Saccharomyces Boulardii and Saccharomyces Cerevisiae Improve Immunity in Broilers Vaccinated Against Pasteurella Multocida and Salmonella Gallinarum

ABSTRACT

This study evaluated the impact of dietary Saccharomyces boulardii and Saccharomyces cerevisiae on the immunity of birds vaccinated against Pasteurella multocida and Salmonella gallinarum. A total of 105 male Cobb 500 broilers were divided into four groups: T1 (vaccinated, no supplement, n = 30), T2 (vaccinated, S. boulardii supplement, n = 30), T3 (vaccinated, S. cerevisiae supplement, n = 30), and T4 (non-vaccinated, no supplement, n = 15). Chickens received a corn-soy diet with 1x10⁷ CFU/g of S. boulardii or S. cerevisiae for 42 days. Immune response was assessed by indirect ELISA and leukocyte counts. At 21 days, both supplemented groups showed significantly higher IgY levels than the vaccinated control (p < 0.05). S. boulardii supplementation increased lymphocytes (p = 0.003) and decreased heterophils (p=0.004), while S. cerevisiae had no significant effect. The heterophil/lymphocyte ratio decreased by respectively 23.4% and 32.8% in the S. cerevisiae and S. boulardii groups at 42 days, with no changes at 21 days. These results indicate that S. boulardii and S. cerevisiae can boost immunity and overall health in broilers.

Keywords:
Broilers; immune response; probiotic supplementation; Saccharomyces boulardii ; Saccharomyces cerevisiae

INTRODUCTION

The ban on antibiotic growth promoters and other antimicrobials in poultry production due to rising concerns over antimicrobial resistance has led to increased use of alternatives, like probiotics (Larsberg et al., 2023).The most appropriate use of the term probiotic is “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host” (Hill et al., 2014). However, in a more contemporary sense, probiotics can be described as microorganisms with beneficial effects for humans and animals (FAO/WHO, 2001), capable of colonizing the intestine and competing with pathogenic bacteria, thereby improving health, immunity, and animal welfare (Chávez et al., 2016; Shojadoost et al., 2022; Jan et al., 2023).

The broiler chicken production system is highly intensive, characterized by high density within aviaries, capable of housing thousands of birds. This scenario highlights the importance and priority of disease control and prevention (Landoni & Albarellos, 2015). The immunity of chickens represents an essential line of defense, given that broiler farming often faces the presence of pathogenic agents on farms. Therefore, enhancing the immunity of these birds is crucial to strengthen resistance against pathogens and optimize poultry production (Adams et al., 2023).

The immunomodulatory effects of probiotics in broilers are a focus of ongoing research by many scientists. The capacity of probiotics to modulate the immune system depends, among other factors, on the specific strains of bacteria or microorganisms used (Huang et al., 2004). A study involving Lactobacillus fermentum and Saccharomyces cerevisiae showed that they could improve immune responses in broilers by stimulating the intestinal T cell immune system (Bai et al., 2013).

Haghighi et al. (2006) found that a probiotic containing Lactobacillus acidophilus, Bifidobacterium bifidum, and Streptococcus faecalis was associated with changes in cytokine expression, particularly IFN-γ and IL-12, in the gut-associated lymphoid tissues of chickens. Recently, Larsberg et al. (2023) demonstrated that two different Bacillus strains enhanced T cell activation and proliferation, indicating an immunomodulatory effect of both strains on chicken immune cells in vitro.

Among the most studied yeasts as probiotics, Saccharomyces boulardii and Saccharomyces cerevisiae stand out, being primarily used against gastrointestinal diseases in humans and animals (Rajput et al., 2013; Banik et al., 2019). Both act as growth promoters in broiler chickens and ensure beneficial colonization of the intestinal microbiota, limiting the survival and growth of pathogenic bacteria (Ahiwe et al., 2021; Roy & Ray, 2023).

Moreover, despite the significant positive effects of probiotics on poultry production, the results are still contradictory regarding their influence on the chicken immune system, and little is known about the response in broilers vaccinated against cholera (Pasteurella multocida) and avian typhoid (Salmonella gallinarum).Thus, the objective of this study was to evaluate the effect of supplementation with Saccharomyces boulardii and Saccharomyces cerevisiae on the immunity and welfare of broiler chickens vaccinated against Pasteurella multocida and Salmonella gallinarum.

