Early life colonization of gut microbiota is vital to animal health, as it influences the microbial profile and intestinal health at later stages of life . Pathogenic protozoan parasites can interfere with the intestinal microbiota [25-28]. In suckling piglets, C. suis commonly parasitizes the epithelial cells of the small intestine and is closely related to Cystoisospora belli of humans  and is considered a major diarrheal pathogen . Its synergism with toxigenic CpA, another important cause of piglet diarrhea, has been previously demonstrated, as was the beneficial effect of early treatment with the anticoccidial drug toltrazuril in disrupting this synergism by effective parasite control . However, little is known about the overall development of the intestinal microbiota of C. suis infected piglets and the influence of antiparasitic treatment.
In this study, 16S rRNA gene sequencing was used to determine the effects of parenteral and oral toltrazuril treatments on the development of the fecal microbiota in C. suis infected piglets during the suckling and early post-weaning period. Alterations in the acute phase of infection were mostly present on dol 11, whereby both toltrazuril treatments prevented the loss of bacterial species and diversity compared to piglets in the control group, which showed typical clinical signs of C. suis infection. Moreover, it was obvious that both toltrazuril treatments, parenteral and oral, affected the fecal taxa composition largely identically. This led to the assumption that alterations on the fecal microbiota after toltrazuril treatments might be due to an indirect effect via interruption of parasite development and subsequent maintenance of small intestinal functions during the acute phase of infection.
The present results confirmed previously reported age-related patterns in the fecal microbiota composition [19, 30, 31] beginning with more aero-tolerant taxa (e.g. Escherichia followed by Bacteroides and Fusobacterium) in the first days of life towards a complex and diverse community post-weaning, thereby contributing to the increase in species richness and evenness with age. As the intestinal colonization mainly takes place post-farrowing, it was expected that the composition on the first dol largely differed from that of the other sampling days. The bacterial community from dol 3 clustered apart from those of dols 5, 11 and 15; however, the shifts in the bacterial community from pre- to post-weaning were more drastic, emphasizing the importance of the type of ingested food. Piglets had access to creep feed from the second week of life but was expected that the intake of solid feed was very low at the beginning and differed among piglets and litters. This was reflected by the greater inter-individual differences before weaning which converged post-weaning showing a greater uniformity among the microbial communities of different piglets. Similar patterns in α- and β-diversities in the gut microbiota of piglets have been reported previously . Therefore, the present results confirmed the progressing microbial maturation during the lactation period, which, after the complete transition to solid feed, led to the gradual establishment of a more stable community post-weaning [19, 31]. A sharp decrease in the relative abundances of Fusobacterium and Bacteroides was found from the suckling to post-weaning period. Bacteroides species utilize milk oligosaccharides and host-derived glycans as carbon sources and often decline post-weaning [32, 33]. Consistent with this, members of Clostridiaceae, Lactobacillus and Prevotella increased in their abundance after weaning, reflecting the dietary change from sow’s milk to a solid cereal-based diet, bacterial substrate preferences and metabolic capabilities [31, 34-36].
The gut microbiota has essential role in resistance to colonization by enteric pathogens, including parasitic protozoa, in the gut [37, 38]. Therefore, disruption in the compositional development of gut microbiota in early life might also alter the pathophysiology of parasitic infection either by promoting infection or by conferring resistance [39-41]. The decrease in diversity on dol 11 and total bacteria on dol 15 may support previous findings that protozoan infections can disrupt the enteric microbiota. As a consequence, the resulting dysbiosis may alter the clinical and pathological outcome of parasitic infections, as previously shown for coccidiosis in chickens [27, 42] and mice  and cryptosporidiosis in mice and humans [28, 43].
