Cattle rearing is one of the important sources of income in Iran, involving both dairy and beef breeds. The sector faces a number of constraints ranging from limited feed resources to diseases. Of the diseases, those caused by infectious agents are of great importance which include bacteria and viruses affecting the respiratory system (Hemmatzadeh et al., 2001; Haji Hajikolaei and Seyfi Abad Shapouri, 2007; Sakhaee et al., 2009). The most important bacteria that play a role in pneumonia include: Mannheimia haemolytica, Pasteurella multocida, and Haemophilus somnus pneumonia, which presence of these bacteria in pneumonia lesions of slaughtered cattle around Iran have also been reported (Haji Hajikolaei et al., 2010).

Bovine respiratory disease (BRD) is a significant cause of morbidity and mortality among beef cattle in the world (Dagleish et al., 2010; Hotchkiss et al., 2010; Portis et al., 2012). Among others, Pasteurella multocida has been identified as a major bacterial etiologic agent for this disease (Confer, 2009; Griffin et al., 2010). It is a zoonotic Gram negative bacterium responsible for a range of infections in domestic animals causing substantial economic losses (Steen et al., 2010). The organism causes fowl cholera in domestic and wild birds, bronchopneumonia and hemorrhagic septicemia in bovids, atrophic rhinitis in porcines and snuffles in rabbits (Mannheim, 1984; Hunt et al., 2000). Most human infections with P. multocida result from dog and cat bites, but infections through the respiratory tract may also occur (Hubbert and Rosen, 1970).

Several host and pathogen-specific attributes do determine the outcome of infections caused by P. multocida (Verma et al., 2013). Of the pathogen factors important ones include the capsular and virulence-associated genes (Katsuda et al., 2013). These virulence factors (VFs) and outer membrane proteins are important for pathogenesis, functionality, protective immunity and vaccine development against P. multocida infections (Harper et al., 2006; Hatfaludi et al., 2010). Based on capsular antigens, P. multocida strains are differentiated into five serogroups i.e., type A causing fowl cholera pathogen and bovine shipping fever, type B causing hemorrhagic fever in ungulates, type D causing atrophic rhinitis in swine, type E, an African serotype, infecting cattle and buffalo; and type F also causing fowl cholera (Carter, 1955, 1961, 1967; Rimler and Rhoades, 1987). Virulence associated genes described for P. multocida isolates and their examples include adherence and colonization factors (ptfA, fimA, hsf-1, hsf-2, pfhA, and tadD), iron-regulated and acquisition proteins(exbB, exbD, tonB, hgbA, hgbB, and Fur), extracellular enzymes such as neuraminidase (nanB and nanH), hyaluronidase (pmHAS) and superoxide dismutases (soda, sodC, and tbpA), toxins (toxA), lipopolysaccharides (LPS), capsule and a variety of outer membrane proteins such as protectins (ompA, omph, oma87, and plpB) (Katoch et al., 2014).

Increased use of antibiotics in modern animal production has been associated with emergence of antimicrobial resistant bacteria with potential for transfer of resistance from animals to humans (Witte, 1998). As a result, antimicrobial resistance among bacterial pathogens has of recent become a big problem in both the veterinary and human medicine fields (Levy, 1998; Caprioli et al., 2000; Kehrenberg et al., 2001; White et al., 2002; Shea, 2003). The implication of the problem is increased treatment cost, prolonged illness due to treatment failure and sometimes death (Kelly et al., 2004).

The present study was conducted with the aim to detect the occurrence of VFs in P. multocida isolated from pneumonic and apparently health lungs of slaughter cattle in Iran. It was also to determine the occurrence of antimicrobial resistance among the isolates.

VFs play a key role in disease production by bacterial pathogens (Nanduri et al., 2009). Among others, their functions include competence, adherence, synthesis, and export of capsules; and evasion of host immune responses (Nanduri et al., 2009). In the present study the factors have been detected in P. multocida isolated from the lungs of slaughter cattle. The higher frequency of the factors among isolates from pneumonic lungs suggests the role of these factors in disease occurrence. It was pointed out that virulence gene occurrence in P. multocida has a strong positive association with the outcome of infection with the organism in cattle (Katsuda et al., 2013). On the other hand occurrence of the factors in apparently healthy lungs could possibly indicate early infection or contained infection which couldn't lead to disease. It was previously reported that this facultative anaerobic bacterium is commonly found in clinically healthy calves (Lainson et al., 2013).

In this study capsular types A and D were detected using PCR among the obtained P. multocida isolates. A small proportion (6.7%; 2/30) of P. multocida strains were untypeable, a similar observation to what was reported by Arumugam et al. (2011). Capsular type A was predominant among the strains accounting for 76.6%. Our observation is similar to a finding by Katsuda et al. (2013) who also detected capsular types A and D among cattle derived P. multocida isolates; with type A occurring at higher frequency. A higher frequency of capsular type A among cattle derived P. multocida isolates has also been reported in a study conducted earlier by Davies et al. (2004) who found that 99.3% of bovine P. multocida strains (n = 153) were of this capsular type. P. multocida isolates of serotype A are common in bovids occurring as normal flora in the nasopharynx; or as causes of disease including BRD and hemorraghic septicemia (Ewers et al., 2004; Dabo et al., 2007). The capsular type A is also most frequently described for rabbits (Ewers et al., 2006) and pigs (García et al., 2011).

