Nonhuman Primate Model for the Study of Respiratory Klebsiella pneumoniae Infection

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1 INFECTION AND IMMUNITY, Oct. 1978, p /78/22275$2./ Copyright C 1978 Americn Society for Microbiology Vol. 22, No. 1 Printed in U.S.A. Nonhumn Primte Model for the Study of Respirtory Klebsiell pneumonie Infection R. F. BERENDT,* G. L. KNUTSEN, AND M. C. POWANDAt U.S. Army Medicl Reserch Institute of Infectious Diseses, Fort Detrick, Frederick, Mrylnd 2171 Received for publiction 2 August 1978 Squirrel mon ere inoculted by the intrtrchel inocultion of 7 Klebsiell pneumonie orgnisms nd developed lobr pneumoni in bout 24 h. Chrcteristic clinicl findings ere fever, norexi, nd coughing. Lbortory findings included leukocytosis or leukopeni (ith the ltter more prominent in ultimtely ftl infections), bcteremi, nd shedding of bcteri into the phrynx. Infected mon shoed incresed plsm lysozyme ctivity s ell s incresed plsm ceruloplsmin, hptoglobin nd 1ntitrypsin. The mortlity rte s 6%, nd the men time of deth s 5.5 h. Pthologiclly, the disese spred by mens of Kohn's pores nd other pthys tht generlly did not involve irys s mens of dissemintion until bout 3 h. Squirrel mon seem to be better models for humn respirtory K. pneumonie infection thn rts or mice. Mice nd rts re the experimentl nimls most commonly employed for the study of respirtory Klebsiell pneumonie infection (2, 3, 1). Clinicl nd biochemicl studies re difficult to crry out on these nimls, hoever, becuse of their smll size. Recently, e hve found tht squirrel mon (Simiri sciureus) developed pneumoni fter intrtrchel instilltion of Streptococcus pneumonie (4), nd lso ere susceptible to K. pneumonie dministered by this route (1). Therefore, in nticiption of studies of erosol therpy, e hve crried out experiments designed to chrcterize the squirrel monkey model in greter detil. This report includes clinicl, histopthologicl, nd selected biochemicl observtions. MATERIALS AND METHODS Test orgnism. Techniques for groing, storing, nd enhncing the virulence of the AD strin of type 1 K. pneumonie hve been described previously (2). Inocul (1.5 ml ech) for the infection of mon contined 6 to 8 vible orgnisms ech nd ere prepred s previously described (1). Test nimls. Helthy, juvenile, mle squirrel mon eighing from.5 to 1. kg ere used. They ere housed individully in irebr cges nd lloed free ccess to commercil monkey cho nd ter. Their diet s supplemented ith fresh fruit severl times eekly. During experiments, fruit s eliminted, nd ech monkey s limited to six biscuits dily to fcilitte estimtion of food consumption. Intrtrchel inocultion. Intrtrchel inocultion s method of infecting mon hs been det Present ddress: Biochemistry Brnch, U.S.A. Institute of Surgicl Reserch, Brooke Army Medicl Center, Fort Sm Houston, TX scribed previously (1). The dose dministered (7 orgnisms) s determined to be pproximtely one 5% lethl dose (1). Preprtion of smples for histologicl exmintion nd estimtion of bcteri concentrtion in tissue. In seprte experiment to determine histopthology, blood smple s obtined, mon ere killed 6, 24, 3, or 48 h fter infection by intrvenous injection of pentobrbitl, nd the bdominl nd thorcic cvities ere opened septiclly. For estimtion of bcteril concentrtion, smples of selected orgns ere removed septiclly, eighed, homogenized in Trypticse soy broth (TSB), nd brought to finl volume of 4. ml. Approprite dilutions ere mde in TSB nd plted on Trypticse soy gr. After incubtion, colonies ere counted nd concentrtions clculted. The remining portion of the orgns s fixed in 1% neutrl buffered Formlin, cut in 6ytm sections, nd stined by hemtoxylin nd eosin or Bron nd Hup