Effects of swab pool size and transport medium on the detection and isolation of avian influenza viruses in ostriches.

BACKGROUND: Rigorous testing is a prerequisite to prove freedom of notifiable influenza A virus infections in commercially farmed ostriches, as is the isolation and identification of circulating strains. Pooling 5 ostrich tracheal swabs in a 50 % v/v phosphate-buffered saline (PBS): glycerol transport medium (without antibiotics) is the current standard practice to increase reverse transcription real time PCR (RT-rtPCR) testing throughput and simultaneously reduce the test costs. In this study we investigated whether doubling ostrich tracheal swabs to 10 per pool would affect the sensitivity of detection of H5N8 high pathogenicity avian influenza virus (HPAIV) and H7N1 low pathogenicity avian influenza virus (LPAIV) by quantitative RT-rtPCR, and we also compared the effect of a protein-rich, brain heart infusion broth (BHI) virus transport media containing broad spectrum antimicrobials (VTM) on the efficacy of isolating the H5N8 and H7N1 viruses from ostrich tracheas, since the historical isolation success rate from these birds has been poor. RESULTS: Increasing the ostrich swabs from 5 to 10 per pool in 3 mls of transport medium had no detrimental effect on the sensitivity of the RT-rtPCR assay in detecting H5N8 HPAIV or H7N1 LPAIV; and doubling of the swab pool size even seemed to improve the sensitivity of virus detection at levels that were statistically significant (p less than or equal to 0.05) in medium and low doses of spiked H5N8 HPAIV and at high levels of spiked H7N1 LPAIV. On virus isolation, more samples were positive when swabs were stored in a protein-rich viral transport medium supplemented with antimicrobials in PBS: glycerol (10/18 vs. 7/18 for H5N8 HPAI); although the differences were not statistically significant, overall higher virus titres were detected (106.7 - 103.0 vs. 106.6 - 103.1 EID50 for H5N8 HPAIV and 105.5 - 101.4 vs. 105.1 - 101.3 EID50 for H7N1 LPAIV); and fewer passages were required with less filtration for both H5N8 HPAI and H7N1 LPAI strains. CONCLUSION: Ostrich tracheal swab pool size could be increased from 5 to 10 in 3mls of VTM with no loss in sensitivity of the RT-rtPCR assay in detecting HPAI or LPAI viruses, and HPAI virus could be isolated from a greater proportion of swabs stored in VTM compared to PBS: glycerol without antibiotics.

Efficacy of a plant-produced virus-like particle vaccine in chickens challenged with Influenza A H6N2 virus.

The efficacy, safety, speed, scalability and cost-effectiveness of producing hemagglutinin-based virus-like particle (VLP) vaccines in plants are well-established for human influenza, but untested for the massive poultry influenza vaccine market that remains dominated by traditional egg-grown oil-emulsion whole inactivated virus vaccines. For optimal efficacy, a vaccine should be closely antigenically matched to the field strain, requiring that influenza A vaccines be updated regularly. In this study, an H6 subtype VLP transiently expressed in Nicotiana benthamiana was formulated into a vaccine and evaluated for efficacy in chickens against challenge with a heterologous H6N2 virus. A single dose of the plant-produced H6 VLP vaccine elicited an immune response comparable to two doses of a commercial inactivated H6N2 vaccine, with mean hemagglutination inhibition titres of 9.3 log2 and 8.8 log2 , respectively. Compared to the non-vaccinated control, the H6 VLP vaccine significantly reduced the proportion of shedders and the magnitude of viral shedding by >100-fold in the oropharynx and >6-fold in the cloaca, and shortened oropharyngeal viral shedding by at least a week. Despite its potency, the cost of the antigenic mismatch between the inactivated H6N2 vaccine and challenge strain was evident not only in this vaccine's failure to reduce viral shedding compared to the non-vaccinated group, but its apparent exacerbation of oropharyngeal viral shedding until 21 days post-challenge. We estimate that a kilogram of plant leaf material can produce H6 VLP vaccines sufficient for between 5000 and 30 000 chickens, depending on the effective dose and whether one or two immunizations are administered.

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.

