Helicobacter pylori and Epstein-Barr Virus Co-Infection in Gastric Disease: What Is the Correlation with p53 Mutation, Genes Methylation and Microsatellite Instability in a Cohort of Sicilian Population?

Genetic predisposition, environmental factors, and infectious agents interact in the development of gastric diseases. Helicobacter pylori (Hp) and Epstein-Barr virus (EBV) infection has recently been shown to be correlated with these diseases. A cross-sectional study was performed on 100 hospitalized Italian patients with and without gastric diseases. The patients were stratified into four groups. Significant methylation status differences among CDH1, DAPK, COX2, hMLH1 and CDKN2A were observed for coinfected (Hp-EBV group) patients; particularly, a significant presence of COX2 (p = 0.0179) was observed. For microsatellite instability, minor stability was described in the Hp-HBV group (69.23%, p = 0.0456). Finally, for p53 mutation in the EBV group, exon 6 was, significantly, most frequent in comparison to others (p = 0.0124), and in the Hp-EBV group exon 8 was, significantly, most frequent in comparison to others (p < 0.0001). A significant positive relationship was found between patients with infection (Hp, EBV or both) and p53 mutation (rho = 0.383, p = 0.0001), methylation status (rho = 0.432, p < 0.0001) and microsatellite instability (rho = 0.285, p = 0.004). Finally, we observed among infection and methylation status, microsatellite instability, and p53 mutation a significant positive relationship only between infection and methylation status (OR = 3.78, p = 0.0075) and infection and p53 mutation (OR = 6.21, p = 0.0082). According to our analysis, gastric disease in the Sicilian population has different pathways depending on the presence of various factors, including infectious agents such as Hp and EBV and genetic factors of the subject.

Retrospective study on transmissible viral proventriculitis and chicken proventricular necrosis virus (CPNV) in the UK.

Chicken proventricular necrosis virus (CPNV) is a recently described birnavirus, which has been proposed to be the cause of transmissible viral proventriculitis (TVP). The understanding of the epidemiology of both the virus and the disease is very limited. A retrospective investigation on TVP and CPNV in broiler chicken submissions from the UK from between 1994 and 2015 was performed with the aims of assessing the longitudinal temporal evolution of TVP and CPNV, and to review the histological proventricular lesions in the studied chickens. Ninety-nine of the 135 included submissions (73.3%) fulfilled the TVP-diagnostic criteria, while the remaining 36 submissions (26.7%) displayed only lymphocytic proventriculitis (LP). The first detection of CPNV by PCR dated from 2009. Results showed a rise in the number of both TVP and positive CPNV RT-PCR submissions from 2009 with a peak in 2013, suggesting that they may be an emerging or re-emerging disease and pathogen, respectively. Twenty-two out of the 99 submissions displaying TVP lesions (22%) and four out of the 36 (11%) submissions with LP gave positive CPNV RT-PCR results, further supporting the association between CPNV and TVP and confirming that CPNV is present in a low proportion of proventriculi that do not fulfil the TVP-diagnostic criteria. In addition, intranuclear inclusion bodies were observed in 22 of the submissions with TVP. The vast majority of these cases (21 of 22, 96%) gave negative CPNV RT-PCR results, raising the question of whether a virus other than CPNV is responsible for some of these TVP-affected cases.RESEARCH HIGHLIGHTSTVP and CPNV have been present in British broilers since at least 1994 and 2009, respectively.TVP and CPNV seem to be an emerging and re-emerging disease and pathogen, respectively.CPNV was detected in proventriculi with both TVP and LP-lesions.Viruses other than CPNV may be responsible for some TVP-affected cases.

Case­control study of Epstein­Barr virus and Helicobacter pylori serology in Latin American patients with gastric disease.

