Electric Field Navigated 1-Hz rTMS for Poststroke Motor Recovery: The E-FIT Randomized Controlled Trial.

BACKGROUND: To determine if low-frequency repetitive transcranial magnetic stimulation targeting the primary motor cortex contralateral (M1CL) to the affected corticospinal tract in patients with hemiparetic stroke augments intensive training-related clinical improvement; an extension of the NICHE trial (Navigated Inhibitory rTMS to Contralesional Hemisphere Trial) using an alternative sham coil. METHODS: The present E-FIT trial (Electric Field Navigated 1Hz rTMS for Post-stroke Motor Recovery Trial) included 5 of 12 NICHE trial outpatient US rehabilitation centers. The stimulation protocol remained identical (1 Hz repetitive transcranial magnetic stimulation, M1CL, preceding 60-minute therapy, 18 sessions/6 wks; parallel arm randomized clinical trial). The sham coil appearance mimicked the active coil but without the weak electric field in the NICHE trial sham coil. Outcomes measured 1 week, and 1, 3, and 6 months after the end of treatment included the following: upper extremity Fugl-Meyer (primary, 6 months after end of treatment), Action Research Arm Test, National Institutes of Health Stroke Scale, quality of life (EQ-5D), and safety. RESULTS: Of 60 participants randomized, 58 completed treatment and were included for analysis. Bayesian analysis of combined data from the E-FIT and the NICHE trials indicated that active treatment was not superior to sham at the primary end point (posterior mean odds ratio of 1.94 [96% credible interval of 0.61-4.80]). For the E-FIT intent-to-treat population, upper extremity Fugl-Meyer improvement ≥5 pts occurred in 60% (18/30) active group and 50% (14/28) sham group. Participants enrolled 3 to 6 months following stroke had a 67% (31%-91% CI) response rate in the active group at the 6-month end point versus 50% in the sham group (21.5%-78.5% CI). There were significant improvements from baseline to 6 months for both active and sham groups in upper extremity Fugl-Meyer, Action Research Arm Test, and EQ-5D (P<0.05). Improvement in National Institutes of Health Stroke Scale was observed only in the active group (P=0.004). Ten serious unrelated adverse events occurred (4 active group, 6 sham group, P=0.72). CONCLUSIONS: Intensive motor rehabilitation 3 to 12 months after stroke improved clinical impairment, function, and quality of life; however, 1 Hz-repetitive transcranial magnetic stimulation was not an effective treatment adjuvant in the present sample population with mixed lesion location and extent. REGISTRATION: URL: https://www. CLINICALTRIALS: gov; Unique identifier: NCT03010462.

Whole-Genome Sequencing and Virulome Analysis of Escherichia coli Isolated from New Zealand Environments of Contrasting Observed Land Use.

Generic Escherichia coli is commonly used as an indicator of fecal contamination to assess water quality and human health risk. Where measured E. coli exceedances occur, the presence of other pathogenic microorganisms, such as Shiga toxin-producing E. coli (STEC), is assumed, but confirmatory data are lacking. Putative E. coli isolates (n = 709) were isolated from water, sediment, soil, periphyton, and feces samples (n = 189) from five sites representing native forest and agricultural environments. Ten E. coli isolates (1.41%) were stx2 positive, 19 (2.7%) were eae positive, and stx1-positive isolates were absent. At the sample level, stx2-positive E. coli (5 of 189, 2.6%) and eae-positive isolates (16 of 189, 8.5%) were rare. Using real-time PCR, these STEC-associated virulence factors were determined to be more prevalent in sample enrichments (stx1, 23.9%; stx2, 31.4%; eae, 53.7%) and positively correlated with generic E. coli isolate numbers (P < 0.05) determined using culture-based methods. Whole-genome sequencing (WGS) was undertaken on a subset of 238 isolates with assemblies representing seven E. coli phylogroups (A, B1, B2, C, D, E, and F), 22 Escherichia marmotae isolates, and 1 Escherichia ruysiae isolate. Virulence factors, including those from extraintestinal pathogenic E. coli, were extremely diverse in isolates from the different locations and were more common in phylogroup B2. Analysis of the virulome from WGS data permitted the identification of gene repertoires that may be involved in environmental fitness and broadly align with phylogroup. Although recovery of STEC isolates was low, our molecular data indicate that they are likely to be widely present in environmental samples containing diverse E. coli phylogroups. IMPORTANCE This study takes a systematic sampling approach to assess the public health risk of Escherichia coli recovered from freshwater sites within forest and farmland. The New Zealand landscape is dominated by livestock farming, and previous work has demonstrated that "recreational exposure to water" is a risk factor for human infection by Shiga toxin-producing Escherichia coli (STEC). Though STEC isolates were rarely isolated from water samples, STEC-associated virulence factors were identified more commonly from water sample culture enrichments and were associated with increased generic E. coli concentrations. Whole-genome sequencing data from both E. coli and newly described Escherichia spp. demonstrated the presence of virulence factors from E. coli pathotypes, including extraintestinal pathogenic E. coli. This has significance for understanding and interpreting the potential health risk from E. coli where water quality is poor and suggests a role of virulence factors in survival and persistence of E. coli and Escherichia spp.

