Vibrio cholerae may be transmitted to humans from bullfrog through food or water

Bullfrog is one of the most important economic aquatic animals in China. It is widely cultured in southern China, and is a key breed recommended as an industry of poverty alleviation in China. During recent years, a fatal bacterial disease has often been found in cultured bullfrogs. The clinical manifestations of the diseased bullfrogs were severe intestinal inflammation and even anal prolapse. A bacterial pathogen was isolated from the diseased bullfrog intestines. The bacterium was identified as Vibrio cholerae using morphological, biochemical and 16S rRNA phylogenetic analysis. In this study, V. cholerae was isolated and identified from diseased bullfrogs for the first time, providing a basis for the diagnosis and control of the disease. At the same time, it was also found that V. cholerae may be transmitted to humans from bullfrogs through bullfrog food and aquaculture water, creating a serious threat for human health. Therefore, society should pay attention to the modes of transmission of Vibrio cholerae from bullfrog and formulate reasonable safety measures to avoid disasters.


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Bullfrog belongs to the Ranae and Rana, family and genus, respectively, of the order Anura industries (Gao, 2016). Therefore, bullfrog has been favored by many consumers since it was first suspension was adjusted to about 10 4 , 10 6 and 10 8 CFU/mL. One hundred and twenty healthy 116 bullfrogs were randomly divided into four groups (A, B, C and D) with 30 in each group. Group A,

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B and C were used as the experimental groups, while group D was used as the control group. The 118 bullfrogs in group A-C were intraperitoneally injected with 0.1 mL of the bacterial suspension, and 119 the concentrations of the bacterial suspensions used were 10 8 , 10 6 and 10 4 CFU/mL respectively, 120 that is that the injection doses were 10 7 , 10 5 and 10 3 CFU/frog, while the bullfrogs in group D were suspension was used to coat MH agar, and the selected drug sensitive paper was pasted on the plate.

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The drug content of the paper is shown in the

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Agglutination at 2 + or more was considered as a positive result.

The epidemic time and clinical symptoms of diseased bullfrog
Epidemiological investigations showed that the disease affected the entire breeding cycle of 146 the bullfrogs, especially during the high temperature season. The increase in feeding intensity ( Fig.   147 1A-C) made the intestinal tract of bullfrogs more prone to inflammation, leading to anal abscission 148 (Fig. 2). The diseased bullfrogs showed no obvious symptoms on the surface of their body.

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Dissection showed that the diseased bullfrogs suffered slight congestion and swelling in its viscera,  The result of Gram staining showed that the isolate NW01 was red, indicating that it was gram 168 negative. The specific physical and chemical characteristics of the bacteria are shown in Table 1.

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The physical and chemical characteristics showed that NW01 was Vibrio cholerae. The 16S rRNA  Table 1 Physiological and biochemical characteristics of the NW01 strain 181 Note: +, positive; -, negative. In the pathogenicity study of the isolate, the experimental groups all died to varying degrees 191 (Fig. 4). The mortality rates of group A, B and C were 100%, 80% and 23.33%, respectively. The The sensitivity of NW01 to 20 antibiotics was determined. The results showed that NW01 was 203 resistant to β -lactams, aminoglycosides, macrolides, tetracyclines and sulfonamides, but sensitive 204 to cephalosporins, quinolones and amido alcohols. Among the sensitive drugs, neomycin,

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doxycycline and florfenicol are allowed to be used in aquaculture (Table 2). Therefore, neomycin 206 can be used for a course of 7 days to control the spread of the disease, but it cannot easily improve 207 bullfrog anal prolapse. Therefore, neomycin was selected to treat bullfrog enteritis. A 208 209  . Therefore this series of findings is worthy of attention.

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Bullfrogs belongs are amphibian but water should not cover its neck during the process of 292 breeding, therefore bullfrog pools contain only a small amount of water. Bullfrogs eat a lot and 293 discharge a lot of feces and urine. Therefore, the water used for breeding bullfrogs is often black,

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with a foul smell. The ammonia nitrogen index is often dozens of times higher than that which 295 should be used for breeding, and is full of organic matter. These observations show that the 296 aquaculture water and living environment of bullfrog provides rich nutrition for the reproduction of 297 V. cholerae. Therefore, the water used in bullfrog breeding is likely to act as a culture medium of 298 V. cholerae and becomes the mother liquor of V. cholerae. The mother liquor is directly discharged 299 into natural waterbodies without treatment and can permeate drinking water sources, resulting in 300 severely detrimental consequences.

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Although only non-O1 and non-O139 V. cholerae were found in bullfrogs in this study, it is  Agricultural Talent Support Plan (2016-139). Thanks are also due to the anonymous reviewers who 316 provided detailed comments that helped to improve the manuscript.

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The authors declare that they have no known competing financial interests or personal 319 relationships that could have appeared to influence the work reported in this paper.

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The authors confirm that the ethical policies of the journal, as noted on the journal's author 322 guidelines page, have been adhered to. No ethical approval was required as human or animal 323 subjects were not involved in this study.

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All data generated or used during the study appear in the submitted article.