MATERIALS AND METHODS

The study was conducted at the animal facility of the Laboratory of Teaching and Zootechnical Experimentation Prof. Dr. Renato Rodrigues Peixoto - Federal University of Pelotas (UFPEL), and was approved by the Animal Experimentation Ethics Committee (CEEA) UFPEL under protocol number 9053. A total of 270 one-day-old Cobb 500 broilers were subjected to three probiotic supplementation schemes (yeast-free diet, diet with S. boulardii, and diet with S. cerevisiae). The broilers were distributed into 45 experimental pens, each measuring 1.20 m x 0.65 m x 0.65 m. Each pen housed 6 birds, comprising 3 males and 3 females.

On the 15th day of the experiment, two male broilers from each pen were vaccinated with a commercial vaccine against cholera and avian typhoid (Pasteurella multocida and Salmonella gallinarum, Labovet®), while the third male and the females were not vaccinated. Thus, to determine the immune response, 105 male Cobb 500 broilers were evaluated under the following treatments: T1. Vaccinated and not supplemented (n=30); T2. Vaccinated and supplemented with S. boulardii (n=30); T3. Vaccinated and supplemented with S. cerevisiae (n=30); T4. Not vaccinated and not supplemented (n=15).

Yeast Cultivation

Strains of Saccharomyces boulardii CNCM I-745 (Floratil) and Saccharomyces cerevisiae YT001 (Yeastech) were cultured from stock plates at the Microbiology Laboratory, Center for Biotechnology, UFPEL. The yeasts were inoculated in a YM (Yeast Malt) medium consisting of 0.3% malt extract, 0.3% yeast extract, 0.5% bacteriological peptone, and 1% glucose. They were then incubated for 24 hours at 28°C in an orbital shaker at 150 rpm. Initially, a yeast colony was introduced into 10 mL of YM medium. Subsequent steps were conducted every 24 hours to scale up production, including inoculations of 100 mL, 1 L, and finally 7 L, all in a bioreactor operating with continuous aeration. Between each step, Gram staining was performed to verify and ensure the purity of the cultures. After this process, the 7 L culture flasks were refrigerated for 72 hours at approximately 4°C. After cell mass sedimentation and supernatant removal, approximately 1 L of culture with a concentration of 1x10⁹ colony-forming units (CFU) per mL was obtained, being determined through serial dilution and plating in YM medium.

Experimental Diets

The diets, based on corn and soybean meal (Table 1), were supplemented with yeasts mixed with the ingredients, with 1 L of yeast for every 100 kg of feed, resulting in a final concentration of 1x10⁷ CFU/g of feed, which was supplemented throughout the experimental period.

Table 1
Nutritional composition of the experimental diets.

Ingredients (Kg)	Initial (1-21 days)	Growth (22-42 days)
Corn	55.080	59.900
Soybean meal	40.920	36.443
L-Lisine	0.154	0.169
DL-Methionine	0.322	0.253
L-Treonine	0.085	0.065
Glycine	0.950	0.871
Serine	1.179	1.082
Calcium	1.095	1.038
Sodium	0.210	0.174
Phytase	0.005	0.005
Total	100.00	100.00
Nutritional Profile
Metabolizable Energy (Kcal/Kg)	2808.024	2856.784
Dry matter (%)	87.839	87.722
Crude protein (%)	23.061	21.214
Crude fiber (%)	3.6480	3.9830
Ethereal Extract (%)	2.2940	2.3860
Lisine (%)	1.2500	1.1500
Treonine (%)	0.8429	0.7587
Total Phosphorus (%)	0.7660	0.5810
Methionine (%)	0.6368	0.5482