Transmission Dynamics of Shiga Toxin-Producing Escherichia coli in New Zealand Cattle from Farm to Slaughter.

Cattle are asymptomatic carriers of Shiga toxin-producing Escherichiacoli (STEC) strains that can cause serious illness or death in humans. In New Zealand, contact with cattle feces and living near cattle populations are known risk factors for human STEC infection. Contamination of fresh meat with STEC strains also leads to the potential for rejection of consignments by importing countries. We used a combination of PCR/matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) and whole-genome sequencing (WGS) to evaluate the presence and transmission of STEC on farms and in processing plants to better understand the potential pathways for human exposure and thus mitigate risk. Animal and environmental samples (n = 2,580) were collected from six farms and three meat processing plants in New Zealand during multiple sampling sessions in spring of 2015 and 2016. PCR/MALDI-TOF analysis revealed that 6.2% were positive for "Top 7" STEC. Top 7 STEC strains were identified in all sample sources (n = 17) tested. A marked increase in Top 7 STEC prevalence was observed between calf hides on farm (6.3% prevalence) and calf hides at processing plants (25.1% prevalence). Whole-genome sequencing was performed on Top 7 STEC bacterial isolates (n = 40). Analysis of STEC O26 (n = 25 isolates) revealed relatively low genetic diversity on individual farms, consistent with the presence of a resident strain disseminated within the farm environment. Public health efforts should focus on minimizing human contact with fecal material on farms and during handling, transport, and slaughter of calves. Meat processing plants should focus on minimizing cross-contamination between the hides of calves in a cohort during transport, lairage, and slaughter.IMPORTANCE Cattle are asymptomatic carriers of Shiga toxin-producing E. coli (STEC) strains, which can cause serious illness or death in humans. Contact with cattle feces and living near cattle are known risk factors for human STEC infection. This study evaluated STEC carriage in young calves and the farm environment with an in-depth evaluation of six farms and three meat processing plants over 2 years. An advanced molecular detection method and whole-genome sequencing were used to provide a detailed evaluation of the transmission of STEC both within and between farms. The study revealed widespread STEC contamination within the farm environment, but no evidence of recent spread between farms. Contamination of young dairy calf hides increased following transport and holding at meat processing plants. The elimination of STEC in farm environments may be very difficult given the multiple transmission routes; interventions should be targeted at decreasing fecal contamination of calf hides during transport, lairage, and processing.

Campylobacter novaezeelandiae sp. nov., isolated from birds and water in New Zealand.

Six isolates of Campylobacter with similar non-standard colonial morphologies were identified during studies isolating Campylobacter from bird faeces and rivers in New Zealand. Genomic (16S rRNA gene sequencing and whole genome analysis) and phenotypic (MALDI-TOF analysis and conventional biochemical tests) showed that the isolates form a monophyletic clade with genetic relationships to Campylobacter coli/Campylobacter jejuni and Campylobacter peloridis/Campylobacter amoricus. They may be distinguished from other Campylobacter by their MALDI-TOF spectral pattern, their florid α-haemolysis, their ability to grow anaerobically at 37 °C, and on 2 % NaCl nutrient agar, and their lack of hippuricase. This study shows that these isolates represent a novel species within the genus Campylobacter for which the name Campylobacter novaezeelandiae sp. nov. is proposed. The presence of C. novaezeelandiae in water may be a confounder for freshwater microbial risk assessment as they may not be pathogenic for humans. The type strain is B423bT (=NZRM 4741T=ATCC TSD-167T).

