates to 14 antimicrobial agents (Hangzhou Microbiology Reagent Co., Ltd., China) from eight classes was determined by the Kirby-Bauer disk diffusion method in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines and the Chinese health industry standard WS/T 639-2018. Escherichia coli ATCC 25922 was used as the quality control strain. After incubation at 37 °C for 18 h, the results were interpreted in accordance with CLSI and WS/T 639-2018 criteria. Isolates resistant to three or more antimicrobial classes were defined as multidrug-resistant isolates.

Three representative A:L3-ST79 isolates from different geographic regions were selected to determine the median lethal dose (LD₅₀) in Kunming mice (SPF (Beijing) Biotechnology Co., Ltd.) and to evaluate pathogenicity. The isolates were inoculated into BHI (Qingdao Hope Biotechnology Co., Ltd.) medium and cultured at 37 °C with shaking at 180 r/min for 8 h, followed by viable colony counting. The bacterial suspensions were washed twice with sterile saline and diluted to five concentration gradients ranging from 1 × 10⁶ to 1 × 10² colony-forming units (CFU)/mL. A total of 80 six-week-old female Kunming mice were randomly divided into 16 groups, including one control group and 15 experimental groups (five groups each for isolates QA-1, TA-1, and SA-1). Each mouse in the challenge groups received an intraperitoneal injection of 0.2 mL of bacterial suspension, whereas the control group received an equal volume of sterile saline. Mice were observed continuously for 7 days. Individuals exhibiting severe respiratory distress, inability to access food or water, or prostration were euthanized humanely by cervical dislocation under isoflurane anesthesia, and the time of death was recorded. The LD₅₀ values were calculated using the Karber method.

Statistical analyses were performed using GraphPad Prism 11.0.0. Fisher's exact test in 2×2 contingency tables was used to analyze the association between A:L3 and ST79, as well as differences in gene carriage and antimicrobial susceptibility among isolates. A p-value of < 0.05 was considered statistically significant. All percentages presented in this study were rounded to one decimal place.

A total of 64 purified bacterial isolates were obtained from 162 clinical samples collected from diseased cattle in seven cities of Hebei Province after isolation on sheep blood agar plates. The isolates formed typical grayish-white, moist, smooth, translucent, drop-like colonies without hemolysis on blood agar plates (Figure 1A). Gram staining showed Gram-negative coccobacilli with clear bipolar staining (Figure 1B). All isolates yielded a specific PCR band of approximately 460 bp for the kmt gene, consistent with the expected fragment size (Figure 1C). Based on colony morphology, staining characteristics, and molecular identification, all 64 isolates were confirmed as P. multocida.

A total of 162 clinical samples were collected from diseased cattle, yielding 64 P. multocida isolates, for an overall isolation rate of 39.5% (Table 4). Samples were collected from seven prefecture-level cities in Hebei Province: Qinhuangdao, Chengde, Tangshan, Shijiazhuang, Hengshui, Xingtai, and Handan. Among them, Qinhuangdao contributed 59 samples, from which 27 isolates were obtained, yielding an isolation rate of 45.8% and accounting for 42.2% of all isolates, making it the major source region (Figure 2). Nine isolates were obtained from Shijiazhuang (42.9%), five from Handan (41.7%), three from Hengshui (37.5%), nine from Xingtai (36.0%), seven from Tangshan (30.4%), and four from Chengde (28.6%). The isolation rates ranged from 28.6% to 45.8% across regions, indicating that P. multocida was distributed in both northern and southern Hebei Province.

Capsular typing of the 64 P. multocida isolates showed that 61 isolates (95.3%) belonged to capsular type A, which was the overwhelmingly predominant serotype (Table 5, Figure 3). Types B, D, and F were each represented by one isolate (1.6% each), whereas type E was not detected. LPS genotyping showed that L3 was the predominant genotype, with 56 isolates (87.5%), followed by L6 with seven isolates (10.9%) and L2 with one isolate (1.6%). L1, L4, L5, L7, and L8 were not detected. Among the 61 capsular type A isolates, 54 (88.5%) were L3 and seven (11.5%) were L6. Taken together, capsular and LPS typing results showed that A:L3 was the predominant circulating type among bovine P. multocida isolates in Hebei Province, accounting for 54 isolates (84.4%) of all isolates.

The 64 P. multocida isolates were classified into eight sequence types (STs) based on amplification and sequencing of seven housekeeping genes and MLST analysis (Table 6). ST79 was the predominant type, comprising 51 isolates (79.7%). ST552 accounted for seven isolates (10.9%), whereas ST5, ST394, ST123, ST122, ST125, and ST9 were each represented by one isolate (1.6% each). Among capsular type A isolates, ST79 accounted for 83.6%. Among the predominant A:L3 isolates, ST79 accounted for 94.4% (51/54). Fisher's exact test indicated a strong association between ST79 and A:L3 (p < 0.0001, Φ = 0.8523), suggesting that this lineage represents the dominant clonal lineage of bovine P. multocida in Hebei Province.

