Publications by authors named "Adrienne L Shircliff"

5 Publications

  • Page 1 of 1

Prediction of Johne's disease state based on quantification of T cell markers and their interaction with macrophages in the bovine intestine.

Vet Res 2021 Apr 13;52(1):55. Epub 2021 Apr 13.

USDA-Agricultural Research Service (ARS), National Animal Disease Center, Ames, IA, USA.

Cell-mediated immune responses to Mycobacterium avium subsp. paratuberculosis (MAP) are regulated by various types of T lymphocytes. The aim of this study was to quantitate T cell subsets in the mid-ileum of cows naturally infected with MAP to identify differences during different stages of infection, and to determine whether these subsets could be used as predictors of disease state. Immunofluorescent labeling of T cell subsets and macrophages was performed on frozen mid-ileal tissue sections archived from naturally infected dairy cows in either subclinical or clinical disease status, and noninfected control cows. Comprehensive IF staining for CD4, CD8α, TcR1-N24 (gamma delta), FoxP3, CXCR3 and CCR9 served to define T cell subsets and was correlated with macrophages present. Clinically affected cows demonstrated significantly higher numbers of CXCR3 (Th1-type) and CCR9 (total small intestinal lymphocytes) cells at the site of infection compared to the subclinical cows and noninfected controls. Further, predictive modeling indicated a significant interaction between CXCR3 and AM3K (macrophages) cells, suggesting that progression to clinical disease state aligns with increased numbers of these cell types at the site of infection. The ability to predict disease state with this model was improved from previous modeling using immunofluorescent macrophage data. Predictive modelling indicated an interaction between CXCR3 and AM3K cells, which could more sensitively detect subclinical cows compared to clinical cows. It may be possible to use this knowledge to improve and develop an assay to detect subclinically infected animals with more confidence during the early stages of the disease.
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http://dx.doi.org/10.1186/s13567-021-00925-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8042692PMC
April 2021

Identification of a reliable fixative solution to preserve the complex architecture of bacterial biofilms for scanning electron microscopy evaluation.

PLoS One 2020 29;15(5):e0233973. Epub 2020 May 29.

Ruminant Diseases and Immunology Research Unit, National Animal Disease Center, United States Department of Agriculture, Agricultural Research Service, Ames, Iowa, United States of America.

Bacterial biofilms are organized sessile communities of bacteria enclosed in extracellular polymeric substances (EPS). To analyze organization of bacteria and EPS in high resolution and high magnification by scanning electron microscopy (SEM), it is important to preserve the complex architecture of biofilms. Therefore, fixation abilities of formalin, glutaraldehyde, and Methacarn (methanol/chloroform/acetic acid-6:3:1) fixatives were evaluated to identify which fixative would best preserve the complex structure of bacterial biofilms. Economically important Gram-negative Mannheimia haemolytica, the major pathogen associated with bovine respiratory disease complex, and Gram-positive Staphylococcus aureus, the major cause of chronic mastitis in cattle, bacteria were selected since both form biofilms on solid-liquid interface. For SEM analysis, round glass coverslips were placed into the wells of 24-well plates and diluted M. haemolytica or S. aureus cultures were added, and incubated at 37°C for 48-72 h under static growth conditions. Culture media were aspirated and biofilms were fixed with an individual fixative for 48 h. SEM examination revealed that all three fixatives were effective preserving the bacterial cell morphology, however only Methacarn fixative could consistently preserve the complex structure of biofilms. EPS layers were clearly visible on the top, in the middle, and in the bottom of the biofilms with Methacarn fixative. Biomass and three-dimensional structure of the biofilms were further confirmed spectrophotometrically following crystal violet staining and by confocal microscopy after viability staining. These findings demonstrate that Methacarn fixative solution is superior to the other fixatives evaluated to preserve the complex architecture of biofilms grown on glass coverslips for SEM evaluation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0233973PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7259777PMC
August 2020

Phenotypes of macrophages present in the intestine are impacted by stage of disease in cattle naturally infected with Mycobacterium avium subsp. paratuberculosis.

PLoS One 2019 23;14(5):e0217649. Epub 2019 May 23.

USDA-Agricultural Research Service (ARS), National Animal Disease Center, Ames, IA, United States of America.