BACKGROUND: Chronic tissue damage induced by Helicobacter pylori (HP)-driven inflammation is considered the main risk of gastric carcinoma (GC). Epstein­Barr virus (EBV) infection has also been associated with GC. In this study, we aim to address the role of EBV in inflammatory GC precursor lesions and its added risk to HP infection. METHODS: Antibodies against EBV, HP and the bacterial virulence factor CagA were measured in sera from 525 Mexican and Paraguayan patients with gastric disease. Gastric samples were characterised according to the updated Sydney classification and associations were estimated between antibody responses and severity of both tissue damage and inflammation. RESULTS: We found significant associations (odd ratios and trends) between EBV and HP copositivity and premalignant lesions and intestinal-type GC. The EBV and HP coinfection was also significantly associated with increased infiltration of immune cells. No association was found between EBV and the less inflammation-driven diffuse-type GC. CONCLUSIONS: Our study suggests that EBV co-participates with HP to induce severe inflammation, increasing the risk of progression to intestinal-type GC.

Runting Stunting Syndrome Associated with Transmissible Viral Proventriculitis in Broiler Chickens.

This report describes an outbreak of transmissible viral proventriculitis (TVP) associated with runting stunting syndrome (RSS) in 25- and 28-day-old broiler chickens, in which chicken proventricular necrosis virus (CNPV) was detected. Clinical signs included poor uniformity, very small birds for their age, increased mortality, and culling of smaller birds. Almost all birds necropsied exhibited moderate to severely enlarged proventriculi with diffusely pale serosa and thickened walls. Microscopically the proventriculi had lesions of degeneration and necrosis of the epithelium of the proventricular glands, accompanied by lymphocytic inflammation and glandular hyperplasia, with occasional formation of lymphoid nodules within the glandular parenchyma. Immunohistochemistry staining for CPNV was positive. Positive staining was generally found in the cytoplasm of glandular epithelial cells in the form of finely granular brown pigment. CPNV RNA was detected in the proventriculi by reverse transcriptase-PCR (RT-PCR). Other findings included mild enteritis in a few birds and small bursa of Fabricius. Direct electron microscopy performed on the intestinal samples was negative for viral particles. RT-PCR analysis of bursae was positive for infectious bursal disease virus (IBDV). In conclusion, this report associates TVP with RSS by describing an outbreak in which TVP attributable to CPNV was the most commonly found lesionin chickens with a clinical history compatible with RSS. Therefore, TVP should be considered as a possible differential diagnosis in cases compatible with RSS.

Equine gamma herpesvirus presence and viral load are not associated with equine glandular gastric disease.

OBJECTIVE: To investigate the role of equine herpesvirus-2 (EHV-2) and equine herpesvirus-5 (EHV-5) in equine glandular gastric disease (EGGD) by visualizing and quantifying these gamma herpesviruses in EGGD-affected and normal glandular gastric mucosa of horses. A secondary objective was to describe the histopathological abnormalities in the equine gastric glandular mucosa in horses with EGGD. ANIMALS: 29 horses (n = 21 postmortem and 8 gastroscopy) categorized as normal (11), EGGD (12), or both EGGD and equine squamous gastric disease (6). METHODS: Glandular gastric mucosal samples were collected from horses by gastroscopy or postmortem. Histopathology and in situ hybridization targeting EHV-2 and EHV-5 were performed on grossly normal and abnormal glandular gastric mucosa. The number of in situ hybridization-positive cells per millimeter squared of tissue was calculated. Evaluators were blinded to groups. RESULTS: Glandular gastric tissues from horses without EGGD had higher viral loads in the mucosa than normal or abnormal tissues from EGGD horses. There was no difference in viral loads for EHV-2 or EHV-5 between grossly or endoscopically normal to abnormal gastric tissues within horses with EGGD. Lymphocytic plasmacytic gastritis was the most common histopathological abnormality, with only 3 horses having mucosal disruption (glandular ulcer or erosion). CLINICAL RELEVANCE: Equine gamma herpesviruses are unlikely to play a role in the pathophysiology of EGGD. EGGD is frequently inflammatory with occasional mucosal disruption (ulcer or erosion).

Adenoviral gizzard erosion in broiler chickens in Germany.

