Abstract
Introduction
Enterohepatic Helicobacter species (EHS) colonize the intestine and liver of humans and animals (Fox, 2002). Helicobacter hepaticus, the prototype EHS, induces chronic active hepatitis and hepatocellular carcinoma in genetically susceptible mouse strains (Fox et al., 1994; Ihrig et al., 1999). Selected EHS, but not Helicobacter pylori, play a role in the pathophysiology of cholesterol gallstone formation in C57L mice fed a lithogenic diet (Maurer et al., 2005, 2006). In addition, Helicobacter species DNA has been identified in bile and gall-bladder tissue of humans with chronic cholecystitis (Fox et al., 1998). In the present study, we describe the isolation of EHS from the ileum, liver and colon of a baboon with pancreatic islet amyloidosis and hepatitis.
Case report
An adult male baboon (Papio anubis) was euthanized due to a chronic wasting disease. Prior to euthanasia with an intravenous barbiturate, blood samples were collected for haematological analyses, and values were compared to clinical reference data for baboons (Hainsey et al., 1993). The complete blood count was within the normal range; however, the biochemical profile revealed hyperglycaemia (192 mg dl1; normal range 65101 mg dl1), hypertriglyceridaemia (315 mg dl1; normal range 3694 mg dl1), hyperlipasaemia (25 U l1; normal range 019 U l1) and hypoproteinaemia (5.7 mg dl1; normal range 6.37.9 mg dl1). On gross examination during necropsy, the liver appeared moderately enlarged. Representative samples were collected from various organs, fixed in 10 % neutral-buffered formalin, and processed for routine histopathological evaluation.
Microbiological and histological studies
Samples that had been collected from the ileum, caecum, colon, gall bladder, bile and liver, and placed in culture media containing 20 %, v/v, glycerol, with brucella broth, and frozen at 70 °C, were subsequently homogenized with PBS for microaerobic culture. Media used for culture included trypticase soy agar with 5 % sheep blood (BAP), medium impregnated with trimethoprim, vancomycin and polymyxin (TVP; Remel), and medium impregnated with cefoperazone, vancomycin and amphotericin B (CVA; Remel). In addition, selective antibiotic medium (ABM) contained the following components: blood agar base no. 2, 5 % horse blood (Remel), 50 µg amphotericin B ml1, 100 µg vancomycin ml1, 3.3 µg polymyxin B ml1, 200 µg bacitracin ml1 and 10.7 µg nalidixic acid ml1 (Sigma). A portion of each homogenized sample was applied directly to TVP, CVA, and ABM media, and filtered through a 0.45 µm pore-size filter and plated on BAP. Plates were incubated at 37 °C under microaerobic conditions for 24 weeks in vented jars containing N2, H2 and CO2 (80 : 10 : 10). Bacterial isolates with characteristic growth on agar and microscopic morphology were subsequently used for biochemical analysis and to obtain genomic DNA for PCR, RFLP and 16S rRNA gene sequencing analyses.
Microaerobic bacteria were isolated from the ileum, liver and colon. The isolates grew at 37 °C, at 42 °C, and in the presence of 1 % glycine. They were oxidase positive, weak catalase positive, urease negative, were unable to reduce nitrate, and were negative for alkaline phosphatase hydrolysis, indoxyl acetate hydrolysis and gamma glutamyl transpeptidase activity. The isolates from the ileum and liver were resistant to nalidixic acid (30 µg disk) but sensitive to cephalothin (30 µg), while the colon isolate was sensitive to nalidixic acid and resistant to cephalothin.
PCR was performed on individual bacterial isolates using Helicobacter genus-specific primers C97 and C05, as previously described (Fox et al., 1998). These primers were used to generate a 1200 bp PCR product of the 16S rRNA gene from Helicobacter species that was then subjected to RFLP (Fox et al., 1998). RFLP analysis revealed that ileal and hepatic isolates had identical banding patterns with AluI and HhaI restriction enzymes, but were distinct from the banding patterns of the colonic isolate. The RFLP patterns from the ileal and hepatic isolates were identical to the pattern of an isolate of Helicobacter macacae (MIT 99-5504) obtained from the colon of a non-diarrheic rhesus monkey with chronic idiopathic colitis (Fox et al., 2001b). The RFLP pattern of the colonic isolate was identical to the pattern of an isolate of Helicobacter cinaedi (MIT 99-10781) obtained from the faeces of a rhesus monkey (J. G. Fox and others, unpublished results) (Fig. 1).