Diversity and emergence of new variants of African swine fever virus Genotype I circulating in domestic pigs in Nigeria (2016-2018).

BACKGROUND: African swine fever (ASF) is the most lethal disease of pigs caused by ASF virus (ASFV) with severe economic implications and threat to the swine industry in endemic countries. Between 2016 and 2018, several ASF outbreaks were reported throughout pig producing states in Nigeria. OBJECTIVES: Thereafter, this study was designed to identify the ASFV genotypes responsible for these outbreaks within the study period (2016-2018). METHODS: Twenty-two ASFV-positive samples by polymerase chain reaction were selected. The samples were collected during passive surveillance in eight states of Nigeria were characterised using 3 partial genes sequences of the virus namely, p72 capsid protein of the B646L, p54 envelope protein of E183L and the central variable region (CVR) within B602L of ASFV. RESULTS: Phylogenetic and sequences analysis based on p72 and p54 revealed ASFV genotype I as the circulating virus. Sequence analysis of the CVR of B602L revealed genetic variations with six ASFV tandem repeat sequence (TRS) variants namely, Tet-15, Tet-20a, Tet-21b, Tet-27, Tet-31 and Tet-34, thus increasing the overall genetic diversity of ASFV in Nigeria. Three of the TRS variants, Tet-21b, Tet-31 and Tet-34, were identified for the first time in Nigeria. The new TRS variants of ASFV genotype I were identified in Enugu, Imo, Plateau and Taraba states, while co-circulation of multiple variants of ASFV genotype I was recorded in Plateau and Benue states. CONCLUSIONS: The high genetic diversity, emergence and increasing recovery of new variants of genotype I in Nigeria should be a concern given that ASFV is a relatively stable DNA virus. The epidemiological implications of these findings require further investigation.

Prevention and Control of African Swine Fever in the Smallholder Pig Value Chain in Northern Uganda: Thematic Analysis of Stakeholders' Perceptions.

African swine fever (ASF) is endemic in Uganda and considered a major constraint to pig production. In the absence of a vaccine, biosecurity is key for ASF prevention and control. To improve prevention and control on farm and community level there is need for more knowledge on current application of biosecurity practises, and better understanding of how pig value chain actors perceive prevention and control. To achieve this, a qualitative interview study involving focus group discussions (FGD) was conducted with actors from the smallholder pig value chain in northern Uganda. Six villages were purposively selected based on previous outbreaks of ASF, preliminary perceived willingness to control ASF, and the representation of several different value chain actors in the village. Results indicated that biosecurity practises such as basic hygiene routines including safe carcass handling, minimising direct and indirect contacts between pigs or between pigs and people, trade restrictions and sharing of disease information were implemented in some of the villages. Thematic analysis based on grounded theory revealed six categories of data relating to ASF prevention and control. Together these categories form a logical framework including both enablers and hindrances for ASF prevention and control. In summary participants mostly had positive perceptions of ASF biosecurity, describing measures as effective. Participants further possessed knowledge of ASF and its transmission, some of which was in line with known scientific knowledge and some not. Nevertheless, participants were hindered from preventing and controlling ASF due to biosecurity costs and a need to prioritise family livelihood over disease transmission risks, incompatibility of current biosecurity practises with local culture, traditions and social contexts and finally lack of access to veterinarians or, occasionally, low-quality veterinary services. The constraints could be addressed by applying participatory processes in designing biosecurity measures to ensure better adaptation to local cultural and social contexts.

Biosecurity aspects of cattle production in Western Uganda, and associations with seroprevalence of brucellosis, salmonellosis and bovine viral diarrhoea.

