Search Results (80)

Bovine Respiratory Disease (BRD) represents one of the largest causes of economic loss and animal morbidity in the global cattle industry, second only to neonatal diarrhea. Its etiology is complex, originating from a multifactorial combination of host susceptibility, environmental stressors, viral infections, and secondary bacterial pathogens. Although viruses are often the initial cause of disease, suppressing the host’s respiratory defense mechanisms, most of the severe pneumonic damage and clinical signs can be attributed to bacterial infections. This review provides an overview of the primary bacterial agents identified within the BRD complex, including Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, and Mycoplasma bovis. We discuss their role as commensals that then become opportunistic pathogens, and further how they interact in a synergistic relationship with a primary viral insult, leading to the resulting pathogenesis and the development of pneumonia. This manuscript discusses in further detail some of the challenges in BRD management, such as the limitations of current diagnostic methodologies, overreliance on antimicrobial therapy, and the growing concern of antimicrobial resistance. Lastly, the need for integrated approaches in management, better husbandry and biosecurity, coupled with the development of novel therapeutic alternatives, is underlined as a means of assuring a sustainable control of this serious syndrome. Full article

Accurate pathogen detection in bovine semen is crucial for animal health surveillance and international trade. Semen presents unique challenges due to the presence of PCR inhibitors from seminal plasma and extender components, reducing nucleic acid extraction efficiency and sensitivity. The two National Animal Health Laboratory Network-approved extraction platforms (MagMAX CORE and IndiMag Pathogen Kits) were evaluated using 88 negative extended semen samples at 200 µL input volume, reduced input volumes, and pretreatment strategies with two influenza A virus (IAV) PCR assays, containing different exogenous internal controls (ICs) to assess PCR inhibition. The ICs yielded overall passing rates from 31.8% to 100.0% and varied greatly based on the extender formulation and extraction protocol. Validation continued with naturally infected semen containing Mycoplasma bovis, bovine viral diarrhea virus, bovine herpesvirus-1, and the limit of detection using Mycoplasma bovis. The IndiMag Pathogen 100-na was then selected for evaluation of diagnostic sensitivity and specificity, reproducibility, and detection limits with IAV-spiked samples, using the two IAV PCR assays and their ICs. Selected archived semen samples used in this study were screened and were negative for IAV by both PCR assays. These findings underscore the importance of tailored extraction methods in overcoming semen-associated inhibition and facilitating reliable pathogen surveillance in bovine germplasm. Full article

Mixed infections of Mannheimia haemolytica and Mycoplasma bovis are relatively common in bovine respiratory diseases, presenting severe respiratory symptoms and high mortality that severely endanger the cattle industry. In this study, a serotype A1 strain of Mannheimia haemolytica, designated as XJCJMh1, was isolated and identified from the lung tissue of a hybrid Simmental calf infected with Mycoplasma bovis. The pathogenicity of this strain was evaluated using Kunming mice as a model. The results indicated that infection with XJCJMh1 caused pathological manifestations such as pulmonary hemorrhage and edema in mice. Subsequently, the genome of this strain was sequenced and assembled using Illumina sequencing to obtain general genomic features. The genome was annotated and analyzed for gene functions using the Swiss-Prot, NR, GO, COG, KEGG, CAZy, TCDB, and Pfam databases. Additionally, the virulence factors and resistance genes of this strain were annotated using the PHI, VFDB, and CARD databases. The genome of Mannheimia haemolytica XJCJMh1 is 2,595,489 base pairs (bp) in length, with a GC content of 40.93%. Notably, this strain exhibits three distinct genomic islands and contains 98 effectors associated with the type III secretion system (T3SS). The XJCJMh1 strain harbors 74 virulence genes and 45 resistance genes. We annotated the proteins, genes, and associated GO and KEGG pathways of the XJCJMh1 strain; exploring the relationship between these annotations and the strain’s pathogenicity is of considerable value. This study is of great significance for clarifying the pathogenic mechanism and genetic characteristics of the Mannheimia haemolytica strain XJCJMh1 in cattle, and its results provide a scientific reference for analyzing the genomic basis of pathogenicity and drug resistance of Mannheimia haemolytica under co-infection conditions. Full article

