Introduction
Suppurative cholangitis or cholangiohepatitis often develops in cats with comorbidities that predispose to bile-borne bacterial infection.1 These comorbidities, as well as clinical features, clinicopathologic findings, and survival, are detailed in a large number of cats with suppurative cholangitis-cholangiohepatitis syndrome (S-CCHS) in the companion report.1 To date, culture or molecular methods of bacterial detection in small numbers of cats support that at least a subset of cats with S-CCHS or obstructive cholangiopathies have bacterial infection.2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22 However, detection of bacteria by culture may be compromised by the common practice of administering broad-spectrum antimicrobials before sample collection.1,11,14 Conversely, highly sensitive molecular methods for bacterial detection such as fluorescent in situ hybridization or PCR assay increase the likelihood of detecting nonrelevant bacteria undergoing enterohepatic transit or tissue contaminants.19,23 Thus, a sensitive method with lower risk for detection of nonrelevant bacteria in cats with S-CCHS would be informative for understanding the involvement of bacteria in this syndrome.
We hypothesized that lipoteichoic acid (LTA [a cell wall component in gram-positive bacteria]) and pathologic expression of toll-like receptor 4 (TLR-4 [reflecting pathologic gram-negative bacterial exposure]) as detected by immunohistochemical (IHC) staining might function as relevant markers of bacterial infection. Lipoteichoic acid is a strongly immunogenic surface reactive antigen that mediates attachment of gram-positive bacteria to host cells.24,25,26,27,28,29 Residual LTA has also been proposed as a mechanistically pathogenic factor in some forms of chronic cholangitis in humans and animals with experimentally induced disease.27,28,29 Expression of TLR-4, a pathogen pattern recognition receptor, reflects pathologic exposure to gram-negative bacterial lipopolysaccharide (LPS [or endotoxin]) and is considered more relevant to disease pathogenesis than detection of lipid-A (biologically active component of LPS).30,31,32,33,34,35 Because endotoxin commonly circulates to the liver in the portal circulation and is removed by hepatic Kupffer cells and sinusoidal endothelium, detection of lipid-A alone does not necessarily reflect a pathologic response.31,32,33,35,36,37,38 In health, tolerance to minor but constant enterohepatic LPS or endotoxin exposure downregulates TLR-4 expression, whereas pathologic exposure provokes TLR-4 upregulation.32,33
The present study was undertaken to investigate the association of bacterial infection with S-CCHS in cats, comparing culture-based detection of bacteria in liver, bile, gallbladder, and choleliths to IHC-detected LTA and TLR-4 expression.
Materials and Methods
Case selection criteria
Cats with S-CCHS were identified by liver biopsy, and clinical data were prospectively cataloged at the time of diagnosis over a 40-year interval (1980 to 2019) by one of the authors (SAC). Details regarding liver sample collection and processing are reported elsewhere.1 All tissue sections were initially inspected by board-certified veterinary anatomic pathologists and pathology residents-in-training and were also independently reviewed by 2 of the authors (SAC and SPM) naïve to case details before data entry to verify histologic characterization. Diagnosis of S-CCHS required predominant portal neutrophilic infiltrates demonstrating a duct-centric orientation, with or without features consistent with extrahepatic bile duct obstruction (EHBDO), cholangiocyte proliferation (ductular reaction), destructive cholangitis (ie, necrotic duct epithelium, irregular duct epithelial stratification or attenuation, and duct collapse or involution), or invasion of the limiting plate by inflammatory infiltrates. Although an attempt was made to apply a previously recommended histologic classification method discriminating between acute and chronic S-CCHS in cats, intraindividual variation among liver sections confounded this effort, similar to previously published observations.39,40 Definitive disease group categorization was assigned after reconciling histologic features and clinical information that included historical and physical examination findings, results of diagnostic imaging, and gross observations at surgery or necropsy.
Medical record review
Clinical records of cats meeting histologic inclusion criteria of S-CCHS were reviewed with pet caretakers and referring veterinarians, as needed, to clarify pre- and postbiopsy treatments, postbiopsy health status, and survival status. An organizational spreadsheet was used to standardized data collection, which included signalment, clinical signs, antecedent or concurrent illness, physical examination findings, and results of clinicopathologic tests, imaging studies, and surgery.1 Results of aerobic and anaerobic bacterial cultures (for samples of liver, gallbladder or common bile duct bile, gallbladder wall, choledochal cyst fluid, or crushed choleliths), treatments (antecedent and postoperative antimicrobials, antecedent glucocorticoid drugs, or other immunosuppressant medications), and survival time after definitive diagnosis (days after definitive diagnosis and age at death) were transcribed. Cats with S-CCHS were subcategorized into designated comorbidities1 including EHBDO, cholelithiasis, cholecystitis, ductal plate malformation (DPM), diabetes mellitus, biopsy-confirmed inflammatory bowel disease (IBD), and biopsy-confirmed pancreatitis and treatment interventions including cholecystectomy and cholecystoenterostomy.
Histologic criteria defining comorbidities
Diagnosis of EHBDO required distention of medium- and large-sized bile ducts with or without evidence of bile duct tortuosity, variable intraluminal biliary debris (mucin or bile-stained secretions and exfoliated epithelial or inflammatory cells), periductal edematous laminating fibrosis, periductal neutrophilic or neutrophilic mixed inflammatory infiltrates, hyperplasia of small bile ducts, and dimensional expansion of portal tracts with edematous extracellular matrix. Ultrasound imaging or gross inspection of extrahepatic biliary structures during surgery or at necropsy confirmed EHBDO in all cases in this category. Diagnosis of cholecystitis required examination of gallbladder sections confirming neutrophilic or mixed inflammatory mural infiltrates, with variable mural edema, hemorrhage or fibrosis, intraluminal inflammatory cells, or biliary concretions (microcholeliths), hemobilia, or bacteria. Gram staining of gallbladder sections was completed in 18 cats. Diagnosis of cholecystitis was reconciled with ultrasound images of the gallbladder and its gross appearance during surgery or necropsy. Diagnosis of DPM was based on finding the characteristic morphology of syndrome phenotypes, as specified in the companion report1 and recently described in 2 other reports.41,42 Pancreatitis was diagnosed histologically on the basis of finding interstitial or periductal suppurative, lymphocytic, or lymphoplasmacytic inflammatory infiltrates. Diagnosis of IBD was based on evaluation of full-thickness intestinal biopsy specimens with consideration given to the inflammatory cellular population as well as degree and distribution of inflammatory infiltrates, presence of intraepithelial lymphocytes, villi length, villi fusion, epithelial cell injury, lacteal dilation, and intestinal crypt depth, tortuosity (hyperplasia), distention, and dropout, and estimated fibrosis (lamina propria of villi and between crypts).43
IHC staining
Immunohistochemical staining for LTA and TLR-4 was completed on liver sections from 151 of 168 (90%) cats with S-CCHS (cases) and 20 cats lacking necroinflammatory liver disease (controls). Controls included clinically healthy cats from unrelated projects (n = 7), cats with congenital portosystemic vascular anomalies (3), cats with hepatic lipidosis (2), cats that died unexpectedly during routine ovariohysterectomy (2), and cats that had liver samples collected during exploratory laparotomy for chronic cystitis (1), chronic gastritis (1), or vomiting related to suspected IBD (1) or immediately after euthanasia because of bacterial otitis and meningitis (1), cardiac disease (1), or noninfectious renal failure (1). Details regarding IHC staining protocols are provided (Supplementary Appendix S1). Negative control slides (processed with primary antibody replaced with a species-matched nonreactive IgG at equivalent concentrations) and positive control slides for LTA (culture-confirmed Streptococcus abscess in a cat) and TLR-4 (liver, spleen, and mesenteric lymph node from a cat with disseminated Escherichia coli) were routinely included.
Localization of TLR-4 expression was verified by prominent staining in inflammatory cells (ie, neutrophils, lymphocytes, and local macrophages) within or adjacent to portal tracts, biliary epithelium of medium- to large-sized bile ducts, and gallbladder, with rare staining in hepatocytes and regional Kupffer cells. Then sections were evaluated in 10 microscopic fields of view at 100× and 200× magnification. Semiquantitative scores (0 to 3) were assigned to characterize TLR-4 expression reflecting the percentage of inflammatory cells and biliary epithelium with positive staining. Per this scoring system, 0 indicated no IHC positivity; 1 indicated rare positive cells (ie, ≤ 10% inflammatory cells, macrophages, or biliary epithelium); 2 indicated > 10% but < 50% positive inflammatory cells, macrophages, or biliary epithelium; and 3 indicated ≥ 50% positive inflammatory cells, macrophages, or biliary epithelium. The types of cells staining positively and their zonal distribution were recorded. For LTA, results were summated as positive or negative for the tissue examined (ie, liver, gallbladder, common bile duct, choledochal cyst wall, or cholelith) and defined as rare single organisms or clusters, chains, or mats of bacteria.
