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lenged), Group 4 (non-treated withMentofin and H9N2 challenged), Group 5(Mentofin treated and H9N2 challenged),Group 6 (non-treated with Mentofin andMG/H9N2 challenged), and Group 7(Mentofin treated and MG/H9N2 chal-lenged). At 1 week of age, an intratrachealchallenge of the birds with MG (2 hemag-glutination units/0.5 mL/bird) and/or H9N2(2 hemagglutination units/0.5 mL/bird) wasgiven to specific groups mentioned previ-ously. Essential oils of Mentofin wereadministered for 6 days, effective 1 daypost-challenge. Histopathological observa-tions were concluded at 6 days post-chal-lenge and revealed a significant reduction (P < 0.05) in microscopic tissue lesions ofbirds treated with Mentofin in comparisonto birds deprived from this treatment but

Evaluation of the Histopathologyof the Respiratory System inEssential Oil-Treated BroilersFollowing a Challenge WithMycoplasma gallisepticum and/orH9N2 Influenza VirusElie K. Barbour, PhD1

Rindala G. El-Hakim, BSc1

Marc S. Kaadi, BSc2

Hussam A. Shaib, MSc1

Danyelle D. Gerges, BSc1

Pia A. Nehme, MSc1

1Department of Animal SciencesFaculty of Agricultural and Food SciencesAmerican University of BeirutBeirut, Lebanon2Department of AgricultureSaint-Joseph UniversityBeirut, Lebanon

KEY WORDS: Avian influenza virus(H9N2), broilers, essential oils, histopathol-ogy, Mycoplasma gallisepticum, respiratorysystem

ABSTRACTThe objective of this work is to evaluate theimpact of eucalyptus and peppermint essen-tial oils (Mentofin®) in the protection of therespiratory system of broilers against con-trolled challenges by Mycoplasma gallisep-ticum (MG) and/or avian influenza virusH9N2. Seventy 1-day-old broilers werereared in 7 groups (10 birds/group) up to 1week of age. Group 1 was the control (non-treated with Mentofin and unchallenged);challenged groups were Group 2 (non-treat-ed with Mentofin and MG challenged),Group 3 (Mentofin treated and MG chal-

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challenged similarly. The significant (P <0.05) reduction in microscopic lesionsincluded a decrease in tracheal deciliation inMG- and MG/H9N2-challenged birds, adecrease in mucosal hypertrophy in MG-,H9N2-, and MG/H9N2-challenged birds, adecrease in goblet cell degeneration in MG-and MG/H9N2-challenged birds, a decreasein mucus accumulation in MG-challengedbirds, and a decrease in heterophil infiltra-tion in MG/H9N2-challenged birds.

INTRODUCTIONMycoplasma gallisepticum (MG) is thecausative agent of chronic respiratory dis-ease in chickens,1 a common disease inmany poultry species around the world.Although the clinical manifestations areusually slow to develop, MG in combina-tion with other respiratory pathogens cancause severe airsacculitis.1,2 Beside feed andegg production reduction, MG problems areof high economic significance since respira-tory tract lesions can cause high morbidity,high mortality, and significant carcass con-demnation and downgrading.2 Consequently,the efforts to limit the losses from MG infec-tions would be of primary importance to thepoultry industry.

Avian influenza virus is one of the mostdevastating viral diseases in the poultryindustry and has a worldwide distribution.Frequent incidences of H9N2 avian influen-za type with high mortality were observedcommonly on broiler farms of the MiddleEast region.3 Mixed infections with otherrespiratory pathogens, particularly MG,were incriminated resulting in high mortali-ty on poultry farms, associated with greateconomic losses.3 Control of this viruswould be of primary importance, especiallywith raising concerns of the potential ofH9N2-AI to induce a pandemic spread.4

The essential oils of eucalyptus and pep-permint present in Mentofin® have proveneffective against respiratory complexes. Infact, Barbour et al 20055 found that theadministration of Mentofin following NDV,

IBV, and IBDV vaccination in MG/H9N2-infected broilers boosted the immuneresponse and ameliorated their performance.Consequently, the administration ofMentofin could have similar alleviatingeffect on signs and lesions of MG- and/orH9N2-infected broilers.

