Master of Science / Department of Animal Sciences and Industry / Terry A. Houser / Nursery age pigs (n=198) were used to evaluate the difference in the occurrence of injection site abscesses between needle-free jet injection and conventional needle-and-syringe injection systems. Pigs were fed for 21 d prior to treatment administration to acclimate the pigs to the environment of the Kansas State University Segregated Early Weaning (SEW) unit. On d 21 each pig was injected with aluminum hydroxide adjuvant in the neck and ham with needle-free jet injection (Pulse Needle-Free Systems, Lenexa, KS) and conventional needle-and-syringe injection. Needle-free and conventional needle-and-syringe injections were randomly assigned to pig side yielding a total of 396 injections per treatment with a total of 792 injections sites. Immediately prior to injection, the external surface of the injection sites were contaminated with an inoculum of Arcanobacterium pyogenes, a bacterium commonly associated with livestock abscesses. The pigs were then fed for a period of 27 or 28 d. On d 27 or d 28 the pigs were humanely euthanized and sent to the Kansas State Veterinary Diagnostics Laboratory where necropsies were performed and the injection sites harvested for histopathological evaluation. The needle-free jet injection system was associated with more injection site abscesses than the conventional needle-and-syringe injection method for both neck (P=0.0625) and ham (P=0.0313) injection sites. Twelve abscesses were found at injection sites administered via needle-free jet injection method while only 1 abscess was found with the conventional needle-and-syringe injection method. 5 abscesses were found at the neck injection sites and 8 abscesses were found at ham injection sites. There were no significant differences seen in tissue granulation resulting from reaction to the adjuvant. In summary, the implementation of needle-free jet injection systems in market hog production will be beneficial to eliminate needles and needle fragments in meat products but, when in the presence of Arcanobacterium pyogenes, it may increase the occurrence of injection site abscesses in pork carcasses that will need to be trimmed in pork processing plants. Although more abscesses were associated with needle-free jet injection, their occurrence was observed at a very low rate given that all injection sites were intentionally contaminated prior to injection.

Master of Science / Department of Animal Sciences and Industry / Michael E. Dikeman / Objectives were to determine the effects of needle-free (NF) versus needle (N) injection methods and/or solutions for enhancement of beef longissimus lumborum muscle (LM) on color, instrumental tenderness, sensory attributes, pump yields, and cooking losses. In experiment 1, LM (n=15) at 9 d postmortem were halved before random assignment to N or NF injection enhancement with a solution containing 2.2% salt, 4.4% sodium tripolyphosphate (STPP), and 1.5% K lactate. Different steaks from each loin half were either placed on a 5 d color display, frozen for later sensory analysis, or aged until d 13 postmortem for LM slice shear force measurements. Pump yields tended (P=0.08) to be higher for NF injection. Needle injected steaks were darker (P<0.05) on day 1, but not after that. Discoloration was not different (P>0.05) between treatments. The NF treatment had greater (P<0.05) instrumental tenderness and intensity of off-flavors but less (P<0.05) cooking loss and beef flavor. In Experiment 2, LM (n=28) at 5 d postmortem were halved before random assignment to one of four treatments: 1) N, or 2) NF injection with a solution containing 2.2% salt, 4.4% STPP, 15% K lactate, and 0.58% rosemary; 3) N, or 4) NF injection with a solution containing 2.4% Ca lactate and 0.58% rosemary. Steaks from each loin half were either frozen for later sensory analysis or aged until d 14 postmortem for LM slice shear force measurements. Loins phosphate enhanced with the NF injector had the highest (P<0.05) pumped yields with no differences (P>0.05) among other treatment combinations. Instrumental tenderness was not different (P>0.05) between N and NF treatments but was higher with the phosphate solution than the Ca lactate solution. The NF treatment had lower (P<0.05) cooking losses when the phosphate solution was used, which resulted in less (P<0.05) cooking loss than the Ca lactate solution. More (P<0.05) off-flavors and abnormal texture resulted from NF injection. The phosphate solution resulted in greater (P<0.05) myofibrillar and overall tenderness, juiciness, off-flavors and abnormal texture with less (P<0.05) connective tissue than the Ca lactate solution. Enhancing beef LM with a phosphate solution and NF injection might improve yields, tenderness, and juiciness while harming texture and flavor.