Macrophages play an important role in the host immune response to Mycobacterium avium subsp. paratuberculosis (MAP) infection, however, MAP is able to disrupt normal macrophage functions to avoid destruction. It is unclear whether the phenotypes of macrophages present in the target tissue play a role in the inability to clear MAP infection. The aim of this study was to identify macrophage phenotypes (host defense or resolution and repair) present within the bovine ileum of naturally infected cattle, as well as to ascertain abundance of each macrophage phenotype present during different stages of MAP infection. Immunofluorescent (IF) labeling was performed on frozen bovine mid-ileal tissue sections collected from 28 Holstein dairy cows. Comprehensive IF staining for cytokines, such as IFN-γ, IL-1Ra, IL-1β, IL-10, TGF-β, TNF-α, and uNOS, along with markers such as CD163, CD206, and TLR4, served to define the macrophage phenotypes. Overall, cows in the clinical stage of disease demonstrated significantly higher numbers of resolution and repair macrophages and lower numbers of host defense macrophages in the ileal tissue. Interestingly, subclinically affected cows with asymptomatic disease had a nearly equal ratio of host defense and resolution and repair macrophage phenotypes, whereas macrophage phenotype was skewed to a host defense macrophage in the tissues of the control noninfected cows. The preponderance of M2-like resolution and repair phenotype for macrophages in the tissues of cows with clinical disease would explain why the host fails to control and/or clear the infection, leading to a higher MAP burden. The results of the current study offer insight into the disparate macrophage phenotypes present in the bovine ileum during different stages of infection.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0217649PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6532939PMC
February 2020

Quantification of Macrophages and in Bovine Intestinal Tissue During Different Stages of Johne's Disease.

Vet Pathol 2019 09 6;56(5):671-680. Epub 2019 May 6.

1 USDA-Agricultural Research Service (ARS), National Animal Disease Center, Ames, IA, USA.

Johne's disease is an enteric disease caused by the intracellular pathogen (MAP). Upon ingestion of MAP, it is translocated across the intestinal epithelium and may be killed by intestinal macrophages, or depending on the bacterial burden and immunological status of the animal, MAP may thwart innate defense mechanisms and persist within the macrophage. This study aimed to determine the numbers of macrophages and MAP present in bovine midileal tissue during different stages of infection. Immunofluorescent (IF) labeling was performed on frozen bovine midileal intestinal tissue collected from 28 Holstein dairy cows. The number of macrophages in midileal tissue sections was higher for clinically affected cows, followed by subclinically affected cows and then uninfected control cows. Macrophages were present throughout the tissue sections in clinical cows, including the tunica muscularis, submucosa, and the lamina propria around the crypts and in the villous tips, with progressively fewer macrophages in subclinically affected and control cows. Clinically affected cows also demonstrated significantly higher numbers of MAP and higher numbers of macrophages with intracellular MAP compared to subclinically affected cows. MAP IF labeling was present within the submucosa and lamina propria around the crypts, progressing into the villous tips in some clinically affected cows. Our findings indicate that number of macrophages increases with progression of infection, but a significant number of the macrophages present in the midileum are not associated with MAP.
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http://dx.doi.org/10.1177/0300985819844823DOI Listing
September 2019

Relationship between the pathology of bovine intestinal tissue and current diagnostic tests for Johne's disease.

Vet Immunol Immunopathol 2018 Aug 23;202:93-101. Epub 2018 Jun 23.

USDA-Agricultural Research Service (ARS), National Animal Disease Center, Ames, IA, United States. Electronic address:

Johne's disease is an enteric disease caused by the intracellular pathogen Mycobacterium avium subsp. paratuberculosis (MAP). Upon translocation from the lumen of the small intestine, mycobacteria have the ability to thwart innate defense mechanisms and persist within the macrophage in the lamina propria. In an effort to understand how the pathology of disease is reflected in current diagnostic tests, immunofluorescent (IFA) labeling was performed to quantitate macrophage and MAP numbers in the ileum of infected cattle and correlate results with common methods for diagnosis of MAP infection; including ELISA, IFN-γ assay, RT-PCR, culture of MAP, and histological classification of tissue sections. Predictive models for clinical and subclinical disease states, histopathology acid-fast (AF), MAP location, granulomatous inflammation and type classifications, as well as macrophage, MAP and macrophages with intracellular MAP IFA labeling were successfully developed. The combination of macrophage number and ELISA were the best predictors of clinical disease state, while macrophage number was the best and only significant predictor of subclinical disease state. Fecal culture and number of MAP were the best predictors of granulomatous inflammation, and of combined AF, MAP location and granuloma type, respectively. Additionally, fecal culture and tissue culture were the best predictors of numbers of macrophages and MAP, respectively, while both ELISA and tissue culture were the best predictors of number of macrophages with intracellular MAP.
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http://dx.doi.org/10.1016/j.vetimm.2018.06.012DOI Listing
August 2018