Outbreak of hepatitis A infection associated with the consumption of frozen berries, Ireland, 2013--linked to an international outbreak.

In May 2013, a European alert was issued regarding a hepatitis A virus (HAV) outbreak in Italy. In June 2013, HAV subgenotype IA with an identical sequence was identified in Ireland in three cases who had not travelled to Italy. The investigation consisted of descriptive epidemiology, a case-control study, microbiological testing of human and food specimens, molecular typing of positive specimens and food traceback. We identified 21 outbreak cases (14 confirmed primary cases) with symptom onset between 31 January and 11 October 2013. For the case-control study, we recruited 11 confirmed primary cases and 42 matched controls. Cases were more likely than controls to have eaten berry cheesecake (matched odds ratio (mOR): 12; 95% confidence interval (CI): 1.3-114), whole frozen berries (mOR: 9.5; 95% CI: 1.0-89), yoghurt containing frozen berries (mOR: 6.6, 95% CI: 1.2-37) or raw celery (mOR: 4; 95% CI: 1.2-16). Among cases, 91% had consumed at least one of four products containing frozen berries (mOR: 12; 95% CI: 1.5-94). Sixteen food samples tested were all negative for HAV. As products containing frozen berries were implicated in the outbreak, the public were advised to heat-treat frozen berries before consumption.

Surveillance of the first 205 confirmed hospitalised cases of pandemic H1N1 influenza in Ireland, 28 April - 3 October 2009.

From 28 April 2009 to 3 October 2009, 205 cases of confirmed pandemic H1N1 influenza were hospitalised in Ireland. Detailed case-based epidemiological information was gathered on all hospitalised cases. Age-specific hospitalisation rates were highest in the age group of 15 to 19 year-olds and lowest in those aged 65 years and over. Nineteen hospitalised cases (9%) were admitted to intensive care units (ICU) where the median length of stay was 24 days. Four hospitalised cases (2%) died. Fifty-one percent of hospitalised cases and 42% of ICU cases were not in a recognised risk group. Asthma was the most common risk factor among cases; however, people with haemoglobinopathies and immunosuppression were the most over-represented groups.

The utilisation and expenditure of medicines for the prophylaxis and treatment of osteoporosis.

The elderly population represent the fastest growing age-group and the incidence of osteoporotic related fractures is likely to increase with continued ageing of the population. This study determined the prescribing patterns on the Health Services Executive's (HSE) Primary Care Reimbursement Services Scheme (HSE-PCRS) of medicines dispensed for the prophylaxis and treatment of osteoporosis in Ireland. The HSE - PCRS prescription databases were analysed for the years 2004/2005. Approximately 65% of patients (total 60,000) were dispensed either Alendronate (Fosamax once weekly) or Risedronate (Actonel once weekly). The majority of the patients (69.3%) were over 70 years. The study identified that the longer a patient was prescribed prednisolone the greater the likelihood of subsequently being prescribed a bisphosphonate. Approximately 50% of patients on long-term steroids did not receive prophylaxis for osteoporosis. There were low levels of co-prescribing (2.5%) with potentially interacting drugs. Levels of co-prescribing with proton pump inhibitors was 22%.

Freeze/thaw-induced embolism depends on nadir temperature: the heterogeneous hydration hypothesis.

Freeze/thaw-induced embolism was studied in leaves of field-grown snow gum (Eucalyptus pauciflora) subject to frequent morning frosts. Juvenile trees were grown in buried pots, brought to the laboratory at different stages of acclimation and subjected to simulated frost-freezes (at 2 degrees C h(-1)) to nadir temperatures of -3 or -6 degrees C, which snow gums commonly experience. Frost-frozen and subsequently thawed leaves were cryo-fixed to preserve the distribution of water and were then examined by cryo-scanning electron microscopy. No embolisms were found in leaves frozen to -3 degrees C and thawed. In contrast, 34% of vessels were embolized in thawed leaves that had been frozen to -6 degrees C. This difference was seen also in the extent of extracellular ice blocks in the mid-vein expansion zones in leaves frozen to -3 and -6 degrees C, which occupied 3 and 14% of the mid-vein area, respectively. While the proportion of embolism depended on nadir temperature, it was independent of season (and hence of acclimation state). From the observation that increased embolism at lower nadir temperature was related to the freeze-induced redistribution of water, we hypothesize that the dehydration of cell walls and cells caused by the redistribution exerts sufficient tension on xylem water to induce cavitation on thawing.

