Pathogenic Species

Taxonomic revision of the Pasteurellaceae family is in progress. Currently, there are 13 validly named species within the genus Pasteurella (Table 10.1). However, 16S rRNA alignments and whole proteome analyses have defined Pasteurella sensu stricto as Pasteurella canis, Pasteurella dagmatis, P. oralis, Pasteurella stomatis, and P. multocida, which has three recognized subspecies, multocida, gallicida, and septica (Christensen and Bisgaard 2018). P. multocida strains can be further differentiated based on their capsule and lipopolysaccharide (LPS) surface antigens. Classically, strains have been typed using serological methods that identify these polysaccharides, but now multiplex polymerase chain reactions (mPCR) are available that target unique genes in the capsule and LPS biosynthetic loci (Townsend et al. 2001; Harper and Boyce 2017). Five different capsule serogroups (A, B, D, E, and F) are known to correlate with five distinct structures. Heddleston LPS serotyping recognizes 16 serologically distinct types (Heddleston et al. 1972), but the species is known to produce many more differentiable LPS structures (> 20). There are a total of eight unique LPS biosynthetic loci (L1–L8) that collectively are responsible for all known P. multocida LPS structures (Harper and Boyce 2017).

Analysis of the 16S rRNA sequences obtained from other current members of the genus suggests that [Pasteurella] aerogenes and [Pasteurella] mairii are the most closely related to, but clearly separate from, the species within Pasteurella sensu stricto. In contrast, [Pasteurella] bettyae and [Pasteurella] caballi cluster close to Bibersteinia trehalosi; [Pasteurella] caecimuris clusters with Rodentibacter spp.; [Pasteurella] langaaensis clusters close to Gallibacterium spp.; [Pasteurella] skyensis clusters with Cavibacterium pharyngocola, and [Pasteurella] testudinis clusters with Chelonobacter oris.

All five species within Pasteurella sensu stricto can cause disease. However, this chapter focuses on P. multocida, as this species is the most problematic with respect to disease in production animals and has been the most widely studied (Table 10.1).

Fowl Cholera

FC can present as an acute or chronic disease in all bird species but is most problematic in poultry and waterfowl. Avian cholera isolates typically belong to capsule serogroup A, F, or D (Smith et al. 2021). In both chronic and acute FC, clinical signs include ruffled feathers, lethargy, depression, fever, sneezing, and nasal discharge. Acute FC is typically of short duration (one to three days) and can be fatal before any signs of disease become apparent (Wilkie et al. 2012). In chronic FC, swelling of wattles and combs can occur but after approximately 14 days of disease clinical signs start to reduce in severity. However, the bacterium can persist in the URT, LRT, liver, and lymphatic system, providing a reservoir for future outbreaks of disease (Mbuthia et al. 2011).

The severity of FC disease can vary greatly and is dependent on the infecting strain, infectious dose, host species, and individual immune response. Turkeys, pheasants, and partridges inoculated with the same virulent FC strain were reported to be more susceptible than chickens (Petersen et al. 2001). A strong immune response, led by the recruitment of heterophils, can lead to hemorrhage and necrosis in birds infected with P. multocida (Bojesen et al. 2004; Mbuthia et al. 2011).

Hemorrhagic Septicemia

HS is an acute systemic disease that affects buffalo, cattle, and wild populations of ungulates in regions of Africa and Asia. Large outbreaks in Asia cause significant livestock losses each year (De Alwis 1992). P. multocida strains that cause HS exclusively belong to the closely related serogroups B and E (Asian and African isolates, respectively) and LPS genotype L2 (Harper and Boyce 2017). The URT of susceptible animals is colonized before the bacterium invades the mucosa at the tonsils and migrates to nearby lymph nodes, most likely inside macrophages (Annas et al. 2014). P. multocida then disseminates throughout the host, causing an overwhelming systemic infection and death. Peracute HS infections develop rapidly (4–12 hours) and death can occur suddenly without prior signs of disease (De Alwis 1992). The lipid A component of LPS, known as endotoxin, is the main driver of severe septic shock observed in peracute cases of HS. In acute HS, the levels of endotoxin rise more slowly, and the disease can last up to five days (Horadagoda et al. 2001). Animals will have a fever, depression/apathy, loss of appetite, and nasal discharge. Late signs of infection include labored breathing, increased respiration rate, a mucopurulent nasal discharge, and swelling of salivary glands, the lower chest, and forelegs. Death is almost certain once the animal becomes recumbent and develops clear signs of septicemia and septic shock. Animals that have survived HS shed P. multocida and some become chronic carriers and may act as a source of future outbreaks (De Alwis 1992).

