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Toxoplasma Toxoplasma gondii gondii in wild boar and roe deer in Northern Italy: in wild boar and roe deer in Northern Italy: serosurvey serosurvey and PCR and PCR- RFLP RFLP Gaffuri, A. 1, Fugazza S. 2, Rota Nodari E. 1, Vicari N. 1a, Barbieri I. 1b, Paterlini F. 1 and Lanfranchi P. 2 1- Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna, Department of Bergamo, Pavia (1a) and Brescia (1b) 2- Università degli Studi di Milano, Dipartimento di Patologia Animale, Igiene e Sanità Pubblica Veterinaria, Italy MATERIALS AND METHODS MATERIALS AND METHODS Samples were collected by hunters in the field during the 2008 and 2009 hunting seasons in Lombardy region, Northern Italy (Fig.1); wild boar sera were tested by IFIT (Toxo-spot ® IF, bio-Meriaux) while roe deer ones by a commercial Elisa kit (ID Screen® Toxoplasmosis Indirect ELISA, IDVET, Montpellier, France); we analysed respectively 281 and 505 sera. Since the muscle wasn’t collected from all the hunted animals, among the seropositive animals we could analyzed the muscular tissue (heart, diaphragm and masseter) of 56 wild boar and the hearts of 69 roe deer. We homogenized 10 g of muscular tissue with saline solution and we transferred 0,25 g of the homogenized sample into an DNA extraction kit (NucleoSpin ® Tissue, Macherey-Nalgen).The detection of the parasite was carried out by a PCR-RFLP assay targeting the 18S small- subunit ribosomal gene of T. gondii (3), using primers that identify also N. caninum and Sarcocystisspp. REFERENCES REFERENCES: 1-Bartova E. et al. 2007. J Parasitol 93 (5):1216-8. 2- Gauss C.B.L. et al, 2006. Vet. Parasitol. 136:193-200. 3- Magnino, S.et al, 1998. Proceedings of the IX International Congress of Parasitology, Makuhari Messe, Chiba, Japan, 24-28 August 1998, pp. 1269–1272. 4- More G. et al, 2008 Parasitol Res 102:671-675. 5- Renzi M. et al, 2009 poster presentation at the III Congresso Nazionale SIEF,Torino, 15-17 ottobre 2009. 6- Richomme C. et al 2009. Vet. Parasitol. 164:296-300. 7- Uggla A. and Buxton D., 1990 Rev.sci.tech.Off.int.Epiz. 9(2) 441- 462. 8- Vikøren T. et al 2004. Vet. Parasitol 120:159-169 RESULTS RESULTS A total of 63 wild boar (22.4%, I.C. 95% 17.77-27.84) and 110 roe deer sera (21.78%, I.C. 95% 18.31-25.69) were positive to T. gondii antibodies. In wild boar samples with a IFIT titres ≥ 40 were considered positive while in red deer the positive reaction was evaluated according to the manufacturer’s instruction of the Elisa test. In wild boar 34 muscular tissues and in roe deer 37 heart samples had a positive reaction to DNA amplification. Successively the restriction enzyme analysis of the amplified products showed that all the samples were positive to Sarcocystis spp., that by sequencing analysis has been identified as S. miescheriana in wild boar and as S. cruzi and S. gracilis in roe deer. 9th CONFERENCE OF THE EWDA, VLIELAND, NETHERLAND, 13 9th CONFERENCE OF THE EWDA, VLIELAND, NETHERLAND, 13- 16 SEPTEMBER 2010 16 SEPTEMBER 2010 INTRODUCTION INTRODUCTION Toxoplasma gondii infects all warm-blooded animals; in Europe several studies carried out in wildlife show seropositivity towards this parasite, in particular in wild ungulates (1,2,5,6,8). In Northern Italy in the last years the culling of wild boar and roe deer is significantly increased and then the venison consumption. As eating of raw or undercooked meat is a risk factor for Toxoplasmosis transmission to humans, we performed a serosurvey for this protozoan and its detection in the muscular tissue with the aim to perform a genetic characterization of the strains and to assess the risk associated with the consumption of game meat . CONCLUSIONS CONCLUSIONS the serological results show a remarkable exposure to T. gondii in both host species and this is an indication that the parasite is widespread in their habitat, that is frequently close to human settlements although a cross-reactivity between antibodies to T.gondii and Sarcocystis spp. cannot be excluded, the serological test used should limited the cross reactivity for the characteristic of the antigen preparation (4,7) a recent study in roe deer (5) show corresponding results in detecting T. gondii antibodies comparing two different assays, the IFIT and ID Screen® Toxoplasmosis Indirect ELISA. We had equivalent results in some wild boar sera tested with both IFIT and Elisa assay the unsuccessful detection of the parasite in muscular tissue could be due to the small size of the analyzed samples and to the low presence of tissue cysts in the muscle. The wild boar tissue samples were also tested by another method (B1 gene PCR) and we obtained the same results a standardisation of the serological and PCR methods for wildlife should be constructive in order to be able to compare the results of different studies and to enhance the research with the same tools a correct information should be done to the game meat consumers for the public health implication, considering that eating undercooked or cured game is a widespread habit. Moreover hunters should be aware that they may also become infected during evisceration and handling of game AKNOWLEDGEMENT AKNOWLEDGEMENT We thank prof. Ezio Ferroglio, University of Turin, for having performed the B1 gene PCR assay and Donatella Ghidotti, IZSLER Bergamo department, for carrying out the PCR analysis. FIGURE 1 1 2 3 4 5 6 7 8 9 10 11 FIGURE 2 1 2 3 4 5 6 7 8 9 10 Lane 1: Molecular weight marker, lanes 2 - 7: samples, lane 8: T. gondii positive control, lane 9: N. caninum positive control, lane 10: Sarcocystis spp. positive control, lane 11: negative control Amplicons obtained by PCR targeting the 18S rDNA RFLP patterns obtained by BseD I digestion. Figure 1: Figure 1: Study Study area area
