Cutaneous antibody-secreting cells and B cells in a teleost fish.

Antibodies in cutaneous mucus and skin of teleosts play a critical role in the protective immune response against infection. We demonstrate by ELISPOT that antibody-secreting cells (ASC), which include LPS-inducible B cells (plasmablasts) and non-replicating plasma cells, reside in low numbers in the skin of channel catfish. Following immunization against the protozoan parasite Ichthyophthirius multifiliis, which infects skin and gills, the number of ASC in skin increased 20-fold, indicating that the number of ASC in skin is dynamic and increases in response to parasite infection. The number of ASC in skin remained elevated for at least 17 weeks after the last parasite exposure. Cutaneous ASC included I. multifiliis-specific ASC, which undoubtedly serve as the primary source of cutaneous antibodies that confer long-term humoral immunity against reinfection. Our demonstration that skin contains B cells and plasma cells suggests that it is an integral component of the teleost immune system.

Rearranged T Cell Receptor Sequences in the Germline Genome of Channel Catfish Are Preferentially Expressed in Response to Infection.

Rearranged V(D)J genes coding for T cell receptor α and ß chains are integrated into the germline genome of channel catfish. Previous analysis of expressed TCR Vß2 repertoires demonstrated that channel catfish express multiple public clonotypes, which were shared among all the fish, following infection with a common protozoan parasite. In each case a single DNA sequence was predominately used to code for a public clonotype. We show here that the rearranged VDJ genes coding for these expressed public Vß2 clonotypes can be amplified by PCR from germline DNA isolated from oocytes and erythrocytes. Sequencing of the Vß2 PCR products confirmed that these expressed public Vß2 clonotypes are integrated into the germline. Moreover, sequencing of PCR products confirmed that all five Vß gene families and Vα1 have rearranged V(D)J genes with diverse CDR3 sequences integrated into the germline. Germline rearranged Vß2 and Vß4 genes retain the intron between the leader and Vß sequence. This suggests that the germline rearranged TCR Vß genes arose through VDJ rearrangement in T cells, and subsequently moved into the germline through DNA transposon mediated transposition. 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.

Vertebrate Adaptive Immunity-Comparative Insights from a Teleost Model.

The channel catfish (Ictalurus punctatus) and the ciliated protozoan parasite Ichthyophthirius multifiliis are used to study pathogen-specific protective immunity. In this review, we briefly describe this host-parasite system and discuss the comparative insights it provides on the adaptive immune response of vertebrates. We include studies related to cutaneous mucosal immunity, B cell memory responses, and analyses of αß T cell receptor (TCR) repertoires. This host-parasite model has played an important role in elucidating host protective responses to parasite invasion and for comparative studies of vertebrate immunity. Recent findings from bioinformatics analyses of TCR ß repertoires suggest that channel catfish preferentially expand specific clonotypes that are stably integrated in the genome. This finding could have broad implications related to diversity in lymphocyte receptors of early vertebrates.

The expressed TCRß CDR3 repertoire is dominated by conserved DNA sequences in channel catfish.

We analyzed by high-throughput sequencing T cell receptor beta CDR3 repertoires expressed by αß T cells in outbred channel catfish before and after an immunizing infection with the parasitic protozoan Ichthyophthirius multifiliis. We compared CDR3 repertoires in caudal fin before infection and at three weeks after infection, and in skin, PBL, spleen and head kidney at seven and twenty-one weeks after infection. Public clonotypes with the same CDR3 amino acid sequence were expressed by αß T cells that underwent clonal expansion following development of immunity. These clonally expanded αß T cells were primarily located in spleen and skin, which is a site of infection. Although multiple DNA sequences were expected to code for each public clonotype, each public clonotype was predominately coded by an identical CDR3 DNA sequence in combination with the same J gene in all fish. The processes underlying this shared use of CDR3 DNA sequences are not clear.

Differences in virulence between two serotypes of Ichthyophthirius multifiliis.

