Evaluation of bird-adapted self-amplifying mRNA vaccine formulations in chickens.

Each year, millions of poultry succumb to highly pathogenic avian influenza A virus (AIV) and infectious bursal disease virus (IBDV) infections. Conventional vaccines based on inactivated or live-attenuated viruses are useful tools for disease prevention and control, yet, they often fall short in terms of safety, efficacy, and development times. Therefore, versatile vaccine platforms are crucial to protect poultry from emerging viral pathogens. Self-amplifying (replicon) RNA vaccines offer a well-defined and scalable option for the protection of both animals and humans. The best-studied replicon platform, based on the Venezuelan equine encephalitis virus (VEEV; family Togaviridae) TC-83 vaccine strain, however, displays limited efficacy in poultry, warranting the exploration of alternative, avian-adapted, replicon platforms. In this study, we engineered two Tembusu virus (TMUV; family Flaviviridae) replicons encoding varying capsid gene lengths and compared these to the benchmark VEEV replicon in vitro. The TMUV replicon system exhibited a robust and prolonged transgene expression compared to the VEEV replicon system in both avian and mammalian cells. Moreover, the TMUV replicon induced a lesser cytopathic effect compared to the VEEV replicon RNA in vitro. DNA-launched versions of the TMUV and VEEV replicons (DREP) were also developed. The replicons successfully expressed the AIV haemagglutinin (HA) glycoproteins and the IBDV capsid protein (pVP2). To assess the immune responses elicited by the TMUV replicon system in chickens, a prime-boost vaccination trial was conducted using lipid nanoparticle (LNP)-formulated replicon RNA and DREP encoding the viral (glyco)proteins of AIV or IBDV. Both TMUV and VEEV replicon RNAs were unable to induce a humoral response against AIV. However, TMUV replicon RNA induced IBDV-specific seroconversion in vaccinated chickens, in contrast to VEEV replicon RNA, which showed no significant humoral response. In both AIV and IBDV immunization studies, VEEV DREP generated the highest (neutralizing) antibody responses, which underscores the potential for self-amplifying mRNA vaccine technology to combat emerging poultry diseases.

Investigating the uses of machine learning algorithms to inform risk factor analyses: The example of avian infectious bronchitis virus (IBV) in broiler chickens.

Infectious bronchitis virus (IBV) is a contagious coronavirus causing respiratory and urogenital disease in chickens and is responsible for significant economic losses for both the broiler and table egg layer industries. Despite IBV being regularly monitored using standard epidemiologic surveillance practices, knowledge and evidence of risk factors associated with IBV transmission remain limited. The study objective was to compare risk factor modeling outcomes between a traditional stepwise variable selection approach and a machine learning-based random forest Boruta algorithm using routinely collected IBV antibody titer data from broiler flocks. IBV antibody sampling events (n = 1111) from 166 broiler sites between 2016 and 2021 were accessed. Ninety-two geospatial-related and poultry-density variables were obtained using a geographic information system and data sets from publicly available sources. Seventeen and 27 candidate variables were screened to potentially have an association with elevated IBV antibody titers according to the manual selection and machine learning algorithm, respectively. Selected variables from both methods were further investigated by construction of multivariable generalized mixed logistic regression models. Six variables were shortlisted by both screening methods, which included year, distance to urban areas, main roads, landcover, density of layer sites and year, however, final models for both approaches only shared year as an important predictor. Despite limited significance of clinical outcomes, this work showcases the potential of a novel explorative modeling approach in combination with often unutilized resources such as publicly available geospatial data, surveillance health data and machine learning as potential supplementary tools to investigate risk factors related to infectious diseases.

Molecular characterization of the meq oncogene of Marek's disease virus in vaccinated Brazilian poultry farms reveals selective pressure on prevalent strains.

Marek's disease virus (MDV) has become an increasingly virulent pathogen in the poultry industry despite vaccination efforts to control it. Brazil has experienced a significant rise of Marek's disease (MD) outbreaks in recent years. Our study aimed to analyze the complete meq gene sequences to understand the molecular epidemiological basis of MD outbreaks in Brazilian vaccinated layer farms. We detected a high incidence rate of visceral MD (67.74%) and multiple circulating MDV strains. The most prevalent and geographically widespread genotype presented several clinical and molecular characteristics of a highly virulent strain and evolving under positive selective pressure. Phylogenetic and phylogeographic analysis revealed a closer relationship with strains from the USA and Japan. This study sheds light on the circulation of MDV strains capable of infecting vaccinated birds. We emphasize the urgency of adopting preventive measures to manage MDV outbreaks threatening the poultry farming industry.

A systematic review and meta-analysis on the efficacy of vaccination against colibacillosis in broiler production.

