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Dolphin morbillivirus (DMV) is a pathogen of great concern in free-ranging cetaceans. Confirmation and staging of morbillivirus infections rely on histology and immunohistochemistry (IHC), following molecular detection. As at the present time no specific antibodies (Abs) against DMV are available, two heterologous Abs have been used worldwide for the examinations of morbillivirus infections of cetaceans. One is a monoclonal Ab (MoAb) prepared against the N protein of canine distemper virus (CDV), whereas the other is a polyclonal Ab raised in rabbits against rinderpest virus (RPV). Both Abs are known to show cross-reactivity with DMV. In this study we compared the labelling quality and the neuroanatomical distribution of staining with these two Abs by means of IHC analysis. To this end, serial sections of the target organs from ten free-ranging stranded cetaceans, previously diagnosed as being infected with DMV by PCR and/or serology, were subjected to IHC. The brain, lungs and lymph nodes of one animal were found to be positive with both Abs. From two other animals, the brain and the spleen, respectively, tested positive only with the polyclonal Ab. In the positive brain tissues, multifocal immunostaining was observed, with similar staining location and extent, with the two antibodies tested. Our results suggest that the polyclonal anti-RPV Ab might have a stronger binding activity to DMV than the anti-CDV MoAb. Nevertheless, the elaboration and use of specific anti-DMV Abs might be essential to guarantee conclusive results in diagnostic and pathogenetic investigations.

This study dealt with the toxicity of inactivated bacteria intended for veterinary autogenous vaccines toward a suitable control assay. Two in vitro methods were used. The [3-(4, 5 -dimethylthiazol-2-yl) -2,5 -diphenyltetrazolium bromide] (MTT) test, based on the metabolic reaction of a tetrazolium salt in vital cells, was adopted on the basis of previous positive results. The Interleukin (IL)-1 beta release assay on monocyte-derived pig macrophages was carried out for comparative purposes, to evaluate the possible role of the inflammatory response. MTT and IL-1 beta responses showed a significant correlation (P 0.05) at defined test dilutions of bacterial antigens, whereas no correlation was demonstrated using MTT responses normalized on bacterial cell concentration. Furthermore, the toxic effects shown in the MTT test were positively correlated to the extracellular protein content. On the whole, the above results could be a useful basis for the development of a toxicity assay on inactivated bacterial vaccines. Also, our data point at bacterial autolysis as a major component underlying toxicity.

Making the zebrafish embryo available to be able to share it between different laboratories groups could pave the way for this animal model with the aim of disseminating and promoting the use and application of zebrafish in scientific research.

According to the 3Rs principle (Replacement, Refinement, Reduction), this study aims to find alternative methods to evaluate the toxicity of autogenous vaccines. Currently in Italy the Istituti Zooprofilattici Sperimentali (II.ZZ.SS.) must perform the in vivo toxicity test for each lot of autogenous vaccine produced as laid down in the Decree of 17 March 1994. This paper describes two in vitro methods for assessing the toxicity of autogenous vaccines. The first is the MTT test based on the metabolic reaction of tetrazolium salt in vital cells. The second method is the test for measurement of IL-1ß production by macrophages, obtained after in vitro differentiation from pig monocytes in peripheral blood mononuclear cells. The two tests were performed on different vaccine antigens dilution: 1:20, 1:100 and 1:500. The results show a positive tendency between the two methods pointing out the potential of these methodologies combined for the replacement of the current in vivo test.