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Comparison between micro-hematocrit centrifugation technique and polymerase chain reaction (PCR) to detect Trypanosoma evansi in experimentally inoculated goats
Small Ruminant Research 96 (2011) 70–72
Contents lists available at ScienceDirect
Small Ruminant Research journal homepage: www.elsevier.com/locate/smallrumres
Short communication
Comparison between micro-hematocrit centrifugation technique and polymerase chain reaction (PCR) to detect Trypanosoma evansi in experimentally inoculated goats M.T. Tejedor-Junco a,∗ , M. González a , N.F. Rodríguez b , J.A. Corbera b , C. Gutiérrez b a b
Department of Clinical Sciences, University of Las Palmas de Gran Canaria, P.O. BOX 550, 35080 Las Palmas de Gran Canaria, Canary Islands, Spain Department of Animal Medicine and Surgery, University of Las Palmas de Gran Canaria, 35413, Arucas, Las Palmas, Canary Islands, Spain
a r t i c l e
i n f o
Article history: Received 26 April 2010 Received in revised form 18 October 2010 Accepted 22 October 2010 Available online 15 December 2010 Keywords: PCR Trypanosoma evansi Micro-hematocrit centrifugation technique Goat
a b s t r a c t Natural Trypanosoma evansi infection in the Canary Islands has only been diagnosed in the camel population, but dissemination of the disease in other hosts has not been excluded. To evaluate the role of the goats in the dissemination of the disease, 8 goats were inoculated and examined during 6 months using a polymerase chain reaction (PCR) with a primer targeting a repetitive region specific for Trypanozoon subgenus used to amplify a 227 bp fragment from the genomic DNA. PCR was able to detect parasitemia in all tested samples; therefore it was considered as gold standard test in this study. The results were compared with those obtained using the micro-hematocrit centrifugation technique showing a sensitivity of 92.7%, specificity of 100%, positive predictive value of 1 and negative predictive value of 0.87. Both techniques seem to be adequate to detect T. evansi from infected goats. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Trypanosoma evansi is the most widely distributed of the pathogenic animal African trypanosomes, affecting domestic livestock and wildlife in Asia, Africa and Latin America (Luckins and Dwinger, 2004). The parasite causes a disease known as surra, and is mechanically transmitted by biting flies other than tsetse and vampire bats in South America. It affects a number of domesticated animals and the principal host species varies among the continents. Buffalo, cattle, camels and horses are particularly susceptible, although other animals, including wildlife, can also be infected (OIE, 2009). Small ruminants are commonly raised in close contact with camel herds in many desert and semi-desert areas in the world. However, it is commonly believed that goats, for example, are highly resistant to infection, that caprine trypanosomosis is only sporadic, and that the disease in
∗ Corresponding author. Tel.: +34 928 454358; fax: +34 928 454358. E-mail address: [email protected] (M.T. Tejedor-Junco). 0921-4488/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.smallrumres.2010.10.014
goats is of little economic consequence (Griffin, 1978). This opinion, however, is currently undergoing a critical reappraisal (Smith and Sherman, 2009). In the Canary Islands, where the camel population is around 2000 animals and the goat census is 363,000 heads (data from 2007, livestock census, Regional Canary Government, http://www2.gobiernodecanarias.org/istac/ estadisticas.html), goats share the same habitat with the camels in those areas where camels inhabit. In the Canarian Archipelago, Trypanosoma evansi infection was detected for the first time in 1998 in an imported dromedary camel and after that, it has only been diagnosed in the camel population (Gutiérrez et al., 2000). However, the possible role that goats could play in the maintenance of the disease in the islands is suspected but not proved, since natural infection has not been demonstrated. At this respect, infected goats could present two important constraints: low and/or alternant parasitemia and no apparent clinical signs of infection, which occurs commonly with many strains of T. evansi, including the Canarian one used in this study (Gutiérrez et al., 2004). Micro-hematocrit centrifugation technique is
M.T. Tejedor-Junco et al. / Small Ruminant Research 96 (2011) 70–72
a simple technique which is easy to perform, even at farm level, quick and at minimum cost; PCR, however, requires a laboratory of molecular biology, is more expensive and is not always available in developing countries. Given that there is considerable variation in the pathogenicity of different strains and the susceptibility of different host species to disease (OIE, 2009), the purpose of this study was to evaluate a PCR with a primer targeting a repetitive region specific for Trypanozoon subgenus used to amplify a 227 bp fragment from the genomic DNA, in comparison with micro-hematocrit centrifugation technique (Woo, 1969), using goats experimentally inoculated with T. evansi Canarian strain. 2. Materials and methods 2.1. Animals Twelve female and adult Canarian goats were acquired for this experiment. The animals were examined using the T. evansi diagnostic methods described below and resulted negative. Eight goats were experimentally inoculated with at least 1 × 105 T. evansi by intravenous route. The strain was obtained from a naturally infected camel in Canaries. The remaining four animals served as control. The goats were placed in fly-proof pens within an experimental area of the Veterinary Faculty and were maintained for 6 months. 2.2. Samples Blood samples were collected every 2 weeks from jugular vein using vacutainer® tubes with EDTA as anticoagulant and stored at 4 ◦ C until processed.
