Pathological changes of highly pathogenic Bacillus cereus on Pelodiscus sinensis

Figures & data

Table 1. Primers used in PCR identification.

Primer	Sequence (5`-3`)	Annealing temperature (°C)	Productive size(bp)	Reference
16S rDNA	AGA GTT TGA TCC TGG CTC AG AAG GAG GTG ATC CAG CC	57	1407	(Li et al. 2020)
gyrB	YGGHTATAAAGTDTCNGGHGG TCNACRTCBGCRTCBGTCATRAT	50	833
SYJ153	CTACAGGTCGCAGATAAAACAGTC GCAAGGGCTAAAAATGAAATCT	53	680	(Yuan et al. 2020)
hblA	GCAAAATCTATGAATGCCTA GCATCTGTTCGTAATGTTTT	54	884	(Song et al. 2019)
hblC	CCTATCAATACTCTCGCAA TTTCCTTTGTTATACGCTGC	54	659
hblD	AATCAAGAGCTCTCACGAAT CACCAATTGACCATGCTAAT	52	430
nheA	TACGCTAAGGAGGGGCA GTTTTATTGCTTCATCGGCT	55	500
nheB	CAAGCTCCAGTTCATGCGG GATCCCATTGTGTACCATTG	58	935
nheC	ACATCCTTTTGCAGCAGAAC CCACCAGCAATGACCATATC	58	618
bceT	TTACATTACCAGGACGTGCTT TGTTTGTGATTGTAATTCAGG	55	428
cytK	CGACGTCACAAGTTGTAACA CGTGTGTAAATACCCCAGTT	52	565
Ces	GCATTTCGTGAAGCAGAGGT CCCTTTATCCCCTTCGATGT	59	699
entFM	AGAATCTAAATCATGCCACTGC AAAGAAATTAATGGACAAACTCAAACTCA	60	596	(Kim et al. 2011)
MT17	CTCCAGGAGAGGATTCGTGG TACCAAGGCAGCGAGCTTTA	56	87	(Park et al. 2017)
16S V3-V4	ACTCCTACGGGAGGCAGCA GGACTACHVGGGTWTCTAAT	55	500

Figure 1. Clinical symptoms of diseased Pelodiscus sinensis. (A) Leaving the water body, the skin was congested (arrow). (B) Neck swelling (sign), eyelid swelling (asterisk). (C) Skin hyperemia (arrow), neck swelling (sign). (D) Redness of oral mucosa and gill-like tissue (triangle) (E) Enlarged liver(arrow). (F) kidney hemorrhage (triangle), ovarian hemorrhage(asterisk), bleeding from the external wall of the gastrointestinal tract (sign). (G)The spleen is hemorrhagic and enlarged (arrow).

Figure 2. Morphological features of strain Y271. (A) Bacterial colony. (B) βhemolytic ring. (C) Gram staining (bar scale: 10 μm). (D) Scanning electron microscope (bar scale: 5 μm).

Figure 3. PCR identification of Bacillus cereus. (A) Phylogenetic tree of strain Y271 based on 16S rDNA gene. (B) Phylogenetic tree of strain Y271 based on gyrb gene. (C) M: 2000 marker; 1: Y271-1 2: Y271-2; 3: Y271-3; 4: Negative control. (D) Y271 toxin gene PCR assays. (E) The bacterial load in tissues of diseased P. sinensis caused by B. cereus. Brain 5.1 ± 0.8 × 10¹ copies/mg, liver 2.58 ± 0.29 × 10³ copies/mg, spleen 1.54 ± 0.12 × 10⁴ copies/mg, kidney 1.17 ± 0.18 × 10³ copies/mg, ovary 1.29 ± 0.04 × 10³ copies/mg, intestine 5.38 ± 0.42 × 10² copies/mg and blood 1.57 ± 0.19 × 10³ copies/mg.

Table 2. Results of Biolog identification of strain Y271.

