Barry J. Blaney

Pyrrolizidine Alkaloids in Crotalaria Taxa from Northern Australia: Risk to Grazing Livestock

Crotalaria species containing hepatotoxic pyrrolizidine alkaloids grow widely in pastures in northern Australia and have sporadically poisoned grazing livestock. The diverse Crotalaria taxa present in these pastures include varieties, subspecies, and chemotypes not previously chemically examined. This paper reports the pyrrolizidine alkaloid composition and content of 24 Crotalaria taxa from this region and assesses the risk of poisoning in livestock consuming them. Alkaloids present in C. goreensis , C. aridicola subsp. densifolia, and C. medicaginea var. neglecta lack the esterified 1,2-unsaturated functionality required for pyrrole adduct formation, and these taxa are not hepatotoxic. Taxa with high levels of hepatotoxic alkaloids, abundance, and biomass pose the greatest risk to livestock health, particularly C. novae-hollandiae subsp. novae-hollandiae, C. ramosissima , C. retusa var. retusa, and C. crispata . Other species containing moderate alkaloid levels, C. spectabilis and C. mitchellii , also pose significant risk when locally abundant.

Alkaloids of the sorghum ergot pathogen (Claviceps africana): assay methods for grain and feed and variation between sclerotia/sphacelia

Assay methods for the alkaloids of sorghum ergot (Claviceps africana) are described and compared. Sorghum ergot bodies (sclerotia/sphacelia) from various regions of Queensland and New South Wales were collected in 1997 and 2001 and assayed by spectrophotometry, thin layer chromatography, or high performance liquid chromatography (HPLC). All contained dihydroergosine (DHES) as the main alkaloid component (about 80%), with smaller amounts of dihydroelymoclavine and festuclavine. The preferred method of assay for infected sorghum and mixed feeds involved extraction into dichloromethane:methanol:ethyl acetate:ammonium hydroxide (50:5:25:1) using an ultrasonic bath. After solvent removal, the extract was dissolved in diethyl ether and partitioned into 0.5 M hydrochloric acid. After adjusting the pH to 8–10 with ammonium hydroxide, the alkaloids were extracted into dichloromethane, the solvent evaporated, and the residue dissolved in methanol. HPLC separation was on a C18 column, 150 × 3.9 mm, run isocratically at 40°C, with acetonitrile:0.1% ammonium acetate:methanol (31:50:20) as the mobile phase. Detection was either by UV at 280 nm or by fluorescence with excitation at 235 nm and absorbance at 340 nm. Levels of quantitation for DHES in sorghum approached 0.1 mg/kg (UV) and 0.01 mg/kg (fluorescence). Method recoveries for DHES in the range of 0.025–7 mg/kg averaged 75%. The total alkaloid content of ergot bodies (sclerotia/sphacelia) from different batches of grain varied from 100 to 7900 mg/kg (0.79%). Within batches, there was much less variation in the alkaloid content of ergot bodies, but larger ergots tended to contain more alkaloid than smaller ergots, and those infected with Cerebella species contained even less; this probably related to the ratio of sclerotial/sphacelial tissue present. Honeydew also contained DHES (1–10 mg/kg) and might contaminate clean grain at significant levels. Tests on 4 farms showed that substantial amounts of ergot bodies and alkaloids were removed during grain harvesting.

Residue Potential of Norsesquiterpene Glycosides in Tissues of Cattle Fed Austral Bracken (Pteridium esculentum).

Austral bracken, Pteridium esculentum , occurs widely in Australian grazing lands and contains both the known carcinogen ptaquiloside and its hydroxy analogue, ptesculentoside, with untested carcinogenic potential. Calves were fed a diet containing 19% P. esculentum that delivered 1.8 mg of ptaquiloside and 4.0 mg of ptesculentoside per kilogram of body weight (bw) per day to explore the carcass residue potential of these compounds. Concentrations of ptaquiloside and ptesculentoside in the liver, kidney, skeletal muscle, heart, and blood of these calves were determined as their respective elimination products, pterosin B and pterosin G, by HPLC-UV analysis. Plasma concentrations of up to 0.97 μg/mL ptaquiloside and 1.30 μg/mL ptesculentoside were found, but were shown to deplete to <10% of these values within 24 h of bracken consumption. Both glycosides were also detected in all tissues assayed, with ptesculentoside appearing to be more residual than ptaquiloside. Up to 0.42 and 0.32 μg/g ptesculentoside was present in skeletal muscle and liver, respectively, 15 days after bracken consumption ended. This detection of residual glycosides in tissues of cattle feeding on Austral bracken raises health concerns for consumers and warrants further investigation.

