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Veterinary Parasitology
journal homepage: www.elsevier.com/locate/vetpar

Effects of supplementation with dietary green tea


polyphenols on parasite resistance and acute phase protein
response to Haemonchus contortus infection in lambs
Rong Zhen Zhong a,b , Hao Yang Li b , Hai Xia Sun a , Dao Wei Zhou a,
a
b

Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, Changchun 130102, Jilin, PR China
College of Animal Science and Technology, Jilin Agricultural University, Changchun 130118, Jilin, PR China

a r t i c l e

i n f o

Article history:
Received 26 October 2013
Received in revised form 6 June 2014
Accepted 17 June 2014
Keywords:
Haemonchus contortus
Tea polyphenols
Resistance
Acute phase proteins
Lambs

a b s t r a c t
The objective of this study was to determine the effects of supplementation with dietary
green tea polyphenols (GTPs) on parasite resistance and acute phase protein (APP) response
to Haemonchus contortus infection in lambs. Thirty male Ujumqin lambs were randomly
assigned to ve treatment groups for an 8-week feeding period. Treatments included: (1)
uninfected as control, (2) infected but not given GTP (INFGTP0) and (3)(5) infected and fed
2, 4, or 6 g GTP/kg feed (dry matter basis; INFGTP2, INFGTP4, and INFGTP6, respectively).
Fecal and blood samples were collected to determine fecal egg count (FEC), packed cell
volume (PCV), and APP concentrations. Live weight was measured once every 2 weeks. At
the end of the feeding period, lambs were slaughtered to determine the adult H. contortus burden. The results demonstrated interaction effects between treatment and sampling
time on the average daily gain (ADG; P = 0.0005), FEC (P < 0.0001), PCV (P = 0.0005), and
concentrations of serum amyloid A (SAA), haptoglobin (Hp), lipopolysaccharide binding
protein (LBP), and 1 -acid glycoprotein (1 AGP) (P < 0.0001). From days 0 to 56, the ADG
values for all infected lambs were lower than that of uninfected lambs, but the ADG values for all GTP-fed lambs were higher than that of INFGTP0 lambs, especially from days
28 to 42. The FECs of all GTP-fed lambs were higher than those of uninfected lambs but
lower than that of INFGTP0 lambs. The PCVs of all infected lambs were lower than those
of uninfected lambs, but PCV increased with increasing amounts of GTP supplementation.
Furthermore, supplementation with different concentrations of GTP signicantly reduced
the numbers of adult H. contortus, including both males and females (P < 0.0001), and the H.
contortus burden in INFGTP6 lambs was reduced to uninfected levels. Overall, the SAA, Hp,
LBP, and 1 AGP concentrations of all infected lambs were higher than those of uninfected
lambs from days 0 to 56. Two peaks in expression were observed from days 0 to 3 and at
day 28, and APP concentrations of all GTP-fed lambs were lower than those of INFGTP0
lambs, except for SAA in INFGTP6 lambs. In conclusion, quantitative measurements of APP
responses to H. contortus infection provide valuable diagnostic information for monitoring
infection progression and treatment responses in lambs. An appropriate dose of dietary GTP

Corresponding author. Tel.: +86 431 85542231; fax: +86 431 85542206.
E-mail address: zhoudaowei@neigae.ac.cn (D.W. Zhou).
http://dx.doi.org/10.1016/j.vetpar.2014.06.022
0304-4017/ 2014 Elsevier B.V. All rights reserved.

Please cite this article in press as: Zhong, R.Z., et al., Effects of supplementation with dietary green tea polyphenols on
parasite resistance and acute phase protein response to Haemonchus contortus infection in lambs. Vet. Parasitol. (2014),
http://dx.doi.org/10.1016/j.vetpar.2014.06.022

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supplementation can increase host resistance by reducing H. contortus burden and weight
loss and suppressing blood APP expression.
2014 Elsevier B.V. All rights reserved.

