Polycarp Nwunuji Tanko, Benjamin O. Emikpe and Yusoff Mohd Sabri

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1 Veterinary Wrld, EISSN: RESEARCH ARTICLE Open Access Evaluatin f the shedding rutes and serlgical patterns in experimentally-induced Brucella melitensis infectin in dexamethasnetreated and transprt-stressed gats Plycarp Nwunuji Tank, Benjamin O. Emikpe and Yusff Mhd Sabri 1. Department f Veterinary Pathlgy and Micrbilgy, Faculty f Veterinary Medicine, Universiti Putra Malaysia, Malaysia; 2. Department f Veterinary Pathlgy, Faculty f Veterinary Medicine, University f Ibadan, Ibadan, Nigeria Crrespnding authr: Yusff Mhd Sabri, sabri@vet.upm.edu.my Received: , Revised: , Accepted: , Published nline: di: /vetwrld Hw t cite this article: Tank PN, Emikpe BO and Sabri MY (2013) Evaluatin f the shedding rutes and serlgical patterns in experimentally-induced Brucella melitensis infectin in dexamethasne-treated and transprt-stressed gats, Veterinary Wrld 6(9): Abstract Aim: T identify and evaluate the shedding rutes and patterns fllwing experimentally-induced Brucella melitensis infectin in dexamethasne-treated and transprt-stressed gats. Materials and Methds: Twenty fur healthy, adult gats were divided int 4 grups: A, B, C and D respectively. Grup A 7 was treated with dexamethasne fr 8 days prir t inculatin with 10 Clny Frming Units f B. melitensis via the intracular rute. Grup B was transprted fr 3 hurs prir t inculatin with a similar dse. Grup C was inculated with a similar dse withut subjecting the animals t any prir treatment, and this grup served as ur psitive cntrl. Grup D was nt inculated with the infective dse and served as ur negative cntrl. Bld samples alng with nasal, cular, and vaginal swabs were cllected n days 0, 3, 7, 10, 14, and weekly thereafter until day 63 pst inculatin (pi) and were analyzed by PCR, Rse Bengal Plate Test (RBPT), and indirect ELISA techniques. Results: The nasal, cular and vaginal swabs tested psitive fr Brucellsis with PCR frm day 7, with nasal rute being the first and mst cnsistent rute t reveal the psitive results. Grup B shwed the earliest nset f shedding the bacterium (day 7) fllwed by grup A which started frm day 10 and shed relatively mre psitive f the bacterium via the rutes examined. Bld samples tested psitive with PCR frm day 7 thrugh 14 and the results were incnsistent subsequently. Sera samples tested psitive with RBPT n day 14 in all the 3 infected grups but mre cnsistent in grup C. On the ther hand, tests using ELISA shwed psitive results frm day 7 pi, with grup C having a 100% sercnvertin while grups A and B shwed nly 50% sercnvertin. Cnclusin: The cnsistent shedding via the nasal, cular, and vaginal rutes in grups A and B implied pssible immunsuppressin in the infected animals. We recmmend that prgrams designed t cntrl Brucellsis shuld cnsider analyzing a larger number f bilgical samples t enhance the accuracy f identificatin f shedders. Keywrds: brucellsis, cnsistent shedding, gats, immunsuppressin, plymerase chain reactin, serlgical tests, swabs. Intrductin f the develping natins that have n effective vaccinatin-based cntrl and slaughter prgrammes, such as the cuntries in Mediterranean, Middle East, Africa, Latin America, and parts f Asia [7]. Gats in the trpics are usually reared under the traditinal extensive management system and are ften transprted ver a cnsiderable distance fr marketing and slaughter purpses [8,9]. This lng range transpr- tatin, an age ld inevitable husbandry practice [10], creates undue stress n the animals. In this regard, it is imprtant t nte that the artificially-induced stress upn administratin f dexamethasne has been reprted t mimic the stress elicited by natural causes [11,12,13]. Studies investigating the effect f stress n the prductivity f small ruminants' have primarily fcused n caprine pneumnia [14,15] with very little emphasis n the elucidating the effect f stress n the transmissin f zntic diseases. With a myriad f stress factrs that gats are cnstantly subjected t, it