Vet Res Commun DOI 10.1007/s11259-014-9601-6

ORIGINAL ARTICLE

In vitro and in vivo pathogenicity studies of Pasteurella multocida strains harbouring different ompA Shailja Katoch & Mandeep Sharma & R. D. Patil & Sandeep Kumar & Subhash Verma

Accepted: 24 February 2014 # Springer Science+Business Media Dordrecht 2014

Abstract Pasteurella multocida is a pathogenic, Gramnegative bacterium that is commonly found as normal flora in nasopharynx of variety of wild and domestic animals. Numerous virulence factors have been described for P. multocida isolates which include adherence and colonization factors, iron-regulated and acquisition proteins, extracellular enzymes such as neuraminidase, lipopolysaccharide (LPS), capsule and a variety of outer membrane proteins (Omp). OmpA has a significant role in stabilizing the cell envelope structure by providing physical linkage between the outer membrane & peptidoglycan. It has been shown to mediate P. multocida -host cells interaction via heparin and/or fibronectin binding and therefore act as an important invasive molecule which could determine the final outcome of initial infection. Comparative nucleotide sequence analysis of ompA gene of P. multocida has revealed that despite extensive genetic diversity in ompA of P. multocida, most sequences could be classified into two major allele classes namely ompA allele (I) and allele (II). The P. multocida recovered from nasal cavity of bovine and belonging to two ompA classes were tested for their differential virulence. In vitro pathogenicity studies on Madin Darby Bovine Kidney (MDBK) cell line employing adhesion and invasion assays indicated that P. multocida strain with ompA (I) is more invasive than P. multocida strain with ompA (II). In vivo studies in mice further reiterated that the

S. Katoch : M. Sharma : S. Kumar : S. Verma (*) Department of Veterinary Microbiology, Dr. G.C. Negi College of Veterinary & Animal Sciences, CSKHPKV, Palampur, Himachal Pradesh 176062, India e-mail: [email protected] R. D. Patil Department of Veterinary Pathology, Dr. G.C. Negi College of Veterinary & Animal Sciences, CSKHPKV, Palampur, Himachal Pradesh 176062, India

isolates harbouring ompA(I) were comparatively more virulent to isolates harbouring ompA (II). Keywords Pasteurella multocida . ompA allele . Pathogenicity studies

Introduction Pasteurella multocida is a Gram-negative, non-motile, nonsporing and facultative coccobacillus bacteria (Carter 1987). It has a broad disease spectrum, known to cause number of primary and secondary infections in a wide range of vertebrate hosts. The major diseases of economic significance include Haemorrhagic Septicemia (HS) in cattle & buffalo, enzootic pneumonia in cattle, sheep & goats, atropic rhinitis in pigs, fowl cholera in poultry and snuffles in rabbits. Virulence factors of P. multocida associated with disease outcome are polysaccharide capsule, endotoxins or lipopolysaccharide (LPS), outer membrane proteins (OMPs), fimbriae, exotoxins, multocidins or siderophores, extracellular enzymes and plasmids (Harper et al. 2006). The OMPs of the gram-negative bacteria have been implicated in conjugation, resistance to the bactericidal effect of serum, adherence to host tissues and contribute to disease process by acting at an interface between the host and pathogen (Lin et al. 2002). In E. coli, OmpA also functions as a multifunctional protein displaying both phage receptor and some porin activity (Nikaido 1983). Further OmpA in E. coli appears to maintain the integrity of the outer membrane and has been shown to be one of the major factors responsible for E. coli invasion of human brain microvascular endothelial cells (Prasadarao et al. 1996). The 35-kDa OmpA protein of E. coli consists of an N-terminal transmembrane domain (19 kDa) and a C-terminal globular periplasmic domain (16 kDa) (Arora et al. 2001). The three dimensional structure

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of the transmembrame domain as determined by X-ray crystallography and nuclear magnetic resonance spectroscopy suggest that it consists of eight membrane-traversing antiparallel β-strands and four relatively long, mobile, hydrophilic surface exposed loop (Popp et al. 1999; Pautsch and Schulz 2000; Arora et al. 2001). The periplasmic domain interacts with the underlying peptidogylcans and confers upon OmpA its role in maintaining the structural integrity of the outer membrane (Mot and Vanderleyden 1994). P. multocida ompA gene was cloned and characterised by Dabo et al. (2003) who found significant similarity between P. multocida and E. coli OmpA protein. The OmpA (or heat modifiable) protein is a major structural component of the outer membrane of P. multocida. The surface exposed loops of P. multocida OmpA play role in P. multocida- host relationship. OMPs of P. multocida have also been shown to play a significant role in the pathogenesis of pasteurellosis (Lübke et al. 1994; Srivastava 1998). Further, it has also been suggested that OMP variation among the P. multocida isolates may also help in epidemiological survey by assessing their inter-strain heterogeneity (Davies et al. 2003). An earlier study emanated from our laboratory (unpublished) has shown that different isolates of P. multocida recovered from apparently healthy and diseased animals harboured different types of ompA variants with particular ompA variant exclusively recovered from either healthy animals or from diseased animals. Keeping in view the above observations and the fact that certain strains isolated from diseased animals possessed a different ompA, in comparison to the isolates associated with apparently healthy animals the present study was carried out. This study aimed at assessing the pathogenic potential of P. multocida isolates bearing different ompA so as to gain more insight into their functions and to determine the differential role of ompA in the disease manifestation.

