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Experimental Parasitology 134 (2013) 109–114
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Trypanosoma evansi: Detection of Trypanosoma evansi DNA in naturally and experimentally infected animals using TBR1 & TBR2 primers Ameen A. Ashour a,1, Tarek R. Abou El-Naga b,2, Safaa M. Barghash b,⇑, Mohamed S. Salama c,1 a
Zoology Department, Faculty of Science, Ain Shams University, P.O. Box 11566, Cairo, Egypt Animal Health Department, Desert Research Center, P.O. Box 11753, Cairo, Egypt c Entomology Department, Faculty of Science, Ain Shams University, P.O. Box 11566, Cairo, Egypt b
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
" TBR1.2 PCR-based assay was
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evaluated for detection of T. evansi DNA in Egypt. PCR detected acute and chronic animals, and the following of drug treatment. It supported the possible role of small ruminants as carrier hosts of T. evansi. PCR was more powerful than other tests used, concerning the infection in mice. This study recommended using PCR for epidemiological studies on T. evansi in Egypt.
a r t i c l e
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Article history: Received 12 October 2009 Received in revised form 23 July 2012 Accepted 5 February 2013 Available online 27 February 2013 Keywords: Trypanozoon Trypanosoma evansi PCR: polymerase chain reaction TBR 1/2 Egypt
a b s t r a c t A polymerase chain reaction (PCR-based) assay was evaluated for detection of Trypanosoma evansi DNA in experimentally infected mice and naturally infected camels, sheep and goats using the set of primers TBr1 & TBr2 that amplified 164 bp DNA fragment. The results revealed that PCR-based assay was able to detect T. evansi directly from the blood during both acute and chronic phase of infection in all tested animals and in the blood and tissues of intraperitoneally infected mice depending upon the level of infection in the test samples. PCR was more powerful than CATT/T. evansi and mouse inoculation tests, when detected the infection in mice (24 h) post infection. Present results show that sheep & goats probably play a role in transmission of T. evansi to camels and supported that PCR could be used as a diagnostic tool for epidemiological studies on T. evansi in Egypt. Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction Trypanosoma evansi is a member of the subgenus Trypanozoon that also comprises Trypanosoma brucei and Trypanosoma equiperdum. T. evansi lacks a mitochondrial genome and its kinetoplast ⇑ Corresponding author. Fax: +20 2 26357858. 1 2
E-mail address:
[email protected] (S.M. Barghash). Fax: +20 2 26842123. Fax: +20 2 26357858.
0014-4894/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.exppara.2013.02.003
contains only a homogeneous set of minicircles, while T. equiperdum contains maxicircle in kDNA (Frasch et al., 1980). Therefore, it is transmitted only mechanically by blood – sucking vectors that lead to wide spreading of this parasite from Africa to the other parts in Asia and South America (Hoare, 1972; Lun et al., 1992). Diagnosis of T. evansi basically relied on conventional Giemsa staining of thin and thick blood smears, which are time consuming and required experienced personnel. Serological tests used for the parasite detection suffer the disadvantages in reproducibility due to the antigenic variation and significant levels of false negative
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and false positive results. In addition, these tests do not distinguish between current infections and residual antibodies from previous vaccination or infection (Chansiri et al., 2002). To overcome the limits of sensitivity and specificity imposed by other diagnostic methods, molecular detection of T. evansi deoxyribonucleic acid (DNA) is preferred; especially it did not need high amount of trypanosome and could be performed with non-sterile blood samples (Büscher, 1999). However, positive amplification products for any set of primers only signal that the target nucleic acid sequences were present in the sample (Aradiab and Majid, 2006). Trypanosomes in rodents found in the heart, liver, lung, spleen and kidney. Liver is one of the most important visceral organs that largely affected by T. evansi infection, PCR does not require an antibody response to the infectious agent and DNA used for PCR obtained from sections of tissues (Biswas et al., 2001). Several reports have suggested that T. evansi and T. equiperdum distinguished from T. brucei by analyses of kinetoplast DNA (kDNA) (Lun et al., 1992), but analysis of kDNA is not possible for the detection of the dyskinetoplastic stocks due to the lack of kDNA (Kaminsky et al., 1997). During the past few years, several research centers have been working on the development of PCR procedures for the detection of minute amount of trypanosomal DNA sequences and detects even dyskinetoplastic T. evansi strains. As an alternative to parasitological tests, DNA detection based on PCR has proposed to distinguish T. evansi from the other members of the Trypanozoon subgenus. Trypanozoon specific primers have designed previously: TBR primers that target a 177 bp