Necrotizing Hepatitis Associated with Clostridium perfringens in Broiler Chicks.

In this retrospective study we describe unusual cases of clostridial hepatitis associated with high mortality in young broiler chicks. Eleven cases of necrotizing hepatitis in broiler chicks from four companies were submitted to the Poultry Diagnostic and Research Center or the Georgia Poultry Laboratory Network between 2017 and 2020. In most flocks, increased 3-day mortality was followed by an elevated 7-day mortality. Gross lesions included green to dark brown discoloration of the liver, congested lungs, serosanguineous fluid in the caudoventral aspect of the abdomen, and emphysema in the yolk sacs. In birds older than a week of age, disease with neurologic signs became evident and consisted of tremors, stargazing, and incoordination. Histopathologic evaluation revealed multifocal to coalescing fibrinoheterophilic and necrotizing hepatitis associated with gram-positive, long, rod-shaped bacteria. Formalin-fixed liver samples from six cases out of eight cases tested were positive for Clostridium perfringens by immunohistochemistry. Liver samples from two cases were culture positive for Clostridium spp., and C. perfringens was isolated from one sample. Toxinotyping by PCR performed in seven samples revealed the presence of the genes that code for alpha toxin phospholipase C (cpa or plc) and necrotic enteritis toxin B-like (netB) in six samples and as well as C. perfringens large cytotoxin (tpeL) in one sample. Broiler breeders are the suspected source of the infection, and testing revealed C. perfringens in hatchery samples and among broiler breeder flocks. Antimicrobial therapy was coupled with enhanced sanitation at the farm and hatchery in that company, markedly decreasing the mortality and clinical signs. This is the first comprehensive evaluation of clostridial necrotizing hepatitis in newly hatched chicks, and the second ever reported in the literature.

Hepatitis necrotizante asociada con Clostridium perfringens en pollos de engorde En este estudio retrospectivo se describen casos inusuales de hepatitis clostridial asociados con una alta mortalidad en pollos de engorde jóvenes. Once casos de hepatitis necrotizante en pollos de engorde de cuatro empresas se enviaron al Centro de Investigación y Diagnóstico Avícola o a la Red de Laboratorios Avícolas del Estado Georgia entre los años 2017 y 2020. En la mayoría de las parvadas, el aumento de la mortalidad a los tres días fue seguido por una mortalidad elevada a los siete días. Las lesiones macroscópicas incluyeron coloración del hígado de verde a marrón oscuro, pulmones congestionados, líquido serosanguinolento en la cara caudoventral del abdomen y enfisema en los sacos vitelinos. En aves mayores de una semana de edad, la enfermedad con signos neurológicos se hizo evidente y consistía en temblores, torticolis (aves como observando a las estrellas) y falta de coordinación. La evaluación histopatológica reveló hepatitis multifocal a fibrinoheterófila coalescente y necrotizante asociada con bacterias grampositivas largas en forma de bastón. Las muestras de hígado fijadas en formalina de seis casos de los ocho casos analizados dieron positivo para Clostridium perfringens por inmunohistoquímica. Las muestras de hígado de dos casos dieron positivo en cultivo para Clostridium spp., y se aisló C. perfringens de una muestra. La tipificación por el tipo de toxina mediante PCR realizado en siete muestras reveló la presencia de los genes que codifican la toxina alfa fosfolipasa C (cpa, plc) y la toxina de enteritis necrótica similar a la toxina B (netB) en seis muestras, así como la citotoxina grande de C. perfringens (tpeL) en una muestra. Se sospecha que las reproductoras de pollos de engorde son la fuente de la infección, y las pruebas revelaron C. perfringens en las muestras de las incubadoras y entre las parvadas de reproductoras de pollos de engorde. La terapia antimicrobiana se combinó con un saneamiento mejorado en la granja y en la incubadora de esa empresa, lo que redujo notablemente la mortalidad y los signos clínicos. Esta es la primera evaluación exhaustiva de la hepatitis necrosante por clostridios en pollitos recién nacidos y la segunda que se ha informado en la literatura.

