Pthology nd Hygiene INFLUENCE OF DIFFERENT STRAINS AND WAYS OF INOCULATION ON THE RABBIT S RESPONSE TO EXPERIMENTAL INFECTION WITH PASTEURELLA MULTOCIDA Kulcsár G. 1, Fáián K. 1 *, Brn T. 1, Virág Gy. 2 1 Centrl Agric. Office, Directorte of Veterinry Medicinl Products, Szállás u. 8, 1107 Budpest, Hungry 2 Reserch Institute for Animl Breeding nd Nutrition, Isszegi ut 200, 2100 Gödöllı, Hungry *Corresponding uthor: fink@oi.hu ABSTRACT Extensively chrcterized Psteurell multocid strins isolted from rits with different signs of psteurellosis were used to study the response of rits triggered y erogen, conjunctivl, intrderml or intrmusculr experimentl infection. Lethlity ws smll (5/45) in generl nd somewht higher tendency (4/17) ws found fter conjunctivl inocultion. Numer of dys with ppernce of illness nd fever within 14 dys p.i., numer of rits with t lest one dy occurrence of fever, P. multocid reisoltion rte, nd verge rectl temperture were influenced oth y the strin inoculted nd y the route of inocultion. Strin 31 presented higher (P<0.05) vlues t more prmeters (ll hd fever, 39.9 C verge rectl temperture, 60% P. multocid reisoltion rte) except numer of dys with illness nd with fever. Intrderml nd intrmusculr inocultion cused douled vlues compred to tht cused y erogen or conjuctivl inocultions, except P. multocid reisoltion rte. Feed intke nd ody weight gin ws lso different y the route of inocultion, rits inoculted intrdermlly or intrmusculrly consuming less feed nd loosing weight insted of gining. Serum ntiody level elevted 2-3 times in rits infected intrmusculrly, nd strin 1 ws found to hve the higher ntigenecity (P<0.05). Key words: Rit, Psteurell multocid, Experimentl infection, Chllenge model. INTRODUCTION Rit psteurellosis is still often enzootic on ig frms (Coudert et l., 2006) nd the most previling chronic forms cuse importnt losses (Rosell et l., 1991). Frm specific vccines hve een nd re to e developed nd their efficcy should e evluted one y one in chllenge experiments. At the sme time when ccepting the strong intention towrds decresing the numer of niml experiments it should e recognized tht chllenge experiments re unvoidle in cse of vccintion studies. The numer of nimls however should e kept t the possile lower numer ut in this wy to keep good experimentl model is necessry. Mking good design is not esy in cse of rit ecuse of the ig individul differences in response given to experimentl infections with P. multocid (Rideud et l., 1999). The purpose of this study ws to compre the rit pthogenecity of five P. multocid (P.m.) strins isolted from rits with different signs of psteurellosis nd pplied on four different wys in order to estlish strong model for chllenge experiments. MATERIALS AND METHODS Prmeters of inoculums used for the experimentl infection P.m. strins used for the infection (Tle 1) hve een isolted from rits with different signs of psteurellosis. Strins mintined in lyophilized stock were grown on TSA pltes. Overnight cultures 981
9 th World Rit Congress June 10-13, 2008 Veron Itly were suspended in PBS nd diluted to pproximtely 10 5 CFU/ml nd 1 ml ws inoculted into ech rit, except the negtive controls. Tle 1: Chrcteristics of strins used for inocultion Strin Mouse LD 50 Lesion of isoltion cpgene production: iovr 1 1.1 10 3 pneumoni cpa+ : 6 5 7.7 10 5 suutneous scess cpa - : n.t. 13 1.9 10 5 rhinitis cpa+ : 1 31 4 10 5 empyem cpa -: 6 32 5.2 10 5 torticollis cpa+ : n.t. Tle 2: Numer of rits inoculted with different strins nd y different routes Inocultion wy Strin 1 5 13 31 32 -cont Totl erosol 0 0 3 3 0 6 id 3 3 0 0 3 9 im 2 2 3 3 2 12 orrkonj 3 3 3 3 3 15 -cont 3 3 Totl 8 8 9 9 8 3 45 Experimentl nimls nd design Forty five 12 weeks old hyrid rits weighing 2860±41 g were used in this experiment (Tle 2). All rits were screened three times for P.m. nsl crrige nd once for serum ntiodies y n ELISA (modified FlockCheck IDEXX P.m. Antiody Test Kit) previously nd ll were negtive. The inocultion ws performed y erogen (ero.), conjunctivl (conj.), intrderml (id.) nd intrmusculr (im.) routes. Negtive control (-cont.) rits were kept without inocultion ut otherwise within the sme conditions. Rits were cged individully