BACKGROUND: Many low-income countries have a human population with a high number of cattle owners depending on their livestock for food and income. Infectious diseases threaten the health and production of cattle, affecting both the farmers and their families as well as other actors in often informal value chains. Many infectious diseases can be prevented by good biosecurity. The objectives of this study were to describe herd management and biosecurity routines with potential impact on the prevalence of infectious diseases, and to estimate the burden of infectious diseases in Ugandan cattle herds, using the seroprevalence of three model infections. RESULTS: Farmer interviews (n = 144) showed that biosecurity measures are rarely practised. Visitors' hand-wash was used by 14%, cleaning of boots or feet by 4 and 79% put new cattle directly into the herd. During the 12 months preceding the interviews, 51% of farmers had cattle that died and 31% had noticed abortions among their cows. Interestingly, 72% were satisfied with the health status of their cattle during the same time period. The prevalence (95% CI) of farms with at least one seropositive animal was 16.7% (11.0;23.8), 23.6% (16.9;31.4), and 53.4% (45.0;61.8) for brucella, salmonella and BVD, respectively. A poisson regression model suggested that having employees looking after the cattle, sharing pasture with other herds, and a higher number of dead cattle were associated with a herd being positive to an increasing number of the diseases. An additive bayesian network model with biosecurity variables and a variable for the number of diseases the herd was positive to resulted in three separate directed acyclic graphs which illustrate how herd characteristics can be grouped together. This model associated the smallest herd size with herds positive to a decreasing number of diseases and having fewer employees. CONCLUSION: There is potential for improvement of biosecurity practices in Ugandan cattle production. Salmonella, brucella and BVD were prevalent in cattle herds in the study area and these infections are, to some extent, associated with farm management practices.

Knowledge, Attitudes and Practices Related to African Swine Fever Within Smallholder Pig Production in Northern Uganda.

Uganda is a low-income country with the largest pig population in East Africa. Pig keeping has a large potential, commercially and as a tool for poverty reduction, but African swine fever (ASF) is a major hurdle for development of the sector. The objective of this study was to evaluate knowledge, attitudes and practices related to ASF in the smallholder pig production value chain in northern Uganda. The study included three separate series of participatory rural appraisals (PRA), comprising purposively selected farmers and other actors in the pig production value chain. In the PRAs, various participatory epidemiology tools were used. A total of 49 PRAs and 574 participants, representing 64 different villages, were included. The results indicate that participants were well aware of the clinical signs of ASF, routes for disease spread and measures for disease control. However, awareness of the control measures did not guarantee their implementation. A majority of middlemen and butchers acknowledged having sold live pigs, carcasses or pork they believed infected with ASF. Outbreaks of ASF had a strong negative impact on participants' socio-economic status with loss of revenue and reversal into more severe poverty. In conclusion, lack of knowledge is not what is driving the continuous circulation of ASF virus in this setting. To control ASF and reduce its impact, initiatives that stimulate changes in management are needed. Because the behaviour of all actors in the value chain is largely influenced by the deep rural poverty in the region, this needs to be combined with efforts to reduce rural poverty.

High performance computation of landscape genomic models including local indicators of spatial association.

With the increasing availability of both molecular and topo-climatic data, the main challenges facing landscape genomics - that is the combination of landscape ecology with population genomics - include processing large numbers of models and distinguishing between selection and demographic processes (e.g. population structure). Several methods address the latter, either by estimating a null model of population history or by simultaneously inferring environmental and demographic effects. Here we present samßada, an approach designed to study signatures of local adaptation, with special emphasis on high performance computing of large-scale genetic and environmental data sets. samßada identifies candidate loci using genotype-environment associations while also incorporating multivariate analyses to assess the effect of many environmental predictor variables. This enables the inclusion of explanatory variables representing population structure into the models to lower the occurrences of spurious genotype-environment associations. In addition, samßada calculates local indicators of spatial association for candidate loci to provide information on whether similar genotypes tend to cluster in space, which constitutes a useful indication of the possible kinship between individuals. To test the usefulness of this approach, we carried out a simulation study and analysed a data set from Ugandan cattle to detect signatures of local adaptation with samßada, bayenv, lfmm and an FST outlier method (FDIST approach in arlequin) and compare their results. samßada - an open source software for Windows, Linux and Mac OS X available at http://lasig.epfl.ch/sambada - outperforms other approaches and better suits whole-genome sequence data processing.

East African pigs have a complex Indian, Far Eastern and Western ancestry.

In this study, we have characterized the mitochondrial diversity of 81 swine from Uganda. Median-joining network analysis of D-loop sequences from these individuals and others characterized in previous studies allowed us to determine that Ugandan pigs cluster with populations from the West (Europe/North Africa), Far East and India. In addition, partial sequencing of the Y-chromosome UTY locus in 18 Ugandan domestic pigs revealed the segregation of a single HY1 lineage that has a cosmopolitan distribution. A Western and Far Eastern ancestry for East African pigs had been already reported, but this is the first study demonstrating an additional contribution from the Indian porcine gene pool. This result is consistent with the high frequency of zebuine alleles in cattle from East Africa. The geographic coordinates of East Africa, at the crossroads of many trading routes that, through the ages, linked Europe, Africa and Asia, might explain the rich and complex genetic heritage of livestock native to this area.

