American Journal of Microbiological Research
ISSN (Print): 2328-4129 ISSN (Online): 2328-4137 Website: Editor-in-chief: Apply for this position
Open Access
Journal Browser
American Journal of Microbiological Research. 2017, 5(3), 59-65
DOI: 10.12691/ajmr-5-3-2
Open AccessArticle

Molecular characterization and Phylogenetic Analysis of Clostridium botulinum Mosaic Type D/C Isolated from Sudan

Dalia. A. Mohamed1, , Mohamed A. abdalla1, Abdallah E. Ahmed2, Mohamed M. Hassan3, Abbas M. Ahmed1 and Mona O. Elhaj1

1Animal Resources Research Corporation, Central Veterinary Research Laboratory, Khartoum, Sudan

2National University Research Institute, Sudan

3Faculty of Medical Laboratory Science, University of Medical Sciences and Technology, Sudan

Pub. Date: June 21, 2017

Cite this paper:
Dalia. A. Mohamed, Mohamed A. abdalla, Abdallah E. Ahmed, Mohamed M. Hassan, Abbas M. Ahmed and Mona O. Elhaj. Molecular characterization and Phylogenetic Analysis of Clostridium botulinum Mosaic Type D/C Isolated from Sudan. American Journal of Microbiological Research. 2017; 5(3):59-65. doi: 10.12691/ajmr-5-3-2


Clostridium botulinum types C and D are related to animal botulism. The disease has been reported in sheep in western Sudan causing economic losses. However, the BoNTs that cause sheep botulism in Sudan have not yet subjected to genetic characterization. The aim of this study is to perform genetic analysis for sheep botulism–related isolates from recent outbreak between January to May 2013 at western Sudan in order to improve the efficiency of control strategies and vaccine development. In this study isolation of Clostridium botulinum from sheep samples was obtained by culture methods and mouse bioassay. Positive samples were confirmed by PCR and DNA sequencing. PCR was used to amplify the BoNTs gene using three sets of primers to differentiate the gene of the mosaic type from the conserved genes of type C and D. The results of polymerase chain reaction with these primers indicated that sheep botulism–related isolates possess the gene for the mosaic form of the neurotoxin. PCR products were sequenced and subjected to genetic analysis. The results provided evidence for close relationships and genetic variation of the isolates and reference strains published on the GenBank. Multiple sequence alignments showed numerous substitutions occurred in heavy chain in the most homologous regions of BoNT/CD and in the light chain of toxin type C and D, respectively. Following sequencing, isolates were compared phylogenetically with reference strains. The close genetic relationship to the strain 193_09 and strain: OFD16 suggests that neurotoxins produced from sheep botulism is BoNT type DC. The present study provides information on genetic classification of BoNTs related to sheep botulism isolates in Sudan.

Clostridium botulinum sheep botulism PCR DNA sequencing Sudan

Creative CommonsThis work is licensed under a Creative Commons Attribution 4.0 International License. To view a copy of this license, visit


