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Browsing by Author "Boes, Katie M."

Now showing 1 - 7 of 7
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    Characterization, toxicity, and biological activities of organometallic compounds and peptide nucleic acids for potential use as antimicrobials
    Ernst, Marigold Ellen Bethany (Virginia Tech, 2019-04-29)
    Bacterial antibiotic resistance is a globally recognized problem that has prompted extensive research into novel antimicrobial compounds. This dissertation describes research focusing on two types of potential antimicrobial molecules, organometallic compounds (OMC) and peptide nucleic acids (PNA). Organometallic compounds show promise as antimicrobial drugs because of their structural difference from conventional antibiotics and antimicrobials, and because of the ability to "tune" their chemical and biological properties by varying ligand attachments. Peptide nucleic acids, when linked to a cell-penetrating peptide (CPP), can suppress bacterial gene expression by an antisense mechanism and are attractive candidates for antimicrobial drugs because they bind strongly to target nucleic acids and are resistant to nucleases. Chapters 1 and 2 of the dissertation provide an introduction and broad literature review to frame the experimental questions addressed. Chapter 3 describes work to test the cytotoxicity and cellular penetration capabilities of novel OMCs by evaluating their effects on J774A.1 murine macrophage-like cells that were either uninfected or were infected with Mycobacterium bovis BCG. Results indicate that OMCs with an iridium (Ir) metal center and an amino acid ligand show minimal cytotoxicity against eukaryotic cells but likely do not penetrate the intracellular compartment in significant amounts. Chapter 4 presents research into in vitro effects of CPP-PNAs targeting the tetA and tetR antibiotic resistance genes (CPP-anti-tetA PNA and CPP-anti-tetR PNA, respectively) in tetracycline-resistant Salmonella enterica ssp. enterica serovar Typhimurium DT104 (DT104). Through the use of modified minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays it was shown that both the CPP-anti-tetA PNA and CPP-anti-tetR PNA increase tetracycline susceptibility in DT104. Chapter 5 explores the molecular mechanism of the CPP-anti-tetA PNA and CPP-anti-tetR PNA through the use of reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Results indicate good specificity of the CPP-anti-tetA PNA for its nucleic acid target as evidenced by suppression of tetA mRNA expression in DT104 cultures treated with a combination of tetracycline and the PNA. Chapter 6 describes the development of a mouse model of DT104 infection using BALB/c mice, followed by implementation of that model to test in vivo antimicrobial effects of the CPP-anti-tetA PNA and the CPP-Sal-tsf PNA, which targets expression of the essential tsf gene. An optimal dose of DT104 was identified that causes clinical illness within 2-4 days. At the doses tested, concurrent treatment of infected mice with tetracycline and the CPP-anti-tetA PNA or with the CPP-Sal-tsf PNA alone did not have a protective effect. Final conclusions are 1) that further research with the OMCs should focus on compounds with an Ir center and an amino acid ligand, and should explore ways to enhance intracellular penetration, 2) that the in vitro results of the PNA studies suggest that PNAs targeting expression of antibiotic resistance genes could allow for repurposing of antibiotics to which bacteria are resistant, and 3) additional study of the behavior of PNAs in vivo is advised.
