The present study evaluated the infection of opossums (Didelphis aurita) by Rickettsia rickettsii and their role as amplifier hosts for horizontal transmission of R. rickettsii to Amblyomma cajennense ticks. Three groups of opossums were evaluated: on day 0, group 1 (G1) was inoculated intraperitoneally with R. rickettsii; group 2 (G2) was infested by R. rickettsii-infected ticks; and group 3 (G3) was the uninfected control group. Opossum rectal temperature was measured daily. Blood samples were collected every 2 to 4 days during 30 days, and used to (1) inoculate guinea pigs intraperitoneally; (2) extract DNA followed by real-time polymerase chain reaction (PCR) targeting the rickettsial gene gltA; (3) study hematology; (4) detect R. rickettsii-reactive antibodies by indirect immunofluorescence assay (IFA). Blood was also collected every 10 days from days 30 to 180, to be tested by serology. Opossums were infested by uninfected A. cajennense larvae and nymphs from days 3 to 15. Engorged ticks were collected and allowed to molt in an incubator. Thereafter, the subsequent flat ticks were allowed to feed on uninfected rabbits, which were tested for seroconversion by IFA. Samples of flat ticks were also tested by real-time PCR. All G1 and G2 opossums became infected by R. rickettsii, as demonstrated by realtime PCR or/and guinea pig inoculation, but they showed no clinical abnormality. Rickettsemia was first detected at days 2 to 8, lasting intermittently till days 1 to 30. Approximately 18% and 5% of the flat ticks previously fed on G1 and G2 opossums, respectively, became infected by R. rickettsii, but only the rabbits infested with G1-derived ticks seroconverted. The study demonstrated that R. rickettsii was capable of infecting opossums without causing illness and developing rickettsemia capable of causing infection in guinea pigs and ticks, although the infection rate in ticks was low.
Objetivo. Aislar Rickettsias mediante cultivo celular a partir de muestras de garrapatas Amblyomma ovale y Amblyomma incisum del estado de São Paulo Materiales y métodos. A. ovale y A. incisum adultas de vida libre fueron colectadas en una área de selva tropical Atlántica en el estado de São Paulo, Brazil. Cada garrapata fue sometida a la prueba de hemolinfa, las garrapatas positivas en esta prueba fueron evaluadas con la técnica de shell vial con el propósito de aislar rickettsias en cultivo de células Vero. Pasajes celulares de los aislados fueron identificados genotípicamente por la reacción en cadena por la polimerasa (PCR) dirigidos a fragmentos de tres genes de rickettsias (gltA, htrA y ompA), seguido por secuenciación de ADN. Resultados. Un total de 388 A. incisum y 50 A. ovale fueron colectadas. Por la prueba de hemolinfa, únicamente una A. incisum y una A. ovale fueron positivas. Las Rickettsias fueron exitosamente aisladas de estas garrapatas. Sin embargo, el cultivo continuo en células Vero fue posible sólo para la garrapata A. ovale, debido a contaminación bacteriana en el primer pasaje celular de la muestra de A. incisum. Los productos de PCR fueron obtenidos con los primers gltA y htrA para los dos aislados, no obstante, ningún producto fue obtenido con los primers ompA. Por análisis BLAST, secuencias parciales de gltA y htrA procedentes de los aislados de A. ovale y A. incisum fueron similares a las secuencias correspondientes a R. bellii. Conclusiones. Este es el primer reporte de R. bellii infectando A. incisum y el primer establecimiento exitoso de un aislado de A. ovale. Palabras clave: Rickettsia bellii, Amblyomma ovale, Amblyomma incisum, Brasil.
