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Pop et al. Parasites Vectors (2019) 12:343
https://doi.org/10.1186/s13071-019-3595-4
RESEARCH
Efficacy of a commercial herbal formula
in chicken experimental coccidiosis
Loredana Maria Pop1, Erzsébet Varga2*, Mircea Coroian1, Maria E. Nedișan1, Viorica Mircean1,
Mirabela Oana Dumitrache1, Lénárd Farczádi3, Ibolya Fülöp4, Mircea Dumitru Croitoru4, Mihaly Fazakas2
and Adriana Gyӧrke1*
Abstract
Background: Coccidiosis represents a serious threat to the poultry industry, affecting production and causing high
morbidity, mortality and significant costs resulting from treatment and prophylaxis. In-feed anticoccidials have been
used for decades for managing avian coccidiosis and were very effective until drug resistance emerged. The use of
natural remedies has become a promising alternative in combating coccidiosis in chickens. Therefore, the purpose
of the present study was to assess the efficiency of a commercial herbal formula (H), as oral liquid preparations, in
experimental chicken coccidiosis.
Methods: Two independent controlled battery experiments (BE1 and BE2) were designed and the product was
tested in 3 different formulas (H1, H2 and H3): H1 contained a propylene glycol extract of Allium sativum and Thymus
serpyllum; H2 contained Origanum vulgare, Satureja hortensis and Chelidonium majus; and H3 contained Allium sativum,
Urtica dioica, Inula helenium, Glycyrrhiza glabra, Rosmarinus officinalis, Chelidonium majus, Thymus serpyllum, Tanace-
tum vulgare and Coriandrum sativum. Chickens were divided into five groups for each BE as follows: (i) uninfected
untreated control (UU1, UU2); (ii) infected untreated control (IU1, IU2); (iii) infected treated with amprolium (ITA1,
ITA2); and (iv, v) two experimental groups infected treated with H1 (ITH1) and H2 (ITH2) formulas in the BE1 and with
H3 (ITH3-5 and ITH3-10) formula in the BE2. The chickens from infected groups were challenged with 5000 (BE1) and
50,000 (BE2) sporulated oocysts of Eimeria spp. (E. acervulina, E. tenella and E. maxima), respectively. The anticoccidial
efficacy was assessed by recording the following: oocysts output (OPG), lesion score (LS), weight gain (WG), feed con-
version ratio (FCR) and anticoccidial index (ACI). Additionally, polyphenolics and flavonoids (caffeic-chlorogenic acid,
apigenin, kaempferol, luteolin, quercitin, quercitrin) from herb extracts found in H3 formula were determined by the
liquid chromatography-tandem mass spectrometry (LC-MS/MS) method.
Results: H1 and H2 reduced the WG, and increased the FCR and OPG compared with controls. H1 reduced the
duodenal lesions, whilst H2 reduced the caecal lesions, compared with control. H3 decreased the OPG of Eimeria spp.,
reduced the total lesion score and improved the zootechnical performance (weight gain and feed conversion ratio).
According to ACI value, H1 and H2 had no efficacy on Eimeria spp. infection, but H3 had good to marked anticoccidial
effect, the ACI being slightly greater in the group ITH3-5. According to the results of LC-MS/MS, the concentration of
polyphenols in H3 formula was the highest, the sum of chlorogenic acid and caffeic acid being 914.9 µg/ml.
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Open Access
Parasites & Vectors
*Correspondence: erzsebet.varga@umftgm.ro;
adriana.gyorke@usamvcluj.ro
1 Department of Parasitology and Parasitic Diseases, Faculty of Veterinary
Medicine, University of Agricultural Sciences and Veterinary Medicine
Cluj-Napoca, Calea Mănăştur 3–5, 400372 Cluj-Napoca, Romania
2 Department of Pharmacognosy and Phytotherapy, Faculty of Pharmacy,
University of Medicine, Pharmacy, Sciences and Technology of Târgu
Mureș, 38 Gheorghe Marinescu, 540139 Târgu Mureș, Romania
Full list of author information is available at the end of the article
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Page 2 of 9
Pop et al. Parasites Vectors (2019) 12:343
Background
Coccidiosis is a parasitic disease caused by seven species
of the genus Eimeria with different localizations within
the intestinal tract of chickens. Eimeria acervulina, E.
