Eubacterial Fluorescence In Situ Hybridization and Histologic Features In 25 Dogs with 1

26 27 Objectives– To detect and localise bacteria in gallbladder mucoceles utilizing fluorescence in 28 situ hybridization (FISH). To report clinical signs, clinicopathologic abnormalities, sonographic 29 findings and histopathological findings in FISH+ and FISHdogs with gallbladder mucoceles. 30 Materials and Methods – Retrospective review of signalment, clinical signs, clinicopathologic 31 and sonographic findings of 25 cases of histopathologically confirmed gallbladder mucocele. 32 Histopathological sections of GBM were evaluated for cystic mucinous hyperplasia, cystic 33 mucinous hyperplasia with cholecystitis and rupture. The number and spatial distribution of 34 bacteria was determined by eubacterial FISH. Gallbladder contents were cultured in 21 dogs. 35 Results –Bacteria were detected within or adherent to the gallbladder in eight of 25 (32%) cases 36 Bacterial culture was positive in one dog. Cystic mucinous hyperplasia with concurrent 37 cholecystitis was found in 17/25 (68%) of dogs with gallbladder mucocele. 38 Clinical significance – FISH was more sensitive for detection of bacteria in gallbladder 39 mucoceles when compared to bacterial culture of bile. Cholecystitis was common in dogs with 40 gallbladder mucocele. Further study is required to elucidate the relationship of cystic mucinous 41 hyperplasia, bacteria and cholecystitis in the aetiopathogenesis and progression of GBM. 42 43

Formalin-fixed paraffin-embedded histological sections (4 μm) were mounted on Probe-On Plus 102 slides (Fisher Scientific) and evaluated by FISH as previously described (Simpson et al. 2006). 103 In short, paraffin-embedded biopsy specimens were de-paraffinized by passage through xylene 104 (3 × 10 mins), 100% alcohol (2 × 5 mins), 95% ethanol (5 mins) and, finally, 70% ethanol (5 105 mins). The slides were air-dried. FISH probes 5'-labeled with either Cy3 or 6-FAM (Integrated 106 DNA Technologies) were reconstituted with sterile water and diluted to a working concentration 107 of 5 ng/µl with a hybridization buffer appropriate to the probe. For evaluation EUB338 Cy-3 was 108 combined with the irrelevant probe non-EUB-338-FAM (ACTCCTACGGGAGGCAGC) to 109 control for non-specific hybridization. Sections were examined on an Olympus BX51 110 epifluorescence microscope and images captured with an Olympus DP-7 camera (Olympus 111 America). The relative number and spatial orientation of bacteria within the section of 112 gallbladder was also recorded. 113 114

Ultrasonographic Data 115
All ultrasound examinations were performed by a board-certified veterinary radiologist or a 116 veterinary radiology resident under the direct supervision of a board-certified veterinary 117 radiologist. Written reports, still images, and video clips of ultrasonographic examinations were 118 then retrospectively reviewed by a board-certified veterinary radiologist (EKR) blinded to the 119 case data. The appearance of the gallbladder, gallbladder wall, adjacent abdominal structures, 120 and free peritoneal fluid was evaluated. Sonographic features of GBM were defined as stellate or 121 finely striated bile patterns that differed from biliary sludge by the absence of gravity-dependent 122 bile movement (Besso et al. 2000). The gall bladder wall was evaluated for echogenicity, 123 presence of oedema, thickening and rupture. A thickened gallbladder wall was defined as more 124 than 2 mm in dogs (Nyland & Hager 1985). GB wall oedema was defined as a thickened GB wall 125 with a hypoechoic layer within the GB wall. 126 127

Histopathological Evaluation 128
Original histopathologic reports (all by board-certified veterinary pathologists) were reviewed to 129 confirm a diagnosis of GBM. Following this, archival formalin-fixed paraffin-embedded tissue 130 blocks were located for 23/25 cases, sectioned at 4 um and stained with hematoxylin and eosin 131 (HE) for blinded review by a board-certified veterinary pathologist (SLP) employing WSAVA 132 criteria for CMH and cholecystitis (Rothuizen 2006). Cholecystitis was defined as the presence 133 of a neutrophilic and/or lymphoplasmacytic infiltrate in the epithelium or wall of the gallbladder 134 +/-fibrosis (Rothuizen 2006) and assigned a grade of mild, moderate or severe. Each case was 135 assigned to one of 4 groups: CMH, CMH with cholecystitis (CMHC), mild, moderate or severe. 136 137

Statistical Analysis 138
Descriptive statistics were calculated for the presence/absence of clinical signs, 139 clinicopathological data, sonographic findings, and histologic findings in FISH+ versus FISH-140 dogs with GBM. 141

143
Twenty-six dogs with a histopathological diagnosis of gallbladder mucocele were identified. One 144 dog was excluded due to concurrent hemolytic anemia and so 25 cases were included. Tissue 145 blocks for 23 of 25 cases were available for blinded histopathological review. 146 147

Patient Demographics, Clinical and Clinicopathologic Characteristics and Outcome 148
The median age was 11 (n=25; range, 6-14), with a near even distribution between castrated 149 male 12 (48%) and spayed females 13 (52%). Breeds included mixed (n=10), Shetland sheepdog 150 dogs were hyperbilirubinemic and hypoalbuminemic. Two of three dogs that died in the peri-179 operative period had cholecystitis and were FISH+ but the cause of death was not determined. 180 The remaining dog suffered respiratory arrest postoperatively and pulmonary thromboembolism 181 was suspected, but not confirmed. 182

FISH analysis of GB mucosa 184
Bacteria that hybridized to the eubacterial FISH probe were detected in eight of 25 (32%) cases. 185 Bacteria were noted adherent to the GB epithelium and/or invasive within the GB mucosa in all 186 dogs, some dogs also had bacteria visualized within the mucus. Three dogs had less than 10 187 bacteria visualized; the remainder of the dogs had bacteria visualized as dense clusters or masses 188 (Table 2; Figure 1). FISH analysis of the dog with E.coli cultured in the bile revealed masses of 189 bacteria within luminal mucus and adhering to the GB wall (Figure 1, D).