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Stingless bee honeys (SBHs) from Australian and Malaysian species were analysed using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) for the presence of pyrrolizidine alkaloids (PAs) and the corresponding N-oxides (PANOs) due to the potential for such hepatotoxic alkaloids to contaminate honey as a result of bees foraging on plants containing these alkaloids. Low levels of alkaloids were found in these SBHs when assessed against certified PA standards in targeted analysis. However, certain isomers were identified using untargeted analysis in a subset of honeys of Heterotrigona itama which resulted in the identification of a PA weed species (Ageratum conyzoides) near the hives. The evaluation of this weed provided a PA profile matching that of the SBH of H. itama produced nearby, and included supinine, supinine N-oxide (or isomers) and acetylated derivatives. These PAs lacking a hydroxyl group at C7 are thought to be less hepatoxic. However, high levels were also observed in SBH (and in A. conyzoides) of a potentially more toxic diester PA corresponding to an echimidine isomer. Intermedine, the C7 hydroxy equivalent of supinine, was also observed. Species differences in nectar collection were evident as the same alkaloids were not identified in SBH of G. thoracica from the same location. This study highlights that not all PAs and PANOs are identified using available standards in targeted analyses and confirms the need for producers of all types of honey to be aware of nearby potential PA sources, particularly weeds. © 2024 by the authors.

Pimelea poisoning of cattle causes distinct symptoms and frequently death, attributable to the toxin simplexin. Pimelea poisoning was induced via addition of ground Pimelea trichostachya plant to the daily feed in a three-month trial with Droughtmaster steers. The trial tested four potential mitigation treatments, namely, biochar, activated biochar, bentonite, and a bacterial inoculum, and incorporated negative and positive control groups. All treatments tested were unable to prevent the development of simplexin poisoning effects. However, steers consuming a bentonite adsorbent together with Pimelea showed lesser rates-of-decline for body weight (P < 0.05) and four hematological parameters (P < 0.02), compared to the positive control group fed Pimelea only. Microbiome analysis revealed that despite displaying poisoning symptoms, the rumen microbial populations of animals receiving Pimelea were very resilient, with dominant bacterial populations maintained over time. Unexpectedly, clinical edema developed in some animals up to 2 weeks after Pimelea dosing was ceased.

Pimelea poisoning of cattle is a unique Australian toxic condition caused by the daphnane orthoester simplexin present in native Pimelea pasture plants. Rumen microorganisms have been proposed to metabolise simplexin by enzymatic reactions, likely at the orthoester and epoxide moieties of simplexin, but a metabolic pathway has not been confirmed. This study aimed to investigate this metabolic pathway through the analysis of putative simplexin metabolites. Purified simplexin was hydrolysed with aqueous hydrochloric acid and sulfuric acid to produce target metabolites for UPLC-MS/MS analysis of fermentation fluid samples, bacterial isolate samples, and other biological samples. UPLC-MS/MS analysis identified predicted hydrolysed products from both acid hydrolysis procedures with MS breakdown of these putative products sharing high-resolution accurate mass (HRAM) fragmentation ions with simplexin. However, targeted UPLC-MS/MS analysis of the biological samples failed to detect the H2SO4 degradation products, suggesting that the rumen microorganisms were unable to produce similar simplexin degradation products at detectable levels, or that metabolites, once formed, were further metabolised. Overall, in vitro acid hydrolysis was able to hydrolyse simplexin at the orthoester and epoxide functionalities, but targeted UPLC-MS/MS analysis of biological samples did not detect any of the identified simplexin hydrolysis products.

Pimelea poisoning of cattle occurs only in arid inland Australia and is caused by the toxin, simplexin found in some native Pimelea plant species. Pimelea-affected cattle show distinctive physical symptoms including subcutaneous oedema under the jaw and brisket, diarrhoea and anaemia. Simplexin was thought to be circulated in the bloodstream to exert its toxic effect, but due to its hydrophobic nature the extent of its blood carriage is unknown. Fletcher et al. (2014) postulated a possible role of rumen microorganisms adapting to detoxify simplexin in cattle fed Pimelea over a prolonged period of time. Anecdotal reports have suggested cattle supplemented with biochar and bentonite showed resistance towards Pimelea poisoning. In this project, a pen trial (QAFFI/QASP/337/20/DAF) was conducted to determine the efficacy of adsorbents (biochar and bentonite) and a bacterial inoculum for reducing the effects of Pimelea poisoning in steers.

