Dietary Supplementation of Usnic Acid, an AntimicrobialCompound in Lichens, Does Not Affect Rumen Bacterial Diversityor Density in Reindeer

Trine Glad • Perry Barboza • Roderick I. Mackie •

Andre-Denis G. Wright • Lorenzo Brusetti •

Svein D. Mathiesen • Monica A. Sundset

Received: 14 June 2013 / Accepted: 17 December 2013

� Springer Science+Business Media New York 2014

Abstract Reindeer (Rangifer tarandus tarandus) may

include large proportions of lichens in their winter diet.

These dietary lichens are rich in phenolic secondary com-

pounds, the most well-known being the antimicrobial usnic

acid. Previous studies have shown that reindeer host rumen

bacteria resistant to usnic acid and that usnic acid is

quickly detoxified in their rumen. In the present study,

reindeer (n = 3) were sampled before, during, and after

usnic acid supplementation to determine the effect on their

rumen microbial ecology. Ad libitum intake of usnic acid

averaged up to 278 mg/kg body mass. Population densities

of rumen bacteria and methanogenic archaea determined

by real-time PCR, ranged from 1.36 9 109 to 11.8 9 109

and 9.0 9 105 to 1.35 9 108 cells/g wet weight, respec-

tively, and the two populations did not change significantly

during usnic acid supplementation (repeated measures

ANOVA) or vary significantly between the rumen liquid

and particle fraction (paired t test). Rumen bacterial com-

munity structure determined by denaturing gradient gel

electrophoresis did not change in response to intake of

usnic acid. Firmicutes (38.7 %) and Bacteriodetes

(27.4 %) were prevalent among the 16S rRNA gene

sequences (n = 62) from the DGGE gels, but representa-

tives of the phyla Verrucomicrobia (14.5 %) and Proteo-

bacteria (1.6 %) were also detected. Rapid detoxification

of the usnic acid or resistance to usnic acid may explain

why the diversity of the dominant bacterial populations and

the bacterial density in the reindeer rumen does not change

during usnic acid supplementation.

Introduction

Usnic acid is a phenolic secondary compound used as a

defense in lichens against bacteria, viruses, fungi, protozoa,

insects, herbivores, and UV-radiation [2]. Reindeer utilize

lichens as an important source of energy and other nutrients

in winter, whereas toxic reactions toward lichens/usnic acid

are reported in other ruminants such as sheep and elk [4,

15]. Ruminants rely on symbiotic anaerobic rumen

microorganisms to digest their herbivorous diet, but rumen

foregut fermentation also allows microbial detoxification

[11]. Recent studies suggest that reindeer harbor rumen

bacteria resistant to usnic acid [19] and that usnic acid and

other phenolic compounds from lichens are rapidly

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00284-014-0534-7) contains supplementarymaterial, which is available to authorized users.

T. Glad � M. A. Sundset (&)

Department of Arctic and Marine Biology, UiT The Arctic

University of Norway, 9037 Tromsø, Norway

e-mail: monica.a.sundset@uit.no

P. Barboza

Institute of Arctic Biology and Department of Biology and

Wildlife, University of Alaska, Fairbanks, AK 99775, USA

R. I. Mackie

Department of Animal Sciences, University of Illinois,

Urbana-Champaign, IL 61801, USA

A.-D. G. Wright

Department of Animal Science, University of Vermont,

Burlington, VT 05405, USA

L. Brusetti

Faculty of Science and Technology, Free University of Bozen,

39100 Bolzano, Bolzano, Italy

S. D. Mathiesen

The Norwegian School of Veterinary Science, Sjøgata 39,

9000 Tromsø, Norway

123

Curr Microbiol

DOI 10.1007/s00284-014-0534-7

detoxified in their rumen [22]. Presuming that the rumen

microbiome is the key site for usnic acid detoxification, we

hypothesize that exposure to usnic acid may select for

bacteria able to tolerate and detoxify this lichen secondary

metabolite. The current study examined the effect of usnic

acid supplementation on the density of ruminal bacteria

and methanogens, and the diversity of rumen bacteria in

reindeer.

