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Helicobacter pullorum is an emerging foodborne pathogen that commonly colonizes the gastrointestinal tract of poultry, causing gastroenteritis. It has been related to several clinically important infections, including colitis and hepatitis, inflammatory bowel disease, recurrent diarrhea, and bacteremia in the human population. The bacterium may be transmitted to humans through undercooked poultry meat. In order to investigate the occurrence of H. pullorum in raw retail chicken meat (thighs and breasts), we analyzed 240 samples: 120 chicken thigh and 120 chicken breast samples. The samples were analyzed by means of an isolation protocol using Steele and McDermott's modified filtration technique on Brucella agar supplemented with 5% of defibrinated sheep's blood. The presumptive colonies were biochemically identified and analyzed using a previously described conventional PCR test based on the 16S rRNA gene. In total, 35% of analyzed samples were positive using the microbiological protocol and 45% were positive by PCR. These results suggest that H. pullorum can be transmitted to humans through the handling and consumption of raw poultry meat, representing a risk for food business operators and consumers. Efforts to control H. pullorum in broiler meat should prioritize the implementation of stringent hygienic practices across all stages of the food chain, from the farm to the consumer. [ABSTRACT FROM AUTHOR]

Helicobacter pullorum is an emerging foodborne pathogen that commonly colonizes the gastrointestinal tract of poultry, causing gastroenteritis. It has been related to several clinically important infections, including colitis and hepatitis, inflammatory bowel disease, recurrent diarrhea, and bacteremia in the human population. The bacterium may be transmitted to humans through undercooked poultry meat. In order to investigate the occurrence of H. pullorum in raw retail chicken meat (thighs and breasts), we analyzed 240 samples: 120 chicken thigh and 120 chicken breast samples. The samples were analyzed by means of an isolation protocol using Steele and McDermott’s modified filtration technique on Brucella agar supplemented with 5% of defibrinated sheep’s blood. The presumptive colonies were biochemically identified and analyzed using a previously described conventional PCR test based on the 16S rRNA gene. In total, 35% of analyzed samples were positive using the microbiological protocol and 45% were positive by PCR. These results suggest that H. pullorum can be transmitted to humans through the handling and consumption of raw poultry meat, representing a risk for food business operators and consumers. Efforts to control H. pullorum in broiler meat should prioritize the implementation of stringent hygienic practices across all stages of the food chain, from the farm to the consumer.

ABSTRACT: Helicobacter pullorum is recognized as an emerging food-borne pathogen that may colonize the intestinal tract and the liver of avian species and humans causing several gastrointestinal and liver diseases. However, not all strains are reported to be capable of causing clinical disease, thus making poultry as reservoir for the zoonotic transmission of the infection through carcass contamination of broilers at slaughter. In poultry, the prevalence of this bacterium could be underestimated and the available data mainly refer to conventional rearing systems, whereas free-range and organic breedings have been poorly investigated. Therefore, this study was aimed to characterize the caecal microbiota community of free-range grown chickens and determine the presence and the relative abundance of H. pullorum by using NGS-based 16S rDNA sequencing. A total of 18 chickens reared for 56 d on a semi-extensive management system were euthanized at two time points: 9 birds at 28 d of age (before have access to outdoor; I = Indoor) and other 9 birds at 56 d of age (before slaughter; O = Outdoor). Cecal contents were collected for microbiota analyses. H. pullorum was detected in the cecum of 16/18 samples and its proportion in indoor was significantly higher than outdoor chickens (2.46 and 0.52%, respectively; P < 0.05), showing 78.8% of decrease with the outdoor access of the chickens. Therefore, it may be assumed that the potential for zoonotic infection is less likely. Moreover, H. pullorum was negatively correlated with 17 bacterial species as significantly more abundant in Outdoor microbial caecal communities. Among these, we highlighted the presence of Mucispirillium schaedleri and Oscillospira, already previously associated with a healthy gut and thus representing promising gut bacterial markers for host health. Our findings suggest that alternative production systems with outdoor access, may play a crucial role in the establishment of a healthy gut microbiota, which in turn might prevent colonization of harmful bacteria such as Helicobacter pullorum.

Background: Helicobacter pullorum can infect the intestinal tracts of both humans and avian species. This study aimed to assess the frequency and antibiotic resistance of H. pullorum isolated from workers in the poultry slaughterhouses, farms, and markets as exposed population and healthy people who referred to the hospital as non-exposed population by culture method and polymerase chain reaction (PCR) test. Methods: Two hundred healthy individuals, including 100 individuals from exposed population and 100 from non-exposed population were selected in Semnan. Fresh stool samples were examined by conventional culture method and biochemical tests. PCR test with 16S rRNA gene was employed to confirm the H. pullorum isolates. Antibiotic resistance test was done using the disk diffusion method and various antimicrobial agents. Results: Generally, 17 (17%) samples from exposed population and 12 (12%) samples from non-exposed population were H. pullorum positive by culture method and biochemical tests. However, PCR test could confirm 10 (10%) and 7 (7%) samples from exposed and non-exposed populations, respectively. Therefore, the frequency of H. pullorum was determined to be 9.5%. Antibiotic resistance test could reveal that most of the isolates were resistant to ciprofloxacin (84.2%), whereas resistance to colistin and fosfomycin was found to be 15.8%. Conclusion: The present study illustrated that H. pullorum can be present among healthy population with the low frequency rate. Moreover, it was indicated that the frequency of this food-borne pathogen is high in the exposed population. Therefore, there is a high demand for good observation for slaughter hygiene and implementation of routine surveillance in the poultry farms and markets.

