Parasitological, Hematological, and Immunological Response of Experimentally Infected Sheep with Venezuelan Isolates of Trypanosoma evansi, Trypanosoma equiperdum, and Trypanosoma vivax

There are three trypanosoma species of veterinary importance in South America: (1) Trypanosoma evansi, the causative agent of derrengadera mechanically transmitted by bloodsucking insects such as tabanids, (2) Trypanosoma vivax, also mechanically transmitted by some dipteras hematophages as tabanids and/or Stomoxys, and (3) T. equiperdum, a tissue parasite adapted to sexual transmission and the causative agent of dourine, a distinctive disease that affects only Equidae. In order to evaluate the parasitological, hematological, and serological response of sheep infected with T. vivax, T. evansi, and T. equiperdum, four female sheep were experimentally infected with Venezuelan trypanosome field isolates: two T. evansi of differing virulences, one T. equiperdum; one T. vivax. Parasitemia and clinical parameters such as hematocrit, red blood cell count, hemoglobin, and body temperature were measured. T. evansi caused a chronic disease with undulant parasitemia alternating with some cryptic periods of at least 54 days, with no clinical signs. T. equiperdum, never described as infectious to ruminants, also caused a chronic disease with low undulant parasitemia. T. vivax caused an acute infection with severe anemia showing a drop of more than 70% of the hematocrit value, high fever, and rapid deterioration of physical condition, for 36 days of infection. Indirect ELISAs using crude extracts of the three species of trypanosomes as antigens were performed for detection of anti-trypanosome antibodies in sheep sera. Cross-reaction was observed between the three parasite species. These results show that sheep are susceptible to the three-trypanosome species and suggest they can act as a reservoir when sheep are raised and managed with other important livestock such as cattle, horses, buffalos, or goats. These findings are especially interesting for T. equiperdum, a species that has not been reported as infective to sheep.


Introduction
Haemoparasitoses represent one of the main limitations for the development of the cattle industry in Africa, Asia, and Latin America. The economic losses caused by parasitic diseases are attributed mainly to reduced weight gain, lower milk production, high mortality, and high costs of veterinary care [1,2]. Only two species of trypanosomes with veterinary implications in South America have been identified due to their pathogenicity in livestock: Trypanosoma vivax and T. evansi [1]. However, Sanchez et al. [3] recently confirmed the first molecular report of T. equiperdum in Latin America.
Trypanosoma evansi is a mechanically transmitted extracellular blood parasite that causes equine trypanosomosis 2 Journal of Parasitology Research or surra, also known as Derrengadera in South America [4,5]. Although it most notably affects horses and camels, it can also affect donkeys, dogs, cats, cattle, and buffalo [6]. Other wild mammals, such as capybaras (Hydrochoerus hydrochaeris), act as reservoirs [7]. Experimental T. evansi infections in sheep have shown a chronic and often cryptic disease, with low parasitemias and some cases of self-cure [8][9][10][11]. Desquesnes et al. [5] review a naturally infected sheep during outbreak in France due to T. evansi. In South America, specifically in Brazil, there are reports of vertical transmission using experimental infection in pregnant sheep [12]. The clinical signs of T. evansi infection include anemia, recurrent fever, weight loss, emaciation, swelling of the hind limbs, and hemostatic abnormalities [6,13].
Trypanosoma vivax is one of the most important pathogenic species of trypanosoma and is cyclically transmitted to domestic livestock by tsetse flies (Glossina spp,) as well as by mechanical means though other bloodsucking insects [1,4,14]. T. vivax cause a critical and often fatal disease in ruminants such as cattle, buffalos, sheep and goats, due to the high fever and induced anemia [1,15]. In Brazil, the infection by T. vivax has also been described in horses [16]. Clinical signs include fever, emaciation, anorexia, and immunosuppression. Thrombocytopenia, microthrombus formation, and hemorrhage suggestive of disseminated intravascular coagulation have also been demonstrated [14,17,18]. This haemoparasite was introduced to South America by cattle imported from Africa, as it can be transmitted by tabanids and stomoxes. Once this haemoflagelar arrived in Latin America, with the absence of Tsetse fly, T. vivax, it initiated an evolutionary differentiation by losing genes from its maxicircle [19]. The parasite has spread in the continent where it is now endemic from Brazil to Costa Rica, including Colombia, Venezuela, Peru, Bolivia, Panama, Paraguay, and Ecuador [1,14,17,20,21].
T. equiperdum is the causative agent of dourine in horses and donkeys. It differs from other mammalian trypanosomes since it is transmitted sexually and is primarily a tissue parasite, very difficult to find in blood [22][23][24][25]. Dourine has a worldwide distribution; it has been reported in Africa, Asia, Southern and Eastern Europe, Mexico, and Venezuela [3,25].
The objective of this preliminary study was to evaluate the effect of experimental infections in sheep with three trypanosomes of veterinary interest in the region: T. evansi, T. vivax, and T. equiperdum.

