Family-specific differences in growth rate and hepatic gene expression in juvenile triploid growth hormone (GH) transgenic Atlantic salmon (Salmo salar).

Growth hormone transgenic (GHTg) Atlantic salmon (Salmo salar) have enhanced growth when compared to their non-transgenic counterparts, and this trait can be beneficial for aquaculture production. Biological confinement of GHTg Atlantic salmon may be achieved through the induction of triploidy (3N). The growth rates of triploid GH transgenic (3NGHTg) Atlantic salmon juveniles were found to significantly vary between families in the AquaBounty breeding program. In order to characterize gene expression associated with enhanced growth in juvenile 3NGHTg Atlantic salmon, a functional genomics approach (32K cDNA microarray hybridizations followed by QPCR) was used to identify and validate liver transcripts that were differentially expressed between two fast-growing 3NGHTg Atlantic salmon families (AS11, AS26) and a slow-growing 3NGHTg Atlantic salmon family (AS25); juvenile growth rate was evaluated over a 45-day period. Of 687 microarray-identified differentially expressed features, 143 (116 more highly expressed in fast-growing and 27 more highly expressed in slow-growing juveniles) were identified in the AS11 vs. AS25 microarray study, while 544 (442 more highly expressed in fast-growing and 102 more highly expressed in slow-growing juveniles) were identified in the AS26 vs. AS25 microarray study. Forty microarray features (39 putatively associated with fast growth and 1 putatively associated with slow growth) were present in both microarray experiment gene lists. The expression levels of 15 microarray-identified transcripts were studied using QPCR with individual RNA samples to validate microarray results and to study biological variability of transcript expression. The QPCR results agreed with the microarray results for 12 of 13 putative fast-growth associated transcripts, but QPCR did not validate the microarray results for 2 putative slow-growth associated transcripts. Many of the 39 microarray-identified genes putatively associated at the transcript expression level with fast-growing 3NGHTg salmon juveniles (including APOA1, APOA4, B2M, FADSD6, FTM, and GAPDH) are involved in metabolism, iron homeostasis and oxygen transport, and immune- or stress-related responses. The results of this study increase our knowledge of family-specific impacts on growth rate and hepatic gene expression in juvenile 3NGHTg Atlantic salmon. In addition, this study provides a suite of putative rapid growth rate-associated transcripts that may contribute to the development of molecular markers [e.g. intronic, exonic or regulatory region single nucleotide polymorphisms (SNPs)] for the selection of GHTg Atlantic salmon broodstock that can be utilized to produce sterile triploids of desired growth performance for future commercial applications.

Goldfish (Carassius auratus L.) possess natural antibodies with trypanocidal activity towards Trypanosoma carassii in vitro.

Natural infection of cyprinids, such as carp, with the extracellular protozoan parasite Trypanosoma carassii can attain up to 100% prevalence and cause significant host morbidity and mortality, particularly in aquaculture settings. Host recovery from T. carassii infection has been shown to be antibody (Immunoglobulin M; IgM)-mediated, conferring long-term immunity in recovered animals upon challenge. To assess the role of IgM in parasite clearance in the goldfish, IgM was purified by PEG-6000 precipitation from goldfish serum collected at 0 (naïve), 21 (peak parasitaemia) and 42 (recovery phase; immune) days post infection (dpi) and used for in vitro assays. Purified IgM from 0, 21, and 42 dpi serum showed dose- and time-dependent trypanocidal activity in vitro. Incubation of T. carassii with 0 dpi IgM showed the greatest reduction in trypanosome numbers after 24 h, followed by 42 dpi IgM, and finally by 21 dpi IgM. The trypanocidal activity of the PEG-purified IgM was abrogated by pre-absorption with parasites in vitro and was affected by temperature. Furthermore, studies using 0 dpi IgM purified using gel permeation chromatography showed increased trypanocidal activity, with complete elimination of parasites after 12 h when incubated with 200 µg of 0 dpi IgM, or by 24 h when incubated with 80 µg or 100 µg of 0 dpi IgM. Lastly, in vivo passive transfer experiments demonstrated that while immune serum or purified IgM from 42 dpi serum conferred protection against a challenge, neither 0 dpi serum or 0 dpi purified IgM conferred protection against challenge with T. carassii.

