Condensed tannins, novel compounds and sources of variation determine the antiparasitic activity of Nordic conifer bark against gastrointestinal nematodes.

The antiparasitic potential of plants could offer a vital solution to alleviating the costs of gastrointestinal nematode (GIN) infections in ruminant production globally. Leveraging known bioactive molecules, however, is complex, where plant species, extraction processes and seasonality impact bioavailability and efficacy. This study assessed the impact of a comprehensive set of factors on the antiparasitic activity of Norwegian conifers to identify bark compounds specific against GIN. Antiparasitic activity was determined using in vitro assays targeting morphologically distinct life stages of ovine GIN: the egg hatch assay and larval motility assay. In depth characterisation of the chemical composition of the bark extracts was carried out using chromatographic separation, UV-absorbance, and molecular mass profiles to identify compounds implicated in the activity. Three key findings emerged: (1) the activity of bark extracts varied markedly from 0 to 100% antiparasitic efficacy, owing to tree species, extraction solvent and seasonality; (2) the GIN exhibited species-and stage-specific susceptibility to the bark extracts; (3) the presence of condensed tannins, amongst other compounds, was associated with anthelmintic activity. These findings add new insights into urgently needed alternative parasite control strategies in livestock.

Administration of spruce bark (Picea abies) extracts in young lambs exhibits anticoccidial effects but reduces milk intake and body weight gain.

BACKGROUND: Eimeria spp. are widespread apicomplexan parasites known to cause coccidiosis in livestock, resulting in reduced animal welfare and productivity, particularly in sheep. The treatment options are limited, and there is an emerging development of resistance against registered pharmaceuticals. Spruce bark is rich in plant secondary metabolites (PSM), such as condensed tannins, which are bioactive compounds previously shown to have antiparasitic activity. Here, we examined the anticoccidial properties of bark extract of Norway spruce (Picea abies) against a field isolate of ovine Eimeria spp. by treating Eimeria-infected pre-ruminant lambs with water-extracted bark daily for 12 days. We hypothesised that the bark extract would reduce the faecal oocyst excretion and, consequently, the severity of diarrhoea. RESULTS: Oral administration of spruce bark extract significantly reduced the excretion of Eimeria oocysts in milk-fed lambs post treatment till the end of the trial 22 days post infection. This difference in oocyst excretion between the treated and the untreated infected animals increased with time. Compared to the untreated and the sham-infected control group, the group treated with bark extract had softer faeces and reduced milk intake during the treatment period. After discontinuing the treatment, the treated animals got a more solid and formed faeces compared to that of the untreated control group, and the milk intake increased to the level of the sham-infected, untreated control group. The bark extract treated animals had a lower body weight and a lower mean daily body weight gain throughout the whole duration of the experiment. CONCLUSIONS: Bark extract from Norway spruce showed marked anticoccidial properties by reducing the faecal oocyst count and associated diarrhoea in young lambs. Simultaneously we experienced detrimental effects of the treatment, displayed as reduced feed intake and daily body weight gain. Therefore, we suggest conducting similar studies with lower bark extract dosage to explore the possibilities of a better trade-off to reduce the negative impact while maintaining the antiparasitic effect.

Overexpression of Two New Acyl-CoA:Diacylglycerol Acyltransferase 2-Like Acyl-CoA:Sterol Acyltransferases Enhanced Squalene Accumulation in Aurantiochytrium limacinum.

Thraustochytrids are heterotrophic marine eukaryotes known to accumulate large amounts of triacylglycerols, and they also synthesize terpenoids like carotenoids and squalene, which all have an increasing market demand. However, a more extensive knowledge of the lipid metabolism is needed to develop thraustochytrids for profitable biomanufacturing. In this study, two putative type-2 Acyl-CoA:diacylglycerol acyltransferases (DGAT2) genes of Aurantiochytrium sp. T66, T66ASATa, and T66ASATb, and their homologs in Aurantiochytrium limacinum SR21, AlASATa and AlASATb, were characterized. In A. limacinum SR21, genomic knockout of AlASATb reduced the amount of the steryl esters of palmitic acid, SE (16:0), and docosahexaenoic acid, SE (22:6). The double mutant of AlASATa and AlASATb produced even less of these steryl esters. The expression and overexpression of T66ASATb and AlASATb, respectively, enhanced SE (16:0) and SE (22:6) production more significantly than those of T66ASATa and AlASATa. In contrast, these mutations did not significantly change the level of triacylglycerols or other lipid classes. The results suggest that the four genes encoded proteins possessing acyl-CoA:sterol acyltransferase (ASAT) activity synthesizing both SE (16:0) and SE (22:6), but with the contribution from AlASATb and T66ASATb being more important than that of AlASATa and T66ASATa. Furthermore, the expression and overexpression of T66ASATb and AlASATb enhanced squalene accumulation in SR21 by up to 88%. The discovery highlights the functional diversity of DGAT2-like proteins and provides valuable information on steryl ester and squalene synthesis in thraustochytrids, paving the way to enhance squalene production through metabolic engineering.

