Diagnosis and features of hospital-acquired pneumonia: a retrospective cohort study.

BACKGROUND: Hospital-acquired pneumonia (HAP) is defined as radiologically confirmed pneumonia occurring ≥48h after hospitalization, in non-intubated patients. Empirical treatment regimens use broad-spectrum antimicrobials. AIM: To evaluate the accuracy of the diagnosis of HAP and to describe the demographic and microbiological features of patients with HAP. METHODS: Medical and surgical inpatients receiving intravenous antimicrobials for a clinical diagnosis of HAP at a UK tertiary care hospital between April 2013 and 2014 were identified. Demographic and clinical details were recorded. FINDINGS: A total of 166 adult patients with a clinical diagnosis of HAP were identified. Broad-spectrum antimicrobials were prescribed, primarily piperacillin-tazobactam (57.2%) and co-amoxiclav (12.5%). Sputum from 24.7% of patients was obtained for culture. Sixty-five percent of patients had radiological evidence of new/progressive infiltrate at the time of HAP treatment, therefore meeting HAP diagnostic criteria (2005 American Thoracic Society/Infectious Diseases Society of America guidelines). Radiologically confirmed HAP was associated with higher levels of inflammatory markers and sputum culture positivity. Previous surgery and/or endotracheal intubation were associated with radiologically confirmed HAP. A bacterial pathogen was identified from 17/35 sputum samples from radiologically confirmed HAP patients. These were Gram-negative bacilli (N = 11) or Staphylococcus aureus (N = 6). Gram-negative bacteria tended to be resistant to co-amoxiclav, but susceptible to ciprofloxacin, piperacillin-tazobactam and meropenem. Five of the six S. aureus isolates were meticillin susceptible and all were susceptible to doxycycline. CONCLUSION: In ward-level hospital practice 'HAP' is an over-used diagnosis that may be inaccurate in 35% of cases when objective radiological criteria are applied. Radiologically confirmed HAP represents a distinct clinical and microbiological phenotype. Potential risk factors were identified that could represent targets for preventive interventions.

Ovine rumen papillae biopsy via oral endoscopy; a rapid and repeatable method for serial sampling.

AIMS: To explore and validate the utility of rumen endoscopy for collection of rumen papillae for gene expression measurement. METHODS: Four adult Coopworth ewes were fasted for either 4 or 24 hours. Animals were sedated, placed in a dorsally recumbent position at 45 degrees with the head upright, and an endoscope inserted via a tube inserted into the mouth. Biopsies of rumen papillae were taken from the ventral surface of the rumen atrium under visual guidance. Two biopsies were collected from one of the animals that had been fasted for 4 hours, and three from one of the animals that had been fasted for 24 hours. Video of the rumen atrium and reticulum was also collected. The animals recovered uneventfully. Biopsies were subsequently used for extraction and sequencing of mRNA. RESULTS: The ventral surface of the rumen atrium was accessible after 4 hours off pasture, but a larger region was accessible after 24 hours of fasting. Sedation allowed access for endoscope use for around 5 to 10 minutes after which increased saliva flow was noted. Rumen papillae biopsies were easily collected, with samples from a variety of sites collected in the ∼10 minute time window. High quality RNA was obtained for stranded mRNA sequencing. Of the resulting reads, 69-70% mapped uniquely to version 3.1 of the ovine genome, and 48-49% to a known gene. The rumen mRNA profiles were consistent with a previously reported study. CONCLUSIONS: This method for obtaining rumenal tissue was found to be rapid and resulted in no apparent short or long term effects on the animal. High quality RNA was successfully extracted and amplified from the rumen papillae biopsies, indicating that this technique could be used for future gene expression studies. The use of rumen endoscopy could be extended to collection of a variety of rumen and reticulum anatomical measurements and deposition and retrieval of small sensors from the rumen. Rumen endoscopy offers an attractive and cost effective approach to repeated rumen biopsies compared with serial slaughter or use of cannulated animals.

