proteins, underlining the complexity of HPAIV Avian Influenza infections. Surveillance and education in Pathobiology of classical H7N1 and Gs/GD commercial but also local chicken holdings are H5N8 highly pathogenic avian influenza viruses required to early detect the circulation of in different chicken breeds and role of Mx HPAIVs. 2032 G/A polymorphism in infection outcome Vaccination Efficacy of Multivalent Kateri Bertran Recombinant Herpesvirus Turkey Vaccines Against Highly Pathogenic Avian Influenza IRTA, Centre de Recerca en Sanitat Animal 1 2 (CReSA, IRTA-UAB) Miria Criado , Jung-hoon Kwon , Mariana Sa e Silva3, David E. Swayne4 The susceptibility to highly pathogenic avian 1 2 influenza virus (HPAIV) infection varies among USDA, aSoutheast Poultry Research chicken breeds/lines. Local chicken breeds are Laboratory, USDA-ARS, 934 College Station Rd., 3 thought to be naturally resistant, but Athens, GA 30605, USA., Boehringer Ingelheim experimental studies seldom support this. To Animal Health USA Inc., 1730 Olympic Drive, GA 4 date, the susceptibility of Spanish local chicken 30601, USA, aSoutheast Poultry Research breeds to HPAIVs is unknown. We evaluated the Laboratory, USA. pathobiology of two HPAIVs (a Spanish Gs/GD Vaccination strategies are important as a control H5N8 clade 2.3.4.4B and a classical H7N1 HPAIV) tool for high pathogenicity avian influenza (HPAI) in local Spanish (n=6), commercial (n=1), and viruses. A single vaccine given in the hatchery experimental (n=1) chicken lines. Both viruses against multiple pathogens is advantageous for were highly lethal in chickens, but H7N1 HPAIV disease control and for cost savings. One-day-old was more virulent based on clinical signs, SPF leghorn chickens received one of three vHVT mortality rates, and mean death times (MDTs). vaccine candidates, which contained avian While the H7N1 HPAIV was shed to high titers by influenza (AI) insert alone (vHVT509-AI) or AI and a higher number of chickens orally and cloacally, additional inserts, as Infectious Bursal Disease the H5N8 HPAIV was shed efficiently only by the Virus (IBDV) (vHVT522-AI-IBD) or Newcastle oral route, possibly decreasing transmission Disease Virus (NDV) (vHVT523-AI-ND). Four- efficiency. Both HPAIVs replicated systemically weeks post-vaccination birds were challenged but displayed tropism differences. Interestingly, against three different H5 HPAI viruses H7N1 HPAIV had a greater neurotropism, [A/Tk/Mn/12582, H5N2, clade 2.3.4.4 explaining the associated higher mortalities. (Minnesota/2015); A/Egypt/N04915/2014, Based on clinical signs, mortality, and virus H5N1, clade 2.2.1 (Egypt/2014); and shedding, 5/6 local breeds were highly A/domestic_turkey/Hungary/54433/2016, susceptible to HPAIV infection, while 1/6 local H5N8, clade 2.3.4.4B (Hungary/2016)]. All sham breed and the commercial and experimental vaccinated birds died between 2 to 3 dpc breeds were more resistant. The analyses of the while100% of vHVT-vaccinated birds lacked chicken Mx gene revealed that AA and AG clinical signs and survived challenge against H5 genotypes at position 2032 were statistically HPAI viruses. The only exception was 90% associated with longer MDTs. In summary, the protection in vHVT523-AI-ND vaccinated birds HPAIV infection outcome is influenced by the and challenged with Egypt/2014 and virus, the genetic background of the host, and Hungary/2016. Independent of the vaccine and particular alleles in genes encoding antiviral challenge virus used the OP shedding was significantly reduced compared to the sham inducing capacities. In addition, the genomic group at 2 dpc (p <0.05). Four weeks after determinants of IFN induction were identified vaccination, most vaccinated birds had HI and this information was used to custom-design antibodies detected when using the HPAI viruses a highly efficacious master LAIV candidate for as antigen, and the titers significantly increased poultry. From one-hundred plaque isolates, we in the post-challenge sera for all survived birds. identified several candidates that induce Therefore, our study demonstrated that vHVT significantly higher levels of IFN in cell cultures vaccine candidates containing AI insert alone or compared to the original LAIV. Vaccination of 2- AI in combination with IBDV or NDV had similar week-old chickens with the top 3 IFN-inducer efficiency in protection against challenge with candidates proved their elite innate immune- homologous or heterologous HPAI viruses. stimulatory nature, while retaining their full Moreover, these vaccine candidates in a single protective efficacies against influenza challenge. dose hold a great promise to provide Genome-wide identification of mutations simultaneous protection against multiple viral revealed additional truncation in the non- diseases affecting poultry worldwide. structural 1 (NS1) and a single amino acid substitution in polymerase basic 2 (PB2) proteins Development of next-generation live as determinants of higher IFN response in these attenuated influenza vaccines with elite strains. A next-generation LAIV candidate, with interferon-inducing capabilities highly enhanced IFN-inducing character, was Amir Ghorbani1, John M. Ngunjiri2, Michael C. reverse-genetically created by combining both Abundo3, Kara J. M. Taylor4 NS1 and PB2 mutations in a single virus. This next-generation LAIV candidate is currently 1 2 The Ohio State University, Food Animal Health being tested for its improved protective efficacy Research Program, Ohio Agricultural Research against early and heterosubtypic influenza virus and Development Center, The Ohio State challenges in chickens. University, Wooster, Ohio, United States of America., 3Food Animal Health Research Evaluation of the cross-antigenicity of two Program, United States of America., 4Food H9N2 vaccine strains assessed by HI-test using Animal Health Research Program, United States sixteen distinct AIV H9N2 antigens of America. Andreas Herrmann Avian influenza outbreaks continue to pose a MERIAL S.A.S. major threat to poultry industry worldwide. There is currently a growing need to develop Immunisation of chicken for protection against effective influenza vaccines against emerging an emerging disease. Insights in chicken post viruses in the field. We previously showed the vaccinal serological immune response against effectiveness of a live attenuated influenza LPAIV H9N2 and cross-reactivity against various vaccine (LAIV) against antigenically distant distinct antigens provide better understanding challenge viruses in chickens. The effectiveness of cross-immunity for better protection. of LAIV was linked to its ability to induce high levels of type I interferon (IFN) in primary chicken embryo cells. The aim of this study was to deconstruct the viral subpopulations of LAIV and further improve its immunogenicity by selecting the viral clones with higher IFN- T Cell Epitope Engineering Strategy to Improve following H7N9 lethal challenge compared to WT the Immunogenicity of Avian Influenza H7N9 vaccinated or control groups. This study confirms Vaccine that T-cell engineered vaccines can improve the immunogenicity and survival against H7N9 Hyesun Jang1, Leonard Moise2, Bethany M. challenge in a humanized mouse model. Biron3, Ted M. Ross4 Characterization of a Novel Avian Influenza 1University of Georgia, 2EpiVax, Inc., Institute for Live Virus Vaccine Based on Disruption of Immunology and Informatics, Department of M2/M42 Gene Expression That Protects Cell and Molecular Biology, University of Rhode Against Clade 0 and 2.3.4.4 Highly Pathogenic Island, University of Rhode Island , RI , USA., Avian Influenza 3cEpiVax, dInstitute for Immunology and Informatics, eUniversity of Rhode Island, USA., Darrell Kapczynski 4University of Georgia USDA-ARS-USNPRC The low immunogenicity of H7 hemagglutinin Vaccine protection of poultry against highly (HA) is a great challenge in developing pathogenic avian influenza virus will be efficacious vaccines against avian-origin H7N9 discussed. In addition, characterization of viral viruses. Previous studies suggest that this low ion-channel proteins (M2 and M42) on immunogenicity could be due to the regulatory T replication, morphology and transmission will be cell (Treg)–stimulating epitopes found on H7 discussed. Attendees interested in influenza HAs. We hypothesized that the immunogenicity virus and immunology should attend this of H7 HAs can be improved by replacing the program. Treg–stimulating sequences with memory CD4+ T cell–stimulating sequences from seasonal H3 Roles of the matrix and nucleoprotein genes strains. These modified H7 HA sequences were on the virulence of H5N8 highly pathogenic expressed as soluble recombinant proteins and avian influenza virus clade 2.3.4.4 were designated as rH7 HA OPT1 or OPT2. To 1 2 evaluate the efficacy of the rH7 HA OPT1 and Christina Leyson , Sungsu Youk , Mary Pantin- 3 OPT2, we designed a novel humanized mouse Jackwood model (HLA-DR3) for the efficacy study to 1Southeast Poultry Research Center, USDA-ARS, emulate the human major histocompatibility 2Southeast Poultry Research Center, USDA-ARS, class II (MHC II) –T cell receptor (TCR) 3Southeast Poultry Research Laboratory interaction. HLA-DR3 mice were intranasally pre- immunized with a H3N2 (A/HK/2014) virus in Highly pathogenic avian influenza viruses order to recall the H3 memory response by the (HPAIV) subtype H5Nx clade 2.3.4.4. have caused CD4+ T cell–stimulating sequences embedded in numerous outbreaks in wild bird populations rH7 HA OPT1 and OPT2. Twenty-four female H3 and in domestic poultry worldwide. Avian pre-immunized HLA-DR3 mice were divided into influenza viruses do not typically cause highly three groups and vaccinated with either rH7 HA mortality in mallards and other wild waterfowl. OPT1, OPT2, or wild-type (WT) H7 HA via the However, some H5Nx viruses have been intra-mauscular route (3 µg/mouse/0.05 ml X implicated in mass die-offs of ducks. We three times). The rH7 OPT1- and OPT2- previously reported clear differences in vaccinated groups showed higher H7 HA– pathogenicity between two H5N8 HPAIV, where specific IgG response, and displayed a lower one strain caused mild clinical disease in mortality, weight loss, and lung viral titer mallards whereas the other strain caused high mortality of up to 80%. In this study, by using more sensitive methods and tests will yield reverse-genetics generated reassortant viruses, higher viral loads within the same level of we found that the matrix and nucleoprotein residual virus. The primary objective of this genes were implicated in the increased virulence study was to compare sampling devices and to in mallards. To further investigate the determine optimal locations for AIV detection on mechanisms behind the disparity in virulence, environmental surfaces in wire cages to provide we examined the characteristics of the general guidance for environmental sample reassortants viruses in vitro, by comparing viral collection and testing for residual AIV after an growth curves in avian fibroblast cells and in liver outbreak. Another objective was to explore the cell lines. Moreover, the role of the possibility of reducing the number of samples nucleoprotein genes in viral mRNA synthesis was collected and yet yield the same results to make examined using a polymerase reporter assay. To the process less laborious. For that purpose, a determine if changes in the matrix gene are laboratory-based, animal trial to simulate the associated with virion morphology, we also environment of real poultry houses containing examined the reassortants viruses under an infected birds was conducted. Three sampling electron microscope. The findings of this study devices and multiple sampling locations, were further our knowledge of pathogenicity of avian evaluated. influenza viruses and help identify genes and Infectivity and transmission in turkeys and mutations associated with increased virulence in chickens of low and highly pathogenic avian avian species. influenza viruses from the 2020 outbreak in Evaluation of environmental sample collection turkey farms in North Carolina and South devices and target sampling areas for Avian Carolina Influenza detection in layer cages Mary Pantin-Jackwood Jongseo Mo1, Erica Spackman2, 3 Southeast Poultry Research Lab, US National 1USDA Agricultural Research Service, Southeast Poultry Research Center, US Dept. of Poultry Research Laboratory, 2USDA Agricultural Agriculture-Agricul Research Service, Southeast Poultry Research The pathobiology of avian influenza virus (AIV) Laboratory changes as it circulates and adapts in different During recovery from an outbreak of avian avian species. In this study we examined the influenza virus (AIV), ensuring that the infected infectivity and transmissibility in chickens of an premises is free of residual virus is critical for early and a more recent Mexican low pathogenic releasing a quarantine and restocking. (LP)AIV subtype H5N2 that has been circulating Environmental testing may also be utilized as an and causing losses in poultry in Mexico since adjunct to testing animals when attempting to 1994. We show that later viruses are better identify infected premises. Currently there is not adapted to chickens which would increase the a uniform approach to testing wire cages. Thus, transmissibility of the virus and it's control. establishing an optimized and validated sampling protocol to ensure the removal of residual virus in such typical environments after an outbreak is essential. Optimization of collection procedures can impact the likelihood of detecting the virus in the environment, as summarized AMR unit of measurement, AMR Bacteriology/Antimicrobial Indicator Index, (AMR Ix for ceftriaxone, Trends in antimicrobial use and antimicrobial gentamicin, and multiclass resistance) and AMU resistance in broiler chickens; impact of indicators (mg/PCUPoultry for ceftriaxone, industry-wide antimicrobial use reduction gentamicin and total AMU) are reflective of the strategy success of the industry-wide AMU reduction strategy. These data highlight the importance of 1 2 3 Agnes Agunos , David F. Leger , Rebecca Irwin , AMU and AMR surveillance for monitoring the 4 Richard Reid-Smith impact of such strategies. 1 2 Public Health Agency of Canada, Public Health Use of AMN to prevent Clostridium perfringens 3 Agency of Canada, Public Health Agency of induced necrotic enteritis in broilers Canada, 4Public Health Agency of Canada David Diez Arias1, Carlos Domenech2, Shivaji In light of the global and national directives to Dey3, Tusar Chowdhury4 reduce the public health risks of antimicrobial 1 2 3 resistance (AMR), the poultry industry in Canada Biovet S.A., Biovet S.A., Agrivet Consultancy 4 has implemented their antimicrobial use (AMU) Ltd., Doctor's Agrovet Ltd. reduction strategy AMN is a natural biocide (botanical origin) (https://www.chickenfarmers.ca/the- effective against bacteria and fungi. An antimicrobial-use-reduction-strategy/; a phased experiment was conducted to determine its approach for reduction from 2014 to 2020). This efficacy to prevent necrotic enteritis (NE) in 35- presentation aims to describe trends in AMU in day-old broilers. broiler chickens and turkeys and to compare with trends in AMR. Data on AMU and AMR in 160 broilers were distributed into four enteric bacteria, collected from 2013 to 2018 treatment groups: non-infected control (T1); from broiler chickens (n = 793 flocks) and from infected control (T2); non-infected group 2016-2018 from turkeys (n = 241) were used. receiving AMN as a prevention at 0.5 kg/t Between 2013 and 2018 in broiler chickens, the through feed, continuously (T3); infected group total AMU in milligrams/population correction receiving AMN as a prevention at the same unit (mg/PCUBr) decreased by 11% and the administration schedule (T4). Birds in T2 and T4 number (n) of defined daily doses for animals were challenged with toxigenic strains of C. using Canadian standards (nDDDvetCA) per perfringens on days 14, 15. All diets contained 1,000 broiler chicken-days decreased by 17%. 23.5% crude protein and no antimicrobials or Between 2016 and 2018 in turkeys, the coccidiostats. mg/PCUTk decreased by 11%, whereas the Results showed that AMN as a preventive in nDDDvetCA/1,000 turkey-days increased by infected birds (T4) was effective in controlling NE 10%. In broiler chickens, the trends in resistance (1.81% ADG increase (P<0.10), 4.72% feed to ceftriaxone paralleled the quantity and conversion improvement, compared to T2). frequency of ceftiofur use (decreasing trends). In AMN in non-infected birds (T3) had a positive both species, trends in resistance to gentamicin effect compared to non-infected birds (T1) as it mirrored the frequency and quantity of improved ADG, feed conversion and livability gentamicin and lincomycin-spectinomycin use (P<0.1). Infected birds in T2 showed the most (decreasing trend). Integration of AMU and AMR severe unspecific lesions and the higher NE data (combined poultry species) using a lesion score (1.7), compared to the other groups. Infected birds with AMN (T4) showed mild S.H. numbers (MPN/g). Censoring all culture unspecific lesions and the lowest NE score (1.3). negative results -0.5 log10/g of ceca a Tobit regression model, the direct challenge of In conclusion, AMN in feed can be used as a treatment 2 had significantly (P = 0.004) reduced preventive for NE in poultry farms. AMN in S.H. numbers in their ceca (-1.05 MPN/g). In unchallenged birds also has a positive effect on summary, the treatment with the DFM (OBL) and performance, thanks to its beneficial activity antibiotic (Flavomycin 3g/ton) with salinomycin towards the balance of the gut flora. (60g/ton) had a significant reduction of S.H. in Evaluation of the Effectiveness of a Direct-fed the birds’ environment and in the ceca. In Microbial with an AGP and an Ionophore addition, all of the DFM and antibiotic Coccidiostat to Reduce Salmonella heidelberg treatments had a significant performance in Broilers improvement by 43 days. Charles Hofacre1, Steve Roney2, Ben Johnson3 Development of a Mass Administered Vaccine for Prevention of Necrotic Enteritis in Broilers 1Southern Poultry Research Group, 2Huvepharma, Inc., 3Huvepharma, Inc Charles Hofacre1, Greg Mathis2, Bob Nordgren3

This study evaluated the effectiveness of 1Southern Poultry Research GroupG, 2Southern modifying the intestinal flora of broilers directly Poultry Research, 3Curtiss Animal Health with a Bacillus licheniformes direct-fed microbial A new generation of a Salmonella typhimurium (OptiBac L?) plus indirectly with the antibiotic antigen delivery vaccine strain expressing Flavomycin? and either salinomycin or the protective antigens of Clostridium perfringens chemical coccidiostat Narasin in a Salmonella type A has been developed. Multiple battery heidelberg challenge study. This study had four cage studies with a Necrotic Enteritis challenge treatments (Treatment 1: infected control with model were performed to 28 days. The vaccine 60 g/ton salinomycin; Treatment 2: Flavomycin 3 strain exhibited significant reduction in N.E. g/ton plus salinomycin 60 gm/ton plus OBL ½ mortality. The challenge model consisted of E. pound/ton; Treatment 3: Flavomycin 3g/ton plus maxima at 14 days and C.P. on days 18, 19, salinomycin 60 g/ton plus OBL 1 pound/ton; and/or 20. In one of the studies, N.E. mortality Treatment 4: Flavomycin 2 g/ton plus Narasin 72 was significantly lower in the vaccinated group g/ton plus OBL ½ pound/ton. The challenge was (8.3%) compared to the unvaccinated challenged on day 3 when ½ of the chicks, 25 chicks per pen group (30.2%) (P < 0.001). The vaccine- of 50 chicks, were tagged and orally inoculated prevented fraction of Necrotic Enteritis mortality with 8.0x107 CFU/chick S. heidelberg (nalidixic was 0.72 (95% CI:0.57, 0.82). Additionally, the acid resistant at 25 µg/ml). On day 43 treatment vaccinated birds had significantly improved body 2 had a significant reduction in bootsock S.H. weight and FCR to the placebo vaccinated enumeration by micro most probable number cohorts. The results of each of the studies will (MPN). At 43 days ten broilers of indirect be presented. In addition, the vaccine candidate challenge and five of direct challenge had ceca is currently under licensing review by USDA and aseptically removed and cultured for S.H. is expected to be field tested soon. prevalence and number by MPN. All treatments had a numerical reduction in S.H. prevalence versus challenge control, and treatment 2 was significantly reduced (44% T2 versus 72.6% T1). Treatments 2 and 3 had numerical reductions in Characterization of bacterial chondronecrosis changes in response to disease; however, more with osteomyelitis (BCO) in broilers in a specific biomarkers such as S. cohnii may provide commercial setting more specific determination of disease as a supplementation to diagnostics and early Elizabeth Kim detection. DuPont Detection of Necrotic Enteritis (NE) B-Like Bacterial chrondronecrosis osteomyelitis (BCO) Toxin in Biological Samples of NE-Afflicted is a leading cause of lameness in broilers and a Chickens by Capture Enzyme-Linked major cause of production losses. With overt Immunosorbent Assay disease visualized as “Kinky Back”, the Kyung-Woo Lee1, Woohyun Kim2, Charles Li3, culmination of disease involves the multi-action Hyun S. Lillehoj4 of both micro-bone lesions and disease along with colonization from opportunistic pathogens. 1ARS-USDA, 2Animal Biosciences and The micro-fissures and lesions of the bones may Biotechnology Laboratory, USDA-ARS, 10300 be related to a complicated pathway of Baltimore Avenue, Bldg. 1043, BARC-East, physiological interactions, while the bacterial Beltsville, MD 20705, 3Animal Biosciences and initiation of disease is more straightforward. To Biotechnology Laboratory, MD 20705, 4Animal better determine dynamics of bacteria within Biosciences and Biotechnology Laboratory, MD general health and BCO scenarios, a field study 20705 was initiated using paired farms within a single Necrotic enteritis (NE) is a devastating enteric complex. Initial investigations first focused on disease caused by Clostridium perfringens type distributions of disease to demonstrate A/G which impacts global poultry industry by sufficient outbreaks and creating statistical compromising performance, health and welfare baselines sufficient for classifying a “sick” farm of the chickens. Although necrotic enteritis B-like compared to a “healthy” farm. From the field (NetB) toxin is considered a major virulent factor samples, a progression of general bacterial in NE development, reports on the detection of isolation/quantification techniques were used NetB protein per se in NE-afflicted chickens are along with 16s sequencing from both sick and lacking. Here, we first report that native NetB healthy farms. From general bacterial isolations, toxin can be detected in biological samples from changes were identified in Avian Pathogenic E. NE-afflicted chickens using NetB-specific coli for their densities and combination of monoclonal antibody-based capture ELISA. virulence factors. Other changes were also identified in C. perfringens as well for their Necrotizing Hepatitis Associated with overall abundance when comparing healthy and Clostridium perfringens in Broiler Chicks sick farm samples. Using 16s sequencing for total 1 2 capture of bacterial communities, it was also Valerie Marcano , Tyler Gamble , Monique 3 found that bacterial changes including Franca Lactobacillus, E. coli, Staphylococcus, and 1Poultry Diagnostic and Research Center, Salmonella were significantly different. Although College of Veterinary Medicine, The University these species tended to change by sample, of Georgia, USA, 2Pilgrim’s Pride Corporation, Staphylococcus cohnii was statistically the 3Poultry Diagnostic and Research Center, USA strongest correlation to disease. Altogether, generally monitored species such as APEC, Five cases of necrotizing hepatitis in broiler Clostridium, and Lactobacillus do show some chicks were submitted to the Poultry Diagnostic and Research Center for histopathology and Characterization and Antimicrobial Resistance bacteriology in 2017. Three additional cases Patterns of Pasteurella multocida Isolates from were submitted to the Georgia Poultry Chickens in Mississippi Laboratory Network. Elevated mortality was Eric Orozco1, Jay Kay Thornton2, Alejandro observed in flocks aged between 1 day to 1.4 Banda3, Martha Pulido-Landinez4 weeks of age with or without neurological signs. In most flocks, increased 3-day mortality was followed by an elevated 7-day mortality. Gross 1 2 lesions included green to dark brown Mississippi State University PRDL, Mississippi discoloration of the liver, congested lungs, Veterinary Research and Diagnostic Laboratory, 3 serosanguineous fluid in the caudoventral aspect Mississippi Veterinary Research and Diagnostic 4 of the abdomen, and emphysema in the yolk Laboratory, Poultry Research and Diagnostic sacs. In birds older than a week of age, disease Laboratory with neurologic signs became evident and consisted of tremors, stargazing, and incoordination. Histopathological evaluation A retrospective study including sixty-seven revealed multifocal to coalescing Pasteurella multocida isolates from commercial fibrinoheterophilic and necrotizing hepatitis poultry obtained from cases submitted to the associated with gram-positive long rod-shaped Poultry Research & Diagnostic Lab (PRDL) at bacteria. Other histopathological lesions Mississippi State University was conducted from observed included fibrinous splenitis, necrotic January 1st, 2015 to June 30th, 2019. The origin enteritis, fibrinoheterophilic pericarditis, and of the submissions included broilers, broiler acute encephalitis with gliosis and cerebellar breeders and commercial layers, being broiler spongiosis. Samples tested were positive for breeders the most frequent. The age of the birds Clostridium perfringens (CP) by ranged from three days old (broilers) up to immunohistochemistry and bacteriology, and CP seventy-nine weeks old (commercial layers). isolates from liver samples harbored netB and Mixed samples from males and females were tpel virulence genes. Broiler breeders are the received in most of the cases with higher suspected source of the infection, and testing incidence during the winter and early spring revealed netB-positive CP among breeder flocks. months. Acute septicemia and chronic lesions Antimicrobial therapy was coupled with were frequently observed in the birds. The hock enhanced sanitation at the farm and hatchery, joints were the samples with the highest rate of markedly decreasing the mortality and clinical successful isolation. Thirty-one isolates were signs. serotyped, nine isolates were classified as serotype 4 and seven isolates as serotype 3x4. Fingerprinting was carried out in fifteen isolates; seven isolates exhibited similar profiles to those observed with attenuated vaccines. Most isolates (90%) exhibited susceptibility to amoxicillin, ceftiofur, enrofloxacin, florfenicol

and trimethoprim/sulfamethoxazole; whereas intermediate susceptibility was observed for erythromycin, gentamycin, neomycin and spectinomycin. Sixty-four isolates showed a multidrug-resistant pattern and one isolate was Genomic landscape of Ornithobacterium pandrug-resistant. rhinotracheale in commercial turkey production in the United States Genomic Analysis of Avibacterium paragallinarum from 2019 infectious coryza Emily Smith1, Elizabeth A. Miller2, Bonnie P. cases in the United States Weber3, Timothy J. Johnson4 Ha-Jung Roh 1University of Minnesota, 2Department of Veterinary and Biomedical Sciences, University Ceva Animal Health of Minnesota, Saint Paul, MN, 55108, Since March of 2019 infectious coryza suspect 3Department of Veterinary and Biomedical cases with symptoms including decreased Sciences, 55108, 4Department of Veterinary and consumption of feed and water, and swollen Biomedical Sciences, 55108 heads and wattles with catarrhal discharge were Ornithobacterium rhinotracheale (ORT) is a submitted from commercial layer farms in bacterium that causes respiratory tract Pennsylvania and nearby states. Avibacterium infections in avian hosts worldwide, but is a paragallinarum was successfully identified and particular problem for commercial turkey isolated from infra-orbital sinus swabs. In production. Little is known about the ecologic addition to classical phenotyping analysis of the and evolutionary dynamics of ORT, which makes isolates, whole genome sequencing (WGS) prevention and control of this pathogen a analysis was performed to identify genetic challenge. The purpose of this study was to gain relationships between the isolates. insight into the genetic relationships between The outer membrane haemagglutinin gene different ORT populations through comparative (hmtP210) showed a high degree of similarity genomics of clinical isolates from different US amongst all regional isolates and clustered with turkey producers. ORT clinical isolates were serotype C reference serovars. However, pan- collected from four major US turkey producers genome analysis of core genes showed distinct and several independent turkey growers and clusters of the isolates and analysis of the whole-genome sequencing was performed. accessory genes further separated them by Genomes were compared phylogenetically using states. single nucleotide polymorphism (SNP)-based analysis, and then assemblies and annotations were performed to identify genes encoding putative virulence factors or antimicrobial resistance determinants. A pangenome approach was also used to establish a core set of genes consistently present in ORT, and to highlight differences in gene content between phylogenetic clades. A total of 1,457 non- recombinant SNPs were identified from 157 ORT

genomes, and four distinct phylogenetic clades were revealed. Isolates clustered by company on the phylogenetic tree. However, each company

had isolates in multiple clades with similar collection dates, indicating that there are multiple ORT strains circulating within each of the companies examined. Additionally, several MG vaccine is applied in the commercial layer antimicrobial resistance proteins, putative industry starting at 8 weeks of age via spray, but virulence factors, and the pOR1 plasmid were it requires intensive labor, results in bird stress, associated with particular clades and multi-locus and provides late protections. In ovo vaccination sequence types, which may explain why the has been effectively used to administer vaccines same strains seem to have persisted in the same in the poultry industry and shows the ability of turkey operations over decades. early protection, and may eliminate labor requirements and stresses associated with Effects of the in ovo Administration of the 6/85 handling poultry. Further, in ovo application may Mycoplasma gallisepticum Vaccine on Layer stimulate the chick’s early immune response and Chicken Embryo Livability and Post-Hatch may result in vaccine-derived in vivo populations Performance that may be statically maintained within Abdulmohsen H Alqhtani1, Jeffrey D. Evans2, vaccinated hosts. Edgar D. Peebles3 Bacterioloty/Antimicrobial: 1Mississippi State University, 2Mississippi State USDA ARS, 3Mississippi State University Salmonella and E. Coli

Mycoplasma gallisepticum (MG) is one of the Exploring the Fitness of Retail Poultry most pathogenic of the avian mycoplasma Escherichia coli to Cause Foodborne Urinary diseases in chickens. It is particularly pathogenic Tract Infections in commercial layers. Mycoplasma gallisepticum has a major impact on the economy of the Ananta Arukha poultry industry worldwide. It causes chronic University of Florida respiratory disease in chickens, and infectious sinusitis in turkeys. It has been reported that Retail poultry has received much attention in the MG affects fallopian tube inflammation, which recent years as a source of urovirulent E. coli causes egg production loss, and egg quality. The colonizing the human colon. In this context, this estimated cost of MG infection is around $150 abstract and the presentation examine various million annually. In the US, commercial layers traits of retail poultry E. coli to determine if they are raised in multiple-age systems and older possess the characteristics required to cause birds can pass MG to younger birds that do not foodborne urinary tract infection. have strong immune systems. To control and prevent MG infection, MG vaccines have been released and used commercially.

