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“Silent” circulation of Trypanosoma spp. in Tabanids (Diptera: Tabanidae) and Cattle in a Tsetse free Range land of Ngaoundere (Adamawa-Cameroon)

The Adamawa region falls within the tsetse belt of Cameroon but harbours isolated pockets of tsetse free range lands like Ngaoundere. There is no report on the occurrence of tsetse and bovine trypanosomosis in Ngaoundere. To provide information on this subject, two Vavoua traps were used to trap vectors of bovine trypanosomiasis and at the same time, blood was collected from cattle. Genomic DNA was extracted from buffy coat of cattle blood (n=42) and biting flies (n=53). The nested PCR was used to screen the samples for Trypanosoma spp. During the 14 days of trapping in November and December 2017 in Ngaoundere, 127 flies were documented and classified under two taxa: Tabanidae and Stomoxyini. Three Trypanosoma spp. DNA was isolated from tabanid (18.9%) samples and identified as T. theileri, T. vivax and T. evansi and two of them that is T. theileri (4%) and T. vivax (3%) were also detected in cattle (7%). There was no case of trypanosome DNA isolated from all the screened Stomoxyini. This result indicates the “silent” transmission of T. theileri and T. vivax by tabanids in the absence of glossines in Ngaoundere. © 2020 International Formulae Group. All rights reserved.


INTRODUCTION
Bovine trypanosomiasis is considered as one of the most important cattle diseases in Adamawa region of Cameroon (Mbahin et al., 2006). The last tsetse eradication of 1994 did not include Ngaoundere because it was considered free of tsetse and trypanosomiasis. Because of fear of the re-introduction of tsetse and trypanosomiasis in tsetse free areas by neighbouring infested areas, deltamethrin coated screen barriers were pitched at the Ngaoundere hill by the special mission for tsetse erradication to prevent the re-infestation of Ngaoundere by wild tsetse from neighbouring and tsetse-infested North region. In Cameroon, the Adamawa plateau harbours several cattle markets (Motta et al., 2017) and receives cattle from neighbouring Chad, Sudan, Central African Republic, Nigeria and Niger which are potential sources for the introduction and re-introduction of new strains of vector-borne livestock diseases. Apart from commercial cattle, cattle of transhumant pastoralists from the aforementioned neighbouring countries come in contact with sedentary cattle thereby introducing different strains of Trypanosoma in local cattle through bites of local vectors. The report of Sevidzem et al. (2015) revealed the occurrence of mechanical vectors (Tabanids and Stomoxys) of bovine trypanosomiasis in the Adamawa plateau. Such vectors have been shown to acyclically and cyclically transmit trypanosomes (Boese et al., 1987;Desquesnes et al., 2003a;Desquesnes et al., 2003b;Taioe et al., 2017;Mounioko et al., 2018). The review document of Wells (1972) highlights the importance of mechanical transmission in the epidemiology of nagana. Wells (1972) stated that transmission of nagana in the absence of glossines is caused by mechanical vectors such as tabanids, stomoxyines and hippoboscids. The study of Mamoudou et al. (2016) reported the occurrence of high tabanid densities in several ecozones of the Far North region of Cameroon. Some authors report that T. evansi is geographically most widespread trypanosome (Desquesnes et al., 2013) and was originally reported in Camels in Africa (Lun and Brun, 1992). In the tsetse free Far north region of Cameroon, Suh et al. (2017) reported 0.67% and 1.46% prevalence of T. vivax. The continuous usage of trypanocides and insecticides in Ngaoundere is an indication of the existence of trypanosomiasis and its mechanical vectors in the apparent absence of glossines. There is need to apply sophisticated tools like PCR to identify the Trypanosoma spp. occurring in cattle and biting flies. Bosson-Vanga et al. (2012) found out that PCR was far more specific than microscopy in trypanosome identification. The goal of the present study is to molecularly screen cattle and flies of trypanosomes in the tsetse free range land of Ngaoundere.

