Electroencephalography Quality and Application Times in a Pediatric Emergency Department Setting: A Feasibility Study.

BACKGROUND: Status epilepticus is the most common neurological emergency presenting to pediatric emergency departments. Nonconvulsive status epilepticus can be extremely challenging to diagnose, however, requiring electroencephalographic (EEG) confirmation for definitive diagnosis. We aimed to determine the feasibility of achieving a good-quality pediatric EEG recording within 20 minutes of presentation to the emergency department. METHODS: Single-center prospective feasibility study in Cork University Hospital, Ireland, between July 2021 and June 2022. Two-channel continuous EEG was recorded from children (1) aged <16 years and (2) with Glasgow Coma Scale <11 or a reduction in baseline Glasgow Coma Scale in the case of a child with a neurodisability. RESULTS: Twenty patients were included. The median age at presentation was 65.8 months (interquartile range, 23.2 to 119.0); 50% had a background diagnosis of epilepsy. The most common reason for EEG monitoring was status epilepticus (85%) followed by suspected nonconvulsive status (10%) and reduced consciousness of unknown etiology (5%). The mean length of recording was 93.1 minutes (S.D. 47.4). The mean time to application was 41.3 minutes (S.D. 11.7). The mean percent of artifact in all recordings was 19.3% (S.D. 15.9). Thirteen (65%) EEGs had <25% artifact. Artifact was higher in cases in which active airway management was ongoing. CONCLUSIONS: EEG monitoring can be achieved in a pediatric emergency department setting within one hour of presentation. Overall, artifact percentage was low outside of periods of airway manipulation. Future studies are required to determine its use in early seizure detection and its support role in clinical decision-making in these patients.

Anatomical Diversity of the Adult Corticospinal Tract Revealed by Single-Cell Transcriptional Profiling.

The corticospinal tract (CST) forms a central part of the voluntary motor apparatus in all mammals. Thus, injury, disease, and subsequent degeneration within this pathway result in chronic irreversible functional deficits. Current strategies to repair the damaged CST are suboptimal in part because of underexplored molecular heterogeneity within the adult tract. Here, we combine spinal retrograde CST tracing with single-cell RNA sequencing (scRNAseq) in adult male and female mice to index corticospinal neuron (CSN) subtypes that differentially innervate the forelimb and hindlimb. We exploit publicly available datasets to confer anatomic specialization among CSNs and show that CSNs segregate not only along the forelimb and hindlimb axis but also by supraspinal axon collateralization. These anatomically defined transcriptional data allow us to use machine learning tools to build classifiers that discriminate between CSNs and cortical layer 2/3 and nonspinally terminating layer 5 neurons in M1 and separately identify limb-specific CSNs. Using these tools, CSN subtypes can be differentially identified to study postnatal patterning of the CST in vivo, leveraged to screen for novel limb-specific axon growth survival and growth activators in vitro, and ultimately exploited to repair the damaged CST after injury and disease.SIGNIFICANCE STATEMENT Therapeutic interventions designed to repair the damaged CST after spinal cord injury have remained functionally suboptimal in part because of an incomplete understanding of the molecular heterogeneity among subclasses of CSNs. Here, we combine spinal retrograde labeling with scRNAseq and annotate a CSN index by the termination pattern of their primary axon in the cervical or lumbar spinal cord and supraspinal collateral terminal fields. Using machine learning we have confirmed the veracity of our CSN gene lists to train classifiers to identify CSNs among all classes of neurons in primary motor cortex to study the development, patterning, homeostasis, and response to injury and disease, and ultimately target streamlined repair strategies to this critical motor pathway.

COVID-19-Related Household Job Loss and Mental Health in a Nationwide United States Sample of Sexual Minority Adolescents.

Household job loss during COVID-19 constitutes a public health crisis. Research suggests associations between household job loss, harsher parenting practices, and mental health challenges in the general population. Sexual minority adolescents (SMA) face high rates of family stress and rejection, but evidence linking household job loss to SMA mental health is lacking. This study evaluated associations between household job loss, family rejection, and mental health with a national sample of SMA who were sheltering in place with families during the pandemic. SMA from an ongoing prospective study completed an online questionnaire between May 13-31, 2020. It was hypothesized that household job loss during the pandemic would be associated with elevated depressive and anxiety symptoms through family rejection. Household job loss during the pandemic was indirectly associated with SMA mental health through family rejection. These findings highlight how socioeconomic change and policy carry implications for SMA health.

