Coinfeccio´n entre el gyrovirus aviar 2 y el virus de la enfermedad de Newcastle avirulento en una parvada de pollo de engorde con signos neurolo´gicos y alta mortalidad

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 112GKQGRQL117. 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.Una enfermedad con signos neurolo´gicos graves causo´ una mortalidad del 100 % en una operacio´n pequen˜a de pollos de engorde en la provincia de Gauteng, en Suda´frica a finales de marzo del 2013. Las pruebas rutinarias de diagno´stico por PCR no lograron identificar una posible causa del brote, por lo que las muestras fueron sometidas al aislamiento viral, serologı´a y bacteriologı´a. Se aislo´ e identifico´ un virus de la enfermedad de Newcastle no virulento (NDV) como una cepa similar al genotipo 1 V4, por secuenciacio´n de ADN, en el sitio de disociacio´n 112GKQGR Q L117. Mediante un me´todo de transcripcio´n reversa y PCR en tiempo real se identifico´ la presencia del virus de Newcastle en el cerebro, pero no en las tonsilas cecales o en las muestras agrupadas de tra´quea, bazo, pulmones, e hı´gado. Una amplificacio´n con secuenciacio´n profunda y aleatoria de una biblioteca de transcriptoma generada a partir de muestras agrupadas de tejidos produjo 927,966 lecturas emparejadas. Se identifico´ un contig de 2309 nucleo´tidos como un genoma casi completo de un Gyrovirus aviar 2 (AGV2). Este es el primer informe en el continente africano de la presencia del AGV2, que se ha reportado hasta el momento en el sur de Brasil, los Paı´ses Bajos y Hong Kong. Un me´todo de PCR en tiempo real para AGV2 so´lo detecto´ al virus en el cerebro, pero no se detecto´ en las tonsilas cecales, o en las muestras agrupadas de tra´quea, bazos, pulmones e hı´gado. Las lecturas de las secuencias tambie´n se relacionaron con el genoma de mycoplasma, Escherichia coli, con el virus de la leucosis aviar subtipo J, y con el virus de la enfermedad de Marek, pero excluyo´ al virus de la influenza A, Ornithobacterium rhinotracheale, al virus de la rinotraqueı´tis aviar, al virus de la encefalomielitis aviar y al virus del Nilo Occidental. Los hisopos de sacos ae´reos fueron positivos para el cultivo bacteriano de E. coli. La posibilidad de un efecto patoge´nico sine´rgico entre el virus de Newcastle avirulento y el AGV2 requiere de ma´s investigacio´n.http://www.aaapjournals.info/loi/avdiam2014ab201

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, sublineage 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.Additional file 1: Table S1a. Percentage nucleotide sequence identity in the HA genes of sub-lineage I viruses isolated since 2015.Additional file 2: Table S1b. Percentage amino sequence identity in the HA proteins of all sub-lineage I viruses.Additional file 3: Table S1c. Amino acid between-group distances.Additional file 4: Figure S1. Alignment of the hemagglutinin protein sequences of South African H6N2 isolates from chickens.Additional file 5: Table S2. Predicted glycosylation patterns in the surface glycoproteins of H6N2 influenza viruses isolated since 2015.Additional file 6: Figure S2. Alignment of the neuraminidase protein sequences of South African H6N2 isolates from chickens.Additional file 7: Figure S3. Alignment of the polymerase B2 (PB2) protein sequences of South African H6N2 isolates from chickens.This work was supported by the South African Department of Science and Technology /National Research Foundation’s South African Research Chair Initiative under grant No. 114612.The South African Department of Science and Technology /National Research Foundation’s South African Research Chair Initiative.https://bmcvetres.biomedcentral.comam2020Production Animal Studie

Dose immunogenicity study of a plant-produced influenza virus-like particle vaccine in layer hens

