RESUMO
Cysteine protease is a metabolic enzyme, whereas metalloprotease is the virulent factor in cryptobiosis caused by Cryptobia salmositica. Recombinant DNA vaccines were produced with the insertion of either the metalloprotease or cysteine protease gene of C. salmositica into plasmid vectors (pEGFP-N). As expected, fishes (Oncorhynchus mykiss and Salmo salar) injected intramuscularly with the metalloprotease-DNA (MP-DNA) vaccine (50 microg/fish) were consistently more anemic (lower packed cell volume, PCV) than controls (injected only with the plasmid) at 3-5 weeks post-inoculation. Also, there were no difference in PCV between fish injected with the cysteine-DNA plasmids and the controls. In addition, agglutinating antibodies against Cryptobia were detected only in the blood of MP-DNA-vaccinated fish at 5-7 weeks post-vaccination and not in cysteine-DNA plasmids and the control groups. MP-DNA-vaccinated fish when challenged with the pathogen had consistently lower parasitemia, delayed peak parasitemia, and faster recovery compared with the controls. All fish vaccinated with attenuated strain were protected when challenged with the pathogen; this positive control group confirmed that the two vaccines operate through different mechanisms.
Assuntos
Doenças dos Peixes/prevenção & controle , Kinetoplastida/imunologia , Metaloproteases/imunologia , Infecções Protozoárias em Animais , Vacinas Protozoárias , Vacinas de DNA , Animais , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/imunologia , Cisteína Endopeptidases/metabolismo , Doenças dos Peixes/parasitologia , Kinetoplastida/enzimologia , Kinetoplastida/genética , Kinetoplastida/patogenicidade , Metaloproteases/genética , Metaloproteases/metabolismo , Oncorhynchus mykiss , Parasitemia/parasitologia , Parasitemia/prevenção & controle , Parasitemia/veterinária , Infecções por Protozoários/parasitologia , Infecções por Protozoários/prevenção & controle , Vacinas Protozoárias/administração & dosagem , Vacinas Protozoárias/genética , Vacinas Protozoárias/imunologia , Salmo salar , Vacinação , Vacinas de DNA/administração & dosagem , Vacinas de DNA/imunologiaRESUMO
The present study describes the identification of a cathepsin L-like cysteine proteinase gene (CYS) from the hemoflagellate Cryptobia salmositica. Genomic DNA sequence of cysteine proteinase was obtained by genome walking using degenerate primers. Specific primers were designed to amplify the cDNA of cysteine proteinase from mRNA by rapid amplification of cDNA ends-PCR. The open reading frame of CYS is 1,329 bp, with 443 deduced amino acids. Based on the sequence analysis, cysteine proteinase of C. salmositica is similar to the cathepsin L-like cysteine proteinase of kinetoplastid parasites such as Leishmania spp. and Trypanosoma spp. The identification of CYS proteinase gene could help to design cysteine proteinase specific inhibitors. Further studies are required to characterize the complete genomic organization of the cysteine proteinase.