This study investigated the possible occurrence of pulmonary disease in beef cattle from 13 municipalities within the State of Mato Grosso (MT), Brazil. The state of MT is a leading player in beef cattle production in Brazil, but with comparatively few data relative to the occurrence of pulmonary disease or ovine gammaherpesvirus 2 (OvGHV2)-related infections in cattle. Pulmonary samples from 44 beef cattle, with ages ranging between 18 and 28 months, were collected during slaughter and processed to determine the patterns of pulmonary lesions. Additionally, duplicate fragments were used in immunohistochemical (IHC) assays designed to detect malignant catarrhal fever (MCFV) antigens and in molecular assays to amplify 10 agents associated with the development of bovine respiratory disease (BRD). Interstitial pneumonia (IP) was diagnosed in most of the lungs (98%; 43/44) evaluated from all municipalities. MCFV antigens were detected in 37% (16/43) of the animals with IP. Only four pathogens were amplified by molecular assays within the lungs of cattle with IP: OvGHV2 (23%; 10/43), bovine viral diarrhea virus (12%; 5/43), bovine coronavirus (7%; 3/43), and Mannheimia haemolytica (2%; 1/43). The nucleic acids of bovine respiratory syncytial virus, bovine alphaherpesvirus 1, bovine parainfluenza virus 3, Pasteurella multocida, Histophilus somni, and Mycoplasmopsis (Mycoplasma) bovis were not amplified. Singular infections in cattle from municipalities were associated with MCFV (n = 3) and OvGHV2 (n = 2), while OvGHV2 occurred in all dual and triple infections. However, in four animals with IP, none of the disease pathogens identified were detected. Statistically, MCFV antigens were more frequently (p = 0.048) detected in the lungs of female (75%; 12/16) cattle with IP relative to males (25%; 4/16). Additionally, there was a positive correlation (p < 0.001) between the IHC detection of MCFV antigens within the bronchial epithelium and the epithelium of peribronchial glands of cattle with IP. This is the first study to statistically demonstrate that female cattle are at greater risk of developing MCFV-related infections as compared to male animals. The detection of OvGHV2 in singular and multiple infections during this investigation supports earlier studies that associate this pathogen with the development of pulmonary disease in cattle, indicating that OvGHV2 can contribute to the etiology of IP. Additionally, the detection of OvGHV2-induced infections in asymptomatic cattle suggests that all animals were subclinically infected, confirming that subclinically OvGHV2-induced infections may be widespread in ruminants from Brazil. Furthermore, the occurrence of atypical interstitial pneumonia cannot be discarded, particularly in animals with IP but without any associated pathogen. These initial findings suggest the need for a more elaborate investigation to understand the dynamics of pulmonary disease within this state. Full article

Mycoplasma bovis (M. bovis) adheres to host cells and persists intracellularly, causing chronic inflammation and significant economic losses in the cattle industry. The role of host cell apoptosis in this host–pathogen interaction remains unclear. This study isolated and identified the M. bovis Xinjiang strain XJ01 from diseased cattle in China. XJ01 exhibited typical “fried egg” colony morphology, distinct biochemical characteristics, and a 1.02 Mb genome (29.33% GC content) encoding 939 genes, including 93 unique genes. Functional analysis under optimal infection conditions (MOI = 1000, 24 h) revealed that XJ01 induced significant apoptosis and reduced viability in bovine macrophages (BoMac). This was accompanied by mitochondrial homeostasis disruption, characterized by increased Bax expression and suppressed Bcl-2 levels. Transcriptome analysis identified 9926 differentially expressed genes. KEGG pathway enrichment indicated significant activation of apoptosis and P53 signaling pathways, with Gadd45 and XIAP identified as key regulators. Mechanistic validation demonstrated that Gadd45 overexpression or XIAP knockdown enhanced Bax expression, inhibited Bcl-2, increased apoptosis rates, and consequently significantly reduced intracellular bacterial load at 24 h post-infection. Conversely, suppressing Gadd45 or overexpressing XIAP promoted pathogen survival. Collectively, this study reveals that M. bovis XJ01 activates host stress signaling to upregulate Gadd45 and suppress XIAP, thereby triggering mitochondrial apoptosis as a mechanism to eliminate intracellular bacteria—illustrating a self-limiting antibacterial mechanism. Full article

