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Newcastle disease is regarded as one of the most important avian diseases throughout the world and continues to be a threat and economic burden to the poultry industry. With no effective treatment, poultry producers rely primarily on stringent biosecurity and vaccination regimens to control the spread of this devastating disease. This concise review provides an historical perspective of Newcastle disease vaccination and how fundamental research has paved the way for the development of instrumental techniques which are still in use today. Although vaccination programmes have reduced the impact of clinical disease, they have historically been ineffective in controlling the spread of virulent viruses and therefore do not always offer an adequate solution to the world’s food security problems. However, the continued development of novel vaccine technology and improved biosecurity measures through education may offer a solution to help reduce the global threat of Newcastle disease on the poultry industry.

Vaccination of livestock plays a major role in improving animal health, welfare and productivity, but also in public health by preventing zoonotic diseases. Advances in bioinformatics and whole-genome sequencing techniques since the 2000s have led to the development of genome-based vaccinology, called reverse vaccinology. Reverse vaccinology is a rapid and competitive strategy that uses pathogen genome sequences to screen for and identify potential vaccine antigens and, unlike conventional methods, does not require culturing the pathogenic microorganism, at least initially. Based on in silico approaches and dedicated software, reverse vaccinology has led to the identification of a wide range of proteins as putative vaccine candidates against human pathogens and has been applied more recently to several animal diseases. After a brief overview of the principle of the approach and its applications in human medicine, this review focuses on the use of reverse vaccinology for the development of vaccines specifically for poultry, a representative example of livestock vaccination, and discusses the important points to consider when using this method. •Poultry vaccines are often composed of inactivated or attenuated pathogens.•The development of subunit vaccines requires knowing which antigens are immunogenic.•Reverse vaccinology capitalises on progress in genome sequencing and bioinformatics.•Vaccine antigens are screened in silico for use against poultry diseases.•Single-strain, pangenomic and subtractive approaches help design new poultry vaccines.

Inactivated poultry vaccines are subject to routine potency testing for batch release, requiring large numbers of animals. The replacement of in vivo tests for cell-based alternatives can be facilitated by the identification of biomarkers for vaccine-induced immune responses. In this study, chicken bone marrow-derived dendritic cells were stimulated with an inactivated vaccine for infectious bronchitis virus and Newcastle disease virus, as well as inactivated infectious bronchitis virus only, and lipopolysaccharides as positive control, or left unstimulated for comparison with the stimulated samples. Next, the cells were lysed and subjected to proteomic analysis. Stimulation with the vaccine resulted in 66 differentially expressed proteins associated with mRNA translation, immune responses, lipid metabolism and the proteasome. For the eight most significantly upregulated proteins, mRNA expression levels were assessed. Markers that showed increased expression at both mRNA and protein levels included PLIN2 and PSMB1. Stimulation with infectious bronchitis virus only resulted in 25 differentially expressed proteins, which were mostly proteins containing Src homology 2 domains. Stimulation with lipopolysaccharides resulted in 118 differentially expressed proteins associated with dendritic cell maturation and antimicrobial activity. This study provides leads to a better understanding of the activation of dendritic cells by an inactivated poultry vaccine, and identified PLIN2 and PSMB1 as potential biomarkers for cell-based potency testing.

•A prime-boost vaccine strategy protects turkeys against a clade 2.3.4.4 H5N2 HPAIV.•A recombinant alphavirus replicon produced protective responses.•An adjuvanted-inactivated influenza virus vaccine produced protection.•HI titers were discernible by 3weeks post-boost.•HI titers were drastically reduced by 13weeks post-boost.•Best overall protective responses were achieved by alternating vaccine strategies. Highly pathogenic avian influenza virus (HPAIV) infections are frequently associated with systemic disease and high mortality in domestic poultry, particularly in chickens and turkeys. Clade 2.3.4.4 represents a genetic cluster within the Asian HPAIV H5 Goose/Guangdong lineage that has transmitted through migratory birds and spread throughout the world. In 2014, clade 2.3.4.4 strains entered the U.S. via the Pacific flyway, reassorted with local strains of the North American lineage, and produced novel HPAIV strains of the H5N1, H5N2, and H5N8 subtypes. By 2015, the H5N2 HPAIVs disseminated eastwards within the continental U.S. and Canada and infected commercial poultry, causing the largest animal health outbreak in recent history in the U.S. The outbreak was controlled by traditional mass depopulation methods, but the outbreak was of such magnitude that it led to the consideration of alternative control measures, including vaccination. In this regard, little information is available on the long-term protection of turkeys vaccinated against avian influenza. In this report, a vaccination study was carried out in turkeys using 3 prime-boost approaches with a combination of 2 different vaccines, an alphavirus-based replicon vaccine and an adjuvanted-inactivated reverse genetics vaccine. Vaccine efficacy was assessed at 6 and 16weeks of age following challenge with a prototypic novel clade 2.3.4.4 H5N2 HPAIV. All three vaccines protocols were protective with significantly reduced virus shedding and mortality after challenge at 6weeks of age. In contrast, significant variations were seen in 16-week old turkeys after challenge: priming with the alphavirus-based replicon followed by boost with the adjuvanted-inactivated vaccine conferred the best protection, whereas the alphavirus-based replicon vaccine given twice provided the least protection. Our study highlights the importance of studying not only different vaccine platforms but also vaccination strategies to maximize protection against HPAIV especially with regards to the longevity of vaccine-induced immune response.