repeat (Moser et al., 1989; Gonzales et al., 2003) and 164 bp repeat (Masiga et al., 1992; Ijaz et al., 1998). Other research groups have used VSG genes as target sequences for PCR detection of T.b. gambiense infections (sleeping sickness). Recently, Ventura et al. (2001) developed a PCR (PCRTe664) for the detection of T. evansi based on a Random Amplified Polymorphic DNA (RAPD) fragment. Urakawa et al. (2001) derived other designed primers from the sequence of VSG cDNA (RoTat 1.2). The objective of this study was to evaluate PCR based-assay using TBr1 & TBr2 primer that amplified 164 bp for the detection of T. evansi DNA in naturally infected camels, goats and sheep, and in experimentally infected mice. 2. Materials and methods 2.1. Study area and animal sampling The study area, Halaib, Shalateen and Abu–Ramad triangle region, situated in Red Sea governorate, at the Southeastern of Egypt, within 22–24 N°, approximates 1100–1200 km South to Cairo. This area is endemic for trypanosomiasis of veterinary importance. It is considered as the major Egyptian Southern entrance from Sudan, through which camels, goats and sheep are imported legally or illegally. In this study, 21 different trypanosome populations isolated from a variety of those host species selected as positive naturally infected with T. evansi by card agglutination test (CATT/T. evansi). Besides, 22 samples of mice experimentally infected by passage with infected blood from the same populations stocks, harvested at peak parasitemia and purified from blood cells using DE-52 column chromatography, were evaluated by PCR and by microscopically examination for the presence of T. evansi. Examined samples originated from a cross sectional survey by Barghash (2005) was carried out during the period between March 2002 and April 2003 to estimate the prevalence of T. evansi in camels, goats and sheep in the study area. 2.2. Blood samples collection For each examined animal, two blood samples were collected by the jugular venipuncture, one on heparin was immediately
diluted 1:3 with ice–cold phosphate-saline-glucose (PSG) pH 7.4 for inoculation into Swiss-bred mice, which were previously immunosuppressed by intraperitoneal injection with cyclophosphamide (5 mg/25 g/mouse) to expand the parasitemia according to El–Sawalhy and Ebeid, 1994. The second was one part on a tube containing the sodium salt of ethylene di-amine tetra-acetic acid (EDTA) for DNA extraction, and the other part in the tube without the anticoagulant for subsequent serum collection. 2.3. Parasitological examination Two blood film slides from each animal were stained with diluted Giemsa‘ stain according to Hoare (1972). Parasitemia level was detected daily post infection in mice by wet smear examination of tail-tip blood. 2.4. Serological test Sera tested for the presence of antibodies against T. evansi in naturally infected camels, goats and sheep and in experimentally infected mice using CATT/T. evansi (Bajyana-Songa and Hamers, 1988). The test performed following the manufacturer’s instructions (Institute of Tropical Medicine, Antwerp, Belgium). Briefly, 25 ll of each animal serum was diluted 1:4 in CATT buffer, and pipette onto a reaction zone of a plastic coated test cards. After adding one drop (about 45 ll) of CATT reagent, the reaction mixture spread out by means of a stirring rod and allowed to react on a card test rotator for 5 min at 70 rpm. Agglutination patterns were scored as (negative), ± (weakly positive), + (positive) and ++ or +++ (strongly positive). Negative and positive controls always used for checking each test. 2.5. Statistical analysis Statistical analysis was performed using SPSS v11. The prevalence of Trypanosoma evansi and the infection between different animals were determined using chi-square test and odds ratios and their 95% confidence intervals. The level of significant difference was set at 0.05. 2.6. Polymerase chain reaction assay 2.6.1. Preparation of samples for PCR Whole blood samples with EDTA from naturally and experimentally infected animals were stored in sterile Eppindorff tubes in deep freeze at 20 °C until used. Liver and spleen tissue samples of experimentally infected mice from camels, goats and sheep origin were selecting before antibodies developed and preserved immediately in 10% formalin until used for DNA preparation. The three T. evansi isolates of camel, goat and sheep origin with PSG pH 8.0 solution as eluting buffer were centrifuged at 12000 rpm for 15 min, then collected cells were dissolved in sterile distilled water and boiled for 5 min at 90 °C to lyse the cells. The clear cell lysate transferred into clean Eppindorff tubes and stored at 80 °C. 2.6.2. Extraction, purification and precipitation of DNA DNAs of T. evansi isolate from whole bloods and from tissue, components were extracted, purified and precipitated directly using commercially available Gentra DNA purification kit (PureGene, USA) according to the manufacturer’s instructions. Some DNA of T. evansi isolates extracted by the same DNA extraction kit from purified parasites of infected mice bloods with di-ethyle-amino-ethyl-cellulose (DEAE) anion-exchange column chromatography according to Lanham and Godfrey (1970).
