The harmful effects of C. perfringens and the growth-promoting effects of B. licheniformis
Before the analysis of the sequencing data, it was necessary to evaluate the effects of C. perfringens and B. licheniformis on broilers. C. perfringens caused damage to the chickens, by reducing their growth performance and causing sudden death. The damaging effects of C. perfringens have been well-documented . By contrast, B. licheniformis mediated pro-growth effects. These results provided the background for the further bacterial community analysis.
Disturbance in the cecal microflora of chickens following the C. perfringens, Eimeria challenge and fishmeal supplementation
Occasionally, even in the absence of C. perfringens infection, improper management of poultry, such as providing high protein feed and leaving parasite infections unchecked, can result in economic losses. It is well established that C. perfringens challenge is associated with altered microflora community structures , and differential responses to the three inducing factors (fishmeal, Eimeria, and C. perfringens) have been evaluated in an NE model . However, the specific differences, including bacterial community structure and composition, in the absence or presence of C. perfringens with predisposing factors have not been thoroughly examined. To determine these differences, we investigated the community structures and taxa present in groups PC and FC.
With regard to the alpha diversity of the cecal microbiota, our findings suggested that predisposing factors, even without C. perfringens, could increase microbial diversity significantly (Shannon index) compared with that in group NC. However, the additional interfering factors (C. perfringens in combination with predisposing factors) in the PC group induced greater changes to the alpha diversity. The observed species and Chao1 indices revealed that predisposing factors alone could affect species richness significantly. Species richness relies only on a count of species and does not take into account the relative abundance of these species. Although the mechanisms underlying the interactions between C. perfringens and predisposing factors are not well understood, there is a popular theory that these factors can disturb the cecal environment, providing nutrients or preferential ecological niches for the proliferation of C. perfringens . When C. perfringens was administered in the context of predisposing factors, the nutrients and niches provided could be utilized by C. perfringens. However, predisposing factors alone provided an opportunity for the proliferation of previously absent microorganisms. Therefore, the species richness of group FC was significantly higher than that of groups NC and PC. Contrastingly, C. perfringens infection might inhibit the proliferation of minor components of the microbiota , which could explain why there were no significant differences between the observed species and Chao1 indices for groups PC and NC.
The PCoA indicated that the samples from group PC were distinct from those of group NC, which is consistent with the findings of a previous study . Whereas groups PC and NC showed distinct microbiota, samples from the FC group were not completely separate from those of either of the other two groups. This might be explained by the fact that the bacterial community structure of group FC, though not completely distinct from that of groups PC and NC, was also disturbed by the predisposing factors. The Venn diagram (S3 Fig) also showed distinct microbiota in the chickens in groups PC and NC.
B. licheniformis treatment prevented the disturbance caused by C. perfringens, Eimeria challenge and fishmeal supplementation in the cecal microbiota
Although probiotics such as B. licheniformis have been applied previously to prevent the harmful effects of C. perfringens challenge in broilers, this is the first time that the intestinal microbiota of the prevention group has been analyzed from an ecological perspective.
There were no significant differences between the BL and the PC or control groups in the three alpha diversity indices for the microbial communities. C. perfringens, Eimeria challenge and fishmeal supplementation is associated with changes in microbial diversity in chicken ceca; the lack of an apparent difference between the diversity in groups BL and NC might imply that, after probiotic pre-treatment, C. perfringens and predisposing factors were unable to induce disorder in the microbial communities, which would be reflected in the alpha diversity measures. However, the UniFrac distance analysis suggested that the microbial structure of group BL was similar to that of group FC. This result is interesting, because it reflects the fact that, after administration of B. licheniformis, C. perfringens could not alter the bacterial community structure significantly and that only the predisposing factors affected the cecal microbiota. We hypothesized that this was because of inhibition of C. perfringens by B. licheniformis. Although the specific mechanism has not been determined, many studies have demonstrated the inhibitory capacity of Bacillus spp. [19, 20].
The top 15 taxa of groups NC, BL, and PC, however, indicated disorder in the microbiota. In studies of normal chickens, cecal communities are mainly colonized by the phyla Firmicutes, Bacteroides, and Proteobacteria [34–36], which agrees with the results of our study. At the genus level, however, there is controversy over the taxa that are predominant in chicken ceca. For instance, in a study by Callaway et al. , the most abundant genera were Bacteroides and Prevotella, whereas Stanley et al.  found that Ruminococcus, Lactobacillus, and Bacteroides were predominant. These discrepancies may reflect differences in the hosts, feeding regimens, and analytical techniques ; hence, it would be inappropriate to define microbiomes in chicken ceca as “healthy” or “unhealthy”. In our study, one of the most interesting findings is that C. perfringens, administered with fishmeal and coccidia, increased the relative abundance of Lactobacillus significantly, such that it became the most abundant taxon in some samples. Considering that the number of replicates was large (n = 15), this discovery about the cecal microbial chaos might be meaningful. Lactobacillus, a widely used probiotic, inhibits the growth and colonization of C. perfringens via the production of organic acids or bacteriocins [39–41]. Nevertheless, in this experiment, an increase in Lactobacillus resulted from infection with C. perfringens. Although this result was unexpected, some previous studies have shown similar results. Research on three breeds of commercial broilers with NE revealed that, compared with controls, the proportion of Lactobacillus in the bacterial community increased with NE in Cobb chickens but decreased in Ross and Hubbard chickens . A study in 2012, which examined experimental NE in Arbor Acre broilers, found higher levels of lactobacilli in the guts of chickens with NE compared with that in the controls . In our research, the mean proportion of Lactobacillus in the group with NE was significantly higher than that in the other three groups. It was not possible for us to determine a consistent pattern in the changes of the Lactobacillus population. Several studies have concluded that the Lactobacillus fraction is not dependent on NE status or the C. perfringens dose [44, 45]. However, the present study showed that C. perfringens together with predisposing factors could cause larger perturbations in the microbiota in chicken ceca in the absence of supplementation with B. licheniformis.
