Third-stage larvae of the anisakid nematode Contracaecum osculatum infecting cod (Gadus morhua) liver elicit a host immune response involving both innate and adaptive factors, but the reactions differ between liver and spleen. Inflammatory reactions occur in both liver and spleen, but a series of immune effector genes are downregulated in liver infected with nematodes whereas these genes in spleen from the same fish are upregulated. A series of novel primer and probe sets targeting cod immune responses were developed and applied in a real-time quantitative polymerase chain reaction set-up to measure the expression of immune-relevant genes in liver and spleen of infected and uninfected cod. In infected liver, 12 of 23 genes were regulated. Genes encoding cytokines associated with inflammatory reactions (IL-1β, IL-6, IL-8) were significantly upregulated, whereas genes encoding effector molecules, assisting the elimination of pathogens, C-reactive protein (CRP)-PII, hepcidin, lysozyme G1, lysozyme G2, C3 and IgDm, were significantly downregulated. The number of downregulated genes increased with the parasite burden. In spleen, 14 of 23 immune genes showed significant regulation and nine of these were upregulated, including genes encoding CRPI, CRPII, C3, hepcidin and transferrin. The general gene expression level was higher in spleen compared to liver, and although inflammation was induced in nematode-infected liver, the effector molecule genes were depressed, which suggests a worm-induced immune suppression locally in the liver.

All organisms encounter pathogens, and birds are especially susceptible to infection by malaria parasites and other haemosporidians. It is important to understand how immune genes, primarily innate immune genes which are the first line of host defense, have evolved across birds, a highly diverse group of tetrapods. Here, we find that innate immune genes are highly conserved across the avian tree of life and that although most show evidence of positive or diversifying selection within specific lineages or clades, the number of sites is often proportionally low in this broader context of putative constraint. Rather, evidence shows a much higher level of negative or purifying selection in these innate immune genes – rather than adaptive immune genes – which is consistent with birds' long coevolutionary history with pathogens and the need to maintain a rapid response to infection. We further explored avian responses to haemosporidians by comparing differential gene expression in wild birds (1) uninfected with haemosporidians, (2) infected with Plasmodium and (3) infected with Haemoproteus (Parahaemoproteus). We found patterns of significant differential expression with some genes unique to infection with each genus and a few shared between ‘treatment’ groups, but none that overlapped with the genes included in the phylogenetic study.