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Article

Effect of a Monoglyceride Blend in Nile Tilapia Growth Performance, Immunity, Gut Microbiota, and Resistance to Challenge against Streptoccocosis and Francisellosis

by
Natália Amoroso Ferrari
1,
Raffaella Menegheti Mainardi
1,
Mayza Brandão da Silva
1,
Gabriel Diogo Guimarães
1,
João Vitor Godoy Takashe
1,
Admilton Gonçalves de Oliveira Junior
2,
Ricardo Mitsuo Hayashi
3,
Giovana Wingeter Di Santis
4 and
Ulisses de Pádua Pereira
1,*
1
Laboratory of Fish Bacteriology, Department of Preventive Veterinary Medicine, State University of Londrina, Londrina 86057-970, Brazil
2
Laboratory of Microbial Biotechnology, Department of Microbiology, State University of Londrina, Londrina 86057-970, Brazil
3
SAN Group Biotech, Campinas 13058-009, Brazil
4
Laboratory of Animal Pathology, Department of Preventive Veterinary Medicine, State University of Londrina, Londrina 86057-970, Brazil
*
Author to whom correspondence should be addressed.
Fishes 2024, 9(9), 351; https://doi.org/10.3390/fishes9090351
Submission received: 31 July 2024 / Revised: 28 August 2024 / Accepted: 29 August 2024 / Published: 6 September 2024
(This article belongs to the Special Issue Fish Diseases Diagnostics and Prevention in Aquaculture)
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Abstract

This study evaluated the effects of supplementing the diet of juvenile Nile tilapia (Oreochromis niloticus), which weighs approximately 20 g, with a blend of monoglycerides (glycerides linked to a fatty acid molecule) for 20 days during a pre-experimental challenge via the intraperitoneal route (IP). Growth performance, immunological parameters, intestinal microbiota, tissue damage, and resistance against the pathogens Streptococcus agalactiae serotypes Ib and III and Francisella orientalis were evaluated. The experimental design included a negative control (NC), a product control (NPC), a positive control for each pathogen (PC), and three groups treated with different doses (0.15, 0.25, and 0.5%). After the challenge, mortality was significantly lower in the groups treated and challenged with S. agalactiae. The treated groups showed better weight gain and food conversion rates. Innate immunity parameters showed no differences between treatments, and there was no good stimulation of diversity in the intestinal microbiota. However, in treated groups, there was a reduction in opportunistic bacteria that could cause secondary infections and increased the presence of beneficial bacteria in the intestinal tract. In this way, it is possible to validate the beneficial effects of monoglycerides as a nutritional additive for tilapia farms against streptoccocosis.
Keywords:
Francisella orientalis; nutritional additive; pisciculture; Streptococcus agalactiae
Key Contribution: The use of the monoglyceride blend improves weight gain and feed conversion of Nile tilapia and is highly effective in reducing mortality against the pathogens S. agalactiae serotypes Ib and III.

1. Introduction

Aquaculture is one of the fastest-growing branches of animal production in recent years and an important alternative to reach the food demand as a protein source, making Brazil the fourth major player worldwide [1]. In Brazil alone, the production of farmed fish has exceeded 860 tons, with a growth of 53.25% since 2014 [2]. Nile tilapia is one of the most cultivated species in the world due to its desirable characteristics of hardiness, growth, and resistance [3]. However, the intensification of production to meet global demands, the search for sustainable development with reduced use of antimicrobials, and the scarcity of effective disease control measures are a limiting challenge for production progress [4].
Among the main pathogens, Streptococcus agalactiae, Streptococcus iniae, Lactococcus spp., Francisella orientalis, Aeromonas spp., Edwardsiella spp., and Flavobacterium columnare stand out as causes of outbreaks with mortality in fish farms [5,6,7]. Streptococcosis is more common in summer and affects various fish species, causing exophthalmia, septicemia, and meningoencephalitis [8]. Meanwhile, francisellosis occurs frequently at lower temperatures and is associated with chronic granulomatous inflammation in the spleen, liver, and kidney, as well as in the musculature of adult fish [9].
Antibiotic therapy remains the main treatment strategy employed in the field for the remission of clinical signs [10]. It was first reported in Brazil the isolation of a highly virulent strain of S. agalactiae serotype III causing outbreaks with high mortality in six tilapia farms. Additionally, the strain showed resistance to all aminoglycosides and fluoroquinolones, tetracycline, and ampicillin tested in vitro, being characterized as a multidrug-resistant strain [11]. It is known that the indiscriminate use of antimicrobials and constant exposure of bacteria present in fish microbiota and water, as well as sub-doses, may favor the selection of multidrug-resistant strains, as well as accumulate residues in meat and pose risks to both animal and human health [12,13].
The use of nutritional additives such as probiotics, prebiotics, and organic acids has been studied as a sustainable alternative to stimulate immune response, improve zootechnical growth parameters, and reduce the use of antimicrobials. In previous studies, our team demonstrated that supplementing the diet of juvenile Nile tilapia (Oreochromis niloticus) with a mixture of organic acids improves feed conversion, immune response, and resistance of the fish against experimental challenge with F. orientalis [14]. The same results were observed against S. agalactiae using Enterococcus faecium [15]. These studies demonstrate the great potential of nutritional additives as alternative ways to reduce the use of antimicrobials in aquaculture. In this way, monoglycerides (MGL), esterified adducts of a fatty acid, and a glycerol molecule have shown either bacteriostatic or bactericidal effects against G+ and G- bacteria with immunomodulatory effects. They are pH-independent, less susceptible to enzymatic breakdown, and active through the intestine and systemically. For other species, such as swine, short-chain glycerides significantly reduced the severity of diarrhea in weaned piglets co-infected with Escherichia coli and a trend toward better weight gain and immune system stimulation in treated animals [16]. Also, sea bream (Acanthopagrus latus) fingerlings supplemented with 1% butyric acid glycerides in the diet showed higher growth rates and better immune and antioxidant responses in the liver [17]. These molecules should be explored for use in aquaculture/fish health due to their antimicrobial activity, improvements in zootechnical performance, and emulsifying function.
Thus, this study aimed to evaluate the effect of dietary supplementation with different doses of a monoglyceride blend on growth, immunity, modulation of microbiota, and response against experimental challenge with S. agalactiae serotypes Ib (SAGA Ib) and III (SAGA III) and F. orientalis.

