Proteinases released in vitro by the parasitic stages of the bovine abomasal nematode Ostertagia ostertagi

Abstract

Host tissue penetration, feeding and immune evasion by helminth parasites may be mediated by both mechanical processes and histolytic products released by the parasite. The aim of this study was to investigate potential histolytic products released during in vitro maintenance of exsheathed third (L3) and 4th larval stage (L4) and adult Ostertagia ostertagi. Therefore, the pH optima, substrate specificity, molecular size and inhibitor sensitivity of in vitro released (IVR) proteinases were analysed by spectrophotometry and substrate gel electrophoresis. It was shown that L3, L4 and adult IVR proteinases degrade a variety of protein substrates in a pH-dependent and stage-specific manner. At alkaline pH, gelatin, casein and fibrinogen were degraded by metallo- and serine proteinases. In contrast, mucin, fibrinogen, albumin and haemoglobin were degraded at acidic pH by aspartyl protease- and cathepsin L-like activity. At pH 3, the heavy chain of bovine IgG was completely degraded by an aspartyl proteinase secreted by all 3 parasitic stages. The specificity of the L3, L4 and adult Ostertagia ostertagi proteinases against the different substrates indicates variable functional requirements.

Vaccination against Ostertagia ostertagi with subfractions of the protective ES-thiol fraction

“…ASPs were visualized by adding 0.05% 3,3 diaminobenzidine tetrachloride in PBS containing 0.01% H 2 O 2 (v/v). Cysteine proteases were monitored by incubating the samples with synthetic Z-Phe-Arg-AMC as was done in a previously described cathepsin assay (Geldhof et al, 2000). Finally, the material was pooled into three different subfractions, i.e.…”

“…The presence of ASP was verified using the same Western blot as described above. Cysteine protease activity was detected by gelatin substrate gel under non-reducing conditions (Geldhof et al, 2000).…”

Gender-enriched transcription of activation associated secreted proteins in Ostertagia ostertagi

“…L3, exsheathed L3, L4 and adult parasites were collected and somatic extract and ES material were prepared as described previously by Geldhof et al (2000). Male and female adult O. ostertagi worms were harvested by opening the abomasum of an infected animal and placing it on a 220 lm mesh sieve in a 0.9% NaCl solution containing penicillin (1,000 U/ml) and streptomycin (1 mg/ml) at 37°C for 5 h. Male and female worms were subsequently separated under a microscope and used to produce gender-specific somatic extract and ES products.…”

“…Somatic extracts were made by suspending the worms in PBS and homogenising them in a glass homogenisator. ES products were made by culturing male and female worms for 3 days in RPMI medium containing the appropriate antibiotics at 37°C and 5% CO 2 (Geldhof et al, 2000). The culture medium was collected and dialysed against PBS and finally concentrated.…”

Expression and purification of an active cysteine protease of Haemonchus contortus using Caenorhabditis elegans

“…Alkaline phosphatase conjugated secondary antibodies (Molecular Probes) were used routinely at 1:3000 dilution. Substrate gel analysis was carried out essentially as described by Geldhof et al (2000), using 0.1% final concentration of gelatin (Heussen and Dowdle, 1980).…”