Recipient Organization
UNIVERSITY OF CALIFORNIA, DAVIS
410 MRAK HALL
DAVIS,CA 95616-8671
Performing Department
Population Health & Reproduction
Non Technical Summary
Since 2017, California has been suffering from the severe consequences of Avibacterium paragallinarum (AP) infections in egg layers and broiler chickens. This bacteria causes infectious coryza in chickens. Since then, several poultry producing States across the U.S. e.g. Texas, Pennsylvania, Arizona, Delaware, Maryland, Virginia and North Carolina have had outbreaks with severe production losses and mortality in affected flocks. While in broilers condemnations up to 70% have been reported (1), in layers severe cases lead to 48% mortality and up to 75% drop in egg production (2). One of the biggest issues in establishing preventative strategies against infectious coryza is the lack of understanding of the environmental persistence of the bacteria. Anecdotally, outbreaks in the CA central valley are associated with high wind and humidity during foggy days. In addition, outbreaks in broilers are associated with spread of raw manure in orchards adjacent to broiler premises. Understanding the persistence of the bacteria in layer manure, broiler litter and water might provide a better understanding of the factors associated with the disease dissemination and provide insights on mechanisms to prevent this disease. In addition, associating humidity and temperature fluctuations with bacterial persistence is crucial. The goal of this proposal is to investigate the persistence of Avibacterium paragallinarum in poultry manure, broiler litter and water at different time points with varying temperatures and humidity.Preliminary experiments in broiler litter have shown that this bacteria is not capable of surviving more than 12 hours (3) in a hypothetically contaminated environment. Even though these results suggest that AP is not a particularly persistent bacteria, a more stringent and controlled experiment including humidity and temperature as variables is needed in order to validate these results. In addition, egg laying hens, which are long-lived birds, may be persistent carriers of AP and act as amplifiers of the infection. Their manure might be a significant source of bacteria which, if improperly managed, may spread the infection to other premises. Finally, water is a common source of bacterial infections in poultry flocks if not treated adequately. While closed systems such as "nipple drinkers" are effective in controlling waterborne infections, if not managed adequately bacteria can create biofilms and be an active infection source for poultry flocks. The PI (Dr. Rodrigo Gallardo) is an Associate Professor in poultry medicine with experience in poultry production, diseases and microbiology. Dr. Simone Stoute is an Associate Clinical Professor, poultry diagnostician and head of our Turlock poultry diagnostic laboratory. Dr. Ana Da Silva has a PhD in molecular microbiology and is the current poultry resident at the CAHFS Turlock lab. The above-mentioned professors have been collaborating to evaluate the protection elicited by commercial and autogenous vaccines and typing the latest infectious coryza outbreaks in California and Arizona. Their findings have been published recently (3). This project fits into three high priority issues listed by CFAH: (a) Alternatives to medically important antimicrobial drugs (through management as prevention strategies); (b) disease prevention, control, and surveillance; and (c) endemic diseases in food production animals. We propose experiments to evaluate the in vitro persistence of AP which may be facilitating transmission of the bacteria among poultry premises. This project is part of the multistate effort NC1180 "Control of endemic, emerging and re-emerging poultry respiratory diseases"
Animal Health Component
80%
Research Effort Categories
Basic
20%
Applied
80%
Developmental
0%
Goals / Objectives
Investigate the ecology of poultry respiratory diseases and their role in poultry flocks The new NC project will continue to build upon the foundation laid during previous NC projects. In the new NC project, state representatives will gather disease surveillance information from each state and share the data among participating institutions in annual meetings looking for further collaboration. A standardized data reporting system will be developed. Since the state representatives and other participants are actively involved in key national committees related to poultry diseases, the NC project will serve by gathering, discussing, and providing critical information on disease status at the state and national level which will help to identify research priorities and develop prevention strategies for the researchers, industry and state and federal government officials. This effort is obviously based on mutually beneficial, collaborative, multi-disciplined approaches among participants and collaborators in state and government agencies. The established surveillance efforts will continue to be coordinated and reassessed as needed at annual meetings and via e-mail communication. In addition to proposed surveillance plan described below, efforts will be made to monitor new or re-emerging respiratory pathogens as needed.
