Virulence Gene Pattern of Pasteurella multocida Isolates of Buffalo in Association to Capsule Biosynthesis Genes

Pasteurella multocida isolates
 
A total of 16 isolates that were isolated from different sources (196 samples) were used in the study. The isolates were confirmed molecularly and also typed for capsular typing in earlier work by Sujatha et al., (2018). 
 
Bacterial DNA preparation
 
Bacterial culture lysates were processed for template preparation. Loopful of bacterial culture was taken from parent colonies with the help of sterile inoculation loop and suspended in 500 µl of sterile PBS  (or) 500 µl broth culture was taken and centrifuged at 10,000 rpm for one minute at room temperature. The pellet was resuspended in 1000 µl of PBS and centrifuged at 10,000 rpm for one minute. Later the pellet suspended in 500 µl of sterile distilled water was boiled  in water bath for 10 min and immediately chilled on ice for 5 min. It was centrifuged at 10,000 rpm for one minute at room temperature. The supernatant containing the DNA was transferred into a sterile vial and used as template for PCR.
 
Virulence associated genes of Pasteurella multocida
 
The virulence associated gene typing of P. multocida was performed using duplex and uniplex PCR. Duplex PCR was used for the detection of type IV fimbrae (ptfA gene) - filamentous haemagglutinin (pfhA), transferrin binding protein (tbpA gene) - outer membrane protein H (ompH gene). Uniplex PCR was used for haemoglobin binding proteins (hgbA, hgbB). The primers of virulence genes and the thermal cycler conditions were given in the Table 1 and 2. Template DNA (0.5 μl) prepared by using boiling method was added to the PCR mixture (total volume of 10μl) containing 3.85μl nuclease free water, 25 mM MgCl₂ (0.4μl) to get 4 mM MgCl₂ concentration in  master mix, 20 pm/μl of each primer (Forward and reverse at 0.125μl) to get 2.5 pm/μl and 2X master mixture (5 μl) for duplex PCR. For hgbA and hgbB additional 25 mM MgCl2 was not added and the nuclease free water was adjusted according to the final volume.
 

 

 
Amplified DNA products (5μl of each sample) were electrophoresed on 1.7% agarose gel containing 0.5 μg/ml Ethidium bromide, in 1× Tris-borate EDTA running buffer (TBE) at 4 V/cm for 90 min. The gel and DNA fragments were viewed by UV transilluminator and photographed. Statistical analyses have been performed with software SPSS 17.0 for Windows (SPSS Inc., Chicago, IL) to conduct Fisher’s exact test.

The 16 P. multocida isolates under study showed 14 Cap A and 2 Cap B isolates. It was found that the cap A isolates were observed from apparently healthy or carrier animals where as Cap B from clinical cases of Haemorrhagic septicaemia in earlier work by Sujatha et al., (2018).
       
The isolates when subjected to the nucleic acid amplification by PCR for virulence gene showed an amplified DNA product of 438 bp, 488 bp, 275 bp, 728 bp, 419 bp and 788 bp was observed for outer membrane protein H (ompH gene), type IV fimbrae (ptfA gene)-filamentous hemagglutinin (pfhA), transferrin binding protein (tbpA gene) and hemoglobin binding proteins (hgbA, hgbB), respectively (Fig 1,2,3). The observation of virulence genes and capsule biosynthesis genes are presented in Table 3.
 

 

 

 

       
The ompH gene that encodes the porins of P. multocida was detected in all isolates (14 Cap A and 2 Cap B isolates) and exhibited 100% prevalence. These findings suggest that the OMPs have a significant role in the host- pathogen interaction, nutrient absorption, importation and exportation of molecules, close interaction with the host tissue. It is a key element in the attachment of the P. multocida to the host epithelial cells and prerequisite for initial infection in the P. multocida (Harper et al., 2006). These results are in agreement with other investigations (Ewers et al., 2006, Katsuda et al., 2013, Jamali et al., 2014, Khamesipour et al., 2014, Ragavendhar et al., 2015 and Aski and Tabatabaei, 2016).
       
The role of iron in pathogenesis of P. multocida is important, two independent non siderophore mediated iron acquisition mechanism have been identified in P. multocida. The first mechanism involves iron binding proteins expressed on the outer membrane of the host cell, interacting directly with host iron binding glycoproteins. The second mechanism includes bacterial proteins that bind hemoglobin and hemoglobin complexed to the host glycoproteins (Cox et al., 2003). The three iron acquisition related genes (tbpA, hgbA and hgbB) were studied in all the isolates irrespective of their clinical status. The tbpA gene (transferring binding protein A) is involved in the expression of iron binding proteins on the outer membrane of the host cell for iron acquisition in P. multocida. Among the different isolates tested for the presence of tbpA gene, the gene was 100% in Cap B isolates and 0% in Cap A isolates. The presence of tbpA in association with disease HS and in non-association with commensal or carrier P. multocida was also supported by Ogunnariwo and Schryvers (2001) and Ewers et al., (2006). The tbpA protein of P. multocida falls in the superfamily of ton B dependent receptor proteins. Veken et al., (1994) observed association of 82 kDa binding protein to the strains B:2, 5 associated with clinical signs of HS in buffalo and cattle whereas serotype B:3,4 strains that were not involved in HS had failed to express this protein, suggesting its virulence association. Ogunnariwo and Schryvers (2001) suggested that tbpA positive P. multocida strains were virulent while receptor negative strains might either be commensals or be associated with some other clinical manifestation in cattle. The studies of Ogunnariwo and Schryvers (2001) also revealed presence of IS element IS 1016 immediately downstream of the tbpA gene a possible insertional element responsible for changes in virulence from non or low pathogenic to highly virulent P. multocida.

