Helicobacter pullorum: A potential hurdle emerging pathogen for public health

Emerging zoonotic pathogens gain more attention due to the adverse effects on human and animal’s health and productivity. One of these zoonotic pathogens is Helicobacter pullorum (H. pullorum) which was firstly diagnosed in 1994. This bacterium is enterpathogenic in poultry and contaminates the carcasses meat during processing or improper handling. Human can get H. pullorum infection mainly through mishandling of contaminated carcasses or consumption of undercooked meat. Infection of H. pullorum in human is associated with gastroenteritis and hepatitis. Diagnosis of H. pullorum is very difficult as misdiagnosis with other enteric zoonotic pathogens like Campylobacter and other Helicobacter species is common. Unlike other types of Helicobacter, there are little information and few researches regarding prevalence, pathogenesis, diagnosis and control of H. pullorum infection either animals or human. Accordingly, this review article was prepared to give more details about H. pullorum sources of infection, pathogenicity, incidence in poultry and human as well as its treatment.


Introduction
Emerging disease is that caused by new etiological agent previously known but now occurring in species or places where the disease was unknown [1]. Infection with Helicobacter species is considered as one of these emerging diseases. Genus Helicobacter is belonging to class Epsilonproteobacteria that was established in 1989. These pathogens are divided into two groups, gastric and enterohepatic, based on their preferred site of colonization [2] and also on 16S rRNA sequence data. More than 30 species of Helicobacter have been recorded in the last two decades [3]. One of these newly identified species is Helicobacter pullorum (H. pullorum). This pathogen is fastidious, microaerophilic, non-sporulated and Gram-negative spirally curved motile bacillus with monopolar flagellae [2]. It has been considered that H. pullorum is an enterohepatic Helicobacter species [4]. This bacterium has been early discovered from the intestine and liver of diarrheic birds [5,6] as well as from the faeces and biopsies of patients with gastroenteritis, chronic liver disease and inflammatory bowel disease [7]. H. pullorum has a zoonotic potential [8][9][10][11][12] as it has been associated with approximately 12% of human zoonotic cases [13]. Consumption of undercooked or surface contaminated chicken is considered as a potential route of Helicobacter transmission to human beings [14]. Susceptibility of avian and animal species as well as human beings to H. pullorum is variable. Infections with H. pullorum were recorded in different avian species like chickens, turkeys, ostriches, Guinea fowl, parrots and psittacine birds [15,16], rabbits and rodents [17][18][19] and human [20]. Limited sources of data concerning H. pullorum infection in different species and their relations to human infection are available. Thus, in this review article, we will investigate H. pullorum sources of infection, pathogenicity, incidence in poultry and human as well as the possible treatment of this pathogen.

Sources of infection
The sources of H. pullorum infection to human is summarized in Table 1. Avian species serve as potent reservoirs for H. pullorum [21]. There is an association between H. pullorum presence in the intestinal tract of poultry with diarrhea and vibrionic hepatitis as well as presence of pathogen in patients with diarrhea, vomiting and liver and gallbladder diseases [2,7,22]. Surface contamination of broiler chickens' carcasses with the caecal contents during processing and handling is common [2,5]. It has been detected that H. pullorum colonizes the caecum of broilers and is excreted in the droppings till slaughtering and this implies that chicken meat constitutes a major source of infection for human [23]. Therefore, H. pullorum is considered as a pathogen of food borne significance [24,25]. In Australia, H. pullorum was isolated from chicken meat in the rate of 13.5% [26]. Furthermore, González et al. [27] identified H. pullorum in 3 types of chicken's meat products with 99% genetic match. Similarly, Borges et al. [10] in Italy demonstrated that the emerging H. pullorum pathogen can be transmitted to humans by chicken meat consumption and/or contact as the organism was isolated from 4 out of 17 (23.5%) fresh chicken meat samples from different producers.
It should be noted that, not only poultry meat is the source of H. pullorum infection for human, but also table eggs are another source. A total of 300 commercial chicken eggs were collected from Assiut and Qena governorates in Egypt for the presence of Helicobacter species detection [4]. The authors found that H. pullorum contamination rate of egg contents was 6.6% in Assiut and 3.3% in Qena governorate. Regarding the sources of chicken's infection in the farms, the study of Wai et al. [6] proved existence of H. pullorum in 17.5% of the house flies and 30% of the house floors in the farms of Malaysia. Moreover, Ceelen et al. [7] isolated H. pullorum from farmers' boots which is regarded as another mechanical source of infection. Concurrent presence of Helicobacter and Campylobacter species in cats has been reported [28] and water contamination with Helicobacter organisms was also demonstrated [29].