Materials and Methods

Three female rumen-fistulated reindeer (88 ± 7.8 kg)

(Rangifer tarandus tarandus) were given ad libitum access

to a pelleted feed (2.7 % N and 34 % neutral detergent

fiber on dry matter (DM) basis). The experimental feed was

coated with a solution of 3 % w/v gelatin (Kraft Foods

Tarrytown, NY) to allow a uniform adherence of usnic acid

(Catalog #026169, Indofine Chemicals, Hillsborough, NJ).

It contained 1 % w/w usnic acid and 0.35 % w/w dry

gelatin (0.05 % N from gelatin), while the control feed was

coated with gelatin only. Animals were acclimated to the

control feed (16 days), fed usnic acid feed (17 days), and

returned to the control feed (8 days). Average daily food

intake was 17 ± 5 g/kg body mass during pre-treatment,

15 ± 6 g/kg the first 10 days of treatment, 28 ± 6 g/kg the

last 7 days of treatment, and 21 ± 6 g/kg post-treatment.

Rumen samples were collected at the end of pre-treatment,

at 9 and 17 days of usnic acid treatment, and day 8 post-

treatment. Samples were taken at the same time of day

(10:06–10:26) to minimize diurnal effects. Whole rumen

contents were collected in prewarmed thermos bottles

(37 �C) and transferred to a glove bag (VWR, Radnor,

USA) filled with CO2. Whole digesta (100 g) was trans-

ferred to 200 mL glass beakers and pressed through clean

stainless-steel filters (Model #1543, Bodum, Zurich, Swit-

zerland) to retain large particulates and extrude fluids.

Fluid-phase microbes were collected from 20 g of extrusa,

and particulate-phase microbes from 20 g of large partic-

ulate digesta, combined with 25 mL anaerobic dilution

solution (ADS) containing buffers with a detergent (Tween

80; 0.15 % w/v) for microbial detachment, then cooled to

10–15 �C in closed bottles under anaerobic conditions.

Fluid collections were centrifuged (5009g for 15 min at

4 �C) under aerobic conditions to remove large particles,

the supernatant subsequently centrifuged (18,0009g for

15 min at 4 �C) to precipitate microbial cells. Microbes

detached from particulate digesta fractions in ADS by

incubation at 4 �C for 2.5 h before centrifugation

(5009g for 15 min, 18,0009g for 15 min) to separate

microbial cells. Microbial cells in centrifuged pellets from

fluid and particulate phases were redissolved in 1.4 mL

stool lysis buffer with a pH of 5.40–5.60 (ASL Buffer;

#19802 Qiagen, Valencia CA, USA) and stored at -80 �C

until further analysis.

Microbial densities were analyzed in triplicate using

real-time PCR [20] with bacterial 16S rRNA gene primers

1114F and 1257R [5] and methanogenic archaea primers

qmcrA-F and qmcrA-R [6]. Statsistical comparisons to

detect any changes in the microbial population (bacteria

and methanogens) densities due to treatment (before, dur-

ing, and after usnic acid supplementation) (repeated mea-

sures ANOVA) or between populations associated with the

two different rumen fractions (liquid and particle fraction)

(paired t-test) was run in STATA 12.0 (StatCorp, College

Station, TX, USA).

Effect of usnic acid on rumen bacterial community

structure was analyzed using DGGE profiling, PCR

amplifying the 16S rRNA gene V3 region with primers 1

and 2 (with GC clamps) [12] generating 220 bp amplicons.

PCR reactions were carried out as described by Simpson

et al. [16, 17]. DGGE was performed using a Bio-Rad

D-Code System (Hercules, CA) and gels were silver-

stained and scanned using a GS-710 calibrated imaging

densitometer (BioRad Inc., Hercules, CA). Principal com-

ponent analysis (primer-E software www.primer-e.com)

was used to analyze the DGGE profiles, and Bray-Curtis

dissimilarity to quantify the compositional dissimilarity

between different samples (liquid vs. particle), sampling

time, and animal.