Naive channel catfish Ictalurus punctatus were infected by 2 isolates of the parasitic ciliate Ichthyophthirius multifiliis that differed in virulence. The isolates, NY1 and G5, Serotypes A and D, respectively, express different surface immobilization-antigens. The virulence of the 2 isolates was compared using tail-fin infections to quantitate parasite numbers and by analysis of the survival of infected fish. Although NY1 infected fish at a lower level than G5, all NY1-infected fish died, but 51% of G5-infected fish survived. The greater virulence of NY1 is apparently a consequence of its shorter life cycle, which results in overwhelming reinfection of fish before they can develop a protective immune response. This report represents the first experimental evidence for differences in virulence between serotypes of I. multifiliis.

Comparing tolerance of Ichthyophthirius multifiliis and Tetrahymena thermophila for new cryopreservation methods.

Ichthyophthirius multifiliis is an obligate protozoan parasite of freshwater fishes that has a complex developmental cycle. It has not been successfully cryopreserved, so management studies are restricted to parasites obtained during outbreaks or perpetuated by passage in live fishes. To overcome this serious limitation, free-swimming I. multifiliis parasites were tested in a cryopreservation protocol routinely used for a related ciliate, Tetrahymena. In this protocol, I. multifiliis theronts retained infectivity for 3 days, although the protocol itself was ultimately lethal. Exposure of I. multifiliis and Tetrahymena thermophila to a battery of media and cryopreservative reagents showed that I. multifiliis was less hardy than T. thermophila and likely had significant biological and cytoskeletal differences. No combination of reagents, media, freezing rates, or dilution media permitted cryopreservation of I. multifiliis parasites that could then undergo development or infect fish. However, a vitrification protocol was formulated using Ficoll, 1,2-propanediol, and N,N-dimethylacetamide from which intact cryopreserved theronts with some motility were recovered. Understanding the effects of these reagents may lead to both a cryopreservation method for I. multifiliis and to improved understanding of the biology of ciliates.

B cell memory following infection and challenge of channel catfish with Ichthyophthirius multifiliis.

B cell responses in channel catfish to infection with the parasitic ciliate Ichthyophthirius multifiliis were followed for 3 years. High titers of serum IgM antibodies recognizing I. multifiliis immobilization antigens were present 5weeks after immunizing infection, but by 1 year titers were at low or undetectable levels. Two to three years after infection the numbers of antibody secreting cells recognizing immobilization antigens in skin and head kidney of immune fish had decreased to the level found in uninfected controls. Challenge of immune fish showed they remained immune and that the numbers of antibody secreting cells recognizing immobilization antigens increased in skin but not head kidney. This suggests that antigen-specific memory B cells persisted for 3 years after infection and upon challenge differentiated into antibody secreting cells that localized in skin. Our results suggest that humoral immunity in channel catfish is maintained through IgM(+) memory B cells.

Transcriptional profiling of stage specific gene expression in the parasitic ciliate Ichthyophthirius multifiliis.

The parasitic ciliate, Ichthyophthirius multifiliis (Ich), is among the most important protozoan pathogens of freshwater fish. Ichthyophthirius cannot be grown in cell culture, and the development of effective prophylactic and therapeutic treatments has been hampered by a lack of information regarding genes involved in virulence, differentiation and growth. To help address this issue, we have generated EST libraries from the two major stages of the parasite life cycle that infect and develop within host tissues. A total of 25,084 ESTs were generated from non-normalized libraries prepared from polyA+ RNA of infective theronts and host-associated trophonts, respectively. Cluster analysis identified 5311 unique transcripts (UniScripts), of which 2091 were contigs and 3220 singletons. Extrapolation of the data based on rates of EST discovery suggests that more than half the expected protein-coding genes of I. multifiliis are represented in this data. BLASTX comparisons against GenBank nr, UniProtKB (SwissProt and TrEMBL), as well as Tetrahymena thermophila, Plasmodium falciparum, and Paramecium tetraurelia protein databases produced 3694 significant (E-value ≤1e(-10)) hits, of which 1178 were annotated using gene ontology (GO) analysis. A high proportion of UniScripts (63%) showed similarity to other ciliate proteins. When combined with expression profiling data, GO ontology analysis of Biological Process, Cellular Component, and Molecular Function revealed interesting differences in gene families expressed in the two stages. Indeed, the most abundant transcripts were highly stage-specific and coincided with the metabolic activities associated with each stage. This work provides an effective genomics resource to further our understanding of Ichthyophthirius biology, and lays the groundwork for the identification of potential drug targets and vaccines candidates for the control of this devastating fish pathogen.