maxima and E. tenella are the most prevalent species in
broilers in the intensive poultry management system [1,
2]. e disease represents a serious threat for the poul-
try industry, affecting the production, and causing high
morbidity, mortality and significant economic loss due to
the associated costs of treatment and prophylaxis. Global
financial losses due to coccidiosis have been estimated
at three billion USD per annum [3]. In-feed anticoccidi-
als have been used for decades for managing avian coc-
cidiosis and they were very effective until drug resistance
emerged. To date, Eimeria strains have gained resistance
to all known coccidiostats, and new anticoccidials are
unlikely to be developed, mainly because of strict legisla-
tive regulations on the use of in-feed drugs and growing
concerns in the general population about the chemical
residues in poultry products [4–6]. Over the past years,
the consumption of poultry meat has grown consistently,
especially because it represents a fairly cheap source
of food with lower production costs and accepted by
all religions [7]. ere is also a higher interest from the
consumer in organic poultry production and a great
demand for natural and healthier products [8]. In this
context, the use of natural remedies has become a prom-
ising alternative to anticoccidial drugs [9]. Numerous
plant-based products have been found to be effective at
treating chicken coccidiosis: Artemisia annua and arte-
misinin [10, 11], oregano [12], garlic [13], neem [14], dif-
ferent species of Aloe [15], green tea [16], sugar cane [17],
turmeric [18] and many others [9, 19–21]. Additionally,
commercially available herbal combinations are already
used in some countries for coccidiosis control [19]. Most
of these natural compounds do not always aim directly
at the parasites but have immunomodulatory effects,
antioxidative or anti-inflammatory properties and act
on the intestinal tract, thus helping the host organism
to fight against the coccidial infection [9, 19]. Moreover,
the plant extracts can have a direct effect on the para-
sites, by altering the process of oocyst wall formation
and inhibiting sporulation [22, 23], or by destroying the
sporozoites [24]. Furthermore, there is a lower risk of
developing resistance to these natural substances com-
pared to anticoccidial drugs [21]. Furthermore, herbal
extracts could improve recovery after coccidiosis [25, 26].
Flavonoids and other polyphenols have been reported to
be responsible for most of the biological properties of the
herbs, including the anticoccidial potential [27].
erefore, the purpose of the present study was to
assess the effect of a commercial multi-plant extract
compound, in experimental coccidiosis in broiler chick-
ens. e composition of the herbal extract was designed
on the basis of a literature search for effective anticoccid-
ial natural compounds.
Methods
Animals and experimental design
Two independent controlled battery experiments (BE)
were designed in order to assess the efficacy of a com-
mercial herbal product (H), in three different formulas of
propylene glycol/alcoholic herbal extracts. In the first BE
(BE1), the anticoccidial effect of the first (H1) and second
(H2) formulas was evaluated. e third formula (H3) was
evaluated during the second BE (BE2).
One hundred one-day-old ROSS 308 hybrid broiler
chickens were purchased from S.C. VIS AVIS S.A. (Vadu
Crişului, Bihor, Romania) for each of the two BE. ey
were housed in batteries in dedicated facilities at the Uni-
versity of Agricultural Sciences and Veterinary Medicine
Cluj-Napoca. At 14-days-old, broiler chickens were ran-
domly divided in five groups, each with three replicates
of five chickens/cage (n = 15). In both BE the experimen-
tal groups were represented by: (i) negative control, unin-
fected and untreated (UU1, UU2); (ii) positive control,
infected and untreated (IU1, IU2); (iii) treatment control,
infected and treated with amprolium (ITA1, ITA2); and
(iv, v) two experimental groups, infected and treated with
H1 (ITH1) and H2 (ITH2) formulas in the BE1and with
H3 (ITH3-5 and ITH3-10) formula in the BE2.
