Study zone
This cross-sectional study was conducted in nine villages (Foumban, Foumbot, Njimom, Massangam, Magba, Malentuen, Koutaba, Bangourain and Kouptamo) of the Noun division and six villages (Lena, Ngoun, Yoko Wankou, Megan, Kong and Kounde) of Yoko. The first survey was performed in November 2017 at Yoko and the second from April to June 2018 in the Noun Division.
Yoko (5°31'60"N; 12° 18′ 57″E) is located in the “Mbam et Kim” Division of the center region of Cameroon (Figure 1). It is situated between the southern green part and the northern Sahelian part of the country and covers about 15 000 km2. Yoko is known for large scale cattle and small ruminant’s production within the center region of Cameroon. The presence of pastures and water favors transhumance of herds not only from the West, East and Adamawa regions of Cameroon, but also from neighboring countries like the Central Africa Republic [31, 32].
The Noun division (4°95’ 6°30’N; 10°30’12°E) is located in the western highlands of Cameroon. It covers about 7687 km2 with a vegetation characterized by the savannah and degraded forest (Figure 1). The Noun division is considered as the main cattle production area of the west region of Cameroon [30]. Animals in this locality move to other regions (Northwest, Center and Adamawa) of Cameroon for trade or transhumance.
In these two localities, the feeding system is essentially free grazing which is sometime associated to stall-feeding. Various animal species including goats, sheep and cattle share the same environment.
Ethics statement
The protocol of this study was approved by the Ministry of livestock, fisheries and animals Industries of Cameroon with the reference number N°015/16/L/DDEPIA.NN. The review board of the molecular parasitology and entomology subunit of the Department of Biochemistry of the Faculty of Science of the University of Dschang gave also its approval. The local administration and traditional authorities of each sampling site were informed and their approvals were obtained. Verbal consent was obtained from each owner, after detailed explanation of the objective of the study.
Sample size estimation
For this study, a stratified sampling strategy was applied to select herds and individual cattle per herd. The sample size was estimated for cattle using a standard formula for cross-sectional studies as described by Thrusfield [33].
See formula 1 in the supplementary files.
where i) N is the minimum sample size required; ii) Z (critical value for a given confidence interval) = 1.96; iii) P (expected prevalence) = 0.052; and iv) L (margin of error) = 0.05.
For this estimation, the bovine brucellosis prevalence of 5.2% previously reported in the North West Region of Cameroon was used [30]. Additionally, a 95% confidence level and a precision of 5% were also used to determine the sample size. In each herd and depending on its size, at least 20% of cattle were sampled. More than 20% of cattle of some herds were sampled for specific reasons like the interests and cooperation of some farmers and advices from veterinarians. Selection of each cattle to be sampled in each chosen herd was done on the basis of systematic random sampling technique as described by Asgedom et al. [34]. A total of 37 farms were selected in 15 villages for a sample size of 597 cattle.
For the sheep, where there is no published data on the brucellosis prevalence in Cameroon, a prevalence of 50% was used for the sampling size estimation. Due to the small number of sheep in each village, all of them were sampled irrespective of the number of animals presented by each household. In consequence 315 sheep were sampled from the two agro-ecological zone of southern Cameroon.
Blood collection and DNA preparation
After approval from each herd owner, the farm characteristics and information regarding each animal including the name of the village (where each sample was collected), the geographical coordinates of each sampling site, the animal species found in farm (cattle, goat, sheep), the origin, sex, age, breed and the feeding system were recorded. Thereafter, about 5 ml of blood were collected, from the jugular vein of sheep and cattle, into EDTA coated tubes by a veterinarian. The tubes were labelled and carefully packed to avoid crossed contamination. In the field, the blood samples were stored at 4°C in an electric cooler before being transported to the laboratory where they were kept at -20°c.
