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The pathophysiological mechanisms associated with immune responses to the opportunistic bacterial pathogen Providencia rettgeri in fish species are unknown. The aim of this study was to evaluate whether purinergic signaling generates anti-inflammatory or pro-inflammatory profiles in lymphoid immune organs of the Nile tilapia (Oreochromis niloticus) experimentally infected by P. rettgeri. Adenosine triphosphate (ATP) and adenosine diphosphate (ADP) hydrolysis activities in the spleen and triphosphate diphosphohydrolase (NTPDase) in the head kidney decreased in experimentally P. rettgeri-infected Nile tilapia on days seven and fourteen post-infection (PI) compared to the control group. At these same time points, splenic and head kidney adenosine deaminase (ADA) activity, and levels of ATP, interleukin-15 (IL-15) and metabolites of nitric oxide (NOx) increased in infected animals compared to their respective control groups; this was also observed for serum ATP levels. No significant difference was observed between groups regarding 5′-nucleotidase activity. These results suggest that purinergic signaling in lymphoid immune organs of Nile tilapia exerts a pro-inflammatory profile in response to P. rettgeri infection. This response contributes to the inhibition of immune and inflammatory responses by reduction of ATP hydrolysis and augmentation of ATP in the extracellular environment as well as augmentation in adenosine deamination and its possible reduction in the extracellular environment, leading to a self-sustained pro-inflammatory cycle, corroborated by excessive content of ATP (extracellular milieu), IL-15 and NOx levels. Purinergic signaling may thereby contribute to the pathophysiology of P. rettgeri infection.

Grape pomace is a by-product of the winemaking process, rich in bioactive compounds such as resveratrol and has antioxidant and antimicrobial properties. Recent evidence has demonstrated the protective effects of grape against impairment of the phosphoryl transfer network, a system linked to energetic homeostasis of high-energy phosphoryl transfer, to maintain energetic balance. Also, Pseudomonas aeruginosa infection in fish has been related to severe impairment of the branchial phosphoryl transfer network via inhibition of creatine kinase (CK), adenylate kinase (AK) and pyruvate kinase (PK) activities. Thus, the aim of this study was to evaluate whether dietary supplementation with grape pomace flour (GPF) is able to reduce or prevent the impairment of cellular energetic homeostasis in grass carp (Ctenopharyngodon idella) experimentally infected by P. aeruginosa. Branchial CK (cytosolic and mitochondrial), AK, and PK activities were lower in infected animals on day 15 post-infection (PI) compared to uninfected fish, while lactate dehydrogenase (LDH) activity was higher. Branchial reactive oxygen species (ROS), thiobarbituric acid reactive substances (TBARS), lipid oxidation (LOOH) and protein carbonylation levels were higher, while the level of non-proteic (NPSH) and proteic (PSH) thiols was lower in infected fish on day 15 PI, as compared to uninfected fish. The use of 300 mg of GPF/kg of feed was able to ameliorate the activities of CK, AK, PK, and LDH as compared to infected fish, but their activities remained different from the control group. On the other hand, dietary supplementation with 300 mg of GPF/kg of feed was able to prevent all alterations linked to oxidative damage elicited by disease. Based on this evidence, inhibition of the phosphoryl transfer network may contribute to the impairment of intracellular energetic communication between cellular ATP synthesis and consumption and is mediated by oxidative damage in the gills of fish infected by P. aeruginosa. The use of 300 mg of GPF/kg of feed exerted protective effects on branchial energetic metabolism linked to ATP metabolism by reducing the impairment in cellular energetic homeostasis; its effects can be mediated by the prevention of SH group oxidation.

It is becoming evident that bacterial infectious diseases negatively affect gill energy metabolism, mainly because it causes alterations of enzymes that belong to the phosphotransfer network, such as creatine kinase (CK), adenylate kinase (AK) and pyruvate kinase (PK). Thus, the aim of this study was to evaluate whether the phosphotransfer network could be one pathway involved in the bioenergetic imbalance during Providencia rettgeri infection, as well as to investigate the bacteria's influence on adenosine triphosphate (ATP)-dependent enzymes. In this study, branchial cytosolic and mitochondrial CK activity decreased in infected animals on day 14 post-infection (PI) when compared to its respective control group. Moreover, branchial sodium-potassium pump (Na+/K+-ATPase) and proton-pump (H+-ATPase) activities decreased in infected animals on day 14 PI compared to the control. Finally, branchial thiobarbituric acid reactive substance levels increased in infected animals on day 14 PI, while non-protein thiol (NPSH) levels decreased. No significant difference was observed between groups regarding AK and PK activities. In summary, the impairment of CK activity elicited by P. rettgeri caused an impairment in branchial energetic homeostasis, reducing ATP availability in the gills. Moreover, inhibition of CK activity provokes an impairment of ATP-dependent enzymes, such as Na+/K+-ATPase and H+-ATPase, which can be mediated by lipid oxidative damage and reduction of NPSH content.

