Introduction
It has been over 130 years since Louis Pasteur used the bacterium Pasteurella multocida in successful production of an attenuated vaccine (Pasteur, Reference Pasteur1880). This versatile pathogen continues to be one of the most fascinating microbes with multiple mysteries deeply buried in its genome (Hunt et al., Reference Hunt, Adler and Townsend2000; Harper et al., Reference Harper, Boyce and Adler2006). P. multocida, a Gram-negative bacterial pathogen associated with a variety of diseases in livestock, causes hemorrhagic septicemia (HS) in cattle and buffaloes, fowl cholera in poultry, atrophic rhinitis in pigs, snuffles in rabbits, pneumonic and septicemic pasteurellosis in sheep and goats, as well as in American bison, yak, deer, hippopotamus, elephants, camels, mink, monkey, cat, horse, elk, tortoise, snow leopard and lion (De Alwis, Reference De Alwis1999). The various serotypes of P. multocida are associated with a wide range of diseases, termed ‘pasteurellosis’. In many instances, P. multocida plays a secondary role in pathogenesis of the disease or acts in combination with other bacterial, viral or parasitic infections. HS, classified as a List B disease by the Office International des Epizooties (OIE), is a primary pasteurellosis with 100% mortality in infected animals in endemic areas of Africa and Asia. This makes HS the most economically important bacterial disease of animals in these countries (De Alwis, Reference De Alwis1999; FAO, 2005; OIE, 2009). The disease has a high global index as an animal health constraint to poor farmers (FAO-WHO-OIE, 1994; Perry et al., Reference Perry, Randolph, McDermott, Sones and Thornton2002).
Several notable achievements in the realm of the organism's characteristics, virulence, pathogenicity, diagnosis and development of vaccines have been reported (Harper et al., Reference Harper, Boyce and Adler2006). The current review highlights the past and most recent advances in etiology, virulence factors, diagnosis and vaccine development with strategic control measures.
HS
HS is a highly fatal, acute septicemic disease of economic importance that occurs mainly in buffalo and cattle, and is caused by two specific serotypes of P. multocida designated B:2 (Asian serotype) and E:2 (African serotype) (De Alwis, Reference De Alwis1999). Various other serotypes, namely, A:1, A:1,3, A:3, A:4, B:1, B:2,5, B:3,4, E:2,5, F:3; F:3,4, have also been isolated from HS outbreaks (De Alwis, Reference De Alwis1999; Biswas et al., Reference Biswas, Shivachandra, Saxena, Kumar, Singh and Srivastava2004; Kumar et al., Reference Kumar, Shivachandra, Biswas, Singh, Singh and Srivastava2004, Reference Kumar, Singh, Agrawal and Singh2009). Following successful control or eradication of rinderpest and continued low mortality from foot-and-mouth disease (FMD), HS emerged as a major disease of livestock (Benkirane and De Alwis, Reference Benkirane and De Alwis2002). Incidence and distribution of HS vary greatly from a few cases to high numbers depending on the type of husbandry practices, geographical area and agro-climatic conditions prevailing in a particular region (De Alwis, Reference De Alwis1999; Benkirane and De Alwis, Reference Benkirane and De Alwis2002; Perry et al., Reference Perry, Randolph, McDermott, Sones and Thornton2002).
Susceptibility and carrier state
Cattle and buffaloes, which are reared mainly for use as draught animals as well as milk production in many developing countries, are mainly affected by HS outbreaks (Benkirane and De Alwis, Reference Benkirane and De Alwis2002). Buffaloes have a shorter course of disease and are generally believed to be more susceptible to HS than cattle (De Alwis, Reference De Alwis1999). Natural infections are generally by inhalation and/or ingestion of contaminated materials due to a clinically infected/carrier animal that comes in contact with a virgin herd. Although P. multocida does not survive outside the host for long periods, moist soil, animal tissues and pasture may prolong its survivability. In endemic areas, most deaths are confined to older calves and young adults. In non-endemic areas, massive epizootics may occur. Case fatality may approach 100%, if treatment is not carried out at an early stage (Benkirane and De Alwis, Reference Benkirane and De Alwis2002). The presence of naturally acquired immunity is observed among buffaloes. It has been opined that HS morbidity and mortality patterns, in endemic and non-endemic areas in a given population, are largely dependent on the proportion of naturally acquired immune to non-immune animals (Bain, Reference Bain1954). The presence of HS-causing P. multocida in small proportion in the nasopharynx of apparently healthy cattle and buffaloes and recent exposure to outbreaks of HS have been documented for a long time (De Alwis et al., Reference De Alwis, Wijewardana, Gomis and Vipulasiri1990). Further investigations were conducted in experimentally exposed buffaloes and induced carrier animals that remained clinically normal. These studies showed that P. multocida persisted in the crypts of tonsils for several months (despite treatment with antibiotics), before they appeared actively in the nasopharynx and were shed intermittently in the nasal secretions (De Alwis et al., Reference De Alwis, Wijewardana, Gomis and Vipulasiri1990; Harper et al., Reference Harper, Boyce and Adler2006). The factors responsible for influencing the latent carriers to become active are still an enigma.
Clinical signs and gross lesions
The disease occurs in acute, sub-acute and chronic forms and is characterized by an initial phase of temperature elevation (often unnoticed), a phase of respiratory involvement and a terminal phase of septicemia and recumbence leading to death (De Alwis, Reference De Alwis1999). The incubation period is usually 1–3 days, and the course of the disease may range from sudden death, with no observable clinical signs, to a protracted course extending up to 5 days (Carter and De Alwis, Reference Carter, De Alwis, Adlam and Rutter1989; De Alwis, Reference De Alwis1999).
The acute form of HS is manifested by sudden onset and death within 24 h. The animals show a high rise in body temperature (41–42°C) and a thin pulse with rapid, shallow respiration and cyanosis of visible mucus membranes (De Alwis, Reference De Alwis1999; Benkirane and De Alwis, Reference Benkirane and De Alwis2002; OIE, 2009). Restlessness, mild colic pain, muscular tremors, lacrimation, nasal discharge and diarrhea are also observed. In the sub-acute form, known as the subcutaneous edematous form, the animal survives for 2–3 days with edematous swelling of the throat and brisket region and bronchopneumonia. In the chronic form, the course of infection is longer with signs of short, rapid painful respiration coupled with mucopurulent or blood-stained nasal discharge (OIE, 2009). At postmortem examination, the first obvious lesion is subcutaneous edema, particularly in the mandibular and brisket regions. Other lesions include petechial-to-echymotic hemorrhages, congestion and/or consolidation of the lungs, fibrinous pneumonia, pleurisy and pericarditis (De Alwis, Reference De Alwis1999; OIE, 2009).
Epidemiology
There is variation in the distribution and occurrence of HS outbreaks in different agro-climatic regions involving various animal species (De Alwis, Reference De Alwis1999). Outbreaks of disease occur throughout the year irrespective of the season (Kumar et al., Reference Gautam, Kumar, Singh, Singh, Dutta and Shivachandra2004). This is in contrast to the perception that the disease is mostly precipitated during the monsoon or post-monsoon period. In India, the incidence of disease outbreaks varies considerably in different states and from year to year in each state (Khera, Reference Khera1979). HS has been reported from most countries of the world, especially in underdeveloped and developing countries of Africa and Asia (Benkirane and De Alwis, Reference Benkirane and De Alwis2002); the developed countries are less affected with the disease. HS has been reported in the Middle East and Southern Europe (FAO-WHO-OIE, 1994).
In the USA, the disease was reported in earlier times among Bison in national parks in 1912, 1922 and 1965–67 (De Alwis, Reference De Alwis1999) and naturally acquired antibodies against P. multocida types B and E were detected in domestic feeder calves (Sawada et al., Reference Sawada, Rimler and Rhoades1985). Outbreaks were also reported among dairy cattle and beef calves in the USA and Canada. However, the disease has not become endemic in North America. Most of the oceanic countries such as Australia, New Zealand and Japan and Western Europe have not reported the disease (FAO-WHO-OIE, 1994; De Alwis, Reference De Alwis1999). Although, the clinical disease has been suspected in other countries, these cases were not confirmed by serology. Isolated cases of HS have been reported from Central and South American states, and from Southern and Eastern European countries (OIE, 2009). The distribution and frequency of disease have been associated with climatic conditions, husbandry practices and types of animals reared (De Alwis, Reference De Alwis1999; Benkirane and De Alwis, Reference Benkirane and De Alwis2002). It is presumed that HS may occur in developed countries with high water buffalo population and where conditions are similar to those of tropical Asia and Africa (Benkirane and De Alwis, Reference Benkirane and De Alwis2002).
