VTechWorks Repository :: Browsing by Author "Mullarky, Isis K."

Browsing by Author "Mullarky, Isis K."

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Ability of Klebsiella spp. mastitis isolates to produce virulence factors for enhanced evasion of bovine innate immune defenses
Nedrow, Alicia (Virginia Tech, 2009-11-24)
Klebsiella spp. are coliform bacteria that cause mastitis in dairy cattle and account for high mortality rates in infected cows leading to a significant financial loss. Recent outbreaks indicate that within farms a single strain can be responsible for clinical signs in multiple animals. Identification of the virulence of factors enabling Klebsiella spp. survival in the mammary glands of multiple animals may provide insight into host adaptation. In this study, Klebsiella spp. strains were evaluated for their ability to evade neutrophil killing, the primary immune defense in the bovine mammary gland. Our research focused on capsule and biofilm production by Klebsiella spp. when strains were grown in Luria Broth or skim milk to examine the effects on evasion of neutrophil killing. Biofilm production was not significantly related to the ability to resist neutrophil killing nor was capsule (P = 0.29). Farm (P < 0.001), media type (P < 0.005), and strain type by cow (P < 0.001) were found to play significant roles in neutrophil evasion. This suggests farm of origin, media type used, and cow all may play a role in evasion of neutrophils by Klebsiella spp. Further evaluation of virulence factor expression in different media types and the role of individual cow immune responses may provide insight into ability of Klebsiella spp. to cause outbreaks of mastitis in multiple animals.
Brucella abortus Strain RB51 Outer Membrane Vesicles as a Vaccine Against Brucellosis in a Murine Model
Cassidy, Clifton Clark (Virginia Tech, 2010-02-03)
Brucella abortus is a zoonotic agent that primarily infects cattle and causes brucellosis. B. abortus strain RB51 is a live, attenuated vaccine licensed for cattle. However, there is no available vaccine to prevent human brucellosis. Outer membrane vesicles have been tested as potential vaccines to prevent diseases caused by bacterial species. OMV are constantly released from Gram-negative bacteria. They are comprised principally of the outer membrane components and periplasmic proteins from the bacterial cell envelope. The research in this thesis examined the adjuvant property of non-replicative, metabolically active irradiated strain RB51 and the protective ability of OMV derived from strain RB51. Irradiated B. abortus strain RB51 was assessed for its ability to act as an adjuvant to induce protection against malaria. It was found that irradiated B. abortus strain RB51 administered along with fasciclin related adhesive protein (FRAP) to mice induced a protective immune response and a significant decrease in parasitemia after challenge with Plasmodium berghei. Strain RB51 and strain RB51 over-producing Cu/Zn superoxide dismutase (Cu/Zn SOD) were used to produce OMV. Western blotting and SDS-PAGE gel staining confirmed the presence of OMV and the over-production of Cu/Zn SOD. OMV were delivered to mice using an intraperitoneal route and, in some cases, with aluminum hydroxide adjuvant. The immune response was assessed by antibody isotyping with respect to OMV and measuring splenic clearance (i.e. protection) from a B. abortus strain 2308 challenge. The results demonstrate that OMV from B. abortus strain RB51 or strain RB51 over producing Cu/Zn SOD produced a Th1 polarized immune response as measured by specific OMV antibodies and cytokines but no statistically significant protection was observed.
