- Open Access
Genome sequence of Haemophilus parasuis strain 29755
Standards in Genomic Sciences volume 5, pages 61–68 (2011)
Haemophilus parasuis is a member of the family Pasteurellaceae and is the etiologic agent of Glässer’s disease in pigs, a systemic syndrome associated with only a subset of isolates. The genetic basis for virulence and systemic spread of particular H. parasuis isolates is currently unknown. Strain 29755 is an invasive isolate that has long been used in the study of Glässer’s disease. Accordingly, the genome sequence of strain 29755 is of considerable importance to investigators endeavoring to understand the molecular pathogenesis of H. parasuis. Here we describe the features of the 2,224,137 bp draft genome sequence of strain 29755 generated from 454-FLX pyrosequencing. These data comprise the first publicly available genome sequence for this bacterium.
H. parasuis is an obligate pathogen of swine . The bacterium is often carried in the nasal passages , but not the lungs , of healthy pigs. Through unknown mechanisms some strains can spread systemically and may be isolated from the meninges, lungs, serosa, joints, and blood. H. parasuis strain 29755 (IA84-29755), though not the type strain, has been used extensively in a variety of investigations [4–8] and is the most fully characterized strain of the species. Originally cultured at Iowa State University from a pig exhibiting Glässer’s disease, 29755 is a serovar 5 isolate , a class recognized as highly virulent and frequently isolated from respiratory and systemic sites [9,10]. Of the 15 recognized serovars, serovar 5 strains are isolated more frequently worldwide than any other . Strain 29755 has been used as a component of at least one commercially available H. parasuis vaccine (Suvaxyn M. hyo – parasuis, Fort Dodge Animal Health).
Classification and features
The genus Haemophilus belongs to the Gammaproteobacteria and is classified in the family Pasteurellaceae  (Table 1). A phylogenetic tree based on 16S ribosomal RNA sequences is depicted in Figure 1 for H. parasuis and related organisms.
H. parasuis is a small, non-motile, rod-shaped bacterium  (Figure 2). The presence of a capsule is variable and may affect colony and cellular morphology . Growth of the bacterium in vitro is dependent on the coenzyme nicotinamide adenine dinucleotide (NAD, or V factor)  but, in contrast to some other members of the genus, does not require porphyrins like hemin (X factor) . Plating on Casman Agar Base (BBL) supplemented with 1% (w/v) NAD (Sigma) and 5% GIBCO filtered horse serum (Invitrogen) or on chocolate agar produces small, translucent colonies that appear within 24 hours and reach full size in approximately two days. Colonies are nonhemolytic when grown on blood agar .
H. parasuis grows under normal atmosphere at 37°C, although added humidity and 5% CO2 may improve growth.
Genome sequencing and annotation
Genome project history
H. parasuis strain 29755 was selected for sequencing because it has long been used in the study of Glässer’s disease. Pyrosequencing (454 Life Sciences) was performed at the State University of New York, University at Buffalo Center of Excellence in Bioinformatics and Life Sciences. The draft genome sequence is deposited in GenBank (NZ_ABKM00000000). Summary project information is shown in Table 2 according to the Minimum Information about a Genomic Sequence (MIGS) recommendations  and the genome content is summarized in Table 3.
Growth conditions and DNA isolation
H. parasuis 29755 was grown from a frozen seed stock for two days under 5% CO2 at 37°C on Casman Agar Base (BBL) supplemented with 1% (w/v) NAD (Sigma) and 5% GIBCO filtered horse serum (Invitrogen). Following growth, a single colony was used to inoculate 5 ml of brain-heart infusion medium supplemented with 10 µg/ml NAD and 10 µg/ml hemin (sBHI) and the culture was incubated overnight at 37°C and 185 rpm. The next day, 2 ml of the culture were added to 100 ml of sBHI and the bacterium was again allowed to grow overnight to stationary phase at 37°C and 185 rpm. Bacterial cells were pelleted by centrifugation at 4000 × g for 10 minutes. The pellet was resuspended and used as the source of genomic DNA purified with the QIAGEN Blood & Cell Culture DNA Kit, as recommended by the manufacturer. The final preparation contained 1.12 µg/ul genomic DNA as determined by UV absorption spectrometry.
Genome sequencing and assembly
Library preparation yielded 9.65 × 108 molecules/µl of DNA with a mean size of approximately 600 nucleotides, as determined with a RNA6000 Pico chip on an Agilent 2100 Bioanalyzer. Emulsion PCR was performed at a concentration of 2 molecules per bead. Following sequencing, contigs were assembled using the 454 Newbler assembler.
Genes were identified manually using GeneMark and automatically using Glimmer as part of the NCBI draft genome submission pipeline. Translated protein sequences were analyzed using PSORTb v.2.0  to predict final location within the cell and assigned to COG functional categories (Table 4).
