Molecular Detection of Human Pathogenic Gastric Helicobacter Species in Wild Rabbits (Oryctolagus cuniculus) and Wild Quails (Coturnix coturnix)

Wildlife plays a major role in the maintenance and transmission of multihost pathogens. Several Helicobacter spp. have been described to have zoonotic potential; thus, human, domestic and wild animal interactions deserve more attention. In this study, the presence of the DNA of human pathogenic gastric Helicobacter species was determined in gastric samples collected from wild rabbits and wild quails during the national hunting campaigns in Portugal. Eleven out of the 12 wild rabbits (91.7%) and all six wild quails tested (100%) were PCR positive for one or more gastric Helicobacter species. In both animal species, H. felis, H. bizzozeronii and H. salomonis DNA were detected. In addition to these non-Helicobacter pylori Helicobacter spp. (NHPH), H. pylori DNA was also identified in gizzard samples of wild quails. These findings might indicate that wild rabbits and wild quails may act as reservoirs and contribute to the H. pylori and NHPH environment dissemination, causing both Public Health and One health concerns to arise.


Introduction
Wildlife plays a major role in the maintenance and transmission of multihost pathogens and the understanding of the role of host species in the epidemiological cycle is essential to prevent diseases caused by zoonotic pathogens [1]. Although both wild and domestic animal reservoirs can be considered important sources of emerging infectious diseases, it is the human impact in the ecological systems that commands the level of risk at the humans/animals interface upon zoonotic disease emergences episodes [2]. In addition, the number of infectious diseases of zoonotic origin has been increasing, and approximately 72% are transmitted from wildlife [3,4].
Helicobacter species are Gram-negative, motile bacteria with a helical form that colonize the gastrointestinal tract of humans and a wide range of animal species [5][6][7][8]. Gastric Helicobacter species have been studied over the years for their association with gastrointestinal diseases and zoonotic potential [9]. These bacteria have been widely described in humans, dogs and cats [8,10,11].
In addition, gastric NHPH have been observed in 0.2-6% of human gastric biopsies and have been associated with a range of gastric pathologies, especially MALT lymphoma, as well as extra digestive diseases [8,[15][16][17]. H. suis is the most common NHPH in humans suffering from gastric disorders, followed by H. salomonis, H. felis, H. heilmannii and H. bizzozeronii [5,8,15]. Humans may acquire these infections through contact with animals since most of NHPH are animal associated bacteria, but only some of them are recognized as potentially zoonotic [9,11,18].
Despite humans being considered the natural reservoir for H. pylori, this Helicobacter species has been detected in other domestic and wild animals [8,11,20,[24][25][26][27]. Nevertheless, to the authors' knowledge, there is a lack of studies regarding the presence of gastric Helicobacter species in both wild rabbits and wild birds.
From an eco-epidemiological perspective, wild animals may have an important role in the spread of several pathogens [28][29][30][31].
Hunting dogs and humans can become infected with game pathogens through direct contact with fresh carcasses and the handling or consumption of raw or undercooked meat; thus, as a result, game wardens, hunters, butchers and other wildlife professionals are at high risk. Therefore, the aim of this study was to screen different regions of wild rabbit stomachs (fundus and corpus) and wild quail gizzards for the presence of DNA from H. pylori and zoonotically important NHPH, mainly associated with pigs, dogs and cats.

Animals and Samples Collection
Wild rabbits (Oryctolagus cuniculus) and wild quails (Coturnix coturnix), shot during three national hunting campaigns held in the centre of Portugal (Coimbra district), were subjected to convenience sampling. All the sampled animals were adults. Dissection of the stomach was performed using disposable scalpels and disposable tweezers. Representative gastric tissues from the fundus and corpus of wild rabbits and from the gizzard of wild quails were collected using a sterile disposable Kruuse ® (Langeskov, Denmark) Biopsy punch 8 mm per sample, rendering cross-contamination between samples unlikely, and then they were stored at −20 • C for further DNA extraction.
The animals were not slaughtered or euthanized in for this investigation and the stomach specimens were obtained as sub-products of the usual meat inspection activity that takes place throughout these campaigns. None of the acts were taken exclusively for the purpose of research, and the researchers had no influence over the campaign's planning or meat inspection methods.

