DETERMINANT HOST FACTORS OF THE ZEBRAFISH MODEL OF STREPTOCOCCAL PATHOGENESIS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0212615
• Project Start Date: Oct 1, 2007
• Project End Date: Sep 30, 2008
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF ILLINOIS
2001 S. Lincoln Ave.
URBANA,IL 61801

Performing Department
VETERINARY RES AND EXTENSION

Non Technical Summary
Horse breeding, racing and shows are an important part of the Illinois and national agricultural landscape and losses due to morbidity, mortality and treatment costs associated with S. equi are significant. In response to the growing concern regarding strangles, the USDA recently listed S. equi as a high priority disease to emphasize the need for increased efforts in this area. Our laboratory has extensive experience in the phenotypic and genotypic characterization of the group C streptococci. Currently we have characterized approximately 500 S. equi isolates which are banked in one of the largest collections of this organism in the country. In addition to these isolates, we have many S. zoo and S. equi strains that have been associated with clinical disease following vaccination of S. zoo infected yearlings, many with atypical or intermediate phenotypes. Our laboratory now has significant experience in the sometimes difficult genetic manipulation of this organism. Our laboratory has a great deal of experience with the zebrafish model having completed one large scale project and significant progress on the current project. The zebrafish model adapted in our laboratory has also been applied to various other Streptococcus spp. including S. pyogenes (a human pathogen) and S. iniae (a zoonotic fish pathogen). This study should provide valuable information into the host response of zebrafish to bacterial pathogens increasing the usefulness of this versatile animal model.

Animal Health Component

25%

Research Effort Categories

Basic

75%

Applied

25%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3810	1100	50%
311	4010	1100	50%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
4010 - Bacteria; 3810 - Horses, ponies, and mules;

Field Of Science
1100 - Bacteriology;

Keywords

streptococcal

pathogenesis

Goals / Objectives
Hypothesis: The morphologic changes in Streptococcus zoo injected fish suggest involvement of both the innate and adaptive immune response. We hypothesize that there are qualitative and quantitative differences in mRNA levels in tissue preparations of spleen, kidney and site of infection of S. zoo infected fish and similar preparations in mock injected (control) fish and fish injected with either WT or VX strains of S. equi. Experimental Design: Whole tissue mRNA will be isolated from the spleens, kidneys and injection sites of groups of 10 fish which will be injected with 1) WT S. equi, 2) VX S. equi, 3) S. zoo, and 4) mock injected control. Isolated mRNA will be sent to NimbleGen for both comparative genomic hybridization (CGH) as well as simple gene expression analysis using commercially available whole zebrafish genome chips. In experiment 1, direct comparison of the transcriptomes of pooled splenic, renal and injection site tissue of WT and S. zoo injected fish using CGH will allow for the rapid identification of those genes uniquely involved in the host response to each organism. In experiment 2, indirect comparisons of the transcriptomes of splenic, renal and injection site tissue from each group will be performed followed by classification of those genes that are active in control fish as well as in the fish injected with WT and VX strains of S. equi and a strain of S. zoo.

Project Methods
WT and VX S. equi strains as well as the laboratory S. zoo strain LBC50 will be intramuscularly injected into groups of 10 anesthetized zebrafish. Fish will be observed every 2 hours for up to 96 hours following injection. Humane euthanasia will be performed using loss of neutral buoyancy as an objective endpoint. Necropsy will immediately follow euthanasia with spleen, kidney and injection site collected separately for mRNA extraction. mRNA will be extracted on pools of 3 to 4 spleens, kidney and dorsal musculature using a Quiagen RNeasy Mini Kit (Cat. No. 74104) according to the manufactures instructions with recommended modifications for tissue. Isolated mRNA will be sent to NimbleGen for microarray analysis using commercially available whole zebrafish genome chips. In experiment 1, CGH will be performed twice on pooled spleen and renal tissue mRNA from WT and S. zoo injected fish. In experiment 2, gene expression analysis will be performed once on each tissue type (total of 14 microarrays). Hybridization data will be compared between groups using software provided by the company.