• ¹
  Initial Diet (1 - 21 days) - The mineral Amino Acid Vitamin nucleus for broilers used was Mig broiler Initial 4% with the following minimum guarantee levels per kg: (Folic Acid (min) 18 mg, Pantothenic Acid (min) 230 mg, Biotin (min) 2.2 mg, Calcium (min - max) 190 - 230 g, Cobalt (min) 4.8 mg, Copper (min) 370 mg, Choline (min) 9800 mg, Iron (min) 1500 mg, Phytase 12500 FTU, Flavomycin 50 mg, Phosphorus (min) 50 g, Iodine (min) 44 mg, Lysine (min) 26 g, Manganese (min) 1600 mg, Methionine (min)70 g, Niacin (min) 970 mg, Salinomycin 1500 mg, Selenium (min) 6.2 mg, Sodium (min) 42 g, Threonine (min)18 g, Vit A (min) 240000 IU, Vit B1 (min) 44 mg, Vit B12 (min) 500 mcg, Vit B2 (min) 136 mg, Vit B6 (min) 60 mg, Vit D3 (min)50000 IU, Vit E (min) 620 IU, Vit K3 (min) 48 mg, Zinc (min) 2200 mg.
• Growth diet (22 - 42 days) - the Mineral Amino Acid Vitamin nucleus for broilers Mig Growing Broilers 4% was used with the following minimum guarantee levels per kg: (Folic Acid (min) 18 mg, Acid Pantothenic (min) 230 mg, Biotin (min) 2.2 mg, Calcium (min - max) 100 - 170 g, Cobalt (min) 4.8 mg, Copper (min) 370 mg, Choline (min) 7300 mg, Iron (min) 1500 mg, Phytase 12500 FTU, Flavomycin 50 mg, Phosphorus (min) 16 g, Iodine (min) 42 mg, Lysine (min) 30 g, Manganese (min) 1800 g, Methionine (min) 58 g, Monensin 2500 mg, Niacin (min) 970 mg, Selenium (min) 6.2 mg, Sodium (min) 32 g, Threonine (min) 14 mg, Vit A (min) 241500 IU, Vit B1 (min) 44 mg, Vit B12 (min) 500 mcg, Vit B2 (min) 138 mg, Vit B6 (min) 60 mg, Vit D3 (min) 50000 IU, Vit E (min) 620 IU, Vit K3 (min) 50 mg, Zinc (min) 2340 mg.

Humoral Immune Response

To evaluate the humoral immune response, a total of 105 blood samples were collected on day 21 of the experiment. Blood samples (1 ml) were collected by puncturing the ulnar vein, incubated for 30 minutes at 37°C, followed by 1 hour at 4°C, and then centrifuged (10 minutes, 4000 x g) to obtain serum.

To assess IgY antibody levels, an in-house enzyme-linked immunosorbent assay (ELISA) was conducted. For this, 96-well plates (CRAL®, Brazil) were coated with 100 μl per well of an inactivated Pasteurella multocida and Salmonella gallinarum suspension from the commercial vaccine (corresponding to 5% of a vaccine dose) diluted (1:1) in carbonate-bicarbonate buffer (0.1 M, pH = 9.8), and incubated for 1 hour and 30 minutes at 37°C. Individual sera samples were diluted (1:100) in phosphate-buffered saline (PBS) and added to the plates in duplicate. Next, anti-chicken IgY antibody conjugated to peroxidase (Sigma Aldrich) diluted 1:4000 in PBS was added. Finally, 100 μl of developing solution (10 ml of citrate phosphate buffer (0.2 M and 0.1 M), 0.004 g of Ortho-Phenylenediamine (OPD, Sigma-Aldrich), and 15 μl of hydrogen peroxide (H2O2) were added. The plates were incubated for 15 minutes in the dark at room temperature, and then 50 μl per well of 3% of sulfuric acid (H2SO4) was added to stop the reaction. Between each step, the plates were washed four times with PBS containing 0.05% Tween20 (PBS-T). Absorbances were measured using an EZ Read 400 microplate reader (Biochrom, UK) with a 492 nm filter.

Leukocytes

For differential leukocyte counting, 105 blood smears were prepared from blood samples collected on days 21 and 42 of the experiment in chickens, totaling 210 samples. After the smear, the slides were fixed with methanol for five minutes and then stained with hematoxylin and eosin. The slides were then washed with distilled water and air-dried. For counting, the smears were observed under an optical microscope with an oil immersion objective (100x), following a zigzag observation pattern. The complete blood cell count was performed based on 100 cells per smear, calculating the proportion of each type (Marín et al., 2003; Tessari et al., 2006; Roll et al., 2010). Leukocyte counting was classified into heterophils, lymphocytes, eosinophils, basophils, and monocytes. The heterophil/lymphocyte ratio was determined according to Gross & Siegel (1983).

Statistical Analysis

Data were subjected to analysis of variance followed by Tukey’s test to compare treatment means (p < 0.05) using the statistical software R (R CORE TEAM, 2023).

