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Genome sequence of Haemophilus parasuis strain 29755

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Abstract

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.

Introduction

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 [48] 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.

Figure 1.
figure1

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.

Table 1. MIGS classification and general features of H. parasuis strain 29755.

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].

Figure 2.
figure2

Scanning electron micrograph of H. parasuis 29755

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

Table 2. Genome sequencing project information
Table 3. Genome statistics

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

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.
figure3

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].

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Acknowledgements

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).

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Correspondence to Karen B. Register.

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Keywords

  • Haemophilus parasuis
  • Glässer’s disease
  • swine