Julia M. Griffith

Rotavirus detection and characterisation in outbreaks of gastroenteritis in aged-care facilities.

BACKGROUND Although rotavirus is a major cause of gastroenteritis in children, its role in adult gastroenteritis and the sensitivity of different methods for its detection in specimens collected from adults are less well understood. OBJECTIVES (1) To examine the frequency and seasonality of rotavirus-associated gastroenteritis outbreaks in aged-care facilities in Victoria, Australia. (2) To determine rotavirus type in these outbreaks. (3) To determine whether other enteropathogenic agents are present in specimens from these outbreaks. (4) To examine the sensitivity of different methods (electron microscopy (EM), reverse transcription-polymerase chain reaction (RT-PCR), enzyme immunoassay (EIA) and latex agglutination (LA)) for the detection of rotavirus in specimens from adults. STUDY DESIGN Specimens from gastroenteritis outbreaks in aged-care facilities forwarded to this laboratory for the years 1997-2000 were tested for enteropathogenic agents by various methods. Epidemiological, clinical and seasonal data from the rotavirus-positive outbreaks were analysed. RESULTS Rotavirus was detected by EM in 18 out of 29 individuals associated with seven out of 53 (13%) gastroenteritis outbreaks in aged-care facilities; norovirus was detected in 22 outbreaks (42%) and astrovirus in one outbreak (2%). No mixed viral infection was found in any outbreak. All rotaviruses were typed as Group A by RT-PCR. The rotaviruses in the seven outbreaks were G-typed as follows: G2 (three outbreaks), G4 (two outbreaks), G1 (one outbreak) and G9 (one outbreak). The rotavirus-associated outbreaks were concentrated in mid-winter to mid-spring. The relative sensitivities of the Group A rotavirus detection methods (for the 29 specimens tested) were EM (18), first-round RT-PCR (11), second-round PCR (19), EIA-visual (19), EIA-photometric (19) and LA (13). CONCLUSIONS In Victoria, Australia, outbreaks of gastroenteritis associated with rotavirus are quite common in aged-care facilities. They involve Group A rotavirus and have a winter/spring seasonality. G-types G1, G2, G4 and G9 were all detected. EIA, second-round PCR and EM proved sensitive methods for rotavirus detection whereas first-round RT-PCR and LA did not.

The mechanism of phosphonic (phosphorous) acid action in Phytophthora

Phosphonates exhibit a complex mode of action in their control of fungi. Using Phytophthora palmivora as a model, we have shown that phosphonate anion can act directly on the fungus, reducing growth, and that growth reduction is associated with a rapid reduction in the total pool of adenylate. At lower concentrations, phosphonate alters fungal metabolism without reduction in growth rate. Under these conditions there is a reduction in the amount of macromolecular materials which can be washed from the surface of the mycelia grown on an agar plate. We suggest that these materials contain suppressors, which normally act to delay the expression of the host defence systems. Therefore, a reduction in their synthesis induced by phosphonate could alter the host response and change the host reaction from compatible to incompatible. Superimposed on these effects is that of orthophosphate, which competes for the same carrier as phosphonate and determines the rate at which phosphonate is absorbed into the fungus.

Calcium efflux associated with encystment of Phytophthora palmivora zoospores

Zoospores of the fungus Phytophthora palmivora, pre-labeled with 45Ca, excreted up to 30% of their total 45Ca when stimulated to encyst. Excretion was essentially completed within 90 sec of the application of the stimulus. Encystment of the population was completed within 5 min. Four different stimuli were used: pectin addition (420 micrograms ml-1), Sr2+ addition (5 mM), cyclic AMP addition (6.7 mM) and mechanical agitation. The kinetics and amount of Ca excretion were essentially the same in each case. The calcium ionophore A23187 increased the rate of 45Ca uptake by motile zoospores, incubated in 100 microM CaCl2, but did not induce encystment under these conditions. The ionophore did not induce 45Ca efflux from pre-labeled zoospores. Incubation in EGTA and in K+ failed to induce either encystment or 45Ca excretion. We conclude that rapid excretion of a significant proportion of the zoospore calcium is linked to the early stage of stimulus-induced encystment, and that this comes from an intracellularly located, non-cytoplasmic source, such as the peripheral vesicles, but that changes in cellular Ca2+ are not necessarily the single controlling factor in the induction of encystment.

A nursing home outbreak of Clostridium perfringens associated with pureed food.

Objective: To investigate and ascertain the source of a nursing home outbreak of gastroenteritis in Melbourne in 1997.

31P NMR studies on the effect of phosphite on Phytophthora palmivora

31P NMR spectra were obtained from perchloric acid (PCA) and KOH extracts of Phytophthora palmivora mycelium. Signals indicating the presence of large amounts of short-chain polyphosphate were observed in the spectra of PCA extracts of mycelia grown under both low (0.1 mM) and high (10 mM) phosphate conditions. The mean chain length of polyphosphate was calculated from the relative areas of signals arising from terminal and internal P nuclei in the polyphosphate chain. The small amount of polyphosphate evident in the KOH extract had an average chain length similar to PCA-soluble polyphosphate. 32P tracer studies indicated that phosphorus in the PCA fraction accounted for between 50 and 60% of total phosphorus, the bulk of the remainder being divided between the lipid and KOH extracts. The presence of the fungicide phosphorous acid markedly reduced the average chain length of acid-soluble polyphosphate. This reduction occurred both under low-phosphate conditions, in which treatment with phosphorous acid retards growth, and under high-phosphate conditions, in which no significant growth retardation is observed. Treatment with phosphorous acid perturbed phosphorus distribution and lipid composition under low-phosphate conditions.

