Abstract Book of EAVLD2012 - eavld congress 2012

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S1 - P - 32 RELIABLE DETECTION AND TYPING OF PRRSV USING MULTIPLEX REAL-TIME RT-PCR Christine Gaunitz, Carsten Schroeder, Marco Labitzke, Eva V. Knoop, Jörg Gabert Labor Diagnostik GmbH Leipzig, Leipzig, Germany PRRSV, High Pathogenic, real-time RT-PCR, simultaneous detection Introduction Infections with the Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) in swine are very prevalent and economically most important for the swine industry. Clinical signs are respiratory disease in piglets and reproductive failure in pregnant sows. PRRSV is a single-stranded RNA virus of the family Arteriviridae, order Nidovirales, and classified into the European (EU/I) and North American (NA/II) genotype. These genotypes show only 55-70 % homology of nucleotides in the different genes. In 2006 in China a highly pathogenic (HP) NA-strain emerged, which shows a deletion of around 30 amino acids in the non-structural protein 2 (Nsp2). Infection with the HP-strain is characterized by high fever and high mortality in pigs of all ages. In the last years there have been detected also new isolates from Eastern Europe. Purpose of this study was to develop a real-time PCR which allows reliable detection and differentiation of PRRSV strains in one test run. Materials & methods To evaluate analytical sensitivity of VIROTYPE ® PRRSV, titration studies with in-vitro RNA were performed. Analytical specificity was proven by testing 12 PRRSV reference strains from cell culture. Diagnostic sensitivity was proven by testing the EPIZONE ring trial panel 2011. Serum, tissue and saliva samples were tested in comparison to other PCR assays. Two commercial PCR kits and VIROTYPE ® PRRSV (Labor Diagnostik GmbH Leipzig, Germany) were used according to the manufacturer´s recommendations. Results The high analytical sensitivity of the VIROTYPE ® PRRSV kit was proven by a titration series of in vitro RNA (PRRSV EU1/EU2/NA/HP [10 8 copies/well to 10 copies/well]), performed in triplicates of ten-fold dilutions. A high correlation between number of copies and amount of amplification product was demonstrated in the range of 10 8 to 10 3 and 10 8 to 10 2 copies, respectively (Fig. 1). PRRSV EU I PRRSV NA Figure 1: Evaluation of analytical sensitivity. PRRSV EU II PRRSV HP The analytical specificity was proven by testing a RNA-panel of 12 PRRSV reference strains comparatively with VIROTYPE ® PRRSV and two commercial available tests (Test I and Test II). All reference strains were detected well with the VIROTYPE ® PRRSV. In comparison to Test II, the VIROTYPE ® PRRSV kit detects the EU strains around two Ct-values and the NA strains around five Ct-values more sensitive. The results of commercial test I and VIROTYPE ® PRRSV are comparable (Tab. 1). Table 1: Evaluation of analytical specificity. VIROTYPE ® PRRSV commercial Test I commercial Test II Strain EU NA HP IC PRRSV EU NA IC EU NA IC Ct FAM Ct TEX Ct Cy5 Ct HEX Ct FAM Ct FAM Ct FAM Ct HEX Ct FAM Ct Cy5 Ct HEX EU Stendal V953 25.22 - - 34.61 24.62 24.08 - 29.89 24.95 - 35.66 Intervet 22.02 - - 33.22 20.41 19.41 - 28.85 24.65 - - Cobbelsdorf 23.86 - - 32.65 22.91 22.20 - 29.32 26.02 - - Stendal V852 23.24 - - 39.96 23.50 21.86 29.68 26.16 - 36.98 Stendal V1904 24.30 - - 39.06 24.48 23.47 29.30 27.63 - 37.41 Stendal V1952/97 26.29 - - 35.41 26.32 24.51 31.32 28.12 - 36.06 Stendal V1445/99 - 21.39 - 34.85 23.80 - 21.17 30.78 - 24.92 34.10 USA VD28775/2 - 21.35 - 29.14 23.94 - 21.61 30.40 - 24.07 39.08 USA VD29949/17 - 22.41 - 28.48 27.72 - 22.18 29.65 - 26.89 - USA 2 - 19.83 - 30.64 23.07 - 20.44 30.81 - 23.64 - USA 18 - 19.06 - 31.48 20.47 - 19.50 30.39 - 26.54 - China - 20.87 22.44 28.36 22.82 - 20.54 29.89 - 25.84 - Mean Value EU 24.16 23.71 22.59 26.26 Mean Value NA 20.82 23.64 20.91 25.32 In comparison to an in-house PCR (Kleiboeker mod.) performed at Friedrich-Loeffler-Institute, VIROTYPE ® PRRSV detects all samples of the Epizone Ring Trial 2011 more sensitive. VIROTYPE ® PRRSV reliably detects the samples of the EU-2, EU-3, EU-4 and the atypical EU genotype 1 (Tab. 2). Table 2: Results from the EPIZONE ring trial panel 2011. VIROTYPE® PRRSV Kleiboeker (mod.) PRRSV-strain Genotyp RNA Dil. EU NA HP IC EU NA EU Stendal V954 EU-1 10 -2 24.96 - - 26.88 28.24 - EU Stendal V954 EU-1 10 -3 28.41 - - 27.06 31.53 - EU Stendal V954 EU-1 10 -4 31.61 - - 26.91 34.12 - EU Stendal V954 EU-1 10 -5 34.63 - - 27.14 37.61 - EU Stendal V954 EU-1 10 -6 - - - 28.11 - - EU Stendal V1952/97 EU-1 10 -1 23.08 - - 27.23 25.82 - EU Stendal V1952/97 EU-1 10 -2 26.57 - - 27.66 29.17 - EU Stendal V1952/97 EU-1 10 -3 29.54 - - 26.99 32.19 - EU Stendal V1952/97 EU-1 10 -4 32.50 - - 26.86 35.25 - EU Stendal V1952/97 EU-1 10 -5 36.92 - - 27.87 40.31 - Cobbelsdorf EU-1 1:500 23.33 - - 27.26 26.13 - Stendal V852 EU-1 1:500 23.55 - - 27.54 25.78 - Stendal V1904 EU-1 1:500 24.37 - - 27.37 26.72 - Aus_LT3 EU-2 01:10 24.80 - - 27.59 25.93 - BY_Bor/59 EU (atypical) 01:10 32.81 - - 27.79 33.63 - BY_231/SZA EU-3 01:10 21.01 - - 27.74 23.78 - BY_8380/OKT_2p EU-4 01:10 25.70 - - 27.59 28.36 - China NA-HP 10 -2 - 20.01 22.14 26.22 - 21.83 China NA-HP 10 -3 - 23.93 25.79 26.89 - 26.06 China NA-HP 10 -4 - 27.61 29.25 27.56 - 28.43 China NA-HP 10 -5 - 29.93 31.71 26.96 - 32.34 China NA-HP 10 -6 - 37.1 34.96 27.50 - 35.20 Stendal V1445/99 NA 1:500 - 21.63 - 27.22 - 23.17 USA VD29949/17 NA 1:500 - 22.74 - 26.75 - 24.44 USA 2 NA 1:500 - 21.12 - 27.51 - 22.43 Mean Value 27.12 25.51 29.62 26.74 Standard Deviation 4.19 5.77 4.17 4.88 Discussion & conclusions To evaluate sensitivity and specificity of VIROTYPE ® PRRSV, titration studies with in-vitro RNA were performed. Serum, tissue and saliva samples were tested in comparison to other commercial PCR assays. Testing the EPIZONE PRRSV ring trial panel, all strains could be detected reliably. East European EU strains and the atypical EU strain scored positive. VIROTYPE ® PRRSV allows the reliable and simultaneously detection of PRRSV EU and NA genotype and the HP strain from porcine blood, serum, tissue, bronchial swabs, bronchial lavage, saliva, semen and also cell culture samples. The assay internal control guarantees the control of extraction as well as amplification. Due to the high sensitivity of VIROTYPE ® PRRSV pools of five samples can be tested. VIROTYPE ® PRRSV is easy to use with only one reaction-mix. Acknowledgements The EPIZONE ring trial panel and the panel of 12 PRRSV reference strains were kindly provided by K. Wernike and B. Hoffmann, Federal Institute for Animal Health, Germany. References 1. Wernike et al., Porcine reproductive and respiratory syndrome virus: inter-laboratory ring trial to evaluate real-time RT-PCR detection methods, submitted – under review
S1 - P - 33 APPLICATION OF A HERD PROGRAMME BASED ON CONTROL MEASURES AND LABORATORY MONITORING TO ERADICATE A LEPTOSPIRA OUTBREAK IN A LARGE DAIRY CATTLE HERD M.T. Scicluna, G. Manna, F. Rosone, A. Caprioli, R. Frontoso, , G.L. Autorino Istituto Zooprofilattico Sperimentale delle Regioni Lazio e Toscana, 00178 Roma Italia Leptospira serovar hardjo, management, laboratory diagnosis Introduction In Italy, the application of severe sanitary measures, including animal movement restriction are mandatory on the diagnosis of Leptospira infection and are lifted only after the removal of all seropositive animals. In the attempt of reducing economic losses deriving from these actions, a herd control programme was applied in a dairy intensive farm, in which the infection had been diagnosed, based on the combination of preventive, treatment and biosecurity measures, aiming at the sterilization of carriers and prevention of new cases of infection (1,2,3,4). The primary objective was to accelerate the control/eradication of the infection which was evaluated by monitoring the absence of its circulation with the aid of serological and molecular techniques and not as requested by the Italian Regulation, through the sole removal of the seropositive animals. Following is the description of the supplementary measures adopted and the actions applied for the assessment of the efficacy of the control protocol. Materials & methods Following the notification of leptospira diagnosis, an extensive serological investigation was undertaken in the farm of 600 producing cows, to verify if the infection was actively circulating: the random sampling scheme (Table 1) adopted, allowed the estimation of a prevalence of at least 50%, with a standard error of 10% and a confidence interval of 95%. The serological method used was the microagglutination test (MAT) described in the OIE manual, employing the serovar hardjo. A sample was considered positive when presenting a titre > to 1/100 (6). The supplementary control plan applied on the farm was based on the following points: - preventive measures, based on vaccination to limit new cases contributing to the further spread of leptospirosis. The vaccine employed, produced by the Istituto