On the same day, the broiler chickens were experimen-
tally infected by the crop-route using insulin syringe, with
1ml of a mixed suspension of fresh sporulated oocysts
(BE1 5 × 103 oocysts/chicken; BE2 5 × 104 oocysts/
chicken) containing E. acervulina, E. tenella and E. max-
ima. e strains were isolated in 2012 from a broiler farm
and the species were identified by PCR [28]. e number
of oocysts per milliliter was determined using a Fuchs-
Rosenthal chamber and adjusted according to sporula-
tion rate.
e control and experimental treatments were given
via drinking water from 14 until 24 days of age, ad libi-
tum. Chickens from ITA1 and ITA2 groups were treated
Conclusions: H3 formula is a promising natural anticoccidial and field trials are recommended in order to validate
the obtained data.
Keywords: Eimeria, Herbal extract, Broiler chickens, Anticoccidial effect, Polyphenols, LC-MS/MS
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Pop et al. Parasites Vectors (2019) 12:343
with amprolium (Amprolium 20%® water-soluble pow-
der; Romvac Company S.A., Voluntari, Ilfov, Romania)
as doses of 2.5 (BE1) and 5g/l water (BE2), respectively.
H1 and H2 herbal formulas were given as doses of 10ml/l
drinking water (BE1) and H3 herbal formula as doses of
5 (ITH3-5) and 10 (ITH3-10) ml/l drinking water (BE2).
e chickens were fed with standard starter (1–13
days-old) and grower (14–24 days-old) feed free of
anticoccidials.
Herbal formulas
e commercial herbal formula was supplied by S.C.
PROMEDIVET S.R.L (Sovata, Mureș, Romania) as oral
liquid preparations. e anticoccidial efficacy in chicken
coccidiosis was evaluated for three different extracts
named H1, H2 and H3. e extracts were obtained from
grounded dried plants after maceration in propylene gly-
col (20kg of dried plants in 120l of propylene glycol) for
14days and cold pressing.
H1 contained extracts from bulbs of Allium sativum
(garlic) and leaves of ymus serpyllum (wild thyme) in
equal proportions. H2 was a mixture of extracts from
leaves of Origanum vulgare (oregano) 40%, Satureja
hortensis (summer savory) 30% and Chelidonium majus
(greater celandine) 30%. H3 contained extracts from nine
herbs: roots of Urtica dioica (nettle) 10%, Inula helenium
(elecampane) 15%, Glycyrrhiza glabra (licorice) 10%,
bulbs of Allium sativum 10%, leaves of Rosmarinus offici-
nalis (rosemary) 10%, Chelidonium majus 10%, ymus
serpyllum 15%, flowers of Tanacetum vulgare (tansy) 10%
and seeds of Coriandrum sativum (coriander) 10%.
Liquid chromatography tandem mass spectrometry
e H3 formula was analyzed by liquid chromatogra-
phy tandem mass spectrometry (LC-MS). One hundred
microliters of H3 formula was mixed with 900µl of puri-
fied water, then centrifuged at 10,000× rpm for 10min
and filtered using nylon micro pore 0.45µm filters. Solu-
tions were transferred to HPLC vials and 5 μl of the
obtained samples were injected into the LC-MS system.
Chromatographic separation of analytes was per-
formed using a NUCLEODUR C18 Gravity, 3 µm,
150 × 3mm (Macherey-Nagel, Düren, Germany) with a
mobile phase consisting of 0.2% formic acid in water and
methanol in gradient elution, with a flow rate of 0.6ml/
min. Detection was carried out in multiple reactions
monitoring mode (SRM). Ionization of analytes was per-
formed using negative electrospray ionization mode. Ion-
ization parameters used for the ionization source were
as follows: spray voltage, 2500V; vaporizer temperature,
350°C; ion gas source 1, 25; ion gas source 2, 25; curtain
gas, 10; declustering potential, 100; ion release delay, 30;
ion release width, 15. Sample run-time was 30 min per
sample.
Standard solutions of each analyte (chlorogenic acid,
caffeic acid, quercitrin, luteolin, quercetin, apigenin and
kaempferol) (100µg/ml) were prepared in methanol. For
analysis, standard solutions were mixed in methanol to a
final concentration of 10µg/ml for each analyte. Further-
more, the mixture was diluted to the following concen-
trations: 0.1 (lower limit of quantification, LLOQ), 0.25,
0.5, 1 and 10µg/ml. e calibration of standard curve
solutions was prepared by diluting 100µl of each stand-
ard solution with 900µl of purified water.
