Earlier onset of flowering and increased reproductive allocation of an annual invasive plant in the north of its novel range.

BACKGROUND AND AIMS: It remains unclear whether invasive species can maintain both high biomass and reproductive output across their invaded range. Along latitudinal gradients, allocation theory predicts that faster flowering onset at high latitudes results in maturation at smaller size and thus reduced reproductive output. For annual invasive plants, more favourable environmental conditions at low latitudes probably result in stronger competition of co-occurring species, potentially driving selection for higher investment in vegetative biomass, while harsher climatic conditions and associated reproductive uncertainty at higher latitudes could reduce selection for vegetative biomass and increased selection for high reproductive investment (stress-gradient hypothesis). Combined, these drivers could result in increased or constant reproductive allocation with increasing latitude. METHODS: We quantified life-history traits in the invasive annual plant Impatiens glandulifera along a latitudinal gradient in Europe. By growing two successive glasshouse generations, we assessed genetic differentiation in vegetative growth and reproductive output across six populations, and tested whether onset of flowering drives this divergence. KEY RESULTS: Trait variation was mainly caused by genetic differentiation. As expected, flowering onset was progressively earlier in populations from higher latitudes. Plant height and vegetative biomass also decreased in populations from higher latitudes, as predicted by allocation theory, but their variation was independent of the variation in flowering onset. Reproductive output remained constant across latitudes, resulting in increased reproductive allocation towards higher latitudes, supporting the stress-gradient hypothesis. We also observed trait genetic differentiation among populations that was independent of latitude. CONCLUSIONS: We show that an annual invasive plant evolved several life-history traits across its invaded range in ~150 years. The evolution of vegetative and reproductive traits seems unconstrained by evolution of flowering onset. This genetic decoupling between vegetative and reproductive traits possibly contributes to the invasion success of this species.

No genetic erosion after five generations for Impatiens glandulifera populations across the invaded range in Europe.

BACKGROUND: The observation that many alien species become invasive despite low genetic diversity has long been considered the 'genetic paradox' in invasion biology. This paradox is often resolved through the temporal buildup genetic diversity through multiple introduction events. These temporal dynamics in genetic diversity are especially important for annual invasive plants that lack a persistent seed bank, for which population persistence is strongly dependent on consecutive seed 're-establishment' in each growing season. Theory predicts that the number of seeds during re-establishment, and the levels of among-population gene flow can strongly affect recolonization dynamics, resulting in either an erosion or build-up of population genetic diversity through time. This study focuses on temporal changes in the population genetic structure of the annual invasive plant Impatiens glandulifera across Europe. We resampled 13 populations in 6 regions along a 1600 km long latitudinal gradient from northern France to central Norway after 5 years, and assessed population genetic diversity with 9 microsatellite markers. RESULTS: Our study suggests sufficiently high numbers of genetically diverse founders during population re-establishment, which prevent the erosion of local genetic diversity. We furthermore observe that I. glandulifera experiences significant among-population gene flow, gradually resulting in higher genetic diversity and lower overall genetic differentiation through time. Nonetheless, moderate founder effects concerning population genetic composition (allele frequencies) were evident, especially for smaller populations. Despite the initially low genetic diversity, this species seems to be successful at persisting across its invaded range, and will likely continue to build up higher genetic diversity at the local scale.

Biotic and abiotic drivers of intraspecific trait variation within plant populations of three herbaceous plant species along a latitudinal gradient.

