Mannheimia haemolytica is an opportunistic pathogen in cattle and the main bacterial agent in bovine respiratory disease. Despite its economic importance, few studies have characterized the genetic diversity of M. haemolytica, particularly from feedlots. Three genotyping techniques (BOX-PCR, (GTG)5-PCR and PFGE) were compared to discriminate M. haemolytica and strains from the family Pasteurellaceae. PFGE was the most discriminating and repeatable, although BOX-PCR was most accurate in clustering isolates together according to species. Mannheimia haemolytica was isolated from nasal swab samples collected from cattle upon entry and exit from two feedlots in southern Alberta. These were characterized by PFGE and antimicrobial susceptibility using a disk-diffusion assay. Select gene determinants were screened for using PCR. PFGE analysis revealed the isolates to be highly diverse. Ten percent of the isolates exhibited resistance. At present, the development and spread of antimicrobial resistance in M. haemolytica observed within the feedlots examined appears to be low. / xi, 116 leaves : ill. ; 29 cm

No description available.

Thesis (D.Tech.: Biotechnology)-Dept. of Biotechnology, Durban Institute of Technology, 2005 xx, 175, [14] leaves : ill. ; 30 cm / Previous studies indicate that a mutation in the non-oncogenic p53 gene is epidemiologically linked to human HCC (Ozturk, 1991; Chan et al., 2003). Hsu et al. (1991) found this link in Chinese, South African and Asian patients and Hollstein et al. (1993) found the same gene mutation in Taiwanese patients. The incidence of these aberrations is reported to be about 20- 50% in HCC’s (Kishimoto et al., 1997). There is sufficient evidence to indicate that carotenoids in addition to their well known antioxidant properties (Paiva and Russel, 1999), also affect intercellular communication, immune responses, neoplastic transformations and growth control, and cellular levels of enzymes that detoxify carcinogens (Zhang et al., 1991; Brockman et al., 1992; Pryor et al., 2000). To date studies carried out have used the rat (Foote et al., 1970; Gradelet et al., 1998) and the mule duckling model (Cheng et al., 2001) to show the protective effect of these carotenoids against AFB1 exposure. Of the well known carotenoids, lycopene and beta- carotene occur in abundance in fruits and vegetables and are safe for human consumption. Aflatoxin B1 frequently induces mutations of the p53 gene which is linked to HCC. Although there is much evidence from epidemiological studies linking the beneficial aspects of carotenoids to the prevention of cancer, the cellular and molecular mechanisms need to be understood in order to implement large scale intervention strategies to prevent AFB1 induced carcinoma. The use of chemical or dietary interventions to alter the susceptibility of humans to the actions of carcinogens and to block, retard or reverse carcinogenesis is an emerging chemoprotective strategy for disease prevention (Abdulla and Gruber, 2000; Kensler et al., 2003; Bingham and Riboli, 2004). Chemoprotection by natural products involves maintaining cellular integrity, preventing DNA alterations, activation of p53 suppressor protein and apoptosis. The aim of this study was thus to investigate the cellular and molecular mechanisms by which beta-carotene and lycopene may prevent the AFB1-induced toxic changes in human hepatocytes. In order to achieve this aim, the following objectives were set out: i. To optimise an in vitro system for the evaluation of AFB1 damage to cultured hepatocytes. ii. To determine the biochemical protection offered by beta-carotene and lycopene to AFB1-exposed hepatocytes, by measuring the mitochondrial activity, cell viability and ROS levels using appropriate enzyme assays and flow cytometry. iii. To determine the cellular protection offered by beta-carotene and lycopene to AFB1-exposed hepatocytes, by studying the morphological changes at the structural and ultrastructural levels using phase contrast light and electron microscopy respectively. iv. To determine the molecular protection offered by beta-carotene and lycopene to AFB1-exposed hepatocytes, by detecting apoptotic bodies as genomic markers and measuring the levels of p53 protein and AFB1-N7-guanine adducts produced.

