Ethical clearance
This study was undertaken as part of the arboviral infection study (AVI) approved by the Ethics Review Committee at the College of Veterinary Medicine, Animal Resources and Biosecurity of Makerere University, Kampala, Uganda (Reference Number: SVARREC/20/20l8) and by the Uganda National Council for Science and Technology (Reference Number: HS 2485). Informed written consent was obtained from all study participants before they, and their animals, were enrolled in the study.
Outbreak area
Lyantonde district is located in the Ankole sub-region in Western Uganda (Fig. 1), bordered by Kiruhura, Sembabule, Rakai and Lwengo districts. Lyantonde occupies 888.1 km2, with an estimated population of 93,753 people [11]. Livestock forms the backbone of the economic activity of people in Lyantonde, with the majority (66.1%) rearing cattle and goats of indigenous and exotic crosses under a semi-intensive production system. Kasagama Sub-county (CCHF outbreak site) houses 80% of Lyantonde’s livestock population.
CCHF case description
On Monday, 29 July 2019, the Kasagama Sub-county One Health team reported the death from suspected viral haemorrhagic fever (VHF) of a 42-year-old male cattle trader, a resident of Kirindimula village, Kisaruwoko Parish Kasagama Sub-county, in Lyantonde district. He had been treated for malaria in Kasagama on 28 July 2019 following a history of fever, headache and vomiting, abdominal pain and general body pains. On Monday, 29 July 2019, he developed haematemesis and epistaxis. He died on Tuesday, 30 July 2019. Samples taken to the Uganda Virus Research Institute (UVRI) Entebbe for analysis and testing on 31 July 2019 indicated that the patient was positive for CCHF and negative for Ebola, Marburg and Rift Valley fever viruses.
Field investigation
A One Health team comprising the UVRI Ministry of Health, Ministry of Agriculture, Animal Industry and Fisheries, and Lyantonde district local government was convened to undertake the CCHF outbreak investigation in Lyantonde. In order to identify the likely source of the outbreak, an outbreak investigation was carried out on farms purposively selected based on the prior-14-day history of farm visitation by the victim before death from CCHF. A total of 10 farms were identified in this category, and the owners consented to participate in the study. All animals on each farm were placed in a restraint crush and one in four of these were sampled based on random selection and farmers’ choice. For this study, we aimed to collect a total of 100 or more animal samples, based on the numbers used in previous CCHF studies in similar settings in Uganda [12, 13].
A semi-structured herd questionnaire was administered to each farm owner to obtain animal demographic data including age, sex, breed, body temperature and tick infestation number. CCHFV herd level risk factors including herd size, animal production system, tick control practices and history of tick-borne infection were also collected.
Farm animals were selected and blood drawn into sterile EDTA and plain Vacutainer tubes (Becton Dickinson, Plymouth, UK) by veterinary professionals, and transported under cold chain for processing at Mbarara Regional Veterinary Laboratory. Samples were then centrifuged and the serum and plasma aliquoted into 2 ml sterile storage vials (Sarstedt, Inc., Newton, NC, USA). Animal sera were heat-inactivated at 56 °C for 2 h and stored at −80 °C until further laboratory investigation was carried out at the arbovirology laboratory based at UVRI, Entebbe, Uganda.
Anti-CCHFV immunoglobulin (IgG) detection
CCHFV IgG antibodies were tested in duplicate using the commercial ID Screen® CCHF double-antigen multi-species enzyme-linked immunosorbent assay (ELISA) kit (IDVet Innovative Diagnostics, France) following the manufacturer’s protocol [14]. This kit correlates highly with other assays that are routinely used in CCHF diagnostics [12]. Briefly, the test sera were diluted and incubated at 25 °C for 45 min. The conjugate and substrate steps were all conducted at 25 °C for 30 and 15 min, respectively, before the reaction was stopped. Absorbance was read at 450 nm on an automated ELISA reader (BioTek ELx800, USA) using Gen5 version 2.06 software. The sample positivity percentage (S/P%) for each sample was calculated by dividing the optical density (OD) value of the sample (ODS) by the OD of the positive control (ODPC), expressed as a percentage. Serum samples were considered positive if the value for their S/P% was greater than 30%.
CCHFV RNA extraction and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) testing
Nucleic acid was extracted from plasma using the Beckman Coulter RNA isolation procedure (Brea, CA, USA), following the manufacturer’s instructions. This kit has been found to yield high-quality RNA, suitable for CCHFV nucleic acid testing [15]. Briefly, equal volumes of 200 µl of phosphate-buffered saline (PBS) and test plasma were added to 330 µl lysis buffer and incubated in a water bath at 56 °C for 15 min. After cooling, 410 µl of bind 1/isopropanol solution was added, pipette-mixed and placed on magnetic beads to separate. The supernatant was removed and the beads washed in two subsequent steps using 800 µl of wash buffer/isopropanol and 80% ethanol. This was followed by a DNase treatment step, and the nucleic acid was eluted in 25 µl of nuclease-free water.
The RT-PCR assay was run using the Applied Biosystems 7500 Fast platform and SuperScript III Platinum One-Step qRT-PCR Kit (Invitrogen) according to methods previously described by Atkinson’s assay [16] with slight modifications. The 20 µl reaction volume comprised 10 µL of 2× reaction mix, 1.7 µL of PCR-grade water, 1 µL each of CCHFV reverse and forward primer (at 18 µM working concentration), 0.5 µL of probe (25 µM working concentration) and 5 µL of RNA template. The assay was set to run under the following cycling conditions: 50 °C for 10 min, 95 °C for 2 min, followed by 45 cycles of 95 °C for 10 s and 55 °C for 40 s. A cycle threshold (CT) value greater than 40 was considered negative.
Livestock ticks were collected as described previously [17] from half of the body of domestic animals, while environmental ticks were collected by both dragging and flagging methods. Briefly, ticks were transported in 70% ethanol for identification at the species level using morphological keys [18, 19]. Tick pools were created by collection site, species, sex, and the host animal. All tick pools were then crushed in 0.5 ml of Agencourt lysis buffer in a Geno/Grinder 2000 (OPS Diagnostics, Lebanon, NJ, USA), followed by downstream RNA extraction as described above for plasma (Beckman Coulter).
Tick pools were investigated for the presence of CCHFV, Nairobi sheep disease virus (NSDV) and Dugbe virus (DUGV) genomes using target enrichment next-generation sequencing (NGS), as described previously, using a probe library (Arbocap) targeting all arboviruses including nairoviruses [20]. Viral genomes were detected by de novo assembly using dipSPAdes and IDBA, followed by BLASTn and mapping to relevant nairovirus reference sequences using Tanoti. Maximum likelihood phylogenetic analysis was carried out using IQ-TREE and 1000 ultrafast bootstrap replicates [21].
Data analysis
Sociodemographic, epidemiological and laboratory data were analysed using Stata software (v15 StataCorp LP, College Station, TX, USA). Demographic and epidemiological characteristics were summarized using frequencies and percentages, stratified by animal species. We estimated the seropositivity of CCHF as the number of samples that tested positive divided by the total tested, expressed as a percentage. We performed both unadjusted and adjusted regression analysis to determine factors associated with CCHFV exposure. For the unadjusted analysis, the association between CCHFV seropositivity in animals and potential risk factors was first assessed using univariate logistic regression analysis. Multicollinearity was examined among different combinations of variables, and where there was a correlation greater than 0.5, we chose a factor most likely to be associated with CCHF exposure. A backward elimination approach was used to remove factors that were not associated with the outcome in the adjusted analysis (P > 0.1).