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Histophilus somni is an important pathogen of the bovine respiratory disease complex, yet the mechanisms underlying its virulence remain poorly understood. It is known that H. somni can incorporate sialic acid into lipooligosaccharide (LOS), and sialylated H. somni is more resistant to phagocytosis and complement-mediated killing by serum compared to non-sialylated bacteria in vitro. However, the virulence of non-sialylated H. somni has not been evaluated in vivo using an animal model. In this study, we investigated the contribution of sialic acid to virulence by constructing an H. somni sialic acid uptake mutant (ΔnanP-ΔnanU) and comparing the parent and mutant strains in a mouse septicemia and mortality model. Intraperitoneal challenge of mice with wildtype H. somni (1 × 108 colony forming units/mouse, CFU) was lethal to all animals. Mice challenged with three different doses (1, 2, or 5 × 108 CFU/mouse) of an H. somni ΔnanP-ΔnanU sialic acid uptake mutant exhibited survival rates of 90 %, 60 %, and 0 % respectively. High-performance anion exchange chromatography analyses revealed that LOS prepared from both parent and the ΔnanP-ΔnanU mutant strains of H. somni were sialylated. These findings suggest the presence of de novo sialic acid synthesis pathway, although the genes associated with de novo sialic acid synthesis (neuB and neuC) were not identified by genomic analysis. The lower attenuation in mice is most likely attributed to the sialylated LOS of H. somni nanPU mutant.
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Modelos Animais de Doenças , Lipopolissacarídeos , Ácido N-Acetilneuramínico , Pasteurellaceae , Sepse , Animais , Camundongos , Ácido N-Acetilneuramínico/metabolismo , Pasteurellaceae/genética , Pasteurellaceae/patogenicidade , Pasteurellaceae/metabolismo , Virulência/genética , Sepse/microbiologia , Sepse/mortalidade , Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/genética , Feminino , Mutação , Bovinos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismoRESUMO
Hematopoietic stem cells (HSCs) reside in a specialized microenvironment in a peculiar anatomic location which regulates the maintenance of stem cells and controls its functions. Recent scientific progress in experimental technologies have enabled the specific detection of epigenetic factors responsible for the maintenance and quiescence of the hematopoietic niche, which has improved our knowledge of regulatory mechanisms. The aberrant role of RNA-binding proteins and their impact on the disruption of stem cell biology have been reported by a number of recent studies. Despite recent modernization in hematopoietic microenvironment research avenues, our comprehension of the signaling mechanisms and interactive pathways responsible for integration of the hematopoietic niche is still limited. In the past few decades, zebrafish usage with regards to exploratory studies of the hematopoietic niche has expanded our knowledge for deeper understanding of novel cellular interactions. This review provides an update on the functional roles of different genetic and epigenetic factors and molecular signaling events at different sections of the hematopoietic microenvironment. The explorations of different molecular approaches and interventions of latest web-based tools being used are also outlined. This will help us to get more mechanistic insights and develop therapeutic options for the malignancies.
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Nicho de Células-Tronco , Peixe-Zebra , Animais , Comunicação Celular , Biologia Computacional , Células-Tronco Hematopoéticas/metabolismoRESUMO
Bacterial secretome is a comprehensive catalog of bacterial proteins that are released or secreted outside the cells. They offer a number of factors that possess several significant roles in virulence as well as cell to cell communication and hence play a core role in bacterial pathogenesis. Sometimes these proteins are bounded with membranes giving them the shape of vesicles called extracellular vesicles (EVs) or outer membrane vesicles (OMVs). Bacteria secrete these proteins via Sec and Tat pathways into the periplasm. Secreted proteins have found to be important as diagnostic markers as well as antigenic factors for the development of an effective candidate vaccine. Recently, the research in the field of secretomics is growing up and getting more interesting due to their direct involvement in the pathogenesis of the microorganisms leading to the infection. Many pathogenic bacteria have been studied for their secretome and the results illustrated novel antigens. This review highlights the secretome studies of different pathogenic bacteria in humans and animals, general secretion mechanisms, different approaches and challenges in the secretome of Mycoplasma sp.
