Deoxynivalenol (DON) and zearalenone (ZEN) are mycotoxins produced by Fusarium fungi, which frequently contaminate maize and grain cereals [1]. These mycotoxins have been widely investigated due to their universal distribution and capacity to cause pathological changes in humans and farm animals. Mycotoxicosis is the term used to refer to diseases caused by these toxins in humans and animals, often presenting in farm animals as reduced feed intake, poor feed conversion, feed refusal, reduced body weight gain, immune suppression, poor reproductive capacity, and long-term chronic effects, ultimately resulting in economic losses.

Numerous mechanisms have been suggested to explain the biological effects elicited by DON-contaminated feed. For instance, long-term exposure to DON may cause anorexia, reduced feed intake and weight gain, decreased nutritional efficiency, and immune modulation [2,3]. According to Pestka and Smolinski [4], DON inhibits protein biosynthesis and induces pro-inflammatory cytokine production. Among farm animals, pigs are the most sensitive to DON with chronic exposure, characterized as 1 to 2 mg DON/kg in feed, resulting in decreased appetite; whereas 3 mg DON/kg reduces body temperature and induces variations in the gastric wall of piglets. Further, according to Reddy et al [3], consumption of a diet containing 8 mg DON/kg for four weeks decreased body weight gain, reduced feed conversion rate, affected inflammatory cytokine production, reduced immunoglobulin (Ig)G, IgM, and serotonin values, and decreased total antioxidant contents in serum samples of pigs, as well as histopathological damage in kidney and liver samples compared to a standard dietary group. Furthermore, the usual growth rate of pigs has been reported to be reduced by approximately 7% for each mg of DON/kg increase in the diet, which may vary depending on numerous factors. Meanwhile, complete refusal of feed occurs at concentrations greater than 12 mg DON/kg. According to Young et al [5], high doses of DON (0.1 to 0.3 mg/kg body weight or 20 mg/kg of feed) in piglets led to vomiting. When pigs consume DON it becomes absorbed in the proximal aspect of the small intestine; and once in the colon de-epoxidation can occur, however, detoxification is not significantly induced [6].

The ZEN is a phytoestrogenic compound with estrogenic effects in farm animals [7]. The ZEN is associated with various dose-dependent mycotoxicoses in farm animals, particularly pigs. Pigs generally show clinical signs at low doses of ZEN (1.5 to 2 mg/kg in diet) including vaginal and vulvar swelling and thickness, increased uterus mass, testicular atrophy, and expansion of the mammary glands, as well as other reproductive effects, such as decreased fertility, increased number of resorptions, and reduced litter size [7]. Typically, hyperestrogenism and mortality are observed in pigs fed high doses of ZEN over an extended period of time [8], and infertility was observed at concentrations higher than 100 mg ZEN/kg [9]. A recent study [3] showed that in pigs fed a 0.8 mg ZEN/kg diet for four weeks, the serum IgG and IgM levels decreased; meanwhile total antioxidant levels decreased in the serum yet increased in urine. At the same ZEN dose, inflammatory cytokine and chemokine marker expression was reduced in the kidney and liver tissues, with microscopic lesions appearing in these tissues.

The gastrointestinal (GI) microbiota plays essential roles in animals, with a strong association reported between the host and its GI microbiota, particularly in immune response, energy uptake from food, activity of important metabolites, resistance to toxins, and metabolic products of fermentation. However, few studies have examined the GI microbiota in farm animals [10], particularly the effects of DON and ZEN mycotoxins on the pig intestinal microbiota communities. Different species colonize various areas of the GI tract to different degrees. Pigs, specifically, are highly sensitive to dietary mycotoxins, which target the mucus and microbiota [11]. For instance, Fusarium toxins can cause gut tissue damage, shorten the height of villi, and cause dysbiosis of gut microbiota [12]. We previously showed that Fusarium mycotoxins primarily influence the composition of pig gut microbiota, ultimately causing activation of intestinal inflammation [10].

Among the various gut microbiota, bacteria play a major role in the GI tract. Next-generation DNA sequencing (NGS) approaches are effective for studying the composition of the host microbiota, with the high read abundance of amplified sequences providing insight into microbial diversity. Advanced molecular biology techniques can be useful in examining complex microbiological communities, such as those in the gut that cannot be cultured outside of the host. Moreover, these approaches have revealed variations in the gut bacterial community between functional sites, individuals, as well as healthy and diseased conditions [13]. Importantly, studies of the gut microbiota have also demonstrated its critical role in maintaining animal and human health.

