Identification of novel small molecule inhibitors of twin arginine translocation (Tat) pathway and their effect on the control of Campylobacter jejuni in chickens.

Introduction: Control of Campylobacter from farm to fork is challenging due to the frequent emergence of antimicrobial-resistant isolates. Furthermore, poultry production systems are known reservoirs of Campylobacter. The twin-arginine translocation (Tat) pathway is a crucial bacterial secretion system that allows Campylobacter to colonize the host intestinal tract by using formate as the main source of energy. However, Tat pathway is also a major contributing factor for resistance to copper sulfate (CuSO4). Methods: Since mammals and chickens do not have proteins or receptors that are homologous to bacterial Tat proteins, identification of small molecule (SM) inhibitors targeting the Tat system would allow the development of safe and effective control methods to mitigate Campylobacter in infected or colonized hosts in both pre-harvest and post-harvest. In this study, we screened 11 commercial libraries (n = 50,917 SM) for increased susceptibility to CuSO4 (1 mM) in C. jejuni 81-176, a human isolate which is widely studied. Results: Furthermore, we evaluated 177 SM hits (2.5 µg/mL and above) that increased the susceptibility to CuSO4 for the inhibition of formate dehydrogenase (Fdh) activity, a Tat-dependent substrate. Eight Tat-dependent inhibitors (T1-T8) were selected for further studies. These selected eight Tat inhibitors cleared all tested Campylobacter strains (n = 12) at >10 ng/mL in the presence of 0.5 mM CuSO4in vitro. These selected SMs were non-toxic to colon epithelial (Caco-2) cells when treated with 50 µg/mL for 24 h and completely cleared intracellular C. jejuni cells when treated with 0.63 µg/mL of SM for 24 h in the presence of 0.5 mM of CuSO4. Furthermore, 3 and 5-week-old chicks treated with SM candidates for 5 days had significantly decreased cecal colonization (up to 1.2 log; p < 0.01) with minimal disruption of microbiota. In silico analyses predicted that T7 has better drug-like properties than T2 inhibitor and might target a key amino acid residue (glutamine 165), which is located in the hydrophobic core of TatC protein. Discussion: Thus, we have identified novel SM inhibitors of the Tat pathway, which represent a potential strategy to control C. jejuni spread on farms.

Concurrent Histomonas meleagridis and Hemorrhagic Enteritis Virus Infection in a Turkey Flock with Recurrent History of Blackhead Disease.

Intestinal health is one of the key factors required for the growth and production of turkeys. Histomoniasis (blackhead disease), caused by a protozoan parasite, Histomonas meleagridis, is a reemerging threat to the turkey industry. Increased incidences of histomoniasis have been reported in recent years due to withdrawal of antihistomonas treatments. H. meleagridis affects ceca and causes cecal inflammation and necrosis. H. meleagridis migrates from ceca to the liver and causes liver necrosis, resulting in high mortalities. Ironically, field outbreaks of histomoniasis are not always associated with high mortalities, while low mortalities have also been documented. There are several exacerbating factors associated with high mortality rates in histomoniasis outbreaks, with concurrent infection being one of them. Recurrent histomoniasis outbreaks in a newly constructed barn were documented, and concurrent infection of H. meleagridis and hemorrhagic enteritis virus was confirmed. Currently, neither commercial vaccines nor prophylactic or therapeutic solutions are available to combat histomoniasis. However, there are treatments, vaccines, and solutions to minimize or prevent concurrent infections in turkeys. In addition to implementing biosecurity measures, measures to prevent concurrent infections are critical steps that the turkey industry can follow to reduce mortality rates and minimize the production and economic losses associated with histomoniasis outbreaks.