Few studies have been done in our lab using a virulent MG vaccine (FMG) using in ovo vaccination. FMG has a promising protection and early life immunity. However, FMG is a virulent strain and resulted in negative impacts on layer hatchability as well as layer performance. Therefore, the goal of this study is to test different in ovo dosages of MG 6/85 vaccine and test its impact on the hatchability and survivability of layer chicken embryo. The 6/85 Colonization of Internal Organs by Salmonella in cage-free indoor housing, including frequent Enteritidis and Salmonella Kentucky in internal organ invasion by S. Enteritidis. Experimentally Infected Laying Hens in Indoor Emergence of a Novel Salmonella enterica Cage-Free Housing Serotype Reading Clone is Linked to its Richard Gast1, Deana R. Jones2, Kenneth E. Expansion in Commercial Turkey Production Anderson3, Darrin M. Karcher4 Timothy Johnson 1USDA, 2USDA-ARS, U.S. National Poultry University of Minnesota Research Center, 3North Carolina State University, 4Purdue University Concurrent separate human outbreaks of Salmonella enterica serotype Reading occurred Salmonella Enteritidis infections in commercial in 2017-2019 in the United States and Canada, egg-laying flocks are an ongoing public health which were both linked to the consumption of concern because reproductive organ raw turkey products. In this study, a colonization leads to deposition inside eggs. comprehensive genomic investigation was Flock housing conditions affect Salmonella conducted to reconstruct the evolutionary persistence and transmission, but the food history of S. Reading from turkeys, and to safety consequences of different housing determine the genomic context of outbreaks systems remain unresolved. The present study involving this rarely isolated Salmonella assessed the invasion of internal organs after serotype. A total of 988 isolates of U.S. origin experimental S. Enteritidis and S. Kentucky were examined using whole genome-based infection of laying hens in indoor cage-free approaches, including current and historical housing. Groups of hens were housed on wood isolates from humans, meat, and live food shavings in isolation rooms simulating animals. Broadly, isolates clustered into three commercial cage-free barns and 1/3 of the hens major clades, with one apparently highly in each room were orally inoculated with 107 cfu adapted turkey clade. Within the turkey clade of either S. Enteritidis or S. Kentucky. At 6 d and isolates clustered into three subclades, including 12 d post-inoculation, half of the hens in each an “emergent” clade that only contained isolates room were euthanized and samples of liver, dated 2016 or later, including many of the spleen, ovary, oviduct, and intestinal tract were isolates from these outbreaks. Genomic removed for bacteriologic culturing. Among hens differences were identified between emergent inoculated with S. Enteritidis, 100% of intestinal and other turkey subclades suggesting that the samples, 100% of livers, and 50% of ovaries were apparent success of currently circulating positive for the pathogen at 6 d post-infection. subclades clade is, in part, attributable to Moreover, 71% of intestines, 42% of livers, and plasmid acquisitions conferring antimicrobial 10% of ovaries from contact-exposed hens were resistance, gain of phage-like sequences with colonized by S. Enteritidis at 12 d post-infection. cargo virulence factors, and mutations in Although 86% of hens inoculated with S. systems that may be involved in beta- Kentucky yielded positive intestinal samples at 6 glucuronidase activity and resistance towards d post-infection, S. Kentucky was found in other colicins. U.S. and Canadian outbreak isolates internal organs of both inoculated and contact- were found interspersed throughout the exposed hens significantly (P < 0.05) less often emergent subclade and the other circulating than S. Enteritidis at both sampling intervals. subclade. The emergence of a novel S. Reading These results demonstrate the potential for turkey subclade, coinciding temporally with Salmonella infection to disseminate among hens expansion in commercial turkey production and The Comparative Analysis of the Salmonella with U.S. and Canadian human outbreaks, enterica subsp. enterica Serovar Infantis IncFIB indicates that emergent strains with higher Plasmid Genetic Sequence Would Aid to potential for niche success were likely vertically Identify Relevant Subtypes for Salmonella transferred and rapidly disseminated from a Control in Poultry common source. Roxana Sanchez Ingunza Transmissible antibiotic resistance genes Private Consulting - Poultry present in Escherichia coli and Salmonella from USA poultry Salmonella enterica subsp. enterica serovar Infantis human infections associated to raw Daniel Karunakaran1, Alexandra Smith2, Tom chicken products has been reported in the Rehberger3, Renae Geier4 United States in 2018. Previously, Salmonella 1Arm and Hammer Animal Nutrition, 2Arm and Infantis was most commonly associated to Hammer Animal and Food Production, 3Arm and human infections linked to contact with live Hammer Animal and Food Production, 4Arm & poultry. The Centers for Disease Control and Hammer Animal and Food Production Prevention (CDC) currently indicates this serovar appears to be widespread in the chicken Alternatives to antibiotics, such as probiotics, are industry. An extended-spectrum ß-lactamase replacing sub-therapeutic levels of antibiotics in (ESBL) producing, multidrug resistant (MDR) the diets of conventional poultry due to clone of Salmonella Infantis was first described regulatory changes limiting the use of in the U.S. in 2017 from isolates recovered from antibiotics. The premise is that this will result in poultry and human infections between 2014 and a reduction of antibiotic resistance genes in the 2015. This megaplasmid also carries genes that environment. Genome data from broiler and provide environmental survival advantage to the turkey Escherichia coli isolates, which we bacteria such as the mer operon. Salmonella collected before 2016, indicated that the most genomes from organisms of different sources in prevalent transmissible resistance genes were to the U.S. containing the plasmid were very closely aminoglycosides, tetracyclines, sulfonamides, related suggesting common origin and and beta-lactams. We developed a multiplex PCR widespread distribution. However, this analysis to detect seven of the antibiotic resistance genes was made on only one PFGE pattern. Additional to monitor transmissible antibiotic resistance PFGE patterns carrying the plasmid has been genes in avian E. coli over time. Our assay identified; therefore, the plasmid characteristics determined that the average number of on other emergent Salmonella Infantis subtypes antibiotic resistance genes per isolate was 1.7 merit further investigation. The overall with a maximum of five of the seven genes in any significance of the presence of this plasmid on one isolate. The number of resistance genes the bacteria phenotypic characteristics is not detected per isolate dropped significantly from clearly understood. This study focuses on the 2.2 genes in 2015 to 1.4 in 2019. As similar genetic analysis of Salmonella Infantis isolates plasmids and therefore resistance genes are recovered from poultry and highlights present in avian Salmonella strains we are also differences in content and distribution of genes using this multiplex PCR assay to monitor and nucleotide polymorphisms observed in the transmissible antibiotic resistance genes in avian IncFIB plasmid. The suggested comparative Salmonella isolates. approach would aid in the selection of antigens for vaccine intervention and in the description of phenotypes of increased environmental An Embryo Co-infection Model for persistence or virulence in poultry. Enterococcus faecalis and Avian Pathogenic E. coli Assesses Pathogenicity and Virulence of Impact of E. coli vaccination on production Polybacterial Infections performance of turkeys and E. coli ecology over time Grayson Walker1,Luke B Borst2, Mitsu Suyemoto3 Katie Stumvoll4, M M Kromm2, E Gerken3, T Johnson4 1North Carolina State University College of Veterinary Medicine 2Population Health and 1Jennie-O Turkey Store, 2Jennie-O Turkey Store, Pathobiology, North Carolina State University 3Jennie-O Turkey Store, 4University of Minnesota College of Veterinary Medicine, Raleigh, NC, Collibacillosis, a disease of poultry caused by 3Population Health and Pathobiology, NC Escherichia coli, is one of the most challenging Enterococcus faecalis (EF) and avian pathogenic bacterial diseases for field practitioners to E. coli (APEC) are frequently co-cultured from address. Colibacillosis is typically a secondary lesions of septicemic birds. Uropathogenic E. disease, therefore primary mitigation strategies coli, which are genetically similar to APEC, often address any primary viral disease demonstrate enhanced pathogenicity during co- challenges and/or inadequate management of infection with EF, including enhanced wound the barn environment. When focus on the colonization and macrophage evasion in murine primary stressors is not sufficient, field disease models. However, the impact of EF practitioners look to additional tools to help coinfection on APEC virulence has not been support the welfare of the birds they are investigated. As virulence factors for both UPEC responsible for. E. coli vaccination has been used and APEC frequently focus on iron acquisition, extensively in layer production and to some our hypothesis is that APEC virulence is extent in broiler production, but it is less enhanced in the presence of EF in iron-limited frequently applied in commercial turkey environments. To this end, field isolates of EF settings. This first talk on the use of E. coli and APEC obtained as infective pairs from yolk vaccination in commercial turkey flocks will sacculitis lesions were assessed with in vitro utilize paired barn data to present data on its growth assays and in vivo with embryo lethality effect on both performance and the overall assays for virulence. For the growth assay, single ecology of E. coli populations in birds and within APEC macrocolonies were grown on iron-limited barns. agar or in mixed culture with EF. APEC in mixed culture exhibited increased growth when compared to APEC in monoculture (P=0.01). For the embryo lethality assay, cultures of APEC, EF, or a mixed culture of APEC and EF were injected into either the yolk sac at day 6 or the allantoic fluid at day 12. Eggs were candled daily to determine survivability and results were compared among groups. Results of in vitro and in vivo assays will be presented which will further characterize the effect of EF co-infection on

APEC virulence and shed light on the significance of mixed infection in colibacillosis. A Case Report of Erysipelothrix rhusiopathiae Case Reports Infection in Turkey Breeder Hens

Diagnosing and managing Inclusion Body Ana Paula Da Silva1 G. Cooper2, C. Jerry3, S. Hepatitis outbreak in primary broiler breeders Stoute4 Emma Castillo 1 University of California, Davis CA, USA 2 Aviagen California Animal Health and Food Safety Laboratory System, Turlock branch, University of In this case study of an Inclusion Body Hepatitis California, Davis CA, USA, 3California Animal outbreak in primary broiler breeders I will begin Health and Food Safety Laboratory System, the presentation by characterizing how the USA, 4California Animal Health and Food Safety disease manifested both in terms of clinical signs Laboratory System, USA and gross necropsy findings in affected male and female chicks. I will include a summary of Erysipelothrix rhusiopathiae is a Gram-positive epidemiological trends including mortality, bacterium of worldwide distribution that affects onset of disease presentation, distribution of multiple avian and mammalian species, causing disease and the search of source flocks. Previous a disease known as erysipelas. Although research confirms that IBH can be seen as early erysipelas has sporadic occurrence, the disease as 4 days of age but most typically at 3 weeks of notably affects turkeys, inducing mortality, age. In this outbreak mortality consistently marketing losses and decrease in fertility in occurred at the beginning of the second week of turkey breeders. The usual port of entry of E. life occasionally blending in with typical first rhusiopathiae is via skin or mucosal lesions, but week of life mortality making detection of the artificial insemination is an important source of disease more difficult. This presentation will infection for turkey breeder hens and is likely discuss important trends and clinical signs the linked to male carriers that shed the bacterium veterinary team used to train specialists for the in their semen. monitoring of IBH in the face of an outbreak. The California Animal Health and Food Safety Because percent mortality in this outbreak had a Laboratory system received 37 case submissions wide range the veterinary team focused on of animals affected with erysipelas from 2007 to specific trends and how daily mortality changed 2019. Of those, 23 cases were in avian species into the second week of life. To conclude, I will and 11 were in turkey breeders. In this study, we cover the extensive vaccination assessment and report a case of erysipelas affecting 36-week-old protocol changes instituted to control the commercial Broad-Breasted White turkey disease outbreak. The longer life span of primary breeder hens from a flock presenting a slight breeders allowed the veterinary team to increase in mortality. At necropsy, two out of compare how IBH affected flocks faired in three birds presented with hepatomegaly and comparison to their non-affected counterparts bronze discoloration of livers and enlarged, after the onset of disease and resolution of mottled spleens. Hepatic and splenic clinical signs. Several of the diagnosed flocks microscopic lesions included fibrinoid necrosis were subject to subsequent disease outbreaks. and mixed inflammatory cell infiltration. Pure cultures of E. rhusiopathiae were isolated from all livers and spleens. Further diagnostics, epidemiology and practical aspects of erysipelas in avian species will be discussed. Case Report: Clinical and Histopathological Salmonella Bacterin Induced Hepatopathy in Findings in First Reported Case of False Layer Broiler Breeder Pullets Syndrome in Georgia Eric Heskett Karen Grogan1 Monique Franca2, Brian Jordan3, Case Farms Jenny Nicholds4 Increases in pullet mortality were observed two 1University of Georgia, PDRC, 2UGA Poultry to three-weeks post autogenous Salmonella Diagnostic and Research Center, 3UGA PDRC, vaccination in two out of three divisions on this 4UGA PDRC vaccine. An extensive diagnostic investigation False Layer Syndrome is currently believed to be was undertaken to discover the root cause of the a delayed clinical sign of infection with infectious mortality. The diagnostic investigation included bronchitis virus (IBV) in pullets at an early age. vaccine handling practices, necropsies, priming Viral infection is believed to impact the post- with other Salmonella bacterins, comparison of embryonic development of the oviduct, which lots of vaccine, ruling out other infectious leads to cystic left oviducts in layer species of conditions, endotoxin level quantification in the birds at peak production, approximately 25-30 autogenous vaccine using the limulus weeks of age. The DMV/1639 strain of IBV is amebocyte lysate assay, and paired house trials currently suspected to cause this syndrome in using different dosages or different commercial the United States and Canada, but other variant Salmonella bacterin products. Necropsies IBV strains cannot be excluded. This disease has revealed large amounts of serous fluid at the been spreading in commercial layer operations injection site as early as two hours post injection. since 2017 throughout Canada and the US, and Necropsies on birds two to three-weeks post in July 2019, a submission was made to PDRC of vaccination revealed birds in good body the first reported case in Georgia in 29-week-old condition with friable, pale livers and a large hens. A clinical work up was completed and amount of unclotted blood in the abdominal tissues were collected for serotype-specific IBV cavity. The level of endotoxin between the two quantitative RT-PCR along with sections of the lots of vaccine revealed a difference in the level uterus of affected hens for histopathology. of endotoxin present. Results of the limulus Choanal swabs and tissues were collected from amebocyte lysate assay, comparisons of vaccine 11-week-old pullets on the source farm of the programs, and results of the paired house trials affected layers for serotype-specific IBV will be presented and discussed. quantitative RT-PCR. Histopathological findings An Interesting Case of Pasteurella multocida showed significant changes to the epithelium of “Like” Clinical Signs with a Slew of Organisms the magnum section of the uterus in affected hens. Special stains were utilized to understand Geoffrey Lossie1 Tsan Long Lin2, Dr. Kristen Hill- the altered function of the changed epithelium. Thimmesch3 Quantitative RT-PCR was positive for DMV/1639 IBV in the cecal tonsil of 11-week-old pullets, and Purdue University College of Veterinary a DMV/1639 virus was isolated in embryos from Medicine/Indiana Animal Disease Diagnostic this tissue. Changes to early IBV vaccination Lab program were instituted for the next pullet A Mycoplasma gallisepticum (MG) and placement. Testing of pullets and monitoring of Mycoplasma synoviae (MS) positive flock of laying flocks has continued. 70,000 White Leghorns in the lower level of a 3 tiered avian style house experienced a sudden affected, but loss was much greater in house increase in mortality. Mortality levels increased one. Initial mortality appeared to be mostly over a two day period and went from females, but eventually both females and males approximately 15 birds a day to 124 birds in a were significantly affected. On walkthrough of a single morning. Mortality was distributed house, 75% of birds were active while 25% were throughout the house. There was a concurrent laying down against the walls of the house and drop in feed and water consumption with an 8% ultimately dying. All birds appeared stunted and drop in production. On-site necropsy revealed many were observed eating litter. On necropsy, non-specific “normal mortality” findings, but birds were dehydrated, and crop and gizzard over time birds were found with swollen faces contents consisted of mostly litter with some and wattles with some showing neurologic signs intact corn kernels in the crop. Bone prior to death. Ten birds were submitted to the development appeared abnormal, as bones Indiana ADDL for diagnostic testing. The results would bend on manipulation rather than snap. of PCR tests for avian influenza, Newcastle No other significant lesions were observed. disease, and infectious laryngotracheitis (ILT) Oropharyngeal swabs were submitted for AI PCR were negative, while those for MG and MS were with negative results. Feed samples were positive. Various bacteria were isolated from submitted for analysis, and mortality was sent to multiple tissues including: Gallibacterium anatis, Iowa State University for histopathological E.coli, Staphylococcus aureus, and an examination. While waiting on these results, Avibacterium sp. (most similar to Avibacterium electrolytes were run in the water and feed was endocarditidis). The case is difficult to interpret replaced. The flock seemed to be improving until as there were many infectious agents isolated, they broke with coccidiosis at day 14. Around with no clear inciting agent. Of particular note is then, additional premises started demonstrating that only the bottom tier house was affected, similar mortality patterns, followed by while the upper two tiers and adjacent houses coccidiosis breaks. This case report will discuss remaining unaffected. Three days prior the final test results, additional investigations, affected house was dewormed. A house in the tentative resolution, and lessons learned. neighboring building was wormed with the same Case Report: A Unique Toxicity in Broiler active ingredient, but a different serial number, Breeders and showed no signs of illness. This case is unique in its clinical presentation, the organisms Eric Shepherd isolated, and possible confounding factors. University of Georgia Poultry Diagnostic and On a Friday afternoon... Research Center

Kayla Niel A case of 39-week old broiler breeders with acute onset of mortality was submitted to the Hy-Line International University of Georgia Poultry Diagnostic and Elevated mortality was reported in a flock of 10- Research Center (PDRC). Mortality was day old layer breeders on a summer day in the approximately 1.5% in the affected house over Midwest. The flock had an excellent start, with the last 24 hours. 1 of the 4 houses on the farm good uniformity at placement and low first-week was primarily affected and coincided with a mortality. The grower did not notice anything recent feed delivery. Birds backed off feed in the abnormal until the increase in mortality on day affected house and water consumption doubled 10. Both houses of the two-house complex were overnight. Egg production fell 15% over the next 5 days and remained 5% below peak production a week later. Feed samples were taken and Immunology/Vaccinology submitted to two separate laboratories for Replication of HVT Single (HVT-ILT) and Double routine mineral analysis. Feed was removed and (HVT-ND-ILT) Constructs and Efficacy against replaced the day that increased mortality was Challenge in SPF Birds. observed. Mortality returned to normal levels 4 1 2 days after the feed was replaced. Birds Ivan Alvarado , Maricarmen Garcia , Daniel 3 presented to PDRC both moribund and dead on Maekawa arrival. Marked dehydration was observed along 1Merck Animal Health,2University of Georgia, with pale, swollen kidneys, regressing follicles, 3University of Georgia and varying degrees of visceral gout. AI rapid test was negative and there was no growth on In this presentation, the level of protection aerobic cultures of bone marrow. provided by two turkey herpesvirus recombinant Histopathological changes were consistent with vaccines against infectious laryngotracheitis a peracute insult including skeletal muscle against challenge will be compared. Other myofiber degeneration and necrosis, criteria used to evaluate HVT constructs will be tubulonephritis and tubular necrosis, and presented. necrotizing interstitial pneumonia with urates. Salmonella Strains Displaying ENhanced Innate Feed analysis from both laboratories Immunity Response Activators (ENIIRA) to consistently showed elevated levels of multiple Stimulate Innate Immunity to Enhance minerals including, but not limited to, potassium, Protective Immunity magnesium, sulfur, iron, and sodium. Feed samples were further submitted for ionophore Roy Curtiss1, Soo-Young Wanda2, Vinicius Lima3, and mycotoxin testing. Samples were saved and Banikalyan Swain4 fed back to birds to recreate the clinical signs and 1University of Florida, 2College of Veterinary lesions observed in this case submission. Medicine, University of Florida, 3College of Investigation of Increased Cases of Erysipelas Veterinary Medicine, University of Florida, in Commercial Turkeys 4College of Veterinary Medicine, University of Florida Stephen Williams We have significantly improved attenuated Butterball Salmonella strains as vectors to deliver Case report covering an increase in the number protective antigens or DNA vaccines encoding of Erysipelas cases in commercial turkeys over a them. We therefore eliminated many means by one-month period in the Fall of 2019. This which Salmonella manipulates induction of presentation will describe the bacteria immunity and have genetically modified them to Erysipelothrix rhusiopathiae, common clinical display regulated delayed attenuation, regulated signs seen in turkeys and how this disease is delayed synthesis of protective antigens and spread. Veterinarians will gain knowledge of the regulated delayed lysis in vivo. As a clinical signs of Erysipelas, interventions and consequence, these mucosally delivered vaccine investigation into this disease challenge. vectors have enhanced abilities compared to the wild-type parent to invade and colonize internal effector lymphoid tissues to maximize induction of mucosal, systematic and cellular immunities and to display complete biological containment The Effective Use of Siderophore Receptor with no persistence in vivo or survival if excreted. Proteins (SRP) as Antigens in Poultry Vaccines. We refer to these strains as Protective Immunity Dan Domingo Enhanced Salmonella Vaccine (PIESV) vectors. Epitopix LLC We have observed that vaccination of both mice and chickens with control PIESV strains with Bacterial pathogenesis requires active empty plasmid vectors not encoding protective acquisition of iron from host reservoirs. This is antigens has conferred low-level protective accomplished by sophisticated iron-gathering immunity to a diversity of bacterial, viral and systems using secreted bacterial siderophores parasite pathogens. We inferred that this was plus outer membrane-bound siderophore due to activation and recruitment of innate receptor proteins (SRP). In the host immunity. We have thus specifically designed environment, bacterial SRP expression is Salmonella strains to serve as superior adjuvants amplified and ultimately provides iron for to activate TLR4, TLR5, Nod1, Nod2 and TLR9. bacterial growth, colonization and disease. These adjuvant strains either with wild-type lipid Given this key role, SRPs are logical targets for A or designed to synthesize the non-toxic MPLA vaccine development and have been shown to are totally avirulent at high doses by all be effective immunogens against Salmonella parenteral and mucosal routes of inoculation. Enteritidis (SE), E. coli (EC), Pasteurella multocida They are superior to other adjuvants in (PM) and other pathogens of veterinary interest. enhancing immune responses to subunit and live attenuated vaccines. We refer to these adjuvant In a series of studies, SRP vaccine effectiveness strains as Enhanced Innate Immunity Response (Fisher’s Exact Test, all P < 0.05) was Activators (ENIIRAs). Further ENIIRA demonstrated using SPF pullets administered improvements are being made and new twice with vaccine containing either SRP-SE, SRP- applications evaluated. EC or SRP-PM then challenged with a pathogenic field strain. Vaccine containing SRP-SE reduced Protective Effects of a Modified Live E. Coli colonization of reproductive tissues 100% in Vaccine Against Homologous And vaccinated birds (0% positive) compared to the Heterologous Challenges In Laying Hens placebo group (24% positive). Vaccine with SRP- EC prevented 100% of mortality in vaccinated Manuel Da Costa1, U. Tri2, CK Mah3, A. Sidna4 pullets (0%) compared to placebo (76%). 1Zoetis, 2Zoetis Inc., 3Zoetis Inc., 4Gadjah Mada Vaccine containing SRP-PM (3x4, 2x5 serotypes) University, Indonesia prevented mortality by 70% after heterologous challenge with serotype 7x9x10 and by 92% with The presentation will explore the effects of live serotype 8x14x15. E. coli vaccination in laying hens when an homologous or heterologous challenge is These studies demonstrate that vaccines using present SRP antigens are effective for prevention of bacterial disease in chickens.