71
2.6. Statistical analysis Data were statistically analyzed following the recommendations of Thrusfield (2007) concerning to the evaluation and interpretation of diagnostic tests. Variables were measured as nominal and dichotomous (positive/negative).
3. Results The inoculated goats showed a subclinical course of the disease. Episodes of fever within the first week postinoculation and arthritis at 6 months post-infection were detected in two inoculated goats. All experimentally inoculated animals were positive in the CATT/T. evansi 1 month post-infection and remained positive with a minimum end-titer of 1/4 throughout the experiment. Parasitemia was detected at 2 weeks post-infection in all inoculated goats, and the animals were then considered infected. From this group (infected) a total of 96 assessments (8 infected goats per 12 checking) were performed. PCR was able to detect parasitemia in all examined samples, while MHCT detected parasitemia in 89 samples; in 7 samples this technique failed to detect trypanosomes. From the control group, a total of 48 assessments (4 goats per 12 checking) were made, being all tested samples negative. Considering the PCR as a gold standard test in this study, comparison between PCR and MHCT showed the following statistical data: sensitivity: 92.7%, specificity: 100%; positive predictive value: 1; negative predictive value: 0.87.
2.3. Serological assessment CATT/T. evansi (Institute of Tropical Medicine, Antwerp, Belgium), a validated serological method referred by OIE (2009) was used to evaluate the presence of antibodies against T. evansi. 2.4. Micro-hematocrit centrifugation technique (MHCT) This technique detects the presence of the motile trypanosomes and was performed using one capillary tube per animal and centrifuging the samples at 9520 × g. Blood samples were maintained under refrigeration and the technique was always done within the next 2 h after the blood was taken. 2.5. DNA extraction and PCR Genomic DNA was extracted from samples of goat blood using commercially available QIAamp DNA mini kit (Qiagen, Hilden, GE) and the purified DNA was stored at −20 ◦ C for later use. PCR was carried out in 25 l reaction mixtures containing 10× reaction buffer (Bioline, London, UK), 2.5 mM MgCl2 , 300 M of each of the four deoxynucleoside triphosphates (dNTPs) (Amersham Bioscience Europe, Barcelona, Spain), primers 100 ng and 0.75 U of Taq DNA polymerase (Bioline). The cycles included an initial step at 94 ◦ C for 4 min, followed by 30 cycles of denaturing at 94 ◦ C for 30 s, annealing at 60 ◦ C for 30 s and extension at 72 ◦ C for 30 s. PCR elongation was continued at 72 ◦ C for 5 min. The amount of DNA template added per reaction was 2 l. Primer targeting a repetitive region specific for Trypanozoon subgenus (Wuyts et al., 1994) pMURTec.F, 5 -TGCAGACGACCTGACGCTACT-3 ; pMURTec.R, 5 -CTCCTAGAAGCTTCGGTGTCCT-3 was used to amplify a 227 bp fragment from the genomic DNA. Seven microlitres of amplification products were resolved in 2% molecular grade agarose gel (Roche Diagnostic, Barcelone, Spain) with a molecular size marker (100 bp ladder). Gels were stained with ethidium bromide (0.5 mg/l) and analyzed on UV transilluminator (Bio-Rad Gel DocTM EQ). Blood from a camel clinical, serological and parasitologically positive for T. evansi was used as positive control.
