Parasitological, Serological and Molecular Survey of Camel Trypanosomosis (Surra) in Northern Nigeria

Surra, a parasitic disease caused by Trypanosoma evansi and transmitted non-cyclically by biting ies signicantly affects the health, productivity and market value of camels thereby constituting a major constraint to food safety, security and economy. This is the rst study on the prevalence of surra in Northern Nigeria, using a broad variety of diagnostic tests along the parasitological-serological-molecular continuum hence, emphasizing it as a major enzootic risk for camels in Nigeria. In this cross-sectional study, 600 blood samples were collected from camels at major abattoirs in Northwestern Nigeria and evaluated for the prevalence of surra due to T. evansi using parasitological (Giemsa staining), serological (CATT/T.evansi) and molecular (VSG-PCR and sequencing) methods. The overall prevalence of surra recorded in this study was 5.3%, 11.5% and 22.5% using Giemsa stained blood smears, CATT/T.evansi and VSG-PCR respectively. However, higher prevalence of 6.0%, 13.7% and 26.7% by Giemsa stained blood smears, CATT/T. evansi and VSG-PCR was recorded in Katsina state when compared with Kano state. A signicantly (p < 0.05) higher prevalence byVSG-PCR was observed when compared with both parasitological and serological methods used. Although age and body condition scores were associated (p < 0.05) with surra prevalence in sampled camels, no seasonal association (p > 0.05) was however recorded. Sequencing of the VSG region of Trypanosoma spp. Further conrmed the presence of T. evansi as the aetiological agent of surra from the sampled camels. Findings from this study call for the implementation of adequate control measures aimed at reducing the impact of T. evansi infections on camel production in Nigeria.


Background
Trypanosomosis in camels also referred to as 'Surra' is a vector-borne disease (VBD) that constitutes a major threat to farm animals in tropical and sub-tropical countries including Nigeria. The disease is caused by Trypanosoma evansi which belongs to the sub-genus Trypanozoon. Microscopically, the organism is long and slender with a prominent undulating membrane, and long free agellum (Getachew, 2005). The disease is endemic in Africa, Asia and South America and it is transmitted mechanically by the bites of haematophagous ies; Tabanids, Stomoxys and Hippoboscids (Eyop and Matios, 2013).
Trypanosomosis in camels is considered the most serious animal protozoan disease in African countries that depend on camels as an important source of food and income for millions of herders (Kamidi et al., 2017). The disease causes production losses, anaemia, weight loss and abortion in a range of domestic species in Africa, Asia and South America. Camel trypanosomosis is usually asymptomatic but can be fatal when not properly diagnosed, This study sought to (1) determine the prevalence of camel trypanosomosis in northern Nigeria using parasitological, serological and molecular methods and (2) to assess the risk factors for camel trypanosomosis in the study areas. Also, genetic characterization and the phylogenetic relationship of sequences in this study will be compared to sequences in the GenBank.

Ethical approval
All experimental protocols and animal work were approved by the Animal Use and Care Committee of the National Veterinary Research Institute (NVRI) Vom, Plateau State, Nigeria with the certi cation ID: NVRI AEC REF No: ACE/02/88/20. All experiments were conducted under strict adherence to the principle of laboratory animal care.

Study area
The study was conducted at the central abattoirs of Katsina (latitudes 11˚08 N and 13˚22 N and longitude 6˚52 E and 9˚20 E) and Kano (latitude 12˚40 N and 10˚31 N and longitude 7˚40 E and 9˚30 E) states located in the Northwestern region of Nigeria. The climate of the two study areas is composed of two major seasons, the dry and wet seasons. The wet season starts from May to ends in September or early October. While the dry season begins in October and ends in April or early May. The mean annual rainfall is about 690mm while the mean annual temperature ranges between a maximum of 33 0 C and a minimum of 19 0 C. The vegetation is mainly savanna; climatically de ne as Sudan savanna, which is characterized by the presence of scattered trees and shrubs in open grassland (Wakawa et al., 2016). The choice of Katsina and Kano States in the Northwestern region was based on (1) the presence of camel international markets in these two states and (2) both states are the major trans-Sahara animal trade routes to Nigeria.

Sample size determination
The sample size was determined based on the prevalence of 27% (Enwezor and Anthony, 2005) with a 95% con dence level and 5% precision as recommended by Thrus eld (1995). A total of 600 camels (Camelus dromedarius) were examined in this survey with 300 camels sampled from each of the two selected states, also exactly 150 each examined during the dry and wet seasons respectively.