Introduction: Helicobacter pullorum has recently emerged as a significant foodborne pathogen. The present study aimed to isolate and determine the antibiotic resistance of H. pullorum from fresh chicken wing using the culture method and a molecular technique in Semnan, Iran. Methods: A total of 60 fresh chicken wings samples were purchased from various local retail markets in different regions of Semnan and processed using the culture method. For initial confirmation, biochemical tests were applied. Suspected colonies were subjected to polymerase chain reaction (PCR) by the 16S rRNA gene. The antibiotic resistance of the isolates was also assessed using the disk-diffusion method. Results: Among 60 samples, 27 (45%) were H. pullorum-positive based on the culture method and biochemical tests. However, the PCR test indicated 18 samples (30%) to be positive for H. pullorum. In the antibiogram, the highest and lowest resistance rates were observed against ciprofloxacin and fosfomycin, respectively. Conclusion: This was the first report in Iran to clearly illustrate that H. pullorum could be found in fresh chicken wings at a moderate level. In addition, the antibiotic resistance of the H. pullorum isolates was confirmed, and the PCR test based on the 16S rRNA gene was considered a reliable and sensitive technique for the detection of this pathogen. However, further investigation is required to explore the life cycle of this novel foodborne pathogen in the other regions of Iran.

Helicobacter pylori (H. pylori) and Helicobacter pullorum (H. pullorum) are frequently reported pathogens in humans and poultry, respectively. Nevertheless, the source of H. pylori is still unclear. This study aimed to detect Helicobacter spp. in chicken carcasses and to assess the antibiogram and the virulence genes of Helicobacter isolates. Three hundred chicken meat samples (100 each of chicken breast, liver, and gizzard), besides 60 swab samples from chicken processing surfaces, were collected from retail shops in Qalyubia Governorate, Egypt, and examined for the prevalence of H. pylori and H. pullorum. The 16S rRNA of three H. pylori and two H. pullorum isolates were sequenced to determine the genetic relationship between these two Helicobacter spp. Of the 300 chicken samples tested, 16 (5.33%) and 14 (4.67%) were positive for H. pylori and H. pullorum, respectively. Multiplex PCR revealed that the virulence genes vacuolating cytotoxin A (vacA)s1, cytotoxin-associated gene A (cagA), and restriction endonuclease-replacing gene A (hrgA) were detected in 66.7%, 77.8%, and 100% of H. pylori strains tested, respectively. H. pylori showed the highest resistance for clarithromycin, while H. pullorum exhibited the highest resistance towards erythromycin and ciprofloxacin. The study concluded that the chicken meat and giblets are potential sources of the virulent and antimicrobial-resistant strains of H. pylori of human origin. [ABSTRACT FROM AUTHOR]

N‐acetylated sugars are often found, for example, on the lipopolysaccharides of Gram‐negative bacteria, on the S‐layers of Gram‐positive bacteria, and on the capsular polysaccharides. Key enzymes involved in their biosynthesis are the sugar N‐acetyltransferases. Here, we describe a structural and functional analysis of one such enzyme from Helicobacter pullorum, an emerging pathogen that may be associated with gastroenteritis and gallbladder and liver diseases. For this analysis, the gene BA919‐RS02330 putatively encoding an N‐acetyltransferase was cloned, and the corresponding protein was expressed and purified. A kinetic analysis demonstrated that the enzyme utilizes dTDP‐3‐amino‐3,6‐dideoxy‐d‐glucose as a substrate as well as dTDP‐3‐amino‐3,6‐dideoxy‐d‐galactose, albeit at a reduced rate. In addition to this kinetic analysis, a similar enzyme from Helicobacter bilis was cloned and expressed, and its kinetic parameters were determined. Seven X‐ray crystallographic structures of various complexes of the H. pullorum wild‐type enzyme (or the C80T variant) were determined to resolutions of 1.7 Å or higher. The overall molecular architecture of the H. pullorum N‐acetyltransferase places it into the Class II left‐handed‐β‐helix superfamily (LβH). Taken together, the data presented herein suggest that 3‐acetamido‐3,6‐dideoxy‐d‐glucose (or the galactose derivative) is found on either the H. pullorum O‐antigen or in another of its complex glycoconjugates. A BLAST search suggests that more than 50 non‐pylori Helicobacter spp. have genes encoding N‐acetyltransferases. Given that there is little information concerning the complex glycans in non‐pylori Helicobacter spp. and considering their zoonotic potential, our results provide new biochemical insight into these pathogens PDB Code(s): 7s3u, 7s3w, 7s41, 7s42, 7s43, 7s44 and 7s45; [ABSTRACT FROM AUTHOR]

Introduction: Helicobacter pullorum has recently emerged as a significant foodborne pathogen. The present study aimed to isolate and determine the antibiotic resistance of H. pullorum from fresh chicken wing using the culture method and a molecular technique in Semnan, Iran. Methods: A total of 60 fresh chicken wings samples were purchased from various local retail markets in different regions of Semnan and processed using the culture method. For initial confirmation, biochemical tests were applied. Suspected colonies were subjected to polymerase chain reaction (PCR) by the 16S rRNA gene. The antibiotic resistance of the isolates was also assessed using the disk-diffusion method. Results: Among 60 samples, 27 (45%) were H. pullorum-positive based on the culture method and biochemical tests. However, the PCR test indicated 18 samples (30%) to be positive for H. pullorum. In the antibiogram, the highest and lowest resistance rates were observed against ciprofloxacin and fosfomycin, respectively. Conclusion: This was the first report in Iran to clearly illustrate that H. pullorum could be found in fresh chicken wings at a moderate level. In addition, the antibiotic resistance of the H. pullorum isolates was confirmed, and the PCR test based on the 16S rRNA gene was considered a reliable and sensitive technique for the detection of this pathogen. However, further investigation is required to explore the life cycle of this novel foodborne pathogen in the other regions of Iran. [ABSTRACT FROM AUTHOR]

Aim: This study aimed to investigate the occurrence of Helicobacter pullorum in broiler chickens and their farm environment. Materials and Methods: The ceca from 100 broiler chickens from ten farms were sampled from processing sites or markets. The cecal contents were aseptically collected from each cecum and cultured. The farms were visited, and environmental samples were collected which included water, house flies, floor swabs and soils in chicken houses. Results: H. pullorum was present in 51% of the broilers; 17.5% of the flies were found to carry H. pullorum and Campylobacter spp., 30% of house floors were positive, while all water samples were negative. Conclusion: Flies could have picked up the organisms from the chickens' feces and/or the environment of the chicken houses or they could be one of the sources in the spread of the organisms. This study also showed that broiler chickens are potential reservoirs for H. pullorum and may serve as a source of infection for humans through the food chain.