2. Materials and Methods

2.1. Inhibition Tests

2.1.1. Solid Media Inhibition Test

Two solid agar inhibition tests were carried out to verify the potential effect of the product against S. agalactiae and F. orientalis. For the first test, the strains S13 of SAGA Ib and S76 of SAGA III were sown using continuous streaks on Mueller Hinton agar (Kasvi, São José dos Pinhais, PR, Brazil), enriched with 5% sheep blood. The F1 strain of F. orientalis was seeded on cystine heart agar, enriched with 1% bovine hemoglobin (Kasvi). Immediately afterward, a 20 µL drop of the 12.5% solution was placed in the center of the plate. After incubation for 24 to 96 h at 28 °C, the growth inhibition zone of each bacterium around the product was measured.
In addition to the first test, the agar disk diffusion technique was also used. Pure cultures of the same pathogens were tested using the Kirby and Bauer methodology (antibiogram using disks impregnated with different concentrations of the product). For this, sterile filter paper disks were impregnated with 10 μL of different concentrations of the product (5%, 2.5%, 1%, 0.5%, 0.25%, 0.1%, and 0.05%). Then, the plates were incubated for 24–96 h at 28 °C, aerobically. During the reading time, the plates were evaluated for the presence of inhibitory halos, which were measured in millimeters.

2.1.2. Minimum Inhibitory Concentration and Minimum Bactericidal Concentration (MBC)

These tests were performed according to a method adapted from the Clinical Laboratory and Standards Institute [18]. The product in the liquid version (containing 21–28% glycerol) has a concentration of 61–68% of the active ingredient. Therefore, a 5% stock solution was made by diluting 2.5 mL in 47.5 mL of sterile distilled water. Then, previously standardized inoculum was prepared using the same bacterial pathogens. The plates were prepared by adding 50 µL of BHI (S. agalactiae) or Eugon (F. orientalis) broth in columns 1, 2, and 3. Then, 50 µL of the stock solution was added in row 1 of these columns and serially diluted in base 2, continuing up to line H. That way, the 8 product concentrations for the microdilution method (MIC/MBC) were: 0.01%, 0.02%, 0.04%, 0.08%, 0.16%, 0.31%, 0.62%, and 1.25%.
The bacterial inoculums were added in triplicate to the 8 dilutions of the product and incubated for 24 and 48 h at 28 °C to evaluate which concentrations would inhibit the growth of the bacteria. The final concentration of the inoculum was 1 × 106 CFU/mL. Wells without growth visible to the naked eye were seeded to determine the minimum bactericidal concentration of each bacterium.

2.2. Fish

A total of 1400 Nile tilapia (Oreochromis niloticus) weighing approximately 20 g, from a local producer in Londrina, Paraná state, Brazil, were allocated to 28 tanks with a volume of 150 L of water, containing 50 fish each. Initially, 10 animals were euthanized, and their health status was confirmed through bacteriological culture and PCR for detection of Tilapia Lake Virus (TiLV) [19] and Infectious Necrosis of the Kidney and Spleen Virus (ISKNV) [20]. The test was conducted in a closed environment, using dechlorinated water, appropriate levels of temperature (18–28 °C, according to the experimental group), ammonia (<0.4 mg L−1), pH (6.8–7.2), and dissolved oxygen (>4 mg L−1), and a photoperiod of 12:12 h of light and dark. This study was carried out following the Ethical Principles in Animal Research of the National Council for the Control of Animal Experimentation and approved by the State University of Londrina’s Ethics Committee on the Use of Animals (approval number CEUA/UEL-111/2022).

2.3. Diet

The blend of monoglycerides used in this trial is composed of monoproprionin, monobutyrine, monovalerin, monocaproin, monoheptanoin, monocaprilin, mononanoin, monocarpic, and monolaurin (Celtz® GH Aqua 128, SAN Group Biotech Brasil, Campinas, Brazil).
After acclimatization of the animals receiving the basal diet (commercial feed), the treatment with the product began, lasting 20 days before experimental infection. The animals were fed 3 times a day, with feed consumption set at 3% of biomass. The product was supplied and incorporated into the feed in the laboratory at concentrations of 0.15%, 0.25%, and 0.5%. Then, the feed was covered with a layer of universal vehicle (Vansil) based on carboxymethylcellulose to waterproof the grain and help fix the product. The control groups received only the basal diet, and the NPC group was fed the highest dose of the product.

