Preventive Veterinary Medicine 176 (2020) 104914

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Preventive Veterinary Medicine

journal homepage: www.elsevier.com/locate/prevetmed

Thymol efficacy against coccidiosis in pigeon (Columba livia domestica) T

a a, b c
Waleed M. Arafa , Shawky M. Abolhadid *, Abeer Moawad , Asmaa S. Abdelaty ,
Usama K. Moawadd, Khaled A.M. Shokiere, Olfat Shehataf, Sahar M. Gadelhaqg
a
Department of Parasitology, Faculty of Veterinary Medicine, Beni-Suef University, Beni-Suef 62511, Egypt
b
Department of Pharmacognosy, Faculty of Pharmacy, Beni-Suef University, Beni-Suef 62511, Egypt
c
Animal Health Institute, Minia Province Lab., Egypt
d
Department of Histology and Cytology, Faculty of Veterinary Medicine, Beni-Suef University, Beni-Suef 62511, Egypt
e
Animal Health Research Institute, Beni-Suef Provincial Lab., Egypt
f
Department of Clinical Pathology, Faculty of Veterinary Medicine, Beni-Suef University, Beni-Suef 62511, Egypt
g
Department of Parasitology, Faculty of Veterinary Medicine, Minia University, El-Minia, Egypt

A R T I C LE I N FO A B S T R A C T

Keywords: Investigation of thymol efficacy to control pigeon coccidiosis was performed using in-vitro and in-vivo studies.
Pigeons The in-vitro experiment was conducted by treatment of unsporulated oocysts of Eimeria species of pigeon by five
Eimeria concentrations (0.625–10%) from either thymol, eucalyptus essential oil or amprolium anticoccidial drug and
Thymol incubation for 72 h. The in-vitro study revealed that thymol concentrations ≥1.25 % caused significant defor-
Biochemical parameters
mity on sporulated and unsporulated oocysts compared to the other two products. Eucalyptus oil was active at
Antioxidant activity
both 5 and 10 % concentrations on unsporulated oocysts but showed non-significant changes on sporulated ones
at all tested concentration. Meanwhile, in-vivo testing of thymol was conducted using 45 squabs which were
equally divided into three groups; untreated uninfected (UU) negative control, untreated infected (UI) positive
control and thymol treated (TT). TT group received 40 mg/kg BWt thymol in feed for 15 days. At day five post
thymol supplementation, the UI and TT groups were orally infected by 25 × 103sporulated oocysts of pigeon
Eimeria labbeana. The in-vivo study showed that thymol minimized the adverse effect of Eimeria infection in
pigeon as observed by less severity of clinical signs, low oocysts count and improvement of body weight when
compared with untreated infected birds. In addition, the biochemical parameters including liver and kidney
functions tests proved thymol safety in pigeon. Moreover, thymol showed excellent antioxidant activity that was
estimated by significantly lower value of malondialdehyde in TT than UI groups. The histopathological findings
of TT group showed intact intestinal villi with mild sloughed epithelium, degenerated coccidian developmental
stages and massive infiltrations of mononuclear cells in lamina propria. In conclusion, thymol can be safely used
to control pigeon coccidiosis as a natural effective compound.

1. Introduction 2015). It affects the intestinal tissue resulting into greenish watery
diarrhea with mucus, dehydration, with other signs such as rough
The domestic pigeon (Columba livia domestica) is distributed feathers and anorexia loss of body weight and sometimes mortality
worldwide. It is usually reared for meat production, gaming, showy along (McDougald, 2003; Latif et al., 2016; Mohammed et al., 2017;
purposes, hobby and recently as laboratory animals (Radfar et al., 2011; Dong et al., 2018). Pigeon coccidiosis is usually sub-clinical; however,
Sood et al., 2017). Coccidiosis in pigeon is caused by an apicomplexan outbreaks of coccidiosis may occur causing mortality among nestlings
protozoan parasite of the genus Eimeria. It is considered an important and young birds (Ali et al., 2015). There are four species of Eimeria in
parasitic disease of pigeons causing high economic losses in the pigeon pigeons: E. columbae, E. columbarum, E. labbeana and E. labbeana-like
industry (Balicka-Ramisz and Pilarczyk, 2014; Mohammed et al., 2017) (Yang et al., 2016). They are characterized by varying degrees of
due to mortality rate and high cost of treatment. It may be seen in virulence.
young squabs aged from 4 weeks to 4 months especially when they are The use of anticoccidial drugs is the most effective control method
reared intensively with poor hygienic measures, while older ones serve but has side effects like; drug residues in meat products, drug resistance
as carrier and remain apparently healthy (Soulsby, 1982; Ali et al., and cost (Chapman, 1997; McEvoy, 2002). Therefore, it is urgent to find

