Epidemiology-driven approaches to surveillance in HPAI-vaccinated poultry flocks aiming to demonstrate freedom from circulating HPAIV.

Incursion pressure of high pathogenicity avian influenza viruses (HPAIV) by secondary spread among poultry holdings and/or from infected migratory wild bird populations increases worldwide. Vaccination as an additional layer of protection of poultry holdings using appropriately matched vaccines aims at reducing clinical sequelae of HPAIV infection, disrupting HPAIV transmission, curtailing economic losses and animal welfare problems and cutting exposure risks of zoonotic HPAIV at the avian-human interface. Products derived from HPAIV-vaccinated poultry should not impose any risk of virus spread or exposure. Vaccination can be carried out with zero-tolerance for infection in vaccinated herds and must then be flanked by appropriate surveillance which requires tailoring at several levels: (i) Controlling appropriate vaccination coverage and adequate population immunity in individual flocks and across vaccinated populations; (ii) assessing HPAI-infection trends in unvaccinated and vaccinated parts of the poultry population to provide early detection of new/re-emerged HPAIV outbreaks; and (iii) proving absence of HPAIV circulation in vaccinated flocks ideally by real time-monitoring. Surveillance strategies, i.e. selecting targets, tools and random sample sizes, must be accommodated to the specific epidemiologic and socio-economic background. Methodological approaches and practical examples from three countries or territories applying AI vaccination under different circumstances are reviewed here.

Molecular characterization of the HMTp210 gene of Avibacterium paragallinarum and the proposition of a new genotyping method as alternative for classical serotyping.

Avibacterium paragallinarum (A. paragallinarum) is the aetiological agent of infectious coryza (IC) in chickens and characterized by acute respiratory distress and severe drop in egg production. Vaccination is important in the control of IC outbreaks and the efficacy of vaccination is dependent on A. paragallinarum serovars included in the vaccine. Classical serotyping of A. paragallinarum is laborious and hampered by poor availability of antigens and antisera. The haemagglutinin, important in classical serotyping, is encoded by the HMTp210 gene. HMTp210 gene analysis has been shown to have potential as alternative to classical serotyping. The aim of the present study was to further investigate the potential of sequence analyses of partial region 1 of the HMTp210 gene, the HMTp210 hypervariable region and the concatenated sequences of both fragments. For this analysis, 123 HMTp210 gene sequences (field isolates, A. paragallinarum serovar reference strains and vaccine strains) were included. Evaluation of serovar references and vaccine strains revealed a need for critical evaluation, especially within Page serovar B and C. Phylogenetic analysis of HMTp210 region 1 resulted in a separation of Page serovar A, B and C strains. Analysis of the HMTp210 HVR alone was not sufficient to discriminate all nine different Kume serovar references. The concatenated sequences of HMTp210 region 1 and HMTp210 HVR resulted in 14 clusters with a high correlation with Page serovar and with the nine currently known Kume serovars and is therefore proposed as a novel genotyping method that could be used as an alternative for classical serotyping of A. paragallinarum.

The DECIDE project: from surveillance data to decision-support for farmers and veterinarians.

Farmers, veterinarians and other animal health managers in the livestock sector are currently missing sufficient information on prevalence and burden of contagious endemic animal diseases. They need adequate tools for risk assessment and prioritization of control measures for these diseases. The DECIDE project develops data-driven decision-support tools, which present (i) robust and early signals of disease emergence and options for diagnostic confirmation; and (ii) options for controlling the disease along with their implications in terms of disease spread, economic burden and animal welfare. DECIDE focuses on respiratory and gastro-intestinal syndromes in the three most important terrestrial livestock species (pigs, poultry, cattle) and on reduced growth and mortality in two of the most important aquaculture species (salmon and trout). For each of these, we (i) identify the stakeholder needs; (ii) determine the burden of disease and costs of control measures; (iii) develop data sharing frameworks based on federated data access and meta-information sharing; (iv) build multivariate and multi-level models for creating early warning systems; and (v) rank interventions based on multiple criteria. Together, all of this forms decision-support tools to be integrated in existing farm management systems wherever possible and to be evaluated in several pilot implementations in farms across Europe. The results of DECIDE lead to improved use of surveillance data and evidence-based decisions on disease control. Improved disease control is essential for a sustainable food chain in Europe with increased animal health and welfare and that protects human health.

