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.

Germline engineering of the chicken genome using CRISPR/Cas9 by in vivo transfection of PGCs.

Development of simple and readily adoptable methods to mediate germline engineering of the chicken genome will have many applications in research, agriculture and industrial biotechnology. We report germline targeting of the endogenous chicken Interferon Alpha and Beta Receptor Subunit 1 (IFNAR1) gene by in vivo transgenic expression of the high-fidelity Cas9 (Cas9-HF1) and guide RNAs (gRNAs) in chickens. First, we developed a Tol2 transposon vector carrying Cas9-HF1, IFNAR1-gRNAs (IF-gRNAs) and green fluorescent protein (GFP) transgenes (pTgRCG) and validated in chicken fibroblast DF1 cells. Next, the pTgRCG plasmid was directly injected into the dorsal aorta of embryonic day (ED) 2.5 chicken embryos targeting the circulating primordial germ cells (PGCs). The resulting chimera roosters generated a fully transgenic generation 1 (G1) hen with constitutive expression of Cas9-HF1 and IF-gRNAs (G1_Tol2-Cas9/IF-gRNA). We detected a spectrum of indels at gRNA-targeted loci in the G1_Tol2-Cas9/IF-gRNA hen and the indels were stably inherited by the G2 progeny. Breeding of the G1_Tol2-Cas9/IF-gRNA hen resulted in up to 10% transgene-free heterozygote IFNAR1 mutants, following null-segregation of the Tol2 insert. The method described here will provide new opportunities for genome editing in chicken and other avian species that lack PGC culture.

The Importance of the Bursa of Fabricius, B Cells and T Cells for the Pathogenesis of Marek's Disease: A Review.

The importance of the bursa of Fabricius (BF) for the pathogenesis of Marek's disease (MD) has been studied since the late 1960's. In this review, the results of these studies are analyzed in the context of the developing knowledge of the immune system of chickens and the pathogenesis of MD from 1968 to 2022. Based on the available techniques to interfere with the development of the BF, three distinct periods are identified and discussed. During the initial period between 1968 and 1977, the use of neonatal bursectomy, chemical methods and irradiation were the main tools to interfere with the B lymphocyte development. The application of these techniques resulted in contradictory results from no effects to an increase or decrease in MD incidence. Starting in the late 1970's, the use of bursectomy in 18-day-old embryos led to the development of the "Cornell model" for the pathogenesis of MD, in which the infection of B lymphocytes is an important first step in MD virus (MDV) replication causing the activation of thymus-derived lymphocytes (T cells). Following this model, these activated T cells, but not resting T cells, are susceptible to MDV infection and subsequent transformation. Finally, B-cell knockout chickens lacking the J gene segment of the IgY heavy chain gene were used to further define the role of the BF in the pathogenesis of MD.

Northeastern Conference on Avian Diseases from 1928-2021: 93 Years of Contributions to Organized Avian Medicine.

Bacillary white diarrhea in chickens was a major disease concern for the poultry industry during the early 1900s. Drs. L. F. Rettger and W. R. Hinshaw organized a meeting in 1928 to discuss methods for controlling this disease. In this meeting, representatives of five northeastern states discussed approaches to test for the presence of the etiological agent of bacillary white diarrhea, namely, Salmonella enterica subsp. enterica serovar Pullorum. Meeting attendees decided to have a yearly meeting of the Northeastern Conference of Laboratory Workers in Bacillary White Diarrhea. The next year, the name was changed to Conference of Laboratory Workers in Pullorum Disease Eradication, which was changed to Northeastern Conference on Avian Diseases (NECAD) in 1957. Not only has NECAD been important for the control of pullorum disease but also, starting with the fifth Annual Conference in 1932, other poultry diseases became an official part of the program. As such, NECAD served for a long time as the premier organization to present new information on avian diseases. The success of NECAD was based on the work of the many committees, which are described in detail in this review. For example, the antigen committee started officially in 1929 and remained active until at least 1987. The main task of this committee was to evaluate Salmonella Pullorum strains to be used by all participants in the pullorum antibody testing programs. NECAD started as a closed organization with participants from universities and government organizations but did not allow representatives from commercial groups until 1968 when all American Association of Avian Pathologists (AAAP) members in the Northeastern United States could participate. The journal Avian Diseases started with discussions by Dr. P. P. Levine with M. S. Cover, H. L. Chute, R. F. Gentry, E. Jungherr, and H. Van Roekel about the idea that NECAD would sponsor a journal dealing specifically with avian diseases. During the first few years of Avian Diseases publication, many articles including abstracts came from the NECAD Annual Conferences. The importance of NECAD as a precursor for other regional meetings and the AAAP and as a forum for graduate students to present their research are described. Several recipients of the award for the best paper presented by a graduate student have continued to work in avian disease research. The decline in the participation of scientists in the late 1990s and early 2000s was discussed extensively in 2006 and led to a merger of the NECAD meeting with the Pennsylvania Poultry Sales and Service Conference. Due to the COVID-19 pandemic, the 92nd Annual Conference was a virtual meeting in 2020. Fortunately, the 93rd Annual Conference in 2021 was an in-person meeting held in State College, PA.

