Leukocytes respiratory burst and lysozyme level in pacu (Piaractus mesopotamicus Holmberg, 1887)

Abstract

Innate immune responses are useful to determine the health status of fish and to evaluate the effect of immunomodulatory substances in fish farming. Leukocytes respiratory burst was measured in pacu (Piaractus mesopotamicus) using chemiluminescence assay and nitroblue tetrazolium (NBT) reduction assay. The nitroblue tetrazolium reduction seemed more adequate than chemiluminescence assay for leukocytes oxidative burst determination, since it was difficult to isolate the blood leucocytes for chemiluminescence assay. Plasma and serum lysozyme were measured using a turbidimetric assay. The heating of serum and plasma samples (56 °C for 30 minutes) for complement system inactivation darkened the plasma samples and interfered in the results. The lysozyme activity in serum was higher than in plasma, suggesting that serum samples are more appropriate for the analysis. This study established protocols that can be useful tools in the study of immune mechanisms of the tropical fish pacu.Keywords: pacu, Piaractus mesopotamicus, innate immunity, respiratory burst, lysozyme. Atividade respiratória de leucócitos e nível de lisozima em pacu (Piaractus mesopotamicus) ResumoRespostas imunológicas inatas são úteis para determinar o estado de saúde de peixes e avaliar o efeito de substâncias imunomoduladoras no cultivo destes animais. A atividade respiratória de leucócitos foi medida em pacu (Piaractus mesopotamicus) através de ensaio de quimioluminescência e ensaio de redução do nitroblue tetrazolium (NBT). O ensaio de redução do nitroblue tetrazolium pareceu mais adequado que o ensaio de quimioluminescência para determinação da atividade respiratória de leucócitos, uma vez que foi difícil isolar com êxito os leucócitos do sangue para o ensaio de quimioluminescência. Lisozima sérica e plasmática foram medidas por meio de ensaio turbidimétrico. Com o objetivo de inativar as proteínas do sistema complemento, as amostras de soro e plasma foram aquecidas (56 °C por 30 minutos). Porém, este procedimento provocou a turvação das amostras de plasma e interferiu nos resultados. A atividade de lisozima no soro foi maior que no plasma, sugerindo que amostras de soro são mais apropriadas para esta análise. Este estudo estabeleceu protocolos que podem ser utilizados como ferramentas no estudo de mecanismos imunológicos do peixe tropical pacu.Palavras-chave: pacu, Piaractus mesopotamicus, imunidade inata, burst oxidativo, lisozima. Abreu, JS. et al.

Leukocytes respiratory burst activity as indicator of innate immunity of pacu Piaractus mesopotamicus

“…A previous study described a different leukocyte respiratory assay for pacu (Abreu et al, 2009) based on chemiluminescence observed in stained blood smears with Leishman's solution in order to determine the percent of cells containing intracellular blue formazan particles under light microscopy. In this new approach, the NBT reduction was measured colorimetrically in a dissolved solution, resulting in an easier, cheaper and more sensitive assay.…”

“…Regarding Brazilian native fish species, very little is known on its innate immune responses (Belo et al, 2005;Abreu et al, 2009;Sado et al, 2010;Biller-Takahashi et al, 2012). However, the appropriate assessment of the immune responses demands a reliable methodology.…”

Proposed method for agglutinating antibody titer analysis and its use as indicator of acquired immunity in pacu, Piaractus mesopotamicus

“…Nevertheless, few studies exist on its immune responses (Abreu et al, 2009;Martins et al, 2009;Sado et al, 2010;Biller-takahashi et al, 2012Biller-takahashi et al, , 2013. In order to provide appropriate analytical methods for future studies on the specific immune system of this native Brazilian fish, the present study aimed to standardize and applied the agglutination assay to measure the serum agglutinating antibody titer produced after immunization with inactive vaccine against A. hydrophila followed by the administration of the immune modulator levamisole.…”

Hemolytic activity of alternative complement pathway as an indicator of innate immunity in pacu (Piaractus mesopotamicus)

“…An alternative practice is the administration of immunostimulants; one example is β-glucan, which acts by increasing the fish resistance against disease outbreaks (Sakai, 1999;Selvaraj et al, 2005). Regarding pacu (Piaractus mesopotamicus), an important farmed Brazilian fish, there is a lack of knowledge of its immune system (Belo et al, 2005;Biller, 2008;Abreu et al, 2009). This study evaluated the methodology to measure the hemolytic activity of the complement system, alternative pathway, as an indicator of the innate immunity in pacu after being experimentally challenged with A. hydrophila and fed β-glucan.…”