AVIAN INFLUENZA VIRUS (AIV). Surveillance will be performed in commercial poultry, backyard poultry, live bird markets and auctions, wild aquatic birds, and swine in collaboration with industry, USDA, US Wildlife Services, and state diagnostic laboratories. AIV will be detected by real-time RT-PCR, virus isolation in eggs or MDCK cells. AIV specimens will be sent to USDA National Veterinary Services Laboratory in Ames, Iowa for HA and NA typing and standard pathotyping tests. The HA genes of the isolates, including all H5 and H7 subtypes will be sequenced. Other genes, depending on the significance of the isolates, may be sequenced. Third generation sequencing efforts are being tested with current surveillance methods in order to update surveillance strategies in the future. This effort is driven by our members in collaboration with the government and diagnostic laboratories.
AVIAN PARAMYXOVIRUS-1 (APMV-1). Virological surveillance testing will be performed in commercial poultry, backyard and auction poultry, and wild birds. Using USDA NAHLN protocols, APMV-1 will be detected by virus isolation and/or real-time RT-PCR. APMV-1 real-time RT-PCR positive samples will be sent to USDA National Veterinary Services Laboratory for confirmation. The fusion (F) genes of recovered isolates will be sequenced and compared to sequences in GenBank. The pathogenicity of APMV-1 isolates will be evaluated in commercial poultry (refer to Objective 2). Likewise, third generation sequencing is being tested as described for AI.
E. COLI. Avian E. coli from respiratory-diseased broiler chickens will be isolated and characterized by O antigen serotyping molecular virulence factors tsh, iss, iucC, IntI and TraT, and pulse field gel electrophoresis (PFGE) to establish diversity.
MYCOPLASMA. Pathogenic avian mycoplasmas in poultry will be isolated and characterized using strain specific PCR, RT-PCR, and culture.
INFECTIOUS BRONCHITIS VIRUS (IBV). Virological surveillance in commercial poultry will be performed. Real time RT-PCR positive samples will undergo virus isolation attempts in SPF embryonated eggs. The S1gene of the isolates will be identified by sequence analysis. Novel variant strains of IBV will be evaluated for their pathogenicity as well as for their ability to break through immunity provided by commercial vaccines (refer to Objective 3).
Genetic changes in the nsp3 gene and in the spike gene of IBV occur at a much higher rate than other genes as the virus adapts to a new host which indicate that monitoring changes in both nsp3 and spike can be a useful indicator of mutations that could potentially lead to emergence of new IBV types. IBV viruses circulating in the field will be examined for genetic changes associated with genetic drift of the virus. This data will be collected for a variety of IBV types isolated over time and be correlated with pathogenicity and emergence of new types.
In addition to commercial poultry, the possibility of a wild bird reservoir for the avian coronaviruses and the potential of those viruses to infect commercial poultry will be investigated. Using a universal avian coronavirus RT-PCR assay, different wild bird species will be monitored. If detected by RT-PCR, efforts will be made to isolate and test wild bird coronavirus isolates for their ability to infect and cause disease in commercial poultry.
INFECTIOUS LARYNGOTRACHEITIS VIRUS (ILTV). ILTV virological surveillance in commercial chickens will be performed. In particular potential sources of infection in the poultry house environment will be identified such as dust, litter, water drinkers, feeders, walls, fans, and darkling beetles. Specimens will be tested by virus isolation and real time (RT)-polymerase chain reaction (PCR). Isolates will be characterized using PCR and restriction fragment length polymorphism (RFLP) to determine if they are wild type or vaccine origin. To evaluate the impact of vaccination in the persistence of the disease, trachea and environmental samples will be collected from ILTV positive and negative farms located in a vaccine buffer zone.
INFECTIOUS BURSAL DISEASE VIRUS (IBDV). Since IBDV significantly affects both the development and outcome of respiratory infection and also vaccine efficacy, IBDV is included in our project. Surveillance for all IBDV strains including vvIBDV will be conducted on commercial and backyard poultry. An RT-PCR assay will be used to detect IBDV strains in bursal tissue from chickens. The nucleotide sequence of positive RT-PCR samples will be determined across the hypervariable sequence region of the VP2 and the VP1 genes. The nucleotide and predicted amino acid sequences will be compared to known strains of the virus. Both VP2 and VP1 sequences are needed to identify vvIBDV strains.
The sharing of surveillance data, sequence information, and isolates will be critical to reduce the overlap of the work among institutions and expedite the selection of strains for in vivo characterization and vaccine development (Objectives 3 and 4).
