Virulence gene profiling of porcine Pasteurella multocida isolates of Assam

Aim: The present study was conducted to detect and identify the virulence genes in Pasteurella multocida isolates of porcine origin from Assam. Materials and Methods: A total of 21 porcine P. multocida isolates were subjected to capsular typing and detection of virulence-associated genes (pfhA, tbpA, hgbB, toxA, oma87, ompH, and nanB) using various polymerase chain reaction (PCR) methods reported elsewhere. Further, pathogenicity of the porcine isolates of P. multocida was studied in mice. For each strain of P. multocida selected for pathogenicity trial, the group of mice was injected intraperitoneally (i/p) with 0.1 ml of the inoculum prepared from respective field isolates, containing 109 organisms per ml. Results: Capsular typing of the isolates by multiplex PCR showed two capsular types, type A (66.66%) and type D (33.33%). All the isolates were positive for outer membrane protein genes, oma87 and ompH genes. Iron acquisition genes, tbpA and hgbB, were detected in 14.28% and 19.04% of the isolates. The dermonecrotoxin encoding gene, toxA, was present in 23.80% of the isolates. Filamentous hemagglutinin encoding gene, pfhA, was detected in 28.57%. The virulence gene distribution pattern of the isolates indicates the important role of the genes in disease pathogenesis. Conclusion: From the present study, it can be concluded that toxA gene is an important marker gene for defining the pathogenic potential of P. multocida strains in swine.


Introduction
Pasteurella multocida belonging to family Pasteurellaceae is a ubiquitous organism affecting multiple host species, thus causing several diseases such as hemorrhagic septicemia in cattle and buffalo, enzootic bronchopneumonia in cattle, sheep, and goats, atrophic rhinitis in swine, fowl cholera in poultry, and snuffles in rabbits [1,2]. It is one of the most fascinating Gramnegative, opportunistic animals and human pathogens with worldwide distribution.
The organism is grouped into 5 capsular serogroups (A, B, D, E, and F) with host specificity and disease induction [3]. Both toxigenic and non-toxigenic strains of serogroups A and D are associated with diseases in swine [4]. The pathogenicity of P. multocida is associated with various virulence factors which include diverse adhesions, dermonecrotic toxin, iron acquisition proteins, sialidases, and outer membrane proteins [3,[5][6][7]. These virulence factors help in colonization and invasion of the host, avoid host defense mechanisms, injury to host tissues, and stimulate host inflammatory response. The association of virulence factors with specific serogroups of P. multocida and its disease status in animals was also reported by Ewers et al. [8]. Since the pathogenic behavior of P. multocida could be predicted both by the virulence factors and the serogroups, evaluation of these virulence factors is important.
The present study investigates the distribution pattern of virulence-associated genes (VGAs) in P. multocida isolates of porcine origin.

Revival and confirmation of P. multocida isolates
The isolates were reconfirmed by following standard bacteriological techniques and by P. multocida species-specific polymerase chain reaction (PM-PCR) as per the method described by Townsend et al. [9] using specific primer pairs (Table-1) [7,[9][10][11][12][13]. PCR was done in 25 μl reaction mixture by mixing 3.0 μl DNA template with 12.5 μl master mix (2X, Qiagen, Germany) and forward and reverse primers (10 pmol each). The PCR amplification was performed in a thermocycler (Applied Biosystems, USA) using the thermal conditions as initial denaturation at 94°C for 4 min, 35 cycles of 94°C for 45 s, 55°C for 45 s, 72°C for 45 s, followed by final extension at 72°C for 6 min.

Capsular typing
Capsular PCR typing for all the isolates was done using a multiplex PCR as per the method described by Townsend et al. [10] with the reaction condition as illustrated in Table-2 [7,11].

Virulence gene detection
P. multocida isolates were tested for the presence of various VGAs, for example, pfhA, tbpA, hgbB, toxA, oma87, ompH, and nanB by simplex PCR as per the method of Ewers et al. [8], using specific primers (Table-1) with the standard thermal conditions (Table-2). The amplified products were electrophoresed in 1.5% agarose gel in 1X tris acetate EDTA buffer at 60 V for 1 h with ethidium bromide stain and visualized with ultraviolet light by Gel Documentation System (Kodak, Biostep, Germany).

Pathogenicity study of P. multocida isolates
Pathogenicity of the porcine isolates of P. multocida was studied in mice following the method described by Curtis [14] with slight modification. For each strain of P. multocida selected for pathogenicity trial, the group of mice was injected intraperitoneally (i/p) with 0.1 ml of the inoculum prepared from respective field isolates, containing 10 9 organisms per ml [15].

Results
All the bacterial isolates were revived in blood agar. The small, smooth, circular, glistening, and dewdrop-like colonies with a very characteristic odor and non-hemolytic colonies on blood agar plate (data not shown) were found to be Gram-negative coccobacilli and identified as P. multocida. The organisms were further confirmed based on specific amplification of KMT1 gene by PCR yielding an expected product size of 460 bp (Figure-1) which was detected in all the isolates.
On capsular PCR typing, of 21 P. multocida isolates, serogroup D-specific gene (657 bp, Figure-2) was detected in seven (33.33%) isolates, while serogroup A-specific gene (1044 bp, Figure-2) was detected in 14 (66.66%) isolates. The reference strain gave an amplified product of 760 bp band size corresponding to capsular type B.
On virulence gene detection PCR (Table-3), it was observed that the outer membrane genes (oma87 and ompH) were found to be present in all the 21 isolates of porcine origin used in the present study and the reference strain giving expected band size of 838  On amplification of filamentous hemagglutinin gene (pfhA), of the 21 isolates, six exhibited the presence of the same by yielding an expected amplicon size of 275 bp (Figure-7), of which 5 isolates belong to serotype A (35.71%) and 1 to serotype D (14.28%). The sialidase coding gene (nanB) was detected only in 2 (28.57%) isolates of serotype D (Figure-8).