Changes in Immune Function during Initial Military Training.

PURPOSE: Initial military training (IMT) is a transitionary period wherein immune function may be suppressed and infection risk heightened due to physical and psychological stress, communal living, and sleep deprivation. This study characterized changes in biomarkers of innate and adaptive immune function, and potential modulators of those changes, in military recruits during IMT. METHODS: Peripheral leukocyte distribution and mitogen-stimulated cytokine profiles were measured in fasted blood samples, Epstein-Barr (EBV), varicella zoster (VZV), and herpes simplex 1 (HSV1) DNA was measured in saliva by quantitative polymerase chain reaction as an indicator of latent herpesvirus reactivation, and diet quality was determined using the healthy eating index measured by food frequency questionnaire in 61 US Army recruits (97% male) at the beginning (PRE) and end (POST) of 22-wk IMT. RESULTS: Lymphocytes and terminally differentiated cluster of differentiation (CD)4+ and CD8+ T cells increased PRE to POST, whereas granulocytes, monocytes, effector memory CD4+ and CD8+ T cells, and central memory CD8+ T cells decreased ( P ≤ 0.02). Cytokine responses to anti-CD3/CD28 stimulation were higher POST compared with PRE, whereas cytokine responses to lipopolysaccharide stimulation were generally blunted ( P < 0.05). Prevalence of EBV reactivation was higher at POST ( P = 0.04), but neither VZV nor HSV1 reactivation was observed. Diet quality improvements were correlated with CD8+ cell maturation and blunted proinflammatory cytokine responses to anti-CD3/CD28 stimulation. CONCLUSIONS: Lymphocytosis, maturation of T-cell subsets, and increased T-cell reactivity were evident POST compared with PRE IMT. Although EBV reactivation was more prevalent at POST, no evidence of VZV or HSV1 reactivation, which are more common during severe stress, was observed. Findings suggest increases in the incidence of EBV reactivation were likely appropriately controlled by recruits and immune-competence was not compromised at the end of IMT.

Explaining behavioral health differences in urban and rural sexual minority adolescents: A longitudinal investigation of minority stress in a diverse national sample of sexual minority adolescents: A longitudinal investigation of minority stress in a diverse national sample of sexual minority adolescents.

PURPOSE: Experiences of sexuality-based discrimination (ie, minority stressors) against youth who identify as nonheterosexual (ie, sexual minority) have been associated with increased symptoms of anxiety, depression, and post-traumatic stress disorder (PTSD) for sexual minority adolescents (SMA; ages 14-17). However, little is known about the experiences of SMA living in rural communities across the United States. Thus, the present study sought to examine differences in mental health patterns between urban and rural dwelling SMA, and to see whether these differences are, at least in part, explained by experiences of lifetime minority stress. METHODS: A nationwide sample of SMA residing in the United States (N = 2,558; aged 14-17, M = 15.90 years, SD = 0.98) was recruited through purposive social media and respondent-driven sampling methods to complete a cross-sectional survey online. Measures included those of minority stress, urbanicity, and symptoms of anxiety, depression, and PTSD. Parallel multiple mediation (PMM) analysis was employed to test whether urbanicity was associated with anxiety, depressive, and PTSD symptoms through reported lifetime minority stress. FINDINGS: On average, SMA living in rural areas significantly reported more lifetime minority stress, depressive, and PTSD symptoms than SMA living in urban settings. Results from our PMM analysis indicated that heightened experiences of lifetime minority stress indirectly linked the effects of living in rural areas on anxiety (b = -0.288, 95% CI = [-0.491, -0.085]), depressive (b = -0.158, 95% CI = [-0.270, -0.047), and PTSD symptoms PTSD (b = -0.349, 95% CI = [-0.596, -0.105]). The model accounted for 16.8%, 18%, and 24.1% of the variability in anxiety symptoms, depressive symptoms, and PTSD symptoms, respectively. CONCLUSIONS: SMA in our study who reside in rural areas reported elevated minority stress, depressive, and PTSD symptoms as compared to their urban dwelling peers. Our study found that lifetime experiences of minority stress fully mediated the relationship between urbanicity and both depressive and PTSD symptoms, and partially mediated the relationship between urbanicity and anxiety. These findings highlight the need to increase support for rural youth who are growing into adulthood and may find continuing challenges in their family, peer, and community relationships.

