J. World Poult. Res., 10(2): ----, 2020

2020, Scienceline Publication

JWPR

J. World Poult. Res. 10(2S): 151-164, June 14, 2020

Journal of World’^s

Poultry Research

Research Paper, PII: S2322455X2000020-10

License: CC BY 4.0

DOI: https://dx.doi.org/10.36380/jwpr.2020.20

The Effect of Bromhexine and Thyme Oil on Enhancement of the

Efficacy of Tilmicosin against Pasteurellosis in Broiler Chickens

Abeer M Radi¹, Nema S Shaban¹, Fatma I Abo El- Ela¹, Elham Ahmed Mobarez², El-Gendy AAM¹and El-Banna HA^3*

¹Department of Pharmacology, Faculty of Veterinary Medicine, Beni-Suef University, 62511, Egypt.

²Department of Pharmacology, Animal Health Research Institute, Dokki, Giza, Egypt.

³Department of Pharmacology, Faculty of Veterinary Medicine, Cairo University, 12211, Giza, Egypt.

^*Corresponding author’s Email: drelbanna3@yahoo.com; ORCID: 0000-0003-4476-7775

Received: 01 Feb. 2020

Accepted: 09 Mar. 2020

ABSTRACT

Pasteurella multocida is one of the commensal flora of the upper respiratory tract. Under stress conditions, it may be

involved as a secondary agent in various respiratory syndromes and caused high mortality as well as significant

economic losses in chickens. This study evaluated the effect of bromhexine or thyme oil on enhancement of efficacy

of tilmicosin in treatment of avian pasteurellosis. A total of 63 adult chickens were infected by Pasteurella multocida

and classified into seven groups and treated as follow; non-infected non-treated group (control negative), infected

non-treated group (control positive), group infected and treated by tilmicosin alone, group infected and treated by

bromhexine alone, group infected and treated by thyme oil alone, group infected and treated by

tilmicosin+bromhexine, and group infected and treated by tilmicosin+thyme oil. Clinical signs, mortality rate,

bacterial re-isolation, hematobiochemical and histopathological parameters were determined. The results showed a

significant decrease in mortality, bacterial re-isolation as well as clinical signs in combined treated groups compared

to tilmicosin group as well as improvement in hematobiochemical and histopathological parameters of combined

treated groups. Furthermore, the combination of tilmicosin and bromhexine or thyme oil was more potent in the

treatment of pasteurellosis in chickens than each treatment alone. Finally, the clinically observed damage in chickens

infected with P. multocida can be ameliorated by a combination of tilmicosin with bromhexine or thyme oil. This

protective effect could improve the use of antibiotics in poultry farms as well as reduce human exposure to antibiotic

residues and bacterial resistance to antibiotics.

Keywords: Bromhexine, Chickens, Efficacy, Pasteurella Multocida, Thyme oil, Tilmicosin

INTRODUCTION

Bromhexine hydrochloride is a quinazoline alkaloid

obtained from Adhatoda vasica plant which increases the

bronchial secretions and reduces their viscosity. Also, it

elevates the immunoglobulin levels in airway secretions,

thins and loosens mucus to help the treatment of chest

congestion thus it is often added to cough syrups (Gubbi et

al., 2009 ). It is used as a mucolytic expectorant, which

increases the production of serous mucus in the respiratory

tract and makes the phlegm thinner thereby easing cough

(Siddappa and Hanamshetty, 2016 ).

Medicinal herbs play an essential role in the fields of

drug development due to their safety, easy accessibility, as

well as restricted side effects (Pathak and Das, 2013). The

essential oils are volatile complex compounds formed by

aromatic plants as secondary metabolites characterized by

a strong odor (Bakkali et al., 2008). Thymol and carvacrol

were the major derived essential oils of Thymus vulgaris

which have shown antioxidant, antibacterial, antifungal,

Fowl cholera is a contagious bacterial disease caused by

Pasteurella multocida affecting domesticated and wild

birds that occur sporadically or enzootically in most

countries of the world with significant economic losses

due to its high mortality (Glisson et al., 2013).

Tilmicosin is a 16-membered ring macrolide, which

penetrates the microbial cell membrane and suppresses the

synthesis of protein by the 50s ribosomal subunit which

leads to the synthesis of incomplete peptide chains (Seiple

et al., 2016). Tilmicosin is characterized by low plasma

concentrations but high and persistent tissue

concentrations, indicating high efficacy of the drug

(Mestorino and Errecalde, 2004). It is an effective remedy

for a wide range of Gram-positive organisms, some Gram-

negative bacteria, as well as atypical bacteria (El-

Mahmoudy et al., 2018 ).