Pathogenesis
After infection of the host with H. pullorum, the bacterium adheres to the microvilli of the intestinal epithelial cells via flagellae for colonization and invasion. Consequently, this invasion induces cellular damage, debris and oedema [30]. In addition, adhesion of the pathogen to the intestinal surface stimulates the production and release of inflammatory substances like IL-8. The inflammatory process is more triggered through production of cytolethal distending toxin and lipopolysaccharide [7]. It has been reported that infection with H. pullorum activates the host's macrophages and secretion of cytokines (TNF-α, IL-1β, IL-6 and MIP-2) as well as production of nitric oxide in murine macrophages [31]. However, Yanagisawa et al. [32] depicted that H. pullorum infected human hepatocytes and bile duct and colon epithelial cells displayed increased expression of matrix metalloproteinases 2, 7 and 9 which help in degradation of extracellular matrix and allowing the pathogen to interact with host cells.

Incidence of infection
The incidence of H. pullorum in poultry and human in different countries is present in Table 2.

Poultry
Early study in Switzerland demonstrated H. pullorum in the caecal contents of 150 apparently healthy broiler chickens (4%) and in 9 out of 18 caeci of layers with vibrionic hepatitis [33]. Also, in broiler chickens with Campylobacters and Archobacters, H. pullorum was identified from 9 out of 15 frozen cecum (60%) and 9 out of 15 fresh carcasses (60%) [34]. The molecular identification results using Polymerase Chain Reaction (PCR) demonstrated presence of H. pullorum in 33.6% of the caecum and in 4.6% of the liver from 110 examined broiler chickens in Belgium [35]. In Italy, Zanoni et al. [36] investigated presence of H. pullorum in the caecal contents of 60 chickens that representing 9 broiler and 6 laying chickens' farms. On the other hand, PCR results showed that 42 out of 55 animals (76.4%) and 11 farms of turkeys were positive for H. pullorum [37]. Chickens reared in free-range Azevedo et al. [29] farms had lower occurrence (57%) of H. pullorum compared to birds reared in conventional (84%) and organic (97%) farms [38]. The work undertaken by Wai et al. [39] in Selangor and Malaysia identified H. pullorum from broiler chickens with 24.72% prevalence rate, where 12.36% of chickens showed concomitant infection with Campylobacter. Recently, the same author recognized H. pullorum in 51% of caeca of 100 chickens collected from processing sites or markets [6]. In Marmara region of Turkey, H. pullorum incidence rate was 55.21% after testing of 12 broiler chicken flocks [40]. Iranian study of Shahram et al. [20] showed that out 120 diarrheic broiler chicken, H. pullorum prevalence rates were 7.5% (intestinal swabs), 5% (liver) and 2.5% (thigh meat). However, higher prevalence rate (61%) of H. pullorum was also detected in Iran from 100 caecal samples of broiler chickens [41]. The highest incidence of H. pullorum in chickens were recorded in many countries where it ranged from 60% in the UK [42] to 78.3% in Czech Republic [43] and 100% in Italy [10] and France [44].
In Egypt, few researches have been conducted to detect the prevalence of H. pullorum among different types of living poultry as well as poultry products. A big study has been done in Assuit Province, where 1800 samples were collected from cloacal swabs, caecal contents and liver of chickens, turkeys and ducks' flocks [45]. The results revealed identification of 100 isolates of H. pullorum from chickens with a percentage of 39.33%. Although the main niche for colonization of H. pullorum is the intestine especially the caecum, but Hassan et al. [16] proved presence of the pathogen also in the liver tissues of the birds. Moreover, the study of Hassan et al. [46] demonstrated that out of 900 cloacal, caecal and liver tissues of broiler chickens, the incidence rate of H. pullorum was 39.33% using species-specific 16S rRNA PCR. Experimentally inoculated broilers with H. pullorum elicited 33.3% mortalities with signs of diarrhea, retardation of growth with poor conversion rate and the pathogen was reisolated from the caecum, liver, yolk sac and air-sacs of dead and sacrificed chickens [46]. H. pullorum was detected in the clinically healthy persons and the patients with gastroenteritis in percentages of 4% and 4.3%, respectively Ceelen et al. [7] Italy H. pullorum was present in the caecal contents of 60 chickens that representing 9 broiler and 6 laying chickens' farms Zanoni et al. [36] H. pullorum was isolated from chickens in incidence of 100% Borges et al. [ Wai et al. [39] H. pullorum in 51% of caeca of 100 chickens collected from processing sites or markets Wai et al. [6] Turkey H. pullorum incidence rate was 55.21% after testing of 12 broiler chicken flocks Beren and Seyyal [40] Iran Out 120 diarrheic broiler chicken, H. pullorum prevalence rates were 7.5% (intestinal swabs), 5% (liver) and 2.5% (thigh meat) Shahram et al. [20] The prevalence rate of H. pullorum was 61% from 100 caecal samples of broiler chickens Jamshidi et al. [41] Six positive cases of H. pullorum was detected in 100 stool samples of patients with gastroenteritis Shahram et al. [20] United Kingdom H. pullorum was isolated from chickens in incidence of 60% Mohamed et al. [45] Out of 900 cloacal, caecal and liver tissues of broiler chickens, the incidence rate of H. pullorum was 39.33% Hassan et al. [46] Experimental infection with H. pullorum in chickens elicited 33.3% mortalities with signs of diarrhea, retardation of growth with poor conversion rate and the pathogen was re-isolated from the caecum, liver, yolk sac and air-sacs of dead and sacrificed chickens