Effects of SARS-CoV-2 Infection on Attention, Memory, and Sensorimotor Performance.

Background: Recovery after SARS-CoV-2 infection is extremely variable, with some individuals recovering quickly, and others experiencing persistent long-term symptoms or developing new symptoms after the acute phase of infection, including fatigue, poor concentration, impaired attention, or memory deficits. Many existing studies reporting cognitive deficits associated with SARS-CoV-2 infection are limited by the exclusive use of self-reported measures or a lack of adequate comparison groups. Methods: Forty-five participants, ages 18-70, (11 Long-COVID, 14 COVID, and 20 No-COVID) underwent behavioral testing with the NIH Toolbox Neuro-Quality of Life survey and selected psychometric tests, including a flanker interference task and the d2 Test of Attention. Results: We found greater self-reported anxiety, apathy, fatigue, emotional dyscontrol, sleep disturbance and cognitive dysfunction in COVID compared No-COVID groups. After categorizing COVID patients according to self-reported concentration problems, we observed declining performance patterns in multiple attention measures across No-COVID controls, COVID and Long-COVID groups. COVID participants, compared to No-COVID controls, exhibited worse performance on NIH Toolbox assessments, including the Eriksen Flanker, Nine-Hole Pegboard and Auditory Verbal Learning tests. Conclusion: This study provides convergent evidence that previous SARS-CoV-2 infection is associated with impairments in sustained attention, processing speed, self-reported fatigue and concentration. The finding that some patients have cognitive and visuomotor dysfunction in the absence of self-reported problems suggests that SARS-CoV-2 infection can have unexpected and persistent subclinical consequences.

Initial military training modulates serum fatty acid and amino acid metabolites.

Initial military training (IMT) results in increased fat-free mass (FFM) and decreased fat mass (FM). The underlying metabolic adaptations facilitating changes in body composition during IMT are unknown. The objective of this study was to assess changes in body composition and the serum metabolome during 22-week US Army IMT. Fifty-four volunteers (mean ± SD; 22 ± 3 year; 24.6 ± 3.7 kg/m2 ) completed this longitudinal study. Body composition measurements (InBody 770) and blood samples were collected under fasting, rested conditions PRE and POST IMT. Global metabolite profiling was performed to identify metabolites involved in energy, carbohydrate, lipid, and protein metabolism (Metabolon, Inc.). There was no change in body mass (POST-PRE; 0.4 ± 5.1 kg, p = 0.59), while FM decreased (-1.7 ± 3.5 kg, p < 0.01), and FFM increased (2.1 ± 2.8 kg, p < 0.01) POST compared to PRE IMT. Of 677 identified metabolites, 340 differed at POST compared to PRE (p < 0.05, Q < 0.10). The majority of these metabolites were related to fatty acid (73%) and amino acid (26%) metabolism. Increases were detected in 41% of branched-chain amino acid metabolites, 53% of histidine metabolites, and 35% of urea cycle metabolites. Decreases were detected in 93% of long-chain fatty acid metabolites, while 58% of primary bile acid metabolites increased. Increases in amino acid metabolites suggest higher rates of protein turnover, while changes in fatty acid metabolites indicate increased fat oxidation, which likely contribute changes in body composition during IMT. Overall, changes in metabolomics profiles provide insight into metabolic adaptions underlying changes in body composition during IMT.

Refusal Rates to Sexual Orientation and Gender Identity Items in the Behavioral Risk Factor Surveillance System, 2014-2019.

Objectives. To explore trends in sexual orientation and gender identity (SOGI) item refusal in the Behavioral Risk Factor Surveillance System (BRFSS). Methods. We used annual data from 7 US states that implemented the SOGI module of the BRFSS from 2014 to 2019 to examine prevalence of sexual orientation (n = 373 332) and gender identity (n = 373 336) item refusal. Analyses included the weighted Wald χ2 test of association between refusal and year and logistic regressions predicting refusal by year. We weighted analyses to account for complex sampling design. Results. We found low SOGI item refusal rates, significant declines in these refusal rates over time, and differences in refusal rates by age, sex, race, education, and language. We also found that Hispanic group membership did not explain SOGI item refusal when accounting for interview language; interview language was strongly associated with both sexual orientation and gender identity item refusal. Conclusions. Our results indicate acceptance of SOGI measurement and empirically support continuation of SOGI in health surveillance surveys. Findings indicate a need to further investigate the association between survey translation and item refusal.