1. Introduction
Gastrointestinal nematodes (GINs) cause considerable
economic losses in small ruminant production worldwide
(Parkins and Holmes, 1989; Miller and Horohov, 2006). The
blood-feeding nematode Haemonchus contortus is especially harmful, causing anemia and even death in host
animals (Miller et al., 1998). In Northern China, approximately 90% of sheep and goats are infected with H. contortus
(Li and Li, 2006). The indigenous Ujumqin sheep, which are
famous for having high quality meat, are listed as a nationally protected domestic animal by the Chinese government.
However, they are susceptible to H. contortus infection,
which decreases productivity (Na et al., 2010; Ren et al.,
2011).
Current prevalent parasitological diagnostic approaches
are mostly based on the fecal egg count (FEC), packed
cell volume (PCV) of whole blood, and FAMACHA scores
(Niezen et al., 1998). These indirect indicators reect GIN
burden. Direct measurements of GIN infection relies on
necropsy procedure which is not feasible in farm conditions. Therefore, if more predictive biomarkers associated
with immune responses can be utilized to monitor GIN burden and disease progression, more accurate therapies can
be developed to improve the health of host animals.
Acute phase proteins (APPs), including serum amyloid A (SAA), haptoglobin (Hp), lipopolysaccharide binding
protein (LBP), and 1 -acid glycoprotein (1 AGP), are a
group of blood proteins that change in concentration when
animals are subjected to external or internal challenges,
such as infection, inammation, or stress (Gonzlez et al.,
2008). Production of APPs is mediated by pro-inammatory
cytokines, such as interleukin (IL)-1, IL-6, interferon (IFN), and tumor necrosis factor (TNF)- (Moshage, 1997).
Some studies have revealed that APPs may be useful as
potential biomarkers for evaluating infection by Plasmodium chabaudi in mice (Taylor-Robinson, 2000), Theileria
annulata in cattle (Glass et al., 2003), and Trypanosoma vivax
in sheep (Sousa Almeida et al., 2012). However, the relationship between APP induction and H. contortus infection
in sheep has not been well described.
An accurate method for clinical diagnosis based on
a variety of parameters is ultimately needed for timely
administration of effective therapies. The current strategy
of using anthelmintics to control H. contortus is in jeopardy
because of prevalent anthelmintic resistance, drug residues
in animal products, and loss of productivity (Waller, 2004;
Sykes, 2010), which has stimulated interest in alternative parasite control approaches that are less reliant on
chemotherapeutics. Several studies have reported that
grazing on tannin-containing plants can reduce GIN infection in sheep and goats, with tannins being the effective
bioactive compounds (Niezen et al., 2002; Min and Hart,
2003; Gujja et al., 2013). Tannins are a heterogeneous
group of high molecular weight phenolic plant secondary

compounds with the capacity to form complexes with proteins, polysaccharides, nucleic acids, etc. (Schoeld et al.,
2001). The proposed mechanism of nematode control by
tannins involves direct binding of tannins to nematode
parasite proteins, which subsequently blocks the parasites physiological processes and/or modies its immune
responses to eliminate infective larvae or adult worms
(Hoste et al., 2006). High levels of galloylated derivatives
of plant polyphenolic extract greatly contribute to the
anthelmintic potency (Azaizeh et al., 2013).
Green tea has been the most popular beverage in China
for thousands of years (Yang and Wang, 1993). Green tea
contains polyphenolic compounds, including avanols, avandiols, avonoids, and phenolic acids, which account
for 30% of the dry weight of green tea leaves. Most of
the polyphenols in green tea are catechins, including
()-epicatechin (EC), ()-epicatechin-3-gallate (ECG), ()epigallocatechin (EGC), and ()-epigallocatechin-3-gallate
(EGCG) (Mukhtar and Ahmad, 2000). Green tea polyphenols (GTPs) have been widely used in animal production
due to their various bioactivities, such as antioxidant
properties (Zhong et al., 2009). Based on the chemical composition of GTPs, similar to other plant-derived
tannins, we hypothesized that GTPs have anthelmintic
bioactivities capable of reducing the H. contortus burden in
sheep.
The objectives of this study were: 1) to determine the
effects of dietary GTP supplementation on host resistance
to H. contortus infection in lambs and 2) to study the APP
response to H. contortus infection in lambs.
2. Materials and methods
2.1. Chemicals and reagents
The green tea leaves (Camellia sinensis L.) used in
the study was supplied by Hunan Tea Group Co., Ltd.,
Hunan, China. GTPs (purity of 98%) were extracted from
green tea leaves using high pressure liquid chromatography (Model Waters 600, Waters Co., Milford, MA,
USA) according to method of Paveto et al. (2004). The
extracted GTPs contained total tannins (8.28%), steroids
(4.12%), and avonoids (85.2%), which included (+)catechin and ()-catechin (DL-C) (1.21%), EC (5.07%), ECG
(11.80%), EGC (0.47%), ()-gallocatechin gallate (GCG)
(1.05%), EGCG (60%), and other avanols (5.6%). All other
chemicals used in the study were obtained from Sigma
Chemical Co. (St. Louis, MO, USA) unless otherwise
stated.
2.2. Animals and management
The use of the animals and the experimental procedure
were approved by the Animal Care Committee, Institute of

Please cite this article in press as: Zhong, R.Z., et al., Effects of supplementation with dietary green tea polyphenols on
parasite resistance and acute phase protein response to Haemonchus contortus infection in lambs. Vet. Parasitol. (2014),
http://dx.doi.org/10.1016/j.vetpar.2014.06.022

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Table 1
Ingredients and chemical composition of the basal experimental diet.
Ingredients (g/kg of DM)1