The direct effects exerted by consumption of highly concentrated and commercially-purified DON and ZEN on the composition of the cecum microbiota have not been reported in pigs. Therefore, this study was performed to determine the effect of commercially-purified Fusarium mycotoxins, DON and ZEN, on individual pigs after four weeks of feeding, as well as to compare mycotoxin-treated pig groups to gain insight into the quantitative and qualitative composition of the pig cecum microbiota. These results may be useful for determining whether disturbances in the intestinal microflora, such as the toxic effects of DON and ZEN, can be ameliorated via modulating the intestinal bacterial flora.

We assessed the effect of diets contaminated with DON and ZEN on the cecum microflora of pigs and evaluated which bacterial strains may reduce the toxicological effects of DON and ZEN. The GI microbiota in pigs has not been completely defined as this dynamic community contains many hundreds of species, comprised primarily of anaerobic bacteria [21]. The DON and ZEN mycotoxins have been shown to negatively affect GI microbiota, affected the GI species composition and bacterial numbers in pigs to varying degrees depending on the mycotoxin concentrations in diet, treatment period, animal age, location of toxins in the GI, and nutritional dietary factors. Although previous studies have examined the effects of feed naturally-contaminated with mycotoxins on the gut microbiota of pigs [22,23], here we sought to analyze the cecum contents of pigs fed commercially-purified DON and ZEN mycotoxins to compare microbial taxonomic abundances in the control, DON, and ZEN dietary treatment groups.

Among the phyla detected, Firmicutes (56.8%) and Bacteroidetes (35.6%) were highly abundant (Figure 3A), occupying more than 92% of the cecum contents from the DON, ZEN, and control dietary groups, however, their abundances showed no significant differences between dietary groups (Figure 2A, Supplementary Table S1). Similarly, in our previous study, the colons of DON and ZEN dietary-treated pigs also showed greater than 90% Firmicutes and Bacteroidetes (p>0.05) [10]. Typically, the compositions of the intestinal microbiota in the cecum and colon are similar among pigs [24]. However, Li et al [23] found that naturally DON-contaminated wheat fed to pigs caused differences in the abundance of Firmicutes and Bacteroidetes in the cecum, colon, and ileum. Further, Isaacson and Kim [24], evaluated naturally weaned pigs and found that Firmicutes and Bacteroidetes accounted for more than 90% of the bacteria detected in the cecum contents. Meanwhile, in another study, Firmicutes and Bacteroidetes comprised more than 90% of bacteria in pigs fed a fuminosin-contaminated diet (12 mg/kg feed) [25]. In the current study, no significant differences were noted at the phylum level in the toxic dietary treatment groups. However, if the dietary treatment period were to be extended, the microbiota may be altered in the toxin treatment groups. Family-level cecum bacterial abundances are shown in Table 2, and significant families in these genera were identified.

Among the cecum contents, specific genera were identified as potentially significant biomarkers for differentiating between the control and the DON and ZEN mycotoxin dietary treatment groups. Specifically, Lactobacillus, Bacteroides, and Megasphaera were significantly more abundant in the cecum microbiota of the DON and ZEN dietary groups compared to the control group (Figure 3b). Lactobacillus accounted for a predominant genus in DON and was moderately abundant in ZEN, meanwhile its levels were very low in the control group (Figure 2A). Similar results were observed in our previous study regarding the pattern of Lactobacillus abundance in the colons of pigs [10], with differences observed between the cecum and colon contents in the DON, ZEN, and control dietary treatments. It has been suggested that a myriad of factors contribute to the microbial shifts occurring between the cecum and colon, including stress resulting from mycotoxin-contaminated feed, chemical composition of the diet, as well as various physiological factors [26]. Recently, the abundance of Lactobacillus was found to be increased by 13% in the cecum of pigs fed DON- contaminated wheat in combination with Clostridium sp. WJ06 [23]. In another study, 15.8% of Lactobacillus sequences were recovered from the pig intestinal samples, indicating their important roles in the gut on host physiology [27]. Similarly, through 16S rRNA analysis, Niu et al [28] demonstrated that Lactobacillus is one of the most prevalent genera in pig intestinal samples, irrespective of age. Furthermore, NGS technology revealed Lactobacillus as a key member of the fecal microbiota in all growth stages of pigs.