To cite this paper: Radi AM, Shaban NS, Abo El- Ela FI, Ahmed Mobarez E, El-Gendy AAM and El-Banna HA (2020). The Effect of Bromhexine and Thyme Oil on Enhancement

of the Efficacy of Tilmicosin against Pasteurellosis in Broiler Chickens. J. World Poult. Res., 10 (2S): 151-164. DOI: https://dx.doi.org/10.36380/jwpr.2020.20

151

Radi et al., 2020

anticoccidial properties and positive effect on growth

(BHIA) and incubated at 37°c for 24 hours. After sub-

culturing of bacteria into Brain heart infusion agar at 37°c

for 24 hours, the uniform-sized colonies were selected and

diluted with sterile physiological saline. The viable count

was adjusted by Macfarland tube No. 1 to obtain 3×10⁸

CFU/ml. The infective dose for each chicken was 0.5 ml/

bird intramuscular into the breast muscle.

performance properties in broilers (Aljabeili et al., 2018 ).

It has been shown that Thymus vulgaris essential oil had

the best antibacterial activity (Santurio et al., 2014).

However, little information is available on the

antibacterial activity of Thymus vulgaris essential oil

against P. multocida, and no study has been conducted to

investigate its synergistic effect with tilmicosin against P.

multocida.

Ethical approval

This work was designed to compare the efficacy of

tilmicosin alone and in combination with bromhexine or

thyme oil in the treatment of experimentally infected

broiler chickens with P. multocida.

The experiment was conducted in accordance with

the principles and guidelines of the Institutional Animal

Care and Use Committee (IACUC) of the faculty of

veterinary medicine, Cairo University.

MATERIALS AND METHODS

Experimental design

The efficacy of tilmicosin alone or in combination

with bromhexine hydrochloride or thyme oil against

experimental infection with P. multocida was studied

according to the method described by Amany and Abd-

Alla (1997). The mature broilers (63 birds) were randomly

divided into 7 groups (9 birds of each). The feed and water

were supplied to the birds ad libitum throughout the

experiment. The birds were kept under investigation for

two weeks to ensure the removal of any antibacterial agent

traces before the onset experiment. The group 1 left as

control non-infected non-treated, whereas other

experimental groups were intramuscularly injected into the

breast muscle with 0.5 ml/birds of 18-24 hours broth

culture of P. multocida containing 3x10⁸CFU/ml.

Drugs

A-Tilmicosin phosphate: It was kindly provided by

Pharma- swede® company, Egypt as a white powder (80

%), well soluble in water. Chemical name: 20-Deoxo-20-

(3, 5-dimethyl-1-piperidinyl) desmycosin.

B- Bromhexine hydrochloride: Pharma- swede®;

Egyptian company; kindly provided it in the form of white

powder (98 %), poorly soluble in water but soluble in N-

methyl pyridine/ propylene glycol (NMP/PG) (50%: 50%)

solvent. Chemical name: 2-amino-3, 5- dibromobenzyl

(cyclohexyl) methylamine hydrochloride.

C-Thyme oil: It was kindly provided by Pharma-

swede® company Egypt as oily solution (100 %), poor

soluble in water. Chemical name: 3, 7-dimethylocta-1, 6-

dien-3-ol; 2-(4-methylcyclohex-3-en-1-yl) propan-2-ol; 1-

Treatment regimens

methyl-4-propan-2-ylbenzene;

ylbicyclo [3.1.0] hexan-4-ol; 5-methyl-2-propan-2-

ylphenol.

Chickens: This study was carried out on 63, 35-day-

old broilers chicken (2.5-3 kg) of both sexes. Balanced

ration and water ad-libitum were provided for birds. Birds

were kept under proper hygienic conditions and left

without treatment for 15 days before the onset of the

experiment for acclimatization and ensuring complete

clearance of any drugs.

4-methyl-1-propan-2-

Group (1): non-infected non-medicated (control

negative).

Group (2): infected non-treated (control positive).

Group (3): infected and treated with tilmicosin

phosphate at a dose level of 20 mg/kg (Amer et al., 2009 )

for 12 hours.

Group (4): infected and treated with bromhexine

hydrochloride (1 mg/kg b.wt) for 12 hours.

Group (5): infected and treated with thyme oil 20%

(0.2 ml/l) (Feizi et al., 2013) for12 hours.

Bacterial strain: P. multocida serotype (A₅) was

obtained from the Microbiology Department, Animal

Health Research, Institute, Dokki, Egypt. Preparation of

the virulent strain was performed by intramuscular

inoculation of mature chicken with 0.5 ml of 18 hour broth

culture of P. multocida containing 3x10⁸Colony Forming

Unit (CFU) viable organism (Amany and Abd-Alla, 1997 ).

