Duration: 10/01/1999 to 09/30/2004

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

Statement of Issues and Justification

Respiratory diseases afflicting poultry in modem commercial production operations are complex entities. Numerous factors including infectious agents, non-infectious agents and environmental factors may contribute to the disease complex. The impetus to raise wholesome food products without (or limited) use of antimicrobial agents has provided new challenges to poultry producers and those responsible for poultry health. The increased globalization of trade has increased the potential for new diseases to emerge or previously controlled diseases to reemerge. The continued need for research to address the complex interactions of disease factors, develop and improve methods for the rapid diagnosis and control of respiratory disease is of paramount importance to the poultry industry as it transcends into the 21st century.

Justification:

The United States Poultry Industry is a growth industry having a reported 1996 production value of $21.76 billion (USDA, National Agricultural Statistics Service (http://www.usda.gov/nass/pubs/agr98/acro98.htm), most recent data). In 1996, 7.6 billion broilers valued at $13.9 billion and 301.4 million turkeys valued at $3.1 billion were slaughtered in the USA. In addition, 303 million layers produced 77.4 billion eggs (Av. 255 eggs/hen) valued at $4.7 billion. Export of broiler and turkey meat has accounted for about 15% and 5.4% respectively, of the total production. Poultry meat production has been continually increasing and consumption has surpassed all other meats. In the meantime, the cost to the consumer of poultry meats has been relatively stable, a result of the efficiency and competitive nature of the industry.

Intensive production is a main feature that contributes to the productivity and efficiency of the poultry industry. A consequence of intensive production is the increased risk of the spread of infectious diseases. Losses caused by infectious diseases are manifested in a variety of ways including mortality, morbidity, condemnations, lowered productivity, decreased efficiency of production and a multiplier effect estimated between 1.3 and 2.6. Losses due to mortality in the US flocks were 4% in broilers, 12% in turkeys and 17% in layers and condemnations were approximately 3% in both broilers and turkeys. Respiratory diseases are a major cause of mortality and approximately 90% of all the condemnations in both chickens and turkeys are due to air sacculitis and septicemia. Most septicemia cases are sequela of respiratory disease (e.g. Escherichia coli infection). Producers and poultry health specialists have continually rated respiratory diseases as the most economically significant group of poultry diseases.

Respiratory diseases are the outcome of a set of interactions contributed by the parasites, host and the environment. The genetic make up of the host could be a major contribution to increased susceptibility or resistance to disease. The environment in the poultry house (temperature, humidity, dust, gases, etc.) could have an equally important effect. Seldom is respiratory disease associated with one pathogen. Indeed, in the majority of cases primary and secondary infections are involved in the disease process and the most serious outcome (mortality) is usually caused by secondary bacterial infections. Causative agents of respiratory diseases are continually undergoing changes because of herd immunity/natural selection and mutations. In addition, newly emerging and reemerging pathogens are encountered with relatively increased frequencies. In the recent past, several viral infections capable of inducing or increasing susceptibility of the host of respiratory diseases were discovered including the avian pneumovirus, paramyxovirus-7, chicken anemia virus and variant (and highly virulent) infectious bursal disease viruses. Newly discovered respiratory bacterial pathogens include Bordetella avium, Omithobacterium rhinotracheale and Neisseria-like bacteria. In addition, influenza viruses continue to re-emerge as a threat to the US poultry industry. The Mexican outbreak of highly virulent avian influenza is a recent reminder of that potential.

The poultry industry is under pressure to limit the use of antibiotics and in response demand for newer vaccines and alternative disease control strategies is increasing. Recent progress in molecular biology is yielding practical and useful information that will have a significant impact on the study, diagnosis, control and prevention of respiratory diseases.

Scientists from the North Central Region are poised to make contributions in both the basic and applied areas of respiratory disease research. Their collective expertise and experience over a wide range of disciplines will enhance their effectiveness and productivity. Respiratory diseases are complex entities that require the cooperation of scientists from different states and the cooperative regional approach provides the forum for initiating and continuing these cooperative research efforts.

