Core Genome Haplotype Diversity and <Emphasis Type="Italic">vacA</Emphasis> Allelic Heterogeneity of Chinese <Emphasis Type="Italic">Helicobacter pylori</Emphasis> Strains

The human gastric pathogen, Helicobacter pylori, has co-evolved with its host and established itself in the human stomach possibly millions of years ago. Therefore, the diversity of this bacterium is important in its clinical manifestations. Our aim has been to evaluate the genetic diversity of 40 H. pylori clinical isolates from four different parts of China. The methods of multi-locus sequence typing and vacA allele genotyping were used to assess their genetic diversity. To discriminate MLST, the vacA genotype method was used to identify strains. Patients from the northern, eastern, southern, and southwestern parts of China were recruited randomly from the cities of Beijing, Shanghai, Guangzhou, and Chongqing, respectively. Most of the sequence types are new and have never been reported in the database of the H. pylori multi-locus sequence typing system. The most prevalent vacA genotype in patients was s1a/m2 (80.0%), followed by s1b/m2 (17.5%). In contrast, the s1a/m1 genotype was scarcely represented (2.5%). The vacA genotype varied for each ST. These results showed that the MLST method offers high resolution of the H. pylori isolates in China when compared to vacA genotyping. The vacA allelic s1a has been correlated with the peptic ulcer. Because of the paucity of data on human isolates due to the absence of systematic investigations of H. pylori in China, the data provide useful information for understanding the epidemiology of H. pylori in China from the viewpoint of nucleotide sequence databases.     

Clinical relevance of cagL gene and virulence genotypes with disease outcomes in a Helicobacter pylori infected population from Iran

Helicobacter pylori infection is common in Iran as in other developing countries. Certain genotypes of H. pylori have been associated with increased occurrence of chronic gastritis, peptic ulcers, and gastric adenocarcinoma. The aim of this study was to investigate the clinical relevance of cagL gene and other virulence genotypes of H. pylori isolates with clinical outcomes in Iranian patients. Totally, 126 symptomatic patients who underwent gastroduodenal endoscopy were enrolled in the study. Sixty-one H. pylori strains were isolated from the patients studied. The presence of the cagL, cagA, vacA, iceA, babA2 and sabA genes in the corresponding H. pylori isolates were determined by polymerase chain reaction and the results were compared with clinical outcomes and histopathology. The cagL, cagA, vacA s1, vacA s2, vacA m1, vacA m2, iceA1, iceA2, babA ₂ , and sabA genotypes were detected in 96.7, 85.2, 75.4, 24.6, 29.5, 70.5, 42.6, 23, 96.7, and 83.6 % of the isolates, respectively. The three genotypic combinations, cagL/cagA/vacAs1m1/iceA1/babA2/sabA, cagL/cagA/vacAs1m2/iceA1/babA2/sabA, and cagL/cagA/vacAs1m2/iceA2/babA2/sabA were determined as the most prevalent combined genotypes. There was a significant correlation between the presence of cagL gene and cagA positivity (P = 0.02). No significant correlation was found between the various genotypes and clinical outcomes (P > 0.05). The present study showed a very high prevalence of cagL genotype among the H. pylori isolates from Iranian patients. Our results demonstrated that neither single genotype nor combination genotypes of virulence-associated genes was significantly helpful markers for predicting the severity of gastroduodenal disease associated with H. pylori infection in Iranian patients.     

Benefits of <Emphasis Type="Italic">Helicobacter pylori</Emphasis><Emphasis Type="Italic">cagE</Emphasis> genotyping in addition to <Emphasis Type="Italic">cagA</Emphasis> genotyping: a Bulgarian study

