Association of Tightly Spiraled Bacterial Infection and Gastritis in Pigs

FULL PAPER Pathology Association of Tightly Spiraled Bacterial Infection and Gastritis in Pigs Jong-Hwan PARK, Beom-Jun LEE 1), Yong-Soon LEE 1) and Jae-Hak PARK* Departments of Laboratory Animal Medicine and 1) Veterinary Public Health, College of Veterinary Medicine and School of Agricultural Biotechnology, Seoul National University, Suwon 441 744, Korea (Received 14 December 1999/Accepted 13 March 2000) ABSTRACT. Tightly spiral bacteria were observed only in the pyloric mucosa of 4 (8.0%) of 50 swine stomachs, mainly in the surface of epithelia, the gastric pits and the lumen of gastric glands. The presence of the spiral bacteria was significantly associated with chronic pyloric gastritis (p<0.05). Mean gastritis score of the bacteria-positive pyloric mucosa was 3.25 ± 0.25, whereas that of the bacterianegative pyloric mucosa was 2.37 ± 0.12. Parakeratosis and hyperkeratosis were spontaneously seen in the mucosa layer of pars oesophagea, regadless of the bacterial infection. Marked infiltration of mononuclear cells and granulocytes were seen in the cardiac mucosa, regardless of the bacterial infection. Mean gastritis score of the bacteria-positive cardiac mucosa was 3.27 ± 0.32, whereas that of the bacteria-negative cardiac mucosa was 2.84 ± 0.13. There was no significant difference between the bacteria-positive and negative cardiac mucosa (p>0.05). Inflammatory response in the fundic mucosa was rare (gastritis score=0.75 ± 0.08). The tightly spiraled bactera were not cultured with various culture media. These results suggest that the presence of tightly spiraled bacteria is associated with only the pyloric gastritis in pigs. KEY WORDS: chronic gastritis, gastric mucosa, swine, tightly spiraled bacteria. J. Vet. Med. Sci. 62(7): 725 729, 2000 Gastrospirillum suis, a tightly spiral bacterium, colonizes the gastric mucosa of swine [1, 4, 7, 8]. By 16S rrna sequencing, the bacterium is known to belong to the genus Helicobacter and is 99.5% similar to G. hominis type 1 [3, 6]. A possible link between such bacteria and gastric diseases of pig has been evoked [1, 12]. Such diseases contribute an important problem in veterinary medicine leading to significant economic consequences, with up to 2.5% of mortality in pigs due to gastrointestinal hemorrhage [14]. Ulceration of the gastric pars esophagus is also a common problem in intensive pig production [1, 12]. Several factors have been suspected in the pathogenesis of these lesions, but the etiology still remains unknown. In the present report, we investigated naturally occurring swine gastritis in Korea. In addition, the gastritis scores in the cardiac, fundic, and pyloric mucosa of swine stomach were calculated and analyzed for an association of the gastritis with the tightly spiraled bacterial infection. MATERIALS AND METHODS *CORRESPONDENCE TO: PARK, J.-H., Department of Laboratory Animal Medicine, College of Veterinary Medicine, Seoul National University, 103 Seodun-dong, Kwonsun-gu, Suwon 441 744, Korea. Sample preparation: Stomach samples were obtained from 50 apparently healthy pigs (about 5 months old) weighing about 100 110 kg after slaughter at a slaughterhouse in Suwon, Korea. The stomach samples were transported to the laboratory in individual ice box (4 C) and cut open along the greater curvature from the pylorus to the diverticulum, and the contents were discarded. The stomachs were then washed gently in tap water, taking care to remove food particles only. The mucosal surfaces were examined and assessed for macroscopic lesions. Histopathological and microbiological examination: Fragments of the pars esophagea, cardia, fundus and pylorus were fixed in 10% neutral formalin for over 24 hr, dehydrated in alcohol-xylene series, and embedded in paraffin wax. From each block, sections 2-µm thick were prepared, and stained with hematoxylin and eosin (H&E) for histological examination. The sections were also stained with modified Steiner s silver for the detection of spiral bacteria. Briefly, after deparaffinization, the slides were soaked in 1% uranyl nitrate for 15 min and then 