Elie K. Barbour, PhD 1 Rindala G. El-Hakim, BSc 1 Marc S. Kaadi, BSc 2 Hussam A. Shaib, MSc 1 Danyelle D. Gerges, BSc 1 Pia A.

Transcription

1 Evlution of the Histopthology of the Respirtory System in Essentil Oil-Treted Broilers Following Chllenge With Myoplsm glliseptium nd/or H9N Influenz Virus Elie K. Brour, PhD Rindl G. El-Hkim, BS Mr S. Kdi, BS Hussm A. Shi, MS Dnyelle D. Gerges, BS Pi A. Nehme, MS Deprtment of Animl Sienes Fulty of Agriulturl nd Food Sienes Amerin University of Beirut Beirut, Lenon Deprtment of Agriulture Sint-Joseph University Beirut, Lenon KEY WORDS: Avin influenz virus (H9N), roilers, essentil oils, histopthology, Myoplsm glliseptium, respirtory system ABSTRACT The ojetive of this work is to evlute the impt of eulyptus nd peppermint essentil oils (Mentofin ) in the protetion of the respirtory system of roilers ginst ontrolled hllenges y Myoplsm glliseptium (MG) nd/or vin influenz virus H9N. Seventy -dy-old roilers were rered in 7 groups ( irds/group) up to week of ge. Group ws the ontrol (nontreted with Mentofin nd unhllenged); hllenged groups were Group (non-treted with Mentofin nd MG hllenged), Group 3 (Mentofin treted nd MG hl- lenged), Group (non-treted with Mentofin nd H9N hllenged), Group 5 (Mentofin treted nd H9N hllenged), Group (non-treted with Mentofin nd MG/H9N hllenged), nd Group 7 (Mentofin treted nd MG/H9N hllenged). At week of ge, n intrtrhel hllenge of the irds with MG ( hemgglutintion units/.5 ml/ird) nd/or H9N ( hemgglutintion units/.5 ml/ird) ws given to speifi groups mentioned previously. Essentil oils of Mentofin were dministered for dys, effetive dy post-hllenge. Histopthologil oservtions were onluded t dys post-hllenge nd reveled signifint redution (P <.5) in mirosopi tissue lesions of irds treted with Mentofin in omprison to irds deprived from this tretment ut 93

2 hllenged similrly. The signifint (P <.5) redution in mirosopi lesions inluded derese in trhel deilition in MG- nd MG/H9N-hllenged irds, derese in muosl hypertrophy in MG-, H9N-, nd MG/H9N-hllenged irds, derese in golet ell degenertion in MGnd MG/H9N-hllenged irds, derese in muus umultion in MG-hllenged irds, nd derese in heterophil infiltrtion in MG/H9N-hllenged irds. INTRODUCTION Myoplsm glliseptium (MG) is the ustive gent of hroni respirtory disese in hikens, ommon disese in mny poultry speies round the world. Although the linil mnifesttions re usully slow to develop, MG in omintion with other respirtory pthogens n use severe irsulitis., Beside feed nd egg prodution redution, MG prolems re of high eonomi signifine sine respirtory trt lesions n use high moridity, high mortlity, nd signifint rss ondemntion nd downgrding. Consequently, the efforts to limit the losses from MG infetions would e of primry importne to the poultry industry. Avin influenz virus is one of the most devstting virl diseses in the poultry industry nd hs worldwide distriution. Frequent inidenes of H9N vin influenz type with high mortlity were oserved ommonly on roiler frms of the Middle Est region. 3 Mixed infetions with other respirtory pthogens, prtiulrly MG, were inriminted resulting in high mortlity on poultry frms, ssoited with gret eonomi losses. 3 Control of this virus would e of primry importne, espeilly with rising onerns of the potentil of H9N-AI to indue pndemi spred. The essentil oils of eulyptus nd peppermint present in Mentofin hve proven effetive ginst respirtory omplexes. In ft, Brour et l 5 5 found tht the dministrtion of Mentofin following NDV, IBV, nd IBDV vintion in MG/H9N- infeted roilers oosted the immune response nd meliorted their performne. Consequently, the dministrtion of Mentofin ould hve similr lleviting effet on signs nd lesions of MG- nd/or H9N-infeted roilers. Although severl experiments were onduted to study the intertion etween MG nd other vin respirtory pthogens,,7 no previous studies on intertion etween ontrolled hllenge y MG nd/or H9N in poultry were done. The ft tht these pthogens re frequently present in the Middle Est region nd mny other prts of the world mkes the study of the effet of ontrolling gent on MG/H9N intertion of prmount importne. The purpose of this projet is to evlute nd understnd, t the ellulr level, the impt of Mentofin on protetion of the respirtory system of roilers ginst ontrolled hllenges y MG nd/or H9N vin influenz. The histopthologil work for understnding the mirosopi hnges t the ellulr level of the trhe inluded the oservtion of the following: trhel deilition, muosl hypertrophy, golet ell degenertion, muus umultion, nd heterophil infiltrtion. MATERIALS AND METHODS Birds Seventy -dy-old roiler hiks were divided eqully into 7 groups ( hiks/group). The 7 groups were put in isoltion rooms supplied with infrred lmps, wterers, nd feeders. All irds were fed with the sme feed s reommended previously. 8 Preprtion of Chllenge Strins MG isolte A trhel sw of ird from n MGinfeted flok ws ultured t 37 C in Frey's roth for dys nd suultured onto Frey's gr for nother dys. One MG-suspeted olony with fried egg olonil morpholo- 9