Characterization of Dendritic Cells in the Bovine Mammary Gland
Maxymiv, Nicolas George (Virginia Tech, 2009-12-03)
Bacterial mastitis is a significant problem for the dairy industry. A vaccine against mastitis pathogens could potentially target dendritic cells (DC). While there has been some research describing bovine DC populations in-vitro, little is known about DC in mammary tissue. In this study, immunohistofluorescence was used to identify and localize bovine mammary DC. DC were found in alveoli, in epithelia, and in interalveolar tissue. Fluorescence-activated cell sorting (FACS) was used to characterize mammary DC as expressing CD11c, MHC-II, CD205, CD11b, and CD8Î±. FACS allowed us to distinguish DC (CD14lo) from macrophages (CD14hi). Two DC subsets, CD11a-, CD11alo, were evident in the mammary gland while an additional CD11ahi population was identified in the supramammary lymph node. After phagocytosis of bacterial components such as lipopolysaccharide (LPS), DC undergo a maturation process, in which they upregulate homing receptors, such as CCR7, and antigen presentation markers, including MHCII and CD80. A primary cell culture model was used to evaluate changes in transcription of CD80 and CCR7 after LPS stimulation. Cell cultures contained digested and Ficoll separated mammary tissue or supramammary lymph node tissue. While the presence of CCR7 and CD80 was confirmed, CD80 and CCR7 transcripts were not upregulated after LPS stimulation. Further, CD11c, CD14, MHCII, CD11b, CD11a, and CD205 protein levels, as assessed by FACS, were similar in LPS stimulated cultures and unstimulated controls. Overall, these studies provide a better understanding of mammary gland immunology, while potentially aiding in the development of novel DC based vaccines.
Characterizing physiological and genetic differences in the early immune response to Haemonchus contortus in resistant and susceptible sheep
Bowdridge, Scott Alexander (Virginia Tech, 2009-10-12)
This dissertation compares immune responses of resistant and susceptible sheep to infection with Haemonchus contortus during the peri-parturient period and larval stage of infection. Identification of immunological events resulting in parasite resistance in St. Croix hair sheep may provide better targets for differential gene expression analysis and eventual discovery of selectable markers for parasite resistance. Antibody levels of hair ewes and composite Dorset x Finnsheep-Rambouillet wool ewes were measured during breeding and again after parturition. Results demonstrated that hair ewes had higher levels immunoglobulin-A after infection and maintained a higher level of circulating antigen-specific antibody when compared to wool ewes. To characterize immune responses to the larval stage of infection, hair and wool lambs were sacrificed at 0, 3, 5, and 7 d after infection with H. contortus. Neutrophil migration to abomasal mucosa and lymph node development were higher in hair sheep than in wool sheep. Gene expression analysis indicated no difference in the abomasal lymph node as both breeds expressed a general T-helper cell type 2 (TH2) response. However, profound differences in TH2 responses were observed in the abomasal mucosa, where hair sheep expressed more IL-4, -13 and -33 than wool sheep. These data thus document the presence of immunological differences between the breeds. Immune responses to larval parasite infection in wool sheep are generally suppressed and may increase the magnitude and duration of infection whereas immune responses to larval infection in hair sheep was more robust and more strongly polarized towards a TH2
Differential expression profiling of proteomes of pathogenic and commensal strains of Staphylococcus aureus using SILAC
Manickam, Manisha (Virginia Tech, 2011-11-29)
Staphylococcus aureus (S. aureus) is the etiological agent of food-borne diseases, skin infections in humans and mastitis in bovines. S. aureus is also known to exist as a commensal on skin, nose and other mucosal surfaces of the host. This symbiotic association is a result of immune dampening or tolerance induced in the host by this pathogen. We proposed the variation in protein expression by commensal and pathogenic strain as an important factor behind the difference in pathogenicity. The identification of differentially expressed proteins was carried out using a quantitative mass spectrometry (MS)-based proteomic approach, known as stable isotope labeling of amino acids in cell culture (SILAC). Four commensal and pathogenic strains each were grown in the SILAC minimal media (RPMI 1640), containing light (12C) and heavy (13C) form of lysine, respectively, until early stationary growth phase. Various protein fractions, including cell wall, membrane and secreted, were extracted from the bacterial cultures and mixed in a 1:1 ratio. The relative abundance of proteins present in light and heavy labeled samples was determined using MS analysis. From a total of 151 differentially expressed proteins, 58 were found to be upregulated in the pathogenic strains. These proteins are involved in a variety of cellular functions, including immune modulation, iron-binding, cellular transport, redox reactions, and metabolic enzymes. The differentially expressed proteins can serve as putative candidates to improve current approach towards development of a vaccine against S. aureus.