Materials and methods Selection of P. multocida isolates and identification of ompA allele classes A total of four isolates with identity number 559, 4358, 224 & 2205 of P. multocida earlier recovered from bovine nasal swabs were used. These have been maintained in 40 % glycerol stock solution in brain heart infusion broth and stored at −80ºC for a period of one year in the Department of Veterinary Microbiology. The isolates bearing identity numbers 559 & 4358 were capsular type B carrying ompA allele (I) and those with identity numbers 224 & 2205 were type A with ompA allele (II). The organisms were revived on 5 % defibrinated sheep blood agar and reconfirmed by conventional bacteriological and biochemical methods described by Muhairwa

et al. 2001. DNA was isolated by Phenol- ChloroformIsoamyl alcohol method (Wilson 1987) and isolates reconfirmed by P. multocida- Polymerase Chain Reaction (PM- PCR) using a pair of P. multocida specific primer used by Townsend et al. (1998). Capsular types were reconfirmed by performing multiplex capsular PCR as described by Townsend et al. (2001). Amplification of ompA gene of P. multocida isolates For PCR reaction, a total of 100 ng DNA template was added to the reaction mixture (25 μl) containing 2.5 μl 10XPCR buffer, 2 μl of 25 mM magnesium chloride, 1 μl of 10 mM dNTPs, 1 μl of each primer pair in a 10 pmol concentration and 1 unit of Pfx DNA polymerase (Promega corporation, Madison, USA). The primer pair PmompA FP: 5′ -ATG AAA AAA ACA GCA ATT GC-3′ and primer PmompA RP: 5′ – TTA TTT GTT ACC TTT AAC AGC-3′ were used. The amplification was carried out using GeneAmp PCR system 9700 (Applied Biosystems, U.S.A) for 35 cycles of denaturation at 95 °C for 20 seconds (s), annealing at 50 °C for 20 s and extension at 72 °C for 1 minute (min) and 20 s, the cycles were followed by a final extension at 72 °C for 10 min. Amplification products were resolved by gel electrophoresis on 2 % agarose gel, stained with ethidium bromide (1 μg/mL) and visualized on UV transilluminator (Alpha Innotech, U.S.A.). Sequencing of the ompA gene The PCR products purified through the QIAquick PCR purification kit (Qiagen) were got sequenced from First BASE Laboratories Sdn Bhd, Selangor, Malaysia. Molecular characterisation of isolates for virulence associated genes The isolates of P. multocida were screened for 11 additional virulence associated genes namely tbpA, pfhA, hgbA, hgbB, toxA, nanH, nanB, sodA, sodC, fim4 and oma87 through PCR. The primer pairs as designed by Lichtensteiger et al. (1996), Lainson et al. (1996), Doughty et al. (2000) and Ewers et al. (2006) were used to confirm the presence of these genes. In vitro pathogenicity studies on Madin Darby Bovine Kidney (MDBK) cell line To study the virulence potential of P. multocida harbouring different ompA, association and invasion assays were carried out. The MDBK cells were seeded onto 12 well tissue culture plates and grown to confluence. Immediately before infection, the monolayers were washed twice with cell culture medium without serum and antibiotics.