BACKGROUND: The importance of intraspecific trait variation (ITV) is increasingly acknowledged among plant ecologists. However, our understanding of what drives ITV between individual plants (ITVBI) at the population level is still limited. Contrasting theoretical hypotheses state that ITVBI can be either suppressed (stress-reduced plasticity hypothesis) or enhanced (stress-induced variability hypothesis) under high abiotic stress. Similarly, other hypotheses predict either suppressed (niche packing hypothesis) or enhanced ITVBI (individual variation hypothesis) under high niche packing in species rich communities. In this study we assess the relative effects of both abiotic and biotic niche effects on ITVBI of four functional traits (leaf area, specific leaf area, plant height and seed mass), for three herbaceous plant species across a 2300 km long gradient in Europe. The study species were the slow colonizing Anemone nemorosa, a species with intermediate colonization rates, Milium effusum, and the fast colonizing, non-native Impatiens glandulifera. RESULTS: Climatic stress consistently increased ITVBI across species and traits. Soil nutrient stress, on the other hand, reduced ITVBI for A. nemorosa and I. glandulifera, but had a reversed effect for M. effusum. We furthermore observed a reversed effect of high niche packing on ITVBI for the fast colonizing non-native I. glandulifera (increased ITVBI), as compared to the slow colonizing native A. nemorosa and M. effusum (reduced ITVBI). Additionally, ITVBI in the fast colonizing species tended to be highest for the vegetative traits plant height and leaf area, but lowest for the measured generative trait seed mass. CONCLUSIONS: This study shows that stress can both reduce and increase ITVBI, seemingly supporting both the stress-reduced plasticity and stress-induced variability hypotheses. Similarly, niche packing effects on ITVBI supported both the niche packing hypothesis and the individual variation hypothesis. These results clearly illustrates the importance of simultaneously evaluating both abiotic and biotic factors on ITVBI. This study adds to the growing realization that within-population trait variation should not be ignored and can provide valuable ecological insights.

Low genetic diversity despite multiple introductions of the invasive plant species Impatiens glandulifera in Europe.

BACKGROUND: Invasive species can be a major threat to native biodiversity and the number of invasive plant species is increasing across the globe. Population genetic studies of invasive species can provide key insights into their invasion history and ensuing evolution, but also for their control. Here we genetically characterise populations of Impatiens glandulifera, an invasive plant in Europe that can have a major impact on native plant communities. We compared populations from the species' native range in Kashmir, India, to those in its invaded range, along a latitudinal gradient in Europe. For comparison, the results from 39 other studies of genetic diversity in invasive species were collated. RESULTS: Our results suggest that I. glandulifera was established in the wild in Europe at least twice, from an area outside of our Kashmir study area. Our results further revealed that the genetic diversity in invasive populations of I. glandulifera is unusually low compared to native populations, in particular when compared to other invasive species. Genetic drift rather than mutation seems to have played a role in differentiating populations in Europe. We find evidence of limitations to local gene flow after introduction to Europe, but somewhat less restrictions in the native range. I. glandulifera populations with significant inbreeding were only found in the species' native range and invasive species in general showed no increase in inbreeding upon leaving their native ranges. In Europe we detect cases of migration between distantly located populations. Human activities therefore seem to, at least partially, have facilitated not only introductions, but also further spread of I. glandulifera across Europe. CONCLUSIONS: Although multiple introductions will facilitate the retention of genetic diversity in invasive ranges, widespread invasive species can remain genetically relatively invariant also after multiple introductions. Phenotypic plasticity may therefore be an important component of the successful spread of Impatiens glandulifera across Europe.

An Evaluation of the Impact of an OPEN Stewardship Generated Feedback Intervention on Antibiotic Prescribing among Primary Care Veterinarians in Canada and Israel.

An interrupted time-series study design was implemented to evaluate the impact of antibiotic stewardship interventions on antibiotic prescribing among veterinarians. A total of 41 veterinarians were enrolled in Canada and Israel and their prescribing data between 2019 and 2021 were obtained. As an intervention, veterinarians periodically received three feedback reports comprising feedback on the participants' antibiotic prescribing and prescribing guidelines. A change in the level and trend of antibiotic prescribing after the administration of the intervention was compared using a multi-level generalized linear mixed-effect negative-binomial model. After the receipt of the first (incidence rate ratios [IRR] = 0.88; 95% confidence interval (CI): 0.79, 0.98), and second (IRR = 0.85; 95% CI: 0.75, 0.97) feedback reports, there was a reduced prescribing rate of total antibiotic when other parameters were held constant. This decline was more pronounced among Israeli veterinarians compared to Canadian veterinarians. When other parameters were held constant, the prescribing of critical antibiotics by Canadian veterinarians decreased by a factor of 0.39 compared to that of Israeli veterinarians. Evidently, antibiotic stewardship interventions can improve antibiotic prescribing in a veterinary setting. The strategy to sustain the effect of feedback reports and the determinants of differences between the two cohorts should be further explored.