nd groups infected with different strins were kept in different rooms. According to the route of the infection the rits were divided further into cge locks to close off ny possiility of the direct contct etween these nimls. Mngement, feeding nd wter supply were the sme for ll groups. Clinicl, pthologicl nd microiologicl oservtions Rits were exmined nd rectl temperture ws recorded dily to dy 14 post infection (p.i.). Sneezing, depression nd forced respirtion were considered s disese signs. Body weight (grm) ws controlled on the dy of inocultion then on dy 7 nd 14 p.i. Feed consumption (grm) ws mesured weekly during two weeks p.i. Blood smples were tken efore inocultion nd t dy 14 p.i. At dy 14 p.i. ll survived rits were euthnized humneously then necropsied. Sws were tken from the nose, phrynx, trche nd lung, hert chmer nd scesses or other lesions if present nd spred onto SBA. P.m. ws isolted nd identified y stndrd microiologicl methods. Sttisticl Anlysis Powerful sttisticl processes in GenStt 8 th softwre (VSN Interntionl Ltd., 2004) GLM module enled for deling with unlnced designs were used for the detection of strin nd inocultion wy effects. Interction of these fctors ws not studied. Feed intke, rectl temperture were verged over rits nd ELISA titers fter inocultion were given s percentge of the sic vlue. The dt were evluted with GLM regression nlysis. Weight gin on susequent weeks ws evluted y REML vrince component nlysis. Occurrence of normlly high ody temperture nd reisoltion of P.m. from different orgns on the level of single niml ws considered s yes or not vrile nd evluted with Bernoulli distriution. Numer of dys with presence of fever or illness nd numer of successful reisoltions of P.m. compred to the numer of totl oservtions on the level of single rits were evluted y modeling of inomil proportions. Results re presented s men±s.e. for groups formed y the two fctors. 982
Pthology nd Hygiene RESULTS AND DISCUSSION One rit infected id. with strin 1 died on dy 2 p.i. nd conjunctivl inocultion with strin 1, 13 nd 31 killed further 4 rits in ll which died etween dy 7 nd 14 p.i. showing tendency (P<0.1) for higher lethlity in cse of conjunctivl inocultion route. Both infective strins (Tle 3) nd inocultion routes (Tle 4) influenced the prevlence of illness nd fever given in numer of dys with positive oservtions, the proportion of feverish rits, P.m. reisoltion rte nd the verge ody temperture. Tle 3: Proportion of oserved nimls nd dys with occurrence of clinicl signs of sickness nd with fever, P. multocid reisoltion rte y strins used for infection Strin Prmeter 1 5 13 31 32 s.e. Pro Sick dys/totl oserved 0.34 0.21 0.16ª 0.12 0.40 0.04 <0.001 Fever dys/totl oserved 0.17 0.26 0.27ª 0.32 0.41 0.04 <0.01 Rits with fever occurence 0.88 1 0.67ª 1 0.63 0.09 <0.05 Rectl temperture 39.0ª 39.4 39.7 c 39.9 d 39.6 c 0.08 <0.001 P.m. reisoltion rte 0.21 0.34 0.38ª 0.60 0.41 0.08 <0.01 Mens with different letters on the sme row differ significntly Tle 4: Proportion of oserved nimls nd dys with occurrence of clinicl signs of sickness nd with fever, P. multocid reisoltion rte y inocultion route pplied Inocultion route Prmeter ero. conj. id. im. s.e. Pro Sick dys/totl oserved 0.08 0.07 0.50 0.34 0.04 <0.001 Fever dys/totl oserved 0.08 0.11 0.50 0.45 0.04 <0.001 Rits with fever occurence 0.67 0.67 1 1 0.08 <0.01 Rectl temperture 39.1 39.3 40.0 d 39.6 c 0.08 <0.001 P.m. reisoltion rte 0.29 0.41 0.41 0.40 0.08 n.s. Mens with different letters on the sme row differ significntly The men rectl temperture ws ove 39.5 C in groups infected id. nd im., while rits in groups infected ero. or conj. were not feverish (P<0.001). All rits hd fever t lest once in groups infected id. or im., ut only 67% in groups infected ero. or conj. (P<0.01). The numer of dys with prevlence of illness nd fever ws low in groups infected ero. or conj. compred to the douled (P<0.001) frequency t groups infected id. or im. P.m. reisoltion rte however did not differ y the wy of inocultion. Oserved prmeters of sickness were influenced y the infective strin too. Rits infected with strin 31 hd the highest P.m. reisoltion rte (P<0.01) nd verge rectl temperture (P<0.001), ll