Mosquitoes of Zika Forest, Uganda: species composition and relative abundance.

Mosquito collections were conducted in Zika Forest near Entebbe, Uganda, from July 2009 through June 2010 using CO2-baited light traps, ovitraps, and human-baited catches. In total, 163,790 adult mosquitoes belonging to 12 genera and 58 species were captured. Of these, 22 species (38%) were captured in Zika Forest for the first time. All the new records found in the forest in this study had previously been captured in other regions of Uganda, implying that they are native to the country and do not represent new introductions. More than 20 species previously collected in Zika Forest were not detected in our collections, and this may suggest a change in the mosquito fauna during the past 40 yr or variation in species composition from year to year. Arboviruses of public health importance have previously been isolated from >50% of the 58 mosquito species captured in Zika Forest, which suggests ahigh potential for transmission and maintenance of a wide range of arboviruses in Zika Forest.

Development of a suspension microarray for the genotyping of African swine fever virus targeting the SNPs in the C-terminal end of the p72 gene region of the genome.

African swine fever virus (ASFV) causes one of the most dreaded transboundary animal diseases (TADs) in Suidae. African swine fever (ASF) often causes high rates of morbidity and mortality, which can reach 100% in domestic swine. To date, serological diagnosis has the drawback of not being able to differentiate variants of this virus. Previous studies have identified the 22 genotypes based on sequence variation in the C-terminal region of the p72 gene, which has become the standard for categorizing ASFVs. This article describes a genotyping assay developed using a segment of PCR-amplified genomic DNA of approximately 450 bp, which encompasses the C-terminal end of the p72 gene. Complementary paired DNA probes of 15 or 17 bp in length, which are identical except for a single nucleotide polymorphism (SNP) in the central position, were designed to either individually or in combination differentiate between the 22 genotypes. The assay was developed using xMAP technology; probes were covalently linked to microspheres, hybridized to PCR product, labelled with a reporter and read in the Luminex 200 analyzer. Characterization of the sample was performed by comparing fluorescence of the paired SNP probes, that is, the probe with higher fluorescence in a complementary pair identified the SNP that a particular sample possessed. In the final assay, a total of 52 probes were employed, 24 SNP pairs and 4 for general detection. One or more samples from each of the 22 genotypes were tested. The assay was able to detect and distinguish all 22 genotypes. This novel assay provides a powerful novel tool for the simultaneous rapid diagnosis and genotypic differentiation of ASF.

Serotype identification and VP1 coding sequence analysis of foot-and-mouth disease viruses from outbreaks in eastern and northern Uganda in 2008/9.

In April 2008, foot-and-mouth disease (FMD) outbreaks were reported in Kamuli district of the eastern region of Uganda. Soon after lifting the quarantines in this area, further FMD outbreaks were reported in northern Uganda, which spread to more than 10 districts. The aim of this study was to identify the serotype and compare the variable protein (VP)1 coding sequences of the viruses responsible for FMD outbreaks during 2008 and 2009, to trace the transmission pathways of the disease in Uganda. Probang and epithelial swab samples were collected from cattle with clinical signs of FMD in the two regions, and the presence of FMDV RNA in these samples was determined using a standard diagnostic RT-PCR assay. From the total of 27 positive samples, the VP1 coding region was amplified and sequenced. Each of these sequences showed >99% identity to each other, and just five distinct sequences were identified. BLAST searches and phylogenetic analysis of the complete variable protein (VP)1 coding sequences revealed that they belonged to serotype O, topotype EA-2. The close similarity between the virus sequences suggested introduction from a single source. We therefore conclude that FMD in the northern region of Uganda was most likely introduced from the outbreak in the eastern region across Lake Kyoga through movement of live animals. This has significant implications for the effectiveness of the current FMD control measures.

Low diversity of foot-and-mouth disease serotype C virus in Kenya: evidence for probable vaccine strain re-introductions in the field.

