Cytokine gene expression assay as a diagnostic tool for detection of Mycobacterium bovis infection in warthogs (Phacochoerus africanus)

Abstract Mycobacterium bovis infection has been described in many wildlife species across Africa. However, diagnostic tests are lacking for many of these, including warthogs (Phacochoerus africanus). Most literature on suids has focused on using serological tools, with few studies investigating the use of cell-mediated immune response (CMI) assays. A recent study showed that warthogs develop measurable CMI responses, which suggests that cytokine gene expression assays (GEAs) may be valuable for detecting M. bovis-infection, as shown in numerous African wildlife species. Therefore, the aim of the study was to develop GEAs capable of distinguishing between M. bovis-infected and uninfected warthogs. Whole blood was stimulated using the QuantiFERON-TB Gold (In-Tube) system, using ESAT-6 and CFP-10 peptides, before determining the relative gene expression of five reference (B2M, H3F3A, LDHA, PPIA and YWHAZ) and five target (CXCL9, CXCL10, CXCL11, IFNG and TNFA) genes through qPCR. The reference gene H3F3A was the most stably expressed, while all target genes were significantly upregulated in M. bovis-infected warthogs with the greatest upregulation observed for CXCL10. Consequently, the CXCL10 GEA shows promise as an ante-mortem diagnostic tool for the detection of M. bovis-infected warthogs.

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Differential Cytokine Gene Expression in Granulomas from Lungs and Lymph Nodes of Cattle Experimentally Infected with Aerosolized Mycobacterium bovis

PLoS ONE ◽

10.1371/journal.pone.0167471 ◽

2016 ◽

Vol 11 (11) ◽

pp. e0167471 ◽

Cited By ~ 9

Author(s):

Mitchell V. Palmer ◽

Tyler C. Thacker ◽

W. Ray Waters

Keyword(s):

Gene Expression ◽

Lymph Nodes ◽

Mycobacterium Bovis ◽

Cytokine Gene Expression ◽

Cytokine Gene

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Analysis of Cytokine Gene Expression using a Novel Chromogenic In-situ Hybridization Method in Pulmonary Granulomas of Cattle Infected Experimentally by Aerosolized Mycobacterium bovis

Journal of Comparative Pathology ◽

10.1016/j.jcpa.2015.06.004 ◽

2015 ◽

Vol 153 (2-3) ◽

pp. 150-159 ◽

Cited By ~ 12

Author(s):

M.V. Palmer ◽

T.C. Thacker ◽

W.R. Waters

Keyword(s):

Gene Expression ◽

In Situ Hybridization ◽

Mycobacterium Bovis ◽

Cytokine Gene Expression ◽

Hybridization Method ◽

Cytokine Gene ◽

Chromogenic In Situ Hybridization ◽

Pulmonary Granulomas

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Cytokine RT-qPCR and ddPCR for immunological investigations of the endangered Australian sea lion (Neophoca cinerea) and other mammals

PeerJ ◽

10.7717/peerj.10306 ◽

2020 ◽

Vol 8 ◽

pp. e10306

Author(s):

María-Ignacia Meza Cerda ◽

Rachael Gray ◽

Damien P. Higgins

Keyword(s):

Gene Expression ◽

Dynamic Range ◽

Quantitative Polymerase Chain Reaction ◽

Ovis Aries ◽

Cytokine Gene Expression ◽

Cytokine Gene ◽

Sea Lion ◽

Wildlife Species ◽

Neophoca Cinerea ◽

Australian Sea Lion

Measurement of cytokine gene expression by reverse transcription quantitative polymerase chain reaction (RT-qPCR) is used widely to assess the immune system of animals and to identify biomarkers of disease, but its application is limited in wildlife species due to a lack of species-specific reagents. The free-ranging endangered Australian sea lion (Neophoca cinerea) experiences significant clinical disease and high pup mortality due to intestinal hookworm infection. Developing immunological tools specific to the species will aid in the assessment of drivers of disease and its impact in population demographics. This study describes the development and validation of cross-reactive RT-qPCR assays to measure five important cytokines involved in innate and Th1/Th2 responses (IL-6, TNFα, IFNγ, IL-4 and IL-10) in unstimulated blood samples from a range of different mammalian species including the Australian sea lion. All RT-qPCR assays efficiencies ranged between 87% (Ovis aries TNFα) and 111% (Bos taurus IL-10) and had strong linearity (R2). IL-4 and IFNγ gene expression for N. cinerea fell below the dynamic range (and therefore quantifiable limits) of RT-qPCR assays but were able to be quantified using the novel droplet digital PCR (ddPCR). This study delivers new immunological tools for eco-immunologists studying cytokine gene expression in wildlife species and is to our knowledge, the first cytokine ddPCR approach to be reported in a pinniped species.

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Associations between cytokine gene expression and pathology in Mycobacterium bovis infected cattle

Veterinary Immunology and Immunopathology ◽

10.1016/j.vetimm.2007.05.009 ◽

2007 ◽

Vol 119 (3-4) ◽

pp. 204-213 ◽

Cited By ~ 45

Author(s):

Tyler C. Thacker ◽

Mitchell V. Palmer ◽

W. Ray Waters

Keyword(s):

Gene Expression ◽

Mycobacterium Bovis ◽

Cytokine Gene Expression ◽

Infected Cattle ◽

Cytokine Gene

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Correlation of Cytokine Gene Expression with Pathology in White-Tailed Deer (Odocoileus virginianus) Infected with Mycobacterium bovis

Clinical and Vaccine Immunology ◽

10.1128/cvi.00024-06 ◽

2006 ◽

Vol 13 (6) ◽

pp. 640-647 ◽

Cited By ~ 12

Author(s):

Tyler C. Thacker ◽

Mitchell V. Palmer ◽

W. Ray Waters

Keyword(s):

Gene Expression ◽

Immune Response ◽

Lymph Nodes ◽

Mycobacterium Bovis ◽

Transforming Growth Factor ◽

Cytokine Gene Expression ◽

Effective Vaccine ◽

Cytokine Gene ◽

Ifn Γ ◽

Severe Pathology

ABSTRACT Mycobacterium bovis-infected white-tailed deer (WTD) in northeast Michigan are a reservoir of mycobacteria that pose a threat to both domestic animals and humans. Relatively little work has been done to characterize the immune response of WTD to M. bovis infection; however, an understanding of the immune response to infection and pathogenesis may be critical to the development of an effective vaccine. Immunological responses to infection were characterized by monitoring cytokine gene expression in M. bovis-infected and uninfected deer. Peripheral blood leukocytes (PBL) from infected WTD expressed more gamma interferon (IFN-γ), interleukin-12p40 (IL-12p40), granulocyte-monocyte colony-stimulating factor, and IL-4 mRNA than did PBL from uninfected deer; however, differences were not detected in expression of IL-10 and transforming growth factor-β mRNA. Infected animals could be divided into two groups based on pathology. Lesions were confined primarily to the lymph nodes of the head in animals with less severe pathology. Animals with more severe pathology had lesions in the lung and associated lymph nodes as well as the lymph nodes of the head. More robust IFN-γ mRNA expression correlated with pathology early in infection. These findings indicate that IFN-γ expression likely plays a role in both protection and pathogenesis.

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