Amount

Aneurolepidium chinense
Ground corn grain
Solid soybean meal (41% CP)
Molasses
Dicalcium phosphate
Limestone
Trace mineral salt and vitamins2
Chemical composition (n = 4)
DM (g/kg of feed)
Crude protein (g/kg of DM)
Fat (g/kg of DM)
Neutral detergent ber (g/kg of DM)
Acid detergent ber (g/kg of DM)
Metabolizable energy (MJ/kg of DM)3

300
475
150
50
10
5
10
902.6 10.65
176.8 9.36
51.2 1.36
584.6 10.23
358.4 7.34
14.2 0.13

DM = dry matter.
Mineral and vitamin salt was purchased from Continental Grain Corp.
(Beijing, China) and contained (per kg) 24,000 IU vitamin A, 4800 IU vitamin E, 120 mg Fe, and 24 mg Cu.
3
Metabolizable energy was calculated according to NRC (1985).
2

Geography and Agroecology, Chinese Academy of Sciences,


Jilin, China.
Thirty male Ujumqin lambs with an average age of
120 10.3 d, an initial body weight (BW) of 24.8 2.4 kg,
and similar genetic background were randomly assigned
to one of ve experimental diets (n = 6/group) for a feeding period of 56 days in a completely randomized design.
Although all lambs were separated from adult nematodeinfected sheep and given GIN-free diets from birth, all
lambs were still dewormed using a combination of moxidectin (0.8 mg/kg BW), levamisole (11.2 mg/kg BW), and
albendazole (21.6 mg/kg BW) by injection to eliminate the
presence of any possible GINs until no fecal eggs were found
using the McMaster technique (Stafford et al., 1994). All
lambs were fed basal diets (Table 1) formulated according
to NRC (1985). Treatments consisted of: (1) uninfected with
H. contortus as control (UNINF), (2) infected with H. contortus but not given GPT supplementation (INFGTP0), (3)
infected with H. contortus and given 2 g GPT/kg feed (dry
matter (DM) basis for all) supplementation (INFGTP2), (4)
infected with H. contortus and given 4 g GPT/kg feed supplementation (INFGTP4), and (5) infected with H. contortus
and given 6 g GPT/kg feed supplementation (INFGTP6).
The third-stage H. contortus larvae were prepared using
the egg-hatch procedure as described previously (Hansen
and Perry, 1990). Briey, approximately 5 kg crushed fecal
pellets collected from mature sheep that were naturally
infected with H. contortus were moistened with tap water
and placed in a pan with two beakers containing tap water
to maintain humidity inside the pan. The pan was kept
at room temperature for 10 days. The fecal samples were
then placed in a strainer lined with four layers of cheesecloth, and the strainer was kept immersed in a pan of
water to allow the larvae to settle to the bottom. The collected larvae were washed with phosphate-buffered saline
solution three times to remove fecal debris. A sample of larvae collection was then stained with iodine to determine
the species by light microscopy (Bausch & Lomb, Scientic Optical Products Div., Rochester, N.Y., USA) according
to the parasitological method described by Foreyt (1997).

The infective larvae of H. contortus (third-stage) were identied according to the feature of tail of sheath ending in
a ne whip-like lament (Hansen and Perry, 1990). Consequently, the percentage of H. contortus in the mixed
larvae was no less than 95%, the other species of larva
were Trichostrongylus colubriformis and Oesophagostomum
columbianum. Each lamb in the infected groups was experimentally drenched with 5000 third-stage H. contortus
larvae in one batch by mouth at day 0 before the morning
feeding.
The feeding experiment lasted for 56 days. At each feeding day, GTP supplement was mixed well with the basal diet
according to the required ratio of GTP to basal diet before
each feeding time. All lambs were fed twice daily at 06:00
and 18:00 h, lambs were fed 0.5 kg feed/head (DM basis)
each feeding time, and the amount of feed daily was maintained for 56 days. The orts for each lamb were recorded
daily to calculate feed intake. Each lamb was assigned
to an individual metabolic cage with access to fresh
water.
2.3. Sampling and analytical procedures
The feed supply and orts for each lamb were recorded
daily to calculate feed intake. Then diet samples were dried
at 105 C for 3 h to determine DM content. The chemical
composition of the orts was not measured.
Before the morning feeding of days 0, 14, 28, 42, and 56
of the experimental period, all lambs were weighed and the
average daily gain (ADG) of lambs was calculated based on
differences in live weight at specic points in time. Fecal
samples were collected directly from the rectum of each
lamb and processed immediately to determine FEC using
the McMaster technique reported by Stafford et al. (1994),
with results expressed as eggs per gram (EPG) of feces.
Before the morning feeding of days 0, 14, 28, 42, and
56 of the experimental period, 2-ml blood samples were
collected from the jugular vein of each lamb by venipuncture into heparin-containing vacutainers (Becton Dickinon,
Vacutainer Systems, Rutherford, NJ, USA) to determine PCV
using a Unico micro-haematocrit centrifuge (182-E, Dayton, NJ, USA) and a micro-capillary reader (Damon/IEC
Division). Before the morning feeding of days 0, 1, 3, 7,
28, 42, and 56 of the experimental period, 6-ml blood
samples were collected from the jugular vein of each
lamb into vacutainers without anticoagulant to harvest
serum for APP determination. The concentration of SAA
was analyzed using a commercially available sandwich
enzyme-linked immunosorbent assay (ELISA) kit (Tridelta
Development, Maynooth, Ireland) according to method of
Piccione et al. (2012). The concentration of serum LBP was
quantied using commercial ELISA kits (Jiancheng Biology
Co., Nanjing, China). Briey, the assay used puried goat
LBP antibody to coat microtiter plate wells. LBP was then
added to wells, where it bound to LBP antibody, which
was then complexed with horseradish peroxidase (HRP)labeled mouse anti-goat antibody. The color change was
measured spectrophotometrically at a wavelength of 450.
The concentrations of serum Hp and 1 AGP were measured
using commercial colorimetric kits (Jiancheng Biology Co.),
which followed a similar principle to that of the LBP assay.