Fifteen virulence genes from nine categories were detected in this study. The results showed that eight genes, ompA, oma87, fimA, nanH, exbB, exbD, tonB, and hgbA, had detection rates of 100% and were stably carried by all isolates (Figure 4). The toxA gene was not detected in any of the 64 isolates. The detection rates of pfhA, tbpA, PmHAS, and tadD were all 96.9%. The overall detection rates of nanB and hgbB were 23.4% and 17.2%, respectively. Notably, the detection rates of nanB and hgbB were only 13.7% and 7.8% in A:L3 isolates, respectively, whereas both genes were detected in 100% of A:L6 isolates. Fisher's exact test revealed significant differences in the carriage of both genes between A:L3 and A:L6 isolates (p < 0.0001), with nanB and hgbB being more prevalent in A:L6 (|Φ| = 0.6597 and |Φ| = 0.7676, respectively). This LPS genotype-associated difference suggests genetic differentiation among LPS types in adhesion, colonization, and iron-utilization capacity.

PCR detection of 12 antimicrobial resistance genes from eight antimicrobial classes identified six resistance genes, whereas the remaining six genes were not detected. The macrolide resistance gene mphE had the highest detection rate, 92.2% (59/64), followed by the sulfonamide resistance gene sul2, 87.5% (56/64), making these the main resistance genes carried by the isolates (Table 7). The beta-lactam resistance gene blaTEM was detected in 35.9% (23/64) of isolates. The aminoglycoside resistance genes strA and strB were detected in 12.5% (8/64) and 9.4% (6/64) of isolates, respectively. The phenicol resistance gene floR was positive in only one isolate, with a detection rate of 1.6%. ermA, tetA, qnrA, qnrB, blaOXA, and lnuA were not detected.

The susceptibility of the isolates to 14 antimicrobial agents from eight classes was determined by the Kirby–Bauer method. The results (Figure 5) showed that the isolates were highly susceptible to doxycycline (DOX, 30 μg), enrofloxacin (ENR, 10 μg), ciprofloxacin (CIP, 5 μg), ceftiofur (CFT, 30 μg), and florfenicol (FFC, 30 μg), with susceptibility rates of 90.6-100%, and no or very few resistant isolates were detected for these agents. In contrast, the isolates showed generally high-level resistance to lincomycin (LIN, 2 μg), erythromycin (E, 15 μg), tylosin (TYL, 30 μg), and sulfadiazine (SUD, 300 μg), with resistance rates ranging from 93.8% to 100%. The overall susceptibility rates to tetracycline (TE, 30 μg), kanamycin (K, 30 μg), and gentamicin (GEN, 10 μg) were relatively high (>70%); however, isolates from Xingtai showed resistance rates exceeding 65% to all three agents, and Fisher's exact test revealed significant differences in resistance to these three antibiotics between Xingtai and other regions (p < 0.0001). Ceftriaxone (CRO, 30 μg) and ampicillin (AMP, 10 μg) showed relatively high intermediate rates (29.7% and 40.6%, respectively), but their resistance rates were low (<25%). Multidrug resistance analysis showed that 61 isolates were multidrug-resistant, corresponding to a multidrug resistance rate of 95.3% (Table 8). Thirty-five isolates (54.7%) were resistant to three antimicrobial classes, 14 (21.9%) to four classes, seven (10.9%) to five classes, and four (7.8%) to six classes. Resistance to three antimicrobial classes was the predominant multidrug resistance pattern.

Three representative A:L3-ST79 isolates, QA-1, TA-1, and SA-1, were selected to determine LD₅₀ values in Kunming mice. All three isolates showed strong pathogenicity. Mice in the control group exhibited normal mental status, smooth and glossy fur, and normal body posture. Mice in the medium- and high-dose groups all developed obvious pathological symptoms, including rough and dull fur, weight loss, reduced activity, dyspnea, and lethargy. The LD₅₀ of QA-1 was 8.06 × 10¹ CFU, indicating extremely high virulence; the LD₅₀ values of TA-1 and SA-1 were 6.44 × 10³ CFU and 2.76 × 10³ CFU, respectively (Table 9). The clear differences in LD₅₀ among different isolates within the same clonal lineage suggest that virulence phenotypes may remain heterogeneous even when genotypes are consistent.

