Modern animal production has been changing in recent years due to the problems of bacterial resistance derived from the overuse of antimicrobials for prophylactic and growth promotion purposes [42,43]. In this regard, many investigations have focused on probiotics, prebiotics, enzymes, acidifiers, plant extracts, and some metals (copper and zinc) as feed additives, given their antimicrobial properties and effects in promoting growth, mainly . In the present study, selection of CA was based on its advantages over inorganic sources since it has been described that inorganic sources tend to dissociate in the upper part of the gastrointestinal tract, causing a decrease in the availability of copper due to its interaction with other metals (chelation) and therefore a reduction in its activity [18,45]. In contrast, the solubility of organic sources of copper is higher in weak acid enviroments, making their dissolution slower and increasing their availability and activity . Furthermore, lower fecal copper excretion rates have been reported in broilers exposed to an organic source of copper compared to inorganic sources . Although the copper ion is known to be more effective against Gram-positive bacteria , dietary supplementation with CA significantly reduced more than 18% the colonization of S. Typhimurium in both trials compared to PC group (Table 2). Copper ion has been reported to cause damage at the bacterial membrane level due to its adhesion to membranes and the generation of reactive oxygen species . Additionally, it can be associated with the functional groups of proteins and enzymes, leading to the inactivation or inhibition of some cellular processes, as well as having a direct negative effect on the genetic material of bacteria [47,48]. In addition, this reduction in the colonization of S. Typhimurium presented a positive effect on BW and BWG since they tend to improve in both experiments (P= 0.081 and P= 0.085, respectively), when compared to PC.
In the case of the group treated with the solid dispersion of curcumin (CR), which was previously described by our research group and is characterized by being more soluble and permeable , the colonization of S. Typhimurium significantly decreased by more than 35% (more than 2 log10) with respect to the PC group after ten days of treatment (Table 2). These results are due to the antimicrobial action of curcumin, which in general, is associated with damage to the bacterial membrane and inhibition of bacterial cell proliferation [49,50]. Furthermore, it has been published that curcumin can induce some physical and mechanical changes of the S. Typhimurium flagellar filament, causing a decrease in motility, adherence, and invasion of the host cells, which results in a reduction or elimination of its virulence . Likewise, curcumin has been reported to decrease bacterial cell division processes since it interacts with the FtsZ protein, a cytoskeleton protein essential for this process . The treatment containing the mixture of CA and CR (CA-CR) reduced 2% and 37% the S. Typhimurium colonization compared to the group treated with CR and the PC group, respectively. These results contrast with those obtained in other articles where the combination of curcumin with heavy metals, including copper showed better effects and even decrease the toxicity of metals [16,53,54].
After oral infection with Salmonella, this pathogen must overcome the conditions of the gastrointestinal tract to interact with the intestinal epithelium . Invasion of epithelial layers by S. Typhimurium is known to increase intestinal permeability in both in vivo and in vitro models since the expression of some markers such as claudin-1, occludin, and mucin-2, mRNA levels of zonula occludens-1 and E-cadherin was reduced [55,56]. In the present study, FITC-d, a large molecule (3–5 kDa) that, under normal intestinal health conditions, does not leak through the epithelium, was used to assess intestinal permeability. However, when there is damage to the epithelium, the permeability of FITC-d increases so that it can be quantified in serum . In the present study, all treated groups showed lower serum FITC-d concentrations compared to the PC group (Table 2). However, only the group treated with CR had significantly lower concentrations when compared to PC and turned out to have serum FITC-d concentrations comparable to the NC group. Perhaps, this result is due to the ability of CR to restore the intestinal barrier function and the expression of proteins associated with the tight junctions, the proliferation-regeneration of the intestinal epithelium, and its antimicrobial action, resulting in decreased paracellular permeability as has been previously reported [58,59]. Regarding the treatments with CA and CA-CR, although the S. Typhimurium counts decreased significantly compared to the PC group, the serum FITC-d concentration only decreased numerically since it has been described that the production of reactive oxygen species by copper affects not only bacteria but also epithelial cells .
The chicken gut microbiota are densely populated with complex microbial communities that are involved in digestion and metabolism, regulation of enterocytes, vitamin synthesis, and development and regulation of the host immune system . Cecum is by far the most densely colonized microbial habitat in chickens . Despite the absence of any clinical signs of Salmonella infection, it has been reported that the composition of the microbiota is affected, but the changes are quite weak at the level of the caecal tonsils [63,64], which supports our results since no significant differences in alpha (measured by the observed OTUs) and beta diversity were observed in the cecal samples at day ten post-S. Typhimurium challenge. This means that there were no changes in the relationship of the number of different species per sample (richness) and in the diversity of the microbial community between different samples, respectively . Notwithstanding the above, the taxonomic composition showed some significant differences at the family and genus levels when the groups were compared.
