The TNF superfamily (TNFSF) of proteins are cytokines involved in diverse immunological and developmental pathways. Little is known about their evolution or expression in lower vertebrate species. Bioinformatic searches of Zebrafish, Tetraodon, and Fugu genome and other teleost expressed sequence tag databases identified 44 novel gene sequences containing a TNF homology domain. This work reveals the following: 1) teleosts possess orthologs of BAFF, APRIL, EDA, TWEAK, 4-1BBL, Fas ligand, LIGHT, CD40L, RANKL, and possibly TL1A; 2) the BAFF-APRIL subfamily is enriched by a third member, BALM, unique to fish; 3) orthologs of lymphotoxins ␣ and ␤ were not clearly identified in teleosts and are substituted by a related ligand, TNF-New; 4) as many as four TRAIL-like genes are present in teleosts, as compared with only one in mammals; and 5) T cell activation ligands OX40L, CD27L, CD30L, and GITRL were not identified in any fish species. Finally, we characterize mRNA expression of TNFSF members CD40L, LIGHT, BALM, APRIL, Fas ligand, RANKL, TRAIL-like, and TNF-New in rainbow trout, Oncorhynchus mykiss, immune and nonimmune tissues. In conclusion, we identified a total of 14 distinct TNFSF members in fishes, indicating expansion of this superfamily before the divergence of bony fish and tetrapods, ϳ360 -450 million years ago. Based on these findings, we extend a model of TNFSF evolution and the coemergence of the vertebrate adaptive immune system.

Selective breeding of animals for increased innate resistance offers an attractive strategy to control disease in agriculture. However, this approach is limited by an incomplete knowledge of the heritability, duration, and mechanism(s) of resistance, as well as the impact of selection on the immune response to unrelated pathogens. Herein, as part of a rainbow trout broodstock improvement program, we evaluated factors involved in resistance against a bacterial disease agent, Flavobacterium psychrophilum. In 2005, 71 full-sibling crosses, weighing an average of 2.4 g, were screened, and resistant and susceptible crosses were identified. Naive cohorts were evaluated at 10 and 800 g in size, and most maintained their original relative resistant or susceptible phenotypes, indicating that these traits were stable as size increased >300-fold. During the course of these studies, we observed that the normalized spleen weights of the resistant fish crosses were greater than those of the susceptible fish crosses. To test for direct association, we determined the spleen-somatic index of 103 fish crosses; created high, medium, and low spleen-index groups; and determined survival following challenge with F. psychrophilum or Yersinia ruckeri. Consistent with our previous observations, trout with larger spleen indices were significantly more resistant to F. psychrophilum challenge; however, this result was pathogen-specific, as there was no correlation of spleen size with survival following Y. ruckeri challenge. To our knowledge, this is the first report of a positive association between spleen size and disease resistance in a teleost fish. Further evaluation of spleen index as an indirect measure of disease resistance is warranted.

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Chemokines play important roles in controlling leukocyte trafficking under normal and inflammatory conditions. Sixteen CXC chemokines have been identified in the human and mouse genomes, while considerably fewer teleost fish CXC chemokines have been reported. Here, we describe a novel clade of trout (Onchorynchus mykiss) CXC chemokines, designated Onmy CXCd, and we identify a novel gene, CXCd1, and a putative duplicate, CXCd2. The trout CXCd proteins contain 112 amino acids and the CXCd1 gene is comprised of four exons and three introns. Constitutive CXCd mRNA expression was detected in skin, gill, visceral fat, and posterior kidney tissues, while low transcript levels were present in the anterior kidney and spleen. Spleen CXCd transcript abundance increased 1 day after bath vaccination (fourfold) and subsided to basal levels by 7 days postvaccination. Challenge with viable Yersinia ruckeri induced expression of trout CXCd RNA up to ninefold in the spleen. The number of viable Y. ruckeri were significantly correlated with CXCd gene transcript abundance (P = 0.0051, Spearman correlation 0.497, n = 30 fish), and fish with the highest bacterial loads had the highest CXCd expression. In contrast, pro-inflammatory cytokine IL-1-beta2 mRNA levels were elevated in fish infected with low numbers of Y. ruckeri, while diminishing in heavily infected fish. CXCd mRNA expression was not increased in rainbow trout infected with infectious hematopoietic necrosis virus, suggesting that up-regulation may be pathogen-specific. Taken together, these results indicate that CXCd transcript elevation follows the pro-inflammatory cytokine response to Y. ruckeri and may be a relevant immunological marker of exposure.