Colibacillosis, a disease caused by Escherichia coli in broiler chickens has serious implications on food safety, security, and economic sustainability. Antibiotics are required for treating the disease, while vaccination and biosecurity are used for its prevention. This systematic review and meta-analysis, conducted under the COST Action CA18217-European Network for Optimization of Veterinary Antimicrobial Treatment (ENOVAT), aimed to assess the efficacy of E. coli vaccination in broiler production and provide evidence-based recommendations. A comprehensive search of bibliographic databases, including, PubMed, CAB Abstracts, Web of Science and Agricola, yielded 2,722 articles. Following a defined protocol, 39 studies were selected for data extraction. Most of the studies were experimental infection trials, with only three field studies identified, underscoring the need for more field-based research. The selected studies reported various types of vaccines, including killed (n = 5), subunit (n = 8), outer membrane vesicles/protein-based (n = 4), live/live-attenuated (n = 16), and CpG oligodeoxynucleotides (ODN) (n = 6) vaccines. The risk of bias assessment revealed that a significant proportion of studies reporting mortality (92.3%) or feed conversion ratio (94.8%) as outcomes, had "unclear" regarding bias. The meta-analysis, focused on live-attenuated and CpG ODN vaccines, demonstrated a significant trend favoring both vaccination types in reducing mortality. However, the review also highlighted the challenges in reproducing colibacillosis in experimental setups, due to considerable variation in challenge models involving different routes of infection, predisposing factors, and challenge doses. This highlights the need for standardizing the challenge model to facilitate comparisons between studies and ensure consistent evaluation of vaccine candidates. While progress has been made in the development of E. coli vaccines for broilers, further research is needed to address concerns such as limited heterologous protection, practicability for application, evaluation of efficacy in field conditions and adoption of novel approaches.

Research Note: Clostridium perfringens NetB and CnaA neutralizing nanobodies in feed reduce the incidence of poultry necrotic enteritis.

Necrotic enteritis is a devastating disease to poultry caused by the bacterium Clostridium perfringens. As a novel approach to combating poultry necrotic enteritis, we identified and characterized several hundred single domain antibody fragments (or nanobodies) capable of binding either the NetB toxin or the collagen-binding adhesin (CnaA) of C. perfringens. Many of the nanobodies could neutralize the in vitro functions of NetB or CnaA with inhibitory concentrations in the nanomolar range. The nanobodies were also screened for proteolytic stability in an extract derived from gastrointestinal tract fluids of chickens. A collection of 6 nanobodies (4 targeting NetB and 2 targeting CnaA) with high neutralizing activity and high gastrointestinal tract extract stability were expressed and secreted by Pichia pastoris or Bacillus subtilis. Chickens were given a feed with 1 of the 2 nanobody-containing groups: 1) nanobody-containing P. pastoris supernatants that were semi-purified, lyophilized, and enterically coated, or 2) B. subtilis spores from strains containing the nanobody genes. Compared to untreated chickens (23.75% mortality), mortality of chickens receiving feed modified with the P. pastoris and B. subtilis products decreased to 11.25 and 7.5%, respectively. These results offer a new opportunity to improve the control of poultry necrotic enteritis by incorporating highly specific nanobodies or bacteria expressing these nanobodies directly into chicken feed.

Bromelain can reduce the negative effects of a subclinical necrotic enteritis in broiler chickens.

This study was conducted to examine the efficacy of a bromelain-based supplementation coded ANR-pf on growth performance and intestinal lesion of broiler chickens under necrotic enteritis (NE) challenge. A total of 540 Ross 308 day-old male chicks were randomly allocated into 6 treatments of 6 replicates. The bromelain formulation was delivered to chickens through gavaging or in drinking water method twice, on d 8 and 13. Nonchallenged groups included 1) without or 2) with the specific bromelain formulation gavaged at 0.8 mL/kg. NE-challenged groups included 3) without the specific bromelain formulation; 4) gavaged with 0.4 mL/kg; 5) gavaged with 0.8 mL/kg and 6) supplemented with 0.8 mL/kg via drinking water. Birds were challenged with Eimeria spp. on d 9 and Clostridium perfringens (NE-18 strain) on d 14 and 15. On d 14 and 19, fresh faecal contents were collected for the determination of oocyst counts. Intestinal lesion scores were determined on d16. Performance and mortality were recorded throughout the entire experiment. Among challenged groups, birds received additive via drinking water had higher weight gain (WG) compared to the remaining groups (P < 0.001) in the grower phase and had lower FCR compared to 0.4 mL/kg inoculated group in the grower and finisher phases (P < 0.001). Bromelain supplementation via drinking water improved the WG of challenged birds, similar to that of the nonchallenged birds (P < 0.001), and lowered FCR compared to other challenged groups (P < 0.001). Nonchallenged birds and birds that received bromelain formulation in drinking water did not have lesions throughout the small intestine whereas challenged birds, either un-supplemented or supplemented with bromelain via inoculation route recorded similar lesion score levels in the jejunum. At d 19, birds received bromelain in drinking water had lower fecal oocyst numbers compared to challenged birds without additive (P < 0.001). In conclusion, bromelain administration via drinking water could ameliorate the negative impacts of NE-infection in broilers by improving performance, lowering the oocyst numbers and lesion scores.

Alpha-sitosterol: a new antiviral agent produced by Streptomyces misakiensis and its potential activity against Newcastle disease virus.