Anticoccidial efficacy evaluation
e efficacy of the herbal formulas in experimental coc-
cidiosis in broiler chickens was assessed by recording and
calculating the number of oocysts shedded per gram of
feces (OPG), lesion score (LS), mortality rate (MR), body
weight gain (BWG), feed conversion ratio (FCR) and
anticoccidial index (ACI).
e feces were collected on days 5, 7 and 10, from all
3 cages/group individually and the OPG was determined
by duplicate counts of duplicate fecal slurries from each
cage by using the McMaster method [29]. e detection
limit was 12oocysts/g feces. e LS was assessed on day
7 post-infection (pi) for eight chickens per group by using
the scoring system of Johnson & Reid [30], according to
the severity of lesions in duodenum, jejunum plus ileum
and caeca. Chickens were weighed individually at the
beginning of the experiments and at 7days post-infection
in order to calculate the body weight gain. e amount
of feed given to the chickens was weighed daily for each
cage, in order to calculate FCR, as the ratio between the
amount of feed consumed per body weight gain of the
chickens.
e ACI was calculated after the formula:
ACI = (%S + %RGW) − (LI + OI) [31], where %S is the
percentage of survival, %RGW is the percentage of rela-
tive weight gain (RWG = BWG × 100/untreated group
BWG), LI is the lesion index as the lesion score multi-
plied by 10 and OI is the oocyst index as (OPG output
of each experimental group/OPG output of the infected-
unmedicated control) × 100. e interpretation of the
results was made as follows: “lack of anticoccidian activ-
ity” when the value was lower than 120, “partially effec-
tive” at values of 120–160 and “very effective” at values
higher than 160 [31].
Statistical analysis
e data were processed with MedCalc Software v.18
(MedCalc Software bvba, Ostend, Belgium; https ://
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Pop et al. Parasites Vectors (2019) 12:343
www.medca lc.org). e arithmetic mean and standard
error were calculated for each assessed parameter and
for each group. ANOVA (repeated measures analysis of
variance) was used for OPG and body weight gain, and
the Kruskal-Wallis test was used for lesion score. Dif-
ferences were considered statistically significant if the
P ≤ 0.05. e experimental groups were compared with
both positive and negative control groups and also with
group A.
Results
First battery experiment
e OPG of groups treated with H1 and H2 formu-
las was significantly higher compared with the positive
control (IU1) or ITA1 groups on day 7 post-infection
(p.i.) (F(3,11) = 28.13, P < 0.001) (Fig. 1). e mortality
rate was 0 for all experimental groups. During the nec-
ropsy, lesions due to Eimeria spp. infection were identi-
fied in duodenum and caecum in all infected groups. In
Fig. 1 Dynamics of mean oocysts number/g of feces in experimental groups of chickens infected with Eimeria spp. (E. acervulina, E. maxima and
E. tenella) and treated with the herbal product in different compositions compared with positive and amprolium control groups. a First battery
experiment when chickens were infected with 5 × 103 sporulated oocysts of Eimeria spp. and treated with H1 and H2 formulas, 10 ml/l water. b
Second battery experiment when chickens were infected with 5 × 104 sporulated oocysts of Eimeria spp. and treated with H3 formula, 5 (ITH3-5)
and 10 ml/l water (ITH3-10)
Table 1 The effect of the herbal product H on lesion score and performance parameters in experimental groups of chickens
challenged with Eimeria spp. (E. acervulina, E. maxima and E. tenella) compared with control groups
*P < 0.05, Mann-Whitney test (independent samples) (MedCalc)
