A Survey on Trypanosoma evansi (Kinetoplastida,Trypanosomatidae) Infection in Domestic Animals in a Surra Endemic Area of Southern Algeria

Abstract

Background:

Trypanosoma (T.) evansi infection is endemic in dromedary camels (Camelus dromedaries) of southern Algeria.

Materials and Methods:

In order to assess the presence of T. evansi in other domestic animals living together with dromedary camels, a study was conducted in the wilayate of Béchar, El Bayadh, Ouargla and Tamanrasset, between 2015 and 2017. Authorisation to conduct the survey was obtained from the Direction des Services Vétérinaires (DSV, Ministry of Agriculture, Rural Development and Fisheries). A total of 190 animals were sampled, including 42 cattle (Bos taurus), 11 dogs (Canis familiaris), 44 horses (Equus caballus), 3 donkeys (Equus asinus) and 1 mule, 49 goats (Capra hircus) and 40 sheep (Ovis aries). These animals were examined by parasitological (Giemsa stained thin smear, GST), serological (card agglutination test for trypanosomosis (CATT/T. evansi), enzyme-linked immunosorbent assay/Variant Surface Glycoprotein/Rode Trypanozoon antigen type 1.2 [ELISA/VSG RoTat 1.2], immune trypanolysis [TL]) and molecular tests (T. evansi type A specific RoTat 1.2 PCR).

Results and Conclusions:

The CATT/T. evansi was positive in 10/42 cattle, 0/11 dogs, 2/48 equids, 27/49 goats and 15/40 sheep. On the other hand, 20/38 cattle, 1/9 dogs, 21/42 equids, 17/44 goats and 31/39 sheep were positive in ELISA/VSG RoTat 1.2. However, no single animal was positive in TL. In addition, the T. evansi parasite could not be demonstrated by either GST or RoTat 1.2 PCR in any of the examined animals. This may suggest cross-reactions of CATT/T. evansi and ELISA/VSG RoTat 1.2 with other pathogenic or commensal trypanosome species such as T. vivax or other parasites. Based on these data, in particular taking into account the high specificity of the TL for T. evansi type A, this study does not support the hypothesis that T. evansi circulates in the studied domestic animal species and that they would act as reservoirs for the parasite that causes trypanosomosis in dromedary camels.

Introduction

T rypanosoma evansi is the most widely distributed of the pathogenic African animal trypanosomes, affecting domestic livestock and wildlife in Asia, Africa, and Latin America (Aregawi et al., 2019). Several species of hematophagous flies, including Tabanids and Stomoxes, are implicated in transmitting the infection from host to host, acting as mechanical vectors (WOAH, 2021).

T. evansi has a wide host range. In Africa, it affects mainly camels (Camelus dromedaries) and horses (Equus caballus) (Aregawi et al., 2019). Other susceptible species are cats (Felis catus), cattle (Bos taurus), dogs (Canis familiaris), donkeys (Equus asinus), goats (Capra hircus), mules, and sheep (Ovis aries) (WOAH, 2021). In many areas of the world, small ruminants are reared near or in close contact to highly susceptible species, in particular cattle, dromedaries, and horses, and could thus play a role as reservoir of the parasite (Gutierrez et al., 2006).

In Algeria, T. evansi is mainly a parasite of dromedary camels, which represents both the main host and reservoir. Indeed, overall prevalences of 2.4% in Giemsa-stained thin smear (GST), 32.4% in card agglutination test for trypanosomosis (CATT/T. evansi), 23.1% in enzyme-linked immunosorbent assay/Variant Surface Glycoprotein/Rode Trypanozoon antigen type 1.2 (ELISA/VSG RoTat 1.2), 21.0% in immune trypanolysis (TL), and 11.2% in polymerase chain reaction (RoTat 1.2 PCR), were observed in dromedary camels in southern Algeria (Boushaki et al., 2019).

