Innate immune markers that distinguish red deer (Cervus elaphus) selected for resistant or susceptible genotypes for Johne's disease

Abstract

While many factors contribute to resistance and susceptibility to infectious disease, a major component is the genotype of the host and the way in which it is expressed. Johne's disease is a chronic inflammatory bowel disease affecting ruminants and is caused by infection with Mycobacterium avium subspecies paratuberculosis (MAP). We have previously identified red deer breeds (Cervus elaphus) that are resistant; have a low rate of MAP infection and do not progress to develop Johne's disease. In contrast, susceptible breeds have a high rate of MAP infection as seen by seroconversion and progress to develop clinical Johne's disease. The aim of this study was to determine if immunological differences exist between animals of resistant or susceptible breeds. Macrophage cultures were derived from the monocytes of deer genotypically defined as resistant or susceptible to the development of Johne's disease. Following in vitro infection of the cells with MAP, the expression of candidate genes was assessed by quantitative PCR as well as infection rate and cell death rate. The results indicate that macrophages from susceptible animals show a significantly higher upregulation of inflammatory genes (iNOS, IL-1α, TNF-α and IL-23p19) than the macrophages from resistant animals. Cells from resistant animals had a higher rate of apoptosis at 24 hours post infection (hpi) compared to macrophages from susceptible animals. The excessive expression of inflammatory mRNA transcripts in susceptible animals could cause inefficient clearing of the mycobacterial organism and the establishment of disease. Controlled upregulation of inflammatory pathways coupled with programmed cell death in the macrophages of resistant animals may predispose the host to a protective immune response against this mycobacterial pathogen.

Figure 1

Resistance and susceptibility to Johne’s disease in a red deer stud herd. (A) Diagnosis of MAP infection by Paralisa™ in animals of distinct pure breeds (anonymised to A – G, n = 1135 animals total). Differences in reactivity rate are statistically significant (p < 0.01) between breeds with the exception of differences between Breeds A, C and G. (B) Deaths from clinical Johne’s disease in the different pure breeds (A – G) recorded over a 6 year period prior to serological testing (n = 1244 animals total). Differences in death rate are statistically significant (p < 0.01) between breeds A – C and breeds E – G. A high level of exposure of all animals confirms that the resistant breeds, with low incidence of MAP infection and no deaths from Johne’s disease (breeds A – C), have a resistant phenotype and do not include unexposed indeterminate animals.

Figure 2

Characteristics of mature deer monocyte-derived macrophages. (A) Monocyte-derived macrophages stained positive (black) for α-naphthyl esterase at day 7 of culture (20X magnification). This enzyme is detected primarily in monocytes and macrophages and is virtually absent from granulocytes and other leukocytes, confirming that these adherent cells derived from deer blood are of a monocytic lineage. (B) Ziehl-Nielsen stain of deer MDM (blue nucleus) which has phagocytosed MAP bacilli; red, acid-fast microorganisms (100X magnification).

Figure 3

Relative mRNA transcript expression of MDM from purebred animals of a resistant or susceptible genotype. MDM were infected with MAP for 24 h before expression of the genes iNOS, IL-1α, TNF-α, IL-23p19, IL-12p35 and IL-10 was measured by Q-PCR. Fold changes (mean ± SEM) are defined as expression upon infection compared to the uninfected control, n = 20 (10 resistant, 3 intermediate and 7 susceptible). Three animals of susceptible genotype were removed from the susceptible group into an “intermediate” category on observing that their macrophage gene expression profiles resembled a resistant-type animal for the iNOS, IL-1α, TNF-α and IL-23p19 molecules. Statistical significance has been calculated using the Mann–Whitney test where ** denotes p < 0.01 and *** denotes p < 0.001.

Figure 4

Relative mRNA transcript expression of MDM from crossbred animals of a resistant or susceptible genotype. MDM were infected with MAP for 24 h before expression of the genes iNOS, IL-1α, TNF-α, IL-23p19, IL-12p35 and IL-10 was measured by Q-PCR. Fold changes (mean ± SEM) are defined as levels of expression after infection compared to the uninfected control, n = 13 (6 resistant and 7 susceptible). No statistically significant differences were observed between the resistant and susceptible crossbred groups.

Figure 5

Fluorescent microscopy images. (A) Hoechst 33342 Stain (Nucleus), (B) TB Auramine M Stain (acid-fast MAP), (C) TUNEL TMR Red Stain (Apoptosis), (D) Overlay of the images, arrow pointing to macrophage containing an acid-fast bacilli (20X magnification).

Figure 6

Infection dynamics of MDM from resistant or susceptible deer, infected with MAP in vitro. (A) Infection rate of macrophages from crossbred red deer of a resistant or susceptible genotype. MDM were isolated from crossbred red deer and cultured for 7 days on slide flasks. At day 7 of MDM culture, the cells were infected with MAP (MOI of 10:1) for 24 or 48 h and the resulting MAP infection rate was determined by auramine staining. The data are expressed as percentages of MAP-infected cells (mean ± SEM) and at least 75 cells per slide were counted, n = 13 (6 resistant and 7 susceptible). (B) Numbers of MAP bacilli per infected macrophage from crossbred red deer of a resistant or susceptible genotype. The data are expressed as percentages of MAP-infected macrophages that contain different numbers (≤ 10, 10 – 20, ≥ 20) of MAP bacilli at 24 and 48 hours after infection (mean ± SEM), n = 13 (6 resistant and 7 susceptible).

Figure 7

Cell death in MDM from resistant or susceptible deer following in vitro MAP infection. (A) Detection of apoptosis by TUNEL TMR red staining of MDM from crossbred animals of resistant or susceptible genotype at 24 or 48 h after MAP infection. Results are shown as mean percentage TUNEL positive cells ± SEM, n = 8 (4 resistant and 4 susceptible). Un = untreated, MAP = MAP-infected, STS = staurosporine at 200 ng/mL for 6 h (positive control). (B) Detection of total cell death by LDH release from MDM isolated from crossbred animals of resistant or susceptible genotype. Following 24 or 48 h MAP infection, supernatants of cultures were collected and analysed for LDH release. Results are given as mean absorbance values ± SEM, n = 13 (6 resistant and 7 susceptible).