Genomic analysis of four strains of Corynebacterium pseudotuberculosis bv. Equi isolated from horses showing distinct signs of infection
© The Author(s). 2017
Received: 24 February 2016
Accepted: 25 January 2017
Published: 31 January 2017
The genomes of four strains (MB11, MB14, MB30, and MB66) of the species Corynebacterium pseudotuberculosis biovar equi were sequenced on the Ion Torrent PGM platform, completely assembled, and their gene content and structure were analyzed. The strains were isolated from horses with distinct signs of infection, including ulcerative lymphangitis, external abscesses on the chest, or internal abscesses on the liver, kidneys, and lungs. The average size of the genomes was 2.3 Mbp, with 2169 (Strain MB11) to 2235 (Strain MB14) predicted coding sequences (CDSs). An optical map of the MB11 strain generated using the KpnI restriction enzyme showed that the approach used to assemble the genome was satisfactory, producing good alignment between the sequence observed in vitro and that obtained in silico. In the resulting Neighbor-Joining dendrogram, the C. pseudotuberculosis strains sequenced in this study were clustered into a single clade supported by a high bootstrap value. The structural analysis showed that the genomes of the MB11 and MB14 strains were very similar, while the MB30 and MB66 strains had several inverted regions. The observed genomic characteristics were similar to those described for other strains of the same species, despite the number of inversions found. These genomes will serve as a basis for determining the relationship between the genotype of the pathogen and the type of infection that it causes.
KeywordsC. pseudotuberculosis Biovar equi Ulcerative lymphangitis Horse Genomic
As of February 2016, thirty-three genomes of the species Corynebacterium pseudotuberculosis had been deposited into the National Center for Biotechnology Information database. This species is an animal pathogen that infects goats and sheep, causing caseous lymphadenitis, as well as horses, which can show distinct signs and symptoms. C. pseudotuberculosis can be classified into two biovars based on its ability to reduce nitrate to nitrite . Non-reducing, i.e., nitrate-negative, strains are grouped into the ovis biovar and are responsible for CL. The reducing, i.e., nitrate-positive, strains are grouped into the equi biovar and mainly infect horses.
Recent increases in the number of infections in horses have led to C. pseudotuberculosis bv. equi being classified as a re-emerging pathogen. In Texas, USA, the number of cases increased 10-fold between 2005 and 2011, with a cumulative increase in annual incidence from 9.3 to 99.5 infections per 100,000 horses over the same period . Kilcoyne et al.  analyzed the number of cultures positive for C. pseudotuberculosis in samples isolated from infected horses in 23 states in the USA. The proportion of positive cultures was higher for the most recent years, 2011 and 2012 (54% of the total number of samples), than for the period spanning 2003 to 2010 (46% of the total number of samples). These current data show the growing numbers of infections caused by this bacterium and emphasize the need for new studies on the genotypic characteristics of the biovar.
C. pseudotuberculosis bv. equi infection is commonly known as “pigeon fever” because it leads to the formation of external abscesses on the chest of the animal, making it expand, similar to a pigeon breast. Despite its common name, the bacteria can also cause other types of infections with distinct signs and symptoms, such as the formation of internal abscesses or ulcerative lymphangitis, which is characterized by the infection of limbs and compromises the lymphatic system . It is currently believed that the major vectors of the disease are domestic flies of the species Haematobia irritans , Stomoxys calcitrans , and Musca domestica .
The pathogenesis of C. pseudotuberculosis is intrinsically linked to its genetic content. Several virulence factors have previously been described in the literature that strongly influence the ability of the bacteria to interact with the host, causing infection. Phospholipase D, the iron uptake system, and pili proteins are examples of these factors . Characterization of these and novel virulence factors depends on the sequencing of new genomes from the biovar, as the vast majority of the genomes in databases belong to the ovis biovar. Therefore, to generate data that allows for a more robust genotypic analysis of the equi biovar, four genomes from strains isolated from horses with distinct signs of infection by C. pseudotuberculosis were sequenced using the next-generation Ion Torrent PGM platform.