After the appearance of clinical signs and before death, the

heart was taken, splitted on Brain Heart Infusion Agar

Group (6): infected and treated with tilmicosin

phosphate (20 mg/kgb.wt) in combination with

bromhexine hydrochloride (1 mg/kg b.wt) for12 hours.

Group (7): infected and treated with tilmicosin

phosphate (20 mg/kg.b.wt) in combination with thyme oil

extract 20% (0.2 ml/l) for 12 hours.

All medications were given orally in drinking water

one-hour post-infection and for 3 successive days. The

birds were examined for 14 days post-infection for the

152

J. World Poult. Res., 10(2S): 151-164, 2020

clinical signs, mortality rate, morbidity, and

isolation to 88.8-100 %. The combination of tilmicosin

with bromhexine reduced the incidence of re-isolation to

11.1-33.3% compared to 44.4-55.5% when used tilmicosin

alone as well as the combination of tilmicosin with thyme

oil reduced bacterial re-isolation to 11.1-22.2% compared

to 44.4-55.5% when used tilmicosin solely as observed in

table 2.

histopathological examination. Blood samples were taken

at 3^rdday and 14^thdays post-infection and divided into 2

parts; one part on EDTA for determination of erythrocytes

(RBCs) count (Feldman et al., 2000 ), Hb concentration

(Varley, 1980 ), total and differential leucocytic count

(Jain, 1986) whereas the second part was collected into

plain centrifuge tube for serum separation and

determination of Aspartate Aminotransferase (AST) and

Alanine Aminotransferase (ALT) activities (Reitaman and

Frankel, 1957), Creatinine (Doolan et al., 1962 ) and uric

acid (Kageyama, 1971). The samples were taken from the

post-mortem examined (liver, lungs, and heart) at 3^rdday

of infection, one and 14^thday post-infection were

incubated on nutrient broth at 37°C for 24 hours, then sub-

cultured on nutrient agar and BHIA plates for 24hours at

37°C, suspected colonies were identified and positive or

negative culture results were recorded.

Hematobiochemical parameters

The results of blood picture revealed anemia

indicated by significant reduction in RBCs count and Hb

concentration; also leukocytosis indicated by considerable

increase in neutrophils in non-treated, bromhexine and

thyme oil-treated groups at 3^rdday and 14^thday of

medication and in tilmicosin treated group at 3^rdday of

treatment, compared to control negative group,

tilmicosin/bromhexine combination and tilmicosin/thyme

oil combination at 3^rdday and 14^thof medication. The

effect of treatment with tilmicosin alone or in combination

with either bromhexine or thyme oil on liver and kidney

functions of chickens inoculated with P. multocida on 3^rd

day and 14^thday of the experiment showed significant

elevation in the values of ALT, AST, uric acid and

creatinine in non-treated, bromhexine and thyme oil-

treated groups and tilmicosin treated group at 3^rdday of

the experiment compared to control negative group,

tilmicosin/bromhexine, and tilmicosin/thyme oil-treated

groups at 3^rdday and 14^thof medication as recorded in

tables 3, 4 and 5.

Statistical analysis

The results are presented in the form of mean ±

standard error of the mean (S.E.M.). Statistical

significance was determined by one-way analysis of

variance (ANOVA) according to (Sndecor and Cochran,

1982), followed by Tukey’s posthoc test for multiple

comparisons using SPSS (version 20.0) software (IBM

SPSS Statistic 20.0, Armonk, NY, USA). The P- values

less than 0.05 were considered statistically significant.

RESULTS

Histopathological findings:

Clinical signs and mortality rate

. Macroscopical findings

Inoculation with P. multocida induced severe

symptoms in non-medicated chickens characterized by

congestion of mucous membranes, depression, ruffled

feathers, off food, greenish diarrhea, cough, and nasal

discharge, gasping, swelling of the wattles, lameness,

sinusitis, and ophthalmia or even sudden death. These

signs appeared on the first day after inoculation. Treatment

with tilmicosin in combination with bromhexine or thyme

oil significantly reduced the prevalence and severity of

clinical signs than each drug alone. A mortality rate was

recorded during the experiment in each group and

calculated as a percent (%) as showed in table 1.