2.4. Experimental Design and Challenge

The experiment had five control groups and nine experimental groups, all in duplicate, as outlined in Table 1.
Groups G1 to G9, which received the product before the experimental infection, continued to receive the same dosage of the product after the experimental infection until the end of the experiment. The same is valid for the NPC, PC, and NC groups, which received the same diet throughout the experiment.
Strains S13 (SAGA Ib) and S76 (SAGA III) of S. agalactiae and F1 of Francisella orientalis from the bacteria bank of the Fish Bacteriology Laboratory at UEL were used in the experimental challenge. The pathogen doses used were: 1 × 107 CFU/fish for F1, 4 × 106 CFU/fish for S13, and 2 × 107 CFU/fish for S76. These dosages are sufficient to cause the death of more than 60% of healthy fish not treated with any product (as already demonstrated by previous studies in our laboratory, a previous experiment with a lethal dose of 50%). The challenge was carried out intraperitoneally, with the inoculation of 100 μL/fish after anesthetizing the animals in a bath containing 100 mg L−1 of benzocaine. All 50 fish contained in each tank were challenged, according to the experimental group to which they belonged. The non-parametric Fisher’s exact test was performed for each treated group concerning its respective positive control group.

2.5. Growth Performance

Fish were counted and weighed at the beginning of this study and before the challenge to evaluate growth performance, which was calculated using the following formulas: weight gain (WG) = final weight (g)—initial weight (g); daily diet consumption = (total feed consumed/final number of individuals)/duration of the experiment; specific growth rate (SGR%) = 100 × (final weight—initial weight)/duration of the experiment; feed conversion rate (FCR) = feed given/weight gain [21]. As weighing is a brief procedure, the animals were not anesthetized.

2.6. Measured Immune Parameters

For these analyses, after 7 days of experimental infection, the fish in each treatment were sedated with 50 mg/mL benzocaine (previously diluted in 1 mL of alcohol and subsequently diluted in water) for blood collection by puncturing the tail vein to obtain serum. Three serum samples were collected from each treatment using one to three animals, varying according to the requirement of 1 mL of blood/sample.

2.6.1. Serum Lysozyme Concentration

Serum lysozyme concentration was determined according to Demers and Bayne [22], with a method based on the lysis of the Gram-positive bacterium Micrococcus lysodeikticus (M3770, Sigma-Aldrich, St. Louis, MA, USA). Standard solutions (0–10 ng µL−1) of chicken egg lysozyme (L6876, Sigma-Aldrich) were prepared at the time of analysis to obtain a standard curve. The absorbance reading was performed on a spectrophotometer at 492 nm, at 0 and 80 s. The results were expressed using the optical density variation values for each sample volume versus the lysozyme volume from the standard curve. The linear regression equation of the lysozyme standard curve was used to determine serum lysozyme levels (ng μL−1). Data were compared separately for each pathogen, along with the NC and NPC groups. The Shapiro–Wilk test was used to verify the normality of the data, which did not present a normal distribution. The Kruskal–Wallis non-parametric test was then performed, and the confidence interval considered was 95%.

2.6.2. Complement System Hemolytic Activity

This test was determined according to the methodology carried out in the work of Kumari and Sahoo [23]. The reading was performed on a plate reader at 450 nm. The results were performed by linear regression and expressed as µL needed to lyse 50% of the red blood cells (AC50). The Shapiro–Wilk test was also used to verify the normality of the data and the Kruskal–Wallis test for non-parametric data for statistical comparison based on the median of the data.

2.6.3. Antibacterial Activity of the Serum

The antibacterial or bactericidal activity of the serum was performed according to Silva et al. [24]. The test was carried out using the bacteria Aeromonas hydrophyla “REF 11”, Escherichia coli ATCC 25922, and the pathogens used for this study. The plates were sealed and incubated at 28 °C in orbital rotation at 80 RPM for 24 h, and the reading was performed visually and corresponds to the titer of the last dilution without bacterial growth.

2.7. Tissues Histology

For histopathological evaluation, animals were sampled (1 fish from each group/treatment, alternating between replicates, every 7 days after infection). As the results of the in vivo test with F. orientalis were not promising, only samples from groups challenged with S. agalactiae were selected for this analysis. Briefly, the proximal intestine, spleen, liver, eye, and brain were collected in a buffered 10% formaldehyde solution for 48 h and subsequently remained in 70% alcohol until macroscopic cleavage. Then, fragments were placed in histological cassettes kept in 70% alcohol and subjected to dehydration, clarification, and paraffinization in an automatic tissue processor using progressive baths of alcohol, xylene, and liquid paraffin, respectively. The samples were embedded in paraffin for subsequent cleavage using a 5 µm thick microtome. Finally, the slides were individually evaluated by optical microscopy [25]. For each organ, characteristic lesions, inflammatory, and apoptotic/necrosis processes were evaluated.

2.8. Fecal Metagenomic Analysis

For metagenomic analysis, four animals from each group were euthanized by an overdose of benzocaine and had their intestinal contents (feces) collected aseptically and under refrigeration 7 days after the experimental challenge. As the results of the in vivo test with F. orientalis were not promising, only samples from groups challenged with S. agalactiae were selected for this analysis. The commercial kit EZNA stool kit DNA (Omega Bio-Tek, Norcross, GA, USA) was used for the total DNA extraction, according to the manufacturer’s instructions. Next, the V4 region of the 16S ribosome subunit gene [26] was amplified with primers containing an overlapping region with primers from the Illumina platform. Chao’s species richness analysis and Shannon’s diversity index were used to analyze the results, which were compared using Dunn’s test [27]. A p-value of 0.05 was adopted for all statistical tests performed.

