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S1 - P - 04 DIAGNOSIS OF MAIN HAEMOPARASITIC DISEASES OF CATTLE BY REAL-TIME PCR. Villa Aleida 1 ; Benito AA 1 ; Arnal JL 1 ; Serrano JD 1 and Baselga R 1 . 1 EXOPOL Autovacunas y Diagnóstico. Pol. Río Gállego, 50840 San Mateo de Gállego Zaragoza, España. Tel 976 69 45 25, exopol@exopol.com Piroplasmosis, Anaplasmosis, Diagnostic, qPCR, cattle Introduction Piroplasmosis and Anaplasmosis are the most important blood parasitic diseases in cattle and responsible for significant economical losses and mortality in livestock from many countries. Although these diseases occur mainly in tropical and subtropical areas, they are also becoming an increasing and serious problem in Europe ( 1 ). Clinical signs in haemoparasitic diseases are quite similar and include anemia, pyrexia, weakness, weight loss and drop in production levels. Bovine Piroplasmosis is caused by pathogenic Babesia and Theileria species. Th. parva and Th. annulata are the most important pathogens in this genus, although only Th. Annulata has been described in Europe ( 2 ). There are several pathogenic Babesia species affecting cattle (B. major, B. bovis, B. divergens and B.bigemina), but B. bigemina and B. bovis are the most frequently described ( 3 ). Bovine Anaplasmosis is caused by rickettsia of genus Anaplasma. A. marginale is considered the most pathogenic specie while A. centrale has been implicated in mild or less severe cases of this disease ( 4 ) . Laboratory diagnosis of these pathogens included their microscopical identification in blood smears; however, this method requires highly qualified personnel and is not reliable for detecting pre-symptomatic or carrier animals. Serological tests can also be used; although problems of cross-reactivity between pathogens of same genus have been reported. Recently, some techniques of polymerase chain reaction (PCR) have been developed and allowed a specific and sensitive detection of these agents ( 5 ) . The main objective of this study was evaluate the feasibility of a Real-time PCR (qPCR) assay for the diagnosis of Th. annulata, Th. parva, B. bigemina, B. bovis, A. marginale, A. centrale and A. phagocitophylum, in a multiparametric test for a specific identification and also quantification of these main tickborne pathogens of cattle. Materials & methods Samples: A total of 122 cases (330 blood, 58 serum and 9 tissue samples) submitted to our laboratory with suspect of haemoparasitic disease were evaluated. Some of these cases (10% aprox) had macroscopical findings and histopathological lesions of haemolytic syndrome. Most of cases 93% (113/122) came from 21 provinces in Spain and the rest from Portugal. Whole blood, serum and tissue samples were subjected to DNA extraction individually or pooled up to five per case. DNA extraction: A commercial kit “Genomic mini Kit LGD 500 LabTurbo” and an automated DNA/RNA extraction System “LabTurbo 36 compact system C3620” (Taigen Bioscience Corporation, Taiwan) were used for DNA extraction following the manufacturer's instructions. DNA purity (260/280) and DNA yields were determined using a micro-volume spectrophotometer (Quawell UV Q5000, software 4.0). Quantitative Real-Time PCR (Genesig Ltd): The qPCR assays used in this study identified the following pathogen-specific targets: Tams1 gene (Merozoite-piroplasm surface antigen) for Th. annulata; p104 gene (Microneme-rhoptry antigen) for Th. Parva; 18S gene (18S ribosomal RNA) for B. bigemina; 18S gene (18S ribosomal RNA) for B. bovis; msp4 gene (Major surface protein 4) for A. marginale; gen; msp2 gene (Major surface protein 2) for A. centrale and msp4 gene (Major surface protein 4) for A. phagocytophylum. A unique qPCR protocol were performed for all these pathogens according manufacturer's instructions, briefly: 10µl of MasterMix, 1µl of pathogen-specific primer/probe (FAM labeled, BHQ quenched), 5µl of RNAse/ DNAse free water and 5µl of DNA sample (> 5ng) or control template conformed the PCR reaction in a final volume of 20 µl. Amplification reactions were carried out on a MiniOpticon RT System CFB3120 thermal cycler (Bio Rad, software CFX manager 2.0) using a unique thermal profile for all qPCRs consisting of an