ISSN 0891-4168, Molecular Genetics, Microbiology and Virology, 2016, Vol. 31, No. 2, pp. 75–81. © Allerton Press, Inc., 2016. Original Russian Text © A.V. Nefedchenko, A.N. Shikov, A.G. Glotov, T.I. Glotova, V.A. Ternovoy, A.P. Agafonov, A.N. Sergeev, N.A. Donchenko, 2016, published in Molekulyarnaya Genetika, Mikrobiologiya i Virusologiya, 2016, No. 2, pp. 62–66.
EXPERIMENTAL WORKS
Development of a Method for Identification and Genotyping of Pasteurella multocida and Mannheimia haemolytica Bacteria Using Polymerase Chain Reaction and Phylogenetic Analysis of Bacterial Cultures Isolated from Cattle A. V. Nefedchenkoa, *, A. N. Shikovb, A. G. Glotova, T. I. Glotovaa, V. A. Ternovoyb, A. P. Agafonovb, A. N. Sergeevb, and N. A. Donchenkoa aInstitute
of Experimental Veterinary of Siberia and the Far East, Federal State Budget Scientific Institution, Novosibirsk, 630501 Russia b Vektor State Science Center for Virology and Biotechnology, Federal Budget Institution of Sciences, Novosibirsk, 630559 Russia *e-mail:
[email protected] Received March 16, 2015
Abstract—The results of developing the identification and genotyping method for the Pasteurella multocida bacteria of five capsule groups and the Mannheimia haemolytica A 1 bacteria, using the multiplex polymerase chain reaction (PCR) with the electrophoretic detection are reported. The diagnostic sensitivity of the developed method came to 103 CFU/mL in the pure culture studies and 105 CFU/g in the biological material studies. The analysis revealed 50% of P. multocida and 11.2% of M. haemolytica in all 260 tested samples of biological material from the infected animals. Circulation of bacteria of P. multocida capsule groups B and E among the susceptible animals was not determined. Group A bacteria were found in the majority of the samples; bacteria of group D were infrequently identified; in one case, group F bacteria were detected. The circulation of capsule group A P. multocida bacteria of two genetic types was determined using phylogenetic analysis. Keywords: Pasteurella multocida, Mannheimia haemolytica, PCR, genes, capsule group, phylogenetic analysis DOI: 10.3103/S0891416816020063
INTRODUCTION
in cattle and buffaloes, and group F strains have been rarely found in septic and respiratory diseases in calves [8, 9]. In M. haemolytica, 12 serological types were revealed, among which only one type (A1) of strain has been identified as a cause of respiratory diseases in cattle [10]. The identification of bacteria, based on their cultural, morphological and biochemical characteristics is a very labor-intensive and time-consuming process. The molecular biology techniques—in particular, polymerase chain reaction (PCR)—offer rapid detection and identification of microorganisms directly from biological material samples and in mixed or pure cultures [11]. Decoding the nucleotide sequence for the second region of the capsule synthesis locus in P. multocida made it possible to identify the genes unique to each capsule group and the encoding proteins involved in the synthesis of group specific polysaccharides. The hyaD gene is responsible for the synthesis of the hyaluronic acid and unique to strains of group A; the fcbD and dcbF genes control the synthesis of chondroitin synthase from type F and the synthesis of glycoside
Respiratory diseases of young cattle stock can cause economic losses in animal husbandry. Bacteria of the Pasteurellaceae family, Mannheimia haemolytica and Pasteurella multocida, play a significant role in the etiology of these diseases [1]. Both agents can cause, as a rule, reinfections under unfavorable environmental conditions or the effects of viral infections. These diseases are found everywhere; they can lead to death and decrease growth rate, weight gain, and milk production in animals [2–5]. M. haemolytica can cause outbreaks of infectious diseases characterized by acute appearance and a severe course, while P. multocida bacteria cause subacute and chronic pneumonia and are less virulent [6, 7]. Five capsule groups (A, B, D, E, and F) that have different epizootiological significance were revealed in P. multocida. Bacterium strains of capsule groups A and D are involved in the occurrence of the respiratory diseases in calves and mature animals, strains of groups B and E can cause the hemorrhagic septicemia 75
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Table 1. Bacterial cultures used in the work and results of genotyping Results of PCR testing Species of bacteria
Strain
Source identification genotyping
M. haemolytica
16
The same '' P. multocida
NK-42 NV-53 1231
The same '' '' '' '' '' '' '' '' '' Streptococcus pneumoniae Clostridium perfringens Klebsiella pneumoniae Salmonella typhimurium Salmonella paratyphimurium Escherichia coli
681 Т-80 Т-14 MSK-13 Sib-13 Omsk-13 UK-59 SR-57 AGM/2013 OV-58
F-50
Collection of the Kovalenko All-Russia Research Institute of Experimental Veterinary Collection of the laboratory The same Collection of the Kovalenko All-Russia Research Institute of Experimental Veterinary The same '' Collection of the laboratory The same '' '' '' '' '' '' '' '' '' '' '' Collection of the Kovalenko All-Russia Research Institute of Experimental Veterinary
M.h.