Genotyping analysis based on nuclear and mitochondrial genetic markers confirmed that these recently reported trypanosome infections in camels in southern Algeria were caused by T. evansi type A (GenBank database accession numbers: ON855347, ON855348, ON855349, ON855350) (Boushaki et al., 2022).

In other animal species, trypanosomosis was reported by Szewczyck in horses of the wilaya of Béchar (Zouzfana), South-West Algeria, in 1903, thus confirming the observations of Chauvrat (1896) who, in 1892, had seen in Algeria an anemic horse carrying a trypanosome and thought it was a case of surra (as cited in Curasson, 1943). Naturally acquired infection with trypanosomes in dogs in south western Algeria was first described by Vialatte (1915) (as cited in Curasson, 1943). Also Donatien and Lestoquard reported in 1923 trypanosome infections in dogs in Algiers (as cited in Curasson, 1943).

The absence of pathognomonic signs of surra necessitates laboratory analysis to confirm the infection, either by microscopy or by molecular tools, to demonstrate the parasite and thus active infection or to suggest active or past infection by serological detection of specific antibodies (Büscher, 2014; Büscher, 2001; Tehseen et al., 2015).

Complementary to an investigation on dromedary camels carried out in Southern Algeria (El Bayadh, Béchar, Ouargla, Tamanrasset) from 2014 to 2016 (Boushaki et al., 2019), and to determine the occurrence of T. evansi in other domestic animals species present in an endemic area, a screening was conducted on cattle, dogs, equids, goats, and sheep.

Materials and Methods

Ethics statement

Authorization to conduct the survey was obtained from the Direction des Services Vétérinaires (DSV, Ministry of Agriculture, Rural Development, and Fisheries). At each wilaya, the study was authorized and supervised by the respective Inspection Vétérinaire de Wilaya (IVW El Bayadh, Béchar, Ouargla, and Tamanrasset), operating under the umbrella of the DSV.

Study area

The study was carried out between 2015 and 2017, in four wilayate in southern Algeria, where T. evansi is endemic in dromedary camels: El Bayadh, Béchar, Ouargla, and Tamanrasset (Fig. 1).

FIG. 1.

Map of the study area showing the four wilayate.

Animals and blood sampling

Domestic animal species, including cattle (B. taurus), dogs (C. familiaris), equids, goats (C. hircus), and sheep (O. aries) were sampled. These animals were sampled around the dromedaries studied in Boushaki et al. (2019). Venous blood was collected on dry tubes and ethylenediaminetetraacetate (EDTA) tubes. The sample on EDTA was used to measure the packed cell volume (PCV) and to extract DNA using the QIAamp DNA Blood Mini Kit 250 from Qiagen, Hilden, Germany according to the manufacturer's instructions. Extracted DNA was preserved at −20°C until analysis. Blood taken in the dry tube was allowed to clot where it was centrifuged for 15 min at 480 g. The obtained serum was tested with CATT/T. evansi and subsequently aliquoted into cryotubes and frozen at −20°C for further serological tests. Some of the sampled animals did not undergo all the available diagnostic tests for technical reasons (loss, deterioration), resulting in variations in the number of samples per diagnostic test.

Diagnostic tests

Packed cell volume

Approximately 70 μL of fresh blood on EDTA was taken up in a microhematocrit capillary tube (75 mm; Hirschmann, Germany). The dry end was closed with plasticine and the tube was centrifuged at 13,000 g for 5 min (microhematocrit centrifuge; Sigma). The PCV was expressed as percentage of the total blood volume (WOAH, 2021) using a reading chart supplied with the microhematocrit centrifuge. The PCV was carried out only on equid samples.

Parasitological examination

Five microliters of blood were placed at one end of a clean microscope slide and a thin film was drawn out. It was air dried and fixed in methanol for 1 min and allowed to dry. The smears were stained with Giemsa (one drop of Giemsa +1 mL phosphate-buffered saline [PBS], pH 7.2) for 25 min, followed by washing of the slides in tap water and drying. The stained preparations were examined microscopically at 400 × magnifications.

