Chicken as a carrier of emerging virulent Helicobacter species: a potential zoonotic risk

Helicobacter is a Gram-negative, microaerophilic, spiral to curve-shaped bacterium isolated from the stomachs of mammals, including humans [1]. Based on phylogenetic analysis and ecological niches, this genus is broadly classified into two major subgroups: Gastric Helicobacter (GH) and Enterohepatic Helicobacter (EHH) species [2]. The most significant among gastric Helicobacter species is Helicobacter pylori (H. pylori), which has received priority attention worldwide due to its association with a variety of illnesses, such as peptic ulcer disease, gastric cancer, type B gastritis [3], and mucosa-associated lymphoid tissue (MALT) lymphoma [4]. The WHO has designated this bacterium as a Class I definite carcinogen [5] due to its significant role in most gastric malignancies. Later, Helicobacter pullorum (H. pullorum), an enterohepatic Helicobacter species [6], emerged and gained significant public health concern [7]. H. pullorum was identified as a new species by Stanley et al. [8] based on 16S rRNA phylogenetic analysis. This organism inhabits the intestinal tract of poultry and has been found in the liver and duodenum of asymptomatic birds, as well as in the liver and cecal contents of broiler chickens and laying hens suffering from vibrionic hepatitis [7, 9, 10]. In poultry slaughterhouses, H. pullorum has been found on chicken carcasses, possibly due to its high concentration in the cecum and subsequent contamination of raw chicken meat during slaughtering and evisceration [11]. Hence, it is considered an emerging foodborne zoonotic pathogen [7, 12]. Moreover, H. pullorum has been isolated from human patients suffering from gastroenteritis [13], chronic liver disorders [14], and even from clinically healthy persons [13].

Subsequently, other Helicobacter species have been recognized in poultry, although they remain beyond the primary research focus. For instance, H. canadensis was isolated from the feces of Barnacle geese (Branta leucopsis) and Canada geese (Branta canadensis) on the Atlantic coast of Europe [15], as well as from diarrheic and bacteremic patients [16, 17]. H. pametensis has been detected in the feces of wild birds [18]. Additionally, Helicobacter anseris (urease-positive) and H. brantae (urease-negative) have been identified in the feces of resident Canada geese in the United States [19]. Notably, several reports indicate that some enterohepatic Helicobacter species, including H. hepaticus, H. bilis, H. cinaedi, and H. pullorum, produce a well-characterized bacterial virulence element, the cytolethal distending toxin (CDT) [20‐23]. CDT induces edema, cytoskeleton anomalies, and G2/M cycle arrest in host cells [24]. It is responsible for symptoms of infection, such as inflammation [25] and the development of diarrhea [22], and it has a potential role in intestinal carcinogenesis [26].

Although the conventional culture method is regarded as the gold standard test for Helicobacter detection, the delicate and fastidious nature of this pathogen makes it a challenging task [27]. This drives the development of molecular techniques like PCR, which do not rely on living bacteria as culture does and provide rapid and reliable results [28‐31]. Since the majority of studies have focused on investigating H. pullorum in avian species [6, 9‐11, 32‐34], and knowledge is scarce regarding other Helicobacter species in poultry, the current study was conducted to investigate the prevalence of emerging Helicobacter species among broiler and layer chicken cloacal swabs, as well as to detect the cdtB virulence gene among the retrieved Helicobacter species to highlight their public health significance.

Currently, understanding the epidemiological aspects of zoonotic Helicobacter species is a subject of great interest among researchers and scholars worldwide [7, 38‐40]. In the present study, the prevalence of Helicobacter species 16S rRNA in cloacal swabs from the examined chickens (25.6%) was higher than that reported by Elrais et al. [6] (12%) in chicken meat in Egypt and García-Amado et al. [41] (5%) in the feces of wild birds in Venezuela, but lower than that detected by Fox et al. [19] (40.2%) in the feces of resident Canada geese in the Greater Boston region. Notably, Helicobacter spp. was found in apparently healthy chickens at a higher prevalence than in diseased ones. There was a significant difference between the two groups, suggesting that apparently healthy chickens may serve as a potential reservoir for Helicobacter species, raising public health concerns.