Leaf water content and palisade cell size.

The palisade cell sizes in leaves of Eucalyptus pauciflora were estimated in paradermal sections of cryo-fixed leaves imaged in the cryo-scanning electron microscope, as a quantity called the cell area fraction (CAF). Cell sizes were measured in detached leaves as a function of leaf water content, in intact leaves in the field during a day"s transpiration as a function of balance pressure of adjacent leaves, and on leaf disks equilibrated with air of relative humidities from 100 to 58%. Values of CAF ranged from 0.82 at saturation to approx. 0.3 in leaves dried to a relative water content (RWC) of 0.5, and in the field to approx. 0.58 at 15 bar (1.5 MPa) balance pressure. At a CAF of 0.58, the moisture content of the cell walls is in equilibrium with air at 90% relative humidity, which is the estimated relative humidity in the intercellular spaces. It is shown that at this moisture content, the cell walls could be exerting a pressure of approx. 50 bar on the cell contents.

The management of extracellular ice by petioles of frost-resistant herbaceous plants.

BACKGROUND AND AIMS: Some frost-tolerant herbaceous plants droop and wilt during frost events and recover turgor and posture on thawing. It has long been known that when plant tissues freeze, extracellular ice forms. Distributions of ice and water in frost-frozen and recovered petioles of Trifolium repens and Escholschzia californica were visualized. METHODS: Petioles of intact plants were cryo-fixed, planed to smooth transverse faces, and examined in a cryo-SEM. KEY RESULTS: With frost-freezing, parenchyma tissues shrank to approx. one-third of their natural volume with marked cytorrhysis of the cells, and massive blocks of extracellular icicles grew under the epidermis (poppy) or epidermis and subepidermis (clover), leaving these layers intact but widely separated from the parenchyma except at specially structured anchorages overlying vascular bundles. On thawing, the extracellular ice was reabsorbed by the expanding parenchyma, and surface tissues again contacted the internal tissues at weak junctions (termed faults). These movements of water into and from the fault zones occurred repeatedly at each frost/thaw event, and are interpreted to explain the turgor changes that led to wilting and recovery. Ice accumulations at tri-cellular junctions with intercellular spaces distended these spaces into large cylinders, especially large in clover. Xylem vessels of frozen petioles were nearly all free of gas; in thawed petioles up to 20 % of vessels were gas-filled. CONCLUSIONS: The occurrence of faults and anchorages may be expected to be widespread in frost-tolerant herbaceous plants, as a strategy accommodating extracellular ice deposits which prevent intracellular freezing and consequent membrane disruption, as well as preventing gross structural damage to the organs. The developmental processes that lead to this differentiation of separation of sheets of cells firmly cemented at determined regions at their edges, and their physiological consequences, will repay detailed investigation.

Evidence for protection of nitrogenase from O(2) by colony structure in the aerobic diazotroph Gluconacetobacter diazotrophicus.

Gluconacetobacter diazotrophicus is an endophytic diazotroph of sugarcane which exhibits nitrogenase activity when growing in colonies on solid media. Nitrogenase activity of G. diazotrophicus colonies can adapt to changes in atmospheric partial pressure of oxygen (pO(2)). This paper investigates whether colony structure and the position of G. diazotrophicus cells in the colonies are components of the bacterium's ability to maintain nitrogenase activity at a variety of atmospheric pO(2) values. Colonies of G. diazotrophicus were grown on solid medium at atmospheric pO(2) of 2 and 20 kPa. Imaging of live, intact colonies by confocal laser scanning microscopy and of fixed, sectioned colonies by light microscopy revealed that at 2 kPa O(2) the uppermost bacteria in the colony were very near the upper surface of the colony, while the uppermost bacteria of colonies cultured at 20 kPa O(2) were positioned deeper in the mucilaginous matrix of the colony. Disruption of colony structure by physical manipulation or due to 'slumping' associated with colony development resulted in significant declines in nitrogenase activity. These results support the hypothesis that G. diazotrophicus utilizes the path-length of colony mucilage between the atmosphere and the bacteria to achieve a flux of O(2) that maintains aerobic respiration while not inhibiting nitrogenase activity.

Contributions to the debate on water transport.

The useful criticisms of my theory of water transport by Comstock (American Journal of Botany 86: 1077-1081) and by Stiller and Sperry (American Journal of Botany 86: 1082-1086) are acknowledged and reviewed. I make the following responses. (1) Tensile stresses to contain tissue pressure are kept within modest limits by the organization of vascular tissues into cylindrical bundles with small ratios of radius/boundary thickness. (2) The balance of pressures within tissues of a nontranspiring leaf is best understood by treating it as a single compartment containing several pressure-generating engines whose resultant is the pressure throughout the compartment. An error in the published notional balances for a transpiring leaf is corrected. (3) The argument against a valve in the transpiring leaf, which allows water out but not in, is not convincing. (4) The "robust and extremely consistent" cohesion theory gains this status by neglecting large bodies of experimental fact, once well known to plant physiologists. (5) The demonstration that living cells are not involved in the refilling of embolisms in birch stems is welcomed as an important advance. However, the major questions remain unresolved. (6) Proof is still needed that embolisms in vessels are not refilled by the collapse of gas bubbles under small positive pressures during conductance measurement. (7) The survival of unbroken water threads in vessels under centrifugal stress has still not been demonstrated. (8) Both questions 6 and 7 can be easily answered by direct observation of gas/liquid volumes in frozen stems in the cryo-scanning electron microscope.

Embolisms and refilling in the maize leaf lamina, and the role of the protoxylem lacuna.

The proportion of embolized vessels in the veins of maize leaf laminas was measured during 24 h by direct counting in snap-frozen samples in the cryo-scanning electron microscope. All vessels were sap filled at night. Vessels of intermediate and small veins, and the small tracheary elements of lateral veins, were sap filled throughout the 24 h. The large metaxylem vessels of lateral veins were embolized during the day. The percentage of these vessels embolized was maximum (>70%) at 1400, and declined during the afternoon to 20% at dusk. Leaf water potential reached a minimum (-1.2 MPa) at dusk. The protoxylem lacuna of the lateral veins was much less embolized than the large vessels, although it was of comparable diameter. The observations are interpreted in terms of the refilling hypothesis that is part of the compensating pressure theory of water transport.

The reliability of cryoSEM for the observation and quantification of xylem embolisms and quantitative analysis of xylem sap in situ.

The reliability of cryoSEM for visualizing gas embolisms in xylem vessels of intact, functioning roots is examined and discussed. The possibility that these embolisms form as a result of freezing water columns under tension is discounted by a double-freeze experiment. Two regions of the same root, one frozen under tension, the other isolated from the tension by the first freeze, had the same percentage of embolisms, as did also long pieces of root frozen simultaneously along their length. The reliability of energy-dispersive X-ray analysis to measure xylem sap concentration in situ in frozen tissue was established by measurement of KCl standard solution frozen on stubs, and within xylem vessels. Solute heterogeneity within the vessels varied with freezing procedure; deep-freeze > LN2 > cryopliers > liquid ethane, but only the deep-freeze method gave unsatisfactory estimates of concentration for the standard solution. It is concluded that cryoanalytical SEM is useful for direct observation of gas and liquid-filled compartments, and for solute analyses at depth within intact plant organs.