Related, Current and Previous Work

Avian Influenza (AI) continues to be a disease of great concern world wide for poultry producers because of increased condemnation at slaughter and marked decrease in egg production and fertility. AI is caused by an orthomyxovirus that can infect a wide range of wild and domestic birds. The existence of many antigenic subtypes of AI virus (AIV) poses a substantial impediment to the control of this disease. Immunization with killed vaccines of homologous hemagglutinin subtypes and subunit/recombinant vaccines (hemagglutinin and neuraminidase based) afford little or no cross-protection against infection with heterologous subtypes.

In contrast to the highly variable surface glycoproteins, the internal viral nucleoprotein (NP) of influenza viruses is highly conserved, and thus may be a suitable candidate for stimulating heterologous protection. This conserved protein can be targeted to generate cell mediated immunity for achieving broad spectrum immunity.

Our overall goal of this project is to develop an effective, safe and broad spectrum vaccine for the prevention and control of AI. The present investigation is aimed at the development of turkey herpes virus-NP (rHVT-NP) live recombinant virus construct and to evaluate its potential as an effective and broad-spectrum live vaccine against AI. Towards the development of a live recombinant vaccine we have made substantial progress. The following points summarize our achievements: (a) A cDNA copy of the NP gene from AI subtype H5N2 (A/Turkey/Minnesota/I 700/82) has been cloned and sequenced; (b) The expression of the recombinant NP has been evaluated in vitro by transfection of chicken embryo fibroblast cells (CEFs) with the plasmid expression vector carrying NP cDNA. The protein expression was detected by western blot and immunofluorescence assays; (c) The recombination plasmid vector carrying nonessential region of HVT genome flanking the NP cDNA was constructed. The expression of NP from the recombination plasmid in CEFs was confirmed by western blot and immunofluorescence assays; (d) The recombinant virus is being generated by site-specific insertion of NP in HVT genome by homologous recombination. The HVT genomic DNA and recombination plasmid were co-transfected into secondary CEFs. NP cDNA was cloned into the Bg/II site of the U_s 10 sequence fragment of the HVT genome; (e) Several recombinant HVT-NP clones have been identified by immunofluorescence assay using NP specific monoclonal antibodies.

Avian Pneumovirus

The United States was considered free of avian pneumovirus infection until the recent recovery of an APV isolate from infected turkey flocks in Colorado in 1997 (by the National Veterinary Services Laboratory). Subsequently, the virus was reported in turkey farms in Minnesota and some adjoining counties in neighboring states and is now widespread in these areas. APV infections are associated with a marked increase (up to 50%) in morbidity and mortality rates on turkey farms as well as in condemnation of carcasses at processing plants. Losses totaling more than $15 million were reported during 1997 due to APV infections, and threaten the viability of turkey farming in Minnesota.

Avian pneumoviruses, including the turkey rhinotracheitis virus (TRTv), belong to the viral family Paramyxoviridae, and have a single-stranded non-segmented RNA genome of approximately 13 - 15,000 nucleotides including 7 structural genes (Randhawa et. al., 1997). Although preliminary information from NVSL and our laboratory shows that the US isolates are distinct from other avian pneumoviruses, there is very little information on the provenance of APV in the US and its relationship to other previously characterized pneumoviruses. Similarly, knowledge of the mechanism of virus evolution and extent of genetic diversity (presence of different viral strains) is also unknown.

Due to the non-pathognomonic nature of the clinical signs of the syndrome, it is important to identify the viral agent in order to make a diagnosis. There are only two methods for detecting the virus in affected flocks: virus isolation or DNA-based diagnostics. Virus isolation has a relatively poor sensitivity, is time-consuming and often difficult to perform. This situation is further complicated with the relatively rapid disappearance (within 3 days) of virus following onset of clinical signs in turkeys (Cook, et. al., 1993; Jones, 1996). Hence, polymerase chain reaction (PCR)-based approaches are the methods of choice for the rapid detection of APV infections in infected birds.

Previous experience with TRTv in Europe, Africa, and Asia and experimental studies suggest that wild birds may play a role in the transmission of avian pneumoviruses. For instance, guinea fowl have been shown to possibly harbor TRTv particles in their respiratory tracts (Picault et. al., 1987), and both guinea fowl and pheasants seroconvert upon intranasal inoculation of TRTv (Gough et. al., 1988). While the recent experience from the US outbreaks also support the role of wild birds in disease spread, the exact mechanism of transmission, natural hosts and potential carriers (including wild birds) of APV remain unknown.

