Chronic cholecystitis: Diagnostic and therapeutic insights from formerly bile-farmed Asiatic black bears (Ursus thibetanus).

Across Southeast Asia and China, more than 17000 Asian bears are kept under suboptimal conditions and farmed for their bile to meet the consumer demand for traditional medicine products. Years of unsterile and repetitive bile extraction contribute to the development of chronic sterile or bacterial cholecystitis, a pathology commonly diagnosed in formerly bile-farmed bears. In both human and veterinary medicine, the diagnostic value of the macroscopic bile examination for assessing gallbladder disease is unclear. The objective of this study is to identify the role of gallbladder bile color, viscosity, and turbidity, while comparing them with established markers of cholecystitis. Moreover, it aims to define the optimal duration of oral antibiotic treatment for chronic bacterial cholecystitis in bears associated with bile farming. Thirty-nine adult, formerly bile-farmed Asiatic black bears (Ursus thibetanus) were examined under anesthesia and underwent percutaneous ultrasound guided cholecystocentesis. A total of 59 bile samples were collected with 20 animals sampled twice to evaluate the therapeutic success. All bile aspirates were assessed macroscopically and microscopically followed by submission for bacterial culture and antimicrobial sensitivity. In the majority of bears, samples with cytological evidence of bactibilia lacked inflammatory cells and did not always correlate with positive bacterial cultures. The most common bacterial isolates were Enterococcus spp, Streptococcus spp and Escherichia coli. Based on our findings, the optimal duration of antibiotic treatment for chronic bacterial cholecystitis is 30 days. Moreover, unlike Gamma-glutamyl Transferase (GGT) and gallbladder wall thickness, the organoleptic properties of bile were found to be reliable markers of chronic gallbladder inflammation with color and turbidity indicating cholestasis. The current study highlights the importance of cholecystocentesis for the management of gallbladder disease and provides initial results on the possible diagnostic value of macroscopic bile examination.

Introduction

Bear bile entered the Chinese pharmacopoeia 3000 years ago [1] to treat various ailments [2]. Bile was obtained by killing wild bears and harvesting their gallbladder. In the late 1970s bile extraction techniques from live bears and bear farming practices were developed in Asia [3] to prevent the loss of wild bears and provide an easy and steady supply of bear bile [4]. However, the growth of bear farms contributed to the increase in numbers of bears illegally sourced from the wild or internationally imported [5]. Currently, more than 17,000 bears are estimated to live on farms, under suboptimal conditions, in Southeast Asia and China [3, 6]. The Asiatic black bear (Ursus thibetanus) is the most commonly used species in farming operations [3, 7] and is classified as vulnerable with a decreasing population [3].

Bile extraction may be performed daily, or every few weeks, depending on the method and demand and is carried out over the lifespan of an individual. In Vietnam, bile is extracted through unsterile ultrasound guided or blind percutaneous cholecystocentesis [8]. Chronic cholecystitis is the most identified pathology in formerly bile-farmed bears [9]. Gallbladder inflammation with or without bacterial infection is caused due to the unsanitary and traumatic bile extraction method (S1 Fig). Specifically, after bile extraction, the irritated gallbladder wall in response to prostaglandin E2 produces and secretes mucin from its epithelium [10]. Additionally, percutaneously introduced bacteria that can survive the antimicrobial environment of bile [11] proliferate causing infection. The bacterial species identified to exhibit bile tolerance are the Gram-negative bacteria Salmonella sp, Escherichia coli, Vibrio cholerae, Campylobacter jejuni and the Gram-positive bacteria Enterococcus faecalis, Listeria monocytogenes and Lactobacillus amylovorus [11]. In general, Gram-positive bacteria seem to be more sensitive to the deleterious effects of bile (bacterial membrane damage, disturbance of bacterial cell macromolecular stability, oxidative and low pH stress) than Gram-negative [11]. Moreover, the increased mucin production promotes the decrease of gallbladder motility and subsequent bile stasis [12]. Prolonged bile stasis will contribute to gallbladder wall ischemia and may allow bacterial colonization (ascending from the gut) and enhancement of the inflammatory response [10, 13]. The optimal duration of antibiotic treatment for chronic cholecystitis in formerly bile-farmed bears is unknown. In some individuals bactibilia resolved after 14 days of antibiotic treatment, while it persisted in most cases. In small animal medicine a minimum of four weeks of antibiotic treatment is recommended for bacterial cholecystitis [14] due to the fact that orally administered antibiotics produce irregular levels in bile resulting in delayed antibacterial activity [15].

The chronicity of the gallbladder inflammation predisposes to cholestasis [16]. The formed mucin strands, cholesterol liquid and monohydrate crystals, bilirubin and calcium salt precipitates will increase the macroscopic turbidity of gallbladder bile and will form sludge or gallstones in moderate to severe cases [17]. We hypothesize that cholestatic gallbladder bile will have a darker color and that dark nuances will positively correlate with macroscopic turbidity.

The objective of this study is to identify the role of the organoleptic properties of bile (color, viscosity and turbidity) while investigating the relationship between these macroscopic characteristics, gallbladder inflammation, and infectious diagnostic markers. Furthermore, we intended to establish the optimal duration of orally administered antibiotic treatment for chronic bacterial cholecystitis in formerly bile-farmed Asiatic black bears.

Materials and methods

Ethics statement

All treatments and sampling were part of essential medical interventions. The collection of data during medical procedures was approved (written consent) by the institutional ethics and animal welfare committee of FOUR PAWS Viet in accordance with the guidelines for good scientific practice from the University of Veterinary Medicine, Vienna and FOUR PAWS International.