stins. Sections ere then exmined by light microscopy. Clinicl determintions. Once dily,.75 ml of heprintreted blood s obtined from the sphenous vein for determintion of totl nd differentil leukocyte concentrtion, hemtocrit, nd bcteremi. Bcteremi s determined by spreding.1 ml of blood on the surfce of Trypticse soy gr plte, incubting for 18 h t 37C, nd counting the colonies produced. Additionl dily determintions or observtions included rectl temperture, respirtory rte, culturing phryngel sbs for Klebsiell, eight, ctivity, sneezing or coughing, dyspne, nd food consumption. Three bseline determintions ere mde on ech of the bove prmeters, nd testing continued for 8 dys fter infection. The volume of the dily blood smple s incresed to 1.5 ml on 2 seprte dys before infection s ell s on dys 1, 2, 3, 6, nd 8 for the determintion of plsm lysozyme, 1ntitrypsin, ceruloplsmin, nd hptoglobin. Prior experi 275

2 276 BERENDT, KNUTSEN, AND POWANDA menttion indicted tht this bleeding schedule ould cuse 2% drop in hemtocrit vlue nd no other chnges. Lysozyme ctivity s mesured by the method of Ossermn nd Llor (8). The concentrtion of three other serum proteins s estimted by rdil immunodiffusion procedures, using kits obtined from Behring Dignostics (Americn Hoechst Corp., Somerville, N.J.). Although these kits ere designed for determintion of humn proteins, preliminry investigtion shoed sufficient crossrectivity ith serum proteins of rhesus, cynomolgus, nd squirrel mon to permit ssy of proteins in these species. Experimentl design. Sixteen mon ere infected nd serilly killed in groups of four t 6, 24, 3, nd 48 h for histopthologicl exmintion nd estimtion of tissue bcteril concentrtions. Three replicte groups of five mon ech ere infected fter three bseline determintions nd then ere ssessed for 8 dys to evlute clinicl course of illness nd mortlity. Simultneously, three groups of five mon ech ere shm inoculted ith sterile Trypticse soy broth. Approximtely 1 dys elpsed beteen the end of one replicte nd the beginning of the next. The results of replicte experiments ere not different for ech of the prmeters under investigtion, so the dt from the three groups ere combined. RESULTS Histologicl nd bcteriologicl studies. The concentrtion of K. pneumonie in selected tissues t vrious times fter infection is presented in Fig. 1. The 6h period s rbitrrily chosen s period erly in infection; t 24 h, clinicl signs ere just becoming pprent, nd t 3 h illness s clerly visible. The 48h time s chosen becuse e nticipted tht the first deths ould occur shortly therefter. One of the four mon reserved for the 48h period died 2 to 3 h erly. The dt from liver nd kidney re not shon becuse concentrtions in these orgns ere lmost identicl to those in the spleen t ll time periods. The dt indicted tht bcteri multiplied in the lungs nd ere crried in smll numbers by the blood to other tissues. Of interest s the reltively smll number of bcteri isolted from blood even t 48 h. At 24 h, fe hundred bcteri ere isolted from the brin nd stomch of ech of the four mon (not shon). Histologicl exmintion of the four mon 6 h fter inocultion ith K. pneumonie reveled miniml to mild lobulr lveolitis tht s restricted, in every instnce, to single diphrgmtic lobe. This lesion s chrcterized by n infiltrtion of lveolr spces by fe neutrophils nd extrvsted red blood cells (Fig. 2A). Upon gross exmintion 24 h fter inocultion, the four mon exmined hd ptchy gryish Z * OROPHARYNX 4 W 6 TRACHEA * SPLEEN A BLOOD 4 E m. 6 2 INFECT. IMMUN HOURS AFTER INFECTION FIG. 1. Concentrtion of K. pneumonie in selected tissues t selected times fter intrtrchel instilltion. Vlues presented indicte the geometric men of results from four