False layer syndrome IBV, pathogenicity period of incubation on bursa and thymus insights and surveillance in layer flocks development. In the experiment, 100 fertile broiler eggs were incubated in individual Rodrigo Gallardo1, Ana Paula da Silva2, tabletop incubators with optimum eggshell Alexandra Mendoza-Reilley3, Corine Giroux4 temperature (approximately 100F°) during the 1University of California, Davis, 2University of whole period of incubation and another 100 California, Davis, 3University of California, Davis, fertile broiler eggs were exposed higher eggshell 4Hickman family farms temperature (approximately 103F°) from Days 15 to 18 of incubation. At hatch, basic chick False layer syndrome (FLS) is a condition in egg quality parameters such as chick body weight, laying flocks (table egg layers, breeders and chick yield, chick length and yolk-free body mass broiler breeders) in which hens do not reach are compared to see the effect of high peak of production due to a significant incubation temperature on embryo percentage of internal layers in a flock. This development. The ratio of heterophil to syndrome has been associated to early exposure lymphocyte in blood which indicate embryonic to infectious bronchitis virus (IBV). We heat stress was analyzed by blood smear. The performed controlled challenges with IBV strains size of bursa and thymus was measured by associated with FLS in the field and compared taking digital pictures and measured its area by them with a wild type M41 IBV strain. Virus using Image J software. The cortical distribution and pathology was evaluated in the lymphocyte/parenchyma ratio was measured respiratory, urinary and reproductive tract at 2, from thymus tissues by H&E staining and 5, 10 and 21 days. In addition, epidemiological morphometric analysis using Image J software surveillance is being performed in flocks with an (Rasband, W.S., ImageJ, U. S. National Institutes IBV induced FLS history to assess if the virus still of Health, Bethesda, Maryland, USA, persists after one day old vaccination was http://imagej.nih.gov/ij/, 1997–2015). The size established to prevent this condition. Results of of bursa and thymus and lymphoid depletion on the experiments and surveillance effort will be thymus will be a distinct indicator of discussed. immunosuppression induced by heat stress Histological Evaluation of the Effects of High during the late incubation period. Incubation Temperature on Embryonic Development of Poultry Immune Reagents Development of Thymus in Broiler Chickens Hyun S. Lillehoj1, Woo H. Kim2, Charles Li3 ChangHee Lee 1USDA-ARS, 2USDA-ARS, 3USDA-ARS Poultry diagnostic and research center The objectives of this project are 1) to identify Providing optimum incubation temperature is chicken immune molecules, particularly critical for successful embryonic development, cytokines, chemokines and cell surface markers, hatchability, and post-hatch growth express them as recombinant proteins, and performance. High incubation temperature, characterize their function, and 2) to develop especially the late stages of incubation will monoclonal antibodies (mAbs) to the target negatively affect embryo development, chicken molecules. Cloning of chicken genes (23 including the immature immune system. The in total) for were carried out the number of sets objective of this study is to examine the of primers which were designed and synthesized influence of embryonic heat stress in the late to amplify based on the chicken genomic and mRNA sequence. The recombinant proteins Two hundred and twenty-five commercial fertile were obtained by transformation into E. coli, eggs were divided in five treatment groups, as transfection into mammalian cells, or expression follows: in yeast in collaboration with Kingfisher Biotech. a) Dual recombinant HVT ND-IBD (Innovax ND- To develop mAbs against them, we immunized IBD®) mice, collected lymphocytes, fused the lymphocytes with myeloma cells, screened, and b) 89/03 generated single-cloned hybridoma. For functional characterization of the recombinant c) Dual recombinant HVT ND-IBD® & 89/03 protein and mAbs, several assays have been d) Non-vaccinated/Challenged conducted including ELISA, immunohistochemistry, Western blot, flow e) Non-vaccinated/Non-challenged cytometry, qPCR, cell proliferation, and nitric Replication of the viral vectored vaccine in oxide assay. All the target we selected have feather follicles of 14-day old vaccinated birds shown to have critical functions in host defense was evaluated by the amplification of specific against pathogens and all recombinant proteins segments of the HVT vector (Viral Flex Seq®). At expressed have met the quality standard for 7, 14 and 21 days of age, twelve birds from each immunization in mice for mAbs production. group were orally challenged with a Variant E Twenty-three target proteins consist of 16 strain. Five days after challenge, body and bursal cytokines (interleukin-4, 7, 10, 12, 13, 17F, 21, weights were registered to calculate bursal/body 22, 23, 26, 34, IFN-a, IFN-?, TNF-a, CSF-1, and weight ratios to determine the degree of bursal TGF-ß), 3 chemokines (CXCLi2, CCL4 and 5), 1 atrophy. Bursal tissues were also collected for surface receptor (CD127), perforin and histopathological evaluation and virological granzyme. The mAbs developed in this study procedures. Data was analyzed by Tukey’s or represent new sets of immune reagents which SNK tests. Replication of the viral vectored are specie-specific for poultry. vaccine was detected in all the HVT ND-IBD Protection by a Novel Dual Recombinant HVT vaccinated groups. ND-IBD Vaccine alone or in combination of an As expected, maternal antibody protection IBD 89/03 Live Attenuated Vaccine against a decreased overtime, as observed in the non- Variant E Strain Challenge of Infectious Bursal vaccinated-challenged group. When compared Disease with non-vaccinated/challenged group, Alexandra Mendoza-Reilley1, Ivan R. Alvarado2, adequate protection against challenge was Alejandro Banda3 observed when the dual recombinant HVT ND- IBD vaccine was applied alone or in combination 1 2 Merck Animal health, Merck Animal Health, of 89/03 live attenuated product. Furthermore, 3 Poultry Research and Diagnostic Laboratory, bursae index scores showed protection starting Mississippi State University at 14 days post-vaccination with the dual The objective of the study was to evaluate the recombinant product, in the groups of broiler level of protection provided by a novel dual- chicks with maternal immunity. recombinant HVT ND-IBD vaccine (Innovax ND- IBD®) and the live attenuated 89/03 vaccine against a challenge with an infectious bursal disease Variant E strain. Resistance to fenbendazole in Ascaridia Parasitology/Enteric Health dissimilis and its economic impact on the Parasitological Surveillance in a Pastured production of turkeys Poultry Farm James Collins Maria Tereza Bethonico Terra University of Georgia, Dept. of Infectious Auburn University Diseases

Alternative systems for poultry rearing cater to Infection with Ascaridia dissimilis, the most an increasing market's demand for products with prevalent and one of the most economically the lowest use of drugs and for environments important gastrointestinal nematodes of with high perceived animal welfare. Pastured turkeys, most often produces reduced feed systems are one example. In this production, the conversion efficiency. Currently, fenbendazole animals are raised in mobile structures that are (FBZ) is the only FDA approved treatment for A. moved every two or three days in a rotation dissimilis. We recently tested the efficacy of FBZ system. This practice can reduce the against 5 field isolates of A. dissimilis. 3 isolates concentration of feces decreasing the chances of (Wi, Ow, Po) demonstrated >99% efficacy. the animals to ingest them. However, there is a However, two (Sn, Ad1018), yielded efficacies of gap in the literature evaluating and quantifying 63.9% and 76.2%, respectively, indicating FBZ the parasitological challenges in pastured resistance. Having proven FBZ resistance in A. production. dissimilis, we wanted to determine the impact of resistant worms on growth and productivity. 384 We collected fecal samples from turkeys, 1-week old turkey poults were infected with the broilers and layers on a pastured farm in two- Sn (resistant) or Ow (susceptible) isolate. For week intervals to determine counts of coccidia each isolate, 8 replicates each of untreated and oocysts and worm eggs. For all three types of treated pens were established with 12 birds, the counts of coccidia and worm eggs were birds/pen. Turkeys were grown to 10 weeks, low compared to published numbers in following commercial growing practices. Birds conventionally reared poultry, indicating that were infected with 25 eggs/bird/week in feed. At the rotation system effectively reduced the 4- and 8-weeks post infection, treated groups infection pressure. We did not find obvious were administered FBZ via water (SafeGuard® shedding patterns related to season and age of Aquasol, 1.25mg/kg) for 5 consecutive days. the flocks. Next-generation sequencing of PCR Weight, weight gain and feed conversion were products showed the presence of most analyzed weekly for differences between described Eimeria spp. in layers as well as in groups. At weeks 7 and 8 post infection, feed broilers. Worm eggs were identified as Ascaridia conversion in Ow-treated was significantly galli. better than the other groups, using 700 grams The study will increase the number of samples less feed/bird. Since this was the only group with and complete a year worth of samples in order an effective treatment, these results strongly to clarify the behavior of those parasites in this suggest that worms infecting the other groups type of production system. (due to either lack of efficacy or lack of treatment) caused a significant decrease in feed efficiency. Further work is needed to determine the full scope of A. dissimilis infections on feed

conversion, but based on these data, there appears to be a large economic impact of A. were altered (p < 0.05) (12 increased, 38 dissimilis on turkey production decreased) with the B. subtilis 747- supplemented diet. Twenty-two metabolites Broiler Breeders: An Assessment of the Enteric were altered (p < 0.05) (18 increased, 4 Parasites Population decreased) in the B. subtilis 1781 vs. B. subtilis Steve Fitz-Coy 747 groups. Changes in the levels of these intestinal biochemicals provided a distinctive Merck AH biochemical signature unique to each B. subtilis- Over 400 chickens from one week of age to over supplemented group, and were characterized 6o weeks were evaluated during a series of by alterations in the levels of dipeptides necropsy sessions over several months. Coccidia (alanylleucine, glutaminylleucine, were prevalent in the younger age groups and phenylalanylalanine, valylglutamine), the helminths were more prevalent in the older nucleosides (N1-methyladenosine, N6- bird populations. methyladenosine, guanine, 2-deoxyguanosine), fatty acids (sebacate, valerylglycine, Dietary supplementation with Bacillus subtilis linoleoylcholine), and carbohydrates (fructose). direct-fed microbials alters chicken intestinal These results provide the foundation for future metabolite levels studies to identify biochemicals that might be Inkyung Park1, Noah P. Zimmerman2, Alexandra used to improve poultry growth performance in H. Smith3 the absence of antibiotic growth promoters. 1Animal Bioscience and Biotechnology In vitro inhibitory effect of a new three-strains Laboratory, Beltsville Agricultural Research selected probiotic on several serovars of Center, Agricultural Research Service, United Salmonella States Department of Agriculture, Beltsville, John Schleifer1, Line Skjoet-Rasmussen2, 2 MD, 20705, Arm & Hammer Animal and Food Christophe Bostvironnois3 Production, Waukesha, WI, 53186, USA, 3Arm & 1 2 3 Hammer Animal and Food Production, USA Chr. Hansen, Inc., Chr. Hansen, A/S, Chr. Hansen, A/S TDirect-fed microbials (DFMs) are dietary supplements containing live microorganisms Bacillus are known to be capable of producing which confer a performance and health benefit antimicrobial substances, including bacteriocins, to the host, but the mechanisms are unclear. able to inhibit Gram-negative bacteria such as Here, a metabolomics approach was used to Salmonella. The objective of the study was to identify changes in intestinal metabolite levels evaluate the in vitro inhibitory effect of a new in chickens fed an unsupplemented diet or a three-strain, bacillus-based probiotic for poultry diet supplemented with B. subtilis strain 1781 on several Salmonella serovars using two or strain 747. Body weight gains of chickens fed different methods. the B. subtilis-supplemented diets were The first method was an agar plate-based increased, compared with chickens fed the method. Separate agar plates were poured unsupplemented diet. Compared with which included selected and differing serovars of unsupplemented controls, the levels of 83 Salmonella enterica. The bacillus-based metabolites were altered (p < 0.05) (25 probiotic was added to wells created into the increased, 58 decreased) in chickens given the plate. After 24 hours of incubation, a zone of B. subtilis 1781-supplemented diet, while 50 inhibition of up to 2mm could be measured. The second method was a feed matrix-based were evaluated. Oocysts were purified by a methodology. The probiotic was added at 10^5 series of washes and centrifugations. To quantify CFU/g of autoclaved feed. The pathogens S. the oocysts, a known amount of beads were Heidelberg or S. Typhimurium were added at added to the suspension. FCM analysis was 10^3 CFU/g of feed. During the 24 hours’ time of performed using the LSRII. The number of oocyst incubation, samples were taken at different time in the suspensions were verified using the points. Samples were added to plates, incubated McMaster modified method. Furthermore the and a counting of pathogens was conducted. species of stock suspension were verified by PCR assay. The LSR II flow cytometry analyzer In these in vitro experiments the new bacillus- simultaneously identified Eimeria maxima, E. based probiotic was able to inhibit the growth of acervulina, and E. tenella oocyst populations all Salmonella serovars belonging to different from vaccine samples. The proportions of serogroups. When testing the probiotic vs. oocysts were similar when using the FCM Salmonella spp. in an agar-based method, a application and the McMaster slide. These radius of the inhibition zones was observed for results suggest that FCM could be used to all serovars. When testing the probiotic vs. identify and quantify coccidia oocysts of chickens Salmonella spp. in a feed matrix, it inhibited the quickly. We will also present FCM results growth of S. Heidelberg (1.3 log inhibition after 4 obtained from litter samples of commercial hours, 4.6 log inhibition after 8 hours) and S. houses. A key advantage of FCM is that it can be Typhimurium (1.6 log inhibition after, 4 hours, done quickly, enabling decisions to be made 2.2 log inhibition after 8 hours). Results of this immediately, whereas other diagnostic methods trial may be an important source of information are slower, labor-intensive, and more expensive. regarding production use of probiotics. Coccidiosis Vaccine Trial in Turkey Poults

Parasitology/Enteric Health: Zachary Adams1, Michelle Kromm2, Jolene Coccidia Tourville3 1 A new method for Eimeria Speciation and Iowa State University College of Veterinary 2 3 Quantification Using Flow Cytometry Medicine, Jennie-O Turkey Store, Jennie-O Turkey Store Daniel Adams Coccidiosis is a term given to describe an North Carolina State University infection of the protozoa Eimeria spp. in the intestinal tract. There are four species of Eimeria An accurate and speedy diagnosis of Eimeria at that are pathogenic in turkeys: E. adenoides, E. the species level are both challenging and necessary. Using a compound microscope, the dispersa, E. gallopavonis, and E. meleagrimitis. oocysts of the different Eimeria sp. can be The objective of this trial was to evaluate the effectiveness of a commercially available anti- discernible by size and shape. A flow cytometer coccidial vaccine in preventing clinical coccidiosis can be described as an “automatic microscope.” The objective of this study was to develop a non- in Jennie-O Turkey Store’s commercial turkey antibody based diagnostic method for rapid, flocks. Eleven farms were selected for this trial, each consisted of one control flock (received automated and objective speciation and coccidiostat) and two treatment flocks (received quantification of Eimeria sp. in poultry, using flow cytometry (FCM). Oocysts of different coccidiosis vaccine). The control flocks were species of Eimeria causing coccidiosis in chicken housed in a separate barn while the two treatment flocks were in a barn with a shared air opportunistically. We determined oocyst counts space but separated by a fence. The vaccine was and investigated the species composition by next administered in barns at poult placement via generation sequencing. Isolates containing E. course gel droplet onto the birds prior to maxima were typed using the developed MLST placement into rings. Fecal samples were scheme. The results will be useful in the collected on days 5-7, 12-14, 19-21, 26-28, and development and evaluation of coccidiosis 33-35 from both the control barn and the prophylaxis programs, by vaccination or use of experimental barns. Fecal samples were anticoccidials. collected via bogus paper and submitted for Zoamix: A Blast from the Past fecal oocyst counts. Oocysts per gram (OPG) were analyzed and data was organized. Further Timothy Cummings analysis of the production statistics including finish barn morbidity and mortality as well as Zoetis harvest weights will help determine the efficacy Zoamix is one of the oldest feed additive of the vaccine over the life of the turkey. anticoccidials that came to the U.S. market in the Typing Eimeria maxima isolates from Alabama 1960's, and following a period of being off the broiler flocks using a multi-locus sequence market, it was relaunched back to the U.S. typing (MLST) scheme market in the fall of 2014 as an aid in the prevention and control of coccicidiosis in broiler Miranda Carrisosa1, Kenneth Macklin2, chickens, pullets, and turkeys. Since that time, Chengming Wang3, Ru¨diger Hauck4 Zoamix usage in the poultry industry increased each year to the point where it has been the 1Auburn University, 2Department of Poultry leading feed additive anticoccicial sold in the U.S. Science, Auburn University, 3Department of for past three years. Although there were some Pathobiology, Auburn University, 4Department general guideline recommendations made for of Poultry Science and Department of Zoamix when it was being relaunched, more Pathobiology, Auburn University specific recommendations for Zoamix will be Eimeria maxima is a eukaryotic parasite of offered based on what has been learned from chickens that can cause coccidiosis and field experiences with Zoamix on performance predispose its host to secondary infection with and cocci control. Clostridium perfringens, the causative agent of Experiences Vaccinating with a Novel Necrotic enteritis (NE). Multi-locus sequence Coccidiosis Vaccine in Long Lived Chickens in typing (MLST) is a molecular typing tool that the United States reflects the population and evolutionary biology of bacterial pathogens. MLST schemes have also Kelli Jones been developed for some eukaryotic pathogens. We developed a MLST scheme for E. maxima Ceva based on six gene loci from MLST schemes of According to surveys conducted recently (2019) several different parasites. Using this scheme, by the American Association of Avian we were able to distinguish two commercial Pathologists (AAAP) Research Priorities vaccines and several types of field strains. Committee, commercial chicken companies in Over the course of one year, we sampled two the United States (US) indicated a “high to very commercial broiler farms in Alabama on a high research need for improved vaccines to regular base and several further farms optimize intestinal health and combat coccidiosis.” More specifically, they would like managing litter moisture in the field, managing work done looking at the most effective non- coccidiosis vaccine reactions, and using oocyst ionophore/antibiotic strategies for controlling counts (OPGs) to optimize the coccidiosis coccidiosis, and to determine best practices for vaccine management program through the using coccidial vaccines. brooding stage.

As a result of this industry interest and demand, The Potential Impact Of Three Phytogenic Feed a novel coccidiosis vaccine, Immucox ® 5, for long Additives On Chicken Eimeria from Coccidiosis lived chickens was released in the United States Vaccine. in late 2019. Leading up to its release, and Chad Malinak1, Steve Fitz-Coy2 immediately thereafter, efforts were made to fine tune and maximize the effectiveness of this 1Merck Animal Health, 2Merck Animal Health vaccine in the industry. In recent years poultry producers have seen an Successful coccidiosis vaccination requires that increase in demand for an antibiotic-free the vaccine be handled and stored product while still dealing with the need for high- appropriately, and that it be administered in a quality animal protein and keeping feed costs way that maximizes the uptake of the vaccine lower. To meet consumers demands, phytogenic oocysts. In addition to ensuring a good initial feed additives are becoming a seemingly more vaccine oocyst uptake, several subsequent popular topic among veterinarians, nutritionists, cycles must take place successfully in the field feed manufactures, producers, farmers and without interruption. This “re-cycling” is entirely scientists. Phytogenic feed additives are plant- dependent on management factors during the based feed additives or botanicals that are used first 4-5 weeks of life in a poultry barn. in animal nutrition as alternatives to antibiotics, ionophores and chemical coccidiostats. Research This paper will focus on experiences in the field on phytogenic alternatives have shown some of related to this vaccine’s use, as well as best them to have antibacterial, anticoccidial, as well management practices in both the hatchery and as, anti-inflammatory properties. These in the field, in order to optimize application, substances can be derived from herbs, spices, uptake, re-cycling, and the development of other plants and their extracts, like essential oils. immunity in long lived chickens against coccidiosis. The aim of this study was to determine the potential impact on coccidiosis vaccine when Lessons learned changing coccidiosis vaccines birds were fed three different phytogenic feed in grandparent broiler breeders additive products: 1.) A 5% oregano essential oil Christina Lindsey1, Kelli Jones2 products; 2.) A proprietary blend of tannic acid extract, phytogenic molecules, beta glucans and 1Aviagen, 2Ceva a Bacillus subtilis; 3) Hops. The potential impact When our previous supplier of coccidiosis of all three phytogenic products are discussed. vaccine announced a limitation in supply, we had to find a different product. In this presentation, we’ll discuss how we handled this transition in the hatchery and in the field for our internal flocks of Grandparent (GP) broiler breeders. Topics will include hatchery administration, Identifying and Quantifying Eimeria Species in convert uninformative OPG counts into precise Mixed Samples with Molecular-Based measures of parasite diversity and relative Technologies abundance is prerequisite for unraveling this disease complex and thereby improve control of Ryan Snyder1, Dr. John R Barta2 coccidiosis in commercial poultry industries. 1University of Guelph, 2University of Guelph Applying Metagenomics to Characterize the Multiple Eimeria species can infect a chicken Eimeria species Composition in Litter from simultaneously with each parasite species Commercial Broiler Farms varying in its pathogenicity and perceived Kate Worthing commercial importance. Therefore, identification of individual Eimeria species in ARS-USDA mixed samples is valuable for understanding this The detection of Eimeria in broiler chickens often disease complex. Recently, molecular tools for relies on examining gut tissue for oocysts and identification have been exploited because schizont stages by light microscopy or on morphological differences of oocysts remains molecular assays such as polymerase chain unreliable. reaction (PCR) that can detect and differentiate Digital droplet PCR (ddPCR) is a sensitive Eimeria species recovered from gut tissue or quantitative method that could be applied to the litter. These diagnostic methods are usually identification and quantification of Eimeria done when there is a spike in mortality at 2-4 species. A ddPCR-based method was developed weeks of age which is often due to necrotic that exploited targets that were genus-specific enteritis (NE), a disease most often associated or species-specific. A multiplexed approach with with a concurrent Eimeria infection. It is now a probe designed to provide DNA copy number well accepted that E. maxima plays an important for all Eimeria spp. combined, and a probe to role in the occurrence of NE in broilers, and provide DNA copy number for the seven control of E. maxima by either vaccination or described Eimeria species was used. Single- anticoccidial drugs is crucial to preventing NE- species samples of known identities were tested associated mortality in young chicks. What alone and in combinations to assess the remains unknown is whether strains of E. sensitivity and repeatability of this experimental maxima exist that are more predisposing to NE assay. Experimentation with this technology and by being more pathogenic, that is causing unique design has demonstrated its ability to greater damage to intestinal tissue. In our provide relative abundance of Eimeria species laboratory, two strains of E. maxima were DNA. Samples analyzed originating from identified, one (E. maxima APU1) being highly commercial coccidiosis vaccine vials and feces pathogenic causes 25% weight gain depression from flocks post-vaccination were tested with with as few as 1000 oocysts. Another strain (E. providing results consistent with expectancies. maxima APU2) requires doses greater than This assay provides a commercially-viable assay 25,000 to cause a similar level of weight gain alternative to Next Generation Sequencing. depression. A series of oligonucleotide primers were made to conserved regions of single copy Obtaining relative species abundance and orthologous genes (SCO) of Eimeria and used in correlating it with oocyst per gram counts will metagenomic analysis (Illumina MiSeq) to create data needed by veterinarians, characterize the relative frequencies and researchers, technical representatives, population structure of Eimeria recovered from nutritionists and producers. Using this assay to litter at various times of growout on commercial experienced an early reduction in BW and poor broiler farms. These primers flanked variable FCR (1-28d, p0.05). At 28d, serum levels of FITC- SCO regions and thus may be helpful in dextran were significantly increased (p<0.05) in identifying different Eimeria strains of interest, the vaccinated birds (V) compared to the such as those whose presence may correlate remaining treatment groups, indicating impaired with mortality at 2-4 weeks of age. gut integrity. The negative impacts of vaccination, both on growth performance and Effects of Coccidiosis Vaccination With or intestinal health, were alleviated (p<0.05) when Without the Use of a Phytogenic Feed Additive treatments included the phytogenic feed in Broilers additive. In conclusion, live coccidiosis Marcos Rostagno1, Meghan Schwartz2, Sergio vaccination does have negative impact on Vieira3 growth performance and intestinal health in broilers. Furthermore, this study demonstrates 1 2 Phytobiotics North America, Phytobiotics the benefits provided by the phytogenic feed 3 North America, Federal University of Rio additive on the intestinal health of broilers. Grande do Sul

The objective of the present study was to Virology determine the effects of coccidiosis vaccination Viral Hepatitis in Turkey Poults with or without a phytogenic in-feed additive (Macleaya cordata extract, MCE) on the growth Megan Lighty performance and intestinal health in broilers. A Jennie-O Turkey Store total of 648 1-day old male Cobb 500 broilers were assigned to 4 treatments in a complete The scientific literature describes turkey viral randomized block design, including 9 replicates hepatitis (TVH) as a generally subclinical disease per treatment. Treatments were: Negative of young turkeys characterized by multifocal Control (NC; No feed additive or vaccine), hepatic necrosis likely caused by a picorna-like Coccidiosis Vaccine (V), V plus phytogenic feed virus. Clinical signs of the disease are not well- additive at 60 ppm (VMCE60), and V plus defined, and the disease is thought to manifest phytogenic feed additive at 90 ppm (VMCE90). when combined with other factors such as Birds were vaccinated for coccidiosis at concurrent infection and/or environmental placement, with a commercially available live stress. Morbidity and mortality can be highly vaccine administered according to the variable and the economic significance of TVH is manufacturer recommendation. The litter used unknown. Within the last year, turkey consisted of pine shavings after 3 cycles. Birds companies in the United States have reported an were fed corn-soybean meal all-vegetable diets increased incidence of multifocal necrotizing ad libitum, and no medication was included in hepatitis in turkey poults. Gross lesions range feed or water throughout the study. Growth from subtle mottling to multifocal performance was determined at 28, 44 and 53 white/gray/tan foci in the livers. Reovirus has days, based on body weight (BW), feed intake been consistently isolated from the livers of (FI) and feed conversion ratio (FCR). Effects on affected poults. Flock history, necropsy findings, intestinal permeability (FITC-dextran and results of diagnostic testing will be reviewed. absorption) and inflammation markers (serum levels of TNF-alpha and IL-10) were evaluated at 28d of age. Results showed that vaccinated birds Isolation of Egg Drop Syndrome (EDS) and fulfil Koch’s postulates. Commercial Adenovirus from Brown Layers in Pennsylvania inactivated EDS vaccines are not currently sold in the USA, therefore attempts were made at Milos Markis1, George Boggan2, Ellie Brimer3, adaptation of EDS adenovirus isolate to cell lines Ha-Jung Roh4 approved for autogenous vaccine production. 1AviServe LLC, 2Ceva Animal Health, 8930 Comparison Of Antibody Response Against Rosehill Rd., Lenexa, KS 66215, 3Ceva Animal Infectious Bursal Disease In Broiler Breeders Health, KS 66215, 4Ceva Animal Health, KS Injected Subcutaneously In The Back Of The 66215 Neck, Inguinal Fold And Intramuscular In The Starting in 2018, several flocks of brown egg Thigh Or Breast laying hens from Pennsylvania presented with Enrique Montiel1, Kelly Sweeney2, Jeanna production of pale, soft-shelled, and shell-less Wilson3 eggs at peak of production, without other apparent clinical signs. The hens tested negative 1Boehringer Ingelheim, 2University of Georgia, for avian influenza, Newcastle disease, and Poultry Science Department, 3University of infectious bronchitis, however, serological Georgia, Poultry Science Department evaluation showed exposure to Egg Drop Broiler breeders were vaccinated as embryos Syndrome (EDS) adenovirus. Avian adenoviruses with a recombinant HVT+IBD vaccine and can be divided into three groups. The first group housed in a breeder facility at the University of comprises of adenoviruses in chickens often Georgia. The recombinant vaccine administered associated with inclusion body hepatitis, the in ovo was the sole Infectious bursal disease second group is hemorrhagic enteritis virus (IBD) primer the flock received. All birds were (HEV) in turkeys, and the third group is EDS virus. raised following broiler breeder company EDS virus is a duck adenovirus that can infect recommendations. The pullets received a chickens through indirect or direct contact with conventional live primer and inactivated duck feces, untreated contaminated water, or vaccination program where the inactivated IBD lapse in biosecurity. The virus replicates in the consisted of two injections of 0.5 ml given at 20 uterus of hens and is transmitted in or on eggs weeks of age intramuscularly on each side of the and droppings contaminated with oviduct breast. When the breeders were 48 weeks of age secretions. they were divided into 5 groups of 20 birds each Each subsequent flock of hens placed on this and received a second inactivated IBD vaccine as multi-age Pennsylvania farm through 2019 follows: 1) subcutaneously in the back of the showed identical presentation of drop in egg neck; 2) Inguinal fold; 3) intramuscular in the production, which can be attributed to EDS virus breast; 4) Intramuscular in the thigh. The fifth becoming endemic on the farm. EDS adenovirus group did not received a second IBD vaccination was isolated from the shell glands of affected to serve as negative control. Serum samples brown hens and propagated in commercial duck were collected from all breeders in the study on embryos inoculated via chorioallantoic sac route the day of vaccination to establish a baseline and and in duck embryo fibroblasts. The virus did not then three and 6 weeks after vaccination. replicate in specific-pathogen-free chicken Antibodies against IBDV were measured with embryos, chicken embryo fibroblasts, or LMH two different ELISA kits. Seroconversion was cells. Isolated virus was inoculated into evaluated by comparing the percent increase on susceptible brown hens to reproduce the disease Geometric mean titer (GMT) and decrease in coefficient of Variation (CV). Vaccine injection in Effect of antibiotic growth promoter the breast produced the highest increase in GMT administration on the intestinal microbiota and biggest decrease of CV values. Detailed and virus shedding post avian influenza virus results will be discussed. infection in chickens