⁎
Corresponding author.
E-mail addresses: drshawky2001@yahoo.com, shawky.abohadid@vet.bsu.edu.eg (S.M. Abolhadid).

https://doi.org/10.1016/j.prevetmed.2020.104914
Received 9 September 2019; Received in revised form 23 January 2020; Accepted 4 February 2020
0167-5877/ © 2020 Elsevier B.V. All rights reserved.
W.M. Arafa, et al. Preventive Veterinary Medicine 176 (2020) 104914

out new natural and safe alternatives (Quiroz-Castaneda and Dantan- powder similarly treated as thymol crystals. Ten percent DMSO was
Gonzalez, 2015)). Essential oils have showed oocysticidal activity used in the negative control wells. The plates were incubated at 28 °C
(Remmal et al., 2011) and specifically thymol (5 mg/mL) caused de- for three days and examined under light microscope to check the
struction of chicken Eimeria oocysts in-vitro (Remmal et al., 2013). So, sporulation percentage by counting the sporulated and unsporulated
the present study is designed to test the potential use of thymol in the oocysts using McMaster technique, and checking the morphological
control of coccidiosis in pigeon through in-vitro and in-vivo experiments. alterations, deformities and destruction percentage in each treatment.
Thymol is a monoterpene phenol abundantly found in certain plants All treatments were carried out in triplicates.
such as Thymus vulgaris (Amiri, 2012). Thymol possesses antibacterial,
anti-microbial (Karpanen et al., 2008) anti-inflammatory (Aeschbach 2.4. In-vivo experiment
et al., 1994), acaricidal (Araujo et al., 2015), antifungal (Mahmoud,
1994), and antioxidant activities (Yanishlieva et al., 1999). Thymol has Forty-five squabs of 30-day-old were purchased from local poultry
no adverse effects on animals and humans according to Environmental shop. The squabs were reared on metal cages and daily examined by
Protection Agency and categorized as ‘Generally Recognized As Safe’ in flotation technique to ensure absence of coccidian oocysts. The birds
United states (NagoorMeeran et al., 2017). It has an oocysticidal ac- were allocated equally in three groups, in separate cages. The groups
tivity against chicken Eimeria(Remmal et al., 2013), therefore, in this were thymol treated (TT), untreated infected (UI, positive control) and
study, we aimed to test efficacy of thymol against Eimeria species of uninfected untreated (UU, negative control) groups receiving feed
pigeon. without treatment. Then each group was subdivided into three re-
plicates; five in each. All birds were fed on artificial feed pellet free
2. Materials and methods from anticoccidial drugs. Birds were provided with feed and water ad
libitum on a 12-12 day/night cycle. TT group was administered the
The experiment was conducted under the ethical standards of calculated dosage 40 mg/kg of body weight/day for 15 days in feed.
Faculty of Veterinary Medicine, Beni-Suef University, Egypt and ap- The dosage was solubilized in absolute ethyl alcohol then dispersed on
proved by Committee of Institutional Animal Care and Use at Beni-Suef the feed and left to dry then introduced to the birds (Robledo et al.,
University (BSUV- 55/2019). 2005). At day five post thymol supplementation, the TT and UI groups
were orally infected by 25 × 103 sporulated oocysts of pigeon E. lab-
2.1. Products beana. All groups were followed up for two weeks post infection (PI).
Blood and serum samples were collected from the different groups.
Purified thymol was purchased from Sigma (Sigma-Aldrich, Intestinal tract was separated, preserved in formalin 10 % for histo-
Germany). Pure eucalyptus oil (99 %) was purchased from local pathological sections.
Company (Diamond for High Purity Chemicals, Cairo, Egypt). A com-
mercial anticoccidial drug Amprolium® 20 % (Uccma, Egypt Masters 2.5. Evaluation of thymol efficacy in control of pigeon coccidiosis