3 gy ws tested ginst MG-speifi hiken ntiody (SPAFOS, Connetiut, USA). A onjugte, whih is got nti-hiken IgG ntiody (hevy + light hin), leled with fluoresin (KPL Lortories, Githerserg, Mrylnd, USA) ws used. The reting ntiodies to the sttioned fixed MG-suspeted ells on mirosopi slide were oserved under fluoresent mirosope (Lei DMLS, Germny) to onfirm the identity of MG. The loned-purified olony ws grown in lrger volume of Frey's roth for dys t 37 C. Pellets of MG were olleted y entrifugtion of the Frey's roth ulture t 5557 g for 5 minutes. The MG pellets were reonstituted in 3 ml of sterile sline nd the hemgglutintion (HA) titer ws determined ginst.% of hiken red lood ell suspension. 9 The reonstituted MG ws further diluted to reh HA units/.5 ml of sline, n djusted MG suspension for hllenge. H9N isolte The H9N strin ws isolted from severe vin influenz outrek in roilers in the summer of. The H9 omponent ws identified t the Amerin University of Beirut, while the omplete sutyping ws ompleted on the isolte t the Centrl Veterinry Lortory of Weyridge, Englnd. A report ws issued y Dr. Ruth Mnvel t Weyridge onfirming the sutype s H9N. The H9N ws preserved t -8 C in our lortory s : rtio of llntoi fluid:tryptose phosphte roth. This mix ws diluted in sterile sline to result in HA units/.5 ml, n djusted virl stok for hllenge. Chllenge nd Tretment The 7 groups of hiks were rered to week of ge. At week of ge, the following hllenges were given s shown in Tle. Mentofin ws diluted in V/V of.5 ml/ ml of drinking wter. Eh treted ird strted reeiving the diluted Mentofin, intr-esophgelly, twie dy (morning nd evening), in volume of ml/ird/time. The tretment ws for dys, strting on the hllenge dy. Tle. Chllenges Administered Esophgelly to the Broilers t Week of Age. Chllenges* Mentofin Tretment None None MG None 3 MG Yes H9N None 5 H9N Yes MG/H9N None 7 MG/H9N Yes *The MG nd H9N were eh given in volume of.5 ml intrtrhelly. The MG nd H9N were eh given s HA units/.5 ml/ird. Mentofin ws dministered on the hllenge dy; the dministrtion ws esophgel, twie per dy (morning nd evening) in volume of ml/ird/eh time dy, nd for period of dys. The dilution of the dministered Mentofin ws s instruted y the mnufturer. Histopthology All irds were srified y CO sphyxition t dys post-hllenge. A -m rosssetionl ut of eh trhe from srified irds ws tken nd kept in % formlin/pbs uffer. The trhel uts were sujeted to ross-setioning of μm nd H&E stining ording to previous proedure. The oservtion of trhel deilition, muosl hypertrophy, golet ell degenertion, muus umultion, nd heterophil infiltrtion from trhel uts/ird were seen mirosopilly t within 3 fields/ut loted t, 8, nd lok positions. A sore of ws given for eh of the following trhel tissue hnges, nmely, deilition, muosl hypertrophy, golet ell degenertion, nd muus umultion, while sore of ws given to the sene of these trhel hnges. The verge sore of irds per group ws used in the histogrms. Cumultive heterophil ount in fields of trhel uts/ird ws reorded, nd the verge ount of irds ws used in the histogrm. Mirosopi imges, mgnified, were tken from seleted histopthologil slides to ompre the tissue hnges in different roiler groups. 95