Abbreviations: BE, battery experiment (5 × 103 oocysts/chicken in BE1 and 5 × 104 oocysts/chicken in BE2); UU1, 2, negative control group; IU1, 2, positive control
group; ITA1,2, Amprolium® 20% (Romvac Company SA, Voluntari, Ilfov, Romania), soluble powder treated group; ITH1, ITH2, ITH3-5 and ITH3-10, experimental
groups treated with the herbal product in different compositions, 10 ml/l water (ITH1, ITH2 and ITH3-10) and 5 ml/l water (ITH3-5); BWG, body weight gain; FCR: feed
conversion ratio
Lesion score BWG FCR
Duodenum Caecum Total
BE1
UU1 0 0 0 55.81 ± 2.54 1.97 ± 0.11
IU1 0.4 ± 0.2 0.5 ± 0.2 0.9 ± 0.3 54.51 ± 1.55 1.77 ± 0.19
ITA1 0.1 ± 0.1 0.1 ± 0.1 0.3 ± 0.2 53.57 ± 2.52 1.64 ± 0.31
ITH1 0 0.5 ± 0.2 0.5 ± 0.2 53.30 ± 0.83 1.99 ± 0.30
ITH2 0.4 ± 0.2 0.1 ± 0.1 0.5 ± 0.2 47.07 ± 1.74* 2.24 ± 0.08
BE2
UU2 0 0 0 46.89 ± 2.68 1.58 ± 0.17
IU2 1.33 ± 0.56 1.0 ± 0.00 2.33 ± 0.56 34.91 ± 4.39 2.04 ± 0.13
ITA2 0.00 ± 0.00* 0.8 ± 0.20 0.8 ± 0.20* 42.36 ± 2.08 1.61 ± 0.02
ITH3-5 0.83 ± 0.54 0.33 ± 0.21* 1.17 ± 0.65 40.57 ± 1.92 1.78 ± 0.03
ITH3-10 0.83 ± 0.48 0.5 ± 0.22 1.33 ± 0.42 39.80 ± 2.83 1.78 ± 0.02
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Pop et al. Parasites Vectors (2019) 12:343
all experimental infected groups, the total lesion score
recorded values below 1 and with no significant dif-
ferences (χ2 = 2.556, df = 3, P = 0.465) between groups
(Table1). Nevertheless, the total lesion score in ITH1 and
ITH2 groups was lower than the positive control group
and higher than in group ITA1.
H1 formula prevented the emergence of E. acervulina
lesions in the duodenum, but had no therapeutic/prophy-
lactic effect on E. tenella; the lesion score in the caecum
was similar with the positive control group. H2 formula
did not prevent the occurrence of duodenal lesions, but
the lesion score in the caecum was lower than in the posi-
tive control group (χ2 = 1.588, df = 1, P = 0.208) (Table1).
All experimental groups presented lower weight gains
compared with the negative control group, with statisti-
cal significance in the case of group ITH2 (F(4,9) = 7.8,
P = 0.007) (Table1). e lowest weight gain was recorded
in the group treated with H2 formula (Table1). e FCR
of negative and experimental groups was higher compar-
ing with positive control group (Table1).
According to the anticoccidial index, the H1 and H2
formulas had no efficacy on Eimeria spp. infection.
Second battery experiment
e OPG value was significantly higher in groups treated
with H3 formula than in the group treated with ampro-
lium but not compared with the positive control group
(F(3,7) = 19.7, P < 0.001) at 5 days p.i. On days 7 and 10 p.i.,
the OPG of chickens treated with H3 decreased under
the values of the positive group, but significantly only
on day 10 p.i. (F(3,7) = 43.44, P < 0.001) (Fig. 1). e group
ITA2 recorded the lowest value of OPG compared with
positive control and H3 treated groups during the entire
recording period (F(3,23) = 6.63, P = 0.003).
No mortality was registered in any experimental group.
e chickens from all groups, with the exception of the
ITA2 group, presented lesions in the duodenum, but all
chickens had lesions in the caecum. Neither 5 nor 10ml/l
water of H3 formula significantly reduced the duodenal
lesions compared with the positive control group. H3
formula at a dose of 10ml/l water significantly reduced
the caecal lesions (Z = 2.162, P = 0.031). Additionally, the
chickens treated with 5ml H3/l drinking water had fewer
lesions than the positive control group, but with no sta-
tistical significance (Table1). e total lesion score was
reduced significantly compared with the positive con-
trol group only in the case of the ITA2 group (Z = 2.106,
P = 0.0352). However, the H3 formula at a dose of 10ml/l
water reduced the total lesion score by 50%.