Infección simultánea por Histomonas meleagridis y el virus de la enteritis hemorrágica en una parvada de pavos con antecedentes recurrentes de enfermedad de la cabeza negra. La salud intestinal es uno de los factores clave necesarios para el crecimiento y producción de los pavos. La histomoniasis (enfermedad de la cabeza negra), causada por un parásito protozoario, Histomonas meleagridis, es una amenaza reemergente para la industria del pavo. En los últimos años se ha informado de un aumento de la incidencia de histomoniasis debido al retiro de los tratamientos con antihistomonas. Histomonas meleagridis afecta los ciegos y causa inflamación y necrosis cecal. Histomonas meleagridis migra desde los ciegos al hígado y causa necrosis hepática, lo que resulta en una alta mortalidad. Irónicamente, los brotes de histomoniasis en el campo no siempre se asocian con una mortalidad elevada, aunque también se han documentado mortalidades bajas. Hay varios factores exacerbantes asociados con altas tasas de mortalidad en los brotes de histomoniasis, siendo la infección concurrente uno de ellos. Se documentaron brotes recurrentes de histomoniasis en un alojamiento avícola recién construido y se confirmó la infección concurrente de H. meleagridis y el virus de la enteritis hemorrágica. Actualmente no se dis-pone de vacunas comerciales ni soluciones profilácticas o terapéuticas para combatir la histomoniasis. Sin embargo, existen tratamientos, vacunas y soluciones para minimizar o prevenir infecciones concurrentes en los pavos. Además de implementar medidas de bioseguridad, las medidas para prevenir infecciones concurrentes son pasos críticos que la industria del pavo puede seguir para reducir las tasas de mortalidad y minimizar las pérdidas económicas y de producción asociadas con los brotes de histomoniasis.

Mortality associated with SARS-CoV-2 in nondomestic felids.

Between September and November 2021, 5 snow leopards (Panthera uncia) and 1 lion (Panthera leo) were naturally infected with severe acute respiratory coronavirus 2 (SARS-CoV-2) and developed progressive respiratory disease that resulted in death. Severe acute respiratory syndrome coronavirus 2 sequencing identified the delta variant in all cases sequenced, which was the predominant human variant at that time. The time between initial clinical signs and death ranged from 3 to 45 days. Gross lesions in all 6 cats included nasal turbinate hyperemia with purulent discharge and marked pulmonary edema. Ulcerative tracheitis and bronchitis were noted in 4 cases. Histologically, there was necrotizing and ulcerative rhinotracheitis and bronchitis with fibrinocellular exudates and fibrinosuppurative to pyogranulomatous bronchopneumonia. The 4 cats that survived longer than 8 days had fungal abscesses. Concurrent bacteria were noted in 4 cases, including those with more acute disease courses. Severe acute respiratory syndrome coronavirus 2 was detected by in situ hybridization using probes against SARS-CoV-2 spike and nucleocapsid genes and by immunohistochemistry. Viral nucleic acid and protein were variably localized to mucosal and glandular epithelial cells, pneumocytes, macrophages, and fibrinocellular debris. Based on established criteria, SARS-CoV-2 was considered a contributing cause of death in all 6 cats. While mild clinical infections are more common, these findings suggest that some SARS-CoV-2 variants may cause more severe disease and that snow leopards may be more severely affected than other felids.

Naturally occurring highly pathogenic avian influenza virus H5N1 clade 2.3.4.4b infection in three domestic cats in North America during 2023.

The Eurasian strain of highly pathogenic avian influenza (HPAI) H5N1 is a devastating pathogen for birds that also has the capacity to infect mammals. This report describes the presentation, clinical case findings (including haemogram and serum biochemistry), gross and microscopic lesions and virus detection in three HPAI H5N1-infected domestic cats from the USA in 2023. All three cats presented with neurological abnormalities and were euthanized due to a poor prognosis within 2 days (two cats) or 10 days (one cat) of known clinical disease onset. Necropsy consistently revealed pulmonary congestion and oedema, and cerebrocortical malacia with haemorrhage was also seen in the cat that survived for 10 days. On histology, all cats had necrotizing encephalitis and interstitial pneumonia with pulmonary congestion, oedema, vasculitis and vascular thrombosis. One cat also had microscopic multifocal necrosis in the liver, pancreas and an adrenal gland. To our knowledge, this report is the first to detail pathological findings in HPAI H5N1 naturally-infected cats during the widespread outbreak in North America beginning in 2021, and that describes a cat surviving for 10 days after onset of HPAI H5N1 encephalitis.

