Detection and molecular characterization of African swine fever virus recovered from Ornithodoros ticks of the Serengeti Ecosystem, Tanzania

Detection and molecular characterization of African swine fever virus recovered from Ornithodoros ticks of the Serengeti Ecosystem, Tanzania | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Detection and molecular characterization of African swine fever virus recovered from Ornithodoros ticks of the Serengeti Ecosystem, Tanzania Baraka Mazengo Mbega, Jean N. Hakizimana, Ester K. Adamson, Mariam R. Makange, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9124525/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract African swine fever (ASF) is a highly lethal hemorrhagic disease of domestic pigs and Eurasian wild boars, caused by ASF virus (ASFV). In Africa, ASFV is maintained in a sylvatic cycle involving wild suids, primarily warthogs ( Phacocherus africanus ) and Ornithodoros ticks. Despite ASF outbreaks in Tanzania, the role of the sylvatic cycle in areas with high wildlife-livestock interactions remains understudied. This study aimed to detect and characterize ASFV in Ornithodoros ticks from warthog burrows in the Serengeti ecosystem. Soft ticks were collected from warthog burrows and screened for ASFV using quantitative polymerase chain reaction (qPCR). A total of 1,003 Ornithodoros ticks were collected from 25 burrows and qPCR detected ASFV in ticks from 9 burrows. Conventional PCR targeting the ASFV B646L (p72) gene, intergenic region (IGR) between I73R and I329L genes, and the central variable region (CVR) of the B602L gene was conducted in positive samples. The amplicons were sequenced and used for phylogenetic analysis. Phylogenetic analysis of the nucleotide sequences of B646L (p72) gene showed that the identified ASFV strains clustered within genotype X, which has been previously associated with outbreaks in domestic pigs in Tanzania and neighboring countries. Analysis of the IGR and the CVR of the B602L gene confirmed the high genetic similarity between the detected strains and those linked to previous ASF outbreaks in Tanzania and neighboring countries. The findings of this study highlight the need to prevent virus spillover from the sylvatic cycle for efficient control of the ASF in Tanzania. African swine fever virus Ornithodoros ticks warthog burrows Serengeti ecosystem phylogenetic analysis Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction African swine fever (ASF) is a contagious and lethal viral disease affecting domestic pigs and Eurasian wild boars, with significant socio-economic implications worldwide (Li et al. 2022 ). The ASF is a notifiable disease to the World Organization for Animal Health (WOAH) due to its severe impact on global pork production and its ability to spread rapidly across regions, posing a substantial challenge to animal health and trade (Plavsik et al. 2019 ). The ASF is a notifiable disease in Tanzania and among the priority diseases listed by the Ministry of Livestock and Fisheries (Cap 1 56 Animal Diseases Act [ R.E 2023 ]). African swine fever virus (ASFV; species Asfivirus haemorrhagiae ), a large, complex, double-stranded DNA virus of the genus Asfivirus , which belongs to the family Asfarvidae , is the aetiological agent for this disease (Alonso et al. 2018 ). The virus is highly stable and can spread efficiently through infected domestic pigs, contaminated domestic pig products, or through the blood-feeding activity of infected Ornithodoros ticks (McKercher et al. 1987 ; Plowright and Parker, 1967 ; Zheng et al. 2023 ). African swine fever virus persists in the wild environment through a complex sylvatic cycle involving wild suids such as warthogs ( Phacochoerus africanus ) and soft ticks of the genus Ornithodoros , which act as reservoirs and vectors, respectively (Jori et al. 2013 ). The virus can persist and replicate in Ornithodoros ticks, enabling transstadial, transovarial, and sexual transmission among tick populations (Kleiboeker and Scoles, 2001 ). It is not very clear if all genotypes that are involved during outbreaks in domesticated pigs are using ticks for persisting in certain areas and if they still depend on ticks for their persistence in pig populations. For instance, genotype II is nowadays circulating in domestic pigs and wild boars worldwide without using ticks as reservoir. It looks like ASFV developed mechanisms to be transmitted without the involvement of ticks. The ease with which genotype II is using the respiratory tract to infect domestic pigs makes it possible for the virus to change its epidemiological behavior (Oh et al. 2023 ) . The ASFV sylvatic cycle serves as a source for emerging new, more diverse, and virulent virus strains within the domestic cycle (Lubisi et al. 2005 ; Nix et al. 2006 ). This is evidenced by the significant genetic diversity of ASFV genotypes found in Eastern and Southern Africa (Lubisi et al. 2005 ; Nix et al. 2006 ), where the ASFV sylvatic cycle exists. The ASFV is transmitted to domestic pigs from the sylvatic cycle via infected Ornithodoros ticks that feed on them (Plowright et al. 1969 ). Infected ticks from the wild can come into contact with domestic pigs when hunted warthogs are transported to households or when warthogs containing the ticks graze on land directly neighboring areas where domestic pigs are kept (Kleiboeker and Scoles, 2001 ). Warthogs are widely distributed in Eastern and Southern Africa, mostly in tropical and subtropical savannas (Katale et al. 2012 ). Studies have shown that a high percentage of warthogs in Kenya and Tanzania and about 50% of warthogs in Uganda had antibodies against ASFV (Katale et al. 2012 ; Peter et al. 2021 ; Plowright et al. 1969 ). The Serengeti ecosystem is a critical area for ASFV studies due to its complex wildlife-livestock and human interface. While ASFV has been characterized in Ornithodoros ticks from isolated environments like Saadani National Park (Peter et al. 2021 ), the Serengeti presents a significant spillover risk. In this ecosystem, warthog burrows are often infested with Ornithodoros ticks, creating a natural environment for the virus to persist. A study conducted in 1968 in a small part of the Serengeti ecosystem, which is the Kirawira area of the Serengeti National Park, Tanzania, revealed a 15% ASFV infection rates of Ornithodoros ticks collected from warthog burrows (Plowright et al. 1969 ). African swine fever outbreaks have been documented in several Tanzanian regions, including Kilimanjaro, Arusha, Mbeya, Iringa, Morogoro, Pwani, and Dar es Salaam (Misinzo et al. 2014 ; Wambura et al. 2006 ; Yona et al. 2020 ). However, surveillance efforts to identify circulating genotypes have primarily focused on outbreaks in domestic pig farms within a limited number of locations. Despite the critical role of Ornithodoros ticks in ASFV transmission, studies on their prevalence and viral detection in interface areas like the Serengeti ecosystem are limited. This study aimed to detect and genetically characterize the ASFV in ` ticks collected from warthog burrows within the Serengeti ecosystem and to evaluate these ticks’ potential role in the ASF epidemiology in Tanzania. Materials and methods Study area The study was conducted in Serengeti National Park and Maswa Game Reserve (Fig. 1 ), the core of the Tanzanian Greater Serengeti Ecosystem (GSE), which forms the southern portion of the larger transboundary Serengeti-Mara ecosystem (Kegamba et al. 2023 ). This portion of the ecosystem is one of the seven important ecosystems for protecting wildlife in Tanzania. The Greater Serengeti Ecosystem in Tanzania is made up of nine administrative areas, including Serengeti National Park (SENAPA), Ngorongoro Conservation Area (NCA), Maswa, Ikorongo, Kijereshi, Grumeti Game Reserves, Loliondo Game Controlled Area, Ikona, and Makao Wildlife Management Area. The GSE in Tanzania spans an area of 25,000 km 2 and is characterized by the annual migration of ungulate herds, which interact with a significant livestock population. The ecosystem is located between 1° and 2° South latitude, 34° and 36° East longitude, featuring a variety of habitats that include expansive open grasslands, acacia-dominated savannas, and rocky outcrops (Katale et al. 2012 ). The sampling was conducted across the Serengeti National Park and Maswa Game Reserve, focusing on specific localities known for high warthog density. In Serengeti National Park, the soft ticks were collected at Seronera, which is the central plain with high wildlife concentration and open grasslands ideal for warthog burrowing. Further, sampling also was conducted at Nyasiroli, the northwestern part of the park near the Grumeti Game Reserve, which is characterized by wooded savanna. In Maswa Game Reserve the sampling was done on northeastern part which is characterized by a mixed woodlands and the central-western part of the reserve, which is covered by denser bushland. Collection of soft ticks from warthog’s burrows A cross-sectional study was conducted in October 2024, where the target population was the active warthog’s burrows for soft tick collection. A total of 24 active warthog burrows were identified within the Serengeti National Park and 6 from Maswa Game Reserve following confirmation of the signs, such as recent warthog footsteps around the burrows before sampling, and then the burrows were thoroughly examined for the presence of Ornithodoros ticks. With the use of a long-handled spade, the soil or sand was scooped from the roofs, walls, and floors of the burrows, then spread on a plastic sheet and exposed to sunlight for three minutes to stimulate movement and identification of ticks (Jori et al. 2013 , 2023 ) (Fig. 2 ). The collected Ornithodoros ticks were put into Falcon tubes with perforated tops to ensure containment and ventilation then they were transported to the laboratory. Once in the laboratory, ticks were preserved in 70% ethanol and stored in a -20°C freezer until analysis. Morphological identification of ticks Under a stereomicroscope (Olympus Sz51, Olympus Corporation, Tokyo, Japan), ticks were examined for morphological characteristics to confirm the genus Ornithodoros. Identification was based on the distinctive traits common to the O. moubata complex group, which included a tough and wrinkled upper surface dotted with nodules, the absence of eyes, the lack of a hard scutum, and the mouth not being visible from the top (Walker and Bouattour, 2003). Viral DNA extraction Following the removal of ethanol in individual Falcon tubes, ticks were pooled according to the sampling site and cleaned with distilled water as previously described (Silaghi et al. 2011 ). The ticks were then ground in a sterile mortar and pestle using phosphate-buffered saline as a suspension medium. A total of 200 µL of homogenized material was used for DNA extraction using a DNeasy blood and tissue kit (QIAGEN, Hilden, Germany), according to the manufacturer's protocol. The steps involved include cell lysis with proteinase K, binding of DNA to a silica membrane, followed by a washing step to remove contaminants, and elution of purified DNA in 100 µL of a low-salt buffer. Viral detection and identification Real-time polymerase chain reaction (qPCR) for screening ASFV was performed as described by King et al. (2003), targeting the viral VP72 gene. The PCR reactions were carried out in a total volume of 50 µL containing 5 µL of extracted DNA, 50 pmol of forward (5'-CTG CTC ATG GTA TCA ATC TTA TCG A-3') and reverse (5'-GAT ACC ACA AGA TCR GCC GT-3') primers, and 5 pmol of a FAM-labeled probe (5'-FAM-CCA CGG GAG GAA TAC CAA CCC AGT G-TAMRA-3'). The reaction mix also contained 50 mM KCl, 10 mM Tris–HCl (pH 9.0), 2 mM MgCl₂, 0.4 mM of each dNTP, and 2.5 U of Taq DNA polymerase (Promega). Amplification was performed on a QuantStudio™ 5 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA) with the following cycling conditions: an initial denaturation at 94°C for 120 s, followed by 40 cycles of 94°C for 15 s and 58°C for 60 s. Samples were run along with positive and negative controls. As per the assay’s analytical sensitivity, all samples with mean cycle quantification (Cq) values ≤ 40 were considered positive, whereas samples with Cq > 40 or with no amplification were classified as being negative. The ASFV-positive samples were subjected to further molecular characterization by amplification and sequencing of the B646L (p72) gene, the intergenic region (IGR) between the I73R and I329L genes, and the central variable region (CVR) of the B602L gene. Amplification of the B646L (p72) gene was performed using primers p72-U (5’-GGCACAAGTTCGGACATGT-3’) and p72-D (5’-GTACTGTAACGCAGCACAG-3’) as described by Bastos et al.