Implications of the ability of Enterococcus spp. to survive the ensiling process and bovine gastrointestinal tract on the risk of bovine mastitis
Masiello, Stephanie Noelle (Virginia Tech, 2010-01-25)
Three studies were conducted to assess if the ability of Enterococcus spp. surviving the ensiling process and bovine gastrointestinal tract could impact risk of bovine mastitis. The first study determined ability of enterococci to survive 3 wk ensiling. Grass and corn crops were divided into 3 treatments: 2 commercial silage inoculants, 1 negative control. After wk 1, 2, and 3 of ensiling, dry matter and bacterial enumeration were performed. Addition of silage inoculant led to greater levels of enterococci in grass silage compared with negative control levels, but showed less difference in inoculated corn silage. The second study quantified enterococci shedding rates in lactating dairy cows. Using a 4 x 4 Latin Square design, lactating, ruminally fistulated Holsteins were inoculated with enterococcal isolates from silage inoculants, ensiled forages, or clinical mastitis cases. Over the 4-period study, each period consisted of rumen and fecal sampling (2 wk) followed by a wash period (10 d). There were no significant differences in rumen or fecal enterococci levels between the 4 treatments. Both rumen and fecal enterococci levels showed significant differences between baseline and treatment periods (period 3, 4). The third study analyzed similarity in enterococcal isolates of silage and bovine origin using pulsed-field gel electrophoresis patterns from SmaI restrictions. Dendogram analysis showed none of the isolates met or were greater than a 75% genetic similarity and produced a genetically diverse population. Results suggest Enterococcus spp. from silage inoculants are not likely to contribute to an increased risk of enterococcal bovine mastitis.
Influence of Growth Factors on Bovine Embryo Development
Lott, Whitney Meghan (Virginia Tech, 2008-07-28)
Many attempts have been made to improve the in vitro production of cattle embryos by refining in vitro maturation (IVM) and culture systems. Cysteine supplementation to IVM media of bovine oocytes increases cellular glutathione production, which reduces reactive oxygen species (ROS). Similarly, beneficial effects of growth factors for improving the rate of blastocyst development have been reported, but combined effects are unknown. This study was conducted to determine the additive effect of the antioxidant cysteine with epidermal growth factor (EGF) and/or insulin-like growth factor-I (IGF-I) on subsequent embryo development. Bovine oocytes from slaughterhouse ovaries were matured in TCM-199 (control), with or without the addition of 0.6 mM cysteine (C) at 0 or 12 h of maturation. After in vitro fertilization, embryos were allocated to culture treatments containing synthetic oviductal fluid medium. Culture treatments included fetal calf serum (FCS, 4%) alone; IGF-I (100 ng/mL); EGF (10 ng/mL); and IGF-I+EGF (100 ng/mL+10 ng/mL) for all IVM treatments. Although rates for blastocysts development were not different among treatments, an increased proportion of embryos attaining morula formation was achieved when cysteine was added to the IVM media (12 h C IGF-I+EGF, 41.4%; 0 h C EGF, 40.0%) as compared to control (FCS: 34.6%). When cysteine treatments were combined, percent cleavage was greater for IGF-I+EGF (70.8%) compared to FCS (61.2%). The abundance of mRNA from the apoptotic genes, Bax and Bcl-2, and the oxidative stress genes, copper (Cu)-zinc (Zn) superoxide dismutase (SOD; SOD1) and manganese (Mn) SOD (SOD2) in embryos was assessed. No significant treatment effect was observed on the expression of apoptotic and oxidative stress genes. Bax was expressed strongly (4-fold) in morulae with the addition of IGF-I, but was less prevalent in all other morula and blastocyst groups relative to FCS. There was slightly less expression of both SOD1 and SOD2 with treatments compared to FCS in morulae and blastocysts, indicative of low mitochondrial activity and/or a low level of oxidative stress in treatments. There was no significant treatment effect on total cell number, apoptotic nuclei, or apoptotic index. In conclusion, supplementation of cysteine during IVM of oocytes, in conjunction with growth factors could effectively be used as a replacement for FCS.