initial enzyme activation step of 10 min a 95º C, followed by 50 cycles of: a denaturing step of 10 sec at 95º C, and an annealing/data collection step of 60 second at 60º C. Positive cut-off value was established in ≤ 45 cycles. Detection limits and quantification were performed by 10 fold dilutions (10 6 a 10 0 ) of specific DNA plasmids for each pathogen. Negative samples were evaluated for DNA integrity by amplification of Beta-actina house-keeping gene. Results This multi-parametric qPCR assay was robust, specific and showed an analytical sensitivity for at less 10 2 copies of the target DNA. The Cq values for positive controls ranged from 25 to 35 and the qPCR efficiencies were around 95% with R 2 values above 0.990. A total of 122 cases were evaluated in this study and 49% (59/122) of them were positive by qPCR for at less one of these pathogens. Haemoparasites were detected in blood samples 52% (171/330), but also in tissue 22% (2/9) and serum 38% (22/58) samples. Nearly 10% of cases had macroscopical and/or histopathological lesions compatible with haemolytic syndrome. Microscopical identification of Piroplasmids and Anaplasma were found in blood and/or tissue samples from 8 of these cases and all of them had one or more positive result for haemoparasites by qPCR. From the overall cases Th. annulata 27% (33/122) and A. marginale 27% (33/122) were the more frequent detected pathogens, followed by B. bigemina 16% (20/122), B. bovis 2% (3/122) and A. phaghocytophylum 2% (2/122). No positive cases were recorded for Th. Parva (0/122) and A. centrale (0/122). Concurrent infections with Th. annulata, A. marginale and B. bigemina were detected in 15% (9/57) of the positive cases. Discussion & conclusions Specific identification of haemoparasites infecting cattle is a crucial step for control of these diseases; because measures and treatment depend of the implicated agent. Although microscopical identification is possible it is not always reliable even for most expertice technicians. Clinical diagnosis is also difficult, by the similar symptomatology in these diseases. Our results clearly demonstrate the value of these qPCRs as a specifics and sensitive diagnostic tools for the rapid detection of main tickborne disease of cattle in blood and tissue sample from infected animals. The use of this qPCR multi-parametric assay allow a complete diagnosis of the main haemoparasitic diseases in a rapid way and reducing cost by pooling samples, up to five, per case. It also has the advantge for no competition of multiple primers by the target DNA, such ocurrs in a qPCR multiplex where a DNA depletion could happen mainly in animals with mixed infections. Our results showed a high percentage of haemoparasitic infections, mainly Th annulata, A. marginale and B. bigemina, in the evaluated cases from the Iberian peninsula and justified the use of specific and sensitive diagnostic test, as this qPCR, to study the real epidemiological situation of these disease in the cattle population from these countries. References 1. Paul Heyman, Christel Cochez, Agnetha Hofhuis, Joke van der Giessen, Hein Sprong, Sarah Rebecca Porter, et al. A clear and present danger: Tick-borne diseases in Europe. Expert Rev Anti Infect Ther. 2010; 8(1):33- 50. 2. R. Cassini, F. Marcer, F. di Regalbono, G. Cancrini, S. Gabrielli, A. Moretti, et al. New insights into the epidemiology of bovine piroplasmoses in Italy. Vet Parasit 2012(184):77-82.2. R. Cassini, F. Marcer, F. di Regalbono, G. Cancrini, S. Gabrielli, A. Moretti, et al. New insights into the epidemiology of bovine piroplasmoses in Italy. Vet Parasit 2012(184):77- 82. 3. S. Almeria, J. Castellà, D. Ferrer, A. Ortuño, A. Estrada-Peña, J.F. Gutiérrez. Bovine piroplasms in Minorca (Balearic Islands, Spain): a comparison of PCR-based and light microscopy detection. Vet Parasitol 2001(99):249–259. 4. L. Ceci, N. Decaro, E. Lorusso, P. Paradies, G. Elia, V. Martella, et al. First report of bovine anaplasmosis by Anaplasma centrale in Europe, molecular identification and phylogenetic analysis. Vet Res commun 2008(32):S236-S266. 5. Oficina Internacional de Epizootias (OIE). Manual de las Pruebas de Diagnóstico y de las Vacunas para los Animales Terrestres. 2008.