Sampling
This study was conducted on apparently healthy camels brought for slaughter in the main abattoirs in Kano and Katsina cities, Northwestern Nigeria. Within the two states, speci c abattoirs were chosen purposively where a high number of camels are largely slaughter for human consumption and also transported to other locations within Nigeria. On every sampling day, apparently healthy camel (n = 600) was selected from the population across the abattoirs and seasons, with the total number of 135male and 165female in Kano state and 103male and 197female in Katsina state being sampled. The camels were examined before slaughter to determine their ages according to their dentition as described by Johnson, (2003). Furthermore, the camels were categorized into; good, fair or poor based on the body conditions score according to Salah (2016).
Blood samples from camels were collected by jugular venipuncture for laboratory diagnosis. About 3ml of blood from each camel were collected into plain tubes and then kept under room temperature (25˚C) until visible cloth retraction was seen. The clothed samples were then centrifuged at 1500rpm for 5min, and the serum was aliquotted and stored at -80˚C until serological analysis was performed. Four (4) ml of blood was placed into a tube containing anticoagulants for parasitological and molecular analysis. In the eld the blood samples were kept in a cold box packed with ice and immediately transported to the Parasitology laboratory of the National Veterinary Research Institute (NVRI), Vom, Plateau State, Nigeria where they were preserved at -80˚C till further analysis.

Parasitological examination
For the parasitological diagnosis of the presence of Trypanosoma spp. from the blood samples collected, microscopic examination of thin smears of bloodstained with Giemsa stain was carried out (OIE, 2012). Brie y, blood samples were processed for microscopic examination according to standard procedures (Soulsby, 1982).
Stained blood smears were examined under the microscope using the oil immersion objective (×100) for the detection of Trypanosoma spp. A minimum of 50 microscopic elds were examined before the result was determined.

Serological examination
Commercially available Card Agglutination Test (CATT/T.evansi) kits were purchased from the Laboratory of Serology, Institute of Tropical Medicine, Antwerp, Belgium and used in this study. Serum samples were tested with Card Agglutination Test for Trypanosomiasis (CATT/T.evansi) following the instruction of the manufacturer with slight modi cations (Ibrahim et al., 2011). Brie y, one drop test serum was diluted 1:4 in CATT-Buffer. The mixture was then pipetted onto a plastic-coated test card. One drop of CATT reagent was added and the reaction mixture was spread out using a clean stirring rod. The reaction mixture was allowed to react on the card with manual rotation for 5min. Positive reactions were interpreted based on the appearance of blue granular agglutinations visible to the naked eye. Positive and negative controls were included in each reaction run.

Molecular characterization
DNA was extracted from the whole blood collected from each of the sampled animals using quick-DNA miniprep kit (Zymo Research) according to the instructions of the manufacturer. The eluted DNA was then stored at -20˚C until PCR analysis. Published primers, TE-FOR-(5 -TGCAGACGACCTGACGCTACT-3 ) and TE-REV-(5 -CTCCTAGAAGCTTCGGTGTCCT-3 ) for the ampli cation of the227bp fragments of T. evansi (Wuyts et al., 1994) was used in this study. The PCR ampli cation of the samples were performed in a 25µl reaction that contained 2.5µl of genomic DNA extract, 0.5µl of 20µM primer (TE-FOR and TE-REV), 12.5µl of one Taq® Quick-Load® 2x Master Mix with Standard Buffer (New England Bio Labs) and the volume made up with 9.0µl Nuclease Free Water (Promega®). This was taken to a pre-set and pre-heated Applied Biosystem®9700 PCR Machine for ampli cation.
The reaction conditions were: initial denaturation at 95˚C for 4minutes followed by 30cycles of denaturation at 95˚C for 1minutes, annealing at 60˚C for 1minutes, extension at 72˚C for 1min and nal extension at 72˚C for 10minutes. The PCR products were visualized in1.5% Agarose gel stained with ethidium bromide. The gel was observed for the appropriate size DNA band under a UV trans-illuminator.
Positive amplicons were sent to a commercial sequencing company (Macrogen Europe, Netherlands) for sequencing in the forward direction. Sequences obtained were manually edited and compared with the sequences available at the GenBank database using the Basic Local Alignment Sequence Techniques (BLAST) programme and databases of the NCBI (National Centre for Biotechnology Information, Bethesda, MD,USA) (www.blast.ncbi.nlm.nih.gov/blast.cgi).The obtained sequences reported in this study were deposited in the GenBank with the accession number (MZ394796 and MZ394795) for T.evansi isolated from Katsina and MZ394797 for T.evansi isolated from Kano. Phylogenetic analysis was constructed by comparing identi ed sequence in this study with the related sequences from GeneBank using the maximum likelihood (ML) method with the distance algorithms available in the Molecular Evolutionary Genetics Analysis package (MEGA 5).

Data analysis
Data generated during the study were entered into excel sheet and analyzed using the R statistical software (R core Team. 2013). The association between prevalence and risk factors was assessed using the chi-square test. The level of signi cance was set at p ≤ 0.05.