Klebsiella spp.: A Practical Summary for Controlling Mastitis
Petersson-Wolfe, Christina S.; Mullarky, Isis K.; Currin, John F. (Virginia Cooperative Extension, 2011-07-29)
Klebsiella spp. are environmental organisms. The most common, mastitis-causing species are Klebsiella pneumoniae and K. oxytoca, but treatment and control is similar for all species of Klebsiella.
Management factors affecting calf growth and health
Machado, Kayla L. (Virginia Tech, 2011-11-29)
Two calf feeding trends are emerging in the dairy industry in the United States. Large herds often find it economical to feed pasteurized waste milk; while smaller herds are embracing technological advancements by utilizing automated calf milk feeders. Housing of calves varies depending on feeding mechanism. Calves fed using autofeeders are grouped together but large herds often find it more labor efficient to house calves individually in elevated wooden crates or polyethylene hutches. Two studies were conducted. The objective of the first field study was to evaluate the influence of diet and housing type on growth and morbidity in 84 Holstein heifer calves in a 2 by 2 factorial experimental design. Calves were housed in either polyethylene hutches or elevated wooden crates with slatted floors. Diets consisted of pasteurized waste milk or the same waste milk supplemented to provide approximately 454 g of milk replacer solids containing 25% protein and 10% fat (LOL Balancer). Calves were randomly placed in 1 of 4 treatment groups 48 h after birth and monitored until weaning (~60 d of age). Body weights and hip heights were measured at time of enrollment and weaning. Milk samples of pasteurized waste milk were obtained five times weekly to measure standard bacteriological plate count, fat, protein and total solids content. All calves were fed 3.3 L of liquid diet via bottle at 0730 and 1530 h. Calves were monitored daily for respiratory and digestive illness and treated according to established protocols. Pasteurized waste milk contained 332,171 ° 733,487 cfu/ mL, 3.51 ° 0.59% fat, 3.13 ° 0.30% protein, and 11.64 ° 1.05% total solids. Housing (P = 0.02) and diet (P = 0.01) affected weight gain, but there was no interaction. Least squares average daily gain for crate and hutches were 0.52 ° 0.024 and 0.59 ° 0.024 kg/d. Least squares average daily gain for waste milk and balancer diets were 0.52 ° 0.024 and 0.60 ° 0.024 kg/d, respectively. Housing or diet did not affect hip height growth/d (0.196 ° 0.007 cm). Health of the calves was not affected by diet or housing. Supplementing waste milk with balancer or housing calves in hutches resulted in higher weight gain. The objective of the second study was to evaluate management, and sanitation and consistency of liquid delivered to calves via automated feeders. Ten herds in Virginia and North Carolina with sophisticated (FÃ¶rster-Technik, Germany) and basic (Biotic Industries Inc., TN, USA) machines completed a 60-question survey concerning calf and autofeeder management. Duplicate milk replacer samples were obtained to measure sanitation, dry matter, and temperature of milk in the autofeeder at the time of the survey. Six dairies from the original 10 were visited monthly for 3 mo for continued evaluation of sanitation, dry matter, and temperature of milk replacer from the autofeeder. Seven herds utilizing basic machines had a mean SPC of 6,925,000 ° 7,371,000 cfu/ml. The mean dry matter and temperature readings were 12.0 ° 2.1 Brix and 38.8 ° 6.7 °C, respectively. Three dairies that used sophisticated autofeeders had a mean SPC of 1,339,000 ° 2,203,000 cfu/ml. Mean dry matter and temperature readings were 10.37 ° 1.68 Brix and 38.6 ° 6.76°C, respectively. Dairies were also categorized based on management strategies. Producers that purchased autofeeders to manipulate feeding rates, refocus labor to sanitation, and care and well-being of calves, or for technological advancements were successful at rearing calves via autofeeders. Dairy producers who purchased an autofeeder to explore feeding options were not as successful because proper time and management was not dedicated to care of calves or to maintenance of the autofeeder.