Bacterial re-isolation: Pasteurella multocida was re-

isolated from all organs of infected non-medicated birds

(100%), whereas non-infected non-medicated chickens

showed no bacterial re-isolation. Similarly, medication

with bromhexine didn’t reduce bacterial re-isolation

100%. Drug with thyme oil 20% reduced bacterial re-

Macroscopically, the liver characterized by

enlargement, severe congestion with friable texture and

pointed edges, pinpoint necrotic foci, fibrinous

perihepatitis. These pathological lesions were markedly

decreased in tilmicosin treated groups at 14^thof the

experiment, whereas they significantly decreased in

tilmicosin/bromhexine and tilmicosin/thyme oil-treated

groups at 3^rdand 14^thdays of the experiment.

. Microscopical lesions

Microscopically examination of the lungs of infected

non-treated (G₂), Tilmicosin (G₃), Bromhexine (G₄) and

Thyme oil (G₅) treated groups during 3^rdday and 14^thpost

medication showed severe congestion, perivascular edema

and lymphocytic infiltrations in comparison to nearly

normal lung in control negative group (G₁) and combined

treated groups (G_{6, 7}) as illustrated in Figures 1 and 2.

The liver of (G_2,

₇) showed severe vacuolar

3, 4, 5,

degeneration in comparison to mild vacuolar degeneration

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Radi et al., 2020

in (G₆) on 3^rdday. At 14^thpost medication, very mild

the myocardium in G₆and slight congestion of coronaries

with focal necrosis in some muscle bundles in G₇on 3^rd

day of experiment whereas at 14^thof the experiment; the

same results were obtained in groups (2, 4 and 5)

compared to normal cardiac muscle in groups (1, 6 and 7)

as shown in Figures 5 and 6 .

congestion, slight leucocytic infiltrations are shown in (G_3,

₆, ). But (G₁, , and₅) gave the same results which

7

2

4

observed on the 3^rdday of the experiment, as shown in

Figures 3 and 4 . The heart of groups (G₂,

₅) showed

3, 4,

severe congestion of coronaries with severe perivascular

edema in comparing with focal lymphocytic infiltrations in

Table 1. Effect of different medications on mortality rate of experimentally infected broiler chickens with Pasteurella

multocida.

Treatment groups

Dead birds/total birds

Mortality (%)

Non-infected non-treated group (Control negative)

Infected non-treated group (Control positive)

Tilmicosin group

0/9

4/9

1/9

3/9

2/9

0/9

0%

44.4%

11.1%

33.3%

22.2%

0%

Bromhexine group

Thyme oil group

Tilmicosin + Bromhexine group

Tilmicosin + Thyme oil group

0%

Table 2. Effect of different medications on bacterial re-isolation from different organs of experimentally infected broiler

chickens with Pasteurella multocida

Number of positive cases

day post-infection

Total No. of positive cases /

total No. of examined

Treatment groups

Organ

%

3^rd

7^th

0

3

2

3

0

1

0

14^th

chickens

0/9

9/9

5/9

4/9

9/9

8/9

9/9

8/9

1/9

3/9

1/9

2/9

1/9

Lung

Liver

Heart

Lung

Liver

Heart

Lung

Liver

Heart

Lung

Liver

Heart

Lung

Liver

Heart

Lung

Liver

Heart

Lung

Liver

Heart

0

3

2

3

1

2

1

2

1

0

3

0

3

2

3

2

0

0%

Non-infected, non-treated

group (Control negative)

100%

55.5%

44.4%

100%

88.8%

100%

88.8%

11.1%

33.3%

11.1%

22.2%

11.1%

Infected, non-treated

group (Control positive)

Tilmicosin group

Bromhexine group

Thyme oil group

Tilmicosin + Brohexine

group

Tilmicosin + Thyme oil

group

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J. World Poult. Res., 10(2S): 151-164, 2020

Table 3. Effect of treatment with tilmicosin alone or with either thyme oil or bromhexine hydrochloride on blood profile of

experimentally infected broiler chickens with Pasteurella multocida.

3^rdday of the experiment

14^thday of the experiment

Treatment groups

RBCs

Hb

WBCs

RBCs

Hb

WBCs

PCV (%)

(gm/dl)

PCV (%)

(gm./dl)

(10⁶/μl)

(10³/μl)

(10⁶/μl)

(10³/μl)

Non-infected, non-treated group

(Control negative)

4.56±0.14^a

14.46±0.07 ^a44.02±1.82 ^a13.52±0.66^b

4.46±0.07^a14.48±0.15^a49.82±2.25^a

15.9±0.69^b

Infected, non-treated group

(Control positive)

2.82±0.14^b

3.31±0.17^b

3.20±0.13^b

7.38±0.08 ^b

8.8±0.29^b

9.1±0.48^b

45.1±1.69

29.06±2.09^a

23.16±1^a

2.58±0.12^b

4.44±0.14^a

2.40±0.12^b

2.56±0.12^b

7.76±0.14^b46.82±1.42^a

35.28±1.7^a

Tilmicosin group

45.26±1.61 ^a

13.6±0.45^a45.36±0.94^a15.18±1.23^b

Bromhexine group

45.98±1.41 ^a24.62±1.31^a

7.94±0.24^b47.06±1.43^a

36.32±1.1^a

Thyme oil group

3.12±0.21^b

4.26±0.16^a

4.26±0.09^a

9.12±0.25 ^b

12.48±0.81^a

43.92±0.92 ^a

48.22±0.94 ^a

27.1±1.45^a

14±2.04^b

7.6±0.17^b44.84±0.96^a

31.4±1.82^a

16.2±1.19^b

Tilmicosin+Brohexine group

Tilmicosin + Thyme oil group

4.38±0.16^a14.22±0.23^a43.58±1.33^a

12.86±0.62 ^a45.06±1.16 ^a16.86±1.29^b

4.44±0.14^a14.26±0.28^a45.40±0.85^a15.92±1.45^b

Means with different superscript letters within the same column are significantly different (p ≤ 0.05).

Table 4. Effect of treatment with tilmicosin alone or with either thyme oil or bromhexine hydrochloride on the differential

leucocyte count of experimentally infected broiler chickens with Pasteurella multocida.

3^rdday of the experiment

14^thday of the experiment

Treatment groups

Neutrophils Lymphocytes Monocytes Eosinophils Neutrophils Lymphocytes Monocytes Eosinophils

(%)

Non-infected non-treated

group (Control negative)

60.0±0.95^b

33.2±1.01^a

3.6±0.40^a

3.2±0.37 ^a

58.80±0.58^c

33.8±.58 ^a

4.0±0.32 ^a

3.4±0.24 ^a

Infected non-treated group

(Control positive)

66.6±0.6^a

29.0±0.63 ^a

2.4±0.24 ^a

2.0±0.32 ^a

63.2.±0.8^a

30.4±1.12 ^a

3.6±0.51 ^a

2.8±0.49^a

Tilmicosin group

Bromhexine group

66.2±1.39^a

66.0±0.63^a

28.2±1.01 ^a

28.4±0.24 ^a

3.0±0.32 ^a

3.0±0.44^a

2.6±0.51^a

2.6±0.24 ^a

61.8±0.97 ^ab

63.2±0.37 ^a

31.4±0.74^a

3.0±0.0 ^a

3.0±0.32 ^a

3.2±0.2 ^a

30.2±0.2^a

3.4 ±0.24 ^a

Thyme oil group

66.4±1.25^a

28.8±1.36 ^a

2.6±0.24 ^a

2.2±0.20 ^a

62.6±0.68^a

30.4±0.68^a

3.6±0.24 ^a

Tilmicosin+Brohexine

group

62.2±2.03^ab

31.8±1.74 ^a

32.2±1.2 ^a

3.4±0.51^a

3.8±0.49 ^a

2.6±0.4 ^a

59.2±.49 ^bc

32.8±1.24 ^a

4.2±0.58 ^a

3.6±0.24 ^a

3.8±0.37 ^a

Tilmicosin + Thyme oil

61.20±0.97 ^ab

2.8±0.37 ^a

59.0±0.55 ^bc

32.6±.93 ^a

3.60±0.51 ^a

Means with different superscript letters within the same column are significantly different (p ≤ 0.05).

Table 5. Effect of treatment with tilmicosin alone or with either thyme oil or bromhexine hydrochloride on liver and kidney

functions of experimentally infected broiler chickens with Pasteurella multocida.

3^rdday of the experiment

14^thday of the experiment

Treatment groups

ALT

AST

Uric acid

Creatinine

ALT

AST

Uric acid

Creatinine

(U/L)

(mg/dl)

(U/L)

(mg/dl)

Non-infected non-treated

group (Control negative)

11.60±0.93^b

180.2±11.57^b

9.00±0.71^c

0.35±0.03^b12.40±0.75^b149.0±8.14^b

8.20±0.73^b

0.36±0.04^b

Infected non-treated group

(Control positive)

27.00±2.07^a252.8±12.53 ^a17.40±0.81^a

0.78±0.08^a31.80±1.16^a287.8±14.63^a17.00±0.71^a0.79±0.04^a

0.58±0.03^a18.60±3.37^b179.6±8.29 ^b12.40±0.81^ab0.56±0.05^b

Tilmicosin group

25.00±1.52^a261.00±17.95^a14.40±1.5^ab

23.80±1.77^a286.00±16.84^a15.60±1.03^a

Bromhexine group

Thyme oil group

0.69±0.05^a31.00±1.76^a297.2±3.72 ^a

16.80±1.5^a

0.68±0.03^a

24.40±1.57^a

14.80±0.86^b

292.80±14.1^a15.00±1.26^ab0.64±0.06^a30.40±0.68^a289.4±7.08 ^a15.40±0.68^a0.73±0.04^a