is imperative t understand the interactins between stress, reprductive zntic disease ccurrence, and transmissin f the disease. Brucellsis is nw recgnized as ne f the mst imprtant amng the cmmn zntic diseases acrss the wrld and is a disease that pses a majr threat t bth human health and animal ppulatins [1]. The Wrld Health Organizatin cnsiders it as a neglected znsis, because adequate cntrl prgrammes were nt planned in numerus cuntries despite its huge impact nt nly n human and animal health, but als n the ecnmy [2]. Brucellsis, especially affecting the fd animals, pses a barrier t the trade f animals and animal prducts between cuntries. This is because Brucellsis causes cnsiderable ecnmic lsses due t abrtins and fertility prblems, encuntered ften in the sheep and gat industry [3, 4, 5]. Brucellsis in small ruminants, especially gats is caused by B. melitensis [6] and the incidences f transmissin t the human ppulatin have significantly increased in mst This article is an pen access article licensed under the terms f the Creative Cmmns Attributin License ( rg/licenses/by/2.0) which permits unrestricted use, distributin and reprductin in any medium, prvided the wrk is prperly cited. Veterinary Wrld, EISSN:

2 Understanding the interactin between the afremen- fr less than 15 minutes under a shade (fr resting tined factrs is critical t enhance ur knwledge base purpses). The gats were neither fed nr watered fr prper planning and swift cntrl f these diseases during the resting perid. The animals were then in gats, especially in thse herds that are cnstantly infected after being ff laded with dses that were subjected t envirnmental and managemental similar t thse administered t grup A. Animals in stresses. T the best f ur knwledge, there is n study grup C were neither treated with dexame-thasne nr that evaluated the relatinship between stressful transprted but were als infected with dses similar t cnditins and the shedding pattern f Brucella melitensis thse administered t grup A and served as psitive infectin in gats. cntrls. Animals in grup D served as the negative Therefre, this study seeks t elucidate the cntrls. shedding rutes f experimentally-infected gats by subjecting them t dexamethasne treatment and Sampling: Bld, cular, nasal and vaginal swabs were transprt stress in rder t evaluate the shedding rutes cllected n days 0, 3, 7, 10, 14 and weekly thereafter and patterns using serlgical tests and PCR f until day 63 pst infectin (pi). The bld samples were samples cllected frm ptential shedders. cllected in duplicates cntaining bth heparinized and nn-heparinized samples. Dry and sterile cttn wl Materials and Methds swabs were used t cllect the cular, nasal and vaginal swabs. Animals: Twenty fur healthy bar gats used fr this experiment were hused in designated pens in the PCR assay: DNA was extracted frm the heparinized Faculty f Veterinary Medicine, experimental unit, bld using Prmega kit by fllwing the manufacturer's Universiti Putra Malaysia. The animals were selected instructins after which PCR was perfrmed using B. after their sera tested negative fr Brucellsis using 1 melitensis specific primers [18] (5 AGTGTTTCG GC Rse Bengal Plate Test (RBPT) and indirect ELISA. 1 1 TCAGAATTAATC 3 and 5 CATGCGCTAT GTCT G Additinally, the cular, nasal and vaginal swabs frm 1 GTTAC 3 ). Briefly, the PCR was carried ut with 2µl these gats were plated in Brucella agar and incubated f the extracted DNA in 48µl f master mix at 37 C fr 4 days. The plates were cnfirmed negative (Fermentas, Germany) in a ttal vlume f 50µl in using PCR using specific primers t B. melitensis. The Eppendrf master cycler (Germany). The Eppendrf animals were fed with cncentrates, grass and water ad master cycler was prgrammed as fllws; Initial libitum. Animals were stabilized fr 3 weeks and then denaturatin at 94 C fr 4 minutes fllwed by 34 randmly divided int 4 grups designated as A (6), B cycles at 94 C fr 1 minute annealing at C fr 1 (6), C (6) and D (6) prir t the nset f the experiment. minute, extensin