The Development of Ovine Gastric and Intestinal Organoids for Studying Ruminant Host-Pathogen Interactions

Gastrointestinal (GI) infections in sheep have significant implications for animal health, welfare and productivity, as well as being a source of zoonotic pathogens. Interactions between pathogens and epithelial cells at the mucosal surface play a key role in determining the outcome of GI infections; however, the inaccessibility of the GI tract in vivo significantly limits the ability to study such interactions in detail. We therefore developed ovine epithelial organoids representing physiologically important gastric and intestinal sites of infection, specifically the abomasum (analogous to the stomach in monogastrics) and ileum. We show that both abomasal and ileal organoids form self-organising three-dimensional structures with a single epithelial layer and a central lumen that are stable in culture over serial passage. We performed RNA-seq analysis on abomasal and ileal tissue from multiple animals and on organoids across multiple passages and show the transcript profile of both abomasal and ileal organoids cultured under identical conditions are reflective of the tissue from which they were derived and that the transcript profile in organoids is stable over at least five serial passages. In addition, we demonstrate that the organoids can be successfully cryopreserved and resuscitated, allowing long-term storage of organoid lines, thereby reducing the number of animals required as a source of tissue. We also report the first published observations of a helminth infecting gastric and intestinal organoids by challenge with the sheep parasitic nematode Teladorsagia circumcincta, demonstrating the utility of these organoids for pathogen co-culture experiments. Finally, the polarity in the abomasal and ileal organoids can be inverted to make the apical surface directly accessible to pathogens or their products, here shown by infection of apical-out organoids with the zoonotic enteric bacterial pathogen Salmonella enterica serovar Typhimurium. In summary, we report a simple and reliable in vitro culture system for generation and maintenance of small ruminant intestinal and gastric organoids. In line with 3Rs principals, use of such organoids will reduce and replace animals in host-pathogen research.

The development of ovine gastric and intestinal organoids for studying ruminant host-pathogen interactions

Gastrointestinal (GI) infections in sheep have significant implications for animal health, welfare and productivity, as well as being a source of zoonotic pathogens. Interactions between pathogens and epithelial cells at the mucosal surface play a key role in determining the outcome of GI infections; however, the inaccessibility of the GI tract in vivo significantly limits the ability to study such interactions in detail. We therefore developed ovine epithelial organoids representing physiologically important gastric and intestinal sites of infection, specifically the abomasum (analogous to the stomach in monogastrics) and ileum. We show that both abomasal and ileal organoids form self-organising three-dimensional structures with a single epithelial layer and a central lumen that are stable in culture over serial passage. We performed RNA-seq analysis on abomasal and ileal tissue from multiple animals and on organoids across multiple passages and show the transcript profile of both abomasal and ileal organoids cultured under identical conditions are reflective of the tissue from which they were derived and that the transcript profile in organoids is stable over at least five serial passages. In addition, we demonstrate that the organoids can be successfully cryopreserved and resuscitated, allowing long-term storage of organoid lines, thereby reducing the number of animals required as a source of tissue. We also report the first published observations of a helminth infecting gastric and intestinal organoids by challenge with the sheep parasitic nematode Teladorsagia circumcincta, demonstrating the utility of these organoids for pathogen co-culture experiments. Finally, the polarity in the abomasal and ileal organoids can be inverted to make the apical surface directly accessible to pathogens or their products, here shown by infection of apical-out organoids with the zoonotic enteric bacterial pathogen Salmonella enterica serovar Typhimurium. In summary, we report a simple and reliable in vitro culture system for generation and maintenance of small ruminant intestinal and gastric organoids. In line with 3Rs principals, use of such organoids will reduce and replace animals in host-pathogen research.

The impact of the COVID-19 pandemic on gastrointestinal infection trends in England, February - July 2020

Objective: To establish the impact of the first six months of the COVID-19 outbreak response of gastrointestinal (GI) infection trends in England. Design: Retrospective ecological study using routinely collected national and regional surveillance data from eight Public Health England coordinated laboratory, outbreak and syndromic surveillance systems using key dates of UK governmental policy change to assign phases for comparison between 2020 and historic data. Results: Decreases in GI illness activity were observed across all surveillance indicators as COVID-19 cases began to peak. Compared to the 5-year average (2015-2019), during the first six months of the COVD-19 response, there was a 52% decrease in GI outbreaks reported (1,544 vs. 3,208 (95% CI: 2,938 - 3,478) and a 34% decrease in laboratory confirmed cases (27,859 vs. 42,495 (95% CI: 40,068 - 44,922). GI indicators began to rise during the first lockdown and lockdown easing, although all remained substantially lower than historic figures. Reductions in laboratory confirmed cases were observed across all age groups and both sexes, with geographical heterogeneity observed in diagnosis trends. Health seeking behaviour changed substantially, with attendances decreasing prior to lockdown across all indicators. Conclusions: There has been a marked change in trends of GI infections in the context of the COVID-19 pandemic. The drivers of this change are likely to be multifactorial; while changes in health seeking behaviour, pressure on diagnostic services and surveillance system ascertainment have undoubtably played a role there has likely been a true decrease in the incidence for some pathogens resulting from the control measures and restrictions implemented. This suggests that if some of these changes in behaviour such as improved hand hygiene were maintained, then we could potentially see sustained reductions in the burden of GI illness.