Mycoplasma bovis is an important pathogen that is associated with respiratory diseases, mastitis, and arthritis in cattle, leading to significant economic losses in the global cattle industry. Most notably in this study, we pioneer the discovery that its secreted effector ENO1 (α-enolase) directly targets host cytoskeletal proteins for metabolic–immune regulation. Using an innovative GST pull-down/mass spectrometry approach, we made the seminal discovery of β-actin (ACTB) as the primary host target of ENO1—the first reported bacterial effector–cytoskeleton interaction mediating metabolic reprogramming. ENO1–ACTB binding depends on a hydrogen bond network involving ACTB’s 117Glu and 372Arg residues. This interaction triggers (1) glycolytic activation via Glut1 upregulation, establishing Warburg effect characteristics (lactic acid accumulation/ATP inhibition), and (2) ROS-mediated activation of dual inflammatory axes (HIF-1α/IL-1β and IL-6/TNF-α). This work establishes three groundbreaking concepts: (1) the first evidence of a pathogen effector hijacking host ACTB for metabolic manipulation, (2) a novel ‘glycolysis–ACTB–ROS-inflammation’ axis, and (3) the first demonstration of bacterial proteins coordinating a Warburg effect with cytokine storms. These findings provide new targets for anti-infection therapies against Mycoplasma bovis. Full article

Bovine respiratory disease complex (BRDC) is one of the primary causes of morbidity, mortality, and economic loss in cattle worldwide. Accurate and rapid identification of causative pathogenic agents is essential for effective disease management and control. In this study, a novel multiplex fluorescence-based quantitative polymerase chain reaction (qPCR) assay was developed for the simultaneous detection of eight major pathogens associated with BRDC. The targeted pathogens included the following: bovine viral diarrhea virus (BVDV), bovine parainfluenza virus type 3 (BPIV3), bovine respiratory syncytial virus (BRSV), bovine coronavirus (BcoV), Mycoplasma bovis (M.bovis), Pasteurella multocida (PM), Mannheimia haemolytica (MH), and infectious bovine rhinotracheitis virus (IBRV). The assay was rigorously optimized to ensure high specificity with no cross-reactivity among targets. The limit of detection (LOD) was determined to be as low as 5 copies per reaction for all target pathogens. The coefficient of variation (CVs) for both intra-assay and inter-assay measurements were consistently below 2%, demonstrating excellent reproducibility. To validate the clinical utility of the assay, a total of 1012 field samples were tested, including 504 nasal swabs from Farm A and 508 from Farm B in Jiangsu Province. BVDV, BcoV, PM, and MH were detected from Farm A, with a BVDV-positive rate of 21.63% (109/504), BcoV-positive rate of 26.79% (135/504), PM-positive rate of 28.77% (145/504), and MH-positive rate of 15.08% (76/504). Also, BcoV, PM, MH, and IBRV were detected from Farm B, with a BcoV-positive rate of 2.36% (12/508), PM-positive rate of 1.38% (7/508), MH-positive rate of 14.76% (75/508), and IBRV-positive rate of 5.51% (28/508). Notably, a significant proportion of samples showed evidence of mixed infections, underscoring the complexity of BRDC etiology and the importance of a multiplex diagnostic approach. In conclusion, the developed multiplex qPCR assay provides a reliable, rapid, and cost-effective tool for simultaneous detection of multiple BRDC-associated pathogens, which will hold great promise for enhancing disease surveillance, early diagnosis, and targeted intervention strategies, ultimately contributing to improved BRDC management and cattle health outcomes. Full article