Successful strategies for including adults with intellectual disabilities in research studies that use interpretative phenomenological analysis.

BACKGROUND: Adults with intellectual disabilities are not often asked to participate in health research. This may be because researchers perceive them as unable to participate meaningfully with or without significant support and anticipate difficulty in obtaining ethical approval because of issues concerning consent and mental capacity. This means that the voices of adults with intellectual disabilities are often missing from health research and their experiences and views are unexplored. AIM: To share successful strategies for accessing, recruiting and collecting data from a purposive sample of adults with intellectual disabilities from a study that used interpretative phenomenological analysis (IPA). DISCUSSION: IPA is a person-centred, flexible and creative approach to research. Meaningful collaboration with people with intellectual disabilities, their families, carers, advocacy group managers, specialists in intellectual disability services and research supervisors was vital to the success of the study. The authors share practical strategies for including people with intellectual disabilities, from the perspective of a novice researcher who is an outsider to the field of intellectual disability. CONCLUSION: It is important to include people with intellectual disabilities in healthcare research. This article presents a framework to support researchers outside the specialist field of intellectual disabilities in achieving this. IMPLICATIONS FOR PRACTICE: Personal views and perceptions of healthcare are important if health services are to meet individual needs. Adults with intellectual disabilities often receive poor healthcare and have poorer outcomes. This will be perpetuated without their input into research. People with intellectual disabilities can make valuable contributions to the evidence base.

A cross-case comparison of the trauma and orthopaedic hospital experiences of adults with intellectual disabilities using interpretative phenomenological analysis.

AIM: To present the cross-case comparison component of a qualitative study exploring and describing the experiences of adults with an intellectual disability who have received trauma and orthopaedic hospital care for musculoskeletal conditions or injuries in the United Kingdom. DESIGN: A qualitative, exploratory study was conducted using 1:1 semi-structured interviews to describe the lived experiences of trauma and orthopaedic hospital care from the perspectives of people with intellectual disabilities and a carer of a person with profound and multiple intellectual disabilities. The data were analysed using interpretative phenomenological analysis. The Standards for Reporting Qualitative Research guidelines were applied. RESULTS: There were common and interconnected experiences across the five participants: communication challenges; lack of person-centred care; issues related to pain management; lack of confidence in hospital care; the valuable support and expertise of carers; and incompetence of hospital staff and isolation and loneliness.

The voices of people with an intellectual disability and a carer about orthopaedic and trauma hospital care in the UK: An interpretative phenomenological study.

INTRODUCTION: People with intellectual disabilities have a greater prevalence of musculoskeletal conditions and injuries than the general population. Orthopaedic and trauma hospital care has not been investigated with this group who seldom have their voices heard or their experiences valued and interpreted. AIM: To understand the orthopaedic and trauma hospital experiences from the perspective of people with intellectual disabilities. METHODS: A qualitative approach, focusing on peoples' lived experiences was utilised. A purposive sample of five participants was recruited and one-to-one, semi-structured interviews were undertaken. Analysis of the interviews employed an interpretative phenomenological analytical framework. FINDINGS: There were communication challenges, a lack of person-centred care, issues with pain management, a lack of confidence in hospital care, valuable support and expertise of carers, incompetence of hospital staff and isolation and loneliness. DISCUSSION AND CONCLUSIONS: There were significant short comings as people with intellectual disabilities and a carer perceived they were unsupported and received poor care. Recommendations for practice: Person-centred care is needed along with specific education and training, including close liaison with the experts by experience - people with intellectual disabilities, their carers as well as the specialists in intellectual disability.

