Abstract Book of EAVLD2012 - eavld congress 2012

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S1 - P - 12 COMPARISON BETWEEN ANALYTICAL SENSITIVITY OF RABIES VIRUS ISOLATION IN NEUROBLASTOMA CELL CULTURE WITH ANALYTICAL SENSITIVITY OF MOUSE INOCULATION TEST E.V. Chernyshova 1 , N.A. Nazarov 1 , A.E. Metlin 1 1 FGBI "Federal Centre for Animal Health"(FGBI "ARRIAH"), Vladimir, Russian FederationRabies, diagnosis, virus isolation in neuroblastoma cell culture, mouse inoculation test, analytical sensitivity Introduction Rabies is an acute viral zoonotic infection characterized by lesions of the central nervous system impairment which almost always results in death. The main scheme of rabies laboratory diagnosis in animals adopted in the Russian Federation (RF) (State Standard 26075- 84) consists of pathological material testing (brain tissue) using fluorescent antibody test (FAT). When FAT is negative mouse inoculation test (MIT) should be carried out (4). In laboratories with appropriate conditions and staff competence the OIE recommends using virus isolation in neuroblastoma cell culture instead of MIT (2). The reasons for such a recommendation are the following: Both methods are of high diagnostic sensitivity and specificity; Minimum and maximum time for diagnosis using virus isolation in neuroblastoma cell culture is from 4 to 12 days respectively and MIT performance takes from 12 to 30 days in average; It is necessary to confirm the results of MIT using FAT in addition; Virus isolation in neuroblastoma cell culture is more humane and less cost-consuming. In the EU countries when FAT is negative the virus isolation in cell culture is performed only in case of animal-human contact (France, Germany). In some countries (Lithuania) in case of absence of such contacts pathological materials are just passaged once in cell culture. Currently, many regional veterinary laboratories of the RF are capable of running diagnostic tests using cultural methods. Introduction the “Methodical instructions on rabies virus isolation in mouse neuroblastoma cell culture” into veterinary laboratory practice in the RF is of great importance. The study is aimed at the comparison of analytical sensitivity between methods of rabies virus isolation in cell culture and rabies virus isolation in white mice (1, 3). neuroblastoma cell culture. As for our experiments, the difference was 1 lg10. Thus, our results provide one more basis to replace a MIT the method of rabies virus isolation in mouse neuroblastoma cell culture in laboratories and research institutions dealing with rabies diagnosis. References 1. Anthony R. Fooks. New Diagnostic Tools for Rabies in Animals (2009) http://www.oie.int/eng/A_RABIES/presentations_rage/S21%20NewDiagno sticTools_ProfFooks.pdf 40 p. 2. OIE. Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. Vol. 1. – 6nd ed. - Paris, (2008). - Chap. 2.1.13. - p. 304-323. 3. Rabies General Aspects &Laboratory DiagnosticTechniques (2007) http://www.whoindia.org/LinkFiles/Communicable_Diseases_Rabies_- General_Aspects_&_Laboratory_Diagnostic_Techniques.pdf 72 p. 4. State Standard 26075-84 Methods of laboratory diagnosis for rabies (1984), 9 p. Materials & methods The following materials were used for the study: mouse neuroblastoma N-2a cell line; white outbred mice (6-8 g); mouse brain-adapted rabies virus (CVS strain; 5.66 lg TCID 50 /ml); antirabies fluorescent immunoglobulin produced by the FGBI “ARRIAH”. Analytical sensitivities of the methods were determined by titration of rabies virus CVS strain in mouse neuroblastoma N-2a cell line and in white mice. The titre was expressed as the highest virus dilution at which specific fluorescence in cell culture or deaths of mice were observed. Specific deaths of mice were confirmed by FAT. Results A 10% suspension of original mouse-brain-adapted CVS strain of rabies virus was prepared in RPMI medium for inoculation. Rabies virus was preliminary titrated using a series of tenfold dilutions of the prepared suspension. The highest dilution of the virus suspension with the specific fluorescence corresponded to 1.66 lg TCID50/ml titer. In order to determine sensitivity more accurately five twofold dilutions of the virus suspension with 1.66 lg TCID50/ml titer were prepared. Test results demonstrated that the last dilution with the specific fluorescence corresponded to 1.358 TCID50/ml titer. Seven twofold dilutions of the virus suspension with 2.66 TCID50/ml titer were prepared to determine sensitivity of the MIT. Test results demonstrated that the last dilution accompanied by specific deaths in mice corresponded to 2.358 TCID50/ml titer. Discussion & conclusions A conclusion was made after comparison of the obtained results that the MIT had a lower analytical sensitivity in comparison to analytical sensitivity of rabies virus isolation in mouse