Genomic Analysis of Fluoroquinolone- and Tetracycline-Resistant Campylobacter jejuni Sequence Type 6964 in Humans and Poultry, New Zealand, 2014-2016.

In 2014, antimicrobial drug-resistant Campylobacter jejuni sequence type 6964 emerged contemporaneously in poultry from 3 supply companies in the North Island of New Zealand and as a major cause of campylobacteriosis in humans in New Zealand. This lineage, not previously identified in New Zealand, was resistant to tetracycline and fluoroquinolones. Genomic analysis revealed divergence into 2 major clades; both clades were associated with human infection, 1 with poultry companies A and B and the other with company C. Accessory genome evolution was associated with a plasmid, phage insertions, and natural transformation. We hypothesize that the tetO gene and a phage were inserted into the chromosome after conjugation, leaving a remnant plasmid that was lost from isolates from company C. The emergence and rapid spread of a resistant clone of C. jejuni in New Zealand, coupled with evolutionary change in the accessory genome, demonstrate the need for ongoing Campylobacter surveillance among poultry and humans.

Use of Genomics to Investigate Historical Importation of Shiga Toxin-Producing Escherichia coli Serogroup O26 and Nontoxigenic Variants into New Zealand.

Shiga toxin-producing Escherichia coli serogroup O26 is an important public health pathogen. Phylogenetic bacterial lineages in a country can be associated with the level and timing of international imports of live cattle, the main reservoir. We sequenced the genomes of 152 E. coli O26 isolates from New Zealand and compared them with 252 E. coli O26 genomes from 14 other countries. Gene variation among isolates from humans, animals, and food was strongly associated with country of origin and stx toxin profile but not isolation source. Time of origin estimates indicate serogroup O26 sequence type 21 was introduced at least 3 times into New Zealand from the 1920s to the 1980s, whereas nonvirulent O26 sequence type 29 strains were introduced during the early 2000s. New Zealand's remarkably fewer introductions of Shiga toxin-producing Escherichia coli O26 compared with other countries (such as Japan) might be related to patterns of trade in live cattle.

Genome-by-genome approach for fast bacterial genealogical relationship evaluation.

Motivation: Large-scale whole-genome sequencing dataset-based studies are becoming increasingly common in pathogen surveillance and outbreak investigations. A highly discriminative and time-efficient bioinformatics tool is needed to transform large amounts of sequencing data into usable biological information. To replace the intuitive, yet inefficient, way of gene-by-gene allele calling algorithm, a new algorithm using genome-by-genome approach was developed. Results: Tests showed that the program equipped with the new algorithm achieved significant improvements in allele calling efficiency compared to a conventional gene-by-gene approach. The new program, Fast-GeP, rendered a fast and easy way to infer high-resolution genealogical relationships between bacterial isolates using whole-genome sequencing data. Availability and implementation: FAST-GeP is freely available from: https://github.com/jizhang-nz/fast-GeP. Supplementary information: Supplementary data are available at Bioinformatics online.

Randomized Sham-Controlled Trial of Navigated Repetitive Transcranial Magnetic Stimulation for Motor Recovery in Stroke.