BACKGROUND: Newcastle Disease Virus (NDV) causes severe economic losses in the poultry industry worldwide. Hence, this study aimed to discover a novel bioactive antiviral agent for controlling NDV. Streptomyces misakiensis was isolated from Egyptian soil and its secondary metabolites were identified using infrared spectroscopy (IR), gas chromatography-mass spectrometry (GC-MS), and nuclear magnetic resonance (NMR) spectroscopy. The inhibitory activity of bioactive metabolite against NDV were examined. Three experimental groups of 10-day-old specific pathogen-free embryonated chicken eggs (SPF-ECEs), including the bioactive metabolite control group, NDV control positive group, and α-sitosterol and NDV mixture-treated group were inoculated. RESULTS: α-sitosterol (Ethyl-6-methylheptan-2-yl]-10,13-dimethyl-dodecahydro-1H-cyclopenta[a]phenanthren-3-ol), a secondary metabolite of S. misakiensis, completely inhibited hemagglutination (HA) activity of the NDV strain. The HA activity of the NDV strain was 8 log2 and 9 log2 for 0.5 and 0.75% RBCs, respectively. The NDV HA activity for the two concentrations of RBCs was significantly (P < 0.0001) inhibited after α-sitosterol treatment. There was a significant (P < 0.0001) decrease in the log 2 of HA activity, with values of - 0.500 (75%, chicken RBCs) before inoculation in SPF-ECEs and - 1.161 (50%, RBCs) and - 1.403 (75%, RBCs) following SPF-ECE inoculation. Compared to ECEs inoculated with NDV alone, the α-sitosterol-treated group showed improvement in histological lesion ratings for chorioallantoic membranes (CAM) and hepatic tissues. The CAM of the α-sitosterol- inoculated SPF-ECEs was preserved. The epithelial and stromal layers were noticeably thicker with extensive hemorrhages, clogged vasculatures, and certain inflammatory cells in the stroma layer in the NDV group. However, mild edema and inflammatory cell infiltration were observed in the CAM of the treated group. ECEs inoculated with α-sitosterol alone showed normal histology of the hepatic acini, central veins, and portal triads. Severe degenerative alterations, including steatosis, clogged sinusoids, and central veins, were observed in ECEs inoculated with NDV. Mild hepatic degenerative alterations, with perivascular round cell infiltration, were observed in the treated group. CONCLUSION: To the best of our knowledge, this is the first study to highlight that the potentially bioactive secondary metabolite, α-sitosterol, belonging to the terpene family, has the potential to be a biological weapon against virulent NDV. It could be used for the development of innovative antiviral drugs to control NDV after further clinical investigation.

Investigating bacteriophages as a novel multiple-hurdle measure against Campylobacter: field trials in commercial broiler plants.

Campylobacter mitigation along the food production chain is considered effective for minimizing the public health burden of human campylobacteriosis. This study is the first combining different measures in a multiple-hurdle approach, using drinking water additives and feed additives in single and combined application schemes in commercial broiler plants. Broiler chickens in the study groups were naturally contaminated with Campylobacter. Application of an organic acid blend via drinking water, consisting of sodium propionate, potassium sorbate, and sodium diacetate, resulted in significant reductions of up to 4.9 log10 CFU/mL in fecal samples and in cecal samples at slaughter. The application of a phage mixture, consisting of Fletchervirus phage NCTC 12673 and Firehammervirus phage vB_CcM-LmqsCPL1/1, resulted in reductions of up to 1.1 log10 CFU/mL in fecal samples 1 day after dosing. The sole administration of curcumin via feed resulted in small and inconsistent reductions. In the group receiving a combination of all tested measures, reductions of up to 1.1 log10 CFU/mL were observed. Based on the results of our field trials, it was shown that both the sole application and the combined application of mitigation measures in primary production can reduce the Campylobacter load in broiler chickens, while no synergism could be observed.

Genetic comparison of the Mycoplasma gallisepticum 6/85 vaccine strain and 6/85-like field isolates.

Mycoplasma gallisepticum infection in poultry leads to disease and pathology that can reduce producer profits. Live attenuated vaccines are available that can limit or completely prevent the effects of infection. Field isolates that are genetically related to the attenuated vaccine strains have been isolated, raising the question of whether the attenuation of the vaccine strains is limited and can lead the strains to revert to more virulent forms. The 6/85 live attenuated vaccine is derived from a field isolate collected in the United States. Analysis of the genome of sequenced M. gallisepticum strains revealed a cluster of 10 6/85-like strains that group with the 6/85 vaccine strain. Four genomic regions were identified that allowed for strain differentiation. The genetic differences between strains points toward nine of the ten strains most likely being sister strains to the 6/85 vaccine strain. Insufficient differences are present in the tenth strain to make a definitive conclusion. These results suggest that most if not all strains similar to the live attenuated vaccine strain are field isolates of the parent strain used to derive the live attenuated vaccine.

Transcriptomic analysis of the effects of tylosin on the protective immunity provided by the Mycoplasma gallisepticum vaccine Vaxsafe MG ts-304.

The antimicrobial tylosin is commonly used to control mycoplasma infections, sometimes in combination with vaccination. However, the efficacy of a live mycoplasma vaccine, when combined with subsequent antimicrobial treatment, against the effects of subsequent infection with a virulent strain is unknown. This study employed differential gene expression analysis to evaluate the effects of tylosin on the protection provided by the live attenuated Vaxsafe MG ts-304 vaccine, which has been shown to be safe and to provide long-term protective immunity against infection with Mycoplasma gallisepticum. The transcriptional profiles of the tracheal mucosa revealed significantly enhanced inflammation, immune cell proliferation and adaptive immune responses in unvaccinated, untreated birds and in unvaccinated birds treated with tylosin 2 weeks after infection with virulent M. gallisepticum. These responses, indicative of the typical immune dysregulation caused by infection with M. gallisepticum, were less severe in the unvaccinated, tylosin-treated birds than in the unvaccinated, untreated birds. This was attributable to the effect of residual levels of tylosin in the tracheal mucosa on replication of virulent M. gallisepticum. These responses were not detected in vaccinated, tylosin-treated birds or in vaccinated, untreated birds after infection. The tracheal mucosal transcriptional profiles of these birds resembled those of unvaccinated, untreated, uninfected birds, suggesting a rapid and protective secondary immune response and effective vaccination. Overall, these results show that, although tylosin treatment reduced the duration of immunity, the initial protective immunity induced by Vaxsafe MG ts-304 lasted for at least 22 weeks after vaccination, even after the administration of tylosin for 16 weeks following vaccination.