Continuing evolution of H6N2 influenza a virus in South African chickens and the implications for diagnosis and control.

BACKGROUND: The threat of poultry-origin H6 avian influenza viruses to human health emphasizes the importance of monitoring their evolution. South Africa's H6N2 epidemic in chickens began in 2001 and two co-circulating antigenic sub-lineages of H6N2 could be distinguished from the outset. The true incidence and prevalence of H6N2 in the country has been difficult to determine, partly due to the continued use of an inactivated whole virus H6N2 vaccine and the inability to distinguish vaccinated from non-vaccinated birds on serology tests. In the present study, the complete genomes of 12 H6N2 viruses isolated from various farming systems between September 2015 and February 2019 in three major chicken-producing regions were analysed and a serological experiment was used to demonstrate the effects of antigenic mismatch in diagnostic tests. RESULTS: Genetic drift in H6N2 continued and antigenic diversity in sub-lineage I is increasing; no sub-lineage II viruses were detected. Reassortment patterns indicated epidemiological connections between provinces as well as different farming systems, but there was no reassortment with wild bird or ostrich influenza viruses. The sequence mismatch between the official antigens used for routine hemagglutination inhibition (HI) testing and circulating field strains has increased steadily, and we demonstrated that H6N2 field infections are likely to be missed. More concerning, sub-lineage I H6N2 viruses acquired three of the nine HA mutations associated with human receptor-binding preference (A13S, V187D and A193N) since 2002. Most sub-lineage I viruses isolated since 2015 acquired the K702R mutation in PB2 associated with the ability to infect humans, whereas prior to 2015 most viruses in sub-lineages I and II contained the avian lysine marker. All strains had an unusual HA0 motif of PQVETRGIF or PQVGTRGIF. CONCLUSIONS: The H6N2 viruses in South African chickens are mutating and reassorting amongst themselves but have remained a genetically pure lineage since they emerged more than 18 years ago. Greater efforts must be made by government and industry in the continuous isolation and characterization of field strains for use as HI antigens, new vaccine seed strains and to monitor the zoonotic threat of H6N2 viruses.

Antimicrobial Resistance Trends in Escherichia coli in South African Poultry: 2009-2015.

Prolonged and widespread in-feed use of antimicrobials as either growth promoters or to treat bacterial infections in commercial poultry production contributed to the emergence of resistant bacterial strains globally. A total of 3544 avian pathogenic Escherichia coli strains isolated from commercial broilers in South Africa between 2009 and 2015 were tested for susceptibility against eight classes of antimicrobials. Time series analyses were conducted to assess seasonal and general trends in antibiotic resistance. Seasonal trends were seen in the tetracyclines, with peaks of resistance in the winter months when respiratory diseases are at their worst. Resistance to quinolones peaked in 2012 after which there was an overall decreasing trend in resistance. Colistin resistance increased gradually from 2009 with a drastic rise to 12.08% in 2015, but its use in feed was stopped in 2016. Florfenicol also showed a sharp increase in resistance from 2.36% in 2009 to 6.63% in 2015. Resistance to trimethoprim-sulphadiazine decreased sharply by the end of 2015, as did spectinomycin and fosfomycin and amoxicillin. The overall prevalence of multidrug resistance (MDR) was 80.6 (95% confidence interval, 0.743-0.819), but the years 2013, 2014, and 2015 showed a significantly lower level of MDR compared with 2009. This study is the first detailed analysis of antimicrobial resistance in poultry production in the country, and constant monitoring of resistance data should be continued to aid in the judicious use of antimicrobial compounds.

Evidence for Multidrug Resistance in Nonpathogenic Mycoplasma Species Isolated from South African Poultry.

