A novel membrane molecule, previously observed to be co-isolated with lipophosphoglycan and called lipophosphoglycan-associated protein, has been detected in Leishmania donovani promastigotes and amastigotes. This kinetoplastid membrane protein (KMP-11) has been purified by preparative SDS/PAGE after organic solvent extraction of promastigote membranes. Isoelectric-focusing experiments indicated that this was an acidic protein with an isoelectric point of 4.8. Immunoblot analysis of subcellular fractions, together with 125I-labelling experiments, showed this molecule to be associated with the promastigote cell surface membrane. KMP-11 was expressed at a copy number similar to that of lipophosphoglycan (1 x 10(6)-2 x 10(6) molecules per cell), making this glycoprotein one of the major features on the parasite cell surface. The primary structure, less a blocked N-terminal region, was determined by automated Edman degradation of peptides derived from CNBr or enzymic fragmentation. Several post-translational modifications were also found during these studies, including an O-linked oligosaccharide and an NG-monomethylarginine functionality which was verified by m.s. Finally, a set of sequential synthetic peptides was made based on the established partial sequence allowing structural determination of two distinct antibody-binding sites for the monoclonal antibodies L98 and L157.

Atlantic salmon, Salmo salar L., were exposed to Kudoa thyrsites (Myxozoa, Myxosporea)-containing sea water for 15 months, and then harvested and assessed for parasite burden and fillet quality. At harvest, parasites were enumerated in muscle samples from a variety of somatic and opercular sites, and mean counts were determined for each fish. After 6 days storage at 4 degrees C, fillet quality was determined by visual assessment and by analysis of muscle firmness using a texture analyzer. Fillet quality could best be predicted by determining mean parasite numbers and spore counts in all eight tissue samples (somatic and opercular) or in four fillet samples, as the counts from opercular samples alone showed greater variability and thus decreased reliability. The variability in both plasmodia and spore numbers between tissue samples taken from an individual fish indicated that the parasites were not uniformly distributed in the somatic musculature. Therefore, to best predict the probable level of fillet degradation caused by K. thyrsites infections, multiple samples must be taken from each fish. If this is performed, a mean plasmodia count of 0.3 mm(-2) or a mean spore count of 4.0 x 10(5) g(-1) of tissue are the levels where the probability of severe myoliquefaction becomes a significant risk.

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Quantitative PCR (QPCR) methods targeting the 18S rDNA gene (DNA QPCR) and cathepsin L mRNA (RNA QPCR) from Kudoa thyrsites (Gilchrist) were developed and compared with histology for determination of K. thyrsites infection levels in Atlantic salmon Salmo salar L. Both QPCR tests were specific, reproducible and sensitive down to 3 copies. DNA QPCR was able to detect lower K. thyrsites infection levels than those detected by RNA QPCR and histology. The higher sensitivity of the DNA-based test compared with the RNA-based test appeared to be biological in nature and suggested that when infection levels were low, there were fewer copies of cathepsin L mRNA than 18S rDNA genes. However, all 3 diagnostic methods were highly correlated. Regression analyses comparing DNA QPCR and histology data from 2 distinct groups of fish showed that the relationship between these 2 diagnostic methods was reproducible. A logistic regression analysis comparing diagnostic data with a visual assessment of post-mortem flesh quality indicated that histology was the single best predictor of flesh quality, followed by DNA QPCR and then RNA QPCR. KEY WORDS: Kudoa thyrsites · Atlantic salmon · Diagnostics · QPCR · RNA test · DNA test · Histology · Myoliquefaction Resale or republication not permitted without written consent of the publisherDis Aquat Org 75: [239][240][241][242][243][244][245][246][247][248][249] 2007 stage, it is difficult to discern biological and environmental factors affecting parasite abundance in the environment and, ultimately, the risk to farmed salmon. Efforts have been made to understand factors affecting the susceptibility of Atlantic salmon to K. thyrsites infections (Funk et al. 2004), but in the absence of a challenge model this type of work is also difficult. At present, management of the infection relies on site selection, optimising smolt quality and monitoring stock for the presence of myxospores. Therefore, development of an efficacious vaccine is arguably the most promising method with which to minimize the impacts of K. thyrsites on fillet quality. To facilitate vaccine development, improved diagnostics are required for measuring K. thyrsites infection levels.Kudoa thyrsites infection levels are routinely determined through examination of stained histological sections, and this measure has been shown to be a relatively good predictor for post-mortem flesh quality (Dawson-Coates et al. 2003). However, histology lacks the sensitivity required to quantify low-level infections and requires a minimum seawater exposure period of 900 degree days before plasmodia can be detected reliably (Moran et al. 1999b). Therefore, histological methods as a measure of vaccine efficacy preclude evaluation of low infection levels and prevent rapid evaluation of newly developed vaccine candidates.Standard PCR is routinely used to detect the Kudoa thyrsites small subunit ribosomal DNA (18S rDNA) (Hervio et al. 1997) and has detected K. thyrsites in smolts after sea water exposures of only 350 degree days (V. Funk unpu...

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