(2003). Thermal profile started with initial denaturation at 96°C for 20s, then 35 cycles of 96°C for 12s denaturation, 50°C for 20s for annealing, and 70°C for 25s for extension. The IGR was amplified using primers ECO1A (5’-CCATTTATCCCCCGCTTTGG-3’) and ECO1B (5’-TCGTCATCCTGAGACAGCAG-3’) (Gallardo et al. 2014 ), with cycling conditions consisting of initial denaturation step at 96°C for 20s followed by 35 cycles each including denaturation at 96°C for 12s, primer annealing at 60°C for 20s, and DNA extension at 70°C for 25s. The CVR of B602L gene was amplified using primers ORF9L-F (5’-AATGCGCTCAGGATCTGTTAAATCGG-3’) and ORF9L-R (5’-TCTTCATGCTCAAAGTGCGTATACCT-3’), with the thermal profile as described by (Nix et al. 2006 ). Amplicons were excised from the agarose electrophoresis gels, purified, and subjected to Sanger sequencing (Craig et al. 2022 ). Phylogenetic reconstruction of ASFV targeting the B646L gene In order to classify viruses characterized in this study among the 24 known ASFV p72 genotypes, the C-terminal end of the B646L gene was amplified and sequenced. The Sequence Scanner software version 2 was used for quality control of the raw nucleotide chromatograms and consensus sequences were generated using BioEdit version 7.2.5. Multiple sequence alignment was performed using the ClustalW algorithm and phylogenetic tree was reconstructed using MEGA 12 (Kumar et al., 2024 ), with the Kimura two-parameter model, which was selected as the optimal model based on the Bayesian Information Criterion (BIC) scores. Branch support was assessed using 1,000 bootstrap replicates. Analysis of intergenic (IGR) and central variable (CVR) regions To further characterize genetic diversity, the amplified and sequenced regions of IGR were aligned with sequences from GenBank using ClustalW in MEGA 12. To get the amino acid signature, the DNA sequences of amplified CVR of the B602L gene were translated using the ExPASy translation tool ( https://web.expasy.org/translate/ , accessed on 15 April 2025 ) and coded as previously described (Lubisi et al. 2007 ; Misinzo et al. 2011 ). Results Collection of soft ticks and ASFV detection A total of 30 warthog burrows were surveyed within the Serengeti National Park and Maswa Game Reserve, of which 25 burrows (83.3%) were infested with soft ticks. From the 25 infested burrows, 1,003 soft ticks were collected, with an average of approximately 40 ticks per infested burrow. The number of ticks collected per burrow varied considerably, with some burrows yielding as few as 2 ticks and others yielding up to 182 ticks ( Table 1 ) . All the collected ticks were morphologically identified as Ornithodoros moubata complex. The morphological features recorded were leathery and wrinkled dorsal surface, eyeless, and non-visible mouthparts from a dorsal view. The DNA of ASFV was detected in soft ticks collected from nine out of 25 barrows (36%) by qPCR analysis. The Serengeti National Park had the highest positive samples of ASFV in ticks (8 out of 9 barrows) as shown in Table 1 . Table 1 Descriptions of location, number of ticks collected per burrow and their African swine fever virus (ASFV) status after real-time polymerase chain reaction (qPCR). Warthog burrow ID Location Coordinate (X) Coordinate (Y) Number of ticks collected ASFV status (qPCR) WB1 Seronera -2.431013 34.854944 182 Negative WB2 Seronera -2.429474 34.851778 157 Negative WB3 Seronera -2.433911 34.868264 83 Negative WB4 Seronera -2.434838 34.867748 75 Negative WB5 Seronera -2.433776 34.865254 32 Positive WB6 Seronera -2.428944 34.850589 94 Negative WB7 Seronera -2.447241 34.851856 19 Positive WB8 Seronera -2.448738 34.85047 2 Positive WB9 Seronera -2.449267 34.850914 55 Positive WB10 Seronera -2.452309 34.851775 26 Negative WB11 Seronera -2.453208 34.851641 13 Negative WB12 Seronera -2.437608 34.813467 17 Negative WB13 Seronera -2.435864 34.814694 8 Negative WB14 Seronera -2.430786 34.813008 3 Positive WB15 Seronera -2.433295 34.814185 4 Positive WB16 Seronera -2.430668 34.811735 0 - WB17 Seronera -2.428590 34.783136 39 Negative WB18 Seronera -2.429077 34.784674 0 Negative WB19 Seronera -2.427280 35.773917 16 Negative WB20 Seronera -2.425280 34.775184 32 Positive WB21 Seronera -2.444415 34.85266 9 Negative WB22 Seronera -2.442913 34.856015 24 Positive WB23 Seronera -2.443750 34.855731 8 Negative WB24 Nyasiroli -2.158943 34.398946 51 Negative WB25 Maswa Game Reserve -3.159760 34.513987 25 Negative WB26 Maswa Game Reserve -3.166753 34.514104 29 Positive WB27 Maswa Game Reserve -134605 34.616974 0 - WB28 Maswa Game Reserve -3.075344 34.656918 0 - WB29 Maswa Game Reserve -3.141809 34.646322 0 - WB30 Maswa Game Reserve -3.147732 34.488759 10 Negative *(-) Represent burrows with no ticks hence no ASFV status Sequencing results Amplicons generated from PCR-positive samples were subjected to Sanger sequencing, and the phylogenetic tree revealed that the isolates ASFV/TAN/2024/Serengeti/SR1 and ASFV/TAN/2024/Serengeti/SR3 clustered within genotype X, which includes other isolates from Tanzania, such as those from Mwanza, Kigoma, Ngara, Geita, Kahama, Katavi, Longido, Arusha, Moshi, Babati, and Manyoni ( Fig. 3 ). At BLASTn, the sequences described in this study showed 99.58 to 99.79% nucleotide identity with ASFV genotype X isolates previously reported in domestic pigs in Burundi, Democratic Republic of the Congo, Kenya, Tanzania and Uganda. Analysis of IGR and CVR genomics regions The tetrameric amino acid repeats within the CVR of the B602L gene had the signature AAABNAAAAAAAAAABA using the code previously described by Lubisi et al. ( 2007 ) and Misinzo et al. ( 2011 ), with high nucleotide identity compared to the ASFV genotype X previously described in Tanzania and neighboring countries. The nucleotide sequence showed 100% identity with isolate Hinde/I (GenBank accession number OK560519) collected from warthog in Kenya in 1954. After BLASTn of the nucleotide sequences of the IGR between the I73R and I329L genes, the most similar tandem repeat sequence (TRS) was that of the isolate TAN/19/Ngara (GenBank accession number MW659865) with 100% nucleotide identity. In addition, the sequences described in this study had 99.77% nucleotide identity to Kenya_1950 (NC_044944.1) reference isolate collected from domestic pig in Kenya in 1950. The sequence described in this study lacked a fragment of 36 bp within the TRS compared to the isolate Ken05/Tk1 (NC_044945.1) collected from ticks in Kenya in 2005. A similar fragment was missing from the ASFV reference genome Kenya_1950 (NC_044944.1) and isolates from domestic pigs in Tanzania ( Fig. 4 ) . Discussion The results of this study provide important insights into the presence of the sylvatic cycle in the Serengeti ecosystem, specifically in warthog burrows. A total of 1,003 soft ticks were collected from 30 warthog burrows, of which five were found to be uninfested. The presence of soft ticks in 25 burrows suggests a widespread distribution of these ASFV vectors in the study area. Detection of ASFV in ticks from nine of the 25 burrows highlights a significant level of viral circulation within the ecosystem. The detection of ASFV in soft ticks associated with warthogs aligns with previous studies that have established warthogs and their associated ticks as key reservoirs in the sylvatic cycle of ASFV (Dixon and Wilkinson, 1988 ; Jori et al. 2013 ; Njau, 2022 ; Quembo et al. 2018 ). The role of Ornithodoros ticks in the maintenance and transmission of ASFV has been widely recognized, as these ticks can acquire the virus from infected warthogs and persistently harbor it for extended periods (Kleiboeker and Scoles, 2001 ). The ability of Ornithodoros ticks to facilitate transstadial and transovarial transmission further enhances their capacity to maintain ASFV in the absence of actively infected hosts (Parker et al., 1969 ; Plowright et al., 1970 a; Plowright et al., 1970 b). The presence of positive ticks from 9 out of 25 infested burrows indicates that ASFV is actively circulating in a significant proportion of the warthog population in the study area. This finding is of concern, given the potential for spillover of the virus to domestic pigs, particularly in areas where warthogs and domestic pigs share common grazing grounds or where ticks are inadvertently transported to farms through human activities such as hunting and bush meat trade, especially on the edges of the ecosystems (Peter et al. 2021 ). The phylogenetic analysis of ASFV sequences obtained from the infected tick samples revealed that the ASFV strains detected clustered within genotype X. This finding aligns with a similar study conducted in Kenya, where ASFV strains isolated from soft ticks and domestic pigs were also classified under genotype X (Gallardo et al. 2011 ), indicating a potential regional circulation of this genotype in East Africa. However, other studies in Kenya and Tanzania have identified ASFV strains from soft ticks belonging to other genotypes, like genotype IX and XV, respectively, highlighting the genetic diversity of ASFV in the region (Obanda et al. 2024 ; Peter et al. 2021 ). The genotype X of ASFV has been previously identified in several regions of Tanzania and other parts of East Africa, suggesting that the virus circulating in the sylvatic cycle is genetically related to strains previously reported in the region (Hakizimana et al. 2021 ; Misinzo et al. 2011 , 2012 ). The close relationship of these sequences with other genotype X isolates, including domestic pig outbreaks in Mwanza, Kigoma, Manyoni, Ngara, Babati, and other places in western and northern Tanzania, highlights the potential risk of spillover from wildlife to domestic animals. The analysis of the CVR of the B602L gene showed a tetrameric amino acid signature consistent with previously reported genotype X isolates from Tanzania and neighboring countries (Lubisi et al. 2007 ; Misinzo et al. 2011 ). The high nucleotide identity with isolates from Burundi, Democratic Republic of the Congo, Kenya, Tanzania and Uganda suggests that there is high relatedness between the detected virus from this study and the circulating viruses in East and Central Africa. The presence of ASFV genotype X in warthog-associated Ornithodoros ticks has several implications. First, it suggests that the sylvatic cycle remains an active reservoir of ASFV in the Serengeti ecosystem. The genetic clustering of the identified sequences from the ticks with already published genotype X strains implies that the virus has likely been maintained in this ecosystem for an extended period, potentially as a source of infection for domestic pigs when conditions allow transmission. In addition, the genetic similarity between these tick-derived isolates and previously identified strains in domestic pigs raises concerns about the role of wildlife in sustaining outbreaks in Tanzania. This observation aligns with previous studies that have demonstrated the genetic overlap between ASFV strains in the sylvatic and domestic cycles, underscoring the importance of surveillance at the wildlife-livestock interface (Gallardo et al. 2011 ; Obanda et al. 2024 ). Moreover, the phylogenetic tree reveals a clear separation between genotype X and other ASFV genotypes circulating in Tanzania, such as genotype II, which has been responsible for major outbreaks in the domestic pig population (Misinzo et al. 2012 ). This distinction highlights the genetic diversity of ASFV within the country and suggests that different transmission dynamics may be at play between the sylvatic and domestic cycles. While genotype II has been associated with severe outbreaks in domestic pig farms, the persistence of genotype X in wildlife may contribute to the long-term maintenance of ASFV in the region, posing a continuous risk to domestic pig production. Our results reinforce the importance of continuous molecular surveillance to track ASFV genotypes within sylvatic cycles. In the absence of a vaccine or treatment, such surveillance is crucial for mapping genetic diversity and transmission pathways, which can inform targeted strategies to mitigate spillover events. Nonetheless, these conclusions are tempered by the study's limited sample size and narrow geographical focus. Therefore, further research is warranted to confirm and broaden these findings. It is recommended to have an atlas of the different genotypes of ASFV that are present in the soft ticks all over Africa. In conclusion, the detection of ASFV in soft ticks from warthog burrows and the phylogenetic clustering of these isolates within genotype X, with genetic relatedness to previously isolated strains from domestic pigs, emphasize