Helicobacter spp. and gastric lesions association in minipigs

Due to their similarity to humans, studies regarding gastric ulcers in pigs have become of great interest. It is known that Helicobacter spp. is related to the occurrence of these ulcers, as they possess a high prevalence in pigs. This study aimed to associate gastric lesions findings naturally occurring in minipigs related to or not related to the presence of Helicobacter spp., through the ultra-rapid urease test and immunohistochemical analysis, reaffirming the disease in swine as a natural biomedical model for human cases. For this, samples of formalin-fixed paraffin-embedded gastric tissues from 40 minipigs were obtained at the Laboratório de Morfologia e Patologia Animal (LMPA) in the Universidade Estadual do Norte Fluminense (UENF). Regarding the ultra-rapid urease test, no animal was positive in all regions. However, 18 were positive in at least one: six (15%) were positive in the aglandular region, eight (20%) in the antrum region, 13 (32.5%) in the region of the cardiac gland, and one (2.5%) in the region of the fundic gland. Regarding immunohistochemical analysis, only one animal was positive in all regions, and 32 animals were positive in at least one: four (10%) were positive in the aglandular region, 10 (25%) in the antrum region, 23 (57.5%) in the region of the cardiac gland, and three (7.5%) in the region of the fundic gland. The gastric lesion findings showed a close relationship with Helicobacter spp., enriching the laboratory animal pathologies list. The immunostaining of the bacteria not associated with gastric lesions in certain regions demonstrated the saprophytic and opportunisitic nature of Helicobacter.


INTRODUCTION
Due to their similarity to humans, studies regarding gastric ulcers in pigs have become of great interest (DE BRUYNE et al., 2012), especially the infectious causes (THOMSON & FRIENDSHIP, 2019). It is known that Helicobacter spp. is related to the occurrence of these ulcers, as they possess Silveira et al. a high prevalence in pigs, which in turn reduced daily weight gain (DE BRUYNE et al., 2012); and subsequently, led to economic losses (KUMAR et al., 2010). Many studies show a high prevalence of gastric ulcers, which deserve further investigation on their risk factors (GOTTARDO et al., 2017;DE WITTE et al., 2019).
The bacteria Helicobacter suis has been associated with gastric lesions in pigs and humans (DE BRUYNE et al., 2016;NAKAGAWA et al., 2018;RIMBARA et al., 2020), This leads to a major concern, since according to the data presented in the literature, people in close contact with pigs are exposed to higher risks of infection, suggesting that this animal is a zoonotic agent (JOOSTEN et al., 2013;LOUGHLIN et al., 2019). Helicobacter suis is one of the most prevalent non-H. pylori sp. in humans (LIANG et al., 2013), and Helicobacter pylori is the most important gastric Helicobacter in human health (HRISTOVA et al., 2017).
There are several similarities regarding the anatomy, physiology, and pathophysiology between pigs and humans, but, due to several limitations such as animal handling, breeding space, and food volume, laboratory dimensions colonies have been developed. These colonies are used in many areas of medical and biological research (MARIANO, 2003) and have replaced other mammalian species, such as animal models (SWINDLE & SMITH, 2016).
This study aimed to associate gastric lesions findings naturally occurring in minipigs related to or not related to the presence of Helicobacter spp. through the ultra-rapid urease test and immunohistochemical (IHC) analysis, reaffirming the disease in swine as a natural biomedical model for human cases.

MATERIALS AND METHODS
The samples were obtained at the Laboratório de Morfologia e Patologia Animal (LMPA). Samples of the aglandular (pars Oesophagea) and glandular (cardia, fundus and pylorus) anatomical regions were collected for ultra-rapid urease test (fresh samples) and immunohistochemical (IHC) evaluations (samples fixed in buffered neutral formalin).
For this study, samples of formalin-fixed paraffin-embedded gastric tissues from 40 minipigs were used (SILVEIRA et al., 2015). The piglets had an average weight of 39.0 Kg and an average age of 17 months, were kept in the colony of the UENF Veterinary Hospital under constant temperature and humidity conditions, and fed twice a day. Aglandular region integrity has been observed in macroscopic evaluation. The ulcerative lesions were classified using the scoring method proposed by SILVEIRA et al. (2014), from zero to five: assigning the score 0 to a normal appearance (smooth, glossy and whitish); score 1 to those with that were partially rough looking, with any yellow pigmentation; score 2 to the ones with fully rough and thickened areas of yellow pigmentation; score 3 to totally rough and thickened aglandular areas associated with yellow pigmentation of the entire surface; score 4 to the ones with totally rough and thickened areas, with the entire surface covered in yellow pigmentation, and eroded areas; and score 5 to those with a rough and thickened appearance, yellow pigmentation on its entire surface, and ulceration areas.
For the ultra-rapid urease test results, we used the criterion of negative (unchanged color) and positive (changed color) during the first five minutes ( Figure 1). With this time, about 80% was already confirmed, but following the laboratory's recommendation, we expected to wait up to two hours for the final result.
To perform the immunohistochemical technique ( Figure 1), 4 µm sections were made in those regions. These, in turn, were deparaffinized in xylene baths, and rehydrated in alcohol and deionized water baths. Then, the process for antigen retrieval was made using citrate buffer in a 96 °C water bath, followed by endogenous peroxidase blocking when the specimens were treated with an alcoholic solution (methanol) of 10% hydrogen peroxide. After carefully drying the slides, sections were circled with a Dako pen ® (Dako, CA, USA), and incubated with 1% bovine serum albumin fatty acid-free solution and 1% skimmed milk powder diluted in Tris-NaCl for blocking nonspecific proteins. After discarding the blocking solution, the sections were incubated overnight in a humid chamber in a 1:200 dilution of rabbit polyclonal anti-Helicobacter pylori Rabbit antibody, Dako, B047. The next day, the slides were washed with brine, treated with HRP-LSAB+ kit (Dako, CA, USA), and developed with DAB chromogen kit (Dako, CA, USA). Finally, the specimens were counterstained with Harris hematoxylin, dehydrated in alcohol baths, and mounted in synthetic resin. To analyze the slides, we used light optical microscopy. The antibody used (Helicobacter spp.) is also used for diagnosis in other species at the UENF university hospital. Samples previously tested were used for the positive and negative controls (SILVEIRA et al., 2014).
To evaluate the frequency of positive animals in relation to gastric lesions, immunohistochemical findings, and ultra-rapid urease tests, the chi-square or Fisher's exact test was performed, considering a 5% probability of error. For all analyzes, the program SPSS ® IBM -v 18.0 was used.