Human
Diarrhea caused by infectious agent is a major cause of worldwide morbidity and mortality in human, especially in children [47]. There are some reports suggesting that H. pullorum is a major pathogen of human. Early, H. pullorum was first isolated from the stool of a male patient with diarrhea and elevated liver enzymes [48]. Later on, this pathogen was discovered from faeces of diarrheic patients, 3 months after the onset of symptoms [49]. Infection of human with H. pullorum is not only associated with gastroenteritis and diarrhea, but also with gall bladder and liver diseases [22]. In addition, H. pullorum was isolated from 35 year old male suffering from bacteraemia, abdominal pain and profuse diarrhea [50]. In 2005, H. pullorum was detected in the clinically healthy Belgium persons and the patients with gastroenteritis in percentages of 4% and 4.3%, respectively [7]. They concluded that presence of H. pullorum in the stool of apparently healthy individuals may indicate that this bacterium is harmless normal inhabitant in the intestine or it proliferates after consumption of contaminated food. They also assumed that certain unknown predisposing factors may change non-pathogenic normal intestinal H. pullorum to highly virulent pathogenic ones. H. pullorum has also been identified by PCR in humans with inflammatory bowel disease [41,51], viral hepatitis C [52][53][54], cholecystitis [55,56] and hepatocellular carcinoma [57,58]. Another study of Shahram et al. [20] recognized 6 positive cases of H. pullorum from 100 stool samples of patients with gastroenteritis in Ardabil province, Iran. It was also found that H. pullorum may have an important role in Crohn's disease caused by Mycobacterium paratuberculosis in inflammatory bowel disease [59,60].

Treatment
Unfortunately, there is no recommended groups of drugs for treatment of H. pullorum infection. The sensitivity or resistance of H. pullorum isolates to different antimicrobials have been studied with variable results. The in-vitro resistance of avian H. pullorum isolates to nalidixic acid revealed percentages of 6% [24] and 26% [5]. Moreover, resistance of H. pullorum to cephalothin and cefoperazone was also recorded [2,24,36]. Recently, tetracycline resistance of H. pullorum mutant strain was recorded [10]. Conversely, H. pullorum was found to be susceptible to polymyxin B [5]. Moreover, human strains of H. pullorum displayed sensitivity to aminoglycosides, third-generation cephalosporins, β-lactams and doxycycline [50]. In Upper Egypt, high incidence of avian H. pullorum resistance to ciprofloxacin, gentamicin and erythromycin followed by tetracycline were observed [45,46]. Nevertheless, the same studies revealed high sensitivity of the pathogen to ampicillin and/or colistin sulfate suggesting them as drugs of choice for treatment of infection in chickens. The study of Abdel Hameed and Sender [4] indicated that H. pullorum isolated from chickens' eggs were resistant to ampicillin, ceftriaxone and sulphamethoxazole trimethoprim in-vitro.

Conclusion
It has been considered that H. pullorum is an emerging pathogen of potential zoonotic importance for both human and animals. Little is known about this bacterium infection. So, more extensive attention and studies should be carried out to increase the knowledge and information about H. pullorum prevalence, infectivity and control measures. The closed-housing system with good biosecurity, management and husbandry practices could reduce and control the presence of H. pullorum in in the farms. It is important to focus on the methods of control of this pathogen at the farm level till retailing. These data will have a public health importance in relation to reducing human exposure associated with the handling and consumption of contaminated processed chicken's meat.