Evaluation of an automated feedback intervention to improve antibiotic prescribing among primary care physicians (OPEN Stewardship): a multinational controlled interrupted time-series study.

Tools to advance antimicrobial stewardship in the primary health care setting, where most antimicrobials are prescribed, are urgently needed. The aim of this study was to evaluate OPEN Stewarship (Online Platform for Expanding aNtibiotic Stewardship), an automated feedback intervention, among a cohort of primary care physicians. We performed a controlled, interrupted time-series study of 32 intervention and 725 control participants, consisting of primary care physicians from Ontario, Canada and Southern Israel, from October 2020 to December 2021. Intervention participants received three personalized feedback reports targeting several aspects of antibiotic prescribing. Study outcomes (overall prescribing rate, prescribing rate for viral respiratory conditions, prescribing rate for acute sinusitis, and mean duration of therapy) were evaluated using multilevel regression models. We observed a decrease in the mean duration of antibiotic therapy (IRR = 0.94; 95% CI: 0.90, 0.99) in intervention participants during the intervention period. We did not observe a significant decline in overall antibiotic prescribing (OR = 1.01; 95% CI: 0.94, 1.07), prescribing for viral respiratory conditions (OR = 0.87; 95% CI: 0.73, 1.03), or prescribing for acute sinusitis (OR = 0.85; 95% CI: 0.67, 1.07). In this antimicrobial stewardship intervention among primary care physicians, we observed shorter durations of therapy per antibiotic prescription during the intervention period. The COVID-19 pandemic may have hampered recruitment; a dramatic reduction in antibiotic prescribing rates in the months before our intervention may have made physicians less amenable to further reductions in prescribing, limiting the generalizability of the estimates obtained.IMPORTANCEAntibiotic overprescribing contributes to antibiotic resistance, a major threat to our ability to treat infections. We developed the OPEN Stewardship (Online Platform for Expanding aNtibiotic Stewardship) platform to provide automated feedback on antibiotic prescribing in primary care, where most antibiotics for human use are prescribed but where the resources to improve antibiotic prescribing are limited. We evaluated the platform among a cohort of primary care physicians from Ontario, Canada and Southern Israel from October 2020 to December 2021. The results showed that physicians who received personalized feedback reports prescribed shorter courses of antibiotics compared to controls, although they did not write fewer antibiotic prescriptions. While the COVID-19 pandemic presented logistical and analytical challenges, our study suggests that our intervention meaningfully improved an important aspect of antibiotic prescribing. The OPEN Stewardship platform stands as an automated, scalable intervention for improving antibiotic prescribing in primary care, where needs are diverse and technical capacity is limited.

Identification of the elements of models of antimicrobial resistance of bacteria for assessing their usefulness and usability in One Health decision making: a protocol for scoping review.

INTRODUCTION: Antimicrobial resistance (AMR) is a complex problem that requires the One Health approach, that is, a collaboration among various disciplines working in different sectors (animal, human and environment) to resolve it. Mathematical and statistical models have been used to understand AMR development, emergence, dissemination, prediction and forecasting. A review of the published models of AMR will help consolidate our knowledge of the dynamics of AMR and will also facilitate decision-makers and researchers in evaluating the credibility, generalisability and interpretation of the results and aspects of AMR models. The study objective is to identify and synthesise knowledge on mathematical and statistical models of AMR among bacteria in animals, humans and environmental compartments. METHODS AND ANALYSIS: Eligibility criteria: Original research studies reporting mathematical and statistical models of AMR among bacteria in animal, human and environmental compartments that were published until 2022 in English, French and Spanish will be included in this study. SOURCES OF EVIDENCE: Database of PubMed, Agricola (Ovid), Centre for Agriculture and Bioscience Direct (CABI), Web of Science (Clarivate), Cumulative Index to Nursing and Allied Health Literature (CINAHL) and MathScinet. Data charting: Metadata of the study, the context of the study, model structure, model process and reporting quality will be extracted. A narrative summary of this information, gaps and recommendations will be prepared and reported in One Health decision-making context. ETHICS AND DISSEMINATION: Research ethics board approval was not obtained for this study as neither human participation nor unpublished human data were used in this study. The study findings will be widely disseminated among the One Health Modelling Network for Emerging Infections network and stakeholders by means of conferences, and publication in open-access peer-reviewed journals.

