Evaluation of truncated LipL32 expressed by Escherichia coli and Pichia pastoris for serodiagnosis of Leptospira infection in rodents

The applicability of the recombinant LipL32 for serodiagnosis of leptospiral infection in field rodents was assessed in this study. An immunodominant region of LipL32 was determined by monoclonal antibodies, and then, truncated LipL32 (tLipL32) was designed to contain the region (87–188th amino acid). The tLipL32 was compared between two recombinant expression hosts Escherichia coli and Pichia pastoris in ELISA. With field rat sera, tLipL32 expressed by P. pastoris (tLipL32p) had high antigenicity without background reactions, while tLipL32 expressed by E. coli (tLipL32e) showed high background reactions, which were reduced by pre-adsorption of sera with E. coli. To evaluate tLipL32-ELISA, field rat sera were tentatively divided into a Leptospira infection positive (12 sera) and a negative group (12 sera) based on the results from flaB gene PCR of kidney samples and WB with whole Leptospira cell. Consequently, the sensitivity of tLipL32p-ELISA for field rat sera was 83% . A similar result was obtained from tLipL32e-ELISA with adsorbed sera, (92%). However, sensitivity of tLipL32e-ELISA using sera without an adsorption treatment was 50%. Regardless of the expression host, tLipL32-ELISA had 100% specificity and sensitivity in experimentally infected laboratory rats. These results suggest that recombinant LipL32 expressed by P. pastoris is more applicable for serodiagnosis in field rats due to a lack of background reaction.     

Determination of plasma vitamin C concentration in fattening cattle

Plasma vitamin C (ascorbic acid + dehydroascorbic acid) concentration is a good index of the nutritional status of vitamin C. However, the methodologies for storage and analyses have not been investigated in bovine plasma. The validity of an analytical method for bovine plasma using high performance liquid chromatography (HPLC) with a spectrophotometric detector was examined. Exogenous dehydroascorbic acid was almost completely converted to ascorbic acid during the preparation for analysis with a reducing reagent, dithioerythritol. The analytical recoveries of ascorbic acid were high. Ascorbic acid was not detected after treatment with ascorbic acid oxidase. Thus, the specificity of this method is considered to be high. Although vitamin C was stable in plasma treated by dithioerythritol at ?20°C for 6 days, vitamin C in untreated plasma significantly decreased during 3‐day storage at ?20°C. These results indicate that the HPLC method is suitable for the determination of plasma vitamin C in cattle and that the storage conditions are important for determination of plasma vitamin C. Plasma vitamin C concentration ranged between 1.49 mg/L and 3.33 mg/L in fattening cattle. This result suggests that fattening cattle show large individual variation in plasma vitamin C concentration.