In order to understand complex upper respiratory diseases, a concerted effort will be carried out to understand the upper respiratory tract virome. Procedures, analysis and bioinformatic pipelines will be shared and discussed in order to expedite the obtention of useful data to understand the ecology of these viruses and how changes can derive into multifactorial diseases.
Develop control and prevention strategies for poultry respiratory diseases We will continue to evaluate the existing control strategies and explore alternative strategies for vaccination and other prevention measures. Different recombinant vaccine technologies are being developed and the techniques described below for specific disease may apply to different respiratory pathogens.
AVIAN INFLUENZA VIRUS (AIV). Broad-spectrum vaccines against influenza A viruses will be developed. In first approach, P particle of norovirus will be used as a vaccine platform to present the highly conserved protein (M2e and HA2 epitope) of viruses and immune-stimulatory proteins. In addition to the structural advantage as a vector, the P particle is highly immunogenic, easily produced in E.coli and extremely stable which may enable reducing production costs and less dependence on the cold - chain distribution which is critical for vaccination programs in remote areas and developing countries. Poultry (chicken), swine and mice challenge models will be used to study the mechanisms of the immune enhancement and develop polyvalent M2e-based vaccines to maximize the protection spectrum against all avian, swine and human flu viruses. In a similar approach, self-assembling peptide nanoparticles will be designed and evaluated. The resemblance of the peptide nanoparticles to virus capsids combines the strong immunogenic effect of live attenuated vaccines with the purity and high specificity in eliciting immune responses of peptide-based vaccines. Two vaccine constructs presenting peptide M2e in monomeric (Mono-M2e) and tetrameric (Tetra-M2e) forms will be evaluated, initially, for their ability to elicit serum antibody responses. Vaccine constructs eliciting antibody will then be tested for the ability to protect against AIV challenge.
In second approach, a system to generate and select NS gene variants (delNS1) of influenza virus with potential use as Live Attenuated Influenza Vaccine (LAIV) will be established. It will demonstrate that most effective LAIV can be systematically selected and vaccine performance can be further potentiated by augmenting the induction of interferon (IFN). The study will include: 1) In vitro analysis of virus particle subpopulations and characterization of delNS1 LAIV candidates for systematic selection of the most effective vaccine; 2) In vivo analysis of selected LAIV candidate and their potential as broad-spectrum vaccines; and 3) Further development of vaccine to ensure the safety of delNS1LAIVs.
INFECTIOUS BRONCHITIS VIRUS (IBV). IBV vaccination methods will be evaluated. The efficacy of IBV vaccines administered by spray at the hatchery will be assessed. Chicks will be examined by real time RT- PCR to assess vaccine coverage following spray administration using vaccines delivered in different volumes.
Studies have shown that vaccinating with two different types of IBV vaccine can provide broad protection against different IBV types. These so called protectotype vaccine combinations will be evaluated for different variant virus in vivo. In addition, different criterion used to evaluate protection will be compared. Vaccine efficacy in the USA is evaluated by detection of the challenge virus as specified by the Code of Federal Regulations, Title 9 whereas in Europe, ciliostasis in the trachea is evaluated as specified by the European Pharmacopeia. To better understand the relationship between protection defined by challenge virus detection and ciliostasis in the trachea, both parameters in vaccinated chickens challenged with homologous and heterologous strains of IBV will be evaluated. Furthermore, clinical signs as well as macroscopic and microscopic lesions in the trachea will be evaluated to generate a more complete picture of parameters used to evaluate vaccine efficacy.
The IBV S2 protein expressed from recombinant NDV has been shown to provide protection against challenge. New recombinant viruses encoding distinct S2 proteins will be produced to optimize protection. In addition, recombinant adenovirus type 5 (rAd) expressing S1 protein will continue to be evaluated for immune responses and protective efficacy in chickens.
Finally, in order to boost the immune response generated by live commercial vaccines we will use adjuvants based on slow release chitosan encapsulated avian interferons (IFN), to boost innate and adaptive immune responses generated by these vaccines and test their efficacy with homoplogous and heterologous challenges.
INFECTIOUS LARYNGOTRACHEITIS VIRUS (ILTV).
Traditional and alternative methods used to attenuate other respiratory viruses will be used to develop new ILTV experimental vaccines. If successful, studies will characterize the attenuated strains in terms of their pathogenicity, transmissibility, and immunogenicity for chickens. The attenuated strains will also be sequenced to identify specific gene(s) mutations arising from the attenuation process.