An integrative review of the hospital experiences of people with an intellectual disability: Lack of orthopaedic and trauma perspectives.

AIMS: To review the empirical literature relating to the orthopaedic and trauma hospital experiences of people with intellectual disabilities (PWID). DESIGN: An integrative review was conducted following the steps advocated by Whittemore and Knafl (2005). Primary research studies from 2007 to 2020 which included the hospital experiences of adults with intellectual disabilities were reviewed. DATA SOURCES: The literature searches were undertaken in 2014, 2015, 2018 and May 2020. REVIEW METHODS: The following electronic databases were searched: Academic Search Complete, Nursing and Allied Health, British Nursing Index and RCN Library archive, Cumulative Index to Nursing and Allied Health Literature (CINAHL), Medical Literature Analysis and Retrieval System Online (MEDLINE) Psychological Information Database (PsychINFO), the Cochrane Collaboration Database and Google Scholar. RESULTS: Despite the high prevalence of musculoskeletal problems for adults with intellectual disabilities, the review found no specific literature related to orthopaedic or trauma hospital experiences. Nine studies related to PWIDs' experiences of general hospital care were included. CONCLUSION: There is a gap in the empirical literature relating to orthopaedic or trauma hospital experiences of PWID. General hospital experiences of adults with an intellectual disability were poor overall.

Updating the UK competence framework for orthopaedic and trauma nurses 2019.

The updated RCN Competence Framework for orthopaedic and trauma practitioners was published in 2019 following completion of a 2 year project undertaken by a working group of representatives from England, Northern Ireland, Scotland and Wales. Expert musculoskeletal practitioners, including an allied health professional and working across the lifespan in varying domains of orthopaedic and trauma practice, collaborated to produce a working document applicable to trauma and orthopaedic (T&O) practitioners from all NHS (UK) pay bands. The 2019 document builds on the original and subsequent versions (2005 and 2012), importing new evidence and reformatting it so that it is contemporary and easily cross referenced with the NMC Code (2018). The restructure includes an example of a learning contract demonstrating how the framework can be applied in practice, whether for self-learning, or in conjunction with the revalidation process. This paper reflects on and describes the process undertaken by the working group in the development and restructuring of the 2019 framework, including its evaluation to date and planned in the future.

Novel Anti-Campylobacter Compounds Identified Using High Throughput Screening of a Pre-selected Enriched Small Molecules Library.

Campylobacter is a leading cause of foodborne bacterial gastroenteritis worldwide and infections can be fatal. The emergence of antibiotic-resistant Campylobacter spp. necessitates the development of new antimicrobials. We identified novel anti-Campylobacter small molecule inhibitors using a high throughput growth inhibition assay. To expedite screening, we made use of a "bioactive" library of 4182 compounds that we have previously shown to be active against diverse microbes. Screening for growth inhibition of Campylobacter jejuni, identified 781 compounds that were either bactericidal or bacteriostatic at a concentration of 200 µM. Seventy nine of the bactericidal compounds were prioritized for secondary screening based on their physico-chemical properties. Based on the minimum inhibitory concentration against a diverse range of C. jejuni and a lack of effect on gut microbes, we selected 12 compounds. No resistance was observed to any of these 12 lead compounds when C. jejuni was cultured with lethal or sub-lethal concentrations suggesting that C. jejuni is less likely to develop resistance to these compounds. Top 12 compounds also possessed low cytotoxicity to human intestinal epithelial cells (Caco-2 cells) and no hemolytic activity against sheep red blood cells. Next, these 12 compounds were evaluated for ability to clear C. jejuni in vitro. A total of 10 compounds had an anti-C. jejuni effect in Caco-2 cells with some effective even at 25 µM concentrations. These novel 12 compounds belong to five established antimicrobial chemical classes; piperazines, aryl amines, piperidines, sulfonamide, and pyridazinone. Exploitation of analogs of these chemical classes may provide Campylobacter specific drugs that can be applied in both human and animal medicine.

