Progress 10/01/12 to 09/30/15
Outputs
Target Audience:Our target audiences include veterinary microbiology researchers, veterinary diagnostic microbiologists and graduate students who are working on this field. Since the data derived from this study may have potential laboratory diagnostic applications, this will be useful for those who are working in a clinical capacity at diagnostic laboratories. We will present the data in the American Association of Veterinary Laboratory Diagnosis (AAVLD) meeting. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Cuilan Ye, Joy Scaria and Ching-Lin have learned most of the techniques proposed in this proposal. This will be important for them to be an independent research scientist when they have their own lab. How have the results been disseminated to communities of interest?We gave a poster in the Cornell Veterinary school graduate student meeting day. What do you plan to do during the next reporting period to accomplish the goals?This is our final report.
Impacts
What was accomplished under these goals?
Our Overall goal in this project was to create a pan-species microarray that can be used for detecting and typing Salmonella enterica and Escherichia coli. We targeted to detect most common disease causing subtypes within these species and probes were designed that determine a strains serotype/pathotype, virulence and antibiotic resistance profile. Our design consists of 414 probes targetting core genes of S. enterica, subspecies I specific genes, fimbrial genes, pathogenicity islands, Gifsy elements and other variable genes. The pan-species array also contained 443 probes for detecting most common disease causing E. coli pathotypes (EHEC, UPEC, EPEC, EAEC, EIEC, and ETEC). Therefore, the pan-species array contained 857 probes. The new array was tested for specificity using known reference strains and strains isolated from clinical cases and food sources. Overall, the pan-species array could differentiate all the targeted Salmonella serovars (14 common disease causing serotypes) and E. coli pathotypes (6 common types). Besides strain typing, pan-species array also could give more resolution for detecting the antibiotic resistance gene profile in the tested strains. Based on these results, a peer-reviewed manuscript is in preparation.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Development of an ELISA using recombinant LigACon4-7.5 as antigen for the diagnosis of equine leptospirosis.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Recombinant antigens rLipL21, rLoa22, rLipL32 and rLigACon4-8 for serological diagnosis of leptospirosis by enzyme-linked immunosorbent assays in dogs.
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Progress 10/01/13 to 09/30/14
Outputs
Target Audience: Our target audience will be those scientists who are working on the diagnosis of food borne pathogens and also the farmers, farm veterinarian and food microbiologist. We will present our finding in the scientific meeting to distribute our data. Eventually, we will submit our manuscript to referee journals for publication. By this way, we can distribute our data in a public domain. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? One of the research associate was working on this project and get training. How have the results been disseminated to communities of interest? Yes, one of the paper is published. What do you plan to do during the next reporting period to accomplish the goals? We will continue to work to finish proposed project.
Impacts
What was accomplished under these goals?
In the previous reporting period, our efforts were focused on developing a more sensitive pathogen detection assay for Salmonella enterica by combining microarray based capture of core genes, serovar specific genes and virulence genes, and sequencing of the captured genes using Illumina next generation sequencing platform. During the current reporting period, we attempted to expand this method to Escherichia coli pathotypes. The following E. coli pathotypes were selected as targets for the new assay; (i) enteropathogenic E. coli (EPEC), which causes diarrhea in children and animals; (ii) enterohemorrhagic E. coli (EHEC), which is responsible for hemorrhagic colitis and hemolytic-uremic syndrome; (iii) enterotoxigenic E. coli (ETEC), which causes traveler's diarrhea and porcine and bovine diarrhea; (iv) enteroaggregative E. coli (EAEC), which causes persistent diarrhea in humans, and diffusely adherent E. coli (DAEC), a subclass of enteroaggregative E. coli which causes diarrhea in children; (v) enteroinvasive E. coli (EIEC). From among the above pathotyes, we have identified 360 genes as candidates strain differentiation and detection. We are in the process of optimizing the gene capture conditions for these E. coli pathotypes. Once we optimize the capture conditions, we anticipate no problems in the sequencing of captured genes as next generation sequencing protocols on Illumina are highly streamlined.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Recombinant Antigens rLipL21, rLoa22,
rLipL32 and rLigACon4-8 for Serological
Diagnosis of Leptospirosis by Enzyme-
Linked Immunosorbent Assays in Dogs
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Progress 10/01/12 to 09/30/13
Outputs
Target Audience: The target of audiences in this period is scientists who are working on this field and farmers who who need to get some of these knowledge. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Joy Scaria is working on this project and get training from this study. How have the results been disseminated to communities of interest? We have a publication in a public domain for those who are interesting in this study. What do you plan to do during the next reporting period to accomplish the goals? We will continue to perform the study as proposed in our proposal.
Impacts
What was accomplished under these goals?
Campylobacter, E. coli, Listeria, Salmonella, Bacillus, Clostridium, Staphylococcus, Streptococcus, Vibrio and Yersinia. For the array design, we use a set of core genes from these genera as positive controls and strain specific genes as species differentiating genes. For each gene, a 60 base region was selected as probes. These probes are then synthesized on Agilent Earray platform. The efficiency of this approach was then tested on a test array targeting to differentiate 14 most common Salmonella enterica serovars. Although this approach could differentiate the targeted serovars, the lower limit of detection was 103 bacteria in the sample. For any robust assay, the lower limit of detection should be at least 10 1 bacteria/sample. To increase the sensitivity of the assay, we have decided to incorporate next generation sequencing based approach. In this method, we will use microarray to capture the target genes and then the captured genes will be linearly amplified and sequenced on an Illumina Miseq platform. We expect that this new approach will enable us to increase the sensitivity of the assay significantly. Since requirements for NGS based assay is different from that of microarray based approach alone, we are designing new probes for target genes and are optimizing the capture and amplification protocols.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Yan, W., M. H. Saleem, P. McDonough, S. P. McDonough,T. J. Divers, Y. F. Chang. 2013. Development of an ELISA using recombinant LigACon4-7.5 as antigen for the diagnosis of equine leptospirosis. Clin Vaccine Immunol. 20:1143-1149
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