At the family level, abundance of Enterococcaceae was lower in all groups supplemented with CA when compared to the PC group. Enterococcaceae, one of the six families of the order Lactobacillales , is comprised of the genera Enterococcus, Bavariicoccus, Catellicoccus, Melissococcus, Pilibacter, Tetragenococcus, and Vagococcus . However, it has been described that the dietary copper supplementation alters the intestinal microbiota, decreasing the abundance of Enterococcaceae due to the total reduction of lactic acid bacteria . In contrast, Salmonella infection is known to increase the relative abundance of Enterococcaceae, Lactobacillaceae, Clostridiaceae, Lachnospiraceae, Erysipelotrichaceae, Peptostreptococcaceae, and Ruminococcaceae, but decrease that of Enterobacteriaceae . Furthermore, the family of Clostridiaceae was significantly lower in chickens whose diet contained CR in common compared to PC. Clostridiaceae is one of the responsible families for converting polysaccharides into short-chain fatty acids (SCFAs) . It has been described that SCFAs such as acetate, propionate, and butyrate, are important in maintaining intestinal homeostasis due to their immunomodulatory capacity, maintenance of metabolism, proliferation, differentiation and promotion at low pH, favoring beneficial bacteria, and reducing the growth and viability of pathogenic bacteria . Therefore, these results support the lower Salmonella counts in CT and the improvement in BW and BGW in the CR treated group.
At the genus level, Salmonella, Coprobacillus, Eubacterium, and Clostridium were significantly enriched in the PC group, which is closely related to the severity of the Salmonella infection process. Coprobacillus, Clostridium, and Eubacterium have an important role in the production of SCFAs essential amino acids and the digestion of non-starch polysaccharides, which stimulate the production of SCFAs for metabolic balance [70,72]. Likewise, it has been reported that the reduction of Clostridium and the maintenance of Eubacterium and Coprobacillus levels could be related to the effectiveness of the treatments since they represent a positive effect in the maintenance of intestinal homeostasis [72–74]. Regarding the high abundance of Salmonella, it has been reported that it is related to its colonization in CT . Although sequencing of the V4 region of 16S rRNA gene is not able to distinguish between Enterobacteriaceae, BLAST analysis using an amplicon sequence variant (ASV) that matched to genus Salmonella strongly supports that the taxonomic assignment of this ASV to genus Salmonella in this study was accurate (see Supplementary Materials). Hence, these results confirm again the effect of the treatments, especially CR, on the decrease in Salmonella counts, the maintenance of intestinal integrity as indirectly measured by the serum FITC-d concentration, and the improvement in BW and BWG.
Furthermore, the genus Faecalibacterium and Enterococcus were significantly enriched in the group treated with CR. After infection with Salmonella, this pathogenic bacteria alters the intestinal microbiota, causing a decrease in bacteria of the genus Blautia, Enorma, Faecalibacterium, Shuttleworthia, Sellimonas, Intestinimonas, and Subdoligranulum, as well as an increase in the abundance of Butyricicoccus, Erysipelatoclostridium, Oscillibacter and Flavonifractor . However, in the case of the group treated with CR, the increase in Faecalibacterium, a genus of bacteria responsible for the production of butyrate and related to health benefits in poultry, could be mainly due to the prebiotic effect of curcumin, like other substances with the same activity . It has been described that CR could act as a factor of promotion, proliferation, growth, and survival for the beneficial bacteria of the intestinal microbiota from its biotransformation . Finally, the bacterial genera that belong to Erysipelotrichaceae and Lachnospiraceae were significantly enriched in the CA-CR and CA groups, respectively. It has been published that in chickens infected with Salmonella this genus of bacteria decreases markedly, which could negatively affect the diversity and development of intestinal bacteria . In the specific case of CA and CA-CR, copper is known to increase the relative abundance of these bacterial genera, which are the most active microbial components in the healthy gut and are responsible for preventing the production of inflammatory cytokines and induce intestinal production of SCFAs by fermenting carbohydrates [78,79]. Although the sample size for microbiome analysis is small, the results are promising and suggestive since there is a close relationship with what was observed in the other determinations.