Classification and features
Classification and general features of the species strain designationT [cite MIGS reference]
Species: C. pseudotuberculosis
strain: MB11, MB14, MB30 and MB66
pH range; optimum
Glucose, fructose, maltose, mannose, and sucrose
Soil and animal pathogens
Up to 2 M NaCl
Intracellular facultative pathogen
MB11 - 38°21′23″
MB14 - 37°00′20″
MB30 - 39°39′32″
MB66 - 38°32′41″
MB11 - 121°59′15″
MB14 - 121°34′05″
MB30 - 121°37′52″
MB66 - 121°44′25″
MB11 - 180 ft
MB14 - 196 ft
MB30 - 351 ft
MB66 - 55 ft
Genome sequencing information
Genome project history
Ion Torrent PGM
842x (MB11); 867x (MB14); 309x (MB30); 658x (MB66).
MIRA4, Lasergene (DNASTAR), GapBlaster.
Gene calling method
Pannotator (FgenesB; Glimmer; tRNAscan; RNAmer)
ATN02_ (MB11); ATN03_ (MB14); ATN04_ (MB30); ATN05_ (MB66)
CP013260 (MB11); CP013261 (MB14); CP013262 (MB30); CP013263 (MB66).
GenBank Date of Release
Gp0131493 (MB11); Gp0131495 (MB14); Gp0131496 (MB30); Gp0131497 (MB66).
Source Material Identifier
Isolated directly from the infected animal
Growth conditions and genomic DNA preparation
After isolation, the bacteria were maintained in 25% glycerol at −80 °C, and the medium was refreshed routinely. To extract genomic DNA, the bacteria were first cultured in liquid brain heart infusion (BHI) medium at 37 °C with shaking. DNA was extracted during the log-phase of cell growth according to the protocol described by Pacheco et al.  for clinical isolates. The extracted DNA was subjected to electrophoresis on a 1% agarose gel to determine the quality of the material.
Genome sequencing and assembly
Number of genes associated with general COG functional categories
Translation, ribosomal structure, and biogenesis
RNA processing and modification
Replication, recombination, and repair
Chromatin structure and dynamics
Cell cycle control, cell division, and chromosome partitioning
Signal transduction mechanisms
Cell wall/membrane biogenesis
Intracellular trafficking and secretion
Posttranslational modification, protein turnover, and chaperones
Energy production and conversion
Carbohydrate transport and metabolism
Amino acid transport and metabolism
Nucleotide transport and metabolism
Coenzyme transport and metabolism
Lipid transport and metabolism
Inorganic ion transport and metabolism
Secondary metabolite biosynthesis, transport, and catabolism
General function prediction only
Not in COGs
% of Total
% of Total
% of Total
% of Total
Genome size (bp)
DNA coding (bp)
DNA G + C (bp)
Protein coding genes
Genes in internal clusters
Genes with function prediction
Genes assigned to COGs
Genes with Pfam domains
Genes with signal peptides
Genes with transmembrane helices
Because of the large number of infections reported for C. pseudotuberculosis biovar equi in recent years, sequencing and analyzing genomes for this biovar is an essential step towards new perspectives that will improve our understanding of pathogen-host interactions and facilitate the development of vaccines to eradicate the disease. The four genomes presented in this study showed structural differences, except for strains MB11 and MB14. The phylogenetic relationship is closer to other strains of the equi biovar, and other genomic characteristics, such as the GC content, number of CDSs, and tRNA and rRNA clusters, are similar to those described for other strains of the same species. Virulence factors that were previously described in the literature were identified in the analyzed genomes. In addition, in silico assembly of the MB11 genome was validated by an optical map of the KpnI restriction sites.
These initial data suggest that differences between types of infection should be analyzed using a reductionist approach, taking into account factors such as pathogenicity islands in each strain, the transmission method, and the entry point of the pathogen for each case, as well as expression levels and use of virulence factors specific to the bacteria, among other factors. Phylogenetic studies and the detection of small genetic changes such as SNPs and INDELs should then be performed because the bacteria have a very high gene density, and therefore, point mutations can strongly affect the biological response of the pathogen.
Brain heart infusion
Coding DNA sequence
Corynebacterium Mycobacterium, Nocardia, Rhodococcus
- Ion Torrent PGM:
Ion torrent personal genome machine
Single nucleotide polymorphism
The authors are thankful for the financial support granted by CNPq and CAPES. The authors also thank the Pró-Reitoria de Pesquisa e Pós-Graduação of Universidade Federal do Pará for the financial support for the publication of the article.
This study was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior.
RAB, RTJR, PHCGS, AAOV, LCG, DAG and ARC conducted the bioinformatics analyses, evaluated the results, and wrote the manuscript. SJS and JJE isolated the strains and designed the project together with VA and AS, in addition to helping to write the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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