Identification of a serine protease gene expressed by Myxobolus cerebralis during development in rainbow trout Oncorhynchus mykiss.
Serine proteases have been recognized as key factors in parasite physiology and disease development. We have identified a serine protease gene from Myxobolus cerebralis, MyxSP-1, the myxozoan parasite causing whirling disease in salmonid fishes. The amino acid sequence, as deduced from the cDNA sequence, included a catalytic residue arrangement similar to that of the chymotrypsin family of serine proteases. A real-time TaqMan polymerase chain reaction (PCR) analysis revealed differences in the transcription levels for the chymotrypsin-like protease as found in early, intermediate, and late developmental stages of the parasite in experimentally-infected rainbow trout Oncorhynchus mykiss. MyxSP-1 transcription differed between individual tissues at each sampling point and in the same tissues over time (p < 0.0001). A nonradioactive mRNA in situ hybridization (ISH) protocol was developed to detect MyxSP-1 transcripts. Using a mixture of 3 digoxigenin-labeled antisense mRNA probes, MyxSP-1 transcription was observed in developmental stages of the parasite during the acute and chronic phases of the disease over a 240 d time period in infected rainbow trout tissues. MyxSP-1 transcription observed by ISH in cartilage and as associated with cartilage destruction was consistent with our real-time TaqMan PCR findings that demonstrated high levels of MyxSP-1 transcription during lesion development. Identifying genes encoding these enzymes and characterization of their functions can lead to the development of new chemotherapeutic protocols and vaccine approaches to control parasitic diseases.
Kelley
Garry O
GO
Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, California 95616, USA. gokelley@ucdavis.edu
Zagmutt-Vergara
Francisco J
FJ
Leutenegger
Christian M
CM
Adkison
Mark A
MA
Baxa
Dolores V
DV
Hedrick
Ronald P
RP
D000595:Amino Acid Sequence; D000704:Analysis of Variance; D000818:Animals; D001483:Base Sequence; D017124:Conserved Sequence / Q000235:genetics; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D056890:Eukaryota / Q000235:genetics* / Q000254:growth & development; D005393:Fish Diseases / Q000469:parasitology*; D006652:Histological Techniques; D017403:In Situ Hybridization; D008969:Molecular Sequence Data; D017686:Oncorhynchus mykiss; D011529:Protozoan Infections, Animal; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D012697:Serine Endopeptidases / Q000235:genetics* / Q000378:metabolism; D013997:Time Factors; D014158:Transcription, Genetic / Q000235:genetics*
D017931:DNA Primers; D018076:DNA, Complementary; D012697:Serine Endopeptidases