S1 - P - 05 DEVELOPMENT AND EVALUATION OF A NEW AND ORIGINAL EXTRACTION PROTOCOL TO DETECT MYCOBACTERIUM AVIUM SUBSP PARATUBERCULOSIS IN BOVINE FECES BY REAL TIME PCR Blanchard Béatrice. 1 , Versmisse Yann. 1 , Rouillard Tony. 2 1 ADIAGENE, 38 rue de Paris 22000 Saint Brieuc, FRANCE 2 AES CHEMUNEX, Rue Maryse Bastié, 35172 Bruz, FRANCE Mycobacterium paratuberculosis (Map), real-time PCR, diagnosis, low shedder. Introduction Mycobacterium avium ssp paratuberculosis (MAP) is the etiological agent of Johne’s disease, a granulomatous enteritis that can affect cattle, sheep and goats and other non ruminant wildlife species. Numerous countries have set up programmes to control the disease. It requires the development of highthroughput sensitive diagnostic methods for the direct detection of infected animals. The performance of the PCR methods was often hampered by low sample input and inhibition. To increase the sensitivity of diagnostic test, we developed an original protocol to concentrate Map from 6g of faecal samples and compared the real time PCR results obtained with classical DNA extraction from 1g. Materials & methods A negative faeces collected from a paratuberculosis-free herd was spiked with serial dilutions of a MAP titrated suspension to 4670, 2333, 1167, 233 and 117 bacteria per g. DNA extraction of each sample spiked was repeat four times using the new protocol (NP) developed by Adiagene. Briefly, 6g +/- 0.2 g of faeces were placed in a bottle with 40 ml of sterile distilled water, mixed 15 seconds and allowed to settle 10 to 20 minutes. 10 ml of the obtained supernatant were transferred on an ADIAFILTER and centrifuged 5 minutes at 3 000 g. The obtained pellet was resupended then disrupted 10 minutes at 30 Hz with a mixer mill and 300 mg of glass beads and centrifuge 5 minutes at 15 000 g. Then DNA was extracted from 200 μl of the supernatant. 5µL of each DNA solution was analyzed with the ADIAVET ® PARATB REALTIME PCR kit (Adiagene). The detection limit of the diagnostic method is the smallest amount of MAP per sample generating 100% positive results. Evaluation of the new protocol on field samples 329 faecal samples were collected from different veterinary diagnostic laboratories. The common point among these samples is that they were sent to the laboratory for MAP analyses; they came from different area, from different herds with or without history of paratuberculosis. All the samples were analysed in parallel with two DNA extraction protocols. This first was performed using Adiapure ® (Adiagene) DNA extraction protocol (SP). The analyses were carried out from 1g of faecal sample as recommended by the manufacturer. The second DNA extraction was realised using the new protocol (NP). It was achieved with 6g +/- 0.2 g of faeces as previously described. 5 µl of the each DNA solution obtained from the 2 different protocols was analysed with ADIAVET ® PARATB REALTIME kit. difficult to obtain a good sensitivity of the test due to the presence of inhibitor and the biology of the bacteria. Foremost, it was important to increase the quantity of treated faecal sample. Most of the works done for MAP detection like culture or PCR treat up to 1g of faeces. Even the new technique combining magnetic beads separation and PCR uses a minimum amount of samples. The originality of our protocol is to be able to treat up to 10g of faeces in order to concentrate the MAP present in the sample. In the standard protocol (SP) the DNA of 1g of faeces are treated. After homogenization and DNA purification, 0.15mg are used for PCR analyse; With the new protocol (NP), 6g are extracted and 15mg of feces are used for PCR analyse. So, we are able to concentrate 100 times the sample and increase the sensitivity of the whole method. Prior study have suggested that high shedders yield > 50CFU/tube and low shedders
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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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antigens or attenuated vaccines can
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conditions, e.g. Staphylococcus aur
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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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