LEfSe indicated that there were smaller differences between the NC and BL groups than between the BL and PC groups with regard to the number of significantly overrepresented bacterial taxa. This finding supports the notion that the disturbances caused by C. perfringens could be alleviated by treatment with B. licheniformis. The chickens in groups BL and NC seemed to have similar overrepresented bacterial taxa, which could also support the preventative effects of B. licheniformis. Most importantly, these bacterial taxa might play a crucial role in the alleviation of the disturbance of the microbiota.
Notably, compared with in the other two groups, the relative abundances of Lactobacillus and Dorea were significantly higher in group PC, which may indicate that these taxa are also important biomarkers of C. perfringens, Eimeria challenge and fishmeal supplementation chickens. Other studies have obtained similar results. For example, in a study on Crohn’s disease, several genera, including Dorea and Lactobacillus, were more abundant in the colons of patients with Crohn’s disease than in patients without inflammatory bowel disease . Dorea has great saccharolytic potential; in a study on children with irritable bowel syndrome, Dorea was overrepresented in the group treated with a low-dose fermentable oligosaccharides, disaccharides, monosaccharides, and polyols diet, and it is believed that this diet could result in decreased microbial fermentation . Therefore, it was hypothesized that, with a decrease in microbial fermentation, taxa with strong saccharolytic metabolic capacity, such as Dorea, become more active. In our study, the relative abundance of Dorea was significantly higher than that of other genera, which suggested that C. perfringens, Eimeria challenge and fishmeal supplementation affected fermentation in the ceca. The cecal microbiota of groups BL and NC showed similar overrepresented taxa compared to those of group PC, based on the LEfSe. These taxa included Bacteroides, Helicobacter, Megamonas, and Akkermansia. The decrease in Bacteroides with C. perfringens, Eimeria challenge and fishmeal supplementation was a novel finding. Many studies have examined Bacteroides, and this taxon is considered an important contributor to the intestinal health of animals. It has also been defined as a probiotic that is beneficial for weight gain and growth performance [48–50]. The fermentation products of Bacteroides inhibit sporulation of C. perfringens, and depletion of the genus Bacteroides may predispose the animal gut to C. perfringens infection and gastroenteritis . To the best of our knowledge, this is the first report of a probiotic treatment resulting in an increase in the abundance of Bacteroides in the ceca of chickens with C. perfringens, Eimeria challenge and fishmeal supplementation. This probiotic treatment could restore the microbial community to its normal status and improve the health of the chickens.
Megamonas and Helicobacter together were confirmed to be hydrogen sinks in the chicken cecum. The higher abundance of these two taxa can be explained partly by their ability to remove hydrogen, which could benefit the other members of the microbial community, as well as improve the host’s ability to recover energy from food . The significant decrease in these two taxa in group PC might indicate an effect on the potential for energy recovery. More importantly, Akkermansia, described as a wound-mucosa-associated microbiota, could contribute to the enhanced repair of mucosal wounds . The overrepresented taxa Akkermansia in group BL indicated the improved intestinal health compared with group PC.
Other taxa should also be explored. Sutterella, for example, is a potential probiotic present in pigeon “milk” that can improve the rate of growth and feed conversion ratio in chickens .
In the PICRUSt analysis, we found larger differences between PC and BL, than between the PC and NC groups (76 and 57 significantly different pathways, respectively). After screening, we found that metabolic pathways were the most common among the significantly differentially represented pathways, which could explain the large metabolic differences between group PC and the other two groups. We also investigated the KEGG pathway types and found that many metabolic pathways associated with lipids (12 out of 45) were shared between groups PC and BL, which agrees with a former study on the expression of lipid metabolism-related genes in broilers with C. perfringens, Eimeria challenge and fishmeal supplementation that were treated with B. licheniformis . Although the expression of lipid metabolism-related genes was not associated with the microbiome in each group, we speculated that changes in the microbiota caused by B. licheniformis treatment played a positive role in the expression of lipid metabolism-related genes. Another KEGG pathway type, glycan metabolism, also warrants further study. This study showed that C. perfringens, Eimeria challenge and fishmeal supplementation leads to the destruction of glycan metabolism in group PC.
Does all of this indicate that B. licheniformis had a positive influence on the cecal microbiota of chickens with C. perfringens, Eimeria challenge and fishmeal supplementation? Given that large differences between individual chickens of the same breed or experimental trial have been reported, even in carefully controlled conditions , it is impossible to define “good” or “bad” microbiota in the chicken cecum. Nevertheless, based on this trial, the microbial communities of normal chickens appeared relatively healthy. The cecal microbiota of group BL chickens was more similar to that of chickens in group NC; therefore, we concluded that disruption of the cecal microbiota of chickens with C. perfringens, Eimeria challenge and fishmeal supplementation was alleviated by B. licheniformis supplementation.