Animals

All animals included in this study were rescued from private bile farms across Vietnam. The majority of the bears were used for bile extraction purposes for a minimum of twelve years, with the exception of four confiscated individuals that were farmed for two to three years. The bears were transferred and housed permanently at BEAR SANCTUARY Ninh Binh (an animal welfare project by FOUR PAWS) in Northern Vietnam.

Thirty-nine (21 female and 18 male), adult, formerly bile-farmed Asiatic black bears (Ursus thibetanus) were examined under anesthesia. A total of 59 bile samples were collected with 20 animals sampled twice to evaluate the therapeutic success.

General health exam

The general health exam included a physical examination, one ventrodorsal and two laterolateral thoracic radiographs, abdominal ultrasound, echocardiography and indirect ophthalmoscopy. Samples collected were blood (20–40 minutes post darting) for complete blood count (mindray BC-2800 Vet Auto Hematology Analyzer, China) and biochemistry (IDEXX VetTest 8008 Chemistry Analyzer, Germany; dry-slide technology), urine for refractometry and culture if indicated, and bile (40–60 minutes post darting) for macroscopic and microscopic examination, bacterial culture and antimicrobial sensitivity. Gamma-glutamyl Transferase (GGT) and all other biochemical parameters were detected and quantified by colorimetric analysis.

The bears were anesthetized with 2.2mg/kg tiletamine-zolazepam (Zoletil 50®, Virbac, Carros, France) + 0.035mg/kg medetomidine (medetomidine 20mg/ml, magistral formulation, Vienna, Austria) + 0.05mg/kg butorphanol (Alvegesic® vet. 10mg/ml, Vienna, Austria), administered intramuscularly via remote projectile. Anesthesia maintenance after 70 minutes from darting was achieved either by intravenous or intramuscular ketamine (1mg/kg ketamine Ket-A-100®, Agrovet Market, Peru) and medetomidine (0.0175mg/kg medetomidine Narcostart® 1mg/ml, Richter pharma, Wels, Austria)(Group I = 22 individuals) every 35 and 80 minutes respectively or by a continuous rate infusion (CRI) with 18mcg/kg/minute of propofol (Fresofol® 1% MCT/LCT, Fresenius-Kabi, Australia) (Group II = 17 individuals) increased to 36mcg/kg/minute at 150 minutes post darting.

Bears with positive bile cultures were treated in most cases according to antibiogram for 14 or 30 days. In case of bacterial resistance to the available agents of the antibiogram, antibiotics were chosen following the recommendations from the human and veterinary medicine literature for the treatment of cholecystitis (bacteria specific) [10, 14, 18–20]. Similarly, cholecystitis treatment duration was decided based on the existing treatment guidelines in other species and the preliminary follow-up gallbladder bile culture results.

All bears received 60 days of oral choleretic treatment consisting of 5mg/kg ursodeoxycholic acid (UDCA) BID, 15mg/kg silymarin SID, 1000mg of artichoke leaf extract SID and 1000mg of Curcuma comosa SID and were re-examined 60 days later. For animals with positive bile cultures, bile was recollected and submitted for bacterial culture and sensitivity to assess treatment efficacy.

Hepatobiliary ultrasonography

Abdominal ultrasound (MyLab™ One Vet- Esaote; SC3421 VET convex probe; 1–8 MHz, The Netherlands) was performed in all 39 bears. Four gallbladder wall measurements were obtained from the anterior, posterior, left lateral and right lateral wall and a mean value was calculated representing the gallbladder wall thickness. Measurements greater than 1mm were considered as increased wall thickness (based on comparison with five Asiatic black bears free of cholecystitis). Moreover, the gallbladder was assessed for its wall echogenicity, presence of wall edema and contents (i.e., sediment, sludge and gallstones). Additionally, when identified, the common bile duct was measured, and the liver was evaluated for its echogenicity and general morphology.

Percutaneous transhepatic ultrasound guided cholecystocentesis

Preanesthetic fasting of 12 to 16 hours was implemented. At the beginning of anesthesia, the bears were injected with 0.2mg/kg meloxicam (Metacam® 20mg/ml, Boehringer Ingelheim) SC and continued with per os 5mg/kg firocoxib (Previcox® 227mg tab, Merial) SID for 5 days post cholecystocentesis to suppress local inflammation. Under general anesthesia, all bears were placed in dorsal recumbency and the right upper abdominal quadrate was aseptically prepared for the aspiration. The ultrasound transducer was disinfected after the localization of the gallbladder. A 20G x 23/4” sterile needle was attached to an IV extension line (Heidelberger, B|BRAUN, Germany) integrated with a 3-way adapter (Discofix®,B|BRAUN, Germany) and a 10ml syringe. The needle was inserted into the gallbladder under ultrasound guidance through a right-sided transhepatic approach including at least 2cm of liver tissue between the abdominal wall and the anterior gallbladder wall [21-24] to prevent leakage into the abdomen. The volume of bile aspirated ranged between 6 to 10ml.

Bile examination

Macroscopic examination

All 59 bile samples were assessed for their color, viscosity, and turbidity. The macroscopic assessment was performed by one examiner (SK Kalogeropoulu). Specifically, color ranging from light amber to amber was characterized as “amber”, “brown” from dark amber to brown and “black” from dark brown to black (S2 Fig). Viscosity was categorized as “mild”, “moderate” and “severe” with “mild” considered as the normal macroscopic viscosity of gallbladder bile (x 2 the relative viscosity of water) [12, 25]. Additionally, turbidity was classified as “non-turbid”, “mild” and “moderate”.

Cytology

Direct smears and cytocentrifuge preparations (1000g for 20minutes; [26]) of the bile were prepared within two hours of sampling and stained using Diff-Quik stain (LT-SYS®, Labor+Technik, Germany).