In-parallel detection of bacteria by combined interpretation of culture and IHC staining
Bacterial isolates were recorded by genus (ie, not all bacteria were speciated) and organized into categories of aerobic or facultative anaerobic and fastidious anaerobic organisms and gram-positive or gram-negative staining. The number of cats with culture-based single and polymicrobial infections was determined. The number of infecting organisms in polymicrobial infections was cataloged. The number of cats with IHC detected gram-positive (LTA IHC positive) or gram-negative (TLR-4 IHC positive) infections and those with both gram-positive and gram-negative infections (ie, LTA IHC positive and TLR-4 IHC positive) were enumerated. Dually positive LTA and TLR-4 IHC samples were deemed IHC polymicrobial even though such classification underestimated polymicrobial status because it could not differentiate multiple different gram-positive or multiple different gram-negative organisms. Frequency of gram-positive or gram-negative bacterial detection by culture and IHC methods was enumerated, and concordance was determined. In-parallel interpretation of culture and IHC test results was used to estimate the maximum number of cats with bacterial infections, gram-positive infections, gram-negative infections, and polymicrobial infections (acknowledging that IHC-detected polymicrobial infection underestimated polymicrobial infections).44 In-parallel test interpretation declared a cat infected if it had either a positive culture or positive IHC staining result. This in-parallel strategy was used to minimize false negative test results.
Single and combined inoculate cultures
The number of inoculate sources for bacterial culture from each cat was enumerated. Samples were designated as combined inoculates if they had 2 or more different origins (eg, liver, bile, gallbladder wall, choleliths, or fluid from cystic malformations). The frequency of positive culture results with single or combined inoculates was also recorded.
Statistical analysis
Data on cat signalment, an absence of sex or breed predilection, spectrum of clinicopathologic abnormalities, and survival status (without details of bacterial infections described herein) are provided in the companion report.1
Because most numerical data were nonparametric, details are reported as median (range) and 95% CIs. The criterion for a positive in-parallel test result required that either test result be abnormal (ie, positive culture or IHC staining result) to avoid false negative results.44 The influence of culture-based, IHC-based, and in-parallel test detection of bacterial infection, gram-positive and gram-negative infection, and polymicrobial infection on clinical features was evaluated by use of 2 × 2 tables and the Fisher exact test. Duration of antecedent clinical illness (days) and WBC count, neutrophil count, fold increase of serum liver enzyme activities, and serum total bilirubin concentrations were compared between cats with and without bacterial infection as deemed by culture, IHC, and in-parallel testing in all cats, cats within each comorbidity group, cats with and without cholecystectomy, cats with and without cholecystoenterostomy, and cats with polymicrobial versus single isolate infections by means of the Wilcoxon rank sum test. The fold increases in liver enzyme activity and total bilirubin concentration were normalized using the upper limit of the reference interval validated for the relevant analytic method. Prevalences were calculated for bacterial infection and gram-positive, gram-negative, aerobic, anaerobic, and polymicrobial isolates among all cats and cats within comorbidity groups. The frequency of bacterial isolation by culture and the number of bacterial isolates per cat from single and combined inoculates were compared using 2 × 2 tables and the Fisher exact test. Differences in the number of aerobic or facultative anaerobic and fastidious anaerobic isolates, proportion of gram-positive cultured isolates versus LTA IHC-positive results, and proportion of gram-negative cultured isolates versus TLR-4 IHC-positive results and culture-based aerobic or facultative anaerobic and fastidious anaerobic isolates were evaluated for cats with single and polymicrobial bacterial infections using 2 × 2 tables and the Fisher exact test.
Survival times were computed for cats with and without bacterial infection and single versus polymicrobial infection for culture-based, IHC-based, and in-parallel test results by means of Kaplan Meier statistics (survival time in days after definitive diagnosis and age at death). The Gehan-Wilcoxon test (short-term survival) and log-rank test (long-term survival) were performed to identify significant (P ≤ 0.05) differences between groups. Discordance and concordance between culture-based and IHC-based bacterial detection were determined and expressed as a percentage. Statistical analyses were performed with a statistical software program (Statistix 9; Analytical Software).
Results
Bacterial culture
Of 168 cats, 135 (80%) had samples submitted for bacterial culture. Of these, 93 (69%) were positive despite common administration of broad-spectrum antimicrobials for several days (sometimes weeks) before culture sample collection.
Among comorbidities, frequencies of bacterial culture submission ranged from 82% to 90% of cats, and frequencies of positive results ranged from 63% to 78% (Table 1). Origins of culture inoculates included liver (n = 119), gallbladder bile (90), choledochal cyst fluid (8), crushed cholelith (10), and gallbladder mucosal scrape or wall section (15). There were fewer single inoculates (n = 52) than combined inoculates (83). Origins of single inoculates included liver (n = 40), gallbladder bile (9), common bile duct bile (1), and crushed cholelith (2). Combined inoculates commonly included liver and gallbladder bile (54/83 [65%]), but also included combinations of liver, bile, and gallbladder wall with or without gallbladder mucosal scrapings (14); liver, bile, and crushed cholelith (4); liver and crushed cholelith (2); bile and crushed cholelith (1); bile and choledochal cyst contents (3); liver, bile, and choledochal cyst contents (4); and liver, bile, choledochal cyst contents, and crushed cholelith (1). Bile often was centrifuged in a sterile vial with bile sediment used as the submitted inoculate. Gram staining of gallbladder sections from 18 cats disclosed gram-positive bacteria in only 3 (17%). Significantly (P < 0.001) more positive cultures were detected with combined inoculates (68/83 [82%]) than with single inoculates (25/52 [48%]). Among 41 cats with culture-based polymicrobial infections, 2 organisms were involved in 27 (66%), 3 organisms in 11 (27%), and ≥ 4 organisms in 3 (7%). One cat with chronic extracorporeal bile drainage had 7 bacterial pathogens isolated from bile. A significantly (P < 0.001) greater number of polymicrobial infections were diagnosed from combined inoculates (33/41 [80%]) than with single inoculates (8/41 [20%]).
Results of bacterial culture for cats with suppurative cholangitis-cholangiohepatitis syndrome (S-CCHS).
| Group | Total No. of cats | No. of cats with bacterial culture | Positive culture | Gram+isolates | Gram–isolates | Polymicrobial isolates |
|---|---|---|---|---|---|---|
| All cats with S-CCHS | 168 | 135 (80) | 93 (69) | 72 (77) | 67 (72) | 41 (44) |
| EHBDO | 89 | 73 (82) | 54 (74) | 42 (78) | 37 (69) | 26 (48) |
| Cholelithiasis | 71 | 62 (87) | 44 (71) | 35 (80) | 31 (71) | 23 (52) |
| Cholecystitis | 68 | 58 (85) | 45 (78) | 36 (80) | 32 (71) | 25 (56) |
| Cholecystectomy | 39 | 35 (90) | 27 (77) | 20 (74) | 19 (70) | 13 (48) |
| Cholecystoenterostomy | 37 | 33 (89) | 21 (64) | 18 (86) | 15 (71) | 12 (57) |
| Ductal plate malformation | 74 | 62 (84) | 39 (63) | 29 (74) | 26 (67) | 18 (46) |
| Biopsy-confirmed IBD | 60 | 52 (87) | 39 (75) | 31 (80) | 23 (59) | 17 (44) |
| Biopsy-confirmed pancreatitis | 41 | 35 (85) | 27 (77) | 18 (67) | 21 (78) | 14 (52) |
EHBDO = Extrahepatic bile duct obstruction. IBD = Inflammatory bowel disease.
Of 93 cats with positive cultures, 69 (74%) had aerobic/facultative anaerobic isolates, 25 (27%) had fastidious anaerobic isolates, and 41 (44%) were polymicrobial. Among positive cultures, the frequency of gram-positive and gram-negative isolates was not significantly different (Table 1); however, aerobic or facultative anaerobic isolates (n = 109) were significantly (P < 0.001) more common than fastidious anaerobic isolates (30; Supplementary Table S1). Single-isolate infections (52/93 [56%]) were not significantly more common than polymicrobial infections (41/93 [44%]). Common aerobic or facultative anaerobic isolates included E coli (n = 41; 44% of positive cultures) and Enterococcus (n = 36; 39% of positive cultures). These organisms also were common in polymicrobial infections (Enterococcus [12/41 {29%}]; E coli [6/41 {15%}]). Among polymicrobial infections, 37 of 41 (90%) involved mixed gram-positive and gram-negative isolates, with 4 cats having only gram-negative isolates. Fastidious anaerobic bacteria were isolated only from cats with polymicrobial infections. The most common fastidious anaerobic isolates were Bacteroides spp (n = 10; 11% positive cultures) and Clostridium spp (n = 7; 8% positive cultures).
Among 69 cats with aerobic or facultative anaerobic infections, 38 (55%) had EHBDO, 31 (45%) had cholelithiasis, 31 (45%) had cholecystitis, 30 (44%) had DPM, 29 (42%) had IBD, 18 (26%) had pancreatitis, and 6 (9%) had diabetes mellitus; 9 of 69 (28%) cats had polymicrobial infections. Among 25 cats with fastidious anaerobic isolates, 16 (64%) had EHBDO, 13 (52%) had cholecystitis, 13 (52%) had cholelithiasis, 9 (36%) had DPM, and none had diabetes mellitus. Among cats with biopsy-confirmed IBD, 75% had positive cultures; 39 positive cultures included 23 (59%) gram-negative, 31 (80%) gram-positive, and 17 (44%) polymicrobial isolates. Bacterial isolates from 39 positive cultures included 17 (44%) E coli and 16 (41%) Enterococcus spp. Among 100 cats without enteric biopsies, positive cultures in 47 of 75 (63%) included 41 (87%) gram-positive, 44 (94%) gram-negative, and 21 (45%) polymicrobial isolates. Bacterial isolates from 47 positive cultures included 24 (51%) E coli and 20 (43%) Enterococcus spp. There were no significant differences in proportions of E coli and Enterococcus spp between cats with and without biopsy-confirmed IBD. There were no significant differences in frequency of bacterial infection, type of infecting organism, or development of culture-based polymicrobial infections among comorbidities.