All the experimental groups presented lower weight
gains than the negative control group (F(4,11) = 1.78,
P = 0.182). However, compared with the positive control
group, the chickens treated with H3 formula had higher
weight gains and only slightly lower than the amprolium
treated chickens.
e best feed conversion ratio was recorded, as
expected, by the negative control group. However, the
groups treated with H3 formula also had good feed con-
version, similar to those of the uninfected chickens. e
positive control group had the lowest use of feed.
e H3 formula was effective for control of experimen-
tal coccidiosis in chickens according to the anticoccidial
index and it was not dose dependent (Fig.2).
Liquid chromatography tandem mass spectrometry
A total ion chromatogram and an extracted ion chroma-
togram of the H3 formula are presented in Additional
file1: Figure S1 and Additional file2: Figure S2, respec-
tively. e presence of seven flavonoids and polyphenols
was tested (Table2). e concentration of polyphenols
was the highest, the sum of chlorogenic acid and caffeic
acid being 914.9µg/ml.
Discussion
Herbal remedies have been used since ancient times in
medicine and have recently gained increasing popularity,
especially because of the declining effectiveness of syn-
thetic compounds and concerns of the general population
about drug side effects and interactions [18]. In chicken
coccidiosis, herbal extracts have been intensively stud-
ied in the recent years in the search for new alternatives
to the traditional anticoccidial drugs [27]. e extensive
use of anticoccidials in the poultry industry may lead to
the occurrence of drug residues in meat and eggs [32]. As
such, consumer interest in organic foods has been rap-
idly increasing in recent years. e organic requirements
restrict the use of chemicals, so natural plant products
may represent an effective solution for pathogen control
in the organic poultry system [28].
In this frame, the present study aimed to assess the
anticoccidial effect of a natural plant product in three
different compositions. H1 formula, which contained
garlic and wild thyme extract, totally reduced the lesions
produced by E. acervulina. H2 formula, which was com-
prised of oregano, summer savory and greater celandine,
reduced the lesions caused by E. tenella. However, the
chickens medicated with these formulas presented higher
OPG output compared with the positive control group.
is aspect can be explained by the over-multiplication of
the uninhibited Eimeria species. According to Dar etal.
[33], garlic administration increases the values of serum
albumin, globulin and total proteins due to its anti-
inflammatory and immunomodulatory action that repair
the organ lesions induced by Eimeria, an aspect observed
in the present study for the duodenum. Arczewska-
Włosek and Świątkiewicz [26] observed an increased
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Pop et al. Parasites Vectors (2019) 12:343
weight gain and better feed conversion in the chickens
highly infected with Eimeria spp. whose diet was sup-
plemented with garlic extract at a level of 750 mg/kg
feed. Similar to our results, the OPG was higher than in
the case of the infected unmedicated group. e authors
stated that the high production performances, despite
of the greater number of oocysts recorded, could be the
effect of reduced damage to intestinal cells or promo-
tion of enterocyte renewal, which can provide the sub-
strate for coccidia multiplication. However, in our study
the first two formulas of the commercial herbal formula
did not improve the chickens’ production performance.
Numerous studies have demonstrated that Origanum
vulgare as aqueous extract at a concentration of 2g/kg
feed [34] or essential oil in concentrations of 600 and
1200mg/kg feed [35] and also Satureja hortensis powder
at 5g/kg feed [36], or 1% powder in feed [37] stimulates
the food intake and growth of chickens. However, as in
the present study, Bozkurt etal. [38] showed that the
administration of an essential oil blend that contained
carvacrol, 1,8-cineole, camphor, and thymol derived
from oregano, laurel leaf and lavender oil did not sig-
nificantly improve the broiler growth performance. e
authors concluded that the magnitude of improvement in
weight gain also depends on other factors like gut micro-
flora, mucus production or host immune response, which
consume part of the nutrients used for growth. In the
present study, because the herbs did not present anticoc-
cidial activity on all Eimeria species used, the coccidian
infection most probably invalidated the growth promot-
ing effects of the plants. Kim etal. [24] found that garlic
metabolites enhance chickens’ production performances
and reduce the oocyst output in chickens challenged
with E. acervulina, due to a direct cytotoxic effect on the
coccidian sporozoites. is probably also occurred in
our study based on the absence of E. acervulina lesions
in the chickens medicated with the first formula. ere
are also studies that demonstrate the anticoccidial effect
of Origanum vulgare essential oil on E. tenella at a level
of 300mg/kg feed [12]. In the present study the formula
which contained oregano (H2) also had a good effect on
reducing the lesions produced by E. tenella. A herbal
extract that contained Allium sativum, Salvia officinalis,
Echinacea purpurea, ymus vulgaris and Origanum
vulgare improved the performance parameters of broil-
ers and reduced the oocyst output [39]. It seems that in
a mixed coccidian infection, the combination of differ-
ent herbs may represent the solution in controlling the
disease.