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Vesículas Extracelulares/fisiologia , Mycoplasma/metabolismo , Mycoplasma/patogenicidade , Percepção de Quorum/fisiologia , Fatores de Virulência/metabolismo , Membrana Externa Bacteriana/fisiologia , Transporte Proteico/fisiologia , Proteoma/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Mycoplasma bovis is a risky pathogen mainly responsible for pneumonia and mastitis in cattle. Up to date, its pathogenesis is not clear. Since secreted proteins have a tricky role in M. bovis pathogenesis, this study was designed to systematically reveal M. bovis secretome and potential role in virulence of the pathogen. By using bioinformatics tools, a total of 246 secreted proteins were predicted based on M. bovis genome. Among them, 14 were classical, 154 non-classical and 78 both pathways. Then by using 2-dimensional gel electrophoresis (2-DE) and Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF- MS), 169 proteins were revealed. Of them, 60 were predicted to be secreted including 3 classical, 43 non-classical, and 14 both classical and non-classical. Further 8 proteins (MbovP0038, MbovP0338, MbovP0341, MbovP0520, MbovP0581, MbovP0674, MbovP0693, MbovP0845) were predicted to be virulence-related factors with VFDB. In addition, MbovP0581 (ABC transporter protein) was validated experimentally as secreted in nature and highly immunogenic reacting with sera of cattle experimentally infected with M. bovis. In conclusion, this study might be a crucial step towards a better understanding of pathogenesis and leading to the development of novel diagnostic marker and potent vaccine against M. bovis.
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Proteínas de Bactérias/metabolismo , Mycoplasma bovis/metabolismo , Anticorpos Antibacterianos/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Sequência Conservada/genética , Eletroforese em Gel Bidimensional , Genoma Bacteriano/genética , Genômica , Espectrometria de Massas , Mycoplasma bovis/genética , Mycoplasma bovis/patogenicidade , Proteômica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , VirulênciaRESUMO
Mycoplasma bovis, one of the major pathogens of bovine respiratory disease, binds to respiratory epithelial cells resulting in severe pneumonia and tissue damage. This study was designed to identify the adhesive function of a putative 27-kDa M. bovis lipoprotein, encoded by the gene MBOV_RS03440 and designated as P27. The gene was cloned and overexpressed to produce antibodies against the recombinant P27 (rP27). The western blot and flow cytometry assay confirmed P27 to be a surface-localized protein, while ELISA confirmed it to be an immunogenic protein. Confocal immunofluorescence microscopy demonstrated that rP27 bound to embryonic bovine lung (EBL) cell monolayers in a dose-dependent manner. Furthermore, anti-rP27 antiserum inhibited the attachment of M. bovis to EBL cells demonstrating the binding specificity of P27 to EBL cells. The attachment of rP27 to EBL cells was mediated by fibronectin (Fn), an extracellular matrix component. The interaction between rP27 and Fn was qualitatively and quantitatively monitored by ligand immunoblot assay, ELISA, and biolayer interferometry. Collectively, these results indicate that P27 is a novel Fn-binding, immunogenic adhesive protein of M. bovis, thereby contributing to the further understanding of the molecular pathogenesis of M. bovis.