Associations of Helicobacter pylori cagE status with complex patient characteristics remain to be elucidated in Eastern Europe. The aim of this study was to assess the frequencies of cagE gene and cagA/cagE combinations in H. pylori strains from symptomatic Bulgarian patients and to improve cagA detection. cagA and cagE genotypes were evaluated in 219 patients with single-strain infections. In total, 84.9% of strains were cagA ⁺, while 68.5% were cagE ⁺. cagA ⁺, cagE ⁺, and cagA ⁺/cagE ⁺ strains were more prevalent in peptic ulcer (93.8%, 84.4%, and 84.4%) compared with nonulcer patients (81.3%, 61.9%, and 61.3%, respectively). In elderly patients, cagE ⁺ and cagA ⁺/cagE ⁺ strains were 1.9-fold more common than in the 12 children evaluated. Only 10% of the elderly subjects harbored low-virulence cagA ⁺/cagE ⁻ strains compared with 16.8% of adults and 41.7% of children. Intriguingly, prevalence of the cagA ⁺/cagE ⁻ genotype was 2.1-fold lower in men than in women, suggesting a higher frequency of more virulent strains in men. The presence of both cag genes and combinations was not linked to strain susceptibility to clarithromycin or metronidazole, place of residence, or prior therapy. Use of an extra primer pair increased cagA detection in 14.7% of 31 cagA ⁻ strains. In conclusion, use of a second primer pair for the cagA gene can be recommended in countries with common cagA ⁺ strains. Although both cag genes were linked to severe diseases in Bulgarian patients, the best discrimination of virulent strains was obtained by the cagA/cagE combination or by the cagE gene alone. cagE prevalence increased gradually with patient age, while the cagA ⁺/cagE ⁻ genotype, implying a disrupted cag pathogenicity island, was associated with both younger age and female gender.     

Prevalence of the Helicobacter pylori babA2 gene and correlation with the degree of gastritis in infected Slovenian children

The aims of our study were to determine the prevalence of the babA2 gene within Helicobacter pylori strains circulating in the Slovenian pediatric population, to further clarify its significance in causing inflammation of gastric mucosa in children and to verify whether cagA, vacA, iceA and babA genes work independently or synergistically in causing gastritis. A total of 163 H. pylori isolates obtained from the same number of children were tested for the presence of cagA, vacA and iceA genes using previously established methods, while the babA2 gene was determined using novel polymerase chain reaction assay targeting a 139-bp fragment of the central region of babA2. The babA2 gene was detected in 47.9 % of H. pylori samples. The presence of the babA2 gene was strongly associated with cagA, vacA s1 and vacA m1 genotype. The babA2 status correlated positively with bacterial density score, activity of inflammation and chronic inflammation of gastric mucosa. No significant correlation was found between the babA2 status and the presence of atrophy or intestinal metaplasia. In addition, the activity of gastric inflammation and density score were significantly associated with the coexpression of the cagA, vacA s1, vacA m1 and babA2 genes. The study, which included the largest number of pediatric H. pylori samples to date, confirmed that babA2 gene plays an important role in the pathogenesis of H. pylori gastritis in children. Furthermore, our results suggest that babA2, cagA and vacA s1 and m1 gene products may work synergistically in worsening the inflammation of gastric mucosa.     

Association between <Emphasis Type="Italic">cagA</Emphasis>, <Emphasis Type="Italic">vacAi</Emphasis>, and <Emphasis Type="Italic">dupA</Emphasis> genes of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> and gastroduodenal pathologies in Chilean patients

In addition to the already known cagA gene, novel genetic markers have been associated with Helicobacter pylori (H. pylori) virulence: the dupA and vacAi genes. These genes might play an important role as specific markers to determine the clinical outcome of the disease, especially the vacAi gene, which has been expected to be a good marker of severe pathologies like gastric adenocarcinoma. In the present study, the association of cagA, dupA, and vacAi genes with gastroduodenal pathologies in Chilean patients was studied. One hundred and thirty-two patients positive for H. pylori were divided into two groups—non-severe and severe gastric pathologies—and investigated for the presence of cagA, dupA, and vacAi H. pylori virulence genes by PCR. The cagA gene was detected in 20/132 patients (15.2%), the vacAi1 gene was detected in 54/132 patients (40.9%), the vacAi2 gene was detected in 26/132 patients (19.7%), and the dupA gene was detected in 50/132 (37.9%) patients. Logistic regression model analysis showed that the vacAi1 isoform gene in the infected strains and the severity of the diseases outcome were highly associated, causing severe gastric damage that may lead to gastric cancer (p < 0.0001; OR = 8.75; 95% CI 3.54–21.64). Conversely, cagA (p = 0.3507; OR = 1.62; 95% CI 0.59–4.45) and vacAi2 (p = 0.0114; OR = 3.09; 95% CI 1.26–7.60) genes were not associated with damage, while the dupA gene was associated significantly with non-severe clinical outcome (p = 0.0032; OR = 0.25; 95% CI 0.09–0.65). In addition, dupA gene exerts protection against severe gastric pathologies induced by vacAi1 by delaying the outcome of the disease by approximately 20 years.     