One hundred seventy-eight mycoplasma strains isolated from South African poultry flocks between 2003 and 2015 were identified by full-genome sequencing and phylogenetic analysis of the 16S rRNA gene and were classified as follows: Mycoplasma gallisepticum (25%), M. gallinarum (25%), M. gallinaceum, (23%), M. pullorum (14%), M. synoviae (10%), and M. iners (3%), as well as one Acheoplasma laidlawii strain (1%). MIC testing was performed on the axenic samples, and numerous strains of each species were resistant to either chlortetracycline or tylosin or both, with variable sensitivity to enrofloxacin. The strains of all species tested remained sensitive to tiamulin, except for one M. gallinaceum sample that demonstrated intermediate sensitivity. The mutation of A to G at position 2059 (A2059G) in the 23S rRNA gene, which is associated with macrolide resistance, was found in the South African M. gallisepticum and M. synoviae strains, as well as a clear correlation between macrolide resistance in M. gallinarum and M. gallinaceum and mutations G354A and G748A in the L4 ribosomal protein and 23S rRNA gene, respectively. No correlation between resistance and point mutations in the genes studied could be found for M. pullorum Only a few strains were resistant to enrofloxacin, apart from one M. synoviae strain with point mutation D420N, which has been associated with quinolone resistance, and no other known markers for quinolone resistance were found in this study. Proportionally more antimicrobial-resistant strains were detected in M. gallinaceum, M. gallinarum, and M. pullorum than in M. gallisepticum and M. synoviae Of concern, three M. gallinaceum strains showed multidrug resistance to chlortetracycline, tylosin, and oxytetracycline.IMPORTANCE Nonpathogenic poultry Mycoplasma species are often overlooked due to their lesser impact on poultry health and production compared to the OIE-listed pathogenic strains M. gallisepticum and M. synoviae The use of antimicrobials as in-feed growth promoters and for the control of mycoplasmosis is common in poultry production across the world. Here, we provide evidence that certain nonpathogenic Mycoplasma species are acquiring multidrug resistance traits. This would have significant implications if these species, for which no vaccines are applied, are able to transfer their antibiotic resistance genes to other mycoplasmas and bacteria that may enter the human food chain.

Assessment of Mycoplasma gallisepticum vaccine efficacy in a co-infection challenge model with QX-like infectious bronchitis virus.

Mycoplasma gallisepticum (MG) is the primary cause of chronic respiratory disease in poultry. We investigated the protective efficacy of the live-attenuated ts-11 and 6/85 MG vaccines against a local MG strain and, in order to enhance signs and mimic a typical field situation, we co-infected birds with a virulent strain of QX-like infectious bronchitis virus (IBV). Both vaccines showed similar ability to protect infected chickens from clinical signs, although ts-11 performed slightly better. Despite the lower protection against clinical disease, 6/85-vaccinated birds had significantly (P ≤ 0.05) lower tracheal lesion scores and mucosal thickness at day 28 post-vaccination (7 days post-challenge [dpc] with MG, 2 dpc IBV) and day 31 post-vaccination (10 dpc MG challenge, 5 dpc IBV) compared to ts-11 vaccinated birds, but these difference was not significant at day 33 (12 dpc MG, 7 dpc IBV). Pathogen infection and replication was assessed by qPCR, and the 6/85 vaccine produced a more significant (P ≤ 0.05) reduction in MG replication in the lungs, kidneys and livers but enhanced late replication in bursae and caecal tonsils. In contrast, the ts-11 vaccine had a more pronounced reductive effect on replication in tracheas, air sacs, bursae and heart at days 28 and 31, yet increased replication in lungs. Interestingly, both vaccines provided non-specific protection against IBV challenge. The co-challenge model provided useful data on vaccine efficacy, especially on days 31 and 33, and tracheas, lungs, air sacs, kidneys, liver and caecal tonsils were the best organs to assess.

Evolution of H5 highly pathogenic avian influenza: sequence data indicate stepwise changes in the cleavage site.

The genetic composition of an H5 subtype hemagglutinin gene quasispecies, obtained from ostrich tissues that had been infected with H5 subtype influenza virus was analysed using a next generation sequencing approach. The first evidence for the reiterative copying of a poly (U) stretch in the connecting peptide region in the haemagglutinin cleavage site (HACS) by the viral RNA-dependent RNA polymerase (RdRp) is provided. Multiple non-consensus species of RNA were detected in the infected host, corresponding to likely intermediate sequences between the putative low pathogenic precursor nucleotide sequence of the H5 influenza strain and the highly pathogenic avian influenza virus gene sequence. In silico analysis of the identified RNA sequences predicted that the intermediary H5 sequence PQREKRGLF plays an important role in subsequent mutational events that relocate the HACS coding region from stable base-paired RNA regions to a single-stranded bulge, thereby priming the connecting peptide coding region for RdRp slippage.

