Impacts of canine distemper virus infection on the giant panda population from the perspective of gut microbiota

Abstract

The recent increase in infectious disease outbreaks has been directly linked to the global loss of biodiversity and the decline of some endangered species populations. Between December 2014 and March 2015, five captive giant pandas died due to canine distemper virus (CDV) infection in China. CDV has taken a heavy toll on tigers and lions in recent years. Here, we describe the first gut microbiome diversity study of CDV-infected pandas. By investigating the influence of CDV infection on gut bacterial communities in infected and uninfected individuals and throughout the course of infection, we found that CDV infection distorted the gut microbiota composition by reducing the prevalence of the dominant genera, Escherichia and Clostridium, and increasing microbial diversity. Our results highlight that increases in intestinal inflammation and changes in the relative abundances of pathogen-containing gut communities occur when individuals become infected with CDV. These results may provide new insights into therapeutics that target the microbiota to attenuate the progression of CDV disease and to reduce the risk of gut-linked disease in individuals with CDV. In addition, our findings underscore the need for better information concerning the dynamics of infection and the damage caused by pathogens in panda populations.

Figure 1. Comparison of the gut microbiota composition between CDV-infected individuals and healthy controls.

(A) Relative contribution of the top 20 dominant genera in each sample. Genera that differed significantly between the CDV-infected individuals and the healthy controls are marked with asterisks (P < 0.01 using one-way ANOVA). The Shannon (B) and Simpson (C) indices were used to estimate diversity (data shown as the mean ± SEM). A Venn diagram illustrating the overlap of OTUs in the gut microbiota between individuals with or without CDV infection (D).

Figure 2. Greater compositional heterogeneity among CDV-infected gut microbiomes than among the uninfected controls.

(A) The difference in the composition of the gut microbiota is illustrated by principal-coordinate plots of the Euclidean distances among samples. All pairwise comparisons involved the first, second and third principal axes. The first, second and third principal axes explained most of the variance (63.1%, 19.9% and 15.6%, respectively). Dots and surrounding areas correspond to gut bacterial communities from the healthy controls (blue) and the CDV-infected individuals (red). (B) The relative abundance of bacterial phylotypes between the CDV-infected samples (red bars) and the healthy control samples (blue bars). Asterisks indicate significant differences (P < 0.01), and the error bars denote 95% confidence intervals for the mean values.

Figure 3. The taxa whose abundances differed between the CDV-infected individuals and the healthy controls were identified by LEfSe.

(A) Taxonomic cladogram obtained from LEfSe sequence analysis (relative abundance ≥0.5%). Biomarker taxa are highlighted by colored circles and shaded areas (CDV-positive samples are shown in red and healthy control samples are shown in blue). Each circle’s diameter reflects the abundance of that taxa in the community. (B) The taxa whose abundance differed between the CDV-infected samples (red bars) and the healthy control samples (blue bars). The cutoff value of ≥2.0 used for the linear discriminant analysis (LDA) is shown.

Figure 4. Increase in the frequency of bacteria from disease-associated genera in the CDV-infected individuals.

Relative abundance of Arcanobacterium, Prevotella, Corynebacterium and Peptoniphilus in the gut microbiota of GP-603 and GP-754 at different time points as the infection progressed. The longitudinal samples (TN) shown in Fig. 4 match the dates of sampling listed in Table 1.

Figure 5. Intestinal clinical signs and pathological changes among the CDV-infected giant pandas, China, 2014.

CDV-infected pandas showed signs of (a) severe enteritis with intestinal hemorrhage of the serosa and (b) necrosis of the intestine. Fixed tissue samples were stained with hematoxylin and eosin (original magnification X100, scale bar = 200 μm).

Figure 6. CDV infection modulates cytokine release and increases the systemic inflammatory response.

Quantitative RT-PCR analysis of the expression of pro-inflammatory TNF-α, IL-1β, IL-6 and IL-18 in plasma from giant pandas at day 7 post CD onset is shown as fold change ± SEM. Asterisks indicate statistically significant differences (P < 0.01 by one-way ANOVA), and the error bars denote 95% confidence intervals for the mean values.