Progress 09/01/13 to 08/31/19
Outputs
Target Audience:Target audiences for this research are immunologists interested in T cell development, comparative immunologists and animal health researchers developing vaccines for farm-raised fish species. Also targeted are extension veterinarians and fish farmers who will ultimately use this new information for strategies to prevent and treat outbreaks of I. multifiliis in fish hatcheries. Research outcomes will also be of interest and use for wildlife biologists developing strategies to prevent or control I. multifiliis infections in wild fish populations in ponds, streams and rivers. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?A veterinary student completed a summer research training project. A graduate student completed a M.S. demonstrating that rearranged IgH V to J sequences are integrated into the channel catfish germline genome. An undergraduate student was trained in laboratory protocols used for the project. A Senior Research Associate developed new computer programs for analysis of CDR3 sequences in our Illumina data. How have the results been disseminated to communities of interest?Four manuscripts have been published, additional manuscripts are in preparation. Posters explaining the TCR data were presented at the annual USDA meeting of PIs. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The major accomplishment was the demonstration for the first time in any vertebrate that rearranged genes for the T Cell Receptor are found in germline DNA. We showed that these germline rearranged genes are preferentially expressed following infection of catfish with the parasite Ichthyophthirius multifiliis. The germline genes for the T Cell Receptor undergo somatic rearrangement during the course of T cell development to form a rearranged TCR gene, which is then expressed in mature T cells. Rearranged TCR genes are only expected to be found in DNA isolated from T cells, but not in germline DNA isolated from oocytes or erythrocytes. Sequencing of PCR products amplified from germline DNA confirmed that all five Vβ gene families and Vα1 have rearranged TCR V(D)J genes with diverse CDR3 sequences integrated into the germline. Germline rearranged Vβ2 and Vβ4 genes retain the intron between the leader and Vβ sequence. This suggests that the germline rearranged TCR Vβ genes arose through VDJ rearrangement in T cells, and subsequently moved into the germline through DNA transposon mediated transposition. We also demonstrated that rearranged genes for the B Cell Receptor (IgH) are also found in germline DNA. These results reveal a new dimension to the adaptive immune system of vertebrates, namely: the expression of evolutionarily conserved, rearranged V(D)J genes from the germline. The objectives of the project were to determine the expressed CDR3 repertoires for the B cell receptor IgM heavy chain (IGH) and the T cell receptor beta chain (TCR β) from clonally expanded catfish B and T cell populations responding to infection with I. multifiliis, or vaccination with an I. multifiliis antigen. We completed sequencing of multiple cDNA libraries using Illumina protocols and are analyzing a total of 31 Gb of data from infected or vaccinated fish. This data is being prepared for publication.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Findly, R. C., Niagro, F. D., Sweeney, R. P., Camus, A. C., & Dickerson, H. W. (2018). Rearranged T Cell
Receptor Sequences in the Germline Genome of Channel Catfish Are Preferentially Expressed in
Response to Infection. Frontiers In Immunology, 9. (doi: 10.3389/fimmu.2018.02117)
- Type:
Theses/Dissertations
Status:
Accepted
Year Published:
2019
Citation:
Rearranged B-cell immunoglobulin heavy chain VDJ genes are integrated into channel catfish germline DNA. Sweeney, R. P
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Progress 09/01/17 to 08/31/18
Outputs
Target Audience:Target audiences for this research are comparative immunologists and animal health researchers developing vaccines for farm-raised fish species. Also targeted are extension veterinarians and fish farmers who will ultimately use this new information for strategies to prevent and treat outbreaks of Ichthyophthirius multifiliis in fish hatcheries and commercial outgrowth farms. Research outcomes will also be of interest and use for wildlife biologists developing strategies to prevent or control Ichthyophthirius infections in wild fish populations in bodies of water such as ponds, streams and rivers. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?A M.S. student is working on the characterization of rearranged TCR and IgH V to J sequences that are integrated into the channel catfish germline genome. An undergraduate student is being trained in all aspects of the project. How have the results been disseminated to communities of interest?A poster presenting the TCR data was presented at annual USDA meeting of PIs in December. A manuscript on the TCR data is currently under review. What do you plan to do during the next reporting period to accomplish the goals?RNA samples from the fish in the study are being prepared, and cDNA libraries of expressed transcripts for both TCRβ and IgH sequences generated for Illumina sequencing. We will then undertake analysis of the Illumina sequencing data, which will provide coverage of CDR3 repertoires expressed by both T cells and B cells before and following vaccination, and after infection and challenge with a natural pathogen. This analysis will define the global patterns of expression of both clonally expanded B and T cell populations responding to vaccination with Ich i-antigen and to Ich infection. As there are rearranged V to J sequences in the germline genome for both TCRβ and IgH genes, we anticipate that a restricted, non-random repertoire of CDR3 sequences may be preferentially expressed. This would represent a novel and significant addition to our understanding of adaptive immunity.