Genomic DNA was extracted from whole blood using the cetyl trimethylammonium bromide (CTAB) method adopted from Navajas et al. [35]. Briefly, frozen samples were thawed and 500µL of whole blood was pipetted into a micro-tube containing 1 mL of sterile water. The micro-tube was vigorously vortexed and then, centrifuged at 10.000 rpm for 5 min. To the resulting pellet, 500 µL of CTAB buffer (CTAB 2%; 1 M Tris, pH 8; 0.5 M EDTA pH 8; 1.4 M NaCl) was added. The pellet was re-suspended and incubated in a water bath at 60°C for 30 min. To the content of each micro-tube, 600 µL of chloroform/isoamyl alcohol (24/1) mixture was added. Each micro-tube was slowly homogenized for 15 min and the upper aqueous phase was removed and transferred to a new 1.5 ml micro-tube. DNA was precipitated with 600 µL of isopropanol. The mixture was gently homogenized for 5 min and then incubated overnight at -20° C. Thereafter, each micro-tube was centrifuged at 13,000 rpm for 15 min. DNA pellet was then washed twice with cold 70% ethanol and dried overnight at room temperature. The resulting DNA pellet was re-suspended in 50 µL of sterile nuclease free water and then stored at -20° C until use.
Detection of Brucella spp
The identification of bacteria of the genus Brucella was performed as described by Mitka etal. [23]. This was done using B4 (5’-TGGCTCGGTTGCCAATATCAA-3’) and B5 (5’-CGCGCTTGCCTTTCAGGTCTG-3’) primers that amplify a 223 bp DNA fragment corresponding to the bcsp31 gene locus of Brucella genus. PCR reactions were performed in a total volume of 25 µL containing 10 pmol of each primer, 2.5 µL of 10X PCR buffer, 2 mM MgCl2, 200 mM of each dNTP, 5 µL of DNA template and 0.5 Unit of Q5 high fidelity Taq polymerase (New England Biolab 5 U/µL). The amplification program comprised an initial denaturation step at 95°C for 5 min followed by 40 amplification cycles made up of a denaturation step at 95°C for 30 s, an annealing step at 60°C for 30 s and an extension step at 72°C for 45s. A final extension step was performed at 72°C for 5 min.
The amplified products were resolved by electrophoresis on 1.5% agarose gel. The separation was done at 100 volts for 30 min. The gel was stained with ethidium bromide and DNA bands were visualized under ultraviolet light and photographed. Each PCR positive sample showing an amplicon of approximately 223 bp was considered as positive or having bacteria of the genus Brucella (Figure 2). These samples were selected and subsequently subjected to the identification of different Brucella species.
Specific identification of different Brucella species
All samples that were positive for the bcsp31 gene locus of Brucella genus (amplification of 223 bp DNA fragment) were further analyzed to determine the specific infecting Brucella species. This identification was done using AMOS PCR as described by Bricker and Halling [36]. For this identification, three primers were used: the IS711-specific primer (5′- TGC-CGA-TCA-CTT-AAG-GGC-CTT-CAT-TGC-3′) hybridizes the IS711 element that is common to all Brucella species while the two other primers hybridize an adjacent region outside the IS711. The primer with sequence 5′-GAC-GAA-CGG-AAT-TTT-TCC-AAT-CCC-3′ was specific to B.abortus while the third (5′- AAA-TCG-CGT-CCT-TGC-TGG-TCT-GA-3′) was specific to B.melitensis. During the identification of the different Brucella species, PCR reactions were carried out in a final volume of 25μL containing 10 pmol of each primer, 2.5µl of 10X PCR buffer, 2 mM MgCl2, 0.5 µL (200 mM) of each dNTPs, 5 µL of DNA template and 0.5 units of Taq DNA polymerase (New England Biolab 5 U/lL). The amplification program comprised an initial denaturation step at 95°C for 5 minutes followed by 40 cycles. Each cycle comprised a denaturation step at 95°C for 30 s, an annealing step at 53°C for 30 s and an extension step at 72°C for 1 min. This was followed by a final extension step at 72°C for 5 min.
The amplified products were separated by electrophoresis on 1.5% agarose gel containing ethidium bromide. This was done at 100 V for 45 min. After the electrophoresis, the gel was visualized under ultraviolet light and photographed. Samples showing an amplicon size of 495 bp were considered as harboring B. abortus while those with 730 bp were considered as positive for B. Melitensis (Figure 3).
Statistical analysis
The statistical analyses were performed using the Statistical Package for Social Sciences (SPSS) for Windows® version 22.0 (SPSS Inc., Chicago, Illinois). Chi-squared tests were used to compare the infection rates of different Brucella species. Significance threshold was set at 0.05