First report and genomic characterization of a bovine-like coronavirus causing enteric infection in an odd-toed non-ruminant species (Indonesian tapir, Acrocodia indica) during an outbreak of winter dysentery in a zoo.

Bovine coronavirus (BCoV) is associated with three distinct clinical syndromes in cattle that is, neonatal diarrhoea, haemorrhagic diarrhoea in adults (the so-called winter dysentery syndrome, WD) and respiratory infections in cattle of different ages. In addition, bovine-like CoVs have been detected in various species including domestic and wild ruminants. However, bovine-like CoVs have not been reported so far in odd-toed ungulates. We describe an outbreak of WD associated with a bovine-like CoV affecting several captive wild ungulates, including Indonesian tapirs (Acrocodia indica) an odd-toed ungulate species (Perissodactyla) which, with even-toed ungulates species (Artiodactyla) form the clade Euungulata. Genomic characterization of the CoV revealed that it was closely related to BCoVs previously reported in America. This case illustrates the adaptability of bovine-like CoVs to new species and the necessity of continued surveillance of bovine-like CoVs in various species.

Detection and genome characterization of bovine kobuvirus (BKV) in faecal samples from diarrhoeic calves in Quebec, Canada.

Kobuviruses are known to infect the gastrointestinal tract of different animal species. Since its discovery in 2003, bovine kobuvirus (BKV) has been identified in faecal samples from diarrhoeic cattle in many countries, but only recently in North America. Although its possible role as an agent of calf diarrhoea remains to be determined, evidence is mounting. Our study reports for the first time the detection of BKV in faecal samples from diarrhoeic calves raised in Quebec, Canada. BKV was more commonly identified than eight known and common enteric calf pathogens. Further sequence analysis revealed that Canada BKV strain 1,043,507 was more closely correlated with the US BKV IL35164 strain than other BKV strains with complete genome. Continued surveillance and genomic characterization are needed to monitor BKV in the cattle around the world.

Influenza D virus in respiratory disease in Canadian, province of Québec, cattle: Relative importance and evidence of new reassortment between different clades.

BACKGROUND: Influenza D virus (IDV), a segmented single-stranded negative-sense ribonucleic acid (RNA) virus, belongs to the new Delta influenza virus genus of the Orthomyxoviridae family. Cattle were proposed as the natural reservoir of IDV in which infection was associated with mild-to-moderate respiratory clinical signs (i.e. cough, nasal discharge and dyspnoea). METHODS AND PRINCIPAL FINDINGS: In order to investigate the role of IDV in bovine respiratory disease, during the period 2017-2020, 883 nasal or naso-pharyngeal swabs from Canadian cattle with respiratory signs (cough and/or dyspnoea) were tested by (RT-)qPCR for IDV and other major bovine viral (bovine herpesvirus 1, bovine viral diarrhoea virus, bovine respiratory syncytial virus, bovine parainfluenza virus 3 and bovine coronavirus) and bacterial (Mannheimia haemolytica, Pasteurella multocida, Histophilus somni and Mycoplasma bovis) respiratory pathogens. In addition, whole-genome sequencing and phylogenetic analyses were carried out on five IDV-positive samples. The prevalence of IDV RT-qPCR (with cut-off: Cq < 38) at animal level was estimated at 5.32% (95% confidence interval: 3.94-7.02). Positive result of IDV was significantly associated with (RT-)qPCR-positive results for bovine respiratory syncytial virus and Mycoplasma bovis. While phylogenetic analyses indicate that most segments belonged to clade D/660, reassortment between clades D/660 and D/OK were evidenced in four samples collected in 2018-2020. CONCLUSIONS AND SIGNIFICANCE: Relative importance of influenza D virus and associated pathogens in bovine respiratory disease of Canadian dairy cattle was established. Whole-genome sequencing demonstrated evidence of reassortment between clades D/660 and D/OK. Both these new pieces of information claim for more surveillance of IDV in cattle production worldwide.

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.

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.

Genome-wide association analysis identified both RNA-seq and DNA variants associated to paratuberculosis in Canadian Holstein cattle 'in vitro' experimentally infected macrophages.