Mycoplasma bovis is the most common mycoplasma associated with cattle diseases worldwide. However, other seemingly less virulent Mycoplasma spp. such as M. bovigenitalium and M. bovirhinis have also been associated with mycoplasmosis. The study objective was to compare the adhesion and cellular invasion characteristics of these bovine Mycoplasma spp. using Madin–Darby Bovine Kidney (MDBK) epithelial cells. MDBK cells were separately infected with 12 M. bovis strains and one strain each of M. bovigenitalium and M. bovirhinis. Following infection, a gentamicin protection assay was performed and the cells lysed at 6 and 54 h post-infection. The MDBK cell lysates were cultured for Mycoplasma spp. and qPCR was used to estimate the average number of Mycoplasma bacterial cells that infected each MDBK cell (Myc/Cell ratio). Confocal and electron microscopy studies using M. bovis mNeonGreen strain were also performed. All 14 Mycoplasma strains multiplied within the MDBK cells, a finding confirmed by microscopy studies of the M. bovis mNeonGreen strain. Unexpectedly, the M. bovis strains, obtained from diseased and asymptomatic cattle and bison, had lower Myc/Cell ratios than M. bovirhinis and M. bovigenitalium strains. These findings suggest that the ability for mycoplasmas to invade and replicate within host cells does not account for the differences in virulence between species. Full article

Mycoplasma bovis is one of the most important pathogens in animal husbandry, and the current infection and morbidity rates are increasing year by year, causing great losses to the farming industry and seriously affecting animal welfare. In this study, we took tracheal tissues from calves infected with M. bovis to make pathological tissue sections for observation, and selected tracheal tissues for transcriptome sequencing to screen differentially expressed genes based on the threshold |log2FoldChange| > 1 and Padjust < 0.05 and functional enrichment, to explore in depth the potential mechanisms of bovine tracheal damage caused by bovine tracheitis. Experiments were conducted to observe the changes in tracheal tissues after M. bovis infection through pathological sections of the trachea of M. bovis-infected calves. From the transcriptome sequencing results, we mined the main differential genes and important metabolic pathways of M. bovis causing damage to the trachea of calves. It was found that the cricoid cartilage tissue of the trachea was congested and hemorrhagic after M. bovis infection in calves, and the pathological sections showed localized necrosis of epithelial cells, disorganization, high inflammatory cell infiltration in the interepithelial and lamina propria, and some epithelial cell detachment. Transcriptome sequencing identified 4199 DEGs, including 1378 up-regulated genes and 2821 down-regulated genes. KEGG enrichment analysis indicated that the differential genes were enriched to 59 significantly differing signaling pathways, and a number of important metabolic pathways related to tracheitis induced by M. bovis-infected calves were unearthed. The major ones included IL-17, the Toll-like receptor, JAK/STAT, the PI3K-Akt signaling pathway, etc. In this study, we found that M. bovis infection of calves caused inflammatory damage to the trachea, and transcriptome sequencing results also showed significant differences in the expression of key genes such as IL-6 inflammatory factor, CASP8, and APOA1. Full article

Bovine respiratory disease (BRD) is a major economic and animal welfare issue in the beef industry. Mycoplasma bovis is one of the main causal organisms, particularly in chronic cases. Due to the difficulty of isolating M. bovis from clinical isolates, there is a lack of information on the genetic diversity of this pathogen in the Texas panhandle region of the United States. Therefore, our objective was to provide genome-level characterization of M. bovis isolated from the lung lesions of beef and dairy cattle in the Texas panhandle. Fifty-four isolates displaying mycoplasma-like growth were recovered from bovine lung lesions by the Texas Veterinary Medical Diagnostic Laboratory in 2021 and 2022. Of these isolates, 32 were determined to be M. bovis via species-specific qPCR using the uvrC gene. Long-read whole-genome sequencing was used to identify key virulence factors, antimicrobial resistance genes, and to assess the genetic diversity of these isolates. Fisher’s exact tests were used to identify associations between isolate characteristics and host metadata, including the state of origin, type of operation, animal age, and animal sex. Our results indicate that there is considerable genetic diversity among the M. bovis isolates, despite their shared geography in the Texas panhandle, though significant clustering based on host metadata was observed. Analysis of the pangenome showed that the M. bovis isolates in this study also harbor a diverse array of virulence genes, but no antimicrobial resistance genes were identified in this study. Full article