at 72 C fr 1 minute, final extensin Animals in grup A were administered dexamethasne at 72 Cfr 10 minutes and hld at 4 C. The PCR (Try Labratries Pvt. Limited, prducts were analyzed by electrphresis n 1% 10kg bdy weight fr eight days fllwing which they agarse gel stained with gel red and visualized and 7 were infected with 10 Clny Frming Units (CFU) f phtgraphed. B. melitensis strain 183. Animals in grup B were The swabs were plated n Brucella Agar and transprted by rad. The mean envirnmental temper- incubated at 37 C fr 4 days after which they were ature ranged between C (Average 38 C) and the tested using PCR with B. melitensis primers as stated relative humidity was between 17 20%. earlier. Briefly, 2µl f DNA Zl was added t 2µl f the clnies in Eppendrf tubes and allwed t stay fr Ethical apprval: The experimental design and animal treatment was in cmpliance with the humane methds 15minutes befre adding the master mix as described recmmended by the Research Ethics Cmmittee, abve. The negative cntrl used was the master mix Faculty f Veterinary Medicine, Universiti Putra withut DNA template and the external psitive Malaysia (AUP N: 12R159). cntrl was DNA templates extracted frm a Brucella 7 The handling, lading and transprtatin f the brth cntaining 10 CFU f B. melitensis strain 183 experimental gats were carried ut humanely in using Prmega Genmic DNA purificatin kit (Prmega, accrdance with the guidelines gverning animal Germany). transprt welfare by rad [16]. A standard Bedfrd, Sera were als harvested frm the nn- pen-rf (Frd, UK), vehicle was used fr the heparinized bld and stred at -20 C until used fr transprtatin. The flr f the van was prvided with RBPT and ELISA. Fr the RBPT, a drp frm each beddings (wd shavings) fr secure fting. The serum sample was laid n a plate and a drp f the 2 gats were stcked at a rate f 0.2 m per animal [17]. RBPT reagent was added, mixed well and allwed t The jurney cmmenced by 1000 hurs and was stay fr 10 minutes. In this test, a classical agglutinatin terminated at 1330 hurs. The vehicle travelled n a reactin was cnsidered psitive fr Brucella. Fr the typical asphalt duble lane rad frm Serdang t indirect ELISA, the antigen was prepared and used t Malaka and back (3 00'N, 'E) Malaysia, cvering cat the wells f a plystyrene plate (Nunc-Immun a ttal distance f abut 350km. The vehicle travelled plate with MaxiSrp surface). After vernight incubatin with an average speed f 60 km/h and had a stp-ver at 4 C, the plate was washed 3 times with the washing Veterinary Wrld, EISSN:

3 Table-1. PCR result fr Nasal, Ocular and Vaginal swabs that were tested fr B. melitensis. Grup Shedding Rute D7 D10 D14 D21 D28 D35 D42 D49 D56 D63 A Nasal 0 0 +(2) +(3) +(2) +(2) +(1) Ocular 0 +(1) +(1) +(2) 0 +(2) +(2) 0 0 +(1) Vagina 0 +(2) +(1) +(3) 0 +(1) B Nasal +(3) +(1) (2) +(1) 0 +(1) 0 Ocular 0 +(2) +(1) +(1) +(1) 0 0 +(1) 0 0 Vagina 0 +(1) C Nasal 0 +(1) +(1) +(1) 0 +(1) 0 0 +(1) 0 Ocular Vagina (1) +(1) +(1) 0 0 D Nasal Ocular Vagina Table-2. Cmparisn f the relatinship between the 3 shedding rutes Grup Shedding rute D7 D10 D14 D21 D28 D42 D49 A NV 0 0 +(1) +(2) 0 +(1) 0 NO (1) 0 +(2) +(1) OV 0 +(1) 0 +(2) 0 +(1) 0 NOV (1) 0 +(1) 0 B NV +(1) (1) 0 NO 0 0 +(1) OV NOV C NV NO OV NOV N: Nasal, V: Vagina and O: Ocular rute buffer (Phsphate Buffered Saline, ph 7.4, with 0.05% Sneezing, ear biting and cughing were als bserved Tween 20). 200 µl f Blcking Buffer was added t t varying degrees within the first 21 days pi in grups each well and incubated at 37 C fr 1hr and washed 3 A and Bnly. times again with the washing buffer. 