The unsafe use of needles propagates cross infections with bloodborne pathogens and reduces the positive impact of vaccinations on global health. While a plethora of needle-free injection devices exist, the reformulation of protein-based vaccines is largely empirical and costly, which presents a barrier to their widespread clinical application. This thesis contributes to the identification of approaches that facilitate rapid vaccine reformulation and enhance the immunogenicity of needle-free dry-powder vaccines with the help of novel antigen delivery platforms. We hypothesised that the thermodynamic stabilisation of diphtheria toxin mutant 197 (CRM197), a glycoconjugate vaccine carrier protein, may enhance its structural preservation during spray-freeze-drying (SFD), and that its formulation in either soluble, surface-adsorbed, or nanoparticle form impacts the elicited immune response. Differential scanning fluorimetry was used to study the effect of excipients on the thermal stability of CRM197. Dry-powder formulation of CRM197 used i) encapsulation into a thermodynamically stabilising excipient matrix by SFD, ii) surface-immobilisation via physisorption onto a novel potassium-doped hydroxyapatite (kHA) carrier microparticle formed by molten salt synthesis, and iii) chemical conjugation and surface presentation on amphiphilic block copolymer nanoparticles that were incorporated into SFD-powders (SFD-NP). The structural integrity of CRM197 was assessed by size separation in addition to various spectral and thermal analysis methods. The immunogenicity of dry-powder CRM197 formulations was subsequently tested in vivo. The results suggest that the thermodynamic stability of CRM197 in solution does not ensure its structural stability during SFD. While needle-free dermal vaccination with kHA-adsorbed CRM197 induced comparable antibody titres to conventional IM injection of alum-adjuvanted CRM197, needle-free SFD and SFD-NP powders were less immunogenic. The highest mean IgG titre and most balanced Th1/Th2 response was achieved with nanoparticle-conjugated CRM197 by IM, which outperformed the current clinical standard. Therefore, future vaccine design should combine thermodynamic and kinetic stability screening, and place special emphasis on the delivery and structural presentation of the antigen to the immune system.

Master of Science / Food Science Institute / Michael E. Dikeman / The objective was to determine the effects of needle-free injection (NF) compared with traditional needle injection (N) on microbial translocation of generic E. coli in beef strip loins. Longissimus muscles (LM) (n=5) from USDA Select carcasses were used in preliminary research to determine the optimal injection pressure required for NF injections. Seven treatments with sterile colored saline solution were administered: 1) 90 psi ; 2) 55 psi ; 3) 50 psi ; 4) 45 psi ; 5) 30 psi ; 6) 25 psi ; or 7) 20 psi . For the second portion of the experiment 15 LM were obtained and halved; the surfaces were inoculated with generic E. coli at a level of 106 CFU/cm2 (three replications of five loins). Matching halves were allocated to NF or N injection treatments with a phosphate, salt solution. Immediately after injection, two cores, 23 cm2 in area, were taken aseptically from each half. A 2-mm thick cross-sectional slice was removed from the inoculated surface of the core and labeled “surface”. Using sterile technique, the two cores from each half were sliced into cross-sectional strips at depths of 1, 3, and 5 cm. Corresponding depth measurements were combined in stomacher bags with 99 ml of peptone water and stomached. Serial dilutions were then plated. From the preliminary study, it was determined that 25 psi was the optimal pressure for NF injection based on dispersion, visual appraisal, and solution retention. Samples taken from the surface of N injected LM had lower (P < 0.05) microbial counts than NF-injected muscles (2.79 versus 3.23 log CFU/g, respectively). The 3 and 5 cm depth samples from N injection had the least (P < 0.05) microbial contamination (1.69 and 2.12 log CFU/g) compared to NF injections. Samples from 1 cm deep of N injected LM had lower (P < 0.05) (2.53 log CFU/g) microbial counts than the 1 cm samples of NF injected LM (3.04 log CFU/g). Traditional N injection resulted in approximately 0.5 log CFU/g less microbial contamination at all depths. N injection posed fewer microbial risks when compared with NF injection using these defined application settings.