Modelling the transmission of dengue, zika and chikungunya: a scoping review protocol.

INTRODUCTION: Aedes mosquitoes are the primary vectors for the spread of viruses like dengue (DENV), zika (ZIKV) and chikungunya (CHIKV), all of which affect humans. Those diseases contribute to global public health issues because of their great dispersion in rural and urban areas. Mathematical and statistical models have become helpful in understanding these diseases' epidemiological dynamics. However, modelling the complexity of a real phenomenon, such as a viral disease, should consider several factors. This scoping review aims to document, identify and classify the most important factors as well as the modelling strategies for the spread of DENV, ZIKV and CHIKV. METHODS AND ANALYSIS: We will conduct searches in electronic bibliographic databases such as PubMed, MathSciNet and the Web of Science for full-text peer-reviewed articles written in English, French and Spanish. These articles should use mathematical and statistical modelling frameworks to study dengue, zika and chikungunya, and their cocirculation/coinfection with other diseases, with a publication date between 1 January 2011 and 31 July 2023. Eligible studies should employ deterministic, stochastic or statistical modelling approaches, consider control measures and incorporate parameters' estimation or considering calibration/validation approaches. We will exclude articles focusing on clinical/laboratory experiments or theoretical articles that do not include any case study. Two reviewers specialised in zoonotic diseases and mathematical/statistical modelling will independently screen and retain relevant studies. Data extraction will be performed using a structured form, and the findings of the study will be summarised through classification and descriptive analysis. Three scoping reviews will be published, each focusing on one disease and its cocirculation/co-infection with other diseases. ETHICS AND DISSEMINATION: This protocol is exempt from ethics approval because it is carried out on published manuscripts and without the participation of humans and/or animals. The results will be disseminated through peer-reviewed publications and presentations in conferences.

Angioma serpiginosum in zosteriform distribution on abdomen: A rare presentation.

Angioma serpiginosum is a rare benign nevoid disorder affecting the small vessels of the upper dermis. We are presenting a case of this rare disease in an 11-year-old girl who presented with this condition on abdomen in zosteriform pattern which is rare presentation and is the first of its type.

Spatio-Temporal Variation in the Prevalence of Major Mastitis Pathogens Isolated From Bovine Milk Samples Between 2008 and 2017 in Ontario, Canada.

An understanding of the spatio-temporal distribution of several groups of mastitis pathogens can help to inform programs for the successful control and management of mastitis. However, in the absence of an active surveillance program such information is not readily available. In this retrospective study we analyzed passive surveillance data from a diagnostic laboratory with an aim to describe the spatio-temporal trend of major mastitis pathogens between 2008 and 2017 in Ontario dairy cattle. Data for all milk culture samples submitted to the Animal Health Laboratory (AHL) at the University of Guelph between 2008 and 2017 was accessed. Descriptive analyses were conducted to identify the major pathogens and Chi-square goodness-of-fit tests were used to compare between multiple proportions. Likewise, univariable logistic regression analysis was performed to determine if there was a change in the probability of isolating the major mastitis pathogens depending on geography or time. Seasonality was assessed by calculating the seasonal relative risk (RR). Of a total of 85,979 milk samples examined, more than half of the samples (61.07%) showed no growth and the proportion of samples that showed no growth almost halved during the study period. Of the samples (36.21%, n = 31,133) that showed any growth, the major bacterial pathogens were Staphylococcus aureus (15.60%), Non-aureus Staphylococci (NAS) (5.04%), Corynebacterium spp. (2.96%), and Escherichia coli (2.00%). Of the NAS, the major species reported were Staphylococcus chromogenes (69.02%), Staphylococcus simulans (14.45%), Staphylococcus epidermidis (12.99%), and Staphylococcus hyicus (2.13%). A temporal change in the prevalence of contagious pathogens like S. aureus and Corynebacterium spp. was observed with an increasing odds of 1.06 and 1.62, respectively. Likewise, except for Trueperella pyogenes, the prevalence of all the major environmental mastitis pathogens increased during the study period. The isolation of most of the pathogens peaked in summer, except for S. aureus, T. pyogenes, and Streptococcus dysgalactiae which peaked in spring months. Interestingly, a regional pattern of isolation of some bacterial pathogens within Ontario was also observed. This study showed a marked spatio-temporal change in the prevalence of major mastitis pathogens and suggests that a regional and seasonal approach to mastitis control could be of value.

