Graduation date: 1997

Endotoxin is a part of the cell wall of gram-negative bacteria, consisting of serotype-specific polysaccharide, core oligosaccharide, and lipid A. Lipid A is responsible for an array of pathophysiologic reactions in animal hosts. Amebocyte lysate originated from Limulus polyphemus (horseshoe crab) has been used extensively in various assay systems to detect endotoxin. One of the assays, a chromogenic Limulus amebocyte lysate (CLAL) test was developed in 1978 and has been used extensively in human clinical fields for its high sensitivity and ease in quantitation. The use of the CLAL test in veterinary fields has been limited to dogs, horses and cattle. The objective of the thesis research was to determine the level of endotoxin by the CLAL assay in broiler chickens. Since gram-negative septicemia is common in broiler chickens, the detection of endotoxemia would help in understanding the pathogenesis and in developing a new treatment or prophylactic mean. By the use of a kinetic method, the CLAL assay detected the standard endotoxin (phenol-water extract from Escherichia coli, 055:B5 strain) in the range between 100 ng and 10 pg/ml. The intra-assay variation was 1.2% and interassay variation was 18.8% based on 1.0 ng standard. The control showed spontaneous release of the chromophore starting around 40 min. after the start of the reaction. This spontaneous release was found not due to contamination of pyrogen-free water (PFW) or substrate by endotoxin. With chicken plasma, various non-specific reactions were detected. Plasma alone released the chromophore in a slow, steady manner, but this reaction was virtually eliminated by heating at 100 C. Chicken plasma contained both inhibitor(s) and enhancer(s) for the test. Endotoxin-free plasma samples were prepared by absorption and reconstituted with 1.0 ng/ml of endotoxin. After 1:10 dilution in PFW, heating (10 min.) at 100 C was found most adequate to inactivate these factors as compared with heating to 70 or 85 C. With plasma samples which had been diluted and heated at 100 C, however, still some nonspecific reaction was detected; the lysate, in the absence of substrate, caused some precipitates with chicken plasma in a nonspecific manner, making it difficult to interpret the OD readings. Because of these nonspecific reactions largely inherent to the state of lysate, sensitivity was judged only to 100 pg endotoxin/ml of chicken plasma. A commercial test kit also showed 100 pg/ml sensitivity with the end point method, but found unreliable since proper controls cannot be evaluated in a similar manner as the kinetic method. Thirty chicken plasma were collected from 3 local broiler farms and was judged to contain less than 100 pg/ml in 29 birds, while one bird showed 12.0 ng/ml of endotoxin. Twenty-three per cent of the chickens showed gram-negative bacteremia without detectable endotoxemia, and the bird with endotoxemia did not have bacteremia. One microgram of the standard endotoxin was injected intravenously to 20 broiler chickens raised in the laboratory, and 5 were sacrificed at 2, 30, 60, and 90 minutes after the injection. The endotoxin was found to be cleared from the blood at the rate of 152 pg/min. To increase the sensitivity and to decrease the cost of the CLAL test, future efforts should be made; 1) to significantly decrease the nonspecific reaction between the lysate and substrate; and 2) to block the precipitation or clotting reaction between the lysate and chicken plasma. If these nonspecific reactions be controlled, the CLAL test could be run in a simple end-point method and/or in an automated manner with chicken plasma. / Graduation date: 1997

O objetivo do presente trabalho foi quantificar, in vitro e in vivo, a endotoxina bacteriana (LPS) aderida a ligaduras ortodônticas elastoméricas estéticas de poliuretano e de silicone, empregando o teste Limulus Amebocyte Lysate (LAL). Para o estudo in vitro foram utilizadas quatro tipos de ligaduras elastoméricas estéticas: Sani-Ties (poliuretano) e Sili-Ties (silicone), da GAC, e Mini Single Case Ligature Stick (poliuretano) e Synergy&reg; Low-friction ligatures (silicone), da Rock Mountain, sendo 5 contaminadas com solução de endotoxina (controle positivo) e 5 não-contaminadas (controle negativo). Réplicas feitas de fio de amarrilho torcido e de aço inox fundido, de mesmo tamanho e formato das ligaduras elastoméricas, contaminadas ou não com endotoxina, foram usadas como controle. A quantificação de endotoxina foi realizada por meio do teste LAL (Kit QCL-1000&trade;), sendo