Etiological agent
The history of the organism dates back to 1782, when it was first studied by Chabert in France. Later, the bacterium was described in 1878–79 by Revolta and Revolee. About the same time, Bollinger from Germany described a disease in cattle and buffaloes (De Alwis, Reference De Alwis1999). In 1887, Trevisan proposed the name ‘Pasteurella’ in recognition of pioneering work done in the early 1880s by Louis Pasteur on the etiology of fowl cholera (Pasteur, Reference Pasteur1880; Harper et al., Reference Harper, Boyce and Adler2006). The bacterium was named initially according to the host species in which it caused disease (such as cattle - boviseptica), but in 1939, Rosenbach and Merchant proposed the name ‘Pasteurella multocida’ (multocida- many killing, referring to pathogenicity for multiple species) (Harper et al., Reference Harper, Boyce and Adler2006). The species is divided into four subspecies; P. multocida subsp. multocida, P. multocida subsp. septica and P. multocida subsp. gallicida (Lehmann and Neumann, Reference Lehmann and Neumann1899) and the recently described P. multocida subsp. tigris (Capitini et al., Reference Capitini, Herrero, Patel, Ishitani and Boyce2002). P. multocida belongs to the large family Pasteurellaceae that includes 15 genera with three core genera namely Pasteurella, Actinobacillus and Haemophilus (Holt et al., Reference Holt, Krieg, Sneath, Staley and Williams1994; Hunt et al., Reference Hunt, Adler and Townsend2000; Kuhnert and Christensen, Reference Kuhnert and Christensen2008), which are obligate parasites or commensals of vertebrate animals, colonizing mainly the mucosal surfaces of the upper respiratory tract, oropharynx and other parts of their hosts.
P. multocida cells are non-motile, small and pleomorphic depending on the growth stage; they occur singly, in pairs or short chains (De Alwis, Reference De Alwis1999). The organisms are Gram-negative and non-endospore forming. Surface colonies on sheep blood agar plates are smooth round, grayish or yellowish, glistening, translucent, non-hemolytic and 1–2 mm in diameter after 24 h of incubation at 37°C (Holt et al., Reference Holt, Krieg, Sneath, Staley and Williams1994). The colony size depends on the capsular type and degree of capsulation. Good growth is achieved in rich broth and there is no growth on MacConkey's agar. The fresh cultures, blood smears, exudates and infected animal tissues show bipolar staining of the organism with Leishman or Methylene Blue or Giemsa stain (OIE, 2009).
The organism is mesophilic, chemo-organotrophic with both oxidative and fermentative types of metabolism (OIE, 2009). Molecular oxygen is used as a terminal electron acceptor but can be replaced by nitrate and fumarate. Oxidase, alkaline phosphatase and catalase tests are positive. Most species are V-factor (beta-nicotinamide adenine dinucleotide) and X-factor (protohemin or protoporphyrin IX) independent, although V-factor requiring species and strains do occur. The organism metabolizes D-glucose, D-galactose, D-fructose, D-mannose, sucrose, L-sorbose, L-rhamnose, m-inositol and adonitol. P. multocida fails to hydrolyze starch, salicin or aesculin. Tests for arginine di-hydrolase, lysine decarboxylase, liquefaction of gelatin and urease are negative (De Alwis, Reference De Alwis1999). Phenotypic variability has been well documented among strains of P. multocida originating from bovine, avian, caprine, leporine and ovine host species (Shivachandra et al., Reference Shivachandra, Kumar, Saxena, Singh and Srivastava2006c; Ekundayo et al., Reference Ekundayo, Odugbo, Olabode and Okewole2008). Biochemical reactions have proven to be useful in epidemiological studies of P. multocida infections (Fegan et al., Reference Fegan, Blackall and Pahoff1995). The differences in the carbohydrate fermentation reactions of the isolates might be due to indiscriminate use of chemotherapeutic agents in a particular geographical region coupled with host and environmental stress factors influencing the enzyme profiles.
The genome of Pasteurella contains 37.7–45.9 mol% G+C, with molecular weight ranging from 1.4×109 to 1.9×109 (Hunt et al., Reference Hunt, Adler and Townsend2000). To date, no attempt has been made to sequence the genome of P. multocida strains involved in causing HS; however, the complete genome sequence of a common avian clone of P. multocida, Pm70 (serotype A:3), causing fowl cholera, has been completed (May et al., Reference May, Zhang, Li, Paustian, Whittam and Kapur2001). The organism has a single circular chromosome with 2,257,487 bp having 2014 predicted coding regions, six ribosomal RNA operons and 57 tRNAs. The entire sequence specifies 2014 potential CDs with an average size of 998 bp, which account for 89% of the chromosome. Genome-scale evolutionary analyses based on pairwise comparisons of 1197 orthologous sequences suggested that P. multocida and H. influenzae diverged approximately 270 million years ago and the γ subdivision of the proteobacteria radiated about 680 million years ago (May et al., Reference May, Zhang, Li, Paustian, Whittam and Kapur2001).
Serotype classifications have been developed by many workers and correlated with specific disease manifestations (Carter, Reference Carter1955; Carter and Chengappa, Reference Carter and Chengappa1981). In 1947, Roberts introduced an immunological typing system based on passive protection of mice by antiserum and four types (I, II, III and IV) were recognized. In 1954, Hudson added type V. In 1961, Namioka and Murata recognized 11 serotypes (1–11) based on a tube agglutination test (De Alwis, Reference De Alwis1999). Currently, the most acceptable and widely used serotype designation system is a combination of Carter's capsular typing and Heddleston's somatic typing, which recognizes five capsular serogroups (A, B, D, E and F) based on an indirect hemagglutination assay (IHA) and 16 (1–16) somatic serotypes based on agar gel precipitation (Carter, Reference Carter1967; Heddleston et al., Reference Heddleston, Gallagher and Rebers1972). The causative agent of HS has been named in different classifications as Roberts type-I, Heddleston type 2, Carter type B and Namioka and Carter types 6:B and 6:E. The Asian serotype for HS is designated as B:2 and the African serotype E:2 by the Carter–Heddleston system that corresponds to 6:B and 6:E of the Namioka−Carter system (De Alwis, Reference De Alwis1999; Benkirane and De Alwis, Reference Benkirane and De Alwis2002; Kumar et al., Reference Gautam, Kumar, Singh, Singh, Dutta and Shivachandra2004).
Virulence factors of P. multocida
P. multocida has several mechanisms or components that evade the host immune system; these are included among the virulence factors (Harper et al., Reference Harper, Boyce and Adler2006). A total of 104 putative virulence-associated genes that account for ~7% of the coding density of the genome have been predicted and about 26% of the CDs analyzed are highly similar to hypothetical proteins (May et al., Reference May, Zhang, Li, Paustian, Whittam and Kapur2001), indicating that a substantial portion of virulence factors remains to be discovered.
Capsule
The capsule determines the serogroup type of P. multocida, namely A, B, D, E and F, whose biosynthetic loci have been identified (Boyce et al., Reference Boyce, Chung and Adler2000; Townsend et al., Reference Townsend, Boyce, Chung, Frost and Adler2001). Polysaccharides constitute the major part of the capsule and in some cases may be associated with lipoproteins. Two genes (bcbA and bcbB) within region 2 of the B:2 cap locus, encode proteins that catalyze the conversion of UDP-N-acetylglucosamine to N-acetyl-D-mannosaminuronic acid, indicating that mannose is the major type B capsular component (Boyce et al., Reference Boyce, Chung and Adler2000). Of nine genes in region 2 of serogroup E, seven are homologs of genes in the B:2 cap locus, and one encodes for a putative glycosyltransferase. Gene expression gives rise to capsular variability of strains (Harper et al., Reference Harper, Boyce and Adler2006). Capsular strains are more virulent than their acapsular variants as the capsule plays a significant role in resistance to phagocytosis that has been demonstrated in vitro by Harmon et al. (Reference Harmon, Glisson, Latimer, Steffens and Nunnally1991) and high by susceptibility of genetically defined acapsular serotype B mutants to uptake by murine macrophages as reported by Boyce and Adler (Reference Boyce and Adler2000). Removal of hyaluronic acid from the capsule increases both adhesiveness of the organism to the animal cell surfaces and its susceptibility to phagocytosis. The production of capsular material of P. multocida is affected by sub-minimal inhibitory concentrations of antibiotics (Boyce et al., Reference Boyce, Chung and Adler2000).
Fimbriae and adhesins
Type IV fimbriae (pili), with repeated subunits of 15–20 kDa and highly conserved 21-amino acid N-terminal sequences, have been isolated and characterized from P. multocida serotypes A, B and D that are able to adhere to mucosal epithelium (Ruffalo et al., Reference Ruffalo, Tennet, Michalski and Adler1997). Fimbriae on P. multocida isolated from calves' nasal and tracheal cells have been identified by electron microscopic studies. The gene ptfA that encodes the subunit has been isolated and sequenced from a number of strains and the predicted protein sequences showed significant variation among strains (Doughty et al., Reference Doughty, Ruffalo and Adler2000). Cloning and sequence analysis of the ptfA gene from P. multocida B:2 (strain p52) indicated 78.4% similarity with that of the A:1 strain (Siju et al., Reference Siju, Kumar, Shivachandra, Chaudhuri, Srivastava and Singh2007). Treatment of the organism with pronase, heat, acidity or homogenization confirmed the involvement of the fimbriae in the attachment process. Moreover, anti-fimbrial antibodies specifically inhibited the adherence capabilities of different serotypes that are known to greatly vary for tracheal epithelial cells from different hosts (Harper et al., Reference Harper, Boyce and Adler2006).