Evaluation of an OPEN Stewardship generated feedback intervention to improve antibiotic prescribing among primary care veterinarians in Ontario, Canada and Israel: protocol for evaluating usability and an interrupted time-series analysis.

INTRODUCTION: Antimicrobial resistance (AMR) impacts the health and well-being of animals, affects animal owners both socially and economically, and contributes to AMR at the human and environmental interface. The overuse and/or inappropriate use of antibiotics in animals has been identified as one of the most important drivers of the development of AMR in animals. Effective antibiotic stewardship interventions such as feedback can be adopted in veterinary practices to improve antibiotic prescribing. However, the provision of dedicated financial and technical resources to implement such systems are challenging. The newly developed web-based Online Platform for Expanding Antibiotic Stewardship (OPEN Stewardship) platform aims to automate the generation of feedback reports and facilitate wider adoption of antibiotic stewardship. This paper describes a protocol to evaluate the usability and usefulness of a feedback intervention among veterinarians and assess its impact on individual antibiotic prescribing. METHODS AND ANALYSIS: Approximately 80 veterinarians from Ontario, Canada and 60 veterinarians from Israel will be voluntarily enrolled in a controlled interrupted time-series study and their monthly antibiotic prescribing data accessed. The study intervention consists of targeted feedback reports generated using the OPEN Stewardship platform. After a 3-month preintervention period, a cohort of veterinarians (treatment cohort, n=120) will receive three feedback reports over the course of 6 months while the remainder of the veterinarians (n=20) will be the control cohort. A survey will be administered among the treatment cohort after each feedback cycle to assess the usability and usefulness of various elements of the feedback report. A multilevel negative-binomial regression analysis of the preintervention and postintervention antibiotic prescribing of the treatment cohort will be performed to evaluate the impact of the intervention. ETHICS AND DISSEMINATION: Research ethics board approval was obtained at each participating site prior to the recruitment of the veterinarians. The study findings will be disseminated through open-access scientific publications, stakeholder networks and national/international meetings.

Evaluation of an automated feedback intervention to improve antimicrobial prescribing among primary care physicians (OPEN Stewardship): protocol for an interrupted time-series and usability analysis in Ontario, Canada and Southern Israel.

INTRODUCTION: Antimicrobial resistance undermines our ability to treat bacterial infections, leading to longer hospital stays, increased morbidity and mortality, and a mounting burden to the healthcare system. Antimicrobial stewardship is increasingly important to safeguard the efficacy of existing drugs, as few new drugs are in the developmental pipeline. While significant progress has been made with respect to stewardship in hospitals, relatively little progress has been made in the primary care setting, where the majority of antimicrobials are prescribed. OPEN Stewardship is an international collaboration to develop an automated feedback platform to improve responsible antimicrobial prescribing among primary care physicians and capable of being deployed across heterogeneous healthcare settings. We describe the protocol for an evaluation of this automated feedback intervention with two main objectives: assessing changes in antimicrobial prescribing among participating physicians and determining the usability and usefulness of the reports. METHODS AND ANALYSIS: A non-randomised evaluation of the automated feedback intervention (OPEN Stewardship) will be conducted among approximately 150 primary care physicians recruited from Ontario, Canada and Southern Israel, based on a series of targeted stewardship messages sent using the platform. Using a controlled interrupted time-series analysis and multilevel negative binomial modelling, we will compare the antimicrobial prescribing rates of participants before and after the intervention, and also to the prescribing rates of non-participants (from the same healthcare network) during the same period. We will examine outcomes targeted by the stewardship messages, including prescribing for antimicrobials with duration longer than 7 days and prescribing for indications where antimicrobials are typically unnecessary. Participants will also complete a series of surveys to determine the usability and usefulness of the stewardship reports. ETHICS AND DISSEMINATION: All sites have obtained ethics committee approval to recruit providers and access anonymised prescribing data. Dissemination will occur through open-access publication, stakeholder networks and national/international meetings.