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Adesinas Bacterianas/metabolismo , Aderência Bacteriana/fisiologia , Fibronectinas/metabolismo , Lipoproteínas/imunologia , Lipoproteínas/metabolismo , Mycoplasma bovis/patogenicidade , Pneumonia/veterinária , Mucosa Respiratória/microbiologia , Adesinas Bacterianas/genética , Animais , Anticorpos Antibacterianos/imunologia , Bovinos , Doenças dos Bovinos/microbiologia , Células Cultivadas , Lipoproteínas/genética , Mycoplasma bovis/genética , Pneumonia/microbiologia , Ligação ProteicaRESUMO
Mycoplasma bovis (M. bovis) is an emerging devastating cause of pneumonia in dairy and feedlot calves around the world, largely due to its increasing resistance to new generation effective antibiotics and lack of efficient vaccine. Failure of protective measures against M. bovis is mainly due to nonspecific targets. Most of the virulent factors of M. bovis and their underlying mechanisms are obscure to devise an effective control strategy. Full genome sequences of M. bovis strains basically provided a useful platform for the accurate identification of novel proteins and understanding their biological value using proteomics tools. Most of the previously documented proteins of M. bovis are involved in adhesion to host cells and are antigenic in nature. However, host immune response to some antigens proved to be non-protective. For the diagnosis of M. bovis infection, a serological assay based on whole cell proteins of M. bovis is commercially available but the specificity is likely to be improved by identifying and targeting the specific proteins. Many of the predicted proteins of M. bovis remain hypothetical, as their functions are yet to be confirmed experimentally. This review mainly focuses on the proteomics analysis of M. bovis and its role in identification of the virulence related factors and antigenic proteins of M. bovis. Future research directions have also been highlighted in this script for the application of important antigenic factors of M. bovis.
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Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Mycoplasma bovis/metabolismo , Proteômica , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/genética , Bovinos , Doenças dos Bovinos/diagnóstico , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/microbiologia , Diagnóstico Diferencial , Genoma Bacteriano , Infecções por Mycoplasma/diagnóstico , Infecções por Mycoplasma/microbiologia , Infecções por Mycoplasma/veterinária , Mycoplasma bovis/genética , Mycoplasma bovis/isolamento & purificação , Domínios e Motivos de Interação entre Proteínas , Fatores de Virulência/metabolismoRESUMO
This study was undertaken to determine the genotypic distribution of Chinese M. bovis strains and their similarity to isolates from other countries. Two multilocus sequence typing (MLST) schemes (MLST-1 and MLST-2) and pulsed field gel electrophoresis (PFGE) were used to compare 44 Chinese strains and the M. bovis type strain PG45. The results showed a high genetic homogeneity of Chinese isolates; 43 of 44 (97.7%) Chinese isolates were identified as ST-10 and as ST-34 by MLST-1, while for MLST-2 42 of 44 (95.5%) were identified as ST-10 with the two remaining isolates of ST-32 and ST43. PFGE clustered 42 of 44 (95.5%) of the Chinese isolates into PT-I. The overall agreement rate between the three typing methods was 97.8% (95% CI:86.8-99.9%). The type strain PG45 was identified as a unique type by all three methods. When the MLST-2 scheme was further used to analyze 16 isolates of Australian and Israeli origin ST-10 was more dominant among Australian isolates (7/8), compared with those from Israel (3/8). The evolutionary relationship of the 60 isolates typed in this study assessed together with 206 additional isolates retrieved from pubmlst/mbovis database analyzed by geoBURST Minimum spanning tree (MST) confirmed that the Chinese, Israeli and Australian M. bovis isolates typed in this study that were predominantly ST-10, were clustered in CC3 with isolates originating from the USA. Our results suggest that ST-10 is an emerging clone of M. bovis population. We hypothesized that the widespread distribution of this type is a result of global livestock movements. These findings will help further the understanding of the global evolution of M. bovis and development of novel vaccines against M. bovis.