Although several experiments were con-ducted to study the interaction between MGand other avian respiratory pathogens,6,7 noprevious studies on interaction between con-trolled challenge by MG and/or H9N2 inpoultry were done. The fact that these 2pathogens are frequently present in theMiddle East region and many other parts ofthe world makes the study of the effect of acontrolling agent on MG/H9N2 interactionof paramount importance.

The purpose of this project is to evaluateand understand, at the cellular level, theimpact of Mentofin on protection of the res-piratory system of broilers against con-trolled challenges by MG and/or H9N2avian influenza. The histopathological workfor understanding the microscopic changesat the cellular level of the trachea includedthe observation of the following: trachealdeciliation, mucosal hypertrophy, goblet celldegeneration, mucus accumulation, and het-erophil infiltration.

MATERIALS AND METHODS

BirdsSeventy 1-day-old broiler chicks weredivided equally into 7 groups (10chicks/group). The 7 groups were put inisolation rooms supplied with infraredlamps, waterers, and feeders. All birds werefed with the same feed as recommendedpreviously.8

Preparation of Challenge Strains

MG isolateA tracheal swab of a bird from an MG-infected flock was cultured at 37°C in Frey'sbroth for 4 days and subcultured onto Frey'sagar for another 4 days. One MG-suspectedcolony with a fried egg colonial morpholo-

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gy was tested against MG-specific chickenantibody (SPAFOS, Connecticut, USA). Aconjugate, which is a goat anti-chicken IgGantibody (heavy + light chain), labeled withfluorescin (KPL Laboratories, Gaithersberg,Maryland, USA) was used. The reactingantibodies to the stationed fixed MG-sus-pected cells on a microscopic slide wereobserved under a fluorescent microscope(Leica DMLS, Germany) to confirm theidentity of MG. The cloned-purified colonywas grown in larger volume of Frey's brothfor 4 days at 37°C. Pellets of MG were col-lected by centrifugation of the Frey's brothculture at 15557 × g for 15 minutes. TheMG pellets were reconstituted in 30 mL ofsterile saline and the hemagglutination (HA)titer was determined against 0.1% of chick-en red blood cell suspension.9 The reconsti-tuted MG was further diluted to reach 2 HAunits/0.5 mL of saline, an adjusted MG sus-pension for a challenge.

H9N2 isolateThe H9N2 strain was isolated from a severeavian influenza outbreak in broilers in thesummer of 2004. The H9 component wasidentified at the American University ofBeirut, while the complete subtyping wascompleted on the isolate at the CentralVeterinary Laboratory of Weybridge,England. A report was issued by Dr. RuthManvel at Weybridge confirming the sub-type as H9N2. The H9N2 was preserved at-80°C in our laboratory as 1:1 ratio ofallantoic fluid:tryptose phosphate broth.This mix was diluted in sterile saline toresult in 2 HA units/0.5 mL, an adjustedviral stock for a challenge.

Challenge and TreatmentThe 7 groups of chicks were reared to 1week of age. At 1 week of age, the follow-ing challenges were given as shown inTable 1. Mentofin was diluted in V/V of0.025 mL/100 mL of drinking water. Eachtreated bird started receiving the dilutedMentofin, intra-esophageally, twice a day(morning and evening), in a volume of 1mL/bird/time. The treatment was for 6 days,starting on the challenge day.

