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Condensed tannins from Norwegian pine and spruce bark - antiparasitic effects and potential commercial exploitation

Alternative title: Kondeserte tanninar i bark frå norsk furu og gran - antiparasittiske effektar og potensiell kommersiell utnytting

Awarded: NOK 10.0 mill.

Gastrointestinal (GIN) parasites of sheep and cattle are common causes of reduced productivity, animal health and welfare. Resistance to pharmaceuticals used to treat sheep against the parasites is increasingly widespread, and there is a need for more sustainable options for treatment and control. Inclusion of small amounts of condensed tannins (CT) in the diet of ruminants may decrease problems with gastrointestinal parasites. Tannin containing forages are not well adapted to Norwegian conditions, but Norway has a forest industry and bark harvested from conifer species that contain CT. In the BarkCure project we 1) evaluated bark as it is produced today with regard to its utilization for industrial production of CT, 2) characterized CT quality and quantity in bark resources 3) tested the biological activity of bark extracts against helminths and unicellular parasites, and 4) developed industrial extraction and processing of bark CT. Six batches of bark from Norway spruce and Scots pine were collected from sawmill and pulp mill, in summer 2017 and winter 2018. Bark extracts rich in CT were prepared using water or the water-miscible solvents acetone and methanol. The solvents resulted in higher CT-yields than water, and highest yields were obtained from spruce. However, the CT-content per dry weight extract was similar or higher for pine, indicating that fewer other compounds were extracted, resulting in a purer CT extract. For spruce, there were no seasonal differences in CT-yield, while for pine, the CT-content was highest in the summer-batch. Adaptations of the extraction process for scale-up to an industrial process have been assessed. Factors such as reduction of the solvent volume by repeated extractions, effect of the particle size on the extraction yields, and extraction from undried bark, were investigated. The experimental results were used as input for a theoretical assessment of possible process designs and production costs for an industrial extraction process. All extracts were tested against the sheep GIN Teladorsagia circumcincta and Trichostrongylus colubriformis using two in vitro laboratory methods, i.e. egg hatch assays and larval motility tests. While several of the extracts were highly effective at inhibiting egg hatching at the highest concentration, acetone extract of pine from winter had the greatest antiparasitic effect at the lowest concentrations for both GIN species. There was a dose-dependent effect against T. circumcincta, while the bark extract maintained its efficacy even at weaker concentrations against T. colubriformis. Preliminary results of the larval motility test suggest that the extracts have less of an impact on larval motility at the concentrations tested. If this outcome is confirmed it will give insights into the possible ways of incorporating bark extracts in sustainable parasite control strategies. All extracts were also tested against two life stages of the cattle GIN Ostertagia ostertagi with similar in vitro methods as for sheep GIN. Seven of the extracts were also tested on freshly hatched larvae. Most extracts at high concentrations succeeded in inhibiting the hatching process and killing the young larvae but not immobilizing the well-developed infective larvae to prevent their migration. The efficiency of each bark extract varied based on dose and the sensitivity of the parasitic life stage upon which it was tested. This indicates that there may be differences in the mode of action of each extract and on each life stage of the nematodes. Using cell cultures, the anti-parasitic effect of the extracts has been tested against the unicellular parasites Cryptosporidium parvum and ovine Eimeria spp. These parasites can cause severe diarrhea in young ruminants and C. parvum may also induce diarrhea in humans. Initial screening of the extracts indicated an effect against C. parvum in vitro. Based on these results, three extracts were selected for additional testing to establish the most efficient extraction method. The results of the C. parvum analyses show a dose-dependent anti-cryptosporidial activity of both the acetone and the methanol extracts compared to their negative controls. There was no difference in parasite growth reducing efficacy between the acetone extract at the highest dose and the positive control, the pharmaceutical paromomycin. Further, we have tested the bark extracts against sheep coccidia (Eimeria spp.) using the sporulation inhibition tests. However, we found no antiparasitic effect of any of the bark extracts against sheep coccidia. Based upon the results from the sheep nematode and C. parvum tests, a pine acetone extract was chosen for testing the antiparasitic properties of the extract against Heligmosomoides bakeri in a mouse model. Preliminary analyses indicate that bark extract mitigate the effect of nematode infection as the bark treated animals tended to have higher live weight gain than the untreated mice.

Arbeidet har gitt oss meir og betre kunnskap om innhaldet av kondenserte tannin i bark frå norsk skogindustri og om antiparasittisk effekt av ekstrakt frå bark. Som ein del av utviklinga mot ein biobasert og sirkulær økonomi er det aukande interesse for utnytting av bark. Sjølv om målet for prosjektet var bruk av barkekstrakt som middel mot parasittar, vil ekstraksjons-resultata, erfaringane frå prosessutviklinga og dei teoretiske vurderingane ha betydeleg nytteverdi også for anna bruk av barkekstrakt.

Condensed tannins (CT) are regarded as anti-nutrients as they may reduce feed intake, nutrient digestion and production in livestock. However, the inclusion of small amounts of CT in the diet of ruminants may decrease problems with gastrointestinal parasites. Tannin containing forages are not well adapted to Norwegian cultivation conditions, but Norway has a strong forest industry and bark harvested from conifer species may have high content of CT. In the proposed project we will: 1) evaluate bark as it is produced today with regard to its utilization in industrial processing of CT; 2) characterize CT quality and quantity in bark resources and their structure-activity relationships; 3) test the biological activity of bark CT against helminths and parasitic protozoa in ruminants in vitro and against helminths in vivo. 4) develop industrial extraction and processing of bark CT. Dissemination of knowledge gains is ensured by integrated activities. The project draws on the coordinated interaction of national and international players in research and enterprises: Norwegian Centre for Organic Agriculture, Norwegian Institute of Bioeconomy Research, SINTEF, Norwegian Institute of Wood Technology, Veterinærinstituttet, Scotland?s Rural College, University of Copenhagen, Bergene Holm AS, Norske Skog Saugbrugs AS. Felleskjøpet Fôrutvikling, Animalia, Norges Bondelag and Norsk sau og geit participate in the Stakeholder advisory group.

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