PESQUISA VETERINÁRIA BRASILEIRA

Abstract in English:

ABSTRACT.- Kim F.J.P., Silva A.E.M., Silva R.V.S., Kim P.C.P., Acosta A.C., Silva S.M.B.C., Sena M.J. & Mota R.A. 2018. [Detection of Aeromonas spp. and virulence gene aerolysin in Nile tilapia (Oreochromis niloticus) using PCR technique.] Detecção de Aeromonas spp. e do gene de virulência aerolisina em tilápias do Nilo (Oreochromis niloticus) com a técnica de PCR. Pesquisa Veterinária Brasileira 38(9):1731-1735. Departamento de Desenvolvimento Educacional, Curso de Tecnologia em Agroecologia, Instituto Federal de Educação, Ciência e Tecnologia de Pernambuco, Campus Barreiros, Fazenda Sapé s/n, Zona Rural, Barreiros, PE 55560-000 Brazil. E-mail: fernando_kim@barreiros.ifpe.edu.br Infections caused by bacteria of the genus Aeromonas are among the most common diseases in fish farming systems worldwide, and this disease occurs in all countries which have Nile tilapia (Oreochromis niloticus) farmed. The present work describes the development of a new multiplex PCR (mPCR) technique that diagnosis the genus Aeromonas and detects aerolysin gene (aerA). Reference strains of several Aeromonas species and other genera were used for standardization of mPCR. Strains of A. hydrophila from “pacaman” fish (Lophiosilurus alexandri) and Aeromonas spp. from Nile tilapia from farming systems were used too. Primers were designed based on the 16S rRNA region and aerA (aerolysin toxin). To verify a better annealing temperature were used gradients between 59°C and 61°C with 40ng of the DNA template. The 16S rRNA gene and the aerA gene amplification products showed 786 and 550 bp, respectively. The mPCR showed better annealing temperature at 57.6°C, and the detection limit for both genes (16S rRNA and aerA) was 10-10g/μL of the DNA. The standardized mPCR is quick, sensitive, and specific for Aeromonas spp. diagnosis and to detect aerolysin gene. This method showed advantages when compared to the conventional diagnostic methods and can be used in Nile tilapia or other fish farming systems. The detection of aerolysin gene is an important tool to determine the potential pathogenicity of Aeromonas spp. isolates.

• Aeromonas gene aerolysin Nile tilapia Oreochromis niloticus PCR technique mPCR virulence factor bacterioses Aeromonas spp. gene de virulência aerolisina tilápias do Nilo Oreochromis niloticus técnica de PCR fator de virulência bacterioses

Abstract in Portuguese:

RESUMO.- Kim F.J.P., Silva A.E.M., Silva R.V.S., Kim P.C.P., Acosta A.C., Silva S.M.B.C., Sena M.J. & Mota R.A. 2018. [Detection of Aeromonas spp. and virulence gene aerolysin in Nile tilapia (Oreochromis niloticus) using PCR technique.] Detecção de Aeromonas spp. e do gene de virulência aerolisina em tilápias do Nilo (Oreochromis niloticus) com a técnica de PCR. Pesquisa Veterinária Brasileira 38(9):1731-1735. Departamento de Desenvolvimento Educacional, Curso de Tecnologia em Agroecologia, Instituto Federal de Educação, Ciência e Tecnologia de Pernambuco, Campus Barreiros, Fazenda Sapé s/n, Zona Rural, Barreiros, PE 55560-000 Brazil. E-mail: fernando_kim@barreiros.ifpe.edu.br As infecções causadas por bactérias do gênero Aeromonas estão entre as doenças mais comuns em peixes cultivados em todo o mundo, com ocorrência de aeromoniose em todos os países que possuem cultivo de tilápia do Nilo (Oreochromis niloticus). O presente trabalho descreve o desenvolvimento de uma nova multiplex PCR (mPCR) para diagnóstico de Aeromonas spp. e identificação do gene aerolisina (aerA). Para padronização da mPCR foram utilizadas cepas de referência de várias espécies do gênero Aeromonas e de outros gêneros. Também foram usadas cepas de campo de A. hydrophila oriundas de cultivos de peixes pacamãs (Lophiosilurus alexandri) e Aeromonas spp. de tilápias do Nilo. Os primers foram desenhados com base na região 16S rRNA e aerA. Para verificar a melhor temperatura de anelamento foram utilizados gradientes entre 59°C a 61°C com 40ng de DNA molde. Os produtos da amplificação da região 16S rRNA e do gene aerA apresentaram 786 e 550pb, respectivamente. A mPCR apresentou melhor temperatura de anelamento a 57,6°C com limite de detecção das concentrações de DNA em ambos genes (16S rRNA and aerA) de 10-10g/µL. A mPCR padronizada é rápida, sensível e específica no diagnóstico de Aeromonas spp. e identificação do gene aerolisina. Esta metodologia apresenta vantagens quando comparada aos métodos de diagnóstico convencionais, podendo ser utilizada em cultivos comerciais de tilápias do Nilo ou outros peixes. A identificação do gene aerolisina é uma importante ferramenta na determinação do potencial patogênico dos isolados de Aeromonas spp. estudados.

