Jully Gogoi-Tiwari

The role of liver progenitor cells during liver regeneration, fibrogenesis, and carcinogenesis.

The growing worldwide challenge of cirrhosis and hepatocellular carcinoma due to increasing prevalence of excessive alcohol consumption, viral hepatitis, obesity, and the metabolic syndrome has sparked interest in stem cell-like liver progenitor cells (LPCs) as potential candidates for cell therapy and tissue engineering, as an alternative approach to whole organ transplantation. However, LPCs always proliferate in chronic liver diseases with a predisposition to cancer; they have been suggested to play major roles in driving fibrosis, disease progression, and may even represent tumor-initiating cells. Hence, a greater understanding of the factors that govern their activation, communication with other hepatic cell types, and bipotential differentiation as opposed to their potential transformation is needed before their therapeutic potential can be harnessed.

Comparative studies of the immunogenicity and protective potential of biofilm vs planktonic Staphylococcus aureus vaccine against bovine mastitis using non-invasive mouse mastitis as a model system

This study was undertaken to compare the immunogenicity and protective potential of biofilm vs planktonic Staphylococcus aureus vaccine for the prevention of mastitis using the mouse as a model system. Mice immunized with formalin-killed whole cell vaccine of S. aureus residing in a biofilm when delivered via an intramammary route produced a cell mediated immune response. Mice immunized with this biofilm vaccine showed significant reductions in colonization by S. aureus in mammary glands, severity of clinical symptoms and tissue damage in mammary glands in comparison with the mice immunized with formalin-killed whole cells of planktonic S. aureus. The planktonic vaccine administered by a subcutaneous route produced a significantly higher humoral immune response (IgG1 and IgG) than the biofilm vaccine. However, considering the host response, tissue damage, the clinical severity and colonization of S. aureus in mammary glands, the biofilm vaccine performed better in immunogenicity and protective potential when administered by the intramammary route.

Relative distribution of virulence-associated factors among Australian bovine Staphylococcus aureus isolates: Potential relevance to development of an effective bovine mastitis vaccine

Staphylococcus aureus is one of the major contagious pathogens causing bovine mastitis worldwide.1 It causes contagious mastitis resulting either clinical or subclinical mastitis with increase in the number of somatic cell count (SCC) in milk. More than

Mammary Gland Pathology Subsequent to Acute Infection with Strong versus Weak Biofilm Forming Staphylococcus aureus Bovine Mastitis Isolates: A Pilot Study Using Non-Invasive Mouse Mastitis Model

130 million is lost by the Australian dairy farmers (

Intramammary Immunization of Pregnant Mice with Staphylococcal Protein A Reduces the Post-Challenge Mammary Gland Bacterial Load but Not Pathology