RESULTS AND DISCUSSION

Supplementation with probiotics enhanced the response to the Pasteurella multocida and Salmonella gallinarum in broiler chickens at 21 days of age (Figure 1). Chickens vaccinated and supplemented with Saccharomyces boulardii (T2) and Saccharomyces cerevisiae (T3) showed an increase of 1.6 and 1.5 times in IgY levels compared to vaccinated and non-supplemented chickens (p < 0.05), and an increase of 4.0 and 3.9 times compared to non-vaccinated and non-supplemented chickens (p < 0.05). The chickens supplemented with S. boulardii and S. cerevisiae did not differ statistically from each other.

Figure 1
Total specific IgY against Pasteurella multocida and Salmonella gallinarum at 21 days of age.

The data represent the mean absorbances (+/- standard error) of individual analyses obtained by indirect IgY ELISA in broiler chickens from the groups supplemented with Saccharomyces boulardii and Saccharomyces cerevisiae, and the vaccinated or non-vaccinated controls against Pasteurella multocida and Salmonella gallinarum. The cut-off was determined as the mean of the values obtained on day 0 plus 2 times the standard deviation. Means followed by different letters are indicative of significant differences (p<0.05).

The results obtained corroborate the results of Nari et al. (2020), who observed an increase in antibody levels against the infectious bronchitis virus in broiler chickens using S. boulardii as dietary supplementation. Similarly, Roy & Ray (2023) found an increase in antibody levels at 35 days of age in broilers vaccinated against the infectious bronchitis virus (IBV) and Newcastle disease. Supplementing broilers with S. cerevisiae also proved to be a promising alternative. Broilers supplemented and vaccinated against Newcastle disease showed an improved humoral immune response throughout the 35-day experimental period (Haldar et al., 2011).

Supplementation of broilers and quails with probiotics represents a promising strategy to enhance the immunogenicity of vaccine antigens. However, there is a lack of studies on the immunomodulatory effects of S. boulardii and S. cerevisiae on vaccination in broilers, particularly against Pasteurella multocida and Salmonella gallinarum.

Saccharomyces cerevisiae is widely used in animal nutrition and has been investigated for its potential as an immunomodulator in vaccination contexts. Chou et al. (2017) demonstrated that supplementation with S. cerevisiae fermentation products in broilers generated faster and stronger antigen-specific humoral immune responses to Newcastle disease virus (NDV) vaccination. The supplementation upregulated important immune genes such as IFN-γ and STAT4, which are involved in antiviral and antibacterial responses.

Saccharomyces cerevisiae has been shown to support immune responses when faced with bacterial infections like Pasteurella multocida. Reuben et al. (2021) demonstrated that multi-strain probiotics, including S. cerevisiae, reduced mortality and improved growth performance in broilers infected with P. multocida. The yeast, in conjunction with other probiotics, modulated intestinal microbiota, improved immune markers, and attenuated the inflammatory response, suggesting its utility in supporting vaccination.

In a study on mice, Roos et al. (2018) investigated the use of the probiotic S. boulardii as a potential adjuvant to enhance vaccine efficacy against bovine herpesvirus 5 (BoHV-5). The results showed that supplemented animals had an improved systemic IgG antibody response, skewed toward a Th1 profile favoring IgG2a. Additionally, there was increased mRNA expression of cytokines IFN-γ, IL-12, IL-17, and IL-10 in the spleen. The study suggested that S. boulardii supplementation could be a promising approach to improving the efficacy of vaccines, particularly those that depend on a cell-mediated immune response.

The potential of other probiotics in poultry farming has also been studied. Among the probiotics evaluated, the probiotic yeast Pichia pastoris X-33, when added “on top” of the feed, improved production indices in laying quails, increased egg weight, and had an immunomodulatory effect, enhancing the humoral response to vaccines against Newcastle disease, infectious bronchitis, and Gumboro (Gaboardi et al., 2019). Immunomodulatory effects were also observed in the responses to vaccines against infectious bursal disease, while also improving feed efficiency (Santos et al., 2018). Additionally, the use of P. pastoris yeast demonstrated antimicrobial activity against Salmonella Typhimurium, a bacterium of great importance due to its negative impacts on broiler farming (França et al., 2015). These results highlight the potential of probiotics in poultry farming to promote not only the growth and health of birds, but also food safety by reducing the prevalence of pathogens.