BACKGROUND: Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of paratuberculosis, or Johne's disease (JD), an incurable bovine disease. The evidence for susceptibility to MAP disease points to multiple interacting factors, including the genetic predisposition to a dysregulation of the immune system. The endemic situation in cattle populations can be in part explained by a genetic susceptibility to MAP infection. In order to identify the best genetic improvement strategy that will lead to a significant reduction of JD in the population, we need to understand the link between genetic variability and the biological systems that MAP targets in its assault to dominate macrophages. MAP survives in macrophages where it disseminates. We used next-generation RNA (RNA-Seq) sequencing to study of the transcriptome in response to MAP infection of the macrophages from cows that have been naturally infected and identified as positive for JD (JD (+); n = 22) or negative for JD (healthy/resistant, JD (-); n = 28). In addition to identifying genetic variants from RNA-seq data, SNP variants were also identified using the Bovine SNP50 DNA chip. RESULTS: The complementary strategy allowed the identification of 1,356,248 genetic variants, including 814,168 RNA-seq and 591,220 DNA chip variants. Annotation using SnpEff predicted that the 2435 RNA-seq genetic variants would produce high functional effect on known genes in comparison to the 33 DNA chip variants. Significant variants from JD(+/-) macrophages were identified by genome-wide association study and revealed two quantitative traits loci: BTA4 and 11 at (P < 5 × 10- 7). Using BovineMine, gene expression levels together with significant genomic variants revealed pathways that potentially influence JD susceptibility, notably the energy-dependent regulation of mTOR by LKB1-AMPK and the metabolism of lipids. CONCLUSION: In the present study, we succeeded in identifying genetic variants in regulatory pathways of the macrophages that may affect the susceptibility of cows that are healthy/resistant to MAP infection. RNA-seq provides an unprecedented opportunity to investigate gene expression and to link the genetic variations to biological pathways that MAP normally manipulate during the process of killing macrophages. A strategy incorporating functional markers into genetic selection may have a considerable impact in improving resistance to an incurable disease. Integrating the findings of this research into the conventional genetic selection program may allow faster and more lasting improvement in resistance to bovine paratuberculosis in dairy cattle.

Transcriptome Profiling of Bovine Macrophages Infected by Mycobacterium avium spp. paratuberculosis Depicts Foam Cell and Innate Immune Tolerance Phenotypes.

Mycobacterium avium spp. paratuberculosis (MAP) is the causative agent of Johne's disease (JD), also known as paratuberculosis, in ruminants. The mechanisms of JD pathogenesis are not fully understood, but it is known that MAP subverts the host immune system by using macrophages as its primary reservoir. MAP infection in macrophages is often studied in healthy cows or experimentally infected calves, but reports on macrophages from naturally infected cows are lacking. In our study, primary monocyte-derived macrophages (MDMs) from cows diagnosed as positive (+) or negative (-) for JD were challenged in vitro with live MAP. Analysis using next-generation RNA sequencing revealed that macrophages from JD(+) cows did not present a definite pattern of response to MAP infection. Interestingly, a considerable number of genes, up to 1436, were differentially expressed in JD(-) macrophages. The signatures of the infection time course of 1, 4, 8, and 24 h revealed differential expression of ARG2, COL1A1, CCL2, CSF3, IL1A, IL6, IL10, PTGS2, PTX3, SOCS3, TNF, and TNFAIP6 among other genes, with major effects on host signaling pathways. While several immune pathways were affected by MAP, other pathways related to hepatic fibrosis/hepatic stellate cell activation, lipid homeostasis, such as LXR/RXR (liver X receptor/retinoid X receptor) activation pathways, and autoimmune diseases (rheumatoid arthritis or atherosclerosis) also responded to the presence of live MAP. Comparison of the profiles of the unchallenged MDMs from JD(+) vs. JD(-) cows showed that 868 genes were differentially expressed, suggesting that these genes were already affected before monocytes differentiated into macrophages. The downregulated genes predominantly modified the general cell metabolism by downregulating amino acid synthesis and affecting cholesterol biosynthesis and other energy production pathways while introducing a pro-fibrotic pattern associated with foam cells. The upregulated genes indicated that lipid homeostasis was already supporting fat storage in uninfected JD(+) MDMs. For JD(+) MDMs, differential gene expression expounds long-term mechanisms established during disease progression of paratuberculosis. Therefore, MAP could further promote disease persistence by influencing long-term macrophage behavior by using both tolerance and fat-storage states. This report contributes to a better understanding of MAP's controls over the immune cell response and mechanisms of MAP survival.