African Swine Fever Diagnosis in Africa: Challenges and Opportunities.

The global spread of African swine fever (ASF) in recent decades has led to the need for technological advances in sampling and diagnostic techniques. The impetus for these has been the need to enable sampling by lay persons and to obtain at least a preliminary diagnosis in the field for early control measures to be put in place before final laboratory confirmation. In rural Africa, rapid diagnosis is hampered by challenges that include lack of infrastructure as well as human and financial resources. Lack of animal health personnel, access to affordable means to transport field samples to a laboratory, and lack of laboratories with the capacity to make the diagnosis result in severe under-reporting of ASF, especially in endemic areas. This review summarizes the challenges identified in gap analyses relevant to low- and middle-income countries, with a focus on Africa, and explore the opportunities provided by recent research to improve field diagnosis and quality of diagnostic samples used. Sampling techniques include invasive sampling techniques requiring trained personnel and non-invasive sampling requiring minimal training, sampling of decomposed carcass material, and preservation of samples in situations where cold chain maintenance cannot be guaranteed. Availability and efficacy of point-of-care (POC) tests for ASF has improved considerably in recent years and their application, as well as advantages and limitations, are discussed. The adequacy of existing laboratory diagnostic capacity is evaluated and opportunities for networking amongst reference and other laboratories offering diagnostic services are discussed. Maintaining laboratory diagnostic efficiency in the absence of samples during periods of quiescence is another issue that requires attention, and the role of improved laboratory networking is emphasized. Early diagnosis of ASF is key to managing the disease spread. Therefore, the establishment of the Africa Chapter of the Global African Swine Fever Research Alliance (GARA) increases opportunities for collaboration and networking among the veterinary diagnostic laboratories in the region.

Target capture sequencing for the first Nigerian genotype I ASFV genome.

African swine fever (ASF) is a contagious viral disease that affects domestic pigs and wild boars, causing significant economic losses globally. After the first Nigerian outbreak in 1997, there have been frequent reports of ASF in pig-producing regions in the country. To facilitate control, it is important to understand the genotype and phylogenetic relationship of ASF viruses (ASFVs). Recent genetic analysis of Nigerian ASFV isolates has revealed the presence of both genotypes I and II; this is based on analysis of a few selected genes. Phylogenetic analysis of ASFV whole genomes highlights virus origins and evolution in greater depth. However, there is currently no information on the ASFV genome from Nigerian isolates. Two ASFV-positive samples were detected during a random survey of 150 Nigerian indigenous pig samples collected in 2016. We assembled near-complete genomes of the two ASFV-positive samples using in-solution hybrid capture sequencing. The genome-wide phylogenetic tree assigned these two genomes into p72 genotype I, particularly close to the virulent Benin 97/1 strain. The two ASFVs share 99.94 and 99.92 % genomic sequence identity to Benin97/1. This provides insight into the origin and relationship of ASFV strains from Nigeria and Italy. The study reports for the first time the determination of near-complete genomes of ASFV using in-solution hybrid capture sequencing, which represents an important advance in understanding the global evolutionary landscape of ASFVs.

Characterization of a Novel African Swine Fever Virus p72 Genotype II from Nigeria.

African swine fever (ASF) is a high-consequence transboundary hemorrhagic fever of swine. It continues to spread across the globe causing socio-economic issues and threatening food security and biodiversity. In 2020, Nigeria reported a major ASF outbreak, killing close to half a million pigs. Based on the partial sequences of the genes B646L (p72) and E183L (p54), the virus responsible for the outbreak was identified as an African swine fever virus (ASFV) p72 genotype II. Here, we report further characterization of ASFV RV502, one of the isolates obtained during the outbreak. The whole genome sequence of this virus revealed a deletion of 6535 bp between the nucleotide positions 11,760-18,295 of the genome, and an apparent reverse complement duplication of the 5' end of the genome at the 3' end. Phylogenetically, ASFV RV502 clustered together with ASFV MAL/19/Karonga and ASFV Tanzania/Rukwa/2017/1 suggesting that the virus responsible for the 2020 outbreak in Nigeria has a South-eastern African origin.

African Suid Genomes Provide Insights into the Local Adaptation to Diverse African Environments.