Evaluation of the immunoprotective effect of the recombinant Eimeria intestinalis rhoptry protein 25 and rhoptry protein 30 on New Zealand rabbits.

BACKGROUND: Rabbit coccidiosis is a parasitism caused by either one or multiple co-infections of Eimeria species. Among them, Eimeria intestinalis is the primary pathogen responsible for diarrhea, growth retardation, and potential mortality in rabbits. Concerns regarding drug resistance and drug residues have led to the development of recombinant subunit vaccines targeting Eimeria species as a promising preventive measure. The aim of this study was to assess the immunoprotective efficacy of recombinant subunit vaccines comprising EiROP25 and EiROP30 (rhoptry proteins (ROPs)) against E. intestinalis infection in rabbits. METHODS: Cloning, prokaryotic expression, and protein purification were performed to obtain EiROP25 and EiROP30. Five groups of fifty 35-day-old Eimeria-free rabbits were created (unchallenged control group, challenged control group, vector protein control group, rEiROP25 group, and rEiROP30 group), with 10 rabbits in each group. Rabbits in the rEiROP25 and rEiROP30 groups were immunized with the recombinant proteins (100 µg per rabbit) for primary and booster immunization (100 µg per rabbit) at a two-week intervals, and challenged with 7 × 104 oocysts per rabbit after an additional two-week interval. Two weeks after the challenge, the rabbits were euthanized for analysis. Weekly collections of rabbit sera were made to measure changes in specific IgG and cytokine level. Clinical symptoms and pathological changes after challenge were observed and recorded. At the conclusion of the animal experiment, lesion scores, the relative weight increase ratio, the oocyst reduction rate, and the anticoccidial index were computed. RESULTS: Rabbits immunized with rEiROP25 and rEiROP30 exhibited relative weight gain ratios of 56.57% and 72.36%, respectively. Oocysts decreased by 78.14% and 84.06% for the rEiROP25 and rEiROP30 groups, respectively. The anticoccidial indexes were 140 and 155. Furthermore, there was a noticeable drop in intestinal lesions. After the primary immunization with rEiROP25 and rEiROP30, a week later, there was a notable rise in specific IgG levels, which remained elevated for two weeks following challenge (P < 0.05). Interleukin (IL)-2 levels increased markedly in the rEiROP25 group, whereas IL-2, interferon gamma (IFN-γ), and IL-4 levels increased substantially in the rEiROP30 group (P < 0.05). CONCLUSION: Immunization of rabbits indicated that both rEiROP25 and rEiROP30 are capable of inducing an increase in specific antibody levels. rEiROP25 triggered a Th1-type immune protection response, while rEiROP30 elicited a Th1/Th2 mixed response. EiROP25 and EiROP30 can generate a moderate level of immune protection, with better efficacy observed for EiROP30. This study provides valuable insights for the promotion of recombinant subunit vaccines targeting rabbit E. intestinalis infection.

Identification and characterization of the receptors of a microneme adhesive repeat domain of Eimeria maxima microneme protein 3 in chicken intestine epithelial cells.

Eimeria maxima microneme protein 3 (EmMIC3) is pivotal in the initial recognition and attachment of E. maxima sporozoites to host cells. EmMIC3 comprises 5 tandem Type I microneme adhesive repeat (MAR) domains, among which MAR2 of EmMIC3 (EmMAR2) has been identified as the primary determinant of EmMIC3-mediated tissue tropism. Nonetheless, the mechanisms through which EmMAR2 guides the parasite to its invasion site through interactions with host receptors remained largely uncharted. In this study, we employed yeast two-hybrid (YTH) screening assays and shotgun LC-MS/MS analysis to identify EmMAR2 receptors in chicken intestine epithelial cells. ATPase H+ transporting V1 subunit G1 (ATP6V1G1), receptor accessory protein 5 (REEP5), transmembrane p24 trafficking protein (TMED2), and delta 4-desaturase sphingolipid 1 (DEGS1) were characterized as the 4 receptors of EmMAR2 by both assays. By blocking the interaction of EmMAR2 with each receptor using specific antibodies, we observed varying levels of inhibition on the invasion of E. maxima sporozoites, and the combined usage of all 4 antibodies resulted in the most pronounced inhibitory effect. Additionally, the spatio-temporal expression profiles of ATP6V1G1, REEP5, TMED2, and DEGS1 were assessed. The tissue-specific expression patterns of EmMAR2 receptors throughout E. maxima infection suggested that ATP6V1G1 and DEGS1 might play a role in early-stage invasion, whereas TMED2 could be involved in middle and late-stage invasion and REEP5 and DEGS1 may participate primarily in late-stage invasion. Consequently, E. maxima may employ a multitude of ligand-receptor interactions to drive invasion during different stages of infection. This study marks the first report of EmMAR2 receptors at the interface between E. maxima and the host, providing insights into the invasion mechanisms of E. maxima and the pathogenesis of coccidiosis.

The Immunological Basis for Vaccination.