Quality improvement project to decrease unnecessary investigations in infants with bronchiolitis in Cork University Hospital.

BACKGROUND: Bronchiolitis is a common reason for infants to present to the emergency department (ED). Clear evidence-based guidelines exist that recommend against routine radiological and laboratory investigations in this cohort. Despite this, preintervention audit showed that children below 12 months of age with bronchiolitis in the ED during November 2018-January 2019 were receiving unnecessary investigations. Our aim was to improve patient care by decreasing unnecessary investigations in bronchiolitis infants. METHODS: Baseline assessment comprised a preintervention audit of children less than 12 months of age with a diagnosis of bronchiolitis that presented to ED during November 2018-January 2019. The outcome measure was average weekly hospital length of stay (LOS), process measures were average weekly chest X-ray (CXR) and laboratory investigation rate. The balancing measure was the average weekly representation rate. INTERVENTION: A multimodal intervention was implemented comprising a locally agreed flowchart enhanced by regular feedback on performance using run charts and in-person sessions. RESULTS: A postintervention audit of November 2019-January 2020 was undertaken. There was a 57% reduction in the mean average weekly CXR rate (from 25% to 11%, p value 0.009974 significant at p<0.05); there was an improvement by 56% in the mean average weekly laboratory investigation rate (from 29% to 13%, p value 0.005475, significant at p<0.05) in the preintervention and postintervention periods, respectively. The mean average weekly representations remained at 4% preintervention and postintervention (p value 0.737). There was no significant difference in hospital LOS (from 25.3 hours to 20.7 hours, p value 0.270549). CONCLUSION: An evidence-based protocol improved physicians' ability in diagnosing and managing infants with bronchiolitis. This led to a reduction in unnecessary and potential harmful investigations, thereby improving patient quality of care. This improvement will contribute to decreased healthcare cost and appropriate use of resources during the high-pressured winter period.

Development of a reference standard for the detection and quantification of Mycobacterium avium subsp. paratuberculosis by quantitative PCR.

Quantitative PCR (qPCR) has become a frequently employed direct method for the detection and quantification of Mycobacterium avium subsp. paratuberculosis (MAP). The quantity of MAP determined by qPCR, however, may be affected by the type of qPCR quantification standard used (PCR product, plasmid, genomic DNA) and the way in which standard DNA quantity is determined (absorbance, fluorescence). In practice, this can be reflected in the inability to properly compare quantitative data from the same qPCR assays in different laboratories. Thus, the aim of this study was to prepare a prototype of an international MAP reference standard, which could be used to calibrate routinely used qPCR quantification standards in various laboratories to promote clinical data comparability. Considering stability, storage and shipment issues, a lyophilised fecal suspension artificially contaminated with a MAP reference strain was chosen as the most suitable form of the standard. The effect of five types of lyophilisation matrices on standard stability was monitored on 2-weeks interval basis for 4 months by F57 qPCR. The lyophilisation matrix with 10% skimmed milk provided the best recovery and stability in time and was thus selected for subsequent comparative testing of the standard involving six diagnostic and research laboratories, where DNA isolation and qPCR assay procedures were performed with the parallel use of the identical supplied genomic DNA solution. Furthermore, the effect of storage conditions on the standard stability was tested for at least 6 months. The storage at room temperature in the dark and under light, at + 4 °C, - 20 °C and - 80 °C showed no significant changes in the stability, and also no substantial changes in MAP viability were found using phage amplification assay. The prepared MAP quantification standard provided homogeneous and reproducible results demonstrating its suitability for utilisation as an international reference qPCR standard.

"Trying My Best": Sexual Minority Adolescents' Self-Care During the COVID-19 Pandemic.