Submitted in partial compliance with the requirements for a Master Degree in Technology: Chiropractic at the Durban University of Technology, 2009. / Background: Research has indicated the majority of bacteria on chiropractic adjustment beds (beds), can persist on dry inanimate surfaces for months. Thus, insufficient disinfection procedures create continuous sources of pathogens endangering patients and healthcare workers alike. This research study aimed to assess the beds as reservoirs for micro-organisms, at a chiropractic teaching clinic (clinic) in South Africa. Method: A selection of samples obtained from the headrests and armrests of the beds were serially diluted, plated in duplicate (using the spread plate technique) and incubated for 24-48 hours at 37°C. After inspection for the presence of micro-organisms, those present were enumerated to determine their quantities, the microbial build-up throughout the day, as well as the degree of the transmission from the patients to the beds during treatment. The incidence of the micro-organisms was established, along with their identities, using microscopic and macroscopic characteristics. These micro-organisms were also used to assess the efficacy of the disinfectant currently in use by the clinic. Results: Microbial growth was present on 89.4% of the beds sampled. The quantities of the micro-organisms increased significantly (p=0,027) from 7:30 am to 16:30 pm, with the median increasing from 25 colony forming units (cfu) / cm2 to 714 792 cfu/ cm2. The microbial build-up was highly significant (p<0.001), with a median of 346 cfu/ cm2 at 7:30 am and 10:30 am; increasing to 162 291 cfu/ cm2 by 13:30 pm and 250 million cfu/ cm2 by 16:30 pm. There was also a significant increase (p<0.001) in the quantity of micro-organisms during treatment with a median of 0 cfu/ cm2 before treatment that rose to 23 479 cfu/cm2 after treatment, indicating that the micro-organisms present on the beds were being deposited by the patient`s skin during the treatment. The most prevalent micro-organisms identified were Staphylococci and Serratia, with an average of 59% and 40% of colonies; while Micrococci and Bacilli were relatively uncommon. No growth was evident after 5 minutes of exposure to the disinfectant during the growth inhibition test. For the Kirby Bauer test, the average size of the zone of inhibition increased as the dilution decreased. The disinfectant is effective but more so against the Gram-positive than the Gram-negative bacteria. The disinfectant was 5,0, 5,5 and 5,6 times more effective than phenol in eradicating Staphylococci, Serratia and Bacilli, respectively. Conclusions and Recommendations: This study showed that micro-organisms were present on the beds. Staphylococci and Serratia have been implicated in many healthcare associated infections. The present disinfectant is effective, but should be used in between every patient. A different or additional disinfectant that is more effective against the Gram-negative bacteria should be considered for future use.

HIV infection has risen to pandemic proportions. Interleukin-10 (IL-10), a potent antiinflammatory cytokine has been shown to enhance the establishment and persistence of chronic viral infections through inactivation of effector antiviral immune responses and it may also directly influence HIV-1 replication in cells of diverse lineages. IL-10 promoter polymorphisms have been shown to affect HIV-1 susceptibility and pathogenesis. However, the underlying mechanisms are poorly understood. We investigated the relationship between IL-10 promoter variants, plasma IL-10 levels, and markers of disease outcome in chronically HIV-1-infected individuals. To investigate the mechanistic role of IL-10 and its genetic variants on HIV pathogenesis, we studied markers of activation on B cells, CD4+ and CD8+ T cells, and assessed effects on CD4+ T cell proliferation with and without blockade of the IL- 10 pathway. We used Taqman genotyping assays to genotype three IL-10 promoter single nucleotide polymorphisms (SNPs) in our study cohort. Baseline plasma IL-10 levels were measured using Luminex technology for 112 individuals. Viral load, CD4+ T cell counts and cytotoxic T lymphocyte (CTL) immune responses were measured at baseline. The rate of CD4+ T cell decrease was calculated in 300 individuals with a median follow-up of 25 months. CD38, CD95, Ki67, IgG and PD-1, markers of activation or exhaustion were measured on B cells, and CD38, CD95, Ki67, HLA-DR and PD-1 were measured on CD4+ and CD8+ T cells in a subset of 63 individuals. CD4+ T cell proliferation was measured using Carboxyfluorescein succinimidyl ester (CFSE) assays, following IL-10 receptor blockade in a subset of 31 individuals. The IL-10 -1082G, -592A and -3575 variants were observed at frequencies of 0.3, 0.34 and 0.23 respectively, in our study cohort. Plasma IL-10 levels were significantly higher in the - 1082GG group than in the combined AA/AG group (p=0.0006). There was a significant association between the 592AA genotype and a greater breadth of CTL responses compared to the CC and CA (p= 0.002 and 0.004 respectively). The -592AA genotype associated significantly with an attenuated loss of CD4 cells (p= 0.0496), with -592AA having the least change in CD4 cells per year. The median expression of HLA-DR, a marker of T cell activation was significantly higher in the-1082AA group for CD8 cells (p= 0.047), and the - 592AA group for CD4 T cells (p= 0.01). The median expression of IgG on the surface of B cells was significantly higher in the -1082GG genotype and the -592CC genotype (p=0.0183 and 0.0659 respectively). Overall, IL-10 variants correlated with IL-10 expression and CD4 decline during chronic HIV-1 infection. IL-10 promoter variants may influence the rate of HIV-1 disease progression by regulating IL-10 levels, which in-turn, may affect the breadth of CTL responses. Furthermore, the increased expression of HLA-DR and PD-1 on CD8+ and CD4+ T cells, indicates that lower IL-10 levels are associated with increased immune activation and immune exhaustion. The increased expression of IgG on B cells, suggests that in a setting of lower IL-10, there is possibly a bias towards a Th2 immune response. These data suggest a significant role for IL-10 genetic variants and IL-10 in HIV pathogenesis. Further studies to determine whether and how the IL-10 pathway may be manipulated for therapeutic or vaccine strategies for HIV are warranted. / Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2012.