Tilmicosin+Brohexine group

192.8±6.57^b

189.2±6.01^b

10.80±0.8^bc

9.40±0.75^c

0.47±0.06^b13.80±0.66^b170.0±6.19^b

0.36±0.02^b14.40±0.1^b153.4±5.53^b

8.00±1.14^b

7.80±1.59^b

0.40±0.03^b

0.31±0.02^b

Tilmicosin + Thyme oil group

14.60±0.5^b

Means with different superscript letters within the same column are significantly different (p ≤ 0.05). AST: Aspartate Aminotransferase, ALT: Alanine

Aminotransferase

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Radi et al., 2020

Figure 1. Lung sections on the 3^rdday of treatment in different broiler chicken groups infected with Pasteurella multocida.

Normal lung with normal bronchi and bronchioles in control negative group (G1); Congestion, hemorrhage, perivascular

edema, and lymphocytic infiltration in the interalveolar cell in control positive group (G2); Vascular thrombosis, perivascular

edema, few leucocyte infiltrations together with mild atelectasis and emphysema. In tilmicosin treated group (G3); Severe

congestion in the vasculature, perivascular edema also obliteration of most alveoli by RBCs and inflammatory cell in

bromhexine treated group (G4); Severe hemorrhage in the tubular septum in Thyme oil treated group (G5); Normal bronchi

and alveoli except slight congestion in the interalveolar capillaries in tilmicosin +bromhexine treated group (G6) and Nearly

normal lung with slight congestion of the vasculature in tilmicosin +thyme oil treated group (G7).

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J. World Poult. Res., 10(2S): 151-164, 2020

Figure 2. Lung sections on the 14^thday of treatment in different broiler chicken groups infected with Pasteurella multocida.

Normal lung with normal bronchi and bronchioles in control negative group (G1); Thrombosis with vacuolation of tunica

media together with slight atelectasis and emphysema in control positive group (G2); Bronchial epithelium with slight

congestion of the blood vessels of lamina propria, especially veins and lymphocytic vessels in tilmicosin treated group (G3);

Severe congestion of the intertubular blood vessels with moderate perivascular edema in bromhexine treated group (g4);

Severe congestion of the vasculature together with focal necrosis surround by giant cells and lymphocytes in thyme oil-treated

group (G5); Normal lung except slight emphysema and atelectasis in tilmicosin +bromhexine treated group (G6) and Normal

lung except slight congestion of the vasculature in tilmicosin +thyme oil treated group (G7).

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Radi et al., 2020

Figure 3: Hepatic sections on 3^rdday of treatment in different broiler chicken groups infected with Pasteurella multocida.

Normal hepatic architecture with normal hepatocytes, centrally located vesicular nucleus, also normal central and veins in

control negative group (G1); Congestion, hemorrhage, focal lymphocytic infiltration, and vacuolar degeneration within

hepatocytes in control positive group (G2); Mild congestion, moderate leucocyte infiltrations, and severe vacuolar

degeneration in tilmicosin treated group (G3); Congestion, mild leucocytic aggregation in the portal area, and severe vacuolar

degeneration in bromhexine treated group (G4); Moderate congestion, disorganization of hepatic cord with focal leucocytic

aggregation in portal triads in thyme oil-treated group (G5); Moderate congestion, moderate inflammatory cell infiltrations

also mild vacuolar degenerations in tilmicosin +bromhexine treated group (G6) and mild congestion, very mild leucocytic

aggregations in the portal area, and severe vacuolar degeneration in tilmicosin +thyme oil treated group (G7).

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Figure 4. Hepatic sections on the 14^thday of treatment in different broiler chicken groups infected with Pasteurella multocida.

Normal hepatic architecture with normal hepatocytes, centrally located vesicular nucleus, normal central and veins in control

negative group (G1); Severe vacuolar degeneration within hepatocytes, congestion of hepatic blood vessels, and few

mononuclear cell infiltrations in the portal area in control positive group (G2); Slight congestion with mild leucocyte

infiltrations in the portal area in tilmicosin treated group (G3); Vacuolar degeneration, thrombosis in the hepatic artery, and

dissociation of hepatocytes in bromhexine treated group (G4); Severe vacuolar degeneration and moderate congestion of the

vasculature also mononuclear cell infiltrations in the portal area in thyme oil-treated group (G5); Mild congestion, very mild

vacuolar degeneration, and few mononuclear cell aggregations in the portal area in tilmicosin +bromhexine treated group (G6)

and slight congestion of the vasculature with mild vacuolar degeneration in tilmicosin +thyme oil treated group (G7).