Virus genome dynamics under different propagation pressures: reconstruction of whole genome haplotypes of West Nile viruses from NGS data.

BACKGROUND: Extensive focus is placed on the comparative analyses of consensus genotypes in the study of West Nile virus (WNV) emergence. Few studies account for genetic change in the underlying WNV quasispecies population variants. These variants are not discernable in the consensus genome at the time of emergence, and the maintenance of mutation-selection equilibria of population variants is greatly underestimated. The emergence of lineage 1 WNV strains has been studied extensively, but recent epidemics caused by lineage 2 WNV strains in Hungary, Austria, Greece and Italy emphasizes the increasing importance of this lineage to public health. In this study we explored the quasispecies dynamics of minority variants that contribute to cell-tropism and host determination, i.e. the ability to infect different cell types or cells from different species from Next Generation Sequencing (NGS) data of a historic lineage 2 WNV strain. RESULTS: Minority variants contributing to host cell membrane association persist in the viral population without contributing to the genetic change in the consensus genome. Minority variants are shown to maintain a stable mutation-selection equilibrium under positive selection, particularly in the capsid gene region. CONCLUSIONS: This study is the first to infer positive selection and the persistence of WNV haplotype variants that contribute to viral fitness without accompanying genetic change in the consensus genotype, documented solely from NGS sequence data. The approach used in this study streamlines the experimental design seeking viral minority variants accurately from NGS data whilst minimizing the influence of associated sequence error.

Avian gyrovirus 2 and avirulent Newcastle disease virus coinfection in a chicken flock with neurologic symptoms and high mortalities.

A disease with severe neurologic symptoms caused 100% mortality in a small broiler operation in the Gauteng Province, South Africa in late March 2013. Routine diagnostic PCR testing failed to identify a possible cause of the outbreak; thus, samples were submitted for virus isolation, serology, and bacteriology. An avirulent Newcastle disease virus (NDV) strain isolated was identified as a V4-like genotype 1 strain, by DNA sequencing, with a cleavage site of 112GKQGR decrease L117. Real-time reverse transcription PCR identified NDV in the brain but not in cecal tonsils or pooled tracheas, spleens, lungs, and livers. A random amplification deep sequencing of a transcriptome library generated from pooled tissues produced 927,966 paired-end reads. A contig of 2,309 nucleotides was identified as a near-complete avian gyrovirus 2 (AGV2) genome. This is the first report on the African continent of AGV2, which has been reported in southern Brazil, The Netherlands, and Hong Kong thus far. A real-time PCR for AGV2 only detected the virus in the brain but not in cecal tonsils or pooled tracheas, spleens, lungs, and livers. Sequence reads also mapped to the genomes of mycoplasma, Escherichia coli, avian leukosis virus subtype J, and Marek's disease virus but excluded influenza A virus, Ornithobacterium rhinotracheale, avian rhinotracheitis virus, avian encephalomyelitis virus, and West Nile virus. Air sac swabs were positive on bacterial culture for E. coli. The possibility of a synergistic pathogenic effect between avirulent NDV and AGV2 requires further investigation.

Extended survival times of Mycoplasma gallisepticum and Mycoplasma synoviae on kanekalon synthetic hair fibres.

The survival times of Mycoplasma gallisepticum (Mg) and Mycoplasma synoviae (Ms) on washed and unwashed natural and synthetic kanekalon hair samples over a 5-d period were evaluated using the color changing unit method for comparison with results of previous studies conducted on natural hair. Regardless of whether synthetic or natural hair samples prewashed with a disinfectant shampoo were spiked with Mg or Ms, all viable organisms rapidly dropped below a count of 1 × 10(1)/mL of culture. Unwashed natural hair seeded with a titer of approximately 1 × 10(6)/mL of viable Mg or Ms decreased to 6 × 10(5)/mL and 6 × 10(3)/mL, respectively, by 4 h postseeding, but no viable Mg or Ms were detected on natural hair from 8 h onwards. By contrast, the titers of Mg and Ms on synthetic hair did not decline from the initial 1 × 10(6)/mL seed dose up to 96 h postseeding, and, in fact, viable Mg and Ms was still detectable at 9 d postinfection. Application of a real-time quantitative single-tube duplex PCR assay confirmed that no proliferation of Mg or Ms had occurred on the synthetic hair samples, the cells simply remained viable. The unexpected finding that Mg and Ms survive for extended periods on synthetic kanekalon hair fibers raises the question of whether attachment to a surface is a prerequisite for the survival and persistence of Mg and Ms in the extra-host environment. Future studies should be aimed at determining whether other synthetic hair types or indeed other types of plastics commonly found in the poultry house offer similar survival advantages to mycoplasmas.