mon exmined t ech time. red res of consolidtion confined to the diphrgmtic lobe on one side. Histologiclly, the inflmmtion hd progressed to n erly moderte lobr pneumoni confined to single lobe. Alveolr spces, ducts, nd some respirtory bronchioles in ffected res ere filled ith fluid, fibrin, nd cellulr infiltrte composed primrily of neutrophils nd fe mcrophges (Fig. 2B). Numerous encpsulted grmnegtive pleomorphic bcilli tht ere consistent ith K. pneumonie ere present in the edemtous exudte (inset). Interlobulr sept ere modertely distended ith fibrinous fluid nd ere infiltrted ith neutrophils. Lymphtic chnnels ithin the sept ere modertely dilted. Mon t 3 h fter inocultion hd totl consolidtion of one diphrgmtic lobe nd ptchy res of involvement in the middle lobes of the sme side. Microscopiclly, these mon hd lobr pneumoni tht involved the entire diphrgmtic lobe nd portions of the middle lobe. The lesion t this time s chrcterized by n intense outpouring of neutrophils, some mcrophges, nd fibrinous edem tht gve the lung prenchym solid ppernce. The lesion s ccompnied by foci of lveolr septl necrosis. Interlobulr sept ere mrkedly distended by n inflmmtory exudte nd con

3 Voi,. 22, 1978 r F lp., t~~~t r }'f X4 J *.; tim..~j.3 p4~~~~~~~~~~~~ 3 4'jJ.; ~~~~~~~~6~~ ~ ~~1!7 *:,%LL' v A;~~~~.4_ k~~~~4~ 4.% r4. A o4 Ad,& C T I A Itt# ( s&) K PNEUMONIAE IN MONKEYS 277 ''~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~i ;*ofhi A ^ S {,*h ^., # m g~~~~~~~~dt~fo# * ~~~~?sj~ r 4~sAn r 44.4:,~~~~~~~~~~~~4 Kf;,;' ''',,*'' ''9 Rt i..or.. i _p. _ Donloded from v4 $* S r * D *W* * 484 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~4 4P v ~~ t on April 19, 219 by guest FIG. 2. (A) Infiltrtion of lveolr spces by fe neutrophils nd extrvsted red blood cells (6 h postinfection). x133. (B) Alveolr spces filled ith edem fluid, fibrin, nd cellulr infiltrte composed primrily of neutrophils nd fe mcrophges (24 h). x133. Inset: Grmnegtive bcteril rods ithin lveolr exudte. x665. (C) Interlobulr sept mrkedly distended by fibrinous edem nd neurophilic infiltrte (24 h). x133. (D) Neutrophilic exudte ithin lveolr spces nd the lumen of bronchiole ithout involvement of the ll of the iry (3 h). x133. tined severely dilted lymphtics (Fig. 2C). The exudte continued onto the pleurl surfce of the ffected lobes. In ddition to the lobr pneumoni, one monkey in this group hd n intense neutrophilic infiltrte ithin the lumen nd lls of bronchioles. The irys in other nimls contined vrible mounts of exudte, but the lls of these irys ere generlly uninvolved (Fig. 2D). The inflmmtory process t 48 h fter inoc

4 278 BEREND)T, KNUTSEN, AND) IPOWANDA ultion s qulittively similr to tht seen t 3 h, but more extensive. A lobr pneumoni involving diphrgmtic nd middle lobes ith ptchy res of inflmmtion extending into the picl lobes s seen in to of the mon. There s mild to moderte splenitis in 5 of 12 mon observed 24 h or more fter infection. Similr splenic chnges re reported in rts experimentlly infected ith K. pneumonie (2). A ptchy interstitil pneumoni s present in 13 of the 16 mon tht ere necropsied. This lesion s subcute to chronic in nture nd s pprently unrelted to the experimentl infection. In severl mon, the interstitil involvement s ssocited ith dult Filroides spp. In only one monkey s K. pneumonie found in ssocition ith this lesion. Clinicl nd lbortory observtions. All of the 15 noninfected control mon survived ithout overt illness, heres 9 of the 15 infected nimls (6%) died ith men time to deth of 5.5 h. Illness, first observed t 24 h, consisted of lethrgy, norexi, nd dyspne. Signs of illness ere pronounced in virtully ll mon ithin 3 h. Principl clinicl nd lbortory findings re