Study area
The study was conducted in a herd located 25Km from the town of Ngaoundere and falls within the following geographical limits: latitude 07° 11, 887' N and longitude 013° 34, 919' E. Ngaoundere is a major livestock production zone and is considered a tsetse free zone but hyper infested with other flies (Lendzele et al., 2019) ( Figure 1). In addition to cattle, other potential hosts in the area include camels that are kept in the Bois de Mardock, a recreational center found some 15 km from the study site. The herd that was used for the study consisted of 42 Zebu Goudali cattle heads.

Entomological prospection
An entomological prospection was carried out for 14 days using Vavoua (n=2) traps. Traps were emptied every 24 hours throughout the prospection days. Traps were placed in potential vector breeding environments, located in cattle pen (open grass savanna) and gallery forest.

Blood collection and buffy coat preparation
Blood was collected through the jugular veino-puncture of the 42 sampled Zebu Goudali cattle, using a vacutainer tube containing ethylenediamine tetraacetic acid (EDTA) as anticoagulant. To prepare the buffy coat, micro-capillary tubes were dipped in the whole blood and centrifuged at 12,000 revolutions per minute for 5 minutes using a micro-haematocrit centrifuge (Hawksley, UK). The capillary tubes were cut with a diamond micro-haematocrit tube cutter, few millimetres below the buffy coat. Buffy coat samples were stored at -20 °C prior to DNA extraction.

Fly preparation
Trapped flies were individually placed in eppendorf tubes containing 1ml of 70% alcohol and stored at -20 °C prior to DNA extraction.

PCR amplification and Gel electrophoresis of amplicons
Genomic DNA from the buffy coat and flies were extracted using the Wizard Genomic DNA Purification Kit (Promega, Germany) according to the manufacturer's instructions, and then stored at -20 °C. Generic primers were used in a nested PCR targeting kinetoplastid ITS-1 as described by Ngomtcho et al. (2017). Briefly, the first 25 µl volume reaction contained 2 µM of each outer primers (ITS1-Out Forward: CTT TGC TGC GTT CTT and ITS1-Out Reverse: TGC AAT TAT TGG TCG CGC) with product size between 180 to 640 bp, 0.2 mM dNTP mix, 0.5 U DreamTaq DNA polymerase (Thermo Scientific, Dreieich, Germany), 1× DreamTaq buffer, and 1 µl of extracted DNA isolates. Water and genomic DNA of T. grayi were added as negative and positive controls respectively.
PCR amplification was carried out as follows: initial denaturation step at 95 °C for 60 s, followed by 30 amplification cycles with 94 °C for 60 s, 52 °C for 60 s, 72 °C for 30 s, and final extension at 72 °C for 5 minutes. After that, the second PCR reaction was carried out with 1 µl of first 1:80 diluted PCR template under the same cycling conditions as described above, except an annealing temperature of 54 °C, and using the inner primer pairs (ITS1-Inner Forward: TAG AGG AAG CAA AAG, ITS1-Inner Reverse : AAG CCA AGT CAT CCA TCG). 20 µl of the resultant amplicons were visualized in Midori green-stained 1.5% agarose gel.

Statistical analysis
The trap index of apparent abundance (ADT) was determined following the already published formula by Sevidzem et al., (2015) as follows: The non-parametric Kruskal Wallis rank sum test of the R-statistical software 3.3.2 (R Core Team, 2016) was used to compare the ADTs of the haematophagous flies caught. The significant level of the statistical test was set at p<0.05. Tsetse data used in this map was provided by the special mission for tsetse eradication (MSEG), Cameroon.

Entomological findings
Entomological prospection resulted in 127 flies grouped under two families notably Tabanidae and Stomoxyini. For Tabanidae, two genera were identified notably Chrysops (deer flies), Tabanus (horse flies) and Haematopota (clegs) with Chrysops highly represented. For the stomoxyini, only one genus was identified as Stomoxys. At the species level, the ADTs of the members of the aforementioned genera were recorded, 21 C.