4 Sttistis The men sore of the histopthologil oservtions of the trhel setions ws done y -wy ANOVA followed y Dunn's test, llowing for sttistil omprisons mong the different groups. Animl Ethis The Institutionl Animl Cre nd Use Committee t the Amerin University of Beirut ides y the interdisiplinry priniples nd guidelines for the use of nimls in reserh, nd this work hs een pproved ording to suh priniples. RESULTS The histopthology of the trhel setions unovered the onsistent signifint redution in mirosopi lesions in the Mentofintreted groups in omprison to the similrly hllenged ut Mentofin-deprived groups (P <.5), nmely, for the mirosopi lesions of trhel deilition, muosl hypertrophy, golet ell degenertion, muus umultion, nd heterophil infiltrtion. A omprison of trhel deilited tissue from MG/H9N-hllenged ird deprived of Mentofin tretment in Group to norml ilition in similrly hllenged nd Mentofin-treted ird of Group 7 is shown in Figure. The men sores of irds in eh of the 7 groups showing deilition re shown in Figure. There ws n pprent redution in trhel deilition in Mentofin-treted 3 nd 7 in omprison to similr respetive hllenged nd tht were deprived of Mentofin; however, the signifine in this redution t P <.5 ws shown in Group 7 in omprison to Group, where oth were hllenged with MG/H9N orgnisms. Birds of the 3 differently hllenged groups showed muosl hypertrophy on the th dy post-hllenge. Figure 3 shows mirosopi slide setion of ird's trhe from Group deprived of Mentofin nd hllenged with MG/H9N in omprison to setion from the trhe of ird from 9 Group 7 hllenged with MG/H9N ut treted with Mentofin. The men sores of muosl hypertrophy in the 7 groups of roilers re shown in Figure. This mirosopi lesion of muosl hypertrophy ws redued signifintly (P <.5) in Mentofin-treted groups in omprison to Mentofin-deprived groups with similr hllenges. The omprison of the mirosopi slide photo of trhe with golet ell-degenertion (ird of Group, MG hllenged nd deprived of Mentofin) versus nother trhe with norml intt golet ells (ird of Group 3, MG hllenged nd Mentofin treted) is shown in Figure 5. The men sores of golet ell degenertion mong the 7 groups of roilers re shown in Figure. There ws n pprent protetion of golet ells ginst degenertion y MG hllenge (Group 3, Mentofin treted) nd MG/H9N hllenge (Group 7, Mentofin treted) in omprison to similrly hllenged nd, respetively, ut deprived of Mentofin (P <.5). Trhel uts with muus umultion versus ler tissues re shown in Figure 7. The men sores of muus umultion in the trhel setions of the 7 groups re shown in Figure 8. There ws derese (P >.5) in muus umultion in trhel setions of irds treted with Mentofin ( 3, 5, nd 7) ompred with Mentofin-deprived nd respetively hllenged,, nd. Lst ut not lest, the heterophil infiltrtion sore ws redued signifintly (P <.5) in the Mentofin-treted irds of Group 7 in omprison to Group irds deprived of tretment, in whih oth groups hd similr hllenge with MG/H9N (Figure 9). An insignifint redution in trhel heterophil ourred in irds of Group 3 (Mentofin treted, MG hllenged) in omprison to those of Group (Mentofin deprived, MG hllenge) (P >.5). On the ontrry, the heterophil sore inresed in Mentofin-treted irds of Group 5 (hllenged with H9N) in omprison to Mentofin-deprived irds of Group (given