Co. for veterinary products) was used.
2.5.1. Clinical signs of coccidiosis and Parasitological findings
2.2. Source of Eimeria species oocysts The signs of coccidiosis infection were recorded in all groups. From
day-four PI, all groups were subjected to daily fecal examination.
Eimeria species were collected from naturally-infected pigeons by Oocysts count per gram was conducted for each group (Lillehoj and
using saturated salt flotation method then sporulated by incubation in Ruff, 1987)
2.5 % potassium dichromate solution in Petri dishes for 72 h at 25–28
°C and 80 % RH (Gadelhaq et al., 2015). Based on the morphological 2.5.2. Body performance
features as descripted by Yang et al. (2016) the collected oocysts were The birds' body weights were measured at the day of infection and
mainly E. labbeana (75–80 %). Continuous source of oocysts was day 8 PI. The body weight gain was calculated (Ma et al., 2011).
maintained through propagation of the obtained sporulated oocyts in
five squabs (30 day-old). The squabs were experimentally infected or- 2.5.3. Determination of biochemical parameters
ally by 25 × 103 purified sporulated oocysts of Eimeria labbeana. The The serum alanine amino-transferase (ALT) and aspartate amino-
fecal samples were collected from day 6 to day 9 post infection (PI) then transferase (AST) activities were estimated according to Reitman and
the pigeons were sacrificed, and the intestinal contents were collected Frankel (1957). Kinetic determination of creatinine and uric acid was
to be used as a source of oocysts in the experimental design. The col- done according to Houot (1985) and Fossati et al. (1980), respectively.
lected oocysts were divided into two groups; the first group was kept as Levels of total protein and albumin were measured according to Peters
unsporulated oocysts at 4 °C and the second group was subjected to (1968) and Dumas and Biggs (1972), respectively. Serum sodium and
sporulation as previously mentioned. At the end of the incubation, the potassium were estimated according to Trinder (1951) and Sunderman
sporualted oocysts were washed twice with phosphate buffered saline, and Sunderman (1958), respectively.
counted by MC Master Technique, stored at 4 °C and kept as continuous
source of pigeon Eimeria species oocysts to be used for in-vitro and in- 2.5.4. Determination of oxidant/antioxidant parameters
vivo studies. Superoxide dismutase (SOD) and Glutathione (GSH) were estimated
in serum according to Misra and Fridovich (1972) and Beutler et al.
2.3. In-vitro effect of thymol, eucalyptus oil and amprolium on unsporulated (1963), respectively. Malondialdehyde (MDA) was determined ac-
E. labbeana oocysts cording to Placer et al. (1966).

Thymol crystals (120 mg) were dissolved in 600 μL DMSO (10 %). 2.5.5. Histopathological examination
Then, 200 μL were transferred in to the first three consecutive wells of Parts of duodenum, ilium and jejunum and large intestine were
the 96-well plates. Double-fold serial dilutions with saline were carried collected from sacrificed pigeons from different groups at day-8 PI. The
out till 0.625 mg/100 μL in the 6th well. About 1 × 104 unsporulated obtained specimens were fixed in 10 % formalin solution and Bouin’s
Eimeria spp. oocysts in 2.5 % potassium dichromate sol. were added to fluid. The fixed samples were washed with tap water to remove fecal
each well to obtain a final concentration ranging from 10.0- 0.625 %. In material then subjected to histopathological processing; dehydration in
the same manner, 120 μL eucalyptus oil was prepared and diluted up to ascending concentrations of ethyl alcohol, clearance in xylene then
600 μL with 10 %DMSO. For positive control, 120 mg of amprolium embedded in paraplast solution. Then tissues were cut into sections of