Reseña histórica- La Conferencia del Noreste sobre Enfermedades Aviaries desde el año 1928 al 2021: 93 años de contribuciones a la medicina aviar organizada. La diarrea blanca bacilar del pollo fue una enfermedad importante para la industria avícola a principios del siglo XX. Los doctores L. F. Rettger y W. R. Hinshaw organizaron una reunión en 1928 para discutir los métodos para controlar esta enfermedad. En esta reunión, representantes de cinco estados del noreste discutieron los enfoques a utilizar para evaluar la presencia del agente etiológico de la diarrea blanca bacilar, Salmonella enterica subsp. enterica serovar Pullorum. Los asistentes a la reunión decidieron tener una reunión anual de la Conferencia del Noreste de Trabajadores de Laboratorio en Diarrea Blanca Bacilar. Al año siguiente, el nombre se cambió a Conferencia de Trabajadores de Laboratorio para la Erradicación de la Enfermedad Pulorosis que se cambió a Conferencia del Noreste sobre Enfermedades Aviares (con las siglas en inglés NECAD) en 1957. La NECAD no solo ha sido importante para el control de la pulorosis, sino también, comenzando con la quinta Conferencia Anual en 1932, otras enfermedades de la avicultura comercial se convirtieron en parte oficial del programa. Como tal, la NECAD sirvió durante mucho tiempo como la principal organización para presentar nueva información sobre enfermedades aviares. El éxito de NECAD se basó en el trabajo de muchos comités, que se describen en detalle en esta reseña. Por ejemplo, el comité de antígenos comenzó oficialmente en 1929 y permaneció activo alrededor de 1987. La tarea principal de este comité fue evaluar las cepas de Salmonella Pullorum para ser utilizadas por todos los participantes en los programas de detección de anticuerpos de pullorum. La NECAD comenzó como una organización cerrada con participantes de universidades y organizaciones gubernamentales y no permitió representantes de grupos comerciales hasta 1968, cuando todos los miembros de la AAAP en el noreste de Estados Unidos pudieron participar. La revista científica Avian Diseases (Enfermedades de las Aves) comenzó con discusiones entre el Dr. P. P. Levine con M. S. Cover, H. L. Chute, R. F. Gentry, E. Jungherr y H. Van Roekel sobre la idea de que la NECAD patrocinaría una revista que se ocupara específicamente de las enfermedades aviares. Durante los primeros años de la publicación de Avian Diseases, muchos artículos, incluidos resúmenes, surgieron de las conferencias anuales de la NECAD. Se describe la importancia de la NECAD como precursor de otras reuniones regionales y de la AAAP y como foro para que los estudiantes de posgrado presentaran sus investigaciones. Varios ganadores del premio al mejor trabajo presentado por un estudiante de posgrado han continuado trabajando en la investigación en enfermedades aviares. La disminución en la participación de científicos a fines de la década de 1990s y principios de la década de los 2000s se debatió ampliamente en año 2006 y llevó a la fusión de la reunión de NECAD con la Conferencia de Servicio y Ventas en Avicultura de Pensilvania. Debido a la pandemia por el COVID-19, la 92a Conferencia Anual fue una reunión virtual en el año 2020. Afortunadamente, la 93a Conferencia Anual en 2021 fue una reunión en persona celebrada en State College, Pensilvania.

Primary Chicken and Duck Endothelial Cells Display a Differential Response to Infection with Highly Pathogenic Avian Influenza Virus.