Background and Purpose- We aimed to determine whether low-frequency electric field navigated repetitive transcranial magnetic stimulation to noninjured motor cortex versus sham repetitive transcranial magnetic stimulation avoiding motor cortex could improve arm motor function in hemiplegic stroke patients when combined with motor training. Methods- Twelve outpatient US rehabilitation centers enrolled participants between May 2014 and December 2015. We delivered 1 Hz active or sham repetitive transcranial magnetic stimulation to noninjured motor cortex before each of eighteen 60-minute therapy sessions over a 6-week period, with outcomes measured at 1 week and 1, 3, and 6 months after end of treatment. The primary end point was the percentage of participants improving ≥5 points on upper extremity Fugl-Meyer score 6 months after end of treatment. Secondary analyses assessed changes on the upper extremity Fugl-Meyer and Action Research Arm Test and Wolf Motor Function Test and safety. Results- Of 199 participants, 167 completed treatment and follow-up because of early discontinuation of data collection. Upper extremity Fugl-Meyer gains were significant for experimental ( P<0.001) and sham groups ( P<0.001). Sixty-seven percent of the experimental group (95% CI, 58%-75%) and 65% of sham group (95% CI, 52%-76%) improved ≥5 points on 6-month upper extremity Fugl-Meyer ( P=0.76). There was also no difference between experimental and sham groups in the Action Research Arm Test ( P=0.80) or the Wolf Motor Function Test ( P=0.55). A total of 26 serious adverse events occurred in 18 participants, with none related to the study or device, and with no difference between groups. Conclusions- Among patients 3 to 12 months poststroke, goal-oriented motor rehabilitation improved motor function 6 months after end of treatment. There was no difference between the active and sham repetitive transcranial magnetic stimulation trial arms. Clinical Trial Registration- URL: https://www.clinicaltrials.gov . Unique identifier: NCT02089464.

Associative Recognition Memory Awareness Improved by Theta-Burst Stimulation of Frontopolar Cortex.

Neuroimaging and lesion studies have implicated specific prefrontal cortex locations in subjective memory awareness. Based on this evidence, a rostrocaudal organization has been proposed whereby increasingly anterior prefrontal regions are increasingly involved in memory awareness. We used theta-burst transcranial magnetic stimulation (TBS) to temporarily modulate dorsolateral versus frontopolar prefrontal cortex to test for distinct causal roles in memory awareness. In three sessions, participants received TBS bilaterally to frontopolar cortex, dorsolateral prefrontal cortex, or a control location prior to performing an associative-recognition task involving judgments of memory awareness. Objective memory performance (i.e., accuracy) did not differ based on stimulation location. In contrast, frontopolar stimulation significantly influenced several measures of memory awareness. During study, judgments of learning were more accurate such that lower ratings were given to items that were subsequently forgotten selectively following frontopolar TBS. Confidence ratings during test were also higher for correct trials following frontopolar TBS. Finally, trial-by-trial correspondence between overt performance and subjective awareness during study demonstrated a linear increase across control, dorsolateral, and frontopolar TBS locations, supporting a rostrocaudal hierarchy of prefrontal contributions to memory awareness. These findings indicate that frontopolar cortex contributes causally to memory awareness, which was improved selectively by anatomically targeted TBS.

High-resolution genomic analysis to investigate the impact of the invasive brushtail possum (Trichosurus vulpecula) and other wildlife on microbial water quality assessments.

Escherichia coli are routine indicators of fecal contamination in water quality assessments. Contrary to livestock and human activities, brushtail possums (Trichosurus vulpecula), common invasive marsupials in Aotearoa/New Zealand, have not been thoroughly studied as a source of fecal contamination in freshwater. To investigate their potential role, Escherichia spp. isolates (n = 420) were recovered from possum gut contents and feces and were compared to those from water, soil, sediment, and periphyton samples, and from birds and other introduced mammals collected within the Makirikiri Reserve, Dannevirke. Isolates were characterized using E. coli-specific real-time PCR targeting the uidA gene, Sanger sequencing of a partial gnd PCR product to generate a gnd sequence type (gST), and for 101 isolates, whole genome sequencing. Escherichia populations from 106 animal and environmental sample enrichments were analyzed using gnd metabarcoding. The alpha diversity of Escherichia gSTs was significantly lower in possums and animals compared with aquatic environmental samples, and some gSTs were shared between sample types, e.g., gST535 (in 85% of samples) and gST258 (71%). Forty percent of isolates gnd-typed and 75% of reads obtained by metabarcoding had gSTs shared between possums, other animals, and the environment. Core-genome single nucleotide polymorphism (SNP) analysis showed limited variation between several animal and environmental isolates (<10 SNPs). Our data show at an unprecedented scale that Escherichia clones are shared between possums, other wildlife, water, and the wider environment. These findings support the potential role of possums as contributors to fecal contamination in Aotearoa/New Zealand freshwater. Our study deepens the current knowledge of Escherichia populations in under-sampled wildlife. It presents a successful application of high-resolution genomic methods for fecal source tracking, thereby broadening the analytical toolbox available to water quality managers. Phylogenetic analysis of isolates and profiling of Escherichia populations provided useful information on the source(s) of fecal contamination and suggest that comprehensive invasive species management strategies may assist in restoring not only ecosystem health but also water health where microbial water quality is compromised.