Monocyte Derived Dendritic Cells: Sentinels and Translators of Immune Response to Staphylococcus aureus
Bharathan, Mini (Virginia Tech, 2010-10-05)
Staphylococcus aureus is a versatile opportunistic pathogen causing a wide spectrum of diseases in both humans and animals. My research focused on characterization of the immune responses of monocyte derived dendritic cells (DC) to S. aureus. We initially evaluated the potential of circulating monocytes to serve as precursors for DC during S. aureus infection. The CD14⁺ monocytes, when stimulated with irradiated (ISA) or live S. aureus (LSA), differentiated into CD11c^high CD11b^high DC (MonoDC) in an autocrine fashion. This was associated with the up- regulation of granulocyte-macrophage colony stimulating factor (GMCSF) and tumor necrosis factor-α (TNF-α) gene transcription. We continued our studies to identify the role of TNF-α in the LSA induced differentiation of monocyte to MonoDC. Blocking TNF-α reduced the expression of CD11c and increased the expression of CD14 on LSA stimulated monocyte derived MonoDC. Stimulated monocytes were able to secrete monocyte chemotactic protein-1 (MCP-1), a chemokine that recruits monocytes to the site of infection/injury and induces the expression of β₂ integrins on DC. Characterization of the response of DC derived from monocytes using GMCSF and IL-4 revealed that, intact S. aureus rather than its purified structural components were efficient in DC activation. In response to ISA or LSA stimulation, DC induced proliferation of T cells collected from the peripheral circulation of cows with a history of S. aureus mastitis. Subsequent characterization of the proliferating T cells identified the presence of memory T cells. Finally, we identified a unique population of DEC205⁺CD8^a+ in monocyte derived DC. We further elucidated the role of DC DEC205, a C-type lectin, in S. aureus uptake. Blocking of receptor mediated endocytosis resulted in reduced uptake of S. aureus by DC. Confocal microscopy confirmed a role for DEC205 in S. aureus internalization and delivery to endosomes. DEC205 DC upon stimulation with S. aureus displayed enhanced maturation and antigen presentation. In conclusion, monocyte derived DC can uptake S. aureus and elicit cell mediated immune responses.
A porcine model for polymicrobial respiratory infections with swine influenza virus and Staphylococcus aureus
Smith, Elizabeth Allison (Virginia Tech, 2010-09-27)
Influenza A virus (IAV) is a significant problem worldwide, and respiratory disease is further complicated by secondary bacterial infection. The emergence of highly pathogenic strains of IAV in conjunction with the increase of antibiotic-resistant bacteria threatens human health. A large-animal model effective for study of polymicrobial infection comparable to humans must therefore be developed. IAV has been studied extensively in small animals, including mice, rats and ferrets. However, these species frequently require IAV adaptation, reducing the capacity of these models to adequately represent human infection. Furthermore, species commonly used lack likeness to humans in both the presentation of symptoms and in lethality of infection. However, pigs are naturally susceptible to unadapted IAV and are considered to be the 'mixing vessel' for the recent pandemic IAV virus. Pigs are also susceptible to infection with Staphylococcus aureus, the most commonly isolated bacteria from IAV-infected human adults. Therefore, the use of pigs in the study of polymicrobial respiratory infections would be ideal for characterizing a host immune response comparable to humans, as well as for the development of diagnostics and therapeutics. Using this novel model, we determined that pigs are susceptible to Staphylococcus aureus, swine IAV, and polymicrobial infection. Furthermore, we showed that IAV infection predisposes pigs to Staphylococcus aureus pneumonia, and this susceptibility is dependent on day post-IAV infection.
