Live Bird Markets in Nigeria: A Potential Reservoir for H9N2 Avian Influenza Viruses.

Since 2006, multiple outbreaks of avian influenza (AI) have been reported in Nigeria involving different subtypes. Surveillance and molecular epidemiology have revealed the vital role of live bird markets (LBMs) in the dissemination of AI virus to commercial poultry farms. To better understand the ecology and epidemiology of AI in Nigeria, we performed whole-genome sequencing of nineteen H9N2 viruses recovered, from apparently healthy poultry species, during active surveillance conducted in nine LBMs across Nigeria in 2019. Analyses of the HA gene segment of these viruses showed that the H9N2 strains belong to the G1 lineage, which has zoonotic potential, and are clustered with contemporary H9N2 identified in Africa between 2016 and 2020. We observed two distinct clusters of H9N2 viruses in Nigeria, suggesting different introductions into the country. In view of the zoonotic potential of H9N2 and the co-circulation of multiple subtypes of AI virus in Nigeria, continuous monitoring of the LBMs across the country and molecular characterization of AIVs identified is advocated to mitigate economic losses and public health threats.

In vitro antibacterial activity of Loxostylis alata extracts and isolated compounds against Salmonella species.

BACKGROUND: Owing to antibiotic resistance, alternative antimicrobials from medicinal plants are receiving attention as leads for anti-infective agents. This study aimed to investigate selected tree species and their constituents for activity against bacterial foodborne pathogens, particularly Salmonella serovars. METHODS: Antibacterial activity of ten plant species was determined by serial microdilution against bacteria implicated in causing gastrointestinal ailments. Active compounds were isolated from Loxostylis alata using bioassay-guided fractionation. Antioxidant activity was determined using free-radical scavenging assays. Cytotoxicity and genotoxicity of the extracts was ascertained on Vero cells, and using the Ames assay respectively. RESULTS: Extracts had low to moderate MIC values from 0.04 to 2.5 mg/mL. Protorhus longifolia and Loxostylis alata were most active and L. alata had the highest selectivity index value (2.51) against Salmonella Typhimurium, as well as high antioxidant activity. Cytotoxicity values ranged from 0.02 to 0.47 mg/mL, while tested extracts were not genotoxic. Bioactive compounds isolated from L. alata included delicaflavone and a polymethoxyflavone. CONCLUSIONS: The Loxostylis alata leaf extract had strong activity against Salmonella serovars but isolated compounds were less active, indicating likely synergistic effects. Extracts of L. alata are promising candidates for development of antimicrobial preparations or food additives against microbial contamination.

First detection of highly pathogenic H5N6 avian influenza virus on the African continent.

Since 2013, highly pathogenic avian influenza (HPAI) subtype H5N6 (clade 2.3.4.4) has been reported in wild birds and poultry in Asia as well as in other parts of the globe. In Africa, information on the presence of this virus subtype is lacking. This study reports the first detection of a HPAI (H5N6) virus (clade 2.3.4.4b) in a duck from a live bird market in Nigeria, whose genome is closely related to the European 2017-2018 H5N6 viruses, indricating a recent virus introduction into the African continent.

Occurrence of infectious bronchitis in layer birds in Plateau state, north central Nigeria.