Xylem feeding by spittlebug nymphs: some observations by optical and cryo-scanning electron microscopy.

The feeding of spittlebug nymphs (Philaenus spumarius) from mature xylem vessels was studied by optical and cryo-analytical scanning electron microscopy. Feeding did not produce xylem embolisms and vessels remained liquid-filled during the day. Saliva secreted by the insect forms a hardened lining (salivary sheath) between the stylet bundle and the plant tissues. This sheath is continuous through the hole made by the stylets as they enter a vessel, and it extends into the vessel and along its periphery beyond the breach. The sheath is heterogeneous, with a thin outer layer adjoining the plant tissues and a thicker layer that contacts the stylet bundle. Both layers give positive histochemical reactions for proteins and, in fresh tissues, contain a red, strongly autofluorescent pigment, possibly condensed tannin derived from the plant (which is lost during tissue preparation), and other phenyl propanoid compounds, which are retained and which may produce the intense reaction of the periodic-acid-Schiff's-positive inner layer. It is concluded that the salivary sheath allows the insects to feed from functioning vessels without embolizing them or losing xylem fluid to the surrounding tissues. These findings and others in the entomological literature indicate low daytime tensions in the xylem conduits of the host plants.

Applications of the compensating pressure theory of water transport.

Some predictions of the recently proposed theory of long-distance water transport in plants (the Compensating Pressure Theory) have been verified experimentally in sunflower leaves. The xylem sap cavitates early in the day under quite small water stress, and the compensating pressure P (applied as the tissue pressure of turgid cells) pushes water into embolized vessels, refilling them during active transpiration. The water potential, as measured by the pressure chamber or psychrometer, is not a measure of the pressure in the xylem, but (as predicted by the theory) a measure of the compensating pressure P. As transpiration increases, P is increased to provide more rapid embolism repair. In many leaf petioles this increase in P is achieved by the hydrolysis of starch in the starch sheath to soluble sugars. At night P falls as starch is reformed. A hypothesis is proposed to explain these observations by pressure-driven reverse osmosis of water from the ground parenchyma of the petiole. Similar processes occur in roots and are manifested as root pressure. The theory requires a pump to transfer water from the soil into the root xylem. A mechanism is proposed by which this pump may function, in which the endodermis acts as a one-way valve and a pressure-confining barrier. Rays and xylem parenchyma of wood act like the xylem parenchyma of petioles and roots to repair embolisms in trees. The postulated root pump permits a re-appraisal of the work done by evaporation during transpiration, leading to the proposal that in tall trees there is no hydrostatic gradient to be overcome in lifting water. Some published observations are re-interpreted in terms of the theory: doubt is cast on the validity of measurements of hydraulic conductance of wood; vulnerability curves are found not to measure the cavitation threshold of water in the xylem, but the osmotic pressure of the xylem parenchyma; if measures of xylem pressure and of hydraulic conductance are both suspect, the accepted view of the hydraulic architecture of trees needs drastic revision; observations that xylem feeding insects feed faster as the water potential becomes more negative are in accord with the theory; tyloses, which have been shown to form in vessels especially vulnerable to cavitation, are seen as necessary for the maintenance of P, and to conserve the supplementary refilling water. Far from being a metastable system on the edge of disaster, the water transport system of the xylem is ultrastable: robust and self-sustaining in response to many kinds of stress.

Retention in situ and spectral analysis of fluorescent vacuole components in sections of plant tissues.

The contents of plant vacuoles vary in different organs and with the health of the plant, but little is known of the cell-to-cell distribution of soluble organic compounds within plant tissues. Soluble fluorescent phenolic compounds can be immobilized in plant tissues using an anhydrous freeze-substitution and resin embedment process. The vacuolar fluorescence can be characterized in fluorescence photomicrographs for variations in color and intensity, or more quantitatively with spectra obtained using a microspectrofluorometer. This is demonstrated here in freeze-substituted roots and leaves of soybean. Excitation and emission spectra of individual vacuoles can be compared with spectra of pure compounds to form profiles of the varied phenolic contents of plant vacuoles. Such analyses will add an important anatomical dimension to the study of plant defense and stress responses.