CATT/T. evansi

The sera were tested for the presence of anti-T. evansi antibodies using the CATT/T. evansi (Institute of Tropical Medicine, Antwerp, Belgium). One drop (∼35 μL) of the antigen suspension was mixed on a test card with 25 μL of serum diluted at 1/4 with PBS pH 7.2 as per the manufacturer's instructions. The card was rocked for 5 min at 70 rpm and the reaction was checked for the presence of blue agglutinates (Bajyana Songa and Hamers, 1988).

ELISA/VSG RoTat 1.2

Indirect ELISA/VSG RoTat 1.2 was performed according to Lejon et al. (2005) and Verloo et al. (2000), at the WOAH Reference Laboratory for Surra at the Institute of Tropical Medicine, Antwerp, Belgium. Sera were diluted 1:150 for goat and 1:200 for bovine, dog, horses, donkeys, mules, and sheep. The following conjugates were used: For cattle: rabbit anti-bovine IgG (A5295, dilution 1:20,000; Sigma); for dog: rabbit anti-dog IgG (H+L) (304-035-003, dilution 1:20,000; Jackson Immunoresearch); for equids: rabbit anti-horse IgG (A9292, dilution 1:20,000; Sigma); and for goat and sheep: donkey anti-goat IgG (H+L) (705-085-008, dilution 1:10,000; Jackson Immunoresearch). ELISA microplates (Nunc MaxiSorp Flat-bottom; Thermo Scientific) were coated with native VSG RoTat 1.2 diluted at 2 μg/mL in phosphate buffer (0.01 M, pH 6.5; NaH₂PO₄·H₂O 0.95 g/L, Na₂HPO₄·2H₂O 0.55 g/L). One hundred fifty microliters of the diluted antigen were added per well in one half of each plate. The other half was left dry as antigen-negative control wells. Plates were incubated at 4°C overnight.

The next day, the antigen was removed and all wells were filled with 350 μL of PBS-Blotto (PBS) (0.01 M, pH 7.4; NaH₂PO₄·H₂O 0.2 g/L, Na₂HPO₄·2H₂O 1.44 g/L, NaCl 11.7 g/L, NaN₃ 0.5 g/L, skimmed milk powder 10 g/L). After 1 h blocking at ambient temperature, the plates were emptied and washed three times with 350 μL/well of PBS-Tween (0.01 M, pH 7.4; NaH₂PO₄·H₂O 0.2 g/L, Na₂HPO₄·2H₂O 1.44 g/L, NaCl 8.2 g/L, Tween 20 0.5 mL/L). Test sera were diluted in PBS-Blotto and 150 μL were added in duplicate to antigen-containing and antigen-free wells.

After 1 h incubation at ambient temperature, the plates were emptied and washed three times with 350 μL/well of PBS-Tween followed by the addition of 150 μL/well of species-specific conjugate diluted in PBS-Tween. Plates were incubated for 1 h at ambient temperature followed by five times washing with 350 μL/well of PBS-Tween. Substrate–chromogen solution (1-Step Ultra TMB; Thermo Scientific) was added at 150 μL/well and incubated for 15 min at ambient temperature, whereafter 150 μL of 2 M H₂SO₄ was added to each well and absorption was read at 450 nm with a Multiskan FC ELISA reader (Thermo Scientific).

Corrected optical density (OD_corr) values for each sample were obtained by subtracting the mean OD of the antigen-free wells from the mean OD of the corresponding antigen-containing wells. Except for equids, these corrected ODs were expressed as percentage positivity (PP) of the OD_corr obtained with a positive control serum included in each plate. The cutoff value for positivity was set at 30% PP. By the absence of a positive control for equids, ELISA results were expressed as OD_corr and a cutoff for positivity was set at 0.5.