Virulence assessment of chicken astrovirus Brittany Seibert (CAstV) isolated from outbreaks of White University of Georgia Chicken Syndrome (WCS) in Western Canada Outbreaks of avian influenza virus (AIV) can have Victor Palomino-Tapia1, Darko Mitevski2, Tom detrimental effects in poultry flocks resulting in Inglis3, Mohamed Faizal Abdul-Careem4 mortality and indirect costs because of 1University of Calgary, 2Poultry Health Services, eradication efforts, control and loss of markets. Airdrie, AB T4A2G8, Canada, 3The Institute of The role of the microbiota in essential Applied Poultry Technologies, AB T4A2G1, physiological processes and disease progression Canada, 4Department of Ecosystem and Public has expanded. Recently, chickens infected with Health, Faculty of Veterinary Medicine, low pathogenicity avian influenza virus (LPAIV) University of Calgary, Calgary, AB T2N4N1, showed an intestinal microbiota population Canada shift, with an increase in Escherichia and decrease in probiotic organisms like In the last 5 years, chicken astrovirus (CAstV), the Lactobacillus. Further, similar studies reported causative agent of a condition known as White that the absence of commensal gut microbiota in Chicken Syndrome (WCS), has emerged as an chickens increased virus shedding and economically important disease across Western compromised immune responses toward LPAIV Canada. CAstVs were isolated from liver and infection. While it is shown that the intestinal intestinal samples of affected chicks and microbiota affects AIV infection, it is not well embryos following inoculation of samples in known how altering the intestinal microbiota will chicken embryo liver cells (CEL). affect AIV replication and shedding. Extensive To evaluate the virulence of a CAstV clinical literature reports showed that a variety of isolate, two ages of embryonated eggs, namely antibiotic growth promoters (AGPs) change the 14 & 18 days of embryonation (DOE) were composition and diversity of the microbiota at infected by the in ovo route at three different different parts of the avian intestinal tract. While titers 101.0, 102.0, 103.0 TCID50 per embryo. At the literature is rich in the relationship between 14 DOE, the site of deposition would be the yolk AGPs and the intestinal microbiota, little is sac, while at 18 DOE, it would be known about the effect of AGP-induced embryo/amniotic sac. Once hatched, the birds microbiota changes on AIV shedding. Therefore, were placed on floor pens and monitored daily we explored the potential effect of a widely used for clinical signs for a 7-day observation period. AGP (Bacitracin methylene disalicylate (BMD)) Cloacal swabs were obtained at day of age from on LPAIV shedding and weight gain in broiler all birds (including unhatched embryos), and at 7 chickens. Results showed that there was no days of age. The samples of liver, gut, and significant difference in virus shedding and kidneys were collected for quantitative weight gain among groups. However, intestinal polymerase reaction (qPCR) and histopathology. microbiota changes were observed. Clinical signs, qPCR, and histology data will be Understanding the relationship between AGP discussed in detail at the conference. induced microbiota changes and AIV infection will further increase our comprehension of the impact that the microbiota plays in AIV infection Comparison of the monitoring methods for in poultry. following the take of immune-complex vaccine against Gumboro disease in commercial layers SARS-CoV-2 and MERS-CoV are unable to replicate in common poultry species and Claudia Carranza1, Luiz Alzamora2, Luiz Sesti3 embryonating chicken eggs 1Ceva Animal Health, 2Animal Health, 3Ceva Erica Spackman Animal Health,

SEPRL-USDA-ARS The control of Infectious bursal disease (IBD, also called Gumboro disease) is extremely important The efficacy of an adenovirus-vectored to prevent losses in layer production. The control Newcastle disease virus (NDV) vaccine of Gumboro disease must include not only the expressing the fusion (F) NDV protein (adeno-F) protection against the clinical signs, but also the was evaluated against challenges with virulent prevention of replication of field IBDV strains, NDV strains containing heterologous and that can introduce and keep disease challenges homologous F proteins. In a preliminary study, for the subsequent flocks. By this way, the virus two different doses (low and high) of adeno-F pressure should decrease cycle after cycle and were tested against a virulent NDV strain the likelihood of emergence of new IBDV strains containing the homologous NDV F protein, CA02. should be reduced. In a second study, at three weeks post vaccination, the efficacy of the high dose of A novel option to control IBD is an immune- adeno-F was compared to a live attenuated NDV complex vaccine (Novamune®), specifically vaccine strain (LaSota) against three developed for egg-type genetic lines and slow antigenically distinct virulent NDV challenge growth chickens. The vaccine strain, a live strains, one homologous (CA02), and two attenuated IBDV, is capable to reach the bursa, heterologous (TZ12, EG11) to the F in the when maternal immunity has waned, it brings a vectored vaccine. In both experiments, clinical high level of protection against various types of signs, mortality, virus shedding, and humoral IBD viruses and it effectively prevents shedding. response were evaluated. In the first To monitor the proper replication of the vaccine experiment, the survival rates from birds strain in the bursa of Fabricius, comparisons of vaccinated with adeno-F at a high and low dose combined methodologies were applied in were 100% and 25%, respectively. As for the parallel. A first vaccinated pullet flock was second experiment, birds vaccinated with the monitored by serology (ELISA) and detection of high dose of adeno-F, had a survival rate of 80%, vaccine virus (RT-PCR). A second vaccinated 75%, and 65% after challenge with the CA02, pullet flock was followed by histology and RT- TZ12, and EG11 viruses, respectively. All of the PCR. LaSota-vaccinated birds survived post challenge no matter the NDV strain used except for one The results of antibody response, vaccine virus bird challenged with CA02 that died from causes detection and histological results will be unrelated to the challenge. disclosed, as well as the comparison between the different methodologies. These data provide

evidence of an attractive, innovative, convenient tool for the control of Gumboro disease in commercial layers. Molecular characterization of field isolates of Virology: ILT and IBV infectious bronchitis virus in Mississippi from Validation of Day of Age Infectious Bronchitis 2013 to 2019 Vaccination Alejandro Banda1, Eric Orozco2, Rebeca 3 4 Matilde Alfonso1, Jose Linares2, Travis Mackey , Scott Baughman 3 4 Cigainero , Ha-Jung Roh 1Mississippi State University, 2Poultry Research 1Ceva Animal Health, 2Veterinary Technical and Diagnostic Laboratory, College of Service, Ceva Animal Health, U.S.A, 3Veterinary Veterinary Medicine, Mississippi State Technical Service, U.S.A, 4Scientific Support and University, 3137 Highway 468 West, Pearl, MS 3 Investigation Unit, Lenexa, U.S.A 39208, Poultry Research and Diagnostic Laboratory, MS 39208, 4Poultry Research and Infectious Bronchitis (IB) vaccination of day-old Diagnostic Laboratory, MS 39208 chicks in the hatchery is a common practice in commercial broilers in the USA. Live attenuated This is a retrospective study about the IB vaccines reconstituted in water are usually presentation of infectious bronchitis virus (IBV) administered using different spray cabinets. genotypes in Mississippi (MS) from January 2013 Dosing and combinations of existing IB vaccine to June 2019. Six hundred and twenty-three IBV strains are often adapted to address the isolates from seven counties (Copiah, Covington, challenges present in each region. However, Jasper, Jones, Neshoba, Pike, and Scott) with despite of vaccinating billions of broilers important poultry production were isolated and annually, validation of IB vaccination has not genotyped at the Poultry Research and been a standard practice in the industry. This Diagnostic Lab (PRDL) in Pearl, MS. The work describes how quantitative Reverse genotyping of these isolates was conducted by Transcriptase Polymerase Chain Reaction (qRT- partial sequence analysis of the hypervariable PCR) was utilized as a validation tool for IB region (HVR) of the S1 gene. Before winter of vaccination of day-old chicks in commercial 2013, the most frequently detected serotype hatcheries. Several field cases utilizing different was Arkansas-Delmarva Poultry Industry (Ark- IB vaccine strain combinations, dosing, spraying DPI) with 76% of the isolations. The second most techniques, and diluents (water vs. gel) were isolated serotype was Connecticut (Conn) with analyzed. Samples consisting of 15 individual 13% of the isolations. Between December 2013 choanal swabs were taken at 5 days post- and the fall of 2014, Georgia 08 (GA08) isolates vaccination from each case. qRT-PCR results emerged, especially in the counties with more showing virus detection and relative dense poultry population (Covington, Jones and quantification across individual birds are Scott). During 2015, isolations of this subtype presented. Opportunities for improvement IB came to an end. However, in spring of 2016 this vaccination of commercial broilers can be subtype re-emerged with limited geographic extracted from these results. presentation (Covington and Jasper) and has been isolated to the present time. On April 2019, the subtype Delmarva (DMV)/1639 was initially isolated in Copiah, Neshoba and Scott, counties, from samples of broiler flocks with respiratory problems. The phylogenetic analysis including several Ark-DPI isolates indicated their very close relation among themselves and distribution in the phylogenetic tree regardless the county or are under way to ascertain the virulence and year of isolation. A similar situation was transmission potential of these Canadian ILTV observed with GA08 isolates. These phylogenetic isolates. analyses may suggest that populations of Ark- Funding from Alberta Agriculture and Forestry is DPI and GA08 field strains circulating throughout acknowledged. the state are very close related. Infectious Bronchitis Challenge Studies in Genotyping of Infectious Laryngotracheitis Broilers—a Comparison of IB Vaccine “Takes” virus (ILTV) isolates from Western Canada and Protection based on partial open reading frame (ORF) a and b Kalen Cookson1, Manuel Da Costa2, John Dickson3, Andrew Barker4 Catalina Barboza Solis 1Zoetis, 2Zoetis, 3Zoetis, 4Zoetis University of Calgary Two studies will be presented. Study 1 compares Infectious Laryngotracheitis (ILT) is an acute different bivalent combinations of IB vaccines upper respiratory disease in chicken caused by (Mass+GA98, Mass+GA08 and GA08+GA98) to the etiological agent Gallid herpesvirus 1 the trivalent combination of these same vaccines (Infectious Laryngotracheitis virus, ILTV). The (Mass+GA98+GA08) given to broilers by day of control of ILT relies on two types of vaccines, age spray and challenged at 25 days with a recombinant and live attenuated vaccines. The DMV/1639 isolate. Study 2 compares the same live attenuated vaccines can be either embryo trivalent combination on protection against passaged (chicken embryo origin, CEO) or tissue either DMV/1639 or Arkansas challenge. Vaccine culture passaged (tissue culture origin, TCO). The takes of the various combinations will be live attenuated vaccines have proven to be very compared by serotype specific PCR probes. effective in disease control however, they have Tracheal protection based on histopathology residual virulence which makes them able to and PCR Ct values will be presented. The results replicate, cause disease and/or revert to the will show that bivalent combinations give original virulent form. Canada lacks information modest protection against heterologous IBV on the molecular nature of ILTV that circulates in challenge but the trivalent combination gives the country. The objective of the study was to significantly better IBV protection and this level determine the molecular nature of ILTV isolates of protection was similar against both that caused ILT in chickens raised in Western DMV/1639 and Arkansas IBV challenge viruses. Canada. From the year 2009 to 2018, samples submitted for the diagnosis of ILT were obtained Evaluation of host genetic resistance against from Alberta (n=46) and British Columbia (n=9). infectious laryngotracheitis Sanger sequencing of ORFa and b was used for 1 2 3 genotyping. We found that most of these John Dunn , Jody Mays , Cari Hearn , James 4 isolates cluster in group V (CEO revertant), some Riggins cluster as grouped VI (wild type) and a few in the 1USDA-ARS-USNPRC, 2USDA-ARS, 3USDA-ARS, TCO group. With this method, TCO related 4USDA-ARS clusters (I, II and III) are grouped together since the USDA strain is likely their parent strain. Infectious laryngotracheitis (ILT) and Marek’s Clusters VII, VIII and IX also group together, and disease (MD) are caused by two closely related are related to Australian strains. Further studies species, Gallid alphaherpesvirus 1 and Gallid alphaherpesvirus 2, respectively. Despite recently, sequence typing of ILT was achieved by similarity of the viruses and 70+ years of work on amplifying and sequencing five large genetic genetic differences in host resistance to MD, segments, which classified ILT into nine there has been very little effort in trying to genotypes. Given the large size of the target control ILT through host genetic resistance. The segments, ILT sequence typing was performed best understood mechanism for the involvement only from viral isolates or from clinical samples of genetic resistance to MD involves the MHC or, with high viral copy numbers. The purpose of this as it is known in the chicken, the B complex. study is to reduce the number and size of target Using ILTV challenge strains 63140 and 1874c5, segments necessary for ILT sequence typing to we utilized our unique poultry genetic resources allow for ILT the sequence typing directly from (including B-congenic lines B*2, B*5, B*12, B*13, clinical samples with relatively lower viral copy B*19 and B*21) to evaluate the effect of MHC on number. Three targets were identified, and ILT disease incidence. In addition, we challenged three sets of primers designed to amplify and Line 6 and Line 7 birds which carry the same MHC sequence these targets. Amplification and but differ in non-MHC genes. Significant sequencing was successful from clinical samples differences between clinical signs and viral load with lower copy number. Two of the original nine were observed between genetic lines, with Lines genotypes, namely tissue-culture vaccines and B*2 and B*5 demonstrating the most resistance. tissue-culture vaccine related genotypes, were Line 6 also demonstrated more resistance not differentiated using this new assay. This compared to Line 7. These results provide a means that the new assay sequence typed ILT basis for genetic selection of breeding flocks that into eight instead of nine genotypes. In spite of are more resistant to ILT. this reduction of discriminatory power, the new assay would allow for the characterization of Infectious Laryngotracheitis Sequence Typing many more ILT cases. This in turn allows for Directly from Clinical Samples better understanding of ILT epidemiology and Mohamed El-Gazzar1, Amro Hashish2, Avanti effective control and eradication efforts. Sinha3, Yuko Sato4 Efficacy of two commercial infectious coryza 1Iowa State University, 2Iowa State University, vaccines after challenge with a serotype C Department of Veterinary Diagnostic and Avibacterium paragallinarum strain isolated Production Animal Medicine, College of from outbreaks in layers and broilers in Veterinary Medicine, Iowa State University, California Ames, IA 50011, 3Iowa State University, IA Rodrigo Gallardo1, Simone Stoute2, Ana paula 50011, 4Iowa State University, IA 50011 Da Silva3, Charles Corsiglia4 Infectious Laryngotracheitis (ILT) is a contagious 1University of California, Davis, 2University of Herpes virus that causes upper respiratory California, Davis, 3University of California, Davis, disease of chicken leading to significant 4Foster Farms economic losses in form of mortality and egg production losses. Identification of ILT virus Infectious coryza is a bacterial upper respiratory strain is important for the disease control and disease caused by Avibacterium paragallinarum. eradication efforts. Identification of ILT viruses In recent years, a significant increase in was historically performed through genotyping infectious coryza cases have been observed in by restriction fragment length polymorphism California, affecting both broiler and layer (RFLP) of multiple sub-genomic segments. More chickens. The strains isolated in California have been classified as serotype C-2. However, euthanized to observe gross lesions and collect genotypic analysis using the hemmaglutinin samples. We found that IBV genome is genes HMTp210 and hagA demonstrated high quantifiable from cloacal and oropharyngeal similarities to strains that belong to serotype C- swabs as well as reproductive tract of infected 1. The aim of this study was to compare the chickens. At the post-mortem examination, we efficacy of two commercial vaccines after found shortened oviductal length and ovarian challenge with a wild-type serotype C strain of regression in some of the infected layers. One of Av. Paragallinarum isolated in California. Vaccine the infected chickens showed pale swollen 1 is a quadrivalent killed bacterin vaccine that kidney with the tubules distended with urates. contains serotype A, B and C-1 strains as well as Histological changes were also noted in the a variant of serotype B. This vaccine is not kidneys and reproductive tracts of infected commercially available in the US. Vaccine 2 is a chickens. We were also able to demonstrate trivalent killed bacterin vaccine that contains anti-IBV antibody response in serum and locally serotype A, B and C-2 strains and is widely used in the reproductive tract washes. We are in the in North America. Despite differences in process of evaluating cytokine response and serotype C strains in vaccines 1 and 2, our results immune cell recruitment in the reproductive indicate that both vaccines, if administered in tract following IBV infection. two doses as recommended by the DMV/1639 type IBV transmits from vaccinated manufacturer, are able to induce appropriate chickens to other vaccinated chickens after protection and reduce bacterial shedding when challenge compared with unvaccinated chickens. Further results from these experiments will be discussed. Brian Jordan1, Jongseo Mo2, Erica Spackman3

Pathogenesis of the Canadian Delmarva 2The University of Georgia, 2Southeast Poultry (DMV/1639) Infectious Bronchitis Virus (IBV) Research Laboratory, 3Southeast Poultry Infection in Layers Research Laboratory

Mohamed S. H. Hassan Infectious bronchitis virus (IBV) is an upper respiratory tract pathogen of commercial University of Calgary poultry that causes relatively mild clinical signs IBV infection may lead to reduced egg but predisposes chickens to secondary bacterial production and poor egg quality in layers. In the infections which can lead to airsacculitis and recent past, one of the most dominant IBV carcass condemnation at processing. Because of variants isolated from Canadian layer flocks with the economic impact of condemnation, nearly egg production problems was DMV/1639. Our every commercial chicken is vaccinated against main hypothesis was that experimental infection IBV. IBV vaccines are serotype specific, however, with DMV/1639 variant will result in pathological so having a perfectly matched vaccine is not changes in the reproductive tract leading to egg always possible. To this end, producers are production and quality issues. To determine combining different IBV vaccine types in attempt whether the isolated Canadian DMV/1639 leads to achieve cross-protection. This strategy has to egg production problems, we infected specific been effective at reducing or preventing clinical pathogen free layers at the peak of lay (26 signs, but field type IBVs are still isolated. Thus, weeks) with an uninfected control group. When it appears that field viruses can still transmit the post-infection egg production dropped to using this vaccine strategy though this has not 40% (10 days post-infection), the chickens were been examined experimentally. To answer this question, we reared vaccinated (Mass and GA08 vaccination strategy against the disease. type vaccines) and non-vaccinated chickens Therefore, the objective of this study was to separately and mixed them together in different evaluate the safety and protection efficacy of an combinations after some were challenged and LMH cell line adapted BDORFC strain when others were not, to evaluate the transmissibility administered in ovo and/or spray with and of a DMV/1639 type field virus. We found that without a rHVT-LT vaccine. Vaccination with the the DMV/1639 virus readily transmits from non- BDORFC strain either alone or in combination vaccinated, challenged chickens to other non- with a rHVT-LT vaccine did not affect hatchability vaccinated chickens as expected. Interestingly, and only minimal signs of respiratory distress the DMV/1639 virus also transmitted from were recorded for groups of birds that received vaccinated, challenged chickens to vaccinated, the BDORFC strain via spray. After challenge, all non-challenged chickens, although it occurred a vaccinated groups showed reduction in clinical week later than in non-vaccinated chickens. As in signs and comparable lymphocyte and monocyte the field experience, clinical signs were infiltration into the trachea. However, a significantly reduced in vaccinated, challenged decrease in challenge virus replication chickens, but challenge virus was still detected. comparable to the non-vaccinated/non- This information is significant because if field challenge group was only observed for groups of virus is able to transmit in vaccinated chickens, it chickens that received dual vaccination of rHVT- can replicate and mutate, leading to a potentially LT (IO) + BDORFC. Consequently, compared to new variant IBV. rHVT-LT or BDORFC when administered alone, the dual vaccination strategy improved Safety and Protection Efficacy of a Cell Line protection against challenge. Adapted Infectious Laryngotracheitis Virus (ILTV) BDORFC Strain Following In ovo and/or Investigating the role of Infectious Bronchitis Spray Vaccination with and without rHVT-LT Virus variant DMV/1639 in incidence of False Vaccine Layer Syndrome

Daniel Maekawa Maeda Adrea Mueller1, Dr. Mark Jackwood2, Dr. Monique Franca3, Dr. Brian Jordan4 University of Georgia 1University of Georgia, 2University of Georgia In an effort to produce more stable live Department of Population Health, 3University of attenuated vaccines for infectious Georgia Poultry Diagnostic and Research laryngotracheitis virus (ILTV), deletion of genes Center, 4University of Georgia Department of related to virulence has been extensively Population Health pursued. Among viral genes associated with virulence but non-essential for viral replication in Infectious Bronchitis virus (IBV) is an avian vitro is the open reading frame C (ORF C). The coronavirus that commonly causes respiratory protection efficacy of an ILTV recombinant strain disease but can also affect the reproductive tract with deletion of the ORF C gene (BDORFC) when of laying type chickens. If infection occurs in administered in ovo was comparable to the young pullets, false layer syndrome can develop tissue culture origin (TCO) vaccine. As rHVT-LT in later in life. False layer syndrome is characterized ovo vaccination is a common practice in the by systemic changes in the oviduct and the poultry industry, the development of live development of large, fluid filled cysts by the attenuated strains that can be also administered time hens come into production. IBV strains QX in ovo is being considered as a possible and Mass have been previously found to cause False Layer Syndrome however, IBV strain challenged with one of two variant ARV strains DMV/1639 in parts of Canada and the U.S. has (pathogenic for tendons or heart), one classical been reported in areas where False Layer (S1133) ARV strain or PBS in the negative control Syndrome is occurring. Because of the delayed group, via footpad. The other half of the chickens presentation of cystic oviducts, the initial IBV remained as contact birds. Clinical signs, body infection is rarely detected and it is not clear if weight-hock joint ratio, histopathology and viral IBV is the sole cause or at what age infection loads in tendon and heart were assessed at must occur for False Layer Syndrome to develop. different time points and compared between Our study investigates the role and timing of IBV groups. Lymphocytic depletion in thymus and infection on development of False Layer bursa was evaluated. Results will be discussed. Syndrome, using both IBV variant DMV/1639 Protection Against 9109-Like Variant IBDV and Mass. For our study, eight groups of 120 SPF Using Dual Recombinant HVT-ND-IBD Vaccine chickens and a control group of 50 chickens were Alone placed separately into colony houses and challenged at either three or seven days of age, Andres Montoya two weeks of age, or sixteen weeks of age with either DMV/1639 or M41 IBV. Post challenge, Merck Animal Health swabs were taken and necropsies were Infectious bursal disease (IBD) is a highly performed where tracheas, kidney, and contagious disease of young chickens caused by reproductive organs were collected for infectious bursal disease virus, characterized by histopathology and immunohistochemistry. The immunosuppression and mortality generally at 3 data collected in this experiment will provide to 6 weeks of age. IBDV is ubiquitous in valuable information in understanding the commercial chicken operations. IBDV causes a pathology and development of False Layer prolonged B-lymphocyte immunodeficiency and Syndrome. increased susceptibility to various viruses and parasites. Both classic and variant strains of IBDV Virology: Marek’s Disease had been isolated in the southeastern United States. 9109 is a variant strain of IBD that has and Reovirus been a problem in the southern states of United Pathogenic and Immunogenic Evaluation of States. 9109 variants have been characterized as Avian Reovirus Variant Strains Isolated from a classical, very virulent, and variant strain. 9109 Clinical Cases had been isolated in commercial broilers that had result in performance issues. An evaluation Sofia Egana of the protection against 9109 variants using a University of California, Davis dual recombinant HVT-ND-IBD vaccine alone or in combination with a live intermediate standard Since 2015, an increase of clinical cases of viral strain and 89/03 strain of IBDV vaccine showed, arthritis/tenosynovitis caused by avian reovirus based on bursa/body weight ratio and variant strains (ARVv) have been diagnosed by histopathology, protection in all the vaccinated the California Health and Food Safety Laboratory groups when it was compared with the non- (CAHFS). In order to better understand the vaccinated/challenged group. Statistical pathobiology of ARVv, four groups (n= 182 ) of difference was observed between the no one-day-old SPF chickens were placed in BSL 2 vax/challenge group when compared to the rooms. Half of the chickens in each room were vaccinated groups. The dual construct rHVT-ND- IBD vaccine alone or in combination with a live 10 weeks of age to collect tendons for IBD vaccine protected against 9109 like variant histopathology and reovirus isolation. The study IBD virus when challenged at 18 days of age. results analyses are in progress. This study will help to determine the origin of TARV infection, Differential Pathogenesis of Turkey Reoviruses to provide information about viral shedding and associated with Arthritis, Enteritis and a sampling scheme for early detection of TARV Hepatitis from infected turkeys. Sunil Mor 1, Rahul Kumar2, Tamer A. Dual ND-IBD HVT Construct Vaccines: Efficacy Sharafeldin3, Sunil K. Mor4 and Onset of Immunity When Challenged with 1University of Minnesota, 2Veterinary Diagnostic IBD Variant Strains Laboratory and Department of Veterinary Linnea Newman1, Holly S. Sellers2, Erich Population Medicine, University of Minnesota, Linnemann3, Vanessa Gauthiersloan4 St. Paul, MN 55108, USA, 3Veterinary Diagnostic Laboratory and Department of Veterinary 1Merck Animal Health, 2PDRC, 953 College Population Medicine, USA, 4Veterinary Station Road, Athens, GA, 30602, 3PDRC, 30602, Diagnostic Laboratory and Department of 4PDRC, 30602 Veterinary Population Medicine, USA HVT construct technology has advanced to a Turkey arthritis reovirus (TARV) has been point where multiple commercial dual-construct associated with lameness in turkey flocks older HVT vaccines (ND-IBD) are now available to the than 10 weeks of age resulting in significant poultry industry. These vaccines differ in how economic losses since 2011. The aim of this each construct was developed. The onset of study is to design a standardized mixed-sampling immunity against classical IBD is generally 2 to 3 scheme for early detection of viral infection. We weeks for HVT construct IBD vaccines. In the hypothesize that use of histopathology, Americas, variant strains of infectious bursal molecular detection and virus isolation will be disease (IBD) are of significant concern, and they helpful in detection of TARV infection at younger often break through maternally derived age which may give us an early opportunity to antibody at an early age. mitigate the problem and to alleviate animal Groups of SPF birds were vaccinated with welfare concerns. To achieve the designated commercially available dual construct HVT-ND- aims, we recruited 10 turkey farms (6 are known IBD vaccines via subcutaneous route. Vaccinates with a repeated history of clinical infection and 4 were challenged with Delaware Variant E, 9109- are clean healthy as negative control) in 2 like IBD variant or AL-2-like IBD variant viruses at different states in the Midwest and the East 14 or 18 days of age. Spleen and feather follicle Coast of the USA. We have collected clean litter samples were submitted for next generation samples and drinker swabs at zero week before sequencing to determine dissemination of the the arrival of turkey poults. Additionally, we have vaccine virus, and bursal histopathology, bursa: collected meconium samples from shipping trays body weight ratio and serology were used to at the arrival time of the turkey poults. After evaluate protection following challenge. Three placement, litter/fecal samples (5 pools) and commercially available dual HVT construct drinker swabs (2 pools) were collected weekly vaccines are compared to see if vaccine for 10 weeks. Samples were processed for construction results in significant differences in reovirus isolation using QT-35 cells. We also vaccine dissemination, protection against received legs of five birds per house at 3, 5 and challenge with variant-type IBD or onset of Evaluation of Attenuated Variant Reoviruses as immunity. Modified Live Vaccines

