Virulence of Pasteurella Multocida Isolated During Pasteurellosis Outbreak among Saigas in Kazakhstan

For the past few years mass deaths of saiga antelopes was recorded in the Republic of Kazakhstan. The outbreaks of Pasteurella infection caused drastic reduction of saiga population. The isolates of P.multocida belong to multocida subspecies. Dedicated from the dead saigas in 2010-2011 gg. Pasteurella multocida isolates contained virulent genes. The study was performed with use of 25 (2010) and 8 (2011) P.multocida field isolates from different organs of dead saigas. Fifteen virulence-associated genes (ptfA, ompA, ompH, oma87, plpB, fimA, hsf-2, pfhA, exbB, tonB, hgbA, fur, nanB, nanH and pmHAS) were detected by multiplex PCR. In isolates of P.multocida from saigas (year 2010) the following genes were detected: tonB (100%), ompA (92%), nanH (92%), fimA (68%), ptfA (68%), exbB (68%), oma87 (48%), plpB (44%), ompH (16%) and pfhA (12%). The genomes of P.multocida isolated from saigas in 2011 contained the following genes: hgbA (100%), ompA (100%), tonB (100%) and nanH (100%); fimA (87.5%), ptfA (87.5%) and exbB (87.5%); pfhA (62.5%), hsf2 (62.5%), fur (62.5%) and none of plpB, oma87, ompH, nanB and pmHAS genes was found in them. All assayed isolates showed high content of virulent hgbA, ompA, tonB and nanH genes. Field isolates of P.multocida (years 2010 and 2011) from saiga antelopes differ in virulence gene profiles.


Introduction
Saiga antelope (Saiga tatarica) is entered in the International Red Book because of drastic decline of its population. Mass death of saigas was observed on the territory of the Republic of Kazakhstan in 2010-2011. About 12000 in 2010 and over 500 animals in 2011 died [1] Taking into account the current low saiga numbers emergence of epizooty can be a real threat to the life story of this animal species. Assay of samples from dead saigas (Borsy village, West Kazakhstan oblast, Republic of Kazakhstan) at the RIBSP in 2010-2011 resulted in isolation of pathogenic organisms that were differentiated as Pasteurella [2]. is a Gram negative bacterium belonging to Рasteurellaceae family. Subject to the specific type of capsular antigens P.multocida has serologic differences and subdivides into capsular serogroups (А, В, D, Е, F) [3,4]. The currently known virulence factor genes include genes encoding the outer membrane proteins (ompA, ompH, oma87, plpB), adhesins (hsf1, hsf2), iron transport genes (fimA, ptfA, exbB, tadD, fur), enzymes (nanH, nanB, pmHAS). It should be also noted that there are genes responsible for high virulence of P.multocida; they are pfhA gene encoding filamentous hemagglutinin, hgbB gene that is responsible for hemoglobin control in a body [5][6][7].
The work was conducted towards determining subspecies and serotyping of P.multocida bacteria isolated from saiga antelopes in 2010 and 2011 in the Republic of Kazakhstan. Pathogenicity of P.multocida strains and isolates is assessed by presence (%) of virulent genes [8,9]. It is clear that high mortality rate among saiga antelopes in the Republic of Kazakhstan is directly related to the virulence factors of P.multocida. On the basis of the above stated the objective of the study was to detect the virulent genes in the genomes of P.multocida isolated from saiga antelopes in 2010 and 2011 in the Republic of Kazakhstan. epidemiological surveillance to determine the cause of saigas illness and death. Suspected pasteurellosis clinical cases were reported, and biological samples were taken from dead saigas for diagnosis. The samples taken from each animal were placed separately in indelibly marked plastic bags with the species name, animal i.d. and tissue name. These were packed in iced refrigerator bags and delivered frozen to the RIBSP. Detailed information on clinical signs and samples taken is shown in Table 1.

DNA Extraction
DNA was extracted with use of "PrepMan Ultra Sample Preparation Reagent" kit (Applied Biosystems) following the manufacturer's guidelines.

