Introduction

Pasteurella multocida is a pathogenic Gram-negative bacterium that has been classified into three subspecies, five capsular serogroups and 16 serotypes. It is non-motile, coccobacillus, nonsporing and facultative anaerobic microbe. It is a part of normal flora of oral cavity and gastrointestinal tract of wild and domestic animals (Krieg and Holt 1984). The stress factors like sudden change in the weather, nutrition, overwork, long and stressful journeys etc. lower down the resistance of the animals and the organisms get upper hand resulting into fulminating infection. Once established inside the immunosuppressed animals, the organism may lead to number of primary and secondary infections in a wide range of vertebrate hosts. P. multocida is responsible for pneumonia in cattle and sheep (Chanter and Rutter 1989; Frank 1989) and HS in cattle and buffaloes (Carter and De Alwis 1989). The organism is also known to be the causative agent of pasteurellosis in American bison, yak, deer, elephants, camels, horses, elk and other wild animals (De Alwis 1996). It causes fowl cholera in poultry, snuffles in rabbits and atropic rhinitis in swines. In India, HS has been quantified as the number one bacterial killer disease among cattle and buffaloes (Dutta et al. 1990; Singh et al. 1996).

Pasteurella multocida possesses a number of virulence factors which include polysaccharide capsule, endotoxins or lipopolysaccharide (LPS), outer membrane proteins (OMPs), fimbriae, exotoxins, multocidins or siderophores, extracellular enzymes and plasmids (Harper et al. 2006). On the basis of variation in the cap loci, the pathogen has been classified into capsule type A, B, D, E and F and a relationship exists between the capsular type and disease predilection (Boyce and Adler 2000). Apart from outer membrane proteins and capsular antigens, the virulence associated genes (tbpA, pfhA, toxA, hgbB, hgbA, nanH, nanB, sodA, sodC, oma87 and ptfA) play important in pathogenesis of P. multocida (Ewers et al. 2006). Many such genes of P. multocida have been suggested as epidemiological markers and PCR-based methods have been used to ascertain their distribution in strains recovered from wide sources and disease conditions (Lainson et al. 1996; Doughty et al. 2000; Ewers et al. 2006). The present study investigated the distribution of some of these important virulence associated genes in P. multocida isolates from dead, diseased or healthy cattle and evaluated the association of these genes in the outcome of disease.

Material and methods

Bacterial isolation and identification

A total of 335 samples were collected from six farms over a period of 1 year. Out of these 335 samples, 50 were collected from 9 animals died from HS suspected disease outbreaks and comprised of whole blood and morbid materials from lung, liver, spleen and heart. Besides these 50 samples, 87 nasal swabs were collected from the live but diseased cattle & 198 nasal swabs were from apparently healthy bovine. Preliminary isolation of the organism was done on 5 % defibrinated sheep blood agar followed by confirmation using traditional bacteriological and biochemical methods as described by Muhairwa et al. 2001 (gram staining, cultural characteristics, oxidase, catalase, methyl red, Voges-Proskauer, sulphide reduction, indole production, motility, triple sugar iron agar, urease production, citrate utilization, nitrate reduction and carbohydrate fermentation reactions). All the biochemical tests were performed in triplicate.

Preparation of chromosomal DNA

Cells from 1.0 ml overnight cultures in Brain Heart Infusion (BHI) broth were harvested by centrifugation for 15 min at 4,000 rpm. DNA was isolated by phenol-chloroform-isoamyl alcohol method (Wilson 1987).

Molecular confirmation of P. multocida by PM-PCR

P. multocida were identified by PM-PCR using a pair of P. multocida specific primers which amplified the KMT1 gene as described by Townsend et al. (1998a). The PM-PCR was performed using a method detailed by Dutta et al. (2001) with slight modifications in the primer annealing conditions of 54 °C for 45 s.

Maintenance of cultures

The culture were maintained on blood agar slants and stored for a week at 4 °C till further use. These cultures were sub-cultured fortnightly to maintain their viability. For long term storage, P. multocida cultures were maintained by suspending them in 20 % glycerol stock solution in BHI broth and storing them at −70 °C.

Capsule typing

The capsular types of the isolates were determined by multiplex capsule-PCR with the capsule- specific primer pairs specific for capA, capB, capD, capE and capF gene as described by Townsend et al. (2001).