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W.M. Arafa, et al. Preventive Veterinary Medicine 176 (2020) 104914

4–5 μ thickness and stained with routine Haematoxylin and Eosin Table 2
method then examined by light microscopy (Bancroft and Gamble, Oocysts per gram (OPG) count in feces of thymol treated pigeon compared to
2008). control groups.
OPG count Group
2.6. Statistical analysis
UU UI TT

Data were analyzed using ANOVA and a Tukey multiple range test Day 5 PI 0.00 ± 0.0a 750.0 ± 28.8 b 103.3 ± 8.8 c
to determine differences between groups. Results are expressed as Day 6 PI 0.00 ± .00 a 2541.6 ± 130.9 b 675.00 ± 43.30 c
means SD. Probability values of less than 0.05 were considered sig- Day 7 PI 0.00 ± .00 a 200458.3 ± 1411.1 b 23475.0 ± 158.7 c
Day 8 PI 0.00 ± .00 a 405583.3 ± 551.1 b 30491.6 ± 108.3 c
nificant.
Day 9 PI 0.00 ± .00 a 128250.0 ± 1419.7 b 10558.3 ± 154.3 c
Day 10 PI 0.00 ± .00 a 34908.3 ± 122.7 b 7825.0 ± 80.3 c
3. Results
Means with different superscripts (a,b,c) within a row are significantly different
at P < 0.05. PI; Post Infection, UU; Uninfected Untreated, UI; Untreated
3.1. In vitro study
Infected, TT; Thymol Treated.

Microscopic examination and counting of the oocysts treated by 5 %

3.2.3. Body performance
and 10 % of thymol, eucalyptus and amprolium caused deformed oo-
The body weight (BW) showed no significant difference at the day of
cysts with cracked wall and lysis (Supplementary Fig. 1). The destruc-
infection in all groups. However, at day 8 PI, a significant difference
tion percentage was 96.00, 39.33, and 97.33 %, respectively in 10 %
(P < 0.05) was recorded between TT group and UI one (321.67 ± 3.52
concentration of thymol, eucalyptus and amprolium respectively. In
g and 260.00 ± 5.00 g, respectively). In addition, BW of UU group was
case of thymol, the destruction of oocysts decreased in the lower con-
significantly higher than other groups. Moreover, the body weight gain
centrations to reach its minimum effect at 0.625 % but still significant
(BWG) was higher in treated group than untreated one (Table 3).
(Table 1). On the other hand, eucalyptus oil and amprolium had no
effect on sporulation process or destruction of oocysts at concentrations
≤2.5 %. 3.2.4. Biochemical analysis
Regarding liver functions, TT birds showed significant decrease in
ALT and AST compared to UI and UU pigeons, (Table 4). Total protein,
3.2. In vivo study globulin, uric acid values in serum concentration in infected pigeons
(UI and TT) were found to decrease significantly compared to negative
3.2.1. Clinical signs of coccidiosis control group. Concerning albumin and creatinine; there were no sig-
The UI birds showed in-appetence, dullness, rough feather, greenish nificant changes among the three groups. Results of electrolytes showed
to blackish diarrhea, and weight loss. However, thymol treated squabs no significant changes in potassium while sodium results revealed sig-
revealed less severity in signs. Negative control group showed none of nificant increase in UI group only.
these signs.
3.2.5. Serum oxidant/antioxidant parameters
3.2.2. Oocysts count (oocysts per gram, OPG) TT birds had significantly lower MDA level and significantly higher
The oocysts shedding appeared at day 5 PI in all groups. From day 6 SOD and GSH levels compared to control groups. In the current study,
PI, it was clear that TT group showed significantly (P < 0.05) lower dietary supplementation of thymol ameliorated the antioxidants (SOD
oocysts count than UI group (Table 2). This significant reduction of and GSH) levels and decreased the oxidant (MDA) level (Table5).
oocysts count was regarded along the course of the experiment till day
10 PI. Additionally, UU group showed no oocysts in feces. 3.2.6. Histopathological findings
The histopathological observations of intestines in UI group reveled
Table 1
sloughed absorptive epithelium in the intestinal lumen leaving the la-
In-vitro effect of thymol, eucalyptus oil and amprolium on Eimeria species oo-
mina propria with congested blood vessels exposed (Fig. 1A). The in-
cysts of pigeon.
testinal mucosa also showed diffuse areas of small hemorrhages. Mature
Concentration/ % Sporulated oocysts % Unsporulated % Destructed oocyst schizonts containing numerous merozoites (Fig. 1B) and different stages
Group Oocyst
of coccidian parasites including microgametocytes, macrogametocytes
Control 80.67 ± 1.45 a
17.67 ± 1.76 a
1.67 ± 0.33 a and oocysts (Fig. 2C) were also detected within the villous epithelium.
Thymol In UU group, the intestine showed normal architecture of the tunica
10 % 4.00 ± 1.53 b 0.00 ± 00.00 b
96.00 ± 1.52 b
mucosa (intestinal villi and underlying lamina propria), submucosa,
5% 12.67 ± 1.45 c 0.00 ± 00.00 b
86.67 ± 0.88 c
musclosa and serosa (Fig. 2A). The intestinal villi showed normal ar-
2.5 47.67 ± 0.66 d 14.67 ± 1.45 ac
37.67 ± 0.88 d