the importance of the investigations on the sylvatic cycle in the ASFV epidemiology in Tanzania and neighboring countries. This finding highlights the urgent need for continued surveillance, improved biosecurity measures, and further research to understand ASFV transmission dynamics at the wildlife-livestock interface. Ethical consideration The President's Office Regional Administration and Local Government of Tanzania (PO-RALG), Sokoine University of Agriculture, Tanzania Wildlife Research Institute (TAWIRI) granted permission to carry out the study under reference numbers AB.307/323/01/51, SUA/DPRTC/MAM/D/2022/0002/06 , and AB.235/325/01/166 , respectively. District Veterinary Officers (DVOs) of each district were duly notified about the study’s objectives and procedures and offered support. Statements & Declarations Data availability statement The nucleotide sequences described in this study have been submitted to the National Center for Biotechnology Information (NCBI) GenBank and assigned accession numbers PX445921 to PX445924 . Conflict of interest Author declare no conflict of interest. Acknowledgement The authors would like to express their sincere gratitude to Mr. Maulid Mdaki for his invaluable assistance in sample collection, Mrs Mariam R. Makange and to Mrs Anna Rogath Masawe for their excellent technical assistance during the laboratory analysis. Funding This work was funded by the Regional Scholarship and Innovation Fund (Rsif) of the Partnership for Skills in Applied Science, Engineering and Technology (PASET) through the Junior Investigator Research Award (JIRA) with grant number RSIF/JIRA/001. The funder had no role in study design, data collection and analysis, decision to publish as well as in the preparation of the manuscript. The findings and conclusions of this study are those of the authors and do not necessarily represent the view of Rsif-PASET References Alonso, C., Borca, M., Dixon, L., Revilla, Y., Rodriguez, F., Escribano, J. M., & ICTV Report Consortium. (2018). ICTV Virus Taxonomy Profile: Asfarviridae. Journal of General Virology , 99 (5), 613–614. https://doi.org/10.1099/jgv.0.001049 Bastos, A. D. S., Penrith, M.-L., Crucière, C., Edrich, J. L., Hutchings, G., Roger, F., Couacy-Hymann, E., & R.Thomson, G. (2003). Genotyping field strains of African swine fever virus by partial p72 gene characterisation. Archives of Virology , 148 (4), 693–706. https://doi.org/10.1007/s00705-002-0946-8 Cap. 156 Animal Diseases Act, R.E 2023—MD, Mendez.pdf . (n.d.). Retrieved August 30, 2025, from https://elibrary.osg.go.tz/bitstream/handle/123456789/2200/Cap.%20156%20Animal%20Diseases%20Act%2c%20R.E%202023%20-%20MD%2c%20Mendez.pdf?sequence=2&isAllowed=y Craig, A. F., Schade-Weskott, M. L., Rametse, T., Heath, L., Kriel, G. J. P., De Klerk-Lorist, L.-M., Van Schalkwyk, L., Trujillo, J. D., Crafford, J. E., Richt, J. A., & Swanepoel, R. (2022). Detection of African Swine Fever Virus in Ornithodoros Tick Species Associated with Indigenous and Extralimital Warthog Populations in South Africa. Viruses , 14 (8), 1617. https://doi.org/10.3390/v14081617 Dixon, L. K., & Wilkinson, P. J. (1988). Genetic Diversity of African Swine Fever Virus Isolates from Soft Ticks (Ornithodoros moubata) Inhabiting Warthog Burrows in Zambia. Journal of General Virology , 69 (12), 2981–2993. https://doi.org/10.1099/0022-1317-69-12-2981 Gallardo, C., Fernández-Pinero, J., Pelayo, V., Gazaev, I., Markowska-Daniel, I., Pridotkas, G., Nieto, R., Fernández-Pacheco, P., Bokhan, S., Nevolko, O., Drozhzhe, Z., Pérez, C., Soler, A., Kolvasov, D., & Arias, M. (2014). Genetic Variation among African Swine Fever Genotype II Viruses, Eastern and Central Europe. Emerging Infectious Diseases , 20 (9), 1544–1547. https://doi.org/10.3201/eid2009.140554 Gallardo, C., Okoth, E., Pelayo, V., Anchuelo, R., Martin, E., Simon, A., Llorente, A., Nieto, R., Soler, A., Martin, R., Arias, M., & Bishop, R. P. (2011). African swine fever viruses with two different genotypes, both of which occur in domestic pigs, are associated with ticks and adult warthogs, respectively, at a single geographical site. Journal of General Virology , 92 (2), 432–444. https://doi.org/10.1099/vir.0.025874-0 Hakizimana, J. N., Yona, C., Kamana, O., Nauwynck, H., & Misinzo, G. (2021). African Swine Fever Virus Circulation between Tanzania and Neighboring Countries: A Systematic Review and Meta-Analysis. Viruses , 13 (2), Article 2. https://doi.org/10.3390/v13020306 (https://web.expasy.org/translate/)—Google Search . (n.d.). Retrieved August 15, 2025, from https://www.google.com/search?q=(https%3A%2F%2Fweb.expasy.org%2Ftranslate%2F)&oq=(https%3A%2F%2Fweb.expasy.org%2Ftranslate%2F)&gs_lcrp=EgZjaHJvbWUyBgg AEEUYOTIICAEQABgWGB7SAQgxMzQ4ajBqN6gCALACAA&sourceid=chrome&ie=UTF-8 Jori, F., Bastos, A., Boinas, F., Van Heerden, J., Heath, L., Jourdan-Pineau, H., Martinez-Lopez, B., Pereira de Oliveira, R., Pollet, T., Quembo, C., Rea, K., Simulundu, E., Taraveau, F., & Penrith, M.-L. (2023). An Updated Review of Ornithodoros Ticks as Reservoirs of African Swine Fever in Sub-Saharan Africa and Madagascar. Pathogens , 12 (3), 469. https://doi.org/10.3390/pathogens12030469 Jori, F., Vial, L., Penrith, M. L., Pérez-Sánchez, R., Etter, E., Albina, E., Michaud, V., & Roger, F. (2013). Review of the sylvatic cycle of African swine fever in sub-Saharan Africa and the Indian ocean. Virus Research , 173 (1), 212–227. https://doi.org/10.1016/j.virusres.2012.10.005 Katale, B., Fyumagwa, R., Mdaki, M., & Hoare, R. (2012). Prevalence of African swine fever virus in Warthogs in the Serengeti ecosystem, Tanzania. Research Opinions in Animal and Veterinary Sciences , 2 , 339-343. Katale, B. Z., Fyumagwa, R. D., Mdaki, M. L., & Hoare, R. (2012). RESEARCH OPINIONS IN ANIMAL & VETERINARY SCIENCES . Kegamba, J. J., Sangha, K. K., Wurm, P. A. S., & Garnett, S. T. (2023). Conservation benefit-sharing mechanisms and their effectiveness in the Greater Serengeti Ecosystem: Local communities’ perspectives. Biodiversity and Conservation , 32 (6), 1901–1930. https://doi.org/10.1007/s10531-023-02583-1 Kleiboeker, S. B., & Scoles, G. A. (2001). Pathogenesis of African swine fever virus in Ornithodoros ticks. Animal Health Research Reviews , 2 (2), 121–128. https://doi.org/10.1079/AHRR200133 Kumar, S., Stecher, G., Suleski, M., Sanderford, M., Sharma, S., & Tamura, K. (2024). MEGA12: Molecular Evolutionary Genetic Analysis Version 12 for Adaptive and Green Computing. Molecular Biology and Evolution , 41 (12), msae263. https://doi.org/10.1093/molbev/msae263 Li, Z., Chen, W., Qiu, Z., Li, Y., Fan, J., Wu, K., Li, X., Zhao, M., Ding, H., Fan, S., & Chen, J. (2022). African Swine Fever Virus: A Review. Life , 12 (8), 1255. https://doi.org/10.3390/life12081255 Lubisi, B. A., Bastos, A. D. S., Dwarka, R. M., & Vosloo, W. (2005). Molecular epidemiology of African swine fever in East Africa. Archives of Virology , 150 (12), 2439–2452. https://doi.org/10.1007/s00705-005-0602-1 Lubisi, B. A., Bastos, A. D. S., Dwarka, R. M., & Vosloo, W. (2007). Intra-genotypic resolution of African swine fever viruses from an East African domestic pig cycle: A combined p72-CVR approach. Virus Genes , 35 (3), 729–735. https://doi.org/10.1007/s11262-007-0148-2 McKercher, P. D., Yedloutschnig, R. J., Callis, J. J., Murphy, R., Panina, G. F., Civardi, A., Bugnetti, M., Foni, E., Laddomada, A., Scarano, C., & Scatozza, F. (1987). Survival of Viruses in “Prosciutto di Parma” (Parma Ham). Canadian Institute of Food Science and Technology Journal , 20 (4), 267–272. https://doi.org/10.1016/S0315-5463(87)71198-5 Misinzo, G., Kasanga, C. J., Mpelumbe–Ngeleja, C., Masambu, J., Kitambi, A., & Van Doorsselaere, J. (2012). African Swine Fever Virus, Tanzania, 2010–2012. Emerging Infectious Diseases , 18 (12), 2081–2083. https://doi.org/10.3201/eid1812.121083 Misinzo, G., Kwavi, D. E., Sikombe, C. D., Makange, M., Peter, E., Muhairwa, A. P., & Madege, M. J. (2014). Molecular characterization of African swine fever virus from domestic pigs in northern Tanzania during an outbreak in 2013. Tropical Animal Health and Production , 46 (7), 1199–1207. https://doi.org/10.1007/s11250-014-0628-z Misinzo, G., Magambo, J., Masambu, J., Yongolo, M. G., Van Doorsselaere, J., & Nauwynck, H. J. (2011). Genetic Characterization of African Swine Fever Viruses from a 2008 Outbreak in Tanzania. Transboundary and Emerging Diseases , 58 (1), 86–92. https://doi.org/10.1111/j.1865-1682.2010.01177.x Nix, R. J., Gallardo, C., Hutchings, G., Blanco, E., & Dixon, L. K. (2006). Molecular epidemiology of African swine fever virus studied by analysis of four variable genome regions. Archives of Virology , 151 (12), 2475–2494. https://doi.org/10.1007/s00705-006-0794-z Njau, E. (2022). Detection and genetic characterization of sylvatic and outbreak African swine fever virus isolates in selected zones of Tanzania [Thesis, NM-AIST]. https://dspace.nm-aist.ac.tz/handle/20.500.12479/1541 Obanda, V., Akinyi, M., King’ori, E., Nyakundi, R., Ochola, G., Oreng, P., Mugambi, K., Waiguchu, G. M., Chege, M., Rosenbaum, W., Ylitalo, E. B., Bäck, A. T., Pettersson, L., Mukunzi, O. S., Agwanda, B., Stenberg-Lewerin, S., & Lwande, O. W. (2024). Epidemiology and ecology of the sylvatic cycle of African Swine Fever Virus in Kenya. Virus Research , 348 , 199434. https://doi.org/10.1016/j.virusres.2024.199434 Oh, D., Han, S., Tignon, M., Balmelle, N., Cay, A. B., Griffioen, F., Droesbeke, B., & Nauwynck, H. J. (2023). Differential infection behavior of African swine fever virus (ASFV) genotype I and II in the upper respiratory tract. Veterinary Research , 54 (1), 121. https://doi.org/10.1186/s13567-023-01249-8 Parker, J., Plowright, W., & Pierce, M. A. (1969). The epizootiology of African swine fever in Africa. The Veterinary Record , 85 (24), 668–674. Peter, E., Machuka, E., Githae, D., Okoth, E., Cleaveland, S., Shirima, G., Kusiluka, L., & Pelle, R. (2021). Detection of African swine fever virus genotype XV in a sylvatic cycle in Saadani National Park, Tanzania. Transboundary and Emerging Diseases , 68 (2), 813–823. https://doi.org/10.1111/tbed.13747 Plavsik, B., Rozstalnyy, A., Park, J. Y., Guberti, V., Depner, K., & Torres, G. (2019). Strategic challenges to global control of African swine fever . O.I.E (World Organisation for Animal Health). https://doi.org/10.20506/TT.2985 Plowright, W., & Parker, J. (1967). The stability of African swine fever virus with particular reference to heat and pH inactivation. Archiv Für Die Gesamte Virusforschung , 21 (3), 383–402. https://doi.org/10.1007/BF01241738 Plowright, W., Parker, J., & Peirce, M. A. (1969). African Swine Fever Virus in Ticks (Ornithodoros moubata, Murray) collected from Animal Burrows in Tanzania. Nature , 221 (5185), 1071–1073. https://doi.org/10.1038/2211071a0 Plowright, W., Perry, C. T., & Peirce, M. A. (1970). Transovarial Infection with African Swine Fever Virus in the Argasid Tick, Ornithodoros moubata porcinus , Walton. Research in Veterinary Science , 11 (6), 582–584. https://doi.org/10.1016/S0034-5288(18)34259-0 Plowright, W., Perry, C. T., Peirce, M. A., & Parker, J. (1970). Experimental infection of the argasid tick, ornithodoros moubata porcinus, with African swine fever virus. Archiv Für Die Gesamte Virusforschung , 31 (1), 33–50. https://doi.org/10.1007/BF01241664 Quembo, C. J., Jori, F., Vosloo, W., & Heath, L. (2018). Genetic characterization of African swine fever virus isolates from soft ticks at the wildlife/domestic interface in Mozambique and identification of a novel genotype. Transboundary and Emerging Diseases , 65 (2), 420–431. https://doi.org/10.1111/tbed.12700 Silaghi, C., Hamel, D., Thiel, C., Pfister, K., & Pfeffer, M. (2011). Spotted Fever Group Rickettsiae in Ticks, Germany. Emerging Infectious Diseases , 17 (5), 890–892. https://doi.org/10.3201/eid1705.101445 Walker, A. R., & Bouattour, A. (n.d.). Ticks of Domestic Animals in Africa: A Guide to Identification of Species . Wambura, P. N., Masambu, J., & Msami, H. (2006). Molecular Diagnosis and Epidemiology of African Swine Fever Outbreaks in Tanzania. Veterinary Research Communications , 30 (6), 667–672. https://doi.org/10.1007/s11259-006-3280-x Yona, C. M., Vanhee, M., Simulundu, E., Makange, M., Nauwynck, H. J., & Misinzo, G. (2020). Persistent domestic circulation of African swine fever virus in Tanzania, 2015–2017. BMC Veterinary Research , 16 (1), 369. https://doi.org/10.1186/s12917-020-02588-w Zheng, W., Xi, J., Zi, Y., Wang, J., Chi, Y., Chen, M., Zou, Q., Tang, C., & Zhou, X. (2023). Stability of African swine fever virus genome under different environmental conditions. Veterinary World , 16 (11), 2374–2381. https://doi.org/10.14202/vetworld.2023.2374-2381 