Helicobacter pylori (H. pylori) and Helicobacter pullorum (H. pullorum) are frequently reported pathogens in humans and poultry, respectively. Nevertheless, the source of H. pylori is still unclear. This study aimed to detect Helicobacter spp. in chicken carcasses and to assess the antibiogram and the virulence genes of Helicobacter isolates. Three hundred chicken meat samples (100 each of chicken breast, liver, and gizzard), besides 60 swab samples from chicken processing surfaces, were collected from retail shops in Qalyubia Governorate, Egypt, and examined for the prevalence of H. pylori and H. pullorum. The 16S rRNA of three H. pylori and two H. pullorum isolates were sequenced to determine the genetic relationship between these two Helicobacter spp. Of the 300 chicken samples tested, 16 (5.33%) and 14 (4.67%) were positive for H. pylori and H. pullorum, respectively. Multiplex PCR revealed that the virulence genes vacuolating cytotoxin A (vacA)s1, cytotoxin-associated gene A (cagA), and restriction endonuclease-replacing gene A (hrgA) were detected in 66.7%, 77.8%, and 100% of H. pylori strains tested, respectively. H. pylori showed the highest resistance for clarithromycin, while H. pullorum exhibited the highest resistance towards erythromycin and ciprofloxacin. The study concluded that the chicken meat and giblets are potential sources of the virulent and antimicrobial-resistant strains of H. pylori of human origin.

Background: It has become established that Helicobacter pullorum could be isolated from raw chicken meat. Aims: This study was aimed to develop a novel culture method (protocol B) to isolate H. pullorum from chicken meat by adding some modifications to the traditional culture method (protocol A), and as a consequence to compare their sensitivity, specificity, and the accuracy of these methods with polymerase chain reaction (PCR) test. Methods: 400 chicken meat samples were collected from various retail markets and supermarkets. Each sample was processed by protocol A, protocol B, and PCR test. Results: Out of 400 samples, 77 (19.25%), and 163 (40.75%) were culture-positive by protocol A and protocol B, respectively. Using PCR test as a gold standard, 196 (49%) samples were identified as H. pullorum. The specificity for both protocols was determined 100%, while the sensitivity of protocol B and protocol A was assessed 83% and 39%, respectively. Also, the higher and lower accuracy belonged to protocol B (92%) and protocol A (70%), respectively. Conclusion: The methodology designed herein can provide a suitable, approximately sensitive, specific, and accurate method to cultivate H. pullorum from chicken meat. [ABSTRACT FROM AUTHOR]

Dietary interventions are a common practice in the poultry industry to promote optimal performance and health of animals. Here, we aim at assessing the influence of supplementing broiler diets with dry whey powder (DWP) and whey protein concentrate (WPC) on nutrient coefficient of apparent ileal digestibility (CAID) and productive performance. Cecal microbiota composition was also determined using Illumina amplicon sequencing. Dietary treatments were control diet (no supplementation of DWP or WPC), 60-DWP (60 g/kg of DWP), and 80-WPC (80 g/kg of WPC). One-day-old male broilers were randomly assigned to one of three treatments, and housed in floor pens. In Trial 1, 90 1-day-old chicks were allocated to three pens/treatment, with 10 birds/pen, during 21 days for CAID evaluation. Diet 60-DWP increased Ca CAID (P=0.041), while diet 80-WPC improved Ca and P CAID (P

Helicobacter pullorum is an emerging zoonotic pathogen that causes gastroenteritis in chickens and inflammatory bowel disease in humans ingesting contaminated meat. However, the mechanism by which the bacterium causes disease is unclear. Type six secretion system (T6SS) plays a major role in bacterial pathogenesis and adaptation. Haemolysin coregulated protein (Hcp) plays a central role in the structure of the T6SS pilus and acts as effector protein in certain bacteria. In this study, H. pullorum isolated from 156 caecal samples of broiler chickens was screened for the presence of T6SS Hcp gene via PCR amplification. 30.7% of caecal and 18.3% of liver samples tested positive for presence of H. pullorum. From these positive samples, 29.7% possessed the T6SS gene. In bacterial co-culture experiments, significant loss of viability (81.6–39.1%) was observed for H. pullorum-infected hepatocytes and presence of Hcp did not contribute to the loss of cell viability. Nevertheless, infection of erythrocytes with Hcp-positive isolates was associated with greater haemolytic activity compared to infection with Hcp-negative isolates. Therefore, presence of T6SS could be indicative of virulent strains meriting further studies to characterize this virulence factor in H. pullorum infection. [ABSTRACT FROM AUTHOR]

Some of the life-threatening, food-borne, and zoonotic infections are transmitted through poultry birds. Inappropriate and irrational use of antimicrobials in the livestock industry has resulted in an increased incidence of multi-drug resistant bacteria of epidemic potentials. The adhesion and invasion properties of 11 free-range and broiler chicken derived Helicobacter pullorum isolates were evaluated. To examine the biofilm formation of H. pullorum isolates, crystal violet assay was performed. A quantitative assay of invasion-associated genes was carried out after infecting HepG2 cells with two different representative (broiler and free-range chicken) H. pullorum isolates, using RT-PCR assay. Furthermore, we investigated the prevalence of H. pullorum , Campylobacter jejuni and Salmonella spp. in chicken caeca and oviducts to determine the possibility of trans -ovarian transmission. All H. pullorum isolates adhered to HepG2 cells significantly but a notable difference towards their invasion potential was observed between free-range and broiler chicken isolates wherein broiler isolates were found to be more invasive compared to free-range isolates. Furthermore, cdt B, flh A and fla B genes of H. pullorum were upregulated post infection of HepG2 cells, in broiler chicken isolates compared to free-range chicken isolates. Moreover, all isolates of H. pullorum were found to form biofilm on the liquid-air interface of the glass coverslips and sidewalls of the wells with similar propensities. Despite presence of H. pullorum and C. jejuni in high concentrations in the caecum, they were completely absent in oviduct samples, thus ruling out the possibility of vertical transmission of these bacterial species. In contrast, Salmonella spp. was found to be present in a significant proportion in the oviduct samples of egg-laying hens suggesting its vertical transmission. Our findings suggest that H. pullorum, an emerging multi-drug resistant (MDR) pathogen could be transmitted from poultry sources to humans. In addition to this, its strong functional similarity with C. jejuni provides a firm basis for H. pullorum to be an emerging food-associated, MDR pathogenic bacterium that could pose risk to public health. • Invasion properties of H. pullorum differ based on source and H. pullorum isolated from broilers is more invasive in nature. • Virulence potential of different bacterial species is changing by the use of antibiotics in livestock farming. • Unlike Salmonella , the vertical route of transmission of H. pullorum has not been observed or proved. [ABSTRACT FROM AUTHOR]