Disruption of the intestinal epithelium caused by C. suis infection can induce diarrhea in the absence of other enteropathogens [11, 44] but is aggravated by co-infection with other enteric pathogens which are also common in conventional pigs . Compared to gnotobiotic piglets, C. suis infection markedly influenced early mortality in piglets that received bacterial flora derived from intestinal contents of conventional piglets with clinical coccidiosis, indicating that gut microbiota have opportunistic rather than synergistic function in C. suis infected piglets . Since the majority of the life cycle of C. suis takes place in intestinal epithelial cells and direct interactions of extracellular parasite stages with the gut microbiota are short and transient, perturbation of the microbial population is likely due to the damage to the intestinal epithelium caused by replicating parasites [5, 45], rather than by direct interaction of the parasite and the bacterial population. For this reason, present relevance networks for OpG excretion and fecal score may support the previously observed correlation between clinical coccidiosis, infection with CpA and early toltrazuril treatment in suckling piglets  and chickens , thereby confirming the assumption that coccidia might create a favorable environment for colonization by opportunistic gut pathogens.
The pig gut microbiota represents a highly complex and dynamic microbial community which is influenced by many factors including the environment, age, diet and breed . Since all animals in the present study were derived from three litters that were fed the same diet and were housed under identical conditions, plus that a random block design was applied to assign piglets to the different treatment groups to account for litter effects, the observed differences in bacterial diversity and taxonomy can be assumed to be the consequences of toltrazuril applications. Following infection and treatment, the greatest treatment-related differences in the gut microbiota occurred on dol 11, when all animals from the control group had shed oocysts at least for one day. Interestingly, before and after that, piglets from the different groups had largely similar fecal microbial patterns. Notably, both toltrazuril treatments prevented the loss of bacterial diversity as was observed in the piglets of the control group on dol 11. This is in accordance with previous studies in which lowest α-diversities were reported in Eimeria challenged chickens [48, 49]. In general, bacterial species richness and diversity often reflect stability and resilience of the gut ecosystem and are therefore considered as potential markers for gut health . Several reasons for our observations are conceivable. First, the missing effect of toltrazuril treatments on the other dols hints at a high plasticity of the fecal microbiota in these young animals. Second, given that dol 11 corresponded to the day of the maximum prevalence of diarrhea and a decline of oocyst excretion in the control group, C. suis infection-related loss of gut integrity and function as well as increased intestinal disruption might have caused the loss of diversity in the control piglets. Conversely, both toltrazuril treatments probably maintained the gut integrity by inhibiting the establishment of C. suis and subsequent dysbiosis associated with the proliferation of specific bacteria and thus maintained a high bacterial diversity in the gastrointestinal tract. Third, microbial transitions following inclusion of pre-starter diet at dol 14 (pre-weaning) and weaning stress might have masked treatment effects at later time points.
Toltrazuril is a coccidiocidal drug that is almost completely absorbed from the intestine following oral administration , and parenteral (intramuscular) administration of toltrazuril resulted in more sustained concentrations in jejunal and ileal tissues and contents compared to oral application . Although drug concentrations at the predilection site of C. suis is crucial for its pharmacological effects, higher drug concentration for longer period might also have an indirect effect on other local gut microbiota. Since, Escherichia rely on other obligate gut anaerobes for mono- and disaccharides needed for their growth , inhibition of these anaerobes might have resulted lower abundances of Escherichia in piglets that received parenteral toltrazuril compared to those who received oral toltrazuril.
Whether the promotion of the phylum Bacteroidetes as a whole in both treatment groups on dol 5 was related to a direct effect of toltrazuril on the gut microbiota or an indirect effect via maintaining epithelial integrity cannot be differentiated by the present results. However, the reduced abundance of the genus Bifidobacterium and the higher abundance of the genus Coprococcus in the parenteral toltrazuril group compared to the oral toltrazuril group on dol 5 indicated other, possibly more direct effects of toltrazuril or its metabolites on the gut microbiota, which needs further investigation.