Zsak, L., Borca, M. V., Risatti, G. R., Zsak, A., French, R. A., Lu, Z., Kutish, G. F., Neilan, J. G., Callahan, J. D., Nelson, W. M., & Rock, D. L. (2005). Preclinical Diagnosis of African Swine Fever in Contact-Exposed Swine by a Real-Time PCR Assay. Journal of Clinical Microbiology , 43 (1), 112–119. https://doi.org/10.1128/jcm.43.1.112-119.2005 Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 06 Apr, 2026 Reviewers invited by journal 04 Apr, 2026 Editor assigned by journal 24 Mar, 2026 First submitted to journal 20 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9124525","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":617628425,"identity":"ad9b59eb-8494-4ace-b447-b1c3d5880ccd","order_by":0,"name":"Baraka Mazengo Mbega","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAElEQVRIiWNgGAWjYBACAyBmbIBzK4CYmbkBh2KsWs6AtCDxCWthbGNANQIbMGc/+/DjjJpt8vLtvYdffJxXG83fDtTyo2IbTi2WPenGkhuO3TZs7DmXZjlz2/HcGYcZGxh7ztzG7bADaQySD9huMzZL5JgZ8247ltsA1MLM2IZHy/lnzD8f/Ltt3yb/BqhlzrHc+QS13Ehjk9zYdjuxR4LH+DFvQ03uBkJaLGc8Y7Oc2Xc7eQZPjhnjjGMHcjcCtRzE5xdz/jTmmz3fbtvObz9j/OFDTV3uvPOHDz74UYFbCzJgk2BgOAxmHSBKPRAwf2BgqCNW8SgYBaNgFIwgAACTeWDX4A6tjwAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0009-0008-0042-8900","institution":"St John's University of Tanzania","correspondingAuthor":true,"prefix":"","firstName":"Baraka","middleName":"Mazengo","lastName":"Mbega","suffix":""},{"id":617628426,"identity":"d7539424-79ac-443e-891c-9bf524be55f5","order_by":1,"name":"Jean N. Hakizimana","email":"","orcid":"","institution":"Southern African Centre for Infectious Disease Surveillance","correspondingAuthor":false,"prefix":"","firstName":"Jean","middleName":"N.","lastName":"Hakizimana","suffix":""},{"id":617628427,"identity":"65a98c03-dfdc-4943-8902-9614161310c7","order_by":2,"name":"Ester K. Adamson","email":"","orcid":"","institution":"Southern African Centre for Infectious Disease Surveillance","correspondingAuthor":false,"prefix":"","firstName":"Ester","middleName":"K.","lastName":"Adamson","suffix":""},{"id":617628428,"identity":"e47994ed-78eb-413a-997c-b35f24489fa7","order_by":3,"name":"Mariam R. Makange","email":"","orcid":"","institution":"Southern African Centre for Infectious Disease Surveillance","correspondingAuthor":false,"prefix":"","firstName":"Mariam","middleName":"R.","lastName":"Makange","suffix":""},{"id":617628429,"identity":"40ad5ce2-553b-49dd-98f6-556f1ac6f929","order_by":4,"name":"Julius Keyyu","email":"","orcid":"","institution":"Tanzania Wildlife Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Julius","middleName":"","lastName":"Keyyu","suffix":""},{"id":617628430,"identity":"6f7be737-1aaf-4f05-9c7c-0e545e3f51b5","order_by":5,"name":"Hans J. Nauwynck","email":"","orcid":"","institution":"Ghent University: Universiteit Gent","correspondingAuthor":false,"prefix":"","firstName":"Hans","middleName":"J.","lastName":"Nauwynck","suffix":""},{"id":617628431,"identity":"1b5d9475-b866-4527-b173-e058306c4841","order_by":6,"name":"Gerald Misinzo","email":"","orcid":"","institution":"Sokoine University of Agriculture College of Veterinary Medicine and Biomedical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Gerald","middleName":"","lastName":"Misinzo","suffix":""}],"badges":[],"createdAt":"2026-03-14 18:42:55","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9124525/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9124525/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106960065,"identity":"c08b036c-1c2f-4f9f-b26f-e25829fdc6ff","added_by":"auto","created_at":"2026-04-15 09:18:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":273178,"visible":true,"origin":"","legend":"\u003cp\u003eMap of Tanzania showing the Serengeti National Park and Maswa Game Reserve where samples were collected, and the distribution of warthog burrows in which the collected samples were positive (red dots) and negative (yellow dots). The map was drawn using QGIS version 3.24.1 and shape file freely available at https://diva-gis.org/.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9124525/v1/17b6f4bd960f4729a4c58cdd.png"},{"id":106960734,"identity":"1b04fbd8-bf69-452d-a307-3dcfb6cf32b2","added_by":"auto","created_at":"2026-04-15 09:22:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1415016,"visible":true,"origin":"","legend":"\u003cp\u003eField sampling of soft ticks from warthog burrows in Serengeti National Park. (A) Warthog burrow, (B) collection of soil sample from the burrow, (C) manual retrieval of soft ticks from the collected soil, and (D) representative soft tick collected from the site.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9124525/v1/8a8f7322bcba3e4386b3fd30.png"},{"id":106747127,"identity":"87bff53a-fa6e-4cc5-9242-cd414038870a","added_by":"auto","created_at":"2026-04-13 06:03:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":309102,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree showing the relationship between sequences from this study compared to publicly available ASFV sequences. The ASFV strains described in this study are indicated by a black square.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9124525/v1/d94ba76998d33a3e354e658f.png"},{"id":106747125,"identity":"93c5c16f-f876-4cf6-9613-8957f961b4d3","added_by":"auto","created_at":"2026-04-13 06:03:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":93920,"visible":true,"origin":"","legend":"\u003cp\u003eNucleotide alignment of the intergenic region (IGR) between the \u003cem\u003eI73R \u003c/em\u003eand \u003cem\u003eI329L\u003c/em\u003egenes of the sequence described in this study along with those retrieved from the NCBI GenBank. The sequence described in this study lacked a 36 bp fragment present within an African swine fever virus isolate collected from ticks in Kenya in 2005.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9124525/v1/f35084b522ce813f8f84e09d.png"},{"id":106964461,"identity":"26b245b5-c090-44c5-91b9-bc0b3755932f","added_by":"auto","created_at":"2026-04-15 09:50:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2941279,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9124525/v1/a96b545c-6789-42be-a359-027c7eafa14c.pdf"}],"financialInterests":"","formattedTitle":"Detection and molecular characterization of African swine fever virus recovered from Ornithodoros ticks of the Serengeti Ecosystem, Tanzania","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAfrican swine fever (ASF) is a contagious and lethal viral disease affecting domestic pigs and Eurasian wild boars, with significant socio-economic implications worldwide (Li et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The ASF is a notifiable disease to the World Organization for Animal Health (WOAH) due to its severe impact on global pork production and its ability to spread rapidly across regions, posing a substantial challenge to animal health and trade (Plavsik et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The ASF is a notifiable disease in Tanzania and among the priority diseases listed by the Ministry of Livestock and Fisheries (Cap \u003cem\u003e1\u003c/em\u003e56 \u003cem\u003eAnimal Diseases Act\u003c/em\u003e [\u003cem\u003eR.E 2023\u003c/em\u003e]). African swine fever virus (ASFV; species \u003cem\u003eAsfivirus haemorrhagiae\u003c/em\u003e), a large, complex, double-stranded DNA virus of the genus \u003cem\u003eAsfivirus\u003c/em\u003e, which belongs to the family \u003cem\u003eAsfarvidae\u003c/em\u003e, is the aetiological agent for this disease (Alonso et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The virus is highly stable and can spread efficiently through infected domestic pigs, contaminated domestic pig products, or through the blood-feeding activity of infected \u003cem\u003eOrnithodoros\u003c/em\u003e ticks (McKercher et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1987\u003c/span\u003e; Plowright and Parker, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1967\u003c/span\u003e; Zheng et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). African swine fever virus persists in the wild environment through a complex sylvatic cycle involving wild suids such as warthogs (\u003cem\u003ePhacochoerus africanus\u003c/em\u003e) and soft ticks of the genus \u003cem\u003eOrnithodoros\u003c/em\u003e, which act as reservoirs and vectors, respectively (Jori et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The virus can persist and replicate in \u003cem\u003eOrnithodoros\u003c/em\u003e ticks, enabling transstadial, transovarial, and sexual transmission among tick populations (Kleiboeker and Scoles, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). It is not very clear if all genotypes that are involved during outbreaks in domesticated pigs are using ticks for persisting in certain areas and if they still depend on ticks for their persistence in pig populations. For instance, genotype II is nowadays circulating in domestic pigs and wild boars worldwide without using ticks as reservoir. It looks like ASFV developed mechanisms to be transmitted without the involvement of ticks. The ease with which genotype II is using the respiratory tract to infect domestic pigs makes it possible for the virus to change its epidemiological behavior (Oh et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) .\u003c/p\u003e \u003cp\u003eThe ASFV sylvatic cycle serves as a source for emerging new, more diverse, and virulent virus strains within the domestic cycle (Lubisi et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Nix et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). This is evidenced by the significant genetic diversity of ASFV genotypes found in Eastern and Southern Africa (Lubisi et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Nix et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), where the ASFV sylvatic cycle exists. The ASFV is transmitted to domestic pigs from the sylvatic cycle via infected \u003cem\u003eOrnithodoros\u003c/em\u003e ticks that feed on them (Plowright et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1969\u003c/span\u003e). Infected ticks from the wild can come into contact with domestic pigs when hunted warthogs are transported to households or when warthogs containing the ticks graze on land directly neighboring areas where domestic pigs are kept (Kleiboeker and Scoles, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Warthogs are widely distributed in Eastern and Southern Africa, mostly in tropical and subtropical savannas (Katale et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Studies have shown that a high percentage of warthogs in Kenya and Tanzania and about 50% of warthogs in Uganda had antibodies against ASFV (Katale et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Peter et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Plowright et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1969\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe Serengeti ecosystem is a critical area for ASFV studies due to its complex wildlife-livestock and human interface. While ASFV has been characterized in \u003cem\u003eOrnithodoros\u003c/em\u003e ticks from isolated environments like Saadani National Park (Peter et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), the Serengeti presents a significant spillover risk. In this ecosystem, warthog burrows are often infested with \u003cem\u003eOrnithodoros\u003c/em\u003e ticks, creating a natural environment for the virus to persist. A study conducted in 1968 in a small part of the Serengeti ecosystem, which is the Kirawira area of the Serengeti National Park, Tanzania, revealed a 15% ASFV infection rates of \u003cem\u003eOrnithodoros\u003c/em\u003e ticks collected from warthog burrows (Plowright et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1969\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAfrican swine fever outbreaks have been documented in several Tanzanian regions, including Kilimanjaro, Arusha, Mbeya, Iringa, Morogoro, Pwani, and Dar es Salaam (Misinzo et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Wambura et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Yona et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, surveillance efforts to identify circulating genotypes have primarily focused on outbreaks in domestic pig farms within a limited number of locations. Despite the critical role of \u003cem\u003eOrnithodoros\u003c/em\u003e ticks in ASFV transmission, studies on their prevalence and viral detection in interface areas like the Serengeti ecosystem are limited. This study aimed to detect and genetically characterize the ASFV in \u003cem\u003e`\u003c/em\u003eticks collected from warthog burrows within the Serengeti ecosystem and to evaluate these ticks\u0026rsquo; potential role in the ASF epidemiology in Tanzania.