Characterization Of Bovine Ruminal And Equine Cecal Microbial Populations Enriched For Enhanced Nitro-toxin Metabolizing Activity

The nitrotoxins3-nitro-l-propionic acid (NPA) and3-nitro-l-propanol (NPOH) are produced by a wide variety of leguminous plants, including over450different species and varieties of Astragalus potentially grazed by livestock. Specific estimates of the number of livestock poisoned by nitrotoxins are not available, but economic losses due to all poisonous plants in the western United States have been estimated to exceed$200million annually. Under the serious situation, United States Department of Agriculture (USD A) has set up many special projects to solve the problem of livestock poisoning caused by toxic plants. These toxins are known to be detoxified by at least one ruminal bacterium but detoxification by bacteria from other gut habitats is not yet known. Therefore, the primary objectives of the present study were to characterize microbial populations from bovine ruminal and equine cecal and thus enriched for enhancing nitro-toxin metabolizing activity.The main results are as follows:1. A rapid and sensitive nitro toxin quantitative method is established to obtain accurate and reliable results, as a rapid quantitative analysis, it is suitable for NPA/NPOH metabolizing bacteria and further study on the degradation of nitro toxin.2. Results from the present study has firstly discovered that the degradation of nitro toxin could also present in monogastric animal digestion tract. In this case, rates of NPA metabolism by the ruminal and equine cecal populations were68and62%more than rates of NPOH metabolism, and metabolism rates by microbes were more rapidly from the bovine rumen than those from the equine cecum. Supplementation of FeSO4and Na2S ions into rumen could significantly enhance rates of NPOH metabolism by rumen microbes. The influences is not present for equine. However, in the present study,rates of NPA metabolism by both the ruminal and equine cecal populations were inhibited45-58%by FeSO4and Na2S supplementation when compared to rates of non-supplemented cultures. 3. As expected, rates of NPA and NPOH metabolism were higher during incubation of2-fold concentrated suspensions of nonenriched ruminal (124.9±13.8and95.4±14.1nmol/mL per h, respectively) and equine cecal microbes (105.7±31.6and107.6±18.5nmol/mL per h, respectively) than during initial culture of the nonenriched populations. Thin layer chromatography revealed β-alanine and3-amino-l-propanol as a product of NPA and NPOH metabolism respectively both in the bovine ruminal and equine cecal suspensions. The metabolic pathways for both in the bovine and equine were probably similar.4. The mixed populations of bovine ruminal and equine cecal microbes were enriched for NPA-metabolizing bacteria via consecutive24-72h culture in a basal minimal rumen fluid-based medium supplemented with4.2mM NPA and H2as the energy source. Rates of NPA disappearance by the respective populations were increased1.5-and5.8-fold. Results from3-tube most probable number tests indicated that numbers of NPA-degrading microbes increased2.1and1.7log10units during enrichment from numbers measured pre-enrichment (3.9×103and4.3×101cells/mL for ruminal and equine cecal populations, respectively).5. Hydrogen was found to be an important energy source during enrichment of NPA-degrading bacteria from the bovine ruminal and equine cecal populations. Formate served as a suitable hydrogen donor in the present experiment. A source of CO2nonetheless appeared to be important for maintaining NPA-metabolizing activity.6. Results reveal that for both bovine ruminal and equine cecal populations, rates of NPA metabolism were inhibited when cultured with the addition of nitrate. Nitrate would be preferred as a more thermodynamically favorable electron acceptor than NPA. While NPA-metabolizing bacteria may be able to use nitrate as an electron acceptor, they were unable to compete with other nitrate-reducing bacteria under these growth conditions. Nitrate-enriched populations in equine cecal content were not able to metabolize NPA.7. Traditional culture method (Plate) and Hungate roll tube method (Roll tube) were applied to isolated the pure separate strains from the enriched equine cecal microbes, and thus the strains were identified by16S rRNA sequences. Although the nitro-toxin metabolizing bacterium were not successfully isolated and identified from equine cecal populations, a unique strain of acetogenic bacteria Sporanaerobacter acetigenes was firstly found in monogastric animal by NPA enrichment cultures. Compared NPA degrading bacteria D. Detoxificans in bovine rumen and different treatments of the enriched microbes (including isolated pure strains and mix populations) from equine cecal content by DGGE showed differences of this two microbes within species.