–
M.h. M.h. P.m.
– – А
P.m. P.m. P.m. P.m. P.m. P.m. P.m. P.m. P.m. P.m. – – – – – –
В В А D А А А А А А – – – – – –
Strains of bacteria from the laboratory collection are isolated from the lungs of calves with respiratory pathology; – indicates a negative PCR result.
heparin from type D, respectively. Genes bcbD and ecbJ encode glycosyltransferases in the strains of capsule groups B and E, respectively. The highly conservative cell wall protein kmt1 gene that is unique to P. multocida was identified as well (12). The sodA gene encoding the superoxide dismutase is highly conservative for M. haemolytica (13). A range of authors have presented data on heterogeneity of the (ompH) gene for the outer membrane protein H in different P. multocida serovariants and the possibility of its use for typing cultures [14, 15]. The objectives of the work are to develop a method for identification and genotyping of both P. multocida bacteria classified into capsule groups A, B, D, and F and M. haemolytica A1 bacteria using multiplex PCRelectrophoresis detection, as well as to perform phylogenetic analysis of isolated cultures of bacteria. MATERIAL AND METHODS Bacteria. Reference strains 1231, 681, and T80 of P. multocida and 16 of M. haemolytica were obtained
from the microorganism culture collections at the Kovalenko All-Russia Research Institute of Experimental Veterinary (Moscow). We isolated the other strains from sick animals (Table 1). Extraction of DNA. DNA was extracted from bacterial cultures and animal viscera samples with commercial RIBO Prep Kits (Central Research Institute of Epidemiology, Russia). PCR performance. PCR was performed with the primers presented in Table 2 in a reaction mixture brought to a volume of 30 μL and composed of 5 μL DNA, 1× Taq buffer without Mg2+ (Medigen Labs LLC, Russia), 3 mM MgCl2 0.4 mM dNTP, 0.15 μM of each primer, 1.5 units Smart-Taq DNA polymerase, and up to 30 μL water; amplification was performed in the following regime: 1 cycle at 95°C for 5 min followed by 45 cycles at 95°C for 20 s, 57°C for 30 s, and 72°C for 40 se and, finally, 1 cycle at 72°C for 7 min. The amplified fragments were divided in 2% agarose gel. Obtaining positive control samples. Positive control samples (PCSs) were obtained by molecular transformation of the top 10 strain of E. coli with pCR® 2.1
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Table 2. Primer sequences for detection and sequencing of P. multocida and M. haemolytica genes Gene
sodA
kmt1
Primers
M.h.-F M.h.-R
Kmt1-F Kmt1-R hyaD P.m.A-F P.m.A-R dcbF P.m.D-F P.m.D-R bcbD P.m.B-F P.m.B-R ecbJ P.m.E-F P.m.E-R fcbD P.m.F-F P.m.F-R ompH* F R
Position in the referent sequence
Sequence of primers
M. haemolytica 5'-GACTACTCGTGTTGGTTCAGGCT-3' 312–334 5'-CGGATAGCCTGAAACGCCT-3' 438–420 P. multocida 5'-TAAGAAACGTAACTCAACATGGAAATA-3' 266–292 5'-GAGTGGGCTTGTCGGTAGTCTT-3' 456–477 5'-CGATAGTCCGTTAGATATTGCAAC-3' 9267–9288 5'-CATAATGGATTTGGCGCCAT-3' 9803–9824 5'-ATCGCATCCAGAATAGCAAACTC-3' 3306–3328 5'-TCCGATGCTTTGGTTGTGC-3' 3661–3643 5'-GCGTGTATAACCTACATCTTCCCA-3' 12541–12564 5'-CGTCCATCAACACCTTTACTGC-3' 12708–12687 5'-TGGGCACATGCTCGCTTA-3' 4539–4556 5'-CTGCTTGATTTTGTCTTTCTCCTAA-3' 4896–4872 5'-CGGAGAACGCAGAAATCAGAA-3' 2885–2905 5'-CAACAACGACTTCAAATGGGTAG-3' 3142–3120 5'-GCGTTTCATTCAAAGCATCTC-3' 1–20 5'-ATGACCGCGTAACGACTTTC-3' 975–996
Size of amplicon, bp
Origin
AY702512
126
This work
FJ986389
211
The same
AF067175
564
''
AF302465
355
''
AF169324
167
''
AF302466
357
''
AF302467
257
''
AY606823
996
[16]
Referent sequence
* Used only for sequencing.