Raising backyard poultry under low biosecurity conditions is a common practice in Egypt. While vaccination is routinely applied in Egypt in commercial settings to curb the spread of avian influenza viruses, it remains less commonly used in backyard settings. We assessed the immunogenicity and protective efficacy of a H5N1 vaccine based on a contemporary Egyptian clade 2.2.1.2 virus among turkeys, ducks, geese, and chickens raised together in a backyard setting. Results showed that this vaccine elicits an immune response in all tested species reaching up to a hemagglutination inhibition titer of 10 log2 after a booster dose. However, this response varied between species. When challenged, vaccinated birds survived and shed less virus in comparison with unvaccinated birds. However, unvaccinated ducks showed no symptoms of infection and survived the duration of the experiment. Moreover, vaccinated ducks shed more virus as compared to vaccinated birds of other species. Hence, we recommend avoiding mixing various species in the backyards of Egypt. Our data indicates that vaccination can be effective in the backyard setting in Egypt, although planning should consider the species covered.

Chickens, turkeys, and other poultry in a production environment can be exposed to stressors and infectious diseases that impair innate and acquired immunity, erode general health and welfare, and diminish genetic and nutritional potential for efficient production. Innate immunity can be affected by stressful physiologic events related to hatching and to environmental factors during the first week of life. Exposure to environmental ammonia, foodborne mycotoxins, and suboptimal nutrition can diminish innate immunity. Infectious bursal disease (IBD), chicken infectious anemia (CIA), and Marek's disease (MD) are major infectious diseases that increase susceptibility to viral, bacterial, and parasitic diseases and interfere with acquired vaccinal immunity. A shared feature is lymphocytolytic infection capable of suppressing both humoral and cell-mediated immune functions. Enteric viral infections can be accompanied by atrophic and depleted lymphoid organs, but the immunosuppressive features are modestly characterized. Some reoviruses cause atrophy of lymphoid organs and replicate in blood monocytes. Enteric parvoviruses of chickens and turkeys merit further study for immunosuppression. Hemorrhagic enteritis of turkeys has immunosuppressive features similar to IBD. Other virulent fowl adenoviruses have immunosuppressive capabilities. Newcastle disease can damage lymphoid tissues and macrophages. Avian pneumovirus infections impair the mucociliary functions of the upper respiratory tract and augment deeper bacterial infections. Recognition of immunosuppression involves detection of specific diseases using diagnostic tests such as serology, etiologic agent detection, and pathology. Broader measurements of immunosuppression by combined noninfectious and infectious causes have not found general application. Microarray technology to detect genetic expression of immunologic mediators and receptors offers potential advances but is currently at the developmental state. Control methods for immunosuppressive diseases rely largely on minimizing stress, reducing exposure to infectious agents through biosecurity, and increasing host resistance to infectious immunosuppressive diseases by vaccination. A longer term approach involves genetic selection for resistance to immunosuppressive diseases, which has shown promising results for MD but equivocal results for IBD and CIA. Abbreviations: BF = bursa of Fabricius; CAV = chicken anemia virus; CIA = chicken infectious anemia; FAV = fowl adenovirus; HE = hemorrhagic enteritis; HEV = hemorrhagic enteritis virus; HVT = turkey herpesvirus; IBD = infectious bursal disease; IBDV = infectious bursal disease virus; IBV = infectious bronchitis virus; MD = Marek's disease; MDV = Marek's disease virus; MHC = major histocompatibility complex; NDV = Newcastle disease virus; ORT = Ornithobacterium rhinotracheale; PEMS = poultry enteritis and mortality syndrome; REV = reticuloendotheliosis virus; RSS = runting stunting syndrome; SE = Salmonella enterica serovar Enteritidis; ST = Salmonella Typhimurium

Abstract Stress is a biological adaptive response to restore homeostasis, and occurs in every animal production system, due to the multitude of stressors present in every farm. Heat stress is one of the most common environmental challenges to poultry worldwide. It has been extensively demonstrated that heat stress negatively impacts the health, welfare, and productivity of broilers and laying hens. However, basic mechanisms associated with the reported effects of heat stress are still not fully understood. The adaptive response of poultry to a heat stress situation is complex and intricate in nature, and it includes effects on the intestinal tract. This review offers an objective overview of the scientific evidence available on the effects of the heat stress response on different facets of the intestinal tract of poultry, including its physiology, integrity, immunology, and microbiota. Although a lot of knowledge has been generated, many gaps persist. The development of standardized models is crucial to be able to better compare and extrapolate results. By better understanding how the intestinal tract is affected in birds subjected to heat stress conditions, more targeted interventions can be developed and applied.