The experiences of orthopaedic and trauma nurses who have cared for adults with a learning disability.

BACKGROUND: There is no published empirical research about the experiences of orthopaedic and trauma nurses who have cared for people with a learning disability. However, adults with a learning disability sustain more injuries, falls and accidents than the general population. Because of their increased health needs, there has been a corresponding increase in their numbers attending general/acute hospitals. The 6 Cs is a contemporary framework and has been used to gauge how orthopaedic and trauma nurses rate the Care, Communication, Competence, Commitment, Courage and Compassion for patients with a learning disability in orthopaedic and trauma hospital settings compared to patients without a learning disability. AIM: The aim of the study was to explore the experiences of orthopaedic and trauma nurses who have cared for people with a learning disability. DESIGN: The study is based on a descriptive survey design and used a questionnaire to elicit data from participants. METHODS: A convenience sample of Registered Nurses completed a questionnaire. The study was explained to delegates attending a concurrent session on the topic of acute hospital care for people with a learning disability at a conference and the questionnaire was left on a table for participants to take if they wished. Questionnaires were returned anonymously. FINDINGS: Of the participants who had completed the questionnaire 100% (n = 13) had cared for a patient with a learning disability. Using the 6 Cs as a framework suggested that care, communication and competence of nurses were worse for people with a learning disability than for people without a learning disability. Three main themes emerged regarding areas of good practices: (1) promoting a positive partnership with patients and carers; (2) modifying care and interventions; (3) supporting the healthcare team. CONCLUSION: There was evidence of good practices within orthopaedic and trauma settings such as the active involvement of family or a paid carer who is known to thepatient and the modification of care and interventions along with specialist advice and support from the Acute Liaison Learning Disability Nurse. There were areas of concern such as the lack of use of Hospital Passports and the inconsistent implementation of reasonable and achievable adjustments. It is unknown if the care for patients with a learning disability is adequate. However, the themes that have emerged accord with the key domains in 'A competency framework for orthopaedic and trauma practitioners' (Royal College of Nursing 2012a, 2012b) and therefore could be considered for inclusion in future orthopaedic and trauma competencies to enable sharing of best practices.

Polyphosphate-mediated modulation of Campylobacter jejuni biofilm growth and stability.

Biofilms increase C. jejuni's resilience to detergents, antibiotics, and environmental stressors. In these investigations, we studied the modulation of biofilm in response to phosphate related stressors. We found that the deletion of ppk1, phoX, and ppk2 (polyphosphate associated [poly P] genes) in C. jejuni modulated different stages of biofilm formation such as attached microcolonies, air-liquid biofilms, and biofilm shedding. Additionally, inorganic phosphate also modulated attached microcolonies, air-liquid biofilms, and biofilm shedding both independently of and additively in the poly P associated mutants. Furthermore, we observed that these different biofilm stages were affected by biofilm age: for example, the adherent microcolonies were maximum on day 2, while biofilm growth at the air-liquid interface and shedding was highest on day 3. Also, we observed altered calcofluor white reactive polysaccharides in poly P-associated mutants, as well as increased secretion of autoinducer-2 (AI-2) quorum sensing molecules in the ∆ppk2 mutant. Further, the polysaccharide and flagellar biosynthesis genes, that are associated with biofilm formation, were altered in these poly P-associated mutants. We conclude that the phosphate limiting condition modulates C. jejuni biofilm formation.

Evaluating the occurrence of host-specific , general fecal indicators, and bacterial pathogens in a mixed-use watershed.