A terapia fotodinâmica (TFD) é uma técnica não invasiva usada no tratamento de lesões de pele, como câncer basocelular, queratose actínica, e doença de Bowen, dentre outros. Basicamente, a combinação da administração de um fotossensibilizador (FS), com a irradiação de luz adequada e o oxigênio celular, gera uma série de reações oxidativas que provocam a morte do tecido. Contudo, o principal efeito colateral desta terapia é a fotossensibilidade prolongada ocasionada pela administração de fotossensibilizadores sistêmicos. Por outro lado, a via tópica não apresenta esta limitação, pois o tratamento é realizado no local da lesão através de pró-drogas. O ácido aminolevulínico, ALA, está entre as pró-drogas mais utilizadas para indução do acúmulo do agente fotossensível na pele, a protoporfirina IX, ou PpIX. Contudo, a via tópica não permite penetração suficiente e homogênea do creme para o tratamento de lesões espessas. Visando a melhoria da TFD, foram realizados estudos in vivo e in vitro. Nos estudos in vivo, técnicas mecânicas - rolos de microagulhas, tape stripping e injeção livre de agulhas foram estudadas buscando encontrar a mais eficiente nos quesitos de: promoção da penetração da pró-droga no tecido, distribuição homogênea e de indução do acúmulo de PpIX. Para isto, foi o utilizado o modelo porcino, in vivo, conhecido como o modelo que possui a pele mais similar à pele humana. Os resultados in vivo mostram que as técnicas têm resultados similares na produção de PpIX e na distribuição de porfirina mais homogênea na superfície. Além disso, todas as técnicas estudadas in vivo têm se destacado em promover uma entrega mais homogênea de ALA também na profundidade da pele quando comparadas ao grupo controle. Nos estudos in vitro, foram examinadas possíveis diferenças na capacidade de formação da PpIX e/ou de resistência de células ao tratamento por TFD entre células expressando diferentes características de transição epitélio-mesenquimal. Os resultados in vitro indicam que as células com características epitélio-mesenquimal mais acentuadas produzem mais PpIX e são mais responsivas à TFD. Estes resultados indicam que a TFD tem maior efetividade no tratamento de células mesenquimais, e os estudos in vivo mostram que no tecido normal há maior seletividade de produção na camada da epiderme e apêndices da pele sugerindo que a terapia pode ser utilizada com maior eficiência em lesões superficiais e, até mesmo diminuir as taxas de recorrência devido a heterogeneidade de distribuição do creme na pele quando umas das técnicas mecânicas são utilizadas. / Photodynamic therapy (PDT) is a noninvasive technique used to treat skin lesions, such as basal cell cancer, actinic keratosis and Bowen\'s disease. Basically, the administration of a photosensitizer (PS), combined with the illumination of adequate light and the cellular oxygen, generate a series of oxidative reactions that cause tissue death. However, the major side effect of the treatment is prolonged photosensitivity caused by the systemic administration of photosensitizers. On the other hand, the topical therapy does not show this limitation, and it is performed at the lesion site via prodrugs. The aminolevulinic acid, ALA, is the most popular pro-drug in topical PDT. This prodrug induces PpIX production that is a photosensitive porphyrin. However, when ALA is used topically, the cream does not provide enough or homogeneous penetration for the treatment of deep lesions. Therefore, with the aim of improving PDT therapy, studies in vivo and in vitro were performed. In the in vivo analysis, mechanical techniques - microneedle roller, tape stripping, and needle-free injection- were studied looking for the most effective regarding to improve the following purposes: promoting penetration of the prodrug into the tissue, homogeneous distribution, and at inducing PpIX accumulation. The evaluations were made by fluorescence spectroscopy, biopsy of skin, and fluorescence images, using the porcine model, in vivo, known as the most similar of human skin tissue. The in vivo results showed that all techniques have similar results in the production of