Impacts
What was accomplished under these goals?
We have now demonstrated that not only are rearranged TCRβ V to J sequences integrated into the germline genome, but have extended our work to include V to J sequences for TCRα, IgH and IgL genes. Thus, we have data indicating that rearranged V to J sequences for the receptors of both αβ T cells and B cells are integrated into the germline genome. These novel discoveries represent the first demonstration that rearranged TCRα and TCRβ V to J sequences are both integrated into the germline genome of a vertebrate and transcriptionally active. A manuscript reporting the TCR data is under review. We were delayed in initiating the long-term vaccination study, as the strain of the Ichthyophthirius multifiliis parasite that we had maintained in our laboratory for years became senescent and could no longer be cultured. However, a similar strain was subsequently established and the long-term vaccination study is now complete.
Publications
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Progress 09/01/16 to 08/31/17
Outputs
Target Audience:Target audiences for this research are comparative immunologists and animal health researchers developing vaccines for farm-raised fish species. Also targeted are extension veterinarians and fish farmers who will ultimately use this new information for strategies to prevent and treat outbreaks of Ichthyophthirius multifiliis in fish hatcheries and commercial outgrowth farms. Research outcomes will also be of interest and use for wildlife biologists developing strategies to prevent or control Ichthyophthirius infections in wild fish populations in bodies of water such as ponds, streams and rivers. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?A M.S. student is working on the characterization ofrearranged TCRβ V to J sequencesthat are integrated into the channel catfish germline genome. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
We have confirmed that rearranged TCRβ V to J sequences are integrated into the channel catfish germline genome. DNA was isolated from channel catfish oocytes and used to determine if sequences corresponding to rearranged TCRβ genes could be amplified from germline DNA by PCR. PCR products of the predicted sizes were amplified using primers specific to Vβ genes for each of the five Vβ gene families and J gene primers. The PCR products were sequenced, confirming that recombined V to J genes containing rearranged CDR3 sequences were present in the germline genome. Our previous high-throughput sequencing of TCR CDR3 repertoires revealed that some Vβ2 clonotypes were expressed by all channel catfish following infection, and that each of these public CDR3 amino acid sequence was predominately coded by an identical CDR3 DNA sequence, respectively, in different fish. The CDR3 DNA sequences coding for six of these public clonotypes were used to design PCR primers. These CDR3 specific primers were paired with Vβ2 gene primers in PCR reactions using oocyte DNA as template. Sequencing of the PCR products demonstrated that the DNA sequences amplified from the germline genome corresponded to the previously defined transcribed Vβ2 to CDR3 sequences. In particular, the germline DNA sequence at the Vβ2-D-J junctions, which specifies the CDR3 sequence of a clonotype, matched the known CDR3 sequences first identified by sequencing of transcribed TCR CDR3 repertoires. This work has demonstrated that sequences coding for rearranged V to J genes for all five Vβ gene families are integrated into the germline genome of channel catfish. This represents the first demonstration in any species that rearranged TCRβ V to J sequences are both integrated into the germline genome of a vertebrate and are transcriptionally active.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Front. Immunol., 26 October 2017 | https://doi.org/10.3389/fimmu.2017.01379
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Progress 09/01/15 to 08/31/16
Outputs
Target Audience:Target audiences for this research are comparative immunologists and researchers developing vaccines for farm-raised fish species. Also targeted are extention veterinarians and farmers who will ultimately use this information for strategies to prevent and treat outbreaks of Ichthyophthirius multifiliis in channel catfish hatcheries and commercial farms. Research outcomes will also be of interest and use for wildlife biologists developing strategies to control Ich infections in wild fish populations in bodies of water such as ponds, streams and rivers. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?A M.S. student is working on a project involving the immune response of channel catfish against I. multifiliis. A 3rd year veterinary student is continuing a project that was initiated last summer on in vitro T cell responses of channel catfish. A technician from the Bozeman Fish Technology Center, Bozeman, MT visited the lab to develop an Ich culture system How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
We completedhigh-throughput sequencing of T cell receptor complementary determining region 3 (CDR3) repertoires expressed by αβ T cells from channel catfish before and following infection with the parasitic protozoan Ichthyophthirius multifiliis. In contrast to mammals, we found that different outbred fish predominately used, respectively, an identical DNA sequence in combination with the same J gene to code for the amino acid sequence of a TCRβ CDR3. This is a novel and unexpected finding. The basic mechanisms governing hematopoiesis and generation of hypervariable sequence diversity in CDR3 domains of TCRs and immunoglobulins are understood to be conserved from teleosts to mammals. As expected we found that the TCRβ CDR3 sequences were formed by rearrangement of variable, diversity and joining genes with nucleotide deletions and additions at the junctions of the rearranged genes. Because of the degeneracy of the genetic code and as addition of nucleotides at the junctions of the rearranged genes is essentially random, we expected that multiple DNA sequences would code for a CDR3 amino acid sequence in individual fish, as well as in different fish. However, we found that each CDR3 amino acid sequence was predominately coded by an identical CDR3 DNA sequence, respectively, in individual fish and in different fish. This suggests that in channel catfish, and perhaps teleosts in general, the processes by which expressed CDR3 repertoires are generated differs in some details from mammals. Our results should be of interest to a wide audience, including those interested in the evolution of adaptive immune responses and in the generation of the expressed TCR CDR3 repertoire.