Calcium control of differentiation in Phytophthora palmivora

A method has been developed for the preparation of zoospores from Phytophthora palmivora which allows the ionic composition of the suspension medium to be closely controlled. Sub-micromolar concentrations of calcium ions have been shown to play a key role in maintaining the zoospore state and in the transition to the cyst stage. Restriction of free Ca2+ to between 0.2 and 1 μM resulted in zoospores which could be maintained for several hours before they finally encysted and germinated. When exposed to citrus-pectin, or 3 mM SrCl2, or to vigorous shaking, these zoospores underwent rapid synchronous encystment. At free Ca2+ concentrations below 0.1 μM, zoospores lysed slowly. If exposed to inducers of encystment before lysis had occurred, the zoospores failed to respond to pectin or to vigorous shaking. However, they did differentiate in response to SrCl2 addition. Provided the free Ca2+ was maintained between 0.02 and 0.2 μM, zoospores survived gentle centrifugation, a procedure which previously had resulted in encystment.

A model to explain ion-induced differentiation in zoospores ofPhytophthora palmivora

Abstract Zoospores of Phytophthora palmivora undergo synchronous encystment followed by germination when exposed to sodium ions at 3–10 m M , calcium ions at 5–30 m M , and strontium ions at 0.3–10 m M . Lithium ions induce encystment at 3–10 m M but do not induce germination. Ferric, manganese, and barium ions.act as chaotropic agents, damaging the zoospore plasma membrane under both isoosmotic and hypoosmotic conditions, at concentrations as low as 30 μ M , ferric ions and 1 m M , barium ions. Cesium and ammonium ions are cytotoxic and induce lysis under hypoosmotic conditions. The capacity of cations to induce zoospore differentiation can be explained in terms of the following model. It is suggested that differentiation in P. palmivora requires the entry of sodium at one or more specific sites. Access to these sites is regulated by a calcium gated monovalent ion translocator with a high specificity for sodium. Calcium not only regulates sodium ion access but also regulates the cells osmoregulatory system. Divalent cations act by competing for the calcium binding site, monovalent cations by restricting fodium ion access.

Accelerated ion fluxes during differentiation in zoospores of Phytophthora palmivora

Zoospores of Phytophthora palmivora show increased fluxes of Na+ and Ca2+ 3-5 min after they have been stimulated to differentiate with pectin. Both spontaneous and pectin-induced encystment are reduced below pH 6 and accelerated above pH 7. The ionophores monensin and A23187 induce slow differentiation when added together, but not when added separately. Ethanol (0.5%) also induces slow differentiation. Amiloride and verapamil inhibit pectin-induced differentiation and also reduce the onset of the Na+ and Ca2+ flux. A requirement for Ca2+ for differentiation is confirmed, but a requirement for Na+ could not be demonstrated.

Properties of the phosphate and phosphite transport systems of Phytophthora palmivora

Germlings of Phytophthora palmivora possess at least two systems for the uptake of inorganic phosphate (Pi). The first is synthesized on germination in medium containing 50 μM Pi and has a Km of approx. 30 μM (Vmax=7–9 nmol Pi/h·106 cells). The second is synthesized under conditions of Pi-deprivation and has a higher affinity for Pi (Km=1–2 μM), but a lower Vmax (0.5–2 nmol Pi/h·106 cells). The fungicide phosphite likewise enters the germlings via two different transport systems, the synthesis of which also depends on the concentration of Pi in the medium. The Km of the lower affinity system is 3 mM (Vmax=20 nmol phosphite/h·106 cells) and that of the higher affinity system is 0.6 mM (Vmax=12 nmol/h·106 cells). Pi and phosphite are competitive inhibitors for each others transport in both systems. However, whereas mM concentrations of phosphite are necessary to inhibit Pi transport, only μM concentrations of Pi are required to inhibit phosphite transport. A third system of uptake for Pi also exists, since when phosphate-deprived cells are presented with mM concentrations of Pi, they transport the anion at a very high rate (around 100 nmol/h·106 cells). High rates of transport of phosphite are also observed when these cells are presented with mM concentrations of this anion.

Phosphonate inhibition as a function of phosphate concentration in isolates of Phytophthora palmivora

SUMMARY: Three isolates of Phytophthora palmivora showing differing sensitivities to phosphonate were grown in the presence of a uniform concentration of the anion (1 mM), together with concentrations of phosphate which varied from zero to 3 mM. The phosphonate-sensitive isolate was inhibited by phosphonate at all levels of phosphate, but the resistant isolates were inhibited by phosphonate only when phosphate was limiting to growth and were able to exclude phosphonate more effectively than the sensitive isolate at higher concentrations of phosphate. The percentage inhibition in each strain was proportional to the internal concentration of phosphonate, but the slope of the plot of inhibition against internal phosphonate varied between strains. Differences in the capacity to discriminate between phosphate and phosphonate therefore provide part of, but not the whole explanation for differences in sensitivity between isolates. There was a significant increase in the internal concentration of orthophosphate in the mycelia which were inhibited by phosphonate.