Haemophilus paragallinarum Strains

Transcription

1 JOURNAL OF CLINICAL MICROBIOLOGY, July 1983, p /83/ $02.00/0 Copyright 1983, Americn Society for Microbiology Vol. 18, No. 1 Reltionships Between Virulence nd Morphologicl or Serologicl Properties of Vrints Dissocited from Serotype 1 Hemophilus prgllinrum Strins AKIRA SAWATA AND KATSUMI KUME* The Kitsto Institute, 5-9-1, Shirokne, Minto-ku, Tokyo 108, Jpn Received 21 September 1982/Accepted 5 April 1983 Reltionships between morphologicl or serologicl properties nd virulence were investigted in seven Hemophilus prgllinrum vrints. The vrints, originted from five serotype 1 strins, were clssified into seven types on the bsis of colony morphology nd iridescence nd the presence of vrint-specific ntigens. Smooth (S) nd encpsulted orgnisms hving vrint-specific ntigens nd forming highly iridescent (ir+) colonies were highly virulent in vivo; slightly encpsulted orgnisms hving vrint-specific ntigens nd forming slightly iridescent (ir+) colonies were modertely virulent; nd nonencpsulted or slightly encpsulted orgnisms with or without vrint-specific ntigens nd forming noniridescent (ir-) or ir+ colonies were virulent. Virulence ws well correlted with the mount of cpsule substnce contining hyluronic cid. The evidence suggests tht the presence of vrint-specific gglutinogen L nd hemgglutinin HA-L seem to be responsible for dherence or coloniztion, but not for virulence, of the orgnisms in chickens. Orgnisms of Hemophilus spp. re well known for wide vrition in both cellulr nd colonil morphologies. Pittmn (12) showed two types of colonies, smooth (S) nd rough (R), in Hemophilus influenze by observtions under obliquely trnsmitted light. Chndler et l. (1) described three colonil forms, mucoid (M), S, nd R, in H. influenze. A correltion between colony type nd pthogenicity hs been reported in H. influenze (1, 12). In preliminry reports (9, 19, 20) on colonil types of Hemophilus prgllinrum, colonies were differentited into S nd R. Hinz (2) ws the first to report the presence of two colony types in reltion to virulence, iridescent M nd noniridescent R, nd noted tht the former is encpsulted nd is virulent in chickens, wheres the ltter, which dissocites from M, is virulent. He lso observed vrition from R to M colonies in vivo. Swt et l. (7, 15) previously reported tht encpsulted orgnisms form iridescent, S colonies nd re virulent to chickens, wheres nonencpsulted colonies form noniridescent, S colonies nd re virulent. It is pprent tht encpsulted H. prgllinrum orgnisms cuse coryz in chickens (2, 7, 15, 17); however, the ntigen relted to the virulence of this orgnism remins to be identified. We hve recently obtined seven vrints, with different morphologicl or serologicl properties, which were dissocited in vitro from five serotype 1 strins; these were used to nlyze the mechnisms of infection with nd immunity to H. prgllinrum. The present study dels with the reltionships between virulence nd the morphologicl or serologicl properties of these vrints. MATERIALS AND METHODS Test orgnisms. Five serotype 1 H. prgllinrum strins, described previously, were used. Of these, strin 221 (5) ws kindly provided by K. Kto, Ntionl Institute of Animl Helth, Hokuriku-Brnch, Niigt, Jpn, nd strins 083 nd W (14) were kindly provided by Richrd B. Rimler, Ntionl Animl Disese Center, Ames, Iow. All the strins were lyophilized nd stored. Strins 7411 nd 7682 (15) were isolted by Swt et l. from prnsl sinuses of chickens with infectious coryz nd were stored in lyophilized stte. Medi. The solid medium used ws S gr (6, 8) consisting of 20% (vol/vol) chicken met infusion, 0.5% (wt/vol) Csmino Acids (Difco Lbortories, Detroit, Mich.), 0.5% (wt/vol) soypeptone (Digo Co., Tokyo, Jpn), 0.5% (wt/vol) NCl, 5% (vol/vol) dry yest extrct (Nippon Beet Sugr Co., Tokyo, Jpn) solution (25%, wt/vol), 0.025% NADH (Orientl Yest Co., Tokyo, Jpn), distilled wter solution (1%, wtlvol), 5% (vol/vol) het-inctivted (560C, 30 min) chicken serum, nd 1.5% (wt/vol) Bcto-gr (Difco). The liquid medium used ws identicl to S gr, except gr ws omitted; this medium ws termed S broth nd ws used for the cultures to prepre gglutinogen, hemgglutinin, nd immunogen. For seril trnsfer of 49

2 50 SAWATA AND KUME the orgnisms, the following three kinds of S broth were used: supplemented with 10% (vol/vol) nti-221 chicken serum (medium