Bacterial culture

The bile specimens were cultured aerobically on trypticase soy supplemented with 5% sheep blood and chocolate agar. The cultures were incubated in 5% CO2 at 36°C and were examined for growth daily for 5 days.

Antimicrobial susceptibility testing

Antimicrobial susceptibility testing was carried out by using an automated microbiology system according to the manufacturer instructions. Interpretations of susceptible, intermediate or resistant were made according to the breakpoints assigned by the National Committee for Clinical and Laboratory Standards [27].

Statistical analysis

Statistical analysis was performed by using the software package R for Mac OS X/Windows (R Foundation for Statistical Computing, Vienna, Austria, 2020). A one-way ANOVA test was carried out between bile organoleptic properties (color, viscosity, and turbidity), plasma GGT, and mean gallbladder wall thickness, followed by a Tukey HSD test for multiple comparison of means. The same statistical tests were used to assess the relationship of GGT, mean gallbladder wall thickness, and positive or negative bile cultures. All models were checked for normality through visual assessment of model assumptions (model residual QQ plots for homoscedasticity and histograms for normal distribution) and a Shapiro-Wilk normality test. Furthermore, the effect of bactibilia on the organoleptic properties of bile was evaluated with a Pearson’s Chi-squared test and the effect of treatment duration on treatment efficacy with a Kruskal-Wallis test with pairwise comparisons, due to normality violation. Additionally, a Pearson’s Chi-squared test was used to investigate if there is a statistically significant association between the three gallbladder bile colors and levels of turbidity, followed by Bonferroni post- hoc analysis for groupwise comparisons. All results were plotted with ggplot2 package boxplots.

Results

All animals were diagnosed with chronic sterile or bacterial cholecystitis and chronic hepatopathy of varying severity. Their mean and standard deviation values of gallbladder wall thickness and GGT were 2.91+/-0.96 mm and 105.89+/-2.91IU/L (108.8–102.98) respectively.

A total of 59 bile samples obtained from 39 Asiatic black bears were examined for their organoleptic properties. The GGT and gallbladder wall thickness means were compared between the categories of organoleptic properties (color, viscosity, turbidity) and all differences were found to be insignificant (p>0.05) (S3–S5 Figs). Moreover, the categorical bile macroscopic characteristics did not differ between bacteria positive and negative bile samples. Specifically, for the comparison of color, viscosity and turbidity with bactibilia, the χ2 and p-value were equal to 3.93 (df = 2) and 0.14, 2.17 (df = 2) and 0.33, and 0.78 (df = 2) and 0.68, respectively. The relationship of gallbladder bile color and turbidity was found to be statistically significant between “black” color and “non-turbid” turbidity (negative relationship) with χ2 and p-value equal to15.95 and 0.003 (df = 4).

Cytology

All 59 bile aspirates were microscopically examined and had a grey background with amorphous aggregates of purple to black material (Fig 1). Golden brown pigment (Fig 2) was present in 8 samples and was interpreted as bilirubin. Microscopic evidence of bactibilia (Fig 2) was shown in 41 cases. Bacterial populations appeared as cocci (N = 25), mixed cocci with bacilli (N = 11) and as bacilli (N = 5). Cholesterol crystals (Fig 3) and hepatobiliary cells (Fig 4) were found in 5 out of 59 bile aspirates. White blood cells (lymphocytes or degenerated neutrophils; Fig 5) were identified in 17 samples and in all cases were consistent with bactibilia. Red blood cells (Figs 4 and 5) were found in one sample out of 59 which was obtained from an individual with chronic calculous cholecystitis with gallstone size equal to 4.45x2.28cm.

Fig 1

Purple to black amorphous aggregates.

40x. Diff-Quik stain, euromex CMEX 5 microscope camera.

Fig 2

Bacteria mixed with golden-brown bile pigment.

40x. Diff-Quik stain, euromex CMEX 5 microscope camera.

Fig 3

Cholesterol crystals.

40x. Diff-Quik stain, euromex CMEX 5 microscope camera.

Fig 4

Red blood cells and hepatobiliary cells.

40x, (A) hepatocytes, (B) biliary columnar epithelial cells, (C) mesothelial cells, and (D) cuboidal epithelial cell. Diff-Quik stain, euromex CMEX 5 microscope camera.

Fig 5

Biliary columnar cells, red blood cells and small lymphocytes (*).

40x. Diff-Quik stain, euromex CMEX 5 microscope camera.

Bacterial culture

Aerobic culture results are presented in Table 1. The most prevalent bacterial genera identified were Enterococcus, Streptococcus spp and Escherichia coli. Furthermore, the comparison of gallbladder wall thickness and GGT means between bacteria positive and bacteria negative bile cultures obtained from chronic cholecystitis patients was insignificant, with p-values equal to 0.39 and 0.15 respectively (S6 Fig).

Table 1

Aerobic culture results.

	Number (%) of Bacterial Cultures
Bacterial Identity	Total
Positive bile cultures	30/59 (50.8%)
Gram-negative aerobes (18.6%)
Escherichia coli	6/59 (10.2%)
Pseudomonas aeruginosa	3/59 (5%)
Acinetobacter spp	1/59 (1.7%)
Klebsiella pneumoniae	1/59 (1.7%)
Gram-positive aerobes (32.2%)
Enterococcus spp	12/59 (20.3%)
E. faecium	1/12
E. faecalis	1/12
E. casseliflavus	1/12
Streptococcus spp	6/59 (10.2%)
Staphylococcus spp	1/59 (1.7%)

Identity and prevalence of bacteria isolates by culture of bile from formerly bile-farmed Asiatic black bears (Ursus thibetanus) with suspected bacterial chronic cholecystitis.

Antimicrobial susceptibility testing

Tables 2 and 3 present the antimicrobial resistance and susceptibility results for both gram-positive and gram-negative microorganisms cultured from the 59 bile samples.