PURPOSE: The COVID-19 pandemic has resulted in major life disruptions for sexual minority adolescents (SMAs), who already face and cope with pervasive and disproportionate rates of social, behavioral, and mental health challenges. Current research suggests that SMAs are struggling with COVID-19-related shelter in place orders navigating family proximity and dynamics and experiencing isolation from SMA-specific supports. Given identified challenges that may exacerbate known mental health disparities in SMAs, this work explores self-care practices among SMAs during the COVID-19 pandemic. METHODS: The present study uses data from open-ended questions to understand SMA experiences of self-care within a nationwide sample of SMAs (N = 770; M = 17.48 years, SD = 1.00) who are part of an ongoing prospective study. Data were collected via online questionnaire between May 13 and 31, 2020. Thematic analysis guided data exploration. RESULTS: Thematic analysis revealed five self-care practices among SMAs: (1) relationships, (2) routines, (3) body and mind, (4) rest and reset, and (5) tuning out. SMAs engaged in many positive coping strategies (i.e., exercise, establishing routine) and often linked these activities to positive well-being. Other SMAs engaged in activities to distract or disengage from stressors (i.e., excessive TV and alcohol and drug use). CONCLUSIONS: These findings highlight the resiliency of SMAs during the current pandemic, opportunities for providers to emphasize adaptive coping skills with youths, and the need for more research on adolescent self-care practices.

Paediatric attendances of the emergency department in a major Irish tertiary referral centre before and after expansion of free GP care to children under 6: a retrospective observational study.

Objectives: To examine the characteristics of paediatric attendances to the emergency department (ED) in Cork University Hospital (CUH) before and after the expansion of free general practitioner (GP) care to children under the age of 6 years. Design: This is a retrospective observational study that used a large administrative dataset. Setting: The study was conducted in major Irish tertiary referral centre that serves a total population of over 1.1 million. It is a public hospital, owned and managed by the health service executive. Participants: Children aged 0-15 years who attended CUH ED during the study period of 6 years (2012-2018) were included in this study (n=76 831). Interventions: Free GP care was expanded to all children aged 0-5 years in July 2015. Main outcome measures: Paediatric attendances to CUH ED were examined before (Time Period 1: July 2012-June 2015) and after (Time Period 2: July 2015-June 2018) the expansion of free GP care to children under 6. Changes in GP referral rates and inpatient hospital admissions were investigated. Results: Paediatric presentations to CUH ED increased from 35 819 during the Time Period 1 to 41 012 during the Time Period 2 (14.5%). The proportion of the CUH ED attendances through GP referrals by children under 6 increased by over 8% in the Time Period 2 (from 10 148 to 14 028). Although the number of all children who attended CUH ED and were admitted to hospital increased in Time Period 2 (from 8704 to 9320); the proportion of children in the 0-5 years group who attended the CUH ED through GP referral and were subsequently admitted to hospital, decreased by over 3%. Conclusion: The expansion of free GP care has upstream health service utilisation implications, such as increased attendances at ED, and should be considered and costed by policy-makers.

Uncoupling Molecular Testing for SARS-CoV-2 From International Supply Chains.

The rapid global rise of COVID-19 from late 2019 caught major manufacturers of RT-qPCR reagents by surprise and threw into sharp focus the heavy reliance of molecular diagnostic providers on a handful of reagent suppliers. In addition, lockdown and transport bans, necessarily imposed to contain disease spread, put pressure on global supply lines with freight volumes severely restricted. These issues were acutely felt in New Zealand, an island nation located at the end of most supply lines. This led New Zealand scientists to pose the hypothetical question: in a doomsday scenario where access to COVID-19 RT-qPCR reagents became unavailable, would New Zealand possess the expertise and infrastructure to make its own reagents onshore? In this work we describe a review of New Zealand's COVID-19 test requirements, bring together local experts and resources to make all reagents for the RT-qPCR process, and create a COVID-19 diagnostic assay referred to as HomeBrew (HB) RT-qPCR from onshore synthesized components. This one-step RT-qPCR assay was evaluated using clinical samples and shown to be comparable to a commercial COVID-19 assay. Through this work we show New Zealand has both the expertise and, with sufficient lead time and forward planning, infrastructure capacity to meet reagent supply challenges if they were ever to emerge.

Estimation of the sensitivity and specificity of four serum ELISA and one fecal PCR for diagnosis of paratuberculosis in adult dairy cattle in New Zealand using Bayesian latent class analysis.