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy area\u003c/h2\u003e \u003cp\u003eThe study was conducted in Serengeti National Park and Maswa Game Reserve (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), the core of the Tanzanian Greater Serengeti Ecosystem (GSE), which forms the southern portion of the larger transboundary Serengeti-Mara ecosystem (Kegamba et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). This portion of the ecosystem is one of the seven important ecosystems for protecting wildlife in Tanzania. The Greater Serengeti Ecosystem in Tanzania is made up of nine administrative areas, including Serengeti National Park (SENAPA), Ngorongoro Conservation Area (NCA), Maswa, Ikorongo, Kijereshi, Grumeti Game Reserves, Loliondo Game Controlled Area, Ikona, and Makao Wildlife Management Area. The GSE in Tanzania spans an area of 25,000 km\u003csup\u003e2\u003c/sup\u003e and is characterized by the annual migration of ungulate herds, which interact with a significant livestock population. The ecosystem is located between 1\u0026deg; and 2\u0026deg; South latitude, 34\u0026deg; and 36\u0026deg; East longitude, featuring a variety of habitats that include expansive open grasslands, acacia-dominated savannas, and rocky outcrops (Katale et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e The sampling was conducted across the Serengeti National Park and Maswa Game Reserve, focusing on specific localities known for high warthog density. In Serengeti National Park, the soft ticks were collected at Seronera, which is the central plain with high wildlife concentration and open grasslands ideal for warthog burrowing. Further, sampling also was conducted at Nyasiroli, the northwestern part of the park near the Grumeti Game Reserve, which is characterized by wooded savanna. In Maswa Game Reserve the sampling was done on northeastern part which is characterized by a mixed woodlands and the central-western part of the reserve, which is covered by denser bushland.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCollection of soft ticks from warthog’s burrows\u003c/h3\u003e\n\u003cp\u003eA cross-sectional study was conducted in October 2024, where the target population was the active warthog\u0026rsquo;s burrows for soft tick collection. A total of 24 active warthog burrows were identified within the Serengeti National Park and 6 from Maswa Game Reserve following confirmation of the signs, such as recent warthog footsteps around the burrows before sampling, and then the burrows were thoroughly examined for the presence of \u003cem\u003eOrnithodoros\u003c/em\u003e ticks. With the use of a long-handled spade, the soil or sand was scooped from the roofs, walls, and floors of the burrows, then spread on a plastic sheet and exposed to sunlight for three minutes to stimulate movement and identification of ticks (Jori et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The collected \u003cem\u003eOrnithodoros\u003c/em\u003e ticks were put into Falcon tubes with perforated tops to ensure containment and ventilation then they were transported to the laboratory. Once in the laboratory, ticks were preserved in 70% ethanol and stored in a -20\u0026deg;C freezer until analysis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eMorphological identification of ticks\u003c/h3\u003e\n\u003cp\u003eUnder a stereomicroscope (Olympus Sz51, Olympus Corporation, Tokyo, Japan), ticks were examined for morphological characteristics to confirm the genus \u003cem\u003eOrnithodoros.\u003c/em\u003e Identification was based on the distinctive traits common to the \u003cem\u003eO. moubata\u003c/em\u003e complex group, which included a tough and wrinkled upper surface dotted with nodules, the absence of eyes, the lack of a hard scutum, and the mouth not being visible from the top (Walker and Bouattour, 2003).\u003c/p\u003e\n\u003ch3\u003eViral DNA extraction\u003c/h3\u003e\n\u003cp\u003eFollowing the removal of ethanol in individual Falcon tubes, ticks were pooled according to the sampling site and cleaned with distilled water as previously described (Silaghi et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The ticks were then ground in a sterile mortar and pestle using phosphate-buffered saline as a suspension medium. A total of 200 \u0026micro;L of homogenized material was used for DNA extraction using a DNeasy blood and tissue kit (QIAGEN, Hilden, Germany), according to the manufacturer's protocol. The steps involved include cell lysis with proteinase K, binding of DNA to a silica membrane, followed by a washing step to remove contaminants, and elution of purified DNA in 100 \u0026micro;L of a low-salt buffer.\u003c/p\u003e\n\u003ch3\u003eViral detection and identification\u003c/h3\u003e\n\u003cp\u003eReal-time polymerase chain reaction (qPCR) for screening ASFV was performed as described by King et al. (2003), targeting the viral VP72 gene. The PCR reactions were carried out in a total volume of 50 \u0026micro;L containing 5 \u0026micro;L of extracted DNA, 50 pmol of forward (5'-CTG CTC ATG GTA TCA ATC TTA TCG A-3') and reverse (5'-GAT ACC ACA AGA TCR GCC GT-3') primers, and 5 pmol of a FAM-labeled probe (5'-FAM-CCA CGG GAG GAA TAC CAA CCC AGT G-TAMRA-3'). The reaction mix also contained 50 mM KCl, 10 mM Tris\u0026ndash;HCl (pH 9.0), 2 mM MgCl₂, 0.4 mM of each dNTP, and 2.5 U of Taq DNA polymerase (Promega). Amplification was performed on a QuantStudio\u0026trade; 5 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA) with the following cycling conditions: an initial denaturation at 94\u0026deg;C for 120 s, followed by 40 cycles of 94\u0026deg;C for 15 s and 58\u0026deg;C for 60 s. Samples were run along with positive and negative controls. As per the assay\u0026rsquo;s analytical sensitivity, all samples with mean cycle quantification (Cq) values\u0026thinsp;\u0026le;\u0026thinsp;40 were considered positive, whereas samples with Cq\u0026thinsp;\u0026gt;\u0026thinsp;40 or with no amplification were classified as being negative.\u003c/p\u003e \u003cp\u003eThe ASFV-positive samples were subjected to further molecular characterization by amplification and sequencing of the \u003cem\u003eB646L\u003c/em\u003e (p72) gene, the intergenic region (IGR) between the \u003cem\u003eI73R\u003c/em\u003e and \u003cem\u003eI329L\u003c/em\u003e genes, and the central variable region (CVR) of the \u003cem\u003eB602L\u003c/em\u003e gene. Amplification of the \u003cem\u003eB646L\u003c/em\u003e (p72) gene was performed using primers p72-U (5\u0026rsquo;-GGCACAAGTTCGGACATGT-3\u0026rsquo;) and p72-D (5\u0026rsquo;-GTACTGTAACGCAGCACAG-3\u0026rsquo;) as described by Bastos et al.(2003). Thermal profile started with initial denaturation at 96\u0026deg;C for 20s, then 35 cycles of 96\u0026deg;C for 12s denaturation, 50\u0026deg;C for 20s for annealing, and 70\u0026deg;C for 25s for extension. The IGR was amplified using primers ECO1A (5\u0026rsquo;-CCATTTATCCCCCGCTTTGG-3\u0026rsquo;) and ECO1B (5\u0026rsquo;-TCGTCATCCTGAGACAGCAG-3\u0026rsquo;) (Gallardo et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), with cycling conditions consisting of initial denaturation step at 96\u0026deg;C for 20s followed by 35 cycles each including denaturation at 96\u0026deg;C for 12s, primer annealing at 60\u0026deg;C for 20s, and DNA extension at 70\u0026deg;C for 25s. The CVR of \u003cem\u003eB602L\u003c/em\u003e gene was amplified using primers ORF9L-F (5\u0026rsquo;-AATGCGCTCAGGATCTGTTAAATCGG-3\u0026rsquo;) and ORF9L-R (5\u0026rsquo;-TCTTCATGCTCAAAGTGCGTATACCT-3\u0026rsquo;), with the thermal profile as described by (Nix et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Amplicons were excised from the agarose electrophoresis gels, purified, and subjected to Sanger sequencing (Craig et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003ePhylogenetic reconstruction of ASFV targeting the\u003c/b\u003e \u003cb\u003eB646L\u003c/b\u003e \u003cb\u003egene\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn order to classify viruses characterized in this study among the 24 known ASFV p72 genotypes, the C-terminal end of the \u003cem\u003eB646L\u003c/em\u003e gene was amplified and sequenced. The Sequence Scanner software version 2 was used for quality control of the raw nucleotide chromatograms and consensus sequences were generated using BioEdit version 7.2.5. Multiple sequence alignment was performed using the ClustalW algorithm and phylogenetic tree was reconstructed using MEGA 12 (Kumar et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), with the Kimura two-parameter model, which was selected as the optimal model based on the Bayesian Information Criterion (BIC) scores. Branch support was assessed using 1,000 bootstrap replicates.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of intergenic (IGR) and central variable (CVR) regions\u003c/h2\u003e \u003cp\u003eTo further characterize genetic diversity, the amplified and sequenced regions of IGR were aligned with sequences from GenBank using ClustalW in MEGA 12. To get the amino acid signature, the DNA sequences of amplified CVR of the \u003cem\u003eB602L\u003c/em\u003e gene were translated using the ExPASy translation tool \u003cem\u003e(\u003c/em\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://web.expasy.org/translate/\u003c/span\u003e\u003cspan address=\"https://web.expasy.org/translate/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, \u003cem\u003eaccessed on\u003c/em\u003e 15 April 2025\u003cem\u003e)\u003c/em\u003e and coded as previously described (Lubisi et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Misinzo et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eCollection of soft ticks and ASFV detection\u003c/h2\u003e \u003cp\u003eA total of 30 warthog burrows were surveyed within the Serengeti National Park and Maswa Game Reserve, of which 25 burrows (83.3%) were infested with soft ticks. From the 25 infested burrows, 1,003 soft ticks were collected, with an average of approximately 40 ticks per infested burrow. The number of ticks collected per burrow varied considerably, with some burrows yielding as few as 2 ticks and others yielding up to 182 ticks \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. All the collected ticks were morphologically identified as \u003cem\u003eOrnithodoros moubata\u003c/em\u003e complex. The morphological features recorded were leathery and wrinkled dorsal surface, eyeless, and non-visible mouthparts from a dorsal view. The DNA of ASFV was detected in soft ticks collected from nine out of 25 barrows (36%) by qPCR analysis. The Serengeti National Park had the highest positive samples of ASFV in ticks (8 out of 9 barrows) as shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDescriptions of location, number of ticks collected per burrow and their African swine fever virus (ASFV) status after real-time polymerase chain reaction (qPCR).