Helicobacter pylori in a poultry slaughterhouse: Prevalence,genotyping and antibiotic resistance pattern

Although Helicobacter pylori (H. pylori) is a highly significant pathogen, its source remains unclear. Many people consume chicken daily as a source of animal protein worldwide; thus, hygienic methods of supplying chickens for consumption are critical for public health. Therefore, our study examined the distribution of the glmM (ureC), babA2, vacA and cagA virulence genes in H. pylori strains in chicken meat and giblets (gizzards and livers) and the resistance of the strains to various antibiotics. Ninety chicken meat, gizzard and liver samples were obtained from a semi-automatic abattoir in Sadat City, Egypt, and were cultured and preliminarily analyzed using biochemical tests. The presence of the ureC, babA2, vacA and cagA genotypes was tested for in samples positive for H. pylori by multiplex polymerase chain reaction (Multiplex-PCR). The resistance of H. pylori to various antimicrobial drugs was tested using the disc diffusion method. In total, 7 of the 90 chicken samples were positive for H. pylori (7.78%); in 3/7 (42.85%) samples, the bacteria were found in the chicken liver, while the bacteria were found in the meat in 2/7 (28.57%) and in the gizzard in 2/7 (28.57%) samples. The total prevalence of both the ureC and babA2 genes in the isolated H. pylori strains was 100%, while the prevalence of the vacA and cagA genes was 57.1% and 42.9%, respectively. The resistance of H. pylori to the antibiotics utilized in our study was 100% for streptomycin; 85.7% for amoxicillin and penicillin; 71.4% for oxytetracycline, nalidixic acid and ampicillin; 57.1% for sulfamethoxazole and erythromycin; and 42.9% for neomycin, chloramphenicol and norfloxacin. In conclusion, the chicken meat and giblets were tainted by H. pylori, with a higher occurrence of the ureC, babA2, vacA and cagA genotypes. Future investigations should investigate the resistance of H. pylori to various antimicrobial agents in Egypt.     

The <Emphasis Type="Italic">GPX1</Emphasis> Pro<Superscript>198</Superscript>Leu polymorphism in gastric cancer patients with and without <Emphasis Type="Italic">Helicobacter pylori</Emphasis> infection

Helicobacter pylori infection could induce oxidative stress. Oxidative stress is involved in the pathogenesis of gastric diseases. Glutathione peroxidase 1 (GPX1), is part of the enzymatic antioxidant defense, preventing oxidative damage. The aim of the present study was to assess the association of GPX1 Pro¹⁹⁸Leu genotypes with gastric cancer in patients with and without H. pylori infection in a population of Northern Iran. The present case-control study consisted of 50 patients with gastric cancer and 78 cancer-free subjects as controls. Extraction of DNA was performed on bioptic samples and the GPX1 genotypes were identified using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The frequencies of GPX1 Pro/Pro, Pro/Leu and Leu/Leu genotypes in controls were 21.8, 71.8 and 6.4%, respectively. However, in gastric cancer patients, the frequencies of 34, 56 and 10% were observed for Pro/Pro, Pro/Leu and Leu/Leu genotypes, respectively (p?=?0.185). In 38 (76%) patients infected with H. pylori, the frequencies of Pro and Leu alleles were 94.7 and 3.3%, respectively. There was a higher frequency of combined genotype of Pro/Leu?+?Leu/Leu (94.7%) in H. pylori positive patients than that in patients without H. pylori infection (75%, p?=?0.047). The presence of this genotype tended to increase the risk of H. pylori related gastric cancer by 5.88–fold (p?=?0.069) in our population. Our findings indicated the absence of association between the GPX1 Pro¹⁹⁸Leu polymorphism and the risk of gastric cancer in an Iranian population. However, we detected an association between H. pylori related gastric cancer with GPX1 Pro/Leu?+?Leu/Leu genotype.     