A flock of 54 wk-old layer birds exhibiting signs of respiratory distress, greenish diarrhea, and drop in egg production was investigated. A marked drop in egg production (55%) was recorded with eggs appearing white and soft-shelled. Mortality was in the range of 1%-2% with post-mortem lesions revealing cloudy air sacs, frothy, and congested lungs. Viral RNA was extracted from pooled tissue samples (trachea, lungs, spleen, and liver) and tested for Avian influenza virus (AIV), Newcastle disease virus (NDV), and infectious bronchitis virus (IBV) by reverse transcriptase-polymerase chain reaction (RT-PCR). In addition, virus isolation was attempted in 9-11 day-old embryonating chicken eggs (ECE). In order to determine the prevalence of IBV serotype(s) in the flock, serum samples were screened by hemagglutination-inhibition (HI) test using IBV antigens and antisera (Arkansas, Connecticut, and Massachusetts). Neither AIV nor NDV but IBV was detected in the tissue samples by RT-PCR. In addition, virus isolate obtained after four serial passages in ECE produced dwarfed, stunted, and hemorrhagic embryos, and the isolate was confirmed by RT-PCR to be IBV. The serum samples were 100% seropositive for three serotypes with HI titres ranging from 5 to 12 Log2. In this study, IBV was confirmed as the causative agent of the observed respiratory distress and drop in egg production. Also, the evidence of co-circulation of multiple IBV serotypes was established, this to the best of our knowledge is the first of such report in Nigeria. We recommend extensive molecular and sero-epidemiology of circulating IBV genotypes and serotypes in Nigeria with the aim of developing better control strategies, including vaccination.

Genomic comparison of Newcastle disease viruses isolated in Nigeria between 2002 and 2015 reveals circulation of highly diverse genotypes and spillover into wild birds.

Newcastle disease virus (NDV) has a wide avian host range and a high degree of genetic variability, and virulent strains cause Newcastle disease (ND), a worldwide concern for poultry health. Although NDV has been studied in Nigeria, genetic information about the viruses involved in the endemicity of the disease and the transmission that likely occurs at the poultry-wildlife interface is still largely incomplete. Next-generation and Sanger sequencing was performed to provide complete (n = 73) and partial genomic sequence data (n = 38) for NDV isolates collected from domestic and wild birds in Nigeria during 2002-2015, including the first complete genome sequences of genotype IV and subgenotype VIh from the African continent. Phylogenetic analysis revealed that viruses of seven different genotypes circulated in that period, demonstrating high genetic diversity of NDV for a single country. In addition, a high degree of similarity between NDV isolates from domestic and wild birds was observed, suggesting that spillovers had occurred, including to three species that had not previously been shown to be susceptible to NDV infection. Furthermore, the first spillover of a mesogenic Komarov vaccine virus is documented, suggesting a previous spillover and evolution of this virus. The similarities between viruses from poultry and multiple bird species and the lack of evidence for host adaptation in codon usage suggest that transmission of NDV between poultry and non-poultry birds occurred recently. This is especially significant when considering that some viruses were isolated from species of conservation concern. The high diversity of NDV observed in both domestic and wild birds in Nigeria emphasizes the need for active surveillance and epidemiology of NDV in all bird species.

South African medicinal plant extracts active against influenza A virus.

BACKGROUND: Influenza infection remains a major health threat for animals and humans which crucially requires effective antiviral remedies. The usage of herbal medications as readily available alternatives for their compatibility with the body and fewer side effects compared to synthetic chemical treatments has become popular globally. The aim of this study was to investigate and screen in vitro anti-influenza activity of extracts of five South African medicinal plants, namely Tabernaemontana ventricosa, Cussonia spicata, Rapanea melanophloeos, Pittosporum viridiflorum and Clerodendrum glabrum, species which are used traditionally for the treatment of several diseases such as inflammatory and respiratory diseases. METHODS: Methanol, ethanol (100% and 30%), acetone, hot and cold water extracts of the powdered plants leaves were obtained by standard methods. The cytotoxicity was determined by the MTT colorimetric assay on MDCK cells. The concentrations below CC50 values were tested for antiviral activity against influenza A virus (IAV) in different combination treatments. The effect of extracts on viral surface glycoproteins and viral titer were tested by HI and HA virological assays, respectively. RESULTS: Based on the applied methods, the most effective results against IAV were obtained from Rapanea melanophloeos methanol leaf extract (EC50 = 113.3 µg/ml) and Pittosporum viridiflorum methanol, 100% and 30% ethanol and acetone leaf extracts (EC50 values = 3.6, 3.4, 19.2, 82.3 µg/ml, respectively) in all types of combined treatments especially in pre- and post-penetration combined treatments with highly significant effects against viral titer (P ≤ 0.01). CONCLUSION: The outcomes offer for the first time a scientific basis for the use of extracts of Rapanea melanophloeos and Pittosporum viridiflorum against IAV. It is worth focusing on the isolation and identification of effective active compounds and elucidating the mechanism of action from these species. However, Tabernaemontana ventricosa, Cussonia spicata and Clerodendrum glabrum leaf extracts were ineffective in vitro in this study.