Table 2

Antibiogram results for the Gram-negative cultured bacteria.

% Resistant / % Susceptible (no. isolates tested)
Microorganism	A	A/C	D	E	IM	ME	CFT	CFZ	AM	NOR	CIP	ENR	COL	NIT
Escherichia coli	100%R (3)	20%R 40%S 40% I (5)	100%S (2)	100%S (4)	100%S (3)	100%S (3)	100%S (1)	66.6%R 33.3%S (3)	66.6%S 33.3%I (3)	75%S 25%I (4)	80%S 20%I (5)	NT	NT	100%S (3)
Pseudomonas aeruginosa	100%R (1)	100%R (1)	100%S (2)	NT	50%R 50%S (2)	100%S (2)	100%R (1)	100%S (2)	100%S (1)	100%S (2)	100%S (3)	NT	100%S (3)	NT
Acinetobacter spp	100%S (1)	100%S (1)	NT	NT	NT	NT	100%I (1)	NT	NT	100%S (1)		100%S (1)	100%S (1)	NT
Klebsiella pneumoniae	100%R (1)	100%I (1)	NT	100%S (1)	100%S (1)	100%S (1)	100%R (1)	NT	100%S (1)	100%S (1)	100%I (1)	NT	NT	100%I (1)

A, ampicillin; A/C, amoxicillin/clavulanate; D, doripenem; E, ertapenem; IM, imipenem; ME, meropenem; CFT, ceftiofur; CFZ, ceftazidime; AM, amikacin; NOR, norfloxacin; CIP, ciprofloxacin; ENR, enrofloxacin; COL, colistin; NIT, nitrofurantoin; NT, not tested; R, resistant; S, sensitive; I, intermediate.

Table 3

Antibiogram results for the Gram-positive cultured bacteria.

% Resistant / % Susceptible (no. isolates tested)
Microorganism	A	A/C	IM	CFT	CHL	VAN	RIM	TET	NOR	CIP	ENR	NIT
Enterococcus spp	82%R	87.5%R	88.9%R	NT	10%R	16.7%R	10%R	63.6%R	100%R	81.8%R	NT	25%R
	18%S	12.5%S	11.1%S		50%S		50%S	18.2%S		18.2%S		50%S
					40%I	83.3%S	40%I	18.2%I				25%I
	(11)	(8)	(9)		(10)	(12)	(10)	(11)	(10)	(11)		(12)
Streptococcus spp	83.4%R	100%R	NT	100%R	100%R	100%S	NT	100%R	100%R	NT	100%R	NT
	16.6%S (6)	(5)		(5)	(1)	(1)		(1)	(4)		(4)
Staphylococcus spp	NT	100%R (1)	NT	100%R (1)	100%R (1)	NT	NT	100%R (1)	100%R (1)	NT	100%R (1)	NT

A, ampicillin; A/C, amoxicillin/clavulanate; IM, imipenem; CFT, ceftiofur; CHL, chloramphenicol; VAN, vancomycin; RIM, rifampicin; TET, tetracycline; NOR, norfloxacin; CIP, ciprofloxacin; ENR, enrofloxacin; NIT, nitrofurantoin; NT, not tested; R, resistant; S, sensitive; I, intermediate.

Antimicrobial treatment duration

The treatment efficacy was evaluated by bacterial culture of bile samples collected following 14 or 30-day antibiotic treatment. Treatment was characterized as effective when culture was negative and ineffective when it was positive. The Kruskal-Wallis test showed a statistically significant difference in treatment efficacy between the different treatment durations of 14 and 30 days, χ2(2) = 10.171 (df = 2), p = 0.006, with mean rank treatment efficacy score of 2.28 for non-effective and 0.75 for effective treatment (Fig 6).

Fig 6

Antibiotic treatment duration and efficacy for chronic bacterial cholecystitis.

Antibiotic treatment duration of 14 and 30 days was compared between Asiatic black bears with positive and negative bile cultures (number of Asiatic black bears in each group is depicted). The comparison (Kruskal-Wallis test with pairwise comparisons) was found to be statistically significant with p-value = 0.006.

Discussion

Gamma-glutamyl transferase is a liver enzyme that binds to the cell membrane of the hepatocytes [28] and the epithelial cells of the bile ducts [29]. Its clinical applications are mainly for hepatobiliary disorders [30, 31]. GGT levels increase due to impaired bile flow, biliary inflammation [32] or necrosis and cholestasis in some species [33]. The cholestatic sensitivity of serum GGT in dogs is more than 90% [33]. However, the half-life of GGT is approximately 3 days in dogs [31], while serum GGT remained almost stable or decreased after 10 days in experimental bile duct ligation in the same species (cholestasis) [30]. In the current study, the majority of animals had GGT values within reference range with the ISIS physiological intervals for captive Asiatic black bears (17–454 IU/L) [34].

The ultrasonographic imaging finding of thickening of the gallbladder wall is considered characteristic of cholecystitis in small animal and human medicine [32, 35, 36]. Unlike gallbladder wall changes described in acute cholecystitis of humans [36] and small animals [37-39], in formerly bile-farmed bears the gallbladder wall is non-uniformly and mildly to moderately thickened due to the chronicity of the disease and the reoccurring focal wall inflammation related to bile extraction. Gallbladder bile viscosity is a marker of cholecystitis since it increases in pathological cases and positively correlates with the gallbladder bile mucin concentration [12]. Macroscopic gallbladder bile turbidity indicates cholestasis [17] and the statistically significant result of the inverse relationship between presence of turbidity and black color therefore suggests that black gallbladder bile color is an indicator of cholestasis. The differences of GGT and gallbladder wall thickness between the organoleptic property levels or categories were insignificant most likely due the chronicity of the hepatobiliary inflammation.