Staphylococcus aureus Mastitis: Cause, Detection, and Control
Petersson-Wolfe, Christina S.; Mullarky, Isis K.; Jones, Gerald M. (Virginia Cooperative Extension, 2010-06-11)
Staphylococcus aureus (S. aureus) mastitis is extremely difficult to control by treatment alone. To date, successful control is gained only through prevention of new infections and culling of infected animals.
Staphylococcus aureus virulence factors dictate host signaling pathways and immune responses
Ortiz Marty, Rebecca Josefina (Virginia Tech, 2011-12-07)
Staphylococcus aureus causes nosocomial- and community- acquired infections. This versatile pathogen expresses virulence factors (VF) that enhance establishment of infection and immune evasion. Our research focused on defining the roles of S. aureus VF on host immune responses during intracellular or extracellular infections. Accessory gene regulator (agr) controls VF expression and intracellular survival. Our goal was to determine mammary epithelial cells (MEC) responses to intracellular infection and subsequent polymorphonuclear leukocyte (PMN) responses. Intracellular S. aureus increased thrombomodulin expression by MEC and activated protein C (APC) production. APC inhibited PMN chemotaxis. Findings depicted an indirect role for VF on PMN responses, so next we determined signaling pathways and cytokine responses of PMN to S. aureus toxins. Live S. aureus infections increased activation of stress signaling pathways and highlighted a role for agr-regulated genes in MAPK p38 phosphorylation and α-hemolysin in ERK phosphorylation and IL-8 expression in PMN. Continuing our studies of VF, chemotaxis inhibitory protein of S. aureus (CHIPS) inhibits monocyte chemotaxis. We hypothesized that CHIPS inhibited C5a receptor (C5aR) signaling. Monocytes pretreated with CHIPS did not inhibit C5aR signaling. Nevertheless, signaling pathways can reduce PMN function in models such as glucocorticoid treatment. Immunosuppressive effects of glucocorticoids on PMN are restored with OmniGen-AF® supplementation. Glucocorticoid receptor and Toll-like receptor signaling potentially crosstalk to restore PMN function. OmniGen-AF® supplementation restored dexamethasone-induced immunosuppression in a MyD88-dependent manner. Overall, this research focused on characterizing immune responses to S. aureus infections and PMN signaling pathways and how it is key to understanding pathogenesis.
Staphylococcus aureus: A Practical Summary for Controlling Mastitis
Petersson-Wolfe, Christina S.; Mullarky, Isis K.; Currin, John F. (Virginia Cooperative Extension, 2011-07-29)
Staphylococcus aureus (S. aureus) mastitis is extremely difficult to control by treatment alone. To date, successful control is gained only through prevention of new infections and culling of infected animals.
A Time Course for Susceptibility to Staphylococcus aureus Respiratory Infection during Influenza in a Swine Model
Smith, E. A.; Kumar, S. R.; Deventhiran, Jagadeeswaran; Cecere, Thomas E.; LeRoith, Tanya; McGilliard, M.; Elankumaran, Subbiah; Mullarky, Isis K. (2011)
Bacterial superinfections following influenza A virus (IAV) are predominant causes of morbidity in humans. The recent emergence of methicillin-resistant Staphylococcus aureus (MRSA) and highly virulent IAV strains has reduced treatment options. Development of an appropriate animal model to study secondary S. aureus infections may provide important information regarding disease pathogenesis. Pigs are natural hosts to both IAV and S. aureus and have respiratory physiology and immune response comparable to humans. To establish a time course of susceptibility to S. aureus after IAV infection, nursery pigs infected intranasally with IAV were challenged with MRSA at different time points. Lung pathology scores and MRSA CFU were evaluated in dual-infected animals after IAV infection. Flow cytometric analysis of bronchoalveolar lavage fluid indicated differences between treatments. These results demonstrate the appropriateness of an intranasal challenge model in nursery pigs for studying the pathogenesis of IAV and S. aureus coinfection and provide insights into the timeframe for susceptibility of IAV-infected pigs to secondary S. aureus infection.

Browsing by Author "Mullarky, Isis K."

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