Turkey Breeders Perspective : How to Achieve Holly Sellers1, Erich G. Linnemann2, Vanessa Reovirus Maternal Antibodies in Poults Gauthiersloan3, Nick Davis-Fields4 through Vaccination of Parents 1Poultry Diagnostic and Research Center, Cynthia Philippe University of Georgia, 2Poultry Diagnostic and Research Center, University of Georgia, 3Poultry Hendrix Genetics Ltd Diagnostic and Research Center, University of Reovirus is a major concern in commercial Georgia, 4Poultry Diagnostic and Research turkeys. To reduce its impact, all partners in the Center, University of Georgia industry need to work together: commercials, Avian reoviruses are causative agents of breeders and researchers. Each entity has its tenosynovitis and viral arthritis in chickens and role. Dr Rosenberger presented in September turkeys. The incidence of clinical disease 2019 data showing that poults are more continues to cause economic and health issues susceptible to reovirus during their first 14 days for commercial poultry. Historically, commercial of age and that reovirus can spread through modified live and inactivated reovirus vaccines contact from infected to naïve birds. A more have provided protection against vertical recent unpublished study from Dr Rosenberger transmission from breeders to broiler progeny showed that maternal antibodies can and clinical disease in breeders and broilers. significantly reduce clinical signs in young poults. However, little protection is provided against As breeders, we can help our customers by challenge with contemporary reoviruses, thus vaccinating against reovirus, to protect poults the use of custom inactivated reovirus vaccines during their most susceptible time and to mute has increased dramatically over the past seven potential vertical transmission. To achieve it, years. Modified live reovirus vaccines contain breeder companies have to look into: vaccine strains that are genetically similar (>97% Sigma C quality and vaccination protocol. Since no amino acid similarity) and were isolated over 2 commercial vaccine is available, turkey breeders decades ago. The lack of similarity between the have to rely on killed autogenous vaccines, current field isolates and vaccine strains is a which come with some limitations, such as no primary reason for lack of adequate protection. proof of efficacy. Also, without live priming, it is Two variant reoviruses belonging to genotype 1 unknown how titers hold during production. This and 5 were plaque-purified and passaged is why, to ensure maternal antibodies in poults, successively in chicken embryos until they we compared different vaccination programs. became attenuated. The attenuated variant Titers (CARV ELISA) will be taken 2 weeks post- strains were found to be safe, pure and vaccination and at 15, 34, 46 and 58 weeks of efficacious per guidelines outlined in the 9 Code age. Maternal antibodies from progeny will also of Federal Regulations. In addition, breeders be monitored. Results will be compared to our were vaccinated with a combination of baseline, which are the titers collected for 3 commercial, modified live and inactivated years prior to vaccination. This will enable to variant vaccines. Protection against clinical select which vaccination program is the most disease was evaluated in broiler progeny from effective to protect hatching poults during their the vaccinated breeders in a series of most susceptible period in our operations. homologous and heterologous challenge studies with reovirus variants belonging to genotypes 1- 7. A high level of protection was observed in entail a comparison of antibody titer production homologous challenge studies, as well as in with the same vaccine but injected in two some of the heterologous challenge studies different locations in the bird (inguinal fold and providing evidence that the attenuated variant intramuscular in the breast muscle). Titer levels strains contribute to cross protection between will determine immunization efficacy. This antigenically dissimilar reoviruses. information will allow broiler companies to know if changes need to be made to the Evaluation of Antibody Response to autogenous vaccination processes in order to Autogenous Reovirus and Infectious Bursal increase coverage and decrease disease Disease Virus Vaccines prevalence. Katharine Shamoun1, Holly Sellers2, Karen Turkey Reovirus Hepatitis: An Emerging Grogan3, Jenny Nicholds4 Disease from a Known Etiological Agent. 1Poultry Diagnostic & Research Center, Andrew Smith University of Georgia, 2Poultry Diagnostic & Research Center, University of Georgia, 3Poultry Butterball Diagnostic & Research Center, University of Reoviruses have been known etiological agents Georgia. 4Poultry Diagnostic & Research Center, of tenosynovitis for many years. While many of University of Georgia these reoviruses are species-specific, they can all Avian reovirus and infectious bursal disease produce similar disease in poultry. In turkeys, virus (IBDV) both cause diseases in poultry lameness is classically seen late in the growout species that have very substantial economic phase leading to excessive mortality and consequences. Currently, there are commercial production losses. vaccines for broiler breeders on the market for In early 2019, periodic cases of poult hepatitis both reovirus and infectious bursal disease were observed in brood hubs across the virus. However, as both of these viruses production complex. Initially believed to be undergo frequent genetic mutations, these cases of Turkey Viral Hepatitis (a picornavirus) commercial vaccines, made up of primary based on gross lesions and histopathology, isolates collected decades ago, do not always further diagnostics and clinical signs in the field protect against clinical disease. Broiler ultimately led to a diagnosis of reovirus hepatitis. companies, therefore, will typically send current This case report will cover the timeline of the field isolates to different biological companies initial diagnostic process as well as current in order to develop autogenous vaccines to knowledge of an emerging presentation of immunize future broiler breeder flocks. USDA turkey reovirus. veterinary biologics regulations require safety and sterility testing for these custom vaccines, Case Report: Runting and Stunting Syndrome but efficacy and potency testing prior to use is in a Flock of Commercial Brown Layers not performed. Our study will analyze, using ELISA and virus neutralization assays, whether Jim Stockam autogenous vaccines used for both reovirus and Merck Animal Health infectious bursal disease virus lead to the production of sufficient antibodies to protect Runting and Stunting Syndrome has been against subtypes of these viruses found in the described in commercial brown laying chickens field. A smaller portion of this study will also and characterized by severe stunting, increased mortality, immunosuppression, and severe safety, enteric diseases, respiratory diseases, cystic enteritis. A flock of commercial brown small flocks, etc.) from the perspectives of layers, housed in a building with a history of early subject matter specialists. Finally, input will be stunting, was clinically evaluated from 3 to 10 sought from federal agencies that need research days of age. Daily clinical observations and to establish science-based policies relating to diagnostics, such as electron microscopy, poultry. The resulting research priorities will histopathology, virus isolation, and PCR on therefore represent the multifaceted intestinal and lymphatic tissues, were perspectives of poultry veterinarians and conducted. When compared with a similar specialists involved in poultry production, clinical case in a previous flock housed in the technical service, extension, research, teaching, same farm, a pattern of comparable pathogens diagnostics, and government, and will provide was identified, including a novel Reovirus and a researchable and specific guidance to novel Astrovirus. Isolated viruses were researchers and research funding agencies. administered to brown chickens alone or in Using geospatial methods to produce a combination to fulfill Koch’s postulate. Georgia risk map for avian influenza Wealth of Knowledge 1 Louise Dufour-Zavala

2020 Research Priorities of the American GPLN Association of Avian Pathologists We are using geospatial methods to determine Natalie Armour the likely interface between waterfowl habitat and poultry growing areas. The final product is Mississippi State University, CVM, DPPM intended to use in grower and industry The Research Priorities Committee (RPC) of the education meetings. American Association of Avian Pathologists Post Vaccination Monitoring and Respiratory (AAAP) exists to advance the application of Disease Monitoring in Broiler Flock by On-Side science-based knowledge in the poultry Fully Automated POCKIT Central PCR system industry, by ensuring that the practical research needs of the industry are communicated to Fu Choong Keat1, Wei-Fen Tsai, Ping-Han Chung, researchers and research funding agencies. The Chuan-Fu Tsai, Nina Chen, and Simon Chung2 RPC will determine the 2020 AAAP research 1Aviagen Inc., Huntsville, AL, USA, 2GeneReach priorities using a three-pronged approach. Biotechnology Corp., Taichung City, Taiwan Firstly, as in previous years, surveys will be conducted of veterinarians in broiler, egg and Respiratory disease causes economic losses in turkey production in North America, facilitating broiler industry. Vaccination at day old of age for the generation of ranked listings of research prevention from field challenge is common priorities for each commodity in the categories: practiced. Serology method is often used for Health/Disease, Vaccines & Pharmaceuticals, post vaccination evaluation. However, Diagnostic Tools, Food Safety, Welfare, monitoring serologically at harvesting age is not Management/Environment, and Top Overall suitable because sero-conversion is still on-going Research Priorities. Secondly, various AAAP task and results often inconclusive. POCKIT Central force committees will be surveyed, to determine provides an efficient way to monitor vaccination research needs in particular fields of poultry effectiveness/uniformity in the early stage with health and medicine (e.g. animal welfare, food direct evidences for vaccine strain replication. 6 batches of 500,000 broiler DOCs were monitored Analytical epidemiology of Spotty Liver Disease from 2018. Tracheal swab samples were Yuanshuo Karen Gao1, Peter Groves2, Mini collected and tested individually with POCKIT Singh3 Central IBV reagent by weeks. S1 gene sequencing was used to confirm the presence of 1Sydney School of Veterinary Science, The the vaccine strain. Generally high positive rate University of Sydney, Australia, 2Sydney School (>80%) were observed on day 7 which indicated of Veterinary Science, Australia,3 Sydney School successful replication of the vaccine strain. of Veterinary Science, Australia Varied positive rate among different suppliers and batches (from 20% to 100%) provided good Since the identification of the causative agent indicator for vaccination quality and vaccination Campylobacter hepaticus, the Australian poultry improvement. industry and researchers have been focussing on the understanding of the pathogenesis of Spotty Mycoplasmosis is common around the world Liver Disease (SLD) and the development of C. which causes down-grade of meat quality, hepaticus vaccines. increases the risk for secondary infection and results in economic losses in broiler industry. It has been found in several field situations that People often use antibiotics for treatment, but presence of C. hepaticus in the animal is not antibiotics resistance now is a problem because always associated with the clinical cases of SLD. of inadequate use. In order to better It has thus been hypothesised that there are management of antibiotics use, POCKITTM potential farm and/or bird risk or protective Central is an effective method for pathogen factor(s) to the onset of SLD, which is the focus monitoring and antibiotic treatment evaluation. of this analytical epidemiology study. Mycoplasmosis monitoring was done by Surveys and observational studies will be used in detection of M. gallisepticum and M. synoviae this study to examine free-range layer farms with specific reagents. 6.67% houses were across different Australian states over a two- mycoplasma positive at day of age and year period. A case-control study in the first year antibiotics were treated immediately. Effective will aim to identify one or more putative risk treatment was found in 75% houses after 1 factor(s) of SLD, using a broad questionnaire will week. Early detection reduced antibiotics use in encompass biosecurity, environment, comparison to treatment in late stage. management, nutrition, bird health and In summary, POCKIT Central allows the physiology factors. This will be followed by a managers to evaluate the vaccination uniformity cohort study in the second year which is and disease monitoring on site and in time which designed to confirm the importance of identified is a health management system to reduce the factor(s) in the development of the disease. At risk of respiratory diseases, improve vaccination, the time of reporting, the first-year study would and cut down the use of antibiotics. have been completed.

The identification of risk/ protective factor(s) of SLD would lead to better understanding of the pathogenesis and control of the disease, improved welfare for the animals, reduced

antibiotic use and potentially improved response to future SLD vaccines. Egg Layer Biosecurity in 2020 – An Assessment Common Misconceptions in Assuming a of Daybreak Foods, Inc. Biosecurity Program Product is a Viable Alternative to an Antibiotic Rosemary A. Marusak1, Caitlin McKenzie2 Greg Mathis

1Daybreak Foods, Inc., 2Daybreak Foods, Inc. Southern Poultry Research, Inc.

The 2015 highly pathogenic avian influenza With the demand for poultry ‘Raised Without outbreak in the upper Midwest had a Antibiotics’ flocks, the number of alternative devastating effect on the egg layer industry, products has greatly increased. This has led to resulting in a loss of approximately 43 M egg the development and use of several products layers and pullets and total national cost such as probiotics, prebiotics, essential oils, expenditures of approximately $950 M. This organic acids, saponins, and tannins. One outbreak led to rigorous company biosecurity question we must consider in determining if the plans, a focus on the new 14 NPIP biosecurity product is a viable alternative; does it provide principles, as well as immediate implementation the same or better disease control as an of demanding on-site biosecurity measures. At antibiotic? Research often demonstrates that Daybreak Foods, Inc., three levels of biosecurity most of the alternatives are not as effective as an - standard, elevated, and critical - were antibiotic. Due to this, poultry producers should established, and accompanying company plans moderate expectations. However, alternatives to address these levels were written and do currently play a major role in maintaining bird implemented. A considerable amount of time health. Unfortunately, not all provide the same and resources have also been invested in benefits. Just as each alternative category varies structural and operational measures and greatly, within each category there can be large procedures, including bio-centers for shower- differences. It would be an error to believe that in/out facilities and truck wash bays, dedicated one product is the same as another. For company clothing, and strict worker site policies. example, there are many types of Probiotics: The lack of an avian influenza introduction feed or water based, non-heat stable or heat suggests these new precautionary policies have stable spore form, single species or mix, one- been successful, to date. time or continuous administration, etc. Even strain variation for one species can be This talk will take a critical look at our company tremendous. Consistency and production of biosecurity program five years post outbreak as Plant based products have a similar issue. Just we begin to evaluate our practices and because a plant extracts claims to be the same procedures from a cost, risk, and compliance product the extracting of active ingredients can standpoint. We will address the following vary from plant source to production facility. questions: What has been effective and what has Like probiotics, dose levels vary with each plant- not? What procedures seem necessary and what based product. Another misconception is that needs revision? Why does non-compliance occur encapsulation processes provide the same and how can we address this? What new, degree of resistance to acid and enzyme emerging biosecurity matters should we start to breakdown. Thus the main misconception is address? that products are the same and will provide similar benefits.

New Features of 14th Edition of Diseases of •The paper copy is published in 2 volumes, thus Poultry reducing the breakage of the binding as seen in prior editions published as single volume David Swayne The electronic version is available in Adobe USDA/ARS/SEPRL Digitals format and has word search function. The first edition of Diseases of Poultry was The electronic version has many more color published in 1943 with 13 additional editions photos than the paper copy. AAAP members will published to date with over 20,000 copies sold receive 25% discount. worldwide. The 14th edition has been extensively revised: Wealth of Knowledge 2

•The beginning of each chapter contains a short Technical and Analytical Considerations concise summary of the most important aspects Performing Retrospective and Prospective of the etiology, clinical features, diagnostic Studies Using Clostridial Dermatitis in Turkeys criteria, and prevention and treatment as a Model strategies Dave Fernandez1, Brian WooMing2 •Provides more clinically relevant information 1FSTAATS, 2Cargill Turkeys on management of specific diseases, contributed by seven clinical poultry veterinarians Investigating diseases with multiple risk factors •Much of the historical information and require study designs that can encompass all the antiquated or historical diagnostic tests were possible variables with the least amount of error. removed, and readers are referred to prior The planning and design of such an investigation editions for in-depth coverage has to be thorough and comprehensive. Retrospective studies allow investigators the •A new subchapter on Disease Prevention and ability to establish possible associations of a Control in Antibiotic-Free Production was added number of risk factors to the outcome. The strength of these associations can be evaluated. •New subchapters were added to Chapter 33: (1) Risk factors with the strongest associations can White Chick Syndrome, (2) Focal Duodenal be tested for causation in prospective studies. Necrosis in Table Egg Layers, (3) Wooden Breast This paper seeks to review both technical and and Other Muscle Abnormalities, and (4) analytical considerations when planning and Idiopathic Egg Production Drops in Brown designing epidemiological investigations based Layers. on an actual, ongoing, field research. •Major revisions were accomplished for Chicken What Happens When You Feed Layer Ration to Infectious Anemia and Circovirus Infections in Chickens From 8 to 14 Weeks of Age. Commercial Flocks, Avian Reovirus Infections, Marek’s Disease, Salmonella Infections, Richard Fulton Mycoplasmosis, and Coccidiosis. Michigan State University •Many figures were updates to color in the electronic version Three (3), reportedly 14-week-old chickens from a flock with birds that were unable to walk were submitted for necropsy. One bird stood with difficulty. One bird was unable to stand, and another bird had valgus angular limb deformity. we developed two TaqMan real-time PCR assays All the birds had atrophy of the breast muscles, targeting unique specific genes only for BA. “S”-shaped keel bones, and collapsed ribs. The These assays showed high level of sensitivity and bones were easily bent. Growth plates of the specificity, that , would allow rapid and accurate proximal tibia and proximal metatarsus were detection of BA directly from clinical specimens thickened grossly. Microscopic examination of and would help in the differential diagnosis of the affected epiphysis revealed elongated avian respiratory diseases. hypertrophic zone of cartilage with normal Comparison of Selected Blood Biochemistry vascular invasion. Alterations were consistent Values in Meat-Type Chickens and Turkeys at with those produced by phosphorus deficiency Critical Ages using the i-STAT and VetScan2 or calcium:phosphorus ratio imbalance. From the case history, birds were fed starter ration Fernando Jimenez1, Maria Correa2, Erika J. until 8 weeks of age at which time they were Gruber3, Rocio Crespo4 switched to an egg layer hen ration. Thus, this 1 was a case of rickets due to a North Carolina State University, College of 2 calcium:phosphorus ratio imbalance. Veterinary Medicine, North Carolina State University, College of Veterinary Medicine, Development And Validation Of Two 3North Carolina State University, College of Diagnostic Real-Time PCR (TaqMan) Assays For Veterinary Medicine, 4North Carolina State The Detection Of Bordetella Avium From University, College of Veterinary Medicine Clinical Samples Clinical pathology is not commonly utilized to Amro Hashish1, Avanti Sinha2, Yuko Sato3, assess the health status or evaluate the effect of Mohamed El-Gazzar4 environmental manipulations or treatments of poultry in the field. Our previous studies showed 1Iowa State University, 2Iowa State University, that most biochemical parameters obtained with Department of Veterinary Diagnostic and handheld analyzers are comparable to the values Production Animal Medicine, 3Iowa State obtained by traditional benchtop analyzers. The University, Department of Veterinary Diagnostic goal of this study was to provide preliminary and Production Animal Medicine, 4Iowa State blood reference values for broilers and meat- University, Department of Veterinary Diagnostic type turkeys, using the i-STAT and VetScan2. We and Production Animal Medicine analyzed a total of 60 blood samples from Each sector of the poultry industry (especially chicken and 60 from turkeys, at 7, 21, and 35 Turkey) in USA and worldwide is challenged by days of age. Using the i-STAT analyzer, we many respiratory pathogens. One of the most measured pH, total CO2, partial pressure CO2 important Pathogens is Bordetella avium (BA). In and O2, bicarbonate, oxygen saturation, glucose 2018, BA was ranked to be the sixth in a list of (GLU), hematocrit (HTC), ionized calcium (iCa), the most threatening health issues for the sodium (Na), and potassium (K) from whole American turkey industry. Interestingly, despite heparinized blood. We used a VetScan VS2 to its major impact and importance, BA laboratory measure aspartate aminotransferase (AST), bile diagnosis still depends on Bacterial isolation and acids (BA), creatine kinase (CK), uric acid (UA), identification. It is noteworthy, that very few total calcium (Ca), phosphorus (P), total protein real-time PCR assays had been developed for the (TP), albumin (ALB), globulin (GLOB), potassium diagnosis of BA. Lack of specificity limits the use (K) and sodium (Na) in whole heparinized blood. of these assays as a diagnostic tool. In our work, All samples were analyzed on-site within 15 minutes of collection. Descriptive statistics and 2. To evaluate current AAAP procedures and graphic representations of the data were used to identify areas of strength and weakness that summarize the values obtained for each species need to be altered/addressed to promote by age. The two most significant differences diversity and inclusion. when compared to other avian reports are 1) 3. To assist in the development of AAAP's higher pH in younger birds (Mean pH= 7.5 for position on diversity and inclusion. both chickens and turkeys, at 7 and 21 days of age), and 2) CK values significantly higher at all 4. To encourage and guide policies to promote ages, particularly in the older group of chickens diversity and inclusion as the demographics of (Mean CK (U/L) = 1104, 4241, and >14,000 in AAAP change. chickens and 1051, 1051, and 1073 in turkeys; at 7, and 35 days of age respectively). While values Comparison of Turkey Biochemical Test Results for gases and plasma ions are published in laying Between a Handheld Blood Analyzer and a and breeder chickens, this is the first study for Conventional Laboratory Blood Analyzer these parameters in growing meat-type chickens Hannah Sather1, Fernando Ruiz Jimenez2, Maria and turkeys. In addition, this is the first report of Correa3, Erika Gruber4 full serum biochemistry values in this type of 1 birds. NC State University College of Veterinary Medicine, 2NC State University College of The Mission and Goals of AAAP’s Diversity and Veterinary Medicine, 3NC State University Inclusion Taskforce College of Veterinary Medicine, 4North Carolina Valerie Marcano State University College of Veterinary Medicine

University of Georgia A reason for the underutilization of clinical pathology in poultry is that rapid and proper The mission of the AAAP’s Diversity and Inclusion sample handling is critical to obtain reliable Taskforce is to foster and embrace a climate measurements. Since many farms are far from a inclusive of all AAAP members regardless of age, diagnostic laboratory, proper sample processing gender, race, sexual orientation, socioeconomic presents a significant challenge to poultry or educational backgrounds to encourage full veterinarians. Therefore, the use of handheld participation in the opportunities the blood analyzers could be a good and cost- organization has to offer. We strive to ensure effective option for on-farm testing. The that those in leadership positions, participating objective of this study was to a) determine the in committees and receiving awards, effect of time on blood chemistry values and b) scholarships, and research support truly reflect evaluate the accuracy of i-STAT and VetScan our membership base. Additionally, the diversity handheld blood analyzers to conventional and inclusion taskforce will maintain a protocol clinical pathology benchtop analyzers. We to adapt as the AAAP membership evolves. collected blood from 60 healthy turkeys. Each sample was tested in triplicate by either a) the Goals: cartridge CD8+ in the Handheld Analyzer i-STAT 1. To collect and provide demographics on the or b) Avian/Reptilian Profile Plus rotor in the AAAP members at large and relay that VetScan2. Blood samples tested with the i-STAT information to the AAAP Board, while following were then analyzed once with a GEM Premier appropriate guidelines for member privacy. 3000 benchtop analyzer, and samples tested with the VetScan2 were analyzed once with the Cobas benchtop analyzer. All samples were activity of SS seems to be comparable to that of analyzed within 60 minutes of collection. aspirin in humans with rheumatoid arthritis. Pearson’s Correlation Coefficient and Mountain The dose of both ASA and SS in poultry is not Plots were used to determine whether there well-established, and has been largely were significant significance between i-STAT extrapolated from other species, which is a risky results. A Bland-Altman assay was used to proposition, as the metabolism of salicylates determine whether results from the handheld varies widely among species, and even among blood analyzers and traditional benchtop different species of birds. The plasma analyzers were comparable. Preliminary results concentration of salicylic acid (SA) above which indicated high (r= 0.7 – 0.89) to very high (r= 0.9 effective antipyretic, anti-inflammatory and – 1.0) correlations in blood electrolytes and analgesic activity can be achieved has been other biochemical parameters, but a poor reported to be 50 µg /mL for several species. correlation for gas analytes. In conclusion, the i- This level was sustained for 8.62 hours after STAT and VetScan2 were reliable and easy to use bolus oral administration of 50 mg/Kg in and had an overall acceptable accuracy in chickens. One would therefore need to give two turkeys. to three bolus doses per day to maintain levels Is Aspirin a Useful Adjunct in the Management about the target of 50 µg/ml, or a total daily dose of Vaccine Reactions and Respiratory Disease of about 100-150 mg/Kg/day. Toxicity was not in Broilers? observed at 100 mg/Kg/day, but was observed at 200 mg/Kg/day given as two oral boluses of 100 John Smith mg/Kg each. Alectryon LLC Oral dosing of 50 mg/Kg SS failed to decrease Salicylates were the first non-steroidal anti- systemic IL-6 levels, had no significant effect on inflammatory drugs (NSAIDs) to be used in any the LPS-induced increase in prostaglandin E2 species. In poultry medicine, aspirin plasma concentration, and did not influence the (acetylsalicylic acid, ASA) and sodium salicylate elevation in body temperature after LPS (SS) have been used for their antipyretic, anti- administration. SS at 100 and 200 mg/kg had an inflammatory, and analgesic properties, effect on temperature in the period from 1 to 5 particularly in the management of vaccine h after LPS administration, whereas at 6 to 8 h reactions and respiratory diseases. ASA is not after LPS administration the effect was reduced. approved by the FDA for use in poultry, including So, it appears that administering about 100-150 in feed. ASA is only poorly soluble in water (3 mg/Kg/d could be relatively safe and may have mg/mL at 25 C (77 F)), and is rapidly hydrolyzed some benefit. However, in an intra-articular to salicylate, so it is not amenable for use in sodium urate crystals model of nociception in drinking water. SS is relatively soluble in water domestic fowl, no analgesic effect was found (1246 mg/mL at 25°C), and preparations are after oral administration of SS at 100 mg/kg, marketed in the US for use in poultry drinking whereas intramuscular (IM) injection at 500 water. Since neither drug is approved by the mg/kg provided analgesia for 1 hour only. FDA, withdrawal times have not been Solutions of 10-12.5% ASA in alcohol (in which established. SS is estimated to have only about the ASA is soluble) are marketed for swine and 60% of the antipyretic potency of aspirin in poultry as unapproved drugs. The humans, on a molar basis. ASA is reputed to be a manufacturer’s instructions call for an initial better analgesic than SS. The anti-inflammatory dose of 4 ounces per gallon of stock solution metered at 1 ounce per gallon of drinking water Evaluation of Factors Correlated with the (1:128) for one day, followed by 1 ounce per Development of Hemorrhagic Hepatopathy in gallon of stock for up to 7 days. The maximum Broiler Breeder Pullets dose of 4 ounces of 12% product per gallon of Linnea Tracy1, Eric Shepard2 stock yields a concentration of 3.75 mg/ml in the stock, which exceeds the solubility of 3 mg/ml at 1University of Georgia Poultry Diagnostic and 25 C (77 F). Disregarding the fact that some of Research Center, 2University of Georgia Poultry the dose will be lost to precipitation, this yields a Diagnostic and Research Center final concentration of about 111 mg/gallon of drinking water when metered at 1:128. If we Hemorrhagic hepatopathy, also known as assume a 5-week-old bird weighing about 1.5 Kg “bacterin-associated hepatopathy”, is an will drink about 0.073 gallon of water a day, this uncommon but severe syndrome of chickens bird will receive roughly 8.1 mg ASA per day, or characterized by the development of increased 5.4 mg/Kg/day, about 1/9th of the suggested mortality and a disseminated intravascular effective dose. coagulation (DIC) -like syndrome in chickens up to 6 weeks post-vaccination. Lesions associated The manufacturer’s instructions for with the syndrome consist of a variable administration of SS recommend 0.8 to 1.6 combination of severe injection site reactions, ounces per 1000 pounds of body weight per day, granulomatous hepatitis and splenitis, visceral which results in 25 to 50 mg/Kg of SS per day, in amyloidosis, intravascular thrombosis, grossly line with suggested doses in the literature. The mottled, darkened, or friable livers, 50 mg/Kg dose is intended as a loading dose for splenomegaly, coagulopathies, and, most one day, followed by 1 ounce per 1000 pounds dramatically, occasional accumulation of or 30/mg/Kg. These doses are closer to the noncoagulated hemorrhagic fluid in the levels recommended in the literature, but may coelomic cavity. While this syndrome has been still be insufficient. A two or three-fold increase qualitatively associated with bacterin in the dose may be necessary to achieve administration in primary breeder technical significant effects, and these doses may be notes and a handful of case reports in table egg approaching toxic levels. layer flocks, the literature lacks information concerning the syndrome and its risk factors in In summary, salicylates may provide some broiler breeders. Nevertheless, personal analgesic, antipyretic, and anti-inflammatory communication with broiler veterinarians effects if given at sufficient doses. It appears suggests that the syndrome occurs at a basal rate that such doses may be approaching toxic levels, in the industry and is often resolved by and considering the questionable efficacy at modifying vaccine protocols in pullets. In this recognized safe doses, the lack of regulatory study, details of reported hemorrhagic approval, and the lack of established withdrawal hepatopathy cases in broiler breeder pullets periods, these products should be used with were collected via veterinarian survey, caution if at all. anonymized, and statistically analyzed to identify risk factors for the syndrome. This evaluation is the first and only quantitative

