Acute inflammatory response in Nile tilapia fed probiotic Lactobacillus plantarum in the diet

The present study evaluated the acute inflammatory response induced by carrageenin (500 μg) injected in the swim bladder of Nile tilapia, after fed or not probiotic supplemented diet. Fifty four fish were distributed in six treatments and three replicates: Group A: Fish fed unsupplemented diet: 0.5 mL saline-injected fish; fish injected with 500 μg carrageenin diluted in 0.5 mL saline; Non-injected. Group B: Fish fed probiotic supplemented diet: saline-injected fish; carrageenin-injected fish; Non-injected. Fifteen days after feeding the fish were injected with carrageenin or saline. After six hours, inflammatory exudate was collected, as well as the blood for hematocrit, red blood cell (RBC) and white blood cell (WBC) counts, differential count of leucocytes and phagocytic activity in the blood. Supplementation with probiotic did not influence the RBC, hematocrit and the numbers of lymphocytes and basophils in the blood. The number of neutrophils was significantly higher in supplemented fish injected with carrageenin. Glucose concentration in supplemented and non-injected fish was higher than that observed in the saline injected ones. Probiotic potentialized the migration of cells to the inflammatory focus in the animals injected with the carrageenin irritant. In fish injected with saline and carrageenin occurred the greatest phagocytic activity in the blood in relation to those treatments.


Introduction
The research on the action of probiotic bacteria in aquaculture is due to continued search for alternative strategies to prevent diseases (GOMEZ-GILL et al., 2000).Schrezenmeir and De Vrese (2001) proposed the use of probiotic term to designate products that contain viable microorganisms in adequate amount responsible for Acta Scientiarum.Biological Sciences Maringá, v. 33, n. 3, p. 239-246, 2011 altering the mucosal microbiota by colonizing of an organ of the host with positive effects on their health.
Probiotics especially from lactic-acid bacteria have been used in diet for fish against infections (VERSCHUERE et al., 2000).Among the microorganisms used as probiotic are Bifidobacterium and Lactobacillus (COLLINS et al., 1998;SANDERS;KLAENHAMMER, 2001;VIEIRA et al., 2008).Lactobacillus is considered the most safety organism by Collins et al. (1998) and Lee et al. (1999).The effects of lactic-acid bacteria against furunculosis caused by Aeromonas salmonicida were related by Gildberg and Mikkelsen (1998) in Atlantic cod Gadus morhua and by Nikoskelainen et al. (2001) in rainbow trout Oncorhynchus mykiss infected by vibriosis.
One of the most important primary defense of fish against pathogens is the inflammatory process.According to Martins et al. (2001Martins et al. ( , 2008)), the inflammatory response in cultivated fish, the knowledge of hematological characteristics and phagocytic activity under stressfull stimuli of infections play an important role for diagnosis of fish health.Inflammatory response in Brazilian freshwater fish was evaluated after carrageenin injection in the swim bladder of Nile tilapia (Oreochromis niloticus) (MATUSHIMA; MARIANO, 1996) and in pacu (Piaractus mesopotamicus) (MARTINS et al., 2000(MARTINS et al., , 2001)).On this view, Bozzo et al. (2007) studied the cellular component of the inflammatory response induced by injection of thioglycolate, Escherichia coli lipopolyssacharide and heat-inactivated Aeromonas hydrophila in the swim bladder of pacu.Other studies tested colloidal carbon in Tautogolabrus adspersus (MACKMULL; MICHELS, 1932) lipopolyssacharide (LPS) of Escherichia coli, complete Freund adjuvant (CFA), turpentine and carrageenin (WHITE et al., 1981;JENKINS;KLESIUS, 1998), Edwardsiella ictaluri (HÉRBERT et al., 2000) and peptidoglican (KONO;SAKAI, 2001).
In the studies of Flores-Quintana and Moraes (2001), the acute inflammatory process is composed by metabolic and cellular changes such as an increase in the vascular permeability, white blood cell count and mainly neutrophils and their precursors.Not only the inflammatory exudate analysis (MARTINS et al., 2001), but also the phagocytic activity (Cai et al., 2004) and the glass cover slip implant in subcutaneous tissue (PETRIC et al., 2003) are techniques to verify the magnification of an inflammatory process when fish are exposed to several conditions.Closely to an inflammatory response analysis, is the knowledge of hematological variables that can reveal the fish health and cell behavior in the blood (BLAXHALL, 1972;REHULKA, 2002;MARTINS et al., 2004;2008;GHIRALDELLI et al., 2006).
The evaluation of inflammatory response in fish associated to probiotic supplementation provides assistance to the development of culture methods actually considered as handling practices ecologically correct by improving the disease resistance (GATESOUPE, 1999).
In order to determine the effects of the addition of probiotic bacteria on the inflammatory and hematological response in Nile tilapia, fish were fed diets containing Lactobacillus plantarum isolated from tilapia after carrageenin or saline injection in the swim bladder.
Fish were fed twice a day, and after 15 days injected with carrageenin or saline in the swim bladder for evaluation 6 hours after injection.During this period the water quality was maintained in pH 7.40 ± 2.50, dissolved oxygen 5.09 ± 3.04 mg L -1 and temperature 21.70 ± 2.80ºC.

