Klebsiella pneumoniae is an important emerging pathogen of humans and animals leading to serious clinical consequences. Increased antibiotic use has promoted the emergence of carbapenem-resistant and extended-spectrum β-lactamase (ESBL)-producing K. pneumoniae strains. Recently, phage therapy has gained momentum as a possible alternative against emerging antimicrobial resistance. This study was performed to assess the therapeutic effects of a novel lytic phage (VTCCBPA43) in a pneumonic mouse model in order to explore the efficacy of phage therapy against virulent K. pneumoniae infection.The tailed phage VTCCBPA43 was assessed for its growth kinetics, in vitro host range, and temperature and pH sensitivity. Protein constituents were analysed by SDS-PAGE and nLC-MS/MS. Therapeutic efficacy was observed 2 h post-challenge with virulent K. pneumoniae in a BALB/c mouse model.Phage VTCCBPA43 was found to be highly temperature-tolerant (up to 80 °C). It was most active at pH 5, had a burst size of 172 PFU/mL and exhibited a narrow host range. It was identified as a KP36-like phage by shotgun proteomics. Following intranasal application of a single dose (2 × 109 PFU/mouse) post-challenge with virulent K. pneumoniae, the presence of biologically active phage in vivo and a significant reduction in the lung bacterial load at all time points was observed. A reduction in lesion severity suggested overall beneficial effects of VTCCBPA43 phage therapy in the pneumonic mouse model.This research represents the first in vivo evidence of effective phage therapy against K. pneumoniae infection by the intranasal route.
Skin is the body's largest organ. The primary function is to act as a shield that protects internal organs from physical and chemical attacks, pathogen invasion and excessive loss of water. The body is rich in immune cells, forming a complex network called as the "skin immune system", as the main immunological barrier to the external environment. Any immune system disruption or defects can lead to immunological skin diseases. Excessive and undesirable immune responses can cause hypersensitivity or autoimmune diseases, while hypoimmunity can lead to infectious diseases and skin tumor. In body, the immune system is located in both major structural compartments: epidermis and dermis, and consists of several essential types of immunocompetent cells. Primary skin-resident immune cells, Langerhans cells (LCs) and melanocytes that produce melanin inhabit epidermis, while other types of immune-specialized cells, such as various subpopulations of dendritic cells (DCs), macrophages, and several types of T cells reside in the deeper layer — dermis. The efficacy of the skin immune system strongly depends on the close interplay and interaction between immune cells and the skin environment, such as adjacent keratinocytes and fibroblasts. The fundamental premise for protective immunity is the ability to distinguish between self and non-self. This ability is acquired during the growth of the fetus. In the normal system, when the above process fails, immune-mediated disease occurs. Some of the most interesting and challenging issues in immune-mediated dermatological diseases are discussed in this book chapter.
Abstract Salmonella enterica serovar Gallinarum biovar Pullorum (bvP) and biovar Gallinarum (bvG) are the etiological agents of pullorum disease (PD) and fowl typhoid (FT) respectively, which cause huge economic losses to poultry industry especially in developing countries including India. Vaccination and biosecurity measures are currently being employed to control and reduce the S . Gallinarum infections. High endemicity, poor implementation of hygiene and lack of effective vaccines pose challenges in prevention and control of disease in intensively maintained poultry flocks. Comparative genome analysis unravels similarities and dissimilarities thus facilitating identification of genomic features that aids in pathogenesis, niche adaptation and in tracing of evolutionary history. The present investigation was carried out to assess the genotypic differences amongst S. enterica serovar Gallinarum strains including Indian strain S. Gallinarum Sal40 VTCCBAA614 with a focus on identification of candidate virulence factors. The comparative genome analysis revealed an open pan-genome consisting of 5091 coding sequence (CDS) with 3270 CDS belonging to core-genome, 1254 CDS to dispensable genome and strain specific genes i.e. singletons ranging from 3 to 102 amongst the analyzed strains. Moreover, the investigated strains exhibited diversity in genomic features such as genomic islands, prophage regions, toxin-antitoxin cassettes, and acquired antimicrobial resistance genes. The identified genetic differences among the S. enterica serovar Gallinarum strains could be used for bacterial typing, design of rapid and reliable diagnostics, structure based inhibitor development and vaccine design for effective infection control as well as eradication and will also form basis for future experimental investigations.