The draft genome is 2,224,137 bp and is likely comprised of one circular chromosome with a G+C content of approximately 39% (Figure 3). For display, contigs were assembled end-to-end with twenty “N” bases between contigs. Orientation and order of contigs will change when the genome sequence is closed.
Rapp-Gabrielson VJ, Oliveira SR, Pijoan C. Haemophilus parasuis. In: Straw BE, Zimmerman JJ, D’Allaire S, Taylor DJ (eds), Diseases of Swine, Ninth Edition, Wiley-Blackwell, Ames, Iowa, 2006, p. 681–690.
Harris DL, Ross RF, Switzer WP. Incidence of certain microorganisms in nasal cavities of swine in Iowa. Am J Vet Res 1969; 30:1621–1624. PubMed
Little TW. Haemophilus infection in pigs. Vet Rec 1970; 87:399–402. PubMed doi:10.1136/vr.87.14.399
Blanco I, Galina-Pantoja L, Oliveira S, Pijoan C, Sanchez C, Canals A. Comparison between Haemophilus parasuis infection in colostrums-deprived and sow-reared piglets. Vet Microbiol 2004; 103:21–27. PubMed doi:10.1016/j.vetmic.2004.06.011
Sgales J, Domingo M, Solano GI, Pijoan C. Immunohistochemical detection of Haemophilus parasuis serovar 5 in formalin-fixed, paraffin-embedded tissues of experimentally infected swine. J Vet Diagn Invest 1997; 9:237–243. PubMed doi:10.1177/104063879700900303
Solano GI, Segalés J, Collins JE, Molitor TW, Pijoan C. Porcine reproductive and respiratory syndrome virus (PRRSv) interaction with Haemophilus parasuis. Vet Microbiol 1997; 55:247–257. PubMed doi:10.1016/S0378-1135(96)01325-9
Oliveira S, Galina L, Blanco I, Canals A, Pijoan C. Naturally-farrowed, artificially-reared pigs as an alternative model for experimental infection by Haemophilus parasuis. Can J Vet Res 2003; 67:146–150. PubMed
Solano-Aguilar GI, Pijoan C, Rapp-Gabrielson V, Collins J, Carvalho LF, Winkelman N. Protective role of maternal antibodies against Haemophilus parasuis infection. Am J Vet Res 1999; 60:81–87. PubMed
Rapp-Gabrielson VJ, Gabrielson DA. Prevalence of Haemophilus parasuis serovars among isolates from swine. Am J Vet Res 1992; 53:659–664. PubMed
Blackall PJ, Rapp-Gabrielson VJ, Hampson DJ. Serological characterisation of Haemophilus parasuis isolates from Australian pigs. Aust Vet J 1996; 73:93–95. PubMed doi:10.1111/j.1751-0813.1996.tb09984.x
Oliveira S, Pijoan C. Haemophilus parasuis: new trends on diagnosis, epidemiology and control. Vet Microbiol 2004; 99:1–12. PubMed doi:10.1016/j.vetmic.2003.12.001
Kilian M. Genus III. Haemophilus. In: Garrity GM, Brenner DJ, Krieg NR, Staley JT (eds), Bergey’s Manual of Systematic Bacteriology, Second Edition, Volume Two, The Proteobacteria, Part B, Springer, New York, 2005, p. 883–904.
Woese CR, Kandler O, Wheelis ML. Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proc Natl Acad Sci USA 1990; 87:4576–4579. PubMed doi:10.1073/pnas.87.12.4576
Garrity GM, Bell JA, Lilburn T. Phylum XIV. Proteobacteria phyl. nov. In: Garrity GM, Brenner DJ, Krieg NR, Staley JT (eds), Bergey’s Manual of Systematic Bacteriology, Second Edition, Volume 2, Part B, Springer, New York, 2005, p. 1.
List Editor. Validation of publication of new names and new combinations previously effectively published outside the IJSEM. List no. 106. Int J Syst Evol Microbiol 2005; 55:2235–2238. doi:10.1099/ijs.0.64108-0
Garrity GM, Bell JA, Lilburn T. Class III. Gammaproteobacteria class. nov. In: Garrity GM, Brenner DJ, Krieg NR, Staley JT (eds), Bergey’s Manual of Systematic Bacteriology, Second Edition, Volume 2, Part B, Springer, New York, 2005, p. 1.
Garrity GM, Bell JA, Lilburn T. Order XIV. Pasteurellales ord. nov. In: Garrity GM, Brenner DJ, Krieg NR, Staley JT (eds), Bergey’s Manual of Systematic Bacteriology, Second Edition, Volume 2, Part B, Springer, New York, 2005, p. 850.
List Editor. Validation List no. 7. Validation of the publication of new names and new combinations previously effectively published outside the IJSB. Int J Syst Bacteriol 1981; 31:382–383. doi:10.1099/00207713-31-3-382
Pohl SPD. Dissertation, Phillips-Universität Marburg, 1979.