Clinical features in cats with and without bacterial cultures were similar, except for a significantly (P < 0.001 each) greater proportion of cats with bacterial cultures having abdominal pain and hepatomegaly (Table 2). Among 135 cats with bacterial cultures, those with positive cultures had a significantly greater incidence of abdominal pain (P = 0.05) and lethargy (P = 0.009). There were no significant differences in clinical features between cats with gram-positive versus gram-negative isolates. Compared with cats with single bacterial isolates, a significantly greater proportion of cats with polymicrobial isolates had pyrexia (P = 0.05), abdominal pain (P = 0.001) and hepatomegaly (P = 0.05). Among clinicopathologic assessments, cats with positive cultures had a significantly (P = 0.01) higher median WBC count (16.4 × 103/μL; range, 4.8 × 103 to 48.1 × 103/μL), compared with cats with negative cultures (9.2 × 103/μL; range, 0.5 × 103 to 49.2 × 103/μL). There were no significant differences between cats with and without positive bacterial cultures in fold-increase of serum total bilirubin concentration or liver enzyme activities, with the exception of alkaline phosphatase (ALP). Although the median fold-increase in ALP activity was modest in each group, ALP activity was significantly (P < 0.002) higher in cats with negative cultures than in cats with positive cultures (median fold increase, 2.4 vs 1.3). Significant differences also were observed in ALP activity between cats without versus with gram-positive isolates (median fold increase, 2.2 and 1.3, respectively; P = 0.05), cats without versus with gram-negative isolates (median fold increase, 2.3 and 1.2, respectively; P = 0.009), and cats without versus with polymicrobial isolates (median fold increase, 2.1 and 1.0, respectively; P = 0.007).
Clinical features of cats with S-CCHS stratified by bacterial culture results, Gram-staining characteristics, infection type, and immunohistochemical (IHC) results.
| Category | Pyrexia | Abdominal pain | Jaundice | Lethargy | Hyporexia | Emesis | Weight loss | Diarrhea | Large liver | Diabetes mellitus |
|---|---|---|---|---|---|---|---|---|---|---|
| Bacterial culture (n = 135) | 61 (45) | 37‡ (27) | 94 (70) | 109 (81) | 111 (82) | 114 (84) | 73 (54) | 29 (21) | 46‡ (34) | 11 (8) |
| No bacterial culture (n = 33) | 13 (39) | 1‡ (3) | 21 (64) | 24 (73) | 27 (82) | 24 (73) | 18 (55) | 3 (9) | 1‡ (3) | 0 (0) |
| Culture positive (n = 93) | 45 (48) | 31* (33) | 62 (67) | 81† (87) | 80 (86) | 80 (86) | 53 (57) | 23 (25) | 32 (34) | 6 (6) |
| Culture negative (n = 42) | 16 (38) | 6* (14) | 32 (76) | 28† (67) | 34 (81) | 32 (76) | 20 (48) | 6 (14) | 14 (33) | 6 (14) |
| Gram-positive (n = 72) | 36 (50) | 26 (36) | 50 (69) | 62 (86) | 62 (86) | 62 (86) | 43 (60) | 18 (25) | 26 (36) | 5 (7) |
| Gram-negative (n = 67) | 32 (48) | 24 (36) | 39 (58) | 55 (82) | 50 (75) | 53 (79) | 34 (51) | 15 (22) | 25 (37) | 3 (5) |
| Single organism infection (n = 52) | 20* (38) | 10† (19) | 32 (62) | 44 (85) | 48 (92) | 43 (83) | 30 (58) | 14 (27) | 13* (25) | 4 (8) |
| Polymicrobial infection (n = 41) | 25* (61) | 21† (51) | 30 (73) | 37 (90) | 32 (78) | 37 (90) | 23 (56) | 9 (22) | 19* (46) | 2 (5) |
| IHC (n = 151) | 66 (44) | 37 (25) | 102 (68) | 125† (83) | 123 (82) | 121 (80) | 80 (53) | 28 (19) | 50 (33) | 13 (9) |
| No IHC (n = 17) | 7 (41) | 2 (12) | 11 (65) | 9† (53) | 15 (88) | 13 (76) | 10 (59) | 4 (24) | 7 (41) | 1 (6) |
| IHC positive (n = 142) | 63 (44) | 35 (25) | 94 (66) | 118 (83) | 116 (82) | 113 (80) | 76 (54) | 26 (18) | 47 (33) | 13 (9) |
| IHC negative (n = 9) | 3 (33) | 2 (22) | 8 (89) | 7 (78) | 7 (78) | 8 (89) | 4 (44) | 2 (22) | 3 (33) | 0 (0) |
| LTA IHC positive (n = 107) | 50 (47) | 30 (28) | 74 (69) | 90 (84) | 88 (82) | 86 (80) | 59 (55) | 23 (21) | 36 (34) | 12 (11) |
| TLR-4 IHC positive (n = 99) | 35 (35) | 28 (28) | 67 (68) | 84 (85) | 77 (78) | 78 (79) | 55 (56) | 16 (16) | 39 (39) | 8 (8) |
| LTA IHC positive and TLR-4 IHC positive (n = 64) | 32 (50) | 23 (36) | 47 (73) | 56 (88) | 49 (77) | 51 (80) | 38 (59) | 13 (20) | 28 (44) | 7 (11) |
| In-parallel bacterial culture and IHC (n = 166) | 68 (41) | 39 (24) | 102 (61) | 127 (77) | 126 (76) | 124 (75) | 84 (51) | 30 (18) | 56 (34) | 14 (8) |
| In-parallel testing: positive (n = 154) | 68 (44) | 37* (24) | 102 (66) | 127† (83) | 126 (82) | 124 (81) | 84 (55) | 30 (20) | 51 (33) | 14 (9) |
| In-parallel testing: negative (n = 12) | 5 (42) | 2 (17) | 10 (83) | 6†** (50) | 10 (83) | 9 (75) | 4 (33) | 2 (17) | 5 (42) | 0 (0) |
| In-parallel testing: polymicrobial (n = 79) | 42 (53) | 29* (37) | 56 (71) | 70** (89) | 62 (79) | 65 (82) | 48 (61) | 19 (24) | 33 (42) | 8 (10) |
LTA = Lipoteichoic acid. TLR-4 = Toll-like receptor-4.
P = 0.05.
P = 0.03
P < 0.01.
P ≤ 0.0001.
Regarding median age at presentation, duration of antecedent clinical illness, survival duration, and last recorded age, there were no significant differences between cats with positive versus negative bacterial cultures, cats with gram-positive versus gram-negative isolates, and cats with polymicrobial versus single isolate cultures (Table 3 and Supplementary Table S2).
Survival duration and age at death for cats with S-CCHS stratified by presence or absence of bacterial culture, bacterial culture results, IHC findings, and in-parallel interpretation of culture and IHC findings.
| Category | Survival (d) after definitive diagnosis | Age at death (y) | ||||
|---|---|---|---|---|---|---|
| Median | Range | 95% CI | Median | Range | 95% CI | |
| Bacterial cultures (n = 135) | 368† | 1–4,015 | 592–886 | 11.3 | 0.5–20.0 | 10.3–11.9 |
| No bacterial cultures (n = 33) | 25† | 1–4,563 | 114–718 | 11.0 | 0.6–20.1 | 8.8–12.2 |
| Positive cultures (n = 93) | 365 | 1–3,468 | 557–915 | 11.9 | 0.7–20.5 | 10.5–12.3 |
| Negative cultures (n = 42) | 490 | 2–4,015 | 464–992 | 10.1 | 0.5–19.0 | 8.8–11.7 |
| Gram positive (n = 72) | 283 | 1–3,468 | 500–910 | 11.3 | 0.7–20.5 | 10.0–12.1 |
| Gram negative (n = 67) | 365 | 1–3,285 | 487–935 | 11.6 | 0.7–10.0 | 10.2–12.5 |
| Polymicrobial culture (n = 41) | 275 | 1–3,285 | 377–928 | 11.2 | 0.7–18.0 | 9.5–12.2 |
| IHC (n = 151) | 330 | 1–4,563 | 556–845 | 11.1 | 0.5–20.5 | 10.2–11.7 |
| Positive IHC (n = 142) | 329 | 1–4,563 | 510–786 | 11.1 | 0.5–20.5 | 10.2–11.7 |
| Negative IHC (n = 9) | 1,460 | 4–4,015 | 411–2,650 | 12.1 | 4.0–19.0 | 7.4–15.6 |
| LTA positive (n = 107) | 292 | 1–4,563 | 453–766 | 11.0 | 0.5–20.5 | 9.8–11.5 |
| TLR-4 positive (n = 99) | 365 | 1–4,563 | 512–859 | 11.1 | 0.6–20.5 | 10.2–12.1 |
| IHC polymicrobial (n = 64) | 348 | 1–4,563 | 425–859 | 11.1 | 0.6–20.5 | 9.6–12.0 |
| In-parallel testing: culture and IHC (n = 166) | 330 | 1–4,563 | 550–817 | 11.2 | 0.5–20.5 | 10.3–11.7 |
| Positive in-parallel testing (n = 154) | 329 | 1–4,563 | 527–792 | 11.2 | 0.5–20.5 | 10.4–11.8 |
| Negative in-parallel testing (n = 12) | 556 | 4–4,015 | 204–1,789 | 6.3 | 4.0–19.0 | 6.1–12.7 |
| Gram-positive ± LTA positive (n = 118) | 310 | 1–4,563 | 494–806 | 11.1 | 0.5–20.5 | 10.0–11.7 |
| Gram-negative ± TLR-4 positive (n = 111) | 348 | 1–4,563 | 505–827 | 11.2 | 0.6–20.5 | 10.5–12.2 |
| Polymicrobial: culture or IHC (n = 79) | 330 | 1–4,563 | 464–868 | 11.2 | 0.6–20.5 | 10.1–12.2 |
P = 0.0007.