Atrophic Rhinitis

AR is a chronic disease that primarily affects the nasal cavities of pigs and rabbits. Clinical signs of infection and turbinate damage include nasal discharge, sneezing, and snuffling (Wilkie et al. 2012). Mild, non‐progressive AR is caused by Bordetella bronchiseptica strains that produce dermonecrotic toxin (DNT). However, a more severe, progressive AR is caused by P. multocida strains that also produce a dermonecrotic toxin, called PMT, and typically belong to capsule serogroup D (Horiguchi 2012). AR starts with colonization of the host URT by toxigenic P. multocida strains, often facilitated by a pre‐existing infection with B. bronchiseptica. P. multocida AR strains colonize the tonsils and continually release PMT that modifies the activity of host G proteins resulting in widespread activation of signaling pathways (for more on PMT see below). In response to this perturbation, osteoclasts disproportionally increase in the nasal cavity, resulting in bone resorption, atrophy of the nasal turbinates, deviation of the nasal septum, and ultimately permanent distortion and twisting of the snout. Untreated animals have an increased chance of later developing LRT infections (Horiguchi 2012).

Lower Respiratory Tract Infections

P. multocida LRT infections can occur in many animal species and are especially common in cattle and other ungulates. Infections range from peracute/acute fulminating pneumonia observed in cattle, to more chronic and localized pneumonia often observed in pigs and sheep. Colonization and persistence of P. multocida in the lungs can occur alone, or more commonly together with a virus or another bacterial species such as Mannheimia haemolytica or Histophilus somni (Wilkie et al. 2012). Clinical signs of Pasteurella pneumonia include inflammation of the nasal mucosa, increased nasal secretions, sneezing, fever, labored breathing, and frothing at the mouth. In severe cases, death occurs from respiratory failure (Wilkie et al. 2012).

Zoonotic Infections in Humans

Pasteurella species can cause a wide range of zoonotic infections in humans, often following a cat or dog bite. Most are caused by P. multocida capsule serogroup A strains, although serogroup F strains have also been isolated (Ujvari et al. 2019; Smith et al. 2021). Data from one extensive study on domestic animal bites found that P. multocida was present in 12% of infected dog‐bite wounds and 62% and infected cat‐bite wounds. Of the other Pasteurella species, P. canis, P. stomatis, and P. dagmatis were present in 13%, 6%, and 2% of infected dog bite wounds, and in 1.5%, 3%, and 5.3% of infected cat bite wounds, respectively (Talan et al. 1999). Edema, cellulitis, and purulent exudate are often present at the site of the wound infection and untreated the infection may lead to necrotizing fasciitis. Cases of bacteremia, meningitis, peritonitis, pneumonia, or septicemia caused by P. multocida have also been reported (Wilson and Ho 2013). Recent bioinformatic analysis has shown strains of P. multocida isolated from human infections share similarities with isolates from commensal strains isolated from domestic animals but are genetically distinct from strains that cause most animal infections, indicating possible human‐specific virulence factors (Smith et al. 2021; our unpublished data).

Capsule

The polysaccharide capsule is a primary virulence factor and can be used to classify strains using serology or mPCR into five serogroups, A, B, D, E, and F (Boyce et al. 2000). Capsules of type A, D, and F strains are closely related and are composed of hyaluronic acid, heparin, and chondroitin, respectively (Figure 10.1). These polymers are also found in the eukaryotic extracellular matrix and are predicted to “mask” the surface components of the bacterium from the host innate immune response. The serogroup B and E main chain disaccharide repeat units both consist of a mannosaminuronic acid derivative linked to an N‐acetylglucosamine but differ with respect to the linkage of the side‐branch fructose and the addition of glycine onto the serogroup B structure (Michael et al. 2021; Figure 10.1).

The P. multocida capsule biosynthesis loci are typical group II‐type capsule biosynthesis loci with three distinct regions (Boyce et al. 2000). Region 1 is highly conserved in all P. multocida capsule loci and contains the genes required for the ATP‐binding cassette (ABC) transport system responsible for exporting the polysaccharide to the bacterial surface. Region 2 genes differ between serogroups and encode proteins required for the synthesis of each type‐specific polysaccharide. This includes the specific synthase required to add the sugar monomers to the growing polymer. In serogroup A, D, and F, these synthases are HyaD, DcbF, and FcbD, respectively (Figure 10.2). The precise function of each gene within region 2 in serogroup B and E is currently unknown. Primers used in the capsule multiplex mPCR anneal to unique sequences within region 2 of each locus (Townsend et al. 2001). Region 3 genes encode proteins predicted to be required for the lipidation and membrane anchoring of the capsular polysaccharide (i.e. PhyA and PhyB in serogroups A, D, and F and LipA and LipB in serogroups B and E). The genes encoding these proteins are co‐located, except for the serogroup B and E lipA and lipB which flank the region 2 genes (Figure 10.2; Townsend et al. 2001).

