RESUMO
By incompletely understood mechanisms, type 2 (T2) inflammation present in the airways of severe asthmatics drives the formation of pathologic mucus which leads to airway mucus plugging. Here we investigate the molecular role and clinical significance of intelectin-1 (ITLN-1) in the development of pathologic airway mucus in asthma. Through analyses of human airway epithelial cells we find that ITLN1 gene expression is highly induced by interleukin-13 (IL-13) in a subset of metaplastic MUC5AC+ mucus secretory cells, and that ITLN-1 protein is a secreted component of IL-13-induced mucus. Additionally, we find ITLN-1 protein binds the C-terminus of the MUC5AC mucin and that its deletion in airway epithelial cells partially reverses IL-13-induced mucostasis. Through analysis of nasal airway epithelial brushings, we find that ITLN1 is highly expressed in T2-high asthmatics, when compared to T2-low children. Furthermore, we demonstrate that both ITLN-1 gene expression and protein levels are significantly reduced by a common genetic variant that is associated with protection from the formation of mucus plugs in T2-high asthma. This work identifies an important biomarker and targetable pathways for the treatment of mucus obstruction in asthma.
Assuntos
Asma , Proteínas Ligadas por GPI , Interleucina-13 , Lectinas , Mucina-5AC , Muco , Criança , Humanos , Asma/genética , Asma/metabolismo , Citocinas , Células Epiteliais/metabolismo , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Interleucina-13/genética , Interleucina-13/metabolismo , Lectinas/genética , Lectinas/metabolismo , Mucina-5AC/genética , Mucina-5AC/metabolismo , Muco/metabolismo , Mucosa Nasal/metabolismo , Polimorfismo Genético , Mucosa Respiratória/metabolismoRESUMO
BACKGROUND: Albuterol is the first-line asthma medication used in diverse populations. Although DNA methylation (DNAm) is an epigenetic mechanism involved in asthma and bronchodilator drug response (BDR), no study has assessed whether albuterol could induce changes in the airway epithelial methylome. We aimed to characterize albuterol-induced DNAm changes in airway epithelial cells, and assess potential functional consequences and the influence of genetic variation and asthma-related clinical variables. RESULTS: We followed a discovery and validation study design to characterize albuterol-induced DNAm changes in paired airway epithelial cultures stimulated in vitro with albuterol. In the discovery phase, an epigenome-wide association study using paired nasal epithelial cultures from Puerto Rican children (n = 97) identified 22 CpGs genome-wide associated with repeated-use albuterol treatment (p < 9 × 10-8). Albuterol predominantly induced a hypomethylation effect on CpGs captured by the EPIC array across the genome (probability of hypomethylation: 76%, p value = 3.3 × 10-5). DNAm changes on the CpGs cg23032799 (CREB3L1), cg00483640 (MYLK4-LINC01600), and cg05673431 (KSR1) were validated in nasal epithelia from 10 independent donors (false discovery rate [FDR] < 0.05). The effect on the CpG cg23032799 (CREB3L1) was cross-tissue validated in bronchial epithelial cells at nominal level (p = 0.030). DNAm changes in these three CpGs were shown to be influenced by three independent genetic variants (FDR < 0.05). In silico analyses showed these polymorphisms regulated gene expression of nearby genes in lungs and/or fibroblasts including KSR1 and LINC01600 (6.30 × 10-14 ≤ p ≤ 6.60 × 10-5). Additionally, hypomethylation at the CpGs cg10290200 (FLNC) and cg05673431 (KSR1) was associated with increased gene expression of the genes where they are located (FDR < 0.05). Furthermore, while the epigenetic effect of albuterol was independent of the asthma status, severity, and use of medication, BDR was nominally associated with the effect on the CpG cg23032799 (CREB3L1) (p = 0.004). Gene-set enrichment analyses revealed that epigenomic modifications of albuterol could participate in asthma-relevant processes (e.g., IL-2, TNF-α, and NF-κB signaling pathways). Finally, nine differentially methylated regions were associated with albuterol treatment, including CREB3L1, MYLK4, and KSR1 (adjusted p value < 0.05). CONCLUSIONS: This study revealed evidence of epigenetic modifications induced by albuterol in the mucociliary airway epithelium. The epigenomic response induced by albuterol might have potential clinical implications by affecting biological pathways relevant to asthma.