Several non-fimbrial adhesins have also been identified as virulence factors in P. multocida. Fibronectin-binding protein ComE1 (12.7 kDa) has recently been identified by panning a phage library of P. multocida (Mullen et al., Reference Mullen, Nair, Ward, Rycroft, Williams, Robertson, Mordan and Henderson2008) and shown to interact with integrin-binding fibronectin type III (FnII) repeats and be involved in bacterial competence and DNA uptake (Harper et al., Reference Harper, Boyce and Adler2006). P. multocida Pm70 genome sequence predicted two genes encoding for the filamentous hemagglutinin proteins (FhaB1 and Fhab2) that appear to increase bacterial dispersion and colonization (Fuller et al., Reference Fuller, Kennedy and Lowery2000; May et al., Reference May, Zhang, Li, Paustian, Whittam and Kapur2001). Mutations in adhesin genes of P. multocida resulted in significantly reduced virulence in mice. In addition, the putative tight adherence (Tad) locus, comprised of genes encoding for outer membrane proteins (OMPs) (TadD, RcpA, RcpB and Flp1), is involved in the Tad macromolecular transport system that is required for biofilm formation and colonization (May et al., Reference May, Zhang, Li, Paustian, Whittam and Kapur2001).
OMPs
Complex OMP profiles of ~15–20 protein bands ranging from 16 to 90 kDa have been demonstrated in HS strains (Tomer et al., Reference Tomer, Chaturvedi, Minakshi, Malik and Monga2002). Correlation between the electrophoretic pattern and serotype properties of isolates and host origin has been established (Wheeler, Reference Wheeler2009), but common OMP bands with molecular weights of 16, 27, 34, 39, 46 and 82–87 kDa have been identified in all isolates regardless of serotype. Comparative protein profiles of HS causing serotypes B and E strains revealed that there was a sharing of major bands (27, 45 and 47 kDa) and a few minor specific bands (32 and 37 kDa) (Johnson et al., Reference Johnson, Dawkins and Spencer1991).
Earlier studies identified proteins of 37 and 39 kDa as protective antigens. Some OMPs were identified as OmpH, OmpA, Oma87, Pm1069 and iron-related proteins, transferrin binding protein A (TbpA), and hemoglobin-binding protein (HgbA) (Wheeler, Reference Wheeler2009). P. multocida strains have several mechanisms for uptake of iron from the host under various conditions (Harper et al., Reference Harper, Boyce and Adler2006). Comparison of P. multocida grown in iron-rich medium, iron-depleted medium, or in vivo has demonstrated that many high-molecular-weight OMPs are regulated by iron levels and are called iron-regulated OMPs (IROMPs). It is considered that IROMPs play a significant role in cross-protective immunity (Srivastava, Reference Srivastava1998). Serum from vaccinated animals contained antibodies reacting with only a few OMP bands. A bioinformatics analysis by Al-Hasani et al. (Reference Al-Hasani, Boyce, McCarl, Bottomley, Wilkie and Adler2007) using P. multocida genome sequence data predicted 129 proteins as secreted and located in the outer membrane, or lipoproteins with a total of 79 proteins as outer membrane localized. Recently, Hatfaludi et al. (Reference Hatfaludi, Al-Hasani, Boyce and Adler2010) published an excellent review of OMPs from P. multocida and their roles in various functions.
Endotoxin (lipopolysaccharide (LPS)) and exotoxins
LPS plays a critical role in pathogenesis of HS in buffaloes as they are absolutely required for in vivo replication (Harper et al., Reference Harper, Boyce and Adler2006). LPS is also responsible for the 16 somatic serotypes. Electrophoretically, LPS of serogroup B and E strains from HS were found to be identical and the same as LPS from serologically related serogroup-A strains (Rimler, Reference Rimler1990). The complete LPS structure of strains VP161 and X73 belonging to Heddleston serotype 1 and PM70, a Heddleston serotype-3 strain, have been determined (St Michael et al., Reference St Michael, Vinogradov, Li and Cox2005). Intravenous inoculation of LPS from serotype B:2 strains reproduced clinical signs of HS in buffaloes (Horadagoda et al., Reference Horadagoda, Hodgson, Moon, Wijewardana and Eckersall2002) and rLPS plays a role in adhesion to bovine neutrophils and transmigration through endothelial cells. LPS is considered to be a protective antigen as it is known to stimulate humoral immunity and generate production of bactericidal antibodies (Ramdani and Adler, Reference Ramdani and Adler1991). Clinical isolates of P. multocida from cattle exhibit serum resistance. Due to its mitogenic action, LPS stimulates the release of many interleukins, TNF-α and other tumor inhibition factors from bovine alveolar macrophages (De Alwis, Reference De Alwis1999; Harper et al., Reference Harper, Boyce and Adler2006).
Most P. multocida strains that cause HS, except for a few bovine isolates, are not known to express any exotoxins (De Alwis, Reference De Alwis1999). The toxA gene encoding for P. multocida toxin (PMT) was frequently detected in porcine and to a lesser extent bovine strains, but an unexpectedly high percentage in strains from small ruminants (Ewers et al., Reference Ewers, Lubke-Becker, Bethe, Kiebling, Filter and Wieler2006).
Multocidin or siderophores
Iron plays a critical role in P. multocida survival inside the host as evidenced by in silico and whole genome microarray analyses that identified more than 50 Pm70 genes involved in iron acquisition and metabolism (Fuller et al., Reference Fuller, Kennedy and Lowery2000; May et al., Reference May, Zhang, Li, Paustian, Whittam and Kapur2001). The organism, grown in iron deprivation conditions, is able to secrete a growth enhancing factor that functions as a siderophore (multocidin) (Harper et al., Reference Harper, Boyce and Adler2006). Siderophores selectively bind ferric iron and are involved in receptor-specific iron transport into the bacterial cell. Several types of siderophores are synthesized by P. multocida (Hu et al., Reference Hu, Felice, Sivanandan and Maheswaran1986). Choi-Kim et al. (Reference Choi-Kim, Maheswaran, Felice and Molitor1991) reported three IROMPs (76, 84 and 96 kDa) which were expressed in iron-restricted medium or in vivo and are believed to be involved in iron−multocidin complex formation. Approximately 1% of the genome encodes large proteins that are filamentous hemagglutinins (May et al., Reference May, Zhang, Li, Paustian, Whittam and Kapur2001). The organism is also capable of obtaining iron for growth by a non-siderophore-mediated mechanism. Such acquisition was associated with the production of high-molecular-weight IROMPs including an 82 kDa receptor protein (TbpA) for bovine transferrin-bound iron (Veken et al., Reference Veken, Shah, Klaasen, Oudega and De Graaf1996; Hatfaludi et al., Reference Hatfaludi, Al-Hasani, Boyce and Adler2010).
Extracellular enzymes
Several lipase enzymes produced by strains of P. multocida from various animals are potential virulence factors. Hyaluronidase is another enzyme produced by P. multocida. Its production appears to be limited to serogroup B virulent strains (B:2) isolated from HS cases (Carter and Chengappa, Reference Carter and Chengappa1980), whereas neuraminidase activity is detected in most serogroups isolated from various species. Hyaluronidase activity test could be used for presumptive identification of B:2 strains causing HS (Rimler and Rhoades, Reference Rimler and Rhoades1994). High neuraminidase production is correlated with high virulence of strains. In P. multocida, a putative pm1426 gene has been predicted to encode OM phospholipase-A which is involved in hydrolysis of phospholipids on the outer surface membrane (Hatfaludi et al., Reference Hatfaludi, Al-Hasani, Boyce and Adler2010). Sialidases (neuraminidases) are produced by several P. multocida strains and are known to be involved in scavenging sialic acid from the host; they increase virulence by blocking the effectiveness of host defenses such as mucin. Sialidase genes encoding for NanH (80 kDa) and NanB (120 kDa) proteins play roles in uptake of sialidic acid through the tripartite ATP-independent periplasmic sialate transporter (Steenbergen et al., Reference Steenbergen, Lichtensteiger, Caughlan, Garfinkle, Fuller and Vimr2005). Many genes for extracellular enzymes have been associated with high virulence potential (Carter and Chengappa, Reference Carter and Chengappa1980).