Spillover of an endemic avian Influenza H6N2 chicken lineage to ostriches and reassortment with clade 2.3.4.4b H5N1 high pathogenicity viruses in chickens.

Prior to 2017, chicken production in South Africa had only ever been affected by an endemic strain of H6N2 low pathogenic avian influenza (LPAI), but since 2017, an outbreak of Goose/Guangdong clade 2.3.4.4b H5N8 high pathogenicity avian influenza (HPAI) introduced by wild birds, followed by clade 2.3.4.4b H5N1 HPAI (2021-present), affected the country. In the present study, the viruses from seven cases of H6N2 LPAI from commercial poultry between October 2019 and August 2020 were genome-sequenced along with an H5N2 HPAI virus, and phylogenetic analysis was performed. The H5N2 HPAI virus caused localized outbreaks in a small-scale chicken farm and a large commercial layer farm in the KwaZulu-Natal province between late October and early December 2022. The phylogenetic results confirmed the first incidence of the chicken-adapted H6N2 lineage in commercial ostriches in the Western Cape province, with a likely epidemiological origin in chickens from the KwaZulu Natal province. The results also showed that the H5N2 HPAI virus was a novel reassortant of PB2, PB1, PA, NP and NA genome segments derived from a parental H6N2 virus that circulated in region, whereas the HA, M and NS genome segments were derived from sub-genotype SA10 H5N1 HPAI parental virus that had circulated in the local wild bird reservoir since July 2021.

Outbreaks of H5N1 High Pathogenicity Avian Influenza in South Africa in 2023 Were Caused by Two Distinct Sub-Genotypes of Clade 2.3.4.4b Viruses.

In 2023, South Africa continued to experience sporadic cases of clade 2.3.4.4b H5N1 high-pathogenicity avian influenza (HPAI) in coastal seabirds and poultry. Active environmental surveillance determined that H5Nx, H7Nx, H9Nx, H11Nx, H6N2, and H12N2, amongst other unidentified subtypes, circulated in wild birds and ostriches in 2023, but that H5Nx was predominant. Genome sequencing and phylogenetic analysis of confirmed H5N1 HPAI cases determined that only two of the fifteen sub-genotypes that circulated in South Africa in 2021-2022 still persisted in 2023. Sub-genotype SA13 remained restricted to coastal seabirds, with accelerated mutations observed in the neuraminidase protein. SA15 caused the chicken outbreaks, but outbreaks in the Paardeberg and George areas, in the Western Cape province, and the Camperdown region of the KwaZulu-Natal province were unrelated to each other, implicating wild birds as the source. All SA15 viruses contained a truncation in the PB1-F2 gene, but in the Western Cape SA15 chicken viruses, PA-X was putatively expressed as a novel isoform with eight additional amino acids. South African clade 2.3.4.4b H5N1 viruses had comparatively fewer markers of virulence and pathogenicity compared to European strains, a possible reason why no spillover to mammals has occurred here yet.

Vaccination of African penguins (Spheniscus demersus) against high-pathogenicity avian influenza.