analysis of the syndrome’s risk factors and the first report of hemorrhagic hepatopathy in broiler breeder pullets in the scientific literature. Results may lead to management changes by integrators to decrease pullet mortality compared to T2=1.21 (p<0.05). The ability of the associated with this condition. phytobiotic to reduce NE symptoms (mortality and lesion scores) resulted in improved gut Posters health and improved feed conversion ratios with the T3=1.563b and the T4=1.455c compared to Bacteriology/Parasitology T2=1.788a, and T1=1.367d (p<0.05). Whole Genome Analysis of Mycoplasma Efficacy of a Hops Extract Phytobiotic gallispeticum Isolates from the United States Administered in the Feed for the Control of Necrotic Enteritis Caused by Clostridium Mohammadreza Ehsan1, Ruth Wooten2, perfringens in Broiler Chickens Marianne Dos Santos3, Naola Ferguson-Noel4 Maria Dashek1, Zsofia Bata2, Viviana Monlar- 1University of Georgia, 2Poultry Diagnostic and Nagy3 Research Center, Athens GA 30605, 3Poultry Diagnostic and Research Center, Athens GA 1BV Science, 2Dr. Bata, 3Dr. Bata 30605, 4Poultry Diagnostic and Research Center, Necrotic Enteritis (NE) is a common sequela to Athens GA 30605 Eimeria infection caused by intestinal dysbiosis Despite attempts to control avian and Clostridium perfringens (Cp) overgrowth. mycoplasmosis through management, With the rise of antibiotic free broiler vaccination, and surveillance, Mycoplasma production, plant extracts are becoming continues to cause significant morbidity, commonly used therapeutics. Study set up was mortality, and economic losses in poultry a randomized complete block design comparing production. Advances have been made in the two doses of a phytobiotic feed additive high throughput sequencing technology, making (Herbanoplex) to a non-medicated feed, to sequencing full genomes more affordable but evaluate its efficacy at relieving the negative unfortunately, the genetic basis for virulence, symptoms of NE caused by Cp. Groups included transmission, host adaption etc., have not been T1: control feed, no Cp challenge; T2: control completely determined for avian Mycoplasma feed, Cp challenge; T3: 750 g phytobiotic/metric spp. The goal of this research was to compare 51 ton, Cp challenge; T4: 1kg phytobiotic/MT, Cp M. gallispeticum isolates from 1990 to 2019 from challenge (n=64). Chicks were orally gavaged the United States in order to identify genome with 5,000 oocysts of E. maxima, followed by differences. The isolates analyzed were from 12 administration of 108 Cp CFU/mL on days 19, 20 states and different production systems and 21 to elicit NE. To assess performance (including broilers, layers, chicken breeders, parameters, feed and bird weights were taken turkeys and turkey breeders). Whole genome on days 0, 14, 21 and 28 of age; and lesion sequencing was performed using Illumina and scoring (scale= 0-3) was performed on d21. Non- the isolates compared to M. gallisepticum Rlow challenged negative control birds had no NE- reference genome and M. gallisepticum live- associated lesions or mortality. Compared to the attenuated vaccine genomes. The collective CP challenged, control fed birds, the phytobiotic contigs for each strain were annotated using reduced mortality by 46.6% at both doses with fully annotated Mycoplasma reference T2 having 23.4% mortality and T3 and T4 groups genomes. The analysis revealed genetic both having 12.5% mortality. The reduced differences among the isolates including mortality is supported by significantly reduced NE lesion scores in T3=0.83 and T4=0.75 presence and absence of genes with potential further spreading and save birds, if possible, by roles in virulence. implementing quarantine procedures, treatments, environment changes, etc. Coccidia dynamics in litter and feces of However, a more challenging aspect of our chickens in a floor pen trial intervention plan is to be able to link the disease Ruediger Hauck1 , Wilmer Pachecco2 event to an infection source with facts. This is problematic, because without facts we can only 1 2 Auburn University, Department of Poultry presume and if the origin isn’t fixed, the Science, Auburn University, Auburn, Al 36849 probability of the problem coming back is high. Counting oocysts in feces or litter is one method This presentation describes an investigation to monitor infection levels with Eimeria spp. in approach to a few Salmonella contaminations chickens in commercial flocks or after identified during the summer of 2019. With time experimental infection. Oocyst counts shed in and resources being limited, being able to focus feces are thought to follow a typical pattern with our efforts is essential. After identifying clear peaks representing infection cycles, while Salmonella, we listed all the possible sources of oocyst counts in litter are representative for a contamination: air, water, feed, supply, human, longer time. The objective of the study was to pest and decontamination process. We based compare oocyst counts in fresh feces and in litter our investigation approach on all those factors to of broilers for 42 days. The birds were vaccinated thoroughly screen each case and used visual against coccidia in the hatchery and were kept in tools to enhance accuracy of our sampling. The pens. Each pen was 150 cm in width, 200 cm in visual tools were also useful to increase the level length and had 25 birds. The birds were fed a of understanding when results were shared with standard three-phase diet without farm personnel. The sampling results showed us anticoccidials. Every two to three days on each locations’ hot spots which allowed us to Mondays, Wednesdays and Fridays, the same focus efforts and enhance biosecurity seven pens were sampled by collecting fresh procedures in these specific areas. Those results feces and litter from at least three sites per pen. in combination with auditing allowed us to Oocysts were counted using a McMaster identify the sources of contamination and chamber. Median oocyst counts were higher in address them accordingly on most locations. We litter samples than in fecal samples. The variance also learnt that timing is paramount. The sooner was lower in litter samples. There were the investigation takes place from the time of significant differences between cages in oocyst identifying the contamination, the higher the counts in feces as well as in litter, and there were chances of findings good answers. no obvious shedding patterns. These results Control of Northern Fowl Mite (Ornithonyssus show that monitoring oocyst counts in feces and sylviarum) on Egg Layer Chickens using a litter is of limited value in floor pen trials. Fluralaner Solution Administered to Birds Salmonella Investigation: Finding the Needle in through Drinking Water the Haystack Faris Jirjis

Genevieve Huard Merck Animal Health

Hendrix Genetics Ltd The northern fowl mite (NFM), Ornithonyssus As veterinarians, we fix problems. After our sylviarum infests a wide variety of domestic fowl diagnostic, we use our resources to prevent and wild birds. These mites are one of the most important and common external parasites of (NARMS) panel including amoxicillin/clavulanic commercial poultry in the United States. Mites acid, ampicillin, cefoxitin, ceftriaxone, ceftiofur, feed on blood, and heavy mite infestations can gentamicin, streptomycin, sulfamethoxazole, irritate and stress poultry, reducing egg and tetracycline. Plasmid replicon analysis production. Fluralaner [(Carbamoyl-Benzamide- detected the presence of a W type replicon. Phenyl-Isoxazoline (CBPI)] is in development by Whole genome sequence analysis using MinION Merck Animal Health as an ectoparasiticide for yielded 57,998 reads generating 146.5 MB of treatment and control of mites in poultry data with average read length of 2,525 bases and (including both northern fowl mite average quality score of 10.14. Preliminary (Ornithonyssus sylviarum) and red mite screening of the sequenced strain using the [Dermanyssus gallinae]). The objective of this comprehensive antibiotic resistance database study was to evaluate the field effectiveness and (CARD) pipeline identified 132 genes associated safety of fluralaner given orally at two single with antimicrobial resistance including genes for doses of 0.5 mg/kg BW, when administered 7 multi-drug efflux pumps, beta-lactamase genes, days apart to control northern fowl mite in tetracycline, spectinomycin, sulfonamides, commercial layers. fosfomycin, kanamycin and others. This strain is Genomic Analysis of Avian Pathogenic of interest as it is a novel APEC due to its Escherichia coli (APEC) Associated with serogroup, possession of a novel plasmid Colibacillosis using Long Read MinION replicon, phylogenetic group and high-level Sequencing. prevalence of antimicrobial resistance by genotype and phenotype analysis. Catherine Logue Identification, serotyping and molecular University of Georgia characterization of Avibacterium Colibacillosis caused by Avian Pathogenic paragallinarum isolates obtained from Escherichia coli (APEC) is a significant cause of outbreaks of infectious coryza in commercial loss to the poultry industry resulting in poultry in the northern area of Mexico, Sonora morbidity, mortality or carcass condemnation at Linda Luna1, Anneke Feberwee2, Rianne Buter3, slaughter. In broilers, the pathogen can cause a Edgardo Soriano Vargas4 range of syndromes including cellulitis, peritonitis, colisepticemia, perihepatitis, 1Universidad Autónoma del estado de México, omphalitis etc. Here, long read MinOIN Facultad de Medicina Veterinaria y Zootecnia, sequence-based technology was used to analyze 2Royal GD, Deventer, the Netherlands, 3Royal the genome of an E. coli strain isolated from the GD, the Netherlands, 4Centro de Investigación y peritoneum of a chicken diagnosed with Estudios Avanzados en Salud Animal, FMVZ, colibacillosis. The strain was identified as a UAEM, México. lactose positive E. coli of phylogenetic group A, Avibacterium paragallinarum (AVP) is the serogroup O:24 and was positive for 5 genes of causative agent of infectious coryza (IC), a severe the minimal predictors multiplex (iroN, aerJ, iss, respiratory disease in chickens. Moreover, AVP ompTp and hlyF), suggesting it is a well- causes egg production losses and increased developed pathogen. culling rates (Blackall and Soriano-Vargas, 2013). Resistance to nine antimicrobials were detected This study describes the investigation of 8 AVP by phenotype analysis using the national isolates obtained from 8 different antimicrobial resistance monitoring system epidemiological unrelated cases of IC in commercial chickens in Sonora and. some farms characteristic whitish-grey spots on the surface had a history of vaccination with trivalent of livers were identified during necropsy and vaccines. liver and bile were collected aseptically.

The isolates were identified by colony growth Campylobacter hepaticus was successfully and biochemical characteristics and isolated and identified from the samples. After conventional PCR (Chen et al., 1998). confirmatory bacteriological tests, whole Additionally classical serotyping (Blackall et al., genome sequencing (WGS) analysis was 1990; Soriano et al., 2004) and molecular performed to identify multiple putative characterization of the partial HPG2 region virulence factors and to characterize the isolates. (Feberwee et al., 2019) was performed. Sequence analysis showed six clusters of the isolates separated by geographical regions. All AVP isolates were NAD dependent, catalase negative, oxidase positive and PCR positive. Five A couple of the isolates were selected for in-vivo isolates belonged to serogroup C, two to virulence evaluation using an animal challenge serogroup A and one to serogroup B. Sequence model and in-vitro virulence evaluation using analysis showed the presence of 3 different LMH cell invasion assays. HPG2 genotypes, with the absence of the single The effect of saponins on the cycling of point mutation at position 1516 (Feberwee et al., commercial coccidiosis vaccines 2019). Luis Gomez These results confirmed the presence of NAD dependent AVP strains with different HPG2 Phibro Animal Health genotypes in the outbreaks of IC in commercial chickens in Sonora. Moreover, the isolates The objective of this floor-pen study is to belonged to different serogroups. There was no determine the effects on performance of a feed correlation between serogroup and genogroup. additive containing two saponins and fed to In previous studies, serovar B-1 was the most coccidia-vaccinated broiler chickens during the prevalent in Sonora (Soriano et al., 2001; 2004), fall in the southeastern United States. The effect now serogroup C seems more relevant. Vaccine- on oocysts per gram (OPG) was measured at challenge studies are important to investigate different intervals with some modulation the effectivity of available vaccines in the control occurring at all stages where there was oocysts l of IC in Sonora. cycling (up to an average of 36%). The relative degree of acquired coccidial immunity was Spotty Liver Disease in the U.S. layers and compared amongst vaccine treatments with no genetic analysis of Campylobacter hepaticus statistical differences between vaccine groups and the groups vaccinated and fed saponins. Ha-Jung Roh There was a statistical significant difference on Ceva Animal Health regards to feed conversion at all stages but specifically at processing age, being better for Campylobacter hepaticus is a causative agent of the saponin-fed group. Spotty Liver Disease (SLD). SLD causes significant health issue and productivity losses in layers but often overlooked.

From 2018-2019 we have received clinical samples from suspected cases of SLD. Its Genetic and Gender influences in a Necrotic between groups. These experiments were Enteritis Challenge Model in Broiler Chickens designed to offer poultry producers and researchers current information on how host Matthew Jones1, Greg Mathis2, Charles Hofacre3 parameters (gender and genetics) influence 1Southern Poultry Research, 2Southern Poultry severity of necrotic enteritis. Research Group, 3Southern Poultry Research Group Case Reports/Pathology

Necrotic enteritis is a multifactorial disease Update on Variant Reovirus Infections and which causes a broad range of mortality and Lameness in Ontario Broilers effects on performance. Infection with Eimeria 1 2 3 sp. paired with exposure to Clostridium Marina Brash , Emily Brouwer , Emily Martin , 4 perfringens is well documented to recreate gross Davor Okjic pathological lesions and mortality, but other 1Animal Health Laboratory, 2Animal Health factors are also reported to influence disease Laboratory, University of Guelph, 3Animal severity, including host factors, environmental Health Laboratory, University of Guelph, conditions, and dietary composition. Genetic 4Animal Health Laboratory, University of Guelp line and gender are host factors which have been implicated in necrotic enteritis pathogenesis. Prior to 2012, reovirus-associated lameness was Males are suspected to be more susceptible due only seen sporadically in Ontario broilers. In to their faster growth rate compared to females. 2012-2013 and 2017-2018, outbreaks of Others have suggested that genetic differences reovirus-associated lameness resulted in also influence resistance or susceptibility to significant animal welfare issues related to pain necrotic enteritis. As genetics are continually and immobility in addition to high economic changing, these observations are likely to change losses due to flock depopulation. Birds were over time. The objective of these studies is to splay-legged and had swollen hocks impairing evaluate susceptibility of broiler gender and their ability to walk normally. Flocks tended to genetics in a controlled necrotic enteritis be of variable age and interestingly, younger challenge. In a pair of 2 x 2 factorial design flocks had more severe clinical signs, higher experiments, each host parameter was mortality, and cull rates. Histologic lesions compared separately. In the first experiment, included non-suppurative epicarditis and genetic line x challenge was assessed. In the tenosynovitis with infiltrates of lymphocytes second experiment, gender was evaluated under and plasma cells with formation of lymphoid the same challenge conditions as the first nodules. Molecular testing identified several experiment. Birds in the challenged group “variant” strains of avian reovirus . Clinical signs received Eimeria oocysts at 14 days and were and severity of histologic lesions were often subsequently inoculated with Clostridium strain dependant, and cull rates ranged from 2- perfringens. To minimize variation in exposure 3% to 50%. In the spring of 2019, autogenous due to differences in feed and water intake, each reovirus vaccination of Ontario broiler breeders bird receiving these pathogens was inoculated was instituted and had a positive impact on by oral gavage. Mortality associated with gross broiler leg health in Ontario. necrotic enteritis lesions was documented within each group. The percent mortality served as the primary metric for the experiments in assessing susceptibility to necrotic enteritis An Unusual Case of IBD in a Commercial Pullet Histopathologic Investigation of Poultry Flock Processing Quality Problems Jessica Higgins1,Eva Wallner-Pendleton2, Linda Frederic Hoerr Michel3, Daral Jackwood4 Veterinary Diagnostic Pathology, LLC 1Kalmbach Feeds, 2Pennsylvania Animal This presentation describes a practical Diagnostic Laboratory System, Animal application of histopathology to define and Diagnostic Laboratory, Penn State University, resolve issues in the quality assurance of 3Food Animal Health Research Program, processed poultry. Department of Veterinary Preventive Medicine, The Ohio State University/OARDC, 4Food Animal Case Study – Can We Accurately Predict Bird Health Research Program, The Ohio State Weights at the Plant? University/OARDC Albert Payne An unusual field case of Infectious Bursal Disease (IBD) was observed in eight week-old caged 1010 Consulting Group pullets. Enlarged, diffusely hemorrhagic bursas As the degree of automation increases in the were observed on gross necropsy examination. broiler processing plant, bird uniformity and This lesion has been frequently described in weight predictability are vital. The dynamic cases of very virulent Infectious Bursal Disease world of poultry sales puts very stringent Virus (vvIBDV). A five-week-old caged pullet requirements on bird size as they compete in the flock, housed in an adjacent building did not market place. The ability of production display hemorrhagic lesions in the bursa. professionals to accurately plan availability and Samples from both flocks were submitted for maximize production efficiency is dependent on further testing, including histopathology, a number of factors. These include but are not serology, virus isolation, molecular diagnostics limited to individual producer profile, disease and challenge studies. Molecular diagnosis status, nutrition program, season of the year, as included RT-PCR and nucleotide sequencing of well as multiple production considerations. the hypervariable region of the VP2 gene (hvVP2). The predicted amino acid sequence of Broiler chickens do not grow in a linear the hvVP2 region indicated the IBDV observed in progression each day. As factors combine with both flocks was identical and was not a vvIBDV. genetic variation, the ability to accurately predict Their sequences were similar to an IBDV from broiler live weights on a consistent basis can Ontario Canada (EF138967). No mortality was prove to be very allusive. The use of simple observed when the virus was orally inoculated averages across the producer profile often into specific-pathogen-free, four-week-old leaves a large number of farms/houses outside pullets. Gross and microscopic lesions in the the acceptable weight range. This narrow bursa tissue were typical of classical IBD. The window may be as small as +/- 0.10 to 0.15 hemorrhagic lesions observed in the original pounds. Given the inherent variation in live field case were not reproduced in the challenged animals, new methods must be used to meet the SPF pullets. ever-important requirement of bird weight accuracy. This case study will review the use of advanced analytics to assist in this production dilemma. Adventures In Seroconversion: Initial Failure Please Keep Your Copper In Pennies, Not My To Respond to Chicken Infectious Anemia Virus Birds; A Field Case Report Of Copper Toxicity In Vaccination 10 Day Old Poults Ian Rubinoff Jolene Tourville1, Dr. Holly Taylor2 Hy-Line International 1Jennie-O Turkey Store, 2Wisconsin Veterinary Diagnostic Laboratory, University of Wisconsin- Chicken Infectious Anemia Virus (CIAV) is a non- Madison enveloped Gyrovirus characterized by marked anemia and immunosuppression due to thymic Case report of three (3) turkey farms atrophy. While all chickens can be infected by experiencing a severe and sudden increase in CIAV, only chicks between 2 and 4 weeks of age mortality. All affected flocks were approximately usually show the clinical signs. Ensuring 100% 10-days old at the time of onset. The initial seroconversion to CIAV in breeder flocks is a clinical presentation of enteritis suggested global standard to provide protection to chicks. Salmonellosis, however poults did not respond Usual vaccination practices around the world to appropriate antibiotic treatment. Further involve one application of live CIAV vaccine via necropsies and diagnostic testing, as well as feed wing-web (WW), intramuscular (IM), or sample results showed a toxic level of copper in subcutaneous (SC) injection between 8 and 15 the feed and the poults. This case report will weeks of age. elaborate on this atypical presentation of copper toxicity, as well as demonstrate the importance In the subject flock, the brown egg breeder hens of communication and teamwork amongst all had 60% seroconversion from wing web CIAV levels of the company. vaccination at 56 days. There was one sister flock of white leghorn parent stock reared on the Histopathologic and Bacteriologic same farm and vaccinated with the same serial Characterization of Femoral Head Separation, of vaccines. This flock seroconverted 100% after Femoral Head Necrosis and Bacterial one wing-web inoculation. The low Chondronecrosis with Osteomyelitis in Broilers seroconversion in the brown PS flock prompted Floyd Wilson1, Craig Wyatt2, Phil Stayer3 an IM injection at 91 days. After the second injection, the seroconversion dropped to 30%. At 1MVRDL, Mississippi State, 2AB Vista, 174 days and 5 vaccinations, the flock was up to 3Sanderson Farms 55% seroconversion. Currently at 52 weeks, the flock has been vaccinated 9 times by 4 methods Histologic and bacteriologic features for groups and 4 different CIAV vaccine strains while of average 31-day-old broilers manifesting with demonstrating titers between 75% and 90% 3 gross categories of femoral head alterations positive. Interestingly, all other vaccine titers were documented. Categories included normal, (bronchitis, Newcastle, avian encephalomyelitis) simple femoral head separation (FHS), are completely normal indicating there is no transitional changes (FHT), and femoral head issue with immunosuppression. necrosis (FHN). Groups having normal femoral heads and “cull-birds” having FHN with gross signs of sepsis (FHN-cull) respectively provided negative and positive control groups. There was

a 9% occurrence of positive bacterial cultures for all birds tested. However, while only a 12% occurrence was seen for the FHS group, and no has been demonstrated previously. positives in the FHT or FHN groups, 29% of the Furthermore, transcriptomic analysis has samples were positive for the FHN-cull group. A revealed different levels of downregulation of 11% total occurrence of femoral bacterial MHC class I antigen and several related genes chondronecrosis with osteomyelitis or simple between two genetically distinct, highly inbred osteomyelitis (BCO-O) was observed. A chicken lines that differ in AIV resistance. In our progressive increase in the group inclusive study, we evaluated antigen presentation occurrence of BCO-O was apparent progressing markers and compared innate and adaptive from normal (0%), FHS (4%), FHN (13%), and components of the immune response to AIV reaching a maximum of 36% in the FHN-cull infection in Fayoumi (more resistant) and group. A 24% overall occurrence and clear Leghorn (less resistant) chickens. relationship between microscopic pathology and Three-week-old Fayoumi and Leghorn chickens gross diagnostic category was also seen for hip were inoculated intranasally and intratracheally synovitis-arthritis. Synovitis was not seen within with 107 50% embryo infectious dose (EID50) of the normal group, was present only at a modest low-pathogenic AIV H6N2. At 4 days post- occurrence within the FHS group, but at a high infection (dpi), clinical signs were recorded, occurrence within the FHT, FHN and the “cull” tracheal swabs were collected for qRT-PCR, and groups. The study underlines the importance of gross pathology was evaluated. Trachea and lung documenting histologic and bacteriologic status were collected for histopathology and cytokine in addition to gross features when conducting mRNA expression, and flow cytometry and studies on femoral head disorder. immunofluorescence were performed to identify MHC class I and II expression. At 7 and Vaccinology/Immunology 14 dpi, tears and serum were collected to Immune responses to avian influenza virus determine local and systemic antibody infection in two genetically distinct, highly responses. inbred chicken lines Fayoumis had significantly lower AIV titers and Emily Aston1, Rodrigo Gallardo2, Huaijun Zhou3 higher AIV-specific serum IgG titers compared to Leghorns. Other samples are currently being 1 2 University of California-Davis, Department of processed. Population Health and Reproduction, School of Veterinary Medicine, University of California- Safety of Fowl Poxvirus-based Vector Vaccines Davis, 3Department of Animal Science, College in Commercial Layers of Agricultural and Environmental Sciences, Jorge Chacón1, Fernando Resende2, Felipe University of California-Davis Pelicioni3, Luiz4 Avian influenza virus (AIV) causes an 1Ceva Animal Health, 2Ceva Animal Health, economically important viral infectious disease Brazil, 3Ceva Animal Health, Brazil, 4Ceva Animal affecting both wild and domestic birds. Infection Health, LATAM results in disease ranging from subclinical infection to respiratory disease and reduced egg Mycoplasma gallisepticum (MG) and Infectious production to severe, systemic disease Laryngotracheitis virus (ILTV) cause severe approaching 100% mortality. MHC class I and II respiratory disease in commercial farms. are involved in antigen presentation following Normally, biosecurity measures are not enough AIV infection, and AIV-induced downregulation to control MG and ILT in multiple-age commercial farms. In addition, early infections vaccines and to knoe if a cmplex antigen demand the establishment of immunity against antibody complex vaccine impacts on this decay these pathogens from the first week of age. Comparison of Serological Response, Then, the safety of Fowl Poxvirus-based vector Persistence and Serological Profile in Progeny vaccines against MG and ILT was tested in one- against Chicken Infectious Anemia Virus in day old commercial layers. Two groups of 70 Three Flocks of Broiler Breeders one-day-old-layers were immunized separately with Fowl Poxvirus-based vector vaccine Bruno Garcia1, Eliana Icochea2, Josue Sanchez3 expressing key immunogenic antigens of MG and 1 2 ILTV. One non-vaccinated group was maintained San Marcos University, School of Veterinary as control. The safety of vaccinated groups was Medicine, Nacional University of San Marcos, 3 evaluated based on clinical aspect, viability, body IDEXX Laboratory weight and microscopic changes. In addition, Chicken Infectious Anemia Virus (CAV) has a seroconversion against MG and ILT was verified worldwide distribution in poultry industry which using commercial indirect ELISA kits (Biocheck is knowledge for its clinical and subclinical and IDvet, respectively). Mortality was not effects. It is knowing the clinical disease has a observed till four weeks post vaccination. low prevalence with current measurements of Vaccinated groups presented body weight control, but the subclinical immunosuppression similar to the non-vaccinated one, with is more complicated for evaluate and control. A uniformity of weight between 83 and 90%. The practical way for monitored the status of histopathological evaluation confirmed the protection is the serology. For this reason, the safety of the Fowl Pox vector vaccines because serological response against (CAV) and the very mild microscopic changes in trachea, liver serological profile in progeny were compared in and spleen were found in the vaccinated groups three flocks of broiler breeders vaccinated with at 10 days post vaccination. Seroconversion a commercial vaccine containing live low- detected at three and four weeks post attenuation strain (CUX-1) applied orally at 14 vaccination demonstrates the vaccine poxvirus weeks old. This study showed that under field replication (until 100% of antibody positivity). conditions with the application of a single live The results of these controlled trials show the vaccine against CAV to broiler breeder flocks was safety of live Poxvirus-based vector vaccines that possible the control of clinical disease but can be used from the first day of age in order to humoral response in the breeders and progeny stimulate early immunity against MG and ILT in was nonuniform, indicating that it did not induce commercial birds. high and uniform levels of antibodies in the Effect of a complex antibody antigen vaccine progeny. Poultry industry professionals on the decay of Infectious bursal disease interested in discussing one of the most used maternal antibodies in broilers programs of control in CAV will find this study very interesting. Alfredo Condemarin