3. Results

3.1. Inhibition Bacterial Growth

The inhibition halos of the product observed in the solid media tests using SAGA Ib, SAGA III, and F. orientalis were 10 mm, 12 mm, and 34 mm, respectively. In the second test using different product concentrations, there was no observation of an inhibition halo for S. agalactiae. For F. orientalis, the concentrations 0.5%, 2.5%, and 1% presented a halo of 18, 13, and 10 mm, respectively. About MIC and MBC, Celtz GH Aqua 128 was able to inhibit the growth of the four pathogens tested. All concentrations were able to inhibit the growth of F. orientalis. It was also possible to determine the bactericidal concentration of S. agalactiae. All concentrations tested were bactericidal for F. orientalis. These results are demonstrated in Table 2.

3.2. Disease Challenge

For SAGA Ib and SAGA III, mortality was 62% and 98% for the positive control group, respectively, and much lower for the treated groups. There was a statistical difference (p < 0.01 Fisher’s exact test) between all treated groups and the positive control group, demonstrating a good efficacy of the product (Figure 1 and Figure 2). As for the pathogen Francisella orientalis, mortality was 100% for the positive control group and very similar for the treated groups, being 98%, 100%, and 100% for dosages 0.15, 0.25, and 0.5 of the product, respectively. In the end of the trial, there was no statistical difference (p > 0.05 Fisher’s exact test) between the treated groups and the positive control group, demonstrating that experimentally the product showed no significant efficacy against francisellosis (Figure 3). The standard deviation can be found in Supplementary Table S1.

3.3. Growth Performance

The treated animals showed statistically significant improvements in weight gain and feed conversion parameters. These groups demonstrated higher averages for weight gain and lower averages for feed conversion when compared to the untreated group (Table 3).

3.4. Innate Immune Analysis

The results obtained in this analysis are shown in Table 4. None of the experimental groups demonstrated significant changes in tilapia serum immunity tests. Comparing the AC50, the higher the value, the lower the complement concentration to lyse 50% of the red blood cells. The results obtained in the test using the strain of Aeromonas hydrophila “REF 11” are represented in Table 4. The tests with the other strains resulted in titer 4. Therefore, there was no variation in the titer obtained for the groups, so there were no statistical differences.

3.5. Histopathological Analysis

In the intestines of the animals, it was possible to observe an increase in the width of the lamina propria and an increased number of caliciform cells and intraepithelial lymphocytes in the PC SIb and PC SIII groups. These changes were observed in lower severity in the treated groups, suggesting a reduction in intestinal damage and preserving intestinal morphology (Figure 4).
In the spleen, moderate to accentuated increases in periarteriolar macrophage sheaths were observed in the PC SIb and PC SIII groups, as well as marked congestion. The treated groups had moderate changes in the organ, which may suggest a positive effect of MGL on the fish’s immune system. The liver showed a difference between the two infections: for SAGA Ib, there was an accentuated reduction in glycogen accumulation in the first collection, with moderate to accentuated inflammation, mild hepatocellular necrosis, and the presence of pigmented macrophages. In the treated groups, there was a marked reduction in glycogen accumulation only in the 0.25 treatment and in the 0.15 treatment in the third collection, with the other changes varying between discrete and moderate. In animals challenged with SAGA III, the PC showed accentuated inflammation with pigmented macrophages in the first collection, progressing to a moderate reduction in glycogen accumulation in the second collection (Figure S1).
The eye was the place where the greatest effect of the treatment could be seen (Figure 4). For SAGA Ib, the PC and 0.15 groups showed marked inflammation with extensive involvement of the ocular structures in the first collection, while the changes in lesion levels in the 0.25 and 0.5 groups varied between moderate and discrete. In SAGA III, lesions were gradually reduced in the treated group according to the date of collection, which did not occur in the PC. Small differences were observed in the brain of the treated group challenged with SAGA Ib compared to the positive control. The main lesion observed was meningitis (Figure 4), with possible ventriculitis and encephalitis.

3.6. Metagenomic Analysis

A total of 1,404,609 sequences were generated, of which 1,019,649 were used after quality control. There was no significant difference between groups in Chao’s species richness analysis. There was a significant difference in the Shannon diversity index, where the groups with the greatest diversity for serotype Ib of S. agalactiae were PC SIb, 0.25 SIb, and 0.25 SIII. For SAGA III, the groups with the greatest diversity were 0.25 SIII, PC SIII, and NPC (Table 5). The abundance of each bacterial general is shown in Figure 5 and Figure 6.