RESULTS AND DISCUSSION
Although, there are differences between naturally occurring gastric ulcers between pigs and humans, the pig represents an interesting animal model to study the repair of gastric ulcers (KOGA et al., 2002;PADRA et al., 2018;BANG et al., 2019;MAEDA et al., 2019), and the pathogenic mechanisms involved in their formation, mainly with Helicobacter pylori (POUTAHIDIS et al., 2001). Pigs have systemic similarities with humans and a suitable profile for biomedical research (GIANOTTI et al., 2010).
Regarding the ultra-rapid urease test, in the four regions, 22 animals (55%) were negative, and 18 animals (45%) were positive in at least one of them. However, none of them were positive in all regions. In According to the authors, further research should be done, comparing several commercially available tests. Concerning the antrum region, eight (20%) were positive, and 32 (80%) were negative. In the aglandular region, six (15%) were positive, and 34 (85%) were negative. For the region of the cardiac gland, 13 (32.5%) were positive, and 27 (67.5%) were negative. Regarding the region of the fundic gland, one animal was positive (2.5%), and 39 (97.5%) were negative. According to these results, there was no statistically significant association between the bacteria and the lesions (Table 1). In an endoscopic Regarding the antrum region, 10 (25%) were positive, and 30 (75%) were negative. In the aglandular region, four (10%) were positive, and 36 (90%) were negative. Concerning the region of the cardiac gland, 23 (57.5%) were positive, and 17 (42.5%) were negative. For the region of the fundic gland, three (7.5%) were positive, and 37 (92.5%) were negative (Table 2). SILVEIRA et al. (2014), in an endoscopic study with piglets, reported 10 (50%) positives in the antrum region, two (10%) positives in the aglandular region, three (15%) positives in the region of the cardiac gland, and two (10%) positives in the region of the fundic gland. According to these results, the impact of bacterial infection on the stomach and on the health of herds associated with the potential to cause disease in humans, justifies the interest of further studies on the subject (TAMIASSO et al., 2017).
In the study presented herein, we observed statistically significant differences regarding the presence of bacteria in the aglandular region and in the region of the cardiac glands (Table 2). In the others, the bacterium was immunostained but not associated with the affected region. This led to the assumption that these agents may dwell saprophytically and opportunistically in the gastric mucosa. In a study with slaughter animals, RODRIGUEZ et al. (2008) also reported an association of the bacteria with the aglandular region. DE WITTE et al. (2017), found a similar result, but unlike this study, the statistical association occurred in the region of the pyloric glands. Furthermore, regarding IHC, SZEREDI et al. (2005) observed that a higher infection rate was found in the cardiac region, followed by the pyloric and fundus regions.  the fact that infection reduces animals weight gain, causing economic losses. YAMASAKI et al. (2009) suggested that the Helicobacter might have an important role in the establishment of early lesions, increasing the possibility of them worsening with increasing age of the pigs. The mechanisms used by the bacteria to produce various pathological conditions are not completely known (SIQUEIRA et al., 2007). The infection consequences reflect the results of the interaction between the agent and the host's immune response. Some authors stated that by understanding this relationship, it should be possible to predict, treat, and even prevent the disease (AEBISCHER et al., 2010). The inflammatory response induced by the bacteria is a key-event related to the pathogenesis (BASSO et al., 2010), as the degeneration of the surface epithelium caused by infection triggers the inflammatory response to an infiltrate, which is composed of leukocytes (neutrophils, lymphocytes, and plasma cells) (SIQUEIRA et al., 2007;BRACARENSE et al., 2013). De WITTE et al. (2017) observed that Helicobacter suis in adult pigs positively affects markers for acid secretion and inflammation, unlike the younger ones.
According to YAMASAKI et al. (2006), the association between Helicobacter spp. and the gastroesophageal ulcer occurrence in pigs has been controversial. Although, the results presented herein and by other authors (MERLINI et al., 2010;DE WITTE et al., 2017) pointed to a correlation between the presence of Helicobacter spp. and the prevalence and the severity of gastric ulcers, other studies do not (SILVEIRA et al., 2014;TAMIASSO et al., 2017).
The possible reason for this inconsistency may be the use of different techniques for determining the bacteria present, the fact that different Helicobacter spp. different strains showed higher or lower pathogenicity, and the fact that some studies were experimental while others were observational (TAYLOR & FRIENDSHIP, 2011;DE WITTE et al., 2018). In our studies; however, IHC proved to be very sensitive to demonstrate the presence of bacteria. One of the advantages of this technique is the diagnosis of predominantly coccoid strains, which are more difficult to observe in routine stains (MCNULTY et al., 2011).