Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Johann F. Coetzee / The Office International des Epizooties (OIE) Animal Health Code categorizes bovine anaplasmosis as a notifiable disease. Many species of the genus Anaplasma cause anaplasmosis. Co-infections with two or more Anaplasma spp. occur in cattle. A competitive ELISA is regarded as a reliable test for identifying A. marginale-infected cattle. However, cross-reactivity among related Anaplasma spp. has been reported when using cELISA. In the absence of effective treatment strategies and vaccine availability, anaplasmosis control strategies are primarily focused on disease identification and prevention and development of chemosterilization strategies. Four studies were completed to improve the diagnosis, treatment, and control of bovine anaplasmosis. In the first study, a real-time qRT-PCR was developed to detect as few as 100 copies of 16S rRNA of both A. marginale and A. phagocytophilum in the same reaction. This detection limit was equitable to the minimum infective unit of one A. marginale bacterium. In the second study, qRT-PCR results determined needle-free injection was superior to needle injection for controlling iatrogenic transmission of A. marginale in cattle. The qRT-PCR demonstrated 100% sensitivity by 21 days post-infection and 21 days prior to 100% sensitivity with cELISA. The third study determined the pharmacokinetic parameters of chlortetracycline in group fed, ruminating Holstein steers: volume of distribution (40.9 L⁄kg); rate constant (0.0478 h-1); dose-normalized area under the curve (0.29 h•µg⁄L); clearance (1.8 L⁄kg⁄h); elimination half-life (16.2 h); maximum concentration/dose (4.5 ng⁄mL); and time of maximum concentration (23.3 h). Dose linearity was confirmed for oral chlortetracycline dosages of 4.4, 11, and 22 mg/kg/day. The final study established an in vivo pharmacokinetic-pharmacodynamic relationship between chlortetracycline and anaplasmosis carrier clearance in bovine plasma (85.3 ng/mL). The qRT-PCR confirmed chemosterilization of all oral chlortetracycline-treated cattle within 49 days of treatment. Furthermore, qRT-PCR was an effective alternative to the subinoculation of splenectomized cattle for accurate and precise disease classification. The diagnosis, treatment, and control of anaplasmosis were enhanced through the application of qRT-PCR. Further studies are necessary for determining the mechanism of action between chlortetracycline binding to the 30S ribosome of A. marginale and carrier clearance.

Processes that regulate site of fat deposition in beef cattle are poorly understood. For the producer to procure the greatest profit, it is ideal to maximize intramuscular fat. Furthermore, to understand the physiological mechanisms affecting fat depots, it is necessary to evaluate hormones involved in growth regulation. Using a 2 x 2 factorial design of treatments, four experiments were conducted to examine two adipogenic compounds, chlortetracycline and dexamethasone. Synovex-S® and Revalor-S® were used to investigate potential interactions between growth implants and adipogenic compound. Growth performance, carcass quality, organ and fat mass and plasma hormone concentrations were measured in these studies. In Exp. 1, 24 steers received either 0 or 350mg chlortetracycline/d, with or without Synovex-S®. On d 30, 56 and 106, steers received a bolus injection of 1 ug/kg BW thyrotropin-releasing hormone and 0.1 ug/kg BW GH-releasing hormone and serial blood samples were collected. Synovex-S® increased (P ≤ 0.009) rate and efficiency of gain and decreased (P = 0.05) time to peak for GH concentrations. Chlortetracycline attenuated (P ≤ 0.05) the effects of implant on triiodothyronine release, slaughter weight and carcass quality. In Exp. 2, 96 steers received either 0 or 39.6 ppm chlortetracycline/d, with or without Revalor-S®. Implant increased (P < 0.0001) ADG; however, efficiency of gain was greater for implanted steers in the absence of chlortetracycline (interaction, P ≤ 0.03). In Exp. 3, 144 steers received either 0 or 0.09 mg dexamethasone/kg BW on d 0, 28 or 56, with or without Revalor-S®. Average daily gain was lower (P = 0.0003) for implanted steers receiving dexamethasone compared to those receiving no dexamethasone (interaction, P = 0.05). Omental fat mass was greater (P = 0.01) for non-implanted steers receiving dexamethasone compared to no dexamethasone (interaction, P = 0.006). In Exp. 4, 96 steers received either 0 or 0.09 mg dexamethasone/kg BW on d 0, 28, 56 or 84, with or without Revalor-S®. Average daily gain and efficiency of gain were both 13% greater (P ≤ 0.05) with implant. Conversely, DEX lowered ADG by 10% (P = 0.007). There were no effects of treatment on fat mass weights.