Avian influenza poses one of the largest known threats to global poultry production and human health, but effective poultry vaccines can reduce infections rates, production losses and prevent mortalities, and reduce viral shed to limit further disease spread. The antigenic match between a vaccine and the circulating field influenza A viruses (IAV) is a critical determinant of vaccine efficacy. Here, an Agrobacterium tumefaciens-mediated transient tobacco plant (Nicotiana benthamiana) system was used to rapidly update an H6 influenza subtype virus-like particle (VLP) vaccine expressing the hemagglutinin (HA) protein of South African H6N2 IAVs circulating in 2020. Specific pathogen free White Leghorn layer hens vaccinated twice with ≥125 hemagglutinating unit (HAU) doses elicited protective antibody responses associated with prevention of viral shedding, i.e. hemagglutination inhibition (HI) mean geometric titres (GMTs) of ≥7 log2, for at least four months before dropping to approximately 5–6 log2 for at least another two months. A single vaccination with a 250 HAU dose induced significantly higher HI GMTs compared lower or higher doses, and was thus the optimal dose for chickens. Use of an adjuvant was essential, as the plant-produced H6 HA VLP alone did not induce protective antibody responses. Plant-produced IAV VLPs enable differentiation between vaccinated and infected animals (DIVA principle), and with sucrose density gradient-purified yields of 20,000 doses per kg of plant material, this highly efficacious, safe and economical technology holds enormous potential for improving poultry health in lower and middle-income countries.DATA AVAILABILITY STATEMENT: Data included in article/supp. material/referenced in article.Department of Science and Innovation/National Research Foundation SARChI grants.https://www.cell.com/heliyon/homeProduction Animal Studie

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.The Technology Innovation Agency-Tshwane Animal Health Innovation Cluster Initiative (grant number TAHC12-00034). A.B. was funded by the Health and Welfare Sector Education and Training Authority, University of Pretoria, and the National Research Foundation.http://aem.asm.orgam2019Production Animal StudiesVeterinary Tropical Disease

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 egggrown 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.Figure S1 Multiple sequence alignment of the hemagglutinin (HA) proteins of the strains used in this study.Figure S2 LC-MS/MS-based peptide sequence analysis for SDSPAGE bands of approximately 62 kDa (A) and 14 kDa (B), respectively.Table S1 Pairwise amino acid distances of the hemagglutinin proteins of H6N2 strains used in the study.Table S2 qRT-PCR results for oropharyngeal swabs as log10 vRNA viral titres/mL, with EID50/mL titres in parenthesis.Table S3 qRT-PCR results for cloacal swabs as log10 vRNA viral titres/mL, with EID50/mL titres in parenthesis.The Department of Science and Technology (DST) – National Research Foundation (NRF) South African Research Chair Initiative Grant No. 114612. TS was funded by the CSIR/DST Inter-bursary fund and a UP doctoral bursary. MA was funded by a NRF postdoctoral bursary.http://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1467-7652am2020Production Animal Studie

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.DGB was a recipient of a University of Pretoria scholarship.http://www.tandfonline.com/loi/cavp202019-03-14hj2018Paraclinical SciencesProduction Animal Studie

Experimental infection of ostriches with H7N1 low pathogenic and H5N8 clade 2.3.4.4B highly pathogenic influenza A viruses

Please read abstract in the article.SUPPLEMENTARY MATERIAL: Figure S1. PCR detection of mycoplasma DNA in the tracheal swabs of bird #12 (a) and bird #16 (b) presented as examples. Complete PCR results are presented in Supplemental tables 2 and 4Supplemental table 1(a). H7N1 LPAI- challenged ostriches. Supplemental table 1(b). H7N1 LPAI-contact ostriches.Supplemental Table 2 (a). Detection of Mycoplasma in the tracheal swabs of ostriches challenged with H7N1 LPAI. Supplemental Table 2 (b). Detection of Mycoplasma in the tracheal swabs of ostriches challenged with H5N8 HPAI.Supplemental table 3(a). H5N8 HPAI- challenged ostriches. Supplemental table 3(b). H5N8 HPAI- contact ostriches.Supplemental table 4(a). Virus detected in the wing feather pulp of ostriches infected with H7N1 LPAI. Supplemental table 4(b). Virus detected in the wing feather pulp of ostriches infected with H5N8 HPAI. Supplemental table 4(c). Virus detected in the tail feather pulp of ostriches infected with H5N8 HPAI.NRF-DSI SARChI grant and a DTI-Exotic Leather Cluster grant- “Healthy flocks- Quality leather”.http://www.elsevier.com/locate/vetmic2022-10-14hj2022Production Animal Studie