Preparing the probiotic supplemented diet
The diet was aspersed with Lactobacillus platarum from the MRS culture medium at a concentration of 1 x 10 9 colony forming units (CFU) mL -1 , in the proportion of 100 mL kg -1 dry ration containing 32% crude protein.The mixture was incubated during 24h at 35ºC, and the bags opened for drying for 24h at 35ºC.Unsupplemented diet was submitted to the same procedure except for the MRS aspersion.The isolation of lactic-acid bacteria and the viability of its use followed the procedures of Vieira et al. (2008) and Jatobá et al. (2008).

Injection and collection of exudate cells
After feeding period the animals were anesthetized by immersion in benzocaine (50 mg L -1 ) (protocol approved by Ethic Commitee no.23080.009404/2006-15/CEUA/UFSC)for injection of 500 g carrageenin diluted in 500 μL sterile saline solution 0.65% or only saline according to Martins et al. (2009).After injection, the fish were maintained in the aquaria for 6h until the sacrifice.The interior of the swim bladder was washed with 0.5 mL complete phosphate buffered saline (PBS) containing 0.01 mL EDTA 5%, and cell suspensions were centrifuged at 150 G gravity for 10 min.before staining.With the aid of a Pasteur pipette, the content was collected into centrifuge tubes maintained in ice, diluted to 1:4 and the total number of leucocytes (number L -1 ) counted in Neubauer chamber.Afterwards, the cells were centrifuged at 150 G for 10 minutes, and the supernatant discarded.A drop of serum obtained from the same fish species was added to the precipitate and smears of the exudate cells made for the differential count of macrophages, lymphocytes, granulocytes and thrombocytes.After drying, the smears were fixed with methyl alcohol (three minutes) and stained with Giemsa according to the method proposed by Martins et al. (2001).

Hematological analysis
Six hours after injection the animals were anesthetized and the blood was withdrawn from the caudal vessel into a syringe containing a drop of 10% EDTA solution.The blood was utilized to measure hematocrit (GOLDENFARB et al., 1971); total count of red blood cells (RBC) in a haemocytometer; the total count of white blood cells (WBC), and total number of thrombocytes by indirect method (ISHIKAWA et al., 2008).For differential counting of leucocytes, the smears were stained by Giemsa/May-Grunwald (ROSENFELD, 1947) in which a hundred cells were counted for the establishment of each cell contents.

Phagocytic activity
Leukocyte phagocytic function followed the method of Cai et al. ( 2004) slightly modified as follows: after blood collection, 0.5 mL of blood was dropped into centrifuge tubes, added 0.25 mL of 1 x 10 6 Enterococcus suspension and shaked.The tubes were kept at 28 o C in a water bath for 30 min., and shaken every 10 min.After this time, in order to centrifuge as recommended by Cai et al. (2004), the blood smears were done in duplicates just after incubation, dried, fixed in methylic alcohol and stained by Giemsa/May-Grunwald (ROSENFELD, 1947).The number of leukocytes that engulfed bacteria was counted in percentage in relation to total leukocyte number in the smear from the phagocytosis assay.

Statistical analysis
The animals were randomly distributed among six treatments (supplemented and saline injected fish, un-supplemented and saline injected, supplemented and carrageenin injected, unsupplemented and carrageenin injected, supplemented non-injected, un-supplemented noninjected) with 3 replicates (3 fish per aquarium forming an experimental unit).The averages were compared by the Tukey test, at 5% probability and the percentage from the differential count of the blood and exudate cells were transformed in arc sin ( 0.5 P  ).

