Genetic Characterization of Corynebacterium pseudotuberculosis Isolates in Egypt

El-Sebay, Nadine A.; Abd Elmonem, Marwah M.; El-Sergany, Elham F.; M. Abbas, Ashraf; M. Osman, Roukaya; Abd El-Moaty, Dalia A. M.

doi:10.21608/javs.2021.154576

Genetic Characterization of Corynebacterium pseudotuberculosis Isolates in Egypt

Document Type : Original Article

Authors

¹ Genetic Engineering Research Department, Veterinary Serum and Vaccine Research Institute (VSVRI), Agriculture Research Center (ARC), Egypt.

² Bacterial diagnostic products department, Veterinary Serum and Vaccine Research Institute (VSVRI), Agriculture Research Center (ARC), Egypt.

³ Anaerobic Bacterial Vaccine Research Department, Veterinary Serum and Vaccine Research Institute (VSVRI), Agriculture Research Center (ARC), Egypt.

10.21608/javs.2021.154576

Abstract

Corynebacterium pseudotuberculosis is a small Gram-positive bacillus containing mycolic acid in the structure of the cell wall. The bacterium is responsible for Caseous Lymphadenitis (CLA) in small ruminants (sheep and goats). The bacteria are also responsible for Ulcerative Lymphangitis in equines. The disease causes great economic loss in the animal industry. This work aimed to check the ability of Quadruplex PCR (Q-PCR) for genotyping and identification of Egyptian isolates of C. pseudotuberculosis and sequence analysis of phospholipase D (PLD) gene of local isolates. Four of C. pseudotuberculosis local isolates previously biochemically identified were tested for narG gene (nitrate reductase gene). Both nitrate negative biovar (ovis) and nitrate positive biovar (equi) showed a positive result for 16S rRNA, rpoB and PLD genes of C. pseudotuberculosis species. The sequence analysis of our local isolates' PLD gene revealed minor changes in PLD proteins between ovine and equine strains compared with other published PLD sequences in GenBank. It was concluded that the Q-PCR method is able to differentiate between C. pseudotuberculosis equi and ovis biovars. Also, the sequence of PLD gene of local isolates representing the two biovars revealed some variation, which leads to an accurate diagnosis of C. pseudotuberculosis biovars and generates a mapping of immerged local isolates. Further, the PCR and sequence of these isolates provide rapid and accurate genotyping, especially with hyperimmune serum's unavailability.

Keywords

Main Subjects

Bacteriology

References

ABDELHAI, M.H.; HASSANIN, H.A.M. AND SUN X., 2016. Comparative Study of Rapid DNA Extraction Methods of Pathogenic Bacteria. American Journal of Bioscience and Bioengineering, 4(1): 1-8.

ALMEIDA, S.; SANDEEP, T.; MARIANO, D.; ROCHA, F.S.; JAMAL, S.B. AND COIMBRA, NAR, 2015. The genome anatomy of Corynebacterium pseudotuberculosis VD57 a highly virulent strain causing Caseous lymphadenitis. Stand Genomic Sci, 57:1-8.

ALMEIDA, S.; DORNELES, E.M.S.; DINIZ, C. AND ABREU, V., 2017(A). Quadruplex PCR assay for identification of Corynebacterium pseudotuberculosis differentiating biovar Ovis and Equi. BMC Veterinary Research, 13:290.

ALMEIDA, S.; SOUSA, C.; ABREU, V.; DINIZ, C.; DORNELES, E.M.S. AND LAGE, A.P., 2017(B). Exploration of nitrate Reductase metabolic pathway in Corynebacterium pseudotuberculosis. Int. J. Genomics 1–12.

AQUINO, M.C.; GOUVEIA, G. V. AND KREWER, CC, 2013. Distribution of PLD and Fag A, B, C and D genes in Corynebacterium pseudotuberculosis isolates from sheep and goats with Caseous lymphadenitis. Genetics and Molecular Biology, 36: 265-268.

BAIRD, G.J. AND FONTAINE, M.C., 2007. Corynebacterium pseudotuberculosis and its role in ovine Caseous lymphadenitis. J. Comp. Pathol., 137:179–210.

BIBERSTEIN, E.L.; KNIGHT, H.D. AND JANG, S., 1971. Two biotypes of Corynebacterium pseudotuberculosis. Vet. Rec., 89:691–692.

BROWN, C.C.; OLANDER, H.J. AND ALVES, S.F., 1987. Synergistic hemolysis inhibition titers associated with Caseous lymphadenitis in a slaughterhouse survey of goats and sheep in northeastern Brazil. Can. J. Vet. Res. Rev., 51:46–9.

CETINKAYA, B.; KARAHAN, M.; ATIL, E.; KALIN, R.; DE BAERE, T. AND VANEECHOUTTE M.,2002. Identification of Corynebacterium pseudotuberculosis isolates from sheep and goats by PCR. Vet. Microbiol., 88:75–83.

DERCKSEN, D.P.; BRINKHOF, J.M.; DEKKER-NOOREN, T.; MAANEN, K.; BODE, C.F.;BAIRD, G.; 2000. A comparison of four serological tests for the diagnosis of Caseous lymphadenitis in sheep and goats. Vet. Microbiol.,75:167–75.

GONÇALVES, J.L.; TOMAZI, T.; BARREIRO, J.R.; BRAGA, P.A.; FERREIRA, C.R.; AND JUNIOR, J.P., 2014. Identification of Corynebacterium spp. isolated from bovine intramammary infections by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Vet. Microbiol., 173:147–151.

GUIMARÃES, A.S.; FILIPE, B.; PAULETTI, R.B.; SEYFFERT, N.; RIBEIRO, D. ;LAGE, A.; HEINEMANN, M.B.; MIYOSHI, A. AND GOUVEIA, A., 2011. Caseous lymphadenitis: epidemiology, diagnosis, and control. IIOAB J., 2: 33–43.