Enterohepatic Helicobacters, such as Helicobacter hepaticus and Helicobacter pullorum, are associated with several intestinal and hepatic diseases. Their main virulence factor is the cytolethal distending toxin (CDT). In the present study, whole genome microarray-based identification of differentially expressed genes was performed in vitro in HT-29 intestinal cells while following the ectopic expression of the active CdtB subunit of H. hepaticus CDT. A CdtB-dependent upregulation of the V-maf musculoaponeurotic fibrosarcoma oncogene homolog B (MAFB) gene encoding the MAFB oncoprotein was found, as well as the CdtB-dependent regulation of several MAFB target genes. The transduction and coculture experiments confirmed MAFB mRNA and protein induction in response to CDT and its CdtB subunit in intestinal and hepatic cell lines. An analysis of MAFB protein subcellular localization revealed a strong nuclear and perinuclear localization in the CdtB-distended nuclei in intestinal and hepatic cells. MAFB was also detected at the cell periphery of the CdtB-induced lamellipodia in some cells. The silencing of MAFB changed the cellular response to CDT with the formation of narrower lamellipodia, a reduction of the increase in nucleus size, and the formation of less γH2AX foci, the biomarker for DNA double-strand breaks. Taken together, these data show that the CDT of enterohepatic Helicobacters modulates the expression of the MAFB oncoprotein, which is translocated in the nucleus and is associated with the remodeling of the nuclei and actin cytoskeleton.

Dietary interventions are a common practice in the poultry industry to promote optimal performance and health of animals. Here, we aim at assessing the influence of supplementing broiler diets with dry whey powder (DWP) and whey protein concentrate (WPC) on nutrient coefficient of apparent ileal digestibility (CAID) and productive performance. Cecal microbiota composition was also determined using Illumina amplicon sequencing. Dietary treatments were control diet (no supplementation of DWP or WPC), 60-DWP (60 g/kg of DWP), and 80-WPC (80 g/kg of WPC). One-day-old male broilers were randomly assigned to one of three treatments, and housed in floor pens. In Trial 1, 90 1-day-old chicks were allocated to three pens/treatment, with 10 birds/pen, during 21 days for CAID evaluation. Diet 60-DWP increased Ca CAID (P=0.041), while diet 80-WPC improved Ca and P CAID (P<0.001 and 0.002, respectively) when compared with control diet. In Trial 2, 810 one-day-old chicks were allocated to nine pens/treatment, with 30 birds/pen, during 42 days. Feeding chickens with 60-DWP and 80-WPC increased their BW, average daily gain (ADG) and feed intake (FI) during the starter (P<0.001 for all variables) and grower-finisher periods (P<0.001 for BW and FI, and P=0.048 for ADG), and during the entire feeding period (P<0.05), when compared with control diet. Diets 60-DWP and 80-WPC reduced the feed conversion ratio of chickens during the starter period (P<0.001 and 0.003, respectively), while 60-DWP reduced this parameter during the entire feeding period (P=0.048), when compared to control diet. At day 42, cecal microbial communities of chickens that were fed with 60-DWP and 80-WPC differed from those fed with control diet (R=0.776, P=0.008; and R=0.740, P=0.008, respectively). The abundance of Bacteroides fragilis, Bacteroides spp., Escherichia coli/Shigella flexneri and Megamonas furniformis increased when 60-DWP and 80-WPC diets were offered, while the presence of Helicobacter pullorum decreased. Lactobacillus salivarius consistently increased in chickens with better feed conversion ratio, which were those fed with 60-DWP. The results obtained in the present study indicate that growth of chickens is improved by DWP and WPC supplementation because of a higher mineral digestibility, increased feed intake and modulation of cecal microbiota communities. [ABSTRACT FROM PUBLISHER]

This work aimed to study the pathogenicity and to investigate the antimicrobial susceptibility and resistance patterns of Helicobacter pullorum (H. pullorum). The minimum inhibitory concentration (MIC) value of ciprofloxacin, ampicillin, gentamycin, erythromycin, colistin sulfate and tetracycline was determined for eight different H. pullorum isolates. H. pullorum resulted into 33.3% mortality of infected chickens with signs of diarrhea, stunted growth and poor conversion rate in survivors. All experimentally infected embryonated chicken eggs showed embryonic mortalities within 48-h post yolk sac inoculation. H. pullorum was re-isolated from cecum, liver, yolk sac and air-sacs of all dead and sacrificed infected chickens. H. pullorum was also re-isolated from dead embryos, embryonic membranes and fluids of infected embryonated chicken eggs. Polymerase Chain Reaction (PCR) assay was used to detect H. pullorum in experimentally infected chickens and embryonated chicken eggs. All tested H. pullorum isolates were resistant to ciprofloxacin, gentamycin and erythromycin, while 7 out of 8 isolates were resistant to tetracycline. All isolates were susceptible to colistin sulfate and ampicillin.