PpIX, and perform a more homogeneous porphyrin distribution in the skin surface. Moreover, all the techniques have excelled in promoting a homogeneous distribution of PpIX in the deep of the skin when compared to the control group. In addition to the skin penetration, studies of PpIX production were performed in vitro in cells expressing different levels of epithelial-mesenchymal transition characteristics. The studies were made in regard to a possible difference in PpIX formation capacity and / or a resistance to the PDT treatment. The in vitro results showed that cells with more epithelial-mesenchymal characteristics produce more PpIX and are more responsive to the PDT therapy. These results indicate that PDT therapy may have a better effectiveness in the treatment of mesenchymal cells and also the results in vivo showed that the ALA-induced PpIX in normal tissue seems to be selective to epidermal and skin appendages, indicating that the topical therapy may be used with a higher efficiency in superficial injuries providing lower recurrence rates when they combine with one of the techniques studied.

A terapia fotodinâmica (TFD) é uma técnica não invasiva usada no tratamento de lesões de pele, como câncer basocelular, queratose actínica, e doença de Bowen, dentre outros. Basicamente, a combinação da administração de um fotossensibilizador (FS), com a irradiação de luz adequada e o oxigênio celular, gera uma série de reações oxidativas que provocam a morte do tecido. Contudo, o principal efeito colateral desta terapia é a fotossensibilidade prolongada ocasionada pela administração de fotossensibilizadores sistêmicos. Por outro lado, a via tópica não apresenta esta limitação, pois o tratamento é realizado no local da lesão através de pró-drogas. O ácido aminolevulínico, ALA, está entre as pró-drogas mais utilizadas para indução do acúmulo do agente fotossensível na pele, a protoporfirina IX, ou PpIX. Contudo, a via tópica não permite penetração suficiente e homogênea do creme para o tratamento de lesões espessas. Visando a melhoria da TFD, foram realizados estudos in vivo e in vitro. Nos estudos in vivo, técnicas mecânicas - rolos de microagulhas, tape stripping e injeção livre de agulhas foram estudadas buscando encontrar a mais eficiente nos quesitos de: promoção da penetração da pró-droga no tecido, distribuição homogênea e de indução do acúmulo de PpIX. Para isto, foi o utilizado o modelo porcino, in vivo, conhecido como o modelo que possui a pele mais similar à pele humana. Os resultados in vivo mostram que as técnicas têm resultados similares na produção de PpIX e na distribuição de porfirina mais homogênea na superfície. Além disso, todas as técnicas estudadas in vivo têm se destacado em promover uma entrega mais homogênea de ALA também na profundidade da pele quando comparadas ao grupo controle. Nos estudos in vitro, foram examinadas possíveis diferenças na capacidade de formação da PpIX e/ou de resistência de células ao tratamento por TFD entre células expressando diferentes características de transição epitélio-mesenquimal. Os resultados in vitro indicam que as células com características epitélio-mesenquimal mais acentuadas produzem mais PpIX e são mais responsivas à TFD. Estes resultados indicam que a TFD tem maior efetividade no tratamento de células mesenquimais, e os estudos in vivo mostram que no tecido normal há maior seletividade de produção na camada da epiderme e apêndices da pele sugerindo que a terapia pode ser utilizada com maior eficiência em lesões superficiais e, até mesmo diminuir as taxas de recorrência devido a heterogeneidade de distribuição do creme na pele quando umas das técnicas mecânicas são utilizadas. / Photodynamic therapy (PDT) is a noninvasive technique used to treat skin lesions, such as basal cell cancer, actinic keratosis and Bowen\'s disease. Basically, the administration of a photosensitizer (PS), combined with the illumination of adequate light and the cellular oxygen, generate a series of oxidative reactions that cause tissue death. However, the major side effect of the treatment is prolonged photosensitivity caused by the systemic administration