Highly pathogenic avian influenza viruses (HPAIVs) in gallinaceous poultry are associated with viral infection of the endothelium, the induction of a 'cytokine storm, and severe disease. In contrast, in Pekin ducks, HPAIVs are rarely endothelial tropic, and a cytokine storm is not observed. To date, understanding these species-dependent differences in pathogenesis has been hampered by the absence of a pure culture of duck and chicken endothelial cells. Here, we use our recently established in vitro cultures of duck and chicken aortic endothelial cells to investigate species-dependent differences in the response of endothelial cells to HPAIV H5N1 infection. We demonstrate that chicken and duck endothelial cells display a different transcriptional response to HPAI H5N1 infection in vitro-with chickens displaying a more pro-inflammatory response to infection. As similar observations were recorded following in vitro stimulation with the viral mimetic polyI:C, these findings were not specific to an HPAIV H5N1 infection. However, similar species-dependent differences in the transcriptional response to polyI:C were not observed in avian fibroblasts. Taken together, these data demonstrate that chicken and duck endothelial cells display a different response to HPAIV H5N1 infection, and this may help account for the species-dependent differences observed in inflammation in vivo.

In Vitro Inhibition of Influenza Virus Using CRISPR/Cas13a in Chicken Cells.

Advances in the field of CRISPR/Cas systems are expanding our ability to modulate cellular genomes and transcriptomes precisely and efficiently. Here, we assessed the Cas13a-mediated targeted disruption of RNA in chicken fibroblast DF1 cells. First, we developed a Tol2 transposon vector carrying the Cas13a-msGFP-NLS (pT-Cas13a) transgene, followed by a stable insertion of the Cas13a transgene into the genome of DF1 cells to generate stable DF1-Cas13a cells. To assess the Cas13a-mediated functional knockdown, DF1-Cas13a cells were transfected with the combination of a plasmid encoding DsRed coding sequence (pDsRed) and DsRed-specific crRNA (crRNA-DsRed) or non-specific crRNA (crRNA-NS). Fluorescence-activated cell sorting (FACS) and a microscopy analysis showed reduced levels of DsRed expression in cells transfected with crRNA-DsRed but not in crRNA-NS, confirming a sequence-specific Cas13a mediated mRNA knockdown. Next, we designed four crRNAs (crRNA-IAV) against the PB1, NP and M genes of influenza A virus (IAV) and cloned in tandem to express from a single vector. DF1-Cas13a cells were transfected with plasmids encoding the crRNA-IAV or crRNA-NS, followed by infection with WSN or PR8 IAV. DF1 cells transfected with crRNA-IAV showed reduced levels of viral titers compared to cells transfected with crRNA-NS. These results demonstrate the potential of Cas13a as an antiviral strategy against highly pathogenic strains of IAV in chickens.

In Vivo Inhibition of Marek's Disease Virus in Transgenic Chickens Expressing Cas9 and gRNA against ICP4.

Marek's disease (MD), caused by MD herpesvirus (MDV), is an economically important disease in chickens. The efficacy of the existing vaccines against evolving virulent stains may become limited and necessitates the development of novel antiviral strategies to protect poultry from MDV strains with increased virulence. The CRISPR/Cas9 system has emerged as a powerful genome editing tool providing an opportunity to develop antiviral strategies for the control of MDV infection. Here, we characterized Tol2 transposon constructs encoding Cas9 and guide RNAs (gRNAs) specific to the immediate early infected-cell polypeptide-4 (ICP4) of MDV. We generated transgenic chickens that constitutively express Cas9 and ICP4-gRNAs (gICP4) and challenged them via intraabdominal injection of MDV-1 Woodlands strain passage-19 (p19). Transgenic chickens expressing both gRNA/Cas9 had a significantly reduced replication of MDV in comparison to either transgenic Cas9-only or the wild-type (WT) chickens. We further confirmed that the designed gRNAs exhibited sequence-specific virus interference in transgenic chicken embryo fibroblast (CEF) expressing Cas9/gICP4 when infected with MDV but not with herpesvirus of turkeys (HVT). These results suggest that CRISPR/Cas9 can be used as an antiviral approach to control MDV infection in chickens, allowing HVT to be used as a vector for recombinant vaccines.

Cytokine Responses in Tracheas from Major Histocompatibility Complex Congenic Chicken Lines with Distinct Susceptibilities to Infectious Bronchitis Virus.