plasmids (Invitrogen Corp., United States) containing specific DNA insertions of genes: M. haemolytica sodA and P. multocida kmt1, hyaD, and bcbF, which were obtained using the primers presented in Table 2. The concentration of the plasmid DNA was determined using reagents of the Quant-iT dsDNA HS assay kit (Invitrogen, United States) and the Qubit Fluorometer (Invitrogen, United States). Determination of analytical and diagnostic sensitivity. The analytical sensitivity of the method was determined specifically for each analysis. Tenfold dilutions of the PCSs were used. The diagnostic sensitivity of the method was determined using tenfold dilutions of the cultures of P. multocida (SR57, 681, and MSK 13) and M. haemolytica [16], concentrations of which were preliminarily determined by standard bacteriological methods. The PCS sensitivity was determined using analysis of tissue material. For that reason, 100 μL material culture dilution was added to 900 μL 10% suspension of a lung or a lymph node; it was mixed and settled and the upper aqueous phase was analyzed. The concentration of bacteria was calculated as colony-forming units (CFU) per 1 g of tissue. The extraction of DNA was carried out as described above. Analysis of biological material samples. Two hundred and sixty samples of biological material (lungs, mediastinal and bronchial lymph nodes, and spleen)
obtained from dead calves of an age of 4 days to 6 months with symptoms of respiratory diseases were analyzed. A 10% suspension was prepared from the organ samples. DNA extraction from the samples was carried out as described above. Determination of nucleotide sequences and phylogenetic analyses. The primers presented in Table 2 were used to sequence the genes of P. multocida and M. haemolytica. The sequencing reaction was performed with Big Dye® Terminator v 3.1 Cycle Sequencing Kits (Applied Biosystems, United States). The products of the sequencing reaction were analyzed using the method of capillary electrophoresis with an ABI PRISM® 3130xl automate sequencer (Applied Biosystems, United States). The sequence alignment was performed by the method for making a NeighborJoining Tree with the MEGA 5 analysis package and the Lasergene 7 Software package. The statistical reliability of the phylogenetic tree topology was determined using the bootstrap test. RESULTS AND DISCUSSION Development of a method for identification and genotyping of P. multocida and M. haemolytica bacteria. To detect genes of P. multocida and M. haemolytica bacteria, primers in the Vector NTI suite 9.0.0 software (InforMax) were selected and designed for gene
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(a) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
564 355 211 126
(b) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
357 257 167
Fig. 1. Results after electrophoresis of the (a) first- and (b) second-round amplification products from genomes of P. multocida and M. haemolytica bacteria. (1 and 18) 100 b.p. molecular weight marker (numerals designate amplicon lengths), (2) M. haemolytica 16, (3—11) P. multocida cultures 1231, 681, T-80, T-14, Sib-13, Omsk-13, UK-59, MSK-13, CP-57, (12) M. haemolytica culture NK-42, (13) S. pneumoniae, (14) Cl. perfringens, (15) K. pneumonia, (16) S. typhimurium, (17) PCS mixture.