The majority of the bears with bactibilia lacked cytological evidence of inflammation which may be associated with transient bacterial colonization [40] due to prolonged gallbladder bile stasis. Only one sample collected from a bear with severe cholelithiasis and decreased gallbladder emptying contained red blood cells. It is assumed that the presence of red blood cells is associated with the traumatic effect of the gallstone within the gallbladder. Even though 41 bile aspirates showed cytological evidence of bactibilia, only 30 had a positive bacterial culture. It is possible that the eleven remaining cultures were negative because they were cultured only aerobically (anaerobic cultures were unavailable in local laboratories), or due to the bacteriostatic effect of bile or the difficulty to culture certain bacterial species. From the 41 samples, 11 presented mixed bacterial populations with only one bacterial culture positive for two microorganisms, while the rest of the samples were positive for a single organism. It is presumed that bacterial culture of a single organism is associated with in vitro overgrowth of one bacterial species [40].

The Gram-positive and Gram-negative bacteria isolated from the 59 bile aspirates were consistent with findings reported in humans [19, 20], small animals [39, 40, 41–44], single cases of a kinkajou (Potos flavus) [45], and a domestic ferret (Mustela putorius) (non-bile farmed) [46], despite the additional infection pathway related to bile extraction. Additionally, in formerly bile-farmed bears rescued from Chinese bile farms the bacteria isolated were E. coli, Enterococcus spp and Pseudomonas spp [9]. In China, bile is extracted continuously by surgically created transabdominal gallbladder fistulas or indwelling catheters [47]. It appears that, independently of the pathogenesis mechanism of cholecystitis and bactibilia, only certain bacterial Genera can survive the bacteriostatic effect of bile. Furthermore, in the current study the bacteria predominantly isolated were Gram-positive aerobes. This finding is not consistent with Begley et al., who suggested that Gram-positive bacteria are more sensitive to the deleterious effects of bile than Gram- negative. The antimicrobial effect of bile is primarily achieved through the bile acids, which alter the membrane integrity and permeability of bacterial cells by interacting with their membrane lipids and causing membrane damage [11]. We hypothesize that chronic gallbladder hypomotility contributes to reduced gallbladder bile acid pool [48, 49] resulting in decreased antimicrobial efficacy of bile and allowing bacteria, that would normally not survive, to proliferate in the gallbladder. The insignificant effect of positive bacterial cultures on GGT and gallbladder wall thickness is most likely also related to the chronic inflammation.

In our study, the majority of the most commonly isolated Gram-positive microorganisms were resistant to beta-lactams (>82%), with the exception of two samples. Specifically, Enterococcus faecalis isolated from one of the samples, was susceptible to ampicillin and to amoxicillin-clavulanate while, Streptococcus spp from the other sample was sensitive to ampicillin. Moreover, Enterococcus spp and Streptococcus spp were also resistant to fluoroquinolones (>81%) and tetracycline (63.6% and 100%, respectively). Vietnam is considered a potential hot-spot for the emergence of antimicrobial drug resistance (AMR) [50]. In human health, the predisposing factors for AMR are inadequate laboratory instrumentation and insufficiently trained staff for bacterial culture and antibiogram in many hospitals across the country. Moreover, market liberation and privatization of health care have resulted in large portions of the population being unable to afford healthcare services, as well as ‘over the counter’ antibiotic availability and financial benefits for the antibiotic prescriber since the majority of the antibiotics are made locally [50-52]. Consequently, self-medicating with antibiotics often in low dosages and prescription of inappropriate antimicrobial agents take place. In animal production, antibiotics are used by farmers excessively without veterinary supervision either “prophylactically” due to the low farm hygiene standards or as growth promoters to increase productivity [53]. Specifically in pig and poultry farms aminoglycosides, beta-lactams, phenicols, tetracyclines, fluoroquinolones, sulphonamides, ionophores and colistin are often utilized [54-56]. In bear bile farming antibiotics are also used “prophylactically” without veterinary supervision and in incorrect dosages and duration to “prevent” further infectious development after unsterile bile extraction. During rescue missions, empty vials of beta-lactams such as penicillin and ampicillin are often found next to the bear cages. Furthermore, the diet of bile-farmed bears consists partly or entirely of pig feeds with medicated feeds being frequently used. AMR in animals is inherently linked to AMR in humans [51]. Our results suggest that the AMR identified in formerly bile-farmed bears is consistent with the general AMR status in the country.

Bear bile farming remains a common practice in Asia, compromising the health and welfare of at least 17000 Asian bears and is one of the main reasons for Asiatic black bear population decline [3]. Morbidities associated with bear bile farming are understudied, making scientific research on this topic essential. The current study provides novel and comprehensive information for the diagnosis and treatment of hepatobiliary disease in formerly bile-farmed bears, facilitating the health management of rescued individuals in Southeast Asia and China.

To conclude, the optimal duration of antibiotic treatment for chronic bacterial cholecystitis in formerly bile-farmed Asiatic black bears based on our findings and considering the AMR status of Vietnam is 30 days, as also recommended in small animal medicine. Furthermore, despite the fact that gallbladder bile color, turbidity and viscosity are inflammatory markers, with color and turbidity indicating cholestasis, they were not associated with GGT and gallbladder wall thickness due to the chronicity of cholecystitis in these animals. Thereby organoleptic properties of bile could be considered a sensitive diagnostic tool of cholecystitis independently of disease chronicity and may be applicable in a wide range of mammalian species.

Minimal dataset included in this study.

(CSV)

Click here for additional data file.

Pathophysiology of chronic cholecystitis in formerly bile-farmed Asiatic black bears.

(TIF)

Click here for additional data file.

Bile color nuances.