Escherichia coli

Escherichia coli (E. coli) is a bacterium that is commonly isolated from commercial poultry flocks that are dying from respiratory disease. It is the ultimate or final cause of disease. In mammals, E. coli isolates are determined to cause disease based upon their serologic or structural characteristics. In poultry however, this is not always the case. Multiple stations have been working to determine which avian E. coli strains cause disease and which strains are relatively harmless. Michigan has been working on determining the effects of a known serotype (078) that causes disease as well as an unknown serotype (serotype not typable) that has been recovered from 3 separate farms in 3 separate cases of E. coli septicemia in an attempt to better understand the differences between E. coli strains. Both serotypes have been found to produce lesions of septicemia when given intravenously as well as pneumonia and septicemia when given intratracheally. It has been found that the unknown serotype was able to make more birds sick and produce more pneumonia than the pathogenic strain 078. This work is being continued to understand the interactions of E. coli with other diseases as well as provide information to poultry producers for timely and strategic antibiotic administration.

Minnesota has concentrated on the molecular aspects of E. coli strains and populations that are have been recovered from turkey flocks with disease. This focus is being undertaken as a prelude to discovery of a single antigenic characteristic that can be utilized in vaccine development.

Minnesota has applied MLEE to 300 isolates from diseased turkeys to detect genetic (allelic) variation among isolates in 20 chromosomally-encoded metabolic enzymes in order to more clearly form a genetic framework for this population. So far, Minnesota has identified on highly pathogenic clone complex (pEc) that has been associated with a vast majority of clinical cases of turkey colibacillosis. At the same time, a non-pathogenic (npEc) isolate was found among the otherwise highly pEc. pEc and npEc were found to differ in characteristics other than at the molecular level. These differences for the pEc include a unique plasmid profile, mucoid colonies when grown on agar and a smoother encapsulation as seen via scanning electron microscopy. These isolates have been analyzed at the molecular level by genomic subtraction hybridization (GSH) and repetitive-sequence PCR banding patterns to identify specific virulence factors. Through GSH, a DNA fragment unique to pEC that is homologous with ColEl plasmid was found both within genomic and plasmid DNA. With repetitive-sequence PCR, 2 bands were found to be unique to pEc. These bands were analyzed by cloning and sequencing and found to have homology with the promoter region of kpsM, a polysialic acid transporter. Polysialic acid is important in capsule formation, which correlates with the mucoid appearance of pEc and the correlation between encapsulation and virulence.

Fowl pox

Fowl poxvirus was isolated in several outbreaks from chickens previously vaccinated with commercial fowl poxvirus vaccines. Although fowl pox is commonly recognized by the development of cutaneous lesions, in these outbreaks a diphtheritic form of the disease was observed. Some of the responsible isolates have undergone minor antigenic changes and are more virulent than the vaccine strains. A genetic comparison of the field and vaccine strains based on restriction fragment length polymorphism (RFLP) did not reveal significant differences. Avian poxviruses have been suggested to be population-limiting factors in endangered Hawaiian forest birds. In this regard we have isolated three avian poxvirus strains from Hawaiian forest birds. Genetic evaluation of two of the isolates revealed marked differences between them indicating that genetically different viruses may exist in this population. Both strains exhibit low pathogenicity for chickens.

Fowl poxvirus has been successfully used as a vector for the expression of foreign genes from poultry pathogens. In this regard recombinant fowl pox virus expressing a cDNA copy of the hemagglutinin gene of avian influenza virus provided protection against challenge with virulent avian influenza virus (Tripathy and Schnitzlein; Beard et. al, 1991; Beard et. al, 1992).

The in vitro and in vivo characteristics of poxvirus from a number of avian species have been studied. It has been demonstrated that currently available vaccines, in certain situations, are ineffective in preventing infection of chickens and turkeys with variant strains of pox. A project developing vaccines to provide a wider range of protection is needed.

See attached "Related, Current and Previous Work" for additional information.

Objectives

1. Determine the pathogenesis and interactions of specific agents
   
2. Surveillance, occurrence and consequences of agents and hosts on disease susceptibility.
   
3. Develop new and improved methods/or the diagnosis, prevention and control of avian respiratory diseases.
   
4. 
   

Methods

Objective 1. Determine the pathogenesis and interactions of specific agents.