Hipra Peru SAC

Evaluate the decay of maternal gumboro antibodies in broiles with the aim to improve the determination of the age of vaccination with live Evaluation of a Novel Autogenous Bacterin Early Onset of Immunity and Duration of Strain for Controlling Escherichia coli Infections Immunity of A Recombinant HVT-IBD Vaccine in Poultry against Virulent, Variant, and Very Virulent IBD in SPF Birds Karen Grogan Angela Hartman1, Amy Brown2, Megan University of Georgia Bosserd3, Lauren Taylor4 Escherichia coli is an important opportunist 1Zoetis, 2Zoetis, 3Zoetis, 4Zoetis pathogen of poultry, causing significant economic loss from carcass condemnations, air A recombinant HVT-IBD vaccine was developed sacculitis, septicemia, peritonitis, and cellulitis. as a bi-valent vaccine for protection against Avian pathogenic E. coli (APEC) exhibit significant infectious bursal disease (IBD) and Marek’s O serogroup diversity and as protective disease, two highly contagious diseases causing immunity is serogroup specific; vaccination has fatality, condemnations, and been a moving target for this pathogen. The O- immunosuppression in broilers. Data supporting antigen represents the terminal part of the LPS an early onset of immunity against four IBD surface molecule and APEC do possess challenges predominant in different global conserved antigens in the LPS. The LPS core regions and duration of immunity will be structure is not diverse and only two types are presented. SPF chickens were vaccinated with found among APEC. Our central hypothesis is the HVT-IBD vaccine in ovo or subcutaneously at that the core LPS structure will produce cross- hatch and challenged with either classical reactive, protective antibodies. virulent, variant E (Del-E), variant AL-2 IDB challenges at 14 days of age or with a very A culture method was developed that inhibits O- virulent IBD challenge at 12 days of age. Duration antigen production, providing an easy method to of immunity was established by challenging HVT- remove the O-antigen from APEC field isolates. IBD in ovo vaccinated SPF birds at 63 days of age This technique offers the industry the ability to with a classical virulent IBD challenge. The acquire cross-protective bacterins from vaccine showed 96-98% efficacy against classical autogenous strains in a short period of time. virulent IBD moratlity and gross lesions, 58% and Autogenous E. coli bacterins were produced by 78% protection against variant AL-2 and DelE a licensed biologics company for evaluation in a challenges based on bursa to body weight ratios, safety study and a field efficacy study. The safety and 90-92.5% protection against very virulent study was performed to evaluate vaccine IBD mortality, clinical signs and histological reactivity. After the completion of the safety lesion scores. The HVT-IBD vaccine also showed study, the vaccine was evaluated in commercial 100% protection against classical virulent IBD production systems, both layer and broiler mortality and gross lesions following a Day 63 breeder pullets. Pullets were vaccinated during challenge. standard handling periods and followed throughout production to assess post- vaccination reaction and production parameters. Paired pullet/hen houses were used, with the unvaccinated house acting as the control. The presentation will present data from the both the safety and field efficacy study. Development and characterization of novel Duration of immunity of ts-11 Mycoplasma mouse monoclonal antibodies against chicken gallisepticum vaccine chemokine CC motif ligand 4 Chris Morrow Mingmin Lu1, Woo H. Kim2, Hyun S. Lillehoj3 Bioproperties 1Animal Biosciences and Biotechnology It seems to have become conventional wisdom Laboratory, Beltsville Agricultural Research that the duration of immunity from MG ts-11 Center, Agricultural Research Service, U.S. vaccination is forty weeks. As far as I can Department of Agriculture, Beltsville, MD determine there is no studies to show this. The 20705, USA, 2Animal Biosciences and original duration of immunity experiments look Biotechnology Laboratory, USA, 3Animal at immunity for upto 40 weeks post vaccination Biosciences and Biotechnology Laboratory, USA but there was no end points demonstrated. Chemokine (C-C motif) ligand (CCL) 4 is a CC Often serology from vaccination increases chemokine subfamily member defined by the around 40 weeks of age but wild strains or sequential positioning of conserved cysteine problems are often not demonstrated. residues. Upon the binding of G-protein-coupled Revaccination with F strain has been advocated receptors on the cell surface, CCL4 mediates a but is it necessary? Antibiotic salesmen in some diverse set of biological processes including countries are reluctant to see sales disappear. chemotaxis, tumorigenesis, homeostasis and Certainly some studies have shown protection to thymopoiesis. Although the physiological roles 57 weeks (Barbour et al 2000) and the ability to of mammalian CCL4s were elucidated > 20 years detect ts-11 populations increasing after 40 ago, there is limited information on the weeks in many flocks. biological activities of chicken CCL4 (chCCL4). In Experience in the field using molecular detection the present study, we developed and does not support a 40 duration of immunity but characterized mouse monoclonal antibodies rather field strains are excluded if ts-11 is (mAbs) against chCCL4 to characterize better the present. immunological properties of chCCL4. Out of initial screening of > 400 clones, two mAbs Compatibility of Procerta HVT-ND Vaccine with detecting chCCL4, 1A12 and 15D9, were Magniplex to Provide Protection Against identified and characterized using western Velogenic NDV and Virulent Classical IBDV blotting and chCCL4-specific antigen-capture Challenge in SPF Birds ELISA, and their neutralizing activity was 4 2 validated by chCCL4-induced peripheral blood Sing Rong , Kelly Turner-Alston , Megan 3 4 mononuclear cell chemotaxis assay. Bosserd , Lauren Taylor Furthermore, the intracellular expression of 1Zoetis VMRD, 2Zoetis VMRD, 3Zoetis VMRD, chCCL4 in various chicken cells by 4Zoetis VMRD immunocytochemistry and flow cytometry was confirmed using 1A12 and 15D9 mAbs. These Due to vaccination routines in the field, there is results collectively indicate that 1A12 and 15D9 a strong desire of administering HVT-ND vaccine mAbs specifically detect chicken CCL4 and they and immune complex IBD vaccine (Magniplex) at will be valuable immune reagents for basic and the same time. Studies were conducted to applied studies in avian immunology. determine the compatibility of HVT-ND and Magniplex when administered together, followed by either an infectious bursal disease HVT parental virus. Efficacy was demonstrated virus (IBDV) or Newcastle disease virus (NDV) after either subcutaneous or in ovo vaccination, challenge. Excellent IBDV and NDV efficacy were against virulent MD, IBD and ILT challenges. The observed. unique vHVT317-IBD-ILT vector vaccine provides an additional tool for poultry veterinarians, Development of a New Trivalent HVT-vectored allowing for control of MD, IBD and ILT with a Vaccine Against Marek’s Disease, Infectious single vaccination, and providing flexibility when Bursal Disease and Infectious Laryngotracheitis designing the optimal vaccination program Mariana Sa e Silva2, Troy Hughes2, Teshome adapted to their specific epidemiological Mebatsion3, Hallie King4 situation

1Boehringer Ingelheim, 2Boehringer Ingelheim Reverse genetic infectious bursal disease virus Animal Health USA Inc., Gainesville, GA, USA, in turkeys 3Boehringer Ingelheim Animal Health USA Inc., Ching Ching Wu Athens, USA, 4Boehringer Ingelheim Animal Health USA Inc., USA School of Veterianry Medicine, National Taiwan University, Taipei, Taiwan Infectious Laryngotracheitis (ILT), Infectious bursal disease (IBD) and Marek’s disease (MD) Infectious bursal disease virus (IBDV) can infect are important diseases in commercial poultry, turkeys and induce neutralizing antibodies but currently controlled with vaccination. This not causes clinical disease and bursal atrophy. control routinely includes the use of The purpose of the present study was to recombinant HVT vaccines, and the use of this determine whether reverse genetic (RG) IBDV vector technology has proven to be an effective could potentially serve as a viral vector for tool for several diseases of poultry, with a well- delivering the genes of interest in turkeys. One- known safety profile, hatchery application and day-old turkey poults were orally inoculated long-term protection. However, due to with 2x107 egg infectious dose (EID50) of wild- interference, administering two or more HVT type variant E (VE) IBDV, 2x107 tissue culture vaccines together creates compatibility issues infectious dose (TCID50) of reverse genetic VE- when designing a vaccination program. Aiming IBDV (RG-IBDV), or 2x105.6 TCID50 of IBDV VP3- to provide a solution to control MD, ILT and IBD deleted RG-IBDV fused with green fluorescence with a single vaccine, a new and unique protein (GFP) (GFP-RG-IBDV). Turkey poults recombinant vHVT317-IBD-ILT construct has were observed for 21 days and clinical signs and been developed. This dual-insert construct has a lesions were recorded. Tissue (thymus, spleen, vHVT013 (VAXXITEK HVT+IBD) backbone with liver, ileum, bursa of Fabricius, and cecal tonsil) one additional insert expressing a glycoprotein D were collected at 1, 3, 10, and 21 days post (gD) gene from ILTV. Both IBDV and ILTV genes inoculation (DPI) for histopathology and are expressed from a single promoter. The immunofluorescence antibody (IFA) assay. Sera vHVT317-IBD-ILT construct was shown to be safe were obtained at 3, and 21 DPI for virus for chickens, non-target avian species and neutralization (VN) tests. Clinical signs and mammalian species. The construct was also grossly visible bursal atrophy were not seen in genetically stable after five in vitro passages, and any of turkey poults in each group. it did not revert to virulence after five back Histopathologic examination of various tissues passages in chickens. The construct had similar did not reveal any microscopic alterations, tissue tropism and environmental safety as the including bursal atrophy, in the turkey poults from each group. The highest percentage of investigated. The study was conducted in two IBDV antigen in the bursa detected by IFA from farms using IC vaccine (W2512 strain) and HVT- each group was 100% for VE-IBDV group at 10 IBD vector vaccine (14 flocks); and IC vaccine DPI, 60% for RG-IBDV group at 3 DPI, and 50% for (W2512 strain) and HVT-IBD-ND vector vaccine GFP-RG-IBDV at 10 DPI. Varying degree of green (22 flocks), respectively. Bursa size and weight, fluorescence for IBDV in bursal cells was microscopic changes, seroconversion, and presented in the bursae of turkey poults in each molecular detection and characterization of group until 21 DPI. The highest VN titer obtained IBDV were used to evaluate vaccine take and from the sera of turkey poults in each group was field virus infection whereas feed conversion, 1/16384 for RG-IBDV group at 10 DPI, 1/1024 for daily body weight gain, mortality and VE-IBDV group at 10 DPI, and 1/16384 for GFP- slaughterhouse condemnations were calculated RG-IBDV group at 10 DPI. The results indicated and compared to evaluate protective reverse genetic IBDV caused infection to turkeys performance of the vaccination programs. 92% without significant clinical signs and lesions and of flocks receiving HVT-IBD vector vaccines elicited neutralizing antibody to IBDV, suggesting showed evidence of field virus infection and that reverse genetic IBDV could potentially serve replication in bursa according to the as a viral vector for delivering the genes of macroscopic and laboratory evaluation (low interest in turkeys. bursa weight and size, lymphoid depletion and high seroconversion). The IBD field viruses were Virology: Non-Respiratory not able to cause clinical disease but they affected sub-clinically the productivity because Viruses the lower productive performance in the farms and the lower carcass yield in the Vaccine Take Monitoring in Broilers Flocks slaughterhouse compared to flocks immunized Immunized against Gumboro Disease with with IC vaccine. This work shows the relevance Immune Complex and Vector Vaccines and its of the vaccine take monitoring that is related to Relationship with Productivity productivity of the flocks. Jorge Chacón1, Aline Sousa2, Graziela Santos3, Luiz Sesti4 Update on IBD Viruses (IBDVs) Infecting Broilers Today and How Inactivated Vaccines 1Ceva Animal Health, 2Ceva Animal Health, Protect Against Different Viruses in the AL2 Brazil, 3Ceva Animal Health, Brazil, 4Ceva Animal and T1 Families Health, LATAM Kalen Cookson1, Manuel Da Costa2, John Immune complex (IC) and HVT-based vector Dickson3, Andrew Barker4 vaccines are used extensively worldwide to 1Zoetis, 2Zoetis, 3Zoetis, 4Zoetis protect farms broilers against infection by Infectious Bursal Disease (IBD) viruses (IBDV). This presentation will summarize the findings These vaccines can be applied in ovo or at day and trends in IBDV isolations from broiler flocks one of age in the hatchery since they are not over the last several years (2014-19). In addition, affected by Maternal Derives Antibodies. In results will be compared to a similar survey order to evaluate the performance of IC and conducted between 2009-11 (Western Poultry HVT-IBD vector vaccines in broiler farms and Disease Conference, 2012) to see how the field under field conditions, vaccine take and challenge picture has changed over the past productivity of 36 broiler flocks were decade. Compared to the 2009-11 survey, AL2 viruses remain the most prevalent type—making monitoring provides a good indicator for up over half of all field virus isolations. T1 viruses vaccination effectiveness/uniformity and re- were again the second most common type boost vaccination might be required as early as recovered. New-types saw a big jump from 2% to possible if the vaccination 15%, perhaps at the expense of Del-E types, effectiveness/uniformity is poor. which fell from 21% to 9%. Finally, two bird On-Side Fully Automated POCKIT Central PCR studies will be presented that compare system is suitable for timely detection of MD protection levels of inactivated vaccines against vaccination effectiveness/uniformity. In this prototypes AL2 and T1, respectively, and their study, spleen and feather pulp were collected for related subtypes. The results will show that AL2 evaluation from day of age, 3, 7, 10, 14, and 21. protection levels varied by commercial vaccines The MD positive rate was up to 100% at day 7 and their antigenic makeup. The T1 results are both in spleen and feather pulp. Comparing the pending but will be presented at the meeting. Ct value between 2 sample types from the same A Serologic Survey for Astrovirus Antibody in chicken showed the MD titer in spleen was Georgia Broilers higher than in feather pulp. Most sample got a Ct value >31, which indicates < 100 copies/reaction James Davis and a high sensitivity method is needed for Georgia Poultry Laboratory Network evaluation.

Astroviruses have been associated with poor In summary, POCKIT Central allows the farmers growth performance in broilers. Our survey was to evaluate MD vaccination conducted to see how prevalent Astrovirus effectiveness/uniformity on-site with simple infections were in broilers. steps and gets the results in 85 minutes. Both spleen and feather pulp are ideal sample type, Post Marek’s Disease Vaccination Monitoring but feather pulp is even better because of less with Feather Pulp and Spleen in invasiveness. Re-boost vaccination can be (Layer/Breeder) by On-Side Fully Automated applied as early as possible if the vaccination POCKIT Central PCR system effectiveness/uniformity is poor. 1 Fu Choong Keat , Wei-Fen Tsai, Ping-Han Chung, Pathogenicity of infectious bursal disease Chuan-Fu Tsai, Nina Chen, and Simon Chung viruses isolated from commercial poultry with 1Aviagen Inc., Huntsville, AL, USA, 2GeneReach immune suppression related disease. Biotechnology Corp., Taichung City, Taiwan Daral J Jackwood1, Linda O. Michel2, Michelle L. 3 Marek’s Disease (MD) causes serious economic Kimber problems especially to the long-living birds 1The Ohio State University/OARDC, 2The Ohio (layers and breeders) and vaccination with State University/OARDC, 3Larad Vaccines CVI988 (Rispens) is considered the “gold standard” for prevention. MD vaccination is Infectious bursal disease virus (IBDV) can be applied by subcutaneous route at day of age at isolated from most commercial broiler and pullet the hatchery. Inappropriate flocks in the US once anti-IBDV maternal storage/reconstitution and incorrect antibodies wane. Genetic characterization of administration of vaccine lead to vaccination these viruses is typically conducted using RT-PCR failure, which results in sub-optimal vaccinal followed by sequence analysis of the VP2 gene. protection of flocks. Post MD vaccination There are specific amino acids in VP2 (253Q and 284A) that have been linked to virulence and the association with virulence in 8 genes UL22, UL36, VP1 polymerase also plays a role in the severity UL37, UL41, UL43, R-LORF8, R-LORF7, and ICP4. of disease caused by very virulent (vv)IBDV but a Using the BAC containing the genome of very complete understanding of the genetic basis for virulent plus MDV strain 686, specific base pair pathogenicity has yet to be realized. Testing changes were introduced in loss-of-function isolates of IBDV for pathogenicity can be done in experiments to investigate the effects of these susceptible specific-pathogen-free (SPF) chicks SNPs in the MDV virulence. Nine SNPs in eight but there are some variables that are difficult to genes within a single BAC genome were control in these studies. In this experiment, we introduced and verified by targeted sequencing. examined the pathogenicity of 7 new IBDV The pathogenicity of the reconstituted virus isolates in SPF pullets and compared them to the from the modified 686-BAC recombinants and 1/chicken/USA/T1/2001 (G2) isolate that of the 686-BAC-derived parental virus will (AF281238.1) from Georgia, USA. Clinical signs be discussed. of disease, bursa/body weight ratios and Genomics and pathogenicity of an Indiana histopathology of the bursa were used to assess turkey coronavirus isolate pathogenicity. The results indicated that all 7 isolates were pathogenic in SPF chicks. This Tsang Long Lin study highlights the problematic issues and Purdue University, CVM variables related to comparing virulence among IBDV isolates. Turkey coronavirus (TCoV) causes atrophic enteritis and uneven growth in the turkey flocks. Investigation of the virulence of Marek's A TCoV isolate was isolated from a turkey flock disease virus with virulence-associated gene modification without significant clinical signs in Indiana (IN) in 2009. The purpose of the present study was to Taejoong Kim analyze and compare the 3’-end genomic sequences of TCoV isolated in IN in 2009 (IN- USDA-ARS-USNPRC 287/09) and in 1994 (IN-540/94) and to Marek’s disease virus (MDV) has evolved toward determine the pathogenicity of TCoV isolate IN- greater levels of virulence, and three virulent 287/09. The 3’-end 7.4kb genome of IN-287/09 pathotypes, virulent, very virulent, and very was sequenced. The two IN TCoV isolates from virulent plus, are currently recognized. Since no different time periods shared the same 3’-end current vaccines provide sterilizing immunity genomic organization. The nucleotide identity of and the implementation of different vaccines 3’-end 7.4kb genomic sequences was 95.1%, parallels the evolution of the higher virulent 92.5%, and 93.5% between IN-287/09 and IN- pathotypes, the specific mutations associated 540/94, MN-ATCC/76 (TCoV isolated in with these changes has been the subject of Minnesota in 1976) and IN-540/94, and MN- comparative genomic studies. These studies ATCC/76 and IN-287/09, respectively. The spike have mainly focus on the genes within the repeat (S) genes of two IN TCoV isolates shared the long region of the MDV genome and its role in highest nucleotide and deduced amino acid the evolution of MDV toward greater virulence. homology. IN-287/09 and IN-540/94 belonged to We recently characterized 70 MDV genomes the same serotype based on the cross- with known virulence by full-genome or targeted neutralization assay. One-week old turkey poults region DNA sequencing and identified single challenged orally with 105 EID50/ml/turkey of nucleotide polymorphisms (SNPs) that showed IN-287/09 did not have watery diarrhea and other gross lesions. Body weight gain in IN- address this question, we isolated CAstVs from 287/09-infected turkey poults was not liver and intestinal samples originated from significantly different (p>0.05) from that in the affected chicks showing clinical signs. Then, the negative control. The jejunum and ileum of extracted nucleic acids were subjected to NGS. IN287/09-infected turkeys only had mild The whole genome sequences obtained in segmental multifocal villous blunting and Canada were compared to available whole irregularity at 3 and 7dpi. The enterocytes lining genome sequences available in public domain the jejunum and ileum of turkey poults and results will be discussed in detail at the challenged with IN287/09 were positive for TCoV conference. The outcomes of the present project by immunofluorescent antibody assay (IFA) at will help clarify the molecular nature of field 3dpi only. In conclusion, the TCoV isolates IN- CAstV-challenge in Western Canada facilitating 287/09 and IN-540/94 shared high genetic sustainable control measures. homology in the 3’-end genome and had high Molecular and pathological characterization of degree of antigenic relatedness. The TCoV avian reovirus from clinical cases of isolate IN-287/09 did not cause significant tenosynovitis in poultry flocks from Costa Rica clinical signs and severe intestinal pathology. María Piche-Ovares1, Alejandro Alfaro-Alarcón2, Next Generation Sequencing (NGS) of chicken Aida Chaves-Hérnandez3, Carlos Jiménez- astrovirus (CAstV) isolated from outbreaks of Sánchez4 White Chicken Syndrome (WCS) in Western Canada 1Universidad Nacional, 2Department of Veterinary Pathology, Faculty of Veterinary Victor Palomino-Tapia1, Darko Mitevski2, Tom Medicine, Universidad Nacional, Costa Rica., Inglis3, Mohamed Faizal Abdul-Careem4 3Department of Poultry Medicine, Costa Rica, 1University of Calgary, 2Poultry Health Services, 4Department of Virology, Costa Rica. Airdrie, AB T4A2G8, Canada, 3The Institute of Avian reovirus (ARV) is the mayor cause of viral Applied Poultry Technologies, AB T4A2G1, arthritis/tenosynovitis in young broiler chickens. Canada, 4Department of Ecosystem and Public It´s characterized by swelling of the foot pad and Health, Faculty of Veterinary Medicine, the hock joint, which leads to lameness, causing University of Calgary, Calgary, AB T2N4N1, an economic impact in the poultry industry. Canada ARV belongs to the Orthoreovirus genus in the In the last 5 years, a condition known as White family Reoviridae. The Sigma C protein is Chicken Syndrome (WCS), has emerged as an expressed by the S1 gene, which is the most economically important disease across Western variable. This gen is used as a genetic marker to Canada. The etiological agent of WCS is chicken be characterized and classify reovirus isolates astrovirus (CAstV). The affected flocks had a into different genotype. history of drop of production in the breeder flock at the time of lay, very poor hatchability of eggs During an eleven months period 32 samples of and poor viability of hatched chicks. These tendon and joint tissues of broiler chickens with affected chicks are very white in colour with lameness, tendon swelling, and inflammation green livers. The present study was directed to were analyzed by RT-PCR for the S1 segment answer one research question: 1) What is the using published P1/P4 primers. A wide variety of genetic diversity of the CAstV obtained from ages were affected ranging from 1 to 42 days. outbreaks of WCS in Western Canada? To Five samples were sequenced by Sanger All birds came from the same breeders and the technique. A phylogenetic analysis was level of maternal antibodies was alike. performed on partial segment of S1. Neighbor- At 1 and 2 weeks of age groups A and B reported joining tree were generated, and robustness was a similar drop in serological titers, but group C tested with 1000 bootstrap replicates. All had significantly higher titers (p<0.01). At 3 analyses were carried out using MEGA 7. Four of weeks group A had lower titers with some them were classified as genotype 5 and one as negative samples in all farms, group B and C had genotype 2. similar titers. At 4 weeks group A had lower titers Histopathological analysis of tendons and than groups B and C, but titers were rising, at 5, synovial bands showed synovial epithelial 6 and 7 weeks all groups had similar titers. hyperplasia and an inflammatory infiltrate The groups vaccinated with rHVT-ND-IBD never composed of heterophils, lymphocytes, plasma had negative samples, whereas the group cells and histiocytes with foci of fibrin. A fibrino- vaccinated with traditional live attenuated heterophilic tenosynovitis with different degrees vaccine had a period of susceptibility to IBD. of severity was diagnosed in all cases. Additionally, lymphoid depletion was found in The group vaccinated with full dose had an the spleen. earlier onset of immunity compared to half dose, this is very important for groups with weak The aim of the study was to characterize the maternal immunity, and can lead to vaccination lesions and to detect/genotype ARVs from an failure in birds vaccinated with partial dose. outbreak of tenosynovitis in broiler poultry flocks in Costa Rica. These data discourage the use of reduced doses of the rHVT-ND-IBD vaccine. Serological Comparison Between Different Vaccination Programs against IBD in the Field Virology: Respiratory Elisa Russo Viruses MSD Animal Health Srl Unfavorable Consequences of Lung Infectious bursal disease (IBD) is one of the most Macrophage Recruitment Following Infectious widespread immunosuppressive poultry Bronchitis Virus (IBV) Infection pathogens. In broilers IBD is controlled through 1 vaccination, with live attenuated vaccines or Mohamed Faizal Abdul-Careem , Aruna 2 3 turkey herpesvirus construct vaccines expressing Amarasinghe , Amber Marie Kameka IBDV VP2 protein. 1University of Calgary, 2University of Calgary, 3 Among a broiler integration serological University of Calgary monitoring program (weekly blood samples) we Infectious bronchitis virus (IBV) infection in compared serological titers of 3 farms chickens is important economically for layer and vaccinated with a traditional live attenuated broiler chicken industries. Macrophages play an vaccine at 21 days (according to Deventer essential role in innate immune responses. The formula, group A), 5 farms vaccinated with rHVT- information is scarce on macrophage ND-IBD half dose (group B) and 5 vaccinated with recruitment to the respiratory tissues. The rHVT-ND-IBD full dose (group C). interaction between macrophages and IBV and the mechanisms which lead to virus clearance and disease pathogenesis are also poorly Comparison of the protection and viral understood. Our main hypothesis was that IBV elimination of a vaccination program against infection will result in increases in macrophage Newcastle disease virus using homologous numbers in respiratory tissues and the recruited vaccines of genotype XII and a conventional macrophages become targets for IBV replication vaccination program in broilers. with negative consequences. In this study, first, Nelly Cribillero1, Eliana Icochea2, Rosa we investigated the ability of IBV to recruit González3, Manolo Fernández4 macrophages in trachea and lungs of chickens. Second, we investigated whether IBV establish 1National University of San Marcos – UNMSM, productive infection in macrophages recruited 2National University of San Marcos, 3National to lungs and trachea in vivo. Then we confirmed University of San Marcos, 4FARVET SAC our data in an in vitro system. When chickens were infected with IBV at 6 days of age, we found Newcastle disease (EN) in Peru is detected in that IBV infection increase macrophage numbers poultry flocks despite of vaccination and in both trachea and lungs as determined by flow biosecurity. Conventional vaccines protect cytometry assay. Using a double against mortality and clinical signs, but do not immunofluorescent technique, we recorded that prevent or decrease the elimination of viruses. In IBV establish productive infection in lung and Peru, the majority of ND outbreaks are caused by tracheal macrophages. This in vivo observation strains of genotype XII. This paper aims to was substantiated by demonstrating IBV compare the protection and viral elimination of antigens and viral particles in macrophages a live and inactivated attenuated homologous following in vitro IBV infection. Evaluation of the vaccine program based on genotype XII and a functions of macrophages following IBV conventional program of live and inactivated infection of macrophages revealed that the genotype II heterologous vaccines, La Sota production of antimicrobial molecule, nitric strain. Percentage of mortality, oral and cloacal oxide is inhibited. Further studies are underway viral elimination, antibody response by HI and to uncover the significance of macrophage clinical signs were evaluated. The statistical infection of IBV in the pathogenesis of IBV analysis was performed with logistic regression infection. analysis, Kruskal Wallis test and Fisher's exact test, with a significance level of 5%. On the 1st Funding acknowledgement: Natural Sciences day of age, two groups of birds were vaccinated and Engineering Council of Canada. with live and inactivated La Sota and homologue of GXII, respectively. A control group was not