[1]  Busharah, I. A., and M. T. Musa (2012). Botulism in Livestock in North Darfur State. Resilience of agricultural systems against crises.
[2]  Critchley, E. M. R. (1991). A comparison of human and animal botulism: a review. Journal of the Royal Society of Medicine, 84: 295-298.
[3]  Espelund, M. and D. Klaveness, (2014). Botulism outbreaks in natural environments–an update. Frontiers in Microbiology, 5: 287.
[4]  Fach, P., D. Hauser, J. P Guillou, and M.R. Popoff, (1993). Polymerase chain reaction for the rapid identification of Clostridium botulinum type A strains and detection in food samples. Journal of Applied Microbiology, 75(3): 234-239.
[5]  Fach, P., S. Perelle, F. Dilasser, J. Grout, C. Dargaignaratz, L. Botella, and V. Broussolle (2002). Detection by PCR-enzyme-linked immunosorbent assay of Clostridium botulinum in fish and environmental samples from a coastal area in northern France. Applied and environmental microbiology, 68(12): 5870-5876.
[6]  Grenda T., E. Kukier and K. Kwiatek (2014). Methods and difficulties in detection of Clostridium botulinum and its toxins, Review. Polish Journal of Veterinary Sciences, 17(1):195-205.
[7]  Hansbauer E, M. Skiba, T. Endermann, J. Weisemann, D. Stern, M. Dorner, F. Finkenwirth, J. Wolf, W. Luginbühl, U. Messelhäußer, L. Bellanger, C. Woudstra, A. Rummel, P. Fach, and B. Dorner., (2016). “Detection, differentiation, and identification of botulinum neurotoxin serotypes C, CD, D, and DC by highly specific immunoassays and mass spectrometry. Analyst, 141: 5281-97.
[8]  Hariharan H, and WR. Mitchell (1976). Observations on bacteriophages of Clostridium botulinum type C isolates from different sources and the role of certain phages in toxigenicity. Applied and Environmental Microbiology, 32: 145-158.
[9]  Hoc, A. (2007). Advisory Committee on the Microbiological Safety of Food.
[10]  Ibrahim, A. A. and M. T. A. Shigidi (2014). An outbreak of botulism among sheep and goats in northern localities of north Kordofan state, Sudan. Journal of Veterinary Medicine and Animal Production, 5: (1).
[11]  Lindström M and H. Korkeala (2006). Laboratory Diagnostics Of Botulism. Clinical Microbiology Reviews, 19 (2): 298-314.
[12]  Masuyer G, Davies JR, Moore K, Chaddock JA, Ravi Acharya K. (2015). Structural analysis of Clostridium botulinum neurotoxin type D as a platform for the development of targeted secretion inhibitors. Scientific Reports., 5:13397.
[13]  Moriishi, K., M. Koura, N. Abe, N. Fujii, Y. Fujinaga, K. Inoue, and K. Ogumad (1996). Mosaic structures of neurotoxins produced from Clostridium botulinum types C and D organisms. Biochimica et Biophysica Acta (BBA)-Gene Structure and Expression, 1307(2): 123-126.
[14]  Moura, H., R. R. Terilli, A.R. Woolfitt, M. Gallegos-Candela, L.G. McWilliams, M.I. Solano, J.L. Pirkle, and J.R. Barr, (2011), Studies on botulinum neurotoxins type /C1 and mosaic/DC using Endopep-MS and proteomics. FEMS Immunology & Medical Microbiology, 61: 288-300.
[15]  Nakamura, K., Kohda, T., Shibata, Y., Tsukamoto, K., Arimitsu, H., Hayashi, M. and Kozaki, S. (2012). Unique biological activity of botulinum D/C mosaic neurotoxin in murine species. Infection and immunity, 80:2886-2893.
[16]  Prevot V., F. Tweepenninckx, E. Nerom, A. Linden, J. Content and A. Kimpe (2007). Optimization of polymerase chain reaction for detection of Clostridium botulinum type C and D in bovine samples. Zoonoses Public Health 54: 320-327.
[17]  Saeed, E. M. A. (2005). Studies on isolation and identification of Clostridium botulinum investigating field samples specially from equine grass sickness cases.
[18]  Sagermann, M., W.A. Baase, and B.W. Matthews (2006). Sequential reorganization of β-sheet topology by insertion of a single strand. Protein Science: A Publication of the Protein Society, 15(5): 1085-1092.
[19]  Sakaguchi Y, T. Hayashi and K. Kurokawa (2005). The genome sequence of Clostridium botulinum type C neurotoxin-converting phage and the molecular mechanisms of unstable lysogeny. Proceedings of the National Academy of Sciences of the United States of America, 102(48):17472-17477.
[20]  Skarin H., T. Håfström, J. Westerberg and B. Segerman (2011). Clostridium botulinum group III: a group with dual identity shaped by plasmids, phages and mobile elements. BMC Genomics, 12: 185.
[21]  Takeda, M., K. Tsukamoto, T. Kohda, M. Matsui, M. Mukamoto, and S. Kozaki, (2005) Characterization of the Neurotoxin Produced by Isolates Associated with Avian Botulism. Avian Diseases, 49: 376-381.
[22]  Vidal, D., Anza, I., Taggart, M. A., Pérez-Ramírez, E., Crespo, E., Hofle, U. and Mateo, R. (2013). Environmental factors influencing the prevalence of a Clostridium botulinum type C/D mosaic strain in nonpermanent Mediterranean wetlands. Applied and environmental microbiology, 79: 4264-4271.
[23]  Wangroongsarb P., T. Kohda, C. Jittaprasartsin, K. Suthivarakom, T. Kamthalang , K. Umeda, P. Sawanpanyalert, S. Kozaki and K. Ikuta (2014). Molecular Characterization of Clostridium botulinum Isolates from Foodborne Outbreaks in Thailand, 2010. PLoS ONE., 9(1): e77792.
[24]  Webb RP, TJ. Smith, PM. Wright, VA. Montgomery, MM. Meagher, and LA. Smith (2007) Protection with recombinant Clostridium botulinum C1 and D binding domain subunit (Hc) vaccines against C and D neurotoxins. Vaccine, 25: 4273-4282.
[25]  Wictome, M., and C.C. Shone, (1998). Botulinum neurotoxins: mode of action and detection. Journal of Applied Microbiology, 84: 87S-97S.
[26]  Woudstra, C., Skarin, H., Anniballi, F., Fenicia, L., Bano, L., Drigo, I., Koene, M., Bäyon-Auboyer, M.H., Buffereau, J.P., De Medici, D. and Fach, P. (2012). Neurotoxin gene profiling of Clostridium botulinum types C and D gathered from different countries within Europe. Applied and environmental microbiology, 78(9): 3120-3127.