RJVP_13_2

Introduction

Pasteurellaceae is a family, initially proposed by Pohl in 1979 (Foster et al., 2000), which represents the commensals or vertebral parasites, unable to survive well outside the natural host. Through the application of advance genetic based taxonomy and identification methods, the family of Pasteurellaceae is now consisting of 28 genera since 1995, including the genus, Gallibacterium. Recently, Actinobacillus, Pasteurella and Haemophilus have also been added in this family as new genera (Eid et al., 2019; Fenwick and Rycroft, 2022; Chong et al., 2021). There are so many devastating and common pathogens that include Actinobacillus pleuropneumoniae, Pasteurella multocida, Avibacterium paragallinarum and Mannheimia haemolytica. Pasteurella genus contains coccobacillary to rod shaped bacteria whose width ranges from 0.3–1.0 μm while their length ranges from 1.0–2.0 μm (Peng et al., 2019; Boyce et al., 2004). They can be grown under aerobic, anaerobic and even under the microaerophilic condition at temperature ranging from 37 ℃ to 41 ℃. This genus is currently containing 12 species (Boyce et al., 2010).

It is a coccobacillus, non-motile and penicillin sensitive gram-negative bacterium (Zhao et al., 2021; Smith et al., 2021). It was first identified in a bird infected by cholera in 1878 and successfully isolated in 1880 by a French microbiologist named Louis Pasteur. This bacterium was named after the name of the scientist (Peng et al., 2019). There are four subspecies of P. multocida which include the type strain: Multocida, septica, gallicida and tigris (Harper et al., 2006). P. multocida strains are classified into serogroups or serotypes (A, B, D, E and F) on the basis of capsular antigens. This system was developed by Carter in 1955 and launched for the classification in 1963 based on haemagglutination (Christensen et al., 2022; Carter, 1955). They are also categorized into 1–16 serotypes which are based primarily on lipopolysaccharide (LPS) antigens (Smith et al., 2021; Michael et al., 2021).

P. multocida is a major pathogen among the members of Pasteurella, responsible for many animal diseases such as bovine hemorrhagic septicemia, swine atrophic rhinitis, and avian fowl cholera and severe economic losses in poultry and cattle industry (Zhao et al., 2021; Wilkie et al., 2012; Xiao et al., 2021). The processes by which these bacteria infect the mucosa, elude the innate immunity, and cause the systemic illnesses are being explained slowly. Capsule and lipopolysaccharide have been identified as the key virulence factors so far (Jiménez-Guerra, 2024). The capsule appears to have a role in bacterial phagocytosis evasion and complement resistance, whereas full lipopolysaccharide is required for bacterial survival in the host (Khamesipour et al., 2014). P. multocida toxin (PMT), putative surface adhesions, and iron acquisition proteins are among the additional virulence components discovered by both directed and random mutagenesis (Hashimi et al., 2023). Many critical virulence factors, including those necessary for adhesion and penetration of host cells, as well as for survival in a poorly nutritious and hostile environment, are likely to remain unknown (Harper et al., 2006). These diseases are associated with the particular serotype/es.

Hemorrhagic Septicemia (HS) majorly affects the livestock animals such as cattle, buffalo, camel and pigs (De Alwis, 1999; Mushtaq et al., 2022) predominantly infected either by serotype B:2 (Asian and European serotype) or serotype E:2 (African serotype) of P. multocida (Almoheer et al., 2022; Boyce et al., 2000). The observed clinical signs in affected animals are the elevation of temperature ranging from 102 to 107oF, chest and neck swelling, salivation, inappetence and protrusion of tongue. Sometimes, development of tympany, eye discharges and oedema have also been observed (Sheikh et al., 1996; Hassan and Mustafa, 1985; Asghar et al., 2023).

Progressive Atrophic Rhinitis (AR) is particularly caused by specific toxins produced by P. multocida serotype A and/or D i.e. PMT (Peng et al., 2019; Boyce et al., 2000; Guan et al., 2023). Major clinical symptoms include: nasal crusting, mucosal atrophy, paradoxical feeling of nasal congestion, decreased growth, facial distortion, enlarged nasal space and fetor while epistaxis, sinusitis and anosmia are also accompanied with these symptoms (Liva et al., 2021).

Fowl Cholera or FC, also known as avian cholera, is a serious septicemic contagious disease of poultry and wild birds that can either be present in acute or chronic form (Omaleki et al., 2022) and its etiological agents are P. multocida strains (especially serovar A:1, A:3, A:4 and serovar D to a lesser extent) (Peng et al., 2019; Saha et al., 2021). Generally, acute FC is caused by the serotype A whose symptoms include anorexia, fever, ruffled feathers, diarrhoea, oral mucous discharge and increase in breathing rate (Harper et al., 2006; Reuben et al., 2021). Apparent condition of the animals suffering from HS, AR and FC has been shown in the Figure 1.

In Figure 2, there is a visualization map of authors from 2010 to 2025 and this map was made by VOS viewer (version 1.6.20). Data was extracted using the Dimensions database using the terms, “Pasteurella multocida AND Capsular polysaccharides AND composition AND genetics AND biosynthesis”. The map shows the clusters of authors who frequently contribute and collaborate in the composition, genetics, and biosynthesis of CPS of P. multocida. Authors in yellow indicate recent active involvement in the research field.

In Figure 3, there is a visualization map of countries that have contributed so far. This map is made by VOSviewer. Data was extracted using the Dimensions database using the terms, “Pasteurella multocida AND Capsular polysaccharides AND composition AND genetics AND biosynthesis”. The map shows the clusters of countries who frequently collaborate in the composition, genetics, and biosynthesis of CPS of P. multocida. Countries in yellow indicate recent active involvement in this area of research. United States stands out as a central node with numerous connections, indicating its significant contribution. Moreover, Canada, Australia, Germany, India, and China are also leading in this research field.

Virulence factors of P. multocida

Virulence is the measure of the pathogenicity of an organism. Virulence factors or pathogenicity factors are the molecules, cellular structures or regulatory systems which enable the microorganisms for colonization, immune-evasion, immunosuppression, getting nutrients and entry/exit causing the virulence in host cell ultimately (Argüello-García and Ortega-Pierres, 2021). There are various virulence factors in P. multocida, involved in its pathogenicity (Casadevall and Pirofski, 2009) including CPS, LPS, toxins, fimbriae, adhesins, iron regulating and iron acquisition proteins, hyaluronidase and several outer membrane proteins (Boyce et al., 2010; Kubatzky, 2012; Dabo et al., 2007; Van Nguyen et al., 2023; Cao et al., 2024). Studies have shown that capsule of P. multocida is very important in the development of vaccines. A lot of research is being carried out to develop the vaccines to prevent the deaths of livestock especially in the developing countries. Due to the importance of capsular polysaccharides (CPS) and their diversity in different serotypes, they are being studied well (Somarajan et al., 2007).