Immune TL

Immune TL was performed at the OIE Reference Laboratory for Surra at the Institute of Tropical Medicine, Antwerp, Belgium, according to Van Meirvenne et al. (1995) and Verloo et al. (2000). TL is an antibody-mediated complement lysis test performed with a cloned trypomastigote population of T. evansi variable antigen type RoTat 1.2. Sera were tested after 1/4 dilution in guinea pig serum (Envigo, The Netherlands). Live trypanosomes suspended in guinea pig serum were incubated for 60 min with test serum. When variant-specific antibodies are present in the serum, lysis of the RoTat 1.2 trypanosomes occurs.

TL test is unable to detect antibodies against trypanosomes other than T. evansi type A (Büscher et al., 2019).

RoTat 1.2 PCR

The RoTat 1.2 PCR test was carried out to detect T. evansi DNA in blood using the following primer pair: ILO7957 (5′- GCC ACC ACG GCG AAA GAC -3′) and ILO8091 (5′-TAA TCA GTG TGG TGT GC-3′), designed by Urakawa et al. (2001). It amplifies a 488 pb fragment within the RoTat 1.2 VSG gene. The amplification parameters were as described by Birhanu et al. (2015), but with a minor modification consisting of the activation of HotStar Taq DNA polymerase at 95°C for 15 min. DNA (10 ng/mL) of T. evansi type A (MCAM/ET/2013/MU/02) and pure water were included in the PCR run as positive and negative control, respectively (Birhanu et al., 2016). The amplified product was visualized under UV after electrophoresis in a 2% agarose gel (Sigma) at 135 V for 30 min and staining with ethidium bromide (Sigma).

Data analysis

Statistical analysis was performed using R version 3.4.1 (R Development Core Team, 2017). The influence of the species on infection rates was assessed by a chi-square test. Pairwise comparisons between animal species infections were performed using pairwise comparison of proportion. The t-test was used to compare between the mean PCV in the group of positive and negative equids according to their status in CATT and ELISA/VSG RoTat 1.2.

The map of the study area showing the four wilayate (Fig. 1), was constructed on QGIS 2.18.15.

Results

A total of 190 animals were sampled, including 42 cattle, 11 dogs, 48 equids (44 horses, 3 donkeys, and 1 mule), 49 goats, and 40 sheep.

The CATT/T. evansi was positive in 10/42 (23.8% [95% CI: 10.9–36.7]) cattle, 0/11 dogs, 0/3 donkeys, 27/49 (55.1% [95% CI: 41.2–69.0]) goats, 2/44 (4.5% [95% CI: 1.4–12.0]) horses, 0/1 mule, and 15/40 (37.5% [95% CI: 22.5–52.5]) sheep. On the other hand, 20/38 (52.6% [95% CI: 36.8–68.5]) cattle, 1/9 dog, 0/3 donkeys, 17/44 (38.6% [95% CI: 24.6–53.0]) goats, 20/38 (52.6% 95% 36.8–68.5]) horses, 1/1 mule, and 31/39 (79.4% [95% CI: 66.8–92.2]) sheep were positive in ELISA/VSG RoTat 1.2 (Table 1). However, no single positive result was observed in TL. In addition, T. evansi could not be demonstrated by either GST or RoTat 1.2 PCR in any of the examined animals.

Table 1.

