The Linear Behaviour of Pathogen Strain of Bacillus anthracis A0843 in Anthrax Subcutaneous Challenge on Rabbit Model

Background: The pathogen strain of Bacillus anthracis A0843, isolated during an anthrax outbreak occurred in Italy, belongs to the Cluster A1a genotype 3. The authors show its activity underlining that the regular behaviour could make it useful as a reference strain for subcutaneous challenge in rabbit model for anthrax vaccines efficacy test. Italy doesn't use Ames strain because the restrictive measures, imposed after the bioterroristic events occurred in October 2001 in USA, reduced the movements of pathogen agents between reference laboratories in the world. It is necessary to adopt new rules that favour the security and the regularity of the research. Method: This study was done, during 3 years, on 50 New Zeeland rabbits, males and females, with a weigh between 1.200 and 1500 grams. The site of injection was back in the space between the two scapulae. It was used 20 LD50 (about 40.000 spores) of the pathogen strain according to the European Pharmacopoeia. Results: It was observed that anthrax begins to kills after 48 hours from the infection. At 72 hours the percentage of survival is 56,66%; at 96 hours is 30%. It was observed that two animals that survived after 120 hours from infection didn't die. Conclusion: The LD50 of B. anthracis strain A0843 in rabbit is 2.000 spores, less virulent then Ames strain which is characterized of a LD50 of about 1.200 spores. The standard amount of 20 DL50 (about 40.000 spores) of B. anthracis strain A0843 injected in subcutaneous area in rabbits shows a linear behaviour. The higher mortality is observed between 72 and 96 hours. All the animals died within 120 hours from the infection. None of the infected animals survived over this time and we consider it the survival line of anthrax subcutaneous challenge in rabbit. Technical support: Angela Aceti and Nicola Nigro Founds: Ricerca Corrente 2005 of Ministry of Health of Italy This research was done in according to the Decreto legislativo n.116/92 on animal welfar

Antigenic, Immunologic and Genetic Characterization of Rough Strains B.abortus RB51, B.melitensis B115 and B.melitensis B18

The lipopolysaccharide (LPS) is considered the major virulent factor in Brucella spp. Several genes have been identified involved in the synthesis of the three LPS components: lipid A, core and O-PS. Usually, Brucella strains devoid of O-PS (rough mutants) are less virulent than the wild type and do not induce undesirable interfering antibodies. Such of them proved to be protective against brucellosis in mice. Because of these favorable features, rough strains have been considered potential brucellosis vaccines. In this study, we evaluated the antigenic, immunologic and genetic characteristics of rough strains B.abortus RB51, B.melitensis B115 and B.melitensis B18. RB51 derived from B.abortus 2308 virulent strain and B115 is a natural rough strain in which the O-PS is present in the cytoplasm. B18 is a rough rifampin-resistan mutant isolated in our laboratory

Brucellosis Vaccines: Assessment of Brucella melitensis Lipopolysaccharide Rough Mutants Defective in Core and O-Polysaccharide Synthesis and Export

Background: The brucellae are facultative intracellular bacteria that cause brucellosis, one of the major neglected zoonoses. In endemic areas, vaccination is the only effective way to control this disease. Brucella melitensis Rev 1 is a vaccine effective against the brucellosis of sheep and goat caused by B. melitensis, the commonest source of human infection. However, Rev 1 carries a smooth lipopolysaccharide with an O-polysaccharide that elicits antibodies interfering in serodiagnosis, a major problem in eradication campaigns. Because of this, rough Brucella mutants lacking the O-polysaccharide have been proposed as vaccines. Methodology/Principal Findings: To examine the possibilities of rough vaccines, we screened B. melitensis for lipopolysaccharide genes and obtained mutants representing all main rough phenotypes with regard to core oligosaccharide and O-polysaccharide synthesis and export. Using the mouse model, mutants were classified into four attenuation patterns according to their multiplication and persistence in spleens at different doses. In macrophages, mutants belonging to three of these attenuation patterns reached the Brucella characteristic intracellular niche and multiplied intracellularly, suggesting that they could be suitable vaccine candidates. Virulence patterns, intracellular behavior and lipopolysaccharide defects roughly correlated with the degree of protection afforded by the mutants upon intraperitoneal vaccination of mice. However, when vaccination was applied by the subcutaneous route, only two mutants matched the protection obtained with Rev 1 albeit at doses one thousand fold higher than this reference vaccine. These mutants, which were blocked in O-polysaccharide export and accumulated internal O-polysaccharides, stimulated weak anti-smooth lipopolysaccharide antibodies. Conclusions/Significance: The results demonstrate that no rough mutant is equal to Rev 1 in laboratory models and question the notion that rough vaccines are suitable for the control of brucellosis in endemic areas.This work was funded by the European Commission (Research Contract QLK2-CT-2002-00918) and the Ministerio de Ciencia y Tecnología of Spain (Proyecto AGL2004-01162/GAN)

Rassegna dei saggi per la standardizzazione dei metodi di isolamento delle salmonelle da substrati diversi. Consuntivo delle prove effettuate sotto l'egida della Commissione scientifica veterinaria presso la CEE, nel periodo 1967-1988

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