Plasmids
Plasmids ranging from 1.3 to 100 kb, with the majority between 2 and 6 kb, have been recovered from P. multocida strains from various animal species (Hirsh et al., Reference Hirsh, Martin and Rhodes1981). These plasmids confer antibiotic resistance or encode for various virulence attributes including toxins. Plasmid profile is considered as a virulence marker for P. multocida as well as an essential epidemiological tool for identifying different strains of the same phenotype (Harper et al., Reference Harper, Boyce and Adler2006). A number of R-plasmids identified were found to be non-conjugative, but readily transferable to other P. multocida and Escherichia coli by transformation (Hirsh et al., Reference Hirsh, Martin and Rhodes1981). Description of antibiotic resistance genes identified on plasmids is mentioned under the chemotherapy section of this review.
Pathogenicity
The rapid onset of clinical signs in affected animals following exposure to low numbers of virulent bacteria (104–107 CFU) during experimental infection is associated with a high rate of bacterial proliferation with the generation time as low as 15 min (Ramdani et al., Reference Ramdani, Dawkins, Johnson, Spencer and Adler1990; De Alwis, Reference De Alwis1999; Harper et al., Reference Harper, Boyce and Adler2006). Most strains of P. multocida are highly virulent to mice and rabbits by experimental inoculation (Chandrasekharan and Yeap, Reference Chandrasekharan and Yeap1982). The minimum lethal dose (LD50) is between 1 and 10 viable organisms by the intravenous route in mice. Guinea pigs are less susceptible to infection. The serotype B:2 strains are not known to cause infection in birds. Strain differences are noticed in terms of lesion severity and time leading to death within 24 h post-inoculation (De Alwis, Reference De Alwis1999; Benkirane and De Alwis, Reference Benkirane and De Alwis2002; OIE, 2009).
Most studies on pathogenesis have used mice as the host animal, with capsular type A or B of P. multocida (Harper et al., Reference Harper, Boyce and Adler2006). A very small dose (20 CFU) of P. multocida organisms is known to proliferate and kill intraperitoneally inoculated mice within 24 h (Ramdani et al., Reference Ramdani, Dawkins, Johnson, Spencer and Adler1990; Biswas et al., Reference Biswas, Shivachandra, Saxena, Kumar, Singh and Srivastava2004). Pathogenesis in mice depends on the virulence characteristics of the organism and the genetic makeup of the host. Although various somatic serovars are reported to be dominant in different geographical areas, there are no indications that any particular serovars is more or less virulent than others (De Alwis, Reference De Alwis1999). Different isolates of the common serovar vary greatly in virulence. Despite the complete genome sequence analysis (May et al., Reference May, Zhang, Li, Paustian, Whittam and Kapur2001), neither the incriminating environmental factors nor the bacterial intrinsic properties responsible for mucosal invasion, evasion of innate immunity, persistence in hostile in vivo conditions and sudden onset of rapid multiplication and the determinants of virulence have been elucidated.
Diagnosis
For diagnosis, specimens to be collected from infected live animals are blood samples and nasal secretions, and from dead animals are heart blood, liver, spleen, lung and bone marrow of long bones (OIE, 2009). The sudden death seen with peracute and acute HS must be differentiated from that due to lightning, snakebites, blackleg, rinderpest or anthrax by both conventional and molecular approaches (De Alwis, Reference De Alwis1999; Benkirane and De Alwis, Reference Benkirane and De Alwis2002; OIE, 2009).
Conventional approaches
The conventional methods of identification and characterization of field isolates of P. multocida are carried out as per the international standards (OIE, 2009). Globally, a large number of P. multocida organisms have been isolated from various outbreaks of HS occurring in different agro-climatic geographical regions (Kumar et al., Reference Gautam, Kumar, Singh, Singh, Dutta and Shivachandra2004). Considerable variation has been observed among serotypes with respect to host predilection, pathogenicity, biochemical, cultural and antigenic specificity (De Alwis, Reference De Alwis1999). Clinical diagnosis is usually done by characteristic signs, gross pathological lesions, herd history, morbidity and mortality pattern, species susceptibility and age group affected (OIE, 2009). Laboratory confirmation is based on demonstration of Gram-negative, bipolar, pleomorphic bacterial cells in fresh blood smears following staining with Gram's, Leishman's or Methylene Blue. Bacterial isolation is based on standard techniques using BHI broth and sheep blood agar culture; subsequent tests may include mice inoculation, identification by colony morphology, pathogenicity testing, biotyping and antibiogram determination (Holt et al., Reference Holt, Krieg, Sneath, Staley and Williams1994; OIE, 2009).
Immunological methods using serum, such as the rapid slide agglutination test (Namioka and Murata, Reference Namioka and Murata1961), IHA (Carter, Reference Carter1955), agar gel immunodiffusion test (Heddleston et al., Reference Heddleston, Gallagher and Rebers1972) and counter immune electrophoresis (Carter and Chengappa, Reference Carter and Chengappa1981), are employed to detect specific antibodies but are not normally used for diagnosis. High IHA titers, from 1/160 up to 1/1280 or higher, among in-contact animals surviving in affected herds are indicative of recent exposure to HS (OIE, 2009). Other serological tests such as passive hemagglutinations, passive mouse protection test (Chandrasekaran et al., Reference Chandrasekaran, Kennett, Yeap, Muniandy, Rani and Mukkur1994a) and ELISA have been used with varying degrees of success in attempts to predict level of immunity (De Alwis, Reference De Alwis1999). The ease of obtaining a definite diagnosis through isolation and identification of the organism precludes the need for sero-diagnosis. Non-serological methods such as the acriflavine flocculation test and hyaluronidase tests could also be employed (Rimler and Rhoades, Reference Rimler and Rhoades1994).
Molecular approaches
Polymerase chain reaction (PCR) assays for the specific identification of P. multocida and serotypes implicated in HS have been developed for identification within 3–4 h (Biswas et al., Reference Biswas, Shivachandra, Saxena, Kumar, Singh and Srivastava2004). The complexities associated with presumptive diagnosis by conventional methods have been overcome by more rapid PCR assays that can be applied directly on bacterial colonies, bacterial culture lysates, mixed bacterial cultures and infected clinical materials to permit rapid disease diagnosis (Lee et al., Reference Lee, Wilkie, Townsend and Frost2000; Choi and Chae, Reference Choi and Chae2001; Shivachandra et al., Reference Shivachandra, Kumar, Gautam, Singh, Chaudhuri and Srivastava2004a, Reference Shivachandra, Kumar, Gautam, Joseph, Saxena, Chaudhuri, Srivastava and Singh2006d). In most countries with endemic disease, molecular assays for primary screening of P. multocida isolates for rapid and specific diagnosis of HS have been introduced in parallel to existing conventional methods (Benkirane and De Alwis, Reference Benkirane and De Alwis2002; Biswas et al., Reference Biswas, Shivachandra, Saxena, Kumar, Singh and Srivastava2004).
A P. multocida-specific PCR assay developed by Townsend et al. (Reference Townsend, Frost, Lee, Papadimitrious and Dawkins1998) has been widely employed worldwide for initial species identification; this test gives a positive signal by an amplicon of ~460 bp with field isolates irrespective of capsular type (A, B, D, E and F). Two independently isolated gene sequences unique to HS-causing serogroup-B P. multocida (Brickell et al., Reference Brickell, Thomas, Long, Panaccio and Widders1998; Townsend et al., Reference Townsend, Frost, Lee, Papadimitrious and Dawkins1998) have been utilized in the development of serogroup B-specific PCR assays. The Haemorrhagic septicaemia serogroup-B developed by Townsend et al. (Reference Townsend, Frost, Lee, Papadimitrious and Dawkins1998) remains highly specific for HS-causing serogroup B serotype 2 or 5, and identifies target strains by the amplification of an approximately 620 bp fragment. For rapid capsular serogrouping, a multiplex capsular PCR typing system has been developed by Townsend et al. (Reference Townsend, Boyce, Chung, Frost and Adler2001) following identification, sequence determination and analysis of the capsular biosynthetic loci of each capsular serogroup. This assay represents a highly specific, rapid and reproducible alternative to the serological and non-serological methods currently used for classification of P. multocida capsular types. Moreover, it is particularly useful in developing countries where conventional capsular serotyping facilities do not exist (Shivachandra et al., Reference Shivachandra, Kumar, Gautam, Singh, Saxena and Srivastava2006a). Later, Gautam et al. (Reference Gautam, Kumar, Singh, Singh, Dutta and Shivachandra2004) developed a P. multocida serogroup A-specific PCR assay based on genes hyaC-hyaD of the region 2 capsule biosynthetic locus of P. multocida A:1; this PCR assay results in an amplified product of ~564 bp and can be employed for rapid detection directly from morbid tissue materials (Shivachandra et al., Reference Shivachandra, Kumar, Gautam, Singh, Chaudhuri and Srivastava2004a). The PCR assays are able to identify acapsular isolates of P. multocida that could not be typed by conventional methods. The PCR assays did not amplify genes from other bacteria and fungi tested in previous studies, and had high specificity as well as high sensitivity (Townsend et al., Reference Townsend, Frost, Lee, Papadimitrious and Dawkins1998, Reference Townsend, Boyce, Chung, Frost and Adler2001).