1.25 68.00 ± 1.15 e 15.33 ± 0.88 ac

16.67 ± 0.88 e rangement and appeared lined with ordinary simple columnar
0.63 77.67 ± 1.45 a 11.33 ± 0.88 c
11.00 ± 1.52 f

Eucalyptus Table 3
b a
10 % 43.00 ± 1.52 17.67 ± 1.45 39.33 ± 2.84 b Body weight and body weight gain of thymol treated pigeon compared to
c a
5% 62.67 ± 1.45 16.33 ± 0.88 21.00 ± 0.58 c control groups.
a a
2.5 85.00 ± 0.57 12.00 ± 0.58 3.00 ± 0.58 a
a a
1.25 81.33 ± 1.76 13.00 ± 1.73 2.00 ± 0.58 a BW at Group
a a
0.63 80.00 ± 1.73 17.00 ± 1.73 3.00 ± 0.58 a
Amprolium UU UI TT
b b
10 % 0.00 ± 00.00 0.00 ± 00.00 97.33 ± 1.20 b
5% 52.33 ± 1.45 c
16.33 ± 1.86 a
31.33 ± 3.17 c day zero of infection 296.00 ± 2.31a 291.33 ± 2.40 a 296.33 ± 3.22a
2.5 83.00 ± 1.73 a
16.00 ± 1.73 a
1.00 ± 0.58 a day 8 PI 332.00 ± 2.08 a 260.00 ± 2.89 b 321.67 ± 2.03 c
1.25 80.00 ± 1.73 a
16.00 ± 1.15 a
2.00 ± 0.58 a BWG 36.00 ± 3.51 a −34.00 ± 1.15b 17.33 ± 1.45 c
a a
0.63 82.00 ± 1.15 15.00 ± 1.73 3.00 ± 0.58 a
Means with different superscripts (a, b, c) within a row are significantly dif-
Means with different superscripts (a–f) within a column are significantly dif- ferent at P < 0.05. PI; Post Infection, UU; Uninfected Untreated, UI; Untreated
ferent at P < 0.05. Infected, TT; Thymol Treated.

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W.M. Arafa, et al. Preventive Veterinary Medicine 176 (2020) 104914