DNA Extraction and PCR Conditions and Sequencing
DNA was extracted from 8 mm of gastric frozen tissue samples, using the EXTRACTME ® DNA tissue Kit (BLIRT S.A., Gdansk, Poland) and according to instructions provided by the supplier.
All the samples were tested for the presence H. pylori, H. felis, H. salomonis, H. bizzozeronii, H. heilmannii, H. suis and H. ailurogastricus DNA through conventional PCR analysis, according to previously described protocols (Table 1). Aliquots of each PCR product were electrophoresed on a 1.5% agarose gel stained with Xpert Green Safe DNA gel stain (GRISP, Porto, Portugal) and examined under UV light for the presence of specific fragments. The size of the DNA fragment was compared to the standard molecular weight, the 100 bp DNA ladder (GRISP, Porto, Portugal) and the molecular weight of the positive controls (Table 1). For the negative control, distilled water was used. For the positive controls, DNA was extracted from pure cultures of each Helicobacter species tested (Table 1).
To exclude false positive samples, the amplicons of each positive sample were sequenced. Bidirectional sequencing was carried out using the Sanger method at the Genomics core facility of the University of Porto's Institute of Molecular Pathology and Immunology. MegaX Molecular Evolutionary Genetic Analysis version 10.1.8 was used for sequence editing and multiple alignments. The sequences obtained were subject to the basic local alignment search tool (BLAST) using the non-redundant nucleotide database (http://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 7 December 2021).

Results and Discussion
A total of 30 gastric samples were evaluated: 24 were collected from 12 wild rabbits and consisted of equal amounts of fundus and corpus mucosa, and six were collected from six wild quails.
Eleven out of the 12 wild rabbits (91.7%) and the six (100%) wild quails were PCR positive for one or more gastric Helicobacter species.
According to the gastric location, nine samples of each fundus and corpus location were PCR positive for at least one Helicobacter species ( Table 2).
The bidirectional sequencing and BLAST analysis of consensus sequences obtained showed a homology of 97% to 100% with the respective species (Table A1).
Co-infections, i.e., the presence of more than one Helicobacter species, were detected in 41.7% of wild rabbits; the most frequent bacterial association was H. salomonis plus H. bizzozeronii (33.3%) and H. salomonis plus H. felis (8.3%). Regarding the Helicobacter species identified, these results are in agreement with those previously described in pets, as well as industrial and laboratory rabbits, which reported H. felis, H. salomonis and H. bizzozeronii DNA in the gastric corpus [19,20], but are here reported for the first time in free range wild rabbits.
Concerning the wild quails: three were H. salomonis-positive, one was H. salomonis plus H. bizzozeronii-positive, one was H. salomonis plus H. pylori-positive and another was H. salomonis plus H. felis plus H. bizzozeronii-positive (Table A1).
The bidirectional sequencing and BLAST analysis of the consensus sequences obtained showed a homology of 97% to 100% with the respective species (Table A1). Previous studies reported the detection of enterohepatic Helicobacter in wild birds' faecal samples using PCR [21][22][23].
To the authors knowledge, this is the first time that gastric NHPH is detected in the fundus/corpus of free range wild rabbits and NHPH and H. pylori DNA in the gizzard of wild quails. This might indicate that wild rabbits and wild quails may act as reservoirs and contribute to the H. pylori and NHPH environment dissemination, causing both Public Health and One health concerns to arise. DNA extraction of the wild rabbit samples and the wild quail samples was performed on different days. Disposable sterile scalpels and tweezers were used to open the stomachs and, for each sample collection, a sterile disposable Kruuse ® Biopsy punch was used, rendering cross-contamination between samples unlikely. It can, however, not be excluded that the presence of Helicobacter DNA in the stomach from some of the animals tested is a consequence of recent contamination from the environment, as described by [32]. Future studies using a larger sample size and including histopathological analysis of gastric tissues is, therefore, necessary to confirm these findings and to associate the presence of Helicobacter spp. with possible gastric alterations.
Wild animals, including leporids and birds, are known to be reservoirs for several infectious and transmissible diseases [30,33], raising concerns regarding zoonotic pathogens cross-species spillover [34]. The close contact of wild animals with domestic animals and humans is continuously increasing due to different factors, which can drive the zoonotic spillovers to become more common [29,34] and directly affect animal and human health [30,31,33,34].
Hunting dogs and humans can be exposed to game pathogens through direct contact with fresh carcasses, the handling or consumption of raw or undercooked meat or indirect contact through contaminated water or the environment; thus, game wardens, hunters, butchers and other wildlife professional duties are at high risk.

Conflicts of Interest:
The authors declare no conflict of interest.