Please cite this article in press as: Zhong, R.Z., et al., Effects of supplementation with dietary green tea polyphenols on
parasite resistance and acute phase protein response to Haemonchus contortus infection in lambs. Vet. Parasitol. (2014),
http://dx.doi.org/10.1016/j.vetpar.2014.06.022

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Table 2
Effect of supplementation with different levels of dietary green tea polyphenols on the total H. contortus counts ( standard error) recovered from abomasum
of lambs infected with H. contortus and control lambs.
Adult H.
contortus

Total
Male
Female

Treatment1

P value

UNINF

INFGTP0

INFGTP2

INFGTP4

INFGTP6

2 1d
1 1c
1 1d

1043 42a
410 21a
633 23a

421 32b
169 15b
252 22b

198 15c
68 8c
130 9c

83 6cd
34 3c
49 5cd

<0.0001
<0.0001
<0.0001

a, b, c, d

Means values with different superscripts in the same row within treatment differ (P < 0.05).
UNINF = uninfected; INFGTP0 = infected and not given GTP; INFGTP2 = infected and given 2 g GTP/kg feed; INFGTP4 = infected and given 4 g GTP/kg feed;
INFGTP6 = infected and given 6 g GTP/kg feed.
1

At the end of the study, all lambs were slaughtered


by exsanguination with previous electrocution, and at
necropsy, the abomasum was isolated, removed, and
washed in plastic buckets with 3 L tap water. Aliquots of
5% (150 ml) of total digesta were collected in 20 duplicates
for each lamb, and 100 ml formalin (10%) was added to each
aliquot. Adult H. contortus from 20 duplicated samples for
each lamb was recovered, sex identied, and counted using
a phase contrast microscope (Leica Inc., Beijing, China),
according to the methods of Minho et al. (2008).

model consisting of dietary treatment, sampling time, and


treatment time interaction as xed effects with animal
as the random effect. The measurements obtained from
each lamb at different sampling times were treated as
repeated measures. The EPG data were log-transformed
(ln (EPG + 1)) prior to statistical analyses. Means were separated using least squares mean and presented with the
standard error of the mean. Statistical signicance was
declared at P 0.05.
3. Results

2.4. Statistical analyses

3.1. Average daily gain

All data were analyzed by SAS (2002) using the MIXED


model procedure described by Littell et al. (1996) with a

The ADG data for all lambs are shown in Fig. 1. There was
no signicant difference of DM intake among ve groups

Table 3
Effects of supplementation with different levels of dietary green tea polyphenols on serum acute phase protein concentration of lambs infected with H.
contortus and control lambs.
Acute phase
protein

Amyloid A (mg/l)

Haptoglobin (g/l)

Lipopolysaccharide
binding protein (g/l)

1 -Acid glycoprotein,
g/L

Day

0
1
3
7
28
42
56
0
1
3
7
28
42
56
0
1
3
7
28
42
56
0
1
3
7
28
42
56

Treatment1

SEM2

UNINF

INFGTP0

INFGTP2

INFGTP4

INFGTP6

1.13
0.963
1.34
6.61
8.54
0.989
1.68
0.038
0.043
0.071
0.045
0.112
0.094
0.051
1.51
1.43
1.62
2.49
2.79
1.20
1.62
0.246
0.285
0.279
0.320
0.470
0.251
0.274

1.14
83.4
74.7
86.2
132
44.0
16.6
0.052
0.511
1.56
0.985
1.78
0.573
1.05
1.81
2.11
9.15
8.78
10.4
4.37
4.17
0.315
0.305
1.26
0.10
1.46
0.842
0.930