See Table 2 for remainder of key.
IHC detection of LTA and TLR-4
None of the 20 control cats had positive LTA or TLR-4 IHC staining. Of 151 cats with S-CCHS that had IHC staining, 142 (94%) were positive. When positive, IHC detection of LTA demonstrated single or multiple organisms within the lumen of bile ducts or phagocytes (ie, macrophages or neutrophils) adjacent to, but external to, bile ducts amid crowded inflammatory infiltrates (Figures 1 and 2). Strong positive LTA staining was especially notable in cats with periductal edema. However, positive staining within bile ducts as well as periportal or portal staining was often inconsistent within and among liver sections from a single cat. Positive LTA IHC staining also was detected within the gallbladder lumen adjacent to gallbladder epithelium, presumably within a mucosal bacterial biofilm (Figure 3). Occasionally, positive LTA IHC staining was seen within the crystalline structure or between laminating layers of choleliths. Immunohistochemical staining for TLR-4 expression demonstrated strong positive staining in neutrophils, macrophages, and biliary epithelium (bile ducts, gallbladder mucosa, and choledochal cyst epithelium) with only occasional staining of regional Kupffer cells and sinusoidal endothelium and rare weak multifocal staining of hepatocytes (Figures 4–7). Among 142 cats with positive IHC staining, 75% were LTA positive and 70% were TLR-4 positive. Among comorbidities, the prevalence of IHC-based bacterial detection ranged from 90% to 97%, with LTA IHC positivity ranging from 72% to 80% and TLR-4 IHC positivity ranging from 68% to 83% of cats (Table 4). Dually positive LTA and TLR-4 IHC staining (ie, IHC-based polymicrobial infection denoting presence of gram-positive and gram-negative bacteria) was observed in 45% of cats and was similar among comorbidities (42% to 56%).
Photomicrographs of serial liver sections from a cat with suppurative cholangitis-cholangiohepatitis (S-CCHS) associated with Clostridium spp. Liver bacterial cultures were negative, whereas cultures of crushed choleliths and gallbladder bile were positive. A—H&E-stained liver section demonstrates neutrophilic and macrophage inflammatory infiltrates in a periductal orientation and intraluminal bile duct plugging (arrow) with inflammatory debris. Bar = 200 µm. B—Immunohistochemical (IHC) staining for lipoteichoic acid (LTA; red signal, hematoxylin counterstain) demonstrates bacteria within a bile duct lumen (arrow) and regional macrophages (asterisk). Bar = 90 µm. C—Fourfold magnification of panel B confirms the rod-form morphology of Clostridium organisms in macrophages (asterisk). Pink stain blush above intraductal bacteria (arrow) indicates additional bacteria in the illustrated field.
Citation: Journal of the American Veterinary Medical Association 260, 2; 10.2460/javma.20.10.0552
Photomicrographs of serial liver sections from a cat with suppurative cholangitis-cholangiohepatitis (S-CCHS) associated with Clostridium spp. Liver bacterial cultures were negative, whereas cultures of crushed choleliths and gallbladder bile were positive. A—H&E-stained liver section demonstrates neutrophilic and macrophage inflammatory infiltrates in a periductal orientation and intraluminal bile duct plugging (arrow) with inflammatory debris. Bar = 200 µm. B—Immunohistochemical (IHC) staining for lipoteichoic acid (LTA; red signal, hematoxylin counterstain) demonstrates bacteria within a bile duct lumen (arrow) and regional macrophages (asterisk). Bar = 90 µm. C—Fourfold magnification of panel B confirms the rod-form morphology of Clostridium organisms in macrophages (asterisk). Pink stain blush above intraductal bacteria (arrow) indicates additional bacteria in the illustrated field.
Citation: Journal of the American Veterinary Medical Association 260, 2; 10.2460/javma.20.10.0552
Photomicrographs of serial liver sections from a cat with suppurative cholangitis-cholangiohepatitis (S-CCHS) associated with Clostridium spp. Liver bacterial cultures were negative, whereas cultures of crushed choleliths and gallbladder bile were positive. A—H&E-stained liver section demonstrates neutrophilic and macrophage inflammatory infiltrates in a periductal orientation and intraluminal bile duct plugging (arrow) with inflammatory debris. Bar = 200 µm. B—Immunohistochemical (IHC) staining for lipoteichoic acid (LTA; red signal, hematoxylin counterstain) demonstrates bacteria within a bile duct lumen (arrow) and regional macrophages (asterisk). Bar = 90 µm. C—Fourfold magnification of panel B confirms the rod-form morphology of Clostridium organisms in macrophages (asterisk). Pink stain blush above intraductal bacteria (arrow) indicates additional bacteria in the illustrated field.
Citation: Journal of the American Veterinary Medical Association 260, 2; 10.2460/javma.20.10.0552
Photomicrographs of serial liver sections from a cat with suppurative cholangitis-cholangiohepatitis (S-CCHS) associated with Clostridium spp. Liver bacterial cultures were negative, whereas cultures of crushed choleliths and gallbladder bile were positive. A—H&E-stained liver section demonstrates neutrophilic and macrophage inflammatory infiltrates in a periductal orientation and intraluminal bile duct plugging (arrow) with inflammatory debris. Bar = 200 µm. B—Immunohistochemical (IHC) staining for lipoteichoic acid (LTA; red signal, hematoxylin counterstain) demonstrates bacteria within a bile duct lumen (arrow) and regional macrophages (asterisk). Bar = 90 µm. C—Fourfold magnification of panel B confirms the rod-form morphology of Clostridium organisms in macrophages (asterisk). Pink stain blush above intraductal bacteria (arrow) indicates additional bacteria in the illustrated field.
Citation: Journal of the American Veterinary Medical Association 260, 2; 10.2460/javma.20.10.0552
Photomicrographs of serial liver sections from a cat with suppurative cholangitis-cholangiohepatitis (S-CCHS) associated with Clostridium spp. Liver bacterial cultures were negative, whereas cultures of crushed choleliths and gallbladder bile were positive. A—H&E-stained liver section demonstrates neutrophilic and macrophage inflammatory infiltrates in a periductal orientation and intraluminal bile duct plugging (arrow) with inflammatory debris. Bar = 200 µm. B—Immunohistochemical (IHC) staining for lipoteichoic acid (LTA; red signal, hematoxylin counterstain) demonstrates bacteria within a bile duct lumen (arrow) and regional macrophages (asterisk). Bar = 90 µm. C—Fourfold magnification of panel B confirms the rod-form morphology of Clostridium organisms in macrophages (asterisk). Pink stain blush above intraductal bacteria (arrow) indicates additional bacteria in the illustrated field.
Citation: Journal of the American Veterinary Medical Association 260, 2; 10.2460/javma.20.10.0552
Photomicrographs of serial liver sections from a cat with suppurative cholangitis-cholangiohepatitis (S-CCHS) associated with Clostridium spp. Liver bacterial cultures were negative, whereas cultures of crushed choleliths and gallbladder bile were positive. A—H&E-stained liver section demonstrates neutrophilic and macrophage inflammatory infiltrates in a periductal orientation and intraluminal bile duct plugging (arrow) with inflammatory debris. Bar = 200 µm. B—Immunohistochemical (IHC) staining for lipoteichoic acid (LTA; red signal, hematoxylin counterstain) demonstrates bacteria within a bile duct lumen (arrow) and regional macrophages (asterisk). Bar = 90 µm. C—Fourfold magnification of panel B confirms the rod-form morphology of Clostridium organisms in macrophages (asterisk). Pink stain blush above intraductal bacteria (arrow) indicates additional bacteria in the illustrated field.
Citation: Journal of the American Veterinary Medical Association 260, 2; 10.2460/javma.20.10.0552
Photomicrographs of serial liver sections from a cat with suppurative cholangitis-cholangiohepatitis (S-CCHS) associated with Clostridium spp. Liver bacterial cultures were negative, whereas cultures of crushed choleliths and gallbladder bile were positive. A—H&E-stained liver section demonstrates neutrophilic and macrophage inflammatory infiltrates in a periductal orientation and intraluminal bile duct plugging (arrow) with inflammatory debris. Bar = 200 µm. B—Immunohistochemical (IHC) staining for lipoteichoic acid (LTA; red signal, hematoxylin counterstain) demonstrates bacteria within a bile duct lumen (arrow) and regional macrophages (asterisk). Bar = 90 µm. C—Fourfold magnification of panel B confirms the rod-form morphology of Clostridium organisms in macrophages (asterisk). Pink stain blush above intraductal bacteria (arrow) indicates additional bacteria in the illustrated field.