African wild suids consist of several endemic species that represent ancient members of the family Suidae and have colonized diverse habitats on the African continent. However, limited genomic resources for African wild suids hinder our understanding of their evolution and genetic diversity. In this study, we assembled high-quality genomes of a common warthog (Phacochoerus africanus), a red river hog (Potamochoerus porcus), as well as an East Asian Diannan small-ear pig (Sus scrofa). Phylogenetic analysis showed that common warthog and red river hog diverged from their common ancestor around the Miocene/Pliocene boundary, putatively predating their entry into Africa. We detected species-specific selective signals associated with sensory perception and interferon signaling pathways in common warthog and red river hog, respectively, which contributed to their local adaptation to savannah and tropical rainforest environments, respectively. The structural variation and evolving signals in genes involved in T-cell immunity, viral infection, and lymphoid development were identified in their ancestral lineage. Our results provide new insights into the evolutionary histories and divergent genetic adaptations of African suids.

Evaluation of a Lateral Flow Assay for Rapid Detection of African Swine Fever Virus in Multiple Sample Types.

Antibody-based lateral flow assay (LFA) is a quick and inexpensive tool used to detect pathogens in field samples, especially in hard-to-reach remote areas that may have limited access to central laboratories during an outbreak or surveillance. In this study, we investigated the ability of a commercially available LFA, PenCheck®, to detect African swine fever virus (ASFV) in clinical samples derived from pigs infected with highly virulent ASFV strains. The assay was specific and positively identified the majority of pigs showing high fever during the early stages (between 3 and 5 days) of infection. PenCheck® LFA also detected ASFV in serum and tissue samples collected from pigs that succumbed to experimental ASFV infection and whole blood, plasma, and tissue samples from the field. The limit of detection of the assay was ASFV titer 107.80 TCID50/mL, corresponding to ASFV real-time PCR values below 23 Ct. Although the sensitivity of the assay is less than that of the laboratory-based real-time PCR assays, the results obtained with the PenCheck® LFA in this study suggest that it can be used as a herd-level, field-deployable, and easy-to-use diagnostic tool to identify ASF-affected farms when access to portable molecular assays or central laboratories is not possible.

Live pig markets are hotspots for spread of African swine fever virus in Nigeria.

Livestock trading through live animal markets are potential pathways for the introduction and spread of economically important pathogens like the African swine fever virus (ASFV) to new areas in several countries. Due to the high demand for live pigs in Nigeria both for restocking and slaughter, live pigs are sold at designated live pig markets (LPM) in the country. This involves movement of pigs over long distances. Despite, reports of ASF outbreaks following restocking of pigs bought from LPMs, there is paucity of information on the role of LPMs in the epidemiology of ASF. In this study, data and pig samples (whole blood, sera, tissue) were collected from 4 selected LPMs in Nigeria (Dawaki, Katsit, Numan and Pandam) between 2019 and 2020. Samples were analysed by polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). Four genes of ASFV positive samples were characterized to identify the circulating genotypes. Results revealed trade activities involving transportation of pigs from these selected markets to 42 major cities and towns in thirteen (13) States of Nigeria. PCR results revealed an overall ASF prevalence of 10.77% (66/613). ASFV was confirmed by PCR in all the selected LPMs with a prevalence rate of 3.13%-23.81%. The phylogeny revealed genotype I and serogroup 4 based on the p72 protein that encodes the B646L gene and the EP402R gene encoding the CD2V. While sequence analysis of CVR of B602L gene revealed 8 tetrameric repeats variants, six of which have never been reported in Nigeria. Analysis of sera samples recorded a seroprevalence of 6.9% (16/217) within the study period. Findings from this study show that LPM are hotspots and channels for transmission and continuous spread of ASFV in Nigeria. Therefore, for ASF to be controlled in Nigeria, disease surveillance and regulation at LPMs are critical.

First-Time Presence of African Swine Fever Virus Genotype II in Nigeria.

A confirmed African swine fever (ASF) outbreak in Nigeria was further investigated by partial sequencing of the B464L and E183L genes of ASF virus (ASFV). Results revealed the first-time presence of ASFV genotype II in Nigeria and West Africa. This finding has serious implications for control measures and food security.

Isolation and identification of Mycoplasma mycoides subsp. mycoides in cattle from south-east Nigeria.