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    Comparison of a Point-of-Care Analyzer With a Chemiluminescent Immunoassay for Serum Progesterone Measurement in Breeding Management of the Bitch
    Zuercher, Julia; Boes, Katie M.; Balogh, Orsolya; Helms, Alyssa B.; Cecere, Julie T. (Frontiers, 2021-05-13)
    Accurate serum progesterone measurements for timing bitches during breeding management is critical for reproductive practice, especially as artificial insemination has become routine to facilitate breeding of animals that are geographically or temporally separated. To measure serum progesterone, chemiluminescent immunoassay (CLIA) has replaced radioimmunoassay as the current standard in the bitch due to its high correlation and increased practicality. In January 2019, a colorimetric point-of-care (POC) immunoassay for quantitative in-clinic canine serum progesterone measurements in <30min was released. This study provides an independent comparison of the POC (Catalyst One, IDEXX) to the current industry standard, CLIA (Immulite-2000, Siemens). To assess inter-assay imprecision of POC and agreement of the POC and CLIA results, 100 canine serum samples were analyzed on three analyzers (POC-1, POC-2, and CLIA), of which, 74 (POC-1) and 75 (POC-2) results were within POCs’ reportable range of 0.2–20 ng/mL and included in the study. To assess intra-assay imprecision, pooled canine serum samples at low (L1), intermediate (L2), and high (L3) progesterone concentrations were analyzed ten times each on POC-1 and CLIA. Relative to CLIA, POC values showed good correlation (POC-1, r² = 0.9366; POC-2, r² = 0.9438, P < 0.0001) and significant positive proportional bias at values >2 ng/mL. The POC inter-assay coefficients of variation (CVs) were 13.2% (0.2–2.9 ng/mL, 0.6–9.2 nmol/L, L1), 10.0% (3.0–9.9 ng/mL, 9.5–31.5 nmol/L, L2), 7.1% (10.0–20.0 ng/mL, 31.8–63.6 nmol/L, L3), and 11.2%(all samples). The intra-assay CVs for POC (L1, 15.3%; L2, 7.0%; L3, 4.7%) were higher than those for CLIA (L1, 5.89%; L2, 4.89%; L3, 3.44%). Based on the more rapid increase in serial serum progesterone concentrations in ovulating bitches and the greater imprecision of the POC, the clinical interpretations of serum progesterone measurements as they relate to canine breeding management should be made with caution.
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    Effects of Levothyroxine Adminstration and Withdrawal on the Hypothalamic-Pituitary-Thyroid Axis in Euthyroid Dogs
    Ziglioli, Vincent (Virginia Tech, 2016-05-17)
    Background: Because of the vague clinical signs and limitations of thyroid function tests, misdiagnosis of hypothyroidism in dogs is common and leads to inappropriate treatment with levothyroxine. Chronic supplementation can suppress the hypothalamic-pituitary-thyroid axis (HPTA) and make it difficult to assess thyroid function following withdrawal of levothyroxine. Objectives: To determine if the HPTA is suppressed following levothyroxine administration in euthyroid dogs and the time required for resolution of any suppression. Animals: Twenty-eight healthy euthyroid dogs Methods: A prospective randomized study administering levothyroxine to euthyroid dogs with levothyroxine, for either 8 weeks (group 1) or 16 weeks (group 2). Serum concentrations of total thyroxine (T4), free thyroxine (fT4) by equilibrium dialysis, thyrotropin (TSH), and 3,5,3'-triiodothyronine (T3) were measured every 4 weeks during supplementation and for 16 weeks after levothyroxine was discontinued. Results: Mean serum T4 and fT4 were significantly higher and TSH was lower in all dogs during levothyroxine administration compared to baseline. Mean serum concentrations of T4 and fT4 in both groups and TSH in group 1, beginning 1 week after levothyroxine was discontinued, were significantly different compared to values during levothyroxine administration but not compared to baseline values. Conclusions and Clinical Importance: Suppression of the HPTA occurred during levothyroxine supplementation and mean serum T4, fT4 and TSH concentrations were not significantly different compared to baseline 1 week after discontinuation in both groups. Assessing thyroid function tests 1 week after cessation of levothyroxine will likely provide an accurate assessment of thyroid function in euthyroid dogs.
