Degradation of the Indospicine Toxin from Indigofera spicata by a Mixed Population of Rumen Bacteria

The leguminous plant species, Indigofera linnaei and Indigofera spicata are distributed throughout the rangeland regions of Australia and the compound indospicine (L-2-amino-6-amidinohexanoic acid) found in these palatable forage plants acts as a hepatotoxin and can accumulate in the meat of ruminant livestock and wild camels. In this study, bovine rumen fluid was cultivated in an in vitro fermentation system provided with Indigofera spicata plant material and the ability of the resulting mixed microbial populations to degrade indospicine was determined using UPLC–MS/MS over a 14 day time period. The microbial populations of the fermentation system were determined using 16S rRNA gene amplicon sequencing and showed distinct, time-related changes occurring as the rumen-derived microbes adapted to the fermentation conditions and the nutritional substrates provided by the Indigofera plant material. Within eight days of commencement, indospicine was completely degraded by the microbes cultivated within the fermenter, forming the degradation products 2-aminopimelamic acid and 2-aminopimelic acid within a 24 h time period. The in vitro fermentation approach enabled the development of a specifically adapted, mixed microbial population which has the potential to be used as a rumen drench for reducing the toxic side-effects and toxin accumulation associated with ingestion of Indigofera plant material by grazing ruminant livestock.

Bioaccumulation and Distribution of Indospicine and Its Foregut Metabolites in Camels Fed Indigofera spicata

In vitro experiments have demonstrated that camel foregut-fluid has the capacity to metabolize indospicine, a natural toxin which causes hepatotoxicosis, but such metabolism is in competition with absorption and outflow of indospicine from the different segments of the digestive system. Six young camels were fed Indigofera spicata (337 µg indospicine/kg BW/day) for 32 days, at which time three camels were euthanized. The remaining camels were monitored for a further 100 days after cessation of this indospicine diet. In a retrospective investigation, relative levels of indospicine foregut-metabolism products were examined by UHPLC-MS/MS in plasma, collected during both accumulation and depletion stages of this experiment. The metabolite 2-aminopimelamic acid could be detected at low levels in almost all plasma samples, whereas 2-aminopimelic acid could not be detected. In the euthanized camels, 2-aminopimelamic acid could be found in all tissues except muscle, whereas 2-aminopimelic acid was only found in the kidney, pancreas, and liver tissues. The clearance rate for these metabolites was considerably greater than for indospicine, which was still present in plasma of the remaining camels 100 days after cessation of Indigofera consumption.

Accumulation and depletion of indospicine in calves (Bos taurus) fed creeping indigo (Indigofera spicata)

Prolonged consumption of Indigofera pasture plants can cause both hepatotoxicosis and reproductive losses in grazing animals with the responsible toxin indospicine forming persistent tissue residues. Separate accumulation and depletion feeding trials were undertaken in calves fed Indigofera spicata (3 mg indospicine/kg bodyweight) to ascertain the appearance and elimination of indospicine from various tissues. In the accumulation trial indospicine concentrations increased throughout the 42-day feeding period with maximum levels of 15 mg/L in plasma and 19 and 33 mg/kg in liver and muscle, respectively. In the depletion trial, calves were fed I. spicata for 35 days, after which the plant was withdrawn from the diet. The rate of elimination was relatively slow with estimates of half-life being 31, 25 and 20 days for muscle, liver and plasma, respectively. Indospicine levels measured in bovine tissues in this trial are comparable with levels in horsemeat and camel meat reported to cause fatal hepatoxicity in dogs, a species known to be susceptible to this toxin. The persistence of indospicine residues in bovine tissues and the widespread distribution of Indigofera species in tropical and sub-tropical grazing lands warrant further investigation, as indospicine has been established as causing reproductive losses and likely contributes to calf losses in these regions.