Assuntos
Asma , Metilação de DNA , Criança , Humanos , Epigenômica , Asma/tratamento farmacológico , Asma/genética , Albuterol/farmacologia , Albuterol/uso terapêutico , Epigênese Genética , Broncodilatadores/farmacologia , Broncodilatadores/uso terapêutico , Células Epiteliais , Estudo de Associação Genômica AmplaRESUMO
To identify genetic determinants of airway dysfunction, we performed a transcriptome-wide association study for asthma by combining RNA-seq data from the nasal airway epithelium of 681 children, with UK Biobank genetic association data. Our airway analysis identified 95 asthma genes, 58 of which were not identified by transcriptome-wide association analyses using other asthma-relevant tissues. Among these genes were MUC5AC, an airway mucin, and FOXA3, a transcriptional driver of mucus metaplasia. Muco-ciliary epithelial cultures from genotyped donors revealed that the MUC5AC risk variant increases MUC5AC protein secretion and mucus secretory cell frequency. Airway transcriptome-wide association analyses for mucus production and chronic cough also identified MUC5AC. These cis-expression variants were associated with trans effects on expression; the MUC5AC variant was associated with upregulation of non-inflammatory mucus secretory network genes, while the FOXA3 variant was associated with upregulation of type-2 inflammation-induced mucus-metaplasia pathway genes. Our results reveal genetic mechanisms of airway mucus pathobiology.
Assuntos
Asma , Transcriptoma , Asma/genética , Asma/metabolismo , Criança , Epitélio/metabolismo , Humanos , Metaplasia/metabolismo , Mucina-5AC/genética , Mucina-5AC/metabolismo , Muco/metabolismoRESUMO
BACKGROUND: Patients with coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demonstrate high rates of co-infection with respiratory viruses, including influenza A (IAV), suggesting pathogenic interactions. METHODS: We investigated how IAV may increase the risk of COVID-19 lung disease, focusing on the receptor angiotensin-converting enzyme (ACE)2 and the protease TMPRSS2, which cooperate in the intracellular uptake of SARS-CoV-2. RESULTS: We found, using single-cell RNA sequencing of distal human nondiseased lung homogenates, that at baseline, ACE2 is minimally expressed in basal, goblet, ciliated and secretory epithelial cells populating small airways. We focused on human small airway epithelial cells (SAECs), central to the pathogenesis of lung injury following viral infections. Primary SAECs from nondiseased donor lungs apically infected (at the air-liquid interface) with IAV (up to 3×105â pfu; â¼1 multiplicity of infection) markedly (eight-fold) boosted the expression of ACE2, paralleling that of STAT1, a transcription factor activated by viruses. IAV increased the apparent electrophoretic mobility of intracellular ACE2 and generated an ACE2 fragment (90â kDa) in apical secretions, suggesting cleavage of this receptor. In addition, IAV increased the expression of two proteases known to cleave ACE2, sheddase ADAM17 (TACE) and TMPRSS2 and increased the TMPRSS2 zymogen and its mature fragments, implicating proteolytic autoactivation. CONCLUSION: These results indicate that IAV amplifies the expression of molecules necessary for SARS-CoV-2 infection of the distal lung. Furthermore, post-translational changes in ACE2 by IAV may increase vulnerability to lung injury such as acute respiratory distress syndrome during viral co-infections. These findings support efforts in the prevention and treatment of influenza infections during the COVID-19 pandemic.