The color samples were obtained from photographs of the collected bile samples.

(TIF)

Click here for additional data file.

Boxplot: Gallbladder wall thickness, GGT and bile color.

Gallbladder wall thickness and GGT means were compared between different bile color categories of Asiatic black bears (number of Asiatic black bears in each group is depicted) with chronic cholecystitis. The comparisons (one-way ANOVA) were found to be statistically insignificant with p-value> 0.05.

(TIF)

Click here for additional data file.

Boxplot: Gallbladder wall thickness, GGT and bile viscosity.

Gallbladder wall thickness and GGT means were compared between different bile viscosity levels of Asiatic black bears (number of Asiatic black bears in each group is depicted) with chronic cholecystitis. The comparisons (one-way ANOVA) were found to be statistically insignificant with p-value> 0.05.

(TIF)

Click here for additional data file.

Boxplot: Gallbladder wall thickness, GGT means and bile turbidity.

Gallbladder wall thickness and GGT means were compared between different bile turbidity levels of Asiatic black bears (number of Asiatic black bears in each group is depicted) with chronic cholecystitis. The comparisons (one-way ANOVA) were found to be statistically insignificant with p-value> 0.05.

(TIF)

Click here for additional data file.

Boxplot: Gallbladder wall thickness, GGT and bactibilia.

Gallbladder wall thickness and GGT means were compared between positive and negative bile cultures of Asiatic black bears (number of Asiatic black bears in each group is depicted) with chronic cholecystitis. The comparisons (one-way ANOVA) were found to be statistically insignificant with p-value> 0.05.

(TIF)

Click here for additional data file.

5 Oct 2021

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript is well written and the study was conducted in rigorous manner. But the only scientific conclusion of the study is the revealing of diagnostic tools and establishing of effective duration of antibiotic therapy of cholecystitis in captured black bears. This is the excellent material for any specialised veterinary journal but not for PLOS ONE that publish only the papers of fundamental scientific importance.

The special notes are as follows:

1. The manuscript is overloaded by the illustrative material. There are 13 figures and three tables in main text. Authors should give the majority of the raw information as supplementary, giving in the text only the most essential data. Box and whiskers on the diagrams should be explained in the legends.

2. The Gamma glutamyl Transferase used in the study as one of the indicators of cholecystitis but no description of the assay is given.

Lines 258-260: "Furthermore, the comparison of gallbladder wall thickness and GGT means between bacteria positive and negative bile cultures was insignificant ...". Was the "comparison" really insignificant? May be difference was?

Lines 285-286: "The Kruskall-Wallis test showed a statistically significant difference in treatment efficacy between the different treatment durations". How the authors estimated the treatment efficacy? It must be clearly explained.

**********

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Reviewer #1: No

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10 Nov 2021

Dear Academic Editor, dear Reviewer,

Thank you very much for considering our publication “Cholecystocentesis: a valuable diagnostic tool for successful treatment of chronic cholecystitis in formerly bile-farmed Asiatic black bears (Ursus thibetanus)”to be reviewed for PLOS ONE. We hope that the new version of our manuscript fulfills the publication requirements of your journal. Please find below the reply to each point raised in the comments. Please also find uploaded a marked-up copy of our revised manuscript entitled “Revised Manuscript with Track Changes”, an unmarked version of our revised paper entitled “Manuscript”, the formatted figures and the supporting information files.

Academic Editor’s comments:

1. Revision of the abstract to highlight the significance and strength of the research. �  Answer (A): the changes were done accordingly.

2. Revision of the text (discussion) to highlight the significant and strength of the research. �  A: the changes were done accordingly.

3. Provision of potential reviewers. �  A: we would like to suggest the following researchers as potential reviewers:

a. Jan S Suchodolski

orcid.org/0000-0002-2176-6932

Email: jsuchodolski@cvm.tamu.edu

Texas A&M University College Station

UNITED STATES

Sections: Veterinary science

Keywords: Biology and life sciences, Microbial ecology, Immunology, Inflammation, Immunity, Clinical immunology, Microbiology, Microbial pathogens, Veterinary science, Veterinary diseases, Veterinary medicine, Small animal care, Veterinary diagnostics, Veterinary microbiology, Biochemistry, Enzymes, Medicine and health sciences, Gastroenterology and hepatology, Infectious diseases, Bacterial diseases, Zoonoses, Leishmaniasis, Pathology and laboratory medicine, Anatomical pathology, Histopathology, Endoscopy, Hepatosplenomegaly, Diagnostic medicine, Retrospective studies, Clinical research design

b. Kristin Mühldorfer

orcid.org/0000-0001-8023-4736

Email: muehldorfer@izw-berlin.de

Leibniz Institute for Zoo and Wildlife Research (IZW)

GERMANY

Sections: Veterinary science, Microbiology - Bacteriology, Infectious diseases - Bacterial and fungal diseases

Keywords: Biology and life sciences, Microbial evolution, Bacterial evolution, Microbial genomics, Bacterial genomics, Bacterial genomes, Microbiome, Bacterial genetics, Microbiology, Bacteriology, Bacterial taxonomy, Gram negative bacteria, Gram positive bacteria, Fungal pathogens, Molecular biology, Molecular biology techniques, Mycology, Veterinary parasitology, Veterinary science, Veterinary diseases, Veterinary medicine, Veterinary diagnostics, Veterinary microbiology, Veterinary pathology, Veterinary virology, Zoonoses, Research and analysis methods

c. Peter Starkel

Email: peter.starkel@uclouvain.be

Cliniques Universitaires Saint-Luc

BELGIUM

Sections: Gastroenterology and hepatology

Keywords: Signal transduction, Signaling cascades, AKT signaling cascade, Primary biliary cirrhosis, Crohn's disease, Medicine and health sciences, Gastroenterology and hepatology, Inflammatory bowel disease, Biliary disorders, Primary sclerosing cholangitis, Hepatitis

d. Maite Garcia Fernández-Barrena

orcid.org/0000-0003-0375-6236

Email: magarfer@unav.es

Centre for Applied Medical Research, University of Navarra

SPAIN

Sections: Gastroenterology and hepatology, Cancer - Genetics and screening, Genetics - Gene expression; Epigenetics; Alternative splicing; RNA splicing; Molecular genetics

Keywords: Biology and life sciences, Biochemistry, Medicine and health sciences, Gastroenterology and hepatology, Inflammatory bowel disease, Hepatocellular

4. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming �  A: The manuscript including the file names was formatted according to the PLOS ONE style requirements.