A new virulence typing system based on PCR was introduced by Ewers et al. (Reference Ewers, Lubke-Becker, Bethe, Kiebling, Filter and Wieler2006) to determine the correlation between P. multocida infection and existence of virulence genes including tbpA, pfhA, toxA and hgbB. Later, Atashpaz et al. (Reference Atashpaz, Shayegh and Hejazi2009) introduced a multiplex PCR assay targeting these four virulence genes in a single reaction. However, these assays were tedious and laborious.
HS can also be rapidly diagnosed by employing PCR assays directly on homogenized infected tissue materials (lung, liver, spleen and heart), bone marrow of long bones, blood and nasal discharge without in vitro cultivation (unpublished results). Storage of morbid materials following detection by PCR also indicated the possibility of detecting up to 3 days at 37°C, 40 days at 4°C and up to 100 days at −20°C especially from bone marrow (Shivachandra et al., Reference Shivachandra, Kumar, Gautam, Singh, Chaudhuri and Srivastava2004a). Despite variable sensitivity for different tissues, the PCR approach has considerable value in early diagnosis. A number of workers have extensively used Pasteurella multocida-PCR, HSB-PCR and multiplex capsular PCR typing systems for rapid identification and capsular serotyping of field isolates from various animal species irrespective of geographical regions of isolation (Lee et al., Reference Lee, Wilkie, Townsend and Frost2000; Biswas et al., Reference Biswas, Shivachandra, Saxena, Kumar, Singh and Srivastava2004; Kumar et al., Reference Gautam, Kumar, Singh, Singh, Dutta and Shivachandra2004; Shivachandra et al., Reference Shivachandra, Kumar, Gautam, Singh, Saxena and Srivastava2006a). High reliability, simplicity and versatility of the PCR approach as a routine basis for diagnosis of HS were noted. Moreover, readily available PCR equipment and experience required to perform these techniques in most laboratories in developing countries provide a marked advantage over other methods of identification.
A novel loop-mediated isothermal amplification (LAMP) method has been developed by Sun et al. (Reference Sun, Wang, Wu, Wang, He, Zheng and Yang2010), targeting the kmt1 gene from the species-specific region of the P. multocida genome. LAMP products generated by incubating the reaction mixture at a fixed temperature are visibly detected using fluorescent dyes at field level without use of any sophisticated equipments (Notomi et al., Reference Notomi, Okayama, Masubuchi, Yonekawa, Watanabe, Amino and Hase2000). Being a simple, rapid, sensitive and highly specific assay, P. multocida specific-LAMP (Pm-LAMP) could be potentially employed as a rapid diagnostic tool at field level in most underdeveloped and developing countries for initial bacterial species identification. However, there is a need to develop a LAMP assay specific for HS strains as well as its application directly on clinical materials without further processing.
Molecular characterization encompasses a large range of methods with variable specificity and discriminatory power, most of which have been used to differentiate phenotypically similar P. multocida isolates from various animal species (Dziva et al., Reference Dziva, Muhairwa, Bisgaard and Christensen2008). Restriction endonuclease analysis (REA) is the simplest method currently being used to differentiate HS isolates. Typically, REA of P. multocida strains has been performed using either a six- or four-base cutter restriction endonuclease, namely, PstI, EcoRI, SmaI, BglII, XhoI, HhaI, HindIII or HpaII (Rimler, Reference Rimler2000). REA using HpaII resulted in a satisfactory number of large, clear and distinct bands that could be appreciated very easily by the unaided eye. REA using HhaI and HpaII enzymes has been successfully employed to compare and differentiate somatic serotypes of HS serogroup B and E isolates (Wilson et al., Reference Wilson, Rimler and Hoffman1992; Rimler, Reference Rimler2000). Pulsed-field gel electrophoresis (PFGE), a specialized form of REA, often considered a ‘gold standard’ of molecular typing methods, uses rare cutter restriction endonucleases to produce a few fragments (5–20) of 10–800 kb in size, larger than REA fragments. Although the technique has been proven to be highly discriminatory for P. multocida isolates (Gunawardana et al., Reference Gunawardana, Townsend and Frost2000; Jablonska and Opacka, Reference Jablonska and Opacka2007), its applicability in routine diagnostic laboratories is limited due to requirements for high-quality DNA and expensive electrophoresis apparatus.
Southern blot-restriction fragment length polymorphism (RFLP) is mainly based on REA coupled with hybridization with one or more labeled probes homologous to the gene to be examined. Ribotyping, a variation of Southern blot-RFLP, uses probes derived either from ribosomal RNA (5s RNA, 16s RNA and 23s RNA) or the genes that code for rRNA (rrf, rrs and rrl), and has been applied for successful typing of P. multocida strains (Townsend et al., Reference Townsend, Dawkins and Papadimitriou1997a; Saxena et al., Reference Saxena, Kumar, Chaudhuri, Shivachandra, Singh, Singh and Sharma2005). Despite its complex procedure, the HpaII enzyme-digested DNA detection with a probe based on 16S+23S rRNA was found to be highly discriminatory (Dziva et al., Reference Dziva, Muhairwa, Bisgaard and Christensen2008). Alternatively, Oligomix5 probes could be used for detection. This technique with additional detection procedures, labeling of probes either with radioactive or chemical compounds and expertise required to use special equipments is less desirable.
PCR amplifications based on repetitive regions using consensus oligonucleotides matching highly conserved extragenic or intergenic repetitive sequences are being used as primers to amplify DNA between successive repetitive elements in various Gram-negative strains including P. multocida (Biswas et al., Reference Biswas, Shivachandra, Saxena, Kumar, Singh and Srivastava2004; Dziva et al., Reference Dziva, Muhairwa, Bisgaard and Christensen2008). Two such techniques, repetitive extragenic palindromic (REP)-PCR and enterobacterial repetitive intergenic consensus (ERIC)-PCR, targeting REP and ERIC elements of the bacterial genome, respectively, have been used for typing (Versalovic et al., Reference Versalovic, Koeuth and Lupski1991). REP- and ERIC-PCR techniques have proved to be valuable tools for rapid epidemiological analysis and differentiation of HS outbreak-related isolates, which phenotypically appear similar by conventional methods (Townsend et al., Reference Townsend, Dawkins and Papadimitriou1997a, Reference Townsend, Dawkins and Papadimitrioub; Biswas et al., Reference Biswas, Shivachandra, Saxena, Kumar, Singh and Srivastava2004; Shivachandra et al., Reference Shivachandra, Kumar and Chaudhuri2008). Unlike REA, ribotyping and PFGE, the PCR-based tests do not require high-quality DNA and the REP-/ERIC-PCR can be performed directly on colonies without any need for extensive DNA extraction (Gunawardana et al., Reference Gunawardana, Townsend and Frost2000), highlighting the time advantage over other DNA-based typing methods for routine analysis (Dziva et al., Reference Dziva, Muhairwa, Bisgaard and Christensen2008). Randomly amplified polymorphic DNA-PCR assay for P. multocida has also been developed as a fast and efficient method of classifying field isolates with high specificity (Dziva et al., Reference Dziva, Christensen, Olsen and Mohan2001; Shivachandra et al., Reference Shivachandra, Kumar and Chaudhuri2007). Commercial kits make it easy to use molecular typing of HS isolates (Dziva et al., Reference Dziva, Muhairwa, Bisgaard and Christensen2008).
A novel PCR-based typing technique known as amplified fragment length polymorphism (AFLP) was developed by Vos et al. (Reference Vos, Heger, Blecker, Reijans, Lee, Hornes, Frigters, Peleman, Kulper and Zabeau1995). This technique selectively amplifies genomic restriction fragments with high resolution coupled with a high degree of reproducibility, sensitivity and good discriminatory power (Dziva et al., Reference Dziva, Muhairwa, Bisgaard and Christensen2008). AFLP analysis is based on variation in restriction sites and could be used to generate fingerprinting without prior sequence knowledge, for identification, epidemiology and taxonomy (Savelkoul et al., Reference Savelkoul, Aarts, Haas, Dijkshoorn, Duim, Otsen, Rademaker, Schouls and Lenstra1999). Single-enzyme AFLP, a variant typing tool with a combination of REA and PCR, is equally discriminatory but time consuming and could be employed for occasional or high stringency typing (Shivachandra et al., Reference Shivachandra, Kumar, Gautam, Joseph, Saxena, Chaudhuri and Srivastava2006b).