Novel Recombinant Newcastle Disease Virus-Based In Ovo Vaccines Bypass Maternal Immunity to Provide Full Protection from Early Virulent Challenge.

Newcastle disease (ND) is one of the most economically important poultry diseases. Despite intensive efforts with current vaccination programs, this disease still occurs worldwide, causing significant mortality even in vaccinated flocks. This has been partially attributed to a gap in immunity during the post-hatch period due to the presence of maternal antibodies that negatively impact the replication of the commonly used live vaccines. In ovo vaccines have multiple advantages and present an opportunity to address this problem. Currently employed in ovo ND vaccines are recombinant herpesvirus of turkeys (HVT)-vectored vaccines expressing Newcastle disease virus (NDV) antigens. Although proven efficient, these vaccines have some limitations, such as delayed immunogenicity and the inability to administer a second HVT vaccine post-hatch. The use of live ND vaccines for in ovo vaccination is currently not applicable, as these are associated with high embryo mortality. In this study, recombinant NDV-vectored experimental vaccines containing an antisense sequence of avian interleukin 4 (IL4R) and their backbones were administered in ovo at different doses in 18-day-old commercial eggs possessing high maternal antibodies titers. The hatched birds were challenged with virulent NDV at 2 weeks-of-age. Post-hatch vaccine shedding, post-challenge survival, challenge virus shedding, and humoral immune responses were evaluated at multiple timepoints. Recombinant NDV (rNDV) vaccinated birds had significantly reduced post-hatch mortality compared with the wild-type LaSota vaccine. All rNDV vaccines were able to penetrate maternal immunity and induce a strong early humoral immune response. Further, the rNDV vaccines provided protection from clinical disease and significantly decreased virus shedding after early virulent NDV challenge at two weeks post-hatch. The post-challenge hemagglutination-inhibition antibody titers in the vaccinated groups remained comparable with the pre-challenge titers, suggesting the capacity of the studied vaccines to prevent efficient replication of the challenge virus. Post-hatch survival after vaccination with the rNDV-IL4R vaccines was dose-dependent, with an increase in survival as the dose decreased. This improved survival and the dose-dependency data suggest that novel attenuated in ovo rNDV-based vaccines that are able to penetrate maternal immunity to elicit a strong immune response as early as 14 days post-hatch, resulting in high or full protection from virulent challenge, show promise as a contributor to the control of Newcastle disease.

A Novel Recombinant Newcastle Disease Vaccine Improves Post- In Ovo Vaccination Survival with Sustained Protection against Virulent Challenge.

In ovo vaccination has been employed by the poultry industry for over 20 years to control numerous avian diseases. Unfortunately, in ovo live vaccines against Newcastle disease have significant limitations, including high embryo mortality and the inability to induce full protection during the first two weeks of life. In this study, a recombinant live attenuated Newcastle disease virus vaccine containing the antisense sequence of chicken interleukin 4 (IL-4), rZJ1*L-IL4R, was used. The rZJ1*L-IL4R vaccine was administered in ovo to naïve specific pathogen free embryonated chicken eggs (ECEs) and evaluated against a homologous challenge. Controls included a live attenuated recombinant genotype VII vaccine based on the virus ZJ1 (rZJ1*L) backbone, the LaSota vaccine and diluent alone. In the first of two experiments, ECEs were vaccinated at 18 days of embryonation (DOE) with either 104.5 or 103.5 50% embryo infectious dose (EID50/egg) and chickens were challenged at 21 days post-hatch (DPH). In the second experiment, 103.5 EID50/egg of each vaccine was administered at 19 DOE, and chickens were challenged at 14 DPH. Chickens vaccinated with 103.5 EID50/egg of rZJ1*L-IL4R had hatch rates comparable to the group that received diluent alone, whereas other groups had significantly lower hatch rates. All vaccinated chickens survived challenge without displaying clinical disease, had protective hemagglutination inhibition titers, and shed comparable levels of challenge virus. The recombinant rZJ1*L-IL4R vaccine yielded lower post-vaccination mortality rates compared with the other in ovo NDV live vaccine candidates as well as provided strong protection post-challenge.