Capsule

Bacterial capsule is a large polysaccharide structure lying outside the cell and can be a cause of many diseases (Peterson, 1996; Hathaway et al., 2016). It is not confined to gram negative bacteria but also present in gram positive bacteria. In contrast to slime, capsule is thicker, tightly bound to the cell wall, well organized, difficult to be washed off and consists only of polysaccharides (Baselga et al., 1994). Capsule and slime are sometimes collectively called as glycocalyx (Weinbaum et al., 2007). Monosaccharides are joined together through glycosidic linkages to make capsular polysaccharide (Weinbaum et al., 2007). Bacteria containing capsule are known as polysaccharide encapsulated bacteria (Yuan et al., 2021) and some of these bacteria make smooth or rough colonies on agar media (Orynbayev et al., 2019). P. multocida also contains capsule whose chemical composition varies in its different serotypes (A, B, D, E and F) (Rimler and Rhoades, 1987).

Capsule is an important virulence factor which means that it helps bacteria to cause disease by protecting them from phagocytosis by macrophages (Daffe and Etienne, 1999). As it contains water so it also protects from desiccation (Tipton et al., 2018; Reckseidler-Zenteno, 2012). Capsule is also important for the structural integrity and controlling the flow of nutrients. It may also act as a signal for the host to produce the needed nutrients. Capsule also helps in the adherence of bacterial cells with each other and to non-living surfaces as well.

Potential for CPS based vaccine development

CPS itself and its conjugate with other proteins have a strong potential for vaccine development. It has been observed that although CPS is essential for virulence, the antibody responses elicited against CPS are not necessarily the primary mediators of protective immunity (Kadioglu et al., 2008). Instead, other surface-exposed proteins, such as outer membrane proteins (OMPs) and lipoproteins, may serve as better vaccine targets (Hara and Nathan, 2022). Indeed, several recombinant protein vaccines targeting OMPs (e.g., PlpE and OmpH) have shown promising results in preclinical studies (Zhou et al., 2023; Jogi et al., 2023).

The challenge in developing an effective CPS-based vaccine for P. multocida lies in the intrinsic T cell-independent nature of polysaccharide antigens. Such antigens typically generate a short-lived immune response with poor immunological memory. To overcome this limitation, conjugate vaccines (chemically linked CPS to a protein carrier) have been developed for other pathogens, significantly improving immunogenicity. However, for P. multocida, the high variability in capsule composition across serogroups (A, B, D, E, and F) poses an additional hurdle for designing a broadly protective CPS-based vaccine (Hara and Nathan, 2022). This serogroup heterogeneity means that a vaccine targeting the capsule of one serogroup might not protect against another. Recent work has identified novel surface lipoproteins such as PmSLP, which are highly conserved among bovine isolates and can elicit robust and long-lasting immune responses in both mice and cattle (Fegan et al., 2024).

Structure and composition of capsule

Although, most of the P. multocida strains produce the capsule made up of polysaccharides, different strains with different chemical composition of capsule have been identified (Rimler and Rhoades, 1987).

Serotype A

Serotype A has capsule primarily made up of hyaluronic acid (HA) or hyaluronan which is a linear polysaccharide consisting of β-D-glucuronic acid (β-D-GlcA) and β-D-N-acetylglucosamine (β-D-GlcNAc) repeating units joined together with the help of alternating β-1,4 and β-1,3 glycosidic linkages (Pasomboon and Chumnanpuen, 2021; Rosner et al., 1992; Smallman et al., 2022). Its chemical structure has been shown in the Figure 4.

Serotypes B and E

Previous studies: The exact chemical composition of capsular polysaccharides of serotype B and E had not been determined till 2021 (Pasomboon and Chumnanpuen, 2021). According to the previous studies it was shown that the capsule of serotype B consists of mannose (Man), arabinose (Ara) and galactose (Gla) with the ratio of 2.0:0.5:0.8. The molecular weight was found to be 90 kDa in ziwittergent buffer by gel filtration and 900 kDa in Phosphate Buffer Saline (PBS) showing that it exists in decamer form in its native state (Pasomboon and Chumnanpuen, 2021). On the other hand, chemical composition of capsule of serotype E remained totally unclear till 2021.

Latest studies: Recently, the capsular polysaccharides of serotypes B and E have been determined and compared with respect to their structures, genetics and serology. Full nuclear magnetic resonance (NMR) spectroscopy revealed that there is a high similarity between CPS repeat units of both serotypes with minor differences. In serotype B, mannosaminuronic acid (ManNAcA) is substituted by fructose (Fruc) at the position 3 and by GlcNAc at position 4. In serotype E, linkages are reversed. At position 3, ManNAcA is substituted by GlcNAc and by fructose at position 4. The presence of glycine in the capsular polysaccharide of serotype B is a unique characteristic (Michael et al., 2021; Richardson et al., 2023). Chemical composition of capsule of serotype B and E has been shown in the Figures 5, 6.

Serotype D

Serotype D has capsule made up of heparosan (Guan et al., 2019) which is a linear polysaccharide consisting of β-D-glucuronic acid (β-D-GlcA) and α-D-N-acetylglucosamine (α-D-GlcNAc) repeating units joined together with the help of 1,4 glycosidic linkages (DeAngelis et al., 2002) as shown in the Figure 7.

Serotype F

Serotype F has capsule made up of chondroitin (Guan et al., 2019) which is a linear polysaccharide consisting of β-D-glucuronic acid (β-D-GlcA) and β-D-N-acetylgalactosamine (β-D-GalNAc) repeating units joined together with the help of 1,3 glycosidic linkages with alternating units linked by 1,4 glycosidic linkages (DeAngelis et al., 2002) as shown in the Figure 8.

Genetics of CPS

Genetic sequences of all genes of all five serotypes have been determined that are involved in the capsular biosynthesis (Chung et al., 1998) and by comparing their genetic organization with other gram-negative bacteria it has been shown that there is similarity of these sequences with group II capsule biosynthetic operons (Boyce et al., 2000). The genes can be grouped into three regions (R1-3) (Guan et al., 2019).