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    Evaluation of hemostasis in hyperthyroid cats
    Keebaugh, Audrey Elizabeth (Virginia Tech, 2020-07-17)
    Background: Hyperthyroid cats are predisposed to thrombus formation. The mechanism for thrombogenesis is currently unknown, but could be associated with altered hemostasis as seen in hyperthyroid humans. Objective: The purpose of this study was to evaluate markers of hemostasis in hyperthyroid cats compared to healthy cats, and in hyperthyroid cats before and after treatments with radioactive iodine (RIT). Methods: Twenty-five cats with hyperthyroidism and 13 healthy euthyroid cats > 8 years of age were studied. Prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen, antithrombin (AT), D-dimers, thrombin-antithrombin complexes (TAT), von Willebrand Factor antigen (vWF:Ag), and activity of factors VIII and IX were measured. An echocardiogram was performed in all cats and healthy cats with abnormal echocardiograms were excluded. Measurements of hemostasis were evaluated again in 7 cats > 6 months after RIT and deemed to have restored euthyroid status. Results: There is a significant likelihood of being in hypercoagulable state based on hyperthyroid state (P = 0.019) and serum T4 level is significantly associated with predicating hypercoagulability (P = 0.043). Hyperthyroidism is associated with significantly higher median fibrinogen concentration (P < 0.0001), higher median AT activity (P < 0.0001), and higher median vWF:Ag level (P = 0.01) with all values decreasing significantly post-RIT. Fibrinogen and AT had a strong positive correlation with serum T4 value (r = 0.79; 95% CI 0.63 - 0.89 and r = 0.70; 95% CI 0.50 - 0.84, respectively). Presence of an abnormal echocardiogram in hyperthyroid cats was associated with a significantly higher median fibrinogen concentration (P = 0.03). Echocardiographic status did not have a significant impact on the remaining hemostatic markers in hyperthyroid cats. Conclusions: These results provide evidence of altered hemostasis and hypercoagulability in hyperthyroid cats that do not appear to be solely attributed to cardiac abnormalities. These differences of altered hemostasis resolved after radioiodine therapy, but further studies are warranted to determine if hypercoagulable state resolves.
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    Platelet Function in Dogs with Chronic Liver Disease
    Wilkinson, Ashley R. (Virginia Tech, 2019-06-10)
    Background: Dogs with acquired chronic liver disease often have hemostatic derangements. It is currently unknown whether dogs with acquired chronic liver disease have decreased platelet function and alterations in von Willebrand factor (vWF) that may contribute to hemostatic abnormalities. Hypothesis: Dogs with chronic liver disease have prolonged platelet closure time (CT), assessed with the PFA-100®, and buccal mucosal bleeding time (BMBT), and increased vWF concentration compared to healthy dogs. Animals: Eighteen dogs with chronic acquired liver disease undergoing ultrasound-guided needle biopsy of the liver or laparoscopic liver biopsy and eighteen healthy age-matched control dogs. Methods: Prospective study. BMBT, CT using the PFA-100®, and vWF antigen were measured in dogs with chronic liver enzyme elevation undergoing ultrasound-guided needle biopsy of the liver or laparoscopic liver biopsy. After undergoing ultrasound-guided needle biopsy, dogs were monitored for hemorrhage with serial packed cell volume measurements and focused assessment with sonography. An unpaired t-test was used for normally distributed data and the Mann-Whitney test was used when non-Gaussian distribution was present. The level of significance was set at P <0.05. Results: The CT was not different between the two groups (P = 0.27). The BMBT was significantly longer in the liver disease group compared to the control group (P = 0.019). There was no difference in the mean vWF antigen of the two groups (P = 0.077). Conclusions and clinical relevance: These results demonstrate mild impairment of primary hemostasis in dogs with chronic liver disease based on prolongation of BMBT.