Fecal contamination of water is very common, and, in the United States, prevention is complicated by the colossal span of waterways (>3.5 million miles), heterogeneous sources of pollution, and competing interests in water monitoring. The focus of this study was the Upper Sugar Creek Watershed, a mixed-use watershed with many headwater streams and one of the most contaminated waterways in Ohio. Quantitative polymerase chain reaction (qPCR) and host-specific PCR for were evaluated for the potential to discern sources of fecal contamination. Pathogen-specific qPCR and culturable by most probable number (MPN) were compared at 21 established water quality monitoring sites in the watershed headwaters. Lower numbers of ruminant-specific markers were detected in the base flow water samples compared with the human-specific marker, suggesting the presence of hotspots of human fecal contamination. qPCR and MPN showed significant correlation ( = 0.57; < 0.001). Correlation between general fecal indicator and pathogen concentrations was weak or nonexistent. Coexistence of and human-specific was common ( = 0.015). qPCR may have a greater potential for predicting fecal contamination due to its sensitivity, rapid analysis, and availability of host-specific assays. However, the lack of a strong correlation between pathogens and general fecal indicators suggests that assessment of health risk associated with fecal contamination will require a complement of approaches.

Contribution of TAT system translocated PhoX to Campylobacter jejuni phosphate metabolism and resilience to environmental stresses.

Campylobacter jejuni is a common gastrointestinal pathogen that colonizes food animals; it is transmitted via fecal contamination of food, and infections in immune-compromised people are more likely to result in serious long-term illness. Environmental phosphate is likely an important sensor of environmental fitness and the ability to obtain extracellular phosphate is central to the bacteria's core metabolic responses. PhoX is the sole alkaline phosphatase in C. jejuni, a substrate of the TAT transport system. Alkaline phosphatases mediate the hydrolytic removal of inorganic phosphate (Pi) from phospho-organic compounds and thereby contribute significantly to the polyphosphate kinase 1 (ppk1) mediated formation of poly P, a molecule that regulates bacterial response to stresses and virulence. Similarly, deletion of the tatC gene, a key component of the TAT system, results in diverse phenotypes in C. jejuni including reduced stress tolerance and in vivo colonization. Therefore, here we investigated the contribution of phoX in poly P synthesis and in TAT-system mediated responses. The phoX deletion mutant showed significant decrease (P<0.05) in poly P accumulation in stationary phase compared to the wild-type, suggesting that PhoX is a major contributor to the inorganic phosphate pool in the cell which is essential for poly P synthesis. The phoX deletion is sufficient for a nutrient stress defect similar to the defect previously described for the ΔtatC mutant. Additionally, the phoX deletion mutant has increased resistance to certain antimicrobials. The ΔphoX mutant was also moderately defective in invasion and intracellular survival within human intestinal epithelial cells as well as in chicken colonization. Further, the ΔphoX mutant produced increased biofilm that can be rescued with 1 mM inorganic phosphate. The qRT-PCR of the ΔphoX mutant revealed transcriptional changes that suggest potential mechanisms for the increased biofilm phenotype.

A quantitative polymerase chain reaction assay for detection and quantification of Lawsonia intracellularis.

Lawsonia intracellularis, a causative agent of porcine proliferative enteropathy, is an obligate intracellular bacterium that is difficult to culture, propagate, and quantify. The intestinal epithelial cell line (IEC-18, derived from rat small intestine crypt cells) has been used to isolate and propagate this pathogen. However, the lack of rapid and simple quantification methods has led to mixed results when using the rat cell line, complicating Lawsonia studies. To overcome these limitations, a SYBR green quantitative polymerase chain reaction (qPCR) assay targeting a unique hypothetical protein was developed for detecting and quantifying L. intracellularis in IEC-18 rat epithelial cells, porcine fecal samples collected from different farms, and experimentally infected pigs. The method was optimized to detect as few as 3 copies per qPCR reaction of the bacterium growing in IEC-18 cells, providing a new and necessary approach to assess the growth of L. intracellularis in these cell lines. Furthermore, the qPCR assay was successful in detecting L. intracellularis in fecal samples collected from pigs with and without a history of Lawsonia infections, as well as in experimentally infected pigs, which further confirmed the suitability of the qPCR assay for studying the epidemiology of this pathogen.