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S1 - P - 10 PRESENTATION OF THE RESULTS OF THE ANNUAL PROFICIENCY TEST FOR THE SEROLOGICAL DIAGNOSIS OF EQUINE INFECTIOUS ANEMIA CONDUCTED IN ITALY BETWEEN 2006-2010. Ricci I 1 , Gasperetti L. 1 , D'Alonzo A. 1 , Nardini R. 1 , Giusti C. 1 , Caprioli A. 1 , Forletta R. 1 . 1 National reference centre for equine infectious Anemia, Istituto Zooprofilattico Sperimentale Lazio e Toscana Keywords: Equine infectious anemia, agar gel immunodiffusion, proficiency test Introduction Equine infection anemia (EIA) is a viral infection of equidae, caused by a virus (EIAV) of the Lentivirus genus, Retroviridae family. Since 2006, Italy has adopted a national surveillance plan based on the serological screening of all animals older than 6 months except for meat horses. According to the Italian Regulations, the official confirmatory test is the agar gel immunodiffusion (AGID), which detects antibodies against the p26, a highly conserved and primary immunogenic structural protein of the virus (1). The serological analysis of the samples, collected for the surveillance plan, can only be performed by the network of the national official laboratories, belonging to the Isitituti Zooprofilattici Sperimentali, mostly operating according to the ISO IEC 17025 and annually participating to proficiency tests. In this context, since 2002, the national reference centre for equine infectious anemia (CRAIE) has conducted inter-laboratory agid proficiency tests (2) with an increase in the number of participating laboratories. The aim of this paper is to present and discuss the results of the proficiency tests conducted in Italy during the years 2006-2010 for the diagnosis of EIA using the AGID test. Materials & methods Over the period 2006-2010, 65 laboratories participated to the annual proficiency test, performing both methods prescribed for AGID for AIE: “the Coggins test” which uses two layers of agar (1.5% and 0.7% respectively and wells of 7mm in diameter and 3 mm apart) and the OIE prescribed by the OIE (3). The panel distributed to each laboratory was each year made up of 10 sera, 3 negatives and 7 positives, with different level of antibodies against the P26 antigen (high to weak positives). Only in 2008, the panel was constituted of 2 negative and 8 positive sera. Before dispatching the samples to each laboratory, they were subjected to homogeneity and stability tests for their reactivity (2), so as to correctly evaluate reproducibility and repeatability parameters. Statistical analysis was carried out using K Cohen statistic, which was calculated for each laboratory and for all laboratories gathered together (4). Results Of the 65 participating laboratories, 27 constantly had a K value equal to 1. The results of the different laboratories per year are reported in table 1. Table 1: K value results per year. Results of the 65 laboratories per year 2006 2007 2008 2009 2010 N. of laboratories 0.21-0.40 8 1 2 1 0 0.42-0.60 5 0 0 0 1 0.61-0.80 7 2 0 2 1 0.81-1 45 62 63 62 63 K Value On the other hand when considering the samples not correctly identified (K < 1) in both methods for all laboratories, those mostly misclassified were weak positive, 7.6%, against the 1.4% of the strong positive sera. When taking into account the concordance of samples for the two different methods, the percentages of error for different AGID methods were respectively 5.2 for the Coggins test and 3.8 for the OIE method. The K analysis conducted to evaluate the concordance among the different laboratories for each year is reported in table 2. The results using the two different AGID methods are not significantly different. Table 2: K value results for all raters per year using both AGID methods. K value results among the raters per year YEAR 2006 2007 2008 2009 2010 AGID Coggins 0.78 0.94 1 0.94 0.95 AGID OIE 0.77 0.98 1 0.97 0.98 Discussion & conclusions The constant increase of participating laboratories to the proficiency tests during the years is an important element for evaluating the efficiency of the diagnostic system at national level for detecting EIA infection. These results stress the importance of performing such trials to achieve efficient diagnostic standards for each official laboratory as well as for the national diagnostic network. Furthermore, the increase in the number of laboratories during the years, having a K value > 0.8, demonstrates an improvement of the diagnostic performances, as also the achievement of good quality standards, with very satisfactory reproducibility and repeatability parameters for both AGID tests, even if better for the AGID OIE. It is important to underline that some difficulties occurred in the interpretation of positive samples with lower levels of antibody against the p26 antigen (weak positive sera). This represents a problem especially because this error during routine