Gene sequencing and multi-locus sequence typing (MLST) have also been used to differentiate strains based on differences in specific gene loci (Dziva et al., Reference Dziva, Muhairwa, Bisgaard and Christensen2008). MLST with high reproducibility and discriminatory power may be a viable option as it targets house-keeping loci and sequence data found to be portable between laboratories and could permit establishment of a global database (Spratt, Reference Spratt1999; Subaaharan et al., Reference Subaaharan, Blackall and Blackall2010). It has been widely accepted that 16S rRNA-based evolutionary phylogeny studies could be effectively employed in understanding the genetic diversity of P. multocida and its classification (Davies, Reference Davies2004). Comparative gene sequence analysis of 16S rRNA genes from isolates of P. multocida serotype B:2 belonging to buffalo, cattle, pig, sheep and goat indicated the close relationships of HS strains of P. multocida serotype B:2 strains from uncommon hosts (Dey et al., Reference Dey, Singh, Kumar, Sharma, Srivastava and Singh2007). It is presumed that comparative gene analysis of various virulence factors could reveal their role in terms of host specificity, predilection site and pathogenesis. Moreover, molecular classification of HS strains would significantly unravel the mysteries at the population level and add to greater understanding of disease and persistence, host associations and host response at the clonal level (Christensen and Bisgaard, Reference Christensen and Bisgaard2010).
Chemotherapy
Treatment is of little use once signs of HS have appeared, but could be effective in the early stages (OIE, 2009). Practically, the procedure employed is to check the rectal temperature of all in-contact animals after the first case has been reported, and to start antibiotic therapy immediately (Benkirane and De Alwis, Reference Benkirane and De Alwis2002). Generally, clinical cases of HS are extensively treated with oxytetracycline, co-trimoxazole, a combination of penicillin and streptomycin or sulphaquinoxaline. However, in the recent times, shifts in antibiotic sensitivity spectrum of P. multocida are evident and there have been increases in incidences of high morbidity and mortality (Biswas et al., Reference Biswas, Shivachandra, Saxena, Kumar, Singh and Srivastava2004; Kumar et al., Reference Gautam, Kumar, Singh, Singh, Dutta and Shivachandra2004, Reference Kumar, Singh, Agrawal and Singh2009). Compromised therapy may be due to emergence of multi-drug-resistant strains as a result of widespread use of antibiotics as a supplement in feed, for prophylaxis and growth promotion, for subtherapeutic use in the feed, and for metaphylactic antibiotic therapy. Our earlier studies identified a rise in multiple drug-resistant strains of P. multocida (Biswas et al., Reference Biswas, Shivachandra, Saxena, Kumar, Singh and Srivastava2004), especially for sulphadiazine, the drug of choice in the field for control of HS, as well as many other commonly used antibiotics such as amikacin, carbencillin, erythromycin and penicillin, where there is gradual development of resistance (Kumar et al., Reference Kumar, Singh, Agrawal and Singh2009). Strains of P. multocida vary in susceptibility to chemotherapeutic agents. Most P. multocida isolates were resistant to sulpha drugs with varied sensitivity to chloramphenicol, gentamicin, tetracycline, kanamycin, penicillin-G, streptomycin, sulphonamides and trimethoprim as assessed by agar dilution and determination of the susceptibility of transconjugants and transformants by disc diffusion assay (De Alwis, Reference De Alwis1999; Shivachandra et al., Reference Shivachandra, Kumar, Biswas, Ramakrishnan, Singh and Srivastava2004b).
The injudicious use of antibiotics in developing countries is probably leading to the severe problem of antibiotic resistance, the genes of which may be on R-plasmids (Hirsh et al., Reference Hirsh, Martin and Rhodes1981). Conjugative R-plasmids are commonly responsible for inter-species and inter-genic spread of multiple drug resistance and transfer of such plasmids among pathogenic strains may give rise to epidemic spread of infection (Stone, Reference Stone1975). Co-existence and the spread of small plasmids are used by P. multocida to become multi-resistant (SanMillan et al., Reference SanMillan, Escudero, Gutierrez, Hidalgo, Garcia, Llagostera, Dominguez and Gonzalez-Zorn2009). Many isolates with R-plasmids carried resistance genes to streptomycin, sulphonomides, tetracycline, kanamycin, chloramphenicol and penicillin (ROB-1 β-lactamase gene), thereby conferring significant resistance to chemotherapy (Silver et al., Reference Silver, Leming, Garon and Hgerpe1979). However, R-plasmids harboring antibiotic resistance genes were not further characterized. Some studies attributed the resistance to chromosomal genes (Tet-B and Tet-M class of tetracycline resistance determinants) (Chalsus-Dancla et al., Reference Chalsus-Dancla, Lesage-Descauses, Leroy-Setrin, Martel and Lafont1995), and hypothesized that they may be borne on the only transposable element (Tn5706) identified in P. multocida. A mandatory check on indiscriminate use of antibiotics and testing for antibiotic sensitivity of P. multocida isolates are warranted in the treatment of HS.
Prophylaxis
Vaccination of susceptible animals in endemic areas is the only practical approach to prevent HS (Benkirane and De Alwis, Reference Benkirane and De Alwis2002; OIE, 2009). In most countries various formulations of HS vaccines are available for vaccination of animals (Verma and Jaiswal, Reference Verma and Jaiswal1998). The current and previous investigations on isolation and serotyping of P. multocida from cases of HS reveal the presence of other serotypes (A:1) as well (Kumar et al., Reference Gautam, Kumar, Singh, Singh, Dutta and Shivachandra2004). Moreover, it has been observed at field level that despite proper vaccination, HS does occur among cattle and buffaloes (Biswas et al., Reference Biswas, Shivachandra, Saxena, Kumar, Singh and Srivastava2004) indicating the imminent need to revamp existing vaccines as well as vaccination strategy against HS.
Conventional approaches in the development of vaccines
Since the beginning of HS vaccination, traditional vaccines are proven to be impeccable with a few exceptions and still continue to dominate in the era of new age vaccines (OIE, 2009). However, there is a need to rejuvenate and/or better modify them in the present context to achieve broader cross-protective and long-lasting immunity (De Alwis, Reference De Alwis1999).
Killed vaccines
Initially, plain bacterins of P. multocida were advocated for prophylactic vaccination of cattle against HS and offered immunity for 6 months (Baldrey, Reference Baldrey1907; De Alwis, Reference De Alwis1999). The basic approach in the development of HS vaccines was to kill the P. multocida organisms using either physical (heat, UV irradiation) or chemical (lysol or formalin) agents that resulted in retention of the antigens responsible for inducing the host's protective immune response (Chorherr, Reference Chorherr1974). Later, several killed vaccines were developed with various adjuvants to enhance the immune response (Iyer et al., Reference Iyer, Gopalkrishnan and Ramani1955; Verma and Jaiswal, Reference Verma and Jaiswal1998). The commonly used vaccines are bacterins, alum precipitated vaccines, aluminum hydroxide gel vaccines, oil adjuvant vaccines (OAV) and multiple emulsion (ME) vaccines (Verma and Jaiswal, Reference Verma and Jaiswal1997) with considerable variation in their duration of induced immunity (Chandrasekaran et al., Reference Chandrasekaran, Kennett, Yeap, Muniandy, Rani and Mukkur1994b) and side effects. Agar-wash heat killed or formalized vaccines using higher concentrations of bacterial cells were developed (Rau and Govil, Reference Rau and Govil1950) to avoid protein shock due to killed broth culture vaccine organisms.
Alum-precipitated HS vaccines have been extensively used in different parts of the world as they are known to protect cattle, sheep and goats against P. multocida infection for 6 months (Nikhiporova, Reference Nikhiporova1958). An aluminum hydroxide gel HS vaccine used in Thailand and Laos provides less immunity despite the incorporation of vitamin E and levamisole as immunomodulators. Simple HS emulsion vaccines (water-in-oil) were developed to enhance the stability of vaccines by using an emulsifying agent such as lanolin, and mannite monooleate (Arlacel-A). Subsequently, oil-adjuvant HS vaccines were developed using small quantities of mineral oil (Bain, Reference Bain1956). In a comparative efficacy study involving OAV and alum vaccines, the OAV was found to be superior in terms of storage at room temperature for 1 year without deterioration, withstanding high temperature (advantage in tropical developing countries), providing protective immunity against challenge after 1 year of vaccination and safety in all ages of cattle and buffaloes (Dhanda et al., Reference Dhanda, Das, Lal and Seth1958; Iyer and Rao, Reference Iyer and Rao1959). In a study by Shah et al. (Reference Shah, Jacobs, Shah and de Graaf2001) an improved oil-adjuvant HS vaccine (serotype B:2,5) was found to provide cross protection against heterologous serotype E:2,5. However, the OAV was difficult to inject due to its high viscosity, and formation of abscess at the site of injection and post-vaccination reactions have often been reported (Verma and Jaiswal, Reference Verma and Jaiswal1998).
