Mycoplasma bovis is a critical bovine pathogen, but its pathogenesis remains poorly understood. Here, the virulent HB0801 (P1) and attenuated HB0801-P150 (P150) strains of M. bovis were used to explore the potential pathogenesis and effect of induced immunity from calves’ differential transcriptomes post infection. Nine one-month-old male calves were infected with P1, P150, or mock-infected with medium and euthanized at 60 days post-infection. Calves in P1 group exhibited other clinical signs and pathological changes compared to the other two groups. Transcriptome profiles of peripheral blood mononuclear cells revealed seven and 10 hub differentially expressed genes (DEGs) in P1 and P150 groups compared with mock-infected group, respectively. Then, P1-induced pathogenesis was predicted to be associated with enhanced Th17, and P150-induced immunity with Th1 response and expression of ubiquitination-associated enzymes. Association analysis showed that 14 and 11 DEGs were positively and negatively correlated with pathological changes, respectively. Furthermore, up-regulated expression in molecules critical to differentiation of pathogenic Th17 cells in lung and peripheral blood mononuclear cells in P1 group was validated at RNA and protein levels. The results confirmed virulent and attenuated strains might be associated with biased differentiation of pro-inflammatory pathogenic Th17 and Th1 subsets respectively.
Mycoplasma bovis is a major pathogen, responsible for bovine respiratory diseases worldwide. The present lack of effective control measures leaves cattle owners at considerable perpetual risk of M. bovis outbreaks. In this study, we identified M. bovis secreted immunogenic proteins in silico as potential candidates for novel diagnostic agents and vaccines. We used immunoinformatics to analyze 438 M. bovis proteins previously identified with a label-free proteomics analysis of virulent M. bovis HB0801 (P1) and its attenuated P150 strains. The subcellular localization of these proteins was preliminarily screened and 59 proteins were found to be secreted extracellular proteins. Twenty-seven of these proteins contained a large number of predictive T-cell epitopes presented by major histocompatibility complex (MHC) class I and II molecules. Twenty-two of these 27 proteins had a high number of conformational B-cell epitopes, predicted from the corresponding 3D structural templates, including one unique to P1, two unique to P150, and 19 common to both strains. Five proteins were selected for further validation, and two of these, MbovP274 and MbovP570, were successfully expressed and purified. Both were confirmed to be secretory and highly immunogenic proteins that induced a mouse antibody response, reacted with cattle serum positive for M. bovis infection, and significantly increased the production of interleukin 8 (IL-8), IL-12 and interferon γ (IFN-γ) during the secretion of these three cytokines by both M. bovis mutants of these genes. These results should be useful in the development of novel immunological agents against M. bovis infection.
Mycoplasma bovis (M. bovis) is one of the major pathogens in the bovine respiratory disease complex, which includes pneumonia, mastitis, and arthritis and causes a great economic loss in the cattle industry. In China, a live-attenuated vaccine strain M. bovis P150 was obtained by a continuous culture of the wild-type strain M. bovis HB0801 (P1) in vitro for 150 passages. Using the infected bovine macrophage cell line BoMac, this work attempted to investigate the mechanism of P150 attenuation and protective immune response. To begin, we show that M. bovis P150 effectively triggered cytotoxicity and apoptosis in BoMac, although with lower intracellular survival than P1. The transcriptomes of BoMac after infection with M. bovis strains P1 and P150 were sequenced, and bioinformatic analysis identified 233 differentially expressed genes (DEGs), with 185 upregulated and 48 downregulated. Further Gene Ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analyses revealed that the majority of the DEGs were linked to CHOP complex, MAP kinase phosphatase activity and were involved in the IL-17 signaling pathway in immune response, MAPK signaling pathway in signal transduction, and p53 signaling pathway in cell growth and death. Among them, the level of C/EBP homologous protein (CHOP) was significantly upregulated in P150-infected BoMac compared to P1-infected cells at different time points, along with its upstream and downstream genes phosphorylated-PERK, phosphorylated-EIF2α, ATF4, and GADD45A increased in the PERK-dependent ER stress response. The role of CHOP in apoptosis was further verified by M. bovis-induced siCHOP knockdown in BoMac cells. The results showed that CHOP knockdown enhanced P150-induced apoptosis and dramatically increased the M. bovis P1 and P150 intracellular survival, particularly for P150. These data suggest that P150 infection upregulates CHOP expression, which can increase apoptosis and mediate a crosstalk between ER stress and apoptosis during infection, and hence, contribute to high cytotoxicity and low intracellular survival.
Eliminating rabies is challenging in many developing countries, especially in rural areas. In contrast to the annual decline of human cases in China in last decade, the incidence of rabies in livestock has been increasingly reported. This paper reports the rabies outbreaks in beef cattle (Angus) in Shaanxi Province, China, which caused 31 and 5 deaths at an attack rate of 19.4% (95% CI: 13.6%-26.4%) and 0.25% (95% CI: 0.1%-0.6%) in a satellite cow farm (farm A) and a core intensive farm (farm B), respectively. The rabies infection was confirmed by several laboratory tests, and rabies virus (RABV) strains SXBJ15 and SXYL15 were isolated and characterized from farm A and B, respectively. The two strains were found to have a high genomic sequence similarity to the dog-associated China clade I strains previously identified in the neighbouring area. SXBJ15 was shown to have a higher mouse pathogenicity (1.07) than SXYL15 (0.45). RABV was also detected in the saliva and salivary glands from the affected cattle. The potential causes were investigated on the farm, and the biosecurity scores were 20 and 64 (a full score of 82) for farms A and B, respectively.The rabies infection is likely to result from rabid free-roaming dogs (FRDs). On farm A with more cow deaths, the rabies transmission between animals can be attributed to the improper disposal of aborted foetuses and placental materials as a food source for rabid FRDs, high stocking density and drinking water sharing. Additionally, vaccinating cattle with a canine vaccine was shown to help stop the spread of rabies in herds. These results indicate that the occurrence of RABV on cattle farms can be prevented by improving biosecurity measures to control the entry of rural FRDs on the farm and immunizing farm cattle against rabies.
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