Comparative genomics of the pathogenic ciliate Ichthyophthirius multifiliis, its free-living relatives and a host species provide insights into adoption of a parasitic lifestyle and prospects for disease control.

BACKGROUND: Ichthyophthirius multifiliis, commonly known as Ich, is a highly pathogenic ciliate responsible for 'white spot', a disease causing significant economic losses to the global aquaculture industry. Options for disease control are extremely limited, and Ich's obligate parasitic lifestyle makes experimental studies challenging. Unlike most well-studied protozoan parasites, Ich belongs to a phylum composed primarily of free-living members. Indeed, it is closely related to the model organism Tetrahymena thermophila. Genomic studies represent a promising strategy to reduce the impact of this disease and to understand the evolutionary transition to parasitism. RESULTS: We report the sequencing, assembly and annotation of the Ich macronuclear genome. Compared with its free-living relative T. thermophila, the Ich genome is reduced approximately two-fold in length and gene density and three-fold in gene content. We analyzed in detail several gene classes with diverse functions in behavior, cellular function and host immunogenicity, including protein kinases, membrane transporters, proteases, surface antigens and cytoskeletal components and regulators. We also mapped by orthology Ich's metabolic pathways in comparison with other ciliates and a potential host organism, the zebrafish Danio rerio. CONCLUSIONS: Knowledge of the complete protein-coding and metabolic potential of Ich opens avenues for rational testing of therapeutic drugs that target functions essential to this parasite but not to its fish hosts. Also, a catalog of surface protein-encoding genes will facilitate development of more effective vaccines. The potential to use T. thermophila as a surrogate model offers promise toward controlling 'white spot' disease and understanding the adaptation to a parasitic lifestyle.

Cross-immunity and antibody responses to different immobilisation serotypes of Ichthyophthirius multifiliis.

Vaccination of channel catfish with either of two serotypes of the parasitic ciliate Ichthyophthirius multifiliis conferred protection against challenge infection by either serotype. Fish were vaccinated by intracoelomic injection with live theronts of isolate G5 (serotype D) or isolate G12 (a new serotype), which express different surface immobilisation antigens. Vaccination with live G12 theronts conferred complete protection against subsequent challenge by both serotypes while vaccination with G5 theronts elicited only partial protection against both serotypes. Vaccination with trophont lysates did not protect against challenge infection. Sera from vaccinated fish were tested in immobilisation assays, ELISAs, and Western blots. Serum antibodies recognised only immobilisation antigens of the serotype used for vaccination in immobilisation assays or on Western blots. No antigens common to both serotypes were identified by Western blots. In contrast, serum antibodies bound antigens in cell lysates from both serotypes by ELISA, demonstrating that antibodies recognising both serotypes are produced in response to infection, which presumably confer observed cross-serotype protection.

Systemic and cutaneous mucus antibody responses of channel catfish immunized against the protozoan parasite Ichthyophthirius multifiliis.

Fish acquire protective immunity against the ciliated protozoan parasite Ichthyophthirius multifiliis following sublethal infection or inoculation with I. multifiliis immobilization antigens (i-antigens). In both cases, parasite-immobilizing antibodies have been identified in sera and mucosal secretions. To investigate the kinetics of this immune response, antibody levels were determined by enzyme-linked immunosorbent assay (ELISA) in the sera and cutaneous mucus of channel catfish (Ictalurus punctatus) that were either infected with parasites or given a single injection of purified i-antigen (5.0 microg/fish) in Freund's incomplete adjuvant. At 5 weeks, infected and inoculated fish had a mean serum (1:80 dilution) antibody absorbance (A405) value of 0.54 +/- 0.17 and 0.35 +/- 0.03, respectively, which were significantly higher (alpha = 0.05) than the pretreatment serum (1:80 dilution) antibody absorbance value of 0.24 +/- 0.05. At 14 weeks, mean serum (1:80 dilution) ELISA absorbance values in the teo groups of fish increased to 0.79 +/- 0.30 and 0.71 +/- 0.24, respectively. In both groups of fish, antibody levels in cutaneous mucus (undiluted) were much lower than those in sera. Infected fish had detectable mucus (undiluted) antibody levels from 3 to 9 weeks, with the highest mean value (0.30 +/- 0.07) occurring at 7 weeks. Although individual inoculated fish produced serum antibody absorbance values comparable to those seen in infected fish, the mean mucus antibody values in this group did not rise above pretreatment levels. I. multifiliis infection induced a transient mucosal antibody response that coincided with the resolution of infection. Whether elicited by infection or intraperitoneal injection of i-antigen, the serum and mucus antibody responses of channel catfish immunized against I. multifiliis did not occur synchronously.