Avian Influenza

No work in pathogenesis and interactions of the avian influenza virus with any other specific agents is planned during the proposed study period.

Avian Pneumoviruses

Iowa and Ohio will collaborate on epidemiologic studies on naturally occurring pneumovirus infections in chicken and turkey flocks. The antigenic relatedness of isolates of pneumovirus from field cases will be studied. Iowa and Ohio have obtained USDA permits to work with the Colorado strain of APV (APV/CO) under BL2 laboratory conditions and in BL3 bio-containment animal facilities for in vivo pathogenesis studies. Iowa will receive selected APV field isolates from Ohio and conduct comparative pathogenesis in vivo studies with APV/CO. Researchers in Ohio will attempt to adapt the virus to tissue culture and produce specific anti sera to be used by Iowa for studies of antigenic relatedness.

The pathogenic mechanisms of Avian Pneumovirus will be examined by investigators at Minnesota and Iowa. Experimental reproduction of the disease in turkeys has already been carried out by investigators in Minnesota and Iowa. Current and future investigations are designed to examine the mechanisms of transmission and pathogenesis of the viral agent in experimentally infected turkeys. Investigators from Minnesota, Ohio and Iowa will continue to collaborate on studies designed to determine the pathogenesis and interactions of the avian pneumovirus in with other agents including 0. rhinotracheale. Minnesota and Iowa will continue to exchange virus stocks, genetic material and primer sequences as relates to the detection and characterization of the avian pneumovirus. Specifically, the collaborations to characterize the virus and emerging strains will be strengthened with continued exchange of visits of scientists between the stations.

Escherichia coli

The pathogenesis of E. coli in commercial poultry is being studied at Alabama, Michigan, and Minnesota. Alabama and Minnesota are addressing E. coli studies on chickens (broiler and egg type). Michigan and Minnesota are studying E. coli in turkeys. Alabama will obtain suspect E. coli isolates from commercial poultry integrators throughout the United States. These isolates will be definitively identified and used in respiratory and non-respiratory challenge models in immature and mature chickens. DNA from challenge isolates will be extracted, subjected to restriction endonuclease digestion and the restriction fragments separated by pulsed field gel electrophoresis (PFGE). Whole cell protein lysates from the challenge strain E.coli will also be analyzed with 2-D electrophoresis to determine protein expression at 35, 27, and 42 C. Results of both the PFGE and 2-D protein analysis will be compared with the results of the same procedures being performed on E. coli isolates recovered from the challenged birds. Recovered challenge isolates that have been verified as being identical to challenge strains will be serotyped, assayed for the production of shiga like toxins, aerobactin, colicins, hemolysins, and the presence of plasmids. Antibiotic sensitivity will also be profiled.

Michigan will be studying the contribution of hemorrhagic enteritis (HE) vaccination in the development of post-vaccinal E. coli septicemia where 4-week-old turkeys will be vaccinated with HE vaccine and challenged with 078 strain of E. coli. To determine the time when birds are most susceptible, turkeys will be infected at time of vaccination, 2 days post vaccination and 7 days post vaccination. Pathogenicity will be determined by comparing mortality rates and gross lesion scores.

Minnesota will continue to examine the population structure of E. coli isolates that are recovered from turkey flocks in an attempt to determine the molecular differences between pathogenic and non-pathogenic E. coli. The overall goal will be to elucidate the population genetic structure of recovered E. coli isolates and use this information in selecting isolates that may be of potential immunoprophylaxis interest. Minnesota will also continue to develop methodologies to detect genetic differences amongst closely related bacterial pathogens by molecular techniques such as serial analysis of gene expression (SAGE), subtraction library generation, as well as more traditional approaches such as transposon mutagenesis. Isolates of E. coli that are identical in MLEE genotype but differ substantially in ability to cause disease in chickens and turkeys will be used as test strains to validate this new methodology. The overall goal is to elucidate the molecular basis of pathogenicity of E. coli isolates in turkey colibacillosis and to utilize this information for selection and design of immunoprophylactic agents.

Minnesota will share with investigators from the other stations E. coli isolates that have been genetically and phenotypically characterized for further studies on virulence and pathogenecity.