An Epidemiologic Study on the Correlation Between Oral <Emphasis Type="Italic">Helicobacter pylori</Emphasis> and Gastric <Emphasis Type="Italic">H. pylori</Emphasis>

The current study was conducted to determine the prevalence of Helicobacter pylori in the oral cavity of a Chinese population. Nested polymerase chain reaction (PCR) was used to investigate whether individuals with oral H. pylori show more possibility of gastric infection and to examine the relationship between gastric H. pylori infection and the presence of the bacteria in the oral cavity. A total of 443 dyspeptic patients participated in the study. Gastric H. pylori infection was detected by the rapid urease test and histology with Giemsa staining, PCR, or smear examination, whereas the presence of the bacteria in the oral cavity was observed by nested PCR from dental plaque. Relevant periodontal and dental parameters were recorded in the process of oral examination. Of the 443 study patients, oral H. pylori was found in the dental plaque of 263 (59.4%) and the stomach of 273 (61.6%). Additionally, in all four age groups, the prevalence of gastric infection was significantly higher among the patients with positive tests for H. pylori in their dental plaque than in the patients with no H. pylori in their dental plaque (P < 0.05). The oral cavity may be a potential reservoir for H. pylori, and the prevalence of oral H. pylori approximated that of gastric H. pylori in the studied population. Furthermore, a close relationship may exist between H. pylori in the oral cavity and bacteria in the stomach or gastric infection, and dyspeptic patients with gastric infection are more likely to harbor H. pylori in their oral cavity. Y. Liu and H. Yue contributed equally to the accomplishment of this study.     

<Emphasis Type="Italic">Helicobacter pylori</Emphasis> isolated from patients with tonsillar cancer or tonsillitis chronica could be of different genotype compared to isolates from gastrointestinal tract

Helicobacter pylori from patients with different diseases, including so-called autoimmune thyroiditis, chronic tonsillitis and tonsillar cancer, was isolated and cultured. It was identified according to the genotype using labeled hybridization probes complementary to six sequences ofcagA andvacA genes. Different types of strains were found in isolates from gastrointestinal tract and patients suffering from thyroiditis. Six out of seven genotyped isolates from patients in ourDepartment of Otorhinolaryngology and Head and Neck Surgery exhibited the same genotype, differing from isolates obtained from other patients; the 7th isolate originated from a patient who had undergone surgery for deviatio septi nasi, at the same time suffering from autoimmune thyroiditis, having confirmed gastric infection byH. pylori from biopsy. This data made it possible to formulate the hypothesis on probable association of specificH. pylori genotype with chronic tonsillitis and tonsillar cancer. We assessed commercial transport media and improved nucleic acid isolation techniques and the RT-PCR-based tests, which allowed us to skip a culture step and to test directly the patients’ samples; however, for ful confirmation of our hypothesis and explanation of possible mechanisms of the contribution ofHelicobacter sp. to the pathogenesis of the disease further data are to be collected and evaluated. The study was supported by grants 8115-3 and 9077-3 of theInternal Grant Agency of the Ministry of Health of the Czech Republic and VZ-MSM of theMinistry of Education, Youth and Sports of the Czech Republic.     

Association of Malaysian Helicobacter pylori Virulence Polymorphisms with Severity of Gastritis and Patients' Ethnicity