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWarthog burrow ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLocation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCoordinate (X)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCoordinate (Y)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNumber of ticks collected\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eASFV status\u003c/p\u003e \u003cp\u003e(qPCR)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWB1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSeronera\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-2.431013\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e34.854944\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e182\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWB2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSeronera\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-2.429474\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e34.851778\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e157\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWB3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSeronera\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-2.433911\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e34.868264\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWB4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSeronera\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-2.434838\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e34.867748\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWB5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSeronera\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-2.433776\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e34.865254\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePositive\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWB6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSeronera\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-2.428944\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e34.850589\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWB7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSeronera\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-2.447241\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e34.851856\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePositive\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWB8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSeronera\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-2.448738\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e34.85047\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePositive\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWB9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSeronera\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-2.449267\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e34.850914\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePositive\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWB10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSeronera\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-2.452309\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e34.851775\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWB11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSeronera\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-2.453208\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e34.851641\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWB12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSeronera\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-2.437608\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e34.813467\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWB13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSeronera\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-2.435864\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e34.814694\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWB14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSeronera\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-2.430786\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e34.813008\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePositive\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWB15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSeronera\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-2.433295\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e34.814185\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePositive\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWB16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSeronera\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-2.430668\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e34.811735\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWB17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSeronera\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-2.428590\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e34.783136\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWB18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSeronera\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-2.429077\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e34.784674\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWB19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSeronera\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-2.427280\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e35.773917\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWB20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSeronera\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-2.425280\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e34.775184\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePositive\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWB21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSeronera\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-2.444415\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e34.85266\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWB22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSeronera\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-2.442913\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e34.856015\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePositive\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWB23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSeronera\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-2.443750\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e34.855731\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWB24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNyasiroli\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-2.158943\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e34.398946\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWB25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaswa Game Reserve\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-3.159760\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e34.513987\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWB26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaswa Game Reserve\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-3.166753\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e34.514104\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePositive\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWB27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaswa Game Reserve\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-134605\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e34.616974\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWB28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaswa Game Reserve\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-3.075344\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e34.656918\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWB29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaswa Game Reserve\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-3.141809\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e34.646322\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWB30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaswa Game Reserve\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-3.147732\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e34.488759\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e*(-) \u003cem\u003eRepresent burrows with no ticks hence no ASFV status\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eSequencing results\u003c/h2\u003e \u003cp\u003eAmplicons generated from PCR-positive samples were subjected to Sanger sequencing, and the phylogenetic tree revealed that the isolates ASFV/TAN/2024/Serengeti/SR1 and ASFV/TAN/2024/Serengeti/SR3 clustered within genotype X, which includes other isolates from Tanzania, such as those from Mwanza, Kigoma, Ngara, Geita, Kahama, Katavi, Longido, Arusha, Moshi, Babati, and Manyoni \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e At BLASTn, the sequences described in this study showed 99.58 to 99.79% nucleotide identity with ASFV genotype X isolates previously reported in domestic pigs in Burundi, Democratic Republic of the Congo, Kenya, Tanzania and Uganda.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of IGR and CVR genomics regions\u003c/h2\u003e \u003cp\u003eThe tetrameric amino acid repeats within the CVR of the \u003cem\u003eB602L\u003c/em\u003e gene had the signature AAABNAAAAAAAAAABA using the code previously described by Lubisi et al. (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) and Misinzo et al. (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), with high nucleotide identity compared to the ASFV genotype X previously described in Tanzania and neighboring countries. The nucleotide sequence showed 100% identity with isolate Hinde/I (GenBank accession number OK560519) collected from warthog in Kenya in 1954. After BLASTn of the nucleotide sequences of the IGR between the \u003cem\u003eI73R\u003c/em\u003e and \u003cem\u003eI329L\u003c/em\u003e genes, the most similar tandem repeat sequence (TRS) was that of the isolate TAN/19/Ngara (GenBank accession number MW659865) with 100% nucleotide identity. In addition, the sequences described in this study had 99.77% nucleotide identity to Kenya_1950 (NC_044944.1) reference isolate collected from domestic pig in Kenya in 1950. The sequence described in this study lacked a fragment of 36 bp within the TRS compared to the isolate Ken05/Tk1 (NC_044945.1) collected from ticks in Kenya in 2005. A similar fragment was missing from the ASFV reference genome Kenya_1950 (NC_044944.1) and isolates from domestic pigs in Tanzania \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe results of this study provide important insights into the presence of the sylvatic cycle in the Serengeti ecosystem, specifically in warthog burrows. A total of 1,003 soft ticks were collected from 30 warthog burrows, of which five were found to be uninfested. The presence of soft ticks in 25 burrows suggests a widespread distribution of these ASFV vectors in the study area. Detection of ASFV in ticks from nine of the 25 burrows highlights a significant level of viral circulation within the ecosystem.\u003c/p\u003e\n\u003cp\u003eThe detection of ASFV in soft ticks associated with warthogs aligns with previous studies that have established warthogs and their associated ticks as key reservoirs in the sylvatic cycle of ASFV (Dixon and Wilkinson, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1988\u003c/span\u003e; Jori et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Njau, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Quembo et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The role of \u003cem\u003eOrnithodoros\u003c/em\u003e ticks in the maintenance and transmission of ASFV has been widely recognized, as these ticks can acquire the virus from infected warthogs and persistently harbor it for extended periods (Kleiboeker and Scoles, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). The ability of \u003cem\u003eOrnithodoros\u003c/em\u003e ticks to facilitate transstadial and transovarial transmission further enhances their capacity to maintain ASFV in the absence of actively infected hosts (Parker et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1969\u003c/span\u003e; Plowright et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1970\u003c/span\u003ea; Plowright et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1970\u003c/span\u003eb).\u003c/p\u003e\n\u003cp\u003eThe presence of positive ticks from 9 out of 25 infested burrows indicates that ASFV is actively circulating in a significant proportion of the warthog population in the study area. This finding is of concern, given the potential for spillover of the virus to domestic pigs, particularly in areas where warthogs and domestic pigs share common grazing grounds or where ticks are inadvertently transported to farms through human activities such as hunting and bush meat trade, especially on the edges of the ecosystems (Peter et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eThe phylogenetic analysis of ASFV sequences obtained from the infected tick samples revealed that the ASFV strains detected clustered within genotype X. This finding aligns with a similar study conducted in Kenya, where ASFV strains isolated from soft ticks and domestic pigs were also classified under genotype X (Gallardo et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), indicating a potential regional circulation of this genotype in East Africa. However, other studies in Kenya and Tanzania have identified ASFV strains from soft ticks belonging to other genotypes, like genotype IX and XV, respectively, highlighting the genetic diversity of ASFV in the region (Obanda et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Peter et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eThe genotype X of ASFV has been previously identified in several regions of Tanzania and other parts of East Africa, suggesting that the virus circulating in the sylvatic cycle is genetically related to strains previously reported in the region (Hakizimana et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Misinzo et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The close relationship of these sequences with other genotype X isolates, including domestic pig outbreaks in Mwanza, Kigoma, Manyoni, Ngara, Babati, and other places in western and northern Tanzania, highlights the potential risk of spillover from wildlife to domestic animals. The analysis of the CVR of the \u003cem\u003eB602L\u003c/em\u003e gene showed a tetrameric amino acid signature consistent with previously reported genotype X isolates from Tanzania and neighboring countries (Lubisi et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Misinzo et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The high nucleotide identity with isolates from Burundi, Democratic Republic of the Congo, Kenya, Tanzania and Uganda suggests that there is high relatedness between the detected virus from this study and the circulating viruses in East and Central Africa.