Assuntos
COVID-19 , Influenza Humana , Células Epiteliais , Humanos , Pandemias , Peptidil Dipeptidase A , SARS-CoV-2RESUMO
Coronavirus disease 2019 (COVID-19) is caused by SARS-CoV-2, an emerging virus that utilizes host proteins ACE2 and TMPRSS2 as entry factors. Understanding the factors affecting the pattern and levels of expression of these genes is important for deeper understanding of SARS-CoV-2 tropism and pathogenesis. Here we explore the role of genetics and co-expression networks in regulating these genes in the airway, through the analysis of nasal airway transcriptome data from 695 children. We identify expression quantitative trait loci for both ACE2 and TMPRSS2, that vary in frequency across world populations. We find TMPRSS2 is part of a mucus secretory network, highly upregulated by type 2 (T2) inflammation through the action of interleukin-13, and that the interferon response to respiratory viruses highly upregulates ACE2 expression. IL-13 and virus infection mediated effects on ACE2 expression were also observed at the protein level in the airway epithelium. Finally, we define airway responses to common coronavirus infections in children, finding that these infections generate host responses similar to other viral species, including upregulation of IL6 and ACE2. Our results reveal possible mechanisms influencing SARS-CoV-2 infectivity and COVID-19 clinical outcomes.
Assuntos
Betacoronavirus/fisiologia , Infecções por Coronavirus/virologia , Interferons/metabolismo , Interleucina-13/metabolismo , Mucosa Nasal/patologia , Peptidil Dipeptidase A/genética , Pneumonia Viral/virologia , Serina Endopeptidases/genética , Enzima de Conversão de Angiotensina 2 , COVID-19 , Criança , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/patologia , Células Epiteliais/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Variação Genética , Interações Hospedeiro-Patógeno , Humanos , Inflamação , Pessoa de Meia-Idade , Mucosa Nasal/metabolismo , Pandemias , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/metabolismo , Pneumonia Viral/patologia , SARS-CoV-2 , Serina Endopeptidases/metabolismo , Internalização do VírusRESUMO
The type 2 cytokine-high asthma endotype (T2H) is characterized by IL-13-driven mucus obstruction of the airways. To further investigate this incompletely understood pathobiology, we characterize IL-13 effects on human airway epithelial cell cultures using single-cell RNA sequencing, finding that IL-13 generates a distinctive transcriptional state for each cell type. Specifically, we discover a mucus secretory program induced by IL-13 in all cell types which converts both mucus and defense secretory cells into a metaplastic state with emergent mucin production and secretion, while leading to ER stress and cell death in ciliated cells. The IL-13-remodeled epithelium secretes a pathologic, mucin-imbalanced, and innate immunity-depleted proteome that arrests mucociliary motion. Signatures of IL-13-induced cellular remodeling are mirrored by transcriptional signatures characteristic of the nasal airway epithelium within T2H versus T2-low asthmatic children. Our results reveal the epithelium-wide scope of T2H asthma and present candidate therapeutic targets for restoring normal epithelial function.
Assuntos
Asma/genética , Epitélio/metabolismo , Análise de Célula Única , Transcriptoma/genética , Transporte Biológico/efeitos dos fármacos , Reprogramação Celular/efeitos dos fármacos , Criança , Cílios/efeitos dos fármacos , Cílios/metabolismo , Regulação para Baixo/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Epitélio/efeitos dos fármacos , Humanos , Imunidade Inata/efeitos dos fármacos , Interferons/metabolismo , Interleucina-13/farmacologia , Metaplasia , Muco/metabolismo , Transcriptoma/efeitos dos fármacosRESUMO
Cigarette smoke first interacts with the lung through the cellularly diverse airway epithelium and goes on to drive development of most chronic lung diseases. Here, through single cell RNA-sequencing analysis of the tracheal epithelium from smokers and non-smokers, we generate a comprehensive atlas of epithelial cell types and states, connect these into lineages, and define cell-specific responses to smoking. Our analysis infers multi-state lineages that develop into surface mucus secretory and ciliated cells and then contrasts these to the unique specification of submucosal gland (SMG) cells. Accompanying knockout studies reveal that tuft-like cells are the likely progenitor of both pulmonary neuroendocrine cells and CFTR-rich ionocytes. Our smoking analysis finds that all cell types, including protected stem and SMG populations, are affected by smoking through both pan-epithelial smoking response networks and hundreds of cell-specific response genes, redefining the penetrance and cellular specificity of smoking effects on the human airway epithelium.