The first group (Region 1) produces a set of proteins that function as a delivery system, transporting essential materials to the cell’s surface. Regions 1 is highly conserved among all the serotypes of P. multocida and encode proteins to make ATP binding cassette transport system (a protein complex) (Boyce et al., 2010). This transport system plays role in the export of polysaccharides to the surface of bacteria. Role of hex in serotype A:1 and role of cexA in serotype B:2 in the transport of polysaccharides to the surface have been determined and confirmed by mutagenesis (Harper et al., 2012).

The second group (Region 2) contains genes that act like construction workers, piecing together the sugar building blocks to form the capsule. Region 2 consists of the genes that encode for the synthases for the polymerization of serotype specific polysaccharides. Synthase PmHAS is responsible for the polymerization of hyaluronic acid, synthase PmHS1 is responsible for the polymerization of heparin and synthase PmCS is responsible for the polymerization of chondroitin in serotypes A, D and F respectively (DeAngelis et al., 1998; Kane et al., 2006). Roles of these synthases have been confirmed by mutagenesis and/or functional analysis. These synthases as bifunctional transferases can add both glucuronic acid and N-acetyl glucosamine or N-acetyl galactosamine in growing polysaccharide molecule. Mutagenesis has also confirmed the role of bcbH encoded protein in type B polysaccharide synthesis (Boyce and Adler, 2001).

The third group (Region 3) makes sure the capsule is properly attached to the bacterial cell, much like an anchoring system. This coordinated process is crucial for the bacterium’s ability to protect itself and cause disease. Region 3 is also highly conserved though all the serotypes of P. multocida consisting of the genes which encode proteins responsible for lipidation as well as surface attachment of polysaccharides (Boyce et al., 2010). R3 has two genes; phyAB and lipAB. These genes encode proteins that are responsible for the substitution of phospholipids for the anchorage of CPS (Roberts, 1996). However, in serotypes B and E, loci lipA is located adjacent to Region 1 for the diagrammatical representation of the synthesis of capsular polysaccharide (Harper et al., 2012). Genetic organization of capsule biosynthetic loci of all serotypes of P. multocida has been shown in the Figure 9.

Later on, outside the capsule biosynthetic region, a cryptic heparin synthase gene was discovered in serotype D which encodes heparin synthase. Homologous genes were also discovered in other A, D and F serotypes. It was proposed without any experimental investigation that these synthases may give variations in capsule composition (DeAngelis and White, 2004).

The development of the typing of P. multocida with the help of multiplex PCR based on the gene sequences present in region 2 was facilitated by the clarification on the genetic basis for capsule biosynthesis (Townsend et al., 2001). As this PCR-based typing is much easier and reliable so this has replaced the conventional serotyping and now is being used as a gold standard throughout the world (Arumugam et al., 2011). Functions of ORFs of different serotypes has been summarized in the Table 1.

Table 1: Function of ORFs of P. multocida.

Serotype	ORFs	Function of encoded proteins
Serotype A, D, F	hexA (R1)	ATP dependent or binding protein (Chung et al., 1998)
	hexB (R1)	Proteins present in inner membrane (Chung et al., 1998)
	hexC (R1)	Proteins present in inner membrane and periplasmic domain (Chung et al., 1998)
	hexD (R1)	Proteins present in outer membrane (lipoprotein) (Chung et al., 1998)
	hyaB/dcbB/fcbB (R2)	Glycosyltransferases (Townsend et al., 2001)
	hyaC/dcbC/fcbC (R2)	Changes the UDP-glucose to UDP-glucuronic acid as UDP glucose dehydrogenase (Boyce et al., 2000; Townsend et al., 2001)
	hyaD/dcbF/fcbD (R2)	Extension of polymer as PmHAS/PmHS/PmCS (Townsend et al., 2001; DeAngelis and White, 2002)
	hyaE/dcbE/fcbE (R2)	Initiation of transfer of enzymes (Boyce et al., 2000; Townsend et al., 2001)
	phyA/phyB (R3)	Glycosyltransferase activity (Boyce et al., 2000, Willis and Whitfield, 2013)
Serotype B, E	cexA (R1)	ATP dependent or binding protein (Boyce et al., 2000)
	cexB (R1)	Proteins present in inner membrane (Boyce et al., 2000)
	cexC (R1)	Proteins present in inner membrane and periplasmic domain (Boyce et al., 2000)
	cexD (R1)	Proteins present in outer membrane (lipoprotein) (Boyce et al., 2000)
	bcbA/ecbA (R2)	UDP N acetylglucosamine 2 epimerase (Townsend et al., 2001)
	bcbB/ecbB (R2)	UDP-N-acetylminnosaminuronic acid dehydrogenase (Townsend et al., 2001)
	bcbC/ecbK (R2)	Extension of polymer as a putative glycosyltransferase (Townsend et al., 2001)
	bcbDEFGI/ecbDEFGI (R2)	Unknown (Townsend et al., 2001)
	bcbH/ecbJ (R2)	Unknown (Townsend et al., 2001)
	lipA/lipB (R3)	Lipidation (Townsend et al., 2001)

Table 2: Summary of CPS composition, genetics and virulence in serotype A, B, D, E, and F.

Serotype	Structure (capsule composition)	Genetics (key genes/regions)	Virulence (associated diseases)
A	Hyaluronic acid (HA): Alternating β-1,4 and β-1,3 linkages of β-D-glucuronic acid (GlcA) and β-D-N-acetylglucosamine (GlcNAc).	R1: Contains conserved hexA-D genes; encoding components of the ABC transport system. R2: Contains PmHAS gene; hyaluronic acid polymerization. R3: Contains phyAB, lipAB genes; responsible for lipidation and surface attachment of CPS.	Atrophic Rhinitis (AR; with PMT toxin), Fowl Cholera (FC; acute form caused by A:1, A:3, A:4).
B	Mannosaminuronic acid (ManNAcA) substituted with fructose (position 3) and GlcNAc (position 4) contains glycine.	R1: Contains cexA-D genes; analogous to the hex genes in serotypes A/D/F. R2: Possesses bcbH gene; polysaccharide synthesis R3: Contains phyAB, lipAB genes; responsible for lipidation and surface attachment of CPS.	Hemorrhagic Septicemia (HS; serotype B:2, Asian/European strains).
D	Heparosan: β-1,4 linkages of β-D-GlcA and α-D-GlcNAc.	R1: Utilizes the conserved hexA–D gene cluster as in serotypes A and F. R2: Contains PmHS1; Heparin polymerization R3: Contains phyAB, lipAB genes for lipidation and surface attachment of CPS.	Atrophic Rhinitis (AR; with PMT toxin), Fowl Cholera (less common).
E	ManNAcA substituted with GlcNAc (position 3) and fructose (position 4); structurally similar to B.	R1: Also contains the cexA–D genes. R2: Possesses a bcb/ecb gene cluster for the altered linkage pattern. R3: Contains phyAB, lipAB genes for lipidation and surface attachment of CPS.	Hemorrhagic Septicemia (HS; serotype E:2, African strains).
F	Chondroitin: Alternating β-1,3 and β-1,4 linkages of β-D-GlcA and β-D-N-acetylgalactosamine (GalNAc).	R1: Shares the conserved hexA–D gene cluster with serotypes A and D. R2: Contains PmCS gene for the chondroitin synthesis. R3: Contains phyAB, lipAB genes for lipidation and surface attachment of CPS.	Less clearly defined; associated with general infections (e.g., avian or livestock diseases).