Vaccination is crucial for health protection of poultry and therefore important to maintaining high production standards. Proper vaccination requires knowledge of the key players of the well-orchestrated immune system of birds, their interdependence and delicate regulation, and, subsequently, possible modes of stimulation through vaccine antigens and adjuvants. The knowledge about the innate and acquired immune systems of birds has increased significantly during the recent years but open questions remain and have to be elucidated further. Despite similarities between avian and mammalian species in their composition of immune cells and modes of activation, important differences exist, including differences in the innate, but also humoral and cell-mediated immunity with respect to, for example, signaling transduction pathways, antigen presentation, and cell repertoires. For a successful vaccination strategy in birds it always has to be considered that genotype and age of the birds at the time point of immunization as well as their microbiota composition may have an impact and may drive the immune reactions into different directions. Recent achievements in the understanding of the concept of trained immunity will contribute to the advancement of current vaccine types helping to improve protection beyond the specificity of an antigen-driven immune response. The fast developments in new omics technologies will provide insights into protective B- and T-cell epitopes involved in cross-protection, which subsequently will lead to the improvement of vaccine efficacy in poultry.

Estudio recapitulativo- Bases inmunológicas de la vacunación. La vacunación es crucial para la protección de la salud de las aves comerciales y por lo tanto, importante para mantener altos estándares de producción. Una vacunación adecuada requiere el conocimiento de los factores clave del sistema inmunológico bien orquestado de las aves, su interdependencia y su delicada regulación y posteriormente, los posibles modos de estimulación a través de antígenos y adyuvantes de las vacunas. El conocimiento sobre los sistemas inmunológicos innato y adquirido de las aves ha aumentado significativamente durante los últimos años, pero quedan preguntas abiertas que deben dilucidarse con profundidad. A pesar de las similitudes entre las especies de aves y mamíferos en la composición de células inmunes y modos de activación, existen diferencias importantes, incluidas las diferencias en la inmunidad innata, pero también en la inmunidad humoral y en la mediada por células, con respecto a las vías de transducción de señales, la presentación de antígenos. y repertorios celulares. Para una estrategia de vacunación exitosa en las aves, siempre se debe considerar que el genotipo y la edad de las aves en el momento de la inmunización, así como la composición de su microbiota pueden tener un impacto y pueden impulsar las reacciones inmunes en diferentes direcciones. Los logros recientes en la comprensión del concepto de inmunidad entrenada contribuirán al avance de los tipos de vacunas actuales que ayudarán a mejorar la protección más allá de la especificidad de una respuesta inmune impulsada por antígenos. Los rápidos avances en las nuevas tecnologías ómicas proporcionarán información sobre los epítopes protectores de las células B y T implicados en la protección cruzada, lo que posteriormente conducirá a la mejora de la eficacia de las vacunas en la avicultura.

Pros and Cons on Use of Live Viral Vaccines in Commercial Chicken Flocks.

The poultry industry is the largest source of meat and eggs for the growing human population worldwide. Key concerns in poultry farming are nutrition, management, flock health, and biosecurity measures. As part of the flock health, use of live viral vaccines plays a vital role in the prevention of economically important and common viral diseases. This includes diseases and production losses caused by Newcastle disease virus, infectious bronchitis virus, infectious laryngotracheitis virus, infectious bursal disease virus, Marek's disease virus, chicken infectious anemia virus, avian encephalomyelitis virus, fowlpox virus, and avian metapneumovirus. These viruses cause direct and indirect harms, such as financial losses worth millions of dollars, loss of protein sources, and threats to animal welfare. Flock losses vary by type of poultry, age of affected animals, co-infections, immune status, and environmental factors. Losses in broiler birds can consist of high mortality, poor body weight gain, high feed conversion ratio, and increased carcass condemnation. In commercial layers and breeder flocks, losses include higher than normal mortality rate, poor flock uniformity, drops in egg production and quality, poor hatchability, and poor day-old-chick quality. Despite the emergence of technology-based vaccines, such as inactivated, subunit, vector-based, DNA or RNA, and others, the attenuated live vaccines remain as important as before. Live vaccines are preferred in the global veterinary vaccine market, accounting for 24.3% of the global market share in 2022. The remaining 75% includes inactivated, DNA, subunit, conjugate, recombinant, and toxoid vaccines. The main reason for this is that live vaccines can induce innate, mucosal, cellular, and humoral immunities by single or multiple applications. Some live vaccine combinations provide higher and broader protection against several diseases or strains of viruses. This review aimed to explore insights on the pros and cons of attenuated live vaccines commonly used against major viral infections of the global chicken industry, and the future road map for improvement.

