Paul Costantino

Trends in therapeutic and prevention strategies for management of bovine mastitis: An overview

Mastitis is one of the most economically significant diseases for the dairy industry for backyard farmers in developing countries and high producing herds worldwide. Two of the major factors impeding reduction in the incidence of this disease is [a] the lack of availability of an effective vaccine capable of protecting against multiple etiological agents and [b] propensity of some of the etiological agents to develop persistent antibiotic resistance in biofilms. This is further complicated by the continuing revolving shift in the predominant etiological agents of mastitis, depending upon a multitude of factors such as variability in hygienic practices on farms, easy access leading to overuse of appropriate or inappropriate antibiotics at suboptimal concentrations, particularly in developing countries, and lack of compliance with the recommended treatment schedules. Regardless, Staphylococcus aureus and Streptococcus uberis followed by Escherichia coli, Streptococcus agalactiae has become the predominant etiological agents of bovine mastitis followed Streptococcus agalactiae, Streptococcus dysagalactiae, Klebsiella pneumonia and the newly emerging Mycoplasma bovis. Current approaches being pursued to reduce the negative economic impact of this disease are through early diagnosis of infection, immediate treatment with an antibiotic found to either inhibit or kill the pathogen(s) in vitro using planktonic cultures and the use of the currently marketed vaccines regardless of their demonstrated effectiveness. Given the limitations of breeding programs, including genetic selection to improve resistance against infectious diseases including mastitis, it is imperative to have the availability of an effective broad-spectrum, preferably cross-protective, vaccine capable of protecting against bovine mastitis for reduction in the incidence of bovine mastitis, as well as interrupting the potential cross-species transmission to humans. This overview highlights the major etiological agents, factors affecting susceptibility to mastitis, and the current status of antibiotic-based therapies and prototype vaccine candidates or commercially available vaccines against bovine mastitis as potential preventative strategies.

Humoral immune responses to systemic Candida albicans infection in inbred mouse strains

The protective role of humoral antibodies in the resolution of systemic candidiasis remains controversial. Investigation of the humoral immune responses in mouse strains of varying susceptibility to infection may demonstrate a link between mouse strain susceptibility, antibody production and specificity, and the ability to resolve an infection. The antibody response in five different strains of mice during a primary imune response to systemic infection with Candida albicans was investigated. Immune sera were fractionated by protein A affinity chromatography to yield fractions containing IgG l, IgG2a and IgG2b immunoglobulins. BALB/c mice of low susceptibility to the infection and DBA/2J mice of high susceptibility produced increased levels of the IgGl isotype and decreased levels of the IgG2a isotype. AKR, CBA/H and C57B1/6J mice of moderate susceptibility produced antibodies predominantly of the IgG2a isotype. The patterns of antigen recognition by antibodies in immune sera and in fractions obtained after protein A chromatography of immune sera were investigated by western blotting and immunostaining. Antibodies from AKR(H‐2K) and CBA/H (H‐2k) mice reacted strongly after immunoblotting with antigens of 87 and 96 kDa. In contrast, immune sera from both the highly susceptible DBA/2J (H‐2d) mice and the resistant BALB/c (H‐2d) mice reacted strongly with an antigen of 48 kDa. C57BI/6J (H‐2b) mice produced variable antibody reactivity to antigens of 48, 65, 66 and 79 kDa depending on the IgG subclass tested. The IgG subclass responses and the patterns of antigen recognition in these mice suggest that humoral responses to C albicans may be restricted by H‐2 haplotype. There was no clear correlation between humoral immunity and resistance or susceptibility to infection with C. albicans.