Highly pathogenic avian influenza (H5N1) in Nigeria in 2015: evidence of widespread circulation of WA2 clade 2.3.2.1c.

Genetic analysis of the complete haemagglutinin (HA) gene of fourteen Nigerian avian influenza isolates showed multiple basic amino acids at the cleavage site (321PQRERRRK del R*GLF333), characteristic of highly pathogenic avian influenza (HPAI). Substitution of Gln to Lys at position 322 (H5-specific numbering) was identified in one isolate. In some isolates, amino acid substitutions were observed across the HA gene, however the receptor binding, antigenic and glycosylation sites were conserved in all. Phylogenetic analysis revealed two clusters of the HPAI H5N1 clade 2.3.2.1c. Cluster I has close genetic relatedness (97.8-99.8%) with viruses circulating in some West Africa countries. Cluster II shared close identity (98.9-100.0%) with isolates from Europe, Côte d'Ivoire and Niger and viruses from this cluster were detected in five of the eleven states investigated in Nigeria. In view of the continuous HPAI outbreaks being recorded in Nigerian poultry and the zoonotic potential of the virus, extensive and continued characterization of HPAI isolates is advocated.

Sero-epizootiological investigation of infectious laryngotracheitis infection in commercial poultry of Plateau State, north central Nigeria.

Infectious laryngotracheitis (ILT) is a highly contagious acute respiratory disease of chickens with outbreaks resulting in high economic losses due to increased mortality and drop in egg production. This study reports a survey of ILT virus antibody conducted in nine local government areas (LGAs) of Plateau State involving 67 randomly selected commercial poultry flocks. In all, 938 sera were tested using the Agar Gel Immuno-diffusion (AGID) technique. Overall prevalence of 1.2% (N = 11) was recorded. ILT virus antibody was found in 2.5% (n = 9) and 7.1% (n = 2) of the tested sera from Jos South and Langtang North LGAs, respectively. No detectable ILT virus antibody was found from the other seven LGAs. This is the first report of ILT infection in poultry from the North central part of Nigeria. It is therefore recommended that the economic implication of ILT infection in Nigerian poultry population be conducted in order to know if vaccination should be adopted for control.

Phylogenetic analysis of Newcastle disease viruses isolated from asymptomatic guinea fowls (Numida meleagris) and Muscovy ducks (Cariana moscata) in Nigeria.

Four Newcastle disease virus isolates were recovered from asymptomatic guinea fowl (Numida meleagris galeata) and Muscovy ducks (Cariana moscata). For the purpose of molecular identification and phylogeny, phylogenetic characterization was performed to identify the pathotypes. All four viruses had a cleavage motif (112)RRQKRF(117) which is characteristic of virulent strains. The isolates grouped with viruses previously reported as sub-lineage 5 g from chickens in Nigeria. This study report for the first time the identification of the virulent sub-lineage 5 g Newcastle disease virus from apparently healthy guinea fowl and domestic ducks in Nigeria, and since infections were sub-clinical, it suggest that these species could play a role in the spread and transmission of virulent Newcastle disease virus that can infect other poultry. The isolation and identification of virulent Newcastle disease virus in these unusual hosts and the high sequence similarity (99.3-100 %) between viruses in this study with strains reported for Niger and Cameroun gives insights into the ecology of virulent Newcastle disease viruses and suggests some cross-border movement and trade in live poultry.