sequences deposited in the international NCBI GeneBank database. At the first stage of the work, the values for several pairs of oligonucleotide primers for each gene were calculated; each set of individual primer pairs and their combination with the other primers was tested for specificity and synthesized. The selected primers are presented in Table 2. PCR parameters, such as the concentrations of dNTP, Mg ions, and primers; the amount of input DNA dilution; the temperature and time regime of a reaction were optimized; and the primer set combinations were determined to increase the sensitivity, specificity, and rates of analysis. In the final version, analysis of each sample was performed using two multiplex reactions. The first reaction involved species-specific primers to detect the P. multocida (Kmt1-F and Kmt1-R) fragment of 211 bp and the M. haemolytica (M.h.-F and M.h.-R) fragment of 126 bp and, moreover, the P.m.A-F and P.m.A-R group-specific primers (564 bp) for genotyping group A and the P.m.D-F and P.m.D-R (355 bp) group-specific primers for group D. For a second-order reaction, genotyping of capsule group B (P.m.B-F and P.m.B-R), group E (P.m.E-F and P.m.E-R), and group F (P.m.F-F and P.m.F-R) containing fragments of 167 bp, 357 bp, and 257 bp, respectively, was performed. The bacterial cultures presented in Table 1 were used to process the parameters of the PCR assay. No
nonspecific reactions were observed. The results of capsule genotyping of cultures coincided with the data obtained during the earlier conducted surveys [17] (Fig. 1). Defining analytical and diagnostic sensitivity. The analytical sensitivity was considered as the final PCS dilution yielding a positive result in the PCR essay. The obtained PCR result was interpreted as positive. The analytical sensitivity of the method was 1.6 × 10 to 5.9 × 102 GE per reaction. The selected primers could be identified with equal efficiency and made it possible to genotype the P. multocida cultures of capsule groups A, B, and D and the M. haemolytica cultures. The diagnostic sensitivity of the test-system comprised 103 CFU per mL in the bacterial suspension and 105 CFU per 1 g of tissue in the tissue material. Analysis of biological material samples. The developed protocol for the PCR assay performance was used to analyze samples from sick animals. The results presented in Table 3 showed that M. haemolytica DNA was identified in 14.3% of samples from lungs and 11.1% of lymph node samples. The DNA of P. multocida was identified in 8.8% of the samples from spleen, in addition to 63.3 and 42.6% of the samples from lungs and lymph nodes, respectively. In addition, 82.6 and 11.5% of the positive samples of P. multocida of capsule groups A and D, respectively, were identified in genotyping. DNA of P. multocida of capsule group F was detected in all analyzed samples from one animal. Nucleotide sequence identification and phylogenetic analysis. The nucleotide sequences of the amplicons produced in the PCR assay were identified and compared with the homologous sequences on the NCBI BAST service (http://www.ncbi.nlm.nih.gov/) to confirm the specificity of typing of the bacterial cultures and genotyping capsule groups in biological material samples. The amplified fragments of genes of the analyzed P. multocida hyaD and dcbF cultures, as well as a fragment of the fcbD gene detected in the sample of biomaterial, had 100% identity with the homologous sequences of the P. multocida genes deposited in the GenBank database. The sequences of the kmt1 gene of different strains had a 97–100% identity, for which reason a phylogenetic analysis of this gene dividing all strains into several clades including cultures of one capsule group was performed (Fig. 2). In addition, the isolated strains of group A were divided into two clades (clades 1 and 2); the strains were genetically different from the 1213 reference strain (of clade 3). According to the phylogenetic analysis of the ompH gene, the analyzed strains were grouped into the same clades (Fig. 3, see second half of region) at high levels of reliability. The M. haemolytica cultures at the sodA gene region were homologous with the published M. hae-
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Table 3. Identification and genotyping of pathogens in the analyzed animal organs Survey results Number of analyzed Kind of biomaterial samples
identification* M. haemolytica
Lungs Lymph nodes pleen Total
161 54 45 260
capsule group**
P. multocida
А
D
102/63.3 24/42.6 4/8.8 130/50.0
89/88.2 20/86.9 3/75.0 112/86.2
12/10.8 3/13.0 0 15/11.5
23/14.3 6/11.1 0 29/11.2
F 1/0.9 1/4.3 1/25.0 3/2.1
*Number of positive results/percentage of the number of analyzed samples. **Number of positive results/percentage of the number of samples with the identified P. multocida DNA.
molytica sequences. In addition, the affinity with analogous sequences of M. glucosida, M. varigena, and M. ruminalis comprised 83–95% (Fig. 4, see second half of region).