5 VolIss.qxp // 8:57 AM Pge 97 Figure. A mirosope slide photogrph of deilited (Group, MG/H9N hllenged nd Mentofin deprived) versus norml ilited trhel tissue (Group 7, MG/H9N hllenged nd Mentofin treted), mgnified. Norml ilition Deilited Figure. Men sores of trhel deilition in the 7 roiler groups. Alphets on the histogrm tht differ mong groups re signifint t P <.5. Men sore of deilition, Figure 3. A mirosopi slide photo of muosl hypertrophy in trhe from MG/H9N-hllenged ird of Group (deprived of Mentofin) versus the sene of muosl hypertrophy of ird treted with Mentofin in Group 7 nd similrly hllenged, mgnified. 8 d the sme hllenge). The mirosopi slide photo in Figure shows the ontrst etween low heterophil infiltrtion in Mentofin-treted Group 7 nd high heterophil infiltrtion in the irds' trhe of Group (deprived of Mentofin). DISCUSSION The redution of deilition y the essentil oil ws more pprent (P <.5) in the MG/H9N-hllenged groups. The tive ingredient in Mentofin seems to protet ginst deilition tht is normlly used y MG nd H9N. This protetion ginst the first line of defense in the poultry host is of prmount importne, sine it will protet ginst oloniztion y seondry teril infetions. It is worth noting tht the iliry tivity of humn respirtory ells is ffeted y exposure to the essentil oil of Eulyptus spp. The thinning of the muus in Muosl hypertrophy Asene of muosl hypertrophy the respirtory trt y the tive ingredients of Eulyptus spp. ould help in its outwrd flow, pushing with it the miroorgnisms, preventing their oloniztion, nd thus proteting the ili from onsequent dmge.3, 97

6 VolIss.qxp // 8:59 AM Pge 98 Figure. Men sores of muosl hypertrophy in the 7 roiler groups. Alphets on the histogrm tht differ mong groups re signifint t P <.5. Figure. Men sores of golet ell degenertion in the 7 roiler groups. Alphets on the histogrm tht differ mong groups re signifint t P <.5. Men sore of muosl hypertrophy Men sore of golet ell degenertion 8 d d Figure 5. A mirosopi slide photo of degenerted golet ells in trhe of MG-hllenged nd Mentofin-deprived hiken (Group ) versus intt golet ells in trhe of MG-hllenged nd Mentofin-treted irds (Group 3), mgnified. 98 Figure 7. A mirosopi slide photo of the trhel setion with muus umultion (Group, MG-hllenged ird deprived of Mentofin) versus muus-ler trhel setion (Group 3, MG-hllenged ird treted with Mentofin), mgnified. Degenerted golet ells Muus umultion Intt golet ells No umultion of muus

7 Figure 8. Men sores of muus umultion in trhel setion of the 7 groups. Alphets on the histogrm tht differ mong groups re signifint t P <.5. Figure 9. The men heterophil numer per fields/ trhel uts/ird. Alphets on the histogrm tht differ mong groups re signifint t P <.5. Men sore of muous umultion Men heterophil sore Figure : A mirosopi slide photo of trhel setion with low heterophil infiltrtion (Group 7, MG/H9N hllenged nd Mentofin treted) versus high heterophil infiltrtion (Group, MG/H9N hllenged nd Mentofin deprived), mgnified. High heterophil infiltrtion Low heterophil infiltrtion Muosl hypertrophy ws signifintly redued y essentil oil in irds exposed to ny of the 3 different hllenges (P <.5). This is ler effet of Mentofin tive ingredients on the integrity of the muosl lyer of hllenged irds keeping this lyer of tissue in nerness to the thikness of ontrol irds (unhllenged, Group ). The prevention of hypertrophy of the muosl lyer y suh ingredients ws doumented in previous work performed on lipopolyshride (LPS)-indued ronhitis in rts, proving the redution of inflmmtory ell infiltrtion into the epithelium of trhe nd ronhioles in rts dministered the essentil oil of Eulyptus gloulus. 5 Degenertion in golet ells ws redued signifintly (P <.5) y essentil oil in MG- nd MG/H9N-hllenged irds. To our knowledge, there is no previous doumenttion on the impt of synergism mong the tive ingredients of Eulyptus spp. nd peppermint for providing protetion of the golet ells in the upper respirtory system of niml or humns. The mintenne of the golet ell struture nd funtion is importnt in respirtory diseses to keep the muus flow in the ir pssges, thus pushing the germs nd other prtiles, with the help of the mintined ili, nteriorly nd outwrds through the mouth or nostrils. The muus umultion ws redued signifintly (P <.5) in MG-hllenged irds dministered the essentil oil. The role of the tive ingredients of Mentofin in 99