A randomised controlled trial of Silirum vaccine for control of paratuberculosis in farmed red deer.

A randomised controlled trial to assess the efficacy of Silirum vaccine in control of paratuberculosis in young farmed deer was carried out in 2008-2009 in six New Zealand herds with a history of clinical disease. Vaccination with Silirum was carried out in four-month-old deer, and vaccinates (n=1671) and controls (n=1664) were weighed at vaccination and at 8 and 12 months old, when faecal samples were collected from 125 vaccinates and 123 controls on five farms. Deer were slaughtered between 11 and 20 months of age, and the incidence of gross visceral lymph node (VLN) pathology typical of paratuberculosis in deer, that is, enlarged and/or granulomatous VLN, was recorded. Clinical disease was confirmed in 18 controls and seven vaccinates, representing a vaccine efficacy estimate of 60 per cent (95% CI 3 per cent to 83 per cent, P=0.04). Forty-seven percent (95% CI 38 per cent to 56 per cent) of faecal samples from vaccinates and 55 per cent (95% CI 46 per cent to 64 per cent) from controls were Mycobacterium avium subspecies paratuberculosis positive (P=0.5). Average daily liveweight gain did not differ between the cohorts. At slaughter, 1.4 per cent of vaccinates and 4.5 per cent of controls had VLN pathology, RR=0.32 (95% CI 0.19 to 0.54, P<0.001). These data indicate that vaccination with Silirum may be useful as an aid to control losses associated with clinical paratuberculosis in young deer.

Prevalence of Mycobacterium avium subsp. paratuberculosis in farmed red deer (Cervus elaphus) with grossly normal mesenteric lymph nodes.

AIM: To estimate the prevalence of Mycobacterium avium subsp. paratuberculosis (Map) in farmed deer with no gross post-mortem evidence of Map infection slaughtered in New Zealand, and to assess predictors of infection. METHODS: Mesenteric lymph node (MLN) samples (n = 251) were collected from 60 lines of deer presented at two slaughterhouses in the North and two in the South Island of New Zealand between October 2008 and January 2009 and cultured for Map. Estimates of individual animal prevalence for each island were adjusted to account for the clustering of individual observations within herds. The national herd prevalence estimate was calculated as a weighted mean, with weights being the proportion of herds from which deer were slaughtered at North and South Island slaughterhouses among all herds slaughtering deer throughout New Zealand. Age, gender, and the presence of other carcasses with enlarged and/or granulomatous MLN in the same line (line status) were assessed as predictors of infection using multivariable logistic regression. RESULTS: A national cluster-adjusted individual animal prevalence of 45 (95% CI = 30-60)% was estimated, with North and South Island prevalences of 29 (95% CI = 16-45)% and 51 (95% CI = 36-66)%, respectively. Line status was a strong predictor of infection in young deer (OR 7.1, 95% CI = 2.4-21.5), but not in older deer. Herd-level prevalence was 44 (95% CI = 24-64)% in the North Island and 67 (95% CI = 49-85)% in the South Island. Weighted adjustment resulted in a national herd-level prevalence estimate of 59 (95% CI = 41-78)%. CONCLUSIONS: This study has provided a national baseline prevalence estimate for Map infection at the individual and herd-level, showing a contrast between the North and South Islands. More research to investigate the factors contributing to the difference in infection prevalence seen between the islands may help to identify suitable control measures for Map in deer herds.

Association between Mycobacterium avium subspecies paratuberculosis and lymph node size in New Zealand farmed deer (Cervus elaphus).