1.03
81.4
56.9
78.7
88.5
15.5
9.83
0.027
0.828
1.05
0.476
0.750
0.184
0.591
1.70
1.76
7.41
5.90
3.10
3.27
3.56
0.250
0.279
0.989
0.779
0.594
0.448
0.419

0.91
85.8
54.3
65.9
55.7
19.4
18.6
0.062
0.720
0.309
0.163
0.331
0.086
0.192
1.56
2.02
6.54
2.50
2.22
1.46
2.15
0.270
0.302
0.599
0.363
0.333
0.399
0.244

0.99
76.3
81.8
91.6
76.1
34.0
36.3
0.045
0.733
0.792
0.341
0.289
0.137
0.170
1.71
2.04
2.98
2.37
1.92
3.87
3.27
0.303
0.316
0.330
0.274
0.314
0.359
0.271

P value

Treatment

Time

Iteractions

6.437

<0.001

<0.001

<0.001

0.125

<0.001

<0.001

<0.001

0.369

<0.001

<0.001

<0.001

0.056

<0.001

<0.001

<0.001

1
UNINF = uninfected; INFGTP0 = infected and not given GTP; INFGTP2 = infected and given 2 g GTP/kg feed; INFGTP4 = infected and given 4 g GTP/kg feed;
INFGTP6 = infected and given 6 g GTP/kg feed.
2
SEM = standard error of the mean.

Please cite this article in press as: Zhong, R.Z., et al., Effects of supplementation with dietary green tea polyphenols on
parasite resistance and acute phase protein response to Haemonchus contortus infection in lambs. Vet. Parasitol. (2014),
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UNINF

300

INFGTP0

INFGTP2

INFGTP4

INFGTP6

UNINF

INFGTP0

INFGTP2

INFGTP4

INFGTP6

29.00

250

Packed cell volume

200
150
100

14

42

56

(0.985, 0.956, 0.946, 0.938, and 0.933 kg/d for the UNINF,
INFGTP0, INFGTP2, INFGTP4, and INGTP6). An interaction
effect on ADG was identied between treatment and sampling time (P = 0.0005). From days 0 to 28, the ADG values
of all infected lambs were lower than those of uninfected
lambs. From days 28 to 42, the ADG of uninfected lambs
decreased markedly (65.60 g/d); the ADG for the INFGTP0
lambs decreased to a lowest point (36.19 g/d), but the
ADG values in all three GTP-fed groups (112.98, 75.71, and
106.19 g/d in the INFGTP2, INFGTP4, and INFGTP6 groups,
respectively) were higher than those in the uninfected
group. From days 42 to 56, the ADG of all lambs except for
those in the INFGTP6 group were higher than the respective
ADG values from days 28 to 42.
3.2. Fecal egg count
The FECs for all lambs are shown in Fig. 2. Overall, an
interaction effect on FEC was identied between treatment
and sampling time (P < 0.0001). There were no differences
in FECs among all groups at day 0 (P > 0.05). The FECs in
INFGTP0 group were higher than those of in the other
UNINF
INFGTP4

INFGTP0
INFGTP6

INFGTP2

6000
5000

a
a
a

2000

1000

0
-1000
-2000

14

ab
ab
b
28 b

b
a

21.00

c
c

b
b

c
c
c

b
b

14

28
42
Day after initiation of feeding

56

Fig. 3. Packed cell volume (PCV standard error)) of lambs infected with
H. contortus and fed a basal diet supplemented with 0 g GTP/kg feed
(INFGTP0), 2 g GTP/kg feed (INFGTP2), 4 g GTP/kg feed (INFGTP4), and 6 g
GTP/kg feed (INFGTP6) (dry matter basis) and uninfected control lambs
(UNINF). a, b, c Means values with different superscripts in the same feeding day within treatments differ (P < 0.05).

four groups at each sampling time. The FECs in all infected


groups were signicantly higher than that of UNINF group
at day 14 (P < 0.0001). Though the FECs in INFGTP0 group
increased by infection at day 28 (P = 0.020), no signicant
differences were observed between UNINF and INFGTP6
groups. At day 42, GTP feeding decreased FECs, but the FECs
in all infected groups were still higher than that of UNINF
group (P < 0.0001), and the similar trend was also observed
at day 56 (P < 0.0001).
3.3. Blood packed cell volume
The PCVs of all lambs are shown in Fig. 3. Overall, an
interaction effect on PCV was identied between treatment
and sampling time (P = 0.0005). Between days 0 to 56, the
PCVs of all infected lambs were lower than those of uninfected lambs. At day 14, the PCVs in UNINF and INFGTP6
groups were signicantly higher than those of the other
three groups (P < 0.0001). At day 28, the PCVs in all infected
groups were signicantly lower than that of the UNINF
group, but feeding 6 GTP/kg of feed increased PCVs compared to other infected groups (P < 0.0001), and the similar
trend was observed at day 42 and 56 (P < 0.0001).
3.4. Adult H. contortus in abomasums

4000
3000

23.00

28

Fig. 1. Average daily gain (ADG standard error) of lambs infected with
H. contortus and fed a basal diet supplemented with 0 g GTP/kg feed
(INFGTP0), 2 g GTP/kg feed (INFGTP2), 4 g GTP/kg feed (INFGTP4), and 6 g
GTP/kg feed (INFGTP6) (dry matter basis) and uninfected control lambs
(UNINF).