Molecularly identified trypanosomes from cattle
By comparing the product size from the present gel electrophoresis with the reference product sizes in literature (Ngomtcho et al., 2017), Trypanosoma theileri (4%) and T. vivax (3%) were detected in the cattle sample with an overall molecular prevalence of 7% (Figure 2).

Molecularly identified trypanosomes from haematophagous flies
Among the 127 haematophagous flies caught, 53 of them were screened, 10 were positive for trypanosomes, resulting in an overall fly infection rate of 18.9%. There was one mix infection of C. longicornis with T. evansi and T. theileri. It was noticed that Haematopota decora harbored all the Trypanosoma spp. identified in the present study. C. longicornis, T. taeniola and T. gratus harbored only T. theileri (Table 1). All the Stomoxys spp screened were negative for trypanosomes.

DISCUSSION
The occurrence of tabanids and stomoxyines, mechanical vectors of bovine trypanosomiasis in the pasture area of Ngaoundere is an indication of the risk of transmission of the disease in the absence of glossines (Lendzele et al., 2019a;Sevidzem et al., 2019a;2019b. In the Far north region of Cameroon, there are no tsetse flies but the area is heavily infested by tabanids and stomoxyines. Suh et al. (2017) reported the occurrence of T. vivax in cattle from the Far North region of Cameroon, indicating the important role of mechanical vectors in transmission. The present study identified one species of the genus Chrysops (Chrysops longicornis), one species of the genus Haematopota (Haematopota decora), two species of Tabanus (Tabanus taeniola and Tabanus gratus) and four species of Stomoxys (S. n. niger, S. omega, S. n. bilineatus and S. inornatus). C. longicornis was infected with all the screened Trypanosoma spp. (T. theileri, T. vivax and T. evansi), but the positive cases of H. decora, T. taeniola and T. gratus were only from T. theileri. From the screening results, H. decora was frequently contaminated with T. theileri as compared to any other infected fly. These findings were not surprising as Taioe et al. (2017) and Mounioko et al. (2018) already reported that some members of the genera Tabanus, Haematopota, Philoliche, Atylotus and Ancala were positive for trypanosomes. Similarly, Boese et al. (1987) reported the cyclical transmission of T. theileri in Haematopota sp. The overall molecular prevalence of trypanosomes in cattle was 7% and was caused by T. theileri and T. vivax. These trypanosomes have been reported to be transmitted cyclically and acyclically (Boese et al., 1987;Desquesnes et al., 2003). There was no T. evansi detected in cattle samples of the sampled animals. This can be explained by the fact that during the sampling, wild animal host like monkeys were common in the area as well as animals from neighbouring Dawa Ranch and Centre Zootechnique that were not sampled. The absence of positive cases of trypanosomes in Stomoxys could be explained by the low number of samples screened in the present study and not their incapacity to be contaminated with trypanosomes. Stomoxys spp. have been shown to be implicated in the acyclical transmission of T. evansi and T. congolense (Sumba et al., 1997). The presence of camels in Ngaoundere might be a source of contamination of local cattle breeds and flies with T. evansi since camels are reservoir host of this species (Lun and Brun, 1992).

Conclusion
In a herd located in the tsetse free belt and trypanosomosis hypo-endemic rangeland of Ngaoundere, two Trypanosoma spp. (T. theileri and T. vivax) circulates 'silently' in cattle and tabanids. Trypanosoma evansi is reported for the first time in tabanids caught from Ngaoundeere. Therefore, the role of Tabanidae and other mechanical vectors in the epizootiology of bovine trypanosomosis in tsetse free areas should not be neglected if control measures are to be taken

COMPETING INTERESTS
The authors declare that they have no competing interests.

AUTHORS' CONTRIBUTIONS
SA and SSL conceived the idea for the work and wrote the proposal; SSL, SA, AMNN, AM, AP, JFM and AZ assisted in the technical aspect of this study and in editing the manuscript. Both parties gave final approval to the manuscript.