S1 - P - 13 IDENTIFICATION OF GLOBICATELLA SANGUINIS ISOLATED FROM PNEUMONIA IN A GOAT M. Cocchi , S. Deotto, T. Di Giusto, M. Bregoli Istituto Zooprofilattico Sperimentale delle Venezie, Sezione territoriale di Udine, Basaldella di Campoformido (UD), Italy Globicatella sanguinis, pneumonia, goat Introduction Globicatella (G.) sanguinis was described in 1992. This bacterium is a catalase-negative, facultative anaerobic, gram positive coccus. It has been associated with various diseases in humans, such as bacteraemia, meningitis and infections of the urinary tract. In veterinary medicine, G. sanguinis was described in meningoencephalitis in lambs (1, 3). The aim of this study was to describe an episode of pneumonia in a goat produced by G. sanguinis. Materials & methods Two 1-year-old Tibetan goats out of a total of four goats belonging to a farm located in Friuli Venezia Giulia region (North east of Italy) showed unspecific clinical signs such as anorexia, depression, and permanent decubitus. These goats died after four days by the onset of the symptoms, and one of them was submitted for a necropsy. Bacteriological examinations were performed from lungs, brain and liver as follows: swabs were streaked onto blood agar base (BAB) and eosin-methylene blue agar (EMB). Brain Heart Infusion broth (BHI) was inoculated, too. Plates and tubes were incubated at 371°C for 48 hrs, in aerobiosis. BAB was incubated in anaerobic condition, too. From intestine a Perfringens agar base (PAB) other than EMB plate was used, incubated in anaerobiosis. It was obtained by using ANAEROGEN System, with an anaerobic indicator (Oxoid). Depending on morphology of the colonies, Gram staining, catalase and oxidase tests were performed. Miniaturized commercial tests were also used for identification. Agar plates and tubes were supplied by the service for media of the Istituto Zooprofilattico Sperimentale delle Venezie. Antimicrobial test was performed with disk diffusion method, in accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines (2). Tested antimicrobials were: penicillin (10 g, Becton Dickinson), ampicillin (10 g, Becton Dickinson), oxacillin (10 g, Becton Dickinson), tetracycline (30 g, Oxoid), sulfamethoxazoletrimethropim (23,75-1,25 g –Oxoid-) and cephalothin (30 g – Oxoid-). Reading was done using the established diameter for Streptococcus (S.) spp other than S. pneumoniae. Results Necropsy. Macroscopic lesions recovered from lungs referred to acute bronchopneumonia and tracheal and laryngeal oedema. Catarrhal enteritis and hepatomegaly were observed, too. Bacteriological tests. Cultures from lungs and liver revealed pure culture of -haemolytic, pin-pointed (2-3 mm of diameter), circular colonies. Subsequently, analyses revealed a gram positive, catalase-negative, coccus-shaped organism. Figure 1 shows the morphology of the bacterium after 24 hrs of incubation, on BAB. Biochemical identification was performed with a commercial kit, API rapid ID 32 Strep (Biomerieux). Both the gram positive strains showed identical biochemical profiles (22274063110), with a percentage of identification of 99.9%, T=0.92. Only fermentation of ribose resulted as an atypical test (negative in our reaction). Cultures from the intestine showed E. coli and Clostridium perfringens. Antimicrobial test. G. sanguinis revealed resistance to oxacillin, and to tetracycline, while it was sensitive to other tested drugs. Discussion & conclusions Pneumonia is one of the most common respiratory problems in small ruminants throughout the world. In goat herds, pneumonia increases production costs associated with expensive treatments. Infectious and non-infectious agents can cause inflammation of the lungs. In goats the most frequent