4. Discussion

Regarding the results obtained in the inhibition tests, it is worth highlighting that, when comparing the agar inhibition tests and the MIC, the solid culture media used present greater nutrient availability compared to the BHI and Eugon broths, which favors the growth of bacteria. Therefore, we speculate that this is the reason why the product did not inhibit the growth of Gram-positive bacteria at concentrations greater than the MIC. Furthermore, organic acids are generally more effective against Gram-negative bacteria [28]. Our result with F. orientalis is in agreement with a previous study, in which the MIC for a blend of organic acids (caprylic acid and capric acid 10%; propionic, sorbic, and formic acid 27%; and silicic acid 63%) against the same bacteria was 0,625 μg mL−1 [14]. The same author also states that this result was expected since facultative intracellular bacteria, such as Francisella spp., tend to have a low value for antimicrobial tests. Another study using organic acids (5% maltodextrin, 1% sodium chloride, 42% citric acid, 18% sodium citrate, 10% silica, 12% malic acid, 9% citrus extract, and 3% olive extract) against S. agalactiae demonstrated a MIC of 0.125% and MBC of 0.50%, values very close to those described in our study [29].
Although the in vitro results were more promising for F. orientalis, the product was not effective in in vivo tests. Treatment of francisellosis is generally difficult, even with antimicrobials, since one of the first clinical signs observed in sick fish is hyporexia/anorexia, and any medication offered orally will have its intake reduced [30]. Furthermore, the bacteria’s intracellular lifestyle can weaken functions such as bacterial death, antigen presentation, and the ability to replicate in macrophages, also affecting the fish’s adaptive immune response [31]. Therefore, even when using dietary supplements to prevent bacterial infections and reduce the use of antimicrobials, there are many immune system escape mechanisms that enable an effective infection in the host. Regarding the challenge with S. agalactiae, the results obtained in this study were very promising in reducing the mortality of animals challenged for both serotypes.
In addition, our results demonstrated improvements in the zootechnical parameters of the treated fish. Similarly, research using butyric acid glycerides (BAG) in the diet of Acanthopagru latus fry also demonstrated better growth and feed conversion in the group supplemented with 1% BAG [17]. Another study with juveniles of the same species fed a diet rich in soybean meal and supplemented with butyrate glycerides demonstrated better growth performance, better integrity and morphology of intestinal cells, and increased antioxidant capacity [32]. Physiologically, acidification of the pH of the digestive tract improves the performance of digestive enzymes and the digestibility of nutrients, which contributes to better zootechnic performance [33]. Despite this, it is worth noting that the effect promoted by supplementation with organic acids may vary according to the fish species, type of acid, dosage, and administration time [14].
In the immunity tests, our results were not promising. The adaptive immunity of fish is not highly developed. Thus, innate immunity mechanisms are the primary barrier against pathogens [14]. Lysozyme is involved in the disruption of the bacterial cell wall [34]. The complement system, in turn, acts in the primary defense against infections [35]. Other studies with different fish species have demonstrated elevated enzyme levels and complement system activity in animals treated with high doses of organic acids [36,37], as well as increased blood cell counts, immunoglobulins, and complement system activity in the skin mucosa of treated tilapia with butyric acid glycerides [17]. Da Silva [14] also found no significant differences in the stimulation of the complement system of fish treated with organic acids and challenged with F. orientalis, despite reporting differences in lysozyme levels and serum antimicrobial activity. Suphoronski [30] also did not observe significant differences in the stimulation of the immune system of fish treated with a phytogenic agent associated with potassium diformate against F. orientalis, and, although our results do not demonstrate significant differences between the groups, Reda [38] suggested that high doses are necessary to maintain an improved immune system.
The action of MGL also suggests a reduction in lesions in animal tissues; however, new data with many samples could reinforce this suggestion. The objective of the histopathological analysis in this study is only to illustrate the lesions presented by the animals, and it was not used as an argument to validate the MGL effect.
In the liver, the reduction in glycogen accumulation in sick fish has been related to hyporexia. Inflammation is more related to the systemic infection of the disease [39]. Few lesions were found in the eyes of treated animals, compared to the PC groups. A septicemia is caused by the bacteria, as observed in both experimental and natural infections [40]. These findings may suggest an indirect action of the product in the stimulation of the immune system that blocked the adhesion and entrance of bacteria in the eye cells. A study using zebrafish (Danio rerio) demonstrated that innate immune responses directly contribute to the survival of ocular cells in the face of injury through the recruitment of macrophages to block inflammation [41]. In the brain, it can be observed that the product helps to delay the development of infection for SAGA III at all doses. This difference may be related to the virulence of the strains, as even different strains of the same serotype may have different virulence [42].
In the microbiota analysis, the predominant bacterial genus was Cetobacterium, an event that has already been seen in different species of fish such as Arapaima gigas and Cyprinus carpio, as well as in other experiments with Oreochromis niloticus [15,43,44]. They are aerotolerant anaerobic bacteria capable of producing a wide range of metabolites, such as vitamin B12, which contribute to the host’s biological processes or act in modulating the immune system. Furthermore, they can play a defense role against pathogens, hindering colonization, producing antimicrobial substances, and competing for nutrients and adhesion sites [45]. In this study, it was possible to observe a significant increase in the abundance of Cetobacterium in the groups supplemented with the highest dose of the product (0.5%) in both serotypes of S. agalactiae. Furthermore, even though there was no increase in the abundance of other dosages, no significant drop was observed, which has already been correlated as an indication of nutritional stress due to changes in eating habits [46]. According to the work of Qi X and team [47], the abundance of Cetobacterium is inversely proportional to the presence of Bacterioides, which may have contributed to the reduction in pathogenic bacteria.
Another group in which it was possible to observe a tendency towards a decrease in abundance in animals challenged with SAGA III was the order Bacteroidales. Conversely, its abundance increased when comparing healthy animals that did not receive the product with animals that received the product (NC vsNPC). Although there is no statistically significant difference between the groups, this observation may indicate that the product also contributes to the development of these bacteria. For serotype Ib, there was a significant difference between the PC group and the 0.25% and 0.5% treatments. Bacteria from the order Bacteroidales assist in the degradation and digestion of organic components and are commonly present in the intestinal microbiome and aquatic environments. However, as this is a very large and heterogeneous group, more studies are needed to understand the role of the product in the dynamics of these bacteria. Another significant increase in the groups that did not undergo experimental challenge and were treated with the product (NPC) were Porphyromonadacea and Burkholderiales, symbiotic bacteria that also assist in the metabolism and digestion of complex carbohydrates. Likewise, more studies are also needed to elucidate these relationships.
About pathogenic bacteria, it was possible to observe the product’s action reducing the abundance of the genus Aeromonas. These bacteria are part of the microbiota of the fish gastrointestinal tract. Under conditions of stress or population imbalance, they can produce various toxins that are harmful to their hosts, becoming opportunistic and thus causing mortality in many fish. Therefore, infections caused by the group are considered one of the most harmful to the aquaculture sector and cause great harm to Nile tilapia farmers [48]. In this study, there was a significant difference between the positive control and all groups treated with product doses in the challenge with SAGA Ib. The NPC group also showed a significant decrease in relation to the NC, which reinforces the effect of the product as well in healthy fish. Something similar was observed in SAGA III, when the highest dosage of the product was used.
However, there was no apparent effect for the 0.15% and 0.25% doses. Even so, the product was able to positively modulate the microbiota of Nile tilapia, increasing or preserving the population of beneficial bacteria and reducing pathogenic bacteria that are harmful to the host’s health, reinforcing the indication for the use of the product.