The chicken major histocompatibility complex (MHC) B locus has been linked to resistance to infectious diseases. We have previously provided evidence that the MHC congenic chicken lines 331/B2 and 335/B19 differ in susceptibility to infectious bronchitis virus (IBV) strains M41 and ArkDPI in in vivo challenge experiments. Innate immune responses can be difficult to measure in vivo because they are nonspecific and can be triggered by environmental factors. In an attempt to address this issue, we used tracheal organ cultures derived from 331/B2 and 335/B19 birds to study local cytokine production after in vitro challenge with IBV M41. Interferon (IFN)-ß, interleukin (IL)-1ß, IL-6, and IL-10 gene expression and production were assessed. Tracheal organ cultures derived from 335/B19 birds presented an increased inflammatory response compared to 331/B2. However, it was not possible to discriminate between cytokine responses in IBV-infected and phosphate-buffered saline-treated tracheal organ cultures. Because tracheal processing entails physical damage to the trachea, it is possible that the tracheal organ cultures presented high levels of inflammation regardless of the IBV challenge. To demonstrate the effects of IBV on innate immune responses in the MHC congenic chicken lines, we performed an additional in vivo experiment that focused on cytokine gene expression and production in tracheas up to 60 hr after a challenge with IBV M41. Our results corroborate previous in vivo observations that suggest that detrimental local inflammatory responses in 335/B19 birds might be associated with their susceptibility to IBV and that inflammation does not necessarily lead to the assembly of an appropriate adaptive immune response. This work provides further insight into the increased susceptibility of 335/B19 birds to infectious bronchitis.

Respuestas de citoquinas en tráqueas de líneas de pollo congénitas para MHC con susceptibilidad distinta al virus de la bronquitis infecciosa. El locus B del complejo principal de histocompatibilidad del pollo (MHC) se ha relacionado con la resistencia a enfermedades infecciosas. Anteriormente se ha proporcionado evidencia de que las líneas de pollo congénitas para MHC tales como 331/B2 y 335/B19 difieren en la susceptibilidad a las cepas del virus de la bronquitis infecciosa M41 y Arkansas DPI en experimentos de desafío in vivo. Las respuestas inmunes innatas pueden ser difíciles de medir in vivo porque son inespecíficas y pueden desencadenarse por factores ambientales. En un intento por abordar este problema, se utilizaron cultivos de órganos de tráquea derivados de aves 331/B2 y 335/B19 para estudiar la producción local de citocinas después de la exposición in vitro con virus de la bronquitis infecciosa serotipo M41. Se evaluaron la expresión y producción de genes de interferón (IFN) -ß, interleucina (IL) -1ß, IL-6, e IL-10. Los cultivos de órganos traqueales derivados de aves 335/B19 presentaron una mayor respuesta inflamatoria en comparación con las aves 331/B2. Sin embargo, no fue posible discriminar entre las respuestas de citocinas en cultivos de órganos traqueales tratados con solución salina amortiguada con fosfato y los infectados con el virus de la bronquitis infecciosa. Debido a que el procesamiento traqueal implica un daño físico a la tráquea, es posible que los cultivos de órganos traqueales presenten altos niveles de inflamación, independientemente del desafío con el virus de la bronquitis infecciosa. Para demostrar los efectos del del virus de bronquitis sobre las respuestas inmunes innatas en las líneas de pollo congénito para MHC, se realizó un experimento in vivo adicional que se centró en la expresión y producción de genes de citocinas en tráqueas hasta 60 horas después de un desafío con el virus de bronquitis M41. Estos resultados corroboran observaciones previas in vivo que sugieren que las respuestas inflamatorias locales perjudiciales en las aves 335/B19 podrían estar asociadas con su susceptibilidad al virus de la bronquitis infecciosa y que la inflamación no necesariamente conduce al establecimiento de una respuesta inmune adaptativa apropiada. Este trabajo proporciona más información sobre la mayor susceptibilidad de las aves 335/B19 contra la bronquitis infecciosa.

A proposed nomenclature for infectious bursal disease virus isolates.