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2.6.3. PCR amplification According to the method described by Masiga et al. (1992) with some modifications, using a commercially prepared specific primer set (TBr1 & TBr2) that yields a 164 bp product for the specific detection of T. evansi. Primer sequences (in 50 -30 direction) were as follows: Forward GAA TAT TAA ACA ATG CGC AG, Reverse CCA TTT ATT AGC TTT GTT GC. These primer sets are five base pairs shorter than the primers used by Moser et al. (1989). Positive control (DNA from blood sample of infected mouse with T. evansi at fulminated parasitemia), and negative control (without DNA) used. PCR amplification reaction was carried out in 1 buffer containing 50 mM KCl, 10 mM Tris-HCl pH 8.3, and 0.1% Triton X-100, enriched with 1.5 mM MgCl2, 0.2 mM of each dATP, dCTP, dGTP and dTTP (Promega, USA), 25 pmol each primer (TBr1 & TBr2), 25 ng of genomic DNA and 1.0 U of Taq DNA polymerase (Promega, USA). All components were mixed and sterile distilled water was added to a final volume of 50 ll. PCR reactions were conducted in (BioRad) thermocycler that was programmed for an initial denaturation and activation step at 94 °C for 10 min. This step followed by 40 cycles of 94 °C for 30 s (denaturation), annealing step at 50 °C for 45 s and extension step at 72 °C for 60 s. Post-extension was by one cycle at 72 °C for 10 min followed by holding at 4 °C. 2.6.4. PCR detection Ten microliters of PCR products were electrophorized on 2% agarose gels with 100 bp markers as size marker (Amersham, Bioscience), at constant voltage (80 V for 90 min). The gels were stained with ethidium bromide and T. evansi PCR products identified following visualization under UV light and photographed using Polaroid camera (Mp4). The molecular sizes of the PCR bands were estimated by comparing with those of a standard molecular size marker (100–2000 bp) DNA ladder, and scored by the Gel work ID advanced software UVP, England.
respectively. PCR assay could be used to detect T. evansi directly from the blood showing the infection in all samples when host DNA was used as template, at the same size of DNA fragment product, despite goats gave very weak or unclear fragment at 164 bp (Figs. 1 and 2). It was noticed that all three methods showed no false-positive results and there was a good correlation between PCR and CATT/T. evansi results in most samples, especially when detected T. evansi in the chronic phase while blood films examination failed to detect it. 3.2. Experimentally infected mice Parasitaemia showed the difference in patterns in all infected groups according to the difference of strain samples injected. However, T. evansi in the blood of experimental mice/camels increased into an acute phase during the course of infection, the highest peak of parasitaemia detected at 9–12 days post infection, and death occurred because of severe anemia and weight loss. On the other hand, T. evansi infection in experimental mice/sheep and mice/goats progressed into a chronic phase. Notably, parasitaemia in mice/ camels could be detected microscopically after 2.4 days post infection until slaughtered; but not in infected mice/sheep and mice/goats, despite the appearance of clinical symptoms (weight loss) on those mice. PCR assay could detect T. evansi DNA in all groups (100%) in acute and chronic phases one day post infection earlier than other tests used giving obvious fragment for the blood of mice/camels and mice/sheep, and very weak bands in mice/ goats at 164 bp (Figs. 1-3). This result correlated to the results of
Table 1 Detection of Trypanosoma evansi in naturally infected animals and experimentally infected mice using parasitological, serological and molecular methods. Tests Animals
3. Results 3.1. Naturally infected animals TBR 1/2 PCR assay used to detect T. evansi infection directly from infected blood samples collected from camels, goats and sheep in different localities of the study area, and in experimental infected mice from those animals. There were significant differences (P < 0.05) between results obtained from each of CATT/T. evansi, PCR with blood film examination tests used, and not between results of CATT/T. evansi and PCR tests (Table 1). Out of 43 DNA samples prepared, 15 (34.9%), 40 (93%) and 43 (100%) were positive by microscopic examination, CATT/T. evansi and PCR assay,
Camels Sheep Goats Total natural Mice/camels Mice/sheep Mice/goats Total Experimental Total
B. film examination
CATT/T. evansi
PCR
n
+Ve
%
n
+Ve
%
n
+Ve
%
7 7 7 21 8 7 7 22 43
4 2 1 7 6 1 1 8 15
57.1 28.6 14.3 33.3 75 14.3 14.3 36.4 34.9
7 7 7 21 8 7 7 22 43
7 7 7 21 6 6 7 19 40
100 100 100 100 75 85.7 100 86.4 93
7 7 7 21 8 7 7 22 43
7 7 7 21 8 7 7 22 43
100 100 100 100 100 100 100 100 100
+Ve = Infected animals, % = Percentage of infected animals. Mice/camels = mice were inoculated from infected camels. Mice/sheep = mice were inoculated from infected sheep. Mice/goats = mice were inoculated from infected goats.