Zooprofillatico Sperimentale of Umbria and Marche, contained serovar hardjo and was administered as prescribed to all animals. - antibiotic treatment was adopted to sterilize carrier animals and eliminate primary source of infection. Two different antibiotics were chosen for economical and management reasons. (1,6) The more economical, Penistrep ®, 25mg/kg b.w., was for 3 consecutive days used in animals managed individually. To avoid any impact of treatment on the sale of milk, antibiotics were administered when the producing animals were dried off. Treatment was conducted so as to ensure that all animals of the same group received it all together, to avoid infection of seronegative subjects, after the disappearance of the effect of treatment. These animals received a single dose of 20 mg/kg b.w., of Duphaciclina 300 LA®. - Biosecurity measures aimed at further limiting the presence of leptospira in the environment, consisted in: isolation of the different units and their drinking systems in relation to whether these had undergone antibiotic treatment and complete vaccination; exclusive use of artificial insemination; systematic draining of paddocks to avoid water and urine stagnation; confinement, feeding and milking last of untreated animals. On conclusion of the control plan, actions were set up to verify its efficacy. These consisted in the microbiological examination of 272 urine samples of the animals in the different productive units, using the same sampling scheme described for the serosurveillance. These were examined in a SYBR Green Real Time PCR, specific for pathogenic Leptospira (7). Advantage of this test is that it can identify the carrier state even in a serologically positive animal and compared to the microbiological isolation, is fast and highly specific. Although reaction inhibition could occur due to the type of sample, using the test at herd level, increases the sensitivity of the method. In addition, a serological control was carried out on 50 animals born after the conclusion of the programme, used also as seronegative sentinels, placed in the productive units for the detection of an active circulation. The number of sentinel animals examined aimed to reveal an infection prevalence of 5% (IC 95%). These animals were retested at the end of their in-contact period, of two months, ensuring exposure, incubation period and also the production of a detectable serological response. Results & discussion The results of the serosurvey (table 1) demonstrate that leptospira infection was actively circulating in the farm. The supplementary measures adopted were considered as successful on the basis of the interruption of the leptospira infection in the different production units, as indicated by the negative results obtained for the urine samples and the seronegativity of the sentinels after their in-contact period with serological positive animals. Further evidence of the efficiency of the control programme was also the seronegativity of all animals born after the conclusion of the adoption of these measures. The adoption of such a herd control programme would be of benefit for both the farmers as well as the Veterinary Authorities, in that they would have more instruments for the control of this infection. Table 1. Sample scheme for serological survey. Productive Consistency Examined Positive N° of Unit (positive) animals (Titres) Heifers 60 34 (5) 5 1(1/100) 1(1/200) 3(1/400) Pregnant Heifers 160 54 (38) 38 27 (1/100) 3(1/400) 8(1/200) Dry cows 140 51 (37) 37 29(1/100) 73 8(1/200) Primipare 120 48 (26) 26 26 (1/100) 54 Pluripare 160 60 (35) 35 34(1/100) 58 1(1/200) Fresh 50 33 (12) 12 10(1/100) 36 Lactating cows 2(1/200) Problem 80 44 (25) 25 25 (1/100) 57 animals Sick bay 20 17 (7) 7 7 (1/100) 41 References 1. Bolin CA, Alt DP. 2001. Am J Vet Res. 62(7):995-1000 2. Cortese VS, et al., 2007. Vet Ther. 8(3):201-8 3. Little TW, et al., 1992. Vet Rec. 1;131(5):90-2 4. Little TW, et al., 1992. Vet Rec. 24;131(17):383-6 5. Levett PN Clin. Microbiol. Rev. 2001 Apr.,: 296–32 6. Alt DP, et al.,2001.J Am Vet Med Assoc.1;219(5):636-9 7. Ahmed A, et. al. 2009. PLoS One 4: Vol 4, Issue 9:1-8 Estimted prevalence % 15 70
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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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advances, the fundamental tools of
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S3-P-06 KELLER SELINA S3-P-07 KÖNI
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