Avian adenovirus infections cause important disease complexes in chickens, but many of the viruses also infect chickens without resulting in overt disease. Previously several outbreaks of gizzard erosions caused by a fowl adenovirus A serotype-1 (FAdV-1) were reported from Japan. Here we report an outbreak of gizzard erosions in 12 broiler flocks in Germany in 2011. Chickens had a reduced daily weight gain and a higher total mortality rate of up to 8%. The birds showed a severe detachment of the koilin layer and ulcerative to necrotizing lesions of the underlying mucosa. Histopathologically, necrotizing ventriculitis with basophilic, intranuclear inclusion bodies in epithelial cells was diagnosed. Immunohistochemistry, egg culture, and electron microscopic examination revealed adenovirus-like particles in the samples. No concurrent infectious agent could be identified. The virus was genotyped as FAdV-1 by PCR and subsequent sequencing. Phylogenetic analysis of the hexon loop L1 gene yielded 100% sequence identity to the chicken embryo lethal orphan strain. These findings suggest that outbreaks of adenoviral gizzard erosion can lead to significant economic losses in Germany and may be caused by an unusual virulent FAdV-1 strain.

Lymphomatoid gastropathy: a distinct clinicopathologic entity of self-limited pseudomalignant NK-cell proliferation.

Diagnostic errors in distinguishing between malignant and reactive processes can cause serious clinical consequences. We report 10 cases of unrecognized self-limited natural killer-cell proliferation in the stomach, designated as lymphomatoid gastropathy (LyGa). This study included 5 men and 5 women (age, 46-75 years) without any gastric symptoms. Gastroscopy showed elevated lesion(s) (diameter, ∼ 1 cm). Histologically, medium-sized to large atypical cells diffusely infiltrated the lamina propria and, occasionally, the glandular epithelium. The cells were CD2(+/-), sCD3(-), cCD3(+), CD4(-), CD5(-), CD7(+), CD8(-), CD16(-), CD20(-), CD45(+), CD56(+), CD117(-), CD158a(-), CD161(-), T cell-restricted intracellular antigen-1(+), granzyme B(+), perforin(+), Epstein-Barr early RNA(-), T-cell receptor αß(-), and T-cell receptor γδ(-). Analysis of the 16 specimens biopsied from 10 patients led to a diagnosis of lymphoma or suspected lymphoma in 11 specimens, gastritis for 1 specimen, adenocarcinoma for 1 specimen, and LyGa or suspected LyGa for 3 specimens. Most lesions underwent self-regression. Three cases relapsed, but none of the patients died. According to conventional histopathologic criteria, LyGa is probably diagnosed as lymphoma, especially as extranodal natural killer/T-cell lymphoma, nasal type. However, LyGa is recognized as a pseudomalignant process because of its clinical characteristics. The concept of LyGa should be well recognized.

Transmissible viral proventriculitis identified in broiler breeder and layer hens.

Transmissible viral proventriculitis (TVP) is a recognized cause of production losses in broiler chickens, but previously it has not been reported in broiler breeder and commercial layer hens. In this study, TVP was identified in broiler breeder and commercial layer hens, 9-20 wk of age, based on histopathologic detection of characteristic microscopic lesions. Microscopic lesions in proventriculi of affected hens consisted of glandular epithelial necrosis, ductal epithelial hyperplasia, replacement of glandular epithelium with ductal epithelium, and diffuse interstitial lymphoid infiltration. Additionally, chicken proventricular necrosis virus (CPNV), a virus previously identified as the etiology of TVP in broiler chickens, was detected in proventriculi of TVP-affected hens using a reverse transcriptase-polymerase chain reaction procedure. The findings identify TVP as a potential cause of production losses in broiler breeder and commercial layer hens and provide additional evidence for etiologic involvement in TVP by CPNV.

Follow-up investigations on different courses of natural avian bornavirus infections in psittacines.