Treatment-associated bacterial shifts were largely evident on dol 11 with a marked increase in the abundance of Fusobacteriaceae and Veillonellaceae in the control group. Interestingly, increased abundance of the genus Fusobacterium coincided with the highest prevalence of diarrhea in control piglets on dol 11. Relevance networking also revealed that, besides Sutterella, Fusobacterium was negatively associated with α-diversity as shown previously [50, 54]. Fusobacterium spp. are a gram-negative bacilli, and increased relative abundance has been reported in piglets with diarrhea [55, 56], calves with cryptosporidiosis  and human with colorectal cancer . The altered composition of glyco-conjugates in villous enterocytes, enhanced mucus secretion by goblet cells and damage to microvilli during acute C. suis infection [59-61] may promote intestinal colonization by Fusobacterium and foster the development of severe diarrhea. Lower abundance of Fusobacterium (as demonstrated by sPLS-DA) and absence of diarrhea in toltrazuril treated piglets compared to the control piglets advocate a significant contribution of this genus to the occurrence of diarrhea in the presence of C. suis. Similarly, significantly higher abundance of Veillonellaceae in the control group might be associated with an intense inflammatory response following destruction of the epithelial lining by C. suis. An increased abundance of Veillonellaceae was positively correlated with the incidence of inflammatory bowel disease in humans , although the exact underlying mechanism is yet to be elucidated.
Loss of intestinal integrity, increased motility and changes in nutrient flows may the reasons for the greatly reduced abundances of some of the major anaerobic commensal bacteria such as Ruminococcaceae, Lachnospiraceae, Bacteroidales S24-7, Clostridiaceae and Erysipelotrichaceae in the control compared to the treated groups. For instance, members of Ruminococcaceae and Lachnospiraceae are main contributors to butyrate biosynthesis , and their increased abundance has been associated with increased cell proliferation and recovery of intestinal morphology . Detailed studies on how toltrazuril assist in preventing C. suis induced gut dysbiosis will help to develop strategies for modulation of gut microbiota to restore homeostasis and promote piglet health in affected litters.
Sparse partial least square-discriminant analysis identified specific genera that could potentially contribute to differentiate the treatment groups. Apart from Fusobacterium, both treated groups displayed a lower relative abundance of the most discriminating genera Dialister, Sutterella and Anaerovibrio. Dialister and Sutterella have been associated with intestinal inflammatory disorders . Therefore, lower relative abundances of these genera in treated groups also advocate the role of toltrazuril in maintaining gut homeostasis in infected piglets, probably indirectly by inhibition of intracellular parasite replication and damage of epithelium. In line with that, relevance networking suggested that the abundances of Sutterella and Dialister were negatively associated with daily body weight gain during the acute phase of infection, potentially supporting a role of these taxa during acute gut inflammation. In contrast, Eubacterium and Oscillospira were positively correlated with body weight gain during the acute phase of infection. Oscillospira is often reported to decline in inflammatory diseases  and both taxa have capacities to utilize resistant starch and are important butyrate producing genera [35, 65], which may promote intestinal integrity and indirectly support the physical development of the piglet itself. Further studies are required to understand how such microbial modulations contribute to body weight development in the suckling piglet.
The area under the curve for the fecal score (indicating diarrhea) was positively related to Clostridium and Turicibacter on dol 11, both of which are commensals in the porcine gut. Nevertheless, Clostridium has been associated with gastrointestinal disorders and diarrhea in several mammalian species, including humans [66-68] and suckling piglets , showing higher relative abundance in diarrheic compared to healthy individuals. Positive associations of the genera Sutterella, Dialister and Clostridiales with the area under the curve for oocyst per gram feces in the present study (indicating the severity of infection) also indicated that the gut microbiota in general is probably considerably affected by C. suis infection. By contrast, repeated supplementation of probiotic bacterial cocktail immediately after birth had no influence on fecal consistency and amount and duration of oocyst excretion in C. suis infected piglets ; therefore, this influence appears largely one-sided. However, further studies are necessary to elucidate the underlying role of specific microbial populations in the establishment and overall consequences of cystoisosporosis.