5. Conclusions

Through the analyses carried out in this study, it can be concluded that the monoglycerides blend was effective against Francisella orientalis and S. agalactiae serotypes Ib and III in in vitro tests. It also presented relevant results in improving weight gain and feed conversion of fish and proved to be highly effective in reducing mortality against the pathogens S. agalactiae serotypes Ib and III. Finally, despite not having improved the stimulation of the fish’s immune system and not promoting greater intestinal diversity, the product was effective in reducing opportunistic bacteria that could cause secondary infections in sick animals, in addition to increasing the presence of bacteria beneficial for the intestinal tract, such as Cetobacterium, Bacteriodales, and Porphyromonadacea. Therefore, monoglycerides are a promising alternative for use as a nutritional additive in the prevention of streptococcosis in Nile tilapia. Future studies can explore its interaction with other molecules or for a longer period to reduce the use of antimicrobials and promote sustainability in fish farms, which directly impact human health.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/fishes9090351/s1. Figure S1: Microscopy of the hepatopancreas of Nile tilapia subjected to different treatments with monoglycerides blend after experimental challenge. Table S1: Standard deviation of the mortality curve of fish treated with different dosages of monoglycerides blended and challenged with Streptococcus agalactiae or Francisella orientalis.

Author Contributions

Conceptualization, N.A.F. and U.d.P.P.; methodology, N.A.F., G.W.D.S., R.M.M. and J.V.G.T.; formal analysis, N.A.F.; investigation, N.A.F., R.M.M. and J.V.G.T.; writing—original draft preparation, N.A.F.; writing—review and editing, U.d.P.P., R.M.H., G.W.D.S., M.B.d.S. and G.D.G.; visualization, A.G.d.O.J., R.M.H. and U.d.P.P.; supervision, U.d.P.P.; project administration, U.d.P.P. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Council for Scientific and Technological Development (CNPq), grant number 306857/2021-9, and Coordination of Superior Level Staff Improvement (CAPES), Brazil, 88887.835189/2023-00.

Institutional Review Board Statement

The animal study protocol was approved by the Ethics Committee on Animal Use of the State University of Londrina (approval number CEUA-UEL 111/2022).

Data Availability Statement

Data are contained within the article and Supplementary Materials.

Acknowledgments

The authors would like to acknowledge the Postgraduate Program in Animal Health and Production Science for its help and support in the research presented in this manuscript. The authors thank the following Brazilian institutes for financial support: the National Council of Technological and Scientific Development (CNPq) and Coordination of Superior Level Staff Improvement (CAPES). Pereira, U.d.P.P., is a recipient of the CNPq Fellowship, and Ferrari, N.A.F., is a recipient of the CAPES Fellowship. The authors would like to thank SAN Group Biotech for providing the inputs.