Citation: Journal of the American Veterinary Medical Association 260, 2; 10.2460/javma.20.10.0552
Photomicrographs of liver sections with IHC staining for LTA (red signal, hematoxylin counterstain) from 2 cats with S-CCHS. Each bar = 3 mm. A—Corynebacterium spp were cultured from gallbladder bile and choleliths, whereas culture results for liver were negative. Positive staining for LTA (red staining) appears in most portal tracts confirming gram-positive bacteria. Gram stain of liver did not identify bacteria. B—Enterococcus spp were cultured from bile, whereas culture results for liver were negative. Note that only 2 larger portal tracts stain positively for LTA (implicating gram-positive bacteria). C—600× magnification of a liver section from the cat in panel A reveals intraluminal inflammatory debris in a centrally located bile duct (asterisk), periductal edema (white spaces), and small aggregates of bacteria (arrows) surrounded by or within macrophages. Gram staining of liver did not identify bacteria.
Citation: Journal of the American Veterinary Medical Association 260, 2; 10.2460/javma.20.10.0552
Photomicrographs of liver sections with IHC staining for LTA (red signal, hematoxylin counterstain) from 2 cats with S-CCHS. Each bar = 3 mm. A—Corynebacterium spp were cultured from gallbladder bile and choleliths, whereas culture results for liver were negative. Positive staining for LTA (red staining) appears in most portal tracts confirming gram-positive bacteria. Gram stain of liver did not identify bacteria. B—Enterococcus spp were cultured from bile, whereas culture results for liver were negative. Note that only 2 larger portal tracts stain positively for LTA (implicating gram-positive bacteria). C—600× magnification of a liver section from the cat in panel A reveals intraluminal inflammatory debris in a centrally located bile duct (asterisk), periductal edema (white spaces), and small aggregates of bacteria (arrows) surrounded by or within macrophages. Gram staining of liver did not identify bacteria.
Citation: Journal of the American Veterinary Medical Association 260, 2; 10.2460/javma.20.10.0552
Photomicrographs of liver sections with IHC staining for LTA (red signal, hematoxylin counterstain) from 2 cats with S-CCHS. Each bar = 3 mm. A—Corynebacterium spp were cultured from gallbladder bile and choleliths, whereas culture results for liver were negative. Positive staining for LTA (red staining) appears in most portal tracts confirming gram-positive bacteria. Gram stain of liver did not identify bacteria. B—Enterococcus spp were cultured from bile, whereas culture results for liver were negative. Note that only 2 larger portal tracts stain positively for LTA (implicating gram-positive bacteria). C—600× magnification of a liver section from the cat in panel A reveals intraluminal inflammatory debris in a centrally located bile duct (asterisk), periductal edema (white spaces), and small aggregates of bacteria (arrows) surrounded by or within macrophages. Gram staining of liver did not identify bacteria.
Citation: Journal of the American Veterinary Medical Association 260, 2; 10.2460/javma.20.10.0552
Photomicrographs of liver sections with IHC staining for LTA (red signal, hematoxylin counterstain) from 2 cats with S-CCHS. Each bar = 3 mm. A—Corynebacterium spp were cultured from gallbladder bile and choleliths, whereas culture results for liver were negative. Positive staining for LTA (red staining) appears in most portal tracts confirming gram-positive bacteria. Gram stain of liver did not identify bacteria. B—Enterococcus spp were cultured from bile, whereas culture results for liver were negative. Note that only 2 larger portal tracts stain positively for LTA (implicating gram-positive bacteria). C—600× magnification of a liver section from the cat in panel A reveals intraluminal inflammatory debris in a centrally located bile duct (asterisk), periductal edema (white spaces), and small aggregates of bacteria (arrows) surrounded by or within macrophages. Gram staining of liver did not identify bacteria.
Citation: Journal of the American Veterinary Medical Association 260, 2; 10.2460/javma.20.10.0552
Photomicrographs of liver sections with IHC staining for LTA (red signal, hematoxylin counterstain) from 2 cats with S-CCHS. Each bar = 3 mm. A—Corynebacterium spp were cultured from gallbladder bile and choleliths, whereas culture results for liver were negative. Positive staining for LTA (red staining) appears in most portal tracts confirming gram-positive bacteria. Gram stain of liver did not identify bacteria. B—Enterococcus spp were cultured from bile, whereas culture results for liver were negative. Note that only 2 larger portal tracts stain positively for LTA (implicating gram-positive bacteria). C—600× magnification of a liver section from the cat in panel A reveals intraluminal inflammatory debris in a centrally located bile duct (asterisk), periductal edema (white spaces), and small aggregates of bacteria (arrows) surrounded by or within macrophages. Gram staining of liver did not identify bacteria.
Citation: Journal of the American Veterinary Medical Association 260, 2; 10.2460/javma.20.10.0552
Photomicrographs of liver sections with IHC staining for LTA (red signal, hematoxylin counterstain) from 2 cats with S-CCHS. Each bar = 3 mm. A—Corynebacterium spp were cultured from gallbladder bile and choleliths, whereas culture results for liver were negative. Positive staining for LTA (red staining) appears in most portal tracts confirming gram-positive bacteria. Gram stain of liver did not identify bacteria. B—Enterococcus spp were cultured from bile, whereas culture results for liver were negative. Note that only 2 larger portal tracts stain positively for LTA (implicating gram-positive bacteria). C—600× magnification of a liver section from the cat in panel A reveals intraluminal inflammatory debris in a centrally located bile duct (asterisk), periductal edema (white spaces), and small aggregates of bacteria (arrows) surrounded by or within macrophages. Gram staining of liver did not identify bacteria.
Citation: Journal of the American Veterinary Medical Association 260, 2; 10.2460/javma.20.10.0552
Photomicrographs of liver sections with IHC staining for LTA (red signal, hematoxylin counterstain) from 2 cats with S-CCHS. Each bar = 3 mm. A—Corynebacterium spp were cultured from gallbladder bile and choleliths, whereas culture results for liver were negative. Positive staining for LTA (red staining) appears in most portal tracts confirming gram-positive bacteria. Gram stain of liver did not identify bacteria. B—Enterococcus spp were cultured from bile, whereas culture results for liver were negative. Note that only 2 larger portal tracts stain positively for LTA (implicating gram-positive bacteria). C—600× magnification of a liver section from the cat in panel A reveals intraluminal inflammatory debris in a centrally located bile duct (asterisk), periductal edema (white spaces), and small aggregates of bacteria (arrows) surrounded by or within macrophages. Gram staining of liver did not identify bacteria.
Citation: Journal of the American Veterinary Medical Association 260, 2; 10.2460/javma.20.10.0552
Photomicrographs of gallbladder and laminated cholelith (yellow-orange color) sections from cat with Escherichia coli and Enterococcus casseliflavus polymicrobial infection, as identified by bacterial culture of combined liver, bile, and crushed cholelith inoculate. A—Overview low magnification shows thick inflamed gallbladder wall, hemobilia, and laminated cholelith (top right). Bar = 9 mm. B—Laminated cholelith. Bar = 3 mm. C—Gram stain demonstrates mat of blue-staining gram-positive bacteria within the cholelith matrix. Bar = 60 µm. D—Immunohistochemical staining for LTA (red signal, hematoxylin counterstain) illustrates chains, mats, doublets, and single gram-positive coccoid bacteria in gallbladder biofilm attached to the cholelith. Bar = 60 µm. Positive LTA IHC staining between lamination layers of the cholelith (not shown) was also evident.
Citation: Journal of the American Veterinary Medical Association 260, 2; 10.2460/javma.20.10.0552
Photomicrographs of gallbladder and laminated cholelith (yellow-orange color) sections from cat with Escherichia coli and Enterococcus casseliflavus polymicrobial infection, as identified by bacterial culture of combined liver, bile, and crushed cholelith inoculate. A—Overview low magnification shows thick inflamed gallbladder wall, hemobilia, and laminated cholelith (top right). Bar = 9 mm. B—Laminated cholelith. Bar = 3 mm. C—Gram stain demonstrates mat of blue-staining gram-positive bacteria within the cholelith matrix. Bar = 60 µm. D—Immunohistochemical staining for LTA (red signal, hematoxylin counterstain) illustrates chains, mats, doublets, and single gram-positive coccoid bacteria in gallbladder biofilm attached to the cholelith. Bar = 60 µm. Positive LTA IHC staining between lamination layers of the cholelith (not shown) was also evident.
Citation: Journal of the American Veterinary Medical Association 260, 2; 10.2460/javma.20.10.0552
Photomicrographs of gallbladder and laminated cholelith (yellow-orange color) sections from cat with Escherichia coli and Enterococcus casseliflavus polymicrobial infection, as identified by bacterial culture of combined liver, bile, and crushed cholelith inoculate. A—Overview low magnification shows thick inflamed gallbladder wall, hemobilia, and laminated cholelith (top right). Bar = 9 mm. B—Laminated cholelith. Bar = 3 mm. C—Gram stain demonstrates mat of blue-staining gram-positive bacteria within the cholelith matrix. Bar = 60 µm. D—Immunohistochemical staining for LTA (red signal, hematoxylin counterstain) illustrates chains, mats, doublets, and single gram-positive coccoid bacteria in gallbladder biofilm attached to the cholelith. Bar = 60 µm. Positive LTA IHC staining between lamination layers of the cholelith (not shown) was also evident.