BACKGROUND: High-pathogenicity avian influenza (HPAI) has become a conservation threat to wild birds. Therefore, suitable vaccine technology and practical application methods require investigation. METHODS: Twenty-four African penguins (Spheniscus demersus) were vaccinated with either a conventional inactivated clade 2.3.4.4b H5N8 HPAI whole virus or a tobacco leaf-produced H5 haemagglutinin-based virus-like particle (VLP). Six birds received a second dose of the inactivated vaccine. Antibody responses were assessed and compared by employing haemagglutination inhibition tests. RESULTS: A second dose of inactivated vaccine was required to induce antibody titres above the level required to suppress virus shedding, while a single dose of VLP vaccine produced these levels by day 14, and one bird still had antibodies on day 430. LIMITATIONS: Bacterial contamination of the VLP vaccine limited the monitoring period and sample size in that treatment group, and it was not possible to perform a challenge study with field virus. CONCLUSION: VLP vaccines offer a more practical option than inactivated whole viruses, especially in logistically challenging situations involving wild birds.

Protective efficacy of a plant-produced beta variant rSARS-CoV-2 VLP vaccine in golden Syrian hamsters.

In the quest for heightened protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants, we engineered a prototype vaccine utilizing the plant expression system of Nicotiana benthamiana, to produce a recombinant SARS-CoV-2 virus-like particle (VLP) vaccine presenting the S-protein from the Beta (B.1.351) variant of concern (VOC). This innovative vaccine, formulated with either a squalene oil-in-water emulsion or a synthetic CpG oligodeoxynucleotide adjuvant, demonstrated efficacy in a golden Syrian Hamster challenge model. The Beta VLP vaccine induced a robust humoral immune response, with serum exhibiting neutralization not only against SARS-CoV-2 Beta but also cross-neutralizing Delta and Omicron pseudoviruses. Protective efficacy was demonstrated, evidenced by reduced viral RNA copies and mitigated weight loss and lung damage compared to controls. This compelling data instills confidence in the creation of a versatile platform for the local manufacturing of potential pan-sarbecovirus vaccines, against evolving viral threats.

Serological investigation of highly pathogenic avian influenza H5N2 in ostriches (Struthio camelus).

An ostrich farm of 929 birds that tested polymerase chain reaction-positive for highly pathogenic avian influenza H5N2 in a single sample was designated for culling, despite no evidence of sero-conversion as assessed by haemagglutination inhibition (HI) tests. A month later and immediately prior to culling, all birds were bled and tested with an IDEXX avian influenza virus (AIV) nucleoprotein (NP)-specific enzyme-linked immunosorbent assay (ELISA) and a high sero-prevalence was detected. To address the question of whether the NP-specific antibodies detected indicated exposure to H5 or non-H5 subtypes (H6N2 and H1N2 strains were also circulating regionally at the time), we developed two H5-specific ELISAs, both based on a recombinant H5 HA1 antigen. The H5 indirect ELISA used a horseradish peroxidase ostrich IgY conjugate that we produced in chicken eggs. The single-chain variable fragment (scFv) competitive ELISA (H5 scFv cELISA) used a scFv derived from an H5-immune chicken scFv library. By comparing IDEXX AIV ELISA results with those of the two H5-specific ELISAs and HI tests, we determined that up to 89% of the flock had been exposed to H5N2 AIV. We also detected evidence of suspected vaccination, since 17% of sera contained antibodies against the H5 glycoprotein but not the NP protein. Comparative analytical sensitivity indicated that HI tests are likely to miss up to 35% of H5-positive samples, and thus we consider that H5/H7-specific ELISAs should replace HI tests for ostrich testing in future.

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.

Serological and molecular investigation of Newcastle disease in household chicken flocks and associated markets in Eastern Shewa zone, Ethiopia.

Cross-sectional survey for Newcastle disease (ND) were conducted in nonvaccinated household flocks of village chickens to assess serological and virological ND status in households and associated live bird markets. In total, 1,899 sera and 460 pools of cloacal and tracheal swabs were sampled and tested using a commercial enzyme-linked immunosorbent assay (ELISA) and real-time reverse transcriptase polymerase chain reaction (rRT-PCR), respectively. Additionally, paired cloacal and tracheal swabs from 1,269 individual chickens were collected from markets and tested using RT-PCR. The prevalence of households with at least one seropositive chicken was higher during the dry season (27.4 %) than during the wet season (17.4 %) (P = 0.003). Viral genome was detected in 14.2 % of households during the wet season using a fusion (F) gene assay and in 24.2 % of households during the dry season using a polymerase (L) gene assay that targets both class I and class II viruses. At the markets sampled, overall bird level prevalence was 4.9 % for period 1 (F gene assay), and 38.2 % and 27.6 % for periods 2 and 3, respectively (L gene assay). Partial sequencing of the F gene (239 bp) cleavage site indicated that the majority of the circulating strains exhibited motifs specific to virulent strains. Seroepidemiology coupled with molecular analysis can be a useful tool to assess the status of NDV infection. The village chicken population in Ethiopia is endemically infected with virulent NDV that pose a significant threat to emerging small- and medium-scale commercial poultry production.