Twenty-three crossbred ewes were utilized during 1995, to investigate the possibility of xenogenous capacitation, using stallion spermatozoa. Ewes were grouped according to endocrine status as anestrus (n = 12) or estrus (n = 11) and were surgically inseminated with either fresh (FR), cooled/extended (FCE) or frozen/thawed (FZ) spermatozoal samples. The capacitation response of spermatozoa recovered from oviductal flushing 4 - 6 hours post-insemination, was assessed using a chlortetracycline (CTC) fluorescent probe. Data were recorded as percentages of spermatozoa exhibiting CTC staining patterns characteristic of capacitated (PCAP), unreacted (PUR) and acrosome reacted (PAR) sperm. Mean PCAP was not significantly different in estrous ewes despite an increasing trend. Mean PUR and PAR were also not different. Differences in capacitated, acrosome reacted and unreacted spermatozoa in inseminated and recovered samples (CAPDIF, ARDIF and URDIF, respectively) were analyzed. CAPDIF was significantly different between treatment groups (p < 0.01). CAPDIF was also significantly greater for the estrus versus anestrus group (p < 0.05). Total number of spermatozoa recovered (RTOTAL) was recorded. More spermatozoa were recovered from estrus ewes, however significance was not established. Mean number of spermatozoa recovered was 37.9 ± 35.9 per ewe. Treatment significantly affected RTOTAL in estrus animals (p = 0.01). FR samples had the highest recovery. Results suggest that xenogenous capacitation of stallion semen may occur in the reproductive tract of the ewe. Implications of these results are discussed in regards to their application of xenogenous gamete intrafallopian transfer (X-GIFT) as a treatment option for certain infertility problems in the mare. / Master of Science

No description available.

Master of Science / Department of Animal Sciences and Industry / Christopher D. Reinhardt / Three experiments were conducted to examine nutritional and management strategies for different segments of the beef industry. The first experiment examined the effects of feeding traditional step-up diets (STEP) vs. limit-feeding (LIMIT) the finishing diet to adapt cattle to high-concentrate diets. When all cattle reached ad libitum intake of the finishing diet there was a trend (P = 0.09) for DMI to be different between treatments. During week 1, STEP cattle had higher total VFA concentrations (P = 0.02), while LIMIT cattle had higher valerate absorption (P = 0.02) and disappearance (P = 0.08). During week 4, LIMIT cattle had higher total VFA concentrations (P = 0.03) and lower valerate disappearance and absorption (P = 0.05) than STEP cattle. These results indicate that limit-feeding the finishing diet may inhibit nutrient absorption from the rumen or this method may cause increased production of valerate by lactate utilizing bacteria due to a more acidotic rumen environment. The second experiment examined the effects of feeding 5% (DM basis) dried, full-fat corn germ (GERM) on feedlot performance and carcass characteristics of naturally raised yearling steers and heifers. Carcass-adjusted ADG was higher for GERM cattle (P = 0.04). There were no other differences in performance or carcass characteristics. Total incidence of liver abscesses and the incidence of severe liver abscesses were decreased by 12 and 8.2% (P = 0.01 and 0.02, respectively) when GERM was added to the diet. Corn germ can be added to finishing diets at 5% without affecting performance and carcass characteristics. Producers raising natural cattle may also be able to benefit from the reduced incidence of liver abscesses. The third experiment examined concurrent metaphylactic treatment of high-risk calves with tulathromycin and chlortetracycline. Calves were placed on 1 of 3 treatments: 1) no top-dress pellets; 2) diet top-dressed with pellets containing chlortetracycline; or 3) diet top-dressed with pellets containing no chlortetracycline. There were no differences in the performance or health of these calves (P > 0.25). There are no additive benefits of concurrent metaphylaxis using both tulathromycin and chlortetracycline. This information could assist producers when designing receiving health protocols for high-risk calves.