Lactobacillus species are considered to be probiotics that play a key role in various physiological functions of their hosts, including microbial interference, antimicrobial properties, supplementary influences on nutrition, antitumor effects, decreasing cholesterol in the host serum, and immunomodulatory influences [29]. Particularly, in pigs, a positive effect was observed on Lactobacillus abundance following feed limitation. According to Yang et al [30], Lactobacillus can support the development of an optimized microbiome by enhancing the richness and number of lactobacilli and other native probiotic bacteria. The primary indigenous probiotic bacteria can promote growth and immunity of piglets through positive cascade signal transduction pathways. The piglet body provides a tolerant habitat and nutrients for bacterial colonization and growth, in return, probiotics generally generate prebiotics such as short-chain fatty acids and bacteriocins that can improve growth, and decrease the risk of enteric diseases caused by pathogens or toxins, while also enhancing the host feed utilization capacity. Furthermore, Walter [31] indicated that autochthonous Lactobacillus are often used as probiotics due to their natural capacity to survive harsh physiological conditions, including the acidic stomach, pancreatic enzymes, and bile salts during their passage through the GI tract [32,33]. These Lactobacillus probiotic bacteria were also selected for their capacity to adhere to mucus and epithelial cells, as these characteristics are required for their effective colonization of the gut’s mucosal and epithelial layer and to increase their competitiveness against pathogens [34]. This competitive profile is likely conferred by autochthonous Lactobacillus, which is used as a biomarker of health in the pig gut microflora.

According to many studies, the large quantity of lactic acid bacteria (LAB) in the digestive tract is associated with the age of pigs and feeding of probiotics. LAB play a crucial role in the host, generating substances such as acetic, butyric, and propionic acid, as well as other similar short-chain fatty acids, B vitamins, and amino acids, including bacteriocins ad antimicrobial metabolites. Colonization of the pig digestive tract by LAB is prevented by various pathogens stimulating the immune system of the host [35]. Moreover, LAB can bind Fusarium mycotoxins in their environment [36]. According to Franco et al [37], the capacity of viable and heat-inactivated Lactobacillus cells was reduced by more than 60% in the presence of 1.5 μg/mL DON in liquid media. However, according to Yang et al [38], Lactobacillus plays a key role in removing ZEN in vitro; meanwhile El-Nezami et al [39] reported the binding affinity of ZEN, and its derivative α-ZEN, with two food-grade strains of Lactobacillus. Still further, although the mechanisms of aflatoxin binding by specific Lactobacillus are unclear, cell wall peptidoglycans and polysaccharides have been suggested as the two most significant elements responsible for binding by Lactobacillus and the absorption of mutagens or carcinogens in the intestine [40]. In this study, we predicted that Lactobacillus concentrations were higher in DON and ZEN dietary treatment groups and that LAB may contribute significantly to the detoxification of DON and ZEN in the pig intestine. However, the intestinal mucus and its resident microbiota are important targets of dietary mycotoxins, particularly DON [11]. Consequently the GI mucus can reduce the ability of probiotics, such as Lactobacillus, to bind mycotoxins, thereby interfering with their adsorption of dietary mycotoxin. Similarly, in the current study, regular administration of the probiotic Lactobacillus during the dietary treatment period may have reduced the effect of mucus. The number of bacterial colony-forming units may have reduced the effects of mucus on the adsorption of DON and ZEN dietary mycotoxins by the Lactobacillus cell wall.

In the current study, Bacteroides accounted for another predominate genus, which was more abundant in the DON and ZEN dietary toxin groups than in the control. Bacteroides spp. are considered as a source of novel useful bacteria for treating gut immune dysfunctions, colitis, and metabolic disorders, as well as for cancer prevention [41]. Bacteroides spp. are dominant and play a key role in pig intestine, making them a major group in pig feces. Similar to our results, Saint-Cyr et al [42] demonstrated that feeding with DON at 100 mg/kg body weight for 4 weeks by oral gavage increased Bacteroides levels in rat intestines. Other studies in pigs supported the use of prebiotics for selecting Bacteroides spp. This capacity to influence the microbiota also involves inclusion of a specific level of fermentable fiber in the pig diet, which stimulates colonic fermentation, as well as the presence of exogenous enzymes, which create oligosaccharides with prebiotic effects from non-starch polysaccharides [43]. Moreover, Bacteroides spp. also help to protect against gut colonization by various potential pathogens. Based on these results, Bacteroides may play a key role in eradicating DON and ZEN from the pig gut. In this study, Megasphaera was approximately three-fold more abundant in the DON group, and two-fold higher in the ZEN group, compared to the control. Conversely in our previous study we reported that Megasphaera was more abundant in the ZEN dietary group, with no significant differences observed between the ZEN and control groups [10]. Megasphaera spp. are abundant and important in the human gut microbiota, and their capacity to produce important metabolites indicates beneficial health effects on the host. Megasphaera elsdenii was reported to comprise approximately 0.12% to 5.9% of bacteria in pig feces [44], where it uses both L- and _D-lactate and increases short-chain fatty acids, which are crucial for pig colonocyte development and proliferation, as well as small intestine growth [45]. We, therefore, hypothesized that Megasphaera abundance increases in the intestines of pigs in response to the lactate produced by abundant Lactobacillus, and may positively influence intestinal disorders or immune responses following DON and ZEN dietary treatment of pigs. However, few studies have examined the effects of dietary mycotoxins on these bacteria in animals.

