FALSE LAYER SYNDROME (FLS) INFECTIOUS BRONCHITIS VIRUS (IBV) IN HENS, PATHOBIOLOGY AND LONG TERM EFFECTS OF CONTROL INTERVENTIONS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: ANIMAL HEALTH
• Reporting Frequency: Annual
• Accession No.: 1025839
• Project Start Date: Feb 4, 2021
• Project End Date: Jun 30, 2021
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF CALIFORNIA, DAVIS
410 MRAK HALL
DAVIS,CA 95616-8671

Performing Department
Population Health & Reproduction

Non Technical Summary
False layer syndrome is a condition in egg laying flocks in which hens do not reach peak of production due to a significant percentage of internal layers in the flock. This syndrome has been associated to early exposure to Infectious bronchitis virus (IBV), which affects oviductal development inducing atrophy of the oviduct early in their lives.We propose to further investigate the FLS-associated agents using molecular techniques and focusing on their full genome and complete the analysis of the samples collected in the abovementioned experiment at 10 and 21 ds post challenge. In addition, samples will be collected from chickens, pullets and layers in commercial flocks that have had FLS and now are using live vaccines as prevention for surveillance of prevalent IBV strains in the field after vaccine intervention. It is important to understand the effect of massive vaccination in the environment and the real presence of FLS IBV viruses in the flocks.

Animal Health Component

20%

Research Effort Categories

Basic

80%

Applied

20%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3299	1101	100%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3299 - Poultry, general/other;

Field Of Science
1101 - Virology;

Keywords

infectious bronchitis

false layer syndrome

Goals / Objectives
iii) Specific objectives:1.Full genome sequence the FLS IBV and check for differences outside the S gene2.Demonstrate the recreation of false layer syndrome in naïve SPF chicks challenged at 3 days of age with DMV1639 isolated from field cases through processing samples obtained at 10 and 21dpc3.Perform epidemiological surveillance in flocks with an IBV-induced FLS history, where early vaccination is used and not used as a preventative strategy

Project Methods
iv) Methodology:Objective 1: viral RNA will be extracted from an isolate of an IBV associated to FLS. Libraries will be prepared, and sequencing will be performed using the MinIon nanopore sequencer at our laboratory.Objective 2: Naïve SPF chicks were challenged at 3 days of age (doa). Samples were collected at 2, 5, 10 and 21 dpi for: viral load by qRT-PCR, histopathology and immune histochemistry (IHC) of ovary, oviduct, sinus, trachea, lungs and kidneys. Results from 2 and 5 dpi suggest respiratory colonization and mild effects in kidneys and oviducts. Analysis of samples at 10 and 21 dpi will help dilucidate the effect of this specific virus in the reproductive tract of these chicks.Objective 3: Tracheas or tracheal swabs will be collected at 1, 7, 14, and 21 doa and every month until lay onset in birds from vaccinated and unvaccinated flocks from a commercial farm that has had IBV associated FLS. In addition, naïve birds will be placed at day of age with the received chicks from one flock, 10 will be euthanized and sampled at the same time points. Tracheas, lungs and cecal tonsils will be collected in PBS, frozen and submitted to the Poultry Medicine lab at UC Davis where RT-qPCR will be performed. Positive samples will be submitted to S1 RT-PCR and sequencing for genotyping.