Genetic organization is divided into three distinct regions: Region 1, which is highly conserved and encodes components of the ATP-binding cassette (ABC) transport system responsible for polysaccharide export; Region 2, which contains the genes encoding serotype-specific synthases that catalyze the polymerization of sugar units into the growing capsule; and Region 3, which comprises genes involved in the lipidation and surface attachment of the capsule.

Summary of CPS composition, Genetics and Virulence in Serotype A, B, D, E, and F has been shown in Table 2 for a holistic understanding.

Mechanism of biosynthesis of capsule

The capsular glycosaminoglycan (GAG) of P. multocida is produced in the inner membrane of bacteria and then transported to outside through the ATP binding cassette (ABC) transporter which belongs to the translocases and is present from prokaryotes to humans (Willis and Whitfield, 2013a). Synthesized glycolipids attach to the cell wall by making hydrogen bonds. The biosynthesis of CPS involves initiation of GAG synthesis, extension of GAG disaccharide units and export of GAG (Willis and Whitfield, 2013b).

In addition to the CPS based vaccine development, we also need to extensively study the CPS biosynthesis and the enzymes involved in this biosynthesis to find potential drug targets. Small molecules blocking PmHAS (serotype A), PmHS (serotype D), or PmCS (serotype F) could prevent capsule assembly. Compounds targeting HexA/CexA (ATP-binding proteins in R1) could halt CPS export, rendering bacteria susceptible to host defenses.

Initiation

In the initiation, GAG glycolipid terminal is synthesized where three steps are involved. (I) PhyB (glycosyltransferase) similar to E. coli protein KpsS, transports the first eight carbon sugar, β-3-deoxy-D-manno-oct-2-ulosonic acid (β-KDO) to lysophosphatidylglycerol (lyso-PG) receptor (Chung et al., 1998; Willis and Whitfield 2013a, b; Ovchinnikova et al., 2016). (II) PhyA (glycosyltransferase) similar to E. coli protein KpsC, adds five to nine β-KDOs to synthesize poly-β-KDO linker chain (Willis and Whitfield 2013a, b). (III) HyaE transports and adds UDP sugar molecules to poly-β-KDO linker chain and this is the first residue of glycolipid terminal (Willis and Whitfield 2013a). So, the bacterium starts by building a “starter anchor” made of sugars and fats (a glycolipid). This anchor acts like a foundation for the capsule. Structure of glycolipid has been shown in the Figure 10.

Extension

The extension or polymerization of GAG disaccharide units is essentially similar in serotypes A, D and F (DeAngelis and White, 2004; DeAngelis, 1996; DeAngelis and Padgett-McCue, 2000). PmHAS in serotype A, PmHS in serotype D and PmCS in serotype F act as bifuntional glycosyltransferase which catalyze the transfer of both β-D-acetylglucosamine (β-D-NGlcNAc) and β-D-N-acetylgalactosamine (β-D-GlcUA) using 2 transferase active sites. These proteins add the activated sugar molecules to the glycolipid terminal, release UDP and form the polysaccharide of the relevant serotype (DeAngelis 1999; Tracy et al., 2007).

Here, n is the degree of polymerization which is 20-100 and 103-104 in serotypes D and F, respectively.

In simple words, enzymes add sugar molecules to the anchor. These sugars are carried into place by UDP molecules acting as a “delivery truck”. The enzymes link them into long chains.

Export

The export of GAG outside the cell surface mainly depends upon the ABC transport system (Willis and Whitfield, 2013a). The encoded protein products of first four genes (Region 1) together make an ATP transport system. HexA encoded by hexA, as an ATP binding protein delivers the energy for the GAG transportation. HexB encoded by hexB, is the protein of inner membrane. HexC encoded by hexC, is the protein of inner membrane that has a periplasmic domain. HexD encoded by hexD, is

the protein of outer membrane (Chung et al., 1998). GAG chain passes through the inner membrane, periplasmic space and outer membrane via a channel made by HexB/C/D with the help of ATP binding domain and ultimately makes the covalent bond with phospholipid at the cell surface (Roberts, 1996). Mechanism of biosynthesis of capsule has been shown in the Figure 11.

The process is depicted in three main stages. Initiation: A glycolipid precursor is synthesized by the sequential addition of β-KDO units to a lipid receptor, laying the foundation for capsule assembly. Extension: Bifunctional glycosyltransferases (such as PmHAS, PmHS, or PmCS, depending on the serotype) add sugar units, forming a polysaccharide chain by polymerizing glycosaminoglycan (GAG) disaccharide units. Export: The fully assembled capsule is translocated to the bacterial surface through the action of the ABC transporter system, which ensures its proper attachment.

Expression and regulation of biosynthetic loci of P. multocida

Regulatory sequences: Expression of capsular biosynthetic loci depends upon the operon. Contrary to R3, R1 and R2 genes make a single transcriptional unit because they undergo the transcription in the same direction. The promoter of both R1 and R2 exists in between R2 and R3 (Boyce et al., 2000, 2010). In serotype A, R2 and R3 promoter is present between phyA and hyaE that is recognized by a transcription factor, σ70. -35 box and -10 box in the promoter are separated from each other by 17bp. R1 and R2 transcription initiation site is present at the 37bp upstream of start codon of hyaE (Steen et al., 2010), while ribosome binding site (RBS) is at 8bp upstream to stop codon of hexA and 4bp downstream of stop codon of hexA. Regulatory sequences of R3 are not well known yet however this is supposed to be not involve σ70 (Guan et al., 2019; Chung et al., 1998).

