Complete Blood Count (CBC) and biochemical analysis results
The complete blood count and biochemical analysis results of the red panda are shown in table 1. Compared with a report which studied the range of blood physiological and biochemical parameters of 28 healthy red pandas, the values of neutrophils, Alanine aminotransferase (ALT), Aspartate aminotransferase (AST) and Blood urea nitrogen (BUN) in this red panda were significantly increased while the other parameters were normal [11].
Table 1 Results of complete blood count and biochemical analysis
Projects
|
Units
|
Value
|
WBC
|
×109/L
|
9.66
|
Neu
|
×109/L
|
6.97
|
Lym
|
×109/L
|
1.69
|
Mon
|
×109/L
|
0.81
|
Eos
|
×109/L
|
0.16
|
Bas
|
×109/L
|
0.03
|
Neu%
|
-
|
72.2
|
Lym%
|
-
|
17.5
|
Mon%
|
-
|
8.4
|
Eos%
|
-
|
1.6
|
Bas%
|
-
|
0.3
|
RBC
|
×109//L
|
6.48
|
HGB
|
g/L
|
91
|
MCH
|
pg
|
14.1
|
MCHC
|
g/L
|
342
|
PLT
|
-
|
571
|
TP
|
g/L
|
83.1
|
TB
|
μmol/L
|
2.37
|
TBA
|
μmol/L
|
7.6
|
ALT
|
U/L
|
1127
|
AST
|
U/L
|
482
|
ALP
|
U/L
|
90
|
CK
|
U/L
|
485
|
TC
|
mmol/L
|
6.22
|
TG
|
mmol/L
|
0.44
|
BUN
|
mmol/L
|
13.64
|
Cr
|
μmol/L
|
110
|
UA
|
μmol/L
|
42.3
|
GLU
|
mmol/L
|
5.01
|
Histopathological observations of the red panda
Histopathological observation showed that glomerular congestion in the renal cortex, necrosis of the renal tubule, disappearance of the luminal structure, and infiltration of neutrophils, lymphocytes, plasma cells and macrophages were seen in the slides(fig.1 A). The cord-like lesions can be seen in the medulla area, shown as part of the normal collecting duct structure were replaced by a large amount of inflammatory cell infiltration (fig.1 B). In the liver, there was vesicular degeneration or steatosis of hepatocytes, mild congestion, and inflammatory cell (neutrophils, macrophages and lymphocytes) infiltration around the blood vessels (fig.1 C). In the lung, significant thickening of the alveolar walls was due to inflammatory cells infiltration and congestion; some of the alveolar space turned narrow, and a few alveolar spaces are dilated showing compensatory emphysema (fig.1 D). The increase in size of the white pulps of the spleen were a result from the expanded germinal center of lymphoid follicles, and the red pulps were congested and hemorrhaged accompanied with the infiltration of inflammatory cells and the increase of nuclear debris (fig.1 E). The lymphoid sinus of the mesenteric lymph nodes was dilated and filled with lymph, in which macrophages and lymphocytes were increased (fig.1 F). The rest of the tissues did not show obvious pathological damage.
Bacterial isolation
Bacteria were isolated from the kidney, liver and lung. On the MacConkey agar, the colonies isolated from these three tissues were pink and round, with a diameter of about 2 mm, and the surface was smooth and moist. After Gram staining, the morphology of the bacteria was short rod-shaped under the oil microscope, indicating Gram-negative bacteria.
16S rRNA
The results of the electrophoresis test illustrated that there was no banding in the negative control, and the isolated strain had a clear band at about 1500 bp, which was consistent with the expected size of the 16S rRNA gene (fig.2). Three strains from different tissues were sequenced. The sequencing results revealed that the three strains were highly consistent. The similarity among three isolated bacteria was 100%, and similarity between the isolated strains and Escherichia coli (E. coli) was 99.45%.
Antimicrobial susceptibility test
The results of antimicrobial susceptibility test showed that the isolated E. coli were sensitive to penicillin drugs (piperacillin), cephalosporins (cefoxitin, ceftriaxone, cefepime) and non-classical β-lactam drugs (Meropenem, piperacillin-tazobactam, ampicillin-sulbactam), aminoglycosides (kanamycin, amikacin, neomycin), tetracycline (minocycline), quinolone (levofloxacin), nitrofuran (furazolidone), peptide (polymyxin B); but resistant to other antibiotics (table 2).