Pasteurella multocida species specific PCR
The species specific primers designed by Townsend et al. [10] KMTIT7 and KMTISP6 were used to amplify the gene sequences in P.multocida. The PCR reaction mixture and the thermal cycle protocol were as follows: initial denaturation-94 o C for 5 min, followed by 30 cycles, each cycle consisting of 3 steps; denaturation at 95 o C for 1 min; annealing at 55 o C for 1 min; extension at 72 o C for 1 min; final extension at 72 o C for 9 min Detection of virulence associated genes by multiplex PCR AccuPrime Taq DNA Polymerase High Fidelity kit manufactured by Invitrogen (USA) was used to produce PCR products of P.multocida virulent genes. Multiplex polymerase chain reaction (PCR) with 15 primer pairs was used for amplification (Table 2) [4,5,11,12].  Table 3: Component formulae of the reaction mixtures for amplification of virulence factor genes.
The parameters of the multiplex PCR that enable to detect several virulence gene profiles at a time have been chosen. While performing multiplex PCR optimal component structures of PCR mixtures and modes for amplification of virulence factor genes were selected. The parameters of the multiplex PCR are shown in Table 3 & 4. DNA electrophoresis in 2% agarose gel was performed with TBE buffer containing 1 µl/ml ethydium bromide in the unit for electrophoresis of nucleic acids GNA-100, Pharmacia (Sweden).

Statistical Analysis
The resulted statistical data were processed with use of SPSS software for Windows 12.0. The diagrams were drawn with the help of Microsoft Excel 2010. Statistical analysis was performed using Fisher's exact test (two tailed) in the GraphPadPrism 6.0 software.

Results
All 25 samples taken from dead saigas in 2010 and 8 samples of year 2011 were identified as P.multocida. It has been shown that KMTI gene is specific for reference strains and all P.multocida isolates [10]. Size of the specific product obtained with use of primers KMTI77 and KMTISP6 is 460 bp. The results of the assay are shown on Figure 1.
As Figure 1 shows PCR fragments of KMTI gene sized 460 bp have been found in DNA of P.multocida isolates under study. So, the assayed isolates are of Pasteurellа genus, multocida species. The mechanisms that take place in virulence factor genes of P.multocida provide a molecular basis for studying the degree of its pathogenicity. Therefore our task was to detect the presence of virulent genes in the genomes of P.multocida isolated from saiga antelopes. The results of the multiplex PCR for detection of virulent genes in P.multocida isolated from saiga antelopes in Kazakhstan are shown on Figure 2 Figure 3A shows that PCR fragment of hgbA gene sized 267 bp has been found in the genomes of the assayed isolates. Product of pfhA gene 286 bp long was detected in genomes of isolates Nos. 4, 5, 6, 7, 8. The results of PCR for presence of ompH, oma87, plpB, hsf2, plpB and omрА genes ( Figure 3B) show that ompH, oma87 and plpB genes are absent in P.multocida isolates. The region of hsf2 gene sized 433 bp was detected in genomes of isolates Nos. 1, 3, 6, 7, 8. OmpA gene encoding the outer membrane protein A was present in genomes of all isolates. Figure 3C shows PCR products of fimA, ptfA, exbB, tonB, fur genes. TonB and exbB genes associated with iron acquisition related factors were present in genomes of all isolates whereas the fimA, ptfA and fur genes were not detected in the genome of isolate No. 2. Only one gene (nanH) sized 287bp, of all genes encoding sialidase proteins was found in the assayed isolates ( Figure 3D). In genomes of P.multocida isolated from dead saigas in 2011 nanB and pmHAS genes were absent. Table 5 shows quantitative content (%) of virulent genes in genomes of P.multocida isolated from saiga antelopes in years 2010 and 2011. The study towards determination of virulent gene profiles of P.multocida isolated from dead saiga antelopes in 2010 Table  5 has shown that hgbA, pfhA, ompA, ompH, oma87, plpB, hsf2, fimA, ptfA, exbB, tonB, fur, nanH genes are present in the genomes of the isolates while nanB and pmHAS genes are absent. On the basis of the obtained data (Figures 2,3  Percentage of virulent genes in the genomes of isolates under study appeared to be different, e.g., content of tonB gene was 100%, of fur gene-72%, of hgbB gene-76%, pfhA-12%, ompA-92%, oma87-48%, plpB-44%, ompH-16%, nanH-80 %, and percentage of fimA, ptfA and exbB genes was 68%. Statistical analysis of data has been performed using Fisher's exact test and the resulted p values are shown in Table 4 and Figure 4. Differences in content of virulent pfhA, oma87 and plpB genes in P.multocida isolated from saiga antelopes in years 2010 and 2011 were considerable (р<0.05). As it concerns virulent hgbA, ompA, ompH, hsf2, fimA, ptfA, exbB, tonbB, fur and nanH genes the differences were insignificant (р>0.1).

