Screening of virulence genes in Pasteurella multocida associated with poultry respiratory diseases

Introduction

Pasteurella multocida is a Gram-negative, facultative anaerobic bacterium that plays a critical role in respiratory infections among a wide range of avian species. As the causative agent of avian pasteurellosis, this pathogen is responsible for significant morbidity and mortality in poultry flocks worldwide, leading to severe economic losses in the poultry industry. The disease presents in both acute and chronic forms, varying in clinical severity and often complicating diagnosis and management. Traditionally, P. multocida classification has relied on serological methods based on capsular and somatic antigens, but these methods are complicated and have been increasingly supplemented by molecular typing techniques that identify 5 (A, B, D, E, andF) distinct capsular (Townsend et al., 2001) [31] and 8(L1-L8) LPS genotypes (Harper et al., 2015) [17] . Molecular studies reveal considerable genetic diversity among avian isolates, indicating differences in virulence and pathogenic potential across strains from different poultry species. This variation poses challenges for diagnosis, treatment, and vaccine development, highlighting the need for comprehensive characterization of P. multocida strains to better understand their role in avian diseases and improve control strategies. The pathogenic potential of P. multocida is largely determined by a complex array of virulence factors, including its polysaccharide capsule, lipopolysaccharides (LPS), outer membrane proteins (OMPs), adhesins, fimbriae, and iron acquisition systems. These components not only facilitate colonization and immune evasion but also play a crucial role in tissue damage and systemic dissemination. Molecular and histopathological investigations have highlighted the strain-specific nature of these factors, particularly in avian hosts, where variations in host immunity, environmental exposure, and geographical area can influence infection dynamics [19,26,27] . This study aims to isolate P. multocida from pulmonary lesions in chickens and analyze the virulence markers responsible for pathogenesis. Understanding these factors is essential to improve diagnostic approaches and inform prevention strategies against this highly adaptable and pathogenic organism.

Materials and Methods

Samples were collected from both clinically ill and deceased poultry birds from farms located in the different districts of Andhra Pradesh. These cases were examined at the Department of Veterinary Pathology, NTR College of Veterinary Science, Gannavaram. During necropsy, tissue samples exhibiting lesions were aseptically collected in sterile phosphate-buffered saline (PBS) for further analysis. The tissue swabs were then cultured in brain heart infusion (BHI) broth and incubated anaerobically at 37 °C for 12-18 hours using a candle jar method. Post-incubation, the samples were streaked on BHI agar, sheep blood agar (5%), and MacConkey agar to observe bacterial growth and colony characteristics. Gram's staining was performed to determine cell wall characteristics. Additionally, blood and tissue impression smears prepared at necropsy were stained with Leishman stain to identify the bipolar-staining characteristic of P. multocida organisms. Bacterial motility was assessed using the hanging drop technique, and biochemical assays were performed for catalase, oxidase, nitrate, methyl red, Voges-Proskauer, sugar fermentation, indole, citrate, gelatin liquefaction, and urease by standard methods [5,22] . For molecular confirmation and strain typing, bacterial DNA was extracted from cultured isolates using a rapid heat lysis method. Culture was suspended in phosphate-buffered saline (PBS), followed by washing, thermal lysis at 95 °C, and centrifugation to collect the DNA-containing supernatant. This DNA was used as a template for PCR-based identification. A multiplex PCR assay targeting speciesspecific and capsular-type genes was performed. Virulence typing of P. multocida was done with the detection of virulence-associated genes pfhA, ptfA, ompH, hgbA, and hgbB. The primers used in this study are given in Table 1, and the reaction conditions are given in Table 2. The DNA amplified by PCR was subjected to agarose gel electrophoresis. The bands were visualized under UV illumination using a Gel documentation system. [9] , 9.6%. by Aboob et al. (2021) [2] 16% by El-Demerdash et al.

(2023) [10] , and 12.4% by El-Tarabili et al. (2025) [11] . The bacterial cultures on Brain Heart Infusion (BHI) agar showed small, whitish, discrete, circular, and translucent colonies, which later became opaque. On blood agar, P. multocida formed small, whitish, circular colonies (Fig. 1) that were initially translucent and gradually turned opaque, with no hemolysis observed. No growth was detected on MacConkey agar plates, consistent with reports from Dogra et al. (2015) [9] and Aboob et al. (2021) [2] . Microscopic evaluation of the isolates using Gram staining revealed small, Gram-negative coccobacilli (Fig. 2) exhibiting a characteristic bipolar appearance, suggesting P. multocida. Impression smears prepared from the liver and heart of deceased birds, stained with Leishman's stain (Fig. 3), demonstrated bipolar organisms displaying a capsule. Leishman's stain proved to be a simple, rapid, and effective method for identifying the distinctive bipolar bacteria in impression smears. These staining characters are in accordance with the Markam et al. (2009) [21] , while some researchers [3,8] have utilized Giemsa and Methylene blue stains to detect P. multocida in hemorrhagic septicaemia Initially, isolates exhibiting characteristic growth on brain heart infusion (BHI) agar and blood agar, no growth on MacConkey agar, and positive results for indole, catalase, oxidase, and nitrate reduction tests were presumptively identified as P. multocida. These isolates were further tested on triple sugar iron (TSI) agar to assess hydrogen sulfide (H₂S) production, with all isolates demonstrating typical reactions consistent with P. multocida. Subsequent sugar fermentation tests showed that all isolates fermented glucose (100%) without gas production; 24 (80%) isolates fermented sucrose, and 30 (62.5%) fermented maltose, both also without gas production. The biochemical test results align with the observations of Charitha Devi et al. (2012) [6] and Penz et al. (2019) [23] . Molecular identification of P. multocida was conducted using species-specific primers KMT1T7 and KMT1SP6, which generated an amplified product of 460 base pairs (bp) in the isolates, as compared to the 100 bp molecular weight marker. Among the 289 samples analyzed, 30 (10.38%) were confirmed positive for P. multocida by PCR. For capsular typing, multiplex PCR was performed employing capsule-specific primers alongside the species-specific primers for confirmation. All isolates (100%) produced an amplified DNA fragment of 1044 bp corresponding to the capA gene, indicating that they belong to capsular group A (Fig. 4). This predominance of capsular type A among poultry P. multocida isolates aligns with previous findings [2,26,30] . Therefore, the geographical region investigated in this study primarily harbors capsular type A strains. Combining molecular identification and capsular typing into a single multiplex PCR reaction proves advantageous by saving time and reagents.