Draft genome sequences of Escherichia spp. isolates from New Zealand environmental sources.

Escherichia coli is often used as a fecal indicator bacterium for water quality monitoring. We report the draft genome sequences of 500 Escherichia isolates including newly described Escherichia species, namely Escherichia marmotae, Escherichia ruysiae, and Escherichia whittamii, obtained from diverse environmental sources to assist with improved public health risk assessments.

Development of a multiplex droplet digital PCR assay for simultaneous detection and quantification of Escherichia coli, E. marmotae, and E. ruysiae in water samples.

Escherichia coli are widely used by water quality managers as Fecal Indicator Bacteria, but current quantification methods do not differentiate them from benign, environmental Escherichia species such as E. marmotae (formerly named cryptic clade V) or E. ruysiae (cryptic clades III and IV). Reliable and specific techniques for their identification are required to avoid confounding microbial water quality assessments. To address this, a multiplex droplet digital PCR (ddPCR) assay targeting lipB (E. coli and E. ruysiae) and bglC (E. marmotae) was designed. The ddPCR performance was assessed using in silico analysis; genomic DNA from 40 local, international, and reference strains of target and non-target coliforms; and spiked water samples in a range relevant to water quality managers (1 to 1000 cells/100 mL). Results were compared to an analogous quantitative PCR (qPCR) and the Colilert method. Both PCR assays showed excellent sensitivity with a limit of detection of 0.05 pg/µL and 0.005 pg/µl for ddPCR and qPCR respectively, and of quantification of 0.5 pg/µL of genomic DNA. The ddPCR allowed differentiation and quantification of three Escherichia species per run by amplitude multiplexing and showed a high concordance with concentrations measured by Colilert once proportional bias was accounted for. In silico specificity testing underlined the possibility to further detect and distinguish Escherichia cryptic clade VI. Finally, the applicability of the ddPCR was successfully tested on environmental water samples where E. marmotae and E. ruysiae potentially confound E. coli counts based on the Most Probable Number method, highlighting the utility of this novel ddPCR as an efficient and rapid discriminatory test to improve water quality assessments.

Transcranial direct current stimulation and aphasia: the case of mr. C.

PURPOSE: To illustrate the ethical challenges that arose from investigating a novel treatment procedure, transcranial direct current stimulation (tDCS), in a research participant with aphasia. METHOD: We review the current evidence supporting the use of tDCS in aphasia research, highlighting methodological gaps in our knowledge of tDCS. Then, we examine the case of Mr. C, a person with chronic aphasia who participated in a research protocol investigating the impact of tDCS on aphasia treatment. We describe the procedures that he underwent and the resulting behavioral and neurophysiological outcomes. Finally, we share the steps that were taken to balance beneficence and nonmaleficence and to ensure Mr. C's autonomy. RESULTS: The objective data show that while Mr. C may not have benefitted from participating in the research, neither did he experience any harm. CONCLUSION: Researchers must consider not only the scientific integrity of their studies, but also potential ethical issues and consequences to the research participants.

A paradox: after stroke, the non-lesioned lower limb motor cortex may be maladaptive.