Discussion
The present paper describes as the first report of virulence gene profiles of porcine P. multocida from Assam, India. Pasteurellosis is a common disease of pigs worldwide with specific serotype and pathotype associated with the respiratory disease [16][17][18]. However, the distribution pattern of the serotypes and pathotypes can vary considerably from region to region and over time in a given region [12,19]. The present study results in the detection of more percentage of serotype A than serotype D. The isolates used in the present study were mainly from cases of the respiratory syndrome. A higher percentage of serotype A than serotype D in P. multocida isolates from indigenous pigs in central Kalorey et al. [20] and Northeast India [19,21] have been reported.
Variable distribution of the virulence genes was observed among the serotypes of P. multocida during the present study (Table-3). However, no correlation could be established with respect to the presence of virulence gene and serotypes of the Wide spread. Widespread distribution of hgbB gene among the porcine P. multocida strains and its regular detection in capD strains compared to capA strains of P. multocida was also reported [7,22]. On the contrary to the present detection of tbpA gene, Ewers et al. [7] could detect the gene exclusively in bovine, sheep, and buffalo isolates and not in P. multocida pig strains. The present observation of 18.18% positive strains of P. multocida isolates possessing the tbpA gene could Shayegh et al. [11] Shayegh et al. [11] Shayegh et al. [11] Ewer et al. [7] Ewer et al. [7] Ewer et al. [7] P. multocida=Pasteurella multocida, PCR=Polymerase chain reaction   Available at www.veterinaryworld.org/Vol.11/March-2018/15.pdf be probably due to interspecies transmission of tbpA gene-positive P. multocida, as also suggested by Kumar et al. [23]. However, further study needs to be conducted before giving conclusive remark on interspecies transmission.
The present finding of low percent positivity of pfhA gene in strains of serogroup D compared with serogroup A also supports the observations of association of filamentous hemagglutinin gene pfhA with serogroups A, B, E, and F [7,12,22].
The occurrence of OMP gene in porcine strains of P. multocida and its equal distribution among capsular serogroups (type A, type D, and other serotypes) was also reported [7,12,22]. P. multocida OMPs have been identified as potent immunogens [24] as reported by Rajkhowa et al. [25] and play a significant role in the pathogenesis of pasteurellosis [26]. Hazarika et al. [27] also reported that the sonicated and bacterin vaccines prepared from pig strains of P. multocida conferred 100% protection against homologous as well as heterologous strains compared to 66.66 and 86.66% protection with vaccine prepared from reference strain (P 52 ). From the present study, it can be opined that the OMP gene has a great      role in pathogenesis of P. multocida infection irrespective of the serotypes. Considering the immunogenic potential of the OMPs, both the genes can be explored in the development of a suitable vaccine against swine pasteurellosis. However, before giving any conclusive remarks on distribution and its applicability as vaccine candidate, a detailed study will be required involving a large number of isolates recovered from pigs of different parts of the Northeastern region.
The 13 (59.09%) pathogenic isolates were found to possess different combinations of virulence genes. Pathogenic serotype A isolates with different virulence gene combinations produce 83.33-100% mortality in inoculated mice when compared to isolates with the presence of only OMP genes (50-83.33%). Similarly, serotype D isolates with different virulence gene combinations produce 33.33-100% mortality in inoculated mice. No reports could be traced out from the available literature with respect to the correlation between presence virulence gene in the P. multocida isolates and their pathogenicity in mice. However, it can be opined from the present observation that OMP alone is not responsible for pathogenesis of the disease and association of other VGAs with OMP gene might play a key role in pathogenesis. The present study indicates that toxA gene plays an important role in pathogenesis of P. multocida in pigs, which is supported by the findings of many workers [28][29][30]. Among the other virulence genes, tbpA gene was found to be closely associated with pathogenesis of the P. multocida. Contrary to the present findings, Ewers et al. [7] reported the presence of tbpA gene only in bovine strains of P. multocida and could not detect in porcine strains. Although they observed a significant association between pfhA and toxA with clinically diseased swine, toxA alone was found to be associated with the disease status independently. A perusal of the literature reveals that there is no report on the detection of tbpA gene from porcine strains, the detection of the gene in highly pathogenic porcine isolate is an important finding, and its role in pathogenesis needs further investigation. During the present study, some of the P. multocida isolates could not produce mortality in mice following inoculation with P. multocida isolate having virulence gene, either alone or in combination. This might be due to repeated subculturing of the isolates in laboratory media that result in the suppression of gene function or due to gene mutation, resulting in non-expression of the genes in vivo [31,32]. Detection of a high proportion of toxigenic capsular type A P. multocida from pigs was also reported by other workers [11,20,30,33]. Detection of toxin gene in both the type A and D isolates of P. multocida of the region in the present study indicates its important role in the disease pathogenesis mechanism.

Conclusion
From the present study, it can be concluded that toxA gene is an important marker gene for defining the pathogenic potential of P. multocida strains in swine. However, other virulence genes are also found to be distributed well among pathogenic strains of P. multocida. Among the other virulence genes, tbpA gene PCR=Polymerase chain reaction was found to be closely associated with the pathogenesis of the P. multocida. The association of the gene in disease producing mechanism needs further evaluation.

Authors' Contributions
This study was a part of LBD's research work during his PhD program. LBD carried out the experiment. SKD, RKS, and DPB designed the experiment. SKD, RKS, DPB, SM, and RAH provided necessary guidelines. DPB drafted the final manuscript. All authors have read and approved the final manuscript.