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Evolução Molecular , Genótipo , Mycoplasma bovis/classificação , Mycoplasma bovis/genética , Mycoplasma bovis/isolamento & purificação , Análise de Variância , Animais , Austrália , Bovinos , Doenças dos Bovinos/microbiologia , China , DNA Bacteriano , Eletroforese em Gel de Campo Pulsado/métodos , Genes Bacterianos/genética , Variação Genética , Israel , Epidemiologia Molecular , Tipagem de Sequências Multilocus/métodos , Análise de Sequência de DNA , Estados Unidos , Sequenciamento Completo do GenomaRESUMO
This study aimed to determine the activity of one Mycoplasma bovis nuclease encoded by MBOV_RS02825 and its association with cytotoxicity. The bioinformatics analysis predicted that it encodes a Ca(2+)-dependent nuclease based on existence of enzymatic sites in a TNASE_3 domain derived from a Staphylococcus aureus thermonuclease (SNc). We cloned and purified the recombinant MbovNase (rMbovNase), and demonstrated its nuclease activity by digesting bovine macrophage linear DNA and RNA, and closed circular plasmid DNA in the presence of 10 mM Ca(2+) at 22-65 °C. In addition, this MbovNase was localized in membrane and rMbovNase able to degrade DNA matrix of neutrophil extracellular traps (NETs). When incubated with macrophages, rMbovNase bound to and invaded the cells localizing to both the cytoplasm and nuclei. These cells experienced apoptosis and the viability was significantly reduced. The apoptosis was confirmed by activated expression of phosphorylated NF-κB p65 and Bax, and inhibition of Iκßα and Bcl-2. In contrast, rMbovNase(Δ181-342) without TNASE_3 domain exhibited deficiency in all the biological functions. Furthermore, rMbovNase was also demonstrated to be secreted. In conclusion, it is a first report that MbovNase is an active nuclease, both secretory and membrane protein with ability to degrade NETs and induce apoptosis.
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Proteínas de Bactérias/metabolismo , Desoxirribonucleases/metabolismo , Mycoplasma bovis/enzimologia , Sequência de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Bovinos , Linhagem Celular , DNA/metabolismo , Desoxirribonucleases/química , Desoxirribonucleases/genética , Armadilhas Extracelulares/genética , Armadilhas Extracelulares/metabolismo , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Nuclease do Micrococo/química , Nuclease do Micrococo/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Dados de Sequência Molecular , Plasmídeos/genética , Plasmídeos/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/toxicidade , Alinhamento de Sequência , Transdução de Sinais/efeitos dos fármacos , Staphylococcus aureus/enzimologia , Fator de Transcrição RelA/metabolismoRESUMO
Pseudomonas aeruginosa is considered an environmental pathogen, and it can cause acute and chronic mastitis in dairy cows. Here, we report the draft genome sequence of a multidrug-resistant P. aeruginosa strain (2011C-S1) isolated from a Holstein cow showing signs of chronic mastitis that was nonresponsive to intramammary antibiotic treatment.
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Mycoplasma bovis (M. bovis) is the etiologic agent of high mortality epizootics of chronic respiratory disease in American bison (Bison bison). Despite the severity of the disease, no efficacious commercial vaccines have been licensed for the prevention of M. bovis infection in bison. Elongation factor thermal unstable (EFTu) and Heat Shock Protein 70 (Hsp70, DnaK) are highly conserved, constitutively expressed proteins that have previously been shown to provide protection against M. bovis infection in cattle. To assess the suitability of EFTu and Hsp70 as vaccine antigens in bison, the immune response to and protection conferred by an injectable, adjuvanted subunit vaccine comprised of recombinantly expressed EFTu and Hsp70 was evaluated. Vaccinates developed robust antibody and cellular immune responses against both EFTu and Hsp70 antigens. To assess vaccine efficacy, unvaccinated control and vaccinated bison were experimentally challenged with bovine herpes virus-1 (BHV-1) 4 days prior to intranasal infection with M. bovis. Vaccinated bison displayed reductions in joint infection, lung bacterial loads, and lung lesions compared to unvaccinated controls. Together, these results showed that this subunit vaccine reduced clinical disease and bacterial dissemination from the lungs in M. bovis challenged bison and support the further development of protein subunit vaccines against M. bovis for use in bison.