Assuntos
Células Epiteliais/metabolismo , Perfilação da Expressão Gênica/métodos , Pulmão/metabolismo , Mucosa Respiratória/metabolismo , Fumar/genética , Traqueia/metabolismo , Animais , Células Cultivadas , Técnicas de Inativação de Genes , Redes Reguladoras de Genes , Humanos , Pulmão/citologia , Camundongos , Células NIH 3T3 , não Fumantes/estatística & dados numéricos , Mucosa Respiratória/citologia , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Fumantes/estatística & dados numéricos , Traqueia/citologiaRESUMO
Air pollution particulate matter <2.5 µm (PM2.5) exposure is associated with poor respiratory outcomes. Mechanisms underlying PM2.5-induced lung pathobiology are poorly understood but likely involve cellular and molecular changes to the airway epithelium. We extracted and chemically characterized the organic and water-soluble components of air pollution PM2.5 samples, then determined the whole transcriptome response of human nasal mucociliary airway epithelial cultures to a dose series of PM2.5 extracts. We found that PM2.5 organic extract (OE), but not water-soluble extract, elicited a potent, dose-dependent transcriptomic response from the mucociliary epithelium. Exposure to a moderate OE dose modified the expression of 424 genes, including activation of aryl hydrocarbon receptor signaling and an IL-1 inflammatory program. We generated an OE-response gene network defined by eight functional enrichment groups, which exhibited high connectivity through CYP1A1, IL1A, and IL1B. This OE exposure also robustly activated a mucus secretory expression program (>100 genes), which included transcriptional drivers of mucus metaplasia (SPDEF and FOXA3). Exposure to a higher OE dose modified the expression of 1,240 genes and further exacerbated expression responses observed at the moderate dose, including the mucus secretory program. Moreover, the higher OE dose significantly increased the MUC5AC/MUC5B gel-forming mucin expression ratio and strongly downregulated ciliated cell expression programs, including key ciliating cell transcription factors (e.g., FOXJ1 and MCIDAS). Chronic OE stimulation induced mucus metaplasia-like remodeling characterized by increases in MUC5AC+ secretory cells and MUC5AC mucus secretions. This epithelial remodeling may underlie poor respiratory outcomes associated with high PM2.5 exposure.
Assuntos
Mucosa Nasal/diagnóstico por imagem , Material Particulado/efeitos adversos , Mucosa Respiratória/efeitos dos fármacos , Poluentes Atmosféricos/efeitos adversos , Poluição do Ar/efeitos adversos , Asma/induzido quimicamente , Asma/genética , Estudo de Associação Genômica Ampla/métodos , Humanos , Inflamação/induzido quimicamente , Inflamação/genética , Mucina-5AC/genética , Mucina-5B/genética , Fatores de Transcrição/genéticaRESUMO
Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of Johne's disease, chronic and ultimately fatal enteritis that affects ruminant populations worldwide. One mode of MAP transmission is oral when young animals ingest bacteria from the collostrum and milk of infected dams. The exposure to raw milk has a dramatic impact on MAP, resulting in a more invasive and virulent phenotype. The MAP1203 gene is upregulated over 28-fold after exposure of the bacterium to milk. In this study, the role of MAP1203 in binding and invasion of the bovine epithelial cells was investigated. By over-expressing the native MAP1203 gene and two clones of deletion mutant in the signal sequence and of missense mutations changing the integrin domain from RGD into RDE, we demonstrate that MAP1203 plays a role in increasing binding in more than 50% and invasion in 35% of bovine MDBK epithelial cells during early phase of infection. Furthermore, results obtained suggest that MAP1203 is a surface-exposed protein in MAP and the signal sequence is required for processing and expression of functional protein on the surface of the bacterium. Using the protein pull-down assay and far-Western blot, we also demonstrate that MAP1203 interacts with the host dihydropyrimidinase-related protein 2 and glyceraldehyde 3-phosphate dehydrogenase proteins, located on the membrane of epithelial cell and involved in the remodeling of the cytoskeleton. Our data suggests that MAP1203 plays a significant role in the initiation of MAP infection of the bovine epithelium.