Helicobacter pullorum is recognized as an emerging food-borne pathogen that may colonize the intestinal tract and the liver of avian species and humans causing several gastrointestinal and liver diseases. However, not all strains are reported to be capable of causing clinical disease, thus making poultry as reservoir for the zoonotic transmission of the infection through carcass contamination of broilers at slaughter. In poultry, the prevalence of this bacterium could be underestimated and the available data mainly refer to conventional rearing systems, whereas free-range and organic breedings have been poorly investigated. Therefore, this study was aimed to characterize the caecal microbiota community of free-range grown chickens and determine the presence and the relative abundance of H. pullorum by using NGS-based 16S rDNA sequencing. A total of 18 chickens reared for 56 d on a semi-extensive management system were euthanized at two time points: 9 birds at 28 d of age (before have access to outdoor; I = Indoor) and other 9 birds at 56 d of age (before slaughter; O = Outdoor). Cecal contents were collected for microbiota analyses. H. pullorum was detected in the cecum of 16/18 samples and its proportion in indoor was significantly higher than outdoor chickens (2.46 and 0.52%, respectively; P < 0.05), showing 78.8% of decrease with the outdoor access of the chickens. Therefore, it may be assumed that the potential for zoonotic infection is less likely. Moreover, H. pullorum was negatively correlated with 17 bacterial species as significantly more abundant in Outdoor microbial caecal communities. Among these, we highlighted the presence of Mucispirillium schaedleri and Oscillospira, already previously associated with a healthy gut and thus representing promising gut bacterial markers for host health. Our findings suggest that alternative production systems with outdoor access, may play a crucial role in the establishment of a healthy gut microbiota, which in turn might prevent colonization of harmful bacteria such as Helicobacter pullorum. [ABSTRACT FROM AUTHOR]

The Helicobacter genus includes Gram negative bacteria which were originally considered to belong to the Campylobacter genus. They have been classified in a separate genus since 1989 because they have different biochemical characteristics, with more than 24 species having been identified and more still being studied. H. pylori is the best known. It has an important etiopathogenic role in peptic ulcer disease and gastric cancer. Enterohepatic Helicobacter s (EHH) other than H. pylori colonize the bowel, biliary tree and liver of animals and human beings with pathogenic potential. The difficulties existing to correctly isolate these microorganisms limit the description of their true prevalence and of the diseases they cause. Many studies have tried to discover the different clinical implications of EHH. Diseases like chronic liver disease, autoimmune hepatitis, hepatocarcinoma, autoimmune hepatobiliary disease, biliary lithiasis, cholangiocarcinoma and gallbladder cancer, Meckel's diverticulum, acute appendicitis and inflammatory bowel disease have been related with different EHH species with different results, although their prevalence is greater than in healthy subjects. However, these data are currently not sufficient to draw definitive conclusions. Finally, the best known role of EHH in bowel disease is production of acute and chronic diarrhea pictures initially referred to as Campylobacter. H. pullorum has been identified in patients with acute gastroenteritis. The correct identification of EHH as producers of infectious gastroenteritis is found in its antibiotic susceptibility. It is generally macrolide-susceptible and quinolone-resistant.

The reports on prevalence of Helicobacter pullorum in broiler chickens are rather limited and lacking in village chickens. This study aimed to determine the occurrence of H. pullorum in broiler and village chickens in Selangor, Malaysia and to report the detection of co-infection of H. pullorum and Campylobacter spp. in these chickens. Village (indigenous) chickens were sampled in five markets and broiler chickens from six farms in different localities. Cecal contents were aseptically obtained from the chickens and subjected to three cultural methods. The isolates were identified by biochemical tests and confirmed using a species-specific PCR assay. Helicobacter pullorum were isolated from 25% village chickens and 24.6% broiler chickens, with an overall occurrence of 24.7%. Eleven (50%) of these positive chickens (nine in broiler and two in village chickens) showed co-infection with Campylobacter spp.

Enterohepatic Helicobacter species are associated with several digestive diseases. Helicobacter pullorum is an emerging human foodborne pathogen, and Helicobacter hepaticus is a mouse pathogen; both species are associated with intestinal and/or hepatic diseases. They possess virulence factors, such as cytolethal distending toxin (CDT). Data indicate that CDT may be involved in chronic inflammatory responses, via its active subunit, CdtB. The proinflammatory properties of the CdtB of H. pullorum and H. hepaticus were assessed on human intestinal and hepatic epithelial cells in vitro. Interleukin 8 expression was evaluated by using wild-type strains and their corresponding CdtB isogenic mutants and by delivering CdtB directly into the cells. Nuclear factor κB nuclear translocation and transcriptomic characteristics in response to CdtB were also evaluated. The CdtB of these Helicobacter species induced nuclear factor κB nuclear translocation and exhibited proinflammatory properties, mainly the expression of T-helper type 17-related genes and genes encoding antimicrobial products also involved in cancer. The Histidine residue in position 265 of the CdtB catalytic site appeared to play a role in the regulation of most of these genes. As for flagellin or lipopolysaccharides, CdtB also induced expression of inflammation-associated genes related to antimicrobial activity. [ABSTRACT FROM AUTHOR]

A total of 110 broilers from 11 flocks were tested for Helicobacter pullorum by polymerase chain reaction; positive samples were reexamined with a conventional isolation method. H. pullorum isolates were examined by amplified fragment length polymorphism (AFLP) fingerprinting for interstrain genetic diversity and relatedness. Sixteen isolates from cecal samples from 2 different flocks were obtained. AFLP analysis showed that these isolates and 4 additional isolates from a different flock clustered according to their origin, which indicates that H. pullorum colonization may occur with a single strain that disseminates throughout the flock. Strains isolated from different hosts or geographic sources displayed a distinctive pattern. H. pullorum is present in approximately one third of live chickens in Belgium and may represent a risk to human health.

According to the World Health Organization, diseases which are naturally transmissible from vertebrate animals to human beings or from humans to vertebrates are defined as the zoonotic diseases. Among the most common zoonotic pathogens, Helicobacter pullorum has earned public recognition regarding its public health significance. This Enterohepatic Helicobacter species has been shown to be a very dangerous and life-threatening microorganism, accounting for several clinically important infections in the human population. However, despite the several studies indicating the significance of H.pullorum in both humans and animals, there is a lack of documented information and reliable statistics about this pathogen throughout the world. Thus, in this review, we would provide a novel knowledge about the general characteristics, isolation methods, host ranges and transmission routes, and occurrences of H.pullorum in poultry, chicken meat, and human in Iran. We would also clarify the status of antimicrobial resistance (AMR) profile of the H.pullorum isolates from various samples in this country. • H.pullorum is a putative food-associated emerging zoonotic pathogen. • This is the first exhaustive report about different aspects of H.pullorum in Iran. • A novel knowledge of H.pullorum in poultry, chicken meat, and human is shown. • Implementation of One Health measures is highly recommended in developing countries. [ABSTRACT FROM AUTHOR]