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Introduction: The antiviral activity of recombinant bovine interferon lambda 3 (bovIFN-λ3) against bovine viral diarrhea virus (BVDV) has been demonstrated in vitro in Madin-Darby bovine kidney cells (MDBK) and in vivo in cattle. However, anti-BVDV activity of bovIFN-λ3 has not been studied in bovine respiratory tract epithelial cells, supposedly a primary target of BVDV infection when entering the host by the oronasal route. Methods: Here we investigated the anti-BVDV activity of bovIFN-λ3 in bovine turbinate-derived primary epithelial cells (BTu) using BVDV infection and immunoperoxidase staining, TCID50, RT-qPCR, DNA and transcriptome sequencing, and transfection with plasmids containing the two subunits, IL-28Rα and IL-10Rß that constitute the bovIFN-λ3 receptor. Results: Our immunoperoxidase staining, RT-qPCR, and TCID50 results show that while BVDV was successfully cleared in MDBK cells treated with bovIFN-λ3 and bovIFN-α, only the latter, bovIFN-α, cleared BVDV in BTu cells. Preincubation of MDBK cells with bovIFN-λ3 before BVDV infection was needed to induce optimal antiviral state. Both cell types displayed intact type I and III IFN signaling pathways and expressed similar levels of IL-10Rß subunit of the type III IFN receptor. Sequencing of PCR amplicon of the IL-28Rα subunit revealed intact transmembrane domain and lack of single nucleotide polymorphisms (SNPs) in BTu cells. However, RT-qPCR and transcriptomic analyses showed a lower expression of IL-28Rα transcripts in BTu cells as compared to MDBK cells. Interestingly, transfection of BTu cells with a plasmid encoding IL-28Rα subunit, but not IL-10Rß subunit, established the bovIFN-λ3 sensitivity showing similar anti-BVDV activity to the response in MDBK cells. Conclusion: Our results demonstrate that the sensitivity of cells to bovIFN-λ3 depends not only on the quality but also of the quantity of the IL-28Rα subunit of the heterodimeric receptor. A reduction in IL-28Rα transcript expression was detected in BTu as compared to MDBK cells, despite the absence of spliced variants or SNPs. The establishment of bovIFN-λ3 induced anti-BVDV activity in BTu cells transfected with an IL-28Rα plasmid suggests that the level of expression of this receptor subunit is crucial for the specific antiviral activity of type III IFN in these cells.
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Interferon lambda , Interferons , Conchas Nasais , Animais , Bovinos , Interferons/metabolismo , Interferons/imunologia , Conchas Nasais/virologia , Conchas Nasais/imunologia , Conchas Nasais/metabolismo , Antivirais/farmacologia , Vírus da Diarreia Viral Bovina/imunologia , Vírus da Diarreia Viral Bovina/fisiologia , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Células Epiteliais/virologia , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Interleucinas/genética , Interleucinas/farmacologia , Interleucinas/imunologia , Interleucinas/metabolismo , Linhagem Celular , Doença das Mucosas por Vírus da Diarreia Viral Bovina/imunologia , Doença das Mucosas por Vírus da Diarreia Viral Bovina/virologia , Proteínas Recombinantes/farmacologia , Subunidade beta de Receptor de Interleucina-10/genética , Subunidade beta de Receptor de Interleucina-10/metabolismo , Receptores de CitocinasRESUMO
Despite numerous efforts, developing recombinant vaccines for the control of M. bovis infections has not been successful. Many factors are contributing to the lack of success including the identification of protective antigens, use of effective adjuvants, and relatively limited information on the quality of immune responses needed for protection. Experimental trials using vaccination with many M. bovis proteins resulted in significant humoral immune responses before and after the challenges, however these responses were not enough to confer protection. We explored the role of complement-fixing antibodies in the killing of M. bovis in-vitro and whether animals vaccinated with proteins that elicit antibodies capable of complement-fixing would be protected against an experimental challenge. We found that antibodies against some of these proteins fixed complement and killed M. bovis in-vitro. Vaccination and challenge experiments with proteins whose cognate antibodies either fixed complement or not resulted in lack of protection against a M. bovis experimental challenge suggesting that complement fixation does not play a role in protection.
