Trypanosoma evansi: A comparative study of four diagnostic techniques for trypanosomosis using rabbit as an experimental model J.R. Ramírez-Iglesias a , M.C. Eleizalde a , E. Gómez-Piñeres a,b , M. Mendoza a,⇑ a Universidad Nacional Experimental Simón Rodríguez, Instituto de Estudios Científicos y Tecnológicos (IDECYT), Centro de Estudios Biomédicos y Veterinarios (CEBIV), Apartado Postal 479245, Caracas 1041A, Venezuela b Universidad de Oriente, Núcleo Monagas, Escuela de Zootecnia, Departamento de Biología y Sanidad Animal, Maturín, Venezuela article info Article history: Received 14 August 2010 Received in revised form 2 February 2011 Accepted 8 February 2011 Available online 12 February 2011 Keywords: Trypanosoma evansi Diagnosis of trypanosomosis PCR Indirect ELISA Parasitological techniques Rabbit as an experimental model abstract The goal of this study was to compare two parasitological diagnostic techniques, such as by Micro- Haematocrit Centrifugation Technique (MHCT) and Direct Microscopic Examination (DME) with a serological method (iELISA), and a molecular procedure PCR, in rabbits experimentally infected with Trypanosoma evansi, in order to determine their sensitivity throughout the course of disease. The parasitological methods were not able of detecting the presence of the parasite during the phases of low parasitemia, the prepatency period and the chronic phase. In contrast, PCR detected T. evansi in the prepatency and chronic phase, when increase the amount of DNA from 100 to 300 ng. 100% detection was observed with iELISA only in the chronic stage of the disease. In the acute phase, all samples were positively diagnosed using either MHCT or PCR, whereas only few samples were diagnosed by DME. Samples obtained from day 15 post infection were also detected by iELISA. The highest diagnostic register during the course of infection was achieved by the PCR technique (93.8%), followed by iELISA (71.1%), MHCT (59%) and DME (13.6%). Therefore, we recommend the use of PCR in epidemiological studies in order to implement sanitary control plans for the improvement of livestock productivity in the country. Ó 2011 Elsevier Inc. All rights reserved. 1. Introduction Trypanosomosis is a disease caused by various protozoan of the Trypanosomatidae family, which can affect either humans or ani- mals. In particular, animal trypanosomosis are of great concern in inter-tropical areas of Asia, Africa and Latin America, where most of the pathogenic Trypanosoma species are present in their hosts, or vectors. In South America, Trypanosoma evansi causes equine trypanomososis, a disease known as ‘‘Derrengadera’’ in Venezuela and ‘‘Mal de Caderas’’ in Argentina, Brazil and Paraguay. This parasitic disease produces a decrease in the productivity of horses used in livestock, which are indispensable for traditional extensive cattle ranching. Despite the great economical losses that this disease generates in affected areas, almost no epidemiological data is available. In Argentina, for example, antibody detection has shown that 20% of the animals are positive in the Formosa region where approximately 57,000 horses are exposed (Monzón et al., 1995). In the Pantanal region of Brazil, about 13% of the horses are lost annually and no appropriate control is used (Seidl et al., 2001). In the Venezuela savannah, it has been reported a seroprev- alence of 80% and 50% for T. evansi in horses (Reyna-Bello et al., 1998) and capybaras (Arias et al., 1997), respectively. There are studies on trypanosomosis caused by T. evansi in rab- bits as experimental models, where characteristic symptoms are reported: elevated levels of parasitemia, descent in haematocrit, weight loss, cellular infiltrations and the development of a marked immune humoral response (Uche and Jones, 1992; Uche et al., 1992). Also, biochemical alterations are described like hyperpro- teinemia, hyperglobulinemia, increment in the levels of urea and decrease of the levels of serum albumin (Da Silva et al., 2007). The implementation of the rabbit, an animal easy to manipulate in the laboratory, as a chronic experimental model for the study of trypanosomosis will allow the development of research in diag- nosis, prevention and possible treatments with direct application in the field. This disease starts with a prepatency period (PP) followed by acute (AP) and chronic (CP) phases. There are diverse diagnostic methods to detect animal trypanosomosis: parasitological, immu- nological and molecular, which show advantages and disadvan- tages according to the stage of the disease in which they are applied. The parasitological methods are based on the direct obser- vation of the parasite under the light microscope. These techniques are simple and of low cost, but have low sensitivity; therefore they are only effective in the AP of the disease when high levels of par- asitemia are present. The immunological methods as the ELISA (en- zyme-linked immunosorbent assay), are based on the detection of antigens (Ag-ELISA) or antibodies (indirect-ELISA) related to the 0014-4894/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2011.02.010 ⇑ Corresponding author. Fax: +58 2126719289. E-mail address: memendoza@cantv.net (M. Mendoza). Experimental Parasitology 128 (2011) 91–96 Contents lists available at ScienceDirect Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr