The Effect of Helicobacter Felis and Helicobacter Bizzozeronii on the Gastric Mucosa in Mongolian Gerbils: a Sequential Pathological Study
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ARTICLE IN PRESS J. Comp. Path. 2006,Vol.135, 226^236 www.elsevier.com/locate/jcpa The Effect of Helicobacter felis and Helicobacter bizzozeronii on the Gastric Mucosa in Mongolian Gerbils: a Sequential Pathological Study à y M. De Bock, K. D’Herde , L. Duchateau , A. Hellemans, A. Decostere, F. Haesebrouck and R. Ducatelle Departments of Pathology,Bacteriologyand Avian Diseases, yPhysiology,Biochemistry and Biometry,Faculty ofVeterinary Medicine,and ÃDepartment of Anatomy, Embryology, Histology and Medical Physics, Faculty of Medicine, Ghent University, Belgium Summary In contrast to Helicobacter(H.)pylori, little is known about the pathogenic mechanisms of gastric non-H. pylori Heli- cobacter species. Mongolian gerbils were inoculated intragastrically with H. felis or H. bizzozeronii and killed at dif- ferent timepoints post-inoculation (p.i.),stomach tissue being taken for light and transmission electron microscopy (TEM) and polymerase chain reaction (PCR) analysis. Parietal cells (PCs), apoptosis, cell proliferation and nu- clear factor-kB (NF-kB) activation were‘‘visualized’’ immunohistochemically. In£ammation consisted of neutro- philic granulocytes, mainly in the antrum, and lymphocytic in¢ltrates around the limiting ridge and throughout the stomach mucosa and submucosa. From day 11p.i. onwards, H. felis-inoculated animals showed moderate to severe loss of PCs extending from the limiting ridge into the fundus. Apoptotic cells, spiral bacteria, cell prolifera- tion, and NF-kB activation were detected at the transition zone between a¡ected and normal PCs.TEM revealed interaction of H. felis £agella with PCs and chief cells. Moreover, H. felis was seen in proximity to, and inside, necrotic cells. At 10 weeks p.i., some H. felis-infected gerbils showed complete loss of fundic glands, and mucous metaplasia of the epithelium. H. bizzozeronii, which made no £agellar contact with epithelial cells, was associated with only mild PC loss.The mechanism by which H. felis induces PC necrosis and apoptosis remains unclear.The observed £agellar contact and NF-kB activation may play an important role in H. felis-associated in£ammation. r 2006 Elsevier Ltd. All rights reserved. Keywords: bacterial infection; gastritis; gerbil; Helicobacter bizzozeronii; Helicobacter felis; Helicobacter pylori; Meriones unguiculatus; parietal cells; stomach Introduction mucosa-associated lymphoid tissue lymphoma (Morgner et al., 2000). These bacteria were provision- Helicobacter felis and Helicobacter bizzozeronii are phylo- ally named ‘‘Helicobacter heilmannii’’,but genetic analysis genetically closely related to the human gastric patho- later revealed that this name did not represent a single gen Helicobacter pylori, yet have a number of species. Instead, it represented a species complex con- characteristics that distinguish them from the latter. sisting of (1) ‘‘Candidatus H. suis’’ (De Groote et al., Their tight helical morphology is clearly di¡erent from 1999), found in the stomach of pigs and referred to as the S-shaped morphology of H. pylori. Moreover, H. felis ‘‘H. heilmannii’’type1and (2) the spiral organisms found possesses periplasmic ¢brils that encase the bacterium. in the gastric mucosa of cats and dogs, referred to as Helical-shaped bacteria, which are occasionally found ‘‘H. heilmannii’’ type 2 (i.e., Helicobacter felis, Helicobacter in the human stomach, are implicated in gastritis and bizzozeronii, Helicobacter salomonis (Jalava et al., 1997), ‘‘Candidatus Helicobacter heilmannii’’ (O’Rourke et al., Correspondence to: M. De Bock, Laboratory of Pathology, Department of 2004) and Helicobactercynogastricus (Van den Bulck et al., Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium 2006). It has been suggested that human gastric infec- (e-mail: [email protected]). tions with spiral-shaped bacteria are zoonotic, arising 0021-9975/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.jcpa.2006.08.003 ARTICLE IN PRESS H. felis and H. bizzozeronii in the Gerbil 227 from reservoirs in various animal species (Yoshimura weeks, were barrier-maintained in a room with con- et al., 2002; Sykora et al., 2003; van Loon et al., 2003). trolled environment and housed in pairs in ¢lter-top Recently, Van den Bulck et al.(2005)subjected human cages on autoclaved (121 1C, 15min) wood shavings. gastric biopsy samples to a multiplex polymerase chain They were fed an autoclaved diet containing18% pro- reaction (PCR), which showed that ‘‘Candidatus tein (Teklad Global Rodent Diet, Harlan NL, Horst, H. suis’’ and H. salomonis were the most prevalent The Netherlands) and received autoclaved water ad Helicobacter species, H. felis and H. bizzozeronii being libitum. The experimental procedure was approved by identi¢ed in14.6% and 4% of the samples, respectively. the Ethical Committee of the Faculty of Veterinary In contrast to H. pylori, little is known about the viru- Medicine, Ghent University. lence and pathogenic mechanisms of these gastric so- called non-H. pylori Helicobacter species. In H. felis, only a Bacterial Strains few virulence genes have been characterized (Josenhans et al.,1999). Nevertheless, a murine H. felis model hasbeen These consisted of H. bizzozeronii CCUG 35545 (kindly used widely to study various aspects of gastric Helicobacter provided by P. Vandamme) and H. felis ATCC 49179 infections such as pathogenesis, antibiotic treatment and (kindly provided by R. Ferrero).They were grown on vaccine development (Czinn et al.,1993; Kobayashi et al., brain^heart infusion (BHI; Oxoid, Basingstoke, UK) agar to which Skirrow supplement (Oxoid), vitamins 1998). It is questionable, however, to what extent the me- s s chanisms that cause H. pylori gastric lesions are compar- (Vitox ; Oxoid), amphotericin B (Fungizone ;Bris- able with those associated with H. felis. tol-Myers Squibb, New York, USA) and horse blood 1 Approximately a decade ago, the Mongolian gerbil 10% were added. The plates were incubated (37 C, (Meriones unguiculatus) was introduced into H. pylori 48 h) in jars under microaerophilic conditions, created research (Hirayama et al., 1996). Severe pathological by evacuating 80% of the normal atmosphere and in- changes are seen in H. pylori-infected Mongolian ger- troducing a gas mixture of N2 84%, H2 8% and CO2 bils, ulcers being formed in most animals (Matsumoto 8%. The bacteria were harvested and suspended in et al., 1997). This animal model provided the ¢rst evi- BHI broth. The ¢nal concentration was adjusted to an optical density of 1.0 at 660 nm, corresponding to dence that H. pylori infection alone could induce gastric 8 tumour formation (Watanabe et al., 1998). Court et al. approximately 10 colony-forming units (cfu) /ml (2002) were the ¢rst to report the successful infection (CortheŁ sy-Theulaz et al.,1995). of gerbils with H. felis. De Bock et al. (2006) recently conducted a prelimin- Experimental Procedure ary study on gastric lesions induced by H. felis and The gerbils were inoculated with H. felis (n ¼ 25) or H. bizzozeronii in gerbils.The results showed loss of par- H. bizzozeronii (n ¼ 25), or were left as uninfected con- ietal cells (PCs) near the cardia after 3 weeks of infec- trols (n ¼ 15). Inoculations were made intragastrically tion, especially in the H. felis-infected gerbils. The with a ball-tipped gavage needle, under iso£urane present study investigated, at di¡erent timepoints after (Iso£os; Abbot, Illinois, USA) anaesthesia. Inocula, experimental infection, the pathogenic e¡ects of H. felis given on three occasions at 48-h intervals, consisted of and H. bizzozeronii on the gastric mucosa of the Mongo- 0 Á 4 ml of bacterial suspension or, for the controls, BHI lian gerbil, with the aim of answering the following broth. At 7,11,21,35 and 70 days after the ¢rst inocula- questions.What is the time of onset of PC destruction? tion, ¢ve H. felis-infected animals, ¢ve H. bizzozeronii-in- Will the observed PC loss progress further to complete fected animals and three control animals were killed by metaplasia of the fundic mucosa in both H. felis-and cervical dislocation after iso£urane anaesthetization. H. bizzozeronii-infected gerbils? Does H. bizzozeronii in- The stomach of each gerbil was resected and samples duce the same pathological changes as those induced were taken for PCR analysis and histopathological by H. felis, possibly with some delay? Is there a distinct and ultrastructural examination, as described below. interaction between the bacteria and the host cells? Answers to these questions might throw light on the PCR Analysis progression from acute to chronic gastric changes in animals and man. From each gerbil, samples of the gastric fundus and antrum (ca 4mm2) were taken for DNA extraction (DNeasy Tissue Kit; Qiagen, Hilden, Germany) and Materials and Methods multiplex PCR analysis (Baele et al., 2004).This PCR test makes possible the distinction between H. felis and Animals H. bizzozeronii based on the transfer RNA intergenic Sixty-¢ve speci¢c pathogen-free female gerbils spacers of Helicobacter species and the urease gene of (Elevage Janvier, Le Genest St Isle, France), aged 6 H. felis. Download English Version: https://daneshyari.com/en/article/2438760 Download Persian Version: https://daneshyari.com/article/2438760 Daneshyari.com.