The spleen is a secondary lymphatic organ that has an important role in fish immune and inflammatory responses. Reports have suggested a role of purinergic signaling in immune and inflammatory responses during bacterial infection; however, the role of this pathway during Pseudomonas aeruginosa infection remains unknown. The aim of this study was to investigate the involvement of purinergic signaling in the pathogenesis of P. aeruginosa infection of grass carp, Ctenopharyngodon idella. In addition, grape pomace flour (GPF), a bio-residue of the wine industry, was assessed as a dietary supplement and whether it was able to modulate purinergic signaling and improve the immune and inflammatory responses of C. idella, experimentally infected with P. aeruginosa. Nucleoside Triphosphate diphosphohydrolase (NTPDase) activity was downregulated in the serum and spleen of fish experimentally infected with P. aeruginosa, compared to the control group. By contrast, serum and splenic adenosine deaminase (ADA) activity was stimulated in fish experimentally infected with P. aeruginosa compared to the control group. Dietary supplementation with GPF was able to prevent all alterations induced by infection. Serum and splenic metabolites of nitric oxide (NOx) were higher in fish experimentally infected with P. aeruginosa compared to the control group, and GPF supplementation was able to prevent this alteration. Serum and splenic myeloperoxidase and catalase activities were also higher in fish experimentally infected with P. aeruginosa compared to the control group, and again, the use of GPF was able to prevent these alterations. Based on these metabolic changes, purinergic signaling contributed to the pro-inflammatory profile of immune lymphatic organs during P. aeruginosa infection and to disease pathophysiology. Dietary supplementation with GPF modulated the inflammatory response by altering serum and splenic purinergic signaling. GPF may be means of improving immune and inflammatory responses resulting from P. aeruginosa infection of grass carp.

It is recognized that phosphoryl transfer network, through the activities of creatine kinase (cytosolic and mitochondrial CK), adenylate kinase (AK) and pyruvate kinase (PK), provides new perspectives to understand the disturbances of energetic metabolism between sites of generation and utilization of adenosine triphosphate (ATP). Thus, the aim of this study was to evaluate the involvement of phosphoryl transfer network in the pathophysiology of Streptococcus agalactiae linked with central nervous system (CNS). Cerebral cytosolic and mitochondrial CK activities decreased in infected animals compared to uninfected animals, similarly to observed by cerebral AK and PK activities. Moreover, the cerebral ATP levels decreased in infected animals compared to uninfected animals. Based on these evidences, the inhibition and absence of efficient nucleocytoplasmic communication between CK isoenzymes caused an impairment on cerebral energetics. Moreover, the inhibition on hepatic AK and PK caused an impairment of communication between sites of ATP generation and ATP utilization, which is corroborated by cerebral ATP depletion. In summary, these alterations contribute to disease pathogenesis linked to CNS during fish streptococcosis.

It has been recognized that the cholinergic and adenosinergic systems, through the enzymes acetylcholinesterase (AChE) and adenosine deaminase (ADA), exert an important role in the immune and inflammatory responses during bacterial fish diseases. The immune response against Aeromonas caviae remains poorly understood, and the involvement of these systems, through the anti-inflammatory molecules acetylcholine (ACh) and adenosine (Ado), has not been studied. Thus, the aim of this study was to evaluate the involvement of cholinergic and adenosinergic systems on the inflammatory and immune responses in peripheric and splenic lymphocytes of fish experimentally infected with A . caviae . AChE and ADA activities in peripheric and splenic lymphocytes increased in infected animals on day 4 post-infection compared to uninfected animals, while the ACh and Ado levels decreased. Moreover, spleen histopathology revealed an increase in the number of melano-macrophage centers, as well as lipid inclusions and cellular debris in the ellipsoids. This evidence indicates that infection by A . caviae alters the cholinergic and adenosinergic systems, suggesting the involvement of AChE and ADA activities in the impairment of the immune system, since infection leads to a reduction in ACh and Ado levels, respectively. In summary, the upregulation of AChE and ADA activities in peripheric and splenic lymphocytes exerts a pro-inflammatory profile, contributing to systemic and tissue inflammatory damage, and consequently to disease pathogenesis.