Enterohepatic Helicobacter species, including H. pullorum, are increasingly recognized as microbial pathogens in humans and animals. Tissue samples comprising 100 muscle, liver and heart (each of them) was collected from seven slaughterhouses in Isfahan province, central of Iran. Furthermore, 100 cecal contents were sampled from 100 broiler chickens which did not have gross pathologic signs suggestive of the disease. DNA was extracted from muscle and liver tissues and PCR was carried out for identification of H. pullorum 16S rRNA gene in the samples. The results showed that the H. pullorum genome was detected in 2 of the 100 (2%) liver samples and in none of muscle or heart samples. The positive samples collected from two different slaughterhouses out of seven slaughterhouses. In this study, 11 out of the 100 cecal content samples (11%) from apparently healthy broiler chickens were found positive for H. pullorum. This study represents that chickens seem to be a source of H. pullorum infection and serve as reservoirs for the H. pullorum. [ABSTRACT FROM AUTHOR]

The presence of Helicobacter pullorum in intestinal tract of broiler chickens may be a potential risk for human health. In this study, a total of 100 caecal samples of broilers carcasses from 20 flocks at a poultry abattoir in Mashhad suburb were tested for the presence of H. pullorum using modified conventional culture method by combination of culturing on Brucella sheep blood agar and a filtering technique. Suspected colonies were determined as H. pullorum using polymerase chain reaction (PCR) by amplifying a 447 bp fragment of the 16SrRNA gene of this bacteria. 41% of caecal content samples and samples from 12 broiler flocks (60%) were determined as positive for the presence of H. pullorum. This is the first report of H. pullorum in Iranian poultry flocks. The results showed high prevalence of this bacterium in broiler chickens in this area of Iran. It seems using combination of conventional culture method and PCR assay based on amplification from conserved genes allows reliable detection and identification of H. pullorum. [ABSTRACT FROM AUTHOR]

Helicobacter pullorum, a bacterium initially isolated from poultry, has been associated with human digestive disorders. However, the factor responsible for its cytopathogenic effects on epithelial cells has not been formally identified. The cytopathogenic alterations induced by several human and avian H. pullorum strains were investigated on human intestinal epithelial cell lines. Moreover, the effects of the cytolethal distending toxin (CDT) were evaluated first by using a wild-type strain and its corresponding cdtB isogenic mutant and second by delivering the active CdtB subunit of the CDT directly into the cells. All of the H. pullorum strains induced cellular distending phenotype, actin cytoskeleton remodeling, and G2/M cell cycle arrest. These effects were dependent on the CDT, as they were (1) not observed in response to a cdtB isogenic mutant strain and (2) present in cells expressing CdtB. CdtB also induced an atypical delocalization of vinculin from focal adhesions to the perinuclear region, formation of cortical actin-rich large lamellipodia with an upregulation of cortactin, and decreased cellular adherence. In conclusion, the CDT of H. pullorum is responsible for major cytopathogenic effects in vitro, confirming its role as a main virulence factor of this emerging human pathogen. [ABSTRACT FROM AUTHOR]

Helicobacter pullorum (H. pullorum) is a bacterium that colonizes the intestines of poultry and causes gastroenteritis. Because these species are known as human and/or animal pathogens, identification of H. pullorum is becoming increasingly necessary. The bacterium has been linked to colitis and hepatitis in humans after being transmitted by infected meat consumption. Misdiagnosis of other enteric zoonotic pathogens such as Campylobacter and other Helicobacter species makes the diagnosis of H. pullorum extremely difficult. This study focused on the molecular detection of H. pullorum from the stomach (proventriculus and gizzard) of different avian species as new target organs for detection and transmission between avian species. Proventriculus and gizzards were obtained from 40 freshly dead chickens and resident wild birds (n=40). Diarrhea was found in the farms that were surveyed. DNA was extracted from all collected samples to conduct PCR amplification. The samples were screened for Helicobacter genus-specific 16s using C97 and C05 primers. To confirm the existence of H. pullorum, the positive samples were sequenced. H. pullorum was recorded in two out of 40 chicken samples. In addition, H. pullorum was recorded in one out of 40 resident wild birds. The 16S rRNA gene sequence for Helicobacter genus-specific in poultry and wild birds showed a 100% homology. In conclusion, broiler chickens and resident wild birds are possible reservoirs for H. pullorum, according to this report, and possibly act as a source of infection for humans via the food supply., (Copyright© by the Polish Academy of Sciences.)

Background Helicobacter pullorum is a putative enterohepatic pathogen that has been associated with hepatobiliary and gastrointestinal diseases in chickens and in humans. The pathogenic potential of H. pullorum NCTC 12826 was investigated. Methods Adherence and gentamicin protection assays and scanning electron microscopy were performed to quantitate and visualise H. pullorum adherence and invasion. Proteomics coupled with mass spectrometry was employed to characterise the secretome of H. pullorum. Results Helicobacter pullorum was able to adhere to the Caco-2 intestinal epithelial cell line with a mean attachment value of 1.98 ± 0.16% and invade Caco-2 cells with a mean invasion value of 0.25 ± 0.02%. The in vitro adherence and invasion assays were confirmed with scanning electron microscopy, which showed that H. pullorum can adhere to host cells through flagellum-microvillus interaction and invade causing a membrane-ruffling effect. One hundred and thirty-seven proteins were identified, of which 33 were bioinformatically predicted to be secreted. Further functional classifications revealed six putative virulence and colonisation factors, which included cell-binding factor 2, flagellin, secreted protein Hcp, valine-glycine repeat protein G, a type VI secretion protein, and a protease. Protein threading of H. pullorum Hcp and subsequent 3 D- Blast searches revealed structural similarities between Hcp and endocytic vesicle coat proteins, suggesting the type VI secretion system of H. pullorum may interact with endocytic vesicles. Conclusions This study has shown that H. pullorum has the ability to adhere to and invade human cells and secrete factors that may contribute to the pathogenic potential of H. pullorum. [ABSTRACT FROM AUTHOR]