Infectious bursal disease virus (IBDV) was initially identified in the USA. For decades, these viruses were not categorized using a typing system because they were considered to be antigenically and pathogenically similar. In the 1980s, a second major serotype, serotype 2, was found in turkeys. Classification of IBDV became more complex with the discovery of antigenic variant strains called "variants" in the United States and a highly virulent strain known as "very virulent" or vvIBDV identified in Europe. To distinguish the IBDV strains identified prior to this time from the antigenic variant viruses, the term "classic viruses" was adopted. Studies over the next three decades produced a wealth of information on the antigenicity, pathogenicity and molecular structure of IBDV isolates. These data made it clear that the descriptive nomenclature used for IBDV was inadequate. For example, not all viruses identified as vvIBDV by genotyping are highly pathogenic; some have reassorted genome segments that result in lower virulence. Furthermore, variant viruses are not an antigenically homogeneous group and the term "classic virus" has been used interchangeably to describe antigenic and pathogenic types of IBDV. These and other issues make the current naming system for strains of IBDV archaic. The lack of uniform testing and standards for antigenicity and pathogenicity makes it difficult to categorize IBDV strains on a global basis. A new nomenclature that includes a genotyping system that can easily be applied worldwide is proposed and serves as a platform to begin discussions on its value to the scientific community.

History of the First-Generation Marek's Disease Vaccines: The Science and Little-Known Facts.

Shortly after the isolation of Marek's disease (MD) herpesvirus (MDV) in the late 1960s vaccines were developed in England, the United States, and The Netherlands. Biggs and associates at the Houghton Poultry Research Station (HPRS) in England attenuated HPRS-16, the first cell-culture-isolated MDV strain, by passaging HPRS-16 in chick kidney cells. Although HPRS-16/Att was the first commercially available vaccine, it never became widely used and was soon replaced by the FC126 strain of herpesvirus of turkeys (HVT) vaccine developed by Witter and associates at the Regional Poultry Research Laboratory (now Avian Disease and Oncology Laboratory [ADOL]) in East Lansing, MI. Ironically, Kawamura et al. isolated a herpesvirus from kidney cell cultures from turkeys in 1969 but never realized its potential as a vaccine against MD. Rispens of the Central Veterinary Institute (CVI) developed the third vaccine. His associate, Maas, had found commercial flocks of chickens with MDV antibodies but without MD. Subsequently, Rispens isolated a very low pathogenic strain from hen number 988 from his MD antibody-positive flock, which was free of avian leukosis virus and clinical MD. This isolate became the CVI-988 vaccine used mostly in The Netherlands. During the late 1970s, HVT was no longer fully protective against some new emerging field strains. The addition of SB-1, isolated by Schat and Calnek, to HVT improved protection against the emerging very virulent strains. In the 1990s CVI-988 became the worldwide vaccine gold standard. This review will present data from published papers and personal communications providing additional information about the exciting 15-yr period after the isolation of MDV to the development of the different vaccines.

Lack of evidence that avian oncogenic viruses are infectious for humans: a review.

Chickens may be infected with three different oncogenic viruses: avian leukosis virus (ALV), reticuloendotheliosis virus (REV), and Marek's disease herpesvirus (MDV). Several epidemiological studies have suggested a link between these viruses and different types of cancer in people working in poultry processing plants and with multiple sclerosis. In this article, we analyze the epidemiological evidence that these viruses are causative agents for human cancer, followed by description of the relevant key characteristics of ALV, REV, and MDV. Finally, we discuss the biological evidence or lack thereof that avian tumor viruses are involved in the etiology of human cancer and multiple sclerosis (MS). The recent primary epidemiologic articles that we reviewed as examples were only hypothesis-generating studies examining massive numbers of risk factors for associations with various imprecise, non-viral-specific outcomes. The studies lacked precise evidence of exposure to the relevant viruses and the statistical methods failed to adjust for the large risks of false-positive claims. ALV subgroups A-D and J have been eradicated in the United States from the pure lines down to the parent stocks by the breeder companies, which have greatly reduced the incidence of infection in layer flocks and broilers. As a consequence, potential exposure of humans to these viruses has greatly diminished. Infection of humans working in processing plants with ALV-A and ALV-B is unlikely, because broilers are generally resistant to infection with these two subgroups. Moreover, these viruses enter cells by specific receptors present on chicken, but not on mammalian, cells. Infection of mammalian cell cultures or animals with ALV-A, ALV-B, and ALV-J has not been reported. Moreover, humans vaccinated with exogenous or endogenous ALV-contaminated vaccines against yellow fever, measles, and mumps did not become antibody- or virus-positive for ALV. The risks for human infection with REV are similarly limited. First of all, REV also has been eradicated from pure lines down to parent stock by breeder companies in the United States. Broilers can still become infected with REV through infection with fowl pox virus containing REV. However, there is no indication that REV can infect human cells. Low levels of antibodies to ALV and REV in human sera have been reported by a few groups. Absorption of sera with chicken antigens reduced the antibody titers, and there was no clear association with contacts with poultry. Possible cross-reactions with human endogenous or exogenous retroviruses were not considered in these publications. MDV is typically associated with infection of chickens, and almost all experimental data show that MDV cannot infect mammalian cells or animals, including nonhuman primates. One study reports the presence of MDV gD DNA in human sera, but this finding could not be confirmed by another group. A Medline search of the term "gene expression in human cancers" was negative for publications with avian retroviruses or MDV. In conclusion, there is no indication that avian oncogenic viruses are involved in human cancer or MS or even able to infect and replicate in humans.