Background: Mycoplasma mycoides subsp. mycoides is the causative organism of Contagious Bovine Pleuropneumonia (CBPP). It is a trans-boundary disease and an endemic in Nigeria having caused serious financial loss for the country's economy. Aim: This study was undertaken to isolate and confirm the presence of M. mycoides subsp. mycoides (Mmm) in cattle, from three selected South-Eastern states of Nigeria. Method: A total of 90 bovine samples (25 pleural fluids and 65 lung tissues) suggestive of CBPP were collected from different abattoirs in the three selected South-eastern states of Nigeria (Anambra, Enugu, and Imo), for the isolation of Mmm by employing cultural method, whereas for confirmation polymerase chain reaction (PCR) approach was used. The collected samples were cultured on Pleuropneumonia like organism (PPLO) agar according to specific protocols. Results: Twenty five of the samples (lungs and pleural fluid) were positive for Mmm on PPLO agar giving an isolation rate of 27.7%. Only 21 of the isolates were further confirmed using PCR. The PCR amplification of the isolates produced a product of 1.1 kbp which is specific for Mmm. No positive isolates were recovered from Imo state. Conclusion: This study confirms the presence of Mmm as the causative organism of CBPP in Southeast Nigeria. It is recommended that active surveillance and vaccination protocol should be undertaken in the region for the control and prevention of this disease.

Questionnaire Survey on Knowledge, Attitude and Practices on COVID 19 Outbreak Among Veterinarians in Nigeria.

Coronavirus Disease 2019 (COVID-19), a viral pneumonia-like disease caused by novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) started in Wuhan, Hubei Province of China on December 31st 2019, and assumed a pandemic status; infecting about 30 million people, with a mortality in excess of 957,000 as at 20st September, 2020. This study assessed the knowledge, attitude, and practices concerning COVID-19 among veterinarians with a view to detecting variables that may hinder their effective contributions towards the management and control of COVID-19 outbreak in Nigeria.The study found that male veterinarians had significantly good knowledge (p=0.012, OR=0.157, CI=0.03-0.66) and attitude (OR=18.415, CI=1.45-5.16, P=0.011) towards COVID-19 than female counterparts. Similar results were also recorded regarding practices (OR=2.941, CI=1.03-8.36, P=0.043). Educational qualification was found to be significantly associated with attitude of veterinarians in respect of COVID-19 prevention (OR=0.473, CI=0.073-0.011, P=0.006). Thus, gender (male) was found to be independent predictors of good knowledge, attitude and practices regarding COVID-19. In addition, educational qualification was also found to be an independent predictor of attitude of veterinarians regarding COVID-19 control. Veterinarians generally had good knowledge about the epidemiology, diagnosis, treatment and prevention of COVID-19. However, despite their knowledge, the level of willingness of veterinarians to support national response activities was still poor. This study advocated for a collaborative efforts (one-health) between medics and vets in tackling future pandemics/infectious diseases like COVID-19. In addition, since most vet knows the basics of infectious diseases like COVID-19, they can be deployed for national response activities.

Laboratory diagnosis of a new outbreak of acute African swine fever in smallholder pig farms in Jos, Nigeria.

African swine fever (ASF) is a highly contagious fatal infectious disease of pigs and wild suids. The disease has a worldwide occurrence and significant impact on pig production. Two adult intensively raised large white boars from two farms in Jos with a history of sudden death were diagnosed of ASF between July and August 2019. Post-mortem examination of carcasses grossly showed splenomegaly, haemorrhagic lymphadenitis and hepatomegaly with severe congestion. The kidneys were enlarged and had generalized petechiae and blood clot in the pelvis. The heart was moderately enlarged. Microscopic examination of the spleen and lymph nodes revealed severe lymphocytic depletion, haemorrhage and severe haemosiderosis. The liver was severely congested with focal coagulative necrosis of the hepatocytes. The kidneys were severely congested and showed renal tubular necrosis with few tubular protein casts. Tissue samples were confirmed to be positive for African swine fever virus (ASFV) by polymerase chain reaction (PCR) assay, and phylogenetic analysis revealed that the isolate belonged to genotype I.

Avian leukosis virus subgroup - J as a contaminant in live commercially available poultry vaccines distributed in Nigeria.