A Case Study of Multiple Parasitisms in a Calf Buffalo (Bubalus bubalis)

Background: Gastrointestinal (GI) parasitism by protozoan and helminth parasites exists as one of the major limiting factors in the buffalo industry, especially in the wellbeing of the calves around the developing countries like Nepal. During a field survey on buffaloes, we encountered a two- and half month ill male calf suffering from various illnesses for 14 days. Methods: We collected its stool sample for three days and processed through a direct wet mount, sedimentation, floatation and acid-fast staining techniques and observed via a compound microscope. Result: We detected the multiple patterns of infections of GI parasites and even counted the oocysts of coccidia and eggs of nematodes released per gram of the feces and discussed that co-infection was associated with the pathologic consequences. Coprological surveys with appropriate egg/oocyst counting techniques are useful in the treatment and preventive options of GI infections in calves.

1339. Antibiotic Prescribing Varies among Patients with a Documented Penicillin or Cephalosporin Adverse Drug Reaction

Background Studies have shown that over half of hospitalized children receive an antibiotic during their encounter, of which between 30-50% is considered inappropriate. Antibiotic prescribing is further complicated as approximately 10% of children are labeled beta-lactam allergic, resulting in the use of either broad-spectrum or suboptimal therapy. The purpose of this study was to compare antibiotic prescribing between patients with a documented ADR vs. those without using a nationwide sample of hospitalized children. Methods We performed a point prevalence study among 32 hospitals between July 2016-December 2017 where data were collected via chart review on pediatric patient and antimicrobial characteristics, including the indication for all antimicrobials. In additional, ADR history data were collected on which antimicrobial(s) were documented (e.g., penicillin, cephalosporins). Patients were mutually assigned into either: 1) no documented ADR; 2) penicillin ADR-only; 3) cephalosporin ADR-only; and 4) ADR for both penicillin and cephalosporin. The distribution of antibiotics were compared between the ADR groups, stratified by the indication for treatment. Results A total of 12,250 pediatric patients (17,929 antibiotic orders) who were actively receiving antibiotics were identified. A history of penicillin and cephalosporin ADR was documented in 5.5% and 2.8% of these patients, respectively. When compared to patients with no documented ADR, penicillin ADR patients were more likely to receive a fluoroquinolone for a SSTI infection (odds ratio [OR]: 5.6), surgical prophylaxis (OR: 18.8) or for surgical treatment (OR: 5.2) (see Figure). Conversely, penicillin ADR patients were less likely to receive first-line agents, such as narrow-spectrum penicillin for bacterial LRTI (OR: 0.08) and piperacillin/tazobactam for GI infections (OR: 0.22). Cephalosporin ADR patients exhibited similar patterns with increased use of carbapenems and fluoroquinolones when compared to patients with no ADR. Figure 1: Odds of Receiving Select Antimicrobials Among PCN ADR Patients When Compared to Non-ADR patients, by Indication Conclusion A large, nationwide sample of pediatric patients who were actively prescribed antibiotics helped identify several diagnoses where comprehensive guidelines for appropriate ADR prescribing and increased ADR de-labeling initiatives are needed to ensure optimal treatment. Disclosures Brian R. Lee, MPH, PhD, Merck (Grant/Research Support) Jason Newland, MD, MEd, FPIDS, Merck (Grant/Research Support)Pfizer (Other Financial or Material Support, Industry funded clinical trial)

Detection and Classification of Gastrointestinal Diseases using Machine Learning

Background: Traditional endoscopy is an invasive and painful method of examining the gastrointestinal tract (GIT) not supported by the physicians and patients. To handle this issue, video endoscopy (VE) or wireless capsule endoscopy (WCE) is recommended and utilized for GIT examination. Furthermore, manual assessment of captured images is not possible for an expert physician because it’s a time taking task to analyze thousands of images thoroughly. Hence, there comes the need for a Computer-Aided-Diagnosis (CAD) method to help doctors in the analysis of images. Many researchers have proposed techniques for automated recognition and classification of abnormality in captured images. Introduction: In this article, existing methods for automated classification, segmentation and detection of several GI diseases are discussed. Paper gives a comprehensive detail about these state-of-the-art methods. Furthermore, literature is divided into several subsections based on preprocessing techniques, segmentation techniques, handcrafted features based techniques and deep learning based techniques. Finally, issues, challenges and limitations are also undertaken. Conclusion: This comprehensive review article combines information related to a number of GI diseases diagnosis methods at one place. Study of this article will facilitate the researchers to develop new algorithms and approaches for early detection of GI diseases detection with more promising results as compared to the existing ones of literature. Results: A comparative analysis of different approaches for the detection and classification of GI infections.

