A novel bacterial pathogen of Biomphalaria glabrata: a potential weapon for schistosomiasis control?

Abstract

Background: Schistosomiasis is the second-most widespread tropical parasitic disease after malaria. Various research strategies and treatment programs for achieving the objective of eradicating schistosomiasis within a decade have been recommended and supported by the World Health Organization. One of these approaches is based on the control of snail vectors in endemic areas. Previous field studies have shown that competitor or predator introduction can reduce snail numbers, but no systematic investigation has ever been conducted to identify snail microbial pathogens and evaluate their molluscicidal effects.

Methodology/principal findings: In populations of Biomphalaria glabrata snails experiencing high mortalities, white nodules were visible on snail bodies. Infectious agents were isolated from such nodules. Only one type of bacteria, identified as a new species of Paenibacillus named Candidatus Paenibacillus glabratella, was found, and was shown to be closely related to P. alvei through 16S and Rpob DNA analysis. Histopathological examination showed extensive bacterial infiltration leading to overall tissue disorganization. Exposure of healthy snails to Paenibacillus-infected snails caused massive mortality. Moreover, eggs laid by infected snails were also infected, decreasing hatching but without apparent effects on spawning. Embryonic lethality was correlated with the presence of pathogenic bacteria in eggs.

Conclusions/significance: This is the first account of a novel Paenibacillus strain, Ca. Paenibacillus glabratella, as a snail microbial pathogen. Since this strain affects both adult and embryonic stages and causes significant mortality, it may hold promise as a biocontrol agent to limit schistosomiasis transmission in the field.

Fig 1. A. Infected Biomphalaria glabrata exhibits white nodules.

B. Dissected snail presents nodules on mantle (Mt), hepato-pancreas (Hp) and ovotestis (Ov) regions.

Fig 2. A. An exophytic nodule contains a homogenous population of bacillus- like bacteria.

B. These rod-shaped bacteria present a mixed pattern of Gram staining and produce some endospores indicated by the black arrow.

Fig 3. Histological sections of Biomphalaria glabrata digestive organs diseased by Paenibacillus (A and B) and control (C).

(A) The hepatic interlobular space is heavily invaded by the bacterial colonies which separate the hepatic lobules widely (d = 159 μm). The compression exerted (arrows) provoke mechanical damages with degeneration and atrophy of the digestive gland cells. (B) The intestine is densely surrounded by big bacterial colonies causing a slight compression. (C) The control shows normal liver tissue with numerous lobules separated by tight interlobular spaces and normal organization of the midintestine. AG: Albumin gland; BC: Bacterial colony; CLF: Collagen-like fibers; d: Interlobular distance; F: Feces; ILCT: Interlobular connective tissue; L: lumen; LL: Liver lobule; MI: Midintestine; ML: Muscle layer; MM: Mucous membrane; S: Serosa; TP: Tunica propria.

Fig 4. Histological sections of reproductive organs from Biomphalaria glabrata infected by Paenibacillus (A and C) and control (B and D).

(A) The ovotestis interacini space is heavily invaded by the bacterial colonies which exert compression (arrows), separating the acini widely (d = 81 μm). (B) The control shows normal acini organisation in the ovotestis with numerous spermatozoa and some ova. (C) Muciparous and albumin glands are moderately invaded. A: Acinus; AG: Albumin gland; BC: Bacterial colony; d: Interacini distance; F: Flagella; FGC: Female germinal cell; IACT: Interacini connective tissue; L: Lumen; MG: Muciparous gland; MGC: Male germinal cells; MO: Mature ova; N: Nucleus; Ov: Oviduct; Pr: Prostate; PTR: Proliferative tissue response; TP: Tunica propria.

Fig 5. Histological analysis of the bacterial invasion within other organs of Biomphalaria glabrata.

(A) The dorsal ridge of the mantle cavity with prominent folds invaded by Paenibacillus colonies. (B) The kidney tubular portion of Biomphalaria glabrata showing Paenibacillus colonies exerting compression on the epithelial tissue. The epithelium, constituted by one layer of cells, shows an irregular wavy appearance and a large vacuole in each cell. (C) Presence of Paenibacillus colony in the heart cavity. (D and E) Many bacterial colonies are present in the headfoot region with no visible damages on the genitalia organs. BC: Bacterial colony; CM: Columnar muscle; DRF: Dorsal ridge folds; EMC: Epithelium of the mantle cavity; MC: Mantle cavity; EC: Epithelial cells; L: Lumen; PV: Pulmonary vein; U: Ureter; V: Vacuole; Ep: Epicardium; PC: Pericardial cavity; SPK: Saccular portion of the kidney; GR: Ganglion ring; Pr: Prostate; PC: Penial complex; S: Statocyst; SRS: Seminal receptacle sac; VD: Vas deferens; BS: Blood space; DCT: Dense connective tissue.

Fig 6. Molecular phylogenies using Bayesian analysis of (A) 16S nucleotide sequences and (B) Rpob aminoacid sequences.

Numbers above nodes represent posterior probabilities recovered by the Bayesian analysis.

Fig 7. Survival time of B. glabrata (BgBRE) exposed to either uninfected (a) or bacteria-infected snails (BgVEN).

A Kaplan-Meier analysis was performed to analyze survival data.

Fig 8. A. Effect of Candidatus Paenibacillus glabratella exposure on egg masses and juvenile snails production.

B and C. Photomicrographs of egg masses from unexposed and exposed snails, respectively. The arrow indicates the presence of Ca. Paenibacillus glabratella inside each egg from exposed snail population.