of photosensitizers. On the other hand, the topical therapy does not show this limitation, and it is performed at the lesion site via prodrugs. The aminolevulinic acid, ALA, is the most popular pro-drug in topical PDT. This prodrug induces PpIX production that is a photosensitive porphyrin. However, when ALA is used topically, the cream does not provide enough or homogeneous penetration for the treatment of deep lesions. Therefore, with the aim of improving PDT therapy, studies in vivo and in vitro were performed. In the in vivo analysis, mechanical techniques - microneedle roller, tape stripping, and needle-free injection- were studied looking for the most effective regarding to improve the following purposes: promoting penetration of the prodrug into the tissue, homogeneous distribution, and at inducing PpIX accumulation. The evaluations were made by fluorescence spectroscopy, biopsy of skin, and fluorescence images, using the porcine model, in vivo, known as the most similar of human skin tissue. The in vivo results showed that all techniques have similar results in the production of PpIX, and perform a more homogeneous porphyrin distribution in the skin surface. Moreover, all the techniques have excelled in promoting a homogeneous distribution of PpIX in the deep of the skin when compared to the control group. In addition to the skin penetration, studies of PpIX production were performed in vitro in cells expressing different levels of epithelial-mesenchymal transition characteristics. The studies were made in regard to a possible difference in PpIX formation capacity and / or a resistance to the PDT treatment. The in vitro results showed that cells with more epithelial-mesenchymal characteristics produce more PpIX and are more responsive to the PDT therapy. These results indicate that PDT therapy may have a better effectiveness in the treatment of mesenchymal cells and also the results in vivo showed that the ALA-induced PpIX in normal tissue seems to be selective to epidermal and skin appendages, indicating that the topical therapy may be used with a higher efficiency in superficial injuries providing lower recurrence rates when they combine with one of the techniques studied.

This study was conducted to compare animal performance, presence of skin reactions and immune response following vaccination of beef calves via needle-free (NF) and needle-syringe (NS) vaccination techniques. Spring-born (Study A) and fall-born (Study B) calves were vaccinated against bovine viral diarrhea virus (BVDV) and Clostridium chauvoei (C. chauvoei) via NF and NS vaccination techniques. The parameters measured in this study included body weight (BW), skin reactions and serum antibodies. Animal performance and antibody levels against BVDV and C. chauvoei did not differ between vaccination techniques. However, NF vaccinated calves had a greater frequency of skin reactions when compared to NS vaccinated calves, except for day 42 of Study B. It can be concluded that a needle-free injection device (NFID) can be used effectively to stimulate an immune response without impacting animal performance, but may cause a greater frequency of skin reactions.

This study was conducted to compare animal performance, presence of skin reactions and immune response following vaccination of beef calves via needle-free (NF) and needle-syringe (NS) vaccination techniques. Spring-born (Study A) and fall-born (Study B) calves were vaccinated against bovine viral diarrhea virus (BVDV) and Clostridium chauvoei (C. chauvoei) via NF and NS vaccination techniques. The parameters measured in this study included body weight (BW), skin reactions and serum antibodies. Animal performance and antibody levels against BVDV and C. chauvoei did not differ between vaccination techniques. However, NF vaccinated calves had a greater frequency of skin reactions when compared to NS vaccinated calves, except for day 42 of Study B. It can be concluded that a needle-free injection device (NFID) can be used effectively to stimulate an immune response without impacting animal performance, but may cause a greater frequency of skin reactions.