Infectious Bronchitis Coronavirus: Genome Evolution in Vaccinated and Non-Vaccinated SPF Chickens.

Infectious Bronchitis virus (IBV) continues to cause significant economic losses for the chicken industry despite the use of many live IBV vaccines around the world. Several authors have suggested that vaccine-induced partial protection may contribute to the emergence of new IBV strains. In order to study this hypothesis, three passages of a challenge IBV were made in SPF chickens sham inoculated or vaccinated at day of age using a live vaccine heterologous to the challenge virus. All birds that were challenged with vaccine heterologous virus were positive for viral RNA. NGS analysis of viral RNA in the unvaccinated group showed a rapid selection of seven genetic variants, finally modifying the consensus genome of the viral population. Among them, five were non-synonymous, modifying one position in NSP 8, one in NSP 13, and three in the Spike protein. In the vaccinated group, one genetic variant was selected over the three passages. This synonymous modification was absent from the unvaccinated group. Under these conditions, the genome population of an IBV challenge virus evolved rapidly in both heterologous vaccinated and non-vaccinated birds, while the genetic changes that were selected and the locations of these were very different between the two groups.

Clinical expression, epidemiology, and monitoring of Mycoplasma gallisepticum and Mycoplasma synoviae: an update.

Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) are of clinical and economic importance for the global poultry industry. Many countries and integrations are involved in monitoring programmes to control both mycoplasma species. This review provides an extensive historic overview of the last seven decades on the development of the knowledge regarding the factors that influence the clinical expression of the disease, the epidemiology, and monitoring of both MG and MS. This includes the detection of new virulent strains, studies unravelling the transmission routes, survival characteristics, and the role of other avian hosts. Also the role of molecular typing tests in unravelling epidemiology and factors that complicate the interpretation of test results is discussed. The latter includes the presence of heterologous mycoplasma infections, the use of heterologous oil-emulsion vaccines, and the use of antibiotic treatments. Also the occurrence of MG and MS strains with low virulence and the use of live and/or inactivated MS and MS vaccines are discussed.

High specificity of the Salmonella Pullorum/Gallinarum rapid plate agglutination test despite vaccinations against Salmonella Enteritidis and Salmonella Typhimurium.

In Europe, monitoring of breeding stock for Salmonella Pullorum (SP) or Salmonella Gallinarum (SG) infections is compulsory at the point of lay. Vaccinations against Salmonella Enteritidis (SE) and Salmonella Typhimurium (ST) are increasingly administered in Europe. These vaccines might induce cross-reactions in the rapid plate agglutination (RPA) SP/SG test due to shared O-antigens, possibly resulting in a lower test specificity. The extent to which the specificity of SP/SG serological tests is influenced by SE and/or ST vaccinations in the field has not been reported. In this paper, we report the diagnostic and flock specificity of the commercially available RPA SP/SG test using 1:2-1:16 serum dilutions on four panels of sera: SPF sera, field sera from flocks of varying age and SE/ST vaccination status, and reference sera from an international proficiency testing scheme. The results showed that the use of live SE/ST vaccines did not influence the specificity of the RPA SP/SG test. Inactivated vaccines showed a drop of the diagnostic specificity to 96.54% and a flock specificity of 34.1% when the 1:2 serum dilution was used. The 1:8 serum dilution showed a diagnostic specificity of 99.41% and a flock specificity of 86.4%. In conclusion, the use of SE/ST vaccines has either no effect or a modest effect on the specificity of the RPA SP/SG test used to monitor flocks. The main factors are the type of vaccine, and the serum dilution used for testing and a cut-off.

Identification, HPG2 Sequence Analysis, and Antimicrobial Susceptibility of Avibacterium paragallinarum Isolates Obtained from Outbreaks of Infectious Coryza in Commercial Layers in Sonora State, Mexico.