Conflicts of Interest

Author Ricardo Mitsuo Hayashi was employed by the company SAN Group Biotech, Campinas, Brazil. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Figure 1. Accumulated mortality curve of different doses of MGL product in fish experimentally challenged with S. agalactiae serotype Ib (SIb). NC: negative control, without challenge and product; NPC: negative control for the pathogen challenge that received the product in the feed; PC: positive control for the pathogen challenge. * After day 7, all treated groups showed a significant difference in the CP group (p < 0.05).
Figure 1. Accumulated mortality curve of different doses of MGL product in fish experimentally challenged with S. agalactiae serotype Ib (SIb). NC: negative control, without challenge and product; NPC: negative control for the pathogen challenge that received the product in the feed; PC: positive control for the pathogen challenge. * After day 7, all treated groups showed a significant difference in the CP group (p < 0.05).
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Figure 2. Accumulated mortality curve of different doses of the MGL product in fish experimentally challenged with S. agalactiae serotype III (SIII). NC: negative control, without challenge and product; NPC: negative control for the pathogen challenge that received the product in the feed; PC: positive control for the pathogen challenge. * After day 2, all treated groups showed a significant difference in the CP group (p < 0.05).
Figure 2. Accumulated mortality curve of different doses of the MGL product in fish experimentally challenged with S. agalactiae serotype III (SIII). NC: negative control, without challenge and product; NPC: negative control for the pathogen challenge that received the product in the feed; PC: positive control for the pathogen challenge. * After day 2, all treated groups showed a significant difference in the CP group (p < 0.05).
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Figure 3. Accumulated mortality curve of different doses of the MGL product in fish experimentally challenged with Francisella orientalis (F). NC: negative control, without challenge and product; NPC: negative control for the pathogen challenge that received the product in the feed; PC: positive control for the pathogen challenge. * After day 12, none of the treated groups showed a significant difference in the CP group (p > 0.05). Between days 5 and 9, treated groups showed a significant difference in the CP group (p < 0.05).
Figure 3. Accumulated mortality curve of different doses of the MGL product in fish experimentally challenged with Francisella orientalis (F). NC: negative control, without challenge and product; NPC: negative control for the pathogen challenge that received the product in the feed; PC: positive control for the pathogen challenge. * After day 12, none of the treated groups showed a significant difference in the CP group (p > 0.05). Between days 5 and 9, treated groups showed a significant difference in the CP group (p < 0.05).
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Figure 4. Microscopy of tissues of Nile tilapia (hematoxylin and eosin stain–H&E), subjected to different treatments with monoglyceride blends, and after experimental challenge; (A) (20x) normal lamina propria width, composed of cells with a thin and delicate nucleus in the NPC group; (B) (20x) increased width of the lamina propria, with an increased number of caliciform cells and intraepithelial lymphocytes in the PC SIb group. The double-direction arrow points the lamina propria width. The arrowhead points a blunt tip of intestinal villi. The circle shows intraepithelial lymphocytes; (C) (20x) absence of inflammation in the optical nerve in the first collection of the group treated with 0.15% of MGL; (D) (20x) accentuated inflammation in the optic nerve in the PC group. The arrow points to the inflammatory cells; (E) (4x) absence of parenchymal brain inflammation and meninges with normal thickness in the group were treated with 0.50% of MGL in the second collection after challenge with Streptococcus agalactiae serotype Ib; (F) (10x) accentuated meningitis and encephalitis, infiltrate of lymphocytes and macrophages in the group were treated with 0.25% of MGL in the third collection after the challenge with Streptococcus agalactiae serotype Ib. The star points to the meninge area, and the double-direction arrows delimit its thickening. The diamond indicates the cerebral cortex.
Figure 4. Microscopy of tissues of Nile tilapia (hematoxylin and eosin stain–H&E), subjected to different treatments with monoglyceride blends, and after experimental challenge; (A) (20x) normal lamina propria width, composed of cells with a thin and delicate nucleus in the NPC group; (B) (20x) increased width of the lamina propria, with an increased number of caliciform cells and intraepithelial lymphocytes in the PC SIb group. The double-direction arrow points the lamina propria width. The arrowhead points a blunt tip of intestinal villi. The circle shows intraepithelial lymphocytes; (C) (20x) absence of inflammation in the optical nerve in the first collection of the group treated with 0.15% of MGL; (D) (20x) accentuated inflammation in the optic nerve in the PC group. The arrow points to the inflammatory cells; (E) (4x) absence of parenchymal brain inflammation and meninges with normal thickness in the group were treated with 0.50% of MGL in the second collection after challenge with Streptococcus agalactiae serotype Ib; (F) (10x) accentuated meningitis and encephalitis, infiltrate of lymphocytes and macrophages in the group were treated with 0.25% of MGL in the third collection after the challenge with Streptococcus agalactiae serotype Ib. The star points to the meninge area, and the double-direction arrows delimit its thickening. The diamond indicates the cerebral cortex.
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Figure 5. Relative abundance of fecal microbiota (genus) of Nile tilapia treated with different dosages of MGL and challenged with Streptococcus agalactiae serotype Ib (SIb). NC: negative control, without challenge and product; NPC: negative control for the pathogen challenge that received the product in the feed; PC: positive control for the pathogen challenge.
Figure 5. Relative abundance of fecal microbiota (genus) of Nile tilapia treated with different dosages of MGL and challenged with Streptococcus agalactiae serotype Ib (SIb). NC: negative control, without challenge and product; NPC: negative control for the pathogen challenge that received the product in the feed; PC: positive control for the pathogen challenge.
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Figure 6. Relative abundance of fecal microbiota (genus) of Nile tilapia treated with different dosages of MGL and challenged with Streptococcus agalactiae serotype III (SIII). NC: negative control, without challenge and product; NPC: negative control for the pathogen challenge that received the product in the feed; PC: positive control for the pathogen challenge.
Figure 6. Relative abundance of fecal microbiota (genus) of Nile tilapia treated with different dosages of MGL and challenged with Streptococcus agalactiae serotype III (SIII). NC: negative control, without challenge and product; NPC: negative control for the pathogen challenge that received the product in the feed; PC: positive control for the pathogen challenge.
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Table 1. Experimental design of groups with Nile tilapia treated with different doses of MGL and challenged with Streptococcus agalactiae and Francisella orientalis.
Table 1. Experimental design of groups with Nile tilapia treated with different doses of MGL and challenged with Streptococcus agalactiae and Francisella orientalis.
GroupMGL Dose (%)ChallengeGroupMGL Dose (%)Challenge
SAGA Ib PC0Streptococcus agalactiae
serotype Ib		SAGA III 0.25%
(G5)		0.25Streptococcus agalactiae
serotype III
SAGA Ib 0.15%
(G1)		0.15Streptococcus agalactiae
serotype Ib		SAGA III 0.50%
(G6)		0.5Streptococcus agalactiae
serotype III
SAGA Ib 0.25%
(G2)		0.25Streptococcus agalactiae
serotype Ib		NPC0.5No
SAGA Ib 0.50%
(G3)		0.50Streptococcus agalactiae
serotype Ib		FRAN PC0.0Francisella orientalis
NC0NoFRAN 0.15%
(G7)		0.15Francisella orientalis
SAGA III PC0Streptococcus agalactiae
serotype III		FRAN 0.25%
(G8)		0.25Francisella orientalis
SAGA III 0.15%
(G4)		0.15Streptococcus agalactiae
serotype III		FRAN 0.50%
(G9)		0.50Francisella orientalis
NC: negative control, without challenge and product; NPC: negative control for the challenge and positive control for the product; PC: positive control for the challenge.
Table 2. Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC) of MGL against S. agalactiae and F. orientalis.
Table 2. Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC) of MGL against S. agalactiae and F. orientalis.
BacteriaMIC (Product %)MBC (Product %)
Streptococcus agalactiae Ib0.160.62
Streptococcus agalactiae III0.310.62
Francisella orientalis<0.01<0.01
Table 3. Zootechnical parameters of fish that received MGL in three different dosages for 20 days.
Table 3. Zootechnical parameters of fish that received MGL in three different dosages for 20 days.
Treatment (% of Product)Weight Gain/Day/Fish (g)Feed Rate Conversion (FRC)Specific Growth Rate (SGR)
MeanStandard DeviationANOVAMeanStandard DeviationANOVAMeanStandard DeviationANOVA
NC0.9488 a0.0458p = 0.001771.299 a0.0667p = 0.00223.14820.167p = 0.57283
0.151.0548 b0.03831.1191 b0.06583.18940.111
0.251.0453 b0.06691.1295 b0.06853.26280.2323
0.51.0652 b0.07031.1418 b0.04853.27980.2644
Different lettes indicates statistic differences between groups.
Table 4. Innate immunity analysis of lysozyme concentration, complement system concentration, and antimicrobial activity present in the serum of fish treated and not treated with different concentrations of MGL.
Table 4. Innate immunity analysis of lysozyme concentration, complement system concentration, and antimicrobial activity present in the serum of fish treated and not treated with different concentrations of MGL.
GroupLysozyme AC50 (µL)AASGroupLysozyme AC50 (µL)AAS
(µg/mL)(Title)(µg/mL)(Title)
SAGA Ib PC20.8524.354SAGA III 0.25%22.0717.224
SAGA Ib 0.15%20.4022.274.3SAGA III 0.50%27.6818.974
SAGA Ib 0.25%21.3129.544NPC17.8217.524
SAGA Ib 0.50%27.8327.434FRAN PC15.0927.024.3
NC17.3721.334FRAN 0.15%16.3123.394
SAGA III PC21.0123.064.3FRAN 0.25%23.4321.374
SAGA III 0.15%13.7323.274FRAN 0.50%14.7927.494
There is no statistical difference between the groups all tests (p-value > 0.05). NC: negative control, without challenge and product; NPC: negative control for the challenge and positive control for the product; PC: positive control for the challenge.
Table 5. Shanon diversity index of Nile Tilapia challenged with Streptococcus agalactiae serotypes Ib and III and supplemented with different dosages of monoglycerides blend.
Table 5. Shanon diversity index of Nile Tilapia challenged with Streptococcus agalactiae serotypes Ib and III and supplemented with different dosages of monoglycerides blend.
GroupShanonGroupShanon
0.15 SIb1.29 a0.15 SIII0.85 ab
0.25 SIb1.35 a0.25 SIII1.40 a
0.50 SIb0.30 b0.50 SIII0.66 ab
PC SIb1.66 aPC SIII1.26 a
NC0.75 abNPC1.06 ab
Different letters in this table indicate statistical differences (p < 0.05) in the Dunn test. NC: negative control, without challenge and product; NPC: negative control for the pathogen challenge that received the product in the feed; PC: positive control for the pathogen challenge.
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Ferrari, N.A.; Mainardi, R.M.; Silva, M.B.d.; Guimarães, G.D.; Takashe, J.V.G.; de Oliveira Junior, A.G.; Hayashi, R.M.; Di Santis, G.W.; Pereira, U.d.P. Effect of a Monoglyceride Blend in Nile Tilapia Growth Performance, Immunity, Gut Microbiota, and Resistance to Challenge against Streptoccocosis and Francisellosis. Fishes 2024, 9, 351. https://doi.org/10.3390/fishes9090351