Estudio recapitulativo- Pros y contras del uso de vacunas virales vivas en parvadas de pollos comerciales. La industria avícola es la mayor fuente de carne y huevos para la creciente población humana en todo el mundo. Las principales preocupaciones en la avicultura son la nutrición, el manejo, la salud de las parvadas y las medidas de bioseguridad. Como parte de la salud de las parvadas avícolas, el uso de vacunas virales vivas juega un papel vital en la prevención de enfermedades virales comunes y de importancia económica. Esto incluye enfermedades y pérdidas en la producción causadas por el virus de la enfermedad de Newcastle, el virus de la bronquitis infecciosa, el virus de la laringotraqueítis infecciosa, el virus de la enfermedad infecciosa de la bolsa, el virus de la enfermedad de Marek, el virus de la anemia infecciosa del pollo, el virus de la encefalomielitis aviar, el virus de la viruela aviar y el metapneumovirus aviar. Estos virus causan daños directos e indirectos, como pérdidas financieras valoradas en millones de dólares, pérdida de fuentes de proteínas y amenazas al bienestar animal. Las pérdidas en las parvadas avícolas varían según el tipo de aves, la edad de los animales afectados, las coinfecciones, el estado inmunológico y los factores ambientales. Las pérdidas en aves de engorde pueden consistir en una alta mortalidad, un pobre aumento de peso corporal, un alto índice de conversión alimenticia y un mayor decomiso de las canales. En las gallinas de postura comerciales y en las parvadas de reproductoras, las pérdidas incluyen una tasa de mortalidad superior a la normal, una escasa uniformidad de la parvada, caídas en la producción y calidad de los huevos, una pobre incubabilidad y una mala calidad de los pollitos de un día. A pesar de la aparición de vacunas de base tecnológica, como las inactivadas, subunitarias, vectoriales, de ADN o ARN, entre otras, las vacunas vivas atenuadas siguen siendo tan importantes como antes. Las vacunas vivas son las preferidas en el mercado mundial de vacunas para uso veterinario y representaron el 24.3% de la cuota de mercado mundial en el año 2022. El 75% restante incluye vacunas inactivadas, de ADN, de subunidades, conjugadas, recombinantes y toxoides. La razón principal de esto es que las vacunas vivas pueden inducir inmunidad innata, de mucosas, celular y humoral mediante aplicaciones únicas o múltiples. Algunas combinaciones de vacunas vivas brindan una protección mayor y más amplia contra varias enfermedades o cepas de virus. Esta revisión tuvo como objetivo explorar ideas sobre los pros y los contras de las vacunas vivas atenuadas comúnmente utilizadas contra las principales infecciones virales de la industria avícola mundial, y las rutas futuras para mejorar.

Vaccines to Control Salmonella in Poultry.

This review is focused on describing and analyzing means by which Salmonella enterica serotype strains have been genetically modified with the purpose of developing safe, efficacious vaccines to present Salmonella-induced disease in poultry and to prevent Salmonella colonization of poultry to reduce transmission through the food chain in and on eggs and poultry meat. Emphasis is on use of recently developed means to generate defined deletion mutations to eliminate genetic sequences conferring antimicrobial resistance or residual elements that might lead to genetic instability. Problems associated with prior means to develop vaccines are discussed with presentation of various means by which these problems have been lessened, if not eliminated. Practical considerations are also discussed in hope of facilitating means to move lab-proven successful vaccination procedures and vaccine candidates to the marketplace to benefit the poultry industry.

Estudio recapitulativo- Vacunas para controlar Salmonella en la avicultura. Esta revisión se centra en describir y analizar los medios mediante los cuales las cepas de serotipo de Salmonella enterica han sido modificadas genéticamente con el propósito de desarrollar vacunas seguras y eficaces para proteger contra la enfermedad inducida por Salmonella en la avicultura y prevenir la colonización de las aves por Salmonella para reducir la transmisión a través de la cadena alimentaria por la contaminación en el interior y exterior del huevo y en los productos cárnicos de origen avícola. Se hace hincapié en el uso de medios desarrollados recientemente para generar mutaciones definidas de deleción para eliminar secuencias genéticas que confieren resistencia contra los antimicrobianos o elementos residuales que podrían conducir a inestabilidad genética. Se analizan los problemas asociados con los medios anteriores para desarrollar vacunas y se presentan diversos medios mediante los cuales estos problemas se han reducido, si no eliminado. También se discuten las consideraciones prácticas para facilitar medios para transferir a condiciones comerciales y de mercado, los procedimientos de vacunación y candidatos a vacunas que han sido exitosos mediante pruebas en el lab-oratorio para beneficiar a la industria avícola.

Vaccination of Poultry Against Influenza.

The complexity of influenza A virus (IAV) infections in avian hosts leads to equally complex scenarios for the vaccination of poultry. Vaccination against avian influenza strains can be used to prevent infections from sources with a single strain of IAV. It has been used as a part of outbreak control strategies as well as a way to maintain production for both low and high pathogenicity outbreaks. Unlike other viral pathogens of birds, avian influenza vaccination when used against highly pathogenic avian influenza virus, is tied to international trade and thus is not freely available for use without specific permission.

Vacunación de aves comerciales contra la influenza aviar. La complejidad de las infecciones por el virus de la influenza A en las aves hospedadoras conduce a escenarios igualmente complejos para la vacunación en la avicultura. La vacunación contra cepas de influenza aviar se puede utilizar para prevenir infecciones provenientes de fuentes con una sola cepa del virus de influenza. Se ha utilizado como parte de las estrategias de control de brotes, así como como una forma de mantener la producción tanto en brotes de baja como de alta patogenicidad. A diferencia de otros patógenos virales de las aves, la vacunación contra la influenza aviar, cuando se usa contra el virus de la influenza aviar altamente patógeno, está vinculada al comercio internacional y por lo tanto, no está disponible para su uso sin un permiso específico.

Advancement of Autogenous Vaccines in the Poultry Industry.