Characteristics of cyprinid herpesvirus 3 in different phases of infection: implications for disease transmission and control.

Koi herpesvirus disease (KHVD) is an emerging and highly contagious viral disease of koi and common carp (Cyprinus carpio), causing mass mortalities and huge economic losses to the carp aquaculture industry. The disease has spread rapidly to 28 countries worldwide. However, mechanisms of koi herpesvirus (species Cyprinid herpesvirus 3; CyHV-3) transmission remain unclear. A potential experimental model of CyHV-3 infection in carp was used to characterise CyHV-3 in different phases of infection and to demonstrate that CyHV-3 persists in survivor fish and has the capacity to reactivate and transmit the disease to healthy fish. During acute infection, which occurred when fish were maintained at 22°C, viral genes were abundantly expressed and infectious virus was produced in association with tissue damage, clinical disease and mortality. In fish maintained at a lower temperature (11°C), viral DNA was present but viral gene expression was absent or greatly restricted, infectious virus was not recovered and there was no evidence of disease. Productive replication was re-initiated following an increase in water temperature to 22°C, resulting in 45% mortality. Shedding of reactivated virus killed 75% of cohabitating naïve fish, suggesting a potential risk for disease transmission.

Importance of differential expression of Marek's disease virus gene pp38 for the pathogenesis of Marek's disease.

The role of pp38 in the pathogenesis of Marek's disease (MD) has not been fully elucidated. Previously, we reported the presence of two splice variants (Spl A and Spl B) for pp38. We also reported that the wild-type pp38 (WT), as well as the mutated pp38 (MUT), altered the oxidative phosphorylation pathway in infected cells. To determine whether the different forms of pp38 are important for the pathogenesis of MD, we generated RB-1B-based bacterial artificial chromosome (BAC) clones expressing pp38MUT, pp38Sp1 A, and pp38Spl B. Infectious viruses were recovered from these BAC clones in chick kidney cells (CKC). The Spl A and Spl B viruses had significantly smaller plaque sizes and replicated to a lesser degree in CKC than the WT and MUT viruses. Two in vivo experiments were conducted by inoculating 7-day-old P2a chicks with 1000 plaque-forming units of each virus. In the first experiment, chicks were sacrificed at 4, 8, 11, and 15 days postinfection (PI). WT and MUT viruses had similar viremia levels using virus isolation and quantitative real-time PCR (qPCR) assays, whereas Spl A and Spl B viruses had significantly lower viremia levels than WT and MUT viruses. In the second experiment, we showed that tumor development and MD mortality were similar in the WT- and MUT-infected chickens, with all birds MD positive at 5 wk PI. In contrast, chickens infected with Spl B and Spl A had a significantly lower MD incidence at 11 wk PI, when the experiment was terminated.

Chronic Mycoplasma conjunctivitis in house finches: host antibody response and M. gallisepticum VlhA expression.

Previous studies have shown that house finch field isolates of Mycoplasma gallisepticum (MG) vary in virulence and ability to induce an antibody response. After experimental inoculation, MG causes persistent, severe disease in a subset of individuals. In this study, we further characterized MG infection using five field isolates, with an emphasis on chronically diseased birds. After experimental inoculation of house finches, MG load was measured by quantitative PCR and anti-MG antibody responses were measured by ELISAs. Birds with chronic disease had significantly higher pathogen loads and antibody responses than did birds without chronic disease. Using a monoclonal antibody (MAb86) specific for a variant of the MG VlhA adhesin and immunodominant surface protein, we show that VlhA expression differs among MG isolates in this study, and that in vivo VlhA changes occur in house finches infected with MG. Overall, our results suggest that chronic MG disease has a strong pathogen-mediated component.