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Association assays The first and second row of the confluent MDBK monolayers on 12 well cell culture plate were infected with 1 mL of bacterial suspension containing 8× 109 cfu of P. multocida harbouring ompA allele (I) & ompA allele (II), respectively. The association was carried out by keeping cells and bacteria in contact for 2 hours (h) at 37 °C. Loosely bound bacteria were then removed from the cell monolayers by five washings with PBS. The infected cells were fixed with methanol and cell associated bacteria were visualized by Giemsa staining and light microscopy under oil immersion or the cells were detached by 0.25 % trypsin for 5 min at 37 °C and the associated bacteria were cultured on brain heart infusion (BHI) agar and colony forming unit (cfu)/mL were counted. Identical concentration of bacteria was inoculated in 12 well plates containing minimum essential medium (MEM) without MDBK cells as a control for nonspecific binding of bacteria. Invasion assays A total of 2×109 cfus of bacteria of ompA allele (I) and ompA allele (II) contained in 1 mL of cell culture medium was inoculated into wells of first and second row respectively of 12 well cell culture plate containing confluent monolayer and incubated for upto 4 h at 37 °C in a humidified atmosphere containing 5 % CO2. After invasion period of 4 h, the monolayers were washed three times with MEM and fresh medium containing 50 μg of gentamicin per mL was added to kill extracellular bacteria. After another 2 h of incubation, the monolayers were washed three times with antibiotic free medium, treated with 0.25 % trypsin for 5 min at 37 °C and lysed by addition of 1 mL of cold 0.1 % Triton X-100 (Sigma, USA) in distilled water for 10 min. The cell lysates were diluted in PBS and plated on BHI agar to allow the quantification of released intracellular bacteria by colony plate count. The invasion ability was expressed as the percentage of the inoculums which survived the gentamicin treatment. Each assay was carried out in triplicate and was independently repeated three times. Results are expressed as averages of all experiments. In vivo pathogenicity studies in mice Experimental animals and their housing Sixty healthy Swiss albino mice of 6 to 8-week age were procured from National Institute of Pharmaceutical Education and Research, Mohali, India and were maintained in a clean experimental animal house of Department of Veterinary Microbiology, Dr. G.C. Negi College

of Veterinary and Animal Sciences, Palampur, H.P. For the first one month, mice were housed in polypropylene cages and fed commercial mice feed @ 5 g/mice/day. Animals were housed in group of five per cage during the study and had free access to water. The 12 h light/ dark cycle was maintained throughout the study period. Before infection 100 μl whole blood was collected from tail of all the mice for bacterial examination to ensure Pasteurella-free status of the mice by culturing on 5 % defibrinated sheep blood agar. Physiological parameters were monitored prior to and after bacterial challenge. Experimental design The mice were divided into two challenge groups of 20 mice each (according to the challenge strain used) and 1 negative control group of 20 mice. Each challenge group was further subdivided into four groups of five mice per group & the mice in a particular group were inoculated with 2,000, 200, 20 and 2 cfu per mouse with P. multocida, respectively. Challenge of mice with P. multocida The two groups of mice were challenged intraperitonially (i.p.) with P. multocida isolates harbouring ompA allele I (isolate- 559) and II (isolate- 224) but carrying common pool of other virulent associated genes with 1 mL of phosphate buffered saline (PBS) containing the indicated number of cfu. Negative control mice were injected i.p. with 1 mL of sterile PBS only. The clinical status of the challenged and control mice, including body weight and behavior pattern were observed and recorded. In mice expressing clinical signs of the disease the monitoring frequency was increased to every 1 h. The mortality pattern was recorded. Control and survived injected animals were humanely euthanized at day eight post-challenge. Gross and histopathology All challenged and control mice were examined for gross and microscopic lesions, if any. Representative tissue samples of lungs, liver, kidneys spleen and intestine were collected from all the mice at the time of death or on sacrifice, fixed in 10 % neutral buffered formalin for 24 h and processed with paraffin wax embedding technique (Luna 1968). Sections (3–4 μm) were cut, stained with hematoxylin and eosin, and examined by light microscopy. Bacteriology For bacteriological examination the swabs were collected aseptically from lungs, liver and kidneys from the dead and sacrificed mice at necropsy. Impression smears

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Results Confirmation of P. multocida and identification of ompA alleles

Fig. 1 ompA gene amplicons (1.1 Kb) of different P. multocida isolates; lane 1 (559), lane 2 (4358), lane 3 (224) & lane 4 (2205), N- negative control and M- Marker (1,000 bp)

of cut sections were made on agar plates and streaked for bacterial isolation. Samples were cultivated on Blood agar (BA). At least one bacterial colony per sample with morphology suggestive of P. multocida was examined by PM-PCR (Townsend et al. 1998).

Titration of bacterial load in organs To measure the bacterial load in different groups of mice, the tissues collected from lung, liver and kidney were homogenized aseptically in normal saline solution (NSS) to make 10 % suspension and tenfold serial dilution were made. These dilutions were cultured in triplicate on BHI agar plates by incubating at 37 °C up to 48 h. The titers of bacteria were expressed as cfu per gram of tissue.

Statistical analysis The significance of mouse challenge experiment in this paper was assessed by Z test for difference of proportion and association & invasion assay by student’s t-test and P

In vitro and in vivo pathogenicity studies of Pasteurella multocida strains harbouring different ompA.

Pasteurella multocida is a pathogenic, Gram-negative bacterium that is commonly found as normal flora in nasopharynx of variety of wild and domestic a...
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