Background and aim: Polymorphisms of Helicobacter pylori cagA and vacA genes do exist and may contribute to differences in H. pylori infection and gastroduodenal diseases among races in the Malaysian population. This study was conducted to characterize the polymorphisms in H. pylori cagA and vacA in Malaysian population. Methods: A total of 110 H. pylori isolates were genotyped by PCR and sequenced for cagA and PCR‐RFLP for vacA. Results: East Asian cagA was predominantly detected (64.5%), whereas vacA s1m1 and s1m2 alleles were detected in 60.9 and 37.3% of strains, respectively. A statistical association between cagA type with patients’ ethnicity (p < .0001) and age group >50 years old (p = .027) was identified. vacA alleles showed significant association with age group >50 years old (p = .017) and increased neutrophil activity in gastric mucosa (p = .028 and p = .016 for moderate and marked activity, respectively). Further identification of vacA polymorphism revealed that 84% of strains from Malays and Indians showed one RFLP pattern (RFLP‐1), whereas more than one RFLP patterns (RFLP‐2, 3, 4, 5, 6, and 8) were predominantly observed in strains from Chinese (82%) (p < .0001). Increasing severity of gastric inflammation was observed in gastric mucosa infected with strains carrying RFLP‐2, 3, 4, 5, and 6 (p = .037). About 86.6% of H. pylori strains with East Asian cagA were vacA RFLP‐2, 3, 4, 5, 6, and 8, and 88% of Western cagA strains were vacA RFLP‐1 (p < .0001). Chinese and Indians are susceptible to different virulence genotypes of H. pylori, whereas Malays showed a mixed virulence genotypes. Conclusion: Marked differences in the polymorphisms of cagA and vacA were observed among strains in Malaysian population. This provides a new insight into the pathogenicity of H. pylori in multiracial population.     

A comparative whole genome analysis of Helicobacter pylori from a human dense South Asian setting

Helicobacter pylori, a Gram-negative bacterium, is associated with a wide range of gastric diseases such as gastritis, duodenal ulcer, and gastric cancer. The prevalence of H pylori and risk of disease vary in different parts of the world based on the prevailing bacterial lineage. Here, we present a contextual and comparative genomics analysis of 20 clinical isolates of H pylori from patients in Bangladesh. Despite a uniform host ethnicity (Bengali), isolates were classified as being part of the HpAsia2 (50%) or HpEurope (50%) population. Out of twenty isolates, eighteen isolates were cagA positive, with two HpEurope isolates being cagA negative, three EPIYA motif patterns (AB, AB-C, and ABC-C) were observed among the cagA-positive isolates. Three vacA genotypes were observed with the s1m1i1dic1 genotype observed in 75% of isolates; the s1m2i1d1c2 and s2m2i2d2c2 genotypes were found to be 15% and 10% of isolates, respectively. The non-virulent genotypes s2m2i2d2c2 was only observed in HpEurope population isolates. Genotypic analysis of oipA gene, present in all isolates, revealed five different patterns of the CT repeat; all HpAsia2 isolates were in “ON” while 20% of HpEurope isolates were genotypically “OFF.” The three blood group antigen binding adhesins encoded genes (bab genes) examined and we observed that the most common genotype was (babA/babB/-) found in eight isolates, notably six were HpAsia2 isolates. The babA gene was found in all HpAsia2 isolates but present in only half of the HpEurope isolates. In silico antibiotic susceptibility analysis revealed that 40% of the strains were multi-drug resistant. Mutations associated with resistance to metronidazole, fluoroquinolone, and clarithromycin were detected 90%, 45%, and 5%, respectively, in H pylori strain. In conclusion, it is evident that two populations of H pylori with similar antibiotic profiles are predominant in Bangladesh, and it appears that genotypically the HpAisa2 isolates are potentially more virulent than the HpEurope isolates.     

<Emphasis Type="Italic">Braf</Emphasis>, <Emphasis Type="Italic">Kras</Emphasis> and <Emphasis Type="Italic">Helicobacter pylori</Emphasis> epigenetic changes-associated chronic gastritis in Egyptian patients with and without gastric cancer