os resultados expressos em unidades de endotoxina (UE/mL). No estudo in vivo, 20 pacientes de ambos os gêneros, com faixa etária entre 15 e 30 anos, que iniciaram tratamento com aparelho ortodôntico fixo na Faculdade de Odontologia de Ribeirão Preto - Universidade de São Paulo receberam, randomicamente, em quadrantes alternados, os mesmos quatro tipos de ligaduras elastoméricas utilizadas no estudo in vitro, sendo uma ligadura de cada marca inserida nos caninos superiores e inferiores (13, 23, 33 e 43), aleatoriamente. Vinte e um dias após, as ligaduras foram removidas e processadas para quantificação da endotoxina bacteriana, utilizando também o Kit QCL-1000&trade;. Todos os dados obtidos foram submetidos à análise estatística apropriada de acordo com a distribuição dos dados, por meio dos testes de Kruskal-Wallis e pós-teste de Dunn (estudo in vitro) e ANOVA de medidas repetidas e pós-teste de Tukey (estudo in vivo). Todas as análises foram efetuadas por meio do programa Graph Pad Prism 4.0, com nível de significância de 5%. De acordo com os resultados obtidos, observou-se que a endotoxina bacteriana aderiu-se a todos os materiais testados. No estudo in vitro, o grupo GAC silicone foi o que apresentou menor mediana de contaminação (1,15 UE/mL), com relação aos outros grupos (p<0,0001), os quais não apresentaram diferença estatisticamente significante quando comparados entre si (p>0,05). No estudo in vivo, de maneira semelhante ao observado no estudo in vitro, o grupo GAC silicone foi o que apresentou menor média de contaminação (0,577±0,017 UE/mL), com diferença estatisticamente significante (p<0,001) em comparação aos demais grupos. Pôde-se concluir, que a endotoxina bacteriana apresentou afinidade pelas ligaduras elastoméricas estéticas à base de silicone e poliuretano testadas. As ligaduras de silicone da marca GAC foram as que apresentaram menor quantidade de endotoxina aderida às suas superfícies / The objective of this study was to quantify, in vitro and in vivo, bacterial endotoxin (LPS) attached to esthetic elastomeric orthodontic ligatures made of polyurethane and silicone using the Limulus Amebocyte Lysate test (LAL). For the in vitro study, were used four types of aesthetic elastomeric ligatures: Sani-Ties (polyurethane) and Sili-Ties (silicone) - GAC, and Mini Single Case Ligature Stick (polyurethane) and Synergy&reg; Low-friction ligatures (silicone) - Rock Mountain; 5 of each were contaminated with endotoxin solution (positive control) and 5 non-contaminated (negative control). Replicas made of twisted wire ligatures and of cast stainless steel, of the same size and shape than the elastomeric ligatures, contaminated or not with endotoxin, were used as control. Endotoxin quantification was performed using the LAL test (QCL-1000&trade; kit), the results being expressed in EU/mL. In the in vivo study, 20 patients of both genders, with ages ranging from 15 to 30 years old, who started treatment with a fixed orthodontic appliance at the School of Dentistry of Ribeirão Preto - University of São Paulo, received randomly the same four types of elastomeric ligatures used in the in vitro study, being a ligature of each brand inserted in the upper and lower canines (UR3, UL3, LR3, LL3), randomly. Twenty-one days later, ligatures were removed and processed for quantification of bacterial endotoxin, using the same test as the in vitro study. All data were submitted to appropriate statistical analysis according to the data distribution, using KruskalWallis tests and Dunn post-test (in vitro study) and ANOVA of repeated measurements and Tukey\'s post-test (in vivo study). All analyzes were performed using the Graph Pad Prism 4.0 program, with a significance level of 5%. According to the results obtained, it was observed that the bacterial endotoxin attached to all materials tested. In the in vitro study, the GAC silicone group had the lowest median contamination (1.15 EU/mL), in relation to the other groups (p<0.0001), which did not present a statistically significant difference when compared to each other (p>0.05). In the in vivo study, similar to that observed in the in vitro study, the GAC silicone group had the lowest mean contamination (0.577 ± 0.017 EU/mL), with a statistically significant difference (p< 0.001) compared to the others groups. It could be concluded that bacterial endotoxin exhibited affinity for the tested silicone and polyurethane aesthetic elastomeric ligatures. The silicone ligatures of GAC brand were the ones that presented less amount of endotoxin attached to their surfaces