Citation: Journal of the American Veterinary Medical Association 260, 2; 10.2460/javma.20.10.0552
Photomicrographs of gallbladder and laminated cholelith (yellow-orange color) sections from cat with Escherichia coli and Enterococcus casseliflavus polymicrobial infection, as identified by bacterial culture of combined liver, bile, and crushed cholelith inoculate. A—Overview low magnification shows thick inflamed gallbladder wall, hemobilia, and laminated cholelith (top right). Bar = 9 mm. B—Laminated cholelith. Bar = 3 mm. C—Gram stain demonstrates mat of blue-staining gram-positive bacteria within the cholelith matrix. Bar = 60 µm. D—Immunohistochemical staining for LTA (red signal, hematoxylin counterstain) illustrates chains, mats, doublets, and single gram-positive coccoid bacteria in gallbladder biofilm attached to the cholelith. Bar = 60 µm. Positive LTA IHC staining between lamination layers of the cholelith (not shown) was also evident.
Citation: Journal of the American Veterinary Medical Association 260, 2; 10.2460/javma.20.10.0552
Photomicrographs of gallbladder and laminated cholelith (yellow-orange color) sections from cat with Escherichia coli and Enterococcus casseliflavus polymicrobial infection, as identified by bacterial culture of combined liver, bile, and crushed cholelith inoculate. A—Overview low magnification shows thick inflamed gallbladder wall, hemobilia, and laminated cholelith (top right). Bar = 9 mm. B—Laminated cholelith. Bar = 3 mm. C—Gram stain demonstrates mat of blue-staining gram-positive bacteria within the cholelith matrix. Bar = 60 µm. D—Immunohistochemical staining for LTA (red signal, hematoxylin counterstain) illustrates chains, mats, doublets, and single gram-positive coccoid bacteria in gallbladder biofilm attached to the cholelith. Bar = 60 µm. Positive LTA IHC staining between lamination layers of the cholelith (not shown) was also evident.
Citation: Journal of the American Veterinary Medical Association 260, 2; 10.2460/javma.20.10.0552
Photomicrographs of gallbladder and laminated cholelith (yellow-orange color) sections from cat with Escherichia coli and Enterococcus casseliflavus polymicrobial infection, as identified by bacterial culture of combined liver, bile, and crushed cholelith inoculate. A—Overview low magnification shows thick inflamed gallbladder wall, hemobilia, and laminated cholelith (top right). Bar = 9 mm. B—Laminated cholelith. Bar = 3 mm. C—Gram stain demonstrates mat of blue-staining gram-positive bacteria within the cholelith matrix. Bar = 60 µm. D—Immunohistochemical staining for LTA (red signal, hematoxylin counterstain) illustrates chains, mats, doublets, and single gram-positive coccoid bacteria in gallbladder biofilm attached to the cholelith. Bar = 60 µm. Positive LTA IHC staining between lamination layers of the cholelith (not shown) was also evident.
Citation: Journal of the American Veterinary Medical Association 260, 2; 10.2460/javma.20.10.0552
Photomicrographs of gallbladder and laminated cholelith (yellow-orange color) sections from cat with Escherichia coli and Enterococcus casseliflavus polymicrobial infection, as identified by bacterial culture of combined liver, bile, and crushed cholelith inoculate. A—Overview low magnification shows thick inflamed gallbladder wall, hemobilia, and laminated cholelith (top right). Bar = 9 mm. B—Laminated cholelith. Bar = 3 mm. C—Gram stain demonstrates mat of blue-staining gram-positive bacteria within the cholelith matrix. Bar = 60 µm. D—Immunohistochemical staining for LTA (red signal, hematoxylin counterstain) illustrates chains, mats, doublets, and single gram-positive coccoid bacteria in gallbladder biofilm attached to the cholelith. Bar = 60 µm. Positive LTA IHC staining between lamination layers of the cholelith (not shown) was also evident.
Citation: Journal of the American Veterinary Medical Association 260, 2; 10.2460/javma.20.10.0552
Photomicrographs of gallbladder and laminated cholelith (yellow-orange color) sections from cat with Escherichia coli and Enterococcus casseliflavus polymicrobial infection, as identified by bacterial culture of combined liver, bile, and crushed cholelith inoculate. A—Overview low magnification shows thick inflamed gallbladder wall, hemobilia, and laminated cholelith (top right). Bar = 9 mm. B—Laminated cholelith. Bar = 3 mm. C—Gram stain demonstrates mat of blue-staining gram-positive bacteria within the cholelith matrix. Bar = 60 µm. D—Immunohistochemical staining for LTA (red signal, hematoxylin counterstain) illustrates chains, mats, doublets, and single gram-positive coccoid bacteria in gallbladder biofilm attached to the cholelith. Bar = 60 µm. Positive LTA IHC staining between lamination layers of the cholelith (not shown) was also evident.
Citation: Journal of the American Veterinary Medical Association 260, 2; 10.2460/javma.20.10.0552
Photomicrographs of gallbladder and laminated cholelith (yellow-orange color) sections from cat with Escherichia coli and Enterococcus casseliflavus polymicrobial infection, as identified by bacterial culture of combined liver, bile, and crushed cholelith inoculate. A—Overview low magnification shows thick inflamed gallbladder wall, hemobilia, and laminated cholelith (top right). Bar = 9 mm. B—Laminated cholelith. Bar = 3 mm. C—Gram stain demonstrates mat of blue-staining gram-positive bacteria within the cholelith matrix. Bar = 60 µm. D—Immunohistochemical staining for LTA (red signal, hematoxylin counterstain) illustrates chains, mats, doublets, and single gram-positive coccoid bacteria in gallbladder biofilm attached to the cholelith. Bar = 60 µm. Positive LTA IHC staining between lamination layers of the cholelith (not shown) was also evident.
Citation: Journal of the American Veterinary Medical Association 260, 2; 10.2460/javma.20.10.0552
Photomicrographs of serial liver sections showing a single portal region from a cat with S-CCHS (A through C) and a midzonal parenchymal liver section from the same cat demonstrating neutrophilic sinusoidal flux (D). E coli and α-hemolytic streptococci were cultured from liver. A—Liver section stained with H&E showing too-numerous-to-count neutrophils and macrophages within portal and periportal regions, and neutrophils invading bile duct epithelium (inset). Bar = 200 µm. B—Serial sections of the same liver region as in panel A IHC stained for toll-like receptor-4 (TLR-4) expression (brown signal, hematoxylin counterstain) showing numerous positive (brown-stained) neutrophils, macrophages, and occasional lymphocytes (implicating gram-negative bacteria endotoxin exposure beyond tolerogenic limits). Bar = 200 µm. C—Magnified (8X) image demarcated by dashed line in panel B highlights positive TLR-4 staining in inflammatory cells and occasionally ductal epithelium. Bar = 70 µm. D—Midzonal (zone 2) hepatic parenchyma demonstrating TLR-4 expression in neutrophils and monocytes fluxing across hepatic sinusoids. Bar = 70 µm.
Citation: Journal of the American Veterinary Medical Association 260, 2; 10.2460/javma.20.10.0552
Photomicrographs of serial liver sections showing a single portal region from a cat with S-CCHS (A through C) and a midzonal parenchymal liver section from the same cat demonstrating neutrophilic sinusoidal flux (D). E coli and α-hemolytic streptococci were cultured from liver. A—Liver section stained with H&E showing too-numerous-to-count neutrophils and macrophages within portal and periportal regions, and neutrophils invading bile duct epithelium (inset). Bar = 200 µm. B—Serial sections of the same liver region as in panel A IHC stained for toll-like receptor-4 (TLR-4) expression (brown signal, hematoxylin counterstain) showing numerous positive (brown-stained) neutrophils, macrophages, and occasional lymphocytes (implicating gram-negative bacteria endotoxin exposure beyond tolerogenic limits). Bar = 200 µm. C—Magnified (8X) image demarcated by dashed line in panel B highlights positive TLR-4 staining in inflammatory cells and occasionally ductal epithelium. Bar = 70 µm. D—Midzonal (zone 2) hepatic parenchyma demonstrating TLR-4 expression in neutrophils and monocytes fluxing across hepatic sinusoids. Bar = 70 µm.
Citation: Journal of the American Veterinary Medical Association 260, 2; 10.2460/javma.20.10.0552
Photomicrographs of serial liver sections showing a single portal region from a cat with S-CCHS (A through C) and a midzonal parenchymal liver section from the same cat demonstrating neutrophilic sinusoidal flux (D). E coli and α-hemolytic streptococci were cultured from liver. A—Liver section stained with H&E showing too-numerous-to-count neutrophils and macrophages within portal and periportal regions, and neutrophils invading bile duct epithelium (inset). Bar = 200 µm. B—Serial sections of the same liver region as in panel A IHC stained for toll-like receptor-4 (TLR-4) expression (brown signal, hematoxylin counterstain) showing numerous positive (brown-stained) neutrophils, macrophages, and occasional lymphocytes (implicating gram-negative bacteria endotoxin exposure beyond tolerogenic limits). Bar = 200 µm. C—Magnified (8X) image demarcated by dashed line in panel B highlights positive TLR-4 staining in inflammatory cells and occasionally ductal epithelium. Bar = 70 µm. D—Midzonal (zone 2) hepatic parenchyma demonstrating TLR-4 expression in neutrophils and monocytes fluxing across hepatic sinusoids. Bar = 70 µm.