1% silver nitrate for 90 min at 60 C. After washing three times with distilled water, the slides were soaked twice in 95% and 100%, respectively, followed by 2.5% gum mastic solution for 5 min at room temperature. After drying the slides in air, they were soaked in a reducing solution (10 ml of 2.5% gum mastic solution, 25 ml of 2% hydroquinone, 5 ml of 100% alcohol, and 0.2 ml of 1% silver nitrate) for 25 min at 45 C and observed by light microscopy. According to the microbiological examination, the stomachs were divided into two groups, bacteria-positive group and bacteria-negative group. The degree of gastritis in the cardiac, fundic and pyloric mucosa was evaluated by the scoring criteria shown in Table 1. Bacterial culture: Fragments of the fundic and pyloric mucosa were cut to very small pieces with a sterilized knife. The tissue pieces were inoculated into chocolate agar (BBL, U.S.A.) plate containing 5% horse serum, GC modified chocolate agar (DIFCO, U.S.A.) plate containing 10% horse serum and antibiotics (amphotericin B, trimethoprim, polymixin B, and vancomycin), or brain heart infusion agar (BBL, U.S.A.) plate. The plates were incubated in an incubator with Gaspak (GasPakPlus', BBL, U.S.A.) for 4 7 days. Statistical analysis: An ANOVA program for statistical analysis was performed on data for the gastritis score and the significant difference between the bacteria-positive and nega-

726 J-H. PARK, B-J LEE Y-S. LEE AND J-H PARK Table 1. Evaluation of gastritis in the cardiac, fundic, and pyloric mucosa of pigs Score Histopathological findings 0 No infiltration of inflammatory cells 1 Mild infiltration of lymphocytes, plasma cells, and some eosinophils 2 Moderately dense infiltration of lymphocytes and plasma cells but no lymphoid follicles 3 Moderately dense infiltration of lymphocytes and plasma cells and presence of lymphoid follicles 4 Very dense infiltration of lymphocytes and plasma cells and presence of lymphoid follicles Fig. 1. The presence of tightly spiral bacteria in the surface of pyloric mucosa (a) and the lumen of gastric glands (b). Steiner s silver stain. (a) 330, (b) 167. tive mucosa was analyzed by the student s t test at the level of p<0.05. RESULTS Macroscopically, proliferation of gastric fold and severe congestion were observed in the fundic region of 5 samples (10%), and focal necrosis of the gastric fold was also observed in the fundic region of 2 samples (4%). Congestion of gland pits and hyperkeratosis of the pars esophagea were seen in 6 samples (12%) and 2 samples (4%), respectively. With Steiner s silver stains, tightly spiraled bacteria were observed only in the pyloric mucosa of 4 (8%) out of a total of 50 stomachs, but not in the pars esophagus, the cardiac mucosa, and the fundic mucosa. The spiral bacteria were mainly seen in the mucus, the gastric pits, and the lumen of gastric glands (Fig. 1a, b). Microorganisms were not seen in lymphoid follicles, macrophages, or the laminar propria. In addition, the cultivation of the tightly spiral bacteria was not successful with various culture media. In the pars esophagus, hyperkeratosis and parakeratosis were spontaneously seen in the mucosa layer of the pars oesophagea, regadless of bacterial infection (Fig. 2a). The epithelial hyperkeratosis and the epithelial parakeratosis were observed in 11 samples (22%) and in 21 samples (42%), respectively (Table 2). However, no glanulomatous lesion and ulcer were seen. In the cardia, a moderate to marked infiltration of lymphocytes, plasma cells, and eosinophils was observed in 47 (94%) of 50 samples (Fig. 2b, Table 2) and the average gastric score from a total of 50 samples was 2.86 ± 0.09 (Table 3). The average gastritis score of the bacteria-positive cardiac mucosa form a total of 4 samples was 3.27 ± 0.32, whereas that of the bacteria-negative cardiac mucosa from a total 46 samples was 2.84 ± 0.13 (Table 3). Epithelial hyperplasia was also seen in 3 samples (6%). In the fundus, the mild inflammatory response, showing a slight to moderate infiltration of lymphocytes and plasma cells, was mainly observed in the gastric pits (Fig. 2c). Epithelial degeneration in 8 samples (16%) and cystic