The status of the Baynespruit bacteriological water quality is very alarming - E-coli concentrations have far exceeded the allowable limit of both local and international guidelines for more than a decade, namely 2000-2010. Concentrations of indicator bacteria have been recorded as high as 2419000 cfu/100 ml, whereas guideline levels of E-coli for recreational contact are about 130 cfu/100 ml. In this study, statistical analyses were carried out on data from two sampling points to clarify the seasonal changes and the variability of the pollution. Cross-correlation analyses showed that there was no significant correlation between E-coli concentrations and rainfall in the uMsunduzi catchment. There was also only a weak correlation between the two sampling points which suggests the existence of unregulated sources of pathogenic water pollution between the sampling locations that are independent of the effect that rainfall has on dilution and dispersion of pollution. The data indicates that the population living along the Baynespruit has about a 2% risk of contracting gastrointestinal illness as a result of the pollution in the stream. / Thesis (M.Sc.Eng.)-University of KwaZulu-Natal, Durban, 2011.

Antibiotics are important to equine medicine, but can cause detrimental side-effects including reduced feed intake, allergic reactions, and diarrhea. Antibiotic-associated diarrhea (AAD) is attributed to disruption of the hindgut microflora, permitting proliferation of pathogenic microbes. The objectives were to evaluate the effects of antibiotics on beneficial fecal bacteria, AAD-associated pathogens, microbial species richness and fermentation. Horses were assigned to treatment groups: control (no antibiotics, n=6), trimethoprim-sulfadiazine (oral, n=6), or sodium ceftiofur (IM, n=6). Fecal samples were taken during adaptation (3 wk), antibiotic challenge (1 wk), and withdrawal (1 wk). Fecal cellulolytics decreased by >99% during challenge and did not recover during withdrawal (P < 0.0001). Lactobacilli decreased by >60% during challenge (P = 0.0453). Salmonella spp. increased 94% with trimethoprim-sulfadiazine challenge (P = 0.0115). There was no detectable Clostridium difficile during adaptation or in any control horse. C. difficile increased (P < 0.0001) when horses were challenged, and remained elevated 7 d after withdrawal. There was no effect of challenge on in vitro digestibility or microbial species richness as evaluated by denaturing gradient gel electrophoresis (P > 0.05). These results indicate that antibiotics can disrupt the normal flora and allow proliferation of pathogens, even without affecting digestibility and causing AAD.

This study explored the in vitro and in situ antilisterial inhibitory activity of 16 essential oils during indirect exposure: Spanish Basil oil (Ocimum basilicum), Bay oil (Pimenta racemosa), Italian Bergamot oil (Citrus bergamia), Roman Chamomile oil (Anthemis nobilis), Sir Lanka Cinnamon oil (Cinnamomum zeylanicum), Citral, Clove Bud oil (Syzygium aromaticum), Cumin Seed oil (cuminum cyminum), Eucalyptus oil (Eucalyptus globulus), Eugenol, Geranium extract (Pelargonium graveolens), Marjoram oil (Origanum majorana), Neroli extract (Citrus aurantium), Peppermint oil (Mentha piperita L.), Rosemary oil (Rosmarinus officinalis L.),and Spanish Sage oil (Salvia officinalis L.). All essential oils were tested against Listeria monocytogenes (ATCC 4644). In vitro inhibitory activity was determined using the microatmosphere method at three temperatures (37°C, 24°C, 4°C) and six possible volumes (0, 10µl, 25µl, 50µl, 100µl, 150µl, or 200µl). In situ inhibitory activity was determined using inoculated bologna slices packaged in Modified Atmosphere Packaging (80% O2, 20% CO2). Essential oils (0, 0.13ml, 1.35ml, or 2.70ml) were injected into the sample packages adjacent, but not touching, the bologna slices and stored at 24°C for 24h. Basil oil displayed the least antilisterial activity across the three temperature applications in vitro. Peppermint, Cumin Seed, and Citral consistently exhibited the greatest antilisterial activity among the temperature applications in vitro. However, only Eugenol applied at 1.35ml achieved a mean one log10 CFU/ml reduction in LM in situ, which could not be replicated. Peppermint (P < 0.048) displayed significant differences between application volumes (0.13ml, 1.35ml) but did not attain a mean one log10 CFU/ml reduction in LM. This study suggests that while various essential oils can display antilisterial activity in vitro, transitioning into a MAP food system warrants further research in mode of actions and application volumes.