Table 4 studied extensively due to the complexity in their composition. Each

Biochemical parameters in serum of thymol treated pigeon compared to control component of the EO imparts an individual effect contributing to a
groups. collective effect depending upon the nature and concentration of each
Group Group component in the specific oil. Moreover, the antiparasitic effect of EOs
Parameter can further be explained as direct and indirect antiparasitic actions,
UU UI TT especially for the control of coccidiosis (Ueda-Nakamura et al., 2006;
Machado et al., 2010).
ALT (U/L) 6.60 ± 0.67 a 6.03 ± 0.35 a 3.09 ± 0.21 b
AST (U/L) 26.11 ± 2.08 a 27.68 ± 2.42 a 18.13 ± 1.90 b Based on the promising findings of thymol of the in-vitro trial, we
T.P (g/dL) 3.50 ± 0.23 a 2.80 ± 0.11 b 2.71 ± 0.93 b designed an experiment to study its efficacy to control of E. labbeana in
Albumin (g/dL) 1.70 ± 0.12 a 1.69 ± 0.17 a 1.66 ± 0.04 a pigeons. The results showed that thymol supplement in pigeon feed
Globulins (g/dL) 1.78 ± 0.46 a 1.15 ± 0.29 b 1.08 ± 0.11 b
showed significant decrease in the adverse effects and clinical signs,
Uric acid (mg/dL) 0.54 ± 0.02 a 0.15 ± 0.01 b 0.16 ± 0.02 b
Creatinine (mg/dL) 4.00 ± 0.11 a 4.23 ± 0.14 a 4.47 ± 0.26 a
lowering oocyst shedding of E. labbeana and maintained tissue integrity
K+ (mEq/L) 4.86 ± 0.15 a 4.59 ± 0.18 a 4.66 ± 0.25 a of treated birds. Thymol possesses great antibacterial and oocysticidal
Na+ (mEq/L) 144.55 ± 0.86 a 161.81 ± 1.67 b 148.45 ± 0.96 a activity against coccidian parasites (Bassolé and Juliani, 2012; Remmal
et al., 2013). In rodents, thymol has been reported to inhibit pro-in-
Means ( ± SE) with different superscripts (a,b,c) within a row are significantly flammatory cytokines, decrease inflammatory cell recruitment and al-
different at P < 0.05.
leviate oxidative damage (Riella et al., 2012).
UU; Uninfected Untreated, UI; Untreated Infected, TT; Thymol Treated.
The oocysts count revealed significant decrease in TT group com-
pared to UI squabs. This significant reduction in the oocyst count may
Table 5
be due to its phenolic properties and monoterpenoid structure.
Oxidant/antioxidant parameters in serum of thymol treated pigeon comparedto
Terpenes in essential oils have proven beneficial characteristics in al-
control groups.
leviation of the injuries resulting from the pathogenicity of the Eimeria
Group Group species (DeGussem, 2011). Decreased fecal oocyst shedding may be due
Parameters
to significant production of interleukins, which increased host protec-
UU UI TT
tion against coccidiosis and reduced intracellular development of Ei-
a b
SOD (U/ml) 12.13 ± 0.41 4.37 ± 0.19 9.07 ± 0.51c meria spp. in chicken (Lillehoj et al., 2004; Zhang et al., 2012). Thymol
a
GSH (nmol/ml) 75.26 ± 1.15 23.18 ± 0.91 b 47.75 ± 1.37 c
anti-inflammatory activity was previously reported. The mechanism
a
MDA (nmol/ml) 28.28 ± 1.06 115.79 ± 2.84 b 53.65 ± 3.49 c
was proposed through inhibiting the T-cell immune response and im-
Means ( ± SE) with different superscripts (a,b,c) within a row are significantly proving T-helper cells-1 (Th1) (interleukin-2 (IL-2) and IFN-γ/T-helper
different at P < 0.05. UU; Uninfected Untreated, UI; Untreated Infected, TT; cells-2 (Th2) (interleukin-4 (IL-4), interleukin-5 (IL-5) and interleukin-
Thymol Treated, SOD; superoxide dismutase, GSH; Glutathione, MDA; 10 (IL-10) ratio(Ku and Lin, 2013).
Malondialdehyde. Infected birds must divert energy from growth to fight the infection,
thus disrupting weight gain (Lin et al., 2015). Regarding body weight,
absorptive epithelium devoid of any parasitic stages (Fig. 2B). In TT TT squabs showed better BWG than UI birds which may be a result of
group, the intestinal villi appeared lined with intact epithelial layer minimal intestinal damage caused by the anti-inflammatory and anti-
