Shifts in the Fecal Microbial Community of Cystoisospora suis Infected Piglets in Response to Toltrazuril

Abstract

The protozoan parasite Cystoisospora suis causes diarrhea and reduced weight gain in suckling piglets. Infections occur in the first days of life; it is transient but can lead to dysbiosis, exacerbating disease and increasing mortality. Cystoisosporosis is effectively controlled by toltrazuril treatment; however, alterations of the gut microbial composition upon infection and treatment have not been investigated. This study evaluated the development of fecal microbiota of C. suis infected piglets in response to treatment with toltrazuril. Thirty-eight conventional piglets were infected with C. suis on the first day of life (dol 1). Twenty-six of them received either parenteral or oral toltrazuril 2 days later. Fecal samples were collected pre- and post-weaning (dol 1-15 and 31-38) for microbiota analysis using 16S rRNA amplicon sequencing and during dol 5-18 to determine fecal consistency and parasite excretion. All control animals shed parasites at least once and the majority developed diarrhea, while toltrazuril-treated piglets did not excrete parasites and only had low levels of diarrhea. Age-related shifts in the fecal microbiota composition and increase in diversity and species richness were seen until after weaning. Parasite infection disrupted bacterial maturation 2 weeks after infection. Irrespective of the route of administration, fecal communities of piglets in the treated groups clustered separately and were more diverse compared to that of control piglets during the acute phase of infection on dol 11. Control piglet feces showed higher levels of Fusobacteriaceae and Veillonellaceae, while Ruminococcaceae, Lachnospiraceae, S24-7, Clostridiaceae, and Erysipelotrichaceae were more abundant in feces of treated piglets on dol 11. Thereafter, treatment-related effects on the microbial communities were small and mainly detectable on dol 34 (5 days post-weaning), potentially indicating that the oral toltrazuril treatment might have had long-term effects on host physiological responses post-weaning. Irrespective of the administration route, toltrazuril prevented C. suis-related dysbiosis and maintained species richness and diversity on dol 11. In addition to cystoisosporosis prevention, toltrazuril seems to contribute to the stabilization of the gut microbial development during the suckling phase and thus may reduce the need for antibiotics to control infections with secondary bacterial enteropathogens in C. suis-infected suckling piglets.

Keywords: 16S rRNA; diarrhea; gut health; microbiota; piglet.

FIGURE 1

Non-metric multidimensional scaling (NMDS) plot of pairwise Bray-Curtis dissimilarities among bacterial communities over time at days of life: 1 (dark gray), 3 (red), 5 (green), 11 (dark blue), 15 (light blue), 31 (pink), 34 (yellow), and 38 (light gray).

FIGURE 2

The most relatively abundant genera (hit counts) in feces in relation to piglet age (day of life 1, 3, 5, 11, 15, 31, 34, and 38).

FIGURE 3

Non-metric multidimensional scaling (NMDS) plot of pairwise Bray-Curtis dissimilarities among bacterial communities at the genus level in feces of suckling and weaned piglets (>0.01% relative abundance) demonstrating treatment effect (green: group C = untreated control), compared to treated groups A (gray = parenteral toltrazuril) and B (red = oral toltrazuril) on day of life 11 (stress value: 0.1273; stress value = approximation for the strength of the pairwise dissimilarities and are in relation to the axes dimensions of the graph).

FIGURE 4

Loading plots of sparse partial least square-discriminant analysis (sPLS-DA) showing the most discriminant bacterial genera (relative genera abundance >0.05%). Positive fold changes indicate lower abundance; negative fold changes indicate higher abundance compared to the treatment means. Comp 1; component 1 of the sPLS-DA.

FIGURE 5

Associations between the most discriminant bacterial genera from dol 11 and (A) α-diversity (Shannon and Simpson) and species richness (S_RichN); (B) daily body weight gain between dol 8 and 15; (C) area under the curve for the fecal score from dols 5–18 (AUC_Feces); and (D) area under the curve of the oocysts per gram of feces from dol 5 to dol 18 (AUC_OpG). For statistical calculation of AUC see Shrestha et al. (2020). Covariations between the most relevant bacterial genera (relative abundance >0.05% of all reads), diversity and species richness, DBWG, AUC_Feces and AUC_OpG were established separately using sparse partial least squares regression and relevance networking. The networks are displayed graphically as nodes (parameters) and edges (biological relationship between nodes). The edge color intensity indicates the level of association: red = positive, green = negative. Only the strongest pairwise associations were projected. Unclass Ruminoc: unclassified Ruminococcaceae genus; Lachnosp: unclassified Lachnospiraceae genus.