Quality control procedures during food processing may involve either lengthy enrichment steps, precluding enumeration of bacteria in contaminated food, or direct inoculation of food samples onto appropriate selective media for subsequent enumeration. However, sublethally injured bacteria often fail to grow on selective media, enabling them to evade detection and intervention measures and ultimately threaten the health of consumers. This study compares traditional selective and nonselective agar-based overlays versus two commercial systems (Petrifilm and Easygel) for recovery of injured Escherichia coli B-41560, originally an isolate from ground beef. Bacteria were propagated in tryptic soy broth (TSB), ground beef, or infant milk formula (IMF) to a density of 106-108 CFU/mL, and stressed for six minutes either in lactic acid (pH of 4.5) or heat-shocked for 3 min. at 60°C. Samples were pour- plated in basal layers of either tryptic soy agar (TSA), Sorbitol MacConkey (SMAC), or Violet Red Bile (VRB) agar and resuscitated for 4h prior to addition of agar overlays. Other stressed bacteria were plated directly onto the commercial media Petrifilm and Easygel. Our results indicate that the use of selective and nonselective agar overlays for sensitive recovery and accurate enumeration of E. coli B-41560 is dependent on the stress treatment and food system. These data underscore the need to implement food safety measures that address sublethally- injured bacteria such as E. coli O157:H7, without the use of enrichment steps, in order to avoid underestimation of true densities for target pathogens. / Department of Biology