Number of Animals Tested, Number (pos) and Percentage (%) of Positives in Different Tests According to Animal Species

Species	Giemsa stained thin smear		CATT/Trypanosoma evansi		ELISA/VSG RoTat 1.2		TL		RoTat 1.2 PCR
Species	Tested	Pos (%)	Tested	Pos (%)	Tested	Pos (%)	Tested	Pos (%)	Tested	Pos (%)
Cattle	42	0 (0.0)	42	10 (23.8)	38	20 (52.6)	38	0 (0.0)	16	0 (0.0)
Dog	11	0 (0.0)	11	0 (0.0)	9	1 (11.1)	9	0 (0.0)	11	0 (0.0)
Equids	48	0 (0.0)	48	2 (4.2)	42	21 (50.0)	42	0 (0.0)	48	0 (0.0)
Donkey	3	0 (0.0)	3	0 (0.0)	3	0 (0.0)	3	0 (0.0)	3	0 (0.0)
Horse	44	0 (0.0)	44	2 (4.5)	38	20 (52.6)	38	0 (0.0)	44	0 (0.0)
Mule	1	0 (0.0)	1	0 (0.0)	1	1 (100)	1	0 (0.0)	1	0 (0.0)
Goat	49	0 (0.0)	49	27 (55.1)	44	17 (38.6)	44	0 (0.0)	24	0 (0.0)
Sheep	40	0 (0.0)	40	15 (37.5)	39	31 (79.4)	38	0 (0.0)	15	0 (0.0)

tested, number of animals tested; Pos, number of positives; %, percentage of positives.

CATT, card agglutination test for trypanosomosis; ELISA/VSG RoTat 1.2, enzyme-linked immunosorbent assay/Variant Surface Glycoprotein/Rode Trypanozoon antigen type 1.2; PCR, polymerase chain reaction; TL, trypanolysis.

A statistically significant difference was observed between species regarding positivity rates for both CATT/T. evansi (chi-squared = 37.454, p < 0.0001) and ELISA/VSG RoTat 1.2 (chi-squared = 21.06, p < 0.001). Pairwise comparisons between animal species of the positivity proportions in CATT/T. evansi revealed significant differences between cattle and equids, cattle and goats, dogs and goats, dogs and sheep, equids and goats, and equids and sheep. Positivity proportions in ELISA/VSG RoTat 1.2 were statistically different between cattle and sheep, dogs and sheep, equids and sheep, and between goats and sheep (Table 2).

Table 2.

p-Values Obtained by Pairwise Comparison of Positivity Proportions Between Animal Species According to Card Agglutination Test for Trypanosomosis/Trypanosoma evansi and Enzyme-Linked Immunosorbent Assay/Variant Surface Glycoprotein Rode Trypanozoon Antigen Type 1.2

	CATT/Trypanosoma Evansi				ELISA/VSG RoTat 1.2
	Cattle	Dog	Equids	Goat	Cattle	Dog	Equids	Goat
Dog	0.17261	—	—	—	0.06011	—	—	—
Equids	0.01535^a	1.00000	—	—	0.99107	0.07725	—	—
Goat	0.00487^a	0.00284^a	1.5e-07^a	—	0.29489	0.22918	0.39893	—
Sheep	0.26867	0.04097^a	0.01535^a	0.14952	0.02443^a	0.00042^a	0.01128^a	0.00040^a

Significant difference.

There was no statistically significant difference in average PCV between groups of equids according to their status in CATT/T. evansi (p = 0.159) and ELISA/VSG RoTat 1.2 (p = 0.070) (Table 3).

Table 3.

Average Packed Cell Volume Values in Equids (in Percentage) ± the Standard Deviation According to the Status in Card Agglutination Test for Trypanosomosis/Trypanosoma evansi and Enzyme-Linked Immunosorbent Assay/Variant Surface Glycoprotein/Rode Trypanozoon Antigen Type 1.2

Test	Negative	Positive	p
CATT/Trypanosoma evansi	44.1 ± 11.8	32 ± 2.8	0.159
ELISA/VSG RoTat 1.2	41.0 ± 12.8	47.9 ± 11	0.070

Discussion

In Africa, Surra is mainly a disease of camels regarding their wide geographical distribution (Aregawi et al., 2019). It is especially pathogenic in camelids and equids (Desquesnes et al., 2013). This study was conducted to determine the occurrence of T. evansi in domestic animals other than dromedary camels in an endemic region of T. evansi infection in southern Algeria. For this purpose, various diagnostic tests were used (parasitological, serological, and molecular).