Results
Probiotic supplementation did not influence (p > 0.05) on the RBC, hematocrit percentage and the numbers of lymphocytes and basophils in the blood (Tabela 1).But an inflammatory reaction in the swim bladder was noted after carrageenin injection (Figura 1).On the other hand, the addition of probiotic provoked decreased (p < 0.05) WBC count in the blood of non-injected (NI) and in saline injected fish (IS).Moreover, fish fed probiotic and saline injected (IS) showed lower number (p < 0.05) of circulating leukocytes than the other treatments.Glucose levels in probiotic supplemented fish and non-injected (NI) were significantly higher (p < 0.05) than that observed in saline injected (IS) (Table 1).The differential counting of leukocytes in carrageenin injected fish (IC) either supplemented or not with probiotic showed an increase (p < 0.05) in the monocyte number when compared to saline injected ones.Interestingly, the number of neutrophils was significantly higher in probiotic supplemented and carrageenin injected fish (IC).
Regarding the inflammatory response, the probiotic supplementation caused an increase (p < 0.05) in WBC and thrombocytes count after carrageenin injection (IC), as well as in macrophage and lymphocyte number on the inflammatory exudate in relation to unsupplemented fish (IC).It was also observed a significant increase in WBC count in supplemented fish both saline and carrageenin injected.Table 2 shows lower WBC number in the exudate of carrageenin (IC) injected fish than saline injected (IS).The number of macrophages on the inflammatory exudate of unsupplemented fish (IS) was lower (p < 0.05) than that related to the other treatments (Table 2).
Probiotic supplementation in the diet in both saline and carrageenin injected fish increased (p < 0.05) the phagocytic activity as shown in Table 2.

Discussion
Hematological parameters are widely used as an important tool for fish health diagnosis under stressful conditions normally found in intensive culture.Moreover, the analysis of inflammatory exudates may help the comprehension of the inflammatory process in fish.
In contrary to an increase in the number of WBC observed by Selvaraj et al. (2005) in carp (Cyprinus carpio) intraperitoneally injected with beta-glican of Saccharomyces cerevisae the present results did not show difference among saline and carrageenin injected fish.It must be emphasized that differently from that related by Selvaraj et al. (2005), unsupplemented fish injected with both saline and carrageenin did not show significant difference in WBC and monocytes in relation to non-injected fish.Similarly to that reported by Selvaraj et al. (2005), it can be thought that the increased number of monocytes in carrageenin-injected tilapia was due to the fact that these cells are directly involved on phagocytosis of foreign particles or organisms.The lack in leukocyte response in supplemented saline/carrageenin-injected tilapia is in agreement with the findings of Selvaraj et al. (2005) in carp orally fed S. cerevisae.As above supported, the feasible way of probiotic supplementation would be via oral.
Enhanced WBC number in indian carp (Labeo rohita) supplemented with Bacillus subtilis and vitamin C (NAYAK et al., 2007) and in tilapia supplemented with L. plantarum (JATOBÁ et al., 2008) differed from the present result, where WBC decreased in carrageenin injected fish when compared to noninjected.Nevertheless, the decreased WBC number in supplemented fish injected with both saline and carrageenin might be explained by the fact that the probiotic supplementation must have favored the migration of cells to the injected place.Based on this fact, it can be inferred that the reduction in monocytes number in saline and carrageenin injected fish were due to cell migration for phagocytosis.Kumar et al. (2008) studying indian carp supplemented with B. subtilis observed increased number of granulocytes and monocytes but a decrease in lymphocyte number.This assay is in agreement with the results of Kumar et al. (2008) on the neutrophill number in supplemented and carrageenin injected fish.Contrarily to that observed by Kumar et al. (2008) andJatobá et al. (2008), in this work circulating lymphocytes were not affected by probiotic supplementation.By supporting the above statements, it can be concluded that probiotic supplementation has enhanced the fish immune system.
This study showed no influence of the probiotic supplementation on RBC and hematocrit as related by Jatobá et al. (2008).However, experimental conditions, fish species and their genetic varieties could influence on the hematological response.