Reaction item	Result	Reaction item	Result
A1 Negative Control	N	E1 Gelatin	P
A2 Dextrin	P	E2 Glycyl-L-Proline	B
A3 D-Maltose	P	E3 L-Alanine	B
A4 D-Trehalose	P	E4 L-Arginine	B
A5 D-Cellobiose	B	E5 L-Aspartic Acid	B
A6 Gentiobiose	B	E6 L-Glutamic Acid	B
A7 Sucrose	P	E7 L-Histidine	B
A8 D-Turanose	P	E8 L-Pyroglutamic Acid	B
A9 Stachyose	P	E9 L-Serine	P
A10 Positive Control	P	E10 Lincomycin	N
A11 PH6	P	E11 Guanidine HCl	B
A12 PH5	N	E12 Niaproof 4	N
B1 D-Raffinose	N	F1 Pectin	B
B2 α-D-Lactose	N	F2 D-Galacturonic Acid	N
B3 D-Melibiose	B	F3 L-Galactonic Acid Lactone	B
B4 β-Methyl-D-Glucoside	B	F4 D-Gluconic Acid	P
B5 D-Salicin	B	F5 D-Glucuronic Acid	B
B6 N-Acetyl-D-Glucosamine	P	F6 Glucuronamide	N
B7 N-Acetyl-β-D-Mannosamine	B	F7 Mucic Acid	B
B8 N-Acetyl-D-Galactosamine	N	F8 Quinic Acid	B
B9 N-Acetyl Neuraminic Acid	B	F9 D-Saccharic Acid	B
B10 1% NaCl	P	F10 Vancomycin	N
B11 4% NaCl	P	F11 Tetrazolium Violet	N
B12 8% NaCl	P	F12 Tetrazolium Blue	N
C1 α-D-Glucose	B	G1 P-Hydroxy-Phenylacetic Acid	B
C2 D-Mannose	B	G2 Methyl Pyruvate	N
C3 D-Fructose	B	G3 D-Lactic Acid Methyl Ester	B
C4 D-Galactose	B	G4 L-Lactic Acid	B
C5 3-Methyl Glucose	B	G5 Citric Acid	N
C6 D-Fucose	B	G6 α-Keto-Glutaric Acid	B
C7 L-Fucose	B	G7 D-Malic Acid	B
C8 L-Rhamnose	B	G8 L-Malic Acid	P
C9 Inosine	B	G9 Bromo-Succinic Acid	P
C10 1% Sodium Lactate	P	G10 Nalidixic Acid	B
C11 Fusidic Acida	N	G11 Lithium Chloride	B
C12 D-Serine	B	G12 Potassium Tellurite	B
D1 D-Sorbitol	N	H1 Tween 40	B
D2 D-Mannitol	B	H2 γ-Amino-Butyric Acid	B
D3 D-Arabitol	B	H3 α-Hydroxy-Butyric Acid	P
D4 Myo-lnositol	B	H4 β-Hydroxy-D, L-Butyric Acid	B
D5 Glycerol	P	H5 α-Keto-Butyric Acid	P
D6 D-Glucose-6-PO4	P	H6 Acetoacetic Acid	P
D7 D-Fructose-6-PO4	B	H7 Propionic Acid	P
D8 D-Aspartic Acid	N	H8 Acetic Acid	P
D9 D-Serine	P	H9 Formic Acid	P
D10 Troleandomycin	N	H10 Aztreonam	P
D11 Rifamycin SV	N	H11 Sodium Butyrate	P
D12 Minocycline	N	H12 Sodium Bromate	B

Notes: “P” = Pos; “N” = Neg; “B” = Borderline.

Figure 4. Percentage survival of P. sinensis attacked by different concentrations of Y271.

Figure 5. Histopathological observation of the healthy and diseased P. sinensis (scale bar: 50 μm and 10 μm). (A) a and b brain of a healthy P. sinensis, c, and brain of a diseased P. sinensis. (B) a and b eyes with healthy P. sinensis, c and d eyes with diseased P. sinensis. (C): a and b the eyelids of healthy P. sinensis, c, and d the eyelids of diseased P. sinensis. (D) a and b gill-like tissues of healthy P. sinensis, c and d gill-like tissues of diseased P. sinensis. (E) a and b spinal cords of healthy P. sinensis, c and d spinal cords of diseased P. sinensis. (F) a and b liver of healthy P. sinensis, c and d liver of diseased P. sinensis. (G) a and b spleen of healthy P. sinensis, c and d spleen of diseased P. sinensis. (H) a and b kidneys with healthy P. sinensis, c and d kidneys with diseased P. sinensis. (I) a and b ovarioles of healthy P. sinensis, c and d ovarioles of diseased P. sinensis. (J) a and b intestine of healthy P. sinensis, c and d intestine of diseased P. sinensis.

Figure 6. Gut microbiota analysis of P. sinensis. (A) Relative abundance of phylum levels. (B) Relative abundance of genus levels. (C) The Venn diagram showing the number of unique and shared operational taxonomic units (OTUs) of gut bacteria of healthy and diseased P. sinensis. (healthy P. sinensis: C; diseased P. sinensis: D).

Table 3. Results of antibiotic sensitivity test for strain Y271.

Medicine name	Content (piece)	Inhibition zone (mm)	Sensitivity
Florfenicol	30μg	28	S
Enrofloxacin	10μg	22	S
Neomycin	30μg	16	S
Doxycycline	30μg	24	S
Ampicillin	10μg	8	R
Penicillin	10U	11	I
Clindamycin	2μg	21	S
Cefradine	30μg	29	S
Midecamycin	30μg	22	S
Gentamicin	10μg	16	S
Minocycline	30μg	22	S
Sulfonamide	300 IU	7	R
Tetracycline	30μg	25	S
Amikacin	30μg	20	S
Erythromycin	15μg	21	S
Streptomycin	10μg	14	I
Carbenicillin	100μg	11	I

Notes: sensitive (S), moderately sensitive (M), and resistant (R).

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