GENOME ANALYSIS OF ACTINOBACILLUS SUIS AS A PLATFORM FOR DISEASE PREVENTION AND INTERVENTION

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: ANIMAL HEALTH
• Reporting Frequency: Annual
• Accession No.: 0216534
• Project No.: MOV-4-FF52
• Project Start Date: Oct 1, 2008
• Project End Date: Sep 30, 2009

Project Director
Calcutt, M. J.

Recipient Organization
UNIVERSITY OF MISSOURI
(N/A)
COLUMBIA,MO 65211

Performing Department
Veterinary Pathobiology

Non Technical Summary
Actinobacillus suis is a bacterial pathogen that causes septicemia in piglets and is an emerging problem for High Health Status swine. As a consequence, there is a significant negative impact on US agriculture. Little is known about how the organism causes disease, or why the bacterium can also be found in swine that are apparently healthy. Accordingly understanding how the bacterium is able to cause disease is important to developing strategies to prevent or treat infections. Determining the DNA sequence of most if not all of the genes of the bacterium Actinobacillus suis will provide the genetic blueprint of this important disease causing organism. New technologies for genome sequencing allow rapid and cost effective generation of this information. Analysis of the data generated will identify candidate targets for diagnostic applications, therapeutic intervention and an unprecedented perspective on how the bacterium causes disease.

Animal Health Component

100%

Research Effort Categories

Basic

80%

Applied

10%

Developmental

10%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3510	1040	50%
311	3510	1100	40%
311	3510	1170	10%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3510 - Swine, live animal;

Field Of Science
1170 - Epidemiology; 1100 - Bacteriology; 1040 - Molecular biology;

Keywords

genome sequencing

actinobacillus

swine pathogen

Goals / Objectives
Actinobacillus suis is a porcine pathogen that is a causative agent of fatal septicemia in piglets and an emerging pathogen of older High Health Status swine. Despite its increasing importance, very little is known about the pathogenesis of A. suis. This is further complicated by the reported isolation of the organism from healthy animals. With the advent of novel DNA sequencing technologies it is now possible to generate high quality draft genome sequences of many bacterial species at substantially reduced costs. Through draft genome sequencing and analysis, the results of this project are expected to have tremendous benefit to translational research applications, particularly in the areas of pathogenesis, surveillance, epidemiology, treatment and prevention, which can now be viewed from the level of the genome. The project will also generate fundamental new insights into the biology and evolution of this important emerging animal pathogen. Comparative analysis with related species will offer a unique perspective of the differential virulence of closely related bacterial species. The sequences generated will be publically accessible and will be a critical adjunct to research by other members of the scientific community.

Project Methods
To generate a draft genome sequence of A. suis within the budget of this application, massively parallel pyrosequencing using the '454' technology will be performed. As originally released, this method is able to generate approx. 20 million bases (Mb) of DNA sequence in a single overnight run. Recent advances have increased this capability to greater than 34 Mb of sequence, representing a 20-fold coverage for the estimated 2.3-Mb genome of A. suis. Because of the relatively short sequence reads (250 bp), the data will likely not produce a complete genome, but rather will provide greater than 99 percent of the genome sequence, in multiple sections. The sequence data will be analyzed to identify open reading frames (ORFs) and non-ORF genome features, annotate ORFs and intra- and intergenomic analyses. Effort will be given to identify putative virulence factors and potential diagnostic targets. Comparison of this genome to closely related pathogens should provide new insights and context concerning their ecology and evolution. The broader impacts of this study will occur on several research fronts. The data will provide a platform from which hypothesis driven federal grant applications can be envisaged and submitted that address aspects of pathogenesis. Candidate diagnostic targets will be identified and strategies for improved control and treatment can be developed. The generation of a genomic perspective through which to study this organism will be of tremendous benefit to the scientific community already studying this pathogen. Availability of genomic resources for A. suis will surely precipitate an expansion in the nature of investigations conducted and the number of researchers interested in its study.

Progress 10/01/08 to 09/30/09

Outputs
OUTPUTS: The goal of this project was to produce a draft genome sequence of Actinobacillus suis, a causative agent of fatal septicemia in piglets and an emerging pathogen of older High Health Status swine. By using the '454' next generation parallel sequencing system (conducted at the Washington University Genome Sequencing Center), a draft genome sequence of the Type strain ATCC 15557 (serotype O1/K1) has been generated. Currently the genome is represented by 31 contigs, with an average size of 80kb. The largest contig is 464 kb and on average the 2.48-Mb genome is covered by 50X coverage. While the sequencing phase of this project was being performed, our group became aware of a parallel project conducted by Dr. Janet MacInnes at the University of Guelph, Ontario, Canada. This group also used the 454 sequencing approach, but used an alternate isolate of serotype O2/K2. This sequence is in 52 contigs. Despite the differences in serotype, it is anticipated that the sequences of the two genomes are very similar and that a comparative approach might facilitate contig joining to generate a complete genome sequence. Accordingly, our group is collaborating with Dr. MacInnes, together with Dr. Andrew Kropinski to integrate optical mapping data and comparative data to close the small number of gaps in the two A. suis genomes. It is our intention that the two genomes will be published together and released into GenBank simultaneously. We are in the process of registering the two projects with the NCBI, with locus tabs ASU1 for the Type strain (sequenced by funding from this project) and ASU2 for the O2/K2 strain sequenced by our colleagues. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The major result of this project period was the generation of an almost complete genome sequence of the Actinobacillus suis Type strain. Advances in the technology (the implementation of the Titanium reagents) and the absence of large numbers of repeated sequences (with the exception of the genes encoding the ribosomal RNAs) enabled the assembly of the data into only 31 contigs. As a result, it should be possible to close the genome with limited resources by either i) comparative analysis with the other A. suis genome; ii) optical mapping or iii) PCR between contig ends. A detailed analysis of the encoded ORFs will be completed once the genome is finally assembled, but an initial survey of the sequence revealed the presence of genes encoding pili (Widespread Colonization Island), hemolysins, urease, acid phosphatase, and components of the mobilome, including bacteriophage and an apparently integrated plasmid. Based on the genome size of 2.48 Mb, it is expected that analysis of the genome will disclose in excess of 2,000 A. suis genes that had not been previously described. Binary Comparison of the two A. suis genomes should reveal the genetic basis that accounts for the serotype differences and should advance our understanding of the 'pan-genome' for the genus and species

Publications

• No publications reported this period