Fowl Pox

Because of the large size of the fowlpox virus genome (300 kb), it is difficult to detect minor genetic changes by some of the routine techniques, e.g. RFLP. It is, however, assumed that some of these alterations may occur as a result of an interaction between more than one pathogen simultaneously infecting the host respiratory system. In this regard, a natural respiratory infection, involving both pox and herpes viruses has been observed (Tripathy et. al 1975; Futanmbi et. al, 1995). Under these circumstances, an exchange of genetic material may occur as suggested by Brunovskis and Velicer (1995) resulting in the possible emergence of antigenically different viruses against which the immune response induced by vaccine viruses would not be adequate. Our recent studies indicate that some of the field strains of fowlpox virus contain integrated sequences of reticuloendothelial virus (REV). Identification of such changes as well as the gene(s) associated with virulence will require the use of more precise molecular tools (e. g., PCR, cloning and DNA sequencing) since such differences cannot be detected by conventional methods. Illinois will focus on this aspect and collaborate with all regions where fowlpox virus infections occur. In addition, cross-protection studies will be done to evaluate these viruses in order to select an appropriate strain for vaccination.

Infectious Bronchitis Virus (IB)

Indiana will compare the nucleotide sequences of S protein gene and leader sequence of enterotropic and respiratory-tropic coronavirus strains. The coronavirus isolated from trachea or digestive tracts will be propagated in chicken embryo and their RNA will be extracted and reverse transcribed to cDNA. The PCR-amplified products generated by 35 cycles of amplification of the region of S gene and leader sequence will be purified using the Magic PCR Preps DNA purification system (Promega) and ligated into the pGEM-T vector (Promega). The ligation product will be used to transform competent Escherichia coli strain DH5a. Plasmid DNA will be isolated from the bacteria by Magic Miniprep purification resin (Promega) and sequenced by an automated sequencer. The nucleotide changes of S gene and leader sequence of enterotropic and respiratory-tropic strains will be compared by DNAsis software program.

A recent isolate of infectious bronchitis virus designated OK 96 has been causing losses in egg production in broiler breeder chickens in Arkansas. The isolate has been partially characterized by Arkansas and by Dr. Mark Jackwood at Georgia. Studies conducted involve serology, pathology, immunization, electron microscopy and molecular biology. IBV OK 96 did not react with a panel of monoclonal antibodies specific for Massachusetts, Connecticut and Arkansas serotypes. A mixture of these monoclonal antibodies did provide a positive test when infected trachea was stained in an indirect fluorescent antibody procedure. IBV OK 96 produced tracheitis in experimentally infected SPF chickens similar to but not as severe as an IBV Massachusetts(Mass) challenge strain. Immunization studies based on cross protection indicated partial immunity to Massachusetts vaccine but not by either Arkansas or Connecticut vaccines. Electron microscopy of purified infected allantoic fluid revealed virus particles typical of IBV to include a size of between 100 to 120 nm in diameter with a rounded, pleomorphic virion with club shaped projections. Dr. Jackwood conducted two different evaluations using reverse transcriptase PCR and then digestion with a series of restriction endonucleases, which produce fragments of DNA of varying lengths. One evaluation indicated that this virus was a unique type while one indicated a similarity to the Delaware IBV Strain 0 72. Arkansas will continue to evaluate IBV OK 96 and to collaborate with other researchers participating in the NC 116project by sharing both virus and antisera for additional characterization. Studies are currently in progress to evaluate the Delaware IBV Strain 0 72 vaccine to provide protection against challenge with IBV OK 96.

Indiana will collaborate with Ohio, and other states in the northeastern region(collaboration through the NE 138 Technical Committee) to study antigenic relationship between IBV and turkey coronavirus (TCV) using antibody-capture ELISA or FA. Indiana will compare nucleotide sequences ofS, M and N protein genes of IBV and TCV isolates from Indiana, Minnesota, Virginia, North Carolina and Texas using PCR, cloning, and sequencing.

Infectious Bursal Disease (IBD)

The pathogenic mechanisms of IBD V strains will be examined at the molecular level by Ohio. The affect of point mutations in the VP2, VP3 and VP4 genes on the pathogenicity of viruses will be examined. These point mutations will be detected using restriction mapping and nucleotide sequencing of PCR amplified genes.