The trypanosome could not be demonstrated by parasitology (GST) in any of the species investigated. This is in accordance with the results of Jacquiet et al. (1993), who could not detect the parasite in 207 goats and 174 sheep in Mauritania. Similarly, during surveys carried out in Mali on 26 cattle, 49 donkeys, 3 horses, and 6 small ruminants, no cases of T. evansi infections could be detected by GST (Diall et al., 1993). Our results are also corroborated by those of Dia (1997) who was unable to observe the presence of trypanosomes in the peripheral blood of cattle, donkeys, goats, and sheep in Mauritania. Similarly, trypanosomes could not be demonstrated in a study conducted on 1228 ruminants on the Canary Islands (Rodríguez et al., 2012). In contrast, in a cross-sectional study conducted in Northern Ethiopia, the parasitology (GST) was positive in cattle, goat, and sheep (Birhanu et al., 2015). The infection with T. evansi was also confirmed in Palestine in goats, equids, and sheep using blood smear microscopy and PCR (Ereqat et al., 2020).

In most hosts, T. evansi can induce mild clinical or subclinical carrier state infections with low parasitemia, in which it is difficult to demonstrate the parasites. In these circumstances, concentration methods are necessary, as they increase the sensitivity of microscopic examination (WOAH, 2021). However, using such more sensitive techniques as the hematocrit centrifugation technique (Woo, 1969) or the dark ground buffy coat examination (Murray et al., 1977) or mouse inoculation was not possible under the field conditions.

Furthermore, the blood smear has a low sensitivity given the lower detection limit of 10⁵ trypanosomes/mL (Desquesnes et al., 2013).

PCR is more sensitive than parasitological examination, but it may give false-negative results when the parasitemia is very low. The sensitivity of the PCR being dependent on the amount of DNA available, it is proportional to the parasitemia. PCR is thus more sensitive in highly susceptible hosts (camels, horses, dogs, etc.) than in hosts of mild or low susceptibility (cattle, buffalo, pigs, etc.). In these cases, suspicion of potential carriers can only be confirmed by serological examination (WOAH, 2021).

Anemia, revealed by low PCV, can be caused by factors other than trypanosomosis (Desquesnes and Desquesnes, 2017). However, in animal African trypanosomisis, the combination of a serological positive result with a low PCV is often considered sufficient to confirm infection (Büscher, 2014). The PCV in equids showed no significant statistical difference between seropositive and seronegative groups using either CATT/T. evansi or ELISA/VSG RoTat 1.2 tests. This can be explained by the good general condition of the investigated individuals, making diagnosis of equine trypanosomosis difficult, as parasitemia levels in infected hosts are generally below the detection limits of parasitological tests and molecular DNA tests (Büscher et al., 2019).

Regarding antibody detection, positive serological responses were observed in all examined species, except in mules, with either CATT/T. evansi and/or ELISA/VSG RoTat 1.2 but no single animal was positive in TL. Given the high specificity of the latter test for contact with T. evansi type A, this may suggest cross-reactions of CATT/T. evansi and ELISA/VSG RoTat 1.2. False positives in CATT/T. evansi and ELISA/VSG RoTat 1.2 can be explained by the exposure of cross-reacting epitopes in the CATT/T. evansi and ELISA antigens to other pathogenic or commensal trypanosome species or to other parasites (Büscher, 2014, Desquesnes et al., 2022, Tehseen et al., 2015). This is all the more true since TL is recognized as highly sensitive (Holland et al., 2002) and parasitology and PCR were negative. However, a negative PCR result may simply indicate the presence of insufficient genomic material or the absence of the VSG gene (Picozzi et al., 2008).