Transcriptional regulation: There is a factor for inversion stimulation (Fis) protein, which performs the transcriptional regulation and regulates the expression of capsule (Steen et al., 2010; Dorman and Deighan, 2003). This nucleoid associated protein is composed of 99 amino acids and has two of its biding sites in promotor region. From 73 to 94 amino acids, there is a DNA binding motif that can bind with promoter region and positively regulate the GAG genes (Steen et al., 2010; Bagchi, 2015). If mutation occurs in Fis gene, it causes production of acapsular strains even in the presence of intact nucleotide sequences. Fis gene is not always present in the capsular gene cluster in all serotypes. This gene also regulates the transcription of some other virulence factors for example LPS, Pasteurella Lipoprotein E (plpE) etc. (Steen et al., 2010).

Translational regulation

There is a protein named as host factor for Qβ (Hfq) which is an RNA chaperone protein (Møller et al., 2002) encoded by hfq gene and regulates the translation of capsule biosynthesis loci (Mégroz et al., 2016; Carmichael, 1975). This protein together with GcvB (a small non-coding RNA molecule) as an Hfq-GcvB complex, targets the mRNA transcribed by capsular biosynthesis gene and positively regulates the gene expression (Gulliver et al., 2018). Hfq-GcvB complex, binds with the target mRNA via specific seed sequence, opens the secondary structure and exposes the RBS and AUG (start codon) (Gulliver et al., 2018; Fröhlich and Vogel, 2009). Ribosome then binds with RBS and AUG, activates the inhibited mRNA and initiates the translation. The seed sequence which is present on mRNA is 5’-AUGUUGUGUU-3′ while the complementary sequence present on GcvB is 5’-AACACAACAU-3′ (Gulliver et al., 2018). Mechanism of regulation of biosynthetic loci of P. multocida has been shown in the Figure 12.

The figure highlights both transcriptional and translational control mechanisms that modulate the expression of capsule biosynthetic genes. The binding of general transcription factors such as σ70 and regulatory proteins like Fis to specific promoter regions initiates and modulates the transcription of the gene clusters. The interaction of the RNA chaperone Hfq with the small RNA GcvB facilitates the exposure of ribosome binding sites on the mRNA, enhancing translation.

Conclusion

P. multocida has many capsular serotypes i.e. A, B, D, E, F whose capsules are made up of hyaluronic acid, mannosaminuronic acid, heparosan, mannosaminuronic acid, and chondroitin respectively. R1 of capsular biosynthetic gene locus encodes proteins to make ATP binding cassette transport system while R2 and R3 are responsible for the polymerization of polysaccharides and lipidation of polysaccharides respectively. CPS is synthesized by the initiation, extension and export of GAG units which is regulated by many genes at transcriptional and translational level.

Novelty Statement

This review article has gathered all the recent informations and studies about the composition, genetics, and biosynthesis of CPS of all serotypes of P. multocida which we do not find in any other article so that we can find the solutions of related diseases in animals.

Author’s Contribution

Waqar Siddique wrote this article under the guidance of Sadia Mahboob and Waqar Rauf. Sobia Jabeen and Zubera Naseem helped in drawing the chemical structures of CPS using ChemDraw. Fiza Shafaqat helped in reviewing the article and mentioned the areas of improvement.

Conflict of interest

The authors have declared no conflict of interest.

REFERENCES

Almoheer R, Abd-Wahid ME, Zakaria HA, Jonet MAB, Al-Shaibani MM, Al-Gheethi A, Addis SNK (2022). Spatial, temporal, and demographic patterns in the prevalence of hemorrhagic septicemia in 41 countries in 2005–2019: A systematic analysis with special focus on the potential development of a new-generation vaccine. Vaccines, 10(2): 315. https://doi.org/10.3390/vaccines10020315

Argüello-García R, Ortega-Pierres G (2021). Giardia duodenalis virulence to be, or not to be. Current tropical medicine reports. pp. 1-11. https://doi.org/10.1007/s40475-021-00248-z

Arumugam N, Ajam N, Blackall P, Asiah N, Ramlan M, Maria J, Yuslan S, Thong KL (2011). Capsular serotyping of Pasteurella multocida from various animal hosts-a comparison of phenotypic and genotypic methods. Trop. Biomed., 28(1): 55-63.

Asghar F, Babar M T, Ali A, Ali M (2023). Hemorrhagic septicemia.

Bagchi A (2015). Structural characterization of Fis. A transcriptional regulator from pathogenic Pasteurella multocida essential for expression of virulence factors. Gene, 554(2): 249-253. https://doi.org/10.1016/j.gene.2014.10.044

Baselga R, Albizu I, Amorena B (1994). Staphylococcus aureus capsule and slime as virulence factors in ruminant mastitis. A review. Vet. Microbiol., 39(3-4): 195-204. https://doi.org/10.1016/0378-1135(94)90157-0

Boyce JD, Adler B (2001). Acapsular Pasteurella multocida B: 2 Can stimulate protective immunity against pasteurellosis. Infect. Immun., 69(3): 1943-1946. https://doi.org/10.1128/IAI.69.3.1943-1946.2001

Boyce JD, Chung JY, Adler B (2000). Genetic organisation of the capsule biosynthetic locus of Pasteurella multocida M1404 (B:2). Vet. Microbiol., 72(1-2): 121-134. https://doi.org/10.1016/S0378-1135(99)00193-5

Boyce JD, Chung JY, Adler B (2000). Pasteurella multocida capsule: Composition, function and genetics. J. Biotechnol., 83(1-2): 153-160. https://doi.org/10.1016/S0168-1656(00)00309-6

Boyce JD, Harper M, Wilkie I, Adler B (2010). Pasteurella. Pathogenesis of bacterial infections in animals: John Wiley and Sons, pp. 325-346. https://doi.org/10.1002/9780470958209.ch17

Boyce JD, Lo RY, Wilkie I, Adler B (2004). Pasteurella and mannheimia. Pathogenesis of bacterial infections in animals. pp. 273-285. https://doi.org/10.1002/9780470344903.ch20

Cao X, Gu L, Gao Z, Fan W, Zhang Q, Sheng J, Zhang Y, Sun Y (2024). Pathogenicity and genomic characteristics analysis of pasteurella multocida serotype a isolated from argali hybrid sheep. Microorganisms, 12(6): 1072. https://doi.org/10.3390/microorganisms12061072

Carmichael GG (1975). Isolation of bacterial and phage proteins by homopolymer RNA-cellulose chromatography. J. Biol. Chem., 250(15): 6160-6167. https://doi.org/10.1016/S0021-9258(19)41172-1

Carter G (1955). Studies on Pasteuvella multocida. I. A hemagglutination test for the identification of serological types.