Ohio will conduct studies to evaluate the value of vaccination of newly hatched and in ovo vaccination for infectious bursal disease. Hatching eggs originating from breeding flocks with different levels of antibodies will be used to vaccinate the late incubation embryo or the chicken immediately after hatch to study the interaction of maternal antibodies with the vaccine virus.

Indiana and Ohio will study the extent of genetic variation of different IBDV strains or isolates in the field, adapted to grow in cell culture, and serially passaged in cell culture. The hypervariable region of VP2 in IBDV genome will be amplified by PCR using published or new primers and sequenced (Wu et. al., 1992; Wu and Lin, 1992; Lin et. al., 1994). This will allow us to have a better understanding of IBDV pathogenicity and immunogenicity as well as to develop new strategies for IBD vaccines. Ohio will supply Indiana with different viruses characterized for antigenicity and immunogenicity to study the molecular basis for their variability.

Ornithobacterium rhinotracheale

Minnesota will continue with investigations on the interactions between other agents such as the avian pneumovirus and 0. rhinotracheale during the proposed study period. The role of these agents in predisposing turkeys for infection will be examined during the proposed study period. Minnesota will also continue to examine the potential role of egg-transmission in0. rhinotracheale infected turkeys.

Pasteurella multocida

Minnesota will continue to study the molecular epidemiology of Pasteurella multocida strains associated with avian species. Minnesota has recently standardized MLEE to detect genetic (allelic) variation among bacterial isolates in 14 chromosomally encoded metabolic enzymes. The information derived from these studies will be used to formulate a molecular population genetic framework of more than 200 P. multocida isolates recovered from poultry. The ongoing investigations are aimed at expanding the number of isolates included in the analysis, and using this information to select isolates for the large-scale automated DNA sequence analysis of P. multocida virulence factor genes. Overall, that these data will provide important information on the molecular basis of P. multocida epidemiology in avian populations. Indiana will determine if the same isolants of Pasteurella multocida have been isolated from the same turkey farms on consecutive years. The farms to be evaluated will have isolants of P. multocida in the bank of isolants collected on consecutive years during the period between 1966 and 1995. The similarities or differences between isolants will be determined by fingerprinting the chromosomal DNA after digesting with the restriction endonucleases Hha I and Hpa II (Wilson et. al, 1993).

Minnesota will make available to scientists at the other stations with plasmids and or DNA-chips containing P. multocida genome for investigation on pathogenicity.

Riemerella anatipestifer

Michigan will collaborate with NADC in collecting and sending isolates from commercial duck flocks for identification and DNA fingerprinting.

See additional "Methods" attached.

Measurement of Progress and Results

Outputs

Outcomes or Projected Impacts

• See attached "Methods continued"

Milestones

(0):0

Projected Participation

View Appendix E: Participation

Outreach Plan

Organization/Governance

The research technical committee shall consist of one technical committee representative from each cooperating agency as appointed or otherwise designated by the respective organization, and administrative advisor appointed by the Association of North Central Experiment Stations Directors, and a representative of the Cooperative State Research, Education and Extension Service (CSREES).

The executive committee shall consist of the project coordinator, chairperson and secretary of the technical committee. The project coordinator is elected by the research technical committee members and shall serve for the duration of the project (i.e. 5 years). The chairperson and secretary are elected annually with the secretary succeeding the chairperson. The chairperson and secretary will serve a one-year term.

Meetings will be held annually at the time and place mutually agreed upon by the technical committee, or designated by the executive committee with the approval of the administrative advisor.

The secretary will normally record the minutes of the annual meeting. Two copies of the minutes with the original signatures of recommendation by the secretary will be sent to the Administrative Advisor with an approval block for her/his signature. The Administrative Advisor will distribute copies to appropriate individuals.

The chairperson will normally prepare the annual report summarized from material supplied to her/him by the technical committee member from each participating station or agency. The chairperson will send the final draft of the annual report to the administrative advisor for her/his approval. The administrative advisor will assume responsibility for its distribution.

The project coordinator will be responsible for continuity and coordination of the project. Coordination of states is identified for the cooperative procedures within each objective. The project coordinator is responsible for an overall coordination of the project including such activities as the annual meetings (especially when in conjunction with another group), special meetings and/or workshops to address various aspects of the project, and communication with the administrative advisor and or administering committees. Such coordination will occur continuously throughout the project period. The annual meeting of the technical committee will serve as a monitoring session for coordination.

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