Of the protectins; OmpA and oma87 were the most frequently detected genes particularly in the isolates from pneumonic lungs. Slightly higher frequencies of the two genes were noted for isolates of the capA serogroup than those belonging to the capD serogroup. The OmpA gene has a significant role in stabilizing the cell envelope structure by providing physical linkage between the outer membrane and peptidoglycan (Katoch et al., 2014). It mediates P. multocida host cells interaction through heparin and/or fibronectin binding and thus acts as an important invasive molecule which could determine the outcome of infection with the organism (Katoch et al., 2014).

The type 4 fimbria (ptfA gene) was described in 92.0% of the isolates tested in the current study. The gene plays a key role of fixing bacterial pathogens on the surface of the epithelial cells of hosts, a phenomenon which is more common in rabbits (Ewers et al., 2006). Consequently a study conducted on rabbits described a high prevalence of the ptfA gene (93.4%; 43/46).

Presence of adhesins on the bacterial surface is usually linked to virulence as these proteins are known to play a crucial role in facilitating host invasion and colonization (Kline et al., 2009). Studies by Ewers et al. (2000) and Tang et al. (2009) have demonstrated that, of the adhesins; fimA, hsf-2, and ptfA are of frequent occurrence among pathogenic isolates of P. multocida. In the current study the three adhesins were demonstrated at higher frequencies than others in both capA and capD serogroup isolates. On the other hand, gene tadD was the least frequently detected adhesin among P. multocida, occurring only in 48.0% of the isolates (n = 30). In these organisms the gene is known to be a putative non-specific tight adherence protein D (May et al., 2001). A more or less similar low frequency (43.3%; 100/233) of tadD was described in a field study involving pigs (Tang et al., 2009). A work on rabbits, however, observed a higher frequency (91.3%; 42/46) of this gene among P. multocida strains.

It is noteworthy that the dermonecrotoxin encoding toxA was the least frequently detected gene among the isolates; demonstrated only in those of capsular type A obtained from pneumonic lungs. Some other researchers indicated that this particular gene is more frequently expressed by strains of serogroup D and is responsible for the clinical symptoms associated with atrophic rhinitis in porcines (Harper et al., 2006; Ferreira et al., 2012). The observation in the current study could be attributed to the small sample size of capsular type D isolates. In a study conducted earlier the gene was detected in P. multocida isolates from avians, swine, shoats and cattle; but was only associated with disease in pigs (Ewers et al., 2006). Pullinger et al. (2004) points out that the toxA gene is not inserted into the bacterial chromosome but in a lysogenic bacteriophage that infects the agent.

The tbpA encoding gene is known to be of common occurrence among ruminant P. multocida strains (Ewers et al., 2006; Atashpaz et al., 2009). Its prevalence was however relatively low when compared to other superoxide dismutases (sodA and sodC) tested in this study. Ferreira et al. (2012) found a low frequency (8.6%; 4/46) of this gene in a study conducted on rabbits. Variable frequencies of the genes encoding proteins with different functions, such as adhesins (fimA, hsf-1, hsf-2, and pfhA), iron acquisition (exbB, exbD, tonB, hgbA, hgbB, and Fur), sialidases (nanB and nanH), hyaluronidase (pmHAS), and protectins (oomph and plpB) were found in the isolates. This finding is similar to what was reported in previous works which involved ruminants, porcine, poultry, and rabbits (Ewers et al., 2006; Tang et al., 2009; Ferreira et al., 2012).

Infections with P. multocida are commonly managed by broad-spectrum antimicrobials (Kehrenberg et al., 2001; Lion et al., 2006; Brogden et al., 2007). Studies have however reported occurrence of resistance to a large number of antimicrobial agents among P. multocida isolates (Hunt et al., 2001; Davies et al., 2004; Arashima and Kumasaka, 2005). In the current study all the P. multocida isolates were susceptible to ciprofloxacin, co-trimoxazole, doxycycline, enrofloxacin, nitrofurantoin, and tetracyclines. Similar observations for ciprofloxacin and co-trimoxazole (Mohamed et al., 2012); and for enrofloxacin, tetracycline, and doxycycline (Ferreira et al., 2012) have also been made earlier. These antibiotics can therefore be used for prevention and treatment of bovine P. multocida infections in the study area. Unlike other authors who reported poor (Gutiérrez Martin and Rodríguez Ferri, 1993; Yoshimura et al., 2001) and moderate (Mohamed et al., 2012) activity of aminoglycoside antibiotics against P. multocida, in the present study kanamycin, gentamicin, amikacin, and streptomycin exhibited high activity against the tested isolates. The frequencies of resistant isolates to other antibiotics varied greatly as reported by other researchers (Salmon et al., 1995; Kehrenberg et al., 2001; Yoshimura et al., 2001; Welsh et al., 2004).

The major limitation in the discussion of the findings of the current study was large differences in the sample sizes of comparison groups as seen for isolates between isolates from pneumonic lungs and those from apparently health lungs; and isolates of different capsular types. This made it difficult to infer on the observed variations as they could be attributed to chance.

In summary, our results reveal presence of VFs in P. multocida strains isolated from the lungs of symptomatic and asymptomatic slaughter cattle. Frequent detection of the factors among isolates from symptomatic study animals may suggest their role in pathogenesis of BRD caused by these organisms. Occurrence of antimicrobial resistance among some isolates is of great concern. Control strategies for this pathogen, which could include development of an effective vaccine, are warranted so as to mitigate the social and economic consequences attributable to natural infections with this bacterium. Further, the use of antimicrobial agents in modern livestock farming need to be controlled so as to minimize the emergence and eventually spread of resistance not only in target microbes but also in other important zoonotic pathogens.