RESUMO
Pathogenic bacteria are responsible for several diseases in humans and in a variety of hosts. Detection of pathogenic bacteria is imperative to avoid and/or fight their potential harmful effects. This work reports on the first amperometric biosensor for the rapid detection of Streptococcus agalactiae (S. agalactiae). The biosensor relies on a single biotinylated antibody that immobilizes the bacteria on a screen-printed carbon electrode while is further linked to a streptavidin-conjugated HRP reporter. The biotinylated antibody provides selectivity to the biosensor whereas serves as an anchoring point to the reporter for further amplification of the electrochemical signal. The resultant immunosensor is simple, responds rapidly, and allows for the selective and highly sensitive quantification of S. agalactiae cells in a concentration range of 101-107CFUml-1, with a detection limit of 10CFUml-1. The approach not only enables a rapid detection and quantification of S. agalactiae in environmental samples but also opens up new opportunities for the simple fabrication of electrochemical immunosensors for different target pathogens.
Assuntos
Anticorpos Imobilizados/química , Técnicas Biossensoriais/métodos , Doenças dos Peixes/diagnóstico , Infecções Estreptocócicas/veterinária , Streptococcus agalactiae/isolamento & purificação , Tilápia/microbiologia , Microbiologia da Água , Animais , Técnicas Biossensoriais/economia , Técnicas Eletroquímicas/economia , Técnicas Eletroquímicas/métodos , Eletrodos , Doenças dos Peixes/microbiologia , Imunoensaio/economia , Imunoensaio/métodos , Limite de Detecção , Infecções Estreptocócicas/diagnóstico , Infecções Estreptocócicas/microbiologiaRESUMO
Rickettsial organisms are well-known fish pathogens in both natural and culture environments. This study reports an outbreak of disease in red tilapia larvae caused by piscirickettsia-like organisms (PLOs), which lasted from June until October 2009. Severe mortality was recorded almost exclusively in larvae and postlarvae aged 1-22 days old. Although clinical or gross findings were not evident in diseased fish, histopathology revealed severe necrosis of the epidermis and gill epithelium, with concomitant changes in the underlying skeletal muscle as being the most relevant microscopic lesions. Although PLOs were visible with the routine hematoxylin eosin technique, they were better observed with Giemsa and toluidine blue stains. Transmission electron microscopy revealed that the bacterium was located within the cytoplasm and phagolysosoma-like structures of epithelial cells from the gills and the skin. The bacteria measured 0.9 ± 0.2 µm × 2.1 ± 0.6 µm and had a double cell membrane (the outer one having undulating projections), with variable electron-dense and electron-lucent areas. Ultrastructurally, abundant myelin figures surrounded the microorganisms within host cell cytoplasm. Results indicated that Piscirickettsia-like organisms can cause massive epithelial cell damage associated with concomitant alteration of the electrolyte balance.