Fig. 2 Anticoccidial index in experimental groups of chickens infected with Eimeria spp. (E. acervulina, E. maxima and E. tenella) and treated with
the herbal product in different compositions compared with control (negative, positive and amprolium) groups. a First battery experiment when
chickens were infected with 5 × 103 sporulated oocysts of Eimeria spp. and treated with H1 and H2 formulas, 10 ml/l water. b Second battery
experiment when chickens were infected with 5 × 104 sporulated oocysts of Eimeria spp. and treated with H3 formula, 5 (ITH3-5) and 10 ml/l water
(ITH310). The line at 120 represents the cut-off value for anticoccidian activity and values under the line means a lack of anticoccidian activity
Table 2 Concentration of the tested compounds in the H3
formula
Compound Concentration
(µg/ml) Amount (µg) in
1 l of water (H3a) Amount (µg)
in 1 l of water
(H3b)
Apigenin 1.04 10.4 5.2
Kaempferol 0.23 2.3 1.1
Quercetin 0.29 2.9 1.4
Luteolin 4.93 49.3 24.6
Quercitrin 0.62 6.2 3.1
Chlorogenic acid 75.87 758.7 379.3
Caffeic acid 15.62 156.2 78.1
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Pop et al. Parasites Vectors (2019) 12:343
As shown in the present study, the multi-herb prod-
uct H3 highly reduced the coccidian multiplication rate
and reduced the severity of intestinal lesions. e prod-
uct had a lower anticoccidial effect on E. acervulina, but
a good effect on E. tenella (markedly reduced OPG out-
put and medium reduced lesion score). e synergistic
effects of the combined herbs enhanced the anticoccidial
activity of broiler chickens medicated with H3. Some of
the herbs from the mixed extract already have proven
anticoccidial activity such as Allium sativum pow-
der, supplemented in broiler feed at 0.1% [13] and y-
mus serpyllum in ducks at a concentration of 2,500mg/
kg [40]. ese two herbs were included in the 1st tested
herbal formula. Other herbs used in H3 composition like
Urtica dioica, Rosmarinus officinalis, Tanacetum vulgare,
Coriandrum sativum or Glycyrrhiza glabra are known to
have immunomodulatory effects [33, 41–44]. Moreover,
many of these plants contain flavonoids, tannins or sapo-
nins that act as antioxidants which reduce the oxidative
stress caused by reactive oxygen species encountered also
in coccidiosis [27]. e antioxidant capacity of a herbal
product is directly linked with its anticoccidial effect
[45]. As shown in LC-MS/MS, the H3 formula is a rich
source of polyphenols. e chlorogenic acid was in high
concentration, followed by the caffeic acid and the lute-
olin. Chlorogenic acid was found to have antibacterial
and antibiofilm properties against nosocomial pathogen
strains [46]. Furthermore, chlorogenic acid and caffeic
acid are powerful antioxidants [47], which neutralize the
reactive oxygen species that are produced during Eimeria
infection, as stated above. e antioxidants can alleviate
the damage to the intestinal tissue during parasite inva-
sion by reducing the cytotoxic effects caused by the reac-
tive oxygen species [48], and thus can explain the lower
lesion score observed in the chickens treated with H3
formula.