In order to overcome the limitations, an ME vaccine was prepared by re-emulsifying the OAV with 2% Tween 80, which was easy to administer and equally effective in protection studies; however, there was a reduced duration of immunity with the multi-emulsion HS vaccine (Mittal et al., Reference Mittal, Jiaswal and Gupta1979; Verma and Jaiswal, Reference Verma and Jaiswal1997, Reference Verma and Jaiswal1998). A double-emulsion vaccine has been shown to be efficient in generation of immunity in buffalo up to 12 months post-vaccination. Generally, vaccination with killed vaccines is practiced in HS endemic areas and found to reduce the incidence (OIE, 2009), but due to the short duration of immunity, outbreaks still occur (Benkirane and De Alwis, Reference Benkirane and De Alwis2002; Biswas et al., Reference Biswas, Shivachandra, Saxena, Kumar, Singh and Srivastava2004). Bacterins are inexpensive to produce and give very limited protection against heterologous serotypes as the immunity generated is serotype specific. Further, the protective potency of different batches of vaccines is always unpredictable due to lack of quality control tests for ascertaining their protective potency. Recently, an experimental low-volume (2 ml) saponified HS vaccine was developed in India with promising protection in cattle and buffaloes up to 12 months post-vaccination (Kumar et al., unpublished results).
Live vaccines
Ever since the attenuation of P. multocida strains using prolonged in-vitro cultivation by Pasteur (Reference Pasteur1880), several modified methods with an attempt to attenuate live P. multocida micro-organisms by aging, serial passage in unnatural hosts (chick embryo, albino rats, pigeons and rabbits), chemical/antibiotics treatment or altering its growth conditions (growth at different temperatures and nutrient-restricted medium) have been described (Wei and Carter, Reference Wei and Carter1978; Kucera et al., Reference Kucera, Wong and Eis1981; De Alwis, Reference De Alwis1999). Several chemical agents such as N-methyl-N-nitro-N-nitrosoguanidine, diamino acridine salts (acriflavin), psoralin treatment and UV irradiation, have been used to develop altered live vaccines (Verma and Jaiswal, Reference Verma and Jaiswal1998), which included streptomycin-dependent mutants of P. multocida type B, 6:B, A:1, A:1,2 and A:3 strain vaccines with varied protection rates in different animal species (Wei and Carter, Reference Wei and Carter1978; Kucera et al., Reference Kucera, Wong and Eis1981). A temperature-sensitive (ts) mutant of P. multocida type B was also used as a live vaccine to protect the calves against homologous challenge (Verma and Jaiswal, Reference Verma and Jaiswal1998).
A fallow deer strain aerosol vaccine (P. multocida strain B: 3,4) developed in Myanmar, is the sole live vaccine that has met with success (Myint and Carter, Reference Myint and Carter1989). Despite the recommendations by FAO for safe use of this vaccine in Asian countries, currently, no country is using it for control of endemic HS cases except for Myanmar, where more than 1.5 million animals were inoculated during 1989–99. Moreover, this vaccine has been found to protect the cattle against subcutaneous challenge with virulent B:2 serotype 1 year after vaccination and the serum of vaccinated cattle also cross-protected passively immunized mice against infection with P. multocida serotypes E:2, F:3,4 and A:3,4 (Myint et al., Reference Myint, Jones and Nyunt2005). Live/attenuated vaccines are often the most successful vaccines with prolonged immunity (Myint and Carter, Reference Myint and Carter1989; Verma and Jaiswal, Reference Verma and Jaiswal1998); however, they always carry a risk of reversion at any time inducing HS outbreaks rather than protection.
Acellular vaccines
Acellular vaccines comprise only antigenic parts of P. multocida, such as capsule (Maslog, Reference Maslog1998), fimbriae, LPS, OMPs (Hatfaludi et al., Reference Hatfaludi, Al-Hasani, Boyce and Adler2010) or part of the cell wall proteins. Previous attempts have yielded conflicting results particularly with regard to the protective role of purified LPS and capsular extract of P. multocida type B (Nagy and Pen, Reference Nagy and Pen1976; Confer, Reference Confer1993). Further, no encouraging results were obtained from repeated attempts to develop conjugate vaccines by using ribosomes or carrier proteins in association with polysaccharides of P. multocida (Verma and Jaiswal, Reference Verma and Jaiswal1998). In contrast, immunization trials with OMPs from bacteria growing in vivo, in vitro, or in iron-restricted media elicited cross-protective antibodies against different serotypes (Pati et al., Reference Pati, Srivastava, Roy and More1996; Srivastava, Reference Srivastava1998). Killed and acellular vaccines, despite being safe in animals (OIE, 2009), induce a less durable immune response requiring a booster dose after every few months.
Adjuvants in vaccines
Many adjuvants, namely, saponin or glucan extract; bacterial components such as monophosphoryl lipid-A, trehalose dymicolate, LPS and their derivatives; chemicals such as aluminum hydroxide gel, surfactants and emulsions have been used for the development of HS vaccines (Verma and Jaiswal, Reference Verma and Jaiswal1998), based on killed/acellular or recombinant proteins or synthetic peptides. A new generation of adjuvants comprising mainly cytokines such as Interferon-gama IFN-γ or Granulocyte macrophage colony stimulating factor GM-CSF and micro/nano-particles, is under trial (Aucouturier et al., Reference Aucouturier, Dupuis and Ganne2001).
Combined vaccines
Several combinations of vaccine formulations (bivalent, trivalent and polyvalent) incorporating two or more disease-causing microbes (bacterial and/or viral) have been used in attempts to reduce the number of vaccine doses and the cost of production and to increase protective immunity of animals against multiple infections (Verma and Jaiswal, Reference Verma and Jaiswal1998; De Alwis, Reference De Alwis1999). For example, with a killed HS vaccine, other formulations targeting black quarter, rinderpest, FMD, black leg, Infectious bovine rhiontracheitis IBR, adenovirus type-3, para-influenza and other pathogens of respiratory diseases with their antigens were incorporated in the development of combined vaccines depending on the target host and disease prevalence in a particular area (Srivastava et al., Reference Srivastava, Harbola and Khera1975; Osman et al., Reference Osman, Abdul, Makeen, Dinvitiri, Monaz and Youswf1990).
Molecular approaches in the development of vaccines
Recently, genetic engineering approaches have resulted in the generation of purified subunits (Hatfaludi et al., Reference Hatfaludi, Al-Hasani, Boyce and Adler2010), synthetic peptides, chemical conjugation of polysaccharides with recombinant proteins, use of recombinant bacteria or defined mutants and bacterial ghosts as vaccines (Adler et al., Reference Adler, Bulach, Chung, Doughty, Hunt, Rajakumar, Serrano, Zanden, Zhang and Ruffolo1999).
Recombinant subunit vaccines
Gene cloning and expression resulted in the generation of various recombinant proteins from P. multocida, which represent only the part of an organism yet stimulate relatively strong immune responses (Hatfaludi et al., Reference Hatfaludi, Al-Hasani, Boyce and Adler2010). Several genes involved in virulence and immunity in P. multocida were localized and targeted as having potential in the development of subunit vaccines (Fuller et al., Reference Fuller, Kennedy and Lowery2000; May et al., Reference May, Zhang, Li, Paustian, Whittam and Kapur2001; Hatfaludi et al., Reference Hatfaludi, Al-Hasani, Boyce and Adler2010). For example, Type 4 fimbriae (PtfA), capsule, OMP87 (Omp87), fragments of exotoxin (PMT), porin (OmpH), lipoprotein E (plpE), heme-binding proteins (HasR, HemR and HgbA), filamentous hemagglutinin (fhaB2), adhesin (OmpA) and transferrin binding protein (TbpA) have been considered as candidate antigens (Veken et al., Reference Veken, Oudega, Luirink and de Graaf1994; Ruffalo et al., Reference Ruffalo, Tennet, Michalski and Adler1997; Paustian et al., Reference Paustian, May and Kapur2001; Bosch et al., Reference Bosch, Garrido, Perez de Rozas, Badiola, Barbe and Llagostera2004; Shivachandra et al., Reference Shivachandra, Kumar, Amaranath, Joseph, Srivastava and Chaudhuri2005; Prado et al., Reference Prado, Dabo and Confer2005; Wu et al., Reference Wu, Shien, Shieh, Chen and Chang2007; Hatfaludi et al., Reference Hatfaludi, Al-Hasani, Boyce and Adler2010). Recombinant proteins used as subunit vaccines were safe for immuno-compromised animals as they did not cause disease (Verma and Jaiswal, Reference Verma and Jaiswal1998; De Alwis, Reference De Alwis1999). The observations that bovine strains of P. multocida regularly harbored several virulence genes (Type 4 fimbrial gene-ptfA, iron acquisition genes-tbpA, hgbA-B, HasR, and genes for multiple hemoglobin-binding proteins), independent of its capsule type and these genes are highly expressed in the host, strongly suggest that their protein products are potential subunit vaccine candidates (Veken et al., Reference Veken, Shah, Klaasen, Oudega and De Graaf1996; Ruffalo et al., Reference Ruffalo, Tennet, Michalski and Adler1997; Bosch et al., Reference Bosch, Garrido, Perez de Rozas, Badiola, Barbe and Llagostera2004). A comprehensive review of various recombinant OMPs and their immunogenicity potential has been written by Hatfaludi et al. (Reference Hatfaludi, Al-Hasani, Boyce and Adler2010).