\u003c/p\u003e\n\u003cp\u003eThe presence of ASFV genotype X in warthog-associated \u003cem\u003eOrnithodoros\u003c/em\u003e ticks has several implications. First, it suggests that the sylvatic cycle remains an active reservoir of ASFV in the Serengeti ecosystem. The genetic clustering of the identified sequences from the ticks with already published genotype X strains implies that the virus has likely been maintained in this ecosystem for an extended period, potentially as a source of infection for domestic pigs when conditions allow transmission. In addition, the genetic similarity between these tick-derived isolates and previously identified strains in domestic pigs raises concerns about the role of wildlife in sustaining outbreaks in Tanzania. This observation aligns with previous studies that have demonstrated the genetic overlap between ASFV strains in the sylvatic and domestic cycles, underscoring the importance of surveillance at the wildlife-livestock interface (Gallardo et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Obanda et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eMoreover, the phylogenetic tree reveals a clear separation between genotype X and other ASFV genotypes circulating in Tanzania, such as genotype II, which has been responsible for major outbreaks in the domestic pig population (Misinzo et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). This distinction highlights the genetic diversity of ASFV within the country and suggests that different transmission dynamics may be at play between the sylvatic and domestic cycles. While genotype II has been associated with severe outbreaks in domestic pig farms, the persistence of genotype X in wildlife may contribute to the long-term maintenance of ASFV in the region, posing a continuous risk to domestic pig production.\u003c/p\u003e\n\u003cp\u003eOur results reinforce the importance of continuous molecular surveillance to track ASFV genotypes within sylvatic cycles. In the absence of a vaccine or treatment, such surveillance is crucial for mapping genetic diversity and transmission pathways, which can inform targeted strategies to mitigate spillover events. Nonetheless, these conclusions are tempered by the study\u0026apos;s limited sample size and narrow geographical focus. Therefore, further research is warranted to confirm and broaden these findings. It is recommended to have an atlas of the different genotypes of ASFV that are present in the soft ticks all over Africa.\u003c/p\u003e\n\u003cp\u003eIn conclusion, the detection of ASFV in soft ticks from warthog burrows and the phylogenetic clustering of these isolates within genotype X, with genetic relatedness to previously isolated strains from domestic pigs, emphasize the importance of the investigations on the sylvatic cycle in the ASFV epidemiology in Tanzania and neighboring countries. This finding highlights the urgent need for continued surveillance, improved biosecurity measures, and further research to understand ASFV transmission dynamics at the wildlife-livestock interface.\u003c/p\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eEthical consideration\u003c/h2\u003e\n \u003cp\u003eThe President\u0026apos;s Office Regional Administration and Local Government of Tanzania (PO-RALG), Sokoine University of Agriculture, Tanzania Wildlife Research Institute (TAWIRI) granted permission to carry out the study under reference numbers \u003cstrong\u003eAB.307/323/01/51, SUA/DPRTC/MAM/D/2022/0002/06\u003c/strong\u003e, and \u003cstrong\u003eAB.235/325/01/166\u003c/strong\u003e, respectively. District Veterinary Officers (DVOs) of each district were duly notified about the study\u0026rsquo;s objectives and procedures and offered support.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Statements \u0026 Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe nucleotide sequences described in this study have been submitted to the National Center for Biotechnology Information (NCBI) GenBank and assigned accession numbers \u003cstrong\u003ePX445921 to PX445924\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthor declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to express their sincere gratitude to Mr. Maulid Mdaki for his invaluable assistance in sample collection, Mrs Mariam R. Makange and to Mrs Anna Rogath Masawe for their excellent technical assistance during the laboratory analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by the Regional Scholarship and Innovation Fund (Rsif) of the Partnership for Skills in Applied Science, Engineering and Technology (PASET) through the Junior Investigator Research Award (JIRA) with grant number RSIF/JIRA/001. The funder had no role in study design, data collection and analysis, decision to publish as well as in the preparation of the manuscript. The findings and conclusions of this study are those of the authors and do not necessarily represent the view of Rsif-PASET\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAlonso, C., Borca, M., Dixon, L., Revilla, Y., Rodriguez, F., Escribano, J. M., \u0026amp; ICTV Report Consortium. (2018). ICTV Virus Taxonomy Profile: Asfarviridae. \u003cem\u003eJournal of General Virology\u003c/em\u003e, \u003cem\u003e99\u003c/em\u003e(5), 613\u0026ndash;614. https://doi.org/10.1099/jgv.0.001049\u003c/li\u003e\n \u003cli\u003eBastos, A. D. S., Penrith, M.-L., Cruci\u0026egrave;re, C., Edrich, J. L., Hutchings, G., Roger, F., Couacy-Hymann, E., \u0026amp; R.Thomson, G. (2003). Genotyping field strains of African swine fever virus by partial p72 gene characterisation. \u003cem\u003eArchives of Virology\u003c/em\u003e, \u003cem\u003e148\u003c/em\u003e(4), 693\u0026ndash;706. https://doi.org/10.1007/s00705-002-0946-8\u003c/li\u003e\n \u003cli\u003e\u003cem\u003eCap. 156 Animal Diseases Act, R.E 2023\u0026mdash;MD, Mendez.pdf\u003c/em\u003e. (n.d.). Retrieved August 30, 2025, from https://elibrary.osg.go.tz/bitstream/handle/123456789/2200/Cap.%20156%20Animal%20Diseases%20Act%2c%20R.E%202023%20-%20MD%2c%20Mendez.pdf?sequence=2\u0026amp;isAllowed=y\u003c/li\u003e\n \u003cli\u003eCraig, A. F., Schade-Weskott, M. L., Rametse, T., Heath, L., Kriel, G. J. P., De Klerk-Lorist, L.-M., Van Schalkwyk, L., Trujillo, J. D., Crafford, J. E., Richt, J. A., \u0026amp; Swanepoel, R. (2022). Detection of African Swine Fever Virus in Ornithodoros Tick Species Associated with Indigenous and Extralimital Warthog Populations in South Africa. \u003cem\u003eViruses\u003c/em\u003e, \u003cem\u003e14\u003c/em\u003e(8), 1617. https://doi.org/10.3390/v14081617\u003c/li\u003e\n \u003cli\u003eDixon, L. K., \u0026amp; Wilkinson, P. J. (1988). Genetic Diversity of African Swine Fever Virus Isolates from Soft Ticks (Ornithodoros moubata) Inhabiting Warthog Burrows in Zambia. \u003cem\u003eJournal of General Virology\u003c/em\u003e, \u003cem\u003e69\u003c/em\u003e(12), 2981\u0026ndash;2993. https://doi.org/10.1099/0022-1317-69-12-2981\u003c/li\u003e\n \u003cli\u003eGallardo, C., Fern\u0026aacute;ndez-Pinero, J., Pelayo, V., Gazaev, I., Markowska-Daniel, I., Pridotkas, G., Nieto, R., Fern\u0026aacute;ndez-Pacheco, P., Bokhan, S., Nevolko, O., Drozhzhe, Z., P\u0026eacute;rez, C., Soler, A., Kolvasov, D., \u0026amp; Arias, M. (2014). Genetic Variation among African Swine Fever Genotype II Viruses, Eastern and Central Europe. \u003cem\u003eEmerging Infectious Diseases\u003c/em\u003e, \u003cem\u003e20\u003c/em\u003e(9), 1544\u0026ndash;1547. https://doi.org/10.3201/eid2009.140554\u003c/li\u003e\n \u003cli\u003eGallardo, C., Okoth, E., Pelayo, V., Anchuelo, R., Martin, E., Simon, A., Llorente, A., Nieto, R., Soler, A., Martin, R., Arias, M., \u0026amp; Bishop, R. P. (2011). African swine fever viruses with two different genotypes, both of which occur in domestic pigs, are associated with ticks and adult warthogs, respectively, at a single geographical site. \u003cem\u003eJournal of General Virology\u003c/em\u003e, \u003cem\u003e92\u003c/em\u003e(2), 432\u0026ndash;444. https://doi.org/10.1099/vir.0.025874-0\u003c/li\u003e\n \u003cli\u003eHakizimana, J. N., Yona, C., Kamana, O., Nauwynck, H., \u0026amp; Misinzo, G. (2021). African Swine Fever Virus Circulation between Tanzania and Neighboring Countries: A Systematic Review and Meta-Analysis. \u003cem\u003eViruses\u003c/em\u003e, \u003cem\u003e13\u003c/em\u003e(2), Article 2. https://doi.org/10.3390/v13020306\u003c/li\u003e\n \u003cli\u003e\u003cem\u003e(https://web.expasy.org/translate/)\u0026mdash;Google Search\u003c/em\u003e. (n.d.). Retrieved August 15, 2025, from https://www.google.com/search?q=(https%3A%2F%2Fweb.expasy.org%2Ftranslate%2F)\u0026amp;oq=(https%3A%2F%2Fweb.expasy.org%2Ftranslate%2F)\u0026amp;gs_lcrp=EgZjaHJvbWUyBgg\u003cbr\u003eAEEUYOTIICAEQABgWGB7SAQgxMzQ4ajBqN6gCALACAA\u0026amp;sourceid=chrome\u0026amp;ie=UTF-8\u003c/li\u003e\n \u003cli\u003eJori, F., Bastos, A., Boinas, F., Van Heerden, J., Heath, L., Jourdan-Pineau, H., Martinez-Lopez, B., Pereira de Oliveira, R., Pollet, T., Quembo, C., Rea, K., Simulundu, E., Taraveau, F., \u0026amp; Penrith, M.-L. (2023). An Updated Review of Ornithodoros Ticks as Reservoirs of African Swine Fever in Sub-Saharan Africa and Madagascar. \u003cem\u003ePathogens\u003c/em\u003e, \u003cem\u003e12\u003c/em\u003e(3), 469. https://doi.org/10.3390/pathogens12030469\u003c/li\u003e\n \u003cli\u003eJori, F., Vial, L., Penrith, M. L., P\u0026eacute;rez-S\u0026aacute;nchez, R., Etter, E., Albina, E., Michaud, V., \u0026amp; Roger, F. (2013). Review of the sylvatic cycle of African swine fever in sub-Saharan Africa and the Indian ocean. \u003cem\u003eVirus Research\u003c/em\u003e, \u003cem\u003e173\u003c/em\u003e(1), 212\u0026ndash;227. https://doi.org/10.1016/j.virusres.2012.10.005\u003c/li\u003e\n \u003cli\u003eKatale, B., Fyumagwa, R., Mdaki, M., \u0026amp; Hoare, R. (2012). Prevalence of African swine fever virus in Warthogs in the Serengeti ecosystem, Tanzania. \u003cem\u003eResearch Opinions in Animal and Veterinary Sciences\u003c/em\u003e, \u003cem\u003e2\u003c/em\u003e, 339-343.\u003c/li\u003e\n \u003cli\u003eKatale, B. Z., Fyumagwa, R. D., Mdaki, M. L., \u0026amp; Hoare, R. (2012). \u003cem\u003eRESEARCH OPINIONS IN ANIMAL \u0026amp; VETERINARY SCIENCES\u003c/em\u003e.