Study area, origin of samples and sample collection
The study was undertaken in five districts of Moroto, Kotido, Mbarara, Sembabule and Hoima (Figure 1). The study districts were located in North East (Moroto, Kotido), Central (Sembabule) and Western (Mbarara, Hoima) regions. Uganda is divided into 121 districts found in four major administrative regions: North, East, Central and West. Each region is primarily characterized by different livestock production systems: Northern region is characterised by agro-pastoral and pastoral system; Eastern region is mainly agro-pastoral; Central and west by agro-pastoral, semi-intensive and ranching (40). We sampled twenty-three cattle suspected to be affected by LSD from six field outbreaks in the 5 districts during the period from August 21, 2017 to December 8, 2018. The sampled animals were not previously vaccinated against LSDV. Samples of skin biopsies and scabs were collected in sterile cryovials containing 1 ml Minimum Essential Medium (MEM), Merck-Sigma, USA and whole blood in EDTA tubes. These samples were collected aseptically as described by the OIE(41). In addition, information on clinical signs of the suspected LSD affected animals was recorded. Each sample was given a unique sample ID, placed in a cooler box with ice and transferred to the molecular biology laboratory, College of Veterinary Medicine Animal Resources and Biosecurity (COVAB) and stored at -80°C for further molecular analysis.
DNA extraction and PCR confirmation of LSDV
The samples (skin biopsies, scabs and whole blood) were thawed at room temperature. Skin biopsy and scab samples were cut with a sterile scalpel blade into small pieces weighing about 400mg and homogenized in 500μl of sterile 1X PBS solution, pH 7.4. Total DNA was extracted from tissue homogenates and 200μl blood aliquots using a DNeasy Blood and tissue kit (Qiagen, Germany) following manufacturer’s instructions. PCR was then performed to confirm presence of LSDV specific nucleic acid by amplifying a 192 bp region in the p32 gene using a pair of primers; forward primer, 5’-TTTCCTGATTTTTCTTACTAT-3’ and reverse primer, 5’-AAATTATATACG TAAATAAC-3’, and PCR conditions as described by Binepal and Ireland (1998). The PCR reaction was set up in a 50 μl final volume containing 25µl of 2X MyTaq™ Red mix (Bioline, United Kingdom), 1.5µl of each 10µM primer concentration, 19.5µl of PCR water, and 2.5µl of extracted DNA. The PCR was performed in a Bio-Rad S1000 ThermoCycler (Bio-Rad, United Kingdom). The PCR conditions had an initial denaturation step of 94°C for 5 minutes, followed by 34 cycles of denaturation at 94°C for 1 minute, annealing at 50°C for 30 seconds, extension at 72°C for 1 minute and a final extension step of 72°C for 5 minutes. The PCR products were viewed on a 1.5% Agarose gel to confirm LSDV positive samples, with a band size of 192bp.
PCR amplification of the GPCR gene
A second PCR was carried out on all positive samples to amplify the GPCR gene for phylogenetic analysis. This was done using primers designed by Le Goff et al. (2009), with the following sequences (5’- TTAAGTAAAGCATAACTCCAACAAAAATG-3’ and 5’-TTTTTTTATTTTTTATCCAATGCTAATACT-3’), that were designed to amplify a fragment between nucleotide 6961–8119 in the LSDV genome (Tulman et al., 2001). An additional primer pair (5’-GATGAGTATTGATAGATACCTAGCTGTAGTT-3’ and 5’-TGAGACAATCCA AACCACCAT-3’) was positioned internally for sequencing (Le Goff et al., 2009). The DNA amplification of the GPCR gene was performed in a 50µl volume in the presence of 25µl of 2X MyTaq™ Red mix (Bioline, UK), 1.5µl of each 10µM primer concentration, 19.5µl of nuclease free water, and 2.5µl of DNA extract. The PCR amplification of the GPCR gene involved an initial denaturation at 96°C for 5 minutes followed by 35 cycles of final denaturation at 95°C for 30s, annealing at 50°C for the 30s, and extension at 72°C for 30s as previously described. All PCR products were resolved on 1.5% agarose gel against HyperLadder™ 100bp DNA ladder (Bioline, United Kingdom) at 125V in 1X Tris-Acetic acid-EDTA (TAE) buffer containing 0.5µg/ml ethidium bromide for 35 minutes. The gels were visualized using the ENDURO™ gel documentation system (LaboNet, USA).
Nucleotide sequencing and analysis
Following agarose gel electrophoresis on a 1.5 % agarose gel, amplification products of the expected size were identified against a molecular weight marker. DNA bands of correct size were excised and purified by gel purification (Qiagen, Germany), as specified by the manufacturer, and sent to Inqaba Biotec (South Africa) for Sanger sequencing. The sequences obtained were checked for quality and the ends of the sequences trimmed using BioEdit software (Ibis Biosciences, Carlsbad, CA, USA). The trimmed sequences were then checked for similarity with other LSDV GPCR sequences in GenBank using the National Center for Biotechnological Information’s (NCBI) web-based Basic Local Alignment Search Tool (BLASTn). These nucleotide sequences were then further checked for LSDV-specific signatures by translating them to amino acid sequences followed by multiple sequence alignment using MUSCLE found at the EMBL-EBI web server. Phylogenetic analysis was done using Molecular Evolutionary Genetics Analysis (MEGA) version 6 (Pennsylvania, USA). Thirty four (34) Capripoxvirus and one Deerpox GPCR sequence (used to root tree) were selected from GenBank to be used for phylogenetic analysis. After BLAST, LSDV sequences were selected based on nucleotide similarity and origin of isolates, so as to have representative sequences from East Africa, the rest of Africa and Eurasia. We also selected sequences from LSDV vaccine strains, goatpox and sheeppox virus. A phylogenetic tree was constructed using the maximum likelihood method based on Tamura 3 parameter model, with 1000 bootstrap replications. The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic trees. All sequences were submitted to GenBank and can be found under the accession numbers MN207136-MN207143.