Identification of Long Non-coding RNA Isolated From Naturally Infected Macrophages and Associated With Bovine Johne's Disease in Canadian Holstein Using a Combination of Neural Networks and Logistic Regression

Abstract

Mycobacterium avium ssp. paratuberculosis (MAP) causes chronic enteritis in most ruminants. The pathogen MAP causes Johne's disease (JD), a chronic, incurable, wasting disease. Weight loss, diarrhea, and a gradual drop in milk production characterize the disease's clinical phase, culminating in death. Several studies have characterized long non-coding RNA (lncRNA) in bovine tissues, and a previous study characterizes (lncRNA) in macrophages infected with MAP in vitro. In this study, we aim to characterize the lncRNA in macrophages from cows naturally infected with MAP. From 15 herds, feces and blood samples were collected for each cow older than 24 months, twice yearly over 3–5 years. Paired samples were analyzed by fecal PCR and blood ELISA. We used RNA-seq data to study lncRNA in macrophages from 33 JD(+) and 33 JD(–) dairy cows. We performed RNA-seq analysis using the “new Tuxedo” suite. We characterized lncRNA using logistic regression and multilayered neural networks and used DESeq2 for differential expression analysis and Panther and Reactome classification systems for gene ontology (GO) analysis. The study identified 13,301 lncRNA, 605 of which were novel lncRNA. We found seven genes close to differentially expressed lncRNA, including CCDC174, ERI1, FZD1, TWSG1, ZBTB38, ZNF814, and ZSCAN4. None of the genes associated with susceptibility to JD have been cited in the literature. LncRNA target genes were significantly enriched for biological process GO terms involved in immunity and nucleic acid regulation. These include the MyD88 pathway (TLR5), GO:0043312 (neutrophil degranulation), GO:0002446 (neutrophil-mediated immunity), and GO:0042119 (neutrophil activation). These results identified lncRNA with potential roles in host immunity and potential candidate genes and pathways through which lncRNA might function in response to MAP infection.

Regionally Distinct Immune and Metabolic Transcriptional Responses in the Bovine Small Intestine and Draining Lymph Nodes During a Subclinical Mycobacterium avium subsp. paratuberculosis Infection

“…Cows identified naturally infected with MAP (MAPinf) and age-matched tested-negative controls (MAPneg) were selected from a cohort of adult cows from 16 commercial dairy herds as part of a companion project ( 23 ). Animals identified as MAPinf or MAPneg were serially tested during the longitudinal study to identify possible uninfected cows and those with persistent MAP infection.…”

“…Animals selected for this study were from companion research studies ( 19 , 22 , 23 ) which includes 16 commercial dairy farms (tie and free stall) with previously diagnosed JD cases in the province of Québec, Canada. Briefly, cows were at least 24 months old at first sampling and paired blood and faecal samples (collected at the same time) were tested every six months over a one to four year period, as described ( 23 ).…”

“…Animals selected for this study were from companion research studies ( 19 , 22 , 23 ) which includes 16 commercial dairy farms (tie and free stall) with previously diagnosed JD cases in the province of Québec, Canada. Briefly, cows were at least 24 months old at first sampling and paired blood and faecal samples (collected at the same time) were tested every six months over a one to four year period, as described ( 23 ). Blood and feces were respectively tested for the presence of MAP-specific antibodies using the Pourquier ELISA assay (IDEXX Laboratories, Markham, Ontario, Canada) and by fecal quantitative PCR as described ( 22 ), following the manufacturer’s instructions.…”

Whole Genome Methylation Analysis Reveals Role of DNA Methylation in Cow’s Ileal and Ileal Lymph Node Responses to Mycobacterium avium subsp. paratuberculosis Infection

“…Animal selection and diagnosis of MAP status has been described in details in our recent publication (Ibeagha-Awemu et al, 2021). Briefly, animals were from companion studies (Ariel et al, 2020;Marete et al, 2021b;Marete et al, 2021c) which includes 23 commercial dairy farms (tie and free stall) positive for JD and located in the provinces of Québec and Ontario, Canada. Blood and fecal samples collected twice a year and over a period of one to 4 years were diagnosed for MAP status as described previously (Fock-Chow-Tho et al, 2017).…”

“…In addition, an in vitro study found that DNA methylation could regulate NLRP3 promoter activity and its gene expression, thereby implicating DNA methylation in NLRP3 inflammasome activation in response to Mycobacterium tuberculosis infection (Wei et al, 2016). Our recent studies reported that MAP impacted the expression of epigenetics genes (Ariel et al, 2019), the lncRNA pattern of macrophage, and the first host immune cell defense encountered while invading cows (Gupta et al, 2019;Marete et al, 2021b). Moreover, comparing the transcriptome landscape of macrophages and small intestinal tissues and draining lymph nodes from JD positive cows with JD negative cows, we observed modifications that show the apparent importance of immune and metabolic pathways hijacked by MAP to subvert the host immune system (Ariel et al, 2020;Ibeagha-Awemu et al, 2021) suggesting a role of epigenetic processes in host response to MAP infection.…”