Managing mycotoxins in maize: case studies

Mycotoxin contamination of Australian maize is neither common nor extensive, but has the capacity to seriously disrupt marketing. Low to moderate levels of aflatoxins and fumonisins can be widespread in some seasons, but zearalenone, nivalenol and deoxynivalenol are usually confined to small growing localities. Possible approaches to such situations were tested by an analysis of several case studies. It is concluded that communication and coordination across the industry, prediction and prevention of contamination, rapid detection and assessment of contamination, effective use of contaminated maize and breeding for resistance comprise a useful set of strategies for managing mycotoxins in maize.

Norsesquiterpene glycosides in bracken ferns (Pteridium esculentum and Pteridium aquilinum subsp. wightianum) from eastern Australia: reassessed poisoning risk to animals

Austral bracken Pteridium esculentum contains three unstable norsesquiterpene glycosides: ptaquiloside, ptesculentoside, and caudatoside, in variable proportions. The concentration of each of the glycosides was determined in this study as their respective degradation products, pterosin B, pterosin G and pterosin A, by HPLC-UV analysis. Samples of P. esculentum collected from six sites in eastern Australia contained up to 17 mg of total glycoside/g DW, with both ptaquiloside and ptesculentoside present as major components accompanied by smaller amounts of caudatoside. Ratios of ptaquiloside to ptesculentoside varied from 1:3 to 4:3, but in all Australian samples ptesculentoside was a significant component. This profile differed substantially from that of P. esculentum from New Zealand, which contained only small amounts of both ptesculentoside and caudatoside, with ptaquiloside as the dominant component. A similar profile with ptaquiloside as the dominant glycoside was obtained for Pteridium aquilinum subsp. wightianum (previously P. revolutum ) from northern Queensland and also P. aquilinum from European sources. Ptesculentoside has chemical reactivity similar to that of ptaquiloside and presumably biological activity similar to that of this potent carcinogen. The presence of this additional reactive glycoside in Australian P. esculentum implies greater toxicity for consuming animals than previously estimated from ptaquiloside content alone.

Alkaloids in Australian rye ergot (Claviceps purpurea) sclerotia: implications for food and stockfeed regulations

Rye ergot (Claviceps purpurea) occasionally causes toxicity (chiefly expressed as hyperthermia) in Australian livestock, either as a result of grazing infected annual (Lolium rigidum) and perennial (L. perenne) rye grasses, or if the ergot sclerotia produced in rye grasses contaminate grain crops used as stockfood. Alkaloids in 30 samples of Australian rye ergot sclerotia taken from rye grasses and grain screenings, and some feed samples contaminated with rye grass ergot sclerotia, were assayed by high performance liquid chromatography. Samples originated from across southern Australia. Ergotamine was the dominant alkaloid in all samples, followed by α-ergocryptine, ergocornine, ergosine and their respective -imine epimers. Ergotamine concentrations in sclerotia ranged up to 2257 mg/kg (as received basis). Ergocristine was a very minor component (<50 mg/kg) in all samples. Total alkaloids in freshly collected sclerotia ranged from 1003 to 3321 mg/kg (0.10 to 0.33%), and up to 3766 mg/kg with epimers included, although lower concentrations were found in samples stored for some time. Alkaloid profiles in sclerotia were all very similar, and concentrations did not appear to be related to size of sclerotia, source region, nor to the rye grass or grain from which they were taken. Previous cases of toxicity in livestock are reviewed and several new cases are reported. The implications of variable alkaloid contents of rye ergot sclerotia are discussed in terms of Australian food and stockfeed regulations.