O presente trabalho tem como objetivo avaliar se o complexo Európio- Clorotetraciclina (EuCTc) ou o corante Tioflavina T (ThT) podem atuar como biossensores precisos e eficientes de colesterol numa fração específica, através de procedimentos simples. Para isso estudaram-se as propriedades ópticas do complexo EuCTc e ThT na presença da Lipoproteína de Baixa Densidade (LDL) em seu estado nativo e em seu estado oxidado. O primeiro estudo realizado verificou a melhor razão molar entre o Európio e a Clorotetraciclina, em seguida verificou-se a influência da diálise da LDL na emissão do complexo EuCTc, para obtenção de um protocolo para quantificação da concentração da LDL. Foram traçadas as curvas de calibração da emissão do complexo EuCTc com várias concentrações da LDL nativa, LDL oxidada com íons de Cobre e LDL oxidada com íons de Ferro. Em seguida obteve-se o tempo de vida do íon Európio no complexo EuCTc na presença de diferentes concentrações de LDL nativa e LDL oxidada por íons de Cobre. Na segunda parte do trabalho estudou-se a emissão do corante Tioflavina T na presença da LDL nativa e LDL oxidada. Na terceira etapa as propriedades ópticas dos biossensores EuCTc e ThT foram investigadas na presença do plasma sanguíneo e foram comparadas às emissões do complexo EuCTc e o corante ThT com a LDL ultracentrifugada, para verificar a possibilidade de quantificar a LDL diretamente no plasma sanguíneo. Na última etapa do trabalho desenvolveu-se uma nova metodologia para oxidar a partícula de LDL a partir da irradiação com laser de pulsos ultracurtos, a fim de produzir uma oxidação branda da partícula de LDL e controlada. / The present study aims at assessing the complex Europium-Clorotetracycline (EuCTc) or the dye Thioflavin T (ThT) can act as biosensors accurate and efficient in a specific fraction of cholesterol through simple procedures.For this purpose, it was studied the optical properties of Europium- Chlortetracycline (EuCTc) complex and the thioflavin T (ThT) dye in the presence of low-density lipoprotein (LDL) in native state and in oxidized state in vitro. First study realized it was verified the influence of dialysis in the emission of complex EuCTc in the presence of LDL, thereby producing a protocol for use of the complex to obtain the concentration of LDL. It was obtained the calibration curves of the complex with various concentrations of native LDL, the oxidized LDL with copper ions and oxidized LDL with iron ions. It was also obtained from the calibration curve of the emission of the complex in the presence of calcium interferent ion with the concentration found in blood plasma with the oxidized LDL with copper ions. It was obtained the lifetime of the europium ion in the complex in the presence of different concentrations of Native LDL and oxidized LDL by copper ions. In the second part of the work it was studied the emission of the dye thioflavin T in the presence of native LDL and oxidized LDL. In the third part the optical properties of biosensors EuCTc and ThT were investigated in the presence of blood plasma and compared to the emission of the complex EuCTc and the dye ThT with LDL ultracentrifugated to verify the possibility of quantifying directly LDL in blood plasma.In the final part of this work it has developed a new methodology for the LDL particles oxidation from the irradiation of ultrashort laser pulses in order to produce mild and controlled oxidation of the LDL particle.