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Doenças dos Bovinos , Infecções por Mycoplasma , Mycoplasma bovis , Animais , Bovinos , Infecções por Mycoplasma/prevenção & controle , Vacinas Bacterianas , Doenças dos Bovinos/prevenção & controle , Anticorpos Antibacterianos , Proteínas do Sistema Complemento , VacinaçãoRESUMO
IMPORTANCE: The Gram-negative coccobacillus Mannheimia haemolytica is a natural inhabitant of the upper respiratory tract in ruminants and the most common bacterial agent involved in bovine respiratory disease complex development. Key virulence factors harbored by M. haemolytica are leukotoxin, lipopolysaccharide, capsule, adhesins, and neuraminidase which are involved in evading innate and adaptive immune responses. In this study, we have shown that CMP-sialic acid synthetase (neuA) is necessary for the incorporation of sialic acid onto the membrane, and inactivation of neuA results in increased phagocytosis and complement-mediated killing of M. haemolytica, thus demonstrating that sialylation contributes to the virulence of M. haemolytica.
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Mannheimia haemolytica , Bovinos , Animais , Mannheimia haemolytica/genética , Mannheimia haemolytica/metabolismo , N-Acilneuraminato Citidililtransferase/genética , N-Acilneuraminato Citidililtransferase/metabolismo , Sorogrupo , Deleção de Genes , FagocitoseRESUMO
Mycoplasma bovis (M. bovis) in cattle causes pneumonia, arthritis, otitis media, and mastitis. In addition, multiple outbreaks have been recorded in North American bison. The genomic data on Canadian M. bovis in bison and cattle to date is limited. Whole-genome sequencing (WGS) was used to assess the degree of genome conservation across four Canadian M. bovis strains recovered from bison and cattle. Whole-genome sequences of four M. bovis isolates (Mb1, Mb160, Mb300, Mb304) and the PG45 reference genome were utilized to identify the M. bovis genomic similarity, whole-genome single nucleotide polymorphism (WGS-SNP), virulence determinants, and genomic islands. The pan-genome analysis showed that M. bovis encodes a minimum of 971 genes, while the core genome contained 637 genes. Comparative genomics revealed limited diversity in gene content between bison and cattle isolates. Whole-genome SNP analysis showed that the four M. bovis isolates differed from each other and to PG45. A total of 40 putative virulence genes associated with adhesion, colonization, and destruction of tissues were found in the bison and cattle isolates using the virulence factors database (VFDB). These putative virulence factors were equally distributed among isolates. Genomic Islands (GIs) ranging from 4 to 9 and associated with transposases, restriction-modification, ribosomal hypothetical proteins, variable surface lipoproteins, and unknowns were also identified. Overall, the genomic characterization of these isolates may provide new insights into the mechanisms of pathogenicity in M. bovis.
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Bison , Infecções por Mycoplasma , Mycoplasma bovis , Animais , Canadá/epidemiologia , Bovinos , Feminino , Genômica , Infecções por Mycoplasma/epidemiologia , Infecções por Mycoplasma/veterinária , Mycoplasma bovis/genética , Fatores de Virulência/genéticaRESUMO
Mycoplasma bovis (M. bovis) is an emerging major bovine pathogen, causing economic losses worldwide in the dairy and beef industry. Whole-genome sequencing (WGS) now allows high resolution for tracing clonal populations. Based on WGS, we developed the core genome multilocus sequence typing (cgMLST) scheme and applied it onto 151 genomes of clonal and non-clonal strains of M. bovis isolated from China, Australia, Israel, Denmark, Canada, and the USA. We used the complete genome of M. bovis PG45 as the reference genome. The pairwise genome comparison of these 151 genome sequences resulted in 478 cgMLST gene targets present in > 99.0 % clonal and non-clonal isolates with 100 % overlap and > 90 % sequence similarity. A total of 478 core genes were retained as cgMLST target genes of which an average of 90.4-99 % were present in 151 M. bovis genomes, while M. agalactiae (PG2) had 17.0 % and M. mycoides subsp. capri (PG3), M. ovipneumoniae (Y98), and M. arginine resulted in 0.0 % of good targets. When tested against the clonal and non-clonal strains, we found cgMLST clusters were congruent with the MLST-defined clonal groups, which had various degrees of diversity at the whole-genome level. Notably, cgMLST could distinguish between clonal and epidemiologically unrelated strains of the same clonal group, which could not be achieved using traditional MLST schemes. Our results showed that ninety-two M. bovis genomes from clonal group isolates had > 10 allele differences and unambiguously differentiated from unrelated outgroup strains. Additionally, cgMLST revealed that there might be several sub-clones of the emerging ST-52 clone. The cgMLST phylogenetic analysis results showed substantial agreement with geographical and temporal information. cgMLST enables the use of next-generation sequencing technology to bovine mycoplasma epidemiology at both the local and global levels. In conclusion, the novel cgMLST scheme not only showed discrimination resolution highly as compared with MLST and SNP cgMLST in sub-typing but also indicated the capability to reveal more population structure characteristics than MLST.