Salmonella gallinarum strains from outbreaks of fowl typhoid fever in Southern Africa closely related to SG9R vaccines.

Introduction: Salmonella enterica subspecies enterica serovar Gallinarum biovar Gallinarum (SG) is associated with fowl typhoid fever, and the attenuated rough strain SG9R is widely used as a vaccine in many regions. Reversion to virulence of vaccine strains was suspected as the cause during recent fowl typhoid fever outbreaks in poultry in South Africa and Eswatini. Methods: To compare nine field isolates with global wild-type SG9 strains and the two commercial SG9R vaccines in use, Nobilis® SG9R and Cevac®-SG, we used whole-genome comparison with single-nucleotide polymorphism (SNP) detection. Results: SNP phylogenic analysis showed that all the southern African field isolates were more closely related to the vaccine strains than wild-type SG9 strains. Furthermore, SNPs in the pyruvate dehydrogenase (aceE) and/or lipopolysaccharide 1,2-glucosyltransferase (rfaJ) genes, which are known markers of attenuation, were found in four of the field isolates along with intact spv, SPI-1, and SPI-2 gene clusters, providing conclusive evidence that these four isolates were originally vaccine strains that reverted to virulence. Five other field isolates lacked the SG9R attenuation markers, but variant analysis identified an SNP in the yihX gene, insertions in the ybjX and hydH genes, and deletions in the ftsK and sadA genes that were shared between the field isolates and vaccine strains but absent in wild-type SG9, indicating that these field isolates were also likely revertant vaccines. Discussion: Overall, this study highlights different mechanisms of reversion of two commercial vaccines, where virulence caused by field isolates closely related to the Nobilis® SG9R vaccine was associated with the restoration of intact virulence gene clusters, and those derived from the Cevac®-SG vaccine were characterized by point mutations resulting in restored aceE and rfaJ genes. A possible new marker of attenuation was identified as a point mutation in the yihX gene, as well as four new candidate genes that could potentially be used to distinguish current vaccine strains from wild-type strains using PCR assays.

The production of Newcastle disease virus-like particles in Nicotiana benthamiana as potential vaccines.

Newcastle disease (ND) is a highly contagious viral respiratory and neurological disease that has a severe impact on poultry production worldwide. In the present study, an expression platform was established for the transient production in N.bethamiana of ND virus-like particles (VLPs) for use as vaccines against ND. The expression of the ND Fusion (F) and/or Hemagglutinin-neuraminidase (HN) proteins of a genotype VII.2 strain formed ND VLPs in planta as visualized under the transmission electron microscope, and HN-containing VLPs agglutinated chicken erythrocytes with hemagglutination (HA) titres of up to 13 log2.The immunogenicity of the partially-purified ND VLPs was confirmed in specific-pathogen-free White leghorn chickens. Birds receiving a single intramuscular immunization with 1024 HA units (10 log2) of the F/HN ND VLPs administered with 20% [v/v] Emulsigen®-P adjuvant, seroconverted after 14 days with F- and HN-specific antibodies at ELISA titres of 5705.17 and HI geometric mean titres (GMTs) of 6.2 log2, respectively. Furthermore, these ND-specific antibodies successfully inhibited viral replication in vitro of two antigenically closely-related ND virus isolates, with virus-neutralization test GMTs of 3.47 and 3.4, respectively. Plant-produced ND VLPs have great potential as antigen-matched vaccines for poultry and other avian species that are highly immunogenic, cost-effective, and facilitate prompt updating to ensure improved protection against emerging ND field viruses.