What are the neuroplastic mechanisms that allow some stroke patients to regain high-quality control of their paretic leg, when others do not? One theory implicates ipsilateral corticospinal pathways projecting from the non-lesioned hemisphere. We devised a new transcranial magnetic stimulation protocol to identify ipsilateral corticospinal tract conductivity from the non-lesioned hemisphere to the paretic limb in chronic stroke patients. We also assessed corticospinal tract degeneration by diffusion tensor imaging, and used an ankle tracking task to assess lower limb motor control. We found greater tracking error during antiphase bilateral ankle movement for patients with strong conductivity from the non-lesioned hemisphere to the paretic ankle than for those with weak or no conductivity. These findings suggest that, instead of assisting motor control, contributions to lower limb motor control from the non-lesioned hemisphere of some stroke survivors may be maladaptive.

Evidence for the clustering of antibacterial resistance phenotypes of enterococci within integrated poultry companies.

From July to December 2006, a panel of 401 enterococci was isolated from carcass rinse samples collected in five poultry processing plants in New Zealand. Agar diffusion assays for nine antibacterial drugs were used to obtain a resistance phenotype for each isolate. Hierarchical clustering techniques and diversity indices showed a high diversity of resistance phenotypes within each plant, with populations of Enterococcus faecalis showing greater heterogeneity than Enterococcus faecium. Bayesian modelling identified three clusters of phenotype patterns within the panel: the E. faecium isolates showed a high probability of containing two distinct clusters, whilst the E. faecalis isolates all grouped together to form the third cluster. The validity of these three clusters was examined using pairwise fixation indices and analysis of variance. Comparing the three clusters to the structure of the participating companies showed that resistance phenotypes for E. faecium isolated from processing plants that were geographically separated but were operated by the same integrated poultry company were more similar than E. faecium isolated from unconnected companies. Company-level management factors, such as the routine use of antibacterial drugs and the genetic line of birds reared, mirrored the structure of these clusters, thus indicating that company-level factors were the dominant selective pressures upon resistance phenotypes across all operating units within these integrated poultry companies.

Functionally navigated transcranial magnetic stimulation to evoke lingual pressure in stroke survivors with dysphagia and healthy adults: a proof of concept trial.

Background: Post-stroke dysphagia is characterized by reduced corticolingual excitability and lingual pressure; however, it remains unknown if transcranial magnetic stimulation (TMS) directly facilitates lingual pressure generation.Objectives: To explore optimal procedures for single pulse TMS using neuronavigation to evoke lingual pressure in intact and disrupted neural networks.Methods: Using co-registered functional magnetic resonance imaging, stimulation sites were determined for five healthy adults (Mage = 67) and four stroke survivors with dysphagia and reduced tongue strength (Mage = 66). Evoked lingual pressures were sampled across 45-65% of maximum stimulator output. Healthy participants repeated TMS with a bite block to isolate lingual pressure from off-target stimulation of mandibular elevators.Results: Only one functionally-guided stimulation site fell within previously reported optimal lateral (8-11cm) and anterior (2-4.25cm) coordinates. Lingual pressure was stable prior to pulse and increased linearly with intensity for both groups (p = .005). Post-stroke active motor thresholds were elevated compared to healthy adults (p = .025). Lingual pressure latency remained stable across intensities (p > .05). Jaw stabilization via bite block reduced the mean magnitude of evoked lingual pressure by approximately 16%.Conclusions: Single pulse TMS directly evokes higher lingual pressure and can define motor thresholds in intact and disordered corticolingual pathways. Stimulation sites using neuronavigation in healthy adults and stroke survivors largely differed from external coordinates in the literature that were predominantly established in young adults. Procedures to investigate motor thresholds for lingual pressure generation are proposed. The therapeutic role of TMS to address post-stroke deficits in lingual pressure and corticolingual excitability warrants continued investigation.

Behavior in Free-Living American Black Bear Dens: Parturition, Maternal Care, and Cub Behavior.