Despite major progress and knowledge related to the application of adult stem cells, finding alternative sources for bone marrow MSCs has remained a challenge in both humans and animals. In the current study, two protocols namely sequential enzymatic tissue digestion and tissue explant techniques were tried for successful establishment of MSC culture. Umbilical tissues were isolated each time of foaling from five sequential foalings of Marwari mares. Total cell yield, their growth potential and cryopreservation potential were studied. Adherent cell colonies could be established using both isolation methods. Both the cell populations yielded from different protocols performed similarly in terms of population doubling and CFU number value. Additionally, the cells proliferated vigourously and displayed a similar morphology of mesenchymal stem cells. The MSCs were plastic adherent, colonogenic and their morphology was polygonal and fibroblast like. During the proliferation, the cells exhibited density dependent inhibition; analysis of microbial contamination from bacteria, mycoplasma and fungi were negative; the population doubling time of the MSCs isolated was 34.8 h and 40.2 h in enzymatic treatment and tissue explant methods respectively, and diploid chromosome number of the cells was 64, and the diploid frequency was higher than 80%. In conclusion, this study reveals that both the techniques proved to be non-invasive, efficient, simple and quick for isolation and establishment of MSC culture of extra embryonic tissues from equines.
Equine influenza (EI) is a highly contagious viral respiratory disease caused by the equine influenza virus (EIV). Frequent antigenic changes of EIV warrants thorough pathological evaluation and in-vivo replication of the circulating viruses in an experimental animal model for selection of vaccine strain. In the present study, EIV (H3N8) was inoculated into BALB/c mice and subsequently, gross, histopathological and immunohistochemical examination of the lungs was performed to evaluate the correlation of virus replication with corresponding pathological changes. Microscopically, at 1dpi, lungs from mice infected with EIV revealed degeneration of epithelial tissues, whereas indirect immunoperoxidase staining (IIPT) results showed high EIV antigen positivity in the bronchial and bronchiolar epithelium and mild positivity in the infiltrating cells. Subsequently from 1 dpi to 3 dpi, the virus replicated and infected other parenchymal cells, which led to recruitment of inflammatory cells. Microscopically moderate peribronchial, peribronchiolar and perivascular cuffing of neutrophils and macrophages were observed, whereas IIPT exhibited, scanty and moderate EIV antigen positive interstitial infiltrating macrophages. At 5 and 7 dpi, severe, diffuse interstitial thickening, bronchial, bronchiolar and alveolar epithelial necrosis and denudation, perivascular and peribronchial mononuclear cells cuffing were observed, which could be correlated with IIPT as high EIV antigen positivity of infiltrating macrophages and type II pneumocytes. However, from 11 dpi onwards, microscopically, moderate interstitium thickening and perivascular and peribronchial cuffing were observed whereas, tissues were negative for EIV antigen in IIPT. Mild, focal inflammation could be observed at 14 dpi. Present findings signify the correlation of virus replication with corresponding histopathological changes, which will be useful for assessing the efficacy of anti-influenza drug molecules and EIV vaccines.
A bacteriophage (VTCCBPA6) against a pathogenic strain of Aeromonas hydrophila was isolated from the sewage of an organized equine breeding farm. On the basis of TEM analysis, phage belonged to family Myoviridae. PCR amplification and sequence analysis of gp 23 gene (encoding for major capsid protein) revealed phylogenetic resemblance to T4 like virus genus. Protein profiling by SDS‐PAGE also indicated its resemblance to T4 like phage group. However, the comparison of its gp 23 gene sequence with previously reported phages showed similarity with T4‐like phages infecting Enterobacteriaceae instead of Aeromonas spp. Thus, to our knowledge, this report points toward the fact that a novel/evolved phage might exist in equine environment against A. hydrophila , which can be potentially used as a biocontrol agent.
Lumpy skin disease (LSD) has devastating economic impact. During the last decade, LSD had spread to climatically new and previously disease-free countries, which also includes its recent emergence in the Indian subcontinent (2019). This study deals with the LSD outbreak(s) from cattle in Ranchi (India). Virus was isolated from the scabs (skin lesions) in the primary goat kidney cells. Phylogenetic analysis based on nucleotide sequencing of LSD virus (LSDV) ORF011, ORF012 and ORF036 suggested that the isolated virus (LSDV/ Bos taurus -tc/India/2019/Ranchi) is closely related to Kenyan LSDV strains. Further, we adapted the isolated virus in Vero cells. Infection of the isolated LSDV to Vero cells did not produce cytopathic effect (CPE) until the 4 th blind passage, but upon adaptation, it produced high viral titres in the cultured cells. The kinetics of viral DNA synthesis and one-step growth curve analysis suggested that Vero cell-adapted LSDV initiates synthesizing its genome at ~24 hours post-infection (hpi) with a peak level at ~96 hpi whereas evidence of progeny virus particles was observed at 36–48 hours (h) with a peak titre at ~120 h. To the best of our knowledge, this study describes the first successful isolation of LSDV in India, besides providing insights into the life cycle Vero cell-adapted LSDV.
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