50 µl f serum Shedding rute: The animals started shedding the frm samples at the dilutin f 1:200 were added t the bacterium frm day 7 pi, first in grup B with 50% f wells and incubated fr anther 1hr and washed 3 times the animals shedding thrugh the nasal rute fllwed with the washing buffer. Hrse Radish Rabbit Anti- by the vaginal rute (Table-1). Significant difference Gat immuncnjugate IgG Perxidase (Nrdic (P<0.05) in the shedding pattern was bserved in Immunlgy, Netherlands) was added and incubated grups A and B when cmpared t grup C. Grup A fr anther hur and washed again 3 times. Then 100µl started shedding thrugh the nasal rute frm day 10 pi. f 3,3,5,5-tetramethylbenzidine (TMB) One slutin As the infectin prgressed, majrity f the animals in substrate (Prmega, USA) was added and incubated fr grup A shed the bacterium via all the rutes examined 10 minutes. The reactin was stpped by the additin and relatively fr a lnger perid with sme f the f 50 µl f 2.5 M sulphuric acid. The ptical density animals shedding cnsistently fr 5 weeks. Grup C, values were measured at 450 nm wavelength in an hwever, did nt shed the bacterium via the vaginal AnthsZenyth 340 st reader (Austria). rute until day 35 pi; while grups A and B shed thrugh this rute as early as day 10 pi. Thrughut the Statistical analysis: Data btained in this study were analyzed using Statistix 9.0 sftware, using repeated experiment, nly 1 animal frm A shed the bacterium in Measure Analysis f Variance and LSD. All-pairwise all the 3 rutes at day 21 and day 35. By day 14 pi, cmparisns was used t test the differences between 100% f animals in grups A and B shed the bacterium the grups. The criterin fr statistical significance was at least nce while nly 33% f animals in grup C set at P < shed the bacterium. All the animals shed the bacterium at least nce in the perid cvered fr the experiment Results with the exceptin f the negative cntrl grup (grup General bservatins: Grup A had a cnsistently D) which remained negative thrughut since grup D higher bdy temperature when cmpared t grups B was nt inculated with the infective dse. and C. The thermal respnse was undulant and differed Cmparisn f the three shedding rutes: Grups A significantly between grups. There was n significant and B shed significantly (P<0.05) higher when difference in the bdy temperature pattern in grups A cmpared t grup C. 100% f the animals in grup A and B, but there was a significant difference between shed the bacterium in at least 2 rutes at a time in this grups A, B and C when cmpared t grup D. A study and they shed mre cnsistently when cmpared generalized inappetence was bserved in all the t grups B and C (Table-2). In grup A, 50% f the infected grups n day 8 pi, but mre severe in grup A. animals shed cncmitantly thrugh the nasal and Veterinary Wrld, EISSN:

4 Figure-1. The pattern f antibdy respnse t experimental B. melitensis infectin in grup A : 1, B : 2 and C : 3; in percentage f psitivity Figure-2. PCR f bld samples n day 7 pi. M: 1-kb standard ladder, Lane 1: negative cntrl, Lane 2: psitive cntrl, Lane 3-10: psitive samples vaginal rutes, 33% thrugh nasal and cular rutes, least 2 ut f the 3 rutes investigated, but the serlgical 50% thrugh cular and vaginal and 17% shed twice tests (RBPT and ielisa) were nt cnsistent in via all the 3 rutes. identifying all the shedders. Animals in grup A started In grup B, hwever, nly 33% f the animals shedding n day 10 pi, where 33% f the animals shed shed the bacterium thrugh at least 2 rutes at a time. the bacterium. These shedders were psitive with 33% f the animals shed thrugh the nasal and cular ELISA (ELISA been mre sensitive), but negative with rutes and 17% shed thrugh nasal and cular rutes RBPT. Hwever, the RBPT was able t identify all the while nne shed thrugh the cular and vaginal and all shedders at day 14 pi and by day 21pi, which was the the 3 rutes. Nne f the animals frm grup C shed the peak shedding perid (100%) fr grup A, bth ELISA bacterium in any 2 rutes at a time thrughut the and RBPT were able t detect nly 17% f the animals study. Even thugh the cular rute was used fr the in grup A that were shedding the bacterium. Even infectin, nne f the animals frm grup C shed thugh the ability f the serlgical tests t identify the thrugh this rute. Hwever, nne f the animals frm shedders increased ver time in this study, they were all the grups shed the bacterium in any 2 rutes at a nt able t detect all the shedders frm this grup. time