Proteinas inmunorreactivas de Trypanosoma vivax.
Bovine (Bos taurus and Bos indicus) trypanosomosis caused by Trypanosoma vivax is a chronic disease of livestock in Africa and Latin America . Cattle infected with T vivax present fever, anemia, lethargy, loss of physical function, reduced milk yields, abortion and death [11, 14]. In Venezuela, the parasite is present all over the Country, affecting cattle with a general seroprevalence of 33.1% . The diagnosis is mostly based in the detection of the parasites in Giemsa-stained blood smears or by microhaematocrit centrifuge technique (HCT) ; however, it is difficult to demonstrate the presence of trypanosomes in this way, since infections are often chronic and few parasites are present . Many serological assays have been developed for the diagnosis of T vivax but few have found practical application in the field . The immunodiagnosis of T vivax is often performed by the ELISA test using soluble antigenic extract of T evansi because of the high antigenic cross-reactivity and the ease of replicating T evansi in rodents to obtain the crude extracts [11, 14, 19], but there have been few attempts to determine the differences in the type of antigen preparation or of the particular isolate from which the antigen is derived. Unfortunately, there is no species-specific ELISA because of cross-reactions between these trypanosomes [19, 23]. However, given that cattle can also be infected by T evansi, and that the clinical and epidemiological importance of this parasite is not clear , it would be extremely desirable to use purified antigens to perform T vivax-specific serodiagnosis because the treatment and control of the disease is trypanosome species-dependent . For this reason, this research was undertaken with the aim of determining the immunoreactive proteins of the soluble extract antigens of Trypanosoma vivax.
MATERIALS AND METHODS
Trypanosoma vivax isolate
T vivax isolate (TvZC1) was obtained from a natural infected heifer in a farm localized in Zulia State, Venezuela. Diagnosis was performed by the microhaematocrit centrifuge technique  and Giemsa-stained blood smears. Then, blood was collected in tubes containing 1 mg/mL of ethylenediaminetetracetic acid (EDTA), and cryopreserved as previously described . Prior to the study, parasitological diagnosis was confirmed using a T vivax-specific polymerase chain reaction (PCR) assay .
Six mixed Barbados Blackbelly sheep (Ovis aries), of approximately one year of age, negative to T vivax infection (determined by serological and molecular tests) were used in the study. Two weeks before and during experimental infection, all animals were maintained under veterinary supervision in order to safeguard health and minimize animal suffering. Protocols used were approved by the Ethical Committee for Laboratory Animal Use (University Simon Rodriguez) under the number 013-11. As well, these ethical procedures were based on Venezuelan law .
T. vivax infection
In order to expand the isolate, one sheep was used as a donor which was immunosuppressed with six intravenous injections of 2 mg/kg dexamethasone every other day, as previously described  and then, 4 mL of cryopreserved blood was inoculated intravenously. This sheep was monitored every day for haematocrit, temperature and parasitemia values. When the parasitemia reached a value of [10.sup.6] trypanosomes/mL, as determined by the hemocytometer method , the other animals (named from 1 to 5) were inoculated intravenously with 2 mL of sheep donor fresh blood (approximately 2 x [10.sup.6] trypanosomes). The donor animal was later treated with 3.5 mg/ kg of 7% diminazene aceturate through a deep intramuscular (i.m.) injection. Animal number 1 was used to obtain soluble extract of proteins at the first parasitemia peak and then it was treated similarly to the donor animal. Infected animals 2 through 5 were monitored during eight weeks in order to obtain sera and perform Western blot analysis (WB). The serum samples of each animal were obtained every two weeks (weeks 2, 4, 6 and 8 post infection). In order to safeguard the health of these animals, clinical aspects such as temperature, haematocrit and general condition were monitored daily.
T. vivax purification
At day 5 after infection, animal 1 presented the first parasitemia peak. In this time, T vivax purification was made as described . The final cell pellet was suspended in 1 mL of phosphate buffered saline (PBS) pH 7.2 containing 1% glucose (PBSG) in the presence of protease inhibitors and was kept frozen at -70 [grados]C (Revco[TM] ULT-1790, Thermo Scientific[TM], USA) until further use.
Preparing of soluble extract of parasites and quantitation of total protein
Parasites pellet was thawed and prepared by sonication (VibraCell[TM] VCX130, Sonics and Materials Inc, USA) three times for 30s on ice and centrifuged at 10.000 g for 10 min at 4[grados]C (CRU5000 Centrifuge, Damon/IEC[TM], USA) to obtain a supernatant rich in soluble trypanosome protein. The protein concentration of this extract was determined using the micro BCA[TM] (Bicinchoninic acid) kit with bovine serum albumin (BSA) as standard (Pierce Laboratories, Illinois, USA).
Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis
Prior to electrophoresis, 30 [micron]g soluble protein extract was diluted with sample loading buffer (0.06 M Tris-CI, pH 6.8, 5% [beta]-mercaptoethanol, 10% glycerol, 2% SDS, 0.01% bromphenol blue) and heated at 100[grados]C for 5 min. For identification of antigenic proteins from soluble extract, electrophoresis was carried out on 12% SDS-PAGE . Standard molecular weight marker protein (BlueRanger[R] Pierce Laboratories, Illinois, USA) was run simultaneously. Following SDS-PAGE, the proteins were electrotransferred (100 V constant for 2 h at 4[grados]C) to a nitrocellulose membrane (0.45 pm pore size) . After the membrane incubation in blocking solution (5% non-fat dry milk diluted in 20 mM PBS, 150 mM NaCl, pH 7.2 for 30 min), sera from infection of animals 2 through 5 were added to nitrocellulose sheets at dilution 1:100 in PBS and incubated over night at ambient temperature. The antigenically reacting proteins were incubated with peroxidase conjugated secondary antibodies against sheep IgG (Jackson ImmunoResearch[R] Laboratories, USA), dilution 1:400 in PBS. Finally, the protein bands were visualized by the addition of 4-chloro-1-naphthol (Horseradish Peroxidase-Color Development Reagent 4 CN, Bio-Rad), according to the provider. To assess cross reactivity with other hemoparasites and recognition of other T vivax isolates, sera from sheep naturally and experimentally infected with T vivax, hemoparasites-negative cattle, cattle experimentally infected with Babesia bigemina and a horse (Equus caballus) naturally infected with T. evansi were used, all of them determined by Immunofluorecence antibody test (IFAT) as described previously . Sera from T vivax-positive (experimentally-infected sheep 1 from this study) and T. vivax-negative sheep were used as control for WB. The relative molecular weight (Mr) of proteins resolved by SDS-PAGE was estimated by Kodak 1D Image Analysis Software .
RESULTS AND DISCUSSION
The pattern of T vivax proteins separated by SDS-PAGE showed eleven bands of approximately Mr 27, 31, 42, 64, 72, 80, 94, 115, 150, 170 and 215 kDa (FIG. 1), of which the most distinctive bands were: 31, 72, 80 and 64 kDa. Proteins of similar molecular weight have received considerable attention and several research studies have been made in regards to their possible utilization in diagnosing bovine trypanosomosis. Using cytosolic antigens of a different T vivax isolate, other researchers  observed in 12% SDS-PAGE several proteins with Mr between 33.9 and 73 kDa, but only one distinct band of Mr approximately 47.5 kDa. Moreover, in a study using the same method of antigen preparation , in soluble extract of T. vivax were observed several proteins, and the most relevant one having an apparent molecular mass of 39 kDa. Most of these proteins are in close conformity with the polypeptides found in the present study; however, discrepancy between findings might be due to differences in isolates of T vivax used and the methods of antigen preparation.
The WB analysis made with sera from four animals during eight weeks of infection is presented in FIG. 2. As observed, sera recognized only three antigenic proteins with apparent Mr 42, 64 and 72 kDa, but some differences were observed among studied animals. The polypeptide of 64 kDa was strongly recognized by all animals during the complete infection period, while the rest of the proteins were weakly recognized in some stages of the infection. The polypeptide with Mr 42 kDa was slightly immunorecognized by animals 2, 3 and 4 (FIGS. 2ABC) during the all infection course, but in animal 5 (FIG. 2D) it was only identified during the second week post infection. On the other hand, 72 kDa protein was weakly detected by sera of animals 2, 3 and 5 (FIGS. 2ABD) during the all infection course, but in animal 4 (FIG. 2C) it could only be detected during weeks 6 and 8 post infection. Likewise, to assess cross reactivity with other hemoparasites and recognition of other T. vivax isolates, proteins were confronted to several sera, as shown in FIG. 3. The 64 kDa protein was recognized by all T vivax infected sheep and by T evansi naturally infected horse; however, there was no detection of 42 and 72 kDa proteins.
