Outbreak of Escherichia coli O157 Phage Type 32 linked to the consumption of venison products.

In September 2015, an outbreak of Escherichia coli Phage Type 32 with an indistinguishable multi locus variable number tandem repeat analysis profile was identified in Scotland. Twelve cases were identified; nine primary cases, two secondary and one asymptomatic case. Extensive food history investigations identified venison products containing wild venison produced by a single food business operator as the most likely source of the outbreak. Of the nine primary cases, eight had consumed venison products, and one case had not eaten venison themselves but had handled and cooked raw venison in the household. This was the first reported outbreak of Shiga toxin-producing Escherichia coli (STEC) linked to venison products in the UK, and was also notable due to the implicated products being commercially produced and widely distributed. In contrast, previous venison outbreaks reported from other countries have tended to be smaller and related to individually prepared carcases. The outbreak has highlighted some important knowledge gaps in relation to STEC in venison that are currently been investigated via a number of research studies.

A cluster of Legionnaires' disease and associated Pontiac fever morbidity in office workers, Dublin, June-July 2008.

In June and July 2008, two office workers were admitted to a Dublin hospital with Legionnaires' disease. Investigations showed that cooling towers in the basement car park were the most likely source of infection. However, positive results from cooling tower samples by polymerase chain reaction (PCR) did not correlate with subsequent culture results. Also, many employees reported Pontiac fever-like morbidity following notification of the second case of Legionnaires' disease. In total, 54 employees attended their general practitioner or emergency department with symptoms of Legionnaires' disease or Pontiac fever. However, all laboratory tests for Legionnaires' disease or Pontiac fever were negative. In this investigation, email was used extensively for active case finding and provision of time information to employees and medical colleagues. We recommend clarification of the role of PCR in the diagnosis of legionellosis and also advocate for a specific laboratory test for the diagnosis of the milder form of legionellosis as in Pontiac fever.

Occurrence of toxigenic cyanobacterial blooms in freshwaters of Sri Lanka.

A previous pioneering study of freshwater bodies in Sri Lanka revealed the presence of toxic cyanobacteria in three out of four water bodies tested. It was therefore important to perform a more detailed investigation into the presence of cyanobacteria and their toxins throughout Sri Lanka. The country has a long history of well-planned water management with the agricultural economy and drinking water supply still dependent on thousands of man-made tanks. Seventeen reservoirs from different user categories and different climatic zones were selected to study variations in phytoplankton communities with relation to major nutrients, with particular emphasis on cyanobacteria. The study was carried out during a two-year period and heavy growths or blooms of cyanobacteria observed during the study period were tested for microcystins. The results clearly categorised the 17 reservoirs into four groups parallel to the classification based on the user categories of water bodies. Biomass of total phytoplankton, the abundance of cyanobacteria, the dominance of Microcystis spp. and concentration of nitrate (N) and total phosphorous (P) were the lowest in drinking water bodies and the highest in aesthetic water bodies. Irrigation water bodies showed the second lowest values for phytoplankton biomass, and concentration of N and P, while hydropower reservoirs showed the second highest values for the same parameters. The fraction of cyanobacteria in irrigation waters was higher than that in hydropower reservoirs, but surprisingly the dominance of Microcystis spp. was reversed. Possible reasons for these variations are discussed. More than half of the bloom material tested contained microcystins up to 81microgl(-1). Our findings indicate the potential for high-risk situations due to toxigenic cyanobacterial blooms in susceptible freshwaters of Sri Lanka.

Can general anaesthesia be used for the Paralytic Shellfish Poison bioassay?

The current method for Paralytic Shellfish Poisoning (PSP) testing in shellfish is based on the mouse bioassay (MBA), which involves injecting shellfish extract into a conscious mouse, and then converting its time to death into PSP toxicity using Sommer's table. To improve animal welfare, the present study investigated the use of anaesthesia. A saxitoxin (STX) calibration study was conducted where known amounts of STX were injected into both unanaesthetised and anaesthetised mice. Death time was approximately doubled when mice were anaesthetised. Both unanaesthetised and anaesthetised animals showed a linear relationship between the inverse death time and log(STX). Based on these data, new calibration curves were developed. This study revealed that the current method employing Sommer's table underestimates toxicity by up to 50% for higher toxin levels. Subsequently, shellfish samples were tested on both unanaesthetised and anaesthetised mice. Using the new calibration curves, the numbers of samples exceeding the field closure limit were similar for unanaesthetised and anaesthetised mice, and were nearly two-fold higher than those obtained with the current method. The studies showed that the bioassay gives variable results for both unanaesthetised and anaesthetised animals. Anaesthesia forms a viable and more ethical alternative to the current bioassay, at least in the short term. A practical summary on how to conduct this method is given.

