Lyn R. Briggs

Toxic marine epiphytic dinoflagellates, Ostreopsis siamensis and Coolia monotis (Dinophyceae), in New Zealand

Abstract Ostreopsis siamensis and Coolia monotis (Ostreopsidaceae) are newly recognised additions to New Zealands toxic marine microflora and occur epiphytically on seaweeds throughout subtropical New Zealand. O. siamensis “blooms” during summer, becoming freely motile; it tolerates temperatures of 15–25°C and salinities of 28–34 ppt. C. monotis also occurs in sediments in Nelson/ Marlborough and Stewart Island, at 10–30°C and salinities of 20–34 ppt. O. siamensis from Rangiputa, Northland, produced activity equivalent to ≤0.3 pg cell∼’ of palytoxin as measured using a haemolysis‐neutralisation assay, and cell extracts killed mice by intraperitoneal injection. Extracts of oysters fed with O. siamensis did not kill mice. C. monotis, from Rangiputa, produced compounds in extracts of cells and of culture supernatant which were also toxic to mice and which are currently being characterised. C. monotis caused positive sodium channel activity in neuroblastoma assays; O. siamensis was cytotoxic, and irreversibly suppressed cell firing in hippocampal brain slices. Lectin probes differentiated the two species, suggesting differential sugar moieties at their cell surfaces.

Effect of irrigation with lake water containing microcystins on microcystin content and growth of ryegrass, clover, rape, and lettuce.

The effect of irrigation with lake water containing a variety of microcystins on accumulation of toxins, or toxin metabolites, and plant growth in ryegrass, clover, rape, and lettuce, was investigated in a glasshouse experiment. The plants were grown in sand culture and received either three or six applications of lake water, which was applied either directly to the sand surface or to the plant shoots. As determined by LC–MS, each plant received 170 μg of a mixture of 10 different microcystins per application. Microcystins in plant samples were extracted with 70% methanol and analyzed by Adda‐specific ELISA. For the shoot application treatment, microcystins were not present at measurable levels in shoots of ryegrass or rape, but were present in lettuce [0.79 mg/kg dry weight (DW)] and clover (0.20 mg/kg DW). Total microcystin concentration in roots did not vary greatly depending on whether treatment water was applied directly to the sand, or reached the roots via run‐off from the shoots. Microcystins in roots were highest in clover (1.45 mg/kg DW), intermediate in lettuce (0.68 mg/kg DW) and low in ryegrass (0.20 mg/kg DW), and rape (0.12 mg/kg DW). There was no evidence for root‐to‐shoot translocation of microcystins. Three applications of microcystins reduced shoot DW of ryegrass, rape and lettuce, and increased root DW of ryegrass and lettuce. Clover DW was not changed by treatment with microcystins. The results show that irrigation with water containing microcystins has the potential to move microcystins into farm animal and human food chains at concentrations that can exceed recommended tolerable limits.

The exploitation of epichloae endophytes for agricultural benefit

Epichloae endophytes of family Clavicipitaceae (comprising genera Epichloë and Neotyphodium) are fungal symbionts of Pooideae grasses. The associations formed, range from mutually beneficial to antagonistic and the nature of this relationship is dependent upon the importance of vertical (via host seeds) versus horizontal (ascospore mediated) transmission of the fungus. These endophytes can enhance their hosts’ survival through protection from abiotic and biotic stresses and can thus be utilized in an agricultural context. Animal-safe grass-endophyte associations that confer bio-protective properties for increased pasture persistence and productivity have been developed and commercialized. One of the crucial drivers underpinning the selection of epichloae strains for commercial development is endophyte derived bioactivity. The potential of next generation endophytes is determined by testing a number of attributes such as agronomic fitness, animal and food safety as well as compatibility with host plants of interest. Strategic research supports these activities by focusing on elucidating mechanisms of compatibility between host and fungal symbiont, as well as investigating other molecular drivers of symbiosis such as siderophore mediated iron-uptake, fungal signalling, fungal growth in host plants and fungal secondary metabolism. This review weaves together the different strands of multidisciplinary research aimed at ultimately exploiting epichloae endophytes for increased pasture performance.

Uptake of palytoxin‐like compounds by shellfish fed Ostreopsis siamensis (Dinophyceae)

Abstract Greenshell™ mussels (Perna canaliculus Gmelin), scallops (Pecten novaezealandiae Reeve), and Pacific oysters (Crassostrea gigas Thunberg) were fed with a New Zealand strain of mass cultured Ostreopsis siamensis Schmidt (for 27 and 84 h and with 1.5 × 106 or 8.6 × 106 cells, respectively) under laboratory conditions. The microalgal cells contained 0.3 pg palytoxin equivalents cell–1 (as determined by the haemolysis neutralisation assay (HNA) of Bignami (1993)) and extracts of these cells were toxic to mice after intraperitoneal injection. No palytoxin‐like material was detected either in the hepatopancreas or the muscle and roe of mussels fed O. siamensis. Oysters contained detectable amounts of toxin in hepatopancreas muscle, and roe while higher concentrations were present in the hepatopancreas of scallops. Extracts of control shellfish (tested biotoxin free and not fed O. siamensis) were toxic to mice, and there was no definitive evidence that feeding shellfish with O. siamensis at the levels employed in the present experiment increased the toxicity of shellfish tissue extracts to mice.