after day 49 and als the cnsistency f shedding There was n significant difference in the ability f decreased after day 49. RBPT and ELISA in the detectin f shedders and nnshedders amng the grup. On the ther hand, PCR was Serlgical tests: Fr the RBPT, Grup C tested able t detect mre accurately in grups A and B than psitive significantly (P<0.05) when cmpared t grup C in all the rutes with significant difference (P < grups A and B n day 14 pi, where 100% f animals in grup C tested psitive and the number f psitives 0.05) between grups A and B cmpared t grup C eventually drpped and remained cnstant at 50% with respect t the cular rute. frm day 21 t day 49 pi. 60% f thse in grup A tested Animals in grup B hwever, started shedding as psitive at day 14 and the number f psitives als early as day 7 pi and unlike grup A, peaked between drpped t 17% frm day 21 nward. 50% f animals day 7 and day 10 pi with 50% f the animals shedding. frm grup B tested psitive n day 14 and then ELISA was able t detect nly 25% f the shedders at drpped t 17% frm day 21 nward. The indirect this perid, while RBPT was negative in all. ELISA (Figure-1) was able t detect the antigen earlier Subsequently, the rate f shedding drpped t 17% at cmpared t the RBPT in this study. 50% f the animals day 14 and ELISA was able t detect all the shedders in grup C tested psitive at day 7 pi and by day 14 pi, while the ability f RBPT t detect the psitives was all the animals in this grup tested psitive. 33% f the relatively less. We als bserved that the ELISA was animals in Grup B tested psitive n day 7 pi and by able t identify mre number f the shedders ver time. day 42 pi, 100% (6/6) tested psitive and remained Animals frm grup C shed less f the bacterium psitive thrughut the study. Only 17% f the animals cmpared t grups A and B, as expected. By day 14, in grup A tested psitive n day 7 pi but 100% by day nne f the animals in grup C was shedding via the 49 pi and remained psitive thrughut the study. rutes examined even thugh sme serlgically tested There was a significant difference (P<0.05) in the psitive with bth ELISA and RBPT. antibdy titres amng the treatment grups frm day 7 Plymerase Chain Reactin (PCR) was carried thrugh day 42 pi. ut using DNA extracted frm the bld samples f the Relatinship between the shedding rutes and the animals and this test detected psitive animals earlier serlgical respnse: In this study, the stressed animals than the serlgical tests. PCR detected 100% f the shwed increased shedding f the bacterium and in at animals in grup C and 50% in grup B as early as day 3 Veterinary Wrld, EISSN:

5 pi. By day 7 pi, bld samples frm all the infected in the stressed grups vs. the cntrl animals (grup C animals tested psitive and thereafter, mst f the infected, but nt subjected t prir stress) further animals were negative. Hwever, grup D (the uninfected implies that stress culd induce pathgen redistributin cntrl grup) remained negative thrughut the study. within the system f the infected animals and thereby cnstituting a high public health risk. This is typified Discussin by the earlier nset f shedding via the vaginal rute as The shedding f B. melitensis by dmesticated bserved in the stressed grups (grups A and B), when animals pses a very serius public health risk. Infected cmpared t the unstressed grup (grup C), despite animals are ften the surce f human infectin with the use f intracular rute fr the inculatin purpse. brucellsis, especially dmesticated animals and the The peak shedding bserved between days 7 and 10 pi cntrl f this disease in humans is nly achievable in grup B shwed the critical perid at which the thrugh the effective cntrl f the disease in animals, transprted animals culd shed; this perid shuld be hence its public health significance. Mst Cntrl taken int accunt while devising the cntrl prgrammes fr Brucellsis ften rely n serlgical strategies. The shedding bserved in grup A shwed tests fr screening and/r identificatin f infected that chrnically-stressed r immunsuppressed gats