Greater migration of leukocytes in carrageenin injected tilapia was in agreement with the inflammatory response found by Martins et al. (2001Martins et al. ( , 2004Martins et al. ( , 2006Martins et al. ( , 2008Martins et al. ( , 2009) ) and Bozzo et al. (2007) in tambacu hybrid (Piaractus mesopotamicus male x Colossoma macropomum female) injected with carrageenin and thioglycolate, in tilapia injected with carrageenin and LPS and in pacu (P.mesopotamicus) injected with carrageenin, thioglycolate or inactivated Aeromonas hydrophila.Contrarily to that reported by Matushima and Mariano (1996) in tilapia, in the differential count of cells in the inflammatory exudate in this assay, macrophages and granulocytes were the main cells found followed by lymphocytes and thrombocytes.Despite the fact that pacu injected with LPS (FLORES-QUINTANA; MORAES, 2001), similar inflammatory response characterized either by macrophages or lymphocytes were here observed.Increased number of macrophage and thrombocyte in carrageenin injected tilapia was in agreement with Martins et al. (2006Martins et al. ( , 2008) ) observations.Similarly to the present result, vitamin C and E supplementation improved the migration of thrombocytes to the inflammatory site (BOZZO et al., 2007).Inflammatory infiltrated cells were also related in vitamin C-supplemented Cirrhinus mrigala injected with Freund Complete Adjuvant (FCA) (SOBHANA et al., 2002).On the other hand, chromium supplementation in pacu did not affect the exudate cell component (FLORES-QUINTANA; MORAES, 2001).The present results showed higher number of thrombocytes, macrophages and lymphocytes in the inflammatory exudate that comprove the probiotic action on fish defense system (VERSCHUERE et al., 2000).The evident involvement of thrombocytes as defense cells was also observed in the swim bladder of pacu after different stimuli (BOZZO et al., 2007).
Although it was not related significant difference in migrated granulocytes to the inflammatory site, their number was higher than lymphocytes and thrombocytes.According to Suzuki and Iida (1992) neutrophils migrate more rapidly to an inflammatory site than the macrophages.Greater migration of macrophages followed by granulocytes in this study was in agreement with the observations of Afonso et al. (1998) in peritoneal cavity of rainbow trout (Oncorhynchus mykiss).
Matsuyama and Iida (1999) reported the highest migration of neutrophils to the inflammatory swim bladder exudate in tilapia 24 hs after injection with formalin-killed E. coli.Contrarily to that observed by Matsuyama and Iida (1999) 6 hs after injection was sufficient to migration of granulocytes to the inflammatory site.As also related by Martins et al. (2004) it was not possible to obtain resident population of leukocytes and thrombocytes in unstimulated or non-injected swim bladder.
Phagocytic activity of circulating blood leukocytes of the present assay varied from that previously found by other authors especially in tilapia (MARTINS et al., 2008). Cai et al. (2004) observed 61 and 39% phagocytosis, respectively in Nile tilapia and blue tilapia (Oreochromis aureus).On the other hand, Casas Solis et al. (2007) verified 74.3 to 88.1% phagocytic activity in three species of tilapia and their hybrids.In the present study lower phagocytosis was observed when compared to 56% phagocytosed bacteria from 1 x 10 6 CFU Enterococcus mL -1 treatment found by Martins et al. (2008).In this work, the highest phagocytic activity in probiotic supplemented fish both injected with saline and carrageenin was clearly observed.The difference in phagocytic response depends on the fish species and experimental conditions.In spite of similar water temperature in this assay and of Martins et al. (2008), there was difference in phagocytic activity.
In the studies of Cai et al. (2004) andCasas Solis et al. (2007) fish were maintained at 28 o C. Differently, in this study the water temperature was near to that normally found in Southern Brazil in fish farm.It can be thought that the lower phagocytosis percentage could be related to this fact.This is especially true when high temperature stimulates more efficiently the inflammatory response as reported by Finn and Nielsen (1971).In probiotic supplemented fish injected with saline, a decrease in WBC number was accompanied by an increase in this cell number in the inflammatory exudate.The results suggested the responsivity of tilapia fed L. plantarum under this experimental conditions.Moreover, this work was able to comprove the highest migration of leukocytes and thrombocytes to the inflammatory site.In order to verify the effects of circulating hormones on the inflammatory response, further studies must be carried out with this animal model under stressfull condition.

Conclusion
This work has demonstrated that tilapia fed probiotic L. plantarum in the diet showed more migration of cells to the inflammatory site, the swim bladder than those not supplemented fish.Besides the reduction in the circulating white blood cell count an increase on these cells on the inflammatory exsudate was found in fish supplemented with probiotic.Probiotic supplementation was also responsible for enhanced phagocytic activity in both saline-injected and carragenin-injected fish.

Figure 1 .
Figure 1.Inflammatory response in the swim bladder of Nile tilapia fed probiotic supplemented diet and injected with 500 g carrageenin six hours after injection.

Table 1 .
Hematological characteristics of Nile tilapia supplemented or not with probiotic in the diet, non-injected (NI) and six hours after injection with saline (IS) or carrageenin (IC) in the swim bladder.Different letters indicate significant difference among the treatments (p < 0.05).RBC: red blood cell, WBC: white blood cell.

Table 2 .
Mean values and standard deviation of white blood cells (WBC) and thrombocytes counts and differential count of leukocytes on the inflammatory exudate of Nile tilapia supplemented or not with probiotic in the diet non-injected (NI) and six hours after injection with saline (IS) or carrageenin (IC) in the swim bladder.Different letters indicate significant difference among the treatments (p < 0.05).