8 lering the muus from the trhel tissue ws reported in other works. 3-8 The heterophil infiltrtion ws redued signifintly (P <.5) y essentil oil in MG/H9N-hllenged irds, while the infiltrtion ws inresed signifintly y essentil oil in irds hllenged y H9N lone. This ontrdition ould e due to the nture of the hllenge in nd 5 tht differed from those in, 3,, nd 7. It is worth noting tht one work doumented the impt of stem-distilled essentil oil from Eulyptus gloulus on redution of inflmmtory ell infiltrtion nd muin hyperseretion in rts exposed to PS hllenge in their respirtory system. 5 In onlusion, histopthologil hnges were oserved in Mentofin-treted versus Mentofin-deprived roilers sujeted to 3 different ntures of hllenges (MG, H9N, nd omintion of MG/H9N). The Mentofin tretment resulted in signifint derese (P <.5) in trhel deilition in MG- nd MG/H9N-hllenged irds, signifint derese (P <.5) in trhel golet ells degenertion in MG- nd MG/H9N-hllenged irds, signifint derese (P <.5) in trhel muus umultion in MG-hllenged irds, nd signifint derese (P <.5) in heterophil infiltrtion in MG/H9N-hllenged irds. These results demonstrte the heling effet of Mentofin ginst MG/H9N infetion, omplex respirtory disese of eonomi importne to the poultry industry. REFERENCES. Kleven SH: Myoplsmosis. In: Swyne DE, Glisson JR, Jkwood MW, Person JE, Reed WM, ed. A Lortory Mnul for the Isoltion nd Identifition of Avin Pthogens. th ed. Kennett Squre, P: Amerin Assoition of Avin Pthologists; 998:7-8.. Ley DH, Yoder HW Jr: Myoplsm glliseptium infetion. In: Clnek BW, Brnes HJ, Berd CW, MDougld LR, Sif YM, eds. Diseses of Poultry. th ed. Ames, Iow: Iow Stte University Press; 997: Nili H, Assi K: Nturl ses nd experimentl study of H9N vin influenz in ommeril roiler hikens of Irn. Avin Pthol ;3:7-5.. Chen H, Suro K, Swyne D, et l: Genertion nd evlution of high-growth ressortnt H9N influenz A virus s pndemi vine ndidte. Vine 3;: Brour EK, Dnkr, S, et l.: Essentil oils of Eulyptus nd peppermint improve the homogeneity of immune responses nd performne in MG/H9N-infeted roilers. J Am Holisti Vet Med Asso 5; :3-7.. Brdury JM: Avin myoplsm infetion: prototype of mixed infetions with myoplsms, teri nd viruses. Ann Miroiol (Institut Psteur) 98;35A: Corstvet RE, Sdler WW: A omprtive study of single nd multiple respirtory infetions with Myoplsm glliseptium, Newstle disese virus, nd infetious ronhitis virus. Am J Vet Res 99;7: Ntionl Reserh Counil. Nutrient Requirement of Poultry. 8th revised ed. Wshington DC: Ntionl Ademy Press; 98: Hierholzer JC: Adenoviruses. In: Shmidt NJ, Lennette DA, Lennette ET, Lennette EH, Emmons RW, eds. Dignosti Proedures for Virl, Rikettsil nd Chlmydil Infetions. 7th ed. Wshington, DC: Amerin Puli Helth Assoition; 995: Rndll JC, Reee LR: Avin Histopthology. Chigo, Ill: Mosy-Wolfe; 9:5-7.. Reynolds HY: Modulting irwy defenses ginst miroes. Curr Opin Pulm Med ;8:5-5.. Riehelmnn H, Brommer C, Hinni M, et l: Response of humn ilited respirtory ells to mixture of menthol, eulyptus oil nd pine needle oil. Arzneimittelforshung. 997;7: Zky-Rones Z, Thom E, Wolln T, et l: Rndomized study of the effiy nd sfety of orl eldererry extrt in the tretment of influenz A nd B virus infetions. J Intern Med Res ;3:3-.. Slri MH, Amine G, Shirzi MH, et l: Antiteril effets of Eulyptus gloulus lef extrt on pthogeni teri isolted from speimens of ptients with respirtory trt disorders. Clin Miroiol Infet ;: Lu XQ, Tng FD, Wng Y, et l: Effet of Eulyptus gloulus oil on lipopolyshrideindued hroni ronhitis nd muin hyperseretion in rts. Zhongguo Zhong Yo Z Zhi ;9:8-7.. Wurges J: Eulyptus. In: Gle Group. Gle Enylopedi of Alterntive Mediine.. Aville t: 7. Gwronski DV: Medil hoies. In: Gle Group. Gle Enylopedi of Mediine.. 8. Pge L: Helthy Heling: A Guide to Self-Heling for Everyone.th ed. Srsot FL: Bookworld Servies; :-. 3