Effect of Cryptosporidium parvum infection on the absorptive capacity and paracellular permeability of the small intestine in neonatal calves.

Cryptosporidium parvum is recognized as one of the most important pathogens causing enteritis and severe diarrhoea in calves up to 1 month of age. Although the infection may be responsible for some mortality, its impact is mainly associated with the impairment of intestinal functions and lower performance of animals. The aim of this study was to determine the effect of cryptosporidiosis on the intestinal functions in neonatal experimentally infected Holstein calves. Absorption tests with d-xylose and retinyl-palmitate, and the lactulose/mannitol test of intestinal permeability were simultaneously performed in 1-week intervals from challenge to full recovery. In infected animals, reduced intestinal absorptive capacity for both D-xylose and retinyl-palmitate was observed on day 7 post-infection (p.i.). At the same time, a more than 100% elevation of intestinal permeability was observed in the infected calves. All intestinal functions, except absorption of retinyl-palmitate, were significantly affected and changes were detected up to day 14 p.i. In contrast, results of all tests obtained on day 21 p.i. suggest full recovery of the infected intestine. Significantly, growth of the calves which had recovered from cryptosporidiosis was still affected between days 14 and 21 p.i.

Synthesis of hapten and conjugates of coumestrol and development of immunoassay.

3-O-Carboxymethylcoumestrol was prepared as the hapten for immunoassay by a partial alkylation of coumestrol with ethyl chloroacetate in acetone alkalized with potassium carbonate. 3-O-Ethoxycarbonylmethylcoumestrol was separated by column chromatography and finally was hydrolyzed with formic acid. 1H and 13C NMR data (APT, COSY, HMQC, and HMBC) revealed that the reaction was regioselective, as 3-O-ethoxycarboxymethylcoumestrol was the only monosubstituted derivative. The hapten was then conjugated to bovine serum albumin and used for immunization of rabbits. A radioimmunoassay (RIA) system was established based on the polyclonal antiserum and a 125I-labeled hapten-tyrosine methyl ester conjugate as the radioligand. Parameters of the RIA: sensitivity: 12 pg per tube, 50% intercept: 140 pg per tube, working range: 20-4000 pg per tube. The cross-reactivity of a panel isoflavonoid and lignan phytoestrogens was either negligible (e.g. formononetin 0.07%; biochanin A 0.06%) or not detectable at all. The major immunoreactive peak in HPLC fractions from an alfalfa extract had the same retention time as coumestrol standard and represented 94.8% of the signal. The remaining 5.2% of immunoreactivity was distributed between five minor peaks. We conclude that after the validation for particular matrices, the method will be a useful tool for analysis of coumestrol, especially in low volume and low concentration samples.