Discussion
This is the first comprehensive study on the possible role of LPMs in spread of ASFV in Nigeria. Field reports revealed that ASF outbreaks occur throughout the year with many unanswered questions on how the ASFV persists and continue to spread in the pig populations in the country. Findings from this study suggest that LPMs in Nigeria may play a key role in the unabated ASF outbreaks in the country. During the duration of this study which was conducted over a period of 13 months involving 34 visits. ASFV was detected by PCR in samples collected during at least one visit in the 4 selected LPMs. In addition, at one sample collected during 28 out of 34 visits was positive and ASFV was also detected in 12 out 13 months that samples were collected within the study period (Table 3). Although, visits to the markets were disrupted by COVID-19 lockdown restrictions in Nigeria between March 2020 and September 2020, however, samples were collected for the first 11 calendar months. The total number of samples collected was 613, with ASFV detected by PCR in 10.72% (66) of samples collected. In addition, ASFV was detected in 3.13% -23.81% of pig samples collected in all selected live pigs markets. Finding from this study indicates that ASF-infected pigs are sold at all the selected pig markets in Nigeria. This is worrisome because pigs from these markets are supplied to several cities and towns in Nigeria (Fig 2) both for slaughtering and restocking. Previously, ASFV has been detected in Katsit pig market, however, this is the first report of detection of ASFV in Dawaki, Numan, and Pandam LPMs (Luka et al., 2016; Owolodun et al., 2010; Owolodun, Yakubu, et al., 2010b). Phylogenetic analysis of characterized sequences B646L andE183L genes selected positive samples collected from the 4 LPMs revealed ASFV genotype I and Ia respectively (Fig 4 & 5). While, analysis of the sequence CD2v gene revealed Nigerian ASFV samples clustered in serogroup 4 (Fig 6). However, analysis of CVR sequences showed 8 TRS variants were recovered from the markets within the study period (Table 4). Six variants (Tet-10, Tet-13, Tet-18, Tet-26, Tet-33, Tet-41) are being reported for the first time in this study. These findings suggest that live pig markets are the most likely locations to recover the current circulating ASFV TRS variants in Nigeria. In addition, adequate attention and regulation of activities at live pig markets are important in the control of the disease in the country. Previously, 5 TRS variants (Tet-15, Tet-17a, Tet-20a, 20b, Tet-48) were recovered at Katsit market, the same variants were detected in outbreak cases in other parts of the country. Particularly, Tet-20b which was widely circulating in Nigeria between 2009 and 2015 (Luka et al., 2016; Owolodun, et al., 2010). In addition to TRS variants recovered in this study, 21 variants have now been are identified in Nigeria, 13 variants of which have been recovered from LPMs in the country. The live animal market trade system plays a crucial role in the introduction of pathogens into new areas particularly long-distance disease spread (Martin et al., 2011; Zhou et al., 2015). Likewise, previous studies have described the important role of the live bird market in the epidemiology of avian influenza in Nigeria, but no report on the role of LPMs in the epidemiology of ASF in the country (Coker et al., 2014; Sulaiman et al., 2021). This study has demonstrated that ASFV is circulating in LPMs in Nigeria. These markets are possibly hotspots for the long-distance spread of the virus via the movement of ASF-infected pigs to several locations in the country from LPMs. Though most of the pigs traded in these markets are for slaughter, however, farmers also buy pigs at these markets for restocking on their farms. In addition, unsold pigs are returned to owners’ farms after the market day because of the unavailability of withholding facilities at these markets. Such practice further complicates the situations of ASF in Nigeria. It is important to note all the markets where this study was carried out, there is limited government regulation of market operators’ activities. The apparent absence of antemortem inspection at market gates, lack of holding pens, close contact of pigs from different locations, and selling of pork within market premises enhances the ease of ASFV spread between pigs at market premises. Other poor biosecurity practices observed were free access to market sites by free-roaming pigs and dogs and unrestricted access of slaughter slab operators. Field reports in Nigeria and previous studies in Zambia have reported outbreaks following the introduction of pigs bought at LPMs (Siamupa et al., 2018; Simulundu et al., 2018). This study also demonstrated the widespread free movement of pigs from surrounding communities to the four live pig markets and the movement of pigs from markets to various destinations in Nigeria (Fig 1 and Fig 2). In total, pigs are sourced from over 70 villages/towns located in 7 States of Nigeria and villages in Cameroon for trading at the market sites (Table 2, Fig 2), while pigs traded at the markets are supplied to 42 towns and cities in 13 States of Nigeria (Table 2, Fig 2). It is interesting to note that all these pig movements are done without movement permits or health certificates issued by the appropriate government agencies. In other countries like Zambia, Indonesia, and China, movement permits or health certificates are required before the movement of pigs between farms, live pig markets, districts, and regions (Gao et al., 2021; Leslie et al., 2016; Siamupa et al., 2018). Movement permits and certificates are important tools to track pig movements and reduce the risk of disease spread, but in Nigeria these movement permits/health certificates were not in use in all the live pig markets selected for this study. Results from this study estimate that nearly 500,000-700,000 pigs transit through these markets yearly, without appropriate restrictions or regulation to mitigate the spread of important transboundary diseases such as ASF. The pigs are transported to live pig markets using motorcycles, cars, buses, trucks, and on foot. The vehicles visit multiple farms, villages or towns, and livestock markets to collect and deliver the pigs to the markets without proper sanitary measures to prevent disease transmission. Also after delivery, the vehicles were washed at market premises leading to possible environmental contamination with the virus.