Regarding H. pylori, all broilers and layers in this study were negative for H. pylori. However, Elrais et al. [6] detected H. pylori in 300 broiler chicken samples (meat and giblets) with a prevalence rate of 5.33% (16/300) and El Dairouty et al. [42] revealed that 5% (1/20) of raw poultry meat samples were positive for H. pylori. Almashhadany et al. [43] found that 18 (13.8%) of 260 raw chicken meat samples tested positive for H. pylori, with 11 (15.7%) and 7 (11.7%) from the thigh and breast, respectively, while Asadi et al. [44] identified H. pylori in raw chicken meat samples at a rate of 15%. In the study conducted by Hamada et al. [45], 7 (7.78%) of 90 chicken samples were positive for H. pylori, including 6.67% of chicken meat and gizzards and 10% of liver. The detection of H. pylori in chicken meat in previous studies might be attributed to contamination by the hands of butchers, veterinarians, and abattoir workers during handling, preparation, and packaging, as well as the use of unclean water for washing chicken carcasses [6]. This could explain why H. pylori was not identified in cloacal swabs from the examined chickens in the present study.

For Helicobacter pullorum, the overall prevalence in the examined chickens was 12.8%. Our findings were higher than those of Hassan et al. [46], who detected H. pullorum in 7% (21 out of 300) of chicken cloacal swabs. Many studies have focused on investigating H. pullorum in chicken meat, breast, thigh, liver, ceca, and wings [6, 9‐11, 46‐48] rather than cloacal swabs. For instance, H. pullorum was detected in 32.29% and 10.15% of broiler chicken caeca and colon, respectively, in Turkey [49]; in caeca (7.5%), liver (5%) and thigh (2.5%) of broiler chickens with gastroenteritis in Aradabil [50]; and in 41% of broiler chicken caeca in Iran [10]. H. pullorum was also identified in the cecum, colon, jejunum, and liver of broiler chickens in Belgium with a prevalence of 33.6%, 31.8%, 10.9%, and 4.6%, respectively [9]; 24.72% of broiler and village chickens in Malaysia [51]; 23.52% of chicken meat in Lisbon [11]; and 30% of tested chicken wings in Iran [52]. Furthermore, the commercial chicken eggs are also believed to be infected with this pathogen [7]. In Egypt, H. pullorum was isolated from the examined baladi hen’s eggshells and egg contents in a percentage of 3.33% for each [53], as well as 10% and 5% of Baladi and poultry farm hen’s eggshells were contaminated with H. pullorum, respectively [54]. The occurrence of H. pullorum in cloacal swabs suggests that this pathogen may be transmitted to chicken carcasses via cross-contamination during the slaughtering process [7], and hens' feces may spread H. pullorum to eggs [54]. As H. pullorum is directly transmitted to humans through fecal contamination [8], poultry excreta represent a potential source of infection to various human populations, particularly slaughterhouse workers, farmers, and housewives [7]. It was noted that the prevalence of H. pullorum was higher in layers (18.9%) than in broilers (7.3%) in this study, whereas a study conducted in Iran found a higher occurrence in broilers (30%) compared to laying hens (13.3%) [55]. From a public health perspective, H. pullorum is an emerging zoonotic pathogen responsible for life-threatening human infections [12]. It has been detected in stool samples from human patients suffering from gastroenteritis, with a prevalence of 6% in Aradabil [50], as well as in the feces from patients with gastrointestinal disease (4.3%) and clinically healthy individuals (4.0%) in Belgium [13]. Furthermore, H. pullorum is associated with recurrent diarrheal illness [56] and it is implicated in cholelithiasis, cirrhosis [14], and gallbladder cancers [57, 58]. This association is attributed to the pathogen's ability to tolerate high bile stress [12]. Additionally, it has been recognized in patients with Crohn's disease [37, 59].