were feverish nd hd high proportion of dys with fever. Contrry to these, signs of illness were oserved with the lowest frequency (<0.001) t this group, wht could e explined y the sence of sneezing or forced rething. The depression s the only sign cused y normlly high ody temperture is often difficult to oserve on rits kept in confinement. Feed intke on the first week p.i. (Figure 1) ws significntly different in the experimentl groups y the wy of inocultion (P<0.005). Uninfected control rits nd rits infected y ero. or conj. route consumed more feed (999, 1067 nd 893 g, respectively) compred to those infected id. or im. (527 nd 673 g, respectively). Strin effect ws not importnt. The decresed feed intke nd weight gin could e prtly explined y the higher ody temperture cused y the infection. On the second week fter inocultion feed intke recovered to the norml level (1184±104 g, P<0.001). The only exception ws the group infected with the strin 31. In line with the decresed feed intke on the first week the rits gined mere 34.4±23.2 g in verge independently from the inocultion strin. The pprently high vriility of the results ws explined gin y the route of the infection (P<0.001) with negtive (i.e. weight loss) vlues t groups infected id. or im. (-80.6 nd -21.7 g, s.e. 56.5 nd 50.2, respectively) nd higher vlues t rits infected ero. or conj. (201.0±63.6 nd 80.9±48.4 g, respectively). Control rits gined 232±69 g with the sme mount of feed consumed. 983
9 th World Rit Congress June 10-13, 2008 Veron Itly ero id im conj negcont feed intke, g 1400 1200 1000 800 600 400 c 200 0 1 5 13 31 32 negcont strin Figure 1: Averge feed intke of rits experimentlly infected with five different P. multocid strins nd four different routes until dy 7 th post inocultion ero id im conj negcont 500 c ody gin, g 400 300 200 100 0-100 1 5 13 31 32 negcont strin Figure 2: Body weight chnge of the rits experimentlly infected with 5 strins of P. multocid y erogen, conjunctivl, intrderml nd intrmusculr route until dy 14 p.i. On the second week the rits gined etter (P<0.001) 156±24 g in verge ut the differences cused y the wy of inocultion remined the sme. Weight gin summrized for the two weeks is demonstrting the sme tendency (Figure 2) nd the highest vlue ws found gin (P<0.05) t the non-infected control group. As shown on Figure 3, serum ntiody level of rits infected y im. route incresed significntly (P<0.05) reching 200-350% of the level efore infection independently from the strin used for inocultion. Inocultion with strin 1 resulted elevted serum ntiody level (P=0.1) independently from the inocultion route. Elevted serum ntiody level ws not found in cse of further strins with ny other inocultion wy nd nor in negtive control rits. erogen id im conj titer, % 450 400 350 300 250 200 150 100 50 0 c 1 5 13 31 32 negcont strin Figure 3: Chnge of serum ntiody titers for rits experimentlly infected with 5 strins of P. multocid y erogen, conjuntivl, intrderml nd intrmusculr route 984
Pthology nd Hygiene CONCLUSIONS The rit virulence of P.m. strins collected from chronic forms of disese on frms with enzootic psteurellosis could e est evluted y im. or id. inocultion, nd oservtion of rectl temperture, pprent clinicl signs of illness, P.m. reisoltion rte nd feed intke. Forceful sttisticl models cn fcilitte chllenge experiments designed with smll numer of experimentl rits. ACKNOWLEDGEMENTS This study ws supported y the grnt 46025/2004 from the Hungrin Ministry of Agriculture nd Rurl Development REFERENCES Coudert P., Rideud P., Virg Gy., Cerrone A. 2006. Psteurellosis in rits. In: Recent Advnces in Rit Sciences (Ed.: Mertens L., Coudert P.), ILVO, Melle, Belgium, pp. 147-162. Rideud P., Coudert P., Roteu D. 1999. Psteurellosis in the rit: pthogenicity of five intrdermlly nd intrnslly inoculted strins. In: Proc. 8 émes Journ. Rech. Cunicole, Pris, Frnce, 13-16. Rosell J.M., Bdiol J.I., De L Fuente L.F., Crmenes P., Bdiol J.J. 1991. Rhinitis of the domestic rit. An epidemiologicl survey during the period 1986-1991. I. Influence of the yer, seson nd type of ritry. J. Applied Rit Res., 15, 1375-1381. 985
9 th World Rit Congress June 10-13, 2008 Veron Itly 986