Extracts of pine bark (Pinus sylvestris) inhibit Cryptosporidium parvum growth in cell culture.

The widespread apicomplexan parasite Cryptosporidium parvum is responsible for severe gastrointestinal disease in humans and animals. The treatment options are limited, and the efficacy of available drugs is low. Bark contains condensed tannins (CT), which are bioactive compounds previously shown to inhibit parasite development. Here, we examined the anti-cryptosporidial properties of bark extract of Scots pine (Pinus sylvestris) against C. parvum by means of an in vitro growth inhibition test. We hypothesised that bark extracts would have dose-dependent inhibitory effects on the development of C. parvum in cell culture.Bark extracts from Scots pine extracted with acetone, methanol, and water as solvents were investigated using human colorectal adenocarcinoma cells infected with C. parvum. Oocysts were inoculated onto the cell monolayer and bark extract was added at seven different concentrations. Parasite growth inhibition was quantified by qPCR.The acetone and methanol extracts demonstrated a sigmoid dose-dependent inhibition of C. parvum. The IC50 values were 244.6 and 279.1 µg dry matter extract/mL, and 25.4 and 24.1 µg CT/mL, for acetone and methanol extracts, respectively. The IC50 for both extracts were similar, both with regard to the dry matter concentration of each extract and to CT concentrations.Given the limited treatment options available for Cryptosporidium spp., the evidence generated in our study encourages further investigation into the in vitro and in vivo effects of pine bark extracts against C. parvum.

A non-canonical Δ9-desaturase synthesizing palmitoleic acid identified in the thraustochytrid Aurantiochytrium sp. T66.

Thraustochytrids are oleaginous marine eukaryotic microbes currently used to produce the essential omega-3 fatty acid docosahexaenoic acid (DHA, C22:6 n-3). To improve the production of this essential fatty acid by strain engineering, it is important to deeply understand how thraustochytrids synthesize fatty acids. While DHA is synthesized by a dedicated enzyme complex, other fatty acids are probably synthesized by the fatty acid synthase, followed by desaturases and elongases. Which unsaturated fatty acids are produced differs between different thraustochytrid genera and species; for example, Aurantiochytrium sp. T66, but not Aurantiochytrium limacinum SR21, synthesizes palmitoleic acid (C16:1 n-7) and vaccenic acid (C18:1 n-7). How strain T66 can produce these fatty acids has not been known, because BLAST analyses suggest that strain T66 does not encode any Δ9-desaturase-like enzyme. However, it does encode one Δ12-desaturase-like enzyme. In this study, the latter enzyme was expressed in A. limacinum SR21, and both C16:1 n-7 and C18:1 n-7 could be detected in the transgenic cells. Our results show that this desaturase, annotated T66Des9, is a Δ9-desaturase accepting C16:0 as a substrate. Phylogenetic studies indicate that the corresponding gene probably has evolved from a Δ12-desaturase-encoding gene. This possibility has not been reported earlier and is important to consider when one tries to deduce the potential a given organism has for producing unsaturated fatty acids based on its genome sequence alone. KEY POINTS: • In thraustochytrids, automatic gene annotation does not always explain the fatty acids produced. • T66Des9 is shown to synthesize palmitoleic acid (C16:1 n-7). • T66des9 has probably evolved from Δ12-desaturase-encoding genes.

Combined Metabolome and Lipidome Analyses for In-Depth Characterization of Lipid Accumulation in the DHA Producing Aurantiochytrium sp. T66.