Skerman VBD, McGowan V, Sneath PHA. Approved Lists of Bacterial Names. Int J Syst Bacteriol 1980; 30:225–420. doi:10.1099/00207713-30-1-225
Winslow CEA, Broadhurst J, Buchanan RE, Krumwiede C, Rogers LA, Smith GH. The Families and Genera of the Bacteria: Preliminary Report of the Committee of the Society of American Bacteriologists on Characterization and Classification of Bacterial Types. J Bacteriol 1917; 2:505–566. PubMed
Zinnemann KS, Biberstein EL. Genus Haemophilus Winslow, Broadhurst, Buchanan, Krumwiede, Rogers and Smith 1917, 561. In: Buchanan RE, Gibbons NE (eds), Bergey’s Manual of Determinative Bacteriology, Eighth Edition, The Williams and Wilkins Co., Baltimore, 1974, p. 364–370.
Biberstein EL, White DC. A proposal for the establishment of two new Haemophilus species. J Med Microbiol 1969; 2:75–78. PubMed doi:10.1099/00222615-2-1-75
Fink DK, St. Geme JW, III. The Genus Haemophilus. In: Dworkin M, Falkow S, Rosenberg E, Schleifer K, Stackebrandt E. (eds), The Prokaryotes, A Handbook on the Biology of Bacteria: Proteobacteria: Gamma Subclass, Third Edition, Volume 6, Springer, New York, 2006, p. 1034–1061.
Kroppenstedt RM, Mannheim W. Lipoquinones in members of the family Pasteurellaceae. Int J Syst Evol Microbiol 1989; 39:304–308.
Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT, et al. Gene Ontology: tool for the unification of biology. Nat Genet 2000; 25:25–29. PubMed doi:10.1038/75556
Ribosomal Database Project. http://rdp.cme.msu.edu/treebuilderpub/treeHelp.jsp
Bruno WJ, Socci ND, Halpern AL. Weighted neighbor joining: a likelihood-based approach to distance-based phylogeny reconstruction. Mol Biol Evol 2000; 17:189–197. PubMed
Som A. Theoretical foundation to estimate the relative efficiencies of the Jukes-Cantor+gamma model and the Jukes-Cantor model in obtaining the correct phylogenetic tree. Gene 2006; 385:103–110. PubMed doi:10.1016/j.gene.2006.03.027
Morozumi T, Nicolet J. Morphological variations of Haemophilus parasuis strains. J Clin Microbiol 1986; 23:138–142. PubMed
Biberstein EL, Gunnarsson A, Hurvell B. Cultural and biochemical criteria for the identification of Haemophilus spp from swine. Am J Vet Res 1977; 38:7–11. PubMed
Biberstein EL, Mini PD, Gills MG. Action of Haemophilus cultures on δ-aminolevulinic acid. J Bacteriol 1963; 86:814–819. PubMed
Lukashin AV, Borodovsky M. GeneMark.hmm: new solutions for gene finding. Nucleic Acids Res 1998; 26:1107–1115. PubMed doi:10.1093/nar/26.4.1107
Field D, Garrity G, Gray T, Morrison N, Selengut J, Sterk P, Tatusova T, Thomson N, Allen MJ, Angiuoli SV, et al. The minimum information about a genome sequence (MIGS) specification. Nat Biotechnol 2008; 26:541–547. PubMed doi:10.1038/nbt1360
Gardy JL, Laird MR, Chen F, Rey S, Walsh CJ, Ester M, Brinkman FS. PSORTb v.2.0: expanded prediction of bacterial protein subcellular localization and insights gained from comparative proteome analysis. Bioinformatics 2005; 21:617–623. PubMed doi:10.1093/bioinformatics/bti057
Grant JR, Stothard P. The CGView Server: a comparative genomics tool for circular genomes. Nucleic Acids Res 2008; 36:W181–W184. PubMed doi:10.1093/nar/gkn179
CGView Server. http://stothard.afns.ualberta.ca/cgview_server/index.html
The authors wish to thank David Alt, USDA/ARS/National Animal Disease Center for technical advice and the State University of New York, University at Buffalo Center of Excellence in Bioinformatics and Life Sciences for performing pyrosequencing. This work was supported, in part, by grants from the NIH/NCRR (D.W. Dyer, Grant #P2PRR016478), National Pork Board (G.J. Phillips and D.W. Dyer) and Iowa Healthy Livestock Initiative (G.J. Phillips and K.B. Register).
Rights and permissions
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. 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.
About this article
Cite this article
Mullins, M.A., Register, K.B., Bayles, D.O. et al. Genome sequence of Haemophilus parasuis strain 29755. Stand in Genomic Sci 5, 61–68 (2011). https://doi.org/10.4056/sigs.2245029
- Haemophilus parasuis
- Glässer’s disease