In New Zealand, a new diagnostic approach for the control of paratuberculosis in mixed aged milking cows has been developed using a combination of ELISA and quantitative fecal PCR (f-qPCR). Our analysis was designed to evaluate performance of these individual tests in infected or infectious mixed aged cows across the prevalence of infection typically encountered on NZ dairy farms and calculate test accuracy when used as a screening test of serological ELISAs for four separate antigens read in parallel followed by a confirmatory quantitative f-qPCR test. Data from a cross-sectional study of 20 moderate prevalence herds was combined with existing data from 2 low and 20 high prevalence herds forming a dataset of 3845 paired serum and fecal samples. Incidence of clinical Johne's disease (JD) was used to classify herds into three prevalence categories. High (≥ 3% annual clinical JD for the last three years), moderate (<3 - 1%) and low (<1% incidence for at least the last five years). Positive tests were declared if> 50 ELISA units and f-qPCR at two cut-points (≥1 × 104 genomes/mL or >1 × 103 genomes/mL). Fixed Bayesian latent class models at both f-qPCR cut-points, accounted for conditional independence and paired conditional dependence. Mixed models at both f-qPCR cut-points, using a different mechanism to account for conditional dependencies between tests were also implemented. Models (24 in number) were constructed using OpenBUGS. The aim was to identify Mycobacterium avium subsp. paratuberculosis (MAP) infected cows that met at least one of two criteria: shedding sufficient MAP in feces to be detected by f-qPCR or mounting a detectable MAP antibody response. The best fit to the data was obtained by modelling pairwise dependencies between tests in a fixed model or by accounting for dependencies in a mixed model at a fecal cut-off of ≥1 × 104 genomes/mL. Test performance differed with prevalence, but models were robust to prior assumptions. For the fixed model, at a prevalence of 0.29 (95 % probability interval (PI) = 0.25-0.33), as a screening plus confirmatory f-qPCR, post-test probability for disease in a positive animal was 0.84 (95 %PI = 0.80-0.88) and 0.16 (95 %PI = 0.15-0.18) for disease in a test negative animal. In low prevalence herds (0.01(95 %PI = 0.00-0.04)) the equivalent figures were 0.84 (95 %PI = 0.08-0.92) and 0.00 (95 %PI = 0.00-0.02). These results suggest this is a useful tool to control JD on dairy farms, particularly in herds with higher levels of infection, where the sampling and testing cost per animal is defrayed across more detected animals.

Bioluminescent flashes drive nighttime schooling behavior and synchronized swimming dynamics in flashlight fish.

Schooling fishes, like flocking birds and swarming insects, display remarkable behavioral coordination. While over 25% of fish species exhibit schooling behavior, nighttime schooling has rarely been observed or reported. This is due to vision being the primary modality for schooling, which is corroborated by the fact that most fish schools disperse at critically low light levels. Here we report on a large aggregation of the bioluminescent flashlight fish Anomalops katoptron that exhibited nighttime schooling behavior during multiple moon phases, including the new moon. Data were recorded with a suite of low-light imaging devices, including a high-speed, high-resolution scientific complementary metal-oxide-semiconductor (sCMOS) camera. Image analysis revealed nighttime schooling using synchronized bioluminescent flashing displays, and demonstrated that school motion synchrony exhibits correlation with relative swim speed. A computer model of flashlight fish schooling behavior shows that only a small percentage of individuals need to exhibit bioluminescence in order for school cohesion to be maintained. Flashlight fish schooling is unique among fishes, in that bioluminescence enables schooling in conditions of no ambient light. In addition, some members can still partake in the school while not actively exhibiting their bioluminescence. Image analysis of our field data and model demonstrate that if a small percentage of fish become motivated to change direction, the rest of the school follows. The use of bioluminescence by flashlight fish to enable schooling in shallow water adds an additional ecological application to bioluminescence and suggests that schooling behavior in mesopelagic bioluminescent fishes may be also mediated by luminescent displays.

Control of Mycobacterium avium subsp. paratuberculosis infection on a New Zealand pastoral dairy farm.