4. Discussion Despite natural T. evansi infection has not been detected in goats living around the infected areas in Canaries, this study demonstrates that goats are susceptible to acquire the infection. Moreover, they seem to develop subclinical form of the disease, which is coincident with the findings of Jacquiet et al. (1993) using a Mauritanian strain, and could act as unapparent reservoirs. Parasitemia was detected at 2 weeks post-infection in all inoculated goats using MHCT. Other authors (Dargantes et al., 2005) have described detection of T. evansi after 2–5 days of experimental infection in goats using a smaller inoculum of a clinical equine strain in Philippines. Differences on virulence among strains of parasite or in susceptibility of Canarian goats could explain this fact. Regarding diagnosis, there is only little information in the available literature regarding diagnostic methods of trypanosomosis in goats. ELISA, IFI, or CATT/T. evansi have successfully been used as serological tests (OIE, 2009); however, parasitological examination is necessary in order to confirm the infection. At this respect, it is commonly assumed that parasitological methods employed in other animal species are also valid for goats, but we need more studies that demonstrate this assumption. The small size of the caprine erythrocytes, for example, must be taken into consideration to obtain valid PCV values (Jain, 1986). Moreover, parasitemia is commonly low in goats under
72
M.T. Tejedor-Junco et al. / Small Ruminant Research 96 (2011) 70–72
natural infection (Gill, 1977), and it could cause false negative cases. To solve this latter, we have evaluated a PCR using a primer targeting a repetitive region specific for Trypanozoon subgenus to amplify a 227 bp fragment from the genomic DNA, and it was able to detect parasitemia in all infected animals throughout the experiment. Probably the high trypanosome concentration inoculated by intravenous route could have contributed to this high sensitivity (100%). Micro-hematocrit centrifugation technique, on the other hand, showed interesting statistical data compared with PCR results. From the epidemiological point of view, for serologically positive goats we would propose the use of MHCT as the first parasitological method due to its high sensitivity (almost 93%) and, if negative, PCR could be used as second method of election. Cost-effectiveness, availability, time consumption and the possibility to perform the test in the field would support our proposal. Given that no serological method shows a sensitivity of 100%, in endemic areas and/or infected herds serologically negative goats should also be examined by those parasitological methods in order to discard/confirm the disease. Conflict interest statement The authors declare that they have no conflict of interest with the contents of this paper in any respect. Ethics All authors declare that animal experiments conducted in the present work meet the International Guiding Principles for Biomedical Research Involving animals.
Acknowledgement This work was supported in part by a grant of Spanish Ministry of Science and Technology (AGL2005-3433). References Dargantes, A.P., Reid, S.A., Copeman, D.B., 2005. Experimental Trypanosoma evansi infection in the goat. I. Clinical signs and clinical pathology. J. Comp. Pathol. 133, 261–266. Gill, B.S., 1977. Trypanosomes and Trypanosomiasis of Indian Livestock. Indian Council of Agriculture Research (ICAR), New Delhi, ICAR, pp. 21–23. Griffin, L., 1978. African trypanosomosis in sheep and goats: a review. Vet. Bull. 48, 819–825. Gutiérrez, C., Corbera, J.A., Doreste, F., Büscher, P., 2004. Use of the miniature anion exchange centrifugation technique to isolate Trypanosoma evansi from goats. Ann. NY Acad. Sci. 1026, 149–151. Gutiérrez, C., Juste, M.C., Corbera, J.A., Magnus, E., Verloo, D., Montoya, J.A., 2000. Camel trypanosomosis in the Canary Islands: assessment of seroprevalence and infection rates using the card agglutination test (CATT/T.evansi) and parasite detection tests. Vet. Parasitol. 90, 155–159. Jain, N.C., 1986. Schalm’s Veterinary Hematology, fourth ed. Lea & Febiger, Philadelphia. Jacquiet, P., Cheikh, D., Thiam, A., Dia, M.L., 1993. Trypanosomiasis caused by Trypanosoma evansi (Steel 1885), Balbiani 1888 in small ruminants in Mauritania: results of experimental inoculation and field surveys. Rev. Elev. Med. Vet. Pays Trop. 46, 574–578. Luckins, A.G., Dwinger, R.H., 2004. Non-tsetse-transmitted animal trypanosomiasis. In: Maudlin, I., Holmes, P.H., Miles, M.A. (Eds.), The Trypanosomiases. Cabi Publishing, Wallingford, U.K., pp. 269–281. OIE, 2009. Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, On line: http://www.oie.int. Smith, M.C., Sherman, D.M., 2009. Goat Medicine, second ed. WileyBlackwell, Ames. Thrusfield, F.M, 2007. Veterinary Epidemiology, third ed. Blackwell Science, Oxford. Woo, P.T.K., 1969. The haematocrit centrifuge for the detection of trypanosomes in blood. Can. J. Zool. 47, 921–923. Wuyts, N., Chokesajjawatee, N., Panyim, S., 1994. A simplified and highly sensitive detection of Trypanosoma evansi by DNA amplification. Southeast Asian J. Trop. Med. Public Health 25, 266–271.