Outcome of immune checkpoint inhibitor (ICI) related diarrhea/colitis (IMDC) in cancer patients with superimposed GI infections.

e15166 Background: ICI therapies have revolutionized the landscape of cancer treatment. However, their increased use has also contributed to adverse events, e.g. IMDC. Clinically it is difficult to distinguish between infectious etiology or IMDC. It is unclear whether antimicrobial treatment substantially impacts the disease course. Here, we evaluated the characteristics and outcome of IMDC in cancer patients with superimposed diarrheagenic GI infections. Methods: We retrospectively evaluated cancer patients who received ICI with clinical symptoms of IMDC and confirmed stool microbiology of E. coli or non-CMV viral infections either at the time of IMDC, or within 60 days after IMDC diagnosis at MD Anderson Cancer Center 01/2011-08/2019. We described the disease course and outcome of IMDC based on their status of GI infection and antimicrobial treatment. Results: Total 72 patients were included, among them, 50 control patients with IMDC and no GI infection, 22 had diarrheagenic infections, composed of 17 E coli pathogens: Enteropathogenic, Enterotoxigenic, Enteroaggregative, and E coli 0157:H7 serotype; and 5 viral etiologies: Adenovirus, Norovirus, and Sapovirus. Patients in the infection group had higher grade of colitis (42.9% vs 18.4%, P = 0.041), and more frequent hospitalization (86.4% vs 62%, P = 0.052). 68.2% patients in the infection group received infliximab/vedolizumab add-on treatment compared to 40% in the no infection group (P = 0.078). Patients with GI infection and antimicrobial treatment had much higher IMDC recurrence rate than no antimicrobial treatment (50% vs 0, P = 0.015). GI infection was not associated with higher IMDC recurrence or worse overall survival of these patients. Conclusions: IMDC can occasionally be complicated by infection from common GI pathogens. Antimicrobial treatment was mostly used in severe IMDC cases and did not circumvent the need for immunosuppressant or improve the clinical outcomes. GI infection was not associated with higher IMDC recurrence or worse overall survival of these patients. [Table: see text]

Evaluation of the Vibrant DNA microarray for the high-throughput multiplex detection of enteric pathogens in clinical samples

ABSTRACT Background Rapid detection of a wide range of etiologic agents is essential for appropriate treatment and control of gastrointestinal (GI) infections. A variety of microbial species including bacteria, viruses, parasites, and fungi have been recognized as diarrheagenic enteric pathogens. However, multiplex testing of various targets in a single reaction needs further improvement because of its limitation in species and throughput. Results This study aims at developing and evaluating a DNA microarray-based qualitative multiplexed polymerase chain reaction (PCR) assay, Vibrant GI pathogen panel (GPP), for simultaneous detection of 27 enteric GI pathogenic targets (16 bacteria, 5 viruses, 4 parasites, and 2 fungi) directly from stool specimens. Limits of detection ranged from 102 to 104 cells/mL for bacteria, 102 to 103 cells/mL for parasites, 102 to 103 RNA copies/mL for viruses, and 102 to 103 cells/mL for fungi. Performance characteristics were determined using 27 Quantitative Genomic DNAs, 212 spiked stool specimens, 1067 clinical and archived stool specimens. Overall sensitivity was 95.9% (95% CI 92.4–98.1) and specificity was 100% (95% CI 99.9–100). Polymicrobial detections contained either two or three organisms was 20.2% (35/173) of positive clinical specimens and 3.3% (35/1055) of all clinical specimens. Conclusion The Vibrant GPP is a comprehensive, high-throughput, and rapid DNA microarray to provide etiologic diagnosis of GI infections in the laboratory setting.

Use of gender distribution in routine surveillance data to detect potential transmission of gastrointestinal infections among men who have sex with men in England

Detecting gastrointestinal (GI) infection transmission among men who have sex with men (MSM) in England is complicated by a lack of routine sexual behavioural data. We investigated whether gender distributions might generate signals for increased transmission of GI pathogens among MSM. We examined the percentage male of laboratory-confirmed patient-episodes for patients with no known travel history for 10 GI infections of public health interest in England between 2003 and 2013, stratified by age and region. An adult male excess was observed for Shigella spp. (annual maximum 71% male); most pronounced for those aged 25–49 years and living in London, Brighton and Manchester. An adult male excess was observed every year for Entamoeba histolytica (range 59.8–76.1% male), Giardia (53.1–57.6%) and Campylobacter (52.1–53.5%) and for a minority of years for hepatitis A (max. 69.8%) and typhoidal salmonella (max. 65.7%). This approach generated a signal for excess male episodes for six GI pathogens, including a characterised outbreak of Shigella among MSM. Stratified analyses by geography and age group were consistent with MSM transmission for Shigella. Optimisation and routine application of this technique by public health authorities elsewhere might help identify potential GI infection outbreaks due to sexual transmission among MSM, for further investigation.