except in few areas which showed sloughed epithelium and the intact oxidant properties of thymol (Arab et al., 2015) leading to increased
villous epithelium showed degenerated Eimerian developmental stages absorption of food. Finally, the stimulation of digestive secretions (e.g.,
(Fig. 3A). Massive infiltrations of mononuclear cells in lamina propria saliva and bile) and enhanced enzyme activity were proposed to be a
results in thickened mucosal layer were noticed (Fig. 3B). The en- core mode of EOs to improve the feed conversion efficiency (Jang et al.,
terocytes showed few numbers of developing Eimerian gametocytes 2007; Zeng et al., 2015).
(Fig. 3C) and degenerated developmental coccidian stages (Fig. 3D). Results of liver function test (ALT and AST) revealed no significant
changes in coccidian infected group in comparison to UU group. This
was in accordance with Abdel-Ghaffar et al. (1990). The significant
4. Discussion decrease in ALT and AST enzymes in thymol treated group give in-
dication that thymol is safe on the liver which is supported by findings
The in-vitro study proved oocysticidal activity of thymol which was showed that thymol has a protective effect on liver (Kim et al., 2014;
concentration-dependent. During the microscopic counting of the oo- Palabiyik et al., 2016) due to its anti-inflammatory and anti-oxidant
cysts treated by thymol, we have noted the presence of damaged and properties which have been documented in many studies.
deformed oocysts with cracked wall. Notably, counting of treated oo- The hypo-proteinemia in coccidial infections may occur due to acute
cysts decreased significantly than control group especially at10 % stress which leads to cortisol secretion and protein catabolism (Mondal
thymol concentration. The latter may be due to the lysis of treated et al., 2011). Marked reduction in total serum protein might be due to
oocysts by highest concentrations of thymol. This lysis effect was ob- nutrient malabsorption, marked haemorrhagic enteritis, and in-appe-
served also by Remmal et al. (2011 and 2013). Similarly, amprolium at tence (Dar et al., 2014). TT pigeons showed significant increase in total
its highest concentration (10 %) led to complete destruction of oocysts. protein compared to UI group.
This supports its effectiveness in chickens infected with E. acervulina, E. It is known that uric acid is the major nitrogenous waste product of
maxima and E. tenella (Ruff et al., 1993). birds. Data showed significant decrease in uric acid in UI and TT
The eucalyptus concentrations; 0.625, 1.25 and 2.50 % had no ef- groups. This might be attributed to coccidiosis which caused anorexia
fect on the oocysts. Meanwhile, 5 % and 10 % concentrations had and starvation. Starvation decreases uric acid levels in plasma
significant effect. This effect supported by published data of Barbour (Quintavalla et al., 2001). Also, Creatinine derived from the breakdown
et al. (2014) who found out that using the essential oils (EOs) of eu- of muscle phosphocreatinine, it is an important index of protein me-
calyptus and peppermint controls Eimeria spp. of broiler chicken, helps tabolism and renal integrity. The significant decrease in serum uric acid
in improvement of their production, alleviation of lesions and reduction and unchanged creatinine levels across the dose level of thymol when
in intestinal oocyst counts. Previous works documented the inhibitory compared to the control shows that thymol was not nephrotoxic. In
effects of terpenes present in EOs of eucalyptus and peppermint against addition, the increase in sodium levels giving further credence to the
avian bacteria (Timbermont et al., 2010) and avian viruses (Barbour safety of thymol on the kidney. Many studies have showed the renal
et al., 2010). Moreover, the mechanism of action of EOs has not been protective effect of thymol (Chang et al., 2014; Hosseinimehr et al.,