performance is expected to be higher in consideration of an expected higher attention in reading a proficiency test. This may constitute a potential bias of the results obtained, generating the calculation of a better proficiency than the effective. In this respect it our opinion that a more sensible technique such as the ELISA should be introduced to improve the efficacy and efficiency of the surveillance system. The introduction of such a screening test, with its characteristic of high sensitivity, rapidity and objectiveness is surely a more useful instrument for the laboratories performing EIA diagnosis, especially within an official surveillance and control programme. Another important issue emerging from our results is that the laboratories having lower level of concordance were those testing fewer samples in their diagnostic routine. This further remarks the importance of the experience in the execution and interpretation of a test such as the AGID. References 1. Ordinanza 8 agosto 2010 Piano di sorveglianza nazionale per l'anemia infettiva degli equidi. G.U. Serie Generale n. 219 del 18 settembre 2010 2. Guidelines OIE 1998. Guidelines of the Office International des Epizooties for laboratory quality evaluation, for international reference standards for antibody assays and for laboratory proficiency testing. Rev. Sci. Tech. Off. Int. Epiz., 17 (2), 600-609 3. Manual of Diagnostic Tests and Vaccines for Terrestrial Animals 2010, Chapter 2 . 5 . 6 .Equine infectious anaemia 4. Langton S.D., Chevennement R., Nagelkerke N., Lombard B., 2002. Analysing collaborative trials for qualitative microbiological methods: accordance and concordance. International Journal of Food Microbiology 79: pp. 175-181 5. Charles J. Issel, R. Frank Cook (1993)REVIEW ARTICLE A review of techniques for the serologic diagnosis of equine infectious anemia J Vet Diagn Invest 5: 137-141
S1 - P - 11 ANTIMICROBIAL SUSCEPTIBILITY OF AVIBACTERIUM (HAEMOPHILUS) PARAGALLINARUM ISOLATES RECOVERED IN THE RUSSIAN TERRITORY A.V. Chernyshov 1 , O.I. Ruchnova 1 , O.P. B’yadovskaya 1 1 FGBI ARRIAH, Vladimir, Russian Federation Avibacterium paragallinarum, Haemophilus paragallinarum, Infectious Coryza, antimicrobial susceptibility Introduction One of the most widely spread respiratory infectious diseases is infectious coryza. The disease is caused by the Gramnegative bacterium of Avibacterium paragallinarum species previously known as Haemophilus paragallinarum (1, 2, 5). The disease belongs to highly contagious diseases. It is manifested by catarrhal inflammation of nasal mucosa and air cells, as well as by the swollen-head-like syndrome and in rare cases by pneumonia. Chicks and chickens are susceptible to infectious coryza. Economic losses are caused by the stunting of chicks (up to (10-40%), drop in egg production (up to 41%) and mortality (up to 10%). (1, 5) Avian infectious coryza is reported from many countries of the world (2, 5). Currently, different antibacterial preparations are used in industrial poultry production but the violation of requirements to their use can lead to the emergence of resistant bacterial strains. Therefore, study of susceptibility of microorganisms to antibiotics has a vital significance for the bacterial infection therapy. The present work was focused on the study of antimicrobial susceptibility pattern A. paragallinarum field isolates from poultry from the RF farms. Materials & methods Pieces of lungs, tracheal and infraorbital sinus washings received from poultry of different age between 2009-2011 were tested for the presence of infectious coryza agent. Thirty-five A. paragallinarum field isolates were used in the given study. A. paragallinarum was identified based on the results of studying morphological, cultural, biochemical and genetic properties of the isolates (1, 2, 5). Susceptibility to antimicrobial preparations was determined using Mueller-Hinton agar supplemented with 5% of sheep red blood cells by Kirby-Bauer disk diffusion test (4) using a kit of disks impregnated with different antibiotics (Research Center of Pharmacotherapy, St. Petersburg). The degree of resistance of the isolates was determined according to the instruction to the kit. Results During 2009-2011 224 samples of pathological materials from broiler chicks and chickens were tested for avian infectious coryza. Total 35 isolates of A. paragallinarum were isolated from chickens and broiler chicks including 57.1% of isolates from infraorbital sinus swabs, 34.3% - from lung and 8.6% - from tracheal swabs. The recovered bacteria possessed tinctorial, morphologic, cultural and biochemical properties typical of A. paragallinarum and consistent with Bergey’s manual of systematic bacteriology and works by P.J. Blackall [et al.] (1, 2). Results of the study of A. paragallinarum susceptibility to antibacterial preparations are presented in Table 1. It was