BACKGROUND: Johne's disease is a major production limiting disease of dairy cows caused by infection with Mycobacterium avium subsp. paratuberculosis in calf-hood. The disease is chronic, progressive, contagious and widespread with no treatment and no cure. Economic losses arise from decreased productivity through reduced growth, milk yield, fertility and also capital losses due to premature culling or death. Control chiefly centers upon removing those animals which actively shed bacteria and protecting calves from infection. A prolonged pre-clinical shedding phase, lack of test sensitivity, organism persistence and abundance in the environment as well as management systems that expose susceptible calves to infection make control challenging, particularly in pastoral, seasonal dairy systems. Combining a novel testing strategy to remove infected cows along with limited measures to protect vulnerable calves at pasture, this study reports the successful reduction over a four-year period of seroprevalence of cows testing positive for MAP infection in a New Zealand pastoral dairy herd. RESULTS: For all age groups considered the apparent seroprevalence of cows testing positive decreased from 297 / 1,122 (26%) in 2013-2014, to 24 / 1,030 (2.3%) in 2016-2017. Over the same period, the apparent seroprevalence in primiparous cows decreased from 39 / 260 (15%) to 7 / 275 (2.5%) and in multiparous cows from 258 / 862 (29.9%) to 17 / 755 (2.3%). The reported proportion of calved cows culled annually from suspected clinical Johne's disease fell from 55 / 1,201 (5%) in the year preceding the control program to 5 / 1,283 (0.4%) in the final year of the study. CONCLUSIONS: On this farm, reduction in the prevalence of infection was achieved by reducing the infectious pressure through targeted culling of heavily shedding animals together with limited measures to protect calves at pasture from exposure to Mycobacterium avium subsp. paratuberculosis. Whilst greater protection of young animals through separation from infected cows and their colostrum and milk would have reduced the risk of neonatal infection, this study demonstrates, in this case, that these management measures while prudent were not essential for effective reduction in the prevalence of MAP infection.

The effect of sub-clinical infection with Mycobacterium avium subsp. paratuberculosis on milk production in a New Zealand dairy herd.

BACKGROUND: Johne's disease is a major production limiting disease of dairy cows. The disease is chronic, progressive, contagious and widespread; there is no treatment and there is no cure. Economic losses arise from decreased productivity through reduced growth, milk yield and fertility and capital losses due to premature culling or death. This study attempts to address the effect of subclinical JD on milk production under New Zealand pastoral dairy farming conditions using a new testing approach. Blood samples were taken from all lactating animals from a single seasonally calving New Zealand dairy herd in the autumn of 2013 and 2014. Samples were subject to serological assay for antibodies to Mycobacterium avium subsp. paratuberculosis using a combination of four ELISA tests in parallel followed by selective quantitative fecal PCR to confirm the fecal shedding characteristics of ELISA positive cows. ELISA status was classified as Not-Detected, Low, Moderate or High and fecal PCR status as Not-Detected, Moderate or High. RESULTS: A mixed generalized regression model indicated that, compared to cows where MAP was not detected, daily milk solids production was 4% less for high ELISA positive cows (p = 0.004), 6% less for moderate fPCR cows (p = 0.036) and 12% less for high fPCR cows (p < 0.001). CONCLUSIONS: This study confirms that sub-clinical JD can have a significant impact on milk production and that the testing methodology used stratified the animals in this herd on their likely impact on production and disease spread. This allowed the farmer to prioritize removal of heavily shedding, less-productive animals and so reduce the risk of infection of young stock. This is the first longitudinal study based in New Zealand looking at the effect of Johne's infection status on daily milk production allowing for intermediary and confounding factors.

Prevalence of Shiga toxin-producing and enteropathogenic Escherichia coli marker genes in diarrhoeic stools in a New Zealand catchment area.

BACKGROUND: Shiga toxin-producing (STEC) and enteropathogenic (EPEC) Escherichia coli are gastrointestinal pathogens causing diarrhoeal and extraintestinal disease. Due to lack of EPEC screening and use of Sorbitol-MacConkey (SMAC) agar in faecal screening, the true prevalence of EPEC and non-O157 STEC in New Zealand diarrhoeal cases is unknown. METHODS: Diarrhoeic stools sourced from Dunedin hospital were pre-enriched, DNA extracted with Chelex-100 resin and screened using a multiplex TaqMan quantitative PCR assay amplifying stx1, sxt2 and EPEC (eae) gene markers. RESULTS: Of the 522 diarrhoeic samples surveyed, 8 (1.53%) were PCR positive for stx1/stx2 and 23 (4.41%) were positive for eae. Six (75%) of the stx+ samples were uncommon non-O157 serotypes, and the remainder were found to be positive for both O103 and O157 STEC somatic antigens. CONCLUSIONS: Results revealed shortcomings in current screening protocols for pathogenic E. coli; SMAC is not sufficiently discriminatory to detect emergent STEC serotypes and EPEC likely has an unappreciated role in cases of diarrhoea in New Zealand.