Conclusions
H3 formula was effective in controlling experimental
coccidiosis in chickens and can be used successfully as
a natural anticoccidial. Field trials are, however, recom-
mended in order to validate the data obtained in experi-
mental studies.
Additional files
Additional file 1: Figure S1. Total ion chromatogram of the H3 formula.
Additional file 2: Figure S2. Extracted ion chromatogram of the H3
formula.
Abbreviations
H: herbal formula; H1: first herbal formula; H2: second herbal formula; H3: third
herbal formula; BE: battery experiment; BE1: first battery experiment; BE2:
second battery experiment; UU1: uninfected untreated control in BE1; UU2:
uninfected untreated control in BE2; IU1: infected untreated control in BE1;
IU2: infected untreated control in BE2; ITA1: infected treated with amprolium
in BE1; ITA2: infected treated with amprolium in BE2; ITH1: infected treated
with H1 10 ml/l water in BE1; ITH2: infected treated with H2 10 ml/l water in
BE1; ITH3-5: infected treated with H3 5 ml/l water in BE2; ITH3-10: infected
treated with H3 10 ml/l water in BE2; PCR: polymerase chain reaction; LC-MS/
MS: liquid chromatography-tandem mass spectrometry; SRM: multiple reac-
tions monitoring mode; OPG: oocysts shed per gram of feces; LS: lesion score;
MR: mortality rate; WG: weight gain; FCR: feed conversion ratio; ACI: anticoc-
cidial index; p.i.: post-infection; %S: percentage survival; %RGW : percentage of
relative weight gain; LI: the lesion index; OI: oocyst index.
Acknowledgements
We would like to especially acknowledge the Center for Advanced Medical
and Pharmaceutical Research (University of Medicine, Pharmacy, Sciences
and Technology of Tîrgu Mureş) and its staff for performing phytochemical
analysis.
Authors’ contributions
LMP wrote the manuscript. EV wrote the manuscript (LC-MS) and prepared
the samples for LC-MS. MC, MN, VM and MOD performed the study. LF
performed MS measurment. IF performed statistical analyses and results
calculation. MDC performed HPLC analysis. MF designed the experimental
protocol. AG designed the experimental protocol and the manuscript, statisti-
cal analysis and reviewed the manuscript. All authors read and approved the
final manuscript.
Funding
This work was supported by the University of Agricultural Sciences and Veteri-
nary Medicine Cluj-Napoca (grant number 6142/10.04.2017), the University of
Medicine, Pharmacy Sciences and Technology of Târgu Mureș (grant number
17972/07.12.2016) and SC Promedivet SRL.
Also, this project was funded by the Ministry of Research and Innova-
tion of Romania, Projects for Financing the Excellence in CDI, Contract no.
37PFE/06.11.2018.
Availability of data and materials
The datasets used and/or analysed during the present study are available from
the corresponding author upon reasonable request.
Ethics approval and consent to participate
The experimental protocol was approved by the Animal Ethics Committee of
the University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca
(approval number 30314/2018).
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Author details
1 Department of Parasitology and Parasitic Diseases, Faculty of Veterinary
Medicine, University of Agricultural Sciences and Veterinary Medicine
Cluj-Napoca, Calea Mănăştur 3–5, 400372 Cluj-Napoca, Romania. 2 Depart-
ment of Pharmacognosy and Phytotherapy, Faculty of Pharmacy, University
of Medicine, Pharmacy, Sciences and Technology of Târgu Mureș, 38 Gheorghe
Marinescu, 540139 Târgu Mureș, Romania. 3 Center for Advanced Medical
and Pharmaceutical Research, Laboratory of Chromatography LC/MS, Faculty
of Pharmacy, University of Medicine, Pharmacy, Sciences and Technology
of Târgu Mureș, 38 Gheorghe Marinescu, 540139 Târgu Mureș, Romania.
4 Department of Toxicology and Biopharmacy, Faculty of Pharmacy, University
of Medicine, Pharmacy, Sciences and Technology of Târgu Mureș, 38 Gheorghe
Marinescu, 540139 Târgu Mureș, Romania.
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 8 of 9
Pop et al. Parasites Vectors (2019) 12:343
Received: 12 February 2019 Accepted: 3 July 2019
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