Experimental Animals.
Four healthy, helminth-free crossbreed female sheep, between 3 and 6 months of age, were obtained locally and maintained for a month prior to experimental infection. For T. evansi and T. equiperdum, fresh 1 x10 6 parasites coming directly from infected rats were inoculated, while, for T. vivax, 1 x 10 6 cryopreserved ones were injected. After inoculation, the course of the infection was monitored for 65 days for T. evansi and T. equiperdum infections, but the T. vivax infected sheep was treated with 0.5 mg/kg isometamidium chloride (Hemoveex 5, Reveex, Venezuela) at 36 days postinfection to prevent animal death. The presence of parasites in the blood, rectal temperature, hematocrit, erythrocyte count, and hemoglobin concentration was monitored every other day during the experimental infection. A noninfected sheep was used as a control and the same clinical parameters were measured.

Detection of Trypanosomes in the Blood.
For detection of parasitemia, 100 microscope fields of wet films were examined using a light microscope at 40 X magnification, as described by Brenner [26]. The parasitemia was expressed as parasites/ml and the hematocrit centrifugation technique (HCT) [27] was used to observe parasites in case of very low levels of parasitaemia.

Body
Temperature. Every other day at the same hour and using a rectal thermometer, temperature was measured and expressed in ∘ C.

Hematocrit.
For determination of packed cell volume (PCV), blood (60 l) was drawn into heparinized micro capillary tubes, which were centrifuged at 10.000 g per 5 min and the PCV was measured using a microhematocrit scale.

Red Blood Cell Count.
To determine hematological data, a sample of sheep blood was drawn, using vacuum tubes containing EDTA as anticoagulant. The blood was diluted 1: 1000 in 3% trisodic Citrate and 1% formol. Red blood cells (RBC) were counted with an optical microscope using a Neubauer chamber.

Hemoglobin.
The cyanmethemoglobin method was used to determine hemoglobin concentration. Blood was diluted in Drabking solution and the absorbance was assessed photometrically at 540 nm on a Milton Roy Spectronic1201.
2.9. Serum Sample. All blood samples were collected from the jugular vein in vacuum tubes without anticoagulant. The tubes were labeled and the blood was allowed to clot overnight at room temperature and the serum was separated by centrifugation. Samples were stored at -20 ∘ C until testing.

Preparation of Antigen for ELISA. Parasites for T. evansi
and T. equiperdum antigen preparation were obtained from experimentally infected rats as describes previously. Parasites were purified by anion exchange chromatography using DEAE-Cellulose by described Lanham and Godfrey [28]. The resultant parasites pellet was resuspended in 2 ml of PBS 20 mM, pH 7.2, and disrupted by thermal shock. The resulting homogenate was stored at -20 ∘ C with proteases inhibitors until used. For T. vivax parasites purification, the protocol described by Gonzalez et al. [29] was used.
2.11. ELISA Procedure. For each trypanosome isolate, an ELISA plate was used, all under the same conditions, with each sample in duplicate. Negative serum samples were obtained from each sheep two weeks prior to infection and used as negative controls. During experimental infection, samples were taken at days -1, 2, 10, 18, 24, 30, 39, 47, and 61. Sensitized plates with 20 g/well of antigen were washed five times with washing buffer (WB) (PBS-Tween 0.05 %, pH 7.2) and blocked with 200 l/well of 5% skim milk in PBS for an hour at 37 ∘ C. Plates were washed five times with WB, and 100 l/well of each sera diluted 1:200 in WB was added. Positive and negative reference sera were included in each ELISA plate. After incubation for an hour at 37 ∘ C, plates were washed five times with WB, and 100 l of conjugate (Rabbit peroxidase conjugated anti-sheep IgG Pierce. Immunopure 5 Antibody) diluted 1:5000 with WB was added and plates were incubated for 60 minutes at 37 ∘ C. After incubation, the plates were washed three times (PBS-Tween 0.05 %, pH 7.2) and 100 l of substrate solution, ABTS 2% H 2 O 2 was added and incubated at 37 ∘ C for 45 min. Absorbance was measured photometrically at 405 nm on an ELISA reader (BioRad 3550).  (Figures 2(b), 2(c), and 2(d)). In the T. vivax infection, significant decreases in these parameters were observed, as compared to the noninfected or infected with the other trypanosomes.

Figure 1: Parasitemia score in experimental ovine infection by Trypanosoma evansi (TeApMantecal01 and R38), Trypanosoma equiperdum (TeAp-N/D1), or Trypanosoma vivax (Tucacas) isolates.
Sheep were inoculated intravenously via jugular vein with 1 x 10 6 parasites obtained from infected rats. Parasitemia was determined by direct counting from blood using a Neubauer chamber. equiperdum antibodies in the experimentally infected sheep are shown in Figure 3. The ELISA tests were either homologous, in which the sera reacted to antigens from the same isolate, or heterologous, whereby sera bound antigens from a different isolate. When the sera were examined against homologous and heterologous antigens of T. evansi, T. vivax, or T. equiperdum, homologues antigens showed a greater recognition. However, for the T. vivax antigen, recognition of the homologous sera is higher than the heterologous system. Note that the increase in the serologic response is continuous throughout the period of the infection when heterologous system is used; however, in homologous systems there is an increase in all antibody kinetics between days 24 and 39.