•HPAI H5N1 viruses undergo antigenic changes.•These antigenic changes may lead to loss of protection against poultry vaccines.•The needs to monitor the H5N1 viruses’ susceptibility to vaccine induced immunity.•A lack of serologic HI reactivity may not always mean lack of vaccine protection.•There is a need for challenge studies in vaccinated poultry. Highly pathogenic avian influenza (HPAI) H5N1 virus continues to circulate in poultry in Asia and Africa posing a threat to both public and animal health. Vaccination, used as an adjunct to improved bio-security and stamping-out policies, contributed to protecting poultry in Hong Kong from HPAI H5N1 infection in 2004–2008 although the virus was repeatedly detected in dead wild birds. The detection of clade 2.3.4 H5N1 viruses in poultry markets and a farm in Hong Kong in 2008 raised the question whether this virus has changed to evade protection from the H5 vaccines in use. We tested the efficacy of three commercial vaccines (Nobilis, Poulvac and Harbin Re-5 vaccine) in specific pathogen free white leghorn chickens against a challenge with A/chicken/Hong Kong/8825-2/2008 (clade 2.3.4) isolated from vaccinated poultry in Hong Kong and A/chicken/Hong Kong/782/2009 (clade 2.3.2). Harbin Re5 vaccine provided the best, albeit not complete protection against challenge with the clade 2.3.4 virus. All three vaccines provided good protection from death and significantly reduced virus shedding following challenge with the clade 2.3.2 virus. Only Harbin Re-5 was able to completely protect chickens from virus shedding as well as mortality. Sera from vaccinated chickens had lower geometric hemagglutination inhibition titers against A/chicken/Hong Kong/8825-2/08, as compared to two other clade 2.3.4 and one clade 0 virus. Alignment of amino-acid sequences of the haemagglutinin of A/chicken/Hong Kong/8825-2/08 and the other H5 viruses revealed several mutations in positions including 69, 71, 83, 95, 133,140, 162, 183, 189, 194 and 270 (H5 numbering) which may correlate with loss of vaccine protection. Our results indicated that the tested HPAI H5N1 (2.3.4) virus has undergone antigenic changes that allow it to evade immunity from poultry vaccines. This highlights the need for continued surveillance and monitoring of vaccine induced immunity, with experimental vaccine challenge studies being done where indicated.

The influenza A virus is of global concern for the poultry industry, especially the H5 and H7 subtypes as they have the potential to become highly pathogenic for poultry. In this study, the hemagglutinin (HA) of a low pathogenic avian influenza virus of the H7N7 subtype isolated from a Swedish mallard Anas platyrhynchos was sequenced, characterized and transiently expressed in Nicotiana benthamiana. Recently, plant expression systems have gained interest as an alternative for the production of vaccine antigens. To examine the possibility of expressing the HA protein in N. benthamiana, a cDNA fragment encoding the HA gene was synthesized de novo, modified with a Kozak sequence, a PR1a signal peptide, a C-terminal hexahistidine (6×His) tag, and an endoplasmic retention signal (SEKDEL). The construct was cloned into a Cowpea mosaic virus (CPMV)-based vector (pEAQ-HT) and the resulting pEAQ-HT-HA plasmid, along with a vector (pJL3:p19) containing the viral gene-silencing suppressor p19 from Tomato bushy stunt virus, was agro-infiltrated into N. benthamiana. The highest gene expression of recombinant plant-produced, uncleaved HA (rHA0), as measured by quantitative real-time PCR was detected at 6 days post infiltration (dpi). Guided by the gene expression profile, rHA0 protein was extracted at 6 dpi and subsequently purified utilizing the 6×His tag and immobilized metal ion adsorption chromatography. The yield was 0.2 g purified protein per kg fresh weight of leaves. Further molecular characterizations showed that the purified rHA0 protein was N-glycosylated and its identity confirmed by liquid chromatography-tandem mass spectrometry. In addition, the purified rHA0 exhibited hemagglutination and hemagglutination inhibition activity indicating that the rHA0 shares structural and functional properties with native HA protein of H7 influenza virus. Our results indicate that rHA0 maintained its native antigenicity and specificity, providing a good source of vaccine antigen to induce immune response in poultry species.

Necrotic enteritis is an economically important poultry disease caused by the bacterium Clostridium perfringens. There are currently no necrotic enteritis vaccines commercially available for use in broiler birds, the most important target population. Salmonella-vectored vaccines represent a convenient and effective option for controlling this disease. We used a single attenuated Salmonella vaccine strain, engineered to lyse within the host, to deliver up to three C. perfringens antigens. Two of the antigens were toxoids, based on C. perfringens α-toxin and NetB toxin. The third antigen was fructose-1,6-bisphosphate aldolase (Fba), a metabolic enzyme with an unknown role in virulence. Oral immunization with a single Salmonella vaccine strain producing either Fba, α-toxoid and NetB toxoid, or all three antigens, was immunogenic, inducing serum, cellular and mucosal responses against Salmonella and the vectored C. perfringens antigens. All three vaccine strains were partially protective against virulent C. perfringens challenge. The strains delivering Fba only or all three antigens provided the best protection. We also demonstrate that both toxins and Fba are present on the C. perfringens cell surface. The presence of Fba on the cell surface suggests that Fba may function as an adhesin.