Administration of recombinant parasite beta-tubulin to goldfish (Carassius auratus L.) confers partial protection against challenge infection with Trypanosoma danilewskyi Laveran and Mesnil, 1904.

The infection of carp and other cyprinid fish with Trypanosoma danilewskyi was reported to cause significant morbidity and mortality in aquaculture. Tubulin is a component of parasite excretory/secretory (ES) products recognized by antibodies present in the serum of recovered hosts. To assess the role of parasite tubulin in the induction of a protective immune response in the goldfish, recombinant T. danilewskyi beta-tubulin was produced in Escherichia coli and used to immunize goldfish against challenge with live parasites. Affinity purified rabbit anti-recombinant tubulin IgG bound to both surface and internal structures of trypanosomes, and when added to parasite cultures caused a dose-dependent inhibition of their growth in vitro. Immunization of goldfish i.p. with either 40 microg or 80 microg of endotoxin-free beta-tubulin+Freund's complete adjuvant (FCA) caused a significant decrease in parasitemia during the establishment phase of the infection (days 3 and 7) and increased the time required to reach the maximal mean number of parasites compared to non-immunized sham-injected control fish. The serum from immune fish contained antibodies that recognized trypanosomes as determined by confocal immunofluorescence microscopy and specific antibodies that recognized recombinant tubulin as measured by ELISA. Thus, the immunization of goldfish with recombinant parasite beta-tubulin conferred partial antibody-mediated protection against a challenge infection with live trypanosomes. This is a first report that parasite tubulin is immunogenic in poikilothermic vertebrates.

Antibodies that recognize alpha- and beta-tubulin inhibit in vitro growth of the fish parasite Trypanosoma danilewskyi, Laveran and Mesnil, 1904.

We have previously demonstrated that immunization of goldfish with excretory/secretory (ES) products of Trypanosoma danilewskyi in conjunction with Freund's complete adjuvant protected naïve goldfish from challenge with live parasites. In the present study, we report that T. danilewskyi alpha- and beta-tubulin, which were identified in ES products, induce an immunological response in cold-blooded hosts such as fish. The cDNA sequences of both the T. danilewskyi alpha- and beta-tubulin subunits were determined and subsequently cloned into a prokaryotic expression vector for production of recombinant proteins in Escherichia coli. The coding sequences for the trypanosome alpha- and beta-tubulin were 1353 and 1326bp, respectively, resulting in production of recombinant proteins of M(r) 55-60kDa. An affinity-purified rabbit IgG raised against the recombinant alpha-tubulin recognized recombinant alpha- and beta-tubulin, and native T. danilewskyi tubulin. Incubation of cultured parasites with greater than 40microg of purified rabbit-anti-recombinant alpha-tubulin IgG in vitro caused a dramatic inhibition of parasite growth after as little as 24h of cultivation. Specific anti-tubulin antibodies were responsible for the inhibition of parasites in culture since incubation with similar concentrations of an isotype control had no effect on parasite growth in vitro after 7 days of cultivation. Our results indicate that T. danilewskyi tubulin was antigenic to goldfish, and since antibodies to trypanosome tubulin have a marked effect on trypanosome growth in vitro, that tubulin may be one of the target molecules for control of trypanosomiasis in fish.

Comparison of select innate immune mechanisms of fish and mammals.

The study of innate immunity has become increasingly popular since the discovery of homologs of many of the innate immune system components and pathways in lower organisms including invertebrates. As fish occupy a key position in the evolution of the innate and adaptive immune responses, there has been a great deal of interest regarding similarities and differences between their defense mechanisms and those of higher vertebrates. This review focuses on describing select mechanisms of the innate immune responses of fish and the implications for evolution of immunity in higher vertebrates.