Publications
- Type:
Journal Articles
Status:
Submitted
Year Published:
2016
Citation:
Findly, R.C., Niagro, F., and Dickerson, H.W. (2016)The expressed TCR? CDR3 repertoire is dominated by conserved DNA sequences in channel catfish
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Progress 09/01/14 to 08/31/15
Outputs
Target Audience:Target audiences for this research are comparative immunologists and researchers developing vaccines for farm-raisedfish species. Also targeted are extention veterinarians and farmerswho will ultimately use this informationfor strategies to prevent and treat outbreaks of Ichthyophthirius multifiliis in channel catfish hatcheries and commercial farms.Research outcomes will also be of interest and use for wildlife biologists developing strategies to control Ich infections inwild fish populationsinbodies of water such as ponds, streams and rivers. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?A M.S. graduate student commenced work ona project involving the immune response of channel catfish against I. multifliis A 2nd-year veterinary student completed a summer training program working on the in vitro T cell response to Ich i-antigens. How have the results been disseminated to communities of interest?Provided information on basic biology and immune response of Ichthyophthirius multifiliis to a wildlife biologist working on an Ich outbreak in the Klamath River in Southern Oregon and Northern California. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Tissue samples from four channel catfish were collected from fins before infection and at day 22 after infection. Samples were subsequently collected from skin, PBL, head kidney and spleen from two of the fish at 7 and 21 weeks after infection.A total of 4 x106 DNA sequences passed quality assurance filters for analysis. The sequences were sorted into Vβ2 and Vβ5 subsets and then by their CDR3 sequences. A unique CDR3 sequence is designated a clonotype. We analyzed these massive data sets at the DNA level and after translation into their predicted amino acid sequences. CDR3 sequences that were found only once in the data set comprised 5% of total sequences. However, these single copy sequences comprised 60% of the unique CDR3 sequences (clonotypes). This suggests that diversity in the anticipatory (i.e. naïve) CDR3 repertoire was maintained by T cells that were present in low numbers in the T cell populations before or after infection. The bulk of the CDR3 sequences in the data set were present in multiple copies, and approximately 50% of CDR3 sequences were present at >100 copies. This suggested that they were expressed by clonally expanded T cells. The data indicate that clonally expanded populations of T cells accumulate at sites of parasite infection (skin), as the most abundant 25 CDR3 clonotypes in samples from surface tissue (fin) at day 22 after infection represent 36% of the total sequences from those samples. In spleen clonally expanded T cell populations predominate, and only 10% of clonotypes account for 90% of all sequences at 7 weeks after infection. The greatest CDR3 clonotype diversity was seen in samples from head kidney and PBL. Public sequences (CDR3 clonotypes present in all fish) were found in all tissues and comprised 10% of CDR3 clonotypes in PBL. We have also sequenced and transcribed Vβ2 and Vβ5 CDR3 TCRβ domains from fish after vaccination with a protective Ich antigen and are beginning analysis of CDR3 repertoires.
Publications
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Progress 09/01/13 to 08/31/14
Outputs
Target Audience:Target audiences for this research are comparative immunologists and researchers developing vaccines against aquacultured fish species.Also targeted are extention veterinarians and fisheries biologists developing strategies to prevent and treat outbreaks ofIchthyophthirius multifiliis in catfish hatcheries,commercial farmsand natural bodies of water such as ponds, streams and rivers. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
We have initiated in depth Illumina sequencing of the transcribed Vβ2 and Vβ5 CDR3 domains of the β-chain of the T cell receptor (TCRβ) from channel catfish before and after Ich infection. The CDR3 domain is the antigen binding site of the TCRβ chain and varies in sequence and size. Tissue samples from four fish were collected from fins before infection and at day 22 after infection. Samples were subsequently collected from skin, PBL, head kidney and spleen from two of the fish at 7 and 21 weeks after infection. A total of 4 x106 DNA sequences passed quality assurance filters for analysis. The sequences were sorted into Vβ2 and Vβ5 subsets and then by their CDR3 sequences. A unique CDR3 sequence is designated a clonotype. We analyzed these massive data sets at the DNA level and after translation into their predicted amino acid sequences.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Dickerson, H W. and Findly, R. C. (2014) Immunity to Ichthyophthirius infections in fish: a synopsis. Dev. Comp. Immunol. 43: 290-299.
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