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Mycoplasma bovis , Animais , Bovinos , Surtos de Doenças , Genoma Bacteriano , Epidemiologia Molecular/métodos , Tipagem de Sequências Multilocus/métodos , Tipagem de Sequências Multilocus/veterinária , Mycoplasma bovis/genética , FilogeniaRESUMO
Multiple outbreaks of Mycoplasma bovis (M. bovis) have been reported in North American bison (Bison bison) in Alberta, Manitoba, Saskatchewan, Nebraska, New Mexico, Montana, North Dakota, and Kansas. M. bovis is mainly spread through direct contact and disseminated via animal movements thus, reliable genotyping is crucial for epidemiological investigations. The present study describes the genotyping of sixty-one M. bovis strains from cattle and bison isolated from different provinces of Canada by multi locus sequence typing (MLST), and multiple-locus variable-number tandem repeat analysis (MLVA). The sixty M. bovis clinical isolates together with the reference strain PG45 were divided into ten sequence types by MLST. Three novel sequence types were identified. Two isolates, one from cattle and one from bison shared the same sequence type, whereas one strain had the same sequence type as PG45. The cattle isolates could be further subdivided in Clade A with two subclades and bison isolates were grouped in Clade B with two subclades. With the exception of one animal, isolates originating from the same animal had the same sequence type. The sixty-one isolates also formed three main clades with several subclades when analyzed by MLVA. A total of 20 VNTR (Variable number tandem repeats) types were distinguished, 8 in cattle and 12 in bison isolates. The results showed multiple sequence types and genotype populations of M. bovis in bison and cattle. The results may further help to understand the evolution of M. bovis and develop strain specific or sequence type diagnostic tools.
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Técnicas de Tipagem Bacteriana/veterinária , Búfalos/microbiologia , Bovinos/microbiologia , Tipagem de Sequências Multilocus/veterinária , Mycoplasma bovis/genética , Filogenia , Animais , Repetições MinissatélitesRESUMO
Mycoplasma bovis is a major bacterial pathogen that causes respiratory diseases in cattle and bison. We report here the complete genome sequences of four Mycoplasma bovis strains isolated in three Canadian provinces. These genome sequences could provide important information on virulence factors and targets for new vaccines against M. bovis.
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The current vaccines to control bovine Babesia bigemina (B. bigemina) infection are not fully protective and vaccination failures incur heavy losses to the cattle industry around the world. Using modified micro-aerophilous stationary phase, we developed a culture-derived attenuated live vaccine against B. bigemina and tested a single subcutaneous inoculation of 2 × 108 infected erythrocytes in calves. The protection was measured after a lethal intravenous challenge with 5 × 108 virulent calf-derived B. bigemina. Our results demonstrated that a single shot of attenuated vaccine was capable of inducing robust humoral and cell-mediated immune responses in calves. We found a significant increase in the IgG antibody titers post-challenge and a strong proliferation of both CD4+ and CD8+ T cells contributing towards the protection. Our vaccine provided complete protection and parasitic clearance, which was followed for more than 100 days post-challenge. This immunity against babesiosis was directly linked to strong humoral responses; however, the parasitic clearance was attributed to significant T cells effector responses in vaccinated calves as compared to the infected control calves. We anticipate that these results will be helpful in the development of more efficient culture-derived vaccines against Babesia infections, thus reducing significant global economic losses to farmers and the cattle industry.