In the current work, partial Helicobacter 16S rRNA gene sequencing revealed other Helicobacter species in layer chickens. H. brantae was the most prevalent species identified in cloacal swabs of layers (16.2%), followed by H. kayseriensis (5.4%), H. winghamensis (2.7%), and Helicobacter sp. TUL (2.7%). To the best of our knowledge, H. brantae, H. kayseriensis, H. winghamensis, and Helicobacter sp. TUL were detected for the first time in layer chickens in this study. Regarding H. brantae, Kaakoush et al. [60] found this species in 64.5% of broiler chicken fecal samples. This urease-negative Helicobacter species was first identified in the feces of seven resident Canada geese within the Greater Boston area [19], and it was detected at a low incidence in tropical terrestrial wild birds in Venezuela [41]. Although the pathogenesis of this bacterium remains unclear, the occurrence of H. brantae in chickens may pose a zoonotic risk, potentially infecting other species of birds and mammals [19]. H. kayseriensis was recognized by Aydin et al. [61] in the feces of urban wild birds in Turkey. Moreover, H. kayseriensis was the most common species (28.57%) isolated from Taiwan's Yanshui and Donggang rivers [2]. H. winghamensis was first discovered in patients with gastroenteritis in Canada, displaying a morphology similar to Campylobacter [62]. Also, it was recovered from wild rodents in China [63] and dogs in Taiwan [28]. Concerning Helicobacter sp. TUL, it is closely related to Helicobacter equorum and classified as an enterohepatic Helicobacter species. This novel species was named after its discovery in a febrile patient with a bloodstream infection in Caesarodunum (Tours, France) [64]. Accordingly, chicken feces may constitute an essential medium for transmitting emerging Helicobacter spp. where fecal droppings can directly or indirectly infect humans through water contamination. Water is a significant vehicle for the dissemination of Helicobacter species [2, 65], and this pathogen can persist in various environments, including soil [66, 67], raising concerns about cross-contamination between birds and the environment. Moreover, wild birds exposed to poultry excreta may transmit Helicobacter spp. [15, 41, 61, 68] to other birds, water sources, and new environments. In the meantime, phylogenetic analysis of the obtained Helicobacter sequences from layer hens in this study showed two distinct clusters. The first cluster demonstrated that H. brantae (PP390176), H. kayseriensis (PP397169), and Helicobacter sp. TUL (PP401975) sequences retrieved in this study were closely related to each other, implying that these Helicobacter spp. share a similar relationship. Furthermore, these sequences were grouped with those isolated from human cases (gastroenteritis and bloodstream infection) and environmental samples (wastewater and drinking water). In the second cluster, H. brantae (PP391550) was similar to H. brantae obtained from the feces of a resident Canada goose in the United States. H. kayseriensis (PP392689) exhibited a genetic relatedness to H. kayseriensis isolated from the feces of wild birds in Turkey. Additionally, H. winghamensis and H. brantae (PP814592 and PP814629, respectively) were grouped in the same clade and showed close relationship with Helicobacter sp. recovered from wild birds in Venezuela. These findings suggest that these Helicobacter spp. may spread from chickens to wild birds, humans, and the environment. Consequently, a comprehensive understanding of the transmission routes of Helicobacter infection can promote One Health approaches and facilitate the development of effective preventive strategies. The prevention and control strategies for Helicobacter spp., particularly H. pullorum, were based on the implementation of biosecurity measures in poultry farms and increasing the resistance of chickens to colonization by introducing organic acid additives to drinking water and/or feed. In addition, improved hygienic measures are required during the transport of live birds, slaughtering, and dressing of carcasses, as carcass contamination may occur through fecal matter spillage or cross-contamination [69]. Control measures should be established to reduce human exposure by minimizing the contamination of chicken meat along the food chain. Furthermore, monitoring and surveillance data would be highly crucial to mitigate the risk of Helicobacter infection through the implementation of One Health policies, especially in developing countries [7].