Most viruses are maintained by complex processes of evolution that enable them to survive but also complicate efforts to achieve their control. In this paper, we study patterns of evolution in foot-and-mouth disease (FMD) serotype C virus isolates from Kenya, one of the few places in the world where serotype C has been endemic and is suspected to remain. The nucleotide sequences encoding the capsid protein VP1 from eight isolates collected between 1967 and 2004 were analysed for patterns of sequence divergence and evolution. Very low nucleotide diversity (π = 0·0025) and remarkably little change (only five segregating sites and three amino-acid changes) were observed in these isolates collected over a period of almost 40 years. We interpret these results as being suggestive of re-introductions of the vaccine strain into the field. The implications of these results for the maintenance of serotype C FMD virus and the use of vaccination as a control measure in Kenya are discussed.

Serotype specificity of antibodies against foot-and-mouth disease virus in cattle in selected districts in Uganda.

Uganda had an unusually large number of foot-and-mouth disease (FMD) outbreaks in 2006, and all clinical reports were in cattle. A serological investigation was carried out to confirm circulating antibodies against foot-and-mouth disease virus (FMDV) by ELISA for antibodies against non-structural proteins and structural proteins. Three hundred and forty-nine cattle sera were collected from seven districts in Uganda, and 65% of these were found positive for antibodies against the non-structural proteins of FMDV. A subset of these samples were analysed for serotype specificity of the identified antibodies. High prevalences of antibodies against non-structural proteins and structural proteins of FMDV serotype O were demonstrated in herds with typical visible clinical signs of FMD, while prevalences were low in herds without clinical signs of FMD. Antibody titres were higher against serotype O than against serotypes SAT 1, SAT 2 and SAT 3 in the sera investigated for serotype-specific antibodies. Only FMDV serotype O virus was isolated from one probang sample. This study shows that the majority of the FMD outbreaks in 2006 in the region studied were caused by FMDV serotype O; however, there was also evidence of antibodies to both SAT 1 and SAT 3 in one outbreak in a herd inside Queen Elizabeth national park area.

Antibodies against foot-and-mouth disease (FMD) virus in African buffalos (Syncerus caffer) in selected National Parks in Uganda (2001-2003).

In East Africa, the foot-and-mouth disease (FMD) virus (FMDV) isolates have over time included serotypes O, A, C, Southern African Territories (SAT) 1 and SAT 2, mainly from livestock. SAT 3 has only been isolated in a few cases and only in African buffalos (Syncerus caffer). To investigate the presence of antibodies against FMDV serotypes in wildlife in Uganda, serological studies were performed on buffalo serum samples collected between 2001 and 2003. Thirty-eight samples from African buffalos collected from Lake Mburo, Kidepo Valley, Murchison Falls and Queen Elizabeth National Parks were screened using Ceditest FMDV NS to detect antibodies against FMDV non-structural proteins (NSP). The seroprevalence of antibodies against non-structural proteins was 74%. To characterize FMDV antibodies, samples were selected and titrated using serotype-specific solid phase blocking enzyme linked immunosorbent assay (ELISAs). High titres of antibodies (> or =1 : 160) against FMDV serotypes SAT 1, SAT 2 and SAT 3 were identified. This study suggests that African buffalos in the different national parks in Uganda may play an important role in the epidemiology of SAT serotypes of FMDV.

Molecular characterization of SAT 2 foot-and-mouth disease virus from post-outbreak slaughtered animals: implications for disease control in Uganda.

In Uganda, limiting the extent of foot-and-mouth disease (FMD) spread during outbreaks involves short-term measures such as ring vaccination and restrictions of the movement of livestock and their products to and from the affected areas. In this study, the presence of FMD virus RNA was investigated in cattle samples 3 months after FMD quarantine measures had been lifted following an outbreak in 2004. Oropharyngeal tissue samples were obtained from 12 cattle slaughtered in a small town abattoir in Kiboga. FMD virus RNA was detected by diagnostic RT-PCR in nine of the 12 tissue samples. Part of the coding region for the capsid protein VP1 was amplified and sequenced. All samples were identified as belonging to the SAT 2 serotype. The implications for FMD control of both virus introduction into Uganda and the presence of carrier animals following outbreaks are discussed.

Prevalence estimates of antibodies towards foot-and-mouth disease virus in small ruminants in Uganda.

