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The innate immune system relies upon a wide range of germ-line encoded receptors including a large number of immunoglobulin superfamily (IgSF) receptors. Different Ig-like immune receptor families have been reported in mammals, birds, amphibians and fish. Most innate immune receptors of the IgSF are type I transmembrane proteins containing one or more extracellular Ig-like domains and their regulation of effector functions is mediated intracellularly by distinct stimulatory or inhibitory pathways. Carp SITR was found in a substracted cDNA repertoire from carp macrophages, enriched for genes up-regulated in response to the protozoan parasite Trypanoplasma borreli. Carp SITR is a type I protein with two extracellular Ig domains in a unique organisation of a N-proximal V/C2 (or I-) type and a C-proximal V-type Ig domain, devoid of a transmembrane domain or any intracytoplasmic signalling motif. The carp SITR C-proximal V-type Ig domain, in particular, has a close sequence similarity and conserved structural characteristics to the mammalian CD300 molecules. By generating an anti-SITR antibody we could show that SITR protein expression was restricted to cells of the myeloid lineage. Carp SITR is abundantly expressed in macrophages and is secreted upon in vitro stimulation with the protozoan parasite T. borreli. Secretion of SITR protein during in vivo T. borreli infection suggests a role for this IgSF receptor in the host response to this protozoan parasite. Overexpression of carp SITR in mouse macrophages and knock-down of SITR protein expression in carp macrophages, using morpholino antisense technology, provided evidence for the involvement of carp SITR in the parasite-induced NO production. We report the structural and functional characterization of a novel soluble immune-type receptor (SITR) in a teleost fish and propose a role for carp SITR in the NO-mediated response to a protozoan parasite.

Like other vertebrate Toll-like receptors (TLRs), the TLRs of teleost fish can be subdivided into six major families, each of which recognize a general class of molecular patterns. However, there also are a number of Tlrs with unknown function, the presence of which seems unique to the bony fish, among which is Tlr20. We identified full-length complementary DNA (cDNA) sequences for tlr20 of zebrafish and common carp, two closely related fish species. Zebrafish have six copies of tlr20 , whereas carp express only a single copy. Both zebrafish Tlr20 (at least Tlr20a–d) and carp Tlr20 have 26 leucine-rich repeats (LRRs). Three-dimensional modeling indicates a best fit to the crystal structure of TLR8. Phylogenetic analyses place Tlr20 in the TLR11 family closest to Tlr11 and Tlr12, which sense ligands from protozoan parasites in the mouse. Conservation of genes on zebrafish chromosome 9, which carries tlr20 , with genes on mouse chromosome 14, which carries tlr11 , indicates Tlr11 could be a possible ortholog of Tlr20. Confocal microscopy suggests a subcellular localization of Tlr20 at the endoplasmatic reticulum. Although in vitro reporter assays could not identify a ligand unique to Tlr20, in vivo infection experiments indicate a role for Tlr20 in the immune response of carp to protozoan parasites ( Trypanoplasma borreli ). Carp tlr20 is mainly expressed in peripheral blood leukocytes (PBL) with B lymphocytes, in particular, expressing relatively high levels of Tlr20 . In vitro stimulation of PBL with T. borreli induces an upregulation of tlr20 , supportive of a role for Tlr20 in the immune response to protozoan parasites.

Genes related to iron metabolism play an important role in inflammatory response. The objective of this study was to investigate the role of ferritin, transferrin receptors 1a and 1b, and transferrin genes in the response to blood parasite infection in common carp ( L.). Two genetically distinct carp groups were used: R3 carp, which are established as being sensitive to parasitic infection, and SA carp ( ) of wild origin. An established challenge model with was applied. Challenged carp were sampled to determine their expression levels of transferrin receptors 1a and 1b, ferritin, and transferrin mRNA. Mortality and serum iron concentration were also measured. The study revealed contrasting differences in the expression profiles of all key iron regulatory genes except the transferrin gene. In the case of other parameters, significant differences were also observed. Our results demonstrate that the level of parasitic infection depends on the blood iron status. This parameter was related to the origin of the fish.