presented in Fig. 3 nd 4. After infection, significnt increse in temperture s observed throughout the experiment ith the exception of dy 5 (Fig. 3). The totl leukocyte concentrtion vried idely during this study (Fig. 3); hoever, the counts obtined from nimls tht ultimtely died ere significntly different thn those of infected mon tht survived (Tble 1). Mon tht survived the infection hd leukocytosis; those tht ultimtely died, leukopeni. Virtully ll infected mon shoed mrked neutrophili on dy 1 (not shon). This vlue returned to ithin norml limits in 2 to 3 dys in mon tht survived, but reltive neutrophili persisted in those tht died. Klebsiell cells ere isolted from the phrynx of ll inoculted mon by the second dy (Fig. 3). The frequency of isoltion declined therefter, nd phryngel cultures ere negtive for K. pneumonie fter dy 5. Bcteri ere isolted from peripherl blood smples from more thn 8% of the mon 24 h fter inocultion nd from ll mon on the third nd fourth dys. Bcteremi s not detected fter dy 6. A greter number of bcteri ere detected in the blood of mon tht died thn in those tht survived (Tble 2). Incresed respirtory rtes ere lso chrcteristic of this infection (Fig. 4), reching pek vlues on dys 2 to 4 nd then sloly declining. All infected mon hd inspirtory nd expirtory dyspne throughout the test period. 15 IL i 11 r 12. o1n1. E 8.O U _c_ z 2 13 I. Yz LL. o g OJ INFECT. IMMUN DAYS AFTER INFECTION FIG. 3. (A) Rectl temperture of infected () nd control () mon. Verticl brs indicte stndrd error of the men nd re shon only here significnt differences exist. Symbols: (*) P <.5, (***) P <.5. (B) Totl leukocyte (WBC) counts for infected () nd control () mon. (C) Isoltion of Klebsiell from blood () nd throt (). Food consumption nd body eight dt re lso shon in Fig. 4. The loss of eight s more pronounced thn the norexi, possibly indicting tht some of the loss of eight s ctbolic in origin. Biochemicl rections. Plots of chnges in four plsm proteins re presented in Fig. 5. Unfortuntely, the vrition beteen mon s very gret, nd sttisticl nlysis often filed to discern differences. When the dt ere normlized to preinfection bse lines, hoever, ceruloplsmin seemed to be the protein most ffected by infection. Concentrtions ere significntly higher (P <.5) on the first dy fter infection nd ere still significntly elevted (P <.5) on dy 8. The pttern for 1ntitrypsin A

5 VOL. 2X2, I x) UA ẕ i In i. (I WA e K. PNEUMONIAE IN MONKEYS A terized by fever, norexi, coughing, leukocytosis or leukopeni, bcteremi, nd shedding of bcteri into the phrynx. The mon lso shoed incresed concentrtions of plsm intitrypsin, ceruloplsmin, nd hptoglobin, s _, ye\ ell s incresed plsm lysozyme ctivity. Mor /\*** * tlity occurred in 6% of infected nimls, ith 1 / ; & men time to deth of bout 5 h. _J,, ~ sse; 5 Pthologiclly, the disese s chrcterized by initil multifocl involvement of the lveoli 9 (6 h) in the diphrgmtic lobe tht spred to involve mjor portions of the sme lobe by 24 h. B The lesion t 24 h s still confined to peripherl 6 tissues ith involvement of lveolr spces, _er ducts, nd fe respirtory bronchioles. This 5\ observtion suggests tht initilly the disese spred by y of Kohn's pores nd other lveo 4 ***lr ductbronchiolr pthys, including Lem 3 bert's cnls, rther thn by the irys. Inflm mtory exudte s not seen in lrger bron 2 chioles nd bronchi until 3 h. Airys themselves ere not involved. It is ssumed tht the I I spred of pneumoni to djcent lobes occurred 5 C hen the inflmmtory exudte s either expelled up irys during respirtion or moved 3],Q^+_Q_5'1 1 1 by mucociliry ction. Usully, involvement s I I 3 _I J **unilterl, ith the disese originting in the DAYS AFTER INFECTION FIG. 4. (A) Respirtory