Other bacterial genera, including Campylobacter, Paludibacter, Turibacter, Peptococcus, Pelosinus, Actinobacillus, Dechloromonas, and Akkermansia, also showed significant differences between the dietary treatment groups. Campylobacter is the most common cause of GI infection in pigs, characterized by inflammation and diarrhea involving cramps, fever, and pain [46]. According to Burrough et al [47], Paludibacter is abundant in the pig intestine and proficiently ferments complex polysaccharides produced by the pig. We found that DON and ZEN influence the composition and fermentation products of the pig intestinal microbiota, thus affecting the health and performance of the pig. However, further studies are needed to determine the functions of these genera.

In the current study, 17 OTUs were identified that differed significantly in abundance between the control, DON, and ZEN dietary treatment groups. Similarly, our previous study of pig colon contents also showed varying OTUs abundances between these groups [10]. However, in the current study, most OTUs in the cecum differed from those in the colon. Specifically, Bacteroides, Lactobacillus, and Campylobacter OTUs were more abundant in the cecum than in the colon content; we also identified three unclassified Lachnospiraceae, and two unclassified Clostridiaceae family OTUs which differed in abundance between the DON and ZEN dietary groups. According to Przybylska-Gornowicz et al [12], the response of the intestinal immune system is unambiguous in the cecum; however, variable and sometimes difficult to interpret results were obtained in the ascending colon and descending colon. Unclassified Lachnospiraceae showed higher abundance in both DON and ZEN dietary treatment groups. Moreover, similar to our results, Gratz et al [48] found that the abundance of Lachnospiraceae was higher in the cecum contents of DON dietary pigs; no previous studies evaluated ZEN dietary pig treatment groups. Lachnospiraceae also showed higher abundance in fecal samples from pigs fed fumonisin in the diet [25]. Lachnospiraceae bacteria may also play a key role in healthy pigs and serve to improve the pig immune system. Compared to the control, unclassified Clostridiaceae was less abundant in DON, however, did not exhibit differences in abundance with the ZEN dietary group. Compared to that reported previously for the colon contents, here we demonstrate that DON OTU abundance was decreased in the cecum, while ZEN did not differ from the control group. According to Piotrowska et al [22], Clostridium abundance is reduced in the colons of both the DON and ZEN dietary groups in gilts, suggesting that the abundance of Clostridiaceae bacteria was inhibited by the highly toxic DON, with no significant effects imposed by ZEN, which has lower toxicity. Prevotella OTU was also significantly decreased in the DON group, without significant differences observed in the ZEN group compared to the control. Conversely, in our previous study we reported significantly higher OTU abundances in both the DON and ZEN dietary groups [15], with a higher Prevotella abundance in pigs fed fumonisin (12 mg/kg feed). The Prevotella-driven enterotype appears to be important in subjects who consumed high levels of carbohydrates and fiber. In this study, due to the high concentrations of DON and ZED, the daily feed intake was reduced in the DON group compared to in the ZEN and control groups, which subsequently reduced the abundance of Prevotella in the DON due to the reduced consumption of carbohydrates.

Since mycotoxins can alter the microbial composition balance of the pig intestine, a complete understanding of the relationship between pigs and their gut microbiota will facilitate the development of new dietary treatments that can increase pig growth, protect piglets from pathogenic bacteria, and enhance host feed utilization. In the current study, specific bacteria genera were highly abundant in both the DON and ZEN dietary treatments compared to in the control; in this case, this microbiota may have positively impacted the host physiology. Other bacteria in the DON and ZEN dietary treatments, particularly in the DON group, showed a lower abundance compared to the control; in these cases, DON and ZEN may have induced lesions in the cecum by disturbing the integrity of the pig intestinal barrier and reducing the abundance of specific microbiota. These results show that the composition and structure of the pig cecum greatly differs compared to that determined in our previous study of microflora in the pig colon. Similarly, a previous study also strongly demonstrated differences between cecum and colon microbial data in pigs [23]. DON and ZEN may alter susceptibility to infectious diseases in humans and animals by affecting gut health as well as the innate and adaptive immune systems. However, the mechanisms by which mycotoxins affect the intestinal microbiota composition remain unclear.

In conclusion, the results of this study indicate that the GI bacterial flora in pigs became disrupted following consumption of feed contaminated with commercial DON and ZEN for four weeks. The genera Lactobacillus (particularly in DON) and Bacteroides dominated the bacterial flora in both the DON and ZEN dietary treatments. In addition, OTUs assigned to unclassified Lachnospiraceae belonging to Firmicutes, were more abundant in both the DON and ZEN dietary treatments than in the control group. Based on the present data, there may be potential opportunities to isolate and characterize useful probiotics that decrease the level of mycotoxins and help restore intestinal microbiota that have been disturbed by mycotoxins.