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W.M. Arafa, et al. Preventive Veterinary Medicine 176 (2020) 104914

Fig. 1. Eimeria spp. in experimentally infected pigeons: Duodenum of control positive group (UI) showed; A). sloughed villous epithelium leaving the glands (G)
exposed with congestion of blood vessels (arrow). B) Mature schizonts (arrows) containing numerous merozoites inside parasitophorous vacuoles. C) Different
developmental stages of coccidian parasites; microgametocytes (Mi), macrogametocytes (Ma) and oocyst (O) inside the intestinal epithelium. H&E stain; X200,
X1000 and X400, respectively.

2015). resulted from diarrhea with hyperosmolarity of which the hyperna-

Regarding the electrolytes, there was hypernatremia which may tremia is a part (Latimer, 2011).
refer profound water depletion due to hypertonic dehydration which Results of the current study revealed significant decrease of

Fig. 2. Eimeria spp. in experimentally infected

pigeon: Duodenum in control negative group
(UU) showed; A) Normal architecture of in-
testinal layers; mucosa with normal intestinal
villi (V), submucosa (S), musclosa (M) and
serosa (SE). B) Intestinal villi (V) lined with
intact epithelial layer of ordinary simple co-
lumnar absorptive epithelium and underlying
lamina propria contained intestinal glands (G).
H&E stain; X100 and X200, respectively.

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W.M. Arafa, et al. Preventive Veterinary Medicine 176 (2020) 104914

Fig. 3. Eimeria spp. in experimentally infected pigeon: Duodenum in thymol treated group (TT) showed; A) intestinal villi lined by more or less intact epithelial layer
contained degenerated parasitic stages (arrows). B) High mononuclear cells infiltration (M) into the tunica mucosa. C) Few numbers of developing gametocytes
(arrow heads) and degenerated coccidian stage (arrow). D) Different degenerated parasitic stages within the intestinal epithelium. H&E stain; X400, X200, X400 and
X400, respectively.

antioxidant enzyme values (GSH and SOD) and increase in MDA value thymol (7.5 mg/kg) inhibited lipid peroxidation, glycation, dyslipi-
in serum of UI birds with values higher than thymol treated and ne- demia, inflammation, ionic homeostasis malfunction and apoptosis by
gative control. This could be caused either by the effect of Eimeria or by virtue of its potent antioxidant property (NagoorMeeran et al., 2014,
the host defense to infection (Min et al., 2004). Botanical products ei- 2015, 2016).
ther interfere directly with parasitic metabolism or indirectly by en- It was obvious that TT group showed less pathological lesion and
hancing the host immune response and antioxidant defense systems for lower number of Eimeria stages in the intestinal tissue. In addition, TT
the effective control and eradication of parasitic invasion (Idris et al., birds showed degenerated parasitic stages in the intestinal tissue. This
2017). The higher MDA levels in serum of infected birds indicates protective effect of thymol on the tissue may be referred to its anti-
oxidative damage generated throughout the immune response against inflammatory (Ku and Lin, 2013) and its lysis effect on parasitic stages
Eimeria infection (Sepp et al., 2012). Coccidian infection in chickens (Remmal et al., 2013).
elicited the production of reactive oxygen species as superoxide O2 − in In conclusion, in vitro, thymol showed excellent oocysticidal activ-
stimulated macrophages that initiated free radical cascades leading to ities against Eimeria species of pigeons. Moreover, In- vivo, thymol
lipid peroxidation (Allen et al., 1996). Thymol treatment in the present minimized the adverse effect of coccidiosis infection in pigeons.
study minimized the effect of coccidian infection depend on its anti-
oxidants activities. In which, thymol effectively scavenges the hydroxyl
free radicals forming major transient species named phenoxyl radicals. Declaration of Competing Interest
The latter undergoes dehydration. So, it increases the activities of en-
dogenous antioxidants levels together with non-enzymatic antioxidants The authors declare that there is no conflict of interests regarding
(NagoorMeeran and Prince, 2012). These results are in accordance with the publication of this article.
Zidan et al.(2016) who found that thymol supplementation elevated the
antioxidant status and attenuated MDA levels in broilers. Moreover,

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Acknowledgments A., Riaz, U., 2017. The potential of antioxidant rich essential oils against avian
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The authors appreciated help of Prof. Abdelnaser Abdelfattah growth performance, digestive enzyme activity and intestinal microflora population
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org/10.1093/jac/dkn325.
Supplementary material related to this article can be found, in the Kim, Y.S., Hwang, J.W., Kang, S.H., Kim, E.H., Jeon, Y.J., Jeong, J.H., 2014. Thymol from
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