RESUMEN
The shelf-life of a culture medium is the maximum period of validity for optimum preparation and preservation. Apart from the composition of the medium, the factors that influence shelf-life are sterilisation method, preservation and packaging procedures, storage temperature and exposure to light. The shelf-life of a culture medium is defined by evaluating its basic chemico-physical characteristics so as to obtain the correct growth and characterisation of a specific microorganism. This research was conducted from March to September 2003 on 12 'critical' culture media, i.e. media that had a coded shelf-life of not more than thirty days. Each medium was produced in three separate batches, with a total of 5 940 samples. The purpose of the study was to define a longer period of validity than that coded for each medium by evaluating weight reduction, pH, fertility and sterility. The shelf-life observed for each medium was longer than those coded. The new shelf-life takes into account both the operational needs of complex organisational structures and the efficiency of the medium, depending on its chemico-physical characteristics and storage and preservation methods.
RESUMEN
In the European Union, RB51 vaccine can be used only under strictly controlled conditions for the immunisation of cattle at risk of infection with Brucella abortus. A test is therefore necessary to distinguish vaccinated from unvaccinated animals. The complement fixation test with RB51 antigen (RB51-CFT), dot-blot and gamma-interferon used to identify vaccinated animals have been described, but sensitivity of the tests has been poor and positivity transient after calfhood vaccination. To avail of a rapid and accurate diagnostic tool, the authors produced, controlled and evaluated an experimental brucellin prepared from strain RB51 (RB51 brucellin). The potency of this brucellin was evaluated in guinea-pigs sensitised with RB51 and compared with a commercially available brucellin. Both allergens produced similar biological activity in guinea-pigs. The RB51 brucellin skin test was performed in 10 cattle 414 days after calfhood vaccination with RB51 when they were negative to the RB51-CFT. The skin test revealed 60% sensitivity (with a confidence interval of 95%, CI 30.8%-83.3%) and 100% specificity (CI 60.7%-100%). These findings limit the use of the skin test only for screening to detect RB51 vaccinated herds, not individual animals. Nevertheless, following intradermal inoculation of RB51 brucellin, a transient antibody increase to the RB51-CFT was observed, from day 9 to day 20 post inoculation with RB51 brucellin. This transient antibody increase, when evaluated in parallel with the RB51 brucellin skin test results, enables detection of individual vaccinated animals (sensitivity 100%; CI 76.2%-100%).
RESUMEN
The occurrence of bluetongue (BT) in Italy prompted an increase in disease surveillance. Thus a competitive enzyme-linked immunosorbent assay (c-ELISA) to detect immunoglobulins to BT virus (BTV) was developed and distributed amongst 27 laboratories comprising the Italian veterinary diagnostic laboratories network to screen field sera. This ring test enabled comparison of the results and the evaluation of the reproducibility of the method. The c-ELISA developed by the National Reference Centre for Exotic Diseases (c-ELISA-IZSA&M) was compared also against a commercially available c-ELISA. In addition, results obtained by the Centre of Athens Veterinary Institutions are presented.
RESUMEN
An inactivated vaccine was produced from an Italian field isolate of bluetongue virus serotype 2 (BTV-2) with a titre of 10(7.8)TCID50/ml. The virus was purified through a molecular cut cassette membrane, inactivated with beta-propriolactone and emulsified with ISA 206 (Seppic) adjuvant. The vaccine was then tested for sterility, toxicity and safety in laboratory and target animals according to European Pharmacopoeia standards. Immunogenicity was assessed by inoculating subcutaneously 10 sheep and 10 goats each with 2 ml of the vaccine and 10 bovines each with 5 ml of the vaccine. A booster dose was inoculated after 14 days and no side-effects were reported following vaccination. Fourteen days after the booster dose, all vaccinated animals developed virus neutralising (VN) bluetongue (BT) antibody titres that on day 60 post vaccination ranged between 1/20 and 1/1 280. After one year, goats still had high VN antibody titres. Sheep were challenged 138 days after vaccination by subcutaneously inoculating 1 ml of 10(5.6)TCID50/ml of an Italian field isolate of BTV serotype 2; four unvaccinated animals were also inoculated and used as controls. Starting from day 6 post challenge, control animals developed a fever, with temperature ranging from 39.9 degrees C to 40.6 degrees C and lasting 48 h on average. BTV-2 was also isolated from the blood of control animals between days 4 and 20 post challenge. Conversely, neither fever nor viraemia were detected in the vaccinated animals that were challenged. A new trial with a larger number of animals, including all target species, has been planned and is in progress.
RESUMEN
Liver regeneration has been studied in necrotic hepatitis of 21 rabbits infected with the haemorrhagic disease virus (VHD). Formalin fixed and paraffin embedded liver sections have been immunostained for the proliferation associated antigen PCNA (Proliferating Cell Nuclear Antigen-clone PC10) and counterstained with toluidine blue that enhances histologic recognition of mitoses. Hepatocyte and bile duct proliferative activity has been quantified, by means of image analysis, both as PCNA reactivity and mitotic activity. The results, compared with a semiquantitative estimation of liver necrosis, showed a positive correlation between hepatocyte proliferative activity and liver necrosis, both in acute and subacute hepatitis. In the chronic phase a residual proliferative activity appeared in bile duct cells.