Contents lists available at ScienceDirect
Small Ruminant Research journal homepage: www.elsevier.com/locate/smallrumres
Short communication
Comparison between micro-hematocrit centrifugation technique and polymerase chain reaction (PCR) to detect Trypanosoma evansi in experimentally inoculated goats M.T. Tejedor-Junco a,∗ , M. González a , N.F. Rodríguez b , J.A. Corbera b , C. Gutiérrez b a b
Department of Clinical Sciences, University of Las Palmas de Gran Canaria, P.O. BOX 550, 35080 Las Palmas de Gran Canaria, Canary Islands, Spain Department of Animal Medicine and Surgery, University of Las Palmas de Gran Canaria, 35413, Arucas, Las Palmas, Canary Islands, Spain
a r t i c l e
i n f o
Article history: Received 26 April 2010 Received in revised form 18 October 2010 Accepted 22 October 2010 Available online 15 December 2010 Keywords: PCR Trypanosoma evansi Micro-hematocrit centrifugation technique Goat
a b s t r a c t Natural Trypanosoma evansi infection in the Canary Islands has only been diagnosed in the camel population, but dissemination of the disease in other hosts has not been excluded. To evaluate the role of the goats in the dissemination of the disease, 8 goats were inoculated and examined during 6 months using a polymerase chain reaction (PCR) with a primer targeting a repetitive region specific for Trypanozoon subgenus used to amplify a 227 bp fragment from the genomic DNA. PCR was able to detect parasitemia in all tested samples; therefore it was considered as gold standard test in this study. The results were compared with those obtained using the micro-hematocrit centrifugation technique showing a sensitivity of 92.7%, specificity of 100%, positive predictive value of 1 and negative predictive value of 0.87. Both techniques seem to be adequate to detect T. evansi from infected goats. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Trypanosoma evansi is the most widely distributed of the pathogenic animal African trypanosomes, affecting domestic livestock and wildlife in Asia, Africa and Latin America (Luckins and Dwinger, 2004). The parasite causes a disease known as surra, and is mechanically transmitted by biting flies other than tsetse and vampire bats in South America. It affects a number of domesticated animals and the principal host species varies among the continents. Buffalo, cattle, camels and horses are particularly susceptible, although other animals, including wildlife, can also be infected (OIE, 2009). Small ruminants are commonly raised in close contact with camel herds in many desert and semi-desert areas in the world. However, it is commonly believed that goats, for example, are highly resistant to infection, that caprine trypanosomosis is only sporadic, and that the disease in
∗ Corresponding author. Tel.: +34 928 454358; fax: +34 928 454358. E-mail address: [email protected] (M.T. Tejedor-Junco). 0921-4488/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.smallrumres.2010.10.014
goats is of little economic consequence (Griffin, 1978). This opinion, however, is currently undergoing a critical reappraisal (Smith and Sherman, 2009). In the Canary Islands, where the camel population is around 2000 animals and the goat census is 363,000 heads (data from 2007, livestock census, Regional Canary Government, http://www2.gobiernodecanarias.org/istac/ estadisticas.html), goats share the same habitat with the camels in those areas where camels inhabit. In the Canarian Archipelago, Trypanosoma evansi infection was detected for the first time in 1998 in an imported dromedary camel and after that, it has only been diagnosed in the camel population (Gutiérrez et al., 2000). However, the possible role that goats could play in the maintenance of the disease in the islands is suspected but not proved, since natural infection has not been demonstrated. At this respect, infected goats could present two important constraints: low and/or alternant parasitemia and no apparent clinical signs of infection, which occurs commonly with many strains of T. evansi, including the Canarian one used in this study (Gutiérrez et al., 2004). Micro-hematocrit centrifugation technique is
M.T. Tejedor-Junco et al. / Small Ruminant Research 96 (2011) 70–72
a simple technique which is easy to perform, even at farm level, quick and at minimum cost; PCR, however, requires a laboratory of molecular biology, is more expensive and is not always available in developing countries. Given that there is considerable variation in the pathogenicity of different strains and the susceptibility of different host species to disease (OIE, 2009), the purpose of this study was to evaluate a PCR with a primer targeting a repetitive region specific for Trypanozoon subgenus used to amplify a 227 bp fragment from the genomic DNA, in comparison with micro-hematocrit centrifugation technique (Woo, 1969), using goats experimentally inoculated with T. evansi Canarian strain. 2. Materials and methods 2.1. Animals Twelve female and adult Canarian goats were acquired for this experiment. The animals were examined using the T. evansi diagnostic methods described below and resulted negative. Eight goats were experimentally inoculated with at least 1 × 105 T. evansi by intravenous route. The strain was obtained from a naturally infected camel in Canaries. The remaining four animals served as control. The goats were placed in fly-proof pens within an experimental area of the Veterinary Faculty and were maintained for 6 months. 2.2. Samples Blood samples were collected every 2 weeks from jugular vein using vacutainer® tubes with EDTA as anticoagulant and stored at 4 ◦ C until processed.