To study the course of natural avian bornavirus (ABV) infection, 63 psittacines of three bird collections where ABV had been demonstrated were investigated over a period of 1 yr. The psittacines were clinically observed and swabs of crop and cloaca as well as serum samples were collected three separate times at intervals of 2-6 mo. According to the results of detection of ABV RNA by reverse transcriptase polymerase chain reaction (RT-PCR) and of anti-ABV antibodies by indirect immunofluorescence assay (IIFA), 43 of the birds were found to be infected with ABV. Based on variations in virus shedding and antibody production in combination with the occurrence of proventricular dilatation disease (PDD) -related clinical signs, pathological findings, and lethal outcome, four different groups of infected psittacines and a fifth group of noninfected psittacines were identified. Group 1 comprised six birds with various courses of ABV infection and forms of clinical PDD. Groups 2-4 included all birds with subclinical ABV infections: Group 2 contained 13 birds that were consistently (subgroup A, 6 birds) or inconsistently (subgroup B, 7 birds) ABV positive by PCR and serology; group 3 was composed of 13 psittacines exhibiting only anti-ABV antibodies; and 8 birds that had positive ABV RNA detection in crop and cloaca, but did not develop anti-ABV specific antibodies, were classified in group 4. Twenty-three out of the 63 psittacines remained free of detectable ABV RNA or anti-ABV antibodies over the whole observation period (group 5). Based on the results, it seems that birds with high ABV RNA load in crop and cloaca combined with high anti-ABV antibodies have a high risk of the development of PDD, indicating that the humoral antibodies do not protect against the disease. The meaning of the detection of ABV RNA and antibodies at a low and inconsistent level for the single bird as well as for the epidemiology of the ABV infection remained unclear in this field study and needs to be further investigated.

Identification of mixed infections with different genotypes of avian bornaviruses in psittacine birds with proventricular dilatation disease.

Proventricular dilatation disease (PDD) is a fatal, progressive neurological disorder of psittacine birds, which is caused by a single-stranded RNA virus, the avian bornavirus (ABV). The disease pattern includes lymphoplasmacytic inflammation of the central, peripheral and autonomic nervous system. Seven avian bornavirus genotypes have been identified during the last years. So far only monoinfections with a single genotype of ABV have been attributed to PDD cases. However, after a recent survey discovered a case of a double infection with two different ABV genotypes, this seemed to indicate the need for a more systematic search for mixed infections. Brain specimens from 21 psittacine birds affected with PDD were examined. Aim of the investigation was to generate partial ABV sequences of a part of the matrix protein (M) gene and to evaluate whether sequences of more than one ABV genotype were present. RNA was extracted, and subjected to reverse transcriptase PCR with primer pairs generating a partial sequence of the matrix protein (M) gene, followed by a cloning procedure. Ten clones per case were sequenced in order to elucidate whether sequences characteristic for one or more than one genotype were present. In 19 of 21 cases clear M gene sequences could be generated; in two cases nucleic acid amplification failed. Seven birds were infected with ABV 2 and nine with ABV 4, representing the predominant genotypes in Europe. Two cases showed a mixed infection with ABV 2 and ABV 4, and one case a mixed infection with ABV 2 and ABV 6. These results suggest that the molecular cloning method is a useful tool for distinguishing between single and multiple infection events by different ABV genotypes.

Cytomegalovirus infection of the upper gastrointestinal tract: a clinical and pathological study of 30 cases.

OBJECTIVE: The study reviews the endoscopic and histological features of human cytomegalovirus (HCMV) infections of the upper gastrointestinal (UGI) tract. MATERIALS AND METHODS: Clinical histories, endoscopic findings and bioptic specimens of 30 cases of HCMV infection of the UGI tract, diagnosed in a University Hospital in a 10-year period, were reviewed. In all cases, viral inclusion bodies were detected in routine histopathological sections and the diagnosis was confirmed with immunohistochemistry. RESULTS: Six patients were HIV+, whereas four had received organ transplantations, one was affected by common variable immunodeficiency and four had a recent history of malignancy. No other pathologic condition was evidenced in the remaining 15 cases. Mucosal alterations were endoscopically observed in the stomach (19 cases), esophagus (9), cardias (6) and duodenum (1), and multiple organs being synchronously affected in five patients (3 HIV+, 2 with history of malignancy). The antropyloric area was the most frequently affected site. Single ulcers were detected in 11 cases and multiple ulcers in 8, whereas mucosal thickenings (in the form of localized thickenings, polyps or rugal hypertrophy) were present in 13 patients. Thickenings of the mucosa were detected only in the stomach. At histology, necrotic material and granulation tissue were associated with moderate or marked lympho-plasmacytic infiltrate and foveolar hyperplasia in ulcerative lesions, whereas lesions labeled as mucosal thickenings showed mild or moderate chronic inflammatory infiltrate and foveolar hyperplasia. CONCLUSIONS: Endoscopic manifestations of UGI tract involvement in HCMV infection are not specific, varying from erythematous mucosa to ulcers to mucosal thickenings.