Dominant bacterial phylotypes were identified using

DGGE and 16S rRNA gene sequencing. Total DNA from the

samples was extracted using FastDNA� SPIN Kit for soil

(Qbiogene, Irvine, California). The V6–V8 region was PCR

amplified using primers F968 with a GC clamp and R1401

[24]. The gels were stained with SYBR� Green I nucleic acid

gel-stain (Invitrogen, California, USA), DGGE bands

excised and reamplified with the primers F968 without the

GC clamp and R1401 as described above, with addition of

2 ll dimethyl sulfoxide (DMSO) in the reaction mixture.

PCR reamplification was initiated by hotstart (95 �C for

3 min), 30 cycles (94 �C 30 s, 64 �C 1 min, and 72 �C

1 min), and a final extension at 72 �C for 10 min. Amplifi-

cation products were purified with QIAquick PCR Purifica-

tion Kit (Qiagen, Solna, Sweden) and cloned using the

pGEM�-T Easy Vector System (Promega, Madison, USA).

Recombinant plasmids were sequenced on a Genetic ana-

lyzer (Applied Biosystems, Foster City, USA) using ABI

BigDye Terminator chemistry using sequencing primers

M13 forward and reverse (Invitrogen). Sequences were

assembled using LasergeneTM Seqman v. 7.1.0. (DNA-

STAR, Madison, USA) and chimeras removed [8]. Sequence

data were deposited in GenBank (Accession numbers

GQ449392—GQ449453). Sequences were identified

using BLAST [1], and assigned to operational taxonomic

units (OTUs) based on a 97 % sequence identity criterion

T. Glad et al.: Usnic acid and reindeer rumen bacteria

123

using the FastGroupII platform [23]. The liquid and particle

fraction clone libraries were compared using The Ribosomal

Database Project [3].

Results and Discussion

Rumen bacterial density did not change significantly, and

diversity remained unchanged during supplementation of

usnic acid (Table 1, Fig. 1), suggesting that usnic acid is

rapidly degraded and detoxified by rumen bacteria in

reindeer, as previously reported by Sundset et al. [22], and

consequently does not influence dominant bacterial popu-

lations. Previous studies by our group have also shown that

reindeer host bacteria resistant to lichen antimicrobial

compounds such as usnic acid [19], and this may offer an

alternative explanation why usnic acid supplementation did

not appear to affect rumen bacterial numbers and diversity

in this current project (Table 1; Fig. 1; Table S1 and S2).