HistopathologyAll birds were sacrificed by CO2 asphyxia-tion at 6 days post-challenge. A 1-cm cross-sectional cut of each trachea from sacrificedbirds was taken and kept in 10%formalin/PBS buffer. The tracheal cuts weresubjected to cross-sectioning of 4 μm andH&E staining according to a previous pro-cedure.10 The observation of tracheal decilia-tion, mucosal hypertrophy, goblet celldegeneration, mucus accumulation, and het-erophil infiltration from 4 tracheal cuts/birdwere seen microscopically at 400× within 3fields/cut located at 4, 8, and 12 clock posi-tions. A score of 1 was given for each ofthe following tracheal tissue changes,namely, deciliation, mucosal hypertrophy,goblet cell degeneration, and mucus accu-mulation, while a score of 0 was given tothe absence of these tracheal changes. Theaverage score of 10 birds per group wasused in the histograms.

Cumulative heterophil count in 12 fieldsof 4 tracheal cuts/bird was recorded, and theaverage count of 10 birds was used in thehistogram. Microscopic images, magnified400×, were taken from selected histopatho-logical slides to compare the tissue changesin different broiler groups.


Table 1. Challenges AdministeredEsophageally to the Broilers at 1 Week of Age.

Groups Challenges* Mentofin Treatment

1 None None

2 MG None

3 MG Yes†

4 H9N2 None

5 H9N2 Yes†

6 MG/H9N2 None

7 MG/H9N2 Yes†

*The MG and H9N2 were each given in volume of 0.5mL intratracheally. The MG and H9N2 were each givenas 2 HA units/0.5 mL/bird.†Mentofin was administered on the challenge day; theadministration was esophageal, twice per day (morningand evening) in a volume of 1 mL/bird/each time a day,and for a period of 6 days. The dilution of the adminis-tered Mentofin was as instructed by the manufacturer.



StatisticsThe mean score of the histopathologicalobservations of the tracheal sections wasdone by 1-way ANOVA followed byDuncan's test, allowing for statistical com-parisons among the different groups.

Animal EthicsThe Institutional Animal Care and UseCommittee at the American University ofBeirut abides by the interdisciplinary princi-ples and guidelines for the use of animals inresearch, and this work has been approvedaccording to such principles.

RESULTSThe histopathology of the tracheal sectionsuncovered the consistent significant reduc-tion in microscopic lesions in the Mentofin-treated groups in comparison to thesimilarly challenged but Mentofin-deprivedgroups (P < 0.05), namely, for the micro-scopic lesions of tracheal deciliation,mucosal hypertrophy, goblet cell degenera-tion, mucus accumulation, and heterophilinfiltration.

A comparison of tracheal deciliated tis-sue from a MG/H9N2-challenged birddeprived of Mentofin treatment in Group 6to a normal ciliation in a similarly chal-lenged and Mentofin-treated bird of Group7 is shown in Figure 1. The mean scores ofbirds in each of the 7 groups showing decil-iation are shown in Figure 2.

There was an apparent reduction in tra-cheal deciliation in Mentofin-treated Groups3 and 7 in comparison to similar respectivechallenged Groups 2 and 6 that weredeprived of Mentofin; however, the signifi-cance in this reduction at P < 0.05 wasshown in Group 7 in comparison to Group6, where both were challenged withMG/H9N2 organisms.

Birds of the 3 differently challengedgroups showed mucosal hypertrophy on the6th day post-challenge. Figure 3 shows amicroscopic slide section of a bird's tracheafrom Group 6 deprived of Mentofin andchallenged with MG/H9N2 in comparisonto a section from the trachea of a bird from

Group 7 challenged with MG/H9N2 buttreated with Mentofin. The mean scores ofmucosal hypertrophy in the 7 groups ofbroilers are shown in Figure 4. This micro-scopic lesion of mucosal hypertrophy wasreduced significantly (P < 0.05) inMentofin-treated groups in comparison toMentofin-deprived groups with similarchallenges.

The comparison of the microscopic slidephoto of a trachea with goblet cell-degener-ation (bird of Group 2, MG challenged anddeprived of Mentofin) versus another tra-chea with normal intact goblet cells (bird ofGroup 3, MG challenged and Mentofintreated) is shown in Figure 5. The meanscores of goblet cell degeneration amongthe 7 groups of broilers are shown in Figure6. There was an apparent protection of gob-let cells against degeneration by MG chal-lenge (Group 3, Mentofin treated) andMG/H9N2 challenge (Group 7, Mentofintreated) in comparison to similarly chal-lenged Groups 2 and 6, respectively, butdeprived of Mentofin (P < 0.05).

Tracheal cuts with mucus accumulationversus clear tissues are shown in Figure 7.The mean scores of mucus accumulation inthe tracheal sections of the 7 groups areshown in Figure 8. There was a decrease (P > 0.05) in mucus accumulation in tra-cheal sections of birds treated withMentofin (Groups 3, 5, and 7) comparedwith Mentofin-deprived and respectivelychallenged Groups 2, 4, and 6.

Last but not least, the heterophil infiltra-tion score was reduced significantly (P <0.05) in the Mentofin-treated birds of Group7 in comparison to Group 6 birds deprivedof treatment, in which both groups had asimilar challenge with MG/H9N2 (Figure9). An insignificant reduction in trachealheterophil occurred in birds of Group 3(Mentofin treated, MG challenged) in com-parison to those of Group 2 (Mentofindeprived, MG challenge) (P > 0.05). On thecontrary, the heterophil score increased inMentofin-treated birds of Group 5 (chal-lenged with H9N2) in comparison toMentofin-deprived birds of Group 4 (given


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the same challenge). The microscopic slidephoto in Figure 10 shows the contrastbetween a low heterophil infiltration inMentofin-treated Group 7 and a high het-erophil infiltration in the birds' trachea ofGroup 6 (deprived of Mentofin).

DISCUSSIONThe reduction of deciliation by the essentialoil was more apparent (P < 0.05) in theMG/H9N2-challenged groups. The activeingredient in Mentofin seems to protectagainst deciliation that is normally causedby MG and H9N2. This protection againstthe first line of defense in the poultry host isof paramount importance, since it will pro-tect against colonization by secondary bac-terial infections.11 It is worth noting that theciliary activity of human respiratory cells isaffected by exposure to the essential oil ofEucalyptus spp.12 The thinning of the mucus in

the respiratory tract by the active ingredientsof Eucalyptus spp. could help in its outwardflow, pushing with it the microorganisms, pre-venting their colonization, and thus protectingthe cilia from consequent damage.13,14


Figure 1. A microscope slide photograph of deciliated (Group 6, MG/H9N2 challenged andMentofin deprived) versus normal ciliated tracheal tissue (Group 7, MG/H9N2 challenged andMentofin treated), magnified 400×.

DeciliatedDeciliated Normal ciliationNormal ciliation

Figure 2. Mean scores of tracheal deciliationin the 7 broiler groups. Alphabets on the his-togram that differ among groups are signifi-cant at P < 0.05.

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Figure 3. A microscopic slide photo ofmucosal hypertrophy in a trachea fromMG/H9N2-challenged bird of Group 6(deprived of Mentofin) versus the absenceof mucosal hypertrophy of a bird treatedwith Mentofin in Group 7 and similarly chal-lenged, magnified 400×.

Mucosal hypertrophyMucosal hypertrophy

Absence of mucosal hypertrophyAbsence of mucosal hypertrophy



Figure 4. Mean scores of mucosal hypertro-phy in the 7 broiler groups. Alphabets on thehistogram that differ among groups are sig-nificant at P < 0.05.

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Figure 5. A microscopic slide photo of degen-erated goblet cells in trachea of MG-chal-lenged and Mentofin-deprived chicken(Group 2) versus intact goblet cells in tra-chea of MG-challenged and Mentofin-treat-ed birds (Group 3), magnified 400×.

Degenerated goblet cellsDegenerated goblet cells

Intact goblet cellsIntact goblet cells

Figure 6. Mean scores of goblet cell degener-ation in the 7 broiler groups. Alphabets onthe histogram that differ among groups aresignificant at P < 0.05.