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Figure 5. Heart sections of on 3^rdday of treatment in different broiler chicken groups infected with Pasteurella multocida.

Normal heart in control negative group (G1); Congestion in coronaries, hemorrhage between cardiac muscles, edema in the

epicardial region, and epicarditis in control positive group (G2); Moderate congestion of coronaries and perivascular edema in

tilmicosin treated group (G3); Moderate congestion in the coronaries, perivascular edema, and lymphocytic infiltrations in

bromhexine treated group (G4); Severe congestion of coronaries, moderate epicardiac edema and mild phagocytic activation

thyme oil treated group (G5); Focal lymphocytic infiltrations in the myocardium in tilmicosin +bromhexine treated group

(G6); Slight congestion of coronaries with focal necrosis of some muscle bundles in tilmicosin +thyme oil treated group (G7).

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Figure 6. Heart sections on 14^thday of treatment in different broiler chicken groups infected with Pasteurella multocida.

Normal heart in control negative group (G1); moderate congestion in coronaries, Zenger necrosis of some muscle bundles

together with perivascular edema in control positive group (G2); mild congestion of coronaries and mild perivascular edema in

tilmicosin treated group (G3); Severe congestion of coronaries and slight perivascular edema in bromhexine treated group

(G4); Severe perivascular edema in thyme oil-treated group (G5); normal cardiac muscle with normal vasculature in tilmicosin

+bromhexine treated group (G6) and normal cardiac muscle in tilmicosin +thyme oil treated group (G7).

(11.1%) in the tilmicosin treated group. The findings

proved that concurrent administration of tilmicosin with

bromhexine or thyme oil for three consecutive days is

highly effective in the treatment of pasteurellosis as

indicated by no mortality during the experiment compared

to groups treated with a single agent. Invasion of lung,

liver, and spleen via P. multocida decreased by treatment

of tilmicosin alone or concurrent with bromhexine or

thyme oil may be due to the antibacterial action of these

drugs. The antibacterial activity of tilmicosin might be

attributed to its accumulation within avian phagocytic

cells. Also, phagocytosis of P. multocida and

DISCUSSION

Experimental infection with P. multocida induced severe

clinical signs included off food, nasal discharge, cough,

ruffled feather, watery green diarrhea, or even sudden

mortality in non-medicated birds and these findings are in

agreement with results of Mohamed (2009). A high

mortality rate (44.4%) was recorded in the non-medicated

group. A similar observation had been recorded in

chickens infected with P. multocida (Mohamed 2009;

Sonone et al., 2011 ). It was clear that experimental

infection with P. multocida induced a low mortality rate

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Radi et al., 2020

lipopolysaccharide exposure increases tilmicosin uptake

values of uric acid and creatinine could be due to the

negative effect of the P. multocida or its endotoxin on the

kidney functions (Mohamed, 2009). The current study

revealed that the birds given combination of tilmicosin

with thyme oil or bromhexine showed amelioration of

serum enzymes in G₆and G₇at 3^rdand 14^thdays of the

experiment and 14^thdays of the experiment in case of

tilmicosin only whereas no changes in cases of thyme oil

or bromhexine groups, this could be due to the

enhancement activities of tilmicosin by bromhexine or

thyme oil.

In the present study, macroscopic lesions in diseased

chickens including congested body, enlarged liver with

pinpoint grayish-white foci, fibrinous pericarditis, and

enlarged congested spleen had come in accordance with

(Srinivasan et al., 2011). The microscopic examination of

different tissues from infected chickens showed vacuolar

degeneration, hemorrhages, and congestion in livers

similar to that recorded by Shivachandra et al. (2005 ),

lymphocytic depletion of the spleen, fibrinous pericarditis

in the cardiac tissue and severe congestion beside

leucocytic infiltration, the lung showed congestion,

hemorrhages and leucocytic infiltrations which might be

attributed to the effect of P. multocida endotoxins

(Christensen and Bisgaard, 2000). These results were

exceedingly compatible with those reported by Afifi and

El-Nesr (2013) and Panna et al., (2015 ). Tilmicosin-

treated birds exhibited less severe histopathological

lesions, probably related to the antibacterial and anti-

inflammatory activities of Tilmicosin. Similarly, infected

chicken treated with Tilmicosin in combination with

bromhexine or thyme oil showed marked reductions in the

severity of histopathological lesions probably due to the

potent antibacterial activity and antioxidant activities of

thyme oil (Aljabeili et al., 2018 ).