Discussion
Experimental Infection with T. evansi, T. equiperdum, and T. vivax. In the present paper, experimental infection of sheep with T. evansi, T. equiperdum, and T. vivax was achieved. Data show a T. vivax acute infection with dramatic decrease of hematocrit and red blood cells counts. In contrast, T. evansi and T. equiperdum isolates caused a chronic disease with slight clinical manifestations.
Interestingly, T. equiperdum is described as the only sexually transmitted trypanosome and not as a vector borne disease. Its host spectrum is very narrow, infecting almost exclusively equine and rabbits after experimental intratesticular inoculation [25,30]. Once these strains have been adapted to the rabbit, other rodents such as rats and mice can be infected (OIE, 2016). A reference author on trypanosomiasis [30] also points out that T. equiperdum, a tissue parasite, rarely invades the blood. In ruminants, experimental infections could produce weak manifestations of Dourine and cryptic or low parasitemias [8,11,12]. In this experimental infection, we observed that T. equiperdum isolate was able to infect sheep and remained by 54 days of the experimental infection. Throughout the infection, the trypanosomes were observed in blood in undulating parasitaemia and the cutaneous edema characteristic of Dourine was never evident. In view of these results TeAp-N/D1 isolate obtained from a horse naturally infected in the Venezuelan savanna shows an unknown evolutionary form of T. equiperdum in the new world.
Trypanosoma evansi has a worldwide geographical distribution; Africa, Asia, and Central and South America are the main endemic areas. In natural infections of horses, various symptoms have been reported, including intermittent fever, severe anemia, limb edema, lethargy, loss of appetite, weight loss, and other symptoms [6,31]. The virulence of a parasite depends on several factors: (a) susceptibility of the host, (b) host ability to control the parasitemia, and (c) intrinsic parasite factors, which could lead to a better adaptation to the host. Perrone et al., 2003, reported nine Venezuelan isolates of Trypanosoma spp. with marked differences in virulence in mice; two of them were used in the present study: TeApMantecal01 (T. evansi) and TeAp-N/D1 (T. equiperdum) [3]. Even though both isolates showed a virulent behavior in mice and rats [32,33], the T. equiperdum isolate appeared extremely virulent in both experimental models, causing an acute disease. These findings agree with observations reported by Onah et al. [8] and Audu et al. [11] Journal of Parasitology Research Our biological data suggest that sheep are susceptible to both T. evansi and T. equiperdum isolates in Venezuela. The presence of the parasites in blood leads to great economic losses and increases the odds of the parasite transmission to other hosts.
The sheep experimentally infected with T. vivax showed an acute disease characterized by undulating parasitemia during the first 24 days after infection, and a rapid deterioration of the physical condition. The hematocrit values and RBC count showed a progressive reduction, until it reached a value of less of 18% at the day of the trypanocidal treatment. The clinical signs confirm the high pathogenicity and virulence of this T. vivax isolate in this ruminant model. This agrees with findings of naturally and experimentally infected sheep and goats reported for other T. vivax field isolates [17,31,34,35].
In Africa and America, cattle and buffalo livestock share their habitats with herds of sheep and goats. Epidemiological data of field-infected herds showed the likelihood of T. vivax transmission between asymptomatic carriers such as buffalo and donkeys to susceptible hosts, in a same area [34]. In Brazil, where sheep and goats are important livestock in the semiarid region, those animals may be severely affected by T. vivax infection, showing anemia, hyperthermia, enlarged lymph nodes, and progressive weight loss [31]. Antigenic Cross-Reactivity by ELISA. The cross-reactivity between T. evansi and T. vivax is well documented (Reyna-Bello et al. [36], Desquesnes et al. [37], Uzcanga et al. [38], and Camargo et al. [39,40]); our results show the same behavior with T. equiperdum. However, a reciprocal cross-reactivity does not occur when T. vivax is used as the antigen to detect antibodies against T. evansi or T. equiperdum. As this is a preliminary account of the infection of T. equiperdum in an ovine model, we can only speculate about the ability of the parasite to stimulate the immune response and more studies are needed to evaluate this phenomenon.

Conclusion
The experimental results of this study suggest the possibility of widespread infection by T. evansi, T. equiperdum, or T. vivax in Venezuela herds sheep. The existence of the disease should be suspected when clinical signs common to any of the three trypanosomes species appear. We state the possibility that sheep act as a reservoir for these three trypanosomes in areas where they live close to equines and cattle. In addition, it was observed in indirect ELISA that cross-reactivity between T. evansi and T. equiperdum versus T. vivax is curiously unidirectional.
Finally, we wish to point out that a sheep infected with a strain of T. equiperdum, described in previous studies, remained infected at least for 54 days, which represents an unusual finding.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Ethical Approval
The authors declare that all the experiments in this paper were carried out in accordance with the legal and ethical standards of Venezuela.