71
2.6. Statistical analysis Data were statistically analyzed following the recommendations of Thrusfield (2007) concerning to the evaluation and interpretation of diagnostic tests. Variables were measured as nominal and dichotomous (positive/negative).
3. Results The inoculated goats showed a subclinical course of the disease. Episodes of fever within the first week postinoculation and arthritis at 6 months post-infection were detected in two inoculated goats. All experimentally inoculated animals were positive in the CATT/T. evansi 1 month post-infection and remained positive with a minimum end-titer of 1/4 throughout the experiment. Parasitemia was detected at 2 weeks post-infection in all inoculated goats, and the animals were then considered infected. From this group (infected) a total of 96 assessments (8 infected goats per 12 checking) were performed. PCR was able to detect parasitemia in all examined samples, while MHCT detected parasitemia in 89 samples; in 7 samples this technique failed to detect trypanosomes. From the control group, a total of 48 assessments (4 goats per 12 checking) were made, being all tested samples negative. Considering the PCR as a gold standard test in this study, comparison between PCR and MHCT showed the following statistical data: sensitivity: 92.7%, specificity: 100%; positive predictive value: 1; negative predictive value: 0.87.
2.3. Serological assessment CATT/T. evansi (Institute of Tropical Medicine, Antwerp, Belgium), a validated serological method referred by OIE (2009) was used to evaluate the presence of antibodies against T. evansi. 2.4. Micro-hematocrit centrifugation technique (MHCT) This technique detects the presence of the motile trypanosomes and was performed using one capillary tube per animal and centrifuging the samples at 9520 × g. Blood samples were maintained under refrigeration and the technique was always done within the next 2 h after the blood was taken. 2.5. DNA extraction and PCR Genomic DNA was extracted from samples of goat blood using commercially available QIAamp DNA mini kit (Qiagen, Hilden, GE) and the purified DNA was stored at −20 ◦ C for later use. PCR was carried out in 25 l reaction mixtures containing 10× reaction buffer (Bioline, London, UK), 2.5 mM MgCl2 , 300 M of each of the four deoxynucleoside triphosphates (dNTPs) (Amersham Bioscience Europe, Barcelona, Spain), primers 100 ng and 0.75 U of Taq DNA polymerase (Bioline). The cycles included an initial step at 94 ◦ C for 4 min, followed by 30 cycles of denaturing at 94 ◦ C for 30 s, annealing at 60 ◦ C for 30 s and extension at 72 ◦ C for 30 s. PCR elongation was continued at 72 ◦ C for 5 min. The amount of DNA template added per reaction was 2 l. Primer targeting a repetitive region specific for Trypanozoon subgenus (Wuyts et al., 1994) pMURTec.F, 5 -TGCAGACGACCTGACGCTACT-3 ; pMURTec.R, 5 -CTCCTAGAAGCTTCGGTGTCCT-3 was used to amplify a 227 bp fragment from the genomic DNA. Seven microlitres of amplification products were resolved in 2% molecular grade agarose gel (Roche Diagnostic, Barcelone, Spain) with a molecular size marker (100 bp ladder). Gels were stained with ethidium bromide (0.5 mg/l) and analyzed on UV transilluminator (Bio-Rad Gel DocTM EQ). Blood from a camel clinical, serological and parasitologically positive for T. evansi was used as positive control.
