Glacier retreat reorganizes river habitats leaving refugia for Alpine invertebrate biodiversity poorly protected.

Alpine river biodiversity around the world is under threat from glacier retreat driven by rapid warming, yet our ability to predict the future distributions of specialist cold-water species is currently limited. Here we link future glacier projections, hydrological routing methods and species distribution models to quantify the changing influence of glaciers on population distributions of 15 alpine river invertebrate species across the entire European Alps, from 2020 to 2100. Glacial influence on rivers is projected to decrease steadily, with river networks expanding into higher elevations at a rate of 1% per decade. Species are projected to undergo upstream distribution shifts where glaciers persist but become functionally extinct where glaciers disappear completely. Several alpine catchments are predicted to offer climate refugia for cold-water specialists. However, present-day protected area networks provide relatively poor coverage of these future refugia, suggesting that alpine conservation strategies must change to accommodate the future effects of global warming.

Screening for Susceptibility of Macadamia to Euwallacea fornicatus and its Fungal Symbiont Fusarium euwallaceae.

The polyphagous shothole borer (Euwallacea fornicatus, PSHB), an ambrosia beetle, with its fungal symbiont, Fusarium euwallaceae, is responsible for Fusarium dieback (FD) in a wide range of woody hosts. In 2019, the first suspected case of E. fornicatus was reported in macadamia in South Africa. The aims of this study were to confirm the E. fornicatus report and thereafter to assess the susceptibility of commercially planted macadamia cultivars to FD caused by F. euwallaceae. The identities of the beetle and associated fungal symbionts were confirmed by means of DNA sequence analysis of the 28S ribosomal large subunit gene for beetles and the internal transcribed spacer region for fungi. Isolates identified as Fusarium species were further characterized by phylogenetic analysis of the translation elongation factor 1α and the ß-tubulin gene regions. Thereafter, Koch's postulates regarding F. euwallaceae were fulfilled on a mature Macadamia integrifolia tree planted at the experimental farm of the University of Pretoria. In order to determine susceptibility against FD, additional cultivar screening was conducted on nine commercially planted cultivars by means of pathogenicity trials using sterilized or inoculated toothpicks inserted into detached branches. Detached branch inoculations showed no significant lesion development six weeks post inoculation, except for cultivar 816. The restricted growth of F. euwallaceae observed in macadamia tissues therefore suggests that macadamia may not be a suitable host for F. euwallaceae and that the threat of FD in macadamia in the event of E. fornicatus infestation is less than for other E. fornicatus hosts. Future work on beetle attraction to macadamia is recommended for a more comprehensive understanding of the interaction between E. fornicatus and its fungal symbionts and macadamia.

Development and validation of an inexpensive and efficient method for the extraction of Theileria orientalis DNA from blood.

Theileria orientalis is an emerging bovine pathogen in Australasia. PCR-based detection methods for this parasite are sensitive but relatively expensive, partly due to costs associated with DNA extraction. An inexpensive and efficient technique was developed for the extraction of T. orientalis DNA from blood based on hypotonic erythrocyte lysis and detergent-proteinase K treatment (DPK method). The DPK method compares favourably to a commercial extraction kit when paired with a T. orientalis multiplex qPCR.

Development and validation of a quantitative PCR assay using multiplexed hydrolysis probes for detection and quantification of Theileria orientalis isolates and differentiation of clinically relevant subtypes.

Theileria orientalis is an emerging pathogen of cattle in Asia, Australia, and New Zealand. This organism is a vector-borne hemoprotozoan that causes clinical disease characterized by anemia, abortion, and death, as well as persistent subclinical infections. Molecular methods of diagnosis are preferred due to their sensitivity and utility in differentiating between pathogenic and apathogenic genotypes. Conventional PCR (cPCR) assays for T. orientalis detection and typing are laborious and do not provide an estimate of parasite load. Current real-time PCR assays cannot differentiate between clinically relevant and benign genotypes or are only semiquantitative without a defined clinical threshold. Here, we developed and validated a hydrolysis probe quantitative PCR (qPCR) assay which universally detects and quantifies T. orientalis and identifies the clinically associated Ikeda and Chitose genotypes (UIC assay). Comparison of the UIC assay results with previously validated universal and genotype-specific cPCR results demonstrated that qPCR detects and differentiates T. orientalis with high sensitivity and specificiy. Comparison of quantitative results based on percent parasitemia, determined via blood film analysis and packed cell volume (PCV) revealed significant positive and negative correlations, respectively. One-way analysis of variance (ANOVA) indicated that blood samples from animals with clinical signs of disease contained statistically higher concentrations of T. orientalis DNA than animals with subclinical infections. We propose clinical thresholds to assist in classifying high-, moderate-, and low-level infections and describe how parasite load and the presence of the Ikeda and Chitose genotypes relate to disease.

Comparison of a commercial ELISA with an indirect fluorescent antibody test to detect antibodies to Lawsonia intracellularis in experimentally challenged pigs.

OBJECTIVE: The ability of a new commercial ELISA to detect pigs with subclinical proliferative enteropathy (PE) was compared with the traditional indirect fluorescent antibody test (IFAT). METHODS: Serum samples were selected from pigs with known Lawsonia intracellularis infection status and clinical signs of PE, but the sample population consisted predominantly of pigs subclinically affected by PE. RESULTS: Significant association and agreement were shown between the ELISA and IFAT assays. ELISA results correlated well with the duration of L. intracellularis shedding as detected by polymerase chain reaction. CONCLUSION: ELISA can be successfully used to monitor L. intracellularis infection in pigs.

Colonisation and shedding of Lawsonia intracellularis in experimentally inoculated rodents and in wild rodents on pig farms.

Lawsonia intracellularis is an intracellular bacterium causing proliferative enteropathy in various animal species, and is considered an economically important pathogen of pigs. Rats and mice have been implicated as external vectors for a wide range of pig pathogens, including L. intracellularis. Previous studies have demonstrated L. intracellularis infection and proliferative enteropathy in rodents, but did not show the duration of shedding or the number of L. intracellularis shed by infected rodents, and therefore the infection risk that rodents pose to pigs. In this study, the number of L. intracellularis shed in the faeces and intestinal mucosa of wild rats trapped on pig farms was determined by a quantitative real time polymerase chain reaction assay. The prevalence of L. intracellularis in wild rats trapped on pig farms with endemic proliferative enteropathy (PE) was very high (≥ 70.6%), and large numbers of L. intracellularis were shed (10(10)/g of faeces) in a small proportion of wild rats. The duration of colonisation in laboratory rats and mice challenged with porcine isolates of L. intracellularis was also shown. Faecal shedding of L. intracellularis persisted for 14-21 days in rats and mice that were mildly affected with histological lesions of PE. The humoral immune response to L. intracellularis persisted for 40 days in both species. This study demonstrates that rodents may be an important reservoir of L. intracellularis on piggeries, and hence rodent control is important in disease eradication programs on pig farms.

Isolation of Mycobacterium avium subspecies paratuberculosis from muscle and peripheral lymph nodes using acid-pepsin digest prior to BACTEC culture.

Meat has received little attention regarding human exposure to Mycobacterium avium subsp. paratuberculosis, a possible infectious trigger of Crohn's disease. Meat has less contamination with other organisms than gut tissues, facilitating modifications to existing decontamination protocols prior to BACTEC culture that could increase analytical sensitivity. Using spiked meat samples we trialled enzymatic and chemical digestion techniques to concentrate larger starting samples, and modifications to existing clinical mycobacteriological decontamination protocols. An acid-pepsin digestion method using a 20 g sample was considerably more sensitive (detection limit 0.88 log(10) viable organisms per gram) than previous techniques. However, it was cumbersome for routine use, and subject to frequent contamination. Modifications to an existing centrifugation protocol yielded a simple, robust technique with slightly improved sensitivity (detection limit 1.77 log(10) per gram). Use of these sensitive tests in parallel identified M. a. paratuberculosis in the muscle of 59% and peripheral lymph nodes (PLN) of 85% of clinically infected sheep. The numbers of M. a. paratuberculosis in these infected tissues were low (1.67+/-0.92 log(10) per gram in muscle and 2.06+/-0.69 log(10) per gram in PLN), such that many would not have been detected by routine methods. Fewer subclinically infected animals with gross lesions harboured M. a. paratuberculosis in meat (4.5%) or PLN (32%), and the numbers of organisms in such infected animals were lower. Because most animals raised specifically for meat production are young and unlikely to be heavily infected, and because meat is usually consumed cooked, the risk of human exposure to viable M. a. paratuberculosis via meat may be small. Measures to prevent heavily infected animals, especially those with clinical signs, from entering the human food chain would further reduce this risk.

Radiometric pooled faecal culture for the detection of Mycobacterium avium subsp paratuberculosis in low-shedder cattle.

OBJECTIVE: To identify the optimum pooling rate for pooled faecal culture (PFC) as a diagnostic tool in bovine Johne's disease control, for detection of cattle shedding low concentrations of Mycobacterium avium subsp paratuberculosis (Map). METHOD: Thirteen target animals were selected by delayed growth of Map from initial individual radiometric faecal cultures (first growth index at 5 weeks or later). A procedure based on radiometric culture and IS900 polymerase chain reaction and restriction endonuclease analysis confirmation was then used for PFC. RESULTS: Eight samples (stored for up to 17 months at -80 degrees C) yielded Map on subsequent culture, either from undiluted faeces or those mixed with normal cattle faeces at dilution rates from 1 in 5 to 1 in 50. From a regression equation, culture-positive animals were considered to be shedding relatively low levels of Map (< 6 x 10(4)/g of faeces). Pooling dilutions of more than 1 in 5 reduced PFC sensitivity. A minimum incubation period of 10 weeks at a dilution of 1 in 5 is recommended to detect such infected cattle. This pooling rate in radiometric culture is probably capable of detecting cattle shedding < or = 5 x 10(3) Map organisms/g of faeces, representing an estimated inoculum per culture vial of fewer than 20 viable organisms. CONCLUSION: Map was detected in more than 50% of the stored faecal samples from cattle shedding low concentrations of the organism. A pooling rate of 5 samples per pool is required to reliably detect infected low-shedder cattle using PFC based on radiometric culture.

Pooled faecal culture for the detection of Mycobacterium avium subsp paratuberculosis in goats.

OBJECTIVE: To evaluate pooled faecal culture for herd diagnosis of caprine Johne's disease and relate these findings to faecal shedding rates of Mycobacterium avium subsp paratuberculosis (Map). DESIGN: Radiometric broth culture was applied to several pooling dilutions, and shedding rates were estimated from a regression equation based on bacterial growth rates and known processing losses during radiometric culture. PROCEDURE: Sixteen faecal samples from goats naturally infected with sheep (n = 3) or cattle (n = 13) strains of Map, were diluted in normal goat faeces from 1 in 5 to 1 in 50. Cultures were confirmed by IS900 polymerase chain reaction and restriction endonuclease analysis, and mycobactin dependency. The numbers of viable Map in the culture inocula were determined by endpoint titration (most probable number) of nine samples and related to a cumulative growth index. RESULTS: A pooling dilution of 1 in 25 with an incubation period of 10 weeks detected 13 of 16 culture positive goats, all shedding > or = 2 x 10(4) Map per gram of faeces. Two samples containing very low numbers of Map (< 2 x 10(3)/g) were only culture positive from undiluted faeces. Thirteen of 16 goats were considered to be shedding low to moderate concentrations of Map (< 2 x 10(5)/g faeces). CONCLUSIONS: These data support a pooling dilution of 1 in 25 for application of pooled faecal culture as a diagnostic tool in caprine Johne's disease control. A test based on this dilution would reduce laboratory costs of whole herd testing in goats by approximately 40% relative to serology and 75 to 90% relative to individual faecal culture.

An assessment of surgeons' abilites to site colostomies accurately.

AIMS: The ability of surgeons to site a colostomy is assessed in a clinical model. In addition, the tuition received by surgical trainees in stoma siting is also reviewed. MATERIALS AND METHODS: Eleven surgeons (trainees--six, colorectal subspeciality interest--two) were asked to site an end colostomy on nine patients using an adhesive disc (diameter--1cm). The position of the stoma was then measured on the 'x' and 'y' axis of a 2cm box grid, (maximum error--1cm) which was placed on the abdomen and centred on the umbilicus. The positions were then compared with that chosen by the stoma nurse who was taken as the gold standard. Results were compared using ANOVA and the Mann Whitney--U test. A telephone questionnaire was then undertaken to review the training of junior surgeons. RESULTS: There was variance present within the group of surgeons studied (p<0.01). Trainees and consultants had a similar accuracy in stoma-siting (p<0.2). Consultants with a colorectal subspeciality interest were better at placing stomas than those with a general interest (p<0.002). Badly placed stomas were three times more likely to be too low than too high (p<0.002). Thirty of 37 trainees reported receiving no undergraduate training from a specialist stoma nurse. Ninety per cent received postgraduate training from another surgeon while a specialist nurse trained only 35%. CONCLUSION: There was no difference between trainees and consultants in their ability to site a colostomy. However, surgeons with a subspeciality interest chose stoma sites that were more consistent with the specialist nurse than general surgeons. Training is haphazard and ability is frequently assumed rather than proven.

Gender may affect the action of garlic oil on plasma cholesterol and glucose levels of normal subjects.

Early trials of garlic preparations on blood lipids mainly supported a lipid-lowering effect, whereas later well-designed garlic tablet trials were mainly entirely null. However, enteric simulation tests suggest that this discordance may result from ineffective delivery of bioactive agents from the brands of garlic powder (GP) and cyclodextrin-bound garlic oil (GO) tablets tested in some recent negative trials. In contrast, enteric simulation tests show that the preformed bioactive agents present in "traditional" gelatin capsules of GO are efficiently released, although such capsules have rarely been investigated in lipid-lowering trials. It was hypothesized that gelatin capsules of GO given to normal subjects would improve specified coronary heart disease risk factors. Effects of a GP preparation were also investigated. Subjects (n = 51; men and women, mean age 27 y) were randomly assigned to receive either 8.2 mg/d of GO (allyl sulfides) or placebo for 11 wk. Another 27 subjects received garlic powder (GP) of similar biopotential (7.8 mg allicin/d). Outcome measures were 95% confidence intervals (CI) between GO and placebo groups for differences between baseline and subsequent sample times. Men and women combined showed no significant differences save for an improved total antioxidant capacity at 6 wk (P = 0.01). Hence, no benefit from GO after 11 wk is one plausible conclusion. However, there were significant differences in effect of GO between men and women for HDL cholesterol (HDL-C) (P = 0.004) and total cholesterol (TC)/HDL-C (P = 0.003). Women showed favorable effects in terms of CHD risk factors (i.e., increases in HDL-C and reductions in TC/HDL-C), whereas men had small adverse effects. There was a significant difference in the GO effect for glucose (P = 0.006), with a reduction seen for men and an increase for women. The gender effects were unexpected and such analyses were not planned in advance. Confirmation of these findings with larger numbers of subjects would have importance for the use of garlic against CHD and for the design of future garlic studies.

A randomized trial of the effects of garlic oil upon coronary heart disease risk factors in trained male runners.

Most trials of bulb garlic and garlic powder tablets indicate reduced coronary heart disease (CHD) risk in elevated-risk subjects. Most persons taking garlic supplements lack overt risk of CHD. However, no trials have tested steam-distilled garlic oil (GO) capsules with healthy subjects. The objectives of the present randomized, double-blind, placebo-controlled study were to determine whether GO capsules reduce CHD risk in trained male runners. Twenty-seven volunteers (mean age, 28.8 years) completed the study. Each took 12.3 mg/day GO (or placebo) capsules for 16 weeks. Main outcome measures were 95% confidence intervals (CIs) between GO and placebo groups for differences in changes of blood pressure (BP), plasma lipids, total antioxidant status (TAS), low-density lipoprotein (LDL) composition and blood clotting factors. Principal results as mean differences (95% CI) between GO and placebo are: pulse, 2.9 beats/min (-0.8 to 6.7), P = 0.12; systolic BP, -4.5 mmHg (-10.8 to 1.9), P = 0.16; plasma total cholesterol, 0.01 mmol/l (-0.34 to 0.37), P = 0.95; plasma triglycerides, -0.20 mmol/l (-0.43 to 0.03), P = 0.09; plasma TAS, 45 micromol Trolox equivalent/l (-35 to 124), P = 0.26; LDL density, 0.0019 g/ml (-0.0005 to 0.0043), P = 0.12; LDL triglycerides/protein, -0.078 mg/mg (-0.149 to -0.007), P = 0.03; LDL cholesterol/protein, -0.24 mg/mg (-0.69 to 0.22), P = 0.3; LDL TAS/triglycerides, 29 nmol/mg (11, 68), P = 0.15; prothrombin time, 0.99 s (-0.36 to 2.35), P = 0.14; partial thromboplastin time, 3.0 s (-1.0 to 7.1), P = 0.13. Results were null statistically. Trends with GO were mostly towards lower CHD risk, and a larger study (approximately 150 subjects) is required to test their validity.

N-1 substituted pyrimidin-4-ones: novel, orally active inhibitors of lipoprotein-associated phospholipase A2.

From two related series of 2-(alkylthio)-pyrimidones, a novel series of 1-((amidolinked)-alkyl)-pyrimidones has been designed as nanomolar inhibitors of human lipoprotein-associated phospholipase A2. These compounds show greatly enhanced activity in isolated plasma. Selected derivatives such as compounds 51 and 52 are orally active with a good duration of action.

Use of pooled fecal culture for sensitive and economic detection of mycobacterium avium subsp. paratuberculosis infection in flocks of sheep.

Ovine Johne's disease, or paratuberculosis, occurs in many countries. In Australia, surveillance using serology is used as part of a control program, but the testing regime is costly relative to its sensitivity. For this reason, culturing of Mycobacterium avium subsp. paratuberculosis in fecal samples pooled from a number of sheep was evaluated. Initially, the effect of pooling on the sensitivity of fecal culture was evaluated using samples from 20 sheep with multibacillary paratuberculosis and 20 sheep with paucibacillary paratuberculosis, each confirmed histologically. All multibacillary cases and 50% of paucibacillary cases were detected by culturing of feces at a pooling rate of 1 infected plus 49 uninfected sheep. In a pilot-scale study in 1997, M. avium subsp. paratuberculosis was detected by pooled fecal culture on 93% of 27 infected farms which were identified originally based on history, clinical signs, and one or more rounds of testing using serologic and histopathologic examinations. Pooled fecal culture was compared with serologic examination for submissions from 335 farms where both tests had been conducted on the same sheep and was significantly more sensitive (P<0.001). Computer simulation of random sampling indicated that the testing of 6 pools of 50 sheep would provide 95% confidence in detecting > or =2% prevalence of infection. The estimated laboratory cost of pooled fecal culture when applied as a flock test is approximately 30% that of serologic examination, and sample collection costs are lower. It is recommended that pooled fecal culture replace serologic examination for detection of M. avium subsp. paratuberculosis infection at the flock level.

A history of pneumonectomy.

Successful pneumonectomy in humans was preceded by the development of techniques for lobectomy. If experience in the animal laboratory had not been ignored, fears regarding sudden occlusion of the pulmonary artery and the fate of the postpneumonectomy pleural space would have been allayed. Precise hilar dissection was facilitated by the development of endotracheal anesthesia, which had been used successfully in animal experiments for at least half a century.

A new pattern of cerebellar hemorrhages in preterm infants.

OBJECTIVE: Posterior fossa hemorrhages may be underdiagnosed in surviving neonates, with cerebellar hemorrhage discovered in 10% to 25% of autopsy specimens from very low birth weight infants. Posterior fossa lesions have been difficult to visualize by the traditional ultrasonography approach through the anterior fontanelle. Late in 1994, routine posterior fossa imaging through the posterolateral fontanelle was instituted to improve the ultrasonographic visualization of the posterior fossa in neonates. METHODS: Infants identified with posterior fossa hemorrhage by cranial ultrasonography between 1994 and 1996 were followed prospectively through discharge and their clinical courses reviewed. Infants diagnosed with posterior fossa hemorrhage between 1991 and 1994 were identified retrospectively from a comprehensive radiology database to use in comparison. All infants surviving to discharge were entered into neurodevelopmental follow-up using standard developmental assessments. RESULTS: Approximately 525 infants underwent cranial sonography during the study period between October 1994 and September 1996, including 250 infants weighing <1500 g. Thirteen infants were identified with posterior fossa hemorrhage using the posterolateral fontanellar approach. In contrast, only 2 infants were identified with posterior fossa hemorrhage between 1991 and 1994 using traditional anterior fontanellar views. Six very low birth weight infants were identified with cerebellar hemorrhages not associated with supratentorial, intraventricular hemorrhage. Each hemorrhage had a clinically silent presentation and, in 5 infants, was not well-appreciated by anterior fontanellar images. Magnetic resonance imaging studies were performed on 5 of the 6 infants and confirmed a cerebellar lesion in the area of previous echo density on ultrasonography. No infant is exhibiting motor abnormalities on neurologic examination, although 4 infants are demonstrating cognitive, developmental delay. Follow-up, however, is limited to a corrected age of

Surgical anatomy of the diaphragm and the phrenic nerve.

In this article, the anatomy of the diaphragm and phrenic nerves is discussed, together with related surgical implications. Since the major cause of phrenic nerve injury is surgery, usually for congenital or acquired heart disease, incisions in the diaphragm that do not injure major branches of the phrenic nerve are also discussed. Diaphragmatic plication is usually required in infants less than 3 months of age, and older children may be managed by ventilatory support if electrophysiologic studies document the possibility of return of nerve function. In adults with normal pulmonary function, unilateral diaphragmatic paralysis is usually asymptomatic.