We aimed to study MLH1 and MGMT methylation status in Helicobacter pylori-associated chronic gastritis in Egyptian patients with and without gastric cancer. 39 patients were included in our study. They were divided into 2 groups; patients without (group I) and with gastric adenocarcinoma (group II). Patients were subjected to clinical examination, abdominal ultrasound and upper endoscopy for gastric biopsy. Biopsies were subjected to urease test, histological examination, and DNA purification. H. pylori, Braf, Kras, MLH1 and MGMT methylation were assessed by quantitative PCR. DNA sequencing was performed to assess Braf and Kras genes mutation. qPCR of H. pylori was significantly higher in patients with adenocarcinoma (group II) than those without adenocarcinoma (group I); with a p < 0.001 as well as in patients with age above 50 years with a p value = 0.008. By applying logistic regression analysis it was reported that the H. pylori qPCR is a significant predictor to the adenocarcinoma with OR = 1.025 (95 % CI: 1. 002–1.048), with sensitivity of 90 % and specificity of 100 %. Adenocarcinoma patients had a significantly higher mean age and levels of H. Pylori, Braf, K-ras, methylated MGMT and methylated MLH1 than those of gastritis patients. DNA sequence analysis of Braf (codon 12) and Kras (codon 600) had genes mutation in gastric adenocarcinoma versus chronic gastritis. Conclusion: H. pylori may cause epigenetic changes predisposing the patients to cancer stomach. Estimation of H. pylori by qPCR can be a good predictor to adenocarcinoma. Braf and Kras genes mutation were reveled in gastritis and adenocarcinoma patients.     

Association between <Emphasis Type="Italic">GSTM1</Emphasis>, <Emphasis Type="Italic">GSTT1</Emphasis>, and <Emphasis Type="Italic">GSTP1</Emphasis> polymorphisms and lung cancer risk in a Turkish population

Several studies focused on investigating genetic polymorphisms in order to estimate genetic contribution to lung cancer often showed conflicting results. In this study, we investigated the role of GSTM1, GSTT1, GSTP1 exon 5 and exon 6 polymorphisms on developing lung cancer and histological subtypes in 213 lung cancer patients and 231 controls. GSTM1 null, GSTT1 null, and GSTP1 exon 5 variant genotypes did not show a significant risk for developing lung cancer overall. Significant association was noted between GSTP1 exon 6 variant genotypes and overall lung cancer risk (OR 2.17, 95% CI 1.25–3.78; P = 0.006). These results show that GSTP1 exon 6 polymorphism might be an important factor in determining lung cancer susceptibility in a Turkish population.     

Phenotype changes inherited by crossing pyrethroid susceptible and resistant genotypes from the cattle tick <Emphasis Type="Italic">Riphicephalus</Emphasis> (<Emphasis Type="Italic">Boophilus</Emphasis>) <Emphasis Type="Italic">microplus</Emphasis>

Dialelic crosses and backcrosses of pyrethroid resistant (RR) and susceptible (SS) Rhipicephalus (Boophilus) microplus tick strains were carried out and the substitution (Phe-Ile) within the sodium channel gene was monitored in order to analyze the effects of the genotype on the pyrethroid resistance phenotype as measured by the larval packet test (LPT). Parental strains: susceptible (SS) and resistant (RR); dialelic crosses: RS (♂RR × ♀SS), and SR (♂SS × ♀RR); and backcrosses: RS × SS, RS × RR, SR × SS and SR × RR were infested on 280 kg calves. Resistance type (monogenic or polygenic) and effective dominance were determined based on the discriminant concentration (DC) for cipermethrine (0.5%), deltamethrine (0.09%) and flumethrine (0.01%). Allele specific PCR (AS-PCR) was used for genotyping, looking at a sodium channel mutation (Phe-Ile substitution). The mortality rates and allele frequency of susceptible and pyrethroid resistant reference strains were 0% mortality and 90% RR alleles for resistant strain, and 100% mortality and 0% RR alleles as measured by the larval packet test (LPT) and allele specific PCR (AS-PCR) respectively. Backcrossed strain SR × RR showed an effective dominance (D_ML) of 0.605 for cypermethrin, 0.639 for deltamethrin and 0.498 for flumethrin, while survival of backcrosses RS × SS, RS × RR and SR × SS showed a significant tendency to recesivity. Backcrossed strain SR × RR (69.4%) also showed a higher RR genotype frequency with regards to RS × SS (25.5%), RS × RR (36.7%) and SR × SS (32.0%), however, susceptible allele was inherited in general as an incomplete dominant trait. Monogenic inheritance hypothesis was tested and the results showed monogenic inheritance for cypermethrin and flumethrin (P < 0.05) but not for deltamethrin (P > 0.05). However, significant correlation was found between RR genotype and the survival rate for all three pyrethroids used (P < 0.05), suggesting that a single substitution on the sodium channel gene can be responsible for resistance to pyrethroids as a class, due to the high frequency for RR genotypes. Combination with different mutations or metabolic resistance mechanisms cannot be excluded.     