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Mycoplasma bovis (M. bovis) is an important pathogen of cattle. An attenuated live vaccine has recently been developed by this laboratory. However, an effective assay for the differentiation of infected from vaccinated animals (DIVA) is still lacking. Therefore, a comparative immunoproteomics study of the membrane and membrane associated proteins (MAPs) of M. bovis HB0801 and its attenuated strain (M. bovis-150) was aimed to identify potential antigens for DIVA assay. Triton-X-114 fractionated liposoluble proteins of both the virulent and attenuated strains were separated with 2-DE and proteins reacting with sera against the virulent M. bovis strain were detected by MS. A total of 19 differently expressed proteins were identified by MS, among them twelve proteins were detected by MALDI-TOF MS and seven antigenic proteins were identified by short-gun LC-MS/MS. Furthermore, these findings were confirmed at mRNA level by qRT-PCR. The results demonstrated that a putative lipoprotein encoded by functionally unknown gene Mbov_0730 (MbovP730) is a sensitive and specific antigen for DIVA assay. MbovP730 is absent in M. bovis-150 confirmed with Western blot assay and also didn't cross-react with other antisera against common pathogens including infectious bovine rhinotracheitis virus and bovine viral diarrhea virus by iELISA. Thereby rMbovP730-based iELISA was established. For clinical samples, this ELISA provided a sensitivity of 95.7% (95% CI: 90.4%, 98.2%) and specificity was 97.8% (95% CI: 88.4%, 99.6%). Antisera from vaccinated calves (n = 44) were found negative with rMbovP730 based iELISA, while positive with assays based on whole cell proteins of M. bovis-150 and M. bovis HB0801, respectively. In conclusion, this study identified the differential antigen MbovP730 between virulent and attenuated strains and established rMbovP730-based iELISA as a new DIVA method.
RESUMO
Mycoplasma bovis is an important cause of bovine respiratory disease worldwide. To understand its virulence mechanisms, we sequenced three attenuated M. bovis strains, P115, P150, and P180, which were passaged in vitro 115, 150, and 180 times, respectively, and exhibited progressively decreasing virulence. Comparative genomics was performed among the wild-type M. bovis HB0801 (P1) strain and the P115, P150, and P180 strains, and one 14.2-kb deleted region covering 14 genes was detected in the passaged strains. Additionally, 46 non-sense single-nucleotide polymorphisms and indels were detected, which confirmed that more passages result in more mutations. A subsequent collective bioinformatics analysis of paralogs, metabolic pathways, protein-protein interactions, secretory proteins, functionally conserved domains, and virulence-related factors identified 11 genes that likely contributed to the increased attenuation in the passaged strains. These genes encode ascorbate-specific phosphotransferase system enzyme IIB and IIA components, enolase, L-lactate dehydrogenase, pyruvate kinase, glycerol, and multiple sugar ATP-binding cassette transporters, ATP binding proteins, NADH dehydrogenase, phosphate acetyltransferase, transketolase, and a variable surface protein. Fifteen genes were shown to be enriched in 15 metabolic pathways, and they included the aforementioned genes encoding pyruvate kinase, transketolase, enolase, and L-lactate dehydrogenase. Hydrogen peroxide (H2O2) production in M. bovis strains representing seven passages from P1 to P180 decreased progressively with increasing numbers of passages and increased attenuation. However, eight mutants specific to eight individual genes within the 14.2-kb deleted region did not exhibit altered H2O2 production. These results enrich the M. bovis genomics database, and they increase our understanding of the mechanisms underlying M. bovis virulence.