Virulence gene detection showed that core virulence factors, including ompA, oma87, fimA, nanH, exbB, exbD, tonB, hgbA, pfhA, tbpA, PmHAS, and tadD were highly prevalent among the predominant isolates. These genes are primarily involved in key pathogenic processes, including bacterial adhesion, colonization, iron acquisition, and host invasion, indicating that circulating isolates in Hebei Province possess a relatively complete molecular basis for pathogenicity [3]. Among them, outer membrane proteins and fimbrial factors may facilitate early adhesion of the pathogen to the respiratory mucosa and persistent colonization, whereas iron acquisition-related genes may help the bacteria maintain growth and survival under iron-limited conditions in the host [1, 36]. Notably, the toxA gene was not detected in any of the isolates in this study. toxA encodes the P. multocida toxin (PMT), which is primarily associated with the development of progressive atrophic rhinitis (PAR) in pigs. This gene is sparsely distributed among P. multocida strains and is mainly found in type D and some type A isolates [17]. Bovine P. multocida primarily causes pneumonia and septicemia, with type A being the predominant serotype [37]; therefore, the absence of toxA in this study is consistent with this host–serotype association and aligns with previous reports on bovine isolates. In addition, although the overall carriage rates of nanB and hgbB in the isolates of this study differed significantly from the reports by Katsuda et al. and Ewers et al. [11, 17], they were respectively consistent with the findings of Verma et al. [12], Ujvári et al. [37], and Calderón et al. [38]. Both nanB and nanH genes encode sialidases; specifically, the NanH sialidase primarily hydrolyzes Neu5Acα (2→3) Gal, whereas NanB exhibits broad-spectrum sialidase activity capable of hydrolyzing both Neu5Acα (2→3) Gal and Neu5Acα (2→6) Gal [39]. The bovine respiratory tract, from the trachea to the alveoli, is predominantly enriched with Neu5Acα (2→3) Gal [40], and the NanH sialidase alone is sufficient to meet the nutritional requirements. Under long-term environmental selective pressure, the nanB gene may be lost, and the differential carriage of nanB and nanH observed in this study reflects the host-adaptive evolution of P. multocida. Both hgbA and hgbB genes encode hemoglobin-binding proteins; previous studies have demonstrated that the hgbA gene is widely distributed among P. multocida strains regardless of capsular type or host origin, whereas the prevalence of the hgbB gene varies with host origin and disease status [41, 42]. Furthermore, the significant differences in the carriage rates of nanB and hgbB among isolates with different LPS genotypes indicate that even within capsular type A isolates, substantial heterogeneity may exist in host preference, adhesion strategies, iron-utilization capacity, and virulence expression patterns [7].

Antimicrobial resistance analysis further indicated that resistance among bovine P. multocida isolates from Hebei Province warrants close attention. In this study, the multidrug resistance rate reached 95.3%. mphE and sul2 were the main resistance genes detected, generally consistent with the high resistance phenotypes observed to macrolides and sulfonamides. This result aligns with global resistance trends; moreover, additional resistance mechanisms to lincosamides beyond those identified in this study may exist in P. multocida [18, 43]. The high resistance rates observed for lincomycin, sulfadiazine, and related agents in this study are consistent with historical and ongoing patterns of lax regulatory oversight and easy access to these agents in veterinary practice. In China, these drugs were historically used as feed additives without prescription requirements until the nationwide withdrawal of growth-promoting drug feed additives in 2020 [44]. Furthermore, local enforcement of veterinary drug regulations has been fragmented, with little monitoring of antibiotic use or resistance in animal populations [45]. Globally, these agents share similar regulatory trajectories [46, 47]. The generic availability and low cost of these older antimicrobial classes further facilitate their widespread and often inappropriate use in intensive farming systems [48]. These factors collectively contribute to the continuous selective pressure that enriches resistant strains and promotes the dissemination of resistance genes such as mphE and sul2 within P. multocida populations.

On the other hand, the high susceptibility observed for doxycycline, enrofloxacin, ceftiofur, and florfenicol in this study may be attributed to the strict regulatory oversight and prescription requirements for these agents in veterinary practice. These drugs are classified as veterinary prescription-only medicines or critically important antimicrobials with restricted use [49, 50], indicating that they may still retain reference value for clinical treatment when used legally and appropriately. The limited use of these agents in cattle production reduces selective pressure, thereby preserving their efficacy against P. multocida. Notably, the high resistance rates of Xingtai isolates to tetracycline, kanamycin, and gentamicin may be attributed to local antimicrobial use practices. National surveillance data on antimicrobial use in Chinese animal husbandry from 2018 to 2020 showed that tetracyclines, despite a declining trend, remained the most frequently used antimicrobial class, while the use of aminoglycosides increased significantly [51]. This long-term and non-standardized use of antimicrobials has created substantial selective pressure, promoting the dissemination of resistance genes among P. multocida populations. Furthermore, although regulatory oversight has been strengthened at the provincial level, challenges persist in grassroots enforcement, particularly in monitoring the off-label use of antimicrobial agents not approved for food-producing animals. To assess whether resistance profiles have expanded beyond approved veterinary drugs, ceftriaxone and ciprofloxacin were included in this study for in vitro resistance profiling and epidemiological comparison only. In addition, the genotypes and phenotypes of some antimicrobial agents were not fully consistent. For example, the detection rate of the β-lactam resistance gene blaTEM was higher than the ampicillin resistance rate, suggesting that resistance phenotypes may be influenced not only by the presence of resistance genes but also by factors such as gene expression levels and enzyme activity. Specifically, even when blaTEM is present, weak promoter activity leading to insufficient expression [52], or amino acid mutations in TEM-1 altering substrate specificity, may result in β-lactamase production or activity inadequate to hydrolyze clinically relevant concentrations of ampicillin [53]. In practice, antimicrobial treatment should be based on approved veterinary drugs, regional susceptibility monitoring, clinical indications, and withdrawal-period requirements, to achieve precise intervention while reducing the risk of further dissemination of resistance.

Animal pathogenicity assays showed that the dominant A:L3-ST79 clone exhibited strong pathogenicity in the mouse model. In this study, the three representative isolates shared an identical core virulence gene profile; however, isolate SA-1 additionally carried the nanB and hgbB genes. Nevertheless, significant differences in LD₅₀ values were observed among the representative isolates, with QA-1 showing a lower LD₅₀ than both SA-1 and TA-1. This indicates that the additional carriage of nanB and hgbB did not confer enhanced virulence to SA-1, suggesting that virulence phenotypes are not simply determined by the number of virulence genes present but may be comprehensively regulated by factors such as gene expression levels, genomic background, and host response status [3, 5]. Therefore, carriage of virulence genes alone is insufficient to fully predict a strain’s actual pathogenicity, and comprehensive evaluation using animal models and phenotypic assays remains necessary.

From a public health perspective, human infections with P. multocida are predominantly associated with bites or scratches from cats and dogs, and no confirmed cases of human infection with bovine P. multocida have been reported in the literature to date [54]. However, given the high multidrug resistance rate (95.3%) of the dominant A:L3-ST79 clone identified in this study and the potential risk of occupational exposure, continued surveillance of the molecular characteristics and antimicrobial resistance trends of bovine isolates remains important for One Health prevention and control strategies.

Based on these findings, prevention and control of bovine P. multocida in Hebei Province should focus on the predominant circulating clone. On the one hand, continuous surveillance of the A:L3-ST79 lineage should be strengthened to dynamically monitor its distribution, antimicrobial resistance changes, and virulence evolution. On the other hand, given the high prevalence, extensive multidrug resistance, and strong pathogenicity of this clone, vaccine development and immunoprophylaxis should be designed to target this major circulating type and its core virulence factor combinations, thereby improving the specificity and coverage of prevention and control strategies. Meanwhile, vaccination can significantly reduce antimicrobial use, alleviate selective pressure driving resistance from the source, decrease treatment costs and veterinary drug residue risks, and align with the One Health framework. In addition, antimicrobial use in clinical treatment should be further standardized, and regional, precision-based medication protocols should be formulated according to susceptibility testing results. These measures should be combined with improved feeding management, environmental control, and biosafety practices to minimize the disease risk associated with stress factors such as transportation, crowding, and mixed herding, thereby reducing the persistent transmission of the dominant clone in cattle populations [27, 29].

This study has several limitations. First, the samples were collected from only seven prefecture-level cities in Hebei Province and therefore may not fully represent the epidemiological situation across the entire province. Second, the study mainly relied on PCR detection of representative virulence and resistance genes and did not integrate whole-genome sequencing or transcriptomic data. Consequently, the evolutionary relationships, virulence regulatory networks, and resistance mechanisms of the predominant clone remain incompletely resolved. Future studies should combine whole-genome sequencing and RNA-seq to systematically elucidate the pathogenic mechanisms, resistance mechanisms, and adaptive evolution of the predominant clone at the genomic and transcriptional regulatory levels, thereby providing a stronger theoretical basis for precise surveillance, targeted prevention and control, and rational clinical medication for bovine respiratory disease.

The novelty of this study lies in its comprehensive, integrated characterization of bovine P. multocida in Hebei Province, the first of its kind in this key cattle-producing region. Unlike previous studies that typically focused on single aspects (e.g., serotyping or resistance alone), we combined molecular epidemiology (capsular/LPS + RIRDC MLST), virulence gene profiling, phenotypic/genotypic antimicrobial resistance analysis, and in vivo pathogenicity testing. This revealed the dominance of the A:L3-ST79 clonal lineage, high MDR prevalence, and strong pathogenicity, findings that significantly advance understanding of regional epidemiology and provide a foundation for targeted interventions. This work fills a critical knowledge gap in northern China and offers original insights into LPS genotype-associated virulence gene variation and the persistence of specific resistance genes under local antimicrobial use patterns.