A new method for producing transgenic birds via direct in vivo transfection of primordial germ cells.

Traditional methods of avian transgenesis involve complex manipulations involving either retroviral infection of blastoderms or the ex vivo manipulation of primordial germ cells (PGCs) followed by injection of the cells back into a recipient embryo. Unlike in mammalian systems, avian embryonic PGCs undergo a migration through the vasculature on their path to the gonad where they become the sperm or ova producing cells. In a development which simplifies the procedure of creating transgenic chickens we have shown that PGCs are directly transfectable in vivo using commonly available transfection reagents. We used Lipofectamine 2000 complexed with Tol2 transposon and transposase plasmids to stably transform PGCs in vivo generating transgenic offspring that express a reporter gene carried in the transposon. The process has been shown to be highly effective and as robust as the other methods used to create germ-line transgenic chickens while substantially reducing time, infrastructure and reagents required. The method described here defines a simple direct approach for transgenic chicken production, allowing researchers without extensive PGC culturing facilities or skills with retroviruses to produce transgenic chickens for wide-ranging applications in research, biotechnology and agriculture.

Immunity to Marek's disease: where are we now?

Marek's disease (MD) in chickens was first described over a century ago and the causative agent of this disease, Marek's disease virus (MDV), was first identified in the 1960's. There has been extensive and intensive research over the last few decades to elucidate the underlying mechanisms of the interactions between the virus and its host. We have also made considerable progress in terms of developing efficacious vaccines against MD. The advent of the chicken genetic map and genome sequence as well as development of approaches for chicken transcriptome and proteome analyses, have greatly facilitated the process of illuminating underlying genetic mechanisms of resistance and susceptibility to disease. However, there are still major gaps in our understanding of MDV pathogenesis and mechanisms of host immunity to the virus and to the neoplastic events caused by this virus. Importantly, vaccines that can disrupt virus transmission in the field are lacking. The current review explores mechanisms of host immunity against Marek's disease and makes an attempt to identify the areas that are lacking in this field.

Can American goldfinches function as reservoirs for Mycoplasma gallisepticum?

We performed experiments to test if American Goldfinches (Spinus tristis) could be a competent reservoir for Mycoplasma gallisepticum and play a role in the epidemic spread of mycoplasmal conjunctivitis among House Finches (Carpodacus mexicanus) in North America. We infected one of two individuals housed together in a cage and determined if transmission occurred to the second bird. Probability of transmission between an American Goldfinch and a House Finch (in either direction) was similar to that between two House Finches. In a second experiment small groups of birds (6-8) were housed in large aviaries. Two source birds were inoculated with M. gallisepticum, and transmission to the naive birds in the aviary was recorded. Transmission occurred among House Finches, among American Goldfinches, and from House Finches to American Goldfinches. Transmission was more likely between House Finches than among American Goldfinches, and between House Finches and American Goldfinches. We conclude that American Goldfinches are a competent reservoir for Mycoplasma gallisepticum and could have played a role in the spread of the epidemic as they are more migratory than House Finches.

Koi herpesvirus encodes and expresses a functional interleukin-10.

Koi herpesvirus (KHV) (species Cyprinid herpesvirus 3) ORF134 was shown to transcribe a spliced transcript encoding a 179-amino-acid (aa) interleukin-10 (IL-10) homolog (khvIL-10) in koi fin (KF-1) cells. Pairwise sequence alignment indicated that the expressed product shares 25% identity with carp IL-10, 22 to 24% identity with mammalian (including primate) IL-10s, and 19.1% identity with European eel herpesvirus IL-10 (ahvIL-10). In phylogenetic analyses, khvIL-10 fell in a divergent position from all host IL-10 sequences, indicating extensive structural divergence following capture from the host. In KHV-infected fish, khvIL-10 transcripts were observed to be highly expressed during the acute and reactivation phases but to be expressed at very low levels during low-temperature-induced persistence. Similarly, KHV early (helicase [Hel] and DNA polymerase [DNAP]) and late (intercapsomeric triplex protein [ITP] and major capsid protein [MCP]) genes were also expressed at high levels during the acute and reactivation phases, but only low-level expression of the ITP gene was detected during the persistent phase. Injection of khvIL-10 mRNA into zebrafish (Danio rerio) embryos increased the number of lysozyme-positive cells to a similar degree as zebrafish IL-10. Downregulation of the IL-10 receptor long chain (IL-10R1) using a specific morpholino abrogated the response to both khvIL-10 and zebrafish IL-10 transcripts, indicating that, despite the structural divergence, khvIL-10 functions via this receptor. This is the first report describing the characteristics of a functional viral IL-10 gene in the Alloherpesviridae.