5. Upload minimal dataset underlying the results- as supporting information file. �  A: We added the minimal dataset.

6. Include ethics statement in the material and methods: �  A: an ethical statement was given in line 95-99.

Reviewer #1 comments:

Thank you very much for taking the time and reviewing our manuscript. Your input is very much appreciated! Please find below our replies to your points:

1. Manuscript overloaded with illustrative material: �  A: We deleted 5 figures and moved them to the supportive information section. Please let us know in case the manuscript still appears overloaded and would like us to delete additional figures. Boxes and whiskers were explained in the legends.

2. Clarification of manuscript’s fundamental scientific importance. �  A: We consider our findings of fundamental scientific importance, since they provide novel and comprehensive information for the diagnosis and treatment of hepatobiliary disease in formerly bile farmed bears, an understudied topic of great concern for wildlife veterinarians, conservationists and bear husbandry related professionals in South East Asia and China. Bear bile farming is still a common practice in Asia, despite being the main threat for population decline of Asiatic black bears and compromising the welfare of at least 17000 individuals, making this manuscript an essential contribution. Moreover, the findings presented in the current study may be applicable in other mammalian species and are not limited only to Asiatic black bears.

3. Provision of description of GGT as an assay: �  A: description of the assay was given in line 271.

4. Lines 258-260: "Furthermore, the comparison of gallbladder wall thickness and GGT means between bacteria positive and negative bile cultures was insignificant ...". Was the "comparison" really insignificant? May be difference was? �  A: The comparison of GGT and gallbladder wall thickness was statistically insignificant between bacteria positive and bacteria negative bile samples collected from individuals with chronic cholecystitis and both p-values were greater than 0.05. The clarification can be found in the lines 235-238 of the revised manuscript.

5. Clear explanation of treatment efficacy estimation (lines 285-286, non-revised MS version)�  A: The clarification of treatment efficacy estimation is given in lines 259-261.

Other corrections

1. The email of the corresponding author was corrected in line 22.

2. The dose of the continuous rate infusion of propofol was corrected in lines 123-125.

3. Escherichia coli was added as one of the most prevalent bacterial genera identified in the bile cultures in line 235.

4. The supportive information captions were added in lines 519-533.

Thank you very much for re-considering our manuscript to be published after major revision.

Kind regards,

Szilvia K. Kalogeropoulu

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

6 Dec 2021

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Mariya Y Pakharukova, Ph.D., D.Sc.

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Authors considered most issues of first review and significantly improved the manuscript. However some questions and notes still remain.

1. In reviewed version authors shifted accents towards the fundamental value of the study. But the title still underlie the practical veterinary aspect.May be authors should correct the title and make it more "neutral". For ex., just "Chronic cholecystitis occasion in formerly bile-farmed 3 Asiatic black bears (Ursus thibetanus).

2. In respond to my note concerning the description of GGT assay authors write: "description of the assay was given

in line 271". There is no any description neither in old, nor in new version on this page.

3. How two different parameters - gallbladder bile turbidity and color can be statistically "compared"? To my mind one can analyse either correlation between two quantitative parameters, or qualitative effects of independent variable on dependent one. Respective figure (fig.2) does not clarify this question. If the turbidity was classified on three categories "mild", "moderate", "no", what do the intermediate points (0.5, 1.5) on Y-axe means? Additionally this figure is too flattened to be perceived correctly. Three mentions on statistical significance of the comparision (in text, in caption and on the diagram) seem to be redundant. Instead, authors should explain the box and whiskers values.

4. What is the total time animal's spent under anesthesia?

5. "Bears with positive bile cultures were treated based on antibiogram or received other antibiotics

in cases of bacterial resistance for 14 or 30 days". If I understand correctly, the exact course of antibiotic treatment was chosen personally, basing of the individual susceptibility to different antibiotics. How can the durations and effectiveness of the courses can be compared and optimal duration established? For me and other readers who are not experts in veterinary the optimal duration of the course seems to be strongly depended on the infection agent and medicines applied.

6. Fig.1 contains no essential information and may be omitted.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Eugene Novikov

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

18 Jan 2022

Rebuttal letter points – PONE-D-21-26096R1

Dear Academic Editor, dear Reviewer,

Thank you very much for taking the time and reviewing our manuscript after the first major revision. We hope that this edited version following your recommendations and comments will fulfill the requirements for publication in PLOS ONE. Please see below the reply to each point raised in the comments. Please also find attached a marked-up copy of our revised manuscript entitled “Revised Manuscript with Track Changes”, an unmarked version of our revised paper entitled “Manuscript”, the formatted figures and the supporting information files.

Academic editor’s comments:

The comments of the reviewer were considered carefully and effort was made to answer every point precisely and modify the text accordingly.

Reviewer comments:

Thank you again for meticulously reviewing our manuscript and helping us to present and communicate better our findings. Your comments truly improved the quality of this manuscript!