This is the first extensive report on the identification and characterization of Avibacterium paragallinarum (AVP) isolates obtained from outbreaks of infectious coryza (IC) in IC-vaccinated layer flocks from Sonora State in Mexico. Isolates obtained from IC outbreaks during the years 2007, 2014, 2015, 2017, and 2019 were identified by conventional PCR test and 16S rRNA gene analysis, serotyped by Page serotyping and genotyped by the recently described partial sequence analysis of the HPG2 region. Furthermore, antimicrobial susceptibility profiles were determined by a recently improved minimal inhibitory concentration (MIC) test. The conventional PCR test and the 16S rRNA analyses confirmed the isolates as AVP. Serotyping results showed the involvement of isolates belonging to serotypes A, B, and C in the IC outbreaks. Genotyping of the HPG2 region revealed the presence of sequence type (ST)1, ST4, and ST11, of which the latter has also been identified in Europe. The MIC susceptibility test showed that all tested isolates were susceptible for the majority of tested antimicrobials, including erythromycin and tetracycline, which are important antibiotics for the treatment of IC. The IC situation in Sonora State, Mexico, is complex because of the presence of serotypes A, B, and C. This finding emphasizes the importance of biosecurity in combination with the application of the most optimal vaccination programs in the control of IC in Sonora State, Mexico.

Nota de investigación­Análisis de secuencias de la región HPG2 y susceptibilidad antimicrobiana de aislamientos de Avibacterium paragallinarum obtenidos de brotes de coriza infecciosa en aves de postura comerciales en el estado de Sonora, México. Este es el primer informe extenso sobre la identificación y caracterización de aislamientos de Avibacterium paragallinarum (AVP) obtenidos de brotes de coriza infecciosa (IC) de parvadas de ponedoras vacunadas con coriza infecciosa en el estado de Sonora en México. Los aislamientos obtenidos de los brotes de coriza infecciosa durante los años 2007, 2014, 2015, 2017 y 2019 se identificaron mediante una prueba de PCR convencional y el análisis del gene de ARNr 16S, se serotipificaron mediante el método de Page y se genotipificaron mediante el análisis parcial de secuencias descrito recientemente de la región HPG2. Además, se determinaron los perfiles de susceptibilidad a los antimicrobianos mediante la prueba de concentración mínima inhibitoria (MIC) que ha sido mejorada recientemente. La prueba de PCR convencional y los análisis de secuencias del gene ARNr 16S confirmaron que los aislados eran A. paragallinarum. Los resultados de la serotipificación mostraron la participación de aislamientos pertenecientes a los serotipos A, B y C en los brotes de coriza infecciosa. La genotipificación de la región HPG2 reveló la presencia de secuencias del tipo (ST) 1, ST4 y ST11, de los cuales este último también ha sido identificada en Europa. La prueba de susceptibilidad por concentración mínima inhibitoria mostró que todos los aislados analizados eran susceptibles a la mayoría de los antimicrobianos analizados, incluida la eritromicina y la tetraciclina, que son antibióticos importantes para el tratamiento contra la coriza infecciosa. La situación de coriza infecciosa en el estado de Sonora, México, es compleja por la presencia de los serotipos A, B y C. Este hallazgo enfatiza la importancia de la bioseguridad en combinación con la aplicación de los programas de vacunación óptimos en el control de la coriza infecciosa en el estado de Sonora, México.

Rate of false positive reactions in 11 M. gallisepticum and M. synoviae serological tests in samples obtained from SPF birds inoculated with heterologous mycoplasma species.

No recent information is available on the specificity of current M. synoviae (Ms) and M. gallisepticum (Mg) serological tests. In this study the performance of a currently available Mg and Ms Rapid Plate Agglutination (RPA) test, and three Mg, three Ms and three Mg/Ms combination ELISAs were evaluated on SPF sera that were obtained from days (D) 0-28 after M. gallinarum, M. imitans or M. gallinaceum inoculation, after sham inoculation and without inoculation. Tracheal swabs for mycoplasma culture were obtained before inoculation (D0), 7 and 28 days post inoculation (d.p.i.) in all groups except the sham inoculated group. The different mycoplasma species colonized well. In the early stage after inoculation (7-14 d.p.i.) with heterologous mycoplasma species, the specificity varied from 85% to 100% in the Mg RPA test and from 70% to 85% in the Ms RPA test. The specificity of both Mg and Ms RPA test was 100% in the sham inoculated samples and ruled out the effect of sham medium. In the late stage (21-28 d.p.i.) specificity was 100% for both RPA tests. The test specificity was 100% for seven ELISAs except for two combination ELISAs where a specificity of 95% was found in the late stage after inoculation. However, this was not significantly different from the specificity of all other tests in the late stage of these groups. These results show that it is not advisable to establish Mg and Ms seromonitoring programmes on the Mg and Ms RPA test alone as other mycoplasma species frequently occur in poultry.