Vessel contents of leaves after excision - a test of Scholander's assumption.

A test was attempted of the assumption that, when a leaf is cut, the xylem still contains water under tension beyond the first vessel cross walls. This assumption enabled Scholander to argue that the balance pressure in his pressure chamber measured the tension in water columns in the vessels before cutting. The numbers of embolized vessels were counted, after rapid freezing of petiole and midrib samples of sunflower leaves, in the cryo-scanning electron microscope. Counts were made on leaves still attached to the plant and at intervals after cutting from the plant (up to 16 min) during a short spring day's transpiration. The lengths of vessels in the leaves, measured by latex particle perfusion, showed that 8% of vessels in the mid-petioles and 0% in the midribs should be opened by cutting. The changing percentages of embolized vessels (E) with time showed that: (1) in intact plants E was close to zero until midday when it rose to ~40%, and then fell progressively to near zero by 1600; (2) in excised leaves there was no detectable change in E immediately after cutting, and, in all but two time courses, no change as large as the 8% of opened vessels within 16 min; (3) but briefly, when E was high (midday), it rose further after cutting to a plateau (_E = 30-40%) in 4 min. From this rate of emptying, the estimated maximum pressure difference between vessels and parenchyma was of the order of 0.05-0.2 MPa (0.5 to 2 bar) at this time. (4) All these changes occurred in the petioles 1 h before they were found in the midribs. The test failed because the expected large pressure difference between vessels and parenchyma was not present. Further, the embolized vessels were refilled at the time of peak transpiration, which would be impossible with any substantial tension in the vessels. Because these results contradict the whole basis of the Cohesion Theory, a second experiment was carried out to test them, and is reported in a companion paper.

Vessel contents during transpiration - embolisms and refilling.

A test was made of the previous unexpected observation that embolized vessels were refilled during active transpiration. The contents of individual vessels in petioles of sunflower plants were examined, after snap-freezing at 2-h intervals during a day's transpiration, in the cryo-scanning electron microscope, and assessed for the presence of liquid or gas (embolism) contents. Concurrent measurements were made of irradiance, leaf temperature, transpiration rate, and leaf water potential (by pressure chamber). Up to 40% of the vessels were already embolized by 0900 (transpiration rate ~5 _g_cm-2_s-1, water potential about -300 J/kg), and the proportion declined to a minimum (as low as 4%) at 1500. This was the time of highest transpiration rate (~25 _g_cm-2_s-1) and most negative water potential (-600 to -700 J/kg). Images of vessels with mixed gas and liquid contents showed water being extruded through pits in the walls of the vessels to refill them. The data indicate that: (1) the water columns are weak and break under quite small tensions; (2) embolisms are repaired by refilling the vessels with water on a short time scale (minutes) throughout the day; (3) the vigor of this refilling process is adjusted by the plant on a longer time scale (hours) to the intensity of the water stress; (4) the pressure chamber balance pressure (P) does not measure tension in the vessels; (5) P is also not a measure of water stress (as measured by vessel embolization); and (6) P is a measure of the plant's response to water stress, i.e., a measure of the vigor of the refilling process. The test confirms the previous observations and negates all the assumptions and evidences of the Cohesion Theory. The data are fully consistent with the Compensating Pressure Theory, which predicted the relations demonstrated in this experiment. Using the assumptions of that theory it is easy to outline a simple mechanism by which the refilling of vessels might be achieved by reverse osmosis, and the adjustment in (3) might be achieved by osmoregulation in the starch sheath.

Formation and Stabilization of Rhizosheaths of Zea mays L. (Effect of Soil Water Content).