One vaccine strategy that has been adopted in several other bacterial species in the Pasteurellaceae, is to target the surface proteins involved in acquisition of iron from the host as vaccine antigens (Paustian et al., Reference Paustian, May and Kapur2001; Hatfaludi et al., Reference Hatfaludi, Al-Hasani, Boyce and Adler2010). In particular, the OMPs and IROMPs have been a focus for the development of subunit vaccines (Choi-Kim et al., Reference Choi-Kim, Maheswaran, Felice and Molitor1991). One of our earlier studies involving analysis of a gene encoding transferrin binding protein (TbpA) from P. multocida serogroup B:2 revealed a high antigenic index of mature TbpA (89 kDa) as well as great sequence similarity with the gene from serogroups A:1 and D:1 organisms, indicating that this protein could be used as a candidate antigen in the development of vaccines against HS (Shivachandra et al., Reference Shivachandra, Kumar, Amaranath, Joseph, Srivastava and Chaudhuri2005). A surface exposed, highly immunogenic lipoprotein (plpE) of P. multocida conferred 80–100% protective immunity in mice and induced cross-serotype protective immunity (Wu et al., Reference Wu, Shien, Shieh, Chen and Chang2007). At present, there is no commercial recombinant HS subunit vaccine and cross-protective antigens still remain elusive.
Synthetic peptides
Several attempts have been made to develop synthetic peptides based on amino acid sequences of predicted loop structures that mimic the epitopes of P. multocida (Choi-Kim et al., Reference Choi-Kim, Maheswaran, Felice and Molitor1991; Luo et al., Reference Luo, Zeng, Glisson, Jackwood, Cheng and Wang1999). For example, synthetic porin (OmpH) peptides have been shown to induce limited protection against P. multocida (Luo et al., Reference Luo, Zeng, Glisson, Jackwood, Cheng and Wang1999). The development of synthetic peptide vaccines was hampered by their low immunogenicity and requirement for powerful adjuvants.
Genetically engineered defined mutant vaccines
Genetic technologies have emerged as powerful tools to identify targets for the development of defined live-attenuated vaccines. For example, in vivo expression technology, subtractive hybridization and signature-tagged mutagenesis have resulted in the identification of many genes responsible for virulence and pathogenesis of P. multocida (Fuller et al., Reference Fuller, Kennedy and Lowery2000; May et al., Reference May, Zhang, Li, Paustian, Whittam and Kapur2001). Thus, a number of attenuated mutants containing transposon insertions in genes encoding biosynthetic enzymes, virulence factors, regulatory components and unknown function have been evaluated at the laboratory level. For example, an acapsular cexA mutant of P. multocida B:2 (PBA875) with impaired capsule export was highly attenuated in mice and elicited protective immunity against a wild-type challenge (Boyce and Adler, Reference Boyce and Adler2001). However, a genetically defined acapsular P. multocida strain (AL18) generated by inactivation of the bcbH gene, predicted to be involved in polysaccharide biosynthesis, failed to confer protection. Another report suggests the use of stable, non-reverting aroA mutants as live vaccines (Ataei et al., Reference Ataei, Burchmore, Christopher, Finucane, Parton and Coote2009). Dagleish et al. (Reference Dagleish, Hodgson, Ataei, Finucane, Finlayson, Sales, Parton and Coote2007) reported the protective efficacy of live P. multocida B:2 wild-type strain-85020 containing a deletion in the aroA gene (strain-JRMT12), following intramuscular vaccination against experimental hemorrhagic septicemia in calves. However, the efficacy of attenuated mutants requires further laboratory investigations before field use.
Bacterial ghosts
Bacterial ghosts from a variety of bacteria are currently being used as non-living candidate vaccines as they possess characteristics of both killed and live cells. These intact bacterial envelope structures devoid of cytoplasmic contents are generated by controlled expression of cloned PhiX174 gene E and protein E-mediated lysis (Kudela et al., Reference Kudela, Koller and Lubitz2010). Marchart et al. (Reference Marchart, Dropmann, Lechleitner, Schlapp, Wanner, Szostak and Lubitz2003) reported the generation of P. multocida ghosts and found 100% protection against homologous challenge in a laboratory model. However, this approach needs further evaluation by generating P. multocida ghosts using HS strains and assessing their protective efficacy against homologous and heterologous challenges in cattle and buffaloes.
Challenges and constraints
Solution of the lingering problem of HS requires that several challenges be met (Khera, Reference Khera1979). Currently, reported HS outbreaks represent only a fraction of actual numbers in most developing countries (Kumar et al., Reference Gautam, Kumar, Singh, Singh, Dutta and Shivachandra2004). As the available HS morbidity and mortality statistics from different parts of world are hardly complete, it is a great challenge to assess the economic losses due to prevalence and distribution of HS globally and to determine the epidemiological characteristics of strains. It appears that the extent and geographic and temporal distribution of rainfall are important factors influencing the incidence and change from enzootic to epizootic occurrence of HS (Dutta et al., Reference Dutta, Rathore, Mullick, Singh and Sharma1990). It has been postulated that the first outbreaks of HS occur with the first rains and the maximum spread of HS is associated with monthly rainfall of 60–270 mm and a total rainfall from 340 to 700 mm in the first 2 months (Khera, Reference Khera1979). Other factors such as bovine population density and highly varying environments in diverse regions include mean air temperature, relative humidity, parasitic infections, carrier rate of P. multocida and herd immunity level (De Alwis, Reference De Alwis1999).
Despite unraveling the complete genome sequence of P. multocida (May et al., Reference May, Zhang, Li, Paustian, Whittam and Kapur2001), the pathogen continues to be an enigma with vague information about the genes involved in virulence factors, pathogenicity, host adaptability, predilection sites, carrier state and cross-protective antigens or immunogens (Harper et al., Reference Harper, Boyce and Adler2006). The elusive determinants of cross-protective immunity pose a substantial challenge to the scientific community.
The occurrence of HS outbreaks despite vaccination (Biswas et al., Reference Biswas, Shivachandra, Saxena, Kumar, Singh and Srivastava2004; Kumar et al., Reference Gautam, Kumar, Singh, Singh, Dutta and Shivachandra2004) generates a new set of questions that remain unanswered because neither the precise mechanism of immunity nor the specific bacterial antigens involved in immunity to HS have been clearly identified. It is also evident that due to genotypic differences among strains of the same serotype, conventional serotyping which is widely used in most developing countries often does not provide sufficiently detailed information to determine the epizootiology of infections. In most developing countries, inefficient para-veterinarians are involved in indiscriminate use of chemotherapeutic agents in the treatment of various animal diseases leading to emergence of multi-drug-resistant strains as well as emergence of mutant strains with high virulence. There are multiple constraints that are natural or anthropogenic, such as:
• Lack of public awareness about the disease and good animal husbandry practices.
• Lack of strong legislation for animal movement and mandatory disease reporting.
• Illegal trafficking of livestock and their products across international borders.
• Indiscriminate use of antibacterial agents.
• Stresses prevalent in HS endemic areas.
• Lack of research data on cross-protective immunogens of P. multocida.
• Lack of an efficient, affordable vaccine for mass application.
Conclusion
Insufficient data on incidence and distribution of HS, coupled with inefficient prophylactic/control measures, contribute to the devastating impact of the disease on the livestock sector. The need is to focus on the key areas enumerated earlier coupled with multi-pronged research and extension activities at rural levels. Incentives to the farmers to adopt modern animal husbandry practices should be provided by giving subsidies. The regular monitoring and surveillance of the general health status of herds would help in control and eradication of HS from any given region. There is also an imminent need for whole genome sequencing and analysis of transcriptional gene expression response during natural infections caused by strains of HS (B:2 or E:2) to get insights into the molecular basis for pathogenesis.
New generation vaccines along with improved conventional approaches are likely to generate a wide range of efficient vaccines against HS. However, these new technology vistas warrant thorough investigations in relation to compatibility of different combinations/formulations of vaccines and their safety in vaccinated animals. There is considerable promise that current frontier areas of research involving new technology on the development of effective new age HS vaccines may prove effective in control of HS.
Acknowledgments
We thank the Director, Indian Veterinary Research Institute (IVRI) and the Indian Council of Agricultural Research (ICAR), New Delhi, for providing the facilities to carry out the research on P. multocida and to prepare the manuscript.