O presente trabalho objetivou verificar o desenvolvimento corpóreo de filhotes de Didelphis albiventris e Didelphis aurita por 52 semanas, possibilitando um maior conhecimento de sua biologia em cativeiro. Os gambás foram separados da mãe a partir da 13ª semana de idade, individualizados e alimentados uma vez ao dia, com ração seca para cães adultos; frutas como manga, banana e laranja; ovos de galinha com casca e água ad libitum. A massa corporal foi mensurada da 13ª à 52ª semana de vida, uma vez por semana, com utilização de balança digital. A média de massa de D. albiventris e D. aurita variou, respectivamente, de 200-270g (média = 232g) e 140-190g (média = 155g) na 13ª semana; e de 1.490-2.300g (média = 1.885g) e 1.310-2.170g (média = 1.651g) na 52ª semana. Os valores obtidos foram comparados com as seguintes funções: linear, logarítmico, quadrático, cúbico e exponencial. Todos os modelos testados foram significativos (p<0,001), porém o modelo que apresentou o melhor coeficiente de determinação (R2) foi o modelo cúbico para D. albiventris, e o modelo exponencial, para D. aurita. Os gambás apresentaram um padrão de crescimento corporal mais lento e uniforme nas primeiras semanas e mais acelerado nas semanas subsequentes, de acordo com a massa corporal inicial de cada indivíduo.
AGRADECIMENTOSAos meus pais Roque e Inêz, e a minha irmã Silvia, por serem tão presentes na minha vida.A minha tia "Dada", pelo carinho e pela ajuda.Aos meus avós Argemiro (in memorian) e Dirce (Lolinha) (in memorian) por todo carinho.Aos animais que sempre farão parte da minha vida: Lana, Nina, Guinho, Xuxa, Pitu, Pretinha, Virinha, Pitoca entre tantos outros. Obrigada por tornarem minha vida melhor.Aos professores do Departamento de Medicina Social da FMRP por contribuírem para minha formação.Aos funcionários do Departamento de Medicina Social da FMRP pela ajuda com todos os procedimentos burocráticos.Aos meus amigos da FMVZ-USP (alunos, funcionários e professores) pelos anos de convivência e de amizade. Aos meus amigos "dinos" da USP Jr pelos ensinamentos e pelo agradável convívio. Aos funcionários Cristina e Robson da Biblioteca Central da USP Ribeirão Preto pela ajuda com as referências. Aos meus colegas de trabalho da Ourofino. À Ourofino pela concessão dos dados. Palavras-chave: período de carência; regressão linear; regressão; resíduo de medicamento. ABSTRACT ROSA SC. Estimation of the withdrawal period for veterinary drugs in edible tissues of animal origin by regression models. 2016. 288p. Dissertation (Master Degree). Faculty of Medicine of Ribeirao Preto, University of São Paulo, Ribeirão Preto.Veterinary drugs residues can be found in foodstuffs of animal origin such as meat, milk, eggs and honey. In order to ensure that the concentration of these residues does not exceed a safe limit (Maximum Residue Limit -MRL) it is necessary to establish a withdrawal period, which is the waiting time necessary for an animal to be sent for slaughtering after having received a veterinary drug. The estimation of the withdrawal period is normally obtained by the fitting of a simple linear regression model, followed by the calculation of a tolerance limit. For this, the assumptions of homoscedasticity and the normality of the errors must be met. However, violations of these assumptions are frequent in the residual depletion studies. In the present study two database of the quantification of veterinary drug residue in bovine tissues were used and the withdrawal period was estimated for liver, fat, muscle and kidneys. The regression models were fitted to the mean value of the results obtained for each animal, to the mean value of the results obtained for each analytical extraction and to the results obtained for the repeated sample measurements, and a linear mixed model was fitted for this later situation.The linear model fitted to the mean value of the results obtained for each analytical extraction showed greater precision in the parameters estimates of the model as well as shorter withdrawal period. However, for this model, more potentially influential points were detected compared to other models fitted. It was not possible to calculate the tolerance limit, and, consequently, to predict the withdrawal period using the mixed effects model. In conclusion, the fitting of the other more robust and flexible statistical m...
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