Table 2 Results of antimicrobial susceptibility test
type
|
Drug
|
dose
(μg/disc)
|
diameter of inhibition zone (mm)
|
sensitivity
|
Penicillin
|
Piperacillin
|
100
|
23
|
S
|
|
Ampicillin
|
10
|
-
|
R
|
|
carbenicillin
|
100
|
-
|
R
|
|
Cephalosporins
|
Cephalexin
|
30
|
-
|
R
|
|
Cefradine
|
30
|
-
|
R
|
|
Cefazolin
|
31
|
-
|
R
|
|
Cefoxitin
|
30
|
26
|
S
|
|
Cefuroxime
|
30
|
-
|
R
|
|
cefoperazone
|
75
|
10
|
R
|
|
Ceftazidime
|
30
|
-
|
R
|
|
Ceftriaxone
|
30
|
31
|
S
|
|
Cefepime
|
30
|
33
|
S
|
|
Nonclassical-
β-lactams
|
Meropenem
|
10
|
33
|
S
|
|
piperacilin-tazobactam
|
100/10
|
26
|
S
|
|
Ampicillin-sulbactam
|
10/10
|
12
|
S
|
|
aminoglycosides
|
Kanamycin
|
30
|
23
|
S
|
|
Amikacin
|
30
|
24
|
S
|
|
Gentamicin
|
10
|
12
|
R
|
|
Neomycin
|
30
|
18
|
S
|
|
tetracyclines
|
acheomycin
|
30
|
-
|
R
|
|
Minocycline
|
30
|
24
|
S
|
|
doxycycline
|
30
|
-
|
R
|
|
Chloramphenicols
|
chloramphenicol
|
30
|
-
|
R
|
|
quinolones
|
Ciprofloxacin
|
5
|
-
|
R
|
|
Norfloxacin
|
10
|
-
|
R
|
|
levofloxacin
|
5
|
33
|
S
|
|
Ofloxacin
|
5
|
-
|
R
|
|
sulfonamides
|
cotrimoxazole
|
23.75/1.25
|
-
|
R
|
|
polypeptid
|
furazolidone
|
300IU
|
19
|
S
|
|
polypeptide
|
Polymyxin B
|
300IU
|
14
|
S
|
|
Note: “S” means sensitive; “R” means resistant.
Pathogenicity test
Mice were injected with different doses of bacterial fluid, while the control group was given the same amount of sterile physiological saline. Six hours after injection, the mice in the high-dose group showed clinical symptoms such as tremors and slowness of movements and died 12 hours post injection. The other test groups of mice died within 72 hours, while no death was noted in the control group. According to the modified Karber method, the LD50 of the median lethal dose was 8.42×107 cfu/mL. The isolated strains of bacteria collected from the heart, liver, spleen, lung and kidney tissues the test mice showed the same morphological characteristics as the isolated strains from the red panda. The isolated strains were identified by 16S rRNA sequencing. The results showed that the similarity between the isolated bacteria and E. coli sequences in different tissues was highly consistent, the similarity was 99.55%. Histopathological observations from the mice showed blue-stained bacterial masses in the heart, liver, and kidney. The edema, congestion, cell degeneration even necrosis, inflammatory cell infiltration and other phenomena were found in many tissues including the heart, liver, kidney, and pancreas.
Whole genome sequencing results
By genomic assembly of strain sequence isolated from the red panda, it was found that the isolated strain had a genome size of 4,990,420 bp. The BLAST alignment of the predicted protein sequence with the Antibiotic Resistance Gene Database (ARDB) showed that the strain contained 20 resistance genes including acra, acrb, mdte, mdtf. These genes can mediate resistance to aminoglycosides, β-lactams, macrolides and other drugs. BLAST alignment with the Virulence Factor Database (VFDB) showed that the measured E. coli contained 713 virulence factors, including outer membrane protein, flagella, P pili, S pili, type I pili, cytotoxic necrosis factor, and hemolysin. In addition, factors related to the iron uptake system and other types of systems are also included. Among these virulence factors, PapA, PapG, OmpA, OmpU and other virulence factors were specific to Uropathogenic Escherichia coli (UPEC) [8], which helped confirm that the bacteria is UPEC.