Casadevall A, Pirofski LA (2009). Virulence factors and their mechanisms of action: The view from a damage–response framework. J. Water Health, 7(S1): S2-S18. https://doi.org/10.2166/wh.2009.036

Chong CWH, Espinosa-Gongora C, Blackall PJ, Sinclair HA, Bojesen AM, Omaleki L, Turni C (2021). Molecular identification of members of the family Pasteurellaceae from the oral cavity of koalas (Phascolarctos cinereus) and their relationship with isolates from koala bite wounds in humans. J. Zoo Wildl. Med., 51(4): 771-779. https://doi.org/10.1638/2020-0041

Christensen H, Sajid SM, Bisgaard M, Magistrali CF, Massacci FR, Liman M, Menke T, Bischoff H, Olsen JE (2022). Prediction of Pasteurella multocida serotypes based on whole genomic sequences. Vet. Microbiol., 271:109492. https://doi.org/10.1016/j.vetmic.2022.109492

Chung JY, Zhang Y, Adler B (1998). The capsule biosynthetic locus of Pasteurella multocida A:1. FEMS Microbiol. Lett., 166(2): 289-296. https://doi.org/10.1111/j.1574-6968.1998.tb13903.x

Dabo SM, Taylor JD, Confer AW (2007). Pasteurella multocida and bovine respiratory disease. Anim. Health Res. Rev., 8(2): 129-150. https://doi.org/10.1017/S1466252307001399

Daffe M, Etienne G (1999). The capsule of Mycobacterium tuberculosis and its implications for pathogenicity. Tuber Lung Dis., 79(3): 153-169. https://doi.org/10.1054/tuld.1998.0200

De-Alwis M (1999). Haemorrhagic septicaemia. Australian Center for International Agricultural Research, Canberra, Australia.

DeAngelis PL (1996). Enzymological characterization of the Pasteurella multocida hyaluronic acid synthase. Biochemistry, 35(30): 9768-9771. https://doi.org/10.1021/bi960154k

DeAngelis PL (1999). Molecular directionality of polysaccharide polymerization by the Pasteurella multocida hyaluronan synthase. J. Biol. Chem., 274(37): 26557-26562. https://doi.org/10.1074/jbc.274.37.26557

DeAngelis PL, Gunay NS, Toida T, Mao WJ, Linhardt RJ (2002). Identification of the capsular polysaccharides of type D and F Pasteurella multocida as unmodified heparin and chondroitin, respectively. Carbohydrate Res., 337(17): 1547-1552. https://doi.org/10.1016/S0008-6215(02)00219-7

DeAngelis PL, Jing W, Drake RR, Achyuthan AM (1998). Identification and molecular cloning of a unique hyaluronan synthase from Pasteurella multocida. J. Biol. Chem., 273(14): 8454-8458. https://doi.org/10.1074/jbc.273.14.8454

DeAngelis PL, Padgett-McCue AJ (2000). Identification and molecular cloning of a chondroitin synthase from Pasteurella multocida type F. J. Biol. Chem., 275(31): 24124-24129. https://doi.org/10.1074/jbc.M003385200

DeAngelis PL, White CL (2004). Identification of a Distinct, cryptic hparosan snthase from Pasteurella multocida types, D, and F. J. Bacteriol., 186(24): 8529-8532. https://doi.org/10.1128/JB.186.24.8529-8532.2004

Dorman CJ, Deighan P (2003). Regulation of gene expression by histone-like proteins in bacteria. Curr. Opin. Genet. Dev., 13(2): 179-184. https://doi.org/10.1016/S0959-437X(03)00025-X

Eid S, Marouf S, Hefny HY, Al-Atfeehy NM (2019). Pasteurellaceae members with similar morphological patterns associated with respiratory manifestations in ducks. Vet. World, 12(12): 2061-2069. https://doi.org/10.14202/vetworld.2019.2061-2069

Fegan JE, Waeckerlin RC, Tesfaw L, Islam EA, Deresse G, Dufera D, Assefa E, Woldemedhin W, Legesse A, Akalu M (2024). Developing a PmSLP3-based vaccine formulation that provides robust long-lasting protection against hemorrhagic septicemia–causing serogroup B and E strains of Pasteurella multocida in cattle. Front. Immunol., 15(1392681). https://doi.org/10.3389/fimmu.2024.1392681

Fenwick BW, Rycroft AN (2022). Pasteurellaceae: Actinobacillus. Vet. Microbiol., pp. 118-128. https://doi.org/10.1002/9781119650836.ch11

Foster G, Ross H, Malnick H, Willems A, Hutson R, Reid R, Collins M (2000). Phocoenobacter uteri gen. nov., sp. nov., a new member of the family Pasteurellaceae Pohl (1979) 1981 isolated from a harbour porpoise (Phocoena phocoena). Int. J. Syst. Evol. Microbiol., 50(1): 135-139. https://doi.org/10.1099/00207713-50-1-135

Fröhlich KS, Vogel J (2009). Activation of gene expression by small RNA. Curr. Opin. Microbiol., 12(6): 674-682. https://doi.org/10.1016/j.mib.2009.09.009

Guan L, Xue Y, Ding W, Zhao Z (2019). Biosynthesis and regulation mechanisms of the Pasteurella multocida capsule. Res. Vet. Sci., 127: 82-90. https://doi.org/10.1016/j.rvsc.2019.10.011

Guan LJ, Yang JQ, Xu QY, Feng YF, Zhang XC, Tang B, Zhao ZQ (2023). Immunogenicity and efficacy of serogroup A and D bacterins against Pasteurella multocida in mice. Front. Vet. Sci., 10: 1132536. https://doi.org/10.3389/fvets.2023.1132536

Gulliver EL, Wright A, Lucas DD, Megroz M, Kleifeld O, Schittenhelm RB, Powell DR, Seemann T, Bulitta JB, Harper M, Boyce JD (2018). Determination of the small RNA GcvB regulon in the Gram-negative bacterial pathogen Pasteurella multocida and identification of the GcvB seed binding region. RNA. 24(5): 704-720. https://doi.org/10.1261/rna.063248.117

Hara Y, Nathan S (2022). Outer membrane proteins as potential candidate vaccine targets. Post-genomic Approaches in Drug and Vaccine Development: River Publishers, pp. 277-322. https://doi.org/10.1201/9781003339090-11