Supplementation with S. boulardii and S. cerevisiae presents benefits in different animal species. The use of S. boulardii in the supplementation of sheep showed an increase in the humoral immune response to vaccines against enteropathogenic Escherichia coli and bovine herpesvirus type 5 (Roos et al., 2010), as well as in the response to the vaccine against Clostridium chauvoei (Santos et al., 2021). Additionally, supplementation with S. cerevisiae also led to modulation of the immune response, increasing serum IgG levels in sheep infected with Haemonchus contortus (Pinto et al., 2020). Thus, there is interest in testing these probiotics due to their potential to strengthen immunity and improve vaccine efficiency in animals.

The immunomodulatory effect of the tested probiotics is suggested to be related to the elements that make up the structure of yeast cell walls (Ding et al., 2019). These walls are composed of β-glucans, which are glucose polymers with β-1,3 and β-1,6 glycosidic bonds, and mannan-oligosaccharides (Abel & Czop, 1992; Griggs & Jacob, 2005). These β-1,3 and β-1,6 glucan bonds are considered immunomodulatory agents in animals, and mannan-oligosaccharides play a role in enhancing the immune response by stimulating increased immunoglobulin production (Cotter et al., 2002; Gurbuz et al., 2011). Studies have also shown that administration of S. boulardii promoted a beneficial effect on the levels of short-chain fatty acids (e.g. acetate, propionate, and butyrate) which are recognized for their immune system modulation properties. Modulation occurs through anti-inflammatory properties, regulation of T and B lymphocytes, cytokine production, and dendritic cell maturation (Sen & Mansell, 2020).

Another aspect evidenced in S. boulardii’s immunomodulation is its ability to activate the complement system by increasing C3, C5, and C3d, which are proteins that form a cascade of reactions performing essential functions in protecting the body against pathogens, from destroying microorganisms to opsonizing particles for phagocytosis (Machado Caetano et al., 1986). Thus, it ensures that invaders are neutralized, contributing to health maintenance. Supplementation with S. boulardii and S. cerevisiae demonstrated an effective capacity to modulate the immune system, enhancing responses and ensuring healthy immune defense in broiler chickens. Optimizing the vaccine response, evidenced by increased antibody levels, highlights the potential benefit of this approach in promoting animal health.

In observing the differential leukocyte count and the heterophil/lymphocyte ratio, no significant differences were found between treatments at 21 days of the experiment (Table 2). The results are consistent with those obtained by Cardoso et al. (2003) and Borsa (2009), showing that the verified hematological values reflect reference values in Cobb broiler chickens, indicating conformity and normality. Additionally, the results align with those found by Al-Dabbagh & Shareef (2009), where supplementation with S. cerevisiae did not influence leukocyte count at 21 days of age in broilers compared to the control treatment.

Table 2
Differential Leukocyte Counts at 21 and 42 Days of Age in Broiler Chickens Receiving Diets Containing Saccharomyces boulardii or Saccharomyces cerevisiae (Means ± Standard Deviation).

	21 days of age					42 days of age
	T1	T2	T3	T4		T1	T2	T3	T4
LYM	68.7±2.06	67.8±2.06	67.6±2.06	66.9±2.92		61.6±1.78B	69.9±1.78A	67.5±1.78AB	62.9±2.61AB
HET	28.6±2.02	30.4±2.02	30.2±2.02	30.6±2.86		36.9±1.70A	29.1±1.70B	31.3±1.70AB	34.7±2.50AB
EOS	0.36±0.11	0.50±0.11	0.43±0.11	0.26±0.15		0.43±0.09	0.30±0.09	0.30±0.09	0.64±0.14
BAS	0.33±0.09	0.10±0.09	0.23±0.09	0.20±0.12		0.03±0.05B	0.16±0.05AB	0.06±0.05B	0.42±0.08A
MON	2.03±0.36	1.26±0.36	1.46±0.36	2.06±0.51		0.96±0.21	0.46±0.21	0.80±0.21	1.28±0.30
H/L	0.44±0.04	0.48±0.04	0.48±0.04	0.50±0.06		0.64±0.04A	0.43±0.04B	0.49±0.04AB	0.58±0.06A

• Means followed by different letters in the rows indicate significant differences (p<0.05). LYM = Lymphocytes; HET = Heterophils; EOS = Eosinophils; BAS = Basophils; MON = Monocytes; H/L = Heterophil/Lymphocyte Ratio. T1 = Vaccinated and not supplemented; T2 = Vaccinated and supplemented with Saccharomyces boulardii; T3 = Vaccinated and supplemented with Saccharomyces cerevisiae; T4 = Not vaccinated and not supplemented.