A200/cow/year) every year due to poor udder health caused by mastitis resulting in reduction of milk production, increase in treatment costs, veterinary consultation fees, and number of cow culls. There are multiple pathogens that have been found to be associated with bovine mastitis in Australia.2 While the relative distribution of the different pathogens causing mastitis may differ in different regions and countries, S. aureus is one of the most significant contagious bacterial pathogens causing bovine mastitis and is of concern to public health because of its potential for transmission to humans. Once the organism enters into the mammary gland, it adheres to epithelial lining and defies the host innate immune defenses by variety of virulence factors such as capsule and protein A which interfere with the process of phagocytosis.3 Once intra-mammary infection is established, damage to the mammary gland epithelial lining is initiated by ulceration and occlusion of lactiferous ducts and alveoli, infiltration of inflammatory cells in the parenchyma.4 S. aureus produces a variety of virulence factors which evade the tissue and host immune system and thereby maintain infection. These virulence factors are capsular polysaccharides, cytotoxins, superantigenic enterotoxins and MSCRAMM (microbial surface components recognizing adhesive matrix molecules). A large number of cytotoxins are produced by S. aureus which form pores in the cell membrane causing osmotic swelling leading to cell death. These cytotoxins include leukocidins, phenol soluble modulins (PSMs) and cytolysins. The cytolysins of S. aureus are α-, β-, γ-, and δ-toxins, of which α-toxin is well characterized for its contribution to biofilm formation and protective potential.5,6 β-toxin is a sphingomyelinaseC and 95% of S. aureus isolates from bovine mastitis cases produce β-toxin7 which causes damage to epithelial lining of mammary gland. Gamma (γ) and delta (δ) toxins, bicomponent toxins are synergohymenotropic toxins that act through the synergistic activity of 2 non-associated secretory proteins creating lytic pores in host cells including neutrophils and are assembled from the 2 components secreted separately by the organism as water-soluble molecules.8 Panton-Valentine Leukocidin (PVL) is encoded by 2 contiguous and cotranscribed genes, LukS-PV and LukF-PV9 and creates lytic pores in neutrophils, monocytes and macrophages adversely affecting their function. Phenol soluble modulins (PSMs) are the peptides produced by S. aureus, which are cytotoxic and proinflammatory agent. Recent finding has demonstrated that it plays a part in the formation of S. aureus biofilm.10 S. aureus produces a number of superantigens including enterotoxins (SEs), Toxic Shock Syndrome toxin and exfoliative toxins. Enterotoxins of S. aureus include the classical enterotoxins A to E and the recently identified and characterized SEG-SEU toxins.11,12 These antigens are considered as superantigens due to their ability to release inflammatory cytokines from both T cells and macrophages by binding to the surface of MHC class II proteins and T cell receptors. 13-16 The first step in establishing infection is the initial attachment of S. aureus to eukaryotic membrane and extracellular matrix proteins which is followed by colonization and subsequent infection.17 Colonization is commonly associated with a variety of adherence factors or adhesins which are known as microbial surface component recognizing adhesive matrix molecules (MSCRAMM). There are over 20 different MSCRAMMs identified, which can be expressed in S. aureus18 that mediate attachment to surface proteins of host cells including collagen, elastin, fibrinogen, thrombospondin, fibronectin, bone sialoprotein and laminin.19 Major adhesins in this group that mediate the initial attachment of S. aureus to the bovine mammary gland, providing the first critical step for establishing infection19 are clumping factors A and B (ClfA and ClfB),20 collagen adhesion (CNA),21 bone sialo binding protein (BBP)22 and the fibronectin binding proteins A and B (FnBPA and FnBPB).23 Besides these major adhesins, biofilm-associated protein (bap) has also been reported to be associated with primary attachment of S. aureus to mammary tissue.24,25 An accessory gene, agr, regulates the production of biofilms including detachment of biofilm that helps in virulence and dissemination of S. aureus in the mammary gland resulting in persistent bovine mastitis,26 whereas penicillin resistance of S. aureus is mediated by blaZ gene.27 Variability in the prevalence of virulence factors in S. aureus may result in various levels of severity and forms of mastitis in cows.28 No studies have been carried out in Australia on the virulence factors of S. aureus isolated from clinical cases of bovine mastitis. Aim of this study was to determine the relative distribution of different virulent factors of bovine S. aureus isolates in Australia including MSCRAMMS and exotoxins using conventional polymerase chain reaction (PCR) and the available serological methods.

Typing of Staphylococcus aureus isolated from bovine mastitis cases in Australia and India

Background Biofilm formation by Staphylococcus aureus is an important virulence attribute because of its potential to induce persistent antibiotic resistance, retard phagocytosis and either attenuate or promote inflammation, depending upon the disease syndrome, in vivo. This study was undertaken to evaluate the potential significance of strength of biofilm formation by clinical bovine mastitis-associated S. aureus in mammary tissue damage by using a mouse mastitis model. Methods Two S. aureus strains of the same capsular phenotype with different biofilm forming strengths were used to non-invasively infect mammary glands of lactating mice. Biofilm forming potential of these strains were determined by tissue culture plate method, ica typing and virulence gene profile per detection by PCR. Delivery of the infectious dose of S. aureus was directly through the teat lactiferous duct without invasive scraping of the teat surface. Both bacteriological and histological methods were used for analysis of mammary gland pathology of mice post-infection. Results Histopathological analysis of the infected mammary glands revealed that mice inoculated with the strong biofilm forming S. aureus strain produced marked acute mastitic lesions, showing profuse infiltration predominantly with neutrophils, with evidence of necrosis in the affected mammary glands. In contrast, the damage was significantly less severe in mammary glands of mice infected with the weak biofilm-forming S. aureus strain. Although both IL-1β and TNF-α inflammatory biomarkers were produced in infected mice, level of TNF-α produced was significantly higher (p<0.05) in mice inoculated with strong biofilm forming S. aureus than the weak biofilm forming strain. Conclusion This finding suggests an important role of TNF-α in mammary gland pathology post-infection with strong biofilm-forming S. aureus in the acute mouse mastitis model, and offers an opportunity for the development of novel strategies for reduction of mammary tissue damage, with or without use of antimicrobials and/or anti-inflammatory compounds for the treatment of bovine mastitis.

Serological versus molecular typing of surface-associated immune evading polysaccharide antigens-based phenotypes of Staphylococcus aureus