dilatation in 2 samples (4%) were also observed (Table 2), and the gastritis score from a total of 50 samples was 0.75 ± 0.08 (Table 3). In the pylorus, a dense infiltration of lymphocytes and lymphoid follicles in the laminar propria were observed in all (100%) of 4 bacteria-positive samples, whereas 19 (41.3%) of the 46 bacteria-negative samples (Fig. 2d, Table 2). The average gastritis scores in the bacteria-positive samples and the bacteria-negative samples were 3.25 ± 0.25 and 2.37 ± 0.12, respectively (Table 3). There was a significant difference between the bacteria-positive and negative samples (p<0.05). DISCUSSION Many spiral bacteria have been demonstrated in the stomachs of many animal species [2, 9, 10]. In this study, tightly spiral bacteria were found only in the pyloric mucosa in pigs, but not in the cardiac and fundic mucosa. In addition, our results reported by the gastritis score showed a significant

BACTERIA INFECTION AND GASTRITIS IN PIGS 727 Fig. 2. Representative histopathological findings in the pars esophagus (a), cardia (b), fundus (c), and pylorus (d). Hyperkeratosis and parakeratosis were seen in the mucosa of the pars oesophagus. A large lymphoid follicle was observed in the cardiac and pyloric mucosa of a pig infected with tightly spiral bacteria. H&E stain, 50. association between the presence of spiral bacteria in the pyloric mucosa and chronic pyloric gastritis in pigs. The histological findings were similar to those reported in man and gnotobiotic piglets colonized by H. pylori [5, 11]. There was also a significant association between the presence of Gastrospirillum suis and lymphoid follicles [8]. In some G. suispositive pigs, the fundic mucosa was normal despite the presence of pyloric gastritis. This is also common in H. pylori infection in man [11]. Gastrospirillum suis is an uncultured tightly spiraled bacterium that colonizes the gastric mucosa of swine. We also tried to culture the bacteria using various culture media under several conditions. However, the spiralled bacteria were not cultured, which is consistent with other reports [8, 13]. Mendes et al. [8] reported that tightly spiraled bacteria were seen in the pyloric mucosa of the stomach of 13 (10.8%) of 120 pigs and in the fundic mucosa of three (5.0%) out of 60 pigs. In this study, the tightly spiraled bacteria were observed only in the pyloric mucosa of the stomach of 4 (8.0%) of 50 pigs that appeared clinically healthy at slaughter. Mendes et al. [8] also reported that chronic gastritis was observed more frequently in the pyloric region than fundic region and that the pyloric gastritis was significantly associated with the presence of the spiral bacteria. In this study, the chronic gastritis was observed most frequently in the cardiac region. However, the tightly spiraled bacteria were not seen in the cardiac region. The inflammatory response was not severe in the fundic region. Barbosa et al. [1] reported that there was no association between the Gastrospirillum suis and gastritis, Nevertheless, the diagnosis of gastritis in each type of gastric mucosa was based on the different areas out of the four examined. In this study we found a more striking inflammatory reaction in cardiac mucosa, in which bacteria were not found, than in the pyloric or fundic mucosa. However, there was no association between the tightly spiral bacteria infection and the cardiac gastritis. Probably there are many causes of chronic gastritis in swine, e.g., dietary habits and parasites such as Hyostrongylus rubidus, Ascarops spp., Physocephalus spp. and Simondsia spp. Chronic gastritis or gastric ulcer in the cardiac region and in the pars esophagus of the stomach is thought to occur sporad-