Therefore, false-positive CATT and ELISA results could be explained by possible T. evansi type B circulating in Algeria. However, this type has not been described in hosts other than camels (Birhanu et al., 2015). Furthermore, Boushaki et al. (2019) found no evidence for the presence of T. evansi type B in dromedary camels using EVAB PCR in the study area. To date, T. evansi type B appears to occur only in camels in Kenya, Chad, Ethiopia, and Sudan (Birhanu et al., 2015, Ngaira et al., 2005, Njiru et al., 2006, Salim et al., 2011) and was recently detected for the first time in Egyptian camels (Behour and Abd El Fattah, 2023).

Similar discrepancies between CATT/T. evansi and TL were also observed in a study in Ethiopia with 37.3% CATT/T. evansi-positive cattle, whereas only 1.9% were positive in TL suggesting cross-reaction of CATT/T. evansi with Trypanosoma vivax (Birhanu et al., 2015).

In a preliminary evaluation of diagnostic tests using horses experimentally infected with T. evansi, it was shown that equine sera have a tendency to give nonspecific reactions in ELISA tests in the measurement of antibodies. Similar results were reported with CATT, with which each of the eight experimental horses tested positive before they were infected, suggesting that horse sera may react to other proteins in the antigen suspension (Wernery et al., 2001). Furthermore, the CATT/T. evansi test is a direct agglutination test that makes use of the same T. evansi RoTat 1.2 clone used in the TL test, but the preparation of the reagent exposes other surface antigens as well, resulting in some cross-reactivity with T. vivax and Trypanosoma equiperdum (Büscher et al., 2019).

Pairwise comparisons between animal species of the positivity proportions in CATT/T. evansi revealed significant differences between goats and the other investigated species. Thus, seroprevalence in goats was 55.1% (27/49) while sheep showed a seroprevalence of 37.5% (15/40). This may support a possible cross-reaction with T. vivax, which can be transmitted to domestic livestock by tsetse flies (Glossina spp., cyclical transmission) and directly (mechanical transmission) by other bloodsucking insects, allowing T. vivax to extend its distribution beyond tropical Africa (Gutierrez et al., 2006).

In conclusion, taking into account the limitations of the diagnostic tests used in this study, we cannot confirm the occurrence of T. evansi in other domestic animals than dromedary camels in an endemic region in Southern Algeria. Whether these animals could play a role as reservoir of T. evansi in the study area remains an open question and deserves further investigations, as well as the hypothesis that T. vivax is also present in Southern Algeria thus causing the apparent false positivity in CATT/T. evansi and ELISA/VSG RoTat 1.2.

With respect to this, further investigations with larger sample sizes may benefit from the use of an ELISA to look for antibodies against T. vivax (Desquesnes et al., 2001). In addition, a combination of molecular tools may be used. Thus, a PCR test with trypanosoma brucei repeat (TBR) primers could be used to confirm the status of a seropositive sample, at least in the first instance. TBR1/2 PCR is considered the gold standard for the detection of Typanozoon DNA (Masiga et al., 1992; WOAH, 2021). For suspected T. vivax, primers that amplify the internal transcribed spacer 1 of ribosomal DNA (Njiru et al., 2005) can be used, as they allow the detection of multiple subgenera and species in a single reaction (Njiru et al., 2005; WOAH, 2021).

An alternative is to use more specific molecular tests for T. vivax, such as TvPRAC PCR (Fikru et al., 2015).

Footnotes

Acknowledgments

The authors would like to thank the Chief Veterinary Officer and the General Director of Institut National de la Médecine Vétérinaire for their assistance, the veterinary doctors of the veterinary services of the Wilayas of El Bayadh, Béchar, Ouargla, and Tamanrasset for their collaboration during the field work. The authors also thank the owners, contacts, and local administrators. They would like to thank the directors of the Central Veterinary Laboratory in Algiers and the Laghouat Regional Veterinary Laboratory for their hospitality and facilitation, as well as all the staff.

Author Disclosure Statement

No conflicting financial interests exist.

Funding Information

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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