Epidemiological investigation of <Emphasis Type="Italic">eaeA</Emphasis>-positive <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Escherichia albertii</Emphasis> strains isolated from healthy wild birds

Escherichia coli has commonly been associated with diarrheal illness in humans and animals. Recently, E. albertii has been reported to be a potential pathogen of humans and animals and to be carried by wild birds. In the present study, the prevalence and genetic characteristics of intimin-producing E. coli and E. albertii strains were evaluated in wild birds in Korea. Thirty one of 790 Enterobacteriaceae strains from healthy wild birds were positive for the intimin gene (eaeA) and twenty two of the 31 strains were identified as atypical enteropathogenic E. coli (aEPEC) that did not possess both EAF and bfpA genes. A total of nine lactose non-fermenting coliform bacterial strains were identified as E. albertii by PCR and sequence analysis of housekeeping genes. A total of 28 (90.3%) eaeA-positive strains were isolated from waterfowl. Fifteen aEPEC (68.2%) and two E. albertii (22.2%) strains had a β-intimin subtype and 14 aEPEC strains harboring β-intimin belonged to phylogenetic group B2. AU eaeA-positive E. albertii and 3 aEPEC strains possessed the cytolethal distending toxin gene (cdtB). The eaeA-positive E. coli and E. albertii strains isolated from healthy wild birds need to be recognized as a potential pathogroup that may pose a potential threat to human and animal health. These findings indicate that eaeA-positive E. coli as well as E. albertii can be carried by wild birds, posing a potential threat to human and animal health.     

Isolation,mapping, SNP detection and association with backfat traits of the porcine <Emphasis Type="Italic">CTNNBL1</Emphasis> and <Emphasis Type="Italic">DGAT2</Emphasis> genes

Both the CTNNBL1 (catenin, β-like1) and DGAT2 (diacylglycerol acyltransferase2) genes play important roles in adipose metabolism. In this study, we cloned these two genes in pigs. Semi-quantitative RT-PCR results showed that both genes were extensively expressed, and CTNNBL1 was at a high level in the heart and spleen, while DGAT2 was most abundant in the liver. In CTNNBL1, one synonymous mutation c.555C>T was identified in the coding region, and association analysis showed that different genotypes of CTNNBL1 were significantly associated with backfat at the shoulder and backfat at the rump (P < 0.05). In 3′-UTR of DGAT2, an A/G variation was detected by the Bcn I PCR-RFLP method, and different genotypes were significantly associated with backfat between the 6th and 7th ribs (P < 0.05). The allele frequency was tested among 188 unrelated pigs from six breeds. The results showed that for CTNNBL1, the Chinese indigenous breeds had higher frequencies of the C allele whereas the western breed had higher frequency of the T allele; and for DGAT2, allele A or G were distributed with no obvious difference in allele frequency. IMpRH was employed to localize these two genes, and CTNNBL1 was assigned to SSC17q21-23 and DGAT2 was assigned to SSC9p23-p24. The results suggest that the porcine CTNNBL1 and DGAT2 genes affect porcine fat deposition and further investigation will be necessary to illustrate the underlying mechanisms.     

Transgenic Peanut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.) Expressing the Urease Subunit B Gene of <Emphasis Type="Italic">Helicobacter pylori</Emphasis>

Helicobacter pylori (H. pylori) has been identified as the main pathogenic factors of chronic gastritis and peptic ulcer, and the Class I carcinogen of gastric cancer by WHO. Vaccine has become the most effective measure to prevent and cure H. pylori infection. The UreB is the most effective and common immunogen of all strains of H. pylori and may stimulate the immunoresponse protecting the human body against the challenge of H. pylori. UreB antigen gene was cloned into the binary vector pBI121 which contains a seed-specific promoter Oleosin of peanut and a kanamycin resistance gene, and then UreB gene was transformed into peanut embryo leaflets by Agrobacter-mediated method. The putative transgenic plants were examined for the presence of UreB in the nuclear genome of peanut plants by PCR analysis. Expression of UreB gene in plants was identified by RT-PCR and Western blot analysis. These results suggest that the UreB transgenic peanut can be potentially used as an edible vaccine for controlling H. pylori.     