AMA Style

Ferrari NA, Mainardi RM, Silva MBd, Guimarães GD, Takashe JVG, de Oliveira Junior AG, Hayashi RM, Di Santis GW, Pereira UdP. Effect of a Monoglyceride Blend in Nile Tilapia Growth Performance, Immunity, Gut Microbiota, and Resistance to Challenge against Streptoccocosis and Francisellosis. Fishes. 2024; 9(9):351. https://doi.org/10.3390/fishes9090351

Chicago/Turabian Style

Ferrari, Natália Amoroso, Raffaella Menegheti Mainardi, Mayza Brandão da Silva, Gabriel Diogo Guimarães, João Vitor Godoy Takashe, Admilton Gonçalves de Oliveira Junior, Ricardo Mitsuo Hayashi, Giovana Wingeter Di Santis, and Ulisses de Pádua Pereira. 2024. "Effect of a Monoglyceride Blend in Nile Tilapia Growth Performance, Immunity, Gut Microbiota, and Resistance to Challenge against Streptoccocosis and Francisellosis" Fishes 9, no. 9: 351. https://doi.org/10.3390/fishes9090351

APA Style

Ferrari, N. A., Mainardi, R. M., Silva, M. B. d., Guimarães, G. D., Takashe, J. V. G., de Oliveira Junior, A. G., Hayashi, R. M., Di Santis, G. W., & Pereira, U. d. P. (2024). Effect of a Monoglyceride Blend in Nile Tilapia Growth Performance, Immunity, Gut Microbiota, and Resistance to Challenge against Streptoccocosis and Francisellosis. Fishes, 9(9), 351. https://doi.org/10.3390/fishes9090351

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