The use of potential live ILTV vaccines attenuated by deletion of virulence determinants for in ovo mass application of broilers will be studied. Recent full genome sequencing analysis of classically attenuated live vaccines indicated that a truncation of the ORF-C gene maybe responsible for the increased attenuation of the tissue culture origin (TCO) vaccine. It is hypothesized that the ORF-C protein is a virulent determinant of ILTV. Therefore, by deleting the ORF-C gene the USDA challenge strain will become attenuated in chickens without affecting in vitro replication. The specific aims of this objective are to determine if deletion of the ORF-C provides attenuation to the virulent USDA strain and whether an ORF-C deleted ILTV strain induce protection in specific pathogen free (SPF) chickens after in ovo vaccination. The development of an effective ILTV vaccine for in ovo vaccination will eliminate the need to vaccinate broilers during the grow-out, consequently reducing the circulation of chicken embryo origin vaccines.
INFECTIOUS BURSAL DISEASE VIRUS (IBDV). DNA vaccination will be optimized for IBD. The chicken cytokine gene or chicken innate immunity gene will be used as the molecular adjuvant in the hope to enhance protective immunity. Specific-pathogen-free chickens or broiler chickens with maternally-derived antibody will be in ovo or intramuscularly injected with a chimeric DNA plasmid (carrying IBDV VP2 gene fused with molecular adjuvant) or a DNA plasmid carrying IBDV VP2 gene that will be co-administered in conjunction with a DNA plasmid carrying molecular adjuvant. Chickens will be challenged with homologous or heterologous classic or variant IBDV strain. ELISA, virus neutralization, gross pathology and histopathology, and IBDV viral RNA load assessment by real-time RT-PCR will be analyzed to determine immune response and protection efficacy. In addition, development of a reverse genetics-based IBDV will serve as a vaccine vector for protection against avian influenza and other emerging poultry respiratory diseases will be explored.
The availability of a single vaccine capable of in ovo administration in broilers that could protect against IB, ND, IBD and Marekâ¿¿s disease (MD) would be major breakthrough in sustainable agriculture and profitability. HVT vector vaccines will be generated by insertion, into one of the 2 genomic sites previously identified, of antigenic genes from IBDV, NDV and IBV into the HVT-BAC genome by the two-step Red recombination technique (86). The genes for the different immunogenic proteins will be cloned under the control of a cytomegalovirus (CMV) immediate early promoter and bovine growth hormone polyA signal sequences. Genes will be cloned in both loci and in different combinations to better identify the configuration that provides the best expression of the different proteins. In vitro characterization of the recombinant HVT viruses will be performed by multi-step growth kinetics in CEF and expression of the antigenic proteins determined by ELISA. In vivo characterization of the recombinant viruses will be performed by in ovo (18 day of embryonation) and at hatch inoculation with the different vaccines. Expression of the immunogenic proteins will be determined by examining levels of antibody production against the immunogenic proteins overtime using ELISA. Protection efficacy of the HVT vector vaccines will be examined by in ovo or day old vaccination with those vaccines that induce the best immune response followed by assessment of antibody levels and/or challenge with pathogenic viruses.
Project Methods
PROJECT DETAILSPersistence in broiler litter and layer manure:Broiler bedding and layer manure will be distributed in three glass containers each. The litter/manure will be humidified until they reach a low (<30%), medium (30 to 50%) and high (>50%) relative humidities. After reaching the humidities, the material will be divided in 2 for experimental replication (total of 12 groups). Each container will be spiked with a culture of Avibacterium paragallinarum with a titer of approximately 105 cfu. Samples of 2 g will be collected at 15 and 30 min, and every 30 min until 12 hours after the spiking. Manure and litter samples will be processed to extract DNA by a commercial kit and qPCR will be performed for the detection of AP. Additional samples will be collected, in transport media, at every time point for bacterial isolation. Isolation will be attempted in chocolate agar plates with a streak of staphylococcus as a nursing colony. Results from isolation and qPCR will be compared. Samples will be processed in small batches until reaching the point in which the bacteria is not alive.Persistence in water at different temperatures: Four beakers with 150 ml of sterile water will be spiked with layer manure infected with 105 cfu of AP. Two beakers will be placed at 10°C and two at 25°C. Water (2 ml) will be collected at 15 and 30 min, and every hour until 24 hours after the spiking. Samples will be processed in a similar fashion as explained above.