Autogenous vaccines, also known as "custom" vaccines, have become an essential instrument in the production veterinarian's toolbox for the control of emerging and evolving diseases. Autogenous vaccines require a reduced burden of U.S. Department of Agriculture licensing, making them rapidly accessible. Autogenous vaccines have made significant advancements in the ability to reduce disease within the poultry industry from a combination of several different advancements in regulation requirements, rapid and accurate diagnostic assessments, and improvements in manufacturing. The use of autogenous vaccines by poultry health professionals has also increased, and these custom-made products have been instrumental in combating diseases resulting from antigenic variants such as salmonellosis, colibacillosis, infectious coryza, infectious bursal disease, inclusion body hepatitis, viral enteritis, and viral arthritis and tenosynovitis.

Estudio recapitulativo- Avance de las vacunas autógenas en la industria avícola Las vacunas autógenas, también conocidas como vacunas "personalizadas, elaboradas de acuerdo con las necesidades del cliente" ("custom"), se han convertido en un instrumento esencial en el inventario de herramientas del veterinario de producción para el control de enfermedades emergentes y en evolución. Las vacunas autógenas requieren un procedimiento reducido para obtener la licencia por parte del Departamento de Agricultura de los Estados Unidos, lo que las hace rápidamente accesibles. Las vacunas autógenas han logrado avances significativos en la capacidad de reducir enfermedades dentro de la industria avícola gracias a una combinación de varios avances diferentes en los requisitos regulatorios, evaluaciones de diagnóstico rápidas y precisas y mejoras en la fabricación. También ha aumentado el uso de vacunas autógenas por parte de los profesionales de la salud avícola, y estos productos hechos a medida han sido fundamentales para combatir enfermedades resultantes de variantes antigénicas como la salmonelosis, la colibacilosis, la coriza infecciosa, la enfermedad infecciosa de la bolsa, hepatitis con cuerpos de inclusión, la enteritis viral y la artritis y tenosinovitis virales.

Licensing Requirements for Avian Vaccines Within the European Union.

Avian vaccines are a key factor when it comes to ensuring the availability of products derived from healthy poultry and preventing the transmission of infections from domestic and wildlife birds to humans. A marketing authorization for veterinary vaccines is granted after the product's quality, safety, and efficacy have been confirmed. During the licensing procedure, the manufacturing process is assessed to guarantee consistent quality and stability of the vaccine components. Furthermore, both the safety for the target species and the risk for the user, the consumer, and the environment must be demonstrated. In addition, specific tests and studies are required to support the efficacy of the vaccine. The authorization procedures and related licensing requirements for avian vaccines to be marketed in the European Union (EU) based on the requirements of Regulation (EU) 2019/6 Article 8 and the Commission Delegated Regulation (EU) 2021/805 amending Annex II to Regulation (EU) No. 2019/6 are explained in the paper.

Requisitos de licencia para vacunas aviares dentro de la Unión Europea. Las vacunas aviares son un factor clave a la hora de garantizar la disponibilidad de productos derivados de aves sanas y prevenir la transmisión de infecciones de aves domésticas y silvestres a los humanos. La autorización de comercialización de vacunas veterinarias se concede una vez confirmada la calidad, seguridad y eficacia del producto. Durante el procedimiento de concesión de licencia, se evalúa el proceso de fabricación para garantizar una calidad y estabilidad constantes de los componentes de la vacuna. Además, se debe demostrar tanto la seguridad para las especies a las que dicha vacuna está destinada, así como el riesgo para el usuario, el consumidor y el medio ambiente. Además, se requieren pruebas y estudios específicos que respalden la eficacia de la vacuna. En este documento se explican los procedimientos de autorización y los requisitos de licencia relacionados para las vacunas aviares que se comercializarán en la Unión Europea (U.E.) con base en los requisitos de la Regulación (U.E.) 2019/6 Artículo 8 y la Regulación Delegada de la Comisión (U.E.) 2021/805 que modifica el Anexo II del Reglamento. (U.E.) No. 2019/6.

Vaccination Against Poultry Parasites.

The complexity of parasites and their life cycles makes vaccination against parasitic diseases challenging. This review highlights this by discussing vaccination against four relevant parasites of poultry. Coccidia, i.e., Eimeria spp., are the most important parasites in poultry production, causing multiple billions of dollars of damage worldwide. Due to the trend of antibiotic-free broiler production, use of anticoccidia vaccines in broilers is becoming much more important. As of now, only live vaccines are on the market, almost all of which must be produced in birds. In addition, these live vaccines require extra care in the management of flocks to provide adequate protection and prevent the vaccines from causing damage. Considerable efforts to develop recombinant vaccines and related work to understand the immune response against coccidia have not yet resulted in an alternative. Leucozytozoon caulleryi is a blood parasite that is prevalent in East and South Asia. It is the only poultry parasite for which a recombinant vaccine has been developed and brought to market. Histomonas meleagridis causes typhlohepatitis in chickens and turkeys. The systemic immune response after intramuscular vaccination with inactivated parasites is not protective. The parasite can be grown and attenuated in vitro, but only together with bacteria. This and the necessary intracloacal application make the use of live vaccines difficult. So far, there have been no attempts to develop a recombinant vaccine against H. meleagridis. Inactivated vaccines inducing antibodies against the poultry red mite Dermanyssus gallinae have the potential to control infestations with this parasite. Potential antigens for recombinant vaccines have been identified, but the use of whole-mite extracts yields superior results. In conclusion, while every parasite is unique, development of vaccines against them shares common problems, namely the difficulties of propagating them in vitro and the identification of protective antigens that might be used in recombinant vaccines.

