High-level production of extracellular chitinase in the absence of substrate is one of the most prominent features of the specialised crayfish-parasite A. astaci [26, 18]. The GH18 family-chitinase Chi1 was the first chitinase described for A. astaci . Here we selected two additional members of this gene family as targets for an A. astaci-specific diagnostic assay. GH18 chitinases can be divided into three clusters, two of which (A and B) differentiated before the appearance of the eukaryotic lineage . For example, fungal GH18 families comprise between one and twenty genes represented by members of all three clusters . We demonstrate the temporally regulated expression
of two novel members of the A. astaci-GH18 family. This functional
constraint was regarded as a basic criterion for the development of a closed-tube diagnostic method for qualitative and quantitative detection selleck Y-27632 mouse of A. astaci. In conclusion, simultaneously targeting multiple chitinase sequences including the novel, functionally constrained chitinase sequences, facilitates a robust analysis of clinical samples with a maximum reduced chance of false-negative detection. Results Strain identification Two putative A. astaci strains were recovered from healthy signal crayfish in two small streams in the GSK3235025 in vivo Austrian province of Burgenland (Gb04 – Ganaubach and Z12 – Zöbernbach). A third strain (GKS07) was isolated from the subabdominal cuticle of a moribund noble crayfish specimen collected during an acute crayfish-plague outbreak in the lake „Gleinkersee” (Austrian province: Upper Austria) in March PtdIns(3,4)P2 2007 (Table 1). ITS-sequence data and constitutive chitinase secretion specific for A. astaci (Additional file 1) confirm the assumed species assignment for all three strains. The strain Gb04 was used to identify two
new chitinase genes, test for their functional constraint and finally to develop the diagnostic assay for A. astaci. Table 1 Biological material used in this work. Species Isolate: reference Origin (year, location) Issue addressed A. astaci type 1 L1 Astacus astacus (1962, Sweden) CHI, MCA, TaqMan A. astaci type 1 Ra A. astacus (1973, Sweden) CHI A. astaci type 1 Sv A. astacus (1970, Sweden) CHI, MCA, TaqMan A. astaci type 2 Hö A. astacus (1974, Sweden) CHI, Chi activity, Western, PCR A. astaci type 2 Ti A. astacus (1970, Sweden) CHI A. astaci type 2 Yx A. astacus (1973, Sweden) CHI A. astaci type 3 Kv1 Pacifastacus leniusculus (1978, Sweden) CHI A. astaci type 4 Pc Procambarus clarkii (1992, Sweden) CHI A. astaci GB04 (CBS 121.537) P. leniusculus (2004, Ganaubach, Austria) CHI, PHYLO, RACE, GX, MCA, TaqMan A. astaci GKS07 (CBS 121.538) A. astacus (2007, Gleinkersee, Austria) PHYLO A. astaci Z12 (CBS 117.160) P. leniusculus (2004, Zöbernbach, Austria) PHYLO A.