Assuntos
Adesinas Bacterianas/metabolismo , Aderência Bacteriana , Endocitose , Células Epiteliais/microbiologia , Mycobacterium avium subsp. paratuberculosis/fisiologia , Fatores de Virulência/metabolismo , Adesinas Bacterianas/genética , Animais , Bovinos , Linhagem Celular , Deleção de Genes , Expressão Gênica , Fatores de Virulência/genéticaRESUMO
The airway epithelium lines the respiratory tract and provides the primary protective barrier against inhalational insults including toxic environmental substances and microorganisms. The airway epithelium also plays a critical role in regulating airway immune responses. The airway epithelial response to the type 2 cytokine, interleukin-13 (IL-13), is critical to airway inflammation, mucus production, and airway hyperresponsiveness present in asthma. Relevant primary cell models of the human airway epithelium are needed to investigate the biology of IL-13-mediated airway epithelial effects. Here, we describe the generation of a differentiated mucociliary human airway epithelium using an in vitro air-liquid interface (ALI) culture model system. We also describe methods to stimulate this culture model with IL-13 and harvest cells and biomolecules to interrogate cellular and molecular aspects of the airway epithelial IL-13 response.
Assuntos
Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Interleucina-13/farmacologia , Mucosa Respiratória/citologia , Animais , Técnicas de Cultura de Células , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Expressão Gênica , Humanos , Técnicas In VitroRESUMO
Mycobacterium avium subsp. hominissuis (M. avium) is a member of the non-tuberculous mycobacteria (NTM), and is a common cause of lung infection in patients with chronic NTM lung conditions. M. avium is an environmental bacterium believed to be transmitted from environmental sources. In this work we used a recently developed model in Caenorhabditis elegans to ask whether M. avium can be transmitted from host-to-host, and the bacterial genes associated with host colonization. Infection of C. elegans was carried out by placing the nematode in cultured with M. avium. Bacteria eliminated from the intestines of infected C. elegans were used to infect naïve nematodes. In parallel experiments, to identify colonization associated genes, a transposon library of M. avium was screened for the ability to bind to HEp-2 mucosal cells. Thirty clones were identified and five selected clones with impaired adherence to HEp-2 epithelial cells were used to infect C. elegans to determine the degree of colonization. It was determined that M. avium eliminated from infected C. elegans were able to colonize a naïve C. elegans with high efficiency. Thirty of the most adherence-deficient M. avium clones obtained from the HEp-2 cell screening were sequenced to identify the location of the transposon. Many of the genes associated with the bacterial cell wall synthesis were shown to be inactivated in the selected mutants. Five out of the 30 bacterial clones were then used to infect C. elegans. All five mutants had impaired ability to colonize C. elegans compared with the wild type bacteria (decrease of 1.5-2.0 logs, p < 0.05). The limitation of this work is that the model can be used for initial screening, but other more complex systems should be used to confirm the findings. C. elegans can be used as a model to test for M. avium adherence/colonization-associated virulence determinants. All the tested adherence-deficient clones that were examined had impaired ability to colonize the host C. elegans, and some can be potentially used to prevent colonization.
Assuntos
Caenorhabditis elegans/microbiologia , Genes Bacterianos/genética , Infecções por Mycobacterium/microbiologia , Infecções por Mycobacterium/transmissão , Mycobacterium avium/genética , Mycobacterium avium/patogenicidade , Animais , Proteínas de Bactérias/genética , Linhagem Celular , Modelos Animais de Doenças , Células Epiteliais/microbiologia , Regulação Bacteriana da Expressão Gênica , Mucosa Intestinal/microbiologia , Infecções Respiratórias/transmissão , Fatores de VirulênciaRESUMO
The adaptation of the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated endonuclease 9 (CRISPR-Cas9) machinery from prokaryotic organisms has resulted in a gene editing system that is highly versatile, easily constructed, and can be leveraged to generate human cells knocked out (KO) for a specific gene. While standard transfection techniques can be used for the introduction of CRISPR-Cas9 expression cassettes to many cell types, delivery by this method is not efficient in many primary cell types, including primary human airway epithelial cells (AECs). More efficient delivery in AECs can be achieved through lentiviral-mediated transduction, allowing the CRISPR-Cas9 system to be integrated into the genome of the cell, resulting in stable expression of the nuclease machinery and increasing editing rates. In parallel, advancements have been made in the culture, expansion, selection, and differentiation of AECs, which allow the robust generation of a bulk edited AEC population from transduced cells. Applying these methods, we detail here our latest protocol to generate mucociliary epithelial cultures knocked out for a specific gene from donor-isolated primary human basal airway epithelial cells. This protocol includes methods to: (1) design and generate lentivirus which targets a specific gene for KO with CRISPR-Cas9 machinery, (2) efficiently transduce AECs, (3) culture and select for a bulk edited AEC population, (4) molecularly screen AECs for Cas9 cutting and specific sequence edits, and (5) further expand and differentiate edited cells to a mucociliary airway epithelial culture. The AEC knockouts generated using this protocol provide an excellent primary cell model system with which to characterize the function of genes involved in airway dysfunction and disease.