Results
The overall prevalence of T. evansi in the study area was 5.3%, 11.5% and 21.5% by microscopy, serology and PCR, respectively (Table 1). However, using the three methods explored in this study, a state-wise comparison indicated a higher prevalence of Surra in Katsina state when compared with Kano state (Table 1). Female camels and those between 1-3 years of age were mostly affected with T. evansi when compared with male and other age groups. In Katsina state camels with good body condition were not infected with T.evansi, but infection with T.evansi was recorded among camels with good body condition in Kano state. (Table 1).
There was a signi cant difference (p<0.05) between the T. evansi prevalence detected by the three diagnostic methods used in this study. PCR was the most sensitive followed by serology where as microscopic detection was the least sensitive (Table 2). Furthermore, the Agarose gel electrophoresis of the ampli ed PCR products from positive samples revealed a 227-bp fragment (Figure 2). Regardless of the diagnostic method used, T. evansi prevalence was associated with body condition score and the age of the animals. Furthermore, the sex of the camels was associated with the prevalence of T. evansi based on serology and PCR methods employed. However, there was no association between the prevalence of T. evansi in camels and seasons within the study area ( Table 2).
The maximum likelihood phylogenetic tree generated a topology showing the T. evansi from Nigeria forming a cluster with high bootstrap with T. evansi from Iran (GenBank: MF188845.1) as well as T.evansi isolated from Dog in India (GenBank: MG600142.1). Other Trypanosoma spp. form distinct clades according to their sequence similarities ( Figure 3).

Discussion
Camels will continue to constitute an important part of the lives and livelihood of subsistent farmers in Nigeria, both as draught animals and a source of protein. However, diseases such T.evansi infection causes a setback to camel productivity and wellbeing with a net socio-economic consequence to the farmers. In this study low to high prevalence (5.3% − 21.5%) of T.evansi was detected in northern Nigeria depending on the diagnostic method used. The relatively low prevalence recorded in this study is consistent with earlier reports from Nigeria The low prevalence recorded by the microscopic examination method could be due to its low sensitivity. However, the CATT/T. evansi serological assay detected higher cases of T. evansi infections from the sampled camels than the microscopy. The disadvantage though is the inability of this test to differentiate between active infection and antibodies from treated animals. It has been reported that following treatment, antibodies from the treated animal remain in blood circulation up to nearly four weeks, thus such animals will be detected as positive cases (  Mohamoud, 2017). Therefore, this fact necessitates getting a reliable history of the animal regarding recent anti-trypanosome treatment before sample collection to make CATT/T.evansi and all other antibody detecting tests more reliable.
The PCR diagnosis gave a higher prevalence than the other two methods used in this study. This nding attests to the ability of this method to detect and amplify low levels of parasite DNA in blood circulation. Unlike the blood smear examination by microscopy where a high level of parasitaemia, as well as the morphology of the parasite, is required to detect positive sample, the PCR is reputed to be sensitive at detecting low parasitaemia (Abdel-Rady, 2006). However, some factors such as the presence of PCR inhibitors during DNA extraction have been reported to limit the sensitivity of PCR (Shyma et al., 2013). Generally, the prevalence recorded by PCR was signi cantly higher than the results from the other methods used in this study. This could be due to the fact that PCR (i) is an accurate, more sensitive and speci c method in the diagnosis of trypanosomes infected camels than the parasitological method. It also overcomes the problem of non-speci c reaction as in the case of the serological method (ii) can also detect low parasitaemic camels in mild as well as chronic cases (Abdel-Rady, 2006 The body condition score is related to the plan of nutrition of the animals, hence, their ability to mount resistance to infections including Surra. Animals with poor body conditions are malnourished and therefore susceptible to disease conditions however, there is often a relationship between the season and body condition score of camels.
The extensive system of camel husbandry and management practiced by farmers in the study area is supported by the readily available pasture for camels during the rainy season compared to the dry season. There is a need for more feed supplements to the camels especially during the dry season when draught is intense and food scarcity is obvious, thereby meeting the high demand in the supply of safe and sustainable animal protein to humans. Also, adequate veterinary care should be provided to the camels to alleviate the effects of different animal diseases including Surra.

Conclusion
This study reports the prevalence of camel Trypanosomiasis in northern Nigeria using three diagnostic methods.
Based on our knowledge, this is the rst report of T.evansi infection in camels using three diagnostic methods along the parasitological-serological-molecular continuum, especially the use of PCR and sequencing to con rm the diagnosis. Taken together, Surra is prevalent in camels in northern Nigeria and constitutes a constraint to camel productivity in the area. Adequate control measures aimed at reducing the impact of trypanosomes on camel production in the study area is recommended.

Declarations
Competing of interest   Phylogenetic relationships of T.evansi detected in this study with trypanosome sequences from different parts of the world in GenBank. Evolutionary history inferred by maximum likelihood method with Trypanosoma brucei as an outgroup.