A team of researchers from several academic institutions in Brazil tracked purinergic signaling and explored the use of grape pomace flour (GPF) as a feed additive with the potential ability to alter purinergic signaling and boost fish immune response.
The group published its work in the journal Aquaculture.
“The aim of this study was to investigate the involvement of purinergic signaling in the pathogenesis of P. aeruginosa infection,” the researchers said. “In addition, we assessed whether dietary grape pomace flour (GPF) was able to modulate purinergic signaling and improve the immune and inflammatory responses of grass carp, Ctenopharyngodon idella, experimentally infected with P. aeruginosa.”
The researchers found that fish facing a disease challenge had reduced nucleoside triphosphate diphosphohydrolase (NTPDase) activity and increased adenosine deaminase (ADA) activity and more metabolites of nitric oxide (NOx) in the spleen and serum compared to control group fish.
However, fish receiving diets with additive grape pomace flour did not see changes from the disease challenge, they said.
"Dietary supplementation with 150 or 300 mg GPF/kg feed can modulate the inflammatory response by increasing serum and splenic purinergic signaling, as well as by preventing the excessive production of NOx,” they added.
“However, we cannot exclude the possibility that [the] antimicrobial effect of GPF controlled the infection and prevented the occurrence of lesions characteristic of the disease,” they said. “GPF may be a valuable approach to improving immune and inflammatory responses during P. aeruginosa infection.”
Disease challenge in aquaculture
Aquatic production accounts for about 15-20% of the global protein intake, the researchers said. Sustainability of capture fisheries and the aquaculture industry is needed to meet the increasing demand for protein.
Beyond protein, fish products also provide nutrients essential for human health, they said.
The pressure to address the demand for fish supports the use of intensive production system, which may stress fish and reduce their ability to fend off pathogens like Pseudomonas aeruginosa.
“P. aeruginosa is a Gram-negative opportunistic bacterium considered to be one of the most common bacterial pathogens in marine and freshwater aquaculture,” the researchers said. “The disease induced by these bacteria is characterized by petechial hemorrhages, darkening of the skin, ascites, exophthalmia, behavioral alterations linked with defective locomotion, and severe impairment of the immune response (Baldissera et al., 2017b); ultimately, causing severe economic losses for fish producers.”
Previous research suggested that stimulating lymphocytic and splenic ADA activity generated a pro-inflammatory profile and was linked to a reduced immune and inflammatory response, they said.
"However, whether the complete purinergic pathway is involved, which is also catalyzed by triphosphate diphosphohydrolase (NTPDase) and 5′-nucleotidase activities, remains unknown,” they added.
“Purinergic signaling, via the extracellular catabolism of purine molecules such as adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine (Ado), acts through positive or negative signals that can modulate immune and inflammatory responses,” they said. “According to Chiu and Freund (2014), this modulation occurs through the interaction of purine nucleotides (ATP, ADP and AMP) and purine nucleosides (Ado) with specific purine receptors (P1 and P2X7) located in the plasma membrane of immune and spleen cells.”
The signaling is controlled by NTPDase and linked with ATP, they added. High levels of ATP can generate a “deleterious” and self-sustained pro-inflammatory pattern.
“Therefore, our hypothesis was that purinergic signaling might be involved in impaired immune and inflammatory responses during P. aeruginosa infection,” the researchers said.
Why grape pomace use?
Finding an ecologically friendly way to improve the immune response in grass carp is of interest to the aquaculture industry especially in intensive production systems as fish face regular bacterial challenges, said the team.
Bio-residues from wine production, including grape pomace, are of interest because they contain bioactive compounds, they said. The by-product may bring antioxidant, antimicrobial and anti-inflammatory elements.
Those elements could stay active as the pomace is added to animal feed, they said.
Recent research in rats linked grape juice to a boost in immune system function through the modulation of purinergic signaling.
Grape juice also has demonstrated an antimicrobial response to P. aeruginosa, the researchers said.
“It is also important to note that resveratrol, the main constituent of GPF, is a potent immunomodulatory phenolic compound that exerts its anti-inflammatory effects by affecting purinergic signaling,” they said. “Twenty, 40 and 80 mg/kg resveratrol was able to modulate ADA activity, which protected brain tissue against damage induced by pentylenetetrazol.”
Feeding and disease challenge
For the feeding trial, a basal diet was developed for grass carp and supplemented with 0, 150 or 300mg/kg GFP, the researchers said. The GFP used was a commercially available product.
About 108 juvenile grass carp were given one of the three diets for a period of 60 days, the researchers said. On day 60, about half of the fish on each diet was infected with P. aeruginosa.
On day 15 post-infection (PI) a selection of fish from each diet was collected and blood and splenic tissue samples were collected for analysis, they said. Fish samples were checked for protein contents and NTPDase along with 5’-nucleotidase activities, ADA activity and NOx levels.
Samples also were assessed for myeloperoxidase (MPO) and catalase (CAT) activities, along with superoxide dismutase (SOD) changes, they said.
Fish on all diets had similar weight gain, length gain and feed intake, the researchers said. No fish mortality was noted during the feeding trial.
Following infection, infected control group fish developed petechial hemorrhages and skin darkening although challenged fish on the supplemented diets and non-challenged fish did not, they said.
Serum and splenic NTPDase activity dropped in infected, control group fish compared to uninfected fish, they said.
Challenged fish on the supplemented diets did not see the drop in activity and all non-challenged fish had similar levels.
No large differences were found for 5’-nucleotidase activities regardless of diet or infection, they added.
ADA activities and NOx levels in the serum and spleen tissue increased in infected, control group fish, the researchers said.
No other fish group saw similar growth in activity or level.
MPO and CAT activity increased in infected control group fish, but did not change in non-infected fish or for infected fish on the supplemented diets, the researchers said.
No significant differences for SOD activities were noted in any of the groups.
“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 a means of improving immune and inflammatory responses resulting from P. aeruginosa infection of grass carp.”
Title: Effects of dietary grape pomace flour on the purinergic signaling and inflammatory response of grass carp experimentally infected with Pseudomonas aeruginosa
Authors: M. Baldissera, C. Souza, S. Descovi, C. Verdi, C. Zeppenfeld, L. Silva, A. Gindri, M. Cunha, R. Santos, B. Baldisserotto, A. da Silva