Trypanoplasma borreli and Trypanosoma carassii are kinetoplastid parasites infecting cyprinid fish. We investigated the role of nitric oxide (NO) in immune modulation during T. borreli and T. carassii infection of carp. Phagocytic cells from different organs produced NO and serum nitrate levels increased, demonstrating that T. borreli activates NO production in vivo. In contrast, T. carassii did not induce NO production in vivo and inhibited LPS-induced NO production in vitro. Production of NO was detrimental to the host as T. borreli-infected carp treated with the inducible NO synthase inhibitor aminoguanidine had a higher survival than infected control carp. This detrimental effect can be explained (in part) by the toxicity of NO to cells in vitro as NO inhibited the proliferative response of blood and spleen leukocytes. Head-kidney phagocytes were resistant to the immunosuppressive effects of NO in vitro. The NO-inducing activity of T. borreli may be an adaptation developed to ensure survival and immune evasion in the fish host. Apparently, T. carassii has adopted another strategy by deactivating specific functions of phagocytes. Both strategies may ensure long-term survival of the parasite.

In this work, we present the sequences and a comparison of the glycosomal GAPDHs from a number of Kinetoplastida. The complete gene sequences have been determined for some species (Crithidia fasciculata, Herpetomonas samuelpessoai, Leptomonas seymouri, and Phytomonas sp), whereas for other species (Trypanosoma brucei gambiense, Trypanosoma congolense, Trypanosoma vivax, and Leishmania major), only partial sequences have been obtained by PCR amplification. The structure of all available glycosomal GAPDH genes was analyzed in detail. Considerable variations were observed in both their nucleotide composition and their codon usage. The GC content varies between 64.4% in L. seymouri and 49.5% in the previously sequenced GAPDH gene from Trypanoplasma borreli. A highly biased codon usage was found in C. fasciculata, with only 34 triplets used, whereas in T. borreli 57 codons were employed. No obvious correlation could be observed between the codon usage and either the nucleotide composition or the level of gene expression. The glycosomal GAPDH is a very well-conserved enzyme. The maximal overall difference observed in the amino acid sequences is only 25%. Specific insertions and extensions are retained in all sequences. The residues involved in catalysis, substrate, and inorganic phosphate binding are fully conserved, whereas some variability is observed in the cofactor-binding pocket. The implications of these data for the design of new trypanocidal drugs targeted against GAPDH are discussed. All available gene and amino acid sequences of glycosomal GAPDHs were used for a phylogenetic analysis. The division of the Kinetoplastida into two suborders, Bodonina and Trypanosomatina, was well supported. Within the letter group, the Trypanosoma species appeared to be monophyletic, whereas the other trypanosomatids form a second clade.

The course of parasitemia of cloned Trypanoplasma borreli in laboratory-infected common carp was investigated. In 25-42-g carp kept at 20 C, the prepatent period was 8 days; after a phase of exponential growth, the parasitemia peaked at day 39 postinjection (PI) at a level of about 103 T. borreli/μl blood. This maximum was followed by a chronic phase of about 6 wk with large numbers of T. borreli. At 20 wk PI, T. borreli was absent in infected carp. In 2.2-g carp kept at 20 C, the prepatent period was 4 days only, and the parasitemia peaked at day 23 PI. At 30 C, T. borreli was present in the blood only for 12 wk, and the number of T. borreli did not exceed 162 trypanoplasms/μl blood. Carp kept at 8 and 15 C showed retarded development of parasitemia. The prepatent period lasted longer and the generation time was increased, but the level of parasitemia was not affected. Carp, inoculated at 8 C and then warmed to 20 C on days 27 and 55 PI, developed a parasitemia of 104 flagellates/μl blood and showed high mortalities. During the prepatent period, T. borreli was found in the muscle tissue of the inoculation area but in no other tissue. In the kidney, T. borreli was found 27 hr PI, whereas in the circulating blood it was manifest at day 3 PI. At the same time it was manifest in the liver and spleen.

The development of Trypanoplasma borreli in the crop of the leech vector Piscicola geometra was characterized by significant changes in morphology. Immediately after ingestion by the leech, stumpy-shaped T. borreli predominated and numerous dividing specimens were found. This led to long and slender trypanoplasms near the end of the infection. The infection was terminated with complete digestion of the blood stored in the crop of the leech. The longest period of infection observed was 11 days. Trypanoplasma borreli was found only in the crop of the leech. At any time during the infection, T. borreli isolated from P. geometra cause a parasitemia when inoculated into parasite-free carp. There was no difference in morphology or infectivity among T. borreli isolated from various crop regions of P. geometra.