Assuntos
Sistemas CRISPR-Cas , Células Epiteliais/metabolismo , Deleção de Genes , Edição de Genes/métodos , Genoma Humano , Mucosa Respiratória/metabolismo , Animais , HumanosRESUMO
Johne's disease, caused by Mycobacterium avium subspecies paratuberculosis (MAP), is a severe chronic enteritis which affects large populations of ruminants globally. Prevention strategies to combat the spread of Johne's disease among cattle herds involve adhering to strict calving practices to ensure young susceptible animals do not come in contact with MAP-contaminated colostrum, milk, or fecal material. Unfortunately, the current vaccination options available are associated with high cost and suboptimal efficacy. To more successfully combat the spread of Johne's disease to young calves, an efficient method of protection is needed. In this study, we examined passive immunization as a mode of introducing protective antibodies against MAP to prevent the passage of the bacterium to young animals via colostrum and milk. Utilizing the infectious MAP phenotype developed after bacterial exposure to milk, we demonstrate that in vitro opsonization with serum from Johne's-positive cattle results in enhanced translocation across a bovine MDBK polarized epithelial cell monolayer. Furthermore, immune serum opsonization of MAP results in a rapid host cell-mediated killing by bovine macrophages in an oxidative-, nitrosative-, and extracellular DNA trap-independent manner. This study illustrates that antibody opsonization of MAP expressing an infectious phenotype leads to the killing of the bacterium during the initial stage of macrophage infection.
Assuntos
Anticorpos Antibacterianos/metabolismo , Translocação Bacteriana , Células Epiteliais/microbiologia , Macrófagos/imunologia , Macrófagos/microbiologia , Viabilidade Microbiana , Mycobacterium avium subsp. paratuberculosis/imunologia , Animais , Bovinos , Imunização Passiva , Mycobacterium avium subsp. paratuberculosis/fisiologia , Proteínas Opsonizantes/metabolismo , Paratuberculose/imunologia , Paratuberculose/prevenção & controleRESUMO
Understanding the pathogenic mechanisms of Mycobacterium avium subspecies paratuberculosis (MAP) and the host responses to Johne's disease is complicated by the multi-faceted disease progression, late-onset host reaction and the lack of available ex vivo infection models. We describe a novel cell culture passage model that mimics the course of infection in vivo. The developed model simulates the interaction of MAP with the intestinal epithelial cells, followed by infection of macrophages and return to the intestinal epithelium. MAP internalization triggers a minimal inflammatory response. After passage through a macrophage phase, bacterial reinfection of MDBK epithelial cells, representing the late phase of intestinal mucosal infection, is associated with increased synthesis of the pro-inflammatory transcripts of IL-6, CCL5, IL-8 and IL-18, paired with decreased levels of TGFß. Transcriptome analysis of MAP from each stage of epithelial cell infection identified increased expression of lipid biosynthesis and lipopeptide modification genes in the inflammatory phenotype of MAP. Total lipid analysis by HPLC-ES/MS indicates different lipidomic profiles between the two phenotypes and a unique set of lipids composing the inflammatory MAP phenotype. The presence of selected upregulated lipid-modification gene transcripts in samples of ileal tissue from cows diagnosed with Johne's disease supports and validates the model. By using the relatively simple cell culture passage model, we show that MAP alters its lipid composition during intracellular infection and acquires a pro-inflammatory phenotype, which likely is associated with the inflammatory phase of Johne's disease.