AIMS: To develop and validate criteria for identification of abnormal lymph nodes (LN) at commercial slaughter, for the purpose of national surveillance for Mycobacterium avium subspecies paratuberculosis (Map) in New Zealand farmed deer. This included estimation of the predictive value of abnormal LN for Map infection; a standard circumference cut-point for measurement of abnormal LN; and identification of risk factors associated with increasing LN circumference. METHODS: In Study 1, official assessors sampled 129 LN with macroscopically visible abnormalities (abnormal LN) from 76 deer herds between May and November 2007. LN samples were cultured for Map, with culture-negative LN further examined for typical histopathological changes. The predictive value of abnormal LN for Map infection was calculated and significance of herd location (North or South Island) assessed. In Study 2, the circumferences of 412 mesenteric LN (MLN) from 79 herds were measured between October 2007 and January 2009, with samples cultured for Map and examined for eight histopathological features. The minimum circumference of an abnormal MLN was defined, based on an arbitrary >95% specificity of a culture-positive Map diagnosis. Associations between the predictor variables Map culture status, carcase weight, animal age and gender, and histopathological features, and increasing MLN circumference were assessed using a linear mixed-effects model. RESULTS: Based solely on culture, the predictive value of abnormal LN for Map infection was 92.2 (95% CI: 86.2-96.2)% with no difference between the North and South Islands (p = 0.09). Inclusion of three culture-negative LN with histopathological changes typical of Map infection increased the predictive value estimate to 94.6 (95% CI: 89.2-97.3)%. The minimum circumference of an abnormal MLN was defined as 55 mm, with a sensitivity of Map detection at this cut-point of approximately 12%. Increasing MLN circumference was positively associated with the presence of moderate follicular hyperplasia (p < 0.01), focal granulomas (p < 0.01) and a synergistic interaction between focal granulomas and Map status (p = 0.03). CONCLUSIONS: Deer MLN with macroscopically visible abnormalities and/or a circumference of >55 mm have >95% likelihood of Map infection. However, sensitivity of Map diagnosis in MLN with circumference of >55 mm was 12%, indicating use of abnormal LN as a sole criterion in national surveillance for Map in slaughtered deer will underestimate animal-level prevalence.

WEE1 accumulation and deregulation of S-phase proteins mediate MLN4924 potent inhibitory effect on Ewing sarcoma cells.

Ewing sarcoma (ES) is an aggressive bone and soft tissue tumor of children and young adults in which finding effective new targeted therapies is imperative. Here, we report an in-depth preclinical study of the investigational cullin-RING ubiquitin ligase (CRL) inhibitor MLN4924 in ES, as we have recently demonstrated the implication of a CRL component in the ES pathogenesis. First, our results support a high sensitivity of ES cells to MLN4924 growth inhibition both in vitro (14 ES cell lines tested, median IC50=81 nM) and in tumor xenografts (tumor regression achieved with 60 mg/kg BID, subcutaneously, n=9). Second, we report a dual mechanism of action of MLN4924 in ES cells: while a wide range of MLN4924 concentrations (∼30-300 nM) trigger a G2 arrest that can only be rescued by WEE1 kinase inhibition or depletion, saturating doses of the drug (>300 nM) cause a delay in S-phase progression concomitant with unbalanced CDK2-Cyclin E and CDK2-Cyclin A relative levels (accumulation of the first and depletion of the latter). The aberrant presence of CDC6 in the nucleus at late S-phase cell cycle stage confirmed the loss of CDK2-Cyclin A-specific functions. Remarkably, other mechanisms explored (P27 accumulation and DNA damage signaling pathways) were found unable to explain MLN4924 effects, strengthening the specificity of our findings and suggesting the absence of functionality of some CRL substrates accumulated in response to MLN4924. This study renders a rationale for clinical trials and contributes molecular mechanisms for a better understanding of this promising antitumoral agent.

Diagnosis of Clostridium difficile-associated disease: examination of multiple algorithms using toxin EIA, glutamate dehydrogenase EIA and loop-mediated isothermal amplification.

The laboratory diagnosis of Clostridium difficile infection (CDI) needs to be accurate and timely to ensure optimal patient management, infection control and reliable surveillance. Three methods are evaluated using 810 consecutive stool samples against toxigenic culture: CDT TOX A/B Premier enzyme immunoassay (EIA) kit (Meridian Bioscience, Europe), Premier EIA for C. difficile glutamate dehydrogenase (GDH) (Meridian Bioscience, Europe) and the Illumigene kit (Meridian Bioscience, Europe), both individually and within combined testing algorithms. The study revealed that the CDT TOX A/B Premier EIA gave rise to false-positive and false-negative results and demonstrated poor sensitivity (56.47%), compared to Premier EIA for C. difficile GDH (97.65%), suggesting this GDH EIA can be a useful negative screening method. Results for the Illumigene assay alone showed sensitivity, specificity, negative predictive value (NPV) and positive predictive value (PPV) of 91.57%, 98.07%, 99.03% and 84.44%, respectively. A two-stage algorithm using Premier EIA for C. difficile GDH/Illumigene assay yielded superior results compared with other testing algorithms (91.57%, 98.07%, 99.03% and 84.44%, respectively), mirroring the Illumigene performance. However, Illumigene is approximately half the cost of current polymerase chain reaction (PCR) methods, has a rapid turnaround time and requires no specialised skill base, making it an attractive alternative to assays such as the Xpert C. difficile assay (Cepheid, Sunnyvale, CA). A three-stage algorithm offered no improvement and would hamper workflow.

1q gain and CDT2 overexpression underlie an aggressive and highly proliferative form of Ewing sarcoma.

Despite extensive characterization of the role of the EWS-ETS fusions, little is known about secondary genetic alterations and their clinical contribution to Ewing sarcoma (ES). It has been demonstrated that the molecular structure of EWS-ETS lacks prognostic value. Moreover, CDKN2A deletion and TP53 mutation, despite carrying a poor prognosis, are infrequent. In this scenario identifying secondary genetic alterations with a significant prevalence could contribute to understand the molecular mechanisms underlying the most aggressive forms of ES.We screened a 67 ES tumor set for copy number alterations by array comparative genomic hybridization. 1q gain (1qG), detected in 31% of tumor samples, was found markedly associated with relapse and poor overall and disease-free survival and demonstrated a prognostic value independent of classical clinical parameters. Reanalysis of an expression dataset belonging to an independent tumor set (n=37) not only validated this finding but also led us to identify a transcriptomic profile of severe cell cycle deregulation in 1qG ES tumors. Consistently, a higher proliferation rate was detected in this tumor subset by Ki-67 immunohistochemistry. CDT2, a 1q-located candidate gene encoding a protein involved in ubiquitin ligase activity and significantly overexpressed in 1qG ES tumors, was validated in vitro and in vivo proving its major contribution to this molecular and clinical phenotype. This integrative genomic study of 105 ES tumors in overall renders the potential value of 1qG and CDT2 overexpression as prognostic biomarkers and also affords a rationale for the application of already available new therapeutic compounds selectively targeting the protein-ubiquitin machinery.

Guidelines on the facilities required for minor surgical procedures and minimal access interventions.

There have been many changes in healthcare provision in recent years, including the delivery of some surgical services in primary care or in day surgery centres, which were previously provided by acute hospitals. Developments in the fields of interventional radiology and cardiology have further expanded the range and complexity of procedures undertaken in these settings. In the face of these changes there is a need to define from an infection prevention and control perspective the basic physical requirements for facilities in which such surgical procedures may be carried out. Under the auspices of the Healthcare Infection Society, we have developed the following recommendations for those designing new facilities or upgrading existing facilities. These draw upon best practice, available evidence, other guidelines where appropriate, and expert consensus to provide sensible and feasible advice. An attempt is also made to define minimal access interventions and minor surgical procedures. For minimal access interventions, including interventional radiology, new facilities should be mechanically ventilated to achieve 15 air changes per hour but natural ventilation is satisfactory for minor procedures. All procedures should involve a checklist and operators should be appropriately trained. There is also a need for prospective surveillance to accurately determine the post-procedure infection rate. Finally, there is a requirement for appropriate applied research to develop the evidence base required to support subsequent iterations of this guidance.

Immunological and pathological responses of red deer resistant or susceptible genotypes, to experimental challenge with Mycobacterium avium subsp. paratuberculosis.

This study aimed to monitor the clinical, immunological and pathological changes in red deer for 49 weeks after experimental oral challenge with Mycobacterium avium subsp. paratuberculosis (MAP) and to assess the heritability of resistance in the offspring of two red stags. Eighteen young deer, which were bred from unselected hinds and sired by two stags resistant (R) or susceptible (S) to paratuberculosis, were challenged with MAP and monitored for 49 weeks. Biopsy samples of the jejunal lymph node were collected at Weeks 4 and 13 and at necropsy after euthanasia of clinically affected animals or when electively killed at Week 49. Three animals (two S and one R) developed clinical disease and were euthanised. The nine S offspring had significantly more severe lesions than the nine R offspring (Mantel-Haenszel Chi-square P=0.017). The average Lesion Severity Score (LSS) of R offspring was 5.9 (mild), and 7/9 had no or very mild lesions. In contrast, the LSS of S offspring averaged 11.7 (severe), and 7/9 had severe lesions. Most of the resistant, but not the susceptible, animals showed evidence of resolving lesions and a reduction in the number of MAP between 13 and 49 weeks after challenge. One R offspring appeared to completely cure itself, and progressed from mild culture-positive paratuberculosis lesions at Week 13 to having no signs of disease or infection 36 weeks later. This study showed significant heritable resistance/susceptibility to paratuberculosis and key differences in immunological responses in the first 3 months after challenge, indicating different paths to relative success or failure to control MAP. In general, R deer had higher IFN-γ levels, low antibody titres and fewer MAP, while S deer had lower IFN-γ levels, higher antibody and more MAP.

Immunoregulatory cytokines are associated with protection from immunopathology following Mycobacterium avium subspecies paratuberculosis infection in red deer.

Although the causative agent of Johne's disease, Mycobacterium avium subsp. paratuberculosis, is well known, the etiology of disease and the immune responses generated in response to infection are still poorly understood. Knowledge of definitive markers of protective immunity, infection, and the establishment of chronic granulomatous Johne's disease is necessary to advance vaccine and diagnostic development. We sought to profile the immune responses occurring within jejunal lymph nodes of experimentally challenged red deer (Cervus elaphus). Quantitative PCR was utilized to measure a range of cytokines, signaling molecules, and transcription factors involved in Th1, Th2, Treg, and Th17 immune responses. Significant differences in gene expression were observed between control, minimally diseased, and severely diseased animals, with severely diseased animals showing elevated proinflammatory transcripts and reduced anti-inflammatory transcripts. We identified a proinflammatory cytokine milieu of gamma interferon, interleukin-1α (IL-1α), and IL-17, which may contribute to the immunopathology observed during clinical Johne's disease and suggest that Th2 and Treg immune responses may play an important role in controlling the development of immunopathology in infected animals.

Histopathology of grossly normal mesenteric lymph nodes of New Zealand farmed red deer (Cervus elaphus) including identification of lipopigment.

This article describes the histopathology of grossly normal mesenteric lymph nodes (MLNs) of New Zealand farmed red deer (Cervus elaphus). Eighty MLNs were sourced from 10 deer from 5 North Island herds and 5 South Island herds classified as low risk and high risk of Mycobacterium avium subspecies paratuberculosis (MAP) infection, respectively. Fixed sections were stained with hematoxylin and eosin; Ziehl-Neelsen; and, selectively, periodic acid-Schiff, Perl's, and Sudan black. Positive Ziehl-Neelsen stain, follicular hyperplasia, capsular eosinophil infiltration, focal granulomas, foci of macrophages containing lipopigment, parasitic granulomas, and calcified foci are described and severity graded where appropriate. Animal age, sex, and herd of origin are variably associated with the presence of one or more features. Trabecular fibrosis and dilated edema-filled sinusoids are described. These observations allow differentiation between likely nonpathologic histologic features in deer MLNs and features possibly attributable to infection with a pathogen such as MAP.

Johne's disease caused by Mycobacterium avium subsp. paratuberculosis infection in red deer (Cervus elaphus): an histopathological grading system, and comparison of paucibacillary and multibacillary disease.

AIM: To describe a grading system for evaluating lesions in the small intestine and mesenteric lymph nodes of red deer (Cervus elaphus) infected with Mycobacterium avium subsp. paratuberculosis (MAP), and report the distribution of granulomas and findings seen in paucibacillary and multibacillary forms of the disease. METHODS: Tissues were examined from red deer either experimentally (n=300) or naturally (n=131) infected with MAP. A disease severity score developed previously was expanded to help provide more sensitivity in assessing severity of disease. The distribution of granulomatous, paucibacillary and multibacillary lesions in sections of jejunum, ileocaecal valve and mesenteric lymph nodes was compared between sites and between animals with mild (severity score< or =7) and severe (severity score> or =8) forms of the disease. RESULTS: Based on the results of three published studies, the severity score related well with the clinical severity and gross lesions associated with the disease. Paucibacillary lesions tended to have smaller macrophages and increased numbers of Langhan's giant cells than multibacillary forms, but this was not a consistent finding. The multibacillary form of the disease had Langhan's giant cells, containing numerous acid-fast organisms (AFO), and in one form sheets of epithelioid-like macrophages with prominent vacuolated cytoplasm and few Langhan's giant cells. In deer experimentally infected with MAP, granulomatous lesions were more evident in mesenteric lymph nodes than in intestinal tissue, especially in the mild form of the disease. In mild cases, granulomas were significantly more evident in Peyer's patches than in the intestinal mucosa, but in severe cases, the difference was not significant. Paucibacillary forms of the disease were more evident in deer with the mild disease, and multibacillary forms were more evident in deer with the severe disease. CONCLUSIONS: The severity score provides an objective measure of the severity of Johne's disease, and is useful for comparing individuals and groups of deer in studies involving experimental or natural infection with MAP. In mild disease, lesions were more evident in mesenteric lymph nodes than in jejunum and ileocaecal tissue, and Langhan's giant cells were present in both paucibacillary and multibacillary forms of the disease. The posterior jejunum and ileocaecal-valve lymph nodes were the best sites for detecting mild lesions, while intestinal samples from the posterior jejunum and ileocaecal valve had a lesser but useful role.

Stable interference of EWS-FLI1 in an Ewing sarcoma cell line impairs IGF-1/IGF-1R signalling and reveals TOPK as a new target.

BACKGROUND: Ewing sarcoma is a paradigm of solid tumour -bearing chromosomal translocations resulting in fusion proteins that act as deregulated transcription factors. Ewing sarcoma translocations fuse the EWS gene with an ETS transcription factor, mainly FLI1. Most of the EWS-FLI1 target genes still remain unknown and many have been identified in heterologous model systems. METHODS: We have developed a stable RNA interference model knocking down EWS-FLI1 in the Ewing sarcoma cell line TC71. Gene expression analyses were performed to study the effect of RNA interference on the genetic signature of EWS-FLI1 and to identify genes that could contribute to tumourigenesis. RESULTS: EWS-FLI1 inhibition induced apoptosis, reduced cell migratory and tumourigenic capacities, and caused reduction in tumour growth. IGF-1 was downregulated and the IGF-1/IGF-1R signalling pathway was impaired. PBK/TOPK (T-LAK cell-originated protein kinase) expression was decreased because of EWS-FLI1 inhibition. We showed that TOPK is a new target gene of EWS-FLI1. TOPK inhibition prompted a decrease in the proliferation rate and a dramatic change in the cell's ability to grow in coalescence. CONCLUSION: This is the first report of TOPK activity in Ewing sarcoma and suggests a significant role of this MAPKK-like protein kinase in the Ewing sarcoma biology.

Potential role of TBC1D4 in enhanced post-exercise insulin action in human skeletal muscle.