Citation: Journal of the American Veterinary Medical Association 260, 2; 10.2460/javma.20.10.0552
Photomicrographs of serial liver sections showing a single portal region from a cat with S-CCHS (A through C) and a midzonal parenchymal liver section from the same cat demonstrating neutrophilic sinusoidal flux (D). E coli and α-hemolytic streptococci were cultured from liver. A—Liver section stained with H&E showing too-numerous-to-count neutrophils and macrophages within portal and periportal regions, and neutrophils invading bile duct epithelium (inset). Bar = 200 µm. B—Serial sections of the same liver region as in panel A IHC stained for toll-like receptor-4 (TLR-4) expression (brown signal, hematoxylin counterstain) showing numerous positive (brown-stained) neutrophils, macrophages, and occasional lymphocytes (implicating gram-negative bacteria endotoxin exposure beyond tolerogenic limits). Bar = 200 µm. C—Magnified (8X) image demarcated by dashed line in panel B highlights positive TLR-4 staining in inflammatory cells and occasionally ductal epithelium. Bar = 70 µm. D—Midzonal (zone 2) hepatic parenchyma demonstrating TLR-4 expression in neutrophils and monocytes fluxing across hepatic sinusoids. Bar = 70 µm.
Citation: Journal of the American Veterinary Medical Association 260, 2; 10.2460/javma.20.10.0552
Photomicrographs of serial liver sections showing a single portal region from a cat with S-CCHS (A through C) and a midzonal parenchymal liver section from the same cat demonstrating neutrophilic sinusoidal flux (D). E coli and α-hemolytic streptococci were cultured from liver. A—Liver section stained with H&E showing too-numerous-to-count neutrophils and macrophages within portal and periportal regions, and neutrophils invading bile duct epithelium (inset). Bar = 200 µm. B—Serial sections of the same liver region as in panel A IHC stained for toll-like receptor-4 (TLR-4) expression (brown signal, hematoxylin counterstain) showing numerous positive (brown-stained) neutrophils, macrophages, and occasional lymphocytes (implicating gram-negative bacteria endotoxin exposure beyond tolerogenic limits). Bar = 200 µm. C—Magnified (8X) image demarcated by dashed line in panel B highlights positive TLR-4 staining in inflammatory cells and occasionally ductal epithelium. Bar = 70 µm. D—Midzonal (zone 2) hepatic parenchyma demonstrating TLR-4 expression in neutrophils and monocytes fluxing across hepatic sinusoids. Bar = 70 µm.
Citation: Journal of the American Veterinary Medical Association 260, 2; 10.2460/javma.20.10.0552
Photomicrographs of serial liver sections showing a single portal region from a cat with S-CCHS (A through C) and a midzonal parenchymal liver section from the same cat demonstrating neutrophilic sinusoidal flux (D). E coli and α-hemolytic streptococci were cultured from liver. A—Liver section stained with H&E showing too-numerous-to-count neutrophils and macrophages within portal and periportal regions, and neutrophils invading bile duct epithelium (inset). Bar = 200 µm. B—Serial sections of the same liver region as in panel A IHC stained for toll-like receptor-4 (TLR-4) expression (brown signal, hematoxylin counterstain) showing numerous positive (brown-stained) neutrophils, macrophages, and occasional lymphocytes (implicating gram-negative bacteria endotoxin exposure beyond tolerogenic limits). Bar = 200 µm. C—Magnified (8X) image demarcated by dashed line in panel B highlights positive TLR-4 staining in inflammatory cells and occasionally ductal epithelium. Bar = 70 µm. D—Midzonal (zone 2) hepatic parenchyma demonstrating TLR-4 expression in neutrophils and monocytes fluxing across hepatic sinusoids. Bar = 70 µm.
Citation: Journal of the American Veterinary Medical Association 260, 2; 10.2460/javma.20.10.0552
Photomicrographs of serial liver sections showing a single portal region from a cat with S-CCHS (A through C) and a midzonal parenchymal liver section from the same cat demonstrating neutrophilic sinusoidal flux (D). E coli and α-hemolytic streptococci were cultured from liver. A—Liver section stained with H&E showing too-numerous-to-count neutrophils and macrophages within portal and periportal regions, and neutrophils invading bile duct epithelium (inset). Bar = 200 µm. B—Serial sections of the same liver region as in panel A IHC stained for toll-like receptor-4 (TLR-4) expression (brown signal, hematoxylin counterstain) showing numerous positive (brown-stained) neutrophils, macrophages, and occasional lymphocytes (implicating gram-negative bacteria endotoxin exposure beyond tolerogenic limits). Bar = 200 µm. C—Magnified (8X) image demarcated by dashed line in panel B highlights positive TLR-4 staining in inflammatory cells and occasionally ductal epithelium. Bar = 70 µm. D—Midzonal (zone 2) hepatic parenchyma demonstrating TLR-4 expression in neutrophils and monocytes fluxing across hepatic sinusoids. Bar = 70 µm.
Citation: Journal of the American Veterinary Medical Association 260, 2; 10.2460/javma.20.10.0552
Photomicrographs of serial liver sections showing a single portal region from a cat with S-CCHS (A through C) and a midzonal parenchymal liver section from the same cat demonstrating neutrophilic sinusoidal flux (D). E coli and α-hemolytic streptococci were cultured from liver. A—Liver section stained with H&E showing too-numerous-to-count neutrophils and macrophages within portal and periportal regions, and neutrophils invading bile duct epithelium (inset). Bar = 200 µm. B—Serial sections of the same liver region as in panel A IHC stained for toll-like receptor-4 (TLR-4) expression (brown signal, hematoxylin counterstain) showing numerous positive (brown-stained) neutrophils, macrophages, and occasional lymphocytes (implicating gram-negative bacteria endotoxin exposure beyond tolerogenic limits). Bar = 200 µm. C—Magnified (8X) image demarcated by dashed line in panel B highlights positive TLR-4 staining in inflammatory cells and occasionally ductal epithelium. Bar = 70 µm. D—Midzonal (zone 2) hepatic parenchyma demonstrating TLR-4 expression in neutrophils and monocytes fluxing across hepatic sinusoids. Bar = 70 µm.
Citation: Journal of the American Veterinary Medical Association 260, 2; 10.2460/javma.20.10.0552
Photomicrographs of serial liver sections showing a single portal region from a cat with S-CCHS (A through C) and a midzonal parenchymal liver section from the same cat demonstrating neutrophilic sinusoidal flux (D). E coli and α-hemolytic streptococci were cultured from liver. A—Liver section stained with H&E showing too-numerous-to-count neutrophils and macrophages within portal and periportal regions, and neutrophils invading bile duct epithelium (inset). Bar = 200 µm. B—Serial sections of the same liver region as in panel A IHC stained for toll-like receptor-4 (TLR-4) expression (brown signal, hematoxylin counterstain) showing numerous positive (brown-stained) neutrophils, macrophages, and occasional lymphocytes (implicating gram-negative bacteria endotoxin exposure beyond tolerogenic limits). Bar = 200 µm. C—Magnified (8X) image demarcated by dashed line in panel B highlights positive TLR-4 staining in inflammatory cells and occasionally ductal epithelium. Bar = 70 µm. D—Midzonal (zone 2) hepatic parenchyma demonstrating TLR-4 expression in neutrophils and monocytes fluxing across hepatic sinusoids. Bar = 70 µm.
Citation: Journal of the American Veterinary Medical Association 260, 2; 10.2460/javma.20.10.0552
Photomicrographs of serial gallbladder sections from a cat with S-CCHS, cholecystitis, and cholelithiasis. Enterococcus spp and E coli were cultured from gallbladder bile, whereas bacterial culture of liver was negative. A—Gallbladder section stained with H&E demonstrates proliferative mucosa and distinct blue-staining lymphoid follicles reflecting antigenic stimulation (infection). Bar = 6 mm. B—Serial section of tissue shown in panel A with IHC staining for TLR-4 expression (brown signal, hematoxylin counterstain) demonstrates diffusely positive staining of gallbladder epithelium and inflammatory cells. Bar = 6 mm. C—Higher magnification of gallbladder wall from panel B illustrating TLR-4–expressing (brown staining) neutrophils infiltrating the proliferative gallbladder mucosa. Bar = 200 µm.
Citation: Journal of the American Veterinary Medical Association 260, 2; 10.2460/javma.20.10.0552
Photomicrographs of serial gallbladder sections from a cat with S-CCHS, cholecystitis, and cholelithiasis. Enterococcus spp and E coli were cultured from gallbladder bile, whereas bacterial culture of liver was negative. A—Gallbladder section stained with H&E demonstrates proliferative mucosa and distinct blue-staining lymphoid follicles reflecting antigenic stimulation (infection). Bar = 6 mm. B—Serial section of tissue shown in panel A with IHC staining for TLR-4 expression (brown signal, hematoxylin counterstain) demonstrates diffusely positive staining of gallbladder epithelium and inflammatory cells. Bar = 6 mm. C—Higher magnification of gallbladder wall from panel B illustrating TLR-4–expressing (brown staining) neutrophils infiltrating the proliferative gallbladder mucosa. Bar = 200 µm.
Citation: Journal of the American Veterinary Medical Association 260, 2; 10.2460/javma.20.10.0552
Photomicrographs of serial gallbladder sections from a cat with S-CCHS, cholecystitis, and cholelithiasis. Enterococcus spp and E coli were cultured from gallbladder bile, whereas bacterial culture of liver was negative. A—Gallbladder section stained with H&E demonstrates proliferative mucosa and distinct blue-staining lymphoid follicles reflecting antigenic stimulation (infection). Bar = 6 mm. B—Serial section of tissue shown in panel A with IHC staining for TLR-4 expression (brown signal, hematoxylin counterstain) demonstrates diffusely positive staining of gallbladder epithelium and inflammatory cells. Bar = 6 mm. C—Higher magnification of gallbladder wall from panel B illustrating TLR-4–expressing (brown staining) neutrophils infiltrating the proliferative gallbladder mucosa. Bar = 200 µm.