Proventricular dilatation disease in cockatiels (Nymphicus hollandicus) after infection with a genotype 2 avian bornavirus.

An isolate of genotype 2 avian bornavirus (ABV) was recovered from a cockatiel (Nymphicus hollandicus) that was euthanatized for an unrelated lesion and showing no clinical evidence of proventricular dilatation disease (PDD). On histopathologic examination, mild inflammatory lesions were present in the heart and brain, but gastrointestinal lesions characteristic of classic PDD were not observed. To investigate if this ABV2 isolate had reduced virulence, the virus was propagated in duck embryo fibroblasts and inoculated into 2 adult cockatiels by the oral and intramuscular routes. One bird developed clinical signs on day 33 and was euthanatized on day 36. The second challenged bird developed clinical signs on day 41 and was euthanatized on day 45. At necropsy, the proventriculus of both birds was slightly enlarged. Histopathologic examination showed lesions typical of PDD in the brain, spinal cord, heart, adrenal gland, and intestine. A control, uninoculated cockatiel was apparently healthy when euthanatized on day 50. These results show that ABV2 is now the second ABV genotype to be formally shown to cause PDD.

Relationship between chicken proventricular necrosis virus prevalence and transmissible viral proventriculitis in broiler chickens in Poland.

Transmissible Viral Proventriculitis (TVP) is a disease of chickens which contributes to significant production losses. Recent reports indicate the role of chicken proventricular necrosis virus (CPNV) in the development of TVP. However, the relationship between CPNV and TVP is inconclusive and it has been addressed in just a few reports. Given the above, a study was conducted to identify the relationship between TVP and CPNV prevalence in broiler chickens in Poland. The study was carried out on 35 proventriculi samples sent for histopathological (HP) examination to the Faculty of Veterinary Medicine in Olsztyn between 2017 and 2019. After HP examination, TVP positive samples were processed for CPNV identification by RT-PCR. TVP was the most common pathological condition of proventriculi (23 cases). CPNV was identified in 10 out of those 23 cases. The average HP score, and the average necrosis and infiltration score for CPNV-positive samples was significantly higher than in CPNV-negative ones. The average age of the CPNV-positive chickens was significantly lower than in CPNV-negative birds. Our study confirms the role of CPNV in TVP pathogenesis and it seems that preservation of the proventriculi in the early stages of the disease, when the lesions are more pronounced, should result in a greater probability of CPNV detection.

Near-infrared Raman spectroscopy for optical diagnosis in the stomach: identification of Helicobacter-pylori infection and intestinal metaplasia.

Raman spectroscopy is a unique vibrational spectroscopic technique which can be used to probe biochemicals and biomolecular structures and conformations of tissues. The main objective of this study is to evaluate the feasibility of applying near-infrared (NIR) Raman spectroscopy for identification of nonneoplastic lesions (Helicobacter-pylori (Hp) infection, and intestinal metaplasia (IM)) highly associated with stomach cancer. A rapid-acquisition NIR Raman spectroscopic system was used for tissue Raman measurements at 785 nm excitation, and a total of 88 gastric tissue samples (57 normal; 11 Hp-infection; 20 IM) from 56 patients were measured. The principal components analysis (PCA) and linear discriminant analysis (LDA) techniques were implemented to develop effective diagnostic algorithms for classification of Raman spectra of different gastric tissue types. High-quality Raman spectra in the range of 800-1800 cm(-1) were acquired from gastric tissue within 5 seconds. Significant spectral differences in Raman spectra were observed among normal, Hp-infection and IM gastric tissue, particularly in the spectral ranges of 848-917, 960-1015, 1088-1133, 1206-1213, 1277-1313, 1395-1445, 1517-1549, 1607-1690, and 1714-1767 cm(-1) which contained signals related to proteins, lipids and porphyrin. PCA-LDA algorithms developed together with leave one patient out, cross validation technique yield diagnostic sensitivities of 91.7%, 80.0%, and 80.0%, and specificities of 80.0%, 100%, and 92.7%, respectively, for classification of normal, Hp-infection and IM gastric tissues. This work demonstrates the utility of NIR Raman spectroscopy for early diagnosis of Hp-infection and IM lesions in the gastric at the molecular level.