7000

17.00

Days after initiation of feeding

Faecal egg count, eggs/g

25.00

19.00

50
0

,%

Average daily gain, g/d

27.00

ab
ab
b
42c

ab
bc
c
d

56

Days after initiation of feeding

Fig. 2. Fecal egg count (eggs per gram (EPG) standard error) of lambs
infected with H. contortus and fed a basal diet supplemented with
0 g GTP/kg feed (INFGTP0), 2 g GTP/kg feed (INFGTP2), 4 g GTP/kg
feed (INFGTP4), and 6 g GTP/kg feed (INFGTP6) (dry matter basis) and
uninfected control lambs (UNINF). a, b, c Means values with different superscripts in the same feeding day within treatments differ (P < 0.05).

Supplementing infected lambs with different levels of


GTP signicantly reduced (P < 0.0001) total adult H. contortus, male H. contortus, and female H. contortus burdens
(Tables 2). Furthermore, with increasing dietary levels of
GTP increased in diets, the numbers of total adult H. contortus, male H. contortus, and female H. contortus decreased.
At a GTP concentration of 6 g/kg feed, the numbers of total
adult H. contortus, male H. contortus, and female H. contortus
were reduced to uninfected levels (P > 0.05).
3.5. Serum acute phase protein concentrations
Interaction effects (P < 0.001) on SAA, Hp, LBP, and
1 AGP concentrations were identied between treatment

Please cite this article in press as: Zhong, R.Z., et al., Effects of supplementation with dietary green tea polyphenols on
parasite resistance and acute phase protein response to Haemonchus contortus infection in lambs. Vet. Parasitol. (2014),
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and sampling time (Tables 3). Overall, at all time points


from days 0 to 56, H. contortus infection increased concentrations of SAA by 10-150-fold, Hp by 1030-fold, LBP by
26-fold, and 1 AGP by 210-fold compared to concentrations in uninfected control lambs. The rst and second
peaks in SAA concentration in the infected groups were
observed at days 1 and 28 post-infection, respectively. The
rst and second peaks in Hp, LBP, and 1 AGP concentrations in the infected groups were all observed at days 3 and
28 post-infection, respectively. At each sampling time, the
concentrations of SAA in the INFGTP2 and INFGTP4 lambs
were lower than those in INFGTP0 lambs, but the concentration of SAA in INFGTP6 lambs was higher than that in
INFGTP0 lambs. Different from the results observed for SAA
response, steady decreases in the mean serum Hp, LBP, and
1 AGP concentrations were observed from days 14 to 56
in all GTP-fed lambs relative to concentrations in INFGTP0
lambs. However, the mean concentrations of these proteins were not reduced to the normal levels observed in
uninfected control lambs.
4. Discussion
Previous studies have demonstrated that parasitism
reduces feed intake and weight gain in small ruminants
(Sykes and Coop, 1976; Coop and Sykes, 2002). However,
a high-protein diet (170 g CP/d) fed to animals infected
with H. contortus can enhance resilience to infection by
increasing feed intake and reducing weight loss (Datta
et al., 1999). In the present study, no signicant reduction
in the DM intake of infected lambs in the four infection
groups (0.956, 0.946, 0.938, and 0.933 kg/d for lambs in the
INFGTP0, INFGTP2, INFGTP4, and INGTP6 groups, respectively) was observed compared with that of uninfected
lambs (0.985 kg/d) (not shown in the tables). This may
because diets with a higher dietary protein level provided
a sufcient supply of metabolizable protein to maintain
feed intake of the animals. However, the ADG values of
all infected lambs were still lower than that of uninfected
lambs. This may be attributed to the portion of dietary
nutrients might lose due to nematodes living in the gastrointestinal tract. A similar hypothesis was veried by
Vaughan et al. (2006). Moreover, H. contortus burden would
induce host immune responses, which would also utilize some portion of the metabolizable protein to produce
antibodies and APPs (Sykes, 2010). During day 28 to 56,
compared with INFGTP0 lambs, higher ADG in GTP feeding
lambs was that GTPs reduced both male and female H. contortus burdens in the abomasums of lambs. Anthelmintic
activity of killing H. contortus larvae of tea extracts have
been veried by Zhong et al. (2014). But the reason of general drop of ADG on day 42 was mainly because of higher
ambient temperature and lower DM intake on July.
The majority of inoculated third-stage H. contortus larvae will establish in the fundic and in the middle of
pyloric thirds of the goat abomasum within 4 days postinoculation and then develop into adults and lay eggs
within 11 days post-inoculation (Rahman and Collins,
1990). The development of H. contortus in sheep is essentially similar to that described in goats. In the current study,
eggs were found in the feces at day 14 after inoculation,