Mycoplasma bovis (M. bovis) has caused huge economic losses to the cattle industry. The interaction between M. bovis and host cells is elucidated by screening and identifying the target protein of M. bovis adhesin on the surface of the host cell membrane. However, the response mechanism of embryonic bovine lung (EBL) cells to M. bovis infection is not yet fully understood. Additionally, it is necessary to further explore whether infection with M. bovis induces oxidative stress and mitochondrial damage in EBL cells. In this study, oxidation reaction, mitochondrial membrane potential, mitochondrial structure, and apoptosis ability of EBL cells infected with M. bovis were assessed at different times (12, 24, 48 h post-infection; hpi). Then, the differential proteomic analysis of M. bovis-infected EBL cells at 12 h and 24 h was performed with uninfected cells as the control. The results showed that M. bovis infection reduced the antioxidant capacity of EBL cells, increased ROS levels, and decreased mitochondrial membrane potential. The mitochondrial membrane of EBL cells was damaged, and the ridge arrangement was disordered after infection by transmission electron microscopy. With the increase in infection time, the mitochondrial matrix partially dissolved and spilled. The apoptosis rate of EBL cells increased with the increase in infection time of M. bovis. Furthermore, proteomic analysis identified 268 and 2061 differentially expressed proteins (DEPs) at 12 hpi and 24 hpi, respectively, compared with the uninfected cells. According to GO analysis, these DEPs were involved in the mitosis and negative regulation of cell growth. Additionally, the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated the following pathways were linked to mitochondrial damage or cell growth regulation, including glycolysis/gluconeogenesis, pentose phosphate pathway, oxidative phosphorylation, AMPK, cGMP-PKG, cAMP, calcium, Wnt, Phospholipase D, apoptosis, MAPK, cell cycle, Ras, PI3K-Akt, mTOR, HIF-1. PPI results indicated that YWHAZ, PIK3CA, HSP90AB1, RAP1A, TXN, RAF1, MAPK1, PKM, PGK1, and GAPDH might be involved in mitochondrial pathway apoptosis induced by M. bovis infection. This study offers helpful data toward understanding the response of mitochondria of EBL cells to M. bovis infection. Full article

The rise in antimicrobial resistance (AMR) in Mycoplasma bovis underscores the urgent need for alternative treatments. This study evaluated the minimal inhibitory concentrations (MICs) of four metal ions (cobalt, copper, silver, and zinc) and colloidal silver against 15 clinical M. bovis isolates, alongside conventional antimicrobials (florfenicol, tetracycline, tulathromycin, and tylosin). Colloidal silver demonstrated the most effective antimicrobial activity, inhibiting 81.25% of isolates at 1.5 mg/L, while silver inhibited 93.7% of isolates at concentrations above 1.5 mg/L. Copper exhibited notable efficacy, inhibiting 37.5% of isolates at 1.5 mg/L, with a small proportion responding at 0.1 mg/L. Cobalt and zinc displayed variable activity, with MIC values ranging from 0.7 to 12.5 mg/L. In contrast, conventional antimicrobials showed limited effectiveness: tetracycline inhibited 31.25% of isolates at ≥16 mg/L, tylosin inhibited 25% at 16 mg/L, and tulathromycin MICs ranged from 0.5 to 8 mg/L. Time–kill assays revealed a reduction in M. bovis viability after eight hours of exposure to silver and colloidal silver, though higher concentrations (4×–8× MIC) were required for complete eradication. These findings highlight the significant potential of colloidal silver and copper as alternatives for treating M. bovis infections and combating AMR. Further research is essential to explore their standalone and synergistic applications for therapeutic use. Full article