animals withut any emphasis n the detectin f culd cntinually shed the bacterium fr cnsiderably apparently animals that are shedding the bacterium. a lnger perid as bserved in diseases such as Q-fever This study evaluates the shedding rutes and the and enteric infectins [19, 22]. diagnstic ptential f analyzing mre than ne Our study als shwed that the shedding rate in bilgical sample in experimentally-infected grup C was relatively lwer and incnsistent (Table-1). (dexamethasne-treated r transprt-stressed) gats as The increase in the number f shedding rutes in the evaluated fr Cxiella burnetti infectin in the earlier stressed animals, as reprted in this study, further studies [19,20]. demnstrates the imprtant rle f stress in inducing All the infected animals in this study shed the nt nly the bacterial redistributin in viv but als in bacterium at least nce via ne f the 3 rutes the disease transmissin. investigated, the earliest being the nasal rute frm the In this study, the tw serlgical techniques emplyed transprt-stressed grup (grup B) at day 7 pi while shwed that stress induced either by dexamethasne r thse belnging t the dexamethasne-treated grup by transprt is capable f causing immune dysregu- (grup A) shed mre cnsistently in all the rutes latin [13, 24], an affect that pssibly can lead t less examined and fr a lnger perid starting frm day 10 sercnvertin bserved in the stressed grups pi when cmpared t animals that were infected shwing nly 50% f the animals being serpsitive withut any prir stress-inducing treatments (grup C). while grup C shwed a 100% (6/6) sercnvertin within the same perid. The use f the serlgical This result is in accrd with earlier studies [21, 22, 23] techniques t identify ptential shedders, especially in which reprted that stress factrs are linked t stressed gats, were nt encuraging as ur study increased pathgen carriage, increased disease shwed that RBPT identified all the animals (100%) susceptibility, and pssible pathgen shedding. Thus, shedding the bacterium in grup C whereas nly 66% stress factrs can lead t immune dysregulatin [24]. and 50% f the animals frm grups A and B tested There is evidence t shw that immunsuppressin psitive, respectively. The indirect ELISA used in this culd accunt fr severe lesins being encuntered in study was able t detect psitive animals at an earlier the respiratry tract in diseases such as peste des petits time when cmpared t the RBPT but it culd nt ruminants [13], and this may be ne f the reasns why identify all the shedders in the curse f this study until we bserved the dminance f the nasal rute in the day 35. The reduced and incnsistent serlgical shedding pattern in ur study. The prximity and the respnses bserved in grups A and B as detected with linkage f the cular and the nasal rutes culd als indirect ELISA and RBPT culd pssibly be assigned explain the dminance f the nasal rute in shedding t stress-induced immune dysregulatin [13]. the bacteria. The PCR perfrmed using the DNA extracted The earlier nset and prlnged shedding f the frm the bld samples identified the infected animals bacterium by the stressed and infected animals in this as early as day 3 pi, and n this day all the animals in study further cnfirmed that the cmbinatin f grup C tested psitive 50% frm grup B and nne infectin and stress culd pse a very serius risk t the frm grup A tested psitive. By day 7 pi, all the bld public health. Our finding als indicates that there is a samples f the animals infected in this study tested high ptential fr the spread f this infectin t humans psitive with PCR. Subsequently, the animals tested wh are invlved in small ruminant husbandry practices, psitive nly spradically. This culd be attributed t especially in the trpics. In agreement with ur the fact that the bacterium culd have migrated frm findings, earlier studies [25, 26] reprted that Brucella the bld and get trapped within the macrphages and infected animals culd serve as surces f infectin t nn-prfessinal phagcytes [27]. Furthermre, it was ther nn-infected animals as well as the human speculated that the incnsistency in the psitive results ppulace. The relatively increased shedding f the culd be due t re-infectin riginating frm intermittent bacterium thrugh the nasal, cular and vaginal rutes shedders. Veterinary Wrld, EISSN:

6 Cnclusin 7. Belay, T. (2008) Develpment f a real-time PCR assay fr Our study prvides vital infrmatin n the rle diagnsis f Brucella melitensis infectin in sheep. University Putra Malaysia Institutinal repsitry, 2008 f stress in mdulating the pattern and rutes f 8. Ay, J.O. and Oladele, S.B. (1996) Natural antixidants and shedding f B. melitensis in dexamethasne-treated their ptential uses in prphylactics and therapy f disease cnditins. W. Af. J. Pharmacl. Drug Res., 12: and transprt-stressed gats. Our significant findings 9. Minka, N.S. and Ay, J.O. (2007) Physilgical respnses f regarding the shedding patterns and the transmissin f transprted gats treated with ascrbic acid during the htbrucellsis in gats call fr actins t alleviate the dry seasn. Anim. Sci. J., 78: serius public health risks impsed by the shedding f 10. Fazi, E and Ferlazz, A. (2003) Evaluatin f stress during transprt. Vet Res Cm, 27(supplement 1): Brucella by the infected gats. Based n ur findings, 11. Zamri-Saad, M., Effendy, W. M., Maswati, M. A., Salim, N. we suggest that strategies aimed fr cntrlling and Sheikh-Omar, A. R. (1996) The gat as a mdel fr brucellsis shuld cnsider cllecting and analyzing studies f pneumnic pasteurellsis caused by Pasteurella mre than just ne bilgical sample. Furthermre, we multcida. Br. Vet. J., 152: recmmend that the cntrl strategies shuld give due 12. Frsberg, N.E. (2004) Recent Insights Int Ruminant Immune Functin: Effects f Stress and Immunstimulatry imprtance t varius methds fr the detectin f Nutritinal Prducts. Flrida Ruminant Nutritin Sympsium. persistent and intermittent shedders rather than just 13. Jagtap, S.P., Rajak, K.K., Garg, U.K., Sen, A.,Bhanuprakash, relying n serlgical tests t identify the infected V., Sudhakar, S.B., Balamurugan, V., Patel, A., Ahuja, A., animals. Singh, R.K. and Vanamayya, P.R. (2012) Effect f immun- suppressin n pathgenesis f peste des petits ruminants Authrs cntributins (PPR) virus infectin in gats, Micrb Pathgen. Di:10: 1016/j.micpath PNT carried ut the experiments and cllected the 14. Zamri, Saad, Jasni, M. S., Nurida, A. B. and Sheikh-Omar, A. samples. BOE carried ut the data analysis. MYS was a R. (1991) Experimental infectin f dexamethasne treated prject leader and carried ut the data analysis. All gats with Pasteurella haemlytica a2 Br. Vet. J. 147:5 65. authrs drafted and revised the manuscript. All authrs 15. Mhamed, R. A. (2002) The effect f irn cmpunds and ther factrs n the pathgenesis f pneumnic pasteurellsis read and apprved the final manuscript. in Nubian gats. Ph.D. Thesis, Faculty f Veterinary Science, Acknwledgements University f Khartum, Sudan. 16. Richardsn, C. (2002) Lwering stress in transprted gats. The authrs remain grateful t all the staff f the Ontari Ministry f Agriculture and Fd Livestck histpathlgy labratry, Necrpsy building cmplex Technlgy.Branch Nrthern Ontari Reginal Office. 17. Zulkifli, Idrus., Bahyuddin, N., Wai, C. Y., Farjam, A. S., f the faculty f Veterinary medicine, Universiti Putra Sazili, A. Q., Rajin, M. A. and Meng, G. Y. (2010) Physil- Malaysia. This prject was funded by the Ministry f gical respnses in gats subjected t rad transprtatin Science, Technlgy and Innvatin (MOSTI), under the ht, humid trpical cnditins, Int. J. Agric. Bil., Malaysia under the Science Fund : Available nline at /MSA/ 2010/ Cmpeting interests 18. Redkar, R., Rse, S., Bricker, B. and Del Vecchi, V. (2001) The authrs declare that they have n cmpeting interests. Real-Time Detectin f Brucella Abrtus, Brucella Melitensis and Brucella Suis. Mlecular and Cellular References Prbes, 15: Guatte, R., Jly, A. and Beaudeau, F. (2012) Shedding and 1. Seleem, M.N., Byle, S.M. and Sriranganathan, N. 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