Three species of Australian native plants, Pimelea trichostachya, P. simplex and P. elongata, are endemic to the arid rangelands of Queensland, New South Wales and South Australia and are responsible for Pimelea poisoning, also known as St George or Marree disease. Pimelea poisoning occurs in cattle ingesting Pimelea plants, with the orthoester simplexin identified as the responsible toxin. There is no effective treatment and economic losses have been estimated at over $50 million during significant Pimelea poisoning events. In a previous feeding trial, animals were fed increasing amounts of Pimelea, and after initially showing signs of poisoning, the animals appeared to adapt to ingesting Pimelea, possibly through rumen microbial degradation of the toxin (Fletcher et al., 2014). Kangaroos, forestomach fermenters, often graze pastures containing Pimelea with no apparent ill effects. To investigate the degradation effect further, a series of 30 day in vitro, anaerobic fermentations were undertaken.

Honey is a popular agricultural product containing mostly sugars and water, but due to its nutritious components and natural production by honeybees (Apis mellifera) from floral nectar, it is marketed as a premium health food item. As environmental monitors, honeybees can potentially transfer environmental contaminants to honey. Whilst pesticides can have ubiquitous presence in agricultural and urban areas, polycyclic aromatic hydrocarbons (PAHs) can be more prevalent in higher density urban/industrial environments. Australian beehives are customarily located in rural areas/forests, but it is increasingly popular to keep hives in urban areas. This study assessed the levels of environmental contaminants in honeys (n = 212) from Queensland/Australian sources including rural, peri-urban and urban areas. Honey samples were analysed by LC-MS/MS and GC-MS/MS for 53 herbicides, 83 pesticides, 18 breakdown products (for certain pesticides/herbicides) and 33 PAHs and showed low/negligible pesticide, herbicide and PAHs contamination, consistent regardless of honey origins.

Animal feeds may contain exogenous compounds that can induce toxicity when ruminants ingest them. These toxins are secondary metabolites originating from various sources including plants, bacteria, algae and fungi. Animal feed toxins are responsible for various animal poisonings which negatively impact the livestock industry. Poisoning is more frequently reported in newly exposed, naïve ruminants while ‘experienced’ ruminants are observed to better tolerate toxin-contaminated feed. Ruminants can possess detoxification ability through rumen microorganisms with the rumen microbiome able to adapt to utilise toxic secondary metabolites. The ability of rumen microorganisms to metabolise these toxins has been used as a basis for the development of preventative probiotics to confer resistance against the poisoning to naïve ruminants. In this review, detoxification of various toxins, which include plant toxins, cyanobacteria toxins and plant-associated fungal mycotoxins, by rumen microorganisms is discussed. The review will include clinical studies of the animal poisoning caused by these toxins, the toxin mechanism of action, toxin degradation by rumen microorganisms, reported and hypothesised detoxification mechanisms and identified toxin metabolites with their toxicity compared to their parent toxin. This review highlights the commercial potential of rumen inoculum derived probiotics as viable means of improving ruminant health and production.

Honey is a widely available natural sweetener containing sugars, and small quantities ofvitamins and minerals, proteins, amino acids and fatty acids. Owing to its nutritious components,commercial honeys are sold in bulk blends or as trendy and premium products. Meanwhile, honeybees are considered as environmental monitors and have the potential to transfer environmentalcontaminants, if present, to honey. In high density urban and industrial environments polycyclicaromatic hydrocarbons (PAHs) and heavy metals can be prevalent, whilst pesticides and mineraland trace elements are ubiquitous. Honey hives are traditionally located in rural and forested areas,but there is a growing trend to locate hives in urban areas. This project has investigated the presenceof environmental contaminants in honey samples from high density urban, peri-urban as well asrural areas. Australian honey samples (n = 211) were purchased between 2016 and 2018, including52 honeys claiming to be of urban origin purchased online. Stingless bee honeys (n = 36) fromQueensland and Malaysia were compared. Processed samples were analysed by UHPLC-MS/MS(herbicides), GC-MS/MS (pesticides and PAHs) and ICP-MS and ICP-OES (elemental analyses). Theresults showed low or negligible pesticide, herbicide, and PAH contamination, and that these lowresults were similar regardless of urban or rural origins. Wide variations of essential trace element(Fe, Zn, Cu, Mo, Co, Mn, Cr) and mineral levels (K, Na, P, Mg, Ca) were found in honey products,which are a good dietary source of K and Zn. Relatively low levels of toxic heavy metals were foundin honeys.