Some studies have demonstrated that xanthine oxidase activity, through the regulation of uric acid levels, exerts a pro-inflammatory profile during infectious diseases. It has been recognized that uric acid induces the release of pro-inflammatory mediators, such as reactive oxygen species (ROS) and nitric oxide (NO), which contribute to inflammatory processes. Considering this, the aim of this study was to evaluate whether upregulation of branchial xanthine oxidase activity may be considered a pathway involved in ROS and nitric oxide (NOx) metabolites production in experimentally infected Rhamdia quelen (silver catfish) with Streptococcus agalactiae. Xanthine oxidase activity, and uric acid, ROS and NOx levels increased in gills of infected animals compared to uninfected animals. Moreover, a positive Pearson correlation was observed between uric acid and ROS levels, and between uric acid and NOx levels. Histopathology revealed the presence of necrosis, hyperplasia and vein congestion in gills of animals infected with S. agalactiae. Based on this evidence, the upregulation of xanthine oxidase activity induces a pro-inflammatory and oxidative profile in the gills of fish infected with S. agalactiae. The excessive uric acid levels induce the release of pro-inflammatory mediators, such as ROS and NOx, which contribute to disease pathogenesis. In summary, the upregulation of xanthine oxidase activity may be considered a pathway involved in ROS and NOx production.

The aim of this study was to evaluate whether infection by Pseudomonas aeruginosa, an important opportunistic Gram-negative bacterium, causes behavioral alterations in Rhamdia quelen, as well as to investigate the involvement of markers of stress response and brain neurotransmitters in experimentally infected fish. For this, twelve animals were divided in two groups: uninfected (negative control) and experimentally infected animals with P. aeruginosa (positive control), in order to assess the behavioral parameters, plasma cortisol levels, as well as the Na+, K+-ATPase and acetylcholinesterase (AChE) activities. Animals infected with P. aeruginosa presented a behavioral impairment linked with hyperlocomotion. Also, the plasma cortisol and reactive oxygen species (ROS) levels increased in infected fish compared to uninfected ones. Cerebral Na+, K+-ATPase activity decreased in infected animals, while cerebral AChE activity increased compared to uninfected animals. In summary, fish experimentally infected with P. aeruginosa presented behavioral impairments, which are associated with stress and hyperlocomotion. Moreover, the impairment of the cerebral Na+, K+-ATPase pump and cholinergic system may directly contribute to this behavior, since dysfunction in the synapse is promoted, and levels of ACh, an important neurotransmitter involved in several behavioral functions, is decreased.

Appropriate control of immune responses is a critical determinant of fish health. The cholinergic and adenosinergic systems have important roles in the immune response, since acetylcholine (ACh) and adenosine (Ado) possess important anti-inflammatory properties and modulate the immune response during infectious diseases. Thus, the aim of this study was to evaluate whether splenic and lymphocytic adenosinergic and cholinergic systems are able to modulate the immune response during Pseudomonas aeruginosa infection. Acetylcholinesterase (AChE) and adenosine deaminase (ADA) activities in spleen tissue and splenic lymphocytes increased in infected animals compared to uninfected animals. Moreover, plasmatic and splenic metabolites of nitric oxide (NOx) increased in infected animals compared to uninfected animals. Therefore, our results support the hypothesis that the cholinergic and adenosinergic systems are not able to modulate the immune response against P. aeruginosa. In summary, increased AChE and ADA activities exert a pro-inflammatory profile, impairing the immune system and contributing to inflammatory damage.

Pseudomonas aeruginosa is one of the most important agent representing high mortality and significant economic losses in aquaculture. Due to growing reports of antimicrobial resistance, it is important to develop studies involving alternative therapies and new technologies, including the use of essential oils and nanotechnology. In this sense, the aim of this study was to evaluate whether Melaleuca alternifolia essential oil (TTO) exerts in vivo bactericidal action against P. aeruginosa strain PA01, as well as to evaluate whether nanotechnology is able to improve this action in experimentally infected Rhamdia quelen. Our results demonstrated that free TTO and nanoencapsulated TTO exert potent bactericidal action against P. aeruginosa. The most important finding is that TTO nanoparticles possess 100% of curative efficacy, while free TTO possesses 70% of curative efficacy. In summary, the nanoencapsulation of TTO was able to improve the bactericidal action of TTO, and may be considered an important approach to prevent and/or treat infected animals with P. aeruginosa.