Doctor of Philosophy / Department of Animal Sciences and Industry / Cassandra K. Jones / Porcine epidemic diarrhea virus (PEDV) is a heat-sensitive virus that devastated the United States swine industry. Because of its heat sensitivity, it was hypothesized that a pellet mill mimicking commercial thermal processing may mitigate PEDV infectivity. From the results, it was determined that a conditioning time of 30 sec or greater and temperatures above 54.4°C were effective point-in-time kill steps to inactive PEDV in a research setting. However, this does not prevent subsequent recontamination after pelleting as it is a point-in-time mitigation step. To further explore this, various mitigation additives were evaluated to prevent or mitigate PEDV post-pellet contamination in swine feed and ingredients. Various additives were examined across 3 experiments and included mitigation additives of medium chain fatty acids (MCFA), organic acids (OA), essential oils (OA), formaldehyde based products, and sodium bisulfate. From Exp. 1, formaldehyde, medium chain fatty acids (MCFA), essential oils (EO), and organic acid (OA) each decreased detectable PEDV RNA compared to the control (P<0.05). Additionally, PEDV stability over time was influenced by matrix as the meat and bone meal and spray-dried animal plasma resulted in a greater (P<0.05) quantity of detectable PEDV RNA over 42 days compared to that of the swine diet and blood meal. In Exp. 2, the 1% MCFA inclusion was equally effective at mitigating PEDV as a commercially available formaldehyde product in the complete swine diet. To further explore the effects of MCFA against PEDV, Exp. 3 was conducted to evaluate lower inclusion levels of MCFA and fat sources containing MCFA. It was noted that formaldehyde, 1% MCFA (1:1:1: of caproic, caprylic, and capric acids), 0.66% caproic, 0.66% caprylic, and 0.66% capric acids enhance the RNA degradation of PEDV in swine feed as determined by a bioassay. The MCFA were also evaluated against Salmonella Typhimurium, Generic Escherichia coli, Enterotoxigenic Escherichia coli, and Campylobacter coli. It was noted that the efficacy of the MCFA varied between each bacteria species with caproic and caprylic being the most effective. Commercial developmental products were also tested and determined that Product A and B provided the lowest MIC values across Salmonella Typhimurium, Generic Escherichia coli, and Enterotoxigenic Escherichia coli (P < 0.05). Product A and B were further tested in an animal disease trial utilizing a strain of enterotoxigenic Escherichia. coli O149:K91: K88. From d 7 to 14, chlortetracycline, 1:1:1 blend, and Product B, all improved G:F compared to the control (P<0.05). This also led to chlortetracycline and Product B having an improvement (P<0.05) over the control diet from d 0 to 14. A treatment × day interaction for the enterotoxigenic E. coli plate scores was observed (P < 0.05), which occurred because of the decrease (P<0.05) in plate scores for Product B from d 1 to d 14 and an increase (P<0.05) in chlortetracycline from d 7 to 14. A decrease (P<0.05) in plasma urea nitrogen and haptoglobin was observed as time increased from d -2 to 14. In summary MCFA have shown to be an effect interventional mitigation strategy against PEDV and various bacteria.

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O presente trabalho tem como objetivo avaliar se o complexo Európio- Clorotetraciclina (EuCTc) ou o corante Tioflavina T (ThT) podem atuar como biossensores precisos e eficientes de colesterol numa fração específica, através de procedimentos simples. Para isso estudaram-se as propriedades ópticas do complexo EuCTc e ThT na presença da Lipoproteína de Baixa Densidade (LDL) em seu estado nativo e em seu estado oxidado. O primeiro estudo realizado verificou a melhor razão molar entre o Európio e a Clorotetraciclina, em seguida verificou-se a influência da diálise da LDL na emissão do complexo EuCTc, para obtenção de um protocolo para quantificação da concentração da LDL. Foram traçadas as curvas de calibração da emissão do complexo EuCTc com várias concentrações da LDL nativa, LDL oxidada com íons de Cobre e LDL oxidada com íons de Ferro. Em seguida obteve-se o tempo de vida do íon Európio no complexo EuCTc na presença de diferentes concentrações de LDL nativa e LDL oxidada por íons de Cobre. Na segunda parte do trabalho estudou-se a emissão do corante Tioflavina T na presença da LDL nativa e LDL oxidada. Na terceira etapa as propriedades ópticas dos biossensores EuCTc e ThT foram investigadas na presença do plasma sanguíneo e foram comparadas às emissões do complexo EuCTc e o corante ThT com a LDL ultracentrifugada, para