Genetic Diversity of a Natural Population of <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> bv. <Emphasis Type="Italic">viciae</Emphasis> Nodulating Plants of <Emphasis Type="Italic">Vicia faba</Emphasis> in the Vesuvian Area

A total of 98 rhizobial strains, isolated during the winter of the years 2003 (35 isolates), 2004 (33 isolates), and 2005 (30 isolates) were analyzed to determine the genetic diversity of the natural population nodulating Vicia faba plants and to identify dominant genotypes. All isolates were identified as Rhizobium leguminosarum bv. viciae by biovar-specific polymerase chain reaction amplification of the nodC gene. Intraspecific DNA polymorphism was evaluated through the restriction endonucleases analysis combined with pulsed-field gel electrophoresis. Four genotypes characterized 53% of the isolates, showing a high occurrence; moreover, they were recovered over the 3 years, thus showing a lasting persistence in the soil, which could mean a high degree of saprophytic competitiveness. The richness, diversity, and dominance indexes of genotypes were calculated to monitor the evolution of the rhizobial population during the 3 years. The genetic diversity of the analyzed strains decreased along the 3 years. In fact, the biodiversity index H′ decreased from 2.6 in the first and second year to 1.9 in the third year; probably, as a result of bean monocropping, specific genotypes of Rh. leguminosarum bv. viciae were naturally selected.     

Analysis of <Emphasis Type="Italic">cry</Emphasis> Gene Profiles in <Emphasis Type="Italic">Bacillus Thuringiensis</Emphasis> Strains Isolated During Epizootics in <Emphasis Type="Italic">Cydia pomonella</Emphasis> L.

The crystal morphology and the profiles of genes encoding protein toxins (Cry and Cyt) were analyzed in 12 Bacillus thuringiensis strains isolated during epizootics in laboratory culture lines of Cydia pomonella, 2 isolates cultured from Leucoma salicis larvae, and 9 reference strains. Epizootic isolates produced crystals of the same bipyramidal shape; however, they revealed a variety of number and type of cry genes. Genes cry1I, cry2Ab, and cry9B were the most frequently observed in epizootic strains. Gene cry1I was noted in of 50% epizootic isolates. Eighty-three percent of them harbored gene cry2Ab. Gene cry9B was found for 42% of strains isolated during epizootics. Three isolates showed the largest number of cry genes and their variety; hence, they were chosen for the toxicity assay of their crystals and spores on C. pomonella larvae. One of them had approximately sixfold higher insecticidal activity than the reference strain B. thuringiensis subsp. kurstaki BTK STANDARD.     

Probiotic <Emphasis Type="Italic">Lactobacillus</Emphasis> strains: <Emphasis Type="Italic">in vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis> studies

Three Lactobacillus strains (LOCK 0900, LOCK 0908, LOCK 0919) out of twenty-four isolates were selected according to their antagonistic activity against pathogenic bacteria, resistance to low pH and milieu of bile salts. Intragastric administration of a mixture of these strains to Balb/c mice affected cytokine T_H1-T_H2 balance toward nonallergic T_H1 response. Spleen cells, isolated from lactobacilli-treated mice and re-stimulated in vitro with the mixture of heat-inactivated tested strains, produced significantly higher amounts of anti-allergic tumor necrosis factor- and interferon-γ than control animals whereas the level of pro-allergic interleukin-5 was significantly lower. Lactobacillus cells did not translocate through the intestinal barrier into blood, liver and spleen; a few Lactobacillus cells found in mesenteric lymph nodes could create antigenic reservoir activating the immune system. The mixture of Lactobacillus LOCK 0900, LOCK 0908 and LOCK 0919 strains represents a probiotic bacterial preparation with possible use in prophylaxis and/or therapy of allergic diseases.