Fig. 1. Detection of Trypanosoma evansi DNA with (TBR1 & TBR2) primers in naturally infected camels (blood), and in experimentally infected mice (blood and tissues). Lane 1 (uninfected camel treated with Berinil). Negative control and positive control (Lanes 1 and 2). Lanes (4, 5, 6, 7 and 8), correspond to PCR products & 2) from blood of camels. Lanes (9, 10 and 11) correspond to PCR products from blood of mice infected with T. evansi from camels (mice/camels). Lanes (12, 13 and 14) correspond to DNA from tissues of mice/camels, mice/goats and mice/sheep, respectively. Lane M, DNA molecular size marker (Amersham, Bioscience).
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Fig. 2. Ethidium bromide stained agarose (2%) gel showing PCR-product of T. evansi DNA from whole bloods of sheep, goats and mouse infected from positive goat. Lane 1: represents ve (negative control). Lane 2: represents +ve (positive control). Lanes (3, 4 and 5): correspond to blood of sheep. Lanes (6, 7 and 8) correspond to blood of goats. Lane 9 represents blood of mouse infected with T. evansi from infected goat. Lanes (M and 10) are DNA molecular size marker (Amersham, Bioscience).
Fig. 3. Results of agarose (2%) gel electrophoresis of 164 bp of PCR-product using (TBR1 & TBR2) primers with T. evansi DNA from blood of mice showing different stages of infection from (24 to 72 h) post infection. Lane 1 represents +ve (positive control), Lanes (2, 3, 4, and 5) correspond to blood of mice infected from positive camels. Lanes (6, 7, 8 and 9) correspond to blood of mice infected from positive goats. Lanes (10, 11, 12 and 13) correspond to blood of mice infected from positive sheep. Lane M, DNA molecular size marker (Amersham, Bioscience).
Fig. 4. Agarose (2%) gel electrophoresis of 164 bp of PCR-product using (TBR1 & TBR2) primers with camel, goat and sheep isolates of T. evansi DNA with and without PSG (Phosphate Saline Glucose Buffer). Lanes 1 and 2 represent ve (negative control) and +ve (positive control), respectively. Lanes (6, 7 and 8) correspond to PCR-products from camel, goat and sheep isolates with PSG buffer. Lanes (9, 10 and 11) correspond to PCR-products from camel, goat and sheep isolates without PSG buffer, respectively. Lane M, DNA molecular size marker (Amersham, Bioscience).
CATT/T. evansi (86.4%) in most samples and not to microscopic examination results showed that out of 22 samples, only eight were positive by microscopic examination (36.4%). Concerning tissues, the spleen was only enlarged as a clinical sign in mice/camels, and slightly enlarged compared to that of the normal and was darker in color in mice/sheep and mice/goats.
At the same time, the liver slightly enlarged and became a bit paler in color than that of negatively infected samples. PCR assay results supported that T. evansi is tissue and blood parasite, however the total DNA from tissues of infected mice amplified giving the same size of DNA fragment (164 bp) in the three samples in spite of weak fragments, especially for tissues of mice/goats.
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Fig. 5. Blood film stained by Giemsa from infected camel showing T. evansi.