Mycoplasma spp. are facultative pathogens that contribute to the pathogenesis of multiple bovine diseases, including the bovine respiratory disease complex, and have been shown to form biofilms. Biofilm formation is associated with increased antibiotic resistance in many organisms, but accurate determination of antimicrobial susceptibility in biofilms is challenging. In Mycoplasma spp., antimicrobial susceptibility is routinely determined using metabolic pH-dependent color change. However, biofilm formation can lead to reduced metabolism, making interpretation of metabolic readouts difficult. Therefore, we developed and optimized a new flow cytometry-based method for antimicrobial susceptibility testing in biofilm-forming Mycoplasma, termed the live/dead antimicrobial susceptibility test (LD-AST). The LD-AST measures the proportion of live bacteria upon exposure to antibiotics, works robustly with both planktonic and biofilm cultures, and enables the determination of the minimum bactericidal concentration (MBC) for a given antibiotic. We used two strains of Mycoplasma bovis (Donetta PG45 and Madison) and two clinical Mycoplasma bovoculi isolates (MVDL1 and MVDL2) to determine the impact of biofilm growth on antimicrobial susceptibility for gentamicin, enrofloxacin, or tetracycline. All Mycoplasma strains were susceptible to all antibiotics when cultured as planktonic cells, with MBCs in the expected range. However, three out of four strains (Donetta PG45, MVDL1, and MVDL2) were completely resistant to all three antibiotics when newly adhered biofilms were analyzed, whereas M. bovis Madison gave variable results. For mature biofilms that were cultured for 4–5 days before antibiotic exposure, results also were variable, with some strains showing an increased resistance with certain antibiotics and a decreased resistance with others. Overall, these results are consistent with earlier reports that biofilms can exhibit increased antimicrobial resistance. Full article

Mycoplasma bovis (M. bovis) is characterized by a reduced genomic size and limited synthetic capacity, including the inability to synthesize nucleotides de novo, relies on nucleases for nutrient acquisition and survival. A number of nucleases have been implicated in M. bovis pathogenicity, facilitating substrate degradation and contributing to DNA repair mechanisms that enhance bacterial persistence. The present study confirmed that the T5.808 mutant, in which a novel nuclease gene (Mbov_0701) was disrupted by the mini-Tn4001 transposon, exhibits a growth defect when co-cultured with EBL cells. However, the restoration of Mbov_0701 resulted in the resumption of growth in the mutant. The characterization of MbovP701 revealed that it had high activity in hydrolyzing dsDNA with 5′- to 3′- polarity. Furthermore, the substrates of MbovP701 were extended to include linear dsDNA, ssDNA, RNA, and plasmid DNA. The exonuclease activity is dependent on the presence of Mn²⁺ and/or Mg²⁺ ions, with an optimal pH and temperature of 8.3 and 43 °C, respectively. The truncation experiments of rMbovP701 revealed that YqaJ (41–185 aa) is the key functional domain of MbovP701 exonuclease. In conclusion, the present study identified a novel nuclease in M. bovis that plays an essential role in the proliferation of this minimal organism. This finding elucidates the survival strategy and pathogenesis of M. bovis, suggesting a potential therapeutic strategy for the treatment of M. bovis through targeting the inhibition of MbovP701. Moreover, it provides a foundation for future investigations into the interactions between MbovP701 and other nucleases involved in M. bovis biology. Full article

Mycoplasma bovis is a global concern for the cattle industry owing to its high rates of infection and resulting morbidity, but there are no reports on the lipid composition and metabolic pathways. This study examined the lipidomics of M. bovis to better investigate the biological characteristics of clinical isolates of M. bovis. The M. bovis strains NX114 and PG45, cultivated to log-phase, underwent untargeted lipidomics via ultra-high-performance liquid chromatography-mass spectrometry for differential analysis. Over 65 lipid types and 1476 lipid molecules were identified. We found that glycerophospholipids and sphingolipids constitute the predominant lipid components of M. bovis, with significant constituents of its cell membrane comprising ceramides, phosphatidylglycerols, sphingomyelins, triacylglycerols, hexosylceramides, ether-linked oxidized phosphatidylcholines, and oxidized phosphatidylglycerols. Secondly, 562 differentially expressed lipid molecules were identified, including 17 lipid subclasses (15 up-regulated and 2 down-regulated) with significant differences in relative content. These findings indicate that distinct M. bovis isolates contain structurally varied lipid molecules, with sphingomyelin, phosphatidylinositol, cardiolipin, and phosphatidylcholine being characteristic lipids. The glycerophospholipid metabolism pathway was identified as a key pathway regulating lipid metabolism in M. bovis by KEGG pathway enrichment analysis. The results indicated alterations in the lipid metabolism of M. bovis, offering insights into its pathogenic mechanisms. Full article