HODGSON, A.L.; BIRD, P. AND NISBET, I.T., 1990. Cloning nucleotide sequence and expression in Escherichia coli of the phospholipase D gene from Corynebacterium pseudotuberculosis. J. Bacteriol., 172(3): 1256-1261.

KHAMIS, A.; RAOULT, D. AND LA SCOLA, B., 2004. rpoB gene sequencing for identification of Corynebacterium species. J Clin. Microbiol., 42:3925–31.

KHAMIS, A., RAOULT, D. AND LA SCOLA, B., 2005. Comparison between rpoB and 16S rRNA gene sequencing for molecular identification of 168 clinical isolates of Corynebacterium. J. Clin. Microbiol., 43:1934–1936.

LANGONI, H.; CAMARGO DA SILVA, C.P.; TRONCARELLI, M.Z.; TATA, A.; BELAZ, K.R.A. AND EBERLIN, M.N., 2017. Short communication: Identification of Corynebacterium Bovis by matrix-assisted laser desorption/ionization mass spectrometry. J Dairy Sci. [Internet]. [cited 2017 Apr 1]; Available from: http://linkinghub.elsevier. com/retrieve/pii/S0022030217302539

MENZIES, P.I. AND MUCKLE, C.A., 1989. The use of a microagglutination assay for the detection of antibodies to Corynebacterium pseudotuberculosis in naturally infected sheep and goat flocks. Can. J. Vet. Res. Rev., 53:313–8.

OLIVEIRA, A.; TEIXEIRA, P.; AZEVEDO, M.; JAMAL, S.B.; TIWARI, S. AND ALMEIDA, S., 2016. Corynebacterium pseudotuberculosis may be under anagenesis and biovar Equi forms biovar Ovis: a phylogenic inference from sequence and structural analysis. BMC Microbiology, 16:100.

PACHECO, L.G.C.; PENA, R.R.; CASTRO, T.L.P. AND DORELLA, F.A., 2007. Multiplex PCR assay for identification of Corynebacterium pseudotuberculosis from pure cultures and for rapid detection of this pathogen in clinical samples. Journal of Medical Microbiology, 56: 480–486.

RAOULT, D., FOURNIER, P.E. AND DRANCOURT, M., 2004. What does the future hold for clinical microbiology? Nat. Rev., Microbiol. 2:151–9.

ROUKAYA, M.O.,1982. Different methods of attenuation Corynebacteriupseudotuberculosis strains possible vaccine production. Thesis for Philosophy in Microbiology, Department of Microbiology Faculty of Veterinary Medicine, Cairo University.

SANKARASUBRAMANIAN, J.; VISHNU, US; GUNASEKARAN, P. AND RAJENDHRAN, J., 2016. A genome-wide SNP-based phylogenetic analysis distinguishes different biovars of Brucellasuis. Infect Genet Evol., 41:213–7.

SELIM, S.A.,2001. Oedematous skin disease of buffalo in Egypt. J. Vet. Med. B infect. Dis. Vet. Public Health., 48:241–58.

SHIGIDI, M.T., 1979. A comparison of five serological tests for the diagnosis of experimental Corynebacterium ovisinfection in sheep. Br. Vet. J., 135:172–7.

SOARES, S.C.; SILVA, A.; TROST, E.; BLOM, J.; RAMOS, R.; CARNEIRO, A.; ALI, A. AND SANTOS, A.R., 2013. The pangenome of the animal pathogen Corynebacterium pseudotuberculosis reveals differences in genome plasticity between the biovar ovis and equi strains. PLoS One., 8: e53818.

SONGER, J.G.; BECKENBACH, K.; MARSHALL, M.M.; OLSON, G.B. AND KELLEY, L.,1988. Biochemical and genetic characterization of Corynebacterium pseudotuberculosis. Am. J. Vet. Res., 49:223–6.

STEINMAN, A.; ELAD, D. AND SHPIGEL, N.Y.,1999. Ulcerative lymphangitis and coronet lesions in an Israeli dairy herd infected with Corynebacterium pseudotuberculosis. Vet. Rec., 145:604–6.

TEJEDOR JUNCO, M.T.; LUPIOLA, P. ; SCHULZ, U. AND GUTIERREZ, C.,2008. Isolation of nitrate reductase positive Corynebacterium pseudotuberculosis from dromedary camels. Trop Anim Health Prod., 40:165–7.

WILLIAMS, R.A. AND SADLER, S.A.,1971. Electrophoresis of glucose-6-phosphate dehydrogenase, cell wall composition and the taxonomy of heterofermentative lactobacilli. J. Gen. Microbiol., 65:351–8.

WILLIAMSON, L.H., 2001. Caseous lymphadenitis in small ruminants. Veterinary Clinics of North America. Food Animal Practice, 17: 359-371.

YERUHAM, I.; BRAVERMAN, Y.; SHPIGEL, N.Y.; CHIZOV-GINZBURG, A.; SARAN, A. AND WINKLER, M., 1996. Mastitis in dairy cattle caused by Corynebacterium pseudotuberculosis and the feasibility of transmission by houseflies. I. Vet. Q.,18:87–9.

YERUHAM, I.; ELAD, D.; VAN-HAM, M.; SHPIGEL, N.Y. AND PERL, S.,1997. Corynebacterium pseudotuberculosis infection in Israeli cattle: clinical and epidemiological studies. Vet. Rec. 140:423–7.

YERUHAM, I.; ELAD, D.; PERL, S. AND RAM, A., 2003. Necrotic-ulcerative dermatitis on the heels of heifers in a dairy herd infected with Corynebacterium pseudotuberculosis. Vet. Rec., 152:598–600.