verificar a possibilidade de quantificar a LDL diretamente no plasma sanguíneo. Na última etapa do trabalho desenvolveu-se uma nova metodologia para oxidar a partícula de LDL a partir da irradiação com laser de pulsos ultracurtos, a fim de produzir uma oxidação branda da partícula de LDL e controlada. / The present study aims at assessing the complex Europium-Clorotetracycline (EuCTc) or the dye Thioflavin T (ThT) can act as biosensors accurate and efficient in a specific fraction of cholesterol through simple procedures.For this purpose, it was studied the optical properties of Europium- Chlortetracycline (EuCTc) complex and the thioflavin T (ThT) dye in the presence of low-density lipoprotein (LDL) in native state and in oxidized state in vitro. First study realized it was verified the influence of dialysis in the emission of complex EuCTc in the presence of LDL, thereby producing a protocol for use of the complex to obtain the concentration of LDL. It was obtained the calibration curves of the complex with various concentrations of native LDL, the oxidized LDL with copper ions and oxidized LDL with iron ions. It was also obtained from the calibration curve of the emission of the complex in the presence of calcium interferent ion with the concentration found in blood plasma with the oxidized LDL with copper ions. It was obtained the lifetime of the europium ion in the complex in the presence of different concentrations of Native LDL and oxidized LDL by copper ions. In the second part of the work it was studied the emission of the dye thioflavin T in the presence of native LDL and oxidized LDL. In the third part the optical properties of biosensors EuCTc and ThT were investigated in the presence of blood plasma and compared to the emission of the complex EuCTc and the dye ThT with LDL ultracentrifugated to verify the possibility of quantifying directly LDL in blood plasma.In the final part of this work it has developed a new methodology for the LDL particles oxidation from the irradiation of ultrashort laser pulses in order to produce mild and controlled oxidation of the LDL particle.

Doctor of Philosophy / Department of Diagnostic Medicine and Pathobiology / Harvey Morgan Scott / The use of antibiotics in food animals is of major concern as a purported cause of antimicrobial resistance (AMR) in human pathogens; as a result, alternatives to in-feed antibiotics such as heavy metals have been proposed. The effect of copper and CTC supplementation in weaned pigs on AMR in the gut microbiota was evaluated. Four treatment groups: control, copper, chlortetracycline (CTC), and copper plus CTC were randomly allocated to 32 pens with five pigs per pen. Fecal samples (n = 576) were collected weekly from three pigs per pen over six weeks and two Escherichia coli isolates per sample were tested phenotypically for antimicrobial and copper susceptibilities and genotypically for the presence of tetracycline (tet), copper (pcoD) and ceftiofur bla[subscript]C[subscript]M[subscript]Y₋₂) resistance genes. CTC-supplementation significantly increased tetracycline resistance and susceptibility to copper when compared with the control group. Copper supplementation decreased resistance to most of the antibiotics, including cephalosporins, over all treatment periods. However, copper supplementation did not affect minimum inhibitory concentrations of copper or detection of pcoD. While tetA and bla[subscript]C[subscript]M[subscript]Y₋₂ genes were associated with a higher multi-drug resistance (MDR), tetB and pcoD were associated with lower MDR. Supplementations of CTC or copper alone were associated with increased tetB prevalence; however, their combination was paradoxically associated with reduced prevalence. These studies indicate that E. coli isolates from the weaned pigs studied exhibit high levels of antibiotic resistance with diverse multi-resistant phenotypic profiles. In a related study, total fecal community DNA (n = 569) was used to detect 14 tet genes and to quantify gene copies of tetA, tetB, pcoD and bla[subscript]C[subscript]M[subscript]Y₋₂. CTC and copper plus CTC supplementation increased both the prevalence and gene copies of tetA, while decreasing both the prevalence and gene copies of tetB, when compared with the control group. The diversity of tet genes were reduced over time in the gut bacterial community. The roles of copper supplementation in pig production and pco-mediated copper resistance in E. coli need to be further explored since a strong negative association of pcoD, with both tetA and bla[subscript]C[subscript]M[subscript]Y₋₂, suggests there exist opportunities to select for a more innocuous resistance profile.