Protein A, encoded by the spa gene, is one of the major immune evading MSCRAMM of S. aureus, demonstrated to be prevalent in a significant percentage of clinical bovine mastitis isolates in Australia. Given its’ reported significance in biofilm formation and the superior performance of S. aureus biofilm versus planktonic vaccine in the mouse mastitis model, it was of interest to determine the immunogenicity and protective potential of Protein A as a potential vaccine candidate against bovine mastitis using the mouse mastitis model. Pregnant Balb/c mice were immunised with Protein A emulsified in an alum-based adjuvant by subcutaneous (s/c) or intramammary (i/mam) routes. While humoral immune response of mice post-immunization were determined using indirect ELISA, cell-mediated immune response was assessed by estimation of interferon-gamma (IFN-γ) produced by protein A-stimulated splenocyte supernatants. Protective potential of Protein A against experimental mastitis was determined by challenge of immunized versus sham-vaccinated mice by i/mam route, based upon manifestation of clinical symptoms, total bacterial load and histopathological damage to mammary glands. Significantly (p<0.05) higher levels of IgG1 isotype were produced in mice immunized by the s/c route. In contrast, significantly higher levels of the antibody isotype IgG2a were produced in mice immunized by the i/mam route (p<0.05). There was significant reduction (p<0.05) in bacterial loads of the mammary glands of mice immunized by Protein A regardless of the route of immunization, with medium level of clinical symptoms observed up to day 3 post-challenge. However, Protein A vaccine failed to protect immunized mice post-challenge with biofilm producing encapsulated S. aureus via i/mam route, regardless of the route of immunization, as measured by the level of mammary tissue damage. It was concluded that, Protein A in its’ native state was apparently not a suitable candidate for inclusion in a cell-free vaccine formulation against mastitis.

An immunological assay for identification of potential biofilm-associated antigens of Staphylococcus aureus

OBJECTIVE To determine the prevalence of the different capsular polysaccharide (CP) and major surface-associated non-CP antigen 336 (SP-336) types among Staphylococcus aureus isolated from bovine mastitis cases in Australia and India. METHODS A total of 414 strains (154 from Australia, 260 from India) isolated from clinical bovine mastitis were included in the study. Mouse antisera raised against CP types (CP1, CP2, CP5, and CP8) or SP-336 were used in slide agglutination tests and compared with detection of cap1, cap5 and cap8 gene fragments by PCR. RESULTS Serological studies revealed the presence of CP2, CP5, CP8 and SP-336 in 9.1%, 23.4%, 31.8%, and 5.8% of the Australian versus 0.8%, 46.9%, 13.1% and 0% of the Indian isolates, respectively. By PCR, CP1, CP5 and CP8 accounted for 0%, 26.6% and 32.4% of the Australian versus 3.9%, 85% and 8.1% of the Indian isolates, respectively. CONCLUSIONS Both PCR and the serological method demonstrated that CP5 and CP8 are the predominant capsular types in Australia, whereas CP5 is the predominant capsular type in India. The study also demonstrated a strong correlation between both methods of typing for CP1, CP5, CP8 and non-typeable S. aureus strains. High-percentage prevalence of non-typeable isolates in both the countries highlights the importance of continued investigations of the identification of unique surface-associated polysaccharide antigens prevalent among S. aureus isolates for the formulation of CP- and SP-based vaccines for bovine mastitis.

Diversity of Virulence Factors Associated with West Australian Methicillin-Sensitive Staphylococcus aureus Isolates of Human Origin

The aim of this study was to compare the performance of serological versus molecular typing methods to detect capsular polysaccharide (CP) and surface-associated polysaccharide antigen 336 phenotypes of Staphylococcus aureus isolates. Molecular typing of CP types 1, 5 and 8 was carried out using PCR, whereas serological typing of CP1, 2, 5, 8 and antigen 336 was carried out by slide agglutination using specific antisera. By genotyping, 14/31 strains were CP8 positive, 12/31 strains were CP5 and the remaining 6/31 isolates were non-typable (NT). One isolate was positive for both CP5 and CP8 by PCR, but was confirmed as CP8 type serologically. Detection of CP2 and type 336 by PCR was not possible because specific primers were either not available or non-specific. Using serotyping, 14/31 strains were CP8 positive, 11/31 CP5 positive and 2/31 positive for antigen 336. The remaining four S. aureus isolates were serologically NT. However, three of four NT and two 336-positive S. aureus isolates were encapsulated as determined by light microscopy after capsular staining. This discovery was surprising and warrants further investigations on the identification and characterization of additional capsular phenotypes prevalent among S. aureus clinical isolates. It was concluded that serological typing was a better method than molecular typing for use in epidemiological investigations based upon the distribution of surface-associated polysaccharide antigens-based phenotypes.

A comparative study of enumeration techniques for Free-roaming dogs in rural Baramati, District Pune, India.

Attachment of bacterial pathogens to the niche tissue in the host is the first step in biofilm formation leading to colonization and establishment of infection in the host. While the most common method used for determining the potential role of a bacterial antigen in biofilm formation has been demonstration of loss of this property using specific knockout mutants, it is an expensive and a laborious procedure. This study describes an alternative immunological assay for identification of attachment antigens of Staphylococcus aureus, potentially important in the development of an effective vaccine against infections caused by this pathogen. The method is based upon the concept of inhibition of attachment of S. aureus to PEGs coated with virulence antigen-specific antibodies. Antibodies used for validation of this assay were specific for ClfA, FnBPA, SdrD, PNAG and α-toxin, accredited biofilm-associated antigens of S. aureus.