A sub-national real-time epidemiological and vaccination database for the COVID-19 pandemic in Canada.

The COVID-19 pandemic has demonstrated the need for real-time, open-access epidemiological information to inform public health decision-making and outbreak control efforts. In Canada, authority for healthcare delivery primarily lies at the provincial and territorial level; however, at the outset of the pandemic no definitive pan-Canadian COVID-19 datasets were available. The COVID-19 Canada Open Data Working Group was created to fill this crucial data gap. As a team of volunteer contributors, we collect daily COVID-19 data from a variety of governmental and non-governmental sources and curate a line-list of cases and mortality for all provinces and territories of Canada, including information on location, age, sex, travel history, and exposure, where available. We also curate time series of COVID-19 recoveries, testing, and vaccine doses administered and distributed. Data are recorded systematically at a fine sub-national scale, which can be used to support robust understanding of COVID-19 hotspots. We continue to maintain this dataset, and an accompanying online dashboard, to provide a reliable pan-Canadian COVID-19 resource to researchers, journalists, and the general public.

Australian Veterinarians' Perceptions Regarding the Zoonotic Potential of Mycobacterium avium Subspecies Paratuberculosis.

Public concerns over exposure to Mycobacterium avium subspecies paratuberculosis (MAP) or MAP components via foods of animal origin could have negative trade consequences, despite the absence of conclusive scientific evidence of a causal association between Mycobacterium avium subspecies paratuberculosis (MAP) and Crohn's disease (CD). This study was conducted among Australian veterinarians to understand (a) their perceptions regarding the role of MAP in the causation of CD (an ordinal outcome), and (b) their consideration of the adoption of the precautionary principle against Johne's disease (JD; a binary outcome). Ordinal and binary logistic regression analyses were performed to evaluate the association of explanatory variables with the above outcomes, respectively. Almost one-third of the respondents (32.2%) considered that MAP was likely to be involved in the causation of CD whereas more than two-thirds (69.8%) agreed with the adoption of the precautionary principle against JD. Veterinarians who were concerned about exposure to and/or getting infected with MAP were more likely to consider MAP as a causative agent of CD (odds ratio: 7.63; 95% CI: 1.55, 37.63) and favor the adoption of the precautionary principle against JD (odds ratio: 6.20; 95% CI: 1.90, 20.25). Those perceiving MAP as a causative agent of CD were also more likely to favor the adoption of the precautionary principle against JD (odds ratio: 13.2; 95% CI: 1.26, 138.90). The results suggest that Australian veterinarians, particularly those who consider MAP as a causative agent of CD are concerned about exposure to MAP and favor the adoption of the precautionary principle against JD. These findings can be useful for animal health authorities for designing JD control programs and policies.

Detection of Mycobacterium avium subspecies paratuberculosis in powdered infant formula using IS900 quantitative PCR and liquid culture media.