AIMS/HYPOTHESIS: TBC1 domain family, member 4 (TBC1D4; also known as AS160) is a cellular signalling intermediate to glucose transport regulated by insulin-dependent and -independent mechanisms. Skeletal muscle insulin sensitivity is increased after acute exercise by an unknown mechanism that does not involve modulation at proximal insulin signalling intermediates. We hypothesised that signalling through TBC1D4 is involved in this effect of exercise as it is a common signalling element for insulin and exercise. METHODS: Insulin-regulated glucose metabolism was evaluated in 12 healthy moderately trained young men 4 h after one-legged exercise at basal and during a euglycaemic-hyperinsulinaemic clamp. Vastus lateralis biopsies were taken before and immediately after the clamp. RESULTS: Insulin stimulation increased glucose uptake in both legs, with greater effects (approximately 80%, p < 0.01) in the previously exercised leg. TBC1D4 phosphorylation, assessed using the phospho-AKT (protein kinase B)substrate antibody and phospho- and site-specific antibodies targeting six phosphorylation sites on TBC1D4, increased at similar degrees to insulin stimulation in the previously exercised and rested legs (p < 0.01). However, TBC1D4 phosphorylation on Ser-318, Ser-341, Ser-588 and Ser-751 was higher in the previously exercised leg, both in the absence and in the presence of insulin (p < 0.01; Ser-588, p = 0.09; observed power = 0.39). 14-3-3 binding capacity for TBC1D4 increased equally (p < 0.01) in both legs during insulin stimulation. CONCLUSION/INTERPRETATION: We provide evidence for site-specific phosphorylation of TBC1D4 in human skeletal muscle in response to physiological hyperinsulinaemia. The data support the idea that TBC1D4 is a nexus for insulin- and exercise-responsive signals that may mediate increased insulin action after exercise.

Spermine and spermidine mediate protection against oxidative damage caused by hydrogen peroxide.

The polyamines spermidine and spermine have been hypothesized to possess different functions in the protection of DNA from reactive oxygen species. The growth and survival of mouse fibroblasts unable to synthesize spermine were compared to their normal counterparts in their native and polyamine-depleted states in response to oxidative stress. The results of these studies suggest that when present at normal or supraphysiological concentrations, either spermidine or spermine can protect cells from reactive oxygen species. However, when polyamine pools are pharmacologically manipulated to produce cells with low levels of predominately spermine or spermidine, spermine appears to be more effective. Importantly, when cells are depleted of both glutathione and endogenous polyamines, they exhibit increased sensitivity to hydrogen peroxide as compared to glutathione depletion alone, suggesting that polyamines not only play a role in protecting cells from oxidative stress but this role is distinct from that played by glutathione.

Primary primitive neuroectodermal tumour of the urinary bladder: a clinico-pathological study emphasising immunohistochemical, ultrastructural and molecular analyses.

Primary primitive neuroectodermal tumours (PNETs) of the bladder are extremely rare and aggressive neoplasms, and only six examples have been reported in the literature. The case of a 21-year-old woman, who remains disease free 3 years after tumour resection, is reported here. Morphological features were found to correspond to a small round blue cell tumour without rosette formation and with extensive areas of necrosis. Strong expression of CD99, vimentin and CD117 (c-kit), and focal reactivity to cytokeratin and S-100 protein was observed in tumour cells. Ultrastructurally, sparse neurosecretory granules were observed. Diagnosis of PNET was supported by molecular genetic analysis, showing the EWS-FLI-1 fusion transcript type 2 by RT-PCR and EWS gene rearrangement by fluorescence in situ hybridisation. A normal genetically balanced genotype was shown by comparative genomic hybridisation, which, together with the expression of c-kit, a known therapeutic target for imatinib, may have prognostic and therapeutic implications.

Mycobacterial diseases of deer.