Foot-and-mouth disease (FMD) is endemic in Uganda with control strategies focusing on vaccination of cattle, while small ruminants are largely ignored. In order for Uganda to establish effective control strategies, it is crucial that the epidemiology of the disease is fully understood. This study summarizes results of serological investigations of sheep and goats for antibodies to FMDV from four districts in 2006 following an FMD outbreak in the region and from an attempted comprehensive random sampling in two districts in 2007. Antibodies were quantified and serotyped using competitive ELISA for antibodies towards non-structural proteins (NSP) and structural proteins towards serotype O, and blocking ELISA for antibodies towards the seven serotypes of FMD virus (FMDV). In 2006, sheep and goats in Bushenyi and Isingiro districts were free from antibodies towards FMDV, while herds in Kasese and Mbarara districts excluding Kahendero village were all positive for antibodies towards NSP and SP-O. In 2007, mean prevalence estimates of antibodies towards FMDV NSP was 14% in goats and 22% in sheep in Kasese district, while Bushenyi was still free. The difference between these two districts probably reflects different levels of FMDV challenge attributed to the variation in exposure rates which again in part may be as a result of the differing husbandry practices. Contrary to 2006, with clear antibodies towards serotype O, the serotype-specificity of the antibodies was less clear in 2007, as antibodies towards both serotype O and SAT serotypes were identified. Our results show that goats and sheep are infected during FMD outbreaks, and that they may be useful for determining the serotype of FMD outbreaks in Uganda, if they are sampled shortly after an outbreak.

Mid-Holocene decline in African buffalos inferred from Bayesian coalescent-based analyses of microsatellites and mitochondrial DNA.

Genetic studies concerned with the demographic history of wildlife species can help elucidate the role of climate change and other forces such as human activity in shaping patterns of divergence and distribution. The African buffalo (Syncerus caffer) declined dramatically during the rinderpest pandemic in the late 1800s, but little is known about the earlier demographic history of the species. We analysed genetic variation at 17 microsatellite loci and a 302-bp fragment of the mitochondrial DNA control region to infer past demographic changes in buffalo populations from East Africa. Two Bayesian coalescent-based methods as well as traditional bottleneck tests were applied to infer detailed dynamics in buffalo demographic history. No clear genetic signature of population declines related to the rinderpest pandemic could be detected. However, Bayesian coalescent modelling detected a strong signal of African buffalo population declines in the order of 75-98%, starting in the mid-Holocene (approximately 3-7000 years ago). The signature of decline was remarkably consistent using two different coalescent-based methods and two types of molecular markers. Exploratory analyses involving various prior assumptions did not seriously affect the magnitude or timing of the inferred population decline. Climate data show that tropical Africa experienced a pronounced transition to a drier climate approximately 4500 years ago, concurrent with the buffalo decline. We therefore propose that the mid-Holocene aridification of East Africa caused a major decline in the effective population size of the buffalo, a species reliant on moist savannah habitat for its existence.

Mitochondrial DNA variation of the common hippopotamus: evidence for a recent population expansion.

Mitochondrial DNA control region sequence variation was obtained and the population history of the common hippopotamus was inferred from 109 individuals from 13 localities covering six populations in sub-Saharan Africa. In all, 100 haplotypes were defined, of which 98 were locality specific. A relatively low overall nucleotide diversity was observed (pi = 1.9%), as compared to other large mammals so far studied from the same region. Within populations, nucleotide diversity varied from 1.52% in Zambia to 1.92% in Queen Elizabeth and Masai Mara. Overall, low but significant genetic differentiation was observed in the total data set (F(ST) = 0.138; P = 0.001), and at the population level, patterns of differentiation support previously suggested hippopotamus subspecies designations (F(CT) = 0.103; P = 0.015). Evidence that the common hippopotamus recently expanded were revealed by: (i) lack of clear geographical structure among haplotypes, (ii) mismatch distributions of pairwise differences (r = 0.0053; P = 0.012) and site-frequency spectra, (iii) Fu's neutrality statistics (F(S) = -155.409; P < 0.00001) and (iv) Fu and Li's statistical tests (D* = -3.191; P < 0.01, F* = -2.668; P = 0.01). Mismatch distributions, site-frequency spectra and neutrality statistics performed at subspecies level also supported expansion of Hippopotamus amphibius across Africa. We interpret observed common hippopotamus population history in terms of Pleistocene drainage overflow and suggest recognising the three subspecies that were sampled in this study as separate management units in future conservation planning.