Open Access

Genome sequence of Haemophilus parasuis strain 29755

  • Michael A. Mullins
  • , Karen B. Register
  • , Darrell O. Bayles
  • , David W. Dyer
  • , Joanna S. Kuehn
  • and Gregory J. Phillips

DOI: 10.4056/sigs.2245029

Received: 23 September 2011

Published: 15 October 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.


Haemophilus parasuisGlässer’s diseaseswine


H. parasuis is an obligate pathogen of swine [1]. The bacterium is often carried in the nasal passages [2], but not the lungs [3], 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 [9], 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 [11]. 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 [12] (Table 1). A phylogenetic tree based on 16S ribosomal RNA sequences is depicted in Figure 1 for H. parasuis and related organisms.

Table 1

MIGS classification and general features of H. parasuis strain 29755.




  Evidence code

   Current classification

   Domain Bacteria

  TAS [13]

   Phylum Proteobacteria

  TAS [14]

   Class Gammaproteobacteria

  TAS [15,16]

   Order Pasteurellales

  TAS [15,17]

   Family Pasteurellaceae

  TAS [18,19]

   Genus Haemophilus

  TAS [20-22]

   Species Haemophilus parasuis

  TAS [20,23]

   Strain 29755

   Serotype 5

   Gram stain


  TAS [1]

   Cell shape

   rods (pleomorphic)

  TAS [1]



  TAS [1]



  TAS [1]

   Temperature range

   mesophile (20°C-37°C)

  TAS [12]

   Optimum temperature


  TAS [12]

   Carbon source


  TAS [24]

   Energy source


  TAS [24]

   Terminal electron receptor


  TAS [25]



   Host, swine upper respiratory tract

  TAS [1]




  TAS [12]


   Oxygen requirement


  TAS [12]


   Biotic relationship

   obligate pathogen of swine

  TAS [1]



   mild to severe

  TAS [1]


   Geographic location




   Sample collection time





   not reported



   not reported



   not reported



   not reported

Evidence codes - NAS: Non-traceable Author Statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from of the Gene Ontology project [26]

Figure 1

Phylogenetic tree based on 16S rRNA of H. parasuis 29755 and type strains of some closely related species and other genera within the Pasteurellaceae. Also included is the only additional H. parasuis strain for which a genome sequence has been reported, SH0165. The tree was generated with the tree-builder available from the Ribosomal Database Project[27] using the Weighbor (weighted neighbor-joining) algorithm [28] with Jukes-Cantor distance correction [29]. Numbers to the left of branches indicate the percentage of trees in which each branch was represented in 100 replicates. An E. coli type strain was used as an outgroup.

H. parasuis is a small, non-motile, rod-shaped bacterium [1] (Figure 2). The presence of a capsule is variable and may affect colony and cellular morphology [30]. Growth of the bacterium in vitro is dependent on the coenzyme nicotinamide adenine dinucleotide (NAD, or V factor) [31] but, in contrast to some other members of the genus, does not require porphyrins like hemin (X factor) [32]. 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 [1]. H. parasuis grows under normal atmosphere at 37°C, although added humidity and 5% CO2 may improve growth.

Figure 2

Scanning electron micrograph of H. parasuis 29755

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 [34] and the genome content is summarized in Table 3.

Table 2

Genome sequencing project information





   Libraries used

   one 454 pyrosequence standard library


   Sequencing platforms

   454 (FLX)





   Finishing quality



   Fold coverage



   Gene calling method

   Glimmer, GeneMark [33]

   Genome Database release

   February 14, 2008

   Genbank ID


   Genbank Date of Release

   February 14, 2008



   Project relevance

   food animal pathogenesis

Table 3

Genome statistics



   % of totala

Size (bp)



G+C content (bp)



Coding region (bp)



Total genes



RNA genes



Protein-coding genes






Genes in paralog clusters



Genes assigned to COGs



PSORT cytoplasmic



PSORT extracellular



PSORT outer membrane



PSORT periplasmic



PSORT unknown



a Based either on the size of the genome in base pairs or the total number of protein coding genes in the annotated genome

b Based on preliminary analysis the of draft genome

c nd = not determined

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.

Genome annotation

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 [35] to predict final location within the cell and assigned to COG functional categories (Table 4).

Table 4

Number of genes associated with the general COG functional categories












   RNA processing and modification








   Replication, recombination and repair




   Chromatin structure and dynamics




   Cell cycle control, mitosis and meiosis




   Nuclear structure




   Defense mechanisms




   Signal transduction mechanisms




   Cell wall/membrane biogenesis




   Cell motility








   Extracellular structures




   Intracellular trafficking and secretion




   Posttranslational modification, protein turnover, 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 metabolites biosynthesis, transport and catabolism




   General function prediction only




   Function unknown




   Not in COGs

a Based on the total number of protein coding genes in the annotated genome

Genome properties

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.

Figure 3

Graphical circular map of the H. parasuis 29755 draft pseudogenome. From the outside to the center: open reading frames (ORFs) on the forward strand (one ring for each reading frame), start and stop codons for forward and reverse strands, ORFs on the reverse strand, GC content, and GC skew. The map was generated using CGView Server [36,37].



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).

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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