Surface immobilization antigen of the parasitic ciliate Ichthyophthirius multifiliis elicits protective immunity in channel catfish (Ictalurus punctatus).

Channel catfish (Ictalurus punctatus) that survive infection with the parasitic ciliate Ichthyophthirius multifiliis acquire immunity to subsequent challenge and produce specific antibodies in serum that immobilize the parasite in vitro. Cellular surface protein antigens targeted by these antibodies are referred to as immobilization antigens (i-antigens). By using an immobilizing mouse monoclonal antibody as a ligand, the i-antigen of I. multifiliis isolate G5 was purified to homogeneity by immunoaffinity chromatography, and its immunogenicity was confirmed by inoculating rabbit and channel catfish to produce immobilizing antisera. To test the purified i-antigen as a subunit vaccine, channel catfish fingerlings were injected intraperitoneally (i.p.) with purified i-antigen at a dose of 10 microg/fish in complete Freund's adjuvant on day 1, followed by a second i.p. injection of the same amount of i-antigen in incomplete Freund's adjuvant on day 15. Negative control fish were immunized similarly with either bovine serum albumin (BSA) or an immobilization-irrelevant I. multifiliis protein. On day 84, the fish were challenged with live I. multifiliis G5 theronts at a dose of 15,000 cells per fish. Seventy-two percent of the fish immunized with i-antigen survived the challenge. All negative control fish died within 16 days of exposure. There was a significant difference in the median days to death between the negative control fish injected with BSA and the fish that died following vaccination with i-antigen. Fish injected with i-antigen developed high immobilizing antibody titers in serum. This is the first demonstration of a direct role for i-antigens in the elicitation of protective immunity, suggesting that these proteins by themselves serve as effective subunit vaccines against I. multifiliis.

Immunisation of channel catfish, Ictalurus punctatus, with Ichthyophthirius multifiliis immobilisation antigens elicits serotype-specific protection.

Surface immobilisation antigens (i-antigens) were purified from two strains of Ichthyophthirius multifiliis (NY1 and G5) that represent different i-antigen serotypes, namely A and D, respectively. The efficacy of the purified antigens as subunit vaccines was then tested in challenge studies using parasites of the homologous or heterologous serotype. Three groups of juvenile channel catfish (70 animals per group) were immunised with i-antigens from either the G5 or NY1 isolates, or with bovine serum albumin (BSA) as a control. Proteins were injected intraperitoneally (i.p.) at a dose of 10 microg/fish with complete Freund's adjuvant on day 1, followed by a second injection in incomplete Freund's adjuvant on day 15. Fish immunised with the purified i-antigens developed high titres of serum immobilising antibodies whereas sera from BSA-injected control fish did not. Fish antisera immobilised parasites of the homologous, but not the heterologous strain, and recognised the corresponding i-antigens on Western blots run under non-reducing conditions. On day 36, each group was divided into two subgroups (n=30). One subgroup was challenged with G5 parasites, and the other was challenged with NY1 parasites. When challenged with G5 parasites, 70% of fish immunised with the G5 i-antigens survived. When challenged with NY1 parasites, 33.3% of fish immunised with the NY1 i-antigens survived. All BSA-injected control fish died, as did all fish injected with the purified antigens and challenged with the non-homologous parasite strain. Statistical analyses indicated significant differences among test and control groups with regard to the mean days to death (MDD). While the results of these studies clearly support a role for i-antigens in protection, active immunity in response to natural infection is not serotype-specific. The utility of i-antigens, as well as the existence of other potential vaccine candidates for the prevention of 'white-spot' disease, are discussed.