Numbers of rumen methanogens were lower than those

previously reported in Norwegian reindeer on natural

pastures [21], and did not change significantly during usnic

acid treatment or vary significantly between the different

rumen fractions investigated (Table 1). Similarly, bacterial

densities associated with the rumen liquid and particle

fractions (Table 1) did not differ either. However, popu-

lation profiling using DGGE gels demonstrated a variation

in the bacterial diversity between the different fractions,

and samples from each animal grouped together when

analyzing the DGGE profiles using cluster analysis and

principal component analysis (Fig. 1). Nine bands from the

DGGE gel of the liquid fraction generated 2–3 different

clones each (25 in total), while 13 DGGE bands from the

particle fraction generated 1–4 different clones each (37 in

total). Among sequences obtained (n = 62), 58 distinct

OTUs were identified. Only one (L9-5) showed a sequence

identity of 97 % to its nearest valid taxon (Selenomonas

ruminantium), while the remaining were novel with only

80–96 % identity to their nearest relative. The high pro-

portion of novel 16S rRNA gene sequences in the rumen of

reindeer is consistent with previous findings [13, 14, 18]. A

meta-analysis of all publicly available 16S rRNA genes of

rumen origin (n = 13,478 bacterial sequences) revealed 19

bacterial phyla, with Firmicutes (57.8 %), Bacteroidetes

(26.7 %), and Proteobacteria (6.9 %) being the most

Table 1 Density (cell numbers/

g wet weight) of bacteria and

methanogenic archaea in the

rumen liquid and particle

fraction of reindeer (Rangifer

tarandus tarandus) pre-

treatment, during usnic acid

supplementation (day 9 and 17),

and 8 days post-treatment

Animal and treatment Bacteria Methanogenic archaea

Liquid Particle Liquid Particle

Prior to treatment

Mean (n = 3) 5.16 9 109 6.08 9 109 1.03 9 108 3.35 9 107

SD 0.79 9 109 3.25 9 109 0.37 9 108 0.96 9 107

Day 9

Mean (n = 3) 2.83 9 109 7.98 9 109 4.48 9 107 2.21 9 107

SD 1.66 9 109 3.09 9 109 4.32 9 107 0.56 9 107

Day 17

Mean (n = 3) 4.27 9 109 5.48 9 109 9.36 9 107 3.06 9 107

SD 1.97 9 109 0.34 9 109 4.64 9 107 2.58 9 107

8 days after treatment

Mean (n = 3) 4.20 9 109 6.50 9 109 5.83 9 107 7.66 9 106

SD 1.24 9 109 3.34 9 109 2.86 9 107 4.83 9 106

-20-10

010

-30-20

-100

10-10

-5

0

5

10

17

PC1 (31%)

1

16

2

13

15

6

10

8

14

129

54

3

7

1119

20

18

21

PC2 (17%)

22

24

23PC

3 (9

%)

Fig. 1 Principal-component analysis of the DGGE profiles (V3

region of the 16S rRNA gene) showing that there is no pattern

related to treatment (pre-, during, and post-dosing of usnic acid).

upper triangle animal no. 151, black circle animal no. 533; asterisk

animal no. 539. Numbering: 1–4 animal no 151 particle fraction; 5–8

animal no 151 liquid fraction; 9–12 animal no 533 particle fraction;

13–16 animal no 533 liquid fraction; 17–20 animal no 539 particle

fraction; 21–24 animal no 539 liquid fraction. For all number series,

the pre-treatment sample has the lowest number, then 9 and 17 days

of usnic acid treatment, and the 8 days post treatment sample has the

highest number

T. Glad et al.: Usnic acid and reindeer rumen bacteria

123

dominant ones [9]. Similarly in this (Table 2), and previous

studies of the reindeer rumen [13, 14, 18], Firmicutes and

Bacteroidetes were found to be the dominant phyla. Liquid

and particle libraries were significantly different for both

Firmicutes and Bacteroidetes clones (Table 2). The large

Clostridial cluster XIVa (Firmicutes) includes typical ru-

minal strains in addition to the novel usnic acid resistant

Eubacterium rangiferina isolated from reindeer [19]. Sev-

eral clones belonged to the Bacteroidetes, clustering with

Prevotella species such as Prevotella ruminocola and

Prevotella salivae, and only 2.7 % of the particle fraction

sequences were Proteobacteria. A notable finding was the

large number of novel clones affiliated with the phylum

Verrucomicrobia, which are poorly represented among

cultivated organisms and little is consequently known

about their phenotypic properties. Only a few Verrucomi-

crobia sequences have been reported in the rumen so far

[9] comprising only 2.2 % of the fecal microbiota in

humans and 59 other mammalian species investigated by

Ley et al. [10]. However, analysis of human intestinal

mucosa biopsies revealed Verrucomicrobia clones ranging

from 5–9 %, and they comprised as much as 17.2 % of the

fecal bacterial community in on wild gorilla examined [7].

In conclusion, real-time PCR and DGGE profiling

revealed no changes in rumen microbial densities or bac-

terial diversity in the presence of usnic acid, suggesting

that the microbes are either resistant to the antimicrobial

properties of the compound usnic acid or that this lichen

secondary compound is rapidly degraded and consequently

do not influence the dominant microbial populations in

reindeer.

Acknowledgments This project is funded by The Reindeer Hus-

bandry Research Fund as part of the International Polar Year con-

sortium # 399 EALAT: Climate change and reindeer husbandry. We

thank A. Falk, J. Edwards, A. Yannarell, and J.-N. Kim for technical

assistance, and R. J. Forster, M. Morrison, and Dr. Zhongtang Yu for

advice on the rumen sampling protocol.

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Phylum Library

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(Number of clones) (25) (37) (62)

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