\u003c/li\u003e\n \u003cli\u003eKegamba, J. J., Sangha, K. K., Wurm, P. A. S., \u0026amp; Garnett, S. T. (2023). Conservation benefit-sharing mechanisms and their effectiveness in the Greater Serengeti Ecosystem: Local communities\u0026rsquo; perspectives. \u003cem\u003eBiodiversity and Conservation\u003c/em\u003e, \u003cem\u003e32\u003c/em\u003e(6), 1901\u0026ndash;1930. https://doi.org/10.1007/s10531-023-02583-1\u003c/li\u003e\n \u003cli\u003eKleiboeker, S. B., \u0026amp; Scoles, G. A. (2001). Pathogenesis of African swine fever virus in Ornithodoros ticks. \u003cem\u003eAnimal Health Research Reviews\u003c/em\u003e, \u003cem\u003e2\u003c/em\u003e(2), 121\u0026ndash;128. https://doi.org/10.1079/AHRR200133\u003c/li\u003e\n \u003cli\u003eKumar, S., Stecher, G., Suleski, M., Sanderford, M., Sharma, S., \u0026amp; Tamura, K. (2024). MEGA12: Molecular Evolutionary Genetic Analysis Version 12 for Adaptive and Green Computing. \u003cem\u003eMolecular Biology and Evolution\u003c/em\u003e, \u003cem\u003e41\u003c/em\u003e(12), msae263. https://doi.org/10.1093/molbev/msae263\u003c/li\u003e\n \u003cli\u003eLi, Z., Chen, W., Qiu, Z., Li, Y., Fan, J., Wu, K., Li, X., Zhao, M., Ding, H., Fan, S., \u0026amp; Chen, J. (2022). African Swine Fever Virus: A Review. \u003cem\u003eLife\u003c/em\u003e, \u003cem\u003e12\u003c/em\u003e(8), 1255. https://doi.org/10.3390/life12081255\u003c/li\u003e\n \u003cli\u003eLubisi, B. A., Bastos, A. D. S., Dwarka, R. M., \u0026amp; Vosloo, W. (2005). Molecular epidemiology of African swine fever in East Africa. \u003cem\u003eArchives of Virology\u003c/em\u003e, \u003cem\u003e150\u003c/em\u003e(12), 2439\u0026ndash;2452. https://doi.org/10.1007/s00705-005-0602-1\u003c/li\u003e\n \u003cli\u003eLubisi, B. A., Bastos, A. D. S., Dwarka, R. M., \u0026amp; Vosloo, W. (2007). Intra-genotypic resolution of African swine fever viruses from an East African domestic pig cycle: A combined p72-CVR approach. \u003cem\u003eVirus Genes\u003c/em\u003e, \u003cem\u003e35\u003c/em\u003e(3), 729\u0026ndash;735. https://doi.org/10.1007/s11262-007-0148-2\u003c/li\u003e\n \u003cli\u003eMcKercher, P. D., Yedloutschnig, R. J., Callis, J. J., Murphy, R., Panina, G. F., Civardi, A., Bugnetti, M., Foni, E., Laddomada, A., Scarano, C., \u0026amp; Scatozza, F. (1987). Survival of Viruses in \u0026ldquo;Prosciutto di Parma\u0026rdquo; (Parma Ham). \u003cem\u003eCanadian Institute of Food Science and Technology Journal\u003c/em\u003e, \u003cem\u003e20\u003c/em\u003e(4), 267\u0026ndash;272. https://doi.org/10.1016/S0315-5463(87)71198-5\u003c/li\u003e\n \u003cli\u003eMisinzo, G., Kasanga, C. J., Mpelumbe\u0026ndash;Ngeleja, C., Masambu, J., Kitambi, A., \u0026amp; Van Doorsselaere, J. (2012). African Swine Fever Virus, Tanzania, 2010\u0026ndash;2012. \u003cem\u003eEmerging Infectious Diseases\u003c/em\u003e, \u003cem\u003e18\u003c/em\u003e(12), 2081\u0026ndash;2083. https://doi.org/10.3201/eid1812.121083\u003c/li\u003e\n \u003cli\u003eMisinzo, G., Kwavi, D. E., Sikombe, C. D., Makange, M., Peter, E., Muhairwa, A. P., \u0026amp; Madege, M. J. (2014). Molecular characterization of African swine fever virus from domestic pigs in northern Tanzania during an outbreak in 2013. \u003cem\u003eTropical Animal Health and Production\u003c/em\u003e, \u003cem\u003e46\u003c/em\u003e(7), 1199\u0026ndash;1207. https://doi.org/10.1007/s11250-014-0628-z\u003c/li\u003e\n \u003cli\u003eMisinzo, G., Magambo, J., Masambu, J., Yongolo, M. G., Van Doorsselaere, J., \u0026amp; Nauwynck, H. J. (2011). Genetic Characterization of African Swine Fever Viruses from a 2008 Outbreak in Tanzania. \u003cem\u003eTransboundary and Emerging Diseases\u003c/em\u003e, \u003cem\u003e58\u003c/em\u003e(1), 86\u0026ndash;92. https://doi.org/10.1111/j.1865-1682.2010.01177.x\u003c/li\u003e\n \u003cli\u003eNix, R. J., Gallardo, C., Hutchings, G., Blanco, E., \u0026amp; Dixon, L. K. (2006). Molecular epidemiology of African swine fever virus studied by analysis of four variable genome regions. \u003cem\u003eArchives of Virology\u003c/em\u003e, \u003cem\u003e151\u003c/em\u003e(12), 2475\u0026ndash;2494. https://doi.org/10.1007/s00705-006-0794-z\u003c/li\u003e\n \u003cli\u003eNjau, E. (2022). \u003cem\u003eDetection and genetic characterization of sylvatic and outbreak African swine fever virus isolates in selected zones of Tanzania\u003c/em\u003e [Thesis, NM-AIST]. https://dspace.nm-aist.ac.tz/handle/20.500.12479/1541\u003c/li\u003e\n \u003cli\u003eObanda, V., Akinyi, M., King\u0026rsquo;ori, E., Nyakundi, R., Ochola, G., Oreng, P., Mugambi, K., Waiguchu, G. M., Chege, M., Rosenbaum, W., Ylitalo, E. B., B\u0026auml;ck, A. T., Pettersson, L., Mukunzi, O. S., Agwanda, B., Stenberg-Lewerin, S., \u0026amp; Lwande, O. W. (2024). Epidemiology and ecology of the sylvatic cycle of African Swine Fever Virus in Kenya. \u003cem\u003eVirus Research\u003c/em\u003e, \u003cem\u003e348\u003c/em\u003e, 199434. https://doi.org/10.1016/j.virusres.2024.199434\u003c/li\u003e\n \u003cli\u003eOh, D., Han, S., Tignon, M., Balmelle, N., Cay, A. B., Griffioen, F., Droesbeke, B., \u0026amp; Nauwynck, H. J. (2023). Differential infection behavior of African swine fever virus (ASFV) genotype I and II in the upper respiratory tract. \u003cem\u003eVeterinary Research\u003c/em\u003e, \u003cem\u003e54\u003c/em\u003e(1), 121. https://doi.org/10.1186/s13567-023-01249-8\u003c/li\u003e\n \u003cli\u003eParker, J., Plowright, W., \u0026amp; Pierce, M. A. (1969). The epizootiology of African swine fever in Africa. \u003cem\u003eThe Veterinary Record\u003c/em\u003e, \u003cem\u003e85\u003c/em\u003e(24), 668\u0026ndash;674.\u003c/li\u003e\n \u003cli\u003ePeter, E., Machuka, E., Githae, D., Okoth, E., Cleaveland, S., Shirima, G., Kusiluka, L., \u0026amp; Pelle, R. (2021). Detection of African swine fever virus genotype XV in a sylvatic cycle in Saadani National Park, Tanzania. \u003cem\u003eTransboundary and Emerging Diseases\u003c/em\u003e, \u003cem\u003e68\u003c/em\u003e(2), 813\u0026ndash;823. https://doi.org/10.1111/tbed.13747\u003c/li\u003e\n \u003cli\u003ePlavsik, B., Rozstalnyy, A., Park, J. Y., Guberti, V., Depner, K., \u0026amp; Torres, G. (2019). \u003cem\u003eStrategic challenges to global control of African swine fever\u003c/em\u003e. O.I.E (World Organisation for Animal Health). https://doi.org/10.20506/TT.2985\u003c/li\u003e\n \u003cli\u003ePlowright, W., \u0026amp; Parker, J. (1967). The stability of African swine fever virus with particular reference to heat and pH inactivation. \u003cem\u003eArchiv F\u0026uuml;r Die Gesamte Virusforschung\u003c/em\u003e, \u003cem\u003e21\u003c/em\u003e(3), 383\u0026ndash;402. https://doi.org/10.1007/BF01241738\u003c/li\u003e\n \u003cli\u003ePlowright, W., Parker, J., \u0026amp; Peirce, M. A. (1969). African Swine Fever Virus in Ticks (Ornithodoros moubata, Murray) collected from Animal Burrows in Tanzania. \u003cem\u003eNature\u003c/em\u003e, \u003cem\u003e221\u003c/em\u003e(5185), 1071\u0026ndash;1073. https://doi.org/10.1038/2211071a0\u003c/li\u003e\n \u003cli\u003ePlowright, W., Perry, C. T., \u0026amp; Peirce, M. A. (1970). Transovarial Infection with African Swine Fever Virus in the Argasid Tick, \u003cem\u003eOrnithodoros moubata porcinus\u003c/em\u003e, Walton. \u003cem\u003eResearch in Veterinary Science\u003c/em\u003e, \u003cem\u003e11\u003c/em\u003e(6), 582\u0026ndash;584. https://doi.org/10.1016/S0034-5288(18)34259-0\u003c/li\u003e\n \u003cli\u003ePlowright, W., Perry, C. T., Peirce, M. A., \u0026amp; Parker, J. (1970). Experimental infection of the argasid tick, ornithodoros moubata porcinus, with African swine fever virus. \u003cem\u003eArchiv F\u0026uuml;r Die Gesamte Virusforschung\u003c/em\u003e, \u003cem\u003e31\u003c/em\u003e(1), 33\u0026ndash;50. https://doi.org/10.1007/BF01241664\u003c/li\u003e\n \u003cli\u003eQuembo, C. J., Jori, F., Vosloo, W., \u0026amp; Heath, L. (2018). Genetic characterization of African swine fever virus isolates from soft ticks at the wildlife/domestic interface in Mozambique and identification of a novel genotype. \u003cem\u003eTransboundary and Emerging Diseases\u003c/em\u003e, \u003cem\u003e65\u003c/em\u003e(2), 420\u0026ndash;431. https://doi.org/10.1111/tbed.12700\u003c/li\u003e\n \u003cli\u003eSilaghi, C., Hamel, D., Thiel, C., Pfister, K., \u0026amp; Pfeffer, M. (2011). Spotted Fever Group Rickettsiae in Ticks, Germany. \u003cem\u003eEmerging Infectious Diseases\u003c/em\u003e, \u003cem\u003e17\u003c/em\u003e(5), 890\u0026ndash;892. https://doi.org/10.3201/eid1705.101445\u003c/li\u003e\n \u003cli\u003eWalker, A. R., \u0026amp; Bouattour, A. (n.d.). \u003cem\u003eTicks of Domestic Animals in Africa: A Guide to Identification of Species\u003c/em\u003e.\u003c/li\u003e\n \u003cli\u003eWambura, P. N., Masambu, J., \u0026amp; Msami, H. (2006). Molecular Diagnosis and Epidemiology of African Swine Fever Outbreaks in Tanzania. \u003cem\u003eVeterinary Research Communications\u003c/em\u003e, \u003cem\u003e30\u003c/em\u003e(6), 667\u0026ndash;672. https://doi.org/10.1007/s11259-006-3280-x\u003c/li\u003e\n \u003cli\u003eYona, C. M., Vanhee, M., Simulundu, E., Makange, M., Nauwynck, H. J., \u0026amp; Misinzo, G. (2020). Persistent domestic circulation of African swine fever virus in Tanzania, 2015\u0026ndash;2017. \u003cem\u003eBMC Veterinary Research\u003c/em\u003e, \u003cem\u003e16\u003c/em\u003e(1), 369. https://doi.org/10.1186/s12917-020-02588-w\u003c/li\u003e\n \u003cli\u003eZheng, W., Xi, J., Zi, Y., Wang, J., Chi, Y., Chen, M., Zou, Q., Tang, C., \u0026amp; Zhou, X. (2023). Stability of African swine fever virus genome under different environmental conditions. \u003cem\u003eVeterinary World\u003c/em\u003e, \u003cem\u003e16\u003c/em\u003e(11), 2374\u0026ndash;2381. https://doi.org/10.14202/vetworld.2023.2374-2381\u003c/li\u003e\n \u003cli\u003eZsak, L., Borca, M. V., Risatti, G. R., Zsak, A., French, R. A., Lu, Z., Kutish, G. F., Neilan, J. G., Callahan, J. D., Nelson, W. M., \u0026amp; Rock, D. L. (2005). Preclinical Diagnosis of African Swine Fever in Contact-Exposed Swine by a Real-Time PCR Assay. \u003cem\u003eJournal of Clinical Microbiology\u003c/em\u003e, \u003cem\u003e43\u003c/em\u003e(1), 112\u0026ndash;119. https://doi.org/10.1128/jcm.43.1.112-119.2005\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"tropical-animal-health-and-production","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"trop","sideBox":"Learn more about [Tropical Animal Health and Production](https://www.springer.com/journal/11250)","snPcode":"11250","submissionUrl":"https://submission.nature.com/new-submission/11250/3","title":"Tropical Animal Health and Production","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"African swine fever virus, Ornithodoros, ticks, warthog burrows, Serengeti ecosystem, phylogenetic analysis","lastPublishedDoi":"10.21203/rs.3.rs-9124525/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9124525/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAfrican swine fever (ASF) is a highly lethal hemorrhagic disease of domestic pigs and Eurasian wild boars, caused by ASF virus (ASFV). In Africa, ASFV is maintained in a sylvatic cycle involving wild suids, primarily warthogs (\u003cem\u003ePhacocherus africanus\u003c/em\u003e) and \u003cem\u003eOrnithodoros \u003c/em\u003eticks. Despite ASF outbreaks in Tanzania, the role of the sylvatic cycle in areas with high wildlife-livestock interactions remains understudied. This study aimed to detect and characterize ASFV in \u003cem\u003eOrnithodoros\u003c/em\u003eticks from warthog burrows in the Serengeti ecosystem. Soft ticks were collected from warthog burrows and screened for ASFV using quantitative polymerase chain reaction (qPCR). A total of 1,003 \u003cem\u003eOrnithodoros\u003c/em\u003e ticks were collected from 25 burrows and qPCR detected ASFV in ticks from 9 burrows. Conventional PCR targeting the ASFV \u003cem\u003eB646L\u003c/em\u003e(p72) gene, intergenic region (IGR) between \u003cem\u003eI73R\u003c/em\u003e and \u003cem\u003eI329L\u003c/em\u003e genes, and the central variable region (CVR) of the \u003cem\u003eB602L\u003c/em\u003e gene was conducted in positive samples. The amplicons were sequenced and used for phylogenetic analysis. Phylogenetic analysis of the nucleotide sequences of \u003cem\u003eB646L\u003c/em\u003e (p72) gene showed that the identified ASFV strains clustered within genotype X, which has been previously associated with outbreaks in domestic pigs in Tanzania and neighboring countries. Analysis of the IGR and the CVR of the \u003cem\u003eB602L\u003c/em\u003e gene confirmed the high genetic similarity between the detected strains and those linked to previous ASF outbreaks in Tanzania and neighboring countries. The findings of this study highlight the need to prevent virus spillover from the sylvatic cycle for efficient control of the ASF in Tanzania.\u003c/p\u003e","manuscriptTitle":"Detection and molecular characterization of African swine fever virus recovered from Ornithodoros ticks of the Serengeti Ecosystem, Tanzania","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-13 06:03:48","doi":"10.21203/rs.3.rs-9124525/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2026-04-06T05:00:24+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-04T19:20:25+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-24T09:03:36+00:00","index":"","fulltext":""},{"type":"submitted","content":"Tropical Animal Health and Production","date":"2026-03-21T03:51:54+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"tropical-animal-health-and-production","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"trop","sideBox":"Learn more about [Tropical Animal Health and Production](https://www.springer.com/journal/11250)","snPcode":"11250","submissionUrl":"https://submission.nature.com/new-submission/11250/3","title":"Tropical Animal Health and Production","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b1ac6587-d72a-4bd7-b9e4-d3dde5ec2432","owner":[],"postedDate":"April 13th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-13T06:03:48+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-13 06:03:48","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9124525","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9124525","identity":"rs-9124525","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}