By using column chromatography, a large number of trypanosomes in mice/camels and mice/sheep, and less number of trypanosomes per field in mice/goats in PSG buffer were obtained. When they detected by PCR mice/camels and mice/sheep samples gave dense specific fragments at 164 bp, while mice/goats also gave very faint PCR fragment as shown in Fig. 4. In contrast, when the three isolates concentrated by removing PSG buffer were subjected to PCR assay, they gave very dense specific bands at 164 bp as represented in Fig. 4. In general, there were two kinds of signals by using TBR1/2 primer pair: one-band signal like or take the form of dimer or trimer-band signal like in other PCR products. (See Fig. 5) 4. Discussion The PCR assay described in the present study has been developed and applied for detection of T. evansi in several animals but the application did not extend to sheep and goats. The primer set (TBR1 & TBR2) was specific for T. brucei, but it would be useful especially outside the tsetse belt in which the kinetoplastic /or akinetoplastic T. evansi were widely prevalent but not for T. brucei as reported by Inoue et al. (1998). TBR1/2 primer set was able to detect 0.01 pg of purified DNA, and a parasitemia below one parasite per ml in rat blood. They presented the highest sensitivity in as well as a high specificity, without non-specific products or false positive reactions out of samples tested (Pruvot et al., 2010; Fernández et al., 2009). Its applicability in actual field studies is of economic concern, especially its suitability as a diagnostic test in rural areas (Baticados et al., 2011). Currently, PCR depends on identifying portions (base pairs) of DNA, from the nucleus or from the kinetoplast, which are specific for T. evansi. The target sequences for TBr1 & TBr2 found in nucleus not in kinetoplast making results independent of the parasite kinetoplastic state and avoid the problem of failure targeting kDNA as reported in South America by Ventura et al. (2000) and Gonzales et al. (2003). In the present study, CATT/T. evansi and PCR assay were used together to confirm the results, especially T. evansi was found to be the common member of the subgenus Trypanozoon that infects camels, goats and sheep at the study area (Barghash, 2005). Besides, the gene encoding RoTat 1.2 VSG is present in all T. evansi but not in T. brucei isolates as reported by Claes et al. (2004). In the present study, the detection of T. evansi DNA in blood samples of camels, goats and sheep using TBR1 & TBR2 specific primer pair, after screening with CATT/T. evansi showed that, all these samples amplified at the same size of DNA fragment product. The intensity of PCR bands was variable in different test samples depending upon the level of infection in the test samples. The amplification signal was strong in camels and sheep, while was un-
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clear or too faint in goats despite the positive results using CATT/T. evansi. The casual appearance of non-specific bands on gel might be due to co-amplification resulted from the primining within the repeated regions of the trypanosome genome. Nature of unclear reaction products might be due to the template contained too much DNA as reported by Pereira de Almeida et al. (1998). The specific band was shorter than 164 bp in lane 13 (Fig. 3), might be a result of infection with another trypanosome like T. brucei. All three tests used were applied to detect parasitaemia in acute phase of infection, but the sensitivity of the PCR was higher (100%), and detected the infection only one-day post infection, earlier than the other tests. On the other hand, parasites during chronic phase could not be demonstrated microscopically in mice/sheep and mice/goats groups. This is due to low level and often-cryptic parasitaemia is characteristic to chronic T. evansi infection as previously observed in rabbits, goats, sheep and camels following experimental and natural infection by Boid et al. (1981) and Audo et al. (1999), despite the changes in the blood picture and weight loss. Significant differences regarding behavior and morbidity patterns in all groups of infected mice were also evident which is in agreement with de Menezes et al. (2004), and Barghash (2005). Generally, goats and mice/goats showed high resistance to early infection with T. evansi and developed a very low level of parasitaemia, which remains almost undetected by common laboratory techniques. This is might be the reason of the unclear or weak fragments produced in blood, tissue samples and in the isolate in PSG buffer, compared to camels, sheep and their infected mice groups that gave stronger positive results. This is could be explained by the shortage of T. evansi numbers that surely influenced by immune system of goats against infection. This explanation coincides with the observation of strong results at 164 bp of the three isolates of T. evansi after samples concentrated by removing PSG before detected by specific PCR. In conclusion, T. evansi is common in the study area and PCR assay could use to detect T. evansi directly from blood and could play an important role in the detection of infected animals harboring a low parasitaemia, which are usually undetectable by smears. Results supported the suggestion of Ijaz et al. (1998) that T. brucei primers TBr1 and TBr2 were able to detect T. evansi, especially in experimental T. evansi infection in mice at 164 bp. However, the majority of domestic animals kept in this endemic area acquires natural infections of trypanosomes, and are therefore important in the transmission cycle, the authors concluded that goats and sheep might play a role in transmission of T. evansi among camels at this area. Acknowledgments The authors gratefully acknowledge the financial support from the Desert Research Center (DRC). We thank the technical staff of Molecular Biology Lab. at Agriculture Genetic Engineering Research Institute (AGERI), Cairo, Egypt for technical assistance.
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