Out of the 11 bands accounted in this study, one (64 kDa) was recognized as a major antigenic protein and two (42 and 72 kDa) were detected as minor polypeptides. Previous studies related with the antigenic proteins of T vivax indicate that 64 kDa is immunodominant in this species [1, 4]. Indeed, it have been found that serum from T vivax-positive cattle detected two bands with apparent Mr 32 and 66 kDa . Similarly, other study reported a strong recognition of a 65 kDa protein by antibodies of ovine which had been experimentally infected with T. vivax . Both of these both immunodominant proteins, 66 and 65 kDa previously described, should be the same as 64 kDa described in this paper. Since T. evansi and T. vivax have shown a very high cross-reactivity, some researchers have performed studies addressing the common antigens found between these two hemoflagellates [2, 3, 23, 24, 26]. The results presented here have demonstrated the high antigenic capacity and cross-reactivity of one polypeptide with apparent Mr of 64 kDa, because it was detected by all infected sheep during the all infection course. It was also recognized by naturally and experimentally T vivax-positive sheep sera and by a horse naturally infected with T evansi, but it was not recognized by the hemoparasites-negative bovine and the Babesia bigemina-positive (T vivax-negative) bovine. Therefore, the 64 kDa polypeptide detected in this experiment may correspond to an antigen that share epitopes with proteins from other trypanosomes, such as T evansi, which means that this protein may be a good antigen for diagnosing infections either with T evansi or with T vivax. Recently, it has been studied the T vivax GM6 (TvGM6) antigen which is an invariant antigen associated with the flagellum and partially conserved in T brucei, T congolense and T vivax . These researchers found some cross-reactions with T congolense-infected animals, therefore the TvGM6 ELISA alone cannot be used for species-specific diagnosis. On the other hand, infected animals in the present study recognized two additional proteins with apparent molecular masses of 42 and 72 kDa. Nonetheless, sheep 4 only recognized the 72 kDa polypeptide in weeks 6 and 8 post infection, while sheep 5 detected 42 kDa protein in the second week of infection. In general, the detection of these two proteins was weaker when compared to the reaction against the 64 kDa protein. These discrepancies in antigen recognition could be due to individual differences among host immune systems or the reduced antigenic capacity of these proteins. Since early biochemical studies showed that the T vivax variable surface glycoprotein (VSG), differed from the T brucei and T congolense counterparts in several features, including its size (~40 kDa), pattern of glycosylation, and C-terminal end , it is likely that the 42 kDa protein found in this study, would correspond to the T vivax VSG. Although it has been demonstrated that VSG in T evansi is the most immunodominant antigen [2, 23, 24], it appears not to be the case in T vivax. These findings are consistent with those of other researchers [9, 21] that have suggested that the VSG from T. vivax is less immunogenic than the VSG from other trypanosomes. In a transcriptome analysis of a different Venezuelan T vivax isolate, LIEM-176, the researchers concluded that the cellular surface of T. vivax is substantially different than that of T brucei and very likely different from those of other African trypanosomes . In fact, the VSG represent 55 % of the externally disposed cell-surface proteins in T vivax, a considerably lower figure than the 95-98 % reported for the T brucei VSG, which could explain the lower antigenic capacity of this molecule in T vivax .
CONCLUSIONS AND RECOMMENDATIONS
In the present study, it was demonstrated three immunoreactive proteins from T vivax, one major with approximate Mr of 64 kDa and two slightly immunorecognized proteins with apparent molecular masses of 42 and 72 kDa. These two proteins should be studied and sequenced in order to test them as antigens for T vivax-specific diagnosis. VSG of T vivax seemed not to be the most antigenic protein detected. Besides, WB analysis indicated cross-reactivity of the 64 kDa protein since it was detected by sera of T evansi positive horse.
Additional detailed studies using WB for the characterization of different isolates of T vivax by antibodies of various host species may enhance the understanding of the antigenic proteins and give some insight about the cross-reactions between T vivax and T. evansi. Additional parasite proteins should be further evaluated by 2D-eletrophoresis for identify specific and nonspecific antigens.
Recibido: 29/05/2015 Aceptado: 15/06/2015
This research was financially supported by the Scientific and Humanistic Development Council (Spanish acronym, CONDES), Universidad del Zulia, Venezuela (Grant CC-0892-07). The authors express their sincere thanks to Veterinary Parasitology Program of Universidad Centroccidental "Lisandro Alvarado" for providing bovine (B. bigemina positive) and equine (T evansi positive) sera used in this experiment.
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Roger Antonio Ramirez-Barrios * (1), Zulayne Valera (1), Omaira Parra (1), Everts Chacin (1), Lucinda Tavares-Marques (2), Philippe Holzmuller (3), Alvaro Martinez-Moreno (4) y Armando Reyna-Bello (2)
(1) Laboratorio de Enfermedades Parasitarias, Facultad de Ciencias Veterinarias, Universidad del Zulia, Maracaibo, Venezuela. (2) Universidad Nacional Experimental Simon Rodriguez-IDECYT Centro de Estudios Biomedicos y Veterinarios. Grupo de Inmunobiologia. Caracas, Venezuela. (3) CIRAD UMR 17 Trypanosomes [UMR 177IRD-CIRAD "Interactions Hotes-Vecteurs-Parasites dans les Trypanosomoses"], TA A-17/G, Campus International de Baillarguet, 34398 Montpellier Cedex 5, France (4) Departamento de Sanidad Animal, Universidad de Cordoba, Cordoba, Espana. * Corresponding author: Roger Antonio Ramirez-Barrios, Laboratorio de Enfermedades Parasitarias, Facultad de Ciencias Veterinarias, Universidad del Zulia. Av. Ziruma, Ciudad Universitaria, Nucleo Agropecuario, Tercer piso, Maracaibo, Venezuela. Phone: +58 261 4126161. E-mail: firstname.lastname@example.org
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|Title Annotation:||texto en ingles|
|Author:||Ramirez-Barrios, Roger Antonio; Valera, Zulayne; Parra, Omaira; Chacin, Everts; Tavares-Marques, Luc|
|Publication:||Revista Cientifica de la Facultad de Ciencias Veterinarias|
|Date:||Jul 1, 2015|
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