The honey bee industry in Australia is small but has a big impact on both producers and consumers. Alarmingly, it has been recently reported that an international laboratory, specializing in honey authentication, found that almost half of the 28 blended and imported honey samples selected from Australian supermarket shelves were "adulterated". The Official method of honey analysis (AOAC 998.12) is based on the stable-isotope ratio mass spectrometry analysis of δ13C value of honey versus δ13C of honey protein to detect the addition of C4 plant derived sugars, such as cane sugar or high fructose corn syrup. This method is used as the primary C4 sugar adulteration test around the world, but honey derived from some Australian plants, particularly Manuka (Leptospermum species), fails this process. Our research aims to examine the characteristics of Australian honey and develop a test that is fit for purpose, particularly where honeys derived from Leptospermum species are concerned. We have focused on the isotopic values of "proteins"; precipitated using the standard AOAC method and “proteins” precipitated after incorporation of a further modification step which removes insoluble material (including pollen) from the honey before precipitation. Our modified method includes the analysis of different isotopes of the precipitated protein, sugar profiles and Manuka markers. A key advantage of the proposed modification is that it does not preclude the detection of residual sugar feeding of bees or extension of honey with C4 sugar. The proposed modification to the AOAC test will reduce false identification of C4 sugars and improve the overall reliability of Australian honey authentication.

Pimelea poisoning affects cattle grazing arid rangelands of Australia, has no known remedy and significant outbreaks can cost the industry $50 million per annum. Poisoning is attributable to consumption of native Pimelea plants containing the toxin simplexin. Charcoal, bentonite and other adsorbents are currently used by the livestock industry to mitigate the effects of mycotoxins. The efficacy of such adsorbents to mitigate Pimelea poisoning warrants investigation. Through a series of in vitro experiments, different adsorbents were evaluated for their effectiveness to bind simplexin using a simple single concentration, dispersive adsorbent rapid screening method. Initial experiments were conducted in a rumen fluid based medium, with increasing quantities of each adsorbent: sodium bentonite (Trufeed®, Sibelco Australia), biochar (Nutralick®Australia) and Elitox® (Impextraco, Belgium). Data showed the unbound concentration of simplexin decreased with increasing quantities of each adsorbent tested. Sodium bentonite performed best, removing ~95% simplexin at 12 mg/mL. A second experiment using a single amount of adsorbent included two additional adsorbents: calcium bentonite (Bentonite Resources, Australia) and a synthetic adsorbent (Waters, USA). The concentration of simplexin remaining in the solution after 1 h, the amount able to be desorbed off the adsorbent-toxin matrix with replacement fresh fluid, and the amount remaining bound to the adsorbent were measured. All samples containing an adsorbent were statistically different compared to the blank (p < 0.05), indicating some binding activity. Future work will explore the binding mechanisms and behaviour of the toxin-adsorbent complex in the lower gastrointestinal tract.

Pimelea poisoning in grazing cattle, also known as St George or Marree Disease, has been a long-time pestilence for the pastoral industry throughout arid regions of inland Australia. The causative species Pimelea (Thymelaeaceae), native to Queensland, New South Wales and South Australia, have been confirmed, with the secondary metabolite simplexin, a daphnane orthoester, being extracted and identified as the principal toxin. Despite the lack of effective prevention or treatment for Pimelea poisoning, naïve calves have previously been demonstrated to develop detoxification capability following prolonged low-dose simplexin intake. A variety of composites are being fabricated by encapsulating Pimelea plant material or a crude extract in biodegradable and biocompatible polyesters, aiming to develop a sustained toxin release mechanism. Studies on screening potential rumen microflora able to decompose simplexin during rumen-fluid fermentation are being conducted simultaneously. In this project, a quantification method for simplexin within these biocomposites was developed and validated utilising solid-phase extraction combined with UHPLC-Q-Orbitrap MS/MS. Reliable simplexin measurement in matrices will allow investigations into the material composition, geometry and rumen microorganism’s effects on the controlled release kinetics of simplexin in vitro. The degradation patterns of toxin delivery systems when exposed to simulated rumen environments will also be thoroughly assessed on both microscopic and chemical scales. Mathematical models of the underlying mass transport mechanisms will ultimately be established through approaches ranging from simple empirical correlations to stochastic simulations, which hold the potential to facilitate future design, optimisation, and prediction of other intra-ruminal devices based on biodegradable polymers.