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 certification ID: NVRI AEC REF No: ACE/02/88/20. All experiments were conducted under strict adherence to the principle of laboratory animal care.
The study was conducted at the central abattoirs of Katsina (latitudes 11˚08ˡN and 13˚22ˡN and longitude 6˚52ˡE and 9˚20ˡE) and Kano (latitude 12˚40ˡN and 10˚31ˡN and longitude 7˚40ˡE and 9˚30ˡE) 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 330C and a minimum of 190C. The vegetation is mainly savanna; climatically define 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% confidence level and 5% precision as recommended by Thrusfield (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.
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, specific 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 field 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.
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). Briefly, 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 fields were examined before the result was determined.
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 modifications (Ibrahim et al., 2011). Briefly, 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.
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-(5ˡ- TGCAGACGACCTGACGCTACT- 3ˡ) and TE-REV-(5ˡ- CTCCTAGAAGCTTCGGTGTCCT- 3ˡ) for the amplification of the227bp fragments of T. evansi (Wuyts et al., 1994) was used in this study. The PCR amplification 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 amplification. 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 final 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 identified 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 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 significance was set at p ≤ 0.05.