Discussion
So far the principles of pathogenicity mechanisms and immune processes in P.multocida are not known. The putative determinants of virulence such as genes encoding the outer membrane proteins, genes participating in iron transport, enzymes, toxins and adhesins play the certain role in these processes and their presence in P.multocida genomes is associated with virulence.
Some authors recommend to use hgbA gene as an epidemiological marker for characterization of bacterial strains and isolates of P.multocida [4,13]. Content of the virulent hgbB gene in genomes of Kazakhstanean isolates from saigas in 2010 was 76% whereas in isolates of year 2011 it was 100%. A number of foreign scientists have shown 100% presence of the virulent hgbA gene in genomes of P.multocida isolated from different animal species [7].
Filamentous hemagglutinin pfhA gene is used as an epidemiological marker in P.multocida isolated from cattle. In our study only 12% of the assayed isolates of year 2010 contained the virulent pfhA gene whereas its content in the isolates of year 2011 was 62.5% (p<0.05). Adler et al. [14] paid special attention to detection of surface antigens participating in pathogenesis of P. multocida. They extracted the protective protein of outer membrane оma87 that participates in export of other virulence factors from a bacterial cell. Our studies show 48% content of оma87 gene in genomes of Kazakhstanean isolates of P.multocida from saiga antelopes in 2010 and its absence in isolates of year 2011 (p<0.05).
Also the assayed Kazakhstanean isolates of P.multocida showed low content of оmpH gene, e.g. only 16% of P.multocida isolated in 2010 contained this gene, and in isolates of year 2011 this gene was not detected. One more gene encoding the outer membrane protein is ompA gene. As Table 3 shows content of this gene in the genomes of Kazakhstanean isolates of P.multocida is high. In isolates of year 2010 the percentage of this gene is 92%, and in isolates of year 2011 it is 100%.
According to Lu YS et al. [15] the protein encoded by ompA gene as well as the protein encoded by оmpH gene is protective, that is why the results of molecular and genetic analysis are significant in studying pathogenesis of P.multocida.
Content of plpB gene encoding outer membrane lipoprotein in the bacteria isolated in 2010 was 44%, whereas in P.multocida isolates of year 2011 it was absent (p<0.05). Adhesins are essential to colonization and invasion of the host by P.multocida and as a rule their presence on bacterial surface is associated with virulence [3]. They include fimA (fimbrial), hsf-1 and hsf-2 (autotransporter adhesins), pfhA (filamentous hemagglutinins), ptfA (subunits of type IV pili) genes. Out of all isolates of year 2010 68% contained virulent fimA gene and it was detected in 87.5% of isolates of year 2011. Occurrence of virulent ptfA gene in genomes of P.multocida isolated in Kazakhstan in years 2010 and 2011 was respectively 68% and 87.5%. Content of the virulent hsf-2 gene in genomes of Kazakhstanean isolates of years 2010 and 2011 was respectively 88% and 62.5%. These data are close to the results of other authors [6].
The virulent tonB gene was detected in all assayed Kazakhstanean isolates of P.multocida. Content of the virulent exbB gene in isolates of year 2010 was 68%, and in isolates of year 2011 it was 87.5%. Virulent fur gene was present respectively in 72% and 62.5% of P.multocida isolated in years 2010 and 2011.
Sialidase proteins encoded by nanH and nanB genes can localize on the surface of host cells and destroy the receptors of the immune system [16,17]. In our study virulent nanH gene was detected respectively in 80% and 100% of bacterial genomes of P.multocida isolated in years 2010 and 2011. Virulent nanB gene was absent in genomes of Kazakhstanean isolates. Virulent pmHAS and nanB genes were not found in genomes of P.multocida isolated from saiga antelopes in Kazakhstan in years 2010 and 2011.