The second mechanism of iron binding in P. multocida was encoded by hgbA and hgbB (haemoglobin binding protein A, B) genes. All the P. multocida isolates revealed hgbB gene, hgbA were observed 100% in Cap B isolates where as 92.9% of hgbA and 100% hgbB in Cap A isolates. These results were in agreement with Ewers et al., (2006), Khamesipour et al., (2014) and Sarangi et al., (2014). However, this is contradictory to the findings of Somashekar et al., (2016) who reported none of the Cap B isolates harboured hgbB gene. In P. multocida the presence or absence of second mechanism of iron acquisition especially in pathogenic serotypes may be compensated by tbpA iron acquisition system (Ogunnariwo and Schryvers, 2001, Cox et al., 2003). Further, the high prevalence of iron acquisition genes in P. multocida plays a crucial role in pathogenesis (Holland et al., 1996, Potter et al., 1999). This observation on iron acquisition genes of P. multocida strongly suggests their products as vaccine candidates by concentrating on the regulation of iron acquisition genes of P. multocida.
       
The adhesion related genes of P. multocida were studied using ptfA (Type IV fimbria) and pfhA (filamentous hemagglutinin A) genes by PCR. All the P. multocida isolates revealed 100% prevalence of ptfA gene irrespective of their capsular type. The fact that P. multocida regularly harbor ptfA gene was also reported by Ewers et al., (2006), Jamali et al., (2014) and Katsuda et al., (2013). Ruffolo et al., (1997) suggested that the higher expression of ptfA gene was observed under microaerophilic conditions in vitro, which is comparable with the environment in the upper respiratory tract of the host, suggests type 4 fimbriae as potential vaccine candidates. However, DNA sequence analyses of ptfA genes from various P. multocida strains showed a high degree of variation that could limit the potency of a vaccine based on this antigen (Doughty et al., 2000).
       
The observations on the other adhesion related gene pfhA, all the Cap B isolates harbored this gene but none of the Cap A isolates contain this gene. The lower prevalence of pfhA gene is in agreement with Shayegh et al., (2010), Katsuda et al., (2013) and Aski and Tabatabaei (2016). Ewers et al., (2006) supported significant association of pfhA to the disease status in bovines. The present investigation is in concurrence with Ewers et al., (2006) as none of the Cap A isolates harbored this gene that were isolated from tonsils, whereas Cap B isolates contain this gene that was isolated from the clinical cases of HS. Although we found a significant association of pfhA to the disease status in bovines and the prevalence of the gene in P. multocida should be investigated more thoroughly in the future using defined sample material including strains representing latent colonizers and those isolated from organ/ tissue of diseased animals.
 
Further, it was observed, there is a clear correlation between capsular type and the disease, with capsular type A and B and was associated with pneumonia and Haemorrhagic septicaemia, respectively in cattle and buffaloes. The Fisher’s exact test results showed significant association of 0.008 at P< 0.05 between tbpA and pfhA. The tbpA and pfhA genes were closely associated with Cap B isolates and they were considered as important epidemiological markers for HS.
 
Virulence factors such as iron sequestering proteins and metabolic enzymes play key roles in acquiring and utilizing substrates for growth within the host and often in relatively nutrient poor and hostile environment. Expression of the type and amount of proteins presented on the bacterial surface also contribute to the disease specificity.

Pasteurella multocida is one of the economically important pathogen of domestic animals. The organism being presents as commensal in animal population and is also responsible for the mortality in buffaloes. In this regard the virulence genes and capsule biosynthesis genes of the Pasteurella multocida was studied using the isolates of buffaloes. It was observed that hgbA, hgbB, ompH and ptfA were associated with Cap A and Cap B isolates of Pasteurella multocida. Whereas the virulence genes tbpA and pfhA were associated with 0.008 at P< 0.05 under Fisher’s exact test. These virulence genes (tbpA and pfhA ) were only associated  with Cap B  isolates which are the possible epidemiological markers.

The authors are highly thankful to Sri Venkateswara Veterinary University, Tirupati for proving necessary facilities and funding for the post-graduation studies. We are also thankful to Dr. K. Sudhakar, Assistant Professor, Department of Animal Genetics and Breeding, NTR CVSc, Gannavaram for providing necessary help in statistical analysis.