Progress 10/01/07 to 09/30/08

Outputs
OUTPUTS: We have engaged two approaches to address differences in virulence mechanisms of Group C Streptococci. Our first goal was to use comparative genomic sequencing (CGS) to examine the Streptococcus equi modified live vaccine (MLV) strain and its progenitor and compare these to S. equi type strain, Sanger 4047. Genomic DNA was amplified by NimbleGen Inc., resulting in overlapping 29 bp segments of the genome, detecting dissimilar sequences as small as single nucleotide polymorphisms (SNPs). These areas of interest are mapped to the sequenced genome of S.equi 4047 and genes with substitutions or deletions were selected for further study. Of ~100 changes between the MLV and its progenitor, 8 have mapped to sites within predicted gene sequences that would alter expression (change to a stop codon or frame shifts within the coding region) or significantly change the resulting predicted amino acid sequence (ignoring silent mutations). A similar CGS approach is being used to study the differences in host-pathogen interactions in a zebrafish model. The complete zebrafish genome has been sequenced. By comparing the mRNA expression differences of unchallenged zebrafish to mRNA of zebrafish that have been challenged with Group C Streptococcus strains, we will identify genes responding to virulence signals of Streptococci. Use of a zebrafish model for virulence screening of a variety of strains of Group C Streptococcus has led us to the finding that there were significant differences in pathogenesis between the highly pathogenic S. equi and the less virulent S. zooepidemicus. Differences in innate immunity were evidenced by macrophage trafficking and other early inflammatory responses observed by histopathology. Migration of numerous leukocytes to the site of injection and markedly increased splenic size and cellularity were typical among S. zoo infected zebrafish as were increased renal macrophages. By comparison there was a poor innate immune response of the fish to S. equi, which may explain the more invasive nature of S. equi infection and more rapid decline in fish survival. Immune function differences might be better assessed by examining mRNA expression, comparing the fish's response to S. equi versus S. zoo challenge to the S. equi MLV challenged and/or mock injected fish. While the model has worked very well for determining attenuating mutations in challenge strains of Streptococcus using survival curves, there have not been significant efforts to discover the mechanisms by which the fish respond locally and systemically to the IM injection, mobilizing immune cells and mediators from the spleen and kidneys. Thus, these tissues were harvested from moribund fish and RNA was extracted using the Qiagen RNA-easy Mini-kit. Recovery of RNA from the spleen and kidneys resulted in sub-optimal concentrations, and a second group of 12 fish were required to attain minimum RNA levels for production, labeling of cDNA and hybridization. Recovery of RNA from the muscle tissue required a bead beater step that increased the RNA yield eight-fold. Hybridization to the zebrafish CHIP is in progress and the sequence data will be analyzed over the next several weeks. PARTICIPANTS: This project was conducted as part of the doctoral thesis research (defending Dec. 19, 2008) of Dr. Luke Borst, DVM. He has put a great deal of time and energy into development of the zebrafish model for screening for attenuation of the Streptococcus strains. As a recently ACVP boarded pathologist, he has added a great deal to evaluating the contribution of virulence genes to pathogenesis. A great deal of effort was also required to generate and characterize mutations in the Streptococcus equi strains. This work was accomplished by postdoctoral research associate Dr. Sheila Patterson. She continues efforts to link changes in the Strep genome to alteration in the zebrafish response to infection. Dr. Carol Maddox, project coordinator, also contributes to study genes affecting innate immune response to Strep infection, which we hope will be part of future gene deletion mutant assays. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The data generated by the CGS of the Strep equi strains has provided several new target genes that may affect virulence based upon their interruption/mutation in a modified live vaccine strain. The comparisons to a sequenced type strain 4047 enabled us to identify many other variants, some surrounding the excision of a phage present in the Sanger 4047 strain and absent from the MLV and its progenitor. The data generated by the RNA expression CHIP analysis of zebrafish challenged with various group C Streptococci will enable us to identify key host/pathogen interactions. Identification of host genes that are over expressed or under expressed in response to infection will enable us to better select target genes of the pathogen that should be attenuated for the next generation MLV. Strangles has been identified by USDA as a high priority area for animal health and well-being research, and this data could contribute greatly to national efforts to eliminate Strep. equi infections that persist in our horse populations.

Publications

• No publications reported this period