Citation: Journal of the American Veterinary Medical Association 260, 2; 10.2460/javma.20.10.0552
Photomicrographs of serial gallbladder sections from a cat with S-CCHS, cholecystitis, and cholelithiasis. Enterococcus spp and E coli were cultured from gallbladder bile, whereas bacterial culture of liver was negative. A—Gallbladder section stained with H&E demonstrates proliferative mucosa and distinct blue-staining lymphoid follicles reflecting antigenic stimulation (infection). Bar = 6 mm. B—Serial section of tissue shown in panel A with IHC staining for TLR-4 expression (brown signal, hematoxylin counterstain) demonstrates diffusely positive staining of gallbladder epithelium and inflammatory cells. Bar = 6 mm. C—Higher magnification of gallbladder wall from panel B illustrating TLR-4–expressing (brown staining) neutrophils infiltrating the proliferative gallbladder mucosa. Bar = 200 µm.
Citation: Journal of the American Veterinary Medical Association 260, 2; 10.2460/javma.20.10.0552
Photomicrographs of serial gallbladder sections from a cat with S-CCHS, cholecystitis, and cholelithiasis. Enterococcus spp and E coli were cultured from gallbladder bile, whereas bacterial culture of liver was negative. A—Gallbladder section stained with H&E demonstrates proliferative mucosa and distinct blue-staining lymphoid follicles reflecting antigenic stimulation (infection). Bar = 6 mm. B—Serial section of tissue shown in panel A with IHC staining for TLR-4 expression (brown signal, hematoxylin counterstain) demonstrates diffusely positive staining of gallbladder epithelium and inflammatory cells. Bar = 6 mm. C—Higher magnification of gallbladder wall from panel B illustrating TLR-4–expressing (brown staining) neutrophils infiltrating the proliferative gallbladder mucosa. Bar = 200 µm.
Citation: Journal of the American Veterinary Medical Association 260, 2; 10.2460/javma.20.10.0552
Photomicrographs of serial gallbladder sections from a cat with S-CCHS, cholecystitis, and cholelithiasis. Enterococcus spp and E coli were cultured from gallbladder bile, whereas bacterial culture of liver was negative. A—Gallbladder section stained with H&E demonstrates proliferative mucosa and distinct blue-staining lymphoid follicles reflecting antigenic stimulation (infection). Bar = 6 mm. B—Serial section of tissue shown in panel A with IHC staining for TLR-4 expression (brown signal, hematoxylin counterstain) demonstrates diffusely positive staining of gallbladder epithelium and inflammatory cells. Bar = 6 mm. C—Higher magnification of gallbladder wall from panel B illustrating TLR-4–expressing (brown staining) neutrophils infiltrating the proliferative gallbladder mucosa. Bar = 200 µm.
Citation: Journal of the American Veterinary Medical Association 260, 2; 10.2460/javma.20.10.0552
Photomicrographs of serial gallbladder sections from a cat with S-CCHS, cholecystitis, and cholelithiasis. Enterococcus spp and E coli were cultured from gallbladder bile, whereas bacterial culture of liver was negative. A—Gallbladder section stained with H&E demonstrates proliferative mucosa and distinct blue-staining lymphoid follicles reflecting antigenic stimulation (infection). Bar = 6 mm. B—Serial section of tissue shown in panel A with IHC staining for TLR-4 expression (brown signal, hematoxylin counterstain) demonstrates diffusely positive staining of gallbladder epithelium and inflammatory cells. Bar = 6 mm. C—Higher magnification of gallbladder wall from panel B illustrating TLR-4–expressing (brown staining) neutrophils infiltrating the proliferative gallbladder mucosa. Bar = 200 µm.
Citation: Journal of the American Veterinary Medical Association 260, 2; 10.2460/javma.20.10.0552
Photomicrographs of gallbladder sections from a cat with S-CCHS, cholelithiasis, and cholecystitis. Corynebacterium spp and E coli were cultured from gallbladder bile and crushed cholelith, whereas bacterial culture of liver was negative. A—Section of gallbladder with IHC staining for TLR-4 (brown signal, hematoxylin counterstain) demonstrates markedly thickened gallbladder wall and papilliferous mucosa (typically associated with feline cholelithiasis) with diffusely positive mucosal TLR-4 expression. Lymphoid follicles (blue circular lesions) reflect response to antigenic stimulation (infection). Bar = 2 mm. B—Higher magnification of panel A highlights diffuse cytosolic TLR-4 expression (brown signal) within the gallbladder mucosa, reflecting pathologic exposure of luminal epithelial surface to gram-negative bacteria (endotoxin). Bar = 60 µm.
Citation: Journal of the American Veterinary Medical Association 260, 2; 10.2460/javma.20.10.0552
Photomicrographs of gallbladder sections from a cat with S-CCHS, cholelithiasis, and cholecystitis. Corynebacterium spp and E coli were cultured from gallbladder bile and crushed cholelith, whereas bacterial culture of liver was negative. A—Section of gallbladder with IHC staining for TLR-4 (brown signal, hematoxylin counterstain) demonstrates markedly thickened gallbladder wall and papilliferous mucosa (typically associated with feline cholelithiasis) with diffusely positive mucosal TLR-4 expression. Lymphoid follicles (blue circular lesions) reflect response to antigenic stimulation (infection). Bar = 2 mm. B—Higher magnification of panel A highlights diffuse cytosolic TLR-4 expression (brown signal) within the gallbladder mucosa, reflecting pathologic exposure of luminal epithelial surface to gram-negative bacteria (endotoxin). Bar = 60 µm.
Citation: Journal of the American Veterinary Medical Association 260, 2; 10.2460/javma.20.10.0552
Photomicrographs of gallbladder sections from a cat with S-CCHS, cholelithiasis, and cholecystitis. Corynebacterium spp and E coli were cultured from gallbladder bile and crushed cholelith, whereas bacterial culture of liver was negative. A—Section of gallbladder with IHC staining for TLR-4 (brown signal, hematoxylin counterstain) demonstrates markedly thickened gallbladder wall and papilliferous mucosa (typically associated with feline cholelithiasis) with diffusely positive mucosal TLR-4 expression. Lymphoid follicles (blue circular lesions) reflect response to antigenic stimulation (infection). Bar = 2 mm. B—Higher magnification of panel A highlights diffuse cytosolic TLR-4 expression (brown signal) within the gallbladder mucosa, reflecting pathologic exposure of luminal epithelial surface to gram-negative bacteria (endotoxin). Bar = 60 µm.
Citation: Journal of the American Veterinary Medical Association 260, 2; 10.2460/javma.20.10.0552
Photomicrographs of gallbladder sections from a cat with S-CCHS, cholelithiasis, and cholecystitis. Corynebacterium spp and E coli were cultured from gallbladder bile and crushed cholelith, whereas bacterial culture of liver was negative. A—Section of gallbladder with IHC staining for TLR-4 (brown signal, hematoxylin counterstain) demonstrates markedly thickened gallbladder wall and papilliferous mucosa (typically associated with feline cholelithiasis) with diffusely positive mucosal TLR-4 expression. Lymphoid follicles (blue circular lesions) reflect response to antigenic stimulation (infection). Bar = 2 mm. B—Higher magnification of panel A highlights diffuse cytosolic TLR-4 expression (brown signal) within the gallbladder mucosa, reflecting pathologic exposure of luminal epithelial surface to gram-negative bacteria (endotoxin). Bar = 60 µm.
Citation: Journal of the American Veterinary Medical Association 260, 2; 10.2460/javma.20.10.0552
Photomicrographs of gallbladder sections from a cat with S-CCHS, cholelithiasis, and cholecystitis. Corynebacterium spp and E coli were cultured from gallbladder bile and crushed cholelith, whereas bacterial culture of liver was negative. A—Section of gallbladder with IHC staining for TLR-4 (brown signal, hematoxylin counterstain) demonstrates markedly thickened gallbladder wall and papilliferous mucosa (typically associated with feline cholelithiasis) with diffusely positive mucosal TLR-4 expression. Lymphoid follicles (blue circular lesions) reflect response to antigenic stimulation (infection). Bar = 2 mm. B—Higher magnification of panel A highlights diffuse cytosolic TLR-4 expression (brown signal) within the gallbladder mucosa, reflecting pathologic exposure of luminal epithelial surface to gram-negative bacteria (endotoxin). Bar = 60 µm.
Citation: Journal of the American Veterinary Medical Association 260, 2; 10.2460/javma.20.10.0552
Photomicrographs of serial liver sections from a cat with Caroli ductal plate malformation (DPM) with severe S-CCHS associated with polymicrobial infection (Enterococcus spp and E coli). Bacteria were cultured from gallbladder bile and liver. A—H&E-stained section of liver demonstrates malformed (irregular, corrugated, and dilated) bile duct profiles with dense purulent intraluminal debris. Bile ducts are embedded in exuberant extracellular matrix (fibrosis) typical of DPM congenital hepatic fibrosis. This cat had acquired portosystemic shunts evolved from presinusoidal portal hypertension caused by DPM. Bar = 200 µm. B—Immunohistochemical staining for TLR-4 (brown signal, hematoxylin counterstain) demonstrates diffusely positive TLR-4 expression in biliary epithelium and inflammatory infiltrates within and surrounding bile ducts. Bar = 200 µm.
Citation: Journal of the American Veterinary Medical Association 260, 2; 10.2460/javma.20.10.0552<