Globally, vaccines are used to prevent and control the menace of infectious diseases in livestock with some reported to be inadvertently contaminated with extraneous agents (EAs). With the aim of screening and characterizing for some selected EAs, 44 live viral poultry vaccines were randomly selected based on availability. The vaccines comprised 14 manufacturers in 10 different countries including Nigeria were screened by Polymerase Chain Reaction. In 9% (4/44) of the vaccines, contamination with only avian leukosis virus (ALV) subgroup J (ALV-J) was recorded. Other exogenous ALV subgroups, chicken infectious anemia and infectious laryngotracheitis viruses were absent. The EAs was found in infectious bursal disease (n = 1), Fowlpox (n = 2) and Mareks disease (n = 1) vaccines. Phylogenetic analysis of the ALV-J env gene showed clustering with contemporary group I and II. The result underscores the importance of screening vaccines to avoid the introduction and spread of EAs that could pose a threat to poultry production.

Molecular Detection of Torque Teno Sus Virus and Coinfection with African Swine Fever Virus in Blood Samples of Pigs from Some Slaughterhouses in Nigeria.

Torque teno sus virus 1 (TTSuV1a/TTSuV1b) infection is present in pig herds worldwide. This study investigated the prevalence of TTSuV1a/TTSuV1b infections in domestic pigs from some slaughterhouses in Nigeria as well as coinfection with African swine fever virus (ASFV) and described the phylogeny in relation to global strains. One hundred and eighty-one (181) blood samples from four slaughterhouses were used for the study and viral nucleic acid detection was carried out by PCR. Comparative sequence analysis was carried out to infer phylogeny. The overall prevalence of TTSuV1a/b was 17.7%. Prevalence of individual genotypes was 10.5% and 7.2% for TTSuV1a and TTSuV1b, respectively. Coinfection of ASFV/TTSuV1a/b was 7.7% while that of TTSuV1a and TTSuV1b was 1.7%. ASFV alone was detected in 11.91% of the total samples. The Nigerian TTSuV1a and TTSuV1b shared a sequence identity of 91-100% and 95-100%, respectively, among each other. The ASFV sequences were 100% identical to members of genotype 1. This is the first report on the presence of TTSuV1a/b in domestic pigs in Nigeria and coinfection with ASFV. Although the prevalence of TTSuV1a/b in Nigeria was low, we recommend further studies to establish the trend and possible role in the pathogenesis of ASFV.

Molecular characterization of peste des petits ruminants virus from the Karamoja region of Uganda (2007-2008).

Antibodies against peste des petits ruminants virus (PPRV) were first detected in goats in East Africa in 1995 without any clinical disease. It was not until during the years 2006 and 2007 that the disease outbreaks were first reported in Kenya and Uganda, respectively. This study was carried out to detect and characterize PPRV from a suspected outbreak in sheep and goats in the Karamoja region in 2007-2008. Oculo-nasal and blood samples were tested using F-gene-based primers, and their genetic relationships to other sequences in the GenBank database were investigated. A total of 383 samples suspected to contain PPRV were randomly collected and tested. Sixty-seven (17.5%) were positive when F protein gene primers were used. During the years 2007 and 2008, 38.1% (26/67) and 13.0% (41/316) of samples were positive by PCR, respectively. The 2007 sequences clustered with Asian sequences in lineage 4 and Cote d'Ivoire 86 (ICV 86) in lineage 2, while all of the 2008 samples clustered in lineage 1. Over the years, the implicated strains were genetically close (88%-91%) to the vaccine strain (Nig 75/1). Based on this study, the circulating PPR strains in Uganda are heterogeneous, and therefore, the disease may have been introduced from different sources.

Molecular characterization and phylogenetic study of peste des petits ruminants viruses from north central states of Nigeria.

BACKGROUND: Peste des petits ruminants is an endemic disease of sheep and goats in Nigeria and vaccination has been the method of control but sporadic outbreaks have been reported. This study was carried out to characterize PPR viruses from outbreaks in 2007 and 2009 from Kaduna and Plateau States. RESULTS: Of the 33 clinical samples analysed, 51.52% (n = 17) were positive for F protein gene primers (F1/F2). All the samples had a sequence similarity of 98-100% among them and 92-97% with the reference vaccine (Nig 75/1) strain. The deduced amino acid homology ranges between 96.3-99.7%. Phylogenetically all the Nigerian sequences cluster with Nig 75/1 and Nig 76/1 in lineage 1. CONCLUSIONS: PPR is still a problem in Kaduna and Plateau States of Nigeria. The strains involved were genetically closely related to the vaccine strain (Nig 75/1) used in the country. Based on this study, the continued outbreaks in the Country is not due to the efficacy of the vaccine. Therefore, to achieve effective control and possibly eradication of PPR in Nigeria, the current control strategies should be revisited.