Identification and characterization of Dutch Avibacterium paragallinarum isolates and the implications for diagnostics.

This study reports the results of diagnostic and molecular typing methods for 18 Avibacterium paragallinarum isolates obtained from outbreaks of infectious coryza in commercial layer flocks in the Netherlands. Isolation, biochemical identification, species-specific PCR tests and classical serotyping were performed. In addition, molecular typing by Enterobacterial Repetitive Intergenic Consensus-Based Polymerase Chain Reaction (ERIC-PCR) and sequence analysis of the partial HPG2 region of A. paragallinarum were applied and results of both techniques were compared. Moreover, the pathogenicity of an isolate of the most common genotype detected in the Netherlands was determined in an animal experiment. All 18 Avibacterium isolates were nicotinamide adenine dinucleotide-dependent. All isolates were detected by the species-specific conventional PCR while 33% of the isolates were missed by the species-specific real-time PCR. Sequence analysis showed a probe mismatch as a result of a single nucleotide polymorphism (G1516A). Modification of the probe of the real-time PCR was necessary to overcome false negative results. Molecular typing showed that sequence analysis of the partial HPG2 region was in concordance with ERIC-PCR results and indicated the presence of two major genotypes. Serotyping showed the presence of serovars A-1, A-2 and B-1. There was no correlation between genotyping results and serotyping results. Inoculation of an isolate of the most prevalent genotype, and belonging to serovar A-1, into brown layer hens demonstrated the pathogenicity of this isolate.

Recombinant Infectious Bronchitis Viruses Expressing Chimeric Spike Glycoproteins Induce Partial Protective Immunity against Homologous Challenge despite Limited Replication In Vivo.

Vaccination regimes against Infectious bronchitis virus (IBV), which are based on a single virus serotype, often induce insufficient levels of cross-protection against serotypes and two or more antigenically diverse vaccines are used in attempt to provide broader protection. Amino acid differences in the surface protein, spike (S), in particular the S1 subunit, are associated with poor cross-protection. Here, homologous vaccination trials with recombinant IBVs (rIBVs), based on the apathogenic strain, BeauR, were conducted to elucidate the role of S1 in protection. A single vaccination of specific-pathogen-free chickens with rIBV expressing S1 of virulent strains M41 or QX, BeauR-M41(S1) and BeauR-QX(S1), gave incomplete protection against homologous challenge, based on ciliary activity and clinical signs. There could be conformational issues with the spike if heterologous S1 and S2 are linked, suggesting a homologous S2 might be essential. To address this, a homologous vaccination-challenge trial incorporating rIBVs expressing full spike from M41, BeauR-M41(S), and S2 subunit from M41, BeauR-M41(S2) was conducted. All chimeric viruses grew to similar titers in vitro, induced virus-specific partial protective immunity, evident by cellular infiltrations, reductions in viral RNA load in the trachea and conjunctiva and higher serum anti-IBV titers. Collectively, these findings show that vaccination with rIBVs primed the birds for challenge but the viruses were cleared rapidly from the mucosal tissues in the head. Chimeric S1 and S2 viruses did not protect as effectively as BeauR-M41(S) based on ciliary activity and clinical signs. Booster vaccinations and an rIBV with improved in vivo replication may improve the levels of protection.IMPORTANCE Infectious bronchitis virus causes an acute, highly contagious respiratory disease, responsible for significant economic losses to the poultry industry. Amino acid differences in the surface protein, spike (S), in particular the S1 subunit, have been associated with poor cross-protection. Available vaccines give poor cross-protection and rationally designed live attenuated vaccines, based on apathogenic BeauR, could address these. Here, to determine the role of S1 in protection, a series of homologous vaccination trials with rIBVs were conducted. Single vaccinations with chimeric rIBVs induced virus-specific partial protective immunity, characterized by reduction in viral load and serum antibody titers. However, BeauR-M41(S) was the only vaccination to improve the level of protection against clinical signs and the loss of tracheal ciliary activity. Growth characteristics show that all of the rIBVs replicated in vitro to similar levels. Booster vaccinations and an rIBV with improved in vivo replication may improve the levels of protection.