1. Title correction and making it more neutral�  Answer (A): the manuscript’s current title is “Cholecystocentesis: a valuable diagnostic tool for successful treatment of chronic cholecystitis in formerly bile-farmed Asiatic black bears (Ursus thibetanus)”. Our suggested new title is: “Chronic cholecystitis: diagnostic and therapeutic insights from formerly bile-farmed Asiatic black bears (Ursus thibetanus)”. Please find it in lines: 1-3.

2. Provision of description of GGT as an assay�  A: Please find GGT assay description in lines 116-117. The GGT analysis was done with the Vettest 8008 machine from IDEXX. The machine uses dry-slide technology (inputted in line 114) and GGT was detected and quantified with colorimetric analysis. We are hoping that our new explanation sufficiently answered your question.

3. How two different parameters - gallbladder bile turbidity and color can be statistically "compared"? To my mind one can analyse either correlation between two quantitative parameters, or qualitative effects of independent variable on dependent one. Respective figure (fig.2) does not clarify this question. If the turbidity was classified on three categories "mild", "moderate", "no", what do the intermediate points (0.5, 1.5) on Y-axe means? Additionally, this figure is too flattened to be perceived correctly. Three mentions on statistical significance of the comparison (in text, in caption and on the diagram) seem to be redundant. Instead, authors should explain the box and whiskers values�  A: Gallbladder bile turbidity and color have been treated as categorical variables with three levels each. A chi-square test can be used to analyze two categorical variables and evaluates whether there is a significant association between the categories of the variables. Usually, box plots help to visualize the distribution of quantitative values in a field. It is recognized that in our case that we have two categorical variables the box plot is not an appropriate visualization method. Moreover, in Figure 2, the categories of turbidity “non-turbid”, “mild” and “moderate” were replaced by “0”, “1” and “2” respectively to allow the visualization of the data as a box-plot and the intermittent points 0.5 and 1.5 did not represent any category of bile turbidity. The Pearson’s Chi- squared test was repeated and followed by a Bonferroni post-hoc analysis for groupwise comparison to identify which categorical pair/s of the variables analyzed was/were significantly associated. The significant p-value of the chi-square test is attributed to the negative association between “black” bile color and “non-turbid” turbidity. Regarding result visualization for small data sets and categorical variables a bar plot is recommended. Despite the statistically significant relationship between “black” and “non-turbid”, this finding was poorly visualized by the bar plot. Bar plots are a descriptive tool that show the distribution of the data and since “amber” was the most common finding led to its “overrepresentation” in the graph suggesting that the relationship with “non-turbid” is statistically significant, while the association of “black” and “non-turbid” appears to be insignificant (please see below). Considering the above points and trying to provide clarity for the reader we would like to remove the former box plot (fig 2) and present our findings only via text. Changes in the “materials and methods” section can be found in lines 195-198. Modifications in the results and discussion are made in lines 212-214 and 296-297 respectively. Thank you for your critical thought, we hope that the statistical changes are conform with you. We believe that your comment has much improved our result communication to the reader!

4. What is the total time animal spent under anesthesia? �  A: Data were collected during essential medical interventions suited to each bear’s medical needs and were not part of a designed experiment, hence the total duration of anesthesia varied between individuals. In an effort to standardize sampling, blood samples were collected within 20-40 minutes post darting and bile samples between 40-60 minutes post darting. This information was added to the manuscript text in lines 112 and 115 respectively.

5. "Bears with positive bile cultures were treated based on antibiogram or received other antibiotics in cases of bacterial resistance for 14 or 30 days". If I understand correctly, the exact course of antibiotic treatment was chosen personally, basing of the individual susceptibility to different antibiotics. How can the durations and effectiveness of the courses can be compared and optimal duration established? For me and other readers who are not experts in veterinary the optimal duration of the course seems to be strongly depended on the infection agent and medicines applied. �  A: The exact course of antibiotic treatment was not chosen personally, but according to the existing literature for the treatment of bacterial cholecystitis. From both human and veterinary medicine there are antibiotic treatment recommendations (agent, treatment duration) against specific microorganisms causing cholecystitis. In cases where bears were resistant to the antibiotics tested in the antibiogram, an antibiotic (not included in the antibiogram) was chosen based on the existing treatment guidelines from other species. The clarification was made in lines 128-133. Literature examples of treatment recommendations were cited and the reference list numbering was adjusted accordingly.

6. Omit Figure 1�  A: Figure 1 was omitted and added to supporting information. Figure numbering and naming was changed accordingly. Additionally, the legend of Figure 1 as a supporting figure was given in lines 534-535.

Thank you very much considering our manuscript for publication after a second major revision.

Kind Regards,

Szilvia K. Kalogeropoulu

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

10 Feb 2022

Chronic cholecystitis: diagnostic and therapeutic insights from formerly bile-farmed Asiatic black bears (Ursus thibetanus).

PONE-D-21-26096R2

Dear Dr. Kalogeropoulu,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Mariya Y Pakharukova, Ph.D., D.Sc.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: N/A

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: The manuscript presents interesting and important research. The authors were aimed to define the optimal treatment for chronic bacterial cholecystitis in Asiatic black bears (Ursus thibetanus). Moreover, their research was also aimed at identification the role of gallbladder bile color, viscosity, and turbidity, while comparing them with established markers of cholecystitis.

After reviewing the previous versions of the text, one may conclude that the authors changed the text and improved the manuscript significantly.

In this version, the authors provide quite sufficient methodological details of their experiments. The details of suggested approach are described well and the approach look appropriate.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

18 Feb 2022

PONE-D-21-26096R2

Chronic cholecystitis: diagnostic and therapeutic insights from formerly bile-farmed Asiatic black bears (Ursus thibetanus).

Dear Dr. Kalogeropoulu:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Mariya Y Pakharukova

Academic Editor

PLOS ONE