groups A, B, D, E, and F and the M. haemolytica bacteria has been developed on the basis of PCR assay with electrophoretic detection of results. It provides high specificity and sensitivity for testing bacterial cultures and samples of biological material from sick animals. P. multocida bacteria were identified more fre-
Therefore, the method for identification and genotyping of both the P. multocida bacteria of capsule
gi|AY225341.1|Pm serovar B2
20 60
gi|DQ233648.1|Pm isolate M13
4
Pm 681 Pm T-80
66
Pm Omsk-13 35
93
gi|AE004439.1|Pm str. Pm70 Pm T-14
5
Pm.MSK-13 Pm1231
50 44
79
2
gi|CP001409|Pm str. 3480
3
gi|CP003313.1|Pm str. HN06 69
gi|EU873317.1|Pasteurella sp. Kmt1 gene gi|AY225313.1|Pm serovar D gi|FJ986389.1|Pm Kmt1 gene gi|AY225344.1|Pm serowar A4
99
gi|DQ233649.1|Pm isolate M14 Pm Sib-13 gi|CP008918.1|Pm str. ATCC 43137 Pm OV-58 40
1
Pm AGM-2013 Pm SR-57 gi|CP003022.1|Pm str. 36950 gi|CP003328.1|Pm str. HB03
gi|DQ871028.1|Pm from sheep 99
gi|DQ871029.1|Pm from poultry
0.001
Fig. 2. Phylogenetic tree for nucleotide sequences of P. multocida kmt1 gene region. Here and in Figs. 3, 4: asterisks designate bacterial strains given in Table1. MOLECULAR GENETICS, MICROBIOLOGY AND VIROLOGY
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PM AGM-2013 PM SR-57
93
PM OV-58 gi|JQ082510.1|Pm XJ121
100
1
gi|DQ054529.1|P.m. gi|DQ417897.1|Pm-17 85
82 gi|DQ417898.1|Pm-Bali gi|DQ417890.1|Pm-454 gi|U52209.1|PMU52209
74
100 gi|U52207.1|PMU52207 PM1231
3
gi|U52202.1|P-1072 gi|U52212.1|PMU52212
100
5
PM MSK-13 gi|JQ082509.1| XJ149 100
gi|GQ914772.1| ATCC:15743 gi|JX473022.1| XJNKY-12-YF1
88 96
2
PM Omsk-13 PM T-14
gi|EU016232.1| P52 PM T-80
4
PM 681 100
gi|EF102481.1| PM1094 gi|AY057870.1| PMI047 100
gi|AY057869.1| PMI032
0.02
Fig. 3. Phylogenetic tree for nucleotide sequences of P. multocida ompH gene region.
M. haemolytica 16 M. haemolytica NK-42 M. haemolytica NV-53 99
gi| NC_020833.1| M. haemolytica USDA-ARS-USMARC-183 gi| CP006619.1| M. haemolytica USMARC_2286
93
gi| NC_021743.1| M. haemolytica D153 gi| CP005383.1| M. haemolytica M42548 gi| AY702510.1| M. glucosida sodA gene 74
gi| AY702510.1| M. glucosida sodA gene gi| CP006943.1| M. varigena USDA-ARS-USMARC-1296 gi| AY702513.1| M. ruminalis sodA gene sodA gene
0.005 Fig. 4. Phylogenetic tree for nucleotide sequences of M. haemolytica sodA gene region. MOLECULAR GENETICS, MICROBIOLOGY AND VIROLOGY
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quently than M. haemolytica bacteria in the organs of animals with respiratory pathology. Identification of the P. multocida bacteria of capsule group A in a greater number of samples can indicate that it played a more significant role in developing the epizootic situation on the studied farms than the P. multocida bacteria of capsule group D did. Bacteria of capsule group F were genotyped in one case. Circulation of capsule group A P. multocida strains related to two genetic types corresponding to the kmt1 and ompH genes was revealed in Siberia. The elaborated PCR protocol may be used as a simple, accessible, and highly reproducible analogue of serological typing in veterinary laboratories that makes it possible to identify and genotype the P. multocida and M. haemolytica strains at each stage of bacteriological surveys and, consequently, to reduce the time taken to reach a diagnosis, thus making it possible to optimize epizootic measures, particularly the development of animal vaccination and immunization programs. ACKNOWLEDGMENTS Funding. The study had no sponsor funding. Conflicts of interest. We declare that there is no conflict of interest. REFERENCES 1. Frank, G.H., Bacteria as etiologic agents in bovine respiratory disease, in Bovine Respiratory Disease, Loan, R.W., Ed., College Station, TX: Texas A & M University Press, 1984, pp. 342–362. 2. Griffin, D., Chengappa, M.M., Kuszak, J., and McVey, D.S., Bacterial pathogens of the bovine respiratory disease complex, Vet. Clin. North Am.: Food Anim. Pract., 2010, vol. 26, pp. 381–394. 3. Shegidevich, E.A., Role of pasteurella in respiratory pathology of sheep and bovines, Doctoral Sci. (Vet.) Dissertation, Moscow, 1993. 4. Glotov, A.G., Petrova, O.G., Glotova, T.I., Nefedchenko, A.V., Tatarchuk, A.T., Koteneva, S.V., et al., Spread of viral bovine respiratory diseases, Veterinariya, 2002, no. 3, pp. 17–21. 5. Glotov, A.G., Glotova, T.I., Nefedchenko, A.V., Koteneva, S.V., Budulov, N.R., and Kungurtseva, O.V., The etiological structure of calves’ mass respiratory diseases in farms producing milk, Sib. Vestn. S-kh. Nauki, 2008, no. 3, pp. 72–78.