causes of respiratory infection and death are Pasteurella multocida and Mannheimia haemolytica. They are commonly found in the upper respiratory tract of healthy goats, too. Transportation stress, viral infections, parasites, poor management conditions, increase goats' susceptibility to these agents of pneumonia. In our case, the identification of G. sanguinis in pure culture from diseased lungs could be indicative of the pathological role of this bacterium. The performed analysis on other organs revealed the presence of G. sanguinis in liver, too. Differences are in reference to the growth in different atmospheres: culture from lungs showed G. sanguinis both in aerobic and in anaerobic conditions. Cultures from liver revealed the presence of the bacterium only in aerobic incubation. The fact that all the goats in the farm showed identical clinical signs is strongly indicative that this bacterium was also responsible for the pathology in the other goats. In veterinary medicine, G. sanguinis was isolated in a case of suppurative meningoencephalitis in lambs (1). In our case macroscopic analysis revealed an acute bronchopneumonia. Any lesion was recovered from the brain. Regarding identification, in this paper we named the bacterium in accordance with the profile given by a miniaturized system. The phenotypic findings are consistent with those described for this bacterium species, except for pyrrolidonil arylamidase test. It is pertinent to note that G. sanguinis belongs to a broad range of Gram-positive, catalase-negative new taxa recently founded in human and animal clinical samples. Some authors revealed that misidentification can occur between Aerococcus viridans and G. sanguinis, because of very similar biochemical profiles (1,5). Moreover, looking into literature only a few number of isolates were tested, with some differences in the results (1,3). For these reasons, subsequent investigations using different methods, such as molecular genetic tools should accompany the identification of this bacterium. The improvement of the identification systems of these unusual gram-positive, catalase-negative bacteria may contribute to making progress in current epidemiological knowledge on host distribution and range of clinical conditions. Data about antimicrobial susceptibility test showed resistance varied significantly among the tested strains (5). In our report resistance to tetracycline does not agree with the data of Seegmuller et al (4). To the author’s knowledge this is the first report about G. sanguinis in an episode of pneumonia in a goat. References 1. Vela, A.I., Fernandez E., Las Heras A., Lawson, P.A., Dominguez L., Collins M.D., and Fernandez-Garayzabal J. (2000). Meningoencephalitis associated with Globicatella sanguinis infection in lambs. J Clin Microbiol vol 38, 4254-4255. 2. Clinical and laboratory standards institute (CLSI). M31-A3. Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals. Vol 28. n 8. 3. Collins, M.D., Aguirre, M., Faclam, R.R., Shallcross J., and Williams A.M. (1992) Globicatella sanguis gen.nov., sp. Nov., a gram-positive catalase-negative bacterium fropm human sources. J Appl. Bacteriol. 73:433-437. 4. Seegmuller I., Van der Linden M., Heeg C., and Reinert R.R. (2007). Globicatella sanguinis is ana etiological agent of ventriculoperitoneal shunt-associated meningitis. J Clinical Microbiol. Febb. 666-667. 5. Shewmaker, P.L., Steigerwalt, A.G., Shealey, L., Weyant, R., and Facklam, R. (2001). DNA relatedness, phenotypic characteristics, and antimicrobial susceptibilities of Globicatella sanguinis strains. J Clinic Microbiol. Nov 4052-4057. Figure 1: G. sanguinis after 24h incubation on blood agar base, in aerobic atmosphere
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2 nd CONGRESSOFTHEEUROPEAN ASSOCIAT
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Welcome Dear colleagues, Welcome to
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16:4518.15 20:0023:00 [S1.]Oralpres
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antigens or attenuated vaccines can
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S1 - O - 1 ORAL FLUIDS - SAMPLE MAT
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List of Abstracts (in order of numb
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