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    Seroprevalence of Anaplasma phagocytophilum in the equine population of Southwest Virginia
    Hinson, Hannah Lee (Virginia Tech, 2021-10-26)
    Background: Equine granulocytic anaplasmosis (EGA), caused by the organism Anaplasma phagocytophilum, is a tick-borne disease of clinical importance in Southwest Virginia. The disease is recognized worldwide and causes pyrexia, anorexia, limb edema, and lethargy. Diagnosis in endemic areas is often based on clinical signs, but confirmation of infection can be made via detection of morulae on a peripheral blood smear or polymerase chain reaction analysis (PCR) at the time of disease or by serologic detection of antibodies 2-4 weeks post infection. There is growing interest in stall-side methods for diagnosis of various equine diseases which has led to an increased use of the SNAP 4DX Plus Test® for vector-borne diseases. Objectives: Determine seroprevalence of antibodies to A. phagocytophilum in the equine population of Southwest Virginia and changes in seroprevalence compared to samples taken 6 years earlier. Determine the percentage of horses with clinical signs consistent with EGA that were positive for A. phagocytophilum infection and assess common presenting clinical signs, hematologic variables, and confirmatory diagnostic test results. Animals: Seroprevalence was evaluated in horses presented for routine annual Coggins testing in 2013 and 2019-2020. Clinical features of disease and diagnostic test results were evaluated in horses presenting with clinical signs compatible with A. phagocytophilum infection from September 2019-August 2020. Methods: Seroprevalence was determined using the IDEXX SNAP 4DX Plus Test® on serum collected from horses presenting for annual Coggins testing in 2013 and 2019-2020. Samples collected in 2013 had been stored at -7580 degrees F since collection. Age, sex, county of residence, and month of sampling were statistically analyzed in the seroprevalence population. Horses presenting with clinical disease consistent with EGA from September 2019-August 2020 had the following diagnostic tests performed: complete blood count (CBC), blood smear for morulae detection, polymerase chain reaction (PCR) analysis, immunofluorescence antibody testing (IFAT), and the IDEXX SNAP 4DX Plus Test®. Results: Seroprevalence of A. phagocytophilum in the equine population of Southwest Virginia increased from 8.5% in 2013 to 11.2% in 2019-2020, although this increase was not statistically significant. In the 2019-2020 population, month of sampling was significantly associated with presence of antibodies to A. phagocytophilum. Positive samples were more common from November-February than other times of the year. When the two sample time periods were combined, sex was significantly associated with presence of antibodies to A. phagocytophilum with geldings more likely to be seropositive. Within the clinical case population, 35% of horses with clinical signs compatible with equine granulocytic anaplasmosis had confirmed infection. The most common hematologic abnormality in affected horses was thrombocytopenia. PCR analysis was the most sensitive diagnostic test to diagnose infection followed by identification of morulae on blood smears. Conclusions: Seroprevalence of A. phagocytophilum is similar to other endemic areas in the United States and appears to be increasing over time. In active clinical cases, diagnosis is best made via PCR or detection of morulae on a blood smear. The SNAP 4DX Plus Test® was not appropriate for diagnosis of active EGA in acute cases. Seroprevalence of Anaplasma phagocytophilum in the equine population of Southwest Virginia
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    Theileria orientalis Ikeda Genotype in Cattle, Virginia, USA
    Oakes, Vanessa J.; Yabsley, Michael J.; Schwartz, Diana; LeRoith, Tanya; Bissett, Carolynn; Broaddus, Charles; Schlater, Jack L.; Todd, S. Michelle; Boes, Katie M.; Brookhart, Meghan; Lahmers, Kevin K. (Centers for Disease Control and Prevention, 2019-09)
    Theileria orientalis Ikeda genotype is a parasite that causes a disease in cattle that results in major economic issues in Asia, New Zealand, and Australia. The parasite is transmitted by Haemaphysalis longicornis ticks, which have recently been reported in numerous states throughout the eastern United States. Concurrently, cattle in Virginia showed clinical signs consistent with a hemoprotozoan infection. We used amplicons specific for the major piroplasm surface protein and small subunit rDNA of piroplasms to test blood samples from the cattle by PCR. Bidirectional Sanger sequencing showed sequences with 100% identity with T. orientalis Ikeda genotype 2 sequences. We detected the parasite in 3 unrelated herds and from various animals sampled at 2 time points. Although other benign T. orientalis genotypes are endemic to the United States, detection of T. orientalis Ikeda genotype might represent a risk for the cattle industry in Virginia.