Detection of virulence associated genes by PCR

The DNA of P. multocida isolated from dead, diseased or apparently healthy bovine was used as a template to amplify virulence associated genes for which PCR-based protocols called virulence genotyping as described by Lainson et al. (1996) for sodC, Doughty et al. (2000) for ptfA and Ewers et al. (2006) for tbpA, pfhA, toxA, hgbB, hgbA, nanH, nanB, sodA and oma87 virulence associated genes were used. The sequences of oligonucleotide primers, amplification conditions and references are listed in Table 1 that has been adapted from Ewers et al. 2006. In brief, for all PCR reactions, a total of 100 ng DNA template was added to the reaction mixture (25 μl) containing 2.5 μl 10XPCR buffer, 2.5 μl of 25 mM magnesium chloride, 0.4 μl of 25 mM dNTPs, 0.5 μl of each primer pair in a 10 pmol concentration and 1unit of Taq-polymerase (Promega corporation, Madison, USA). The samples were then subjected to 25 cycles of amplification in a thermal cycler GeneAmp PCR System 9700 (Applied Biosystems, U.S.A). Amplification products were resolved by gel electrophoresis on 1.2 % agarose gel, stained with ethidium bromide (1 μg/ml) and visualized on UV transilluminator (Alpha Innotech, USA).

Table 1 Details of primers, PCR conditions and citations used for the detection of virulence associated genes
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Statistical analyses

A statistical analysis was performed using GraphPad QuickCales software to establish association between virulence associated genes, P. multocida isolates and origin of samples i.e. diseased or apparently healthy animals.

Results

Bacterial isolation and identification

A total of 23 (6.87 %) P. multocida isolates were recovered from 335 samples collected from dead, diseased or apparently healthy bovine. The origin, source and capsule type of P. multocida isolates have been summarized in Table 2. All the isolates of P. multocida showed positive reactions for biochemical tests: catalase, oxidase and nitrate reduction; while negative reaction for urease production, sulphur reduction, motility, MR and VP test. These isolates yielded variable patterns for indole production and citrate utilization. In triple sugar iron agar slants, acid slant and acid butt was produced by all the isolates. Besides this, all were positive for glucose fermentation, however variable results were obtained for other sugars viz. sucrose, lactose, maltose, mannitol, galactose, dulcitol, sorbitol, salicin, arabinose and trehalose.

Table 2 Animal number, clinical status, capsule type and presence (+) or absence (−) of virulence associated genes in P. multocida isolates
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Molecular confirmation by PM-PCR

All 23 isolates were found positive for PM-PCR. The primer pair KMT1SP6 and KMT1T7 amplified KMT1 gene fragment from P. multocida that was electrophoresed to approximately 460 bp (Fig. 1).

Fig. 1
figure 1

Confirmation of P. multocida using PM-PCR ; Lanes 1–4 460 bp KMT gene amplicons, N- negative control, P-positive control and L- 100 bp Ladder

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Distribution of capsular types

The isolates of P. multocida belonging to capsular type A were obtained from 4 (17.4 %) animals and those of capsular type B were obtained from 19 (82.6 %) animals. Capsular type D, E and F were not detected in the population sampled.

Prevalence of virulence associated genes in P. multocida isolates

The distribution of virulence associated genes in relation to the origin of isolates (healthy vis-a vis diseases) is shown in Fig. 2.

Fig. 2
figure 2

Distribution of virulence associated genes in P. multocida based on the health status of animals

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Their distribution in P. multocida isolates ranged from 26.09 % (hgbB) to 100 % (tbpA, pfhA, hgbA & nanH). All the isolates possessed tbpA, pfhA, hgbA and nanH gene. The gene hgbB, sodA, sodC, oma87 and ptfA were present in 26.09 %, 39.13 %, 91.30 %, 91.30 % and 86.95 % isolates, respectively. The nanB and toxA were absent in all 23 isolates. P. multocida isolates belonging to capsular type B carried virulence genes tbpA, pfhA, hgbA, sodC and nanH whereas those belonging to capsular type A were harbouring tbpA, pfhA, hgbA and nanH genes as detailed in Table 3. Only 50 % of capsular type A isolates had sodC gene while 100 % of capsular type B isolates contained sodC gene. In capsular type A isolates, either sodA or sodC gene was present and these genes did not occur concurrently.

Table 3 Percent distribution of virulence associated genes in P. multocida isolates
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Discussion and conclusion

The present study was carried out to isolate P. multocida from samples collected from apparently healthy, diseased and dead cattle from suspected HS disease outbreaks and to study the prevalence of virulence associated genes among these isolates. Traditional methods depends on using biochemical characterization as one of the means to confirm P. multocida identity but variable reactions shown by some of the strains with some of these biochemical tests create difficulties in arriving at conclusive decisions. This means additional assays have to be carried out to ensure fool-proof identity. Many such variations have been described by Fegan et al. 1995; Blackall et al. 1997; Townsend et al. 1998b and Ekundayo et al. 2008. In the present study, the results of catalase, oxidase and sugar-fermentation were very similar to Flavobacterium spp. and Neisseria spp. whose members are oxidase and catalase positive and were fermentative (Kim et al. 2006; Parija 2009). Molecular confirmation by PCR therefore, proved to be quick, specific and sensitive assay for the confirmation of P. multocida. Higher percentages of isolates belonging to capsule-type B as compared to capsule-type A were recovered from bovines.