which means some of the inoculated larvae had developed


into adult worms.
The main ingredients of GTPs are catechins, which are
mostly condensed tannins. The consumption of high concentrations of condensed tannins (>7% of DM) has been
associated with a number of detrimental effects in ruminants, such as reduction in food intake, growth inhibition,
and interference with rumen fermentation. However, consumption of low concentrations of condensed tannins (<6%
DM) was shown to have positive effects on ruminants,
such as anthelmintic bioactivity (Min et al., 2003; Waghorn
and McNabb, 2003). The proposed mechanisms of the
anthelmintic bioactivity of tannins involve direct binding of
tannins to nematode proteins, which subsequently blocks
the parasites physiological processes and/or modies its
host immune responses to eliminate infective larvae or
adult worms (Hoste et al., 2006, 2012).
Joshi et al. (2011) reported that feeding tannincontaining forages reduces the fecundity of female worms
and lowers worm burdens in goats. Tea is very popular and
produced with high yield in China. Its utilization as a feed
additive for ruminants has positive effects mainly against
oxidative stress (Zhong et al., 2009, 2011). In the current
study, H. contortus burden, including male and female H.
contortus, was reduced in GTP-fed lambs compared with
INFGTP0 lambs, with higher levels of GTP having greater
efcacy against abomasal H. contortus, which persisted
until the end of the experimental period. Similar results
were reported by Gujja et al. (2013) for goats fed Lespedeza
leaf. Consistent with the report of Azaizeh et al. (2013), a
high concentration of galloylated derivatives may explain
the anthelmintic bioactivity of GTPs.
The EPG count is a recognized phenotypic marker for
evaluating worm burdens in ruminants. A positive correction between fecal H. contortus egg counts and total
H. contortus worm burdens have been shown (Roberts
and Swan, 1981). While an adult female can lay thousands of eggs daily, but the eggs may not release and their
excretion in feces is not synchronized (Zajac and Moore,
1993). Some daily variation in egg counts occurs in individual animals, relative differences are consistent overall.
This is why EPG counts usually uctuate daily even in
the same animals. Dietary tannins decreases egg excretion, but reductions in EPG counts are associated with a
reduced number of worms, a signicant decrease in female
fecundity, or combined effects (Paolini et al., 2003; Joshi
et al., 2011). Reductions in EPG counts in tannin-containing
forage-fed sheep have been reported (Niezen et al., 2002;
Valderrbano et al., 2010), and these inhibitory effects were
attributed to polyphenols present in the plants (Lorimer
et al., 1996). However, little was known about such effects
in lambs fed tea-derived polyphenols. The results of the
present study suggest that the decreased EPG counts were
positively correlated with the reduced H. contortus burden
in all GTP-fed lambs.
The lower PCV values of infected lambs were, in turn,
attributed to the greater H. contortus burden. Both adult
and fourth larval stage H. contortus suck host blood and
cause hemorrhage into the abomasum. An adult laying
female can consume 200 l of blood daily. The normal total
blood volume of sheep ranges from 55 to 147 ml/kg live

Please cite this article in press as: Zhong, R.Z., et al., Effects of supplementation with dietary green tea polyphenols on
parasite resistance and acute phase protein response to Haemonchus contortus infection in lambs. Vet. Parasitol. (2014),
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weight depending on the physiological state of the animals (Durotoye and Oyewale, 2000). In the current study,
H. contortus infection of lambs decreased PCV by approximately 57%. The reason for reduction of PCV is to the
reduction in H. contortus burden. Although supplementation with GTPs reduced this blood loss, PCV was not
restored to original uninfected level. To date, the cost and
speed of an accurate diagnostic method remain barriers to
controlling GIN infection and disease. Worm burden is a
direct parameter that represents the grade of GIN infection. However, animals must be slaughtered to determine
worm burden, which is not always economically feasible
for producers. The FEC is easy to measure, but it does not
always reect the true worm burden. Hence, multiple relevant parameters such as serological biomarkers should be
screened.
Acute inammation is an organisms normal homeostatic and immediate physiological response to injury or
infection. Blood protein proles are valuable for assessing the physiological status of animals, because peripheral
blood monocytes are the cells most likely to initiate the
acute phase responses when a foreign organism such as
a parasite is encountered (Suffredini et al., 1999; Ceciliani
et al., 2012). The APPs are mediated by pro-inammation
cytokines when animals are subjected to external or internal challenges, such as infection, inammation, or stress
(Gonzlez et al., 2008). The APPs play important roles in
the innate defense mechanisms (Eckersall, 2000). The most
important cytokines include IFN-, IL-2, IL-12, and TNF-
for T helper (Th)1 type immunity and IL-4, IL-5, IL-6, IL10, and IL-13 for Th2 type immunity. In addition, levels
of some pro-inammatory cytokines are enhanced by H.
contortus infection in sheep (Gill et al., 2000; Alba-Hurtado

