Viral infection risks for patients using the finished product Hirudo verbana (medicinal leech).

The virological safety of medicinal leeches has to be ensured prior to their use on patients. While leeches can be kept and bred under standardized conditions, feeding them horse blood adds a non-standardized component, which poses some risk of infection of the treated patients. Here, we investigated the speed at which blood-borne viruses are degraded by the microbial flora in the leech intestine, in order to define the safety of the product and the length of the necessary quarantine period prior to its administration to patients. Feeding blood was spiked with bovine viral diarrhea virus (BVDV), reovirus, and murine parvovirus (10(7) ID50 ml(-1)). The virus titer in the intestinal contents of the leeches was determined using permissive cell cultures and compared to that of the original virus titer at the following time points: immediately after feeding; after 3, 14, and 30 days; and monthly thereafter until the 7th month. The BVDV titer was below the detection limit of 10(1) TCID50 ml(-1) after 3 months, while reovirus and murine parvovirus titers were undetectable after 4 months. No positive virus findings were obtained at later time points. Thus, when fed the blood of vertebrates, the finished product "Medicinal leech, Hirudo verbana" can be considered virologically safe if the animals are maintained at 20 °C, which corresponds to their natural habitat conditions and ensures a high metabolic rate. Therefore, after the last feeding, a quarantine period of 4-6 months and appropriate care at room temperature, which supports microbial degradation and digestive processes, are recommended.

Heat stability of prion rods and recombinant prion protein in water, lipid and lipid-water mixtures.

Prion rods, i.e. insoluble infectious aggregates of the N-terminally truncated form of the prion protein, PrP 27-30, and the corresponding recombinant protein, rPrP(90-231), were autoclaved in water, bovine lipid or lipid-water mixtures for 20 min at temperatures from 100 to 170 degrees C. A protocol was developed for the quantitative precipitation of small amounts of protein from large excesses of lipid. PrP remaining undegraded after autoclaving was quantified by Western blot and degradation factors were calculated. The Arrhenius plot of the rate of degradation vs temperature yielded linear relationships for prion rods in water or lipid-water as well as for rPrP(90-231) in lipid-water. The presence of lipids increased the heat stability of prion rods, especially at lower temperatures. Prion rods had a much higher thermal stability compared to rPrP. Autoclaving of prion rods in pure lipid gave different results - not simple degradation but bands indicative of covalently linked dimers, tetramers and higher aggregates. The heat stability of prion rods in pure lipid exceeded that in lipid-water mixtures. Degradation factors larger than 10(4) were reached at 170 degrees C in the presence of lipids and at 150 degrees C in the absence of lipids. The linear correlation of the data allows cautious extrapolation to conditions not tested, i.e. temperatures higher than 170 degrees C. A factual basis for assessing the biological safety of industrial processes utilizing potentially BSE-or scrapie-contaminated animal fat is provided.