Newcastle Disease Virus Infection in Quail.

Newcastle disease (ND), caused by virulent strains of Newcastle disease virus (NDV), is a devastating disease of poultry worldwide. The pathogenesis of ND in quail is poorly documented. To characterize the ability of virulent NDV strains to replicate and cause disease in quail, groups of 14 two-week-old Japanese quail ( Coturnix japonica) were experimentally inoculated with 108 EID50 (embryo infectious dose 50%) units of 1 of 4 virulent NDV strains: 2 isolated from quail ( N2, N23) and 2 from chickens ( Israel, Pakistan). At day 2 postinfection, noninfected quail (contact group) were added to each infection group to assess the efficacy of virus transmission. Tested NDV strains showed moderate pathogenicity, with highest mortality being 28% for the N2 strain and below 10% for the others. Two N2-inoculated birds showed neurological signs, such as head tremor and ataxia. Microscopic lesions were present in N2-, Israel-, and Pakistan-inoculated birds and consisted of nonsuppurative encephalitis. Contact birds showed no clinical signs or lesions. In both inoculated and contact birds, virus replication was moderate to minimal, respectively, as observed by immunohistochemistry in tissues and virus isolation from oropharyngeal and cloacal swabs. Strains originally isolated from quail resulted in higher numbers of birds shedding in the inoculation group; however, transmission appeared slightly more efficient with chicken-derived isolates. This study shows that virulent NDV strains have limited replicative potential and mild to moderate disease-inducing ability in Japanese quail.

Intracellular fixation buffer inactivates Newcastle disease virus in chicken allantoic fluid, macrophages and splenocytes.

Inactivation of Newcastle disease virus (NDV) has been routinely achieved with heat, ß-propiolactone, binary ethylenimine, ultraviolet light and formalin. However, these strategies have not been tested for cell surface ligand or receptor phenotype in viral-infected chicken immune cells. To study the capacity of fixation buffers to preserve surface markers while inactivating NDV, a primary splenocyte culture was infected with NDV and incubated with a commercial intracellular fixation buffer (ICB), formulated with 4% formaldehyde. Splenocytes were fixed with a 1:2 dilution of ICB in phosphate buffered saline (PBS) for 45min at 23°C or 4°C and inactivation of NDV was tested in addition to recognition of antigens by antibodies in fixed and non-fixed splenocytes via flow cytometric analysis. The binding and percentage of splenic CD4+ and CD8+ cells were not affected. In addition, NDV titers as high as 109.5 and 107.6 EID50 in allantoic fluid (AF) and macrophages, respectively, were successfully inactivated after 45min at 23°C and 4°C, confirming the ICB's effectiveness in inactivating high concentrations of NDV. In conclusion, high concentrations of NDV in AF, chicken splenocytes, and macrophages can be inactivated using ICB. Additionally, this method did not compromise cell phenotyping of enriched chicken splenocytes.

Effects of Chicken Interferon Gamma on Newcastle Disease Virus Vaccine Immunogenicity.