by the avian phagocytes and the presence of opsonized P.

multocida enhances the release of tilmicosin from the

phagocytes. Also, intracellular tilmicosin increases cellular

lysosomal production in all three chicken phagocyte types

(Scorneaux and Shryock, 1998 ). According to McKellar et

al. (2004), the release of drug from intracellular sites

during phagocytosis can help to maintain sustained tissue

concentrations and subject bacteria to prolonged exposure

appropriate for

a

time-dependent drug. Moreover,

tilmicosin has an active post-antibiotic effect and post-

antibiotic sub-MIC effect on P. multocida, which may

impede disease progression by enhancing the animal’s

immune response to eliminate a weakened bacterial

population (Lim and Yun, 2001). Thyme oil exhibit a

significant antibacterial effect against many Gram-

negative and Gram-positive microorganisms (Amat et al.,

2019 ).

The results of hematology in the present study

showed anemia indicated by significant decrease in RBCs

count and Hb concentration in groups (2, 4 and 5) at 3^rd

day of infection and group (2) at the end of experiment

which could be attributed to the hemolytic effect of the P.

multocida endotoxin as stated by Diallo and Frost (2000).

The leukogram in groups (2,

4 and 5) showed

leukocytosis, due to neutrophilia which might be attributed

to the endotoxin of P. multocida (Sonone et al., 2011 ) as

well as elevated heterophils and WBCs in diseased birds

may be recorded as the primary defense mechanism

against any pathogen to prevent the spread of infection

(Ahamefule et al., 2006). Treatment of infected chicken

with bromhexine or thyme oil alone showed non-

significant changes in the blood picture. On the other

hand, concurrent medication with Tilmicosin and thyme

oil or bromhexine revealed improvement of blood picture

as shown by the significant increase in the RBCs and Hb

concentration in the G_6,G₇as compared with G₂at 3^rdday

and 14^thpost medication, that might be due to the

enhancement of antibacterial activity of tilmicosin by

bromhexine in addition to the antioxidant activity of

thyme oil (Aljabeili et al., 2018; Saricaoglu and Turhan

2018 ).

The values of ALT, AST, uric acid, and creatinine in

the serum of chicken in G₂were significantly (P ≤ 0.05)

increased in comparison to the control group at 3^rdand 14^th

days post-infection. The increased value in ALT and AST

could be due to hepatopathy as well as degenerative

changes caused by P. multocida or its endotoxins

(Campbell and Coles, 1986 ). Similar results obtained by

(Sonone et al., 2011 ) in broiler chickens. The increased

CONCLUSION

This study showed that the clinically observed damage in

chickens infected with P. multocida could be ameliorated

by the combination of tilmicosin with bromhexine or

thyme oil. This protective effect could reduce the use of

antibiotics in livestock, human exposure to antibiotic

residues and bacterial resistance to antibiotics.

Furthermore, tilmicosin can be used for the effective

treatment of pasteurellosis in chicken. The combination of

tilmicosin and bromhexine or thyme oil was more potent

against Pasteurella multocida infection than each

treatment alone.

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J. World Poult. Res., 10(2S): 151-164, 2020

Doolan PD, Alen EL and Theil GB (1962). A clinical appraisal

DECLARATIONS

Competing interests

of the plasma concentration and endogenous clearance of

creatinine, The American Journal of Medicine, 32(1): 65-79.

El-Mahmoudy A M, Gheith IM, Elmajdoub AA and Awidat SK

(2018). In-vivo assessment of the antipyretic activity of

The authors have no competing interest to declare.

tilmicosin,

African

Journal

of

Pharmacy

and

Pharmacology, 12(14): 176-182.

Author s’ contribution

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volatile oils on performance of broiler chickens. European

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Hematology. 5^thEdition, Lippincott Williams & Wilkins,

1120-1124.

Glisson JR, Hofacre CL and Christensen JP (2013). In: Diseases

of Poultry; Thirteenth Edition, Swayne D.E., Editor in

Chief, Glisson J.R., McDougald L.R., Nolan L.K., Suarez

D.L. & Nair V., Associate Editors. Wiley-Blackwell, Ames,

Iowa, USA and Oxford, UK, pp. 807–823.

Dr. Abeer, Dr. Nema, Prof. Dr. Hossny, and Prof.

Dr. Abd El Nasser involved in the collection of data,

developed the idea of research and design, analysis and

interpretation of data; also involved in drafting and

revising the manuscript. Prof. Dr. Elham revised the

article. Dr. Fatma designed the figures.

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