Progress 02/04/21 to 06/30/21

Outputs
Target Audience:Poultry veterinarians and scientists working on disease prevention and health Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One post doc and two graduate students were trained in this project How have the results been disseminated to communities of interest?Results have been disseminated through journal publications, presentation in scientific and outreach meetings and during the outreach work of the PD What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? This project focused on the investigation of the basis of false layer syndrome in laying hens and how preventative measures e.g., vaccination or maternal antibodies, act in ameliorating the outcomes of early IBV infection. We investigated the whole genome of strains previously associated with FLS in laying hens finding information suggesting common evolutionary patterns between IBV QX and IBV DMV1639 both strains causing FLS in Europe, Asia, South America, and North America, respectively. This evolution allows the virus to acquire pathogenicity to diverse organs (kidneys / reproductive tract) on its way to become a merely respiratory infection. We also performed challenge experiments with AZ/FLS/17, a strain associated with FLS in Arizona, and M41 were pathological and replication outcomes were compared. This was performed in 3wk old chicks. We did not find any histopathology or viral load differences between the two challenges. We also performed IBV surveillance in flocks where interventions (day old IBV vaccination) were in place still finding the FLS/like strains (DMV 1639) usually after 3 weeks of age and in production. These results suggest that the vaccine displaces the early challenge but does not eliminate the FLS-associated IBV strain from the environment. Finally, we designed an experiment in which we proved the real effects of early vaccination and maternal antibodies against early IBV challenges in hens. In this experiment we found a clear protective effect provided by maternal antibodies against both early vaccination and challenge. We also observed that vaccination at early ages, in absence of maternal antibodies, can induce reproductive issues such as reduced egg production and FLS associated lesions like cystic oviducts and egg yolk coelomitis. These results suggested that maternal antibodies and timing of IBV infection are more important in the generation of FLS than the IBV strain itself, and while vaccines can displace the challenge reducing the incidence of FLS in the flock they can cause FLS if the vaccinated birds don't have enough maternal antibodies.

Publications

• Type: Journal Articles Status: Published Year Published: 2021 Citation: 1. A.P. da Silva, C. Giroux, H. S. Sellers, A. Mendoza-Reilley, S. Stoute and R.A. Gallardo. Characterization of an IBV isolated from commercial layers suffering from false layer syndrome. 2021. Avian Diseases 65:3 373-380
• Type: Journal Articles Status: Published Year Published: 2022 Citation: 2. Gallardo, R.A., da Silva, A.P., Gilbert, R., Alfonso, M., Conley, A., Jones, K., Stayer, P.A. and Hoerr, F.J., 2022. Testicular Atrophy and Epididymitis-Orchitis Associated with Infectious Bronchitis Virus in Broiler Breeder Roosters. Avian Diseases, 66(1), pp.112-118
• Type: Journal Articles Status: Published Year Published: 2022 Citation: 3. A.P. Da Silva, R. Jude, R.A. Gallardo. Infectious bronchitis virus: A comprehensive multilocus genomic analysis to compare DMV/1639 and QX strains. Viruses. 2022
• Type: Journal Articles Status: Published Year Published: 2023 Citation: 4. R. Jude, B. Jordan, A. Muller-Slay, R. Luciano, A. P da Silva, R. A. Gallardo. Mitigation of False Layer Syndrome Through Maternal Antibodies Against Infectious Bronchitis Virus. 2023. Avian Diseases

Progress 02/04/21 to 06/30/21

Outputs
Target Audience:Poultry bussiness owners and poultry scientists and veterinarians Changes/Problems:We found that we couldnt proof the pathigenicity of the virus in chicks, so we had to work with old hens challenged as chicks What opportunities for training and professional development has the project provided?This project has trained one graduate student, four undergrads and one postdoctoral scholar How have the results been disseminated to communities of interest?Journal publications Talks in conferences Talks with producers and veterinarians in the field What do you plan to do during the next reporting period to accomplish the goals?this project is comopleted

Impacts
What was accomplished under these goals? All these objectives were accomplished

Publications

• Type: Journal Articles Status: Published Year Published: 2021 Citation: 24. A.P. da Silva, C. Giroux, H. S. Sellers, A. Mendoza-Reilley, S. Stoute and R.A. Gallardo. Characterization of an IBV isolated from commercial layers suffering from false layer syndrome. 2021. Avian Diseases. Accepted
• Type: Journal Articles Status: Submitted Year Published: 2023 Citation: R. Jude, B. Jordan, A. Muller-Slay, R. Luciano, A. P da Silva, R. A. Gallardo. Mitigation of False Layer Syndrome Through Maternal Antibodies Against Infectious Bronchitis Virus. 2023. Avian Diseases. Submitted