vaccinated. The challenge was performed at day 30 of age with a virulent VEN genotype XII. Groups with the homologous vaccine

suppressed or reduced the viral elimination of the challenge virus. Protection against mortality and clinical signs was more than 94% in both groups. However, the levels of antibodies specific for the homologous vaccine were not increased. These results demonstrate that homologous vaccines are capable of controlling the elimination of virulent virus, in a homologous challenge, to the environment. Molecular Characterization of Newcastle Evaluation of the impact of the temperature of Disease Viruses Isolated from Chickens in water used for the reconstitution of Infectious Tanzania Bronchitis (IB) live attenuated vaccines Ana Paula Da Silva1, Amandus Muhairwa2, Andrea Delvecchio1, Steve Crussard2 Huaijun Zho3, Rodrigo Alejandro Gallardo4 1Boehringer Ingelheim, 2Boehringer Ingelheim 1California Animal Health and Food Safety Infectious Bronchitis is one of the major diseases Laboratory - Turlock branch, 2Department of in the poultry industry at global level. Veterinary Medicine and Public Health, Sokoine Vaccination is widely used in order to protect the University of Agriculture, Morogoro, Tanzania, chickens from clinical disease and reduce 3Department of Animal Science, University of economic losses due to primary or secondary California, Davis, Davis CA, USA, 4Department of infections. In recent years, the temperature of Population Health and Reproduction, School of water used for the IB vaccines reconstitution has Veterinary Medicine, USA been questioned and temperatures superior to Newcastle disease is a highly transmissible 20°C have been correlated with a consequent disease that affects many avian species, and significant loss of IB vaccine titres. The including domestic poultry. Infections with objective of our trials was to evaluate, under velogenic Newcastle disease virus (vNDV) have experimental conditions, the impact that water been reported worldwide. In Africa, vNDV is temperature could have on the Boehringer endemic and a major burden in poultry Ingelheim IB live vaccines. production. To investigate NDV genotypes In the present trial two commercial IB vaccines circulating in Tanzania, the goal of this study was belonging to different genotype were tested: to molecularly characterize NDV isolates H120 strain (Mass-type vaccine) and CR88 strain originated from village indigenous flocks and (793B-type vaccine). Each vaccine was chickens obtained at live bird markets. reconstituted with drinking water at different Twenty-four vNDV strains were isolated temperature from 5 up to 30° C and the titers of between 2011 and 2017. A RT-PCR reaction was the vaccine solutions were measured on Chicken performed to amplify a 788-bp fragment that Kidney Cells (CKC) immediately after the included the 3’ end of the M gene (173 bp) and reconstitution. Furthermore, the stability of the the 5’ end of the F gene, which includes the F vaccine solution titers for each vaccine were gene hypervariable region (615 bp). Sequencing evaluated up to 120 minutes after the was performed on a MinION. This third- reconstitution on CKC. generation sequencing method allowed us to The results showed that vaccine titers over time amplify the F gene hypervariable region of all were comparable and at the expected level isolates. whatever the vaccine used and the Phylogenetic analysis of the partial F gene temperatures tested: the temperature of water sequences showed that, out of the 24 isolates up to 30°C had a no significant impact on IB sequenced, 15 clustered with sequences from vaccine titers after reconstitution and its stability genotype XIII, 8 with genotype V and one with during at most 2 hours. However, it is genotype VII. These results will be further recommended to use any vaccine in a period of discussed. time as short as possible after reconstitution as expected by best practises. Live Attenuated Vaccine-like Viruses are Infectious Bronchitis Surveillance via RT-PCR Involved in the Evolution of the Canadian Jose Linares1, HaJung Roh2, Matilde Alfonso3, Delmarva (DMV/1639) Infectious Bronchitis Kelli Jones4 Virus 1Ceva Animal Health, 2Ceva Animal Health, Mohamed S. H. Hassan 3Ceva Animal Health, Ceva Animal Health University of Calgary Animal disease surveillance is the systematic Infectious bronchitis (IB) results in significant collection, management and analysis of health economic losses in the global poultry industry. status data. It serves as a tool to monitor trends The causative agent, infectious bronchitis virus and perform a risk analysis. (IBV), shows marked genetic diversity due to A wide range of infectious bronchitis virus (IBV) mutations and genomic recombination. Control variants circulate globally and understanding of IB relies on vaccination. The impact of genetic their geographical prevalence is a key factor for diversity of the IBV is failure of vaccination. Live successful prevention. While prevalence data attenuated vaccines belong to two serotypes, obtained from clinical cases submitted to Massachusetts (Mass) and Connecticut (Conn), diagnostic laboratories is useful, additional are commercially available in Canada. In the past active monitoring for circulating IBV in non- few years, a number of US variant-like IBVs, clinical flocks can provide a baseline status to predominantly DMV/1639 strain, have been compare. increasingly isolated from IB vaccinated broiler and layer flocks in Eastern Canada. We received During the last 3 years, we have performed five archived clinical samples that had been surveillance to monitor circulating IBV in sick and genotyped based on partial S1 gene sequences healthy broilers and layers, using highly sensitive as DMV/1639 variants for further virus isolation real-time PCR genotyping assays. The and detailed molecular characterization. In order information generated has been helpful to to understand the evolutionary origin of the document the spread of certain IBV strains such Canadian DMV/1639 isolates, we examined the as DMV1639 and GA13 into new areas and whole genome sequences from the five IBVs evaluate the effectiveness of vaccination retrieved after serial passages in specific programs. Active surveillance also has the pathogen-free (SPF) chicken embryonated eggs. potential to help detect novel strains, which is Phylogenetic analyses, nucleotide sequence critically important for rapidly changing viruses identities, and SimPlot analyses confirmed that such as IBV. recombination events involving a Conn vaccine- like strain, a 4/91 vaccine-like strain, and one Examples and data from our IBV surveillance will strain that is yet-unidentified have led to the be presented. emergence of the Canadian DMV/1639 isolates. We are in the process of studying the pathogenesis of Canadian DMV/1639 variants both in young and adult laying chickens and optimizing a vaccination strategy to control IB caused by this IBV variant.

Highly pathogenic avian influenza H5N8 virus the virus and discovered a novel presentation of infection: pathological presentation of game diphtheric plaques on the oropharyngeal bird cases during the 2016/2017 epizootic in mucosa, which was associated with a Britain. nercrotising stomatitis and viral tropism for the oropharyngeal mucosa and salivary glands. This Alejandro Núñez1, Christopher Poulos2, Rowena presentation aims to increase awareness of the Hansen3 potential pathological presentations of HPAIV in 1Pathology Department, Animal and Plant pheasants for future disease recognition in the Health Agency (APHA-Weybridge) UK, field. Improved detection could help prevent the 2Pathology Department Animal and Plant distribution of virus between farms via the Health Agency (APHA-Lasswade) UK, 3Avian movement of birds and mitigate the substantial Virology & Mammalian Influenza Research economic impacts for farmers, the wider poultry (APHA-Weybridge) UK industry and trade.

Thirteen outbreaks of high pathogenic avian The Effect of Eurasian Lineage H9N2 Low influenza virus (HPAIV) H5N8 were detected in Pathogenicity Avian Influenza Virus on Chicken poultry in England and Wales from December Egg Production and Contamination of Eggs 2016 to June 2017. Amongst the outbreaks in with Virus England were a cluster of three infected game Erica Spackman1, Mary J. Pantin-Jackwood2, bird rearing farms. The initial infection of Christopher B. Stephens3 pheasants on one of these farms most likely came from the large populations of wild birds in 1SEPRL-USDA-ARS, 2US National Poultry the surrounding area, especially migratory Research Center, USDA-Agricultural Research waterfowl. Following serological and virological Service, 3US National Poultry Research Center, testing, it was determined that the virus was USDA-Agricultural Research Service spread to another two linked premises by the The effects of infection of hens in production transfer of birds between sites, a common with an unusually virulent strain of low practice in the rearing process. Disease pathogenic avian influenza virus will be suspicions were reported to the Department of presented. Environment, Food and Rural affairs, which obtained official samples in according with Reconstitution and Mutagenesis of Avian standard EU protocols. These samples were Infectious Laryngotracheitis Virus from Cosmid submitted to the Animal and Plant Health and Yeast Centromeric Plasmid Clones Agency for diagnosis as official statutory disease 1 2 investigations. H5N8 was confirmed with the Stephen Spatz , Maricarmen García , Walter 3 molecular detection of viral RNA and virus Fuchs isolation. Additional carcases from the three 1US National Poultry Research Center, Athens, premises were submitted to APHA for an GA, USA, 2Poultry Diagnostic and Research extensive investigation of gross pathology, Center, College of Veterinary Medicine, histopathology and viral distribution by University of Georgia, USA, 3Institute of immunohistochemistry. Whilst gross Molecular Virology and Cell Biology, Friedrich observations in some birds were consistent with Loeffler-Institut, Federal Research HPAIV, these finds were inconsistent within and between premises . The investigation yielded Infectious laryngotracheitis is an economically important information regarding the tropism of important pathogen of chicken with morbidity of 100% and mortality rates as high as 70%. the viral subtypes circulating in the migratory Factoring in decreased production, mortality, birds present in the Peruvian territory and to vaccination and medication, a single outbreak determine the variability by comparison with the can cost producers over a million dollars (1). strains previously detected in our environment, Current live attenuated and vectored vaccines as well as in Latin America. lack safety and efficacy, respectively, leaving a Avian influenza virus surveillance in need for better vaccines. Since infectious columbiform birds of Huarmey, Ancash-Peru bacterial artificial chromosome-based clones of infectious laryngotracheitis virus (ILTV) Eliana Icochea1 Gina Castro2, Ana P. Apaza3, containing intact origins of replication are not Rosa Gonzalez4 feasible, we present the reconstitution of 1 2 infectious ILTV from a collection of both yeast National University of San Marcos, National 3 and bacterial clones and the identification of a University of San Marcos, National University 4 nonessential insertion site within a redundant of San Marcos, National University of San packaging site (ipac2). These constructs and the Marcos methodology necessary to manipulate them will The avian influenza (AI) virus has been isolated in facilitate the development of improved live virus many species of birds around the world. In wild vaccines through modification of genes encoding birds the infection is normally asymptomatic, but virulence factors and establish ILTV based viral H5 and H7 subtypes of virus can become highly vectors for the expression of immunogens of pathogenic when they are transmitted from wild other avian pathogens. birds to domestic birds. In Peru the presence of Detection and Molecular Characterization of any type of avian influenza virus in birds was Strains of Avian Influenza Virus Isolated From never detected, however, the introduction of Migratory Birds of The Coast of Peru, 2018- the virus in many areas has been demonstrated, 2019 principally due to wild bird’s migration. The objective of this study is to detect the presence Rosa Gonzalez1, Eliana Icochea2, Nelly of AIV in wild birds that live surrounding of Cribillero3, Gina Castro4 poultry farms in Huarmey, Ancash-Perú, located 300 km north of Lima, where there are several 1San Marcos University, Lima-peru, 2San Marcos flocks of broiler breeders and where are University, 3San Marcos University, 4San Marcos prevalent columbiform birds. Fresh fecal University samples are being collected since october 2019 The main objective of this study was the to march 2020 because during this time occurs identification and molecular characterization of greatest bird migration in Perú. The samples will strains isolated of the Avian Influenza virus (AI) be processed in the Avian Pathology Laboratory in migratory birds from 04 wetlands of the at the Veterinary School, San Marcos University central coast of Peru, during the 2018-2019 by virus isolation in SPF embryonated eggs. period. A total of 400 samples were taken from Samples will be analyzed in pools of five samples, stool swabs and were processed by viral isolation according to specie and date and following the in SPF chicken embryonated eggs and standard methods. The allantoid fluid will be confirmation by RT-PCR test. Six samples were tested by hemagglutinating inhibition assay. All positive for influenza A virus, which are being positive samples will be molecularly processed for characterization by sequencing. characterized. This study will allow us to determine and analyze Characterization of variant infectious B, and C have been recognized to cause clinical bronchitis virus from a small scale poultry disease in poultry, but only subtype C has been holding in Egypt identified in the United States. Detection of subtype A in Mexico it was reported in 2015. Ibrahim Moharam1, Hesham Sultan2, Hafez M. Next generation sequencing using a random Hafez3, Christain Grund4 detection approach was used on samples from 1Friedrich-Loeffler-Institut, Germany, 2Faculty of chicken flocks in Mexico in 2019. Although this Veterinary medicine, Unversity of Sadat City random sequence approach has limits in Egypt, 3Faculty of Veterinary medicine, FU-Berlin sensitivity, both subtype A and B AmPV was University, Germany, 4Friedrich-Loeffler-Institut, detected was samples. The use of real-time RT- Germany PCR targeted to either A or B subtypes confirmed detection of both viruses. Sequence analysis Attending of the AAAP meeting will help me to showed the subtype A virus was highly similar to discus my work about infectious bronchitis virus the published 2015 virus. The subtype B virus characterization and evolution in Egypt through was a new detection and sequence analysis is full genome sequence of IBV isolate that was ongoing. The presence of AmPV in commercial isolated in 2016 from small scale production and poultry in Mexico represents an additional comparing the sequence with other related respiratory pathogen of concern and a potential sequences in the gene bank. For better threat to U.S. poultry. understand and comparison, we did full genome sequence for an IBV isolate from 2012 isolated Wealth of from large scale production. Moreover we followed up the evolution in GI-23 IBV viruses in Knowledge/Epidemiology/D Egypt over time period of seven years through identification of a recombination break point iagnostics including gene 3ab in 26 samples from 2012 until Assessing perceived risks and benefits for 2019 participation in poultry disease monitoring Protection against different genotypes of projects Newcastle disease viruses (NDV) afforded by Andreia Goncalves Arruda1, Geoffrey Lossie2, an adenovirus-vectored fusion protein and Chang-Won Lee3 live NDV vaccines 1The Ohio State University, 2Purdue University, 1 2 3 David Suarez , Josh Parris , Iryna Goraichuk , 3The Ohio State University Henry Kariithi4 The development and implementation of 1Southeast Poultry Research Laboratory, ARS, disease monitoring projects is useful for rapid USDA, 2Southeast Poultry Research Laboratory, communication of outbreaks and improvement USDA, 3Southeast Poultry Research Laboratory, of the understanding of endemic and epidemic USDA, 4Southeast Poultry Research Laboratory, diseases that affect animal health and producer Agricultural Research Service, U.S. Department profitability. However, producers have of Agriculture traditionally been reluctant to share information Avian metapneumovirus (AmPV) is an infectiou related to animal health, challenging the RNA virus associated with respiratory disease in implementation of such programs. chickens and turkeys. Three unique subtypes, A, The objective of this study was to develop a from a breeder flock that experienced a recent national survey aimed at poultry professionals in drop in egg production from 1,700 to 1,000 eggs order to describe their beliefs toward produced per day were submitted for necropsy. participation in poultry disease mapping and During the drop, the mortality rate increased, monitoring projects; and to identify main and some birds appeared sick. All four hens perceived benefits and risks for participation. An were out of egg production. One bird had a pale anonymous online survey was developed using cardiac ventricle. Two ducks had fungal Qualtrics software and distributed to poultry airsacculitis and pneumonia. Microscopic professionals through industry associations. examination of lungs and air sacs revealed fungal hyphae that were consistent with Aspergillus sp. A total of 60 participants filled out the survey, Cerebral blood vessels were cuffed by representing 25 states for poultry production. lymphocytes with lymphocytes within the Layers, breeders, and pullets were the most vascular walls. The heart had multifocal necrosis represented commodities; followed by broilers, with random foci of lymphocytes and turkeys and game birds/backyard/small flock mononuclear cells within the myocardium. poultry. The majority of responders (75%) Polymerase chain reaction (PCR) testing reported being familiar with such projects, with revealed the presence of West Nile virus RNA. 30% stating already participating in one. Approximately half of the responders were Comparative Analysis of Two New Multilocus poultry veterinarians, with the other half being Sequence Typing (MLST) Schemes for represented by system owners, production Characterization of Mycoplasma gallisepticum managers, and administrative positions. The strains main perceived benefits by responders were up- Mostafa Ghanem3, Mohamed Elgazzar2, Yosra to-date visualization of disease outbreaks and Helmy3 planning of truck routes; and the main risks were the potential for increased attention and 1University of Maryland-College Park, 2Iowa misinformation from animal rights groups and state University, 3University of Maryland, the release of premise locations in the event of a public records request. Results from our survey Mycoplasma gallisepticum (MG) is the most highlight areas that need to be considered for pathogenic avian mycoplasma species. It affects future development of these programs. commercial, non-commercial poultry and wild birds. MG strain identification is essential for West Nile Virus Infection in Commercial Duck epidemiological outbreak investigation, Breeder Flocks prevention and control programs. Recently, two new MLST schemes have been developed Richard Fulton1, Matti Kiupel, D.V.M., Ph.D.2, independently for typing MG isolates and clinical Stephen Bolin, Ph.D.3 samples. Both schemes are available for public 1Michigan State University, 2Michigan State use at pubMLST database. No direct head to University, 3Michigan State University head comparison has been performed to evaluate how their results are compared. A commercial duck company had a recurring Moreover, one of the two schemes has not been problem with drops in breeder flock egg evaluated against a reference method. The aim production over the past few years. The egg of this study was to compare the characteristics production drop occurred around September. of the two schemes to make a recommendation Egg drops had been dramatic with 10, 20, 35, and about their usage. In order to compare the 40 percent drops in some flocks. Four ducks results of these two schemes and evaluate their Design and evaluation of a device to teach results, we have typed a diverse collection of 81 euthanasia of chickens by cervical dislocation isolates. In addition, we have compared the Brian Ladman1, Matthew Symes2, Gianmarco results of the two schemes to a whole genome Vasquez3 based typing approach core genome multilocus sequence typing (cgMLST). All schemes were 1University of Delaware, 2University of assessed based on the discriminatory power Delaware, 3University of Delaware index and degree of congruency with the epidemiological information of the typed strains. In conjunction with Engineering students The results of this study have confirmed that enrolled in the University of Delaware's (UD) cgMLST had a superior discriminatory power to Senior Design Capstone Program, a device was both MLST schemes and both MLST schemes are developed to train Animal Science students to valuable tools for population-based studies. In perform cervical dislocation (CD) on avian addition, the higher discriminatory power of the species. This method of euthanasia is a humane cgMLST scheme is better and recommended for and approved (AVMA) method to dispatch many outbreak investigation. We recommend using types of research animals. Institutional Animal only one MLST scheme for typing isolates Care and Use Committees require training and globally in order to build the global database for proof of proficiency prior to granting approval to MG and maintain its usefulness for perform the procedure. UD’s Avian Virology understanding the global MG population group currently trains students on animals that dynamics and epidemiology. have been euthanized by other methods rather than a trial by fire approach that can be MinION Sequencing for Selective and Rapid traumatic to both the animals and student. The Detection of Avian Influenza A Virus goal of this project was to capture the forces used by experienced scientists while performing Iryna Goraichuk CD to euthanize chickens. The data was then USDA/ARS/SEPRL used to design a training device that would prevent the sacrificing of additional animals Until recently, sequencing technologies have strictly for training purposes. The training device been providing data through short-read simulates the mechanics of cervical dislocation, technique. A relatively new sequencing including proper location and use of force, and technology, the MinION nanopore sequencer, provides visual feedback to the trainee and provides a platform that is much cheaper than instructor as to the successful execution of CD to other existing sequencing platforms with the humanely euthanizing the chicken. potential of rapid sequence and analysis. The MinION allows the sequencing of individual strands of DNA and can produce millions of reads

which places it in the Next Generation Sequences category. The Nanopore MinION sequencer can provide a useful and inexpensive method to obtain influenza virus genomic sequences for screening, diagnostics, and research purposes.

The Mission and Goals of AAAP’s Diversity and Expression and Characterization of Avian Inclusion Taskforce Reovirus SigmaC-MBP Fusion Proteins as ELISA Antigens Valerie Marcano1, Ellen Lapuck2, Christina Leyson3 Linda Michel 1Poultry Diagnostic and Research Center, The Ohio State University College of Veterinary Medicine, The University Avian reoviruses (ARV) belong to the of Georgia, 953 College Station Road, Athens, Orthoreovirus genus in the family Reoviridae. Georgia, 30602, USA, 2Boehringer Ingelheim They are widespread in chickens and turkeys and Animal Health USA Inc., 3239 Satellite Blvd NW, have been linked to several diseases in poultry; Duluth, GA 30096, 3Southeast Poultry Research most notably tenosynovitis in chickens. ARV Laboratory (SEPRL, ARS, USDA) 934 College strains form at least six distinct genotype Station Road Athens, GA 30605 clusters. The ARV protein primarily responsible The mission of the AAAP’s Diversity and Inclusion for this diversity is sigma C, which induces the Taskforce is to foster and embrace a climate formation of neutralizing antibodies that protect inclusive of all AAAP members regardless of age, chickens from disease. The presence of these gender, race, sexual orientation, socioeconomic antibodies can be detected with commercial or educational backgrounds to encourage full ELISA kits. Increasing antigenic diversity of the participation in the opportunities the virus, however, may decrease the effectiveness organization has to offer. We strive to ensure of this assay for detection of ARV antibodies. To that those in leadership positions, participating circumvent this problem, we used a platform in committees and receiving awards, expression system to produce the sigma C scholarships, and research support truly reflect protein fused to a maltose binding protein (MBP) our membership base. Additionally, the diversity tag to facilitate purification. ARV strains were and inclusion taskforce will maintain a protocol isolated from infected broilers and the sigma C to adapt as the AAAP membership evolves. gene was sequenced. Genes from different genogroups were cloned into a MBP fusion Goals: plasmid transfer vector and then transfected 1. To collect and provide demographics on the into the genome of baculovirus under the AAAP members at large and relay that control of the polyhedron promotor. Sigma C- information to the AAAP Board, while following MBP expressed from baculovirus cultures was appropriate guidelines for member privacy. purified over amylose resin and used to coat ELISA plates. An ELISA was performed using 2. To evaluate current AAAP procedures and serum from chickens infected with ARV from identify areas of strength and weakness that different genogroups. The results indicate the need to be altered/addressed to promote different sigma-C MBP antigens reacted stronger diversity and inclusion. to homologous ARV antibodies.

3. To assist in the development of AAAP's position on diversity and inclusion.

4. To encourage and guide policies to promote diversity and inclusion as the demographics of AAAP change. Trends in Mycotoxin Contamination in the have influenced sample submission and the risk United States Corn Crop profile of the harvest is likely to shift as additional samples are analyzed. Chasity Pender1, Shelby Ramirez2, Ursula Hofstetter -Schähs4, Raj Murugesan4 Characterization of Reoviral Hepatitis and Splenitis in Commercial Turkey Poults 1Biomin America Inc., 2BIOMIN America, Inc., Overland Park, KS, USA, 3BIOMIN Holding Robert Porter1, Saad Gharaibeh2, Sunil Mor3, GmbH, Getzersdorf, Austria, 4BIOMIN America, Anibal Armien4 USA 1Univ of Minnesota, 2Minnesota Poultry Testing Mycotoxins are secondary fungal metabolites Laboratory, 622 Business Hwy 71NE, Willmar, that are detrimental to animal health and MN, 3MN Veterinary Diagnostic Laboratory, performance even at low levels. The classic signs 1333 Gortner Avenue, St. Paul, MN, 4MN such as reduced feed intake and oral and Veterinary Diagnostic Laboratory, MN intestinal lesions used as indicators for exposure For the past year, a spiking mortality (2-8% loss often underestimate other costs of in less than one week) affecting 10 to 25-day-old mycotoxicosis, including increasing frequency commercial poults has been observed in flocks and severity of disease via immunosuppression, throughout the Midwest. Poults are generally inflammation, and modulating the found dead without premonitory signs. The most gastrointestinal environment. Corn samples consistent gross lesion is hepatosplenomegaly. were submitted and analyzed for aflatoxins The enlarged livers are often pale and contain (Afla), type A trichothecenes, type B numerous 1-3 mm white foci. Crops and trichothecenes (B-Trich), fumonisins (FUM), intestines are often devoid of ingesta. Gizzards zearalenone (ZEN), and ochratoxin-A utilizing LC- are filled with litter and have linear erosions in MS/MS. The 2019 data were compared to the koilin lining. Histopathology reveals previous years to examine contamination necrotizing hepatitis and splenitis. Liver and trends. Preliminary results are limited due to spleen have discrete, multifocal to coalescing harvest delays reducing sample submission, but islands of necrosis with infiltrates of plump suggest a high proportion of samples contain at macrophages containing cellular debris, and least one mycotoxin (96%), similar to 2018 varying numbers of lymphocytes, plasma cells (97%). The co-occurrence thus far is lower than and heterophils. The lung often contains 2018 (2019: 51%, 2018: 75%). Currently, B-Trich interstitial infiltrates of mononuclear cells. Viral is the most prevalent mycotoxin group and inclusion bodies are not observed. There is no appears to have lower prevalence, but similar significant bacterial growth from liver, spleen, average contamination levels compared to 2018 intestine and lung. Negative contrast electron (2019: 75%, 1,376 ppb; 2018: 83%,686 ppb). microscopy of ground liver and spleen reveals FUM has lower prevalence and similar average virus particles resembling reovirus. Livers are levels compared to last year (2019: 54%, 2968 positive for reovirus by RT-PCR and by ppb; 2018: 68%, 2,905 ppb), while ZEN levels and inoculation of embryonated chicken eggs and prevalence have decreased (2019: 42%, 386 ppb; LMH cells without any indication of picornavirus 2018: 54%, 604 ppb). To date, no Alfa has been or adenovirus. Initial Sanger sequencing of the detected; however, corn stored with higher reoviral gene fragments indicates identity with moisture has increased risk for storage toxins. turkey enteric and arthritis reoviruses. These Thus far, the 2019 harvest has a continued risk due to mycotoxins; however, harvest delays findings indicate a novel presentation for GIS Tools for Poultry Health Reporting: reovirus in turkeys. Development of an Interactive Web Map for BREWS Fecal Parasite and Select Pathogen ELISA Testing on Wild Turkeys from Middle Nicki Smith Tennessee Georgia Poultry Laboratory Network Sierra Slautterback1, Dr. Richard Gerhold- DVM, Each month, Georgia Poultry Laboratory PhD, MS, Diplomat ACVM (parasitology)2, Dr. Network (GPLN) produces a Bronchitis Early Sawsan Ammar- BVsC, PhD3, Tania Dawant4 Warning System (BREWS) report to keep 1University of Tennessee CVM, 2University of Georgia’s commercial poultry industry informed Tennessee College of Veterinary Medicine, about cases of infectious bronchitis virus (IBV) 3University of Tennessee College of Veterinary within the state. Provided via email to key Medicine, 4University of Tennessee College of individuals, a map of Georgia showing county- Veterinary Medicine level IBV isolations serves as an important component of the monthly BREWS report. This study is part of an on-going 6-year Today’s web-mapping technologies make it investigation on the health of wild turkeys in possible not only to deliver this map product in middle Tennessee. Fecal exams and ELISA an online environment, but to encode additional screening on wild turkeys was preformed to information using interactive elements. This determine the prevalence of fecal parasites and project explores the development and utilization select pathogens and to determine the baseline of an interactive BREWS web map as a tool to health of the wild turkey population in middle support Georgia’s commercial industry. Tennessee.