established that most isolates of A. paragallinarum were characterized by a wide range of resistance to antibacterial preparations. In particular, to lincosamide group (clindamycin) (94.5%), tetracycline (tetracycline (82.9%), doxycycline (87.9%)), co-trimoxazole (trimethoprim) (82.9%), natural penicillins (benzylpenicillin) (82.9%), third generation cephalosporins (cefixime) (71.4 %), second generation fluoroquinolones (ofloxacin (60 %), ciprofloxacin (intermediate susceptibility 42.9 % of isolates), norfloxacin (51.4 %)) and macrolide group (erythromycin) (62.9 %). A. paragallinarum isolates had high susceptibility mainly to inhibitor-protected penicillins (amoxicillin clavulanate/amoxiclav) – 77.1% of strains, first and second generation aminoglycosides (gentamicin, amikacin respectively) and nitrofuran group (furadonin) – more than 60 %. Table 2. Antimicrobial susceptibility of A, paragallinarum isolates. Number of isolates in % of total number. R – resistant, S – susceptible, I – intermediate susceptibility. No. Antibiotic name S I R 1 Ofloxacin 34,3 5,7 60,0 2 Ciprofloxacin 31,4 42,9 25,7 3 Norfloxacin 34,3 14,3 51,4 4 Levofloxacin 34,3 22,9 42,9 5 Tetracycline 0,0 17,1 82,9 6 Doxycycline 12,1 0,0 87,9 7 Clindamycin 0,0 5,7 94,3 8 Trimethoprim 17,1 0,0 82,9 9 Benzylpenicillin 17,1 0,0 82,9 10 Amoxiclav 77,1 5,7 17,1 11 Ampicillin 28,6 25,7 45,7 12 Laevomycetin 62,9 17,1 20,0 13 Erythromycin 5,7 31,4 62,9 14 Gentamicin 60,0 22,9 17,1 15 Amikacin 65,7 22,9 11,4 16 Furadonin 57,1 25,7 17,1 17 Cefuroxime 48,6 17,1 34,3 18 Cefoperazone 48,6 22,9 28,6 19 Сefixime 11,4 17,1 71,4 Discussion & conclusions A high rate of resistance to erythromycin (62,9%) in our research according with Yuan-Man Hsu [et. al.] (1) results. He also marked the resistance of the majority of tested field strains to this preparation. Data on tetracycline resistance are similar to P. J. Blackall (4) investigations. Our investigation, on the contrary, demonstrated high percentage of isolates resistant to aminopenicillin, erythromycin and benzylpenicillin in contrast to the results presented by E.S. Vargas and H.R. Terzolo (10). In conclusion, A. paragallinarum isolates have wide resistance range to large spectrum of antimicrobials, in particular tetracyclines (82.9 %), lincosamides (94.3 %), co-trimoxazole (82.9 %), natural penicillines (82.9 %). The majority of bacteria were susceptible to aminoglycosides (57.1 %), laevomycetin (62.9 %), inhibitor-protected penicillins (77.1 %) and nitrofurans (57.1 %). These data on antibiotic resistance should be helpful in planning strategies for the control of A. paragallinarum infection in poultry. References 1. Blackall, P.J. (1989) The Avian Haemophili. Clin. Microbiology Reviews. Vol.2(3). P. 270-277. 2. Blackall, P.J. (1999) Infectious Coryza: Overview of the disease and new diagnostic options. Clin. Microbiology Reviews. Vol. 12, №4. P. 627- 632. 3. Hsu, Y.-M., Shieh, H.K., Chen, W.-H. [et al.] (2007) Antimicrobial susceptibility, plasmid profiles and haemocin activities of Avibacterium paragallinarum strains. Vet. Microbiology. Vol.124. P. 209–218. 4. Jorgensen, J.H., Turnidge, J.D. (2007) Susceptibility test methods: dilution and disk diffusion methods. In P. R. Murray, E. J. Baron, J. H. Jorgensen, M. L. Landry, and M. A. Pfaller (ed.), Manual of clinical microbiology, 9th ed. ASM Press, Washington, D.C. P. 1152–1172. 5. Vargas, E.S., Terzolo, H.R. (2004) Haemophilus paragallinarum: Etiology of infectious coryza. Vet. Mex. Vol. 35, №3. P. 245–259.
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2 nd CONGRESSOFTHEEUROPEAN ASSOCIAT
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Welcome Dear colleagues, Welcome to
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worked with many diagnostic compani
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Products and services for scientist
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16:4518.15 20:0023:00 [S1.]Oralpres
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Day4 Wednesday,4July2012 4 th sessi
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Oral presentations “General Sessi
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antigens or attenuated vaccines can
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conditions, e.g. Staphylococcus aur
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S1 - O - 1 ORAL FLUIDS - SAMPLE MAT
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S1 - O - 03 INTRODUCTION RATE OF A
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S4 - P - 01 MOLECULAR CHARACTERIZAT
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List of Abstracts (in order of numb
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S1-P-03 BALLAGI ANDREA S1-P-04 BENI
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S3-P-06 KELLER SELINA S3-P-07 KÖNI
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