The most significant mycobacterial diseases of free-living, captive and farmed deer are bovine tuberculosis, caused by Mycobacterium bovis, Johne's disease (paratuberculosis), caused by Mycobacterium avium subsp paratuberculosis (basonym M. paratuberculosis), and avian tuberculosis, caused principally by M. avium subsp avium. The first case of M. bovis infection in farmed deer was identified in New Zealand in 1978. In 1983, a voluntary scheme was introduced in New Zealand to control tuberculosis in farmed deer, followed by a compulsory tuberculosis control scheme in 1990. The primary control measure is the slaughter of infected animals, detected by skin testing and blood testing, together with movement control and vector control. The number of infected deer herds peaked in the mid 1990s at over 160 herds, but by 30 June 2002 this had been reduced to 79 (1.45%), and to 67 (1.23%) by June 2003. Deer-to-deer transmission occurs, but the majority of herd breakdowns are believed to be from infected vectors. Factors likely to affect the susceptibility of deer include age, environment, population density, exposure and genetics. Avian tuberculosis occasionally causes clinical disease in wild, captive and farmed deer in New Zealand and overseas. Mycobacterium intracellulare, and subspecies of M. avium other than M. paratuberculosis, are widespread throughout New Zealand and are thought to be largely responsible for the high level of sensitisation to avian purified protein derivative (PPD), which is used for comparison purposes in tuberculosis skin testing of deer in this country. Infections with these organisms are usually subclinical in farmed deer, although M. avium subsp avium commonly causes lesions in retropharyngeal, mesenteric and ileocaecal lymph nodes. These lesions cause problems because of their gross and microscopic similarity to those due to M. bovis infection. Birds and domestic animals are most likely to become infected via environmental contamination of food, water, bedding litter or soil, while carnivores or scavengers may also become infected by ingesting infected carcasses. Johne's disease has been reported in deer in the wild and in zoos, especially in North America, the United Kingdom (UK) and Europe. Since first being confirmed in farmed deer in New Zealand in 1979, the incidence of Johne's disease has increased steadily. To date, M. paratuberculosis has been identified in >600 farmed deer on 300 properties. The majority of cases have been identified from suspected tuberculous lesions submitted from deer slaughter plants. Clinically, Johne's disease in deer is similar to the disease in sheep and cattle, with typical signs of loss of weight and condition, and diarrhoea. However, outbreaks of Johne's disease frequently occur in young red deer, 8-15 months of age, whereas the clinical disease in sheep and cattle is sporadic and usually affects adults 3-5 years of age. The disease is characterised by a chronic granulomatous enteritis and lymphadenitis, especially affecting the jejunum and ileum and the mesenteric lymph nodes. Deer affected subclinically may have lesions in these lymph nodes at slaughter, which are grossly indistinguishable from those due to bovine tuberculosis. Because of the antigenic similarity between M. intracellulare and all the subspecies of M. avium, including M. paratuberculosis, the diagnostic tests for Johne's disease lack sensitivity and specificity, making control difficult.

Viral, parasitic and prion diseases of farmed deer and bison.

The most important viral disease of farmed deer and bison is malignant catarrhal fever. The other herpesviruses which have been isolated from these species are briefly described. Other viral agents that are recognised in these animals, including adenovirus, parapox, foot and mouth disease, bluetongue, epizootic haemorrhagic disease, bovine virus diarrhoea, rotavirus and coronavirus, are also discussed. Ectoparasites of importance in this group in various parts of the world include a variety of ticks, as well as lice, keds, Oestridae, mange mites and fire ants. Helminth parasites include liver flukes (Fascioloides and Fasciola), gastrointestinal nematodes of the family Trichostrongylidae, pulmonary lungworms of the genus Dictyocaulus and extra-pulmonary lungworms of the family Protostrongylidae. Chronic wasting disease is principally important in North America, where the disease occurs in wild cervids in a limited area and has been reported in farmed deer in a small number of states in the United States of America and one province in Canada. These diseases are summarised in terms of their classification, epidemiology, clinical signs, pathology, diagnosis, treatment and control.