Denning behavior has long remained the least observed aspect of bear behavior. During 2010-2013, we used webcams, microphones, the internet, and 14,602 h of archived video to document the denning behaviors of two adult wild black bears (Ursus americanus) as they gave birth and cared for four litters through six winters in northeastern Minnesota. Observations included types of dens, labor, pre-parturient genital swelling, birthing positions, post-partum vocalizations, mothers removing amniotic tissues and warming newborn cubs in sub-freezing temperatures, frequency of nursing, cubs establishing nipple order, yearlings suckling, the ingestion of snow and icicles, the ingestion of foot pads, urination and defecation in latrine areas, toilet-licking, eye opening, reciprocal tongue-licking, play, rapid eye movement (REM) sleep and possible dreaming, and reactions to wildlife intruders. The use of this new method for observing natural bear dens allowed the identification of many behaviors undescribed for any species of wild bear in dens. We also discuss the need for future studies and how the depth and duration of black bear hibernation varies with body condition and geographic region.

Draft Whole-Genome Sequences of Campylobacter Strains Isolated from Brushtail Possums in New Zealand.

Draft genomes of five Campylobacter isolates recovered from New Zealand brushtail possums are described. Genome sizes ranged from 1.591 Mbp to 1.594 Mbp, with G+C contents of 29.9% to 29.95%. Comparison to Australian Campylobacter 16S rRNA gene sequences suggests that the species may be common to possums.

Pup mortality in New Zealand sea lions (Phocarctos hookeri) at Enderby Island, Auckland Islands, 2013-18.

New Zealand sea lions (Phocarctos hookeri) are an endemic and endangered species. Pup mortality at Enderby Island (50.5°S, 166.28°E) in the New Zealand sub-Antarctic has been well studied, with subsequent investigations yielding more intricate detail of the causes of mortality, as new diagnostic methods become available. Klebsiella pneumoniae was first reported in 2001-02 at this site, causing a pup mortality epizootic and is now known to be present at several colonies. This bacterium is a common mucosal commensal of humans and animals, however the agent found in pups at necropsy is a hypervirulent strain, readily recognised in microbial culture as being hypermucoviscous. Infection causes septicaemia with a common syndrome of subsequent meningitis and polyarthritis. This investigation uses histopathology and microbiology, with new modalities such as matrix assisted laser desorption/ionisation-time of flight mass spectrometry to show that Klebsiella septicaemia could have historically been, and continues to be, the most important cause of pup mortality, but has been previously underrepresented due to the often cryptic presentation and sometimes peracute course of disease. Hypermucoviscous K. pneumoniae should be considered a serious threat to pup survival in the species, causing on average 60.2% of pup deaths annually at Enderby Island between 2013 and 2018, with likely more continuing mortality following pup dispersal and the cessation of the summer monitoring season. Less common causes of death included starvation (14.8%), trauma/asphyxiation (9.9%) and other infections (7%). This study forms the basis for further evaluation of risk factors for pup mortality in the species, with a view to developing active mitigation.

Pathology and molecular epidemiology of Mycobacterium pinnipedii tuberculosis in native New Zealand marine mammals.

Mycobacterium pinnipedii causes tuberculosis in a number of pinniped species, and transmission to cattle and humans has been reported. The aims of this study were to: characterize the pathology and prevalence of tuberculosis in New Zealand marine mammals; use molecular diagnostic methods to confirm and type the causal agent; and to explore relationships between type and host characteristics. Tuberculosis was diagnosed in 30 pinnipeds and one cetacean. Most affected pinnipeds had involvement of the pulmonary system, supporting inhalation as the most common route of infection, although ingestion was a possible route in the cetacean. PCR for the RD2 gene confirmed M. pinnipedii as the causal agent in 23/31 (74%) cases (22 using DNA from cultured organisms, and one using DNA from formalin-fixed paraffin-embedded (FFPE) tissue), including the first published report in a cetacean. RD2 PCR results were compared for 22 cases where both cultured organisms and FFPE tissues were available, with successful identification of M. pinnipedii in 7/22 (31.8%). In cases with moderate to large numbers of acid-fast bacilli, RD2 PCR on FFPE tissue provided a rapid, inexpensive method for confirming M. pinnipedii infection without the need for culture. VNTR typing distinguished New Zealand M. pinnipedii isolates from M. pinnipedii isolated from Australian pinnipeds and from common types of M. bovis in New Zealand. Most (16/18) M. pinnipedii isolates from New Zealand sea lions were one of two common VNTR types whereas the cetacean isolate was a type detected previously in New Zealand cattle.