Rapid selection of anti-hapten antibodies isolated from synthetic and semi-synthetic antibody phage display libraries expressed in Escherichia coli.

Antibody phage display libraries (Griffin and Tomlinson I) displaying antibody genes and maintained and amplified in Escherichia coli were used to isolate antibodies to the hapten target microcystin LR (1000 Da) conjugated to either bovine serum albumin or keyhole limpet haemocyanin. In competition enzyme-linked immunosorbent assay, bacterially expressed antibodies selected via the Griffin library showed at least 300 times greater sensitivity than those isolated from the Tomlinson library, for free microcystin. Bacterially expressed phage antibody libraries provide a rapid and relatively easy route for the selection of monoclonal antibodies specific for even the most difficult of antigenic targets such as free haptens.

Investigations into the inhibitory effects of microcystins on plant growth, and the toxicity of plant tissues following exposure.

The cyanobacterial toxins microcystins are known to affect a number of processes in plant tissues, and their presence in water used for irrigation may have considerable impact on the growth and development of crop plants. In this study, two plant bioassays were employed to investigate the phytotoxic effects of microcystins. A plant tissue culture assay revealed that the growth and chlorophyll content of Solanum tuberosum L. cultures was inhibited at microcystin-LR concentrations of 0.005 and 0.05 microg x cm(-3), respectively. A previously developed bioassay was also employed to determine the effects of three commonly occurring microcystin variants on the growth of Synapis alba L. seedlings. Microcystins-LR, -RR, and -LF inhibited the growth of seedlings, with GI50 values of 1.9, 1.6 and 7.7 microg x ml(-1), respectively. The growth of Phaseolus vulgaris was also examined in the presence of microcystin-LR. The toxin was found to have little effect on growth for up to 18 days, but impaired the development of the roots of exposed plants, causing them to take up approximately 30% less growth medium than those grown in the absence of toxin. Microcystin was also detected in the tissues of exposed plants using a commercially available ELISA kit, suggesting that the uptake of these toxins by edible plants may have significant implications for human health.

Detection and quantification of microcystins (cyanobacterial hepatotoxins) with recombinant antibody fragments isolated from a naïve human phage display library.

Single-chain antibody fragments against the cyanobacterial hepatotoxin microcystin-LR were isolated from a naive human phage display library and expressed in Escherichia coli. In competition enzyme-linked immunosorbent assay (ELISA), the most sensitive antibody clone selected from the library detected free microcystin-LR with an IC(50) value of 4 microM. It was found to cross react with three other microcystin variants - microcystin-RR, microcystin-LW and microcystin-LF - and detected microcystins in extracts of the cyanobacterium Microcystis aeruginosa, found to contain the toxins by high-performance liquid chromatography (HPLC). The quantification of microcystins in these extracts by ELISA and HPLC showed good correlation. Although the antibody isolated in this study was considerably less sensitive than the polyclonal and monoclonal antibodies already available for microcystin detection, phage display technology represents a cheaper, more rapid alternative for the production of anti-microcystin antibodies than the methods currently in use.

Development of a bioassay employing the desert locust (Schistocerca gregaria) for the detection of saxitoxin and related compounds in cyanobacteria and shellfish.

Monitoring paralytic shellfish toxins (PST) in shellfish and freshwater cyanobacteria is largely dependent on the mouse bioassay. An alternative assay was devised using the desert locust Schistocerca gregaria. The bioassay successfully identified the optimum extraction procedure for PST from cyanobacterial cells and was also suitable for screening acid extracts of shellfish flesh. These results demonstrate the potential of the locust bioassay for the routine screening of PST in a range of sample matrices.