Field observations have shown that rhizosheaths of grasses formed under dry conditions are larger, more coherent, and more strongly bound to the roots than those formed in wet soils. We have quantified these effects in a model system in which corn (Zea mays L.) primary roots were grown through a 30-cm-deep prepared soil profile that consisted of a central, horizontal, "dry" (9% water content) or "wet" (20% water content) layer (4 cm thick) sandwiched between damp soil (15-17% water content). Rhizosheaths formed in dry layers were 5 times the volume of the subtending root. In wet layers, rhizosheaths were only 1.5 times the root volume. Fractions of the rhizosheath soil were removed from individual roots by three successive treatments; sonication, hot water, and abrasion. Sonication removed 50 and 90% of the soil from rhizosheaths formed in dry and wet soils, respectively. After the heat treatment, 35% of the soil still adhered to those root portions where rhizosheaths had developed in dry soil, compared with 2% where sheaths had formed in wet soil. Root hairs were 4.5 times more abundant and were more distorted on portions of roots from dry layers than from wet layers. Drier soil enhanced adhesiveness of rhizosheath mucilages and stimulated the formation of root hairs; both effects stabilize the rhizosheath. Extensive and stable rhizosheaths may function in nutrient acquisition in dry soils.

A Nitrogen-Fixing Endophyte of Sugarcane Stems (A New Role for the Apoplast).

The intercellular spaces of sugarcane (Saccharum officinarum L.) stem parenchyma are filled with solution (determined by cryoscanning microscopy), which can be removed aseptically by centrifugation. It contained 12% sucrose (Suc; pH 5.5.) and yielded pure cultures of an acid-producing bacterium (approximately 104 bacteria/mL extracted fluid) on N-poor medium containing 10% Suc (pH 5.5). This bacterium was identical with the type culture of Acetobacter diazotrophicus, a recently discovered N2-fixing bacterium specific to sugarcane, with respect to nine biochemical and morphological characteristics, including acetylene reduction in air. Similar bacteria were observed in situ in the intercellular spaces. This demonstrates the presence of an N2-fixing endophyte living in apoplastic fluid of plant tissue and also that the fluid approximates the composition of the endophytes's optimal culture medium. The apoplastic fluid occupied 3% of the stem volume; this approximates 3 tons of fluid/ha of the crop. This endogenous culture broth consisting of substrate and N2-fixing bacteria may be enough volume to account for earlier reports that some cultivars of sugarcane are independent of N fertilizers. It is suggested that genetic manipulation of apoplastic fluid composition may facilitate the establishment of similar symbioses with endophytic bacteria in other crop plants.

Planing frozen hydrated plant specimens for SEM observation and EDX microanalysis.

A procedure is described for forming a flat face on a frozen piece of plant tissue, which may then be observed fully-hydrated or lightly etched, and coated or uncoated with a metal film, in scanning electron microscopy (SEM). The frozen sample was planed with a glass knife at -80 degrees C in a cryo-ultramicrotome. The sections were discarded, and the planed block face placed on the cold stage in the microscope column, either for observation uncoated at low kV, or for light etching (-90 degrees C) to reveal the cell outlines. If a higher accelerating voltage was needed, the face was given an evaporative coating of Al in the cryo-preparation chamber and returned to the column. The advantages of the planed face over the usual fracture face are illustrated: imaging at a chosen rather than a chance position; clearer cellular and subcellular detail; preservation of hydrated gels like mucilage and swollen cell walls; the possibility of making serial parallel sections through the same piece of tissue; opportunities for accurate morphometric analyses on the planed face; capacity to produce longitudinal sections; preservation of very delicate structures that are destroyed by fixation and drying. A major advantage of the Al-coated planed face is the increased accuracy of energy-dispersive X-ray (EDX) microanalyses on a smooth rather than a rough surface. Tests are included which show that neither the light etching employed, nor successive planing, interferes with the analyses of elements in the frozen face.