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6. Griffin, D., Bovine pasteurellosis and other bacterial infections of the respiratory tract, Vet. Clin. North Am.: Food Anim. Pract., 2010, vol. 26, pp. 57–71. 7. Dabo, S.M., Taylor, J.D., and Confer, A.W., Pasteurella multocida and bovine respiratory disease, Anim. Health Res. Rev., 2007, vol. 8, pp. 129–150. 8. Hemorrhagic septicemia, in Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (Mammals, Birds and Bees), vol. 1, chap. 2.4.14: 2012, pp. 732–745. 9. Wilkie, I.W., Harper, M., Boyce, J.D., and Adler, B., Pasteurella multocida: diseases and pathogenesis, Curr. Top. Microbiol. Immunol., 2012, vol. 361, pp. 1–22. 10. Katsuda, K., Kamiyama, M., Kohmoto, M., Kawashima, K., Tsunemitsu, H., and Eguchi, M., Serotyping of Mannheimia haemolytica isolates from bovine pneumonia: 1987–2006, Vet. J., 2012, vol. 178, pp. 146–148. 11. Terent’yeva, T.E., Glotova, T.I., Glotov, A.G., and Donchenko, N.A., Comparative efficiency of different methods for diagnosticating bovine diseases caused by Pasteurella multocida, Sib. Vestn. S-kh. Nauki, 2014, no. 3, pp. 90–95. 12. Townsend, K.M., Boyce, J.D., Chung, J.Y., Frost, A.J., and Adler, B., Genetic organization of Pasteurella multocida cap loci and development of a multiplex capsular PCR typing system, J. Clin. Microbiol., 2001, vol. 39, pp. 924–929. 13. Guenther, S., Schierack, P., Grobbel, M., LübkeBecker, A., Wieler, L.H., and Ewers, C., Real-time PCR assay for the detection of species of the genus Mannheimia, J. Microbiol. Methods, 2008, vol. 75, pp. 75–80. 14. Luo, Y., Zeng, Q., Glisson, J.R., Jackwood, M.W., Cheng, I.H., and Wang, C., Sequence analysis of Pasteurella multocida major outer membrane protein (OmpH) and application of synthetic peptides in vaccination of chickens against homologous strain challenge, Vaccine, 1999, vol. 17, pp. 821–831. 15. Singh, R., Tewari, K., Packiriswamy, N., Marla, S., Rao Ingh, V.D.P., and Tewari, R.K., Molecular characterization and computational analysis of the major outer membrane protein (ompH) gene of Pasteurella multocida P52, Vet. Arh., 2011, vol. 81, pp. 211–222. 16. Antony, P.X., Nair, G.K., Jayaprakasan, V., Mini, M., and Aravindakshan, T., Nucleic acid based differentiation of Pasteurella multocida serotypes, Internet J. Vet. Med., 2007, vol. 2, pp. 85–89. 17. Glotov, A.G., Terent’yeva, T.E., Nefedchenko, A.V., Glotova, T.I., Shikov, A.N., and Agafonov, A.P., Heterogeneity of Pasteurella isolated from bovines in dairy units, Veterinariya, 2014, no. 12, pp. 23–26.
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