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Figure 7. A microscopic slide photo of the tra-cheal section with mucus accumulation(Group 2, MG-challenged bird deprived ofMentofin) versus a mucus-clear tracheal sec-tion (Group 3, MG-challenged bird treatedwith Mentofin), magnified 400×.

Mucus accumulationMucus accumulation

No accumulation of mucusNo accumulation of mucus


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Mucosal hypertrophy was significantlyreduced by essential oil in birds exposed toany of the 3 different challenges (P < 0.05).This is a clear effect of Mentofin activeingredients on the integrity of the mucosallayer of challenged birds keeping this layerof tissue in nearness to the thickness of con-trol birds (unchallenged, Group 1). The pre-vention of hypertrophy of the mucosal layerby such ingredients was documented in aprevious work performed on lipopolysac-charide (LPS)-induced bronchitis in rats,proving the reduction of inflammatory cellinfiltration into the epithelium of tracheaand bronchioles in rats administered theessential oil of Eucalyptus globulus.15

Degeneration in goblet cells wasreduced significantly (P < 0.05) by essential

oil in MG- and MG/H9N2-challenged birds.To our knowledge, there is no previous doc-umentation on the impact of synergismamong the active ingredients of Eucalyptusspp. and peppermint for providing protec-tion of the goblet cells in the upper respira-tory system of animal or humans. Themaintenance of the goblet cell structure andfunction is important in respiratory diseasesto keep the mucus flow in the air passages,thus pushing the germs and other particles,with the help of the maintained cilia, anteri-orly and outwards through the mouth ornostrils.

The mucus accumulation was reducedsignificantly (P < 0.05) in MG-challengedbirds administered the essential oil. The roleof the active ingredients of Mentofin in


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Figure 8. Mean scores of mucus accumula-tion in tracheal section of the 7 groups.Alphabets on the histogram that differamong groups are significant at P < 0.05.

Figure 9. The mean heterophil number per 12fields/4 tracheal cuts/bird. Alphabets on thehistogram that differ among groups are sig-nificant at P < 0.05.

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Figure 10: A microscopic slide photo of tracheal section with low heterophil infiltration (Group 7,MG/H9N2 challenged and Mentofin treated) versus high heterophil infiltration (Group 6,MG/H9N2 challenged and Mentofin deprived), magnified 400×.

High heterophil infiltrationHigh heterophil infiltration Low heterophil infiltrationLow heterophil infiltration



clearing the mucus from the tracheal tissuewas reported in other works.13-18

The heterophil infiltration was reducedsignificantly (P < 0.05) by essential oil inMG/H9N2-challenged birds, while the infil-tration was increased significantly by essen-tial oil in birds challenged by H9N2 alone.This contradiction could be due to thenature of the challenge in Groups 4 and 5that differed from those in Groups 2, 3, 6,and 7. It is worth noting that one work doc-umented the impact of steam-distilled essen-tial oil from Eucalyptus globulus onreduction of inflammatory cell infiltrationand mucin hypersecretion in rats exposed toPS challenge in their respiratory system.15

In conclusion, histopathological changeswere observed in Mentofin-treated versusMentofin-deprived broilers subjected to 3different natures of challenges (MG, H9N2,and a combination of MG/H9N2). TheMentofin treatment resulted in a significantdecrease (P < 0.05) in tracheal deciliation inMG- and MG/H9N2-challenged birds, a sig-nificant decrease (P < 0.05) in tracheal gob-let cells degeneration in MG- andMG/H9N2-challenged birds, a significantdecrease (P < 0.05) in tracheal mucus accu-mulation in MG-challenged birds, and a sig-nificant decrease (P < 0.05) in heterophilinfiltration in MG/H9N2-challenged birds.These results demonstrate the healing effectof Mentofin against MG/H9N2 infection, acomplex respiratory disease of economicimportance to the poultry industry.