Investigation of the cdtB virulence gene among the Helicobacter species retrieved from broilers and layers in this work showed that it was present in 10 H. pullorum, 5 H. brantae, 1 H. winghamensis, and 1 Helicobacter sp. TUL; however, none of H. kayseriensis had cdtB. The cdtB gene appears to be the most conserved gene amongst all cdt genes in terms of differences between bacterial species [70]. For instance, Ceelen et al. [71] and Qumar et al. [33] detected cdtB in all H. pullorum strains obtained from poultry, while Mohamed et al. [72] observed cdtB in H. pullorum isolates from clinically healthy and diseased chickens at a prevalence rate of 32.9% and 67%, respectively. Yet, there is limited data regarding the occurrence of this virulence gene in H. brantae, H. winghamensis, H. kayseriensis, and Helicobacter sp. TUL, which requires further study. The cdtB is an important virulence factor that induces edema, cytoskeletal anomalies, and G2/M cycle arrest in the host cell. It causes cellular and nuclear enlargement, accompanied by profound remodelling of the actin cytoskeleton, resulting in the formation of large actin-rich cortical lamellipodia and membrane ruffle structures. Furthermore, disturbance of focal adhesion and the microtubule network were also observed. These effects may have significant consequences on bacterial adhesion and intestinal barrier integrity [22, 25]. The presence of cdtB in H. pullorum may play a significant role in various complications associated with human infections, such as gastroenteritis [22] and Crohn's disease [37]. Moreover, previous reports have shown that chronic infection by CDT-producing H. pullorum might lead to malignant transformation and cancer [73]. Detailed explanations of cdtB pathogenesis, interaction with its natural host, and factors contributing to the expression of Helicobacter cdtB remain unclear [21, 22, 74]. The findings of experimental infection carried out by Pratt et al. [75] suggested that CDT expression may reflect a bacterial adaptation that influences the interaction between the pathogen and the host immune system. CDT has been shown to induce apoptosis in primary human peripheral blood mononuclear cells and cultured T-cell lines [76, 77]. In addition to its direct effect on T cells, CDT may be able to interfere with immune responses via interfering with antigen-presenting cells [75]. Moreover, the bacterial adaptation of CDT production allows long-term persistence within the mammalian host and modifies the development of host immunity, resulting in specific immune responses which fail to clear the organism. In a host with an altered immune system, this modification of the specific immune response leads to the development of dysregulated immunity and colitis [75]. In this study, we provided partial sequences of H. brantae and H. pullorum cdtB from layer and broiler chickens, respectively, where these sequences exhibited a high identity percentage (98.68%-99.07%) to H. pullorum cdtB strains isolated from patients suffering from gastroenteritis, diarrhea, and liver cirrhosis, highlighting the public health significance of such sequences.

The occurrence of emerging virulent Helicobacter species in broiler and layer chickens highlights the potential zoonotic role of chickens as a reservoir of Helicobacter infection, which raises a public health concern. Establishing zoonotic links of Helicobacter spp. requires a variety of ways to determine how this pathogen can be transmitted between animals and humans. There are several important methods to identify zoonotic links, including molecular and genetic analysis such as multi-locus sequence typing and whole genome sequencing; epidemiological studies like cross-sectional studies, case–control studies, surveys of animal populations, and risk factor analysis. In addition, experimental animal models should not be ruled out. When these methods are combined, they can provide compelling evidence of zoonotic linkages of Helicobacter species since the findings of these studies help to improve our understanding of the transmission dynamics and potential public health risks posed by these bacteria. From a One Health perspective, the interconnection between human, animal, and environmental health sectors is crucial, necessitating continuous monitoring and surveillance of Helicobacter infections to mitigate their public health threat.