The persistence of biofilm-associated antibiotic resistance of Staphylococcus aureus isolated from clinical bovine mastitis cases in Australia

The aim of this investigation was to determine the persistence of biofilm-associated antibiotic resistance developed by methicillin-sensitive Staphylococcus aureus (MSSA), of different capsular types, during biofilm formation. Because of superiority of the tissue culture plate (TCP) over the Congo Red Agar (CRA) method for measuring biofilm formation, it was used to determine the persistence of the antibiotic resistance developed by the isolates in biofilms. The antibiotic resistance was found to persist for 3–4 wk post-propagation as planktonic subcultures. Interestingly, some strains even developed resistance to vancomycin and/or teicoplanin. However, no association of either biofilm formation or persistent antibiotic resistance with the major capsular phenotype was observed. These observations highlight the potential significance of (a) determining the antibiograms of S. aureus subcultured from biofilms developed in vitro using the TCP method as well as from planktonic cultures for formulation of an optimal therapeutic strategy, and (b) continuing to identify predominant non-capsular antigens contributing to biofilm formation, regardless of the capsular phenotype for the development of an effective potentially broad-spectrum vaccine for prevention of bovine mastitis caused by S. aureus.

Human methicillin-sensitive Staphylococcus aureus biofilms: potential associations with antibiotic resistance persistence and surface polysaccharide antigens

The development of persistent antibiotic resistance by human methicillin‐sensitive Staphylococcus aureus (MSSA) strains and substantial association with poly‐N‐acetyl glucosamine (PNAG) in biofilms is reported in this investigation. Sixteen of 31 MSSA strains under study were found to have developed resistance to one or more antibiotics, with four strains, two of which did not produce biofilms, showing resistance to cefoxitin, undetectable by mecA amplification. Antibiotic resistance displayed by 13/14 biofilm‐forming S. aureus isolates remained persistent for 4 weeks prior to reverting back to the original antibiotic susceptibility, prompting a suggestion of determining antibiograms for clinical S. aureus isolates subcultured from biofilms developed in vitro as well as planktonic subcultures prepared from the site of infection. While there was correlation of antibiotic resistance with biofilm formation confirming previous reports, this is the first time that persistence of the biofilm‐associated antibiotic resistance by S. aureus as planktonic cells is reported. Among the two methods used for assessment of biofilm formation, the tissue culture plate (TCP) method revealed that almost all strains were strong or moderate biofilm producers whereas only 19/31 strains were biofilm producers using the Congo Red agar (CRA) method indicating the superiority of the TCP method in detecting biofilm producers. We also observed no association between biofilm formation and major capsule types. However, substantial, although not absolute, association of biofilm formation with PNAG was observed, warranting continued identification of additional surface‐associated polysaccharide and/or protein antigens associated with biofilm formation for development of an effective vaccine against S. aureus infections regardless of capsular phenotype.

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.

Differentiation of Staphylococcus aureus and Staphylococcus epidermidis by PCR for the fibrinogen binding protein gene.

Mastitis is one of the most common and burdensome diseases afflicting dairy animals. Among other causes of mastitis, staphylococci are frequently associated with clinical and subclinical mastitis. Although Staphylococcus aureus is the predominant species involved, Staphylococcus epidermidis and other coagulase-negative staphylococci are increasingly being isolated from cases of bovine mastitis. Although Staph. aureus and Staph. epidermidis can be easily differentiated based on their biochemical properties, such phenotypic identification is time consuming and laborious. This study aimed to rapidly identify Staph. aureus and Staph. epidermidis. Accordingly, a multiplex PCR was developed and we found that a single gene encoding the adhesin fibrinogen binding protein could be used to identify and differentiate the two species. Consequently, a multiplex reaction combining a triplex PCR for Staph. aureus and a duplex PCR for Staph. epidermidis was standardized, first using bacterial cultures and then with pasteurized milk spiked with live organisms or DNA extracted from the organisms. The test could specifically detect Staph. aureus and Staph. epidermidis even in the presence of a dozen other organisms. The limit of detection for detecting Staph. aureus and Staph. epidermidis separately was 10 to 100 cfu/mL for simplex PCR and 10(4)cfu/mL for multiplex PCR. Conversely, the limit was 10(6)cfu/mL by multiplex PCR for simultaneous detection of both the organisms when spiked into culture medium or pasteurized milk. Overnight enrichment enhanced the assay sensitivity 100-fold. The assay had a high diagnostic sensitivity and specificity. The application of the test was verified on 602 field isolates of staphylococci that had been characterized earlier by phenotypic methods. Importantly, 25 coagulase-negative isolates were identified as Staph. aureus by the multiplex PCR. The test could be adapted for use in clinical diagnostic laboratories.

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.

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

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.