Isoquinoline alkaloids and keto-fatty acids of Argemone ochroleuca and A. mexicana (mexican poppy) seed. I. An assay method and factors affecting their concentration

An assay for the isoquinoline alkaloids of Argemone ochroleuca and A. mexicana seeds is described. The method consists of extraction into weakly acidified methanol and ion-pair high performance liquid chromatography with sodium lauryl sulfate and tartaric acid in acetonitri1e:water as eluent. Analysis of A. ochroleuca seed showed it to contain dihydrosanguinarine and dihydrochelerythrine (c. 3 : 2) as major alkaloid components with minor amounts of protopine, sanguinarine, berberine and chelerythrine. A single sample of authenticated A. mexicana seed contained dihydrosanguinarine as the major alkaloid with minor amounts of sanguinarine and berberine, in agreement with earlier studies. Dihydrosanguinarine and dihydrochelerythrine were measured as their oxidized products, sanguinarine and chelerythrine, after U.V. irradiation. A crystalline solid which separates from A. ochroleuca seed oil was shown to contain 11-oxo-octacosanoic acid and 11-oxo-triacontanoic acid, which are also the major components of a similar solid from A. mexicana seed oil. Mexican poppy seed (probably A. ochroleuca) collected from various regions of Queensland during 1987-89 showed that place of origin and length of storage of intact seed had little effect on alkaloid levels. Exposure of crushed seed to light, however, caused a rapid decrease in the concentration of dihydro-alkaloids. Total alkaloid content correlated with seed maturity, with immature seed containing much less than mature seed.

Effect of Feeding Sorghum ergot (Claviceps africana) to sows during mid-lactation on plasma prolactin and litter performance

Diets containing 3% sorghum ergot (16 mg alkaloids/kg, including 14 mg dihydroergosine/kg) were fed to 12 sows from 14 days post-farrowing until weaning 14 days later, and their performance was compared with that of 10 control sows. Ergot-fed sows displayed a smaller weight loss during lactation of 24 kg/head vs. 29 kg/head in control sows (p > 0.05) despite feed consumption being less (61 kg/head total feed intake vs. 73 kg/head by control sows; p < 0.05). Ergot-fed sows had poorer weight gain of litters over the 14-day period (16.6 kg/litter vs. 28.3 kg/litter for controls; p < 0.05) despite an increase in consumption of creep feed by the piglets from the ergot-fed sows (1.9 kg/litter compared with 1.1 kg/litter by the control; p > 0.05). Sow plasma prolactin was reduced with ergot feeding after 7 days to 4.8 microg/l compared with 15.1 microg/l in the control sows (p < 0.01) and then at weaning was 4.9 microg/l compared with 8.0 microg/l (p < 0.01) in the control sows. Two sows fed ergot ceased lactation early, and the above sow feed intakes, body weight losses with litter weight gains and creep consumption indirectly indicate an ergot effect on milk production.

Spontaneous fermentation of traditional sago starch in Papua New Guinea.

Sago starch is an important dietary carbohydrate in lowland Papua New Guinea (PNG). An investigation was conducted to determine whether microbes play a role in its preservation using traditional methods. In 12 stored sago samples collected from PNG villages, lactic acid bacteria (LAB) were present (> or = 3.6 x 10(4)cfu/g) and pH ranged from 6.8 to 4.2. Acetic and propionic acids were detected in all samples, while butyric, lactic and valeric acids were present in six or more. In freshly prepared sago, held in sealed containers in the laboratory at 30 degrees C, spontaneous fermentation by endogenous microflora of sago starch was observed. This was evident by increasing concentrations of acetic, butyric and lactic acids over 4 weeks, and pH reducing from 4.9 to 3.1: both LAB and yeasts were involved. Survival of potential bacterial pathogens was monitored by seeding sago starch with approximately 10(4)/g of selected organisms. Numbers of Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus fell to < 30/g within 7 days. Salmonella sp. was present only in low numbers after 7 days (< 36/g), but Escherichia coli was still detectable after three weeks (> 10(2)/g). Fermentation appeared to increase the storability and safety of the product.

Risk management for mycotoxin contamination of Australian maize

Recent incidents of mycotoxin contamination (particularly aflatoxins and fumonisins) have demonstrated a need for an industry-wide management system to ensure Australian maize meets the requirements of all domestic users and export markets. Results of recent surveys are presented, demonstrating overall good conformity with nationally accepted industry marketing standards but with occasional samples exceeding these levels. This paper describes mycotoxin-related hazards inherent in the Australian maize production system and a methodology combining good agricultural practices and the hazard analysis critical control point framework to manage risk.