Estudio recapitulativo- Vacunación contra los parásitos de las aves de corral. La complejidad de los parásitos y sus ciclos de vida hace que la vacunación contra las enfermedades parasitarias sea un desafío. Esta revisión destaca este concepto al discutir la vacunación contra cuatro parásitos relevantes en la avicultura. Las coccidias, como, Eimeria spp., son los parásitos más importantes en la producción avícola y causan daños por miles de millones de dólares en todo el mundo. Debido a la tendencia de la producción de pollos de engorde sin antibióticos, el uso de vacunas anticoccidianas en pollos de engorde se está volviendo mucho más importante. Por el momento, sólo hay en el mercado vacunas vivas y casi todas ellas deben producirse en aves. Además, estas vacunas vivas requieren un cuidado especial en el manejo de las parvadas para brindar una protección adecuada y evitar que las vacunas causen daños. Los esfuerzos considerables para desarrollar vacunas recombinantes y los trabajos relacionados para comprender la respuesta inmune contra coccidias aún no han dado como resultado una alternativa. Leucozytozoon caulleryi es un parásito sanguíneo que prevalece en el este y el sur de Asia. Es el único parásito de las aves de corral para el que se ha desarrollado y comercializado una vacuna recombinante. El parásito Histomonas meleagridis causa tiflohepatitis en pollos y pavos. La respuesta inmune sistémica después de la vacunación intramuscular con parásitos inactivados no es protectora. El parásito se puede cultivar y atenuar in vitro, pero sólo junto con bacterias. Esto y la necesaria aplicación intracloacal dificultan el uso de vacunas vivas. Hasta el momento no ha habido intentos de desarrollar una vacuna recombinante contra H. meleagridis. Las vacunas inactivadas que inducen anticuerpos contra el ácaro rojo de las aves Dermanyssus gallinae tienen el potencial de controlar las infestaciones por este parásito. Se han identificado antígenos potenciales para vacunas recombinantes, pero el uso de extractos completos de ácaros produce resultados superiores. En conclusión, si bien cada parásito es único, el desarrollo de vacunas contra ellos comparte problemas comunes, por ejemplo, las dificultades de propagarlos in vitro y la identificación de antígenos protectores que podrían usarse en vacunas recombinantes.

Veterinary Autogenous Vaccines for Poultry in Europe-Many Ways to Crack an Egg.

In the past decade, European animal farming has increasingly used autogenous vaccines for the prevention of nonnotifiable diseases. In Europe, these vaccines are exclusively inactivated bacterial and viral vaccines, with a set of specific regulations that differentiate them from conventional vaccines. The highest number of applications most likely occurs in poultry, as these animal species are farmed in the highest numbers compared with other types of food-producing animals. In 2019, autogenous vaccines came within the scope of harmonized European regulation for the first time, although many important aspects are still missing and need to be further developed. Consequently, several important legal provisions remain in national legislations and can vary tremendously between different member states of the European Union. The inclusion of autogenous vaccines in the management of certain diseases of poultry is justified by the nonavailability of licensed vaccines and the evolution and diversity of antigens in the field that are not covered by licensed vaccines. In addition, these vaccines aid in reducing the use of antibiotics. The methods for isolating and typing pathogenic isolates to obtain relevant antigens are pathogen specific and require a careful approach based on clinical evidence. Manufacturing processes are optimized according to regulatory standards, and they represent the most critical factor influencing the quality of autogenous vaccines and their placement on the market. This review presents the important requirements for manufacturing autogenous vaccines for poultry in addition to the relevant regulatory considerations. The results from a survey of several European Union member states regarding specific provisions within their national legislations are also presented.

Vacunas veterinarias autógenas para la avicultura en Europa: "Varias formas de romper un huevo". En la última década, la ganadería europea ha utilizado cada vez más vacunas autógenas para la prevención de enfermedades no declarables. En Europa, estas vacunas son exclusivamente vacunas inactivadas bacterianas y virales, con un conjunto de regulaciones específicas que las diferencian de las vacunas convencionales. El mayor número de aplicaciones probablemente se produce en la avicultura, ya que estas especies animales se crían en mayor número en comparación con otros tipos de animales productores de alimentos. En el año 2019, las vacunas autógenas entraron por primera vez en el ámbito de aplicación de una regulación europea armonizada, aunque todavía faltan muchos aspectos importantes y es necesario desarrollarlos más. En consecuencia, varias disposiciones legales importantes permanecen en las legislaciones nacionales y pueden variar enormemente entre los diferentes estados miembros de la Unión Europea. La inclusión de vacunas autógenas en el manejo de determinadas enfermedades avícolas se justifica por la falta de disponibilidad de vacunas autorizadas y la evolución y diversidad de antígenos en el campo que no están cubiertos por vacunas con licencia. Además, estas vacunas ayudan a reducir el uso de antibióticos. Los métodos para aislar y tipificar aislados patógenos para obtener antígenos relevantes son específicos de cada patógeno y requieren un enfoque cuidadoso basado en evidencia clínica. Los procesos de fabricación se optimizan de acuerdo con las normas reglamentarias y representan el factor más crítico que influye en la calidad de las vacunas autógenas y su comercialización. Esta revisión presenta los requisitos importantes para la fabricación de vacunas autógenas en la avicultura, además de las consideraciones regulatorias relevantes. También se presentan los resultados de una encuesta realizada en varios estados miembros de la Unión Europea sobre disposiciones específicas dentro de sus legislaciones nacionales.