4. Discussion Despite natural T. evansi infection has not been detected in goats living around the infected areas in Canaries, this study demonstrates that goats are susceptible to acquire the infection. Moreover, they seem to develop subclinical form of the disease, which is coincident with the findings of Jacquiet et al. (1993) using a Mauritanian strain, and could act as unapparent reservoirs. Parasitemia was detected at 2 weeks post-infection in all inoculated goats using MHCT. Other authors (Dargantes et al., 2005) have described detection of T. evansi after 2–5 days of experimental infection in goats using a smaller inoculum of a clinical equine strain in Philippines. Differences on virulence among strains of parasite or in susceptibility of Canarian goats could explain this fact. Regarding diagnosis, there is only little information in the available literature regarding diagnostic methods of trypanosomosis in goats. ELISA, IFI, or CATT/T. evansi have successfully been used as serological tests (OIE, 2009); however, parasitological examination is necessary in order to confirm the infection. At this respect, it is commonly assumed that parasitological methods employed in other animal species are also valid for goats, but we need more studies that demonstrate this assumption. The small size of the caprine erythrocytes, for example, must be taken into consideration to obtain valid PCV values (Jain, 1986). Moreover, parasitemia is commonly low in goats under
72
M.T. Tejedor-Junco et al. / Small Ruminant Research 96 (2011) 70–72
natural infection (Gill, 1977), and it could cause false negative cases. To solve this latter, we have evaluated a PCR using a primer targeting a repetitive region specific for Trypanozoon subgenus to amplify a 227 bp fragment from the genomic DNA, and it was able to detect parasitemia in all infected animals throughout the experiment. Probably the high trypanosome concentration inoculated by intravenous route could have contributed to this high sensitivity (100%). Micro-hematocrit centrifugation technique, on the other hand, showed interesting statistical data compared with PCR results. From the epidemiological point of view, for serologically positive goats we would propose the use of MHCT as the first parasitological method due to its high sensitivity (almost 93%) and, if negative, PCR could be used as second method of election. Cost-effectiveness, availability, time consumption and the possibility to perform the test in the field would support our proposal. Given that no serological method shows a sensitivity of 100%, in endemic areas and/or infected herds serologically negative goats should also be examined by those parasitological methods in order to discard/confirm the disease. Conflict interest statement The authors declare that they have no conflict of interest with the contents of this paper in any respect. Ethics All authors declare that animal experiments conducted in the present work meet the International Guiding Principles for Biomedical Research Involving animals.
Acknowledgement This work was supported in part by a grant of Spanish Ministry of Science and Technology (AGL2005-3433). References Dargantes, A.P., Reid, S.A., Copeman, D.B., 2005. Experimental Trypanosoma evansi infection in the goat. I. Clinical signs and clinical pathology. J. Comp. Pathol. 133, 261–266. Gill, B.S., 1977. Trypanosomes and Trypanosomiasis of Indian Livestock. Indian Council of Agriculture Research (ICAR), New Delhi, ICAR, pp. 21–23. Griffin, L., 1978. African trypanosomosis in sheep and goats: a review. Vet. Bull. 48, 819–825. Gutiérrez, C., Corbera, J.A., Doreste, F., Büscher, P., 2004. Use of the miniature anion exchange centrifugation technique to isolate Trypanosoma evansi from goats. Ann. NY Acad. Sci. 1026, 149–151. Gutiérrez, C., Juste, M.C., Corbera, J.A., Magnus, E., Verloo, D., Montoya, J.A., 2000. Camel trypanosomosis in the Canary Islands: assessment of seroprevalence and infection rates using the card agglutination test (CATT/T.evansi) and parasite detection tests. Vet. Parasitol. 90, 155–159. Jain, N.C., 1986. Schalm’s Veterinary Hematology, fourth ed. Lea & Febiger, Philadelphia. Jacquiet, P., Cheikh, D., Thiam, A., Dia, M.L., 1993. Trypanosomiasis caused by Trypanosoma evansi (Steel 1885), Balbiani 1888 in small ruminants in Mauritania: results of experimental inoculation and field surveys. Rev. Elev. Med. Vet. Pays Trop. 46, 574–578. Luckins, A.G., Dwinger, R.H., 2004. Non-tsetse-transmitted animal trypanosomiasis. In: Maudlin, I., Holmes, P.H., Miles, M.A. (Eds.), The Trypanosomiases. Cabi Publishing, Wallingford, U.K., pp. 269–281. OIE, 2009. Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, On line: http://www.oie.int. Smith, M.C., Sherman, D.M., 2009. Goat Medicine, second ed. WileyBlackwell, Ames. Thrusfield, F.M, 2007. Veterinary Epidemiology, third ed. Blackwell Science, Oxford. Woo, P.T.K., 1969. The haematocrit centrifuge for the detection of trypanosomes in blood. Can. J. Zool. 47, 921–923. Wuyts, N., Chokesajjawatee, N., Panyim, S., 1994. A simplified and highly sensitive detection of Trypanosoma evansi by DNA amplification. Southeast Asian J. Trop. Med. Public Health 25, 266–271.