Survey of cyanotoxins in New Zealand water bodies between 2001 and 2004

Abstract Contamination of drinking and recreational water bodies by toxic cyanobacteria is a significant water management issue in many countries. Until recently, knowledge of the occurrence of cyanotoxins and species responsible for cyanotoxin production in New Zealand was limited. In this study a total of 266 water and cyanobacterial mat samples collected from 227 different water bodies between 2001 to 2004 were analysed for cyanotoxins. Enzyme‐linked immunosorbent assays, liquid chromatography mass spectrometry, high performance liquid chromatography, and neuroblastoma assays resulted in the identification of microcystins/nodularins (102 samples from 54 different water bodies), anatoxin‐a (three samples from three different water bodies), and saxitoxins (48 samples from 41 different water bodies). The highest microcystin concentration was 36.5 mg litre‐1. The detection of anatoxin‐a is the first definitive report for New Zealand. Only low concentrations of saxitoxins were detected. Results indicate that cyanotoxins are more widespread in New Zealand water bodies than previously reported, in particular when cyanobacterial blooms or benthic cyanobacterial mats are present.

Development of immunodiagnostic field tests for the detection of the mycotoxin, sporidesmin A

Three rapid immunoassays—immunofiltration, immunochromatography and dipstick—were developed for the detection of sporidesmin A, the mycotoxin responsible for facial eczema’, a disease which causes liver injury and photosensitization of livestock in New Zealand. Each assay format exhibited particular advantages and disadvantages with regard to assay sensitivity, working range and ease of use. The immunofiltration assay proved to be suitable for the detection of sporidesmin A in pasture due to its sensitivity and its potential to be used outside a laboratory environment. Semi‐quantitative scores of spiked pasture samples using the immunofiltration assay were in good agreement with the amount of sporidesmin A added to the samples.

Development of an ELISA for the Detection of Azaspiracids

Azaspiracids (AZAs) are a group of biotoxins that cause food poisoning in humans. These toxins are produced by small marine dinoflagellates such as Azadinium spinosum and accumulate in shellfish. Ovine polyclonal antibodies were produced and used to develop an ELISA for quantitating AZAs in shellfish, algal cells, and culture supernatants. Immunizing antigens were prepared from synthetic fragments of the constant region of AZAs, while plate coating antigen was prepared from AZA-1. The ELISA provides a sensitive and rapid analytical method for screening large numbers of samples. It has a working range of 0.45-8.6 ng/mL and a limit of quantitation for total AZAs in whole shellfish at 57 μg/kg, well below the maximum permitted level set by the European Commission. The ELISA has good cross-reactivity to AZA-1-10, -33, and -34 and 37-epi-AZA-1. Naturally contaminated Irish mussels gave similar results whether they were cooked or uncooked, indicating that the ELISA also detects 22-carboxy-AZA metabolites (e.g., AZA-17 and AZA-19). ELISA results showed excellent correlation with LC-MS/MS analysis, both for mussel extract spiked with AZA-1 and for naturally contaminated Irish mussels. The assay is therefore well suited to screening for AZAs in shellfish samples intended for human consumption, as well as for studies on AZA metabolism.

Food quality on the farm: Immunological detection of mycotoxins in New Zealand pastoral agriculture

Mycotoxin research throughout the world focuses primarily on the problems associated with stored grains, legumes and the commodities produced from these feed stocks. While contaminated grains and legumes represent a direct threat of major importance to both human and animal consumers, they are not the only source of mycotoxins important to the agricultural sector. Grazing animals are exposed to mycotoxins produced by fungi resident in the pastures they graze, thus, in pastoral agricultural systems, a different spectrum of mycotoxicoses takes prominence. The analysis of toxins from herbage samples by conventional means (such as liquid chromatography and high‐pressure liquid chromatography) is often difficult, slow and expensive. Consequently, we have developed immunoassays which permit analysis of simple extracts and allow the rapid quantification of mycotoxins in large numbers of herbage samples. We detail enzyme‐linked immunosorbent assays using both polyclonal and monoclonal antibodies for the measureme...

Development and evaluation of an enzyme immunoassay for sporidesmin in pasture

Abstract An enzyme immunoassay was developed which could detect sporidesmin in pasture samples. The assay had a linear working range of 0.4–40 ng/ml sporidesmin A while validation using pasture extracts gave mean percentage recoveries of 124% for samples containing 1 ng/ml sporidesmin and 97% for extracts containing 20 ng/ml sporidesmin. The sporidesmin levels and numbers of Pithomyces chartarum spores from many pasture samples were compared. The amount of sporidesmin detected generally corresponded to the number of spores found in the sample.

Development of an enzyme-linked immunosorbent assay for the detection of lolines in pastures

ABSTRACT Polyclonal antibodies were produced in sheep for the development of a competitive enzyme-linked immunoassay for use in quantifying loline alkaloids in pasture samples. Lolines are aminopyrrolizidine secondary metabolites produced by fungal endophytes present in tall fescue and meadow fescue grasses, and confer increased resistance to a range of grass pests. Immunizing and plate coating antigens were prepared with derivatized loline dihydrochloride. Cross-reactivity studies indicated that the assay developed has broad specificity and antibody binding to loline analogues is affected by structural changes in the side chain of the loline molecule. Results obtained using the optimized immunoassay were compared with those determined by gas chromatography. The assay provides a sensitive and rapid analytical method that detects the loline analogues of interest and has been applied in pasture breeding programmes. The assay limit of quantitation for lolines in pasture is 3 µg/g in dried herbage.