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E S T R AT T I R E L A Z I O N I COMUNICAZIONI BREVI • POSTERS E S T R AT T I R E L A Z I O N I A Z I E N D A L I
CONGRESS PROCEEDINGS S H O R T C O M M U N I C AT I O N S • P O S T E R S C O M PA N Y R E S E A R C H A B S T R A C T S
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INTERNATIONAL SCIVAC CONGRESS CANINE LEISHMANIOSIS AND OTHER VECTOR-BORNE DISEASES: OUR CURRENT STATE OF KNOWLEDGE MARCH 8TH -10TH 2013 - PISA - ITALY
RETRIEVAL OF LEISHMANIAL DNA IN A COMMERCIALLY AVAILABLE LIESP/QA-21 VACCINE T. Furlanello, Dr Med Vet, PhD, ECVCP Dip 1,2, M. Trotta, Dr Med Vet, PhD 1, M. Caldin, Dr Med Vet, PhD, ECVCP Dip 2 1 Laboratorio d’Analisi Veterinarie San Marco, Padova, Italia 2 Clinica Veterinaria San Marco, Padova, Italia Tipologia: Ricerca Originale Area di interesse: Leishmaniosi Purpose of the work. Due to the limited effectiveness of most of the available methods for canine visceral leishmaniasis treatment and control, the development of a dog vaccine is most desirable, practical and an ef?cient control tool. This development reduces the dog-sand?y-dog peridomestic transmission cycle and lowers the risk of transmission to humans. The protective potential of the excreted/secreted antigens of L. infantum promastigotes (LiESAP) was demonstrated in dogs (Lemesre et al., 2007). A LiESAP/QA-21 vaccine has been recently authorized in the European Union under the trade name CaniLeish (Virbac, France). It is composed of purified excreted-secreted proteins of Leishmania infantum, produced by means of a patented cell-free, serum-free culture system (Moreno et al., 2012). According to European public assessment reports (EMA, 2011a; EMA, 2011b), only purified excreted secreted antigens of Leishmania infantum promastigotes and an adjuvant should be present in the vaccine. This purification is of crucial importance on the grounds that the secreted and surface proteins that are exposed, can be processed by the host immune system, unlike the intracellular proteins which are not. Furthermore, the release of internal proteins may function in conjugation with the secreted and surface proteins acting as a transient “smoke screen” that enables the onset of the initial infection by viable parasites (Santarém et al., 2007). The aim of the study was to investigate the presence of unexpected DNA from Leishmania in vaccine vials. Materials and used methods. Real-time PCR for L. infantum DNA detection was performed in 7 vials belonging to two different lots and acquired through standard commercial veterinary suppliers. We used primer and probe sequences specific for the L. infantum kinetoplast mini-circle (TIBMOLBiol, Genua, Italy) as previously described (Solano-Gallego et al., 2007; SolanoGallego et al., 2012). Real-time PCR was performed by using LightCycler
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INTERNATIONAL SCIVAC CONGRESS CANINE LEISHMANIOSIS AND OTHER VECTOR-BORNE DISEASES: OUR CURRENT STATE OF KNOWLEDGE MARCH 8TH -10TH 2013 - PISA - ITALY
FastStart DNA Master, Hybridization Probes (Roche, Mannheim, Germany) using LightCycler version 3.5.17 instrument (Roche, Mannheim, Germany). Positive and negative controls were used in all runs. A conventional PCR for ITS gene and direct sequencing were performed on each samples tested by real-time PCR (Schoenian et al., 2003). Sequences were compared with sequences deposited in GenBank using BLAST hosted by the National Center for Biotechnology Information, National Institutes of Health, USA (http://www.ncbi.nlm.nih.gov). Outcomes. High concentrations of Leishmania infantum DNA were found during real-time qPCR based on kinetoplast multilocus gene on all samples (7/7). Direct sequencing from conventional PCR for ITS gene revealed the presence of Leishmania infantum with 100% homology with sequences deposited in GenBank database (FN398341). Conclusions. Although, to our knowledge, very limited data is available in the current veterinary literature related to the only commercially available vaccine against canine leishmaniasis, the official documentation clearly states that the product is highly purified and protein-free other than LiESAp (EMA, 2011). Utilizing a protocol validated for samples other than canine whole blood, enabled us to obtain significant amounts of Leishmania infantum DNA in all samples. Upon confirmation by others, our finding could raise some concern about the efficacy of the purification process of the product. If at all the presence of Leishmania DNA in the vial is considered to be an expected finding, this would greatly emphasize the necessity to provide every detail of pertinent information to the scientific community before or concurrently with the marketing of any innovative veterinary drug or vaccine. The presence of kDNA in the vaccine could cause further debate, because we can not exclude, almost theoretically, a false positive PCR result in a sample from a recently immunizated dog. Bibliography Lemesre JL et al., Long-lasting protection against canine visceral leishmaniasis using the LiESAp-MDP vaccine in endemic areas of France: Double-blind randomized efficacy field trial, Vaccine 25 (2007) 4223–4234. European Medicines Agency [EMA] (2011a), CVMP summary of positive opinion for Canileish, www.ema.europa.eu, accessed January 10th, 2013. European Medicines Agency [EMA] (2011b), CaniLeish: EPAR – Product Information (EMEA/V/C/002232), www.ema.europa.eu, accessed January 10th, 2013. Moreno J et al. (2012), Use of a LiESP/QA-21 Vaccine (CaniLeish) Stimulates an Appropriate Th1-Dominated Cell-Mediated Immune Response in Dogs, PLoS Negl Trop Dis 6(6): e1683.
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INTERNATIONAL SCIVAC CONGRESS CANINE LEISHMANIOSIS AND OTHER VECTOR-BORNE DISEASES: OUR CURRENT STATE OF KNOWLEDGE MARCH 8TH -10TH 2013 - PISA - ITALY
Santarém N et al. (2007), Immune Response Regulation by Leishmania Secreted and Nonsecreted Antigens, Journal of Biomedicine and Biotechnology, Volume 2007, Article ID 85154, 10 pages. Solano-Gallego L et al. (2007), Detection of Leishmania infantum DNA by fret-based real-time PCR in urine from dogs with natural clinical leishmaniosis. Vet Parasitol, 147(3–4):315 Solano-Gallego L et al. (2012), Detection of Leishmania infantum DNA mainly in Rhipicephalus sanguineus male ticks removed from dogs living in endemic areas of canine leishmaniosis, Parasites & Vectors 2012, 5:98, 6 pages. Schoenian G et al. (2003), PCR diagnosis and characterization of Leishmania in local and imported clinical samples. Diagn Microbiol Infect Dis. 2003 Sep;47(1):349-58.
Corresponding Address: Dott. Tommaso Furlanello - Clinica Veterinaria San Marco, Via Sorio, 114/C, 35141 Padova (PD), Italia - Tel. ++390498561098 - E-mail: tf@sanmarcovet.it
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