More effective vaccines are needed to control avian diseases. The use of chicken interferon gamma (chIFNγ) during vaccination is a potentially important but controversial approach that may improve the immune response to antigens. In the present study, three different systems to co-deliver chIFNγ with Newcastle disease virus (NDV) antigens were evaluated for their ability to enhance the avian immune response and their protective capacity upon challenge with virulent NDV. These systems consisted of: 1) a DNA vaccine expressing the Newcastle disease virus fusion (F) protein co-administered with a vector expressing the chIFNγ gene for in ovo and booster vaccination, 2) a recombinant Newcastle disease virus expressing the chIFNγ gene (rZJ1*L/IFNγ) used as a live vaccine delivered in ovo and into juvenile chickens, and 3) the same rZJ1*L/IFNγ virus used as an inactivated vaccine for juvenile chickens. Co-administration of chIFNγ with a DNA vaccine expressing the F protein resulted in higher levels of morbidity and mortality, and higher amounts of virulent virus shed after challenge when compared to the group that did not receive chIFNγ. The live vaccine system co-delivering chIFNγ did not enhanced post-vaccination antibody response, nor improved survival after hatch, when administered in ovo, and did not affect survival after challenge when administered to juvenile chickens. The low dose of the inactivated vaccine co-delivering active chIFNγ induced lower antibody titers than the groups that did not receive the cytokine. The high dose of this vaccine did not increase the antibody titers or antigen-specific memory response, and did not reduce the amount of challenge virus shed or mortality after challenge. In summary, regardless of the delivery system, chIFNγ, when administered simultaneously with the vaccine antigen, did not enhance Newcastle disease virus vaccine immunogenicity.

Inhibition of influenza virus infection and hemagglutinin cleavage by the protease inhibitor HAI-2.

Influenza virus remains a significant concern to public health, with the continued potential for a high fatality pandemic. Vaccination and antiviral therapeutics are effective measures to circumvent influenza virus infection, however, multiple strains have emerged that are resistant to the antiviral therapeutics currently on the market. With this considered, investigation of alternative antiviral therapeutics is being conducted. One such approach is to inhibit cleavage activation of the influenza virus hemagglutinin (HA), which is an essential step in the viral replication cycle that permits viral-endosome fusion. Therefore, targeting trypsin-like, host proteases responsible for HA cleavage in vivo may prove to be an effective therapeutic. Hepatocyte growth factor activator inhibitor 2 (HAI-2) is naturally expressed in the respiratory tract and is a potent inhibitor of trypsin-like serine proteases, some of which have been determined to cleave HA. In this study, we demonstrate that HAI-2 is an effective inhibitor of cleavage of HA from the human-adapted H1 and H3 subtypes. HAI-2 inhibited influenza virus H1N1 infection in cell culture, and HAI-2 administration showed protection in a mouse model of influenza. HAI-2 has the potential to be an effective, alternative antiviral therapeutic for influenza.

Plasmin-mediated activation of pandemic H1N1 influenza virus hemagglutinin is independent of the viral neuraminidase.

Influenza virus is well recognized to modulate host tropism and pathogenesis based on mutations in the proteolytic cleavage site of the viral hemagglutinin (HA), which activates HA and exposes the fusion peptide for membrane fusion. Instead of the conventional trypsin-mediated cleavage event, modification of the cleavage site allows extended use of host cell proteases and enhanced spread in vivo. For H1N1 influenza viruses, the mouse-adapted A/WSN/33 strain is known to replicate in the brain based on recruitment of plasminogen by the viral neuraminidase (NA), as well as a Ser-Tyr substitution at the P2 position of the HA cleavage site. Here, we show that an equivalent Ser-Tyr substitution has occurred in the HA of naturally occurring human H1N1 influenza viruses. We characterize one of these viruses (A/Beijing/718/2009), as well as the prototype A/California/04/2009 with a Ser-Tyr substitution in the cleavage site, and show that these HAs are preferentially cleaved by plasmin. Importantly, cleavage activation by plasmin/plasminogen was independent of the viral NA, suggesting a novel mechanism for HA cleavage activation. We show that the viral HA itself can recruit plasminogen for HA cleavage. We further show that cellular factors, as well as streptokinase from bacteria commonly coinfecting the respiratory tract of influenza patients, can be a source of activated plasminogen for plasmin-mediated cleavage of influenza virus HAs that contain a Ser-Tyr substitution in the cleavage site.