Genomic characterization of a proventriculitis-associated infectious bronchitis coronavirus.

Transmissible proventriculitis associated with infectious bronchitis virus (IBV) was at first seen in eastern China in mid-1995, and is now endemic in China. Herein, the complete genome sequence of a proventiculitis-associated infectious bronchitis coronavirus (ZJ971) was sequenced and analyzed. Compared with the genome of the vaccine strain H120, ZJ971 had 54 nucleotide substitutions and a deletion in the 3'-UTR. The substitutions were in the regions of nsp2-nsp5, nsp7, nsp12, nsp13, nsp15, S and N genes, and the untranslating region. The results indicated that ZJ971 could be a variant of IBV strain H120.

Avian bornavirus is present in many tissues of psittacine birds with histopathologic evidence of proventricular dilatation disease.

Proventricular dilatation disease (PDD) is a neurologic disease of psittacine birds suspected to be caused by a recently identified Avian bornavirus (ABV). In the current report, data supporting the causal association of ABV with PDD are presented. Immunohistochemistry (IHC) with rabbit polyclonal antiserum raised against ABV nucleocapsid protein was used to identify cell and organ distribution of viral antigen. The ABV antigen was most consistently detected in brain, spinal cord, adrenal gland, pancreas, and kidney. Histopathologic evaluation was correlated with ABV-specific polymerase chain reaction (PCR) and immunohistochemical tests in multiple tissues from 16 psittacine birds with and without PDD. Using histopathologic diagnosis as the gold standard, the sensitivity and specificity of IHC for ABV antigens were found to be 100% and 100%, respectively. Many more tissues were positive for ABV RNA by reverse transcription PCR than were positive for pathologic changes or viral antigens by IHC, indicating the presence of subclinical or asymptomatic infection at many sites.

Equus caballus papillomavirus type 2 (EcPV2) in co-occurring vulvar and gastric lesions of a pony.

In horses, squamous cell carcinomas (SCCs) are the most common malignant tumors developing on non-pigmented skin, muco-cutaneous areas, like external genitalia, and, less frequently, in the stomach. Growing evidence suggests Equus caballus papillomavirus type 2 (EcPV2) as causative agent of genital SCCs. Our case report describes a 20-year-old, female, mixed-breed pony with co-occurring vulvar papilloma and in situ carcinoma and gastric SCC. Both lesions were positive for the same EcPV2, as confirmed by DNA sequencing. E6 mRNA expression was observed both in vulvar lesions and gastric SCC, while L1 mRNA was expressed in the vulvar tumor. To the best of the Authors' knowledge, this is the first report of an association between EcPV2 and equine gastric squamous cell carcinoma, with co-occurring EcPV2-positive genital lesions. Further studies are required to assess the real prevalence and the possible role of this viral type in these equine tumors.

Histopathology and the detection of avian bornavirus in the nervous system of birds diagnosed with proventricular dilatation disease.

Avian bornavirus (ABV) is currently considered a probable etiologic agent of proventricular dilatation disease (PDD) of psittacines. We tested 24 stored avian brain samples, processed for histopathology and retained following their submission for necropsy or histopathology to the Schubot Exotic Bird Center diagnostic laboratory in 1992. Thirteen of these samples were from birds diagnosed at that time as suffering from PDD. The remaining 11 samples were diagnosed as suffering from diseases other than PDD. Immunohistochemistry was performed using an antiserum directed against the ABV nucleoprotein (N-protein). Stained slides were read by an investigator unaware of their prior histopathology results. Cells containing ABV N-protein were present in the nervous tissues of all 13 PDD cases. One bird not previously diagnosed with PDD also had ABV N-protein in its brain. A review of this bird's necropsy report indicated that it was, most probably, also suffering from PDD. The remaining 10 non-PDD birds had no detectable N-protein in their brains. The N-protein was present in the cerebrum, cerebellum and spinal cord. These findings support other studies that indicate that ABV is an etiological agent of PDD.