rte in infected () nd control () mon. Verticl brs indicte stndrd error of the men. (B) Appetite s mesured by biscuit consumption. (C) Body eight during infection. Symbols: (*) P <.5, (**) P <.25, (***) P <.5. seems to be bout the sme s tht of ceruloplsmin, but infected nimls differed significntly from controls only on dys 2, 3, nd 6. The vlues for hptoglobin nd lysozyme ctivity lso ere quite vrible, but differed from controls on dys 2 nd 3. When the plsm protein vlues of surviving nd dying mon ere compred, only lysozyme exhibited significnt difference (Tble 3), nd this difference s seen only t one time period. DISCUSSION Intrtrchelly instilled K. pneumonie (7 cells) cused n cute lobr pneumoni in squirrel mon. Cliniclly, the disese s chrc TABLE 1. Totl leukocyte concentrtion in dying nd surviving mon during K. pneumonie infection Leukocytes/mm3 (x 13) Dy fter pb infection Surviving mon Dying mon Bse line 6,275 (6) 6,955 (9) 1 12,633 6,522 (9) < ,75 2,233 (3) <.1 3 8,117 2,9 (1) 4 7, 6,5 (1) Vlues in prentheses indicte number of mon tested. b Clculted by t test. TABLE 2. Dy Bcteremi in K. pneumonieinfected mon No. of bcteri/ml of blood pb fection Surviving mon Dying mon x 11 (6) 5.6 x 12 (9) < x 11 (6) 1.5 x 13 (3) < x 12 (6) 5.4 x 12 (1) x 1 (6) 5. x 13 (1) 5 (6) No survivors Vlues represent geometric men. Vlues in prentheses indicte number of mon tested. b Clculted by t test, computed on log trnsformtion.

6 28 BERENI)T, KNUTSEN, ANI) POWANI)A diphrgmtic lobes nd extending in time to the picl lobe on the sme side. The most importnt question concerning this ork is ho ell the disese in the model simultes tht in humns. Cliniclly, the lobr form of the disese in humns s described by Julinelle (6) is chrcterized by brupt onset, vrible fever (frequency lo), cough, extensive rusty sputum production, neutrophili, nd bcteremi in bout 6% of ptients. Extension of infection to other tissues is infrequent. Generlly, our clinicl nd lbortory findings re consistent ith these observtions. We hve not seen sputum production, but this lck my be due to difficulties in observtion. Conversely, the observtion tht 1% of mon hve bcteremi in contrst to 6% of humn ptients my be the result of more frequent blood smpling in the mon. The ssocition of leukopeni nd persistent bcteremi ith mortlity, s seen in mon, hs lso been reported in humns (6, 7). When pthologicl lesions re considered, the similrity beteen humn nd simin models is even more stiking. In both species, the lesions my be described s lobr pneumoni ith severe edem of interlobulr sept nd distended lymphtic chnnels. Airys re reltively free of involvement erly in the disese in humns (5, 11). The principl difference beteen the to species seems to be the lo frequency of bscess formtion in the squirrel monkey. Although the necrosis of lveolr sept, believed to be pre z (IV U l ANTITRYPSIN HAPTOGLOBIN * * SHAM CONTROL INFECTED * P<O.5 ** PcO.O25 *** P<O.OO5 *5** *** DAYS AFTER INFECTION Fl(;. 5. Vl(Ues of four selected plsm proteins duiing infection. Stndrd error brs omitted for cltit,. INFF.CT. IMMUN. TABLE 3. Lysozyme concentrtion in K. pneumonieinfected mon Plsm lysozyme concn (,ug/ml)" Dy fter Ph infection Surviving mon Dying mon Bse line 1.6 (6) 1.8 (9) (6) 2.4 (9) (6) 5.5 (3) < (6) 4.6 (1) (6) No survivors (6) "Vlues in prentheses indicte number of mon tested. h Clculted by t test. requisite for bscess formtion in humns (5, 11), lso occurs in mon, bscess formtion s seen very rrely in our studies, even in mon in hich the infection hd cused deth. The bsence of bscess formtion, hoever, my be due to the rpidity ith hich deth occurred in mon. Specific plsm proteins ere mesured in the expecttion of using