728 J-H. PARK, B-J LEE Y-S. LEE AND J-H PARK Table 2. Histopathological findings of pig stomachs infected with and without tightly spiral bacteria (TSB) Number of pigs Histological findings TSB-negative TSB-positive Pars esophagus hyperkeratinization 10 1 parakeratosis 20 1 lymphocytes 19 0 eosinophils 12 0 lymphoid follicles 8 0 Cardia epithelial degeneration 14 1 erosion 10 0 lymphocytes 43 4 eosinophils 40 2 lymphoid follicles 34 2 epithelial hyperplasia 3 0 eosinophils 2 1 Muscularis lymphocytes 1 0 eosinophils 1 0 Fundus epithelial degeneration 7 1 erosion 2 1 lymphocytes 27 1 eosinophils 7 0 lymphoid follicles 3 0 cystic dilatation 1 1 eosinophils 10 0 Muscularis lymphocytes 2 0 eosinophils 5 0 Pylorus epithelial degeneration 26 3 erosion 8 1 lymphocytes 41 4 eosinophils 36 3 lymphoid follicles 19 4 epithelial hyperplasia 3 0 eosinophils 3 0 The infiltration of lymphocytes and eosinophils was not seen in the muscularis of the pylorus. Table 3. Gastritis score of cardiac and pyloric mucosa Portion of stomach No. of stomach Gastritis score Cardiac mucosa (total) 50 2.86 ± 0.09 Bacteria-positive 4 3.27 ± 0.32 Bacteria-negative 46 2.84 ± 0.13 Pyloric mucosa (total) 50 2.44 ± 0.11 Bacteria-positive 4 3.25 ± 0.25* Bacteria-negative 46 2.37 ± 0.12 Fundic mucosa (total) 50 0.75 ± 0.08 The gastritis score in the fundic mucosa was calculated only on the base of a total of 50 stomachs because there is no enough number of the bacteria-positive samples. *Significantly different from the average score of bacteria-negative pyloric mucosa (p<0.05). ically in pigs, but the cause remains unclear [1]. Although several functions are involved in the pathogenesis, the presence of the spiral bacteria is also considered as a possible factor for swine gastric ulcer [12]. They found Gastrospirillum spp. in the most regions of the stomach including antral, oxyntic, fundic, cardiac mucosa. In this study, the presence of tightly spiralled bacteria was not associated with the gastric ulcer in the pars esophagus of the stomach. In conclusion, although an inflammatory response was found most frequently in the cardiac mucosa, there was no association between tightly spiral bacteria infection and cardiac gastritis, whereas there was a close association between the presence of tightly spiral bacteria and pyloric gastritis. REFERENCES 1. Barbosa, A. J. A., Silva, J. C. P., Nogueira, A. M. M. F., Paulino, E. and Miranda, C. R. 1995. Higher incidence of Gastrospirillum sp. in swine with gastric ulcer of the Pars oesophagea. Vet. Pathol. 32: 134 139. 2. Cantet, F., Magras, C., Marais, A., Federighi, M. and Megraud, F. 1999. Helicobacter species colonizing pig stomach: molecular characterization and determination of prevalence. Appl. Environ. Microbiol. 65: 4672 4676. 3. De Groote, D., Van Doorn, L. J., Ducatelle, R., Verschuuren, A., Haesebrouck, F., Quint, W. G., Jalava, K. and Vandamme, P. 1999. Candidatus Helicobacter suis, a gastric helicobacter from pigs, and its phylogenetic relatedness to other gastrospirilla. Int. J. Syst. Bacteriol. 49: 1769 1777. 4. Grasso, G. M., Ripabelli, G., Sammarco, M. L., Ruberto, A. and Iannitto, G. 1996. Prevalence of Helicobacter-like organisms in porcine gastric mucosa: a study of swine slaughtered in Italy. Comp. Immunol. Microbiol. Infect. Dis. 19: 213 217. 5. Krakowka, S., Morgab, D. R., Kraft, W. G. and Lunk, R. D. 1987. Establishment of gastric Campylobacter pylori infection in the neonatal gnotobiotic piglet. Infect. Immun. 55: 2789 2796. 6. Mendes, E. N., Queiroz, D. M. M., Coimbra, R. S., Moura, S. B., Barbosa, A. J. A. and Rocha, G. A. 1996. Experimental infection of Wistar rats with Gastrospirillum suis. J. Med. Microbiol. 44: 105 109. 7. Mendes, E. N., Queiroz, D. M. M., Rocha, G. A., Moura, S. B., Leite, V. H. R. and Fonseca, M. E. F. 1990. Ultrastructure of a spiral micro-organism from pig gastric mucosa ( Gastrospirillum suis ). J. Med. Microbiol. 33: 61 66. 8. Mendes, E. N., Queiroz, D. M. M., Rocha, G. A., Nogueira, A. M. M. F., Carvalho, A. C. T., Lage, A. P. and Barbosa, A. J. A. 1991. Histopathological study of porcine gastric mucosa with and without a spiral bacterium ("Gastrospirillum suis"). J. Med. Microbiol. 35: 345 348. 9. Neiger, R., Dieterich, C., Burnens, A., Waldvogel, A., Corthesy-Theulaz, I., Halter, F., Lauterburg, B. and Schmassmann, A. 1998. Detection and prevalence of Helicobacter infection in pet cats. J. Clin. Microbiol. 36: 634 637. 10. Otto, G., Hazell, S. H., Fox, J. G., Howlett, C. R., Murphy, J. C., O Rourke, J. L. and Lee, A. 1994. Animal and public

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