Abstract in English:

ABSTRACT.- Bezerra F.S.B., Garcia H.A., Alves H.M., Oliveira I.R.S., Silva A.E., Teixeira M.M.G. & Batista J.S. 2008. [Trypanosoma vivax in testicular and epidydimal tissues of experimentally infected sheep.] Trypanosoma vivax nos tecidos testicular e epididimário de ovinos experimentalmente infectados. Pesquisa Veterinária Brasileira 28(12):575-582. Laboratório de Patologia Veterinária, Departamento de Ciências Animais, Universidade Federal Rural do Semi-árido, BR 110 Km 47, Cx. Postal 147, Mossoró, RN 59625-900, Brazil. E-mail: jaelsbatista@hotmail.com Four adult sheep (number 1, 2, 3 and 4), all males, were inoculated intravenously with 1ml of blood containing 1.25x105 trypomastigotes of Trypanosoma vivax, and Sheep 5, 6, 7 and 8 were used as control. After infection, clinical exams considering rectal temperature, respiratory and cardiac frequencies, and parasitaemia were recorded daily for a 30-day experiment period. Blood samples were obtained for 5-day intervals to hematocrit analysis. At the end of the experimental period, the sheep were orquiectomized. Testes and epididymides from these animals were studied anatomopathologically. Samples from these tissues of Sheep 1, 4 and 5 were taken to polymerase chain reaction (PCR). Clinical parameters remained for the infected group above the values observed in the control group during the experimental period. Parasitaemia was observed on day 3 post-infection, and the highest values occurred between day 6 and 10, and day 15 and 18 post-infection. Sheep 1 and 4 showed severe anemia on day 25 post-infection. All sheep of the infected group showed flabby and palid testes. Histologically, moderate to severe testicular degeneration, multifocal epididymitis and hyperplasia of epididymal epithelium were observed. The result of T. vivax PCR analysis in the testes and epididymal tissues was positive in 100% of the samples of the experimentally infected sheep. Epididymal and testicular lesions associated with the presence of the parasite in these tissues, shown by PCR, suggest the participation of T. vivax in the pathophysiological mechanism of reproductive damage.

• rypanosomosis small ruminant PCR anatomopathology testicular degene-ration epididymitis extravascular foci

Abstract in Portuguese:

ABSTRACT.- Bezerra F.S.B., Garcia H.A., Alves H.M., Oliveira I.R.S., Silva A.E., Teixeira M.M.G. & Batista J.S. 2008. [Trypanosoma vivax in testicular and epidydimal tissues of experimentally infected sheep.] Trypanosoma vivax nos tecidos testicular e epididimário de ovinos experimentalmente infectados. Pesquisa Veterinária Brasileira 28(12):575-582. Laboratório de Patologia Veterinária, Departamento de Ciências Animais, Universidade Federal Rural do Semi-árido, BR 110 Km 47, Cx. Postal 147, Mossoró, RN 59625-900, Brazil. E-mail: jaelsbatista@hotmail.com Four adult sheep (number 1, 2, 3 and 4), all males, were inoculated intravenously with 1ml of blood containing 1.25x105 trypomastigotes of Trypanosoma vivax, and Sheep 5, 6, 7 and 8 were used as control. After infection, clinical exams considering rectal temperature, respiratory and cardiac frequencies, and parasitaemia were recorded daily for a 30-day experiment period. Blood samples were obtained for 5-day intervals to hematocrit analysis. At the end of the experimental period, the sheep were orquiectomized. Testes and epididymides from these animals were studied anatomopathologically. Samples from these tissues of Sheep 1, 4 and 5 were taken to polymerase chain reaction (PCR). Clinical parameters remained for the infected group above the values observed in the control group during the experimental period. Parasitaemia was observed on day 3 post-infection, and the highest values occurred between day 6 and 10, and day 15 and 18 post-infection. Sheep 1 and 4 showed severe anemia on day 25 post-infection. All sheep of the infected group showed flabby and palid testes. Histologically, moderate to severe testicular degeneration, multifocal epididymitis and hyperplasia of epididymal epithelium were observed. The result of T. vivax PCR analysis in the testes and epididymal tissues was positive in 100% of the samples of the experimentally infected sheep. Epididymal and testicular lesions associated with the presence of the parasite in these tissues, shown by PCR, suggest the participation of T. vivax in the pathophysiological mechanism of reproductive damage.