Candidate Animal Disease Model of Elizabethkingia Spp. Infection in Humans, Based on the Systematic Pathology and Oxidative Damage Caused by E. miricola in Pelophylax nigromaculatus

Abstract

Most species of the genus Elizabethkingia are pathogenic to humans and animals, most commonly causing meningitis. However, our understanding of the pathogenic mechanisms involved is poor and there have been few pathological studies of Elizabethkingia spp. in animals. To understand the host injury induced by Elizabethkingia spp., we established a model of E. miricola infection in the black-spotted frog (Pelophylax nigromaculatus). The systematic pathology in and oxidative damage in the infection model were investigated. Our results show that recently isolated E. miricola is a bacterium that mainly parasitizes the host brain and that neurogenic organs are the predominant sites of damage. Infection mainly manifested as severe brain abscesses, meningoencephalitis, necrotic spondylitis, and necrotic retinitis. The liver, spleen, kidney, gastrointestinal tract, and lung were also affected to varying degrees, with bacterial necrotic inflammation. P. nigromaculatus also suffered enormous damage to its oxidative system during E. miricola infection, which may have further aggravated its disease state. Our results provide a preliminary reference for the study and treatment of Elizabethkingia spp.-induced neurological diseases in animals.

Figure 1

The phylogenetic tree was generated with MEGA 7.0 based on an alignment of the 16S rRNA gene sequences of the isolated strains (fy-70815 1–3) and related species.

Figure 2

Gross lesions in E. miricola-infected P. nigromaculatus. (a) Head biased to one side; (b) abdomen and thigh swelling; (c) congestion of the lungs, hypoplasia of the ovary; (d) left: normal eyes and right: cataracts; (e) top: normal spine and bottom: rachiocampsis; (f) left: normal thigh and right: redness on frog thigh. (g) Morbidity (%) among P. nigromaculatus during E. miricola infection. (h) Symptom types in frogs of different groups. (i) Survival (%) of P. nigromaculatus during E. miricola infection. P values and hazard ratios (HRs) are relative to the control group.

Figure 3

Dynamic systematic pathology of P. nigromaculatus caused by E. miricola infection. (a) Histopathological changes induced by E. miricola (▲: inflammatory cell infiltration; →: necrotic cells; E: edema; C: congestion; H: hyperplasia). (b) Overall health status (total index) of frogs in different groups, based on histopathological lesions. (c) Ultrastructural changes induced by E. miricola (N: nucleus; M: mitochondria; ER: endoplasmic reticulum; L: lysosome). P < 0.05 or ^∗∗P < 0 01 represents a significant difference or highly significant difference between groups. Dynamic systematic pathology of P. nigromaculatus caused by E. miricola infection.

Figure 4

Organs parasitized by E. miricola in P. nigromaculatus. (a) PCR identification of bacteria in diseased frogs. Up: identification of E. miricola with 16S-rRNA-specific primers; 1: Escherichia coli; 2: Klebsiella pneumoniae; 3: Aeromonas veronii; 4: A. hydrophila; 5: Pseudomonas plecoglossicida; 6: Stenotrophomonas sp. Down: confirmation of strains of P. nigromaculatus in the experimental group. (b) Tissue distribution of E. miricola in P. nigromaculatus. Different lowercase letters indicate significant differences between groups.

Figure 5

Antioxidation capacity of P. nigromaculatus in different groups. (a–c) Assessment of TAC, SOD, and MDA in P. nigromaculatus in different groups. (d–f) Expression of SOD, CAT, and GPX mRNAs in P. nigromaculatus. P < 0.05 or ^∗∗P < 0 01 represents a significant difference or highly significant difference between groups.

Figure 6

Systemic infection model of E. miricola against P. nigromaculatus.