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The objective of the study was to investigate the occurrence of H. pullorum in chicken hens' eggs and to determine their in vitro susceptibility to various antimicrobial agents. A total of 300 fresh commercial chicken eggs of Balady native breed and poultry farms (PF) were collected from both Assiut and Qena Governorates in Egypt. Every five eggs represented one sample (mixed contents of five eggs). The eggshell and eggshell contents were examined for the presence of Helicobacter species by conventional methods, next confirmed with PCR. The highest incidence of contamination with Helicobacter species was recorded in eggs from Assiut Governorate poultry farms. The obtained results of Helicobacter pullorum (H. pullorum) isolation revealed that poultry farms egg shells and contents were less contaminated in both Governorates than Balady ones. Additionally, fresh egg contents of poultry farms in both Governorates were free from H. pullorum. All 12 isolates that were conventionally classified as carrying H. pullorum occurred free from the organism when tested with PCR method by detection of one PCR product on agarose gel that matched the predicted size of 477 bp that corresponding to 16S rRNA region of the gene. H. pullorum show in vitro susceptibility to almost all tested types of antibiotics except for ampicillin, ceftriaxone, and sulphamethoxazole trimethoprim. [ABSTRACT FROM AUTHOR]

Abstract: In order to investigate the occurrence of Helicobacter pullorum in turkeys, caecum contents collected at the slaughterhouse from 55 animals intensively reared in 11 farms were sampled. Gram-negative curved rod bacteria were isolated by a modified Steele and McDermott filter technique and further identified as H. pullorum by polymerase chain reaction (PCR). Eleven and 31 isolates, randomly selected from each positive farm, underwent phenotypic (biochemical and antibiotic susceptibility tests) and genotypic characterization (PFGE and AFLP analysis), respectively. Forty-two out of 55 animals (76.4%) and all the 11 farms sampled were positive for H. pullorum. Isolates showed similar biochemical characteristics and whole cell protein profiles but showed a high degree of genetic heterogeneity. Ten out of 11 isolates were resistant to one or more antibiotics with erythromycin, ciprofloxacin and nalidixic acid resistance being the most frequently detected. This is the first description of H. pullorum in turkeys. H. pullorum is a frequent intestinal colonizer in turkeys; therefore, attention should be given to clarify the food-borne risk linked to carcass contamination. Antibiotic resistance is a concern since high values of resistance rates were observed. [Copyright &y& Elsevier]

A novel real-time PCR assay was developed for the direct detection in food of Helicobacter pullorum-like bacteria, which are occasionally associated with human enteric disease. Experiments using control strains showed that the real-time PCR assay was specific and reproducible, with a detection level of 1 colony-forming unit (CFU)/g. The assay was then applied to determine contamination rates in 30 samples of three types of chicken-meat products obtained from five retail outlets in Spain (Valencia); all of the samples were initially considered to be culture-negative for Helicobacter even after an enrichment period. H. pullorum-like DNA was detected in seven out of ten chicken carcasses and in one chicken-burger sample (without enrichment), as well as in one liver sample (after enrichment). Sequencing of three randomly selected PCR products confirmed concordance (99% homology) with the H. pullorum 16S rDNA gene. The advantages of real-time PCR over conventional PCR assays are the improved detection level, speed of testing, and validation of specificity by melting-point analysis. The fact that bacteria are frequently present in chicken carcasses sold in retail stores highlights the importance of more widely monitoring contamination rates. The novel assay described herein allows better assessment of potential human health risks posed by H. pullorum. [ABSTRACT FROM AUTHOR]

Abstract: To assess the molecular basis of nalidixic acid and ciprofloxacin resistance in Helicobacter pullorum, the gyrA gene of H. pullorum CIP 104787T was sequenced. In addition, 9 isolates (2 susceptible to ciprofloxacin and resistant to nalidixic acid, 3 susceptible and 4 resistant to both antibiotics) were selected from 44 poultry isolates and the nucleotide sequences of their quinolone resistance-determining regions (QRDRs) were compared. The 2490bp gyrA gene showed an open reading frame encoding a polypeptide of 829 amino acids. The deduced amino acid sequence of gyrA showed ≥72% identity to Helicobacter hepaticus, Helicobacter pylori and Wolinella succinogenes. Moreover, ≥98% amino acid sequence identity was found comparing the QRDR of the H. pullorum type strain with the QRDRs of the aforementioned bacterial species. All ciprofloxacin-resistant poultry isolates showed an ACA→ATA (Thr→Ile) substitution at codon 84 of gyrA, corresponding to codons 86, 87 and 83 of Campylobacter jejuni, H. pylori and Escherichia coli gyrA genes, respectively. This substitution was functionally confirmed to be associated with the ciprofloxacin-resistant phenotype of poultry isolates. This is the first report describing the complete 2490bp nucleotide sequence of H. pullorum gyrA and confirming the involvement of the Thr84Ile substitution of GyrA in ciprofloxacin resistance of H. pullorum. [Copyright &y& Elsevier]

Abstract: Four groups of 23 one-day-old broiler chickens were each inoculated by gavage with a different Helicobacter pullorum strain isolated from humans or poultry. As a control, a fifth group of eight animals was inoculated with phosphate-buffered saline. Faecal samples were collected weekly and tested for the presence of H. pullorum DNA using PCR. At 1, 8, 15, 22 and 42 days postinoculation, birds were euthanized and samples from the liver and intestinal tract were histologically, immunohistochemically and bacteriologically examined. The samples were also tested for the presence of H. pullorum DNA by PCR. All animals remained clinically healthy throughout the experiment although mild lesions in the caeca were present in animals inoculated with H. pullorum. In all H. pullorum-inoculated groups, DNA of this bacterium was detected in faecal samples until 42 days postinoculation. The main site of colonization was the caecum. Immunohistochemical examination revealed that the bacterium was closely associated with the caecal epithelial cells. It was concluded that H. pullorum may colonize the caecum of broilers and is excreted in their faeces until slaughter age. This implies that chicken meat might constitute a source of infection for human beings. [Copyright &y& Elsevier]