ltertions in concentrtion s prognostic indices, s e hve done erlier ith rts (2). Too much vrition s encountered mong mon, hoever, to permit use of concentrtion estimtes for this purpose, except ith lysozyme. The chnges tht ere observed, hoever, re consistent ith the presence of inflmmtory disese; the possible significnce of these metbolic chnges is discussed in detil elsehere (9). Although squirrel mon pper to be good models for cute pneumoni due to K. pneumonie, the question of their utility compred to mice nd rts rises. The lo price nd smll size of smll rodents is prticulrly ppeling becuse more nimls cn be employed nd, thus, sttisticl nlysis cn be more redily utilized. Hoever, intrnsl injection of orgnisms into mice nd rts results in bronchopneumoni (2, 4) rther thn in lobr pneumoni. Intrtrchel instilltion hs lso been reported to produce lobr pneumoni in rodents (1). Hoever, the use of gstric mucin s n djuvnt in the rodent study (1) my hve hd profound effects. Squirrel mon, in contrst, mimic humns in the form tht the Klebsiell pneumoni tkes nd, in ddition, more redily llo the sequentil mesurement of conventionl clinicl signs such s fever, ppetite, respirtory rte, eight chnge, blood nd throt cultures, nd hemtology in the sme niml thn do rodents. Also, the crossrectivity beteen certin humn nd monkey plsm proteins not only llos mesurement of chnges in specific cutephse rectnts, s opposed to frctions

7 VOL. 22, 1978 thereof, but my, ssuming tht the right protein or combintion of proteins cn be found, llo for dditionl quntittion of the severity of disese nd the success of therpy. With subhumn primte models for lobr pneumoni, e cn no more thoroughly study the pthogenesis of such pneumoni, evlute the effective uses of chemotherpy, nd devise ne pproches to therpy, both ntimicrobil nd supportive, ith more confidence tht our findings ill be relevnt to humns. ACKNOWLEDGMENTS We cknoledge ith thnks the technicl ssistnce of Mrk Schneider, Robert Mgruder, nd Frnk Frol. We lso thnk Alexnder DePoli for revieing histopthologicl dt. LITERATURE CITED 1. Berendt, R. F Reltionship of method of dministrtion of respirtory virulence of Klebsiell pneumonie for mice nd squirrel mon. Infect. Immun. 2: Berendt, R. F., G. G. Long, F. B. Abeles, P. G. Cnonico, M. R. Elell, nd M. C. Pond Pthogenesis of respirtory Klebsiell pneumonie infection in rts: bcteriologicl nd histologicl findings K. PNEUMONIAE IN MONKEYS 281 nd metbolic ltertions. Infect. Immun. 15: Berendt, R. F., G. G. Long, nd J. S. Wlker Tretment of respirtory Klebsiell pneumonie infection in mice ith erosols of knmycin. Antimicrob. Agents Chemother. 8: Berendt, R. F., G. G. Long, nd J. S. Wlker Influenz lone nd in sequence ith pneumoni due to Streptococcus pneumonie in the squirrel monkey. J. Infect. Dis. 132: Hopps, H. C Bcteril diseses, p In W. A. D. Anderson (ed.), Pthology, vol. 1, 6th ed. C. V. Mosby Co., St. Louis, Mo. 6. Julinelle, L. A The pneumoni of Friedlnder's bcillus. Ann. Intern. Med. 15: Limson, B. M., M. J. Romnsky, nd J. G. She An evlution of tentyto ptients ith cute nd chronic pulmonry infection ith Friedlnder's bcillus. Ann. Intern. Med. 44: Ossermn, E. F., nd D. P. Llor Serum nd urinry lysozyme (murmidse) in monocytic nd monomyelocytic leukemi. J. Exp. Med. 124: Pond, M. C Chnges in body blnces of nitrogen nd other key nutrients: description nd underlying mechnisms. Am. J. Clin. Nutr. 3: Sle, L. J., nd W. B. Wood, Jr Studies on the mechnism of recovery in pneumoni due to Friedlnder's bcillus. I. The pthogenesis of experimentl Friedlnder's bcillus pneumoni. J. Exp. Med. 86: Spencer, H Pthology of the lung, p , 2nd ed. Pergmon Press, Oxford. Donloded from on April 19, 219 by guest