Mycobacterium avium subspecies paratuberculosis (MAP) has been implicated in Crohn's disease in humans resulting in public concern over the presence of MAP in powdered infant formula, which could contribute towards early human exposure to MAP or MAP components. Testing of representative powdered infant formula samples using effective tests is required to provide information on contamination of infant formula with MAP, so that consumers can make informed decisions. This study aimed to test representative powdered infant formula samples for the presence of MAP using a quantitative PCR and liquid culture method. For this purpose, an efficient DNA extraction method was developed and an optimum decontamination protocol for culture method was identified. A total of 122 powdered infant formula samples were tested, comprising 72 brands produced by 12 manufacturers from 9 countries. Powdered infant formula samples were reconstituted and centrifuged to separate the casein pellet, cream layer and whey fraction. A sensitive qPCR test was performed on DNA extracted from the casein pellet. In addition, the cream layer and casein pellet were cultured in liquid media, following decontamination with the optimum protocol. Of the 122 samples tested, 6 were positive for MAP DNA but none were positive for growth in culture at 12 and 20 weeks. The limit of detection of the quantitative PCR was less than 5 MAP organisms per 1.5g milk powder. The methods developed in the study could be used for quality assurance testing for infant formula and calf milk replacers. The low contamination level of MAP and absence of viable forms in our study suggests a relatively low risk of exposure of infants to MAP components.

PCR Inhibition of a Quantitative PCR for Detection of Mycobacterium avium Subspecies Paratuberculosis DNA in Feces: Diagnostic Implications and Potential Solutions.

Molecular tests such as polymerase chain reaction (PCR) are increasingly being applied for the diagnosis of Johne's disease, a chronic intestinal infection of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). Feces, as the primary test sample, presents challenges in terms of effective DNA isolation, with potential for PCR inhibition and ultimately for reduced analytical and diagnostic sensitivity. However, limited evidence is available regarding the magnitude and diagnostic implications of PCR inhibition for the detection of MAP in feces. This study aimed to investigate the presence and diagnostic implications of PCR inhibition in a quantitative PCR assay for MAP (High-throughput Johne's test) to investigate the characteristics of samples prone to inhibition and to identify measures that can be taken to overcome this. In a study of fecal samples derived from a high prevalence, endemically infected cattle herd, 19.94% of fecal DNA extracts showed some evidence of inhibition. Relief of inhibition by a five-fold dilution of the DNA extract led to an average increase in quantification of DNA by 3.3-fold that consequently increased test sensitivity of the qPCR from 55 to 80% compared to fecal culture. DNA extracts with higher DNA and protein content had 19.33 and 10.94 times higher odds of showing inhibition, respectively. The results suggest that the current test protocol is sensitive for herd level diagnosis of Johne's disease but that test sensitivity and individual level diagnosis could be enhanced by relief of PCR inhibition, achieved by five-fold dilution of the DNA extract. Furthermore, qualitative and quantitative parameters derived from absorbance measures of DNA extracts could be useful for prediction of inhibitory fecal samples.

Culture-Independent Identification of Mycobacterium avium Subspecies paratuberculosis in Ovine Tissues: Comparison with Bacterial Culture and Histopathological Lesions.

Johne's disease is a chronic debilitating enteropathy of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). Current abattoir surveillance programs detect disease via examination of gross lesions and confirmation by histopathological and/or tissue culture, which is time-consuming and has relatively low sensitivity. This study aimed to investigate whether a high-throughput quantitative PCR (qPCR) test is a viable alternative for tissue testing. Intestine and mesenteric lymph nodes were sourced from sheep experimentally infected with MAP and the DNA extracted using a protocol developed for tissues, comprised enzymatic digestion of the tissue homogenate, chemical and mechanical lysis, and magnetic bead-based DNA purification. The extracted DNA was tested by adapting a previously validated qPCR for fecal samples, and the results were compared with culture and histopathology results of the corresponding tissues. The MAP tissue qPCR confirmed infection in the majority of sheep with gross lesions on postmortem (37/38). Likewise, almost all tissue culture (61/64) or histopathology (52/58) positives were detected with good to moderate agreement (Cohen's kappa statistic) and no significant difference to the reference tests (McNemar's Chi-square test). Higher MAP DNA quantities corresponded to animals with more severe histopathology (odds ratio: 1.82; 95% confidence interval: 1.60, 2.07). Culture-independent strain typing on tissue DNA was successfully performed. This MAP tissue qPCR method had a sensitivity equivalent to the reference tests and is thus a viable replacement for gross- and histopathological examination of tissue samples in abattoirs. In addition, the test could be validated for testing tissue samples intended for human consumption.