REFERENCES1. Kleven SH: Mycoplasmosis. In: Swayne DE,

Glisson JR, Jackwood MW, Pearson JE, ReedWM, ed. A Laboratory Manual for the Isolationand Identification of Avian Pathogens. 4th ed.Kennett Square, Pa: American Association ofAvian Pathologists; 1998:74-80.

2. Ley DH, Yoder HW Jr: Mycoplasma gallisep-ticum infection. In: Calnek BW, Barnes HJ, BeardCW, McDougald LR, Saif YM, eds. Diseases ofPoultry. 10th ed. Ames, Iowa: Iowa StateUniversity Press; 1997:194-207.

3. Nili H, Asasi K: Natural cases and experimentalstudy of H9N2 avian influenza in commercialbroiler chickens of Iran. Avian Pathol2002;31:247-252.

4. Chen H, Subbaro K, Swayne D, et al: Generationand evaluation of a high-growth reassortant H9N2influenza A virus as a pandemic vaccine candi-date. Vaccine 2003;21:1974-1979.

5. Barbour EK, Dankar, S, et al.: Essential oils ofEucalyptus and peppermint improve the homo-geneity of immune responses and performance inMG/H9N2-infected broilers. J Am Holistic VetMed Assoc 2005; 24:23-27.

6. Bradbury JM: Avian mycoplasma infection: pro-totype of mixed infections with mycoplasmas,bacteria and viruses. Ann Microbiol (InstitutPasteur) 1984;135A:83-89.

7. Corstvet RE, Sadler WW: A comparative study ofsingle and multiple respiratory infections withMycoplasma gallisepticum, Newcastle diseasevirus, and infectious bronchitis virus. Am J VetRes 1996;27:1703-1720.

8. National Research Council. Nutrient Requirementof Poultry. 8th revised ed. Washington DC:National Academy Press; 1984:11-15.

9. Hierholzer JC: Adenoviruses. In: Schmidt NJ,Lennette DA, Lennette ET, Lennette EH,Emmons RW, eds. Diagnostic Procedures forViral, Rickettsial and Chlamydial Infections. 7thed. Washington, DC: American Public HealthAssociation; 1995:169-188.

10. Randall JC, Reece LR: Avian Histopathology.Chicago, Ill: Mosby-Wolfe; 1966:145-170.

11. Reynolds HY: Modulating airway defensesagainst microbes. Curr Opin Pulm Med2002;8:154-165.

12. Riechelmann H, Brommer C, Hinni M, et al:Response of human ciliated respiratory cells to amixture of menthol, eucalyptus oil and pine nee-dle oil. Arzneimittelforschung. 1997;47:1035-1039.

13. Zakay-Rones Z, Thom E, Wollan T, et al:Randomized study of the efficacy and safety oforal elderberry extract in the treatment of influen-za A and B virus infections. J Intern Med Res2004;32:132-140.

14. Salari MH, Amine G, Shirazi MH, et al:Antibacterial effects of Eucalyptus globulus leafextract on pathogenic bacteria isolated from speci-mens of patients with respiratory tract disorders.Clin Microbiol Infect 2006;12:194-196.

15. Lu XQ, Tang FD, Wang Y, et al: Effect ofEucalyptus globulus oil on lipopolysaccharide-induced chronic bronchitis and mucin hypersecre-tion in rats. Zhongguo Zhong Yao Za Zhi2004;29:168-171.

16. Wurges J: Eucalyptus. In: Gale Group. GaleEncyclopedia of Alternative Medicine. 2001.Available at: http://www.findarticles.com/p/arti-cles/mi_g2603.

17. Gawronski DV: Medical choices. In: Gale Group.Gale Encyclopedia of Medicine. 2002.

18. Page L: Healthy Healing: A Guide to Self-Healingfor Everyone.12th ed. Sarasota FL: BookworldServices; 2004:42-44.


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