Abstract: In 2005, in order to investigate the occurrence of Helicobacter pullorum in poultry, the caecal contents collected from a total of 60 animals intensively reared in Italy on 15 different farms (9 farms of broiler chicken and 6 of laying hens) were examined at the slaughterhouse. A modified Steele–McDermott membrane filter method was used. Small, greyish-white colonies of Gram-negative, gently curved, slender rod bacteria were preliminarily identified as H. pullorum by a Polymerase Chain Reaction (PCR) assay based on 16S rRNA and were then subjected to an ApaLI digestion assay to distinguish H. pullorum from Helicobacter canadensis. One isolate from each farm was phenotypically characterized by biochemical methods and 1D SDS-PAGE analysis of whole cell proteins; antibiotic susceptibility was also tested. According to the PCR and PCR-RFLP results, all the animals examined were positive for H. pullorum. The 1D SDS-PAGE whole protein profile analysis showed high similarity among the 15 isolates tested. A monomodal distribution for the Minimum Inhibitory Concentrations (MICs) was found for ampicillin, chloramphenicol, gentamicin and tetracycline. For erythromycin and ciprofloxacin, a bimodal trend having a second peak at >128 μg ml−1 and 32 μg ml−1 was found. The isolation method used in this study seems to be highly suitable for isolating H. pullorum from chicken caecal contents. Moreover, the detection of a high number of colonies phenotypically similar to H. pullorum suggests that this microorganism, when present, colonizes the caecum at high concentration. [Copyright &y& Elsevier]

Abstract: Helicobacter pullorum has been associated with diarrhoea, gastroenteritis and liver disease in humans and with hepatitis and enteritis in poultry. The purpose of the present study was to examine whether cytolethal distending toxin was present among 10 poultry and three human H. pullorum isolates and whether a different level of cytolethal distending toxin activity was noted. A PCR assay was performed to detect the cdtB gene. In addition, epithelial Hep-2 cells inoculated with sonicate from all strains were observed microscopically and DNA analysis of these cells was done by flow cytometry. All H. pullorum isolates harboured the cdtB gene, but functional cytolethal distending toxin activity was only demonstrated in the human H. pullorum strain CCUG 33839. A significant number of cells treated with sonicate from this strain were enlarged. The nuclei were distended proportionally. Giant cells and multinucleated cells were observed as well. In addition, stress fibers accumulated. DNA analysis by flow cytometry revealed 31.0% of these cells at the S/G2 stage of the cell cycle. The tested poultry and human H. pullorum isolates all possess the cdtB gene, but under the circumstances adopted in this study only the human strain CCUG 33839 seems to show biological activity typically for CDT in vitro. [Copyright &y& Elsevier]

Abstract Aims: To validate a phenotypic Campylobacter species identification method employed to identify campylobacters in broilers by comparison with campylobacterial species identification using various species-specific PCR analyses. Methods and Results: From a collection of 2733 phenotypically identified campylobacterial cultures, 108 Campylobacter jejuni cultures and 351 campylobacterial cultures other than Camp. jejuni were subjected to various species-specific PCR assays. On the basis of the genotypic tests, it was demonstrated that Camp. jejuni and Camp. coli constituted approx. 99% of all cultures, while other species identified were Helicobacter pullorum , Camp. lari and Camp. upsaliensis . However, 29% of the 309 Camp. coli cultures identified by phenotypic tests were hippurate-variable or negative Camp. jejuni cultures, whereas some Camp. lari cultures and unspeciated campylobacter cultures belonged to H. pullorum . It was also notable that 2–6% of the cultures were, in fact, mixed cultures. Conclusions: The phenotypic identification scheme employed failed to appropriately differentiate Campylobacter species and particularly to identify the closely related species, H. pullorum . Significance and Impact of the Study: Future phenotypic test schemes should be designed to allow a more accurate differentiation of Campylobacter and related species. Preferably, the phenotypic tests should be supplemented with a genotypic strategy to disclose the true campylobacterial species diversity in broilers. [ABSTRACT FROM AUTHOR]

For evaluation of pet birds as a resource of Helicobacter pullorum, 100 cecal content samples were collected from 50 finches and 50 Australian parrots, with no clinical signs suggestive of systemic and/or enteric disease, in Iran. DNA was extracted from cecal samples and PCR was carry out for identification of H. pullorum 16S rRNA gene. Results showed, H. pullorum DNA was detected in 3 of the 50 (6%) cecal samples of finch and 6 out of 50 (12%) cecal samples of Australian parrot. This study represents that finch and Australian parrot can be a source of H. pullorum infection and serve as reservoirs for the H. pullorum. [ABSTRACT FROM AUTHOR]

N-acetylated sugars are often found, for example, on the lipopolysaccharides of Gram-negative bacteria, on the S-layers of Gram-positive bacteria, and on the capsular polysaccharides. Key enzymes involved in their biosynthesis are the sugar N-acetyltransferases. Here, we describe a structural and functional analysis of one such enzyme from Helicobacter pullorum, an emerging pathogen that may be associated with gastroenteritis and gallbladder and liver diseases. For this analysis, the gene BA919-RS02330 putatively encoding an N-acetyltransferase was cloned, and the corresponding protein was expressed and purified. A kinetic analysis demonstrated that the enzyme utilizes dTDP-3-amino-3,6-dideoxy-d-glucose as a substrate as well as dTDP-3-amino-3,6-dideoxy-d-galactose, albeit at a reduced rate. In addition to this kinetic analysis, a similar enzyme from Helicobacter bilis was cloned and expressed, and its kinetic parameters were determined. Seven X-ray crystallographic structures of various complexes of the H. pullorum wild-type enzyme (or the C80T variant) were determined to resolutions of 1.7 Å or higher. The overall molecular architecture of the H. pullorum N-acetyltransferase places it into the Class II left-handed-β-helix superfamily (LβH). Taken together, the data presented herein suggest that 3-acetamido-3,6-dideoxy-d-glucose (or the galactose derivative) is found on either the H. pullorum O-antigen or in another of its complex glycoconjugates. A BLAST search suggests that more than 50 non-pylori Helicobacter spp. have genes encoding N-acetyltransferases. Given that there is little information concerning the complex glycans in non-pylori Helicobacter spp. and considering their zoonotic potential, our results provide new biochemical insight into these pathogens., (© 2021 The Protein Society.)