Characterization of chicken thrombocyte responses to Toll-like receptor ligands.

Thrombocytes are the avian equivalent to mammalian platelets. In addition to their hemostatic effects, mammalian platelets rely in part on pattern recognition receptors, such as the Toll-like receptors (TLR), to detect the presence of pathogens and signal the release of certain cytokines. Ligands for TLRs include lipopolysaccharide (LPS), which is bound by TLR4, as well as unmethylated CpG DNA motifs, which are bound by TLR9 in mammals and TLR21 in chickens. Similar to mammalian platelets, avian thrombocytes have been shown to express TLR4 and secrete some pro-inflammatory cytokines in response to LPS treatment. However, the full extent of the contributions made by thrombocytes to host immunity has yet to be elucidated. Importantly, the mechanisms by which TLR stimulation may modulate thrombocyte effector functions have not been well characterized. As such, the objective of the present study was to gain further insight into the immunological role of thrombocytes by analyzing their responses to treatment with ligands for TLR4 and TLR21. To this end, we quantified the relative expression of several immune system genes at 1, 3, 8 and 18 hours post-treatment using real-time RT-PCR. Furthermore, production of nitric oxide and phagocytic activity of thrombocytes was measured after their activation with TLR ligands. We found that thrombocytes constitutively express transcripts for both pro- and anti-inflammatory cytokines, in addition to those associated with anti-viral responses and antigen presentation. Moreover, we found that both LPS and CpG oligodeoxynucleotides (ODN) induced robust pro-inflammatory responses in thrombocytes, as characterized by more than 100 fold increase in interleukin (IL)-1ß, IL-6 and IL-8 transcripts, while only LPS enhanced nitric oxide production and phagocytic capabilities. Future studies may be aimed at examining the responses of thrombocytes to other TLR ligands.

Understanding the origin of seasonal epidemics of mycoplasmal conjunctivitis.

1. Many host-pathogen systems show regular seasonal oscillations. 2. Seasonal variation in mycoplasmal conjunctivitis prevalence in house finches is an example of such oscillations. 3. An annual pulse of Mycoplasma gallisepticum-naïve juveniles increasing the number of susceptibles, seasonal changes in flocking behaviour increasing transmission rate and a gradual loss of resistance to reinfection with time are sufficient to model the observed seasonal variation in disease prevalence. Nevertheless, experiments are needed to test the underlying mechanisms. 4. We carried out an 18-month experiment with small groups of birds in large aviaries to test two hypotheses. 5. To test the first hypothesis that an influx of naïve juveniles in a group of recovered adults is sufficient to cause an outbreak, we added eight juveniles to a group of 11 adults that had recovered from an earlier infection. In all, three replicates juveniles became infected, but only after some of the adults relapsed. 6. To test the second hypothesis that reintroduction of M. gallisepticum into a multiage group of previously exposed but fully recovered house finches causes a new outbreak, we inoculated two birds in each group in March of the 2nd year. Contrary to what happens in the wild at that time disease prevalence increased rapidly after reintroduction of M. gallisepticum. 7. We conclude that asymptomatic, recovered adults can initiate an epidemic and transmit M. gallisepticum to naïve house finches and that the reintroduction of M. gallisepticum is sufficient to cause a new outbreak, even at a time of the year when mycoplasmal conjunctivitis is low in free-living birds. Date, as such, seems to be less important to explain seasonal variation in conjunctivitis than the presence of naïve juveniles or the introduction on M. gallisepticum. 8. Seasonality in outbreaks is most likely tightly linked to seasonal variation in bird movements and behaviour.