Although the molecular basis of the pathogenicity and host specificity of P. multocida is not well understood, the organism is known to possess a number of virulence factors which have integrated role in pathogenesis (Hunt et al. 2000a & Harper et al. 2006). The present study was thus carried out to study the prevalence of virulence associated genes in the bovine isolates of P. multocida. The prevalence of 11 virulence associated genes which included genes coding for iron acquisition factors (hgbA, hgbB & tbpA), adhesion related genes (ptfA, nanB, nanH and pfhA), outer membrane and porin proteins (oma87), superoxide dismutases (sodA & sodC) and dermonecrotoxin (toxA) were studied.

Iron acquisition and uptake are essential for bacterial survival and as a result pathogenic bacteria have developed different strategies for their uptake. P. multocida produces both iron chelating siderophores and outer membrane receptors such as transferring binding protein and haemoglobin binding protein for the iron binding host molecules, transferrin and haemoglobin (Choi-Kim et al. 1991; Ogunnariwo et al. 1991; Ogunnariwo and Schryvers 2001; Cox et al. 2003; Bosch et al. 2004). P. multocida grown under iron depleted media or in vivo expressed three iron regulated OMPs with molecular masses of 76, 84 and 94 kDa, respectively with all three having affinity for siderophore binding (Choi-Kim et al. 1991). Haemoglobin binding proteins A and B help bacteria by using haemin as iron source. Although the bacterium does not produce a classical haemolysin, it carries esterase gene causing a haemolytic phenotype as seen in E. coli under anaerobic conditions (Cox et al. 2000; Hunt et al. 2000b). By inducing lysis of erythrocytes, haemoglobin is released and is thought to be bound by P. multocida haemoglobin binding proteins. The high prevalence of iron acquisition genes in the P. multocida as well as their significant role in pathogenesis suggests that their presence in P. multocida provide the bacterium an added advantage for enhanced pathogenicity (Venken et al. 1994; Ogunnariwo and Schryvers 2001). Veken et al. (1994) reported the presence of tbpA in bovine isolates of P. multocida associated with pneumonia and haemorrhagic septicaemia. In another study, Ogunariwo and Schryvers found that tbpA (−) strains are commensal or at least cause other diseases. Many previous studies have shown that iron acquisition related gene tbpA is an epidemiological marker (Cox et al. 2003; Bosch et al. 2004; Ewers et al. 2006) in addition to an important virulence factor in P. multocida isolates of cattle. Current study also found high occurrence of pfhA and tbpA among P. multocida isolates from diseased as well as healthy cattle. These findings are although at variance with results obtained in previous studies advocating tbpA and pfhA as virulence factors (Ewers et al. 2006) but are in agreement with the results of Shayegh et al. 2010.

The adhesion related genes nanH was found to be regularly distributed in P. multocida irrespective of the capsular type A or B or the health status of cattle. In this study, the presence of ptfA and an association between its distribution and bovine disease (P < 0.05) was observed. High prevalence of the ptfA gene (type 4 fimbriae) in isolates from diseased bovine (100 %;16/16) as compared to isolates from healthy bovine (57.14 %; 4/7) was expected given the fact that this gene is supposed to be a key element in fixing bacteria on the surface of the epithelial cells (Ewers et al. 2006).

Components of bacterial outer membrane such as transmembrane proteins and lipoproteins play key role in the interaction of pathogen with host environment and in the host immune response to infection. OMPs of gram negative bacteria have a role in disease processes as they act at an interface between the host and pathogen (Lin et al. 2002). High prevalence of the gene coding for oma87 in the isolates points towards their important role in host-pathogen interaction. Since many genes were also harboured by isolates originated from healthy animals; how and under what circumstances the gene or its pathways and products contribute to pathogenesis is a matter of investigation.

Superoxide dismutases (SODs) are virtually ubiquitous in bacteria, catalysing the conversion of O -2 generated by macrophages & neutrophils into hydrogen peroxide and oxygen (McCord and Fridovich 1969). In the present study, the virulence associated genes sodA & sodC were found in a higher percentage among isolates from diseased animals as compared to isolates recovered from apparently healthy animals. However, the association of these genes with disease in bovine was not statistically significant.

All isolates of P. multocida from dead, diseased or apparently healthy bovine were carrying the virulence associated genes tbpA, pfhA, hgbA, sodC and nanH. Among the five virulence associated genes; tbpA and pfhA have been considered as epidemiological markers in the past studies and supposed to have strong positive association to the outcome of disease in cattle. However, their presence in isolates arising from healthy bovines warrants detailed investigation about their role in disease outcome. The virulence associated genes sodA, sodC, oma87 and pfhA were found in a higher percentage in isolates from diseased animals as compared to isolates from apparently healthy animals; however strong association between genes and the isolates recovered from diseased animals was confirmed only for ptfA. Future studies should focus on the role of these genes in health & disease and how their expression is influenced and regulated under immunosuppression.