The first definitive evidence that P. multocida capsule was a major virulence factor came from experiments that showed a cexA capsule export mutant was strongly attenuated for virulence in mice compared with the highly virulent serogroup B parent strain M1404. Murine peritoneal macrophages could also readily phagocytose the M1404 cexA mutant but not the wild‐type parent (Boyce and Adler 2000). Similar experiments using the serogroup A FC isolate X‐73 and an isogenic capsule (hexA) mutant showed the capsule mutant was strongly attenuated for disease in both mice and chickens and was highly sensitive to complement‐mediated killing (Chung et al. 2001). Taken together, these data support the critical role of capsule in virulence and avoidance of the host innate immune response.

Lipopolysaccharide

Like most Gram‐negative bacteria, the external surface of the P. multocida outer membrane is predominantly comprised of LPS molecules, the lipid A component of which is a potent endotoxin (Horadagoda et al. 2001). Each LPS molecule consists of a hydrophobic lipid A membrane anchor, a conserved inner core oligosaccharide, and a highly variable outer core oligosaccharide. The lipid A structure of the LPS from a P. multocida HS isolate has recently been reported (Tawab et al. 2020) but it is not known whether this structure is conserved across the species. The complete repertoire of inner and outer core oligosaccharide structures produced by P. multocida has also been determined (Harper and Boyce 2017; Figure 10.1). Strains are classified into eight LPS genotypes (L1 to L8) using mPCR analysis with primers that anneal to unique regions within each LPS outer core biosynthetic locus (Harper and Boyce 2017). Each outer core locus contains a range of genes, the majority of which encode sugar transferases, each responsible for the transfer of a specific sugar to a specific position on the LPS structure (Harper and Boyce 2017). The LPS produced by each P. multocida strain initiates a specific antibody response that historically was used to serologically classify strains into 16 Heddleston LPS serotypes (Heddleston et al. 1972). However, many more LPS types exist due to the existence of truncated LPS structures in addition to the full‐length “parent” structures. Truncated LPS structures typically result from inactivating mutations within sugar transferase genes, resulting in the arrest of LPS assembly and truncation of the full‐length structure (Harper and Boyce 2017).

Virulence studies have shown that a full‐length LPS structure is required for the P. multocida A:L1 strain VP161 to cause systemic disease in chickens. However, some VP161 mutants expressing truncated LPS molecules can persist at the site of intramuscular injection but not in the blood (Harper and Boyce 2017). Many isolates recovered from natural infections have mutations within the LPS loci and express shortened LPS structures, suggesting that strains lacking full‐length LPS may reside naturally in tissues other than blood, such as the tonsils or other niches within the URT. Interestingly, some LPS mutants are highly attenuated for systemic disease in chickens but can still cause systemic disease in mice, demonstrating that different animal species respond differently to this important component of the P. multocida cell surface (Harper and Boyce 2017).

Pasteurella multocida Toxin

The P. multocida dermonecrotic toxin PMT is encoded by toxA, a gene located within a lysogenic bacteriophage that is integrated into the genome of P. multocida AR isolates that typically belong to capsular serogroup D or A (Pullinger et al. 2004). The toxin is passively released from P. multocida cells colonizing the URT, especially within the turbinates. It belongs to the A‐B family of toxins; the B domain facilitates binding to the host cells while the A domain exerts a toxic effect on the heterotrimeric G class of GTPases following transport of the toxin into the cytosol (Wilson and Ho 2012). The eukaryotic heterotrimeric GTPases regulate the amount of available GTP to control multiple cell‐signaling pathways. In the inactive state, the G protein complex consists of GDP‐bound alpha, beta, and gamma subunits. When GTP displaces GDP, the trimer dissociates into two active effectors, GTP‐bound alpha G protein and the beta/gamma complex (Wilson and Ho 2012). At the molecular level, PMT deamidates a specific glutamine on the alpha subunit of the G protein subunits Gαq, Gαi, and Gα12/13 (Orth et al. 2013). This stops the conversion of the active GTP‐bound Gα protein to the GDP‐bound inactive state, which subsequently results in accumulation of the active GTP‐Gα protein and the beta/gamma G protein complex. The abundance of activated Gα protein affects many cell‐signaling pathways and results in increased levels of inflammatory cytokines. One of the most profound effects of PMT is the proliferation of osteoclasts, responsible for bone remodeling/degradation and resorption, and a deficiency of bone‐forming osteoblasts (Wilson and Ho 2012). This imbalance leads to the distortion of the nasal turbinate bones, as observed in animals with AR disease. PMT permanently activates the Gαq proteins, which ultimately affects the expression of genes involved in osteoblast formation (Siegert et al. 2013). Normal osteoclastogenesis is also affected. Unlike the normal process, PMT‐induced osteoclast formation is receptor activator of nuclear factor κB ligand‐independent and leads to an increase in the master transcription factor that controls osteoclast differentiation (Chakraborty et al. 2017). Osteocytes (derived from osteoblasts) are the main cells within mature bone and are also affected by the toxin (Heni et al. 2018). Thus, PMT exerts its effect on the cells within the nasal turbinates in a myriad of ways. Current data indicates the toxA

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