Harper M, Boyce JD, Adler B (2006). Pasteurella multocida pathogenesis: 125 years after Pasteur. FEMS Microbiol. Lett., 265(1): 1-10. https://doi.org/10.1111/j.1574-6968.2006.00442.x

Harper M, Boyce JD, Adler B (2012). The key surface components of Pasteurella multocida: Capsule and lipopolysaccharide. Curr. Top. Microbiol. Immunol., 361: 39-51. https://doi.org/10.1007/82_2012_202

Hashimi T, Joyce D, Nasir SM, Masarudin MJ, Salleh A, Othman S (2023). Characterisation of the putative antigenic genes of the outer membrane proteins of Pasteurella multocida B: 2 Strain PMTB2. 1 through in silico Analysis. Pertanika J. Trop. Agric. Sci., 46(1): 277-313. https://doi.org/10.47836/pjtas.46.1.16

Hassan AK, Mustafa AA (1985). Isolation of Pasteurella multocida type B from an outbreak of haemorrhagic septicaemia in camels in the Sudan. Rev. Elev. Med. Vet. Pays. Trop., 38(1): 31-33.

Hathaway LJ, Grandgirard D, Valente LG, Tauber MG, Leib SL (2016). Streptococcus pneumoniae capsule determines disease severity in experimental pneumococcal meningitis. Open Biol., 6(3): 150269. https://doi.org/10.1098/rsob.150269

Jiménez-Guerra G (2024). Six years of Pasteurella spp. in the Pitiusas. https://doi.org/10.20944/preprints202305.0941.v1

Jogi J, Nayak A, Rai A, Dubey A (2023). Immunoprotection in mice immunized with native OmpH, recombinant OmpH and HS alum precipitated vaccine of Pasteurella multocida P52 against P. multocida challenge.

Kadioglu A, Weiser JN, Paton JC, Andrew PW (2008). The role of Streptococcus pneumoniae virulence factors in host respiratory colonization and disease. Nat. Rev. Microbiol., 6(4): 288-301. https://doi.org/10.1038/nrmicro1871

Kane TA, White CL, DeAngelis PL (2006). Functional characterization of PmHS1, a Pasteurella multocida heparosan synthase. J. Biol. Chem., 281(44): 33192-33197. https://doi.org/10.1074/jbc.M606897200

Khamesipour F, Momtaz H, Azhdary Mamoreh M (2014). Occurrence of virulence factors and antimicrobial resistance in Pasteurella multocida strains isolated from slaughter cattle in Iran. Front. Microbiol., 5: 536. https://doi.org/10.3389/fmicb.2014.00536

Kubatzky KF (2012). Pasteurella multocida and immune cells. Curr. Top Microbiol. Immunol., 361: 53-72. https://doi.org/10.1007/82_2012_204

Liva GA, Karatzanis AD, Prokopakis EP (2021). Review of rhinitis: Classification, types, pathophysiology. J. Clin. Med., 10(14): 3183. https://doi.org/10.3390/jcm10143183

Mégroz M, Kleifeld O, Wright A, Powell D, Harrison P, Adler B, Harper M, Boyce JD (2016). The RNA-binding chaperone Hfq is an important global regulator of gene expression in pasteurella multocida and plays a crucial role in production of a number of virulence factors, including hyaluronic acid capsule. Infect. Immun., 84(5): 1361-1370. https://doi.org/10.1128/IAI.00122-16

Michael FS, Cairns CM, Fleming P, Vinogradov EV, Boyce JD, Harper M, Cox AD (2021). The capsular polysaccharides of Pasteurella multocida serotypes B and E: Structural, genetic and serological comparisons. Glycobiology, 31(3): 307-314. https://doi.org/10.1093/glycob/cwaa069

Møller T, Franch T, Højrup P, Keene DR, Bächinger HP, Brennan RG, Valentin-Hansen P (2002). Hfq: A bacterial Sm-like protein that mediates RNA-RNA interaction. Mol. Cell., 9(1): 23-30. https://doi.org/10.1515/HF.2002.081

Mushtaq A, Singh S, Gazal S, Jadhao A, Kantale R, Basak G, Singh P (2022). Haemorrhagic septicemia: A persistent nuisance in Indian livestock review. Differentiation, 71: 156.

Omaleki L, Blackall PJ, Cuddihy T, White RT, Courtice JM, Turni C, Forde BM, Beatson SA (2022). Phase variation in the glycosyltransferase genes of Pasteurella multocida associated with outbreaks of fowl cholera on free-range layer farms. Microb. Genom., 8(3): 000772. https://doi.org/10.1099/mgen.0.000772

Orynbayev M, Sultankulova K, Sansyzbay A, Rystayeva R, Shorayeva K, Namet A, Fereidouni S, Ilgekbayeva G, Barakbayev K, Kopeyev S, Kock R (2019). Biological characterization of Pasteurella multocida present in the Saiga population. BMC Microbiol., 19(1): 37. https://doi.org/10.1186/s12866-019-1407-9

Ovchinnikova OG, Mallette E, Koizumi A, Lowary TL, Kimber MS, Whitfield C (2016). Bacterial beta-Kdo glycosyltransferases represent a new glycosyltransferase family (GT99). Proc. Natl. Acad. Sci. U.S.A., 113(22): E3120-9. https://doi.org/10.1073/pnas.1603146113

Pasomboon P, Chumnanpuen PTEK (2021). Comparison of hyaluronic acid biosynthetic genes from different strains of Pasteurella multocida. Bioinf. Biol. Insights, 15:11779322211027406. https://doi.org/10.1177/11779322211027406

Peng Z, Wang X, Zhou R, Chen H, Wilson BA, Wu B (2019). Pasteurella multocida: Genotypes and genomics. Microbiol. Mol. Biol. Rev., 83(4): e00014-19. https://doi.org/10.1128/MMBR.00014-19

Peterson JW (1996). Bacterial pathogenesis. In: th, Baron S, editors. Med Microbiol. Galveston (TX): University of Texas Medical Branch at Galveston. https://doi.org/10.1128/jb.178.14.4189-4199.1996

Reckseidler-Zenteno SL (2012). Capsular polysaccharides produced by the bacterial pathogen Burkholderia pseudomallei. The complex world of polysaccharides. pp. 128-152.