At 42 days, significant differences between the treatments were observed in lymphocyte, basophil, and heterophil counts, as well as in the heterophil/lymphocyte ratio (Table 2). The lymphocyte percentage increased in the group supplemented with S. boulardii (p = 0.003) compared to the control group. The group that received S. cerevisiae did not differ from the other treatments (p = 0.05). Lymphocytes play a crucial role in specific immunity, initiating the body’s adaptive responses, and an increase in lymphocyte count indicates a positive physiological state in broilers under appropriate conditions (Roll et al., 2010; Mardhotillah et al., 2021).

The increase in lymphocyte count is important for the birds’ immunomodulation, contributing to disease resistance through the production of antibodies and cytokines responsible for humoral and cellular responses (Levkut et al., 2009; Hidanah et al., 2018). A robust immune system is essential for ensuring broiler welfare, as housing conditions often promote disease emergence (Wegner et al., 2023).

Regarding heterophils, the group supplemented with S. boulardii (T2) showed lower levels (p = 0.004) compared to the control group (T1), which had higher levels; while the group that received S. cerevisiae (T3) did not differ from either group. Heterophils are phagocytic cells of the innate immune system, constituting the first line of defense against pathogenic microorganisms (Minias, 2019).

The basophil count was higher in the non-vaccinated and non-supplemented treatment (T4) compared to S. cerevisiae (p=0.01) and the control treatment (T1), but it did not differ from S. boulardii (p = 0.21). The absence of changes in monocyte and eosinophil percentages in the S. boulardii and S. cerevisiae treatments suggests no infection or influence from pathogenic bacteria (Mardhotillah et al., 2021). This suggests that S. boulardii and S. cerevisiae plays a role in modulating the immune system, contributing to leukocyte level regulation and possibly positively influencing the immune response.

The heterophil/lymphocyte ratio was lower in treatments without probiotic supplementation (T1 and T4) compared to the treatment supplemented with S. boulardii, but it did not differ from S. cerevisiae. This result is similar to those found by Gheisari & Kholeghipour (2006), who observed that broilers fed with the probiotic S. cerevisiae had a lower heterophil/lymphocyte ratio due to a higher lymphocyte population compared to the control treatment. A lower heterophil/lymphocyte ratio indicates reduced stress levels (Vleck et al., 2000).

Elevated heterophil/lymphocyte ratios are associated with chronic stress in broilers and are a reliable variable for assessing bird welfare levels (Gross & Siegel, 1983; Maxwell, 1993). During stress, corticosterone is released, and as its levels increase, a rise in the heterophil to lymphocyte (H/L) ratio in the blood is also observed (Post et al., 2003; Blas, 2015; Scanes, 2016). Yeast supplementation can be effective in reducing circulating corticosterone levels and preventing immune system depression, which is crucial during the final life stage when antibody production generally decreases (Haldar et al., 2011).

CONCLUSION

Saccharomyces boulardii and Saccharomyces cerevisiae increased the level of specific vaccine antibodies. Additionally, supplementation with Saccharomyces boulardii significantly reduced stress levels in broilers, as higher heterophil/lymphocyte (H/L) ratios were observed in the control groups. These results suggest the potential of these yeasts as agents promoting health and welfare in broiler chickens. However, further studies are necessary to explore their role as immunomodulators in poultry production, and future research should focus on validating these findings for broader application.

ACKNOWLEDGEMENTS

The authors are grateful to the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES Foundation), for providing a scholarship to Von Mühlen C. The authors Leite FPL, Corcini CD, Varela Junior AS and Roll VFB were supported by a grant from Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil (CNPq/Produtividade em Pesquisa).

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• FUNDING
  This work presents partial results of the research project (19/2551-0001985-8) supported by the Rio Grande do Sul State Research Support Foundation - FAPERGS.
• DATA AVAILABILITY STATEMENT
  The data that support the findings of this study are available from the corresponding author upon reasonable request.
• DISCLAIMER/PUBLISHER’S NOTE
  The published papers’ statements, opinions, and data are those of the individual author(s) and contributor(s). The editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions, or products referred to in the content.

Edited by

• Section Editor:
  Maria Fernanda Burbarelli

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Publication Dates

• Publication in this collection
  10 Feb 2025
• Date of issue
  2024

History

• Received
  14 Aug 2024
• Accepted
  03 Dec 2024