Thraustochytrids are marine heterotrophic microorganisms known for their potential to accumulate docosahexaenoic acid (DHA)-enriched lipids. There have been many attempts to improve thraustochytrid DHA bioprocesses, especially through traditional optimization of cultivation and media conditions. Nevertheless, thraustochytrid-based bioprocesses are still not commercially competitive for high volume-low cost production of DHA. Thus, it is realized that genetic and metabolic engineering strategies are needed for the development of commercially competitive thraustochytrid DHA cell factories. Here, we present an analytical workflow for high resolution phenotyping at metabolite and lipid levels to generate deeper insight into the thraustochytrid physiology, with particular focus on central carbon and redox metabolism. We use time-series sampling during unlimited growth and nitrogen depleted triggering of DHA synthesis and lipid accumulation (LA) to show-case our methodology. The mass spectrometric absolute quantitative metabolite profiling covered glycolytic, pentose phosphate pathway (PPP) and tricarboxylic acid cycle (TCA) metabolites, amino acids, complete (deoxy)nucleoside phosphate pools, CoA and NAD metabolites, while semiquantitative high-resolution supercritical fluid chromatography MS/MS was applied for the lipid profiling. Interestingly, trace amounts of a triacylglycerols (TG) with DHA incorporated in all three acyl positions was detected, while TGs 16:0_16:0_22:6 and 16:0_22:6_22:6 were among the dominant lipid species. The metabolite profiling data indicated that lipid accumulation is not limited by availability of the acyl chain carbon precursor acetyl-CoA nor reducing power (NADPH) but rather points to the TG head group precursor glycerol-3-phosphate as the potential cause at the metabolite level for the gradual decline in lipid production throughout the cultivation. This high-resolution phenotyping provides new knowledge of changes in the central metabolism during growth and LA in thraustochytrids and will guide target selection for metabolic engineering needed for further improvements of this DHA cell factory.

Seaweed and Seaweed Bioactives for Mitigation of Enteric Methane: Challenges and Opportunities.

Seaweeds contain a myriad of nutrients and bioactives including proteins, carbohydrates and to a lesser extent lipids as well as small molecules including peptides, saponins, alkaloids and pigments. The bioactive bromoform found in the red seaweed Asparagopsis taxiformis has been identified as an agent that can reduce enteric CH4 production from livestock significantly. However, sustainable supply of this seaweed is a problem and there are some concerns over its sustainable production and potential negative environmental impacts on the ozone layer and the health impacts of bromoform. This review collates information on seaweeds and seaweed bioactives and the documented impact on CH4 emissions in vitro and in vivo as well as associated environmental, economic and health impacts.

Lipid and DHA-production in Aurantiochytrium sp. - Responses to nitrogen starvation and oxygen limitation revealed by analyses of production kinetics and global transcriptomes.

Thraustochytrids of the genera Schizochytrium and Aurantiochytrium accumulate oils rich in the essential, marine n3 fatty acid docosahexaenoic acid (DHA). DHA production in Aurantiochytrium sp T66 was studied with the aim to provide more knowledge about factors that affect the DHA-productivities and the contributions of the two enzyme systems used for fatty acid synthesis in thraustochytrids, fatty acid synthetase (FAS) and PUFA-synthase. Fermentations with nitrogen starvation, which is well-known to initiate lipid accumulation in oleaginous organisms, were compared to fermentations with nitrogen in excess, obtained by oxygen limitation. The specific productivities of fatty acids originating from FAS were considerably higher under nitrogen starvation than with nitrogen in excess, while the specific productivities of DHA were the same at both conditions. Global transcriptome analysis showed significant up-regulation of FAS under N-deficient conditions, while the PUFA-synthase genes were only marginally upregulated. Neither of them was upregulated under O2-limitation where nitrogen was in excess, suggesting that N-starvation mainly affects the FAS and may be less important for the PUFA-synthase. The transcriptome analysis also revealed responses likely to be related to the generation of reducing power (NADPH) for fatty acid synthesis.

Draft genome sequence of the docosahexaenoic acid producing thraustochytrid Aurantiochytrium sp. T66.

Thraustochytrids are unicellular, marine protists, and there is a growing industrial interest in these organisms, particularly because some species, including strains belonging to the genus Aurantiochytrium, accumulate high levels of docosahexaenoic acid (DHA). Here, we report the draft genome sequence of Aurantiochytrium sp. T66 (ATCC PRA-276), with a size of 43 Mbp, and 11,683 predicted protein-coding sequences. The data has been deposited at DDBJ/EMBL/Genbank under the accession LNGJ00000000. The genome sequence will contribute new insight into DHA biosynthesis and regulation, providing a basis for metabolic engineering of thraustochytrids.