and Munoz-Guzmn,
2013). Therefore, it is reasonable
to investigate APP responses to H. contortus infection in
sheep.
Although Gnheim et al. (2004) reported that lungworm
infection cannot be detected on the basis of APP measurements, this may vary with different species of parasites
and hosts. Screening for blood protein proles has been
used to identify ruminants that are infected with parasites.
Previous studies reported that H. contortus infection promotes increased globulin and decreased total protein and
albumin concentrations in sheep (Ahmad et al., 1990) and
goats (Diogenes et al., 2010). However, very few reports
have been published on APP induction by H. contortus in
sheep. The results of the present study indicate that the
levels of these APPs in uninfected lambs were within the
normal ranges (Ceciliani et al., 2012), and H. contortus
infection induced primary elevated concentrations of SAA,
Hp, LBP, and 1 AGP within 3 days post-inoculation. The
SAA response was the fastest (24 h post-infection) among
those of the four measured APPs. Generally, the pattern of
protein synthesis by the liver is drastically altered upon
infection, resulting in increased production of some blood
proteins, the positive APPs, within a few hours after infection (Blackburn, 1994). The second peak of concentrations
for the four APPs was observed at day 28 post-infection,
which may be the result of combined effects of H. contortus
larvae and adult H. contortus worms. Several studies have
reported that H. contortus infection to ruminants trigger

an immune response and changes of cytokine proles (Gill


et al., 2000; Zhong et al., 2014). In this case, such marked
increases in APP concentration within days of infection
demonstrate that APPs can be used as serological biomarkers to monitor early H. contortus infection in sheep.
Polyphenols are abundant micronutrients in many
species of plants. Tannins are bitter plant polyphenolic
compound with molecular weights ranging from 500 to
more than 3000 and as high as 20,000 Da. Forage-derived
condensed tannins have been shown to be able to reduce
H. contortus burden in small ruminants and to play a dual
role in the host by both immobilizing the parasitic larvae and up-regulating immune responses associated with
parasite elimination (Hoste et al., 2006). The antioxidant,
anticancer, antiproliferative, and anti-inammatory properties of GTPs have been widely reported in humans and
animals (Stangl et al., 2007; Zhong et al., 2009, 2011). However, little is known about their anthelmintic bioactivity.
Rein et al. (2006) reported that avonoid intake reduces
C-reactive protein levels in humans, but not brinogen levels. The mechanisms by which APP production is regulated
may be attributed to the role played by polyphenols in
suppressing pro-inammatory cytokines (Schroecksnadel
et al., 2007). The present results show that supplementation with GTPs steadily decreased serum Hp, LBP, and
1 AGP levels, but supplementation with a higher dose of
GTP (6 g/kg feed) was not able to reduce SAA concentrations to normal levels. These results indicate that GTPs
regulate SAA production in a dose-dependent manner. The
higher level of SAA observed in INFGTP6 lambs might be
the result of oxidative stress caused by supplementation
with higher GTP concentration as the exogenous polyphenols have dual effects on cellular redox state, exhibiting
pro-oxidant or antioxidant activities intimately dependent
on the dosage administered (Bouayed and Bohn, 2010).
Hence, supplementation with GTPs at an appropriate dose
should be considered to balance the physiological functions
of polyphenols in lambs.

5. Conclusions
In addition to worm burden, FEC, and PCV, our results
strongly suggest that quantitative measurements of SAA,
Hp, LBP, and 1 AGP responses provide valuable diagnostic
information and that these APPs can be used as potential
biomarkers for monitoring H. contortus infection and treatment responses in lambs. Dietary GTP supplementation
increased host resistance to H. contortus infection by reducing worm burdens and weight loss, and the mechanisms of
the anthelmintic bioactivity of GTPs may be associated with
the suppression of APP expression.

Acknowledgments
This work was supported by the National Natural Science Foundation of China (Grant no. 31201820) and the
Excellent Young Scientists Foundation of the Northeast
Institute of Geography and Agroecology, Chinese Academy
of Sciences, Changchun, China (Grant no. DLSYQ12008).

Please cite this article in press as: Zhong, R.Z., et al., Effects of supplementation with dietary green tea polyphenols on
parasite resistance and acute phase protein response to Haemonchus contortus infection in lambs. Vet. Parasitol. (2014),
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