Reuben RC, Sarkar SL, Ibnat H, Setu MAA, Roy PC, Jahid IK (2021). Novel multi-strain probiotics reduces Pasteurella multocida induced fowl cholera mortality in broilers. Sci. Rep., 11(1): 8885. https://doi.org/10.1038/s41598-021-88299-0

Richardson NI, Ravenscroft N, Kuttel MM (2023). Conformational comparisons of Pasteurella multocida types B and E and structurally related capsular polysaccharides. Glycobiology, 33(9): 745-754. https://doi.org/10.1093/glycob/cwad049

Rimler RB, Rhoades KR (1987). Serogroup F, a new capsule serogroup of Pasteurella multocida. J. Clin. Microbiol., 25(4): 615-618. https://doi.org/10.1128/jcm.25.4.615-618.1987

Roberts IS (1996). The biochemistry and genetics of capsular polysaccharide production in bacteria. Annu. Rev. Microbiol., 50: 285-315. https://doi.org/10.1146/annurev.micro.50.1.285

Rosner H, Grimmecke HD, Knirel YA, Shashkov AS (1992). Hyaluronic acid and a (1-4) beta-D-xylan, extracellular polysaccharides of Pasteurella multocida (Carter type A) strain 880. Carbohydr Res., 223: 329-333. https://doi.org/10.1016/0008-6215(92)80032-V

Saha O, Islam MR, Rahman MS, Hoque MN, Hossain MA, Sultana M (2021). First report from Bangladesh on genetic diversity of multidrug-resistant Pasteurella multocida type B:2 in fowl cholera. Vet. World, 14(9): 2527-2542. https://doi.org/10.14202/vetworld.2021.2527-2542

Sheikh MA, Anzam M, Shakoori AR (1996). Observations on haemorrhagic septicaemia in Pakistan livestock. Zentralbl Vet. B, 43(5): 293-304. https://doi.org/10.1111/j.1439-0450.1996.tb00317.x

Smallman TR, Williams GC, Harper M, Boyce JD (2022). Genome-wide investigation of Pasteurella multocida identifies the stringent response as a negative regulator of hyaluronic acid capsule production. Microbiol. Spect., 10(2): e00195-22. https://doi.org/10.1128/spectrum.00195-22

Smith E, Miller E, Aguayo JM, Figueroa CF, Nezworski J, Studniski M, Wileman B, Johnson T (2021). Genomic diversity and molecular epidemiology of Pasteurella multocida. PLoS One, 16(4): e0249138. https://doi.org/10.1371/journal.pone.0249138

Somarajan S, Chaturvedi V, Kumar S, Kumar B, Nair B, Gupta P, Pandey K (2007). Immunogenicity of capsular polysaccharide of Pasteurella multocida B: 2 Conjugated to protein carrier in rabbits. Indian J. Comp. Micobiol. Immunol. Infect. Dis., 27:14.

Steen JA, Steen JA, Harrison P, Seemann T, Wilkie I, Harper M, Adler B, Boyce JD (2010). Fis is essential for capsule production in Pasteurella multocida and regulates expression of other important virulence factors. PLoS Pathog., 6(2): e1000750. https://doi.org/10.1371/journal.ppat.1000750

Tipton KA, Chin CY, Farokhyfar M, Weiss DS, Rather PN (2018). Role of capsule in resistance to disinfectants, host antimicrobials, and desiccation in Acinetobacter baumannii. Antimicrob. Agents Chemother., 62(12): e01188-18. https://doi.org/10.1128/AAC.01188-18

Townsend KM, Boyce JD, Chung JY, Frost AJ, Adler B (2001). Genetic organization of Pasteurella multocida cap Loci and development of a multiplex capsular PCR typing system. J. Clin. Microbiol., 39(3): 924-929. https://doi.org/10.1128/JCM.39.3.924-929.2001

Tracy BS, Avci FY, Linhardt RJ, DeAngelis PL (2007). Acceptor specificity of the Pasteurella hyaluronan and chondroitin synthases and production of chimeric glycosaminoglycans. J. Biol. Chem., 282(1): 337-344. https://doi.org/10.1074/jbc.M607569200

Van Nguyen P, Le CT, Nguyen XH, Nguyen TM, Nguyen KCT (2023). First study on capsular serotypes and virulence factors of Pasteurella multocida isolates from Phan Rang sheep in Vietnam. Vet. World, 16(2): 281. https://doi.org/10.14202/vetworld.2023.281-290

Weinbaum S, Tarbell JM, Damiano ER (2007). The structure and function of the endothelial glycocalyx layer. Annu. Rev. Biomed. Eng., 9: 121-167. https://doi.org/10.1146/annurev.bioeng.9.060906.151959

Wilkie IW, Harper M, Boyce JD, Adler B (2012). Pasteurella multocida: Diseases and pathogenesis. Curr. Top. Microbiol. Immunol., 361: 1-22. https://doi.org/10.1007/82_2012_216

Willis LM, Whitfield C (2013). KpsC and KpsS are retaining 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) transferases involved in synthesis of bacterial capsules. Proc. Natl. Acad. Sci. U.S.A., 110(51): 20753-20758. https://doi.org/10.1073/pnas.1312637110

Willis LM, Whitfield C (2013). Structure, biosynthesis, and function of bacterial capsular polysaccharides synthesized by ABC transporter-dependent pathways. Carbohydr Res., 378: 35-44. https://doi.org/10.1016/j.carres.2013.05.007

Xiao J, Li Y, Hu Z, Zhang Y, Chang Y-F, Zhou Q, Yan Z, Zhang X, Chen L, Li W (2021). Characterization of Pasteurella multocida isolated from ducks in China from 2017 to 2019. Microb Pathogen., 160: 105196. https://doi.org/10.1016/j.micpath.2021.105196

Yuan L, Wei H, Yang XY, Geng W, Peterson BW, van der Mei HC, Busscher HJ (2021). Escherichia coli colonization of intestinal epithelial layers in vitro in the presence of encapsulated bifidobacterium breve for its protection against gastrointestinal fluids and antibiotics. ACS Appl. Mater. Interfaces, 13(14): 15973-15982. https://doi.org/10.1021/acsami.0c21790

Zhao G, Li P, Mu H, Li N, Peng Y (2021). L-ascorbic acid shapes bovine Pasteurella multocida serogroup A infection. Front. Vet. Sci., 8: 687922. https://doi.org/10.3389/fvets.2021.687922

Zhou G, Tian J, Tian Y, Ma Q, Li Q, Wang S, Shi H (2023). Recombinant-attenuated Salmonella enterica serovar Choleraesuis vector expressing the PlpE protein of Pasteurella multocida protects mice from lethal challenge. BMC Vet. Res., 19(1): 128. https://doi.org/10.1186/s12917-023-03679-0