Thraustochytrids as production organisms for docosahexaenoic acid (DHA), squalene, and carotenoids.

Thraustochytrids have been applied for industrial production of the omega-3 fatty acid docosahexaenoic (DHA) since the 1990s. During more than 20 years of research on this group of marine, heterotrophic microorganisms, considerable increases in DHA productivities have been obtained by process and medium optimization. Strains of thraustochytrids also produce high levels of squalene and carotenoids, two other commercially interesting compounds with a rapidly growing market potential, but where yet few studies on process optimization have been reported. Thraustochytrids use two pathways for fatty acid synthesis. The saturated fatty acids are produced by the standard fatty acid synthesis, while DHA is synthesized by a polyketide synthase. However, fundamental knowledge about the relationship between the two pathways is still lacking. In the present review, we extract main findings from the high number of reports on process optimization for DHA production and interpret these in the light of the current knowledge of DHA synthesis in thraustochytrids and lipid accumulation in oleaginous microorganisms in general. We also summarize published reports on squalene and carotenoid production and review the current status on strain improvement, which has been hampered by the yet very few published genome sequences and the lack of tools for gene transfer to the organisms. As more sequences now are becoming available, targets for strain improvement can be identified and open for a system-level metabolic engineering for improved productivities.

Selection of candidate probionts by two different screening strategies from Atlantic cod (Gadus morhua L.) larvae.

Two primary selection criteria were used to collect a pool of nearly 500 candidate probiotic bacteria from Atlantic cod (Gadus morhua L.) larvae, i.e. the dominant intestinal bacterial flora and isolates with antagonistic activity against Vibrio anguillarum. Bacteria were isolated from cod larvae from five rearing groups with variable rearing technologies. The bacteria were brought to pure culture and characterized phenotypically. Based on properties such as uniqueness, dominance and fermentative ability, a selection of approximately 10% of the isolates were chosen from the initial pool of bacteria to reduce the number of candidates. These 55 isolates were characterized further in vitro regarding antagonism, adhesion to mucus, growth in mucus, production of extracellular enzymes, fish bile resistance and haemolytic properties. Based on the results of the in vitro tests, the number of probiotic candidates was further reduced to seven isolates. To evaluate the probiotic potential and to assure that the seven isolates were not harmful to the host, yolk sac larvae of cod were exposed to the isolates in a small-scale in vivo experiment. The in vivo experiment excluded two of the candidate bacteria due to increased mortality of cod larvae, whereas three isolates from the dominant (Vibrio and two different strains of Microbacterium) and two from the antagonistic (Ruegeria and Pseudoalteromonas) group improved the survival of larvae compared to the positive control. Thus, a combination of the two screening methods was suited for making multistrain probiotics with complementary modes of action.

Interactions of the bacteriocins sakacin P and nisin with food constituents.

Bacteriocins are amphiphilic peptides susceptible to adsorption to food macromolecules and proteolytic degradation. These properties may limit their use as preservation agents. The aim of the present work has been to elucidate the fate of the bacteriocin sakacin P in food. Nisin was used in a few experiments for comparison. Recovery of bacteriocins was studied in homogenates of cold-smoked salmon, chicken cold cuts and raw chicken, with verification of the results in the corresponding food products. More than 80% of the added sakacin P and nisin were quickly adsorbed to proteins in the food matrix. In foods that had not been heat-treated, proteolytic activity caused a rapid degradation of the bacteriocins, with less than 1% of the total activity left after 1 week in cold-smoked salmon, and even less in raw chicken. In heat-treated foods, the bacteriocin activity was stable for more than 4 weeks. The high fat content in salmon compared to chicken had no adverse effect on bacteriocin recovery or activity. However, mixing of triglyceride oils and bacteriocin solutions caused a considerable loss of activity. No principal differences were observed between sakacin P and nisin, but less nisin was adsorbed to muscle proteins at low pH, and the negative effect of oils was less pronounced for nisin. Growth of Listeria monocytogenes was completely inhibited for at least 3 weeks in both chicken cold cuts and cold-smoked salmon by addition of sakacin P (3.5 microg/g), despite the proteolytic degradation in the salmon.