Foodborne diseases are one among the diseases with high morbidity and mortality rate. The concern is raised with the emergence of pathogenic strains that are resistant to the available set of antibiotics. Conventional regimens fail to treat the infections caused by these pathogens prolonging the sickness leading to increased morbidity and mortality. The situation can get further complicated with the dietary intake of the host. Of late it has been understood that the dietary flavonoids play an important role in regulating the immune system. Curcumin, a pigment from turmeric, is one among such bioflavonoid with an immunomodulatory potential. Curcumin has been a front-line topic of mainstream scientific research for a variety of diseases from cancer to Alzheimer’s to infectious diseases. Curcumin being considered as a spicy panacea is not a remedy for all diseases. Its ability to act differentially as an antioxidant or pro-oxidant can be either beneficial or harmful for the host. It exhibits antioxidant properties at concentrations achievable in the body; this can make the host vulnerable to infections due to the suppression of innate immune responses. Curcumin also suppresses the type 1 immune response, which might lead to alleviation of type 1 immune response disorders. However, the inhibition of type 1 immune response might invite infections with opportunistic pathogens. We have chosen curcumin to assess the effect of diet on the regulation of pathogenesis of Salmonella along with few medically important pathogens like Yersinia enterocolitica, Staphylococcus aureus, Shigella flexneri and Listeria monocytogenes. The thesis is divided into five chapters. As the main focus of the thesis is on Salmonella, in Chapter 1 we introduce diverse aspects of curcumin and the basic biology of Salmonella. Initially the properties of curcumin, the molecule of interest are introduced followed by brief overview to Salmonella biology and pathogenesis. Various activities of curcumin dealing with the variety of diseases are discussed. Further, the introduction to the intricate underlying mechanisms and the functional determinants of curcumin is given. The subsequent sections give an overview of different phases of Salmonella pathogenesis and the molecular mechanisms of Salmonella virulence and host defense. Towards the end of the chapter we discuss the strength, limitations and the distinctive characteristics of the murine model of typhoid fever. Curcumin has gained immense importance for its vast therapeutic and prophylactic applications. Its anti-bacterial effect has been demonstrated in bacteria, like B. subtilis, H. pylori and E. coli. Contrary to this, the results of the Chapter 2 reveals that curcumin at a nontoxic concentration to both host and pathogen, regulates the defense pathways of Salmonella enterica serovar Typhimurium (S. Typhimurium) to enhance its pathogenicity. In a murine model of typhoid fever, we observed higher bacterial load in reticuloendothelial organs when infected with curcumin-treated Salmonella. Curcumin increased the resistance of S. Typhimurium against antimicrobial agents like antimicrobial peptides, reactive oxygen and nitrogen species. It up-regulated the genes involved in resistance against antimicrobial peptides - pmrD and pmrHFIJKLM and genes with antioxidant function - mntH, sodA and sitA. We implicate that the iron chelation property of curcumin has a role in regulating mntH and sitA. Interestingly, we see that the curcumin-mediated modulation of pmr genes is through the PhoPQ two-component regulatory system (TCS). Curcumin downregulates SPI-1 genes required for entry into epithelial cells and upregulates SPI-2 genes required for intracellular survival, through PhoPQ TCS. Thus, this common regulator (PhoPQ) could explain curcumin's mode of action. Another important factor for the pathogen’s success is its ability to counteract the action of antibiotics. Almost all the bactericidal antibiotics act via production of reactive oxygen species in the bacteria. Curcumin has anti-oxidant property that might interfere with the action of antibiotics. Ciprofloxacin is a commonly used anti-typhoidal drug. It kills the bacteria by inhibiting DNA replication and increasing reactive oxygen species in bacterial cell. In Chapter 3 we present the results obtained after the investigation of the interference of curcumin with the anti-bacterial action of ciprofloxacin against Salmonella. We found that curcumin indeed increased the proliferation of Salmonella Typhi and Salmonella Typhimurium in ciprofloxacin treated macrophages by reducing the ciprofloxacin-induced reactive oxygen species. It also inhibited ciprofloxacin mediated DNA damage and the resultant SOS response and filamentation. However, curcumin was unable to rescue the ciprofloxacin induced gyrase inhibition. The reduced antibiotic (ciprofloxacin) efficacy against Salmonella by curcumin might aggravate the disease. Thus, the results of chapter 1 and 2 urge us to rethink the indiscriminate use of curcumin especially during Salmonella outbreaks. Bacteria modulate its virulence determinants in response to the environmental cues. Salmonella being a foodborne pathogen has a very likely chance of getting exposed to turmeric and hence curcumin. In Chapter 4 we have assessed the modulation of motility of S. Typhimurium, an important virulence determinant, by curcumin. We show that curcumin reduced the motility of the S. Typhimurium by decreasing the flagellar density around it. Surprisingly, this was achieved without affecting the expression of the flagellin gene and protein. Curcumin physically adhered to the flagella making it fragile and breaking it into fragments. This can hinder bacterial motility, chemotaxis, adherence and invasion into the host cells. However, aflagellate bacteria are hypervirulent as is the case with our experimental results with curcumin treated bacteria. Curcumin regulates myriad of bacterial (Salmonella) activities increasing its pathogenicity. Curcumin is known to regulate the host defenses in response to the disease. In Chapter 5 we have sought to address the effect of curcumin treatment of host cells on the outcome of infection by different pathogens. Pathogens have evolved different strategies to evade the host innate immune system, one of them being avoiding lysosome mediated degradation. Pathogens like Salmonella, Yersinia, Mycobacterium and Staphylococcus have acquired molecular machinery to inhibit the fusion of the pathogen containing vacuole with lysosomes and multiply within the vacuole whereas other pathogens like Shigella, Listeria and Rickettsia escape into and multiply in the cytosol. In our study we show that pretreatment of macrophage with curcumin increased the fold proliferation of S. Typhimurium, S. aureus and Y. enterocolitica whereas decreased that of S. flexneri and L. monocytogenes. From the results obtained, we can state that curcumin differentially regulates the pathogenesis of vacuolar and cytosolic pathogen. We hypothesized that curcumin pretreatment stabilizes the membrane of pathogen containing vacuole retarding the lysis of the phagolysosome harboring the cytosolic pathogen and hence facilitating its clearance. We indeed observed that the membrane stabilizing effect of curcumin led to increased fusion of cytosolic pathogen with the lysosome, decreasing its proliferation in the cells. As the vacuolar pathogens have an inherent ability to inhibit this fusion, they proliferate better in curcumin treated cells. In a nutshell curcumin can have multiple and sometimes unexpected effects not only on a pathogen’s potential to successfully cause infection but also on the host’s ability to counter it. A brief summary of the study that does not directly deal with the modulation of bacterial pathogenesis by curcumin is included in the Appendix. In this study a novel, simple, sensitive and efficient PCR based assay was devised to detect Salmonella contamination in milk, fruit juice and ice-cream without any pre-enrichment.