ABSTRACT: Along with the prevalence of edible frog farming in China, the outbreak of a deadly infectious frog diseased, called frog meningitis (or cataracts and torticollis), has increased in frequency and geographical range dramatically. More than 10 bacterial species, belonging to 8 genera, has been reported as its potential pathogens. Diseased frogs typically manifest as torticollis, cataracts, edema and finally death, resulting in huge economic loss. Currently, the pathogenesis of this disease has not been investigated systematically. Here, we summarized the pathological stages of infected black-spotted frogs (Pelophylax nigromaculata) in Sichuan province according to their symptoms, typically progressing of pathological stage with only torticollis to stage with both torticollis and cataracts. On the basis, we analyzed the pathogenesis by a combination of comparative environmental analysis, microbiomics and transcriptomics. Results showed that more severely infected frog ponds tended to have lower water alkalinity. Elizabethkingia miricola was the only bacteria, whose abundance was positively correlated with the disease degree, and it has absolute dominance in the eyeball and brain of some torticollis-cataracts frogs. E. miricola and several other bacterial species, which belonged to pathogenic genera of meningitis, might be constitutively existed in the resident microbiome in frogs or their environment. Activations of infectious processes and immune responses related pathways were the major difference between health and diseased frogs at transcriptional level. Despite transcriptional activation of immunoglobulins was observed in both torticollis-only and torticollis-cataracts frogs, transcriptional activation of innate immune system (including MHC, toll-like receptor, and cathelicidins) in brain, inflammation system (including interleukins and receptors) in brain, and acute phase proteins (including transferrins and fibrinogens) in both liver and brain was only observed in torticollis-cataracts frogs. Activation of inflammation and the resulting higher vascular permeability in torticollis-cataracts frogs could explain the severe brain infection, cooccurrence of torticollis and cataracts, and systemic edema in torticollis-cataracts frogs. In addition, meningitis could also result in reduction in energy production in liver, and this was more severe in torticollis-cataracts frogs. In conclusion, our results suggested environment might have a role in susceptibility of frog meningitis. E. miricola was the most likely pathogen of meningitis of black-spotted frogs in Sichuan. Refer to the pathogenesis of human meningitis, excessive inflammation likely played a critical role in the progress of frog meningitis, and its resulted sepsis and organ failure might be the direct cause of infected frogs.