The pathogenic potential of P. multocida is largely determined by a complex array of virulence factors. The major virulence factors identified in P. multocida are capsular proteins, lipopolysaccharide, fimbriae, bacterial adhesins, and outermembrane proteins (OMPs). In the present study, the occurrence of virulence genes, namely, hgbA and hgbB (iron uptake proteins), ptfA (Type IV fimbrial protein), pfhA (haemagglutinin protein), and ompH (porin) were screened with all the isolates. The five virulence-associated genes, namely ompH, ptfA, pfhA, hgbA, and hgbB were detected using PCR in the presence of specific primers, which yielded PCR products of 438 bp, 488 bp, 275 bp, 419 bp, and 778 bp, respectively. The virulence-associated genes, namely ompH, ptfA, and hgbA were present in all isolates (100%). The ompH gene encodes an outer membrane porin protein that functions as a general transporter facilitating the diffusion of nutrients and molecules, crucial for bacterial survival and adaptation within the host. Additionally, OmpH is involved in nutrient acquisition and possibly immune evasion, supporting bacterial colonization and persistence during infection [18] . The ptfA encodes a type IV fimbrial protein, an adhesion factor that enables the bacterium to attach to host mucosal surfaces. Adhesion is a critical first step for colonization and establishment of infection, allowing the bacteria to resist clearance by host defenses [24] . The hgbA codes for a hemoglobin binding protein involved in iron acquisition from the host. Iron uptake is critical for bacterial growth and virulence because iron is a limited but essential nutrient within the host environment. The presence of hgbA ensures that P. multocida can efficiently scavange iron from host hemoglobin, promoting survival and multiplication [13] . Filamentous hemagglutinins encoded by the pfhA gene have an important role in the colonization of bacteria in the upper respiratory tract initially and this gene in several avian species varies frequently [16,20] . Together, these genes facilitate key steps of infection-adherence to host cells (ptfA), nutrient acquisition including iron uptake (hgbA), and maintenance of bacterial integrity and nutrient exchange via outer membrane functions (ompH) essential for bacterial pathogenesis and successful infection. The virulence-associated genes hgbB and pfhA were 75% in Cap A isolates. The hgbB gene encodes a hemoglobinbinding outer membrane protein that helps in acquiring iron from host hemoglobin, a critical factor for bacterial survival and virulence within the host [7] . The pfhA is commonly, though not universally, found in P. multocida isolates, and its presence is linked with enhanced ability to colonize and initiate infection, especially in respiratory and oral environments [28] . Among all the 40 isolates, 30 isolates possessed all the virulent genes, and the other 10 isolates lacked some virulent genes (hgbB and ptfH), suggesting that those two genes might play a less critical role in disease causation compared to others. Similar findings were reported by Ewers et al., (2006) [12] while working with virulence genes of P. multocida, reporting 100% of ompH and ptfA genes, 90% of hgbA, 85% of hgbB, and 45% pfhA from poultry.

Previous studies showed ptfA is present in 100% of isolates irrespective of their capsular type [15,29,33] . In a study by El-Demerdash et al. (2023) [10] , ptfA is present in 86.2% of isolates. Aski and Tabatabaei (2016) [4] reported the 100% occurrence of ptfA, hgbA, and ompH genes, 93% occurrence of hgbB, and 46% of pfhA genes and various degrees of other virulence genes. In contrast to the present study, Furian et al. (2013) [14] observed 100% occurrence of the hgbB virulence gene among all the isolates, whereas 92% occurrence of the ptfA gene. These results are also in agreement with the present study. Sarangi et al. (2015) [29] reported the presence of multiple virulence-associated genes ptfA, hgbA, hgbB, and ompH, which were frequently detected among Indian strains.

It is noteworthy that the presence of virulence-associated genes in P. multocida isolates varies widely across different geographical areas and study populations [25,29] . Differences in virulence profiles between investigations revealed that various P. multocida isolates may have different pathogenic mechanisms [1,20] . This study concludes that Complete presence (100%) of ptfA, hgbA, and ompH virulence genes, coupled with variable presence of other virulence genes such as hgbB and pfhA highlights the regional genetic diversity of P. multocida in Andrapradeh. These genes collectively contribute to the pathogen's ability to cause disease and represent crucial molecular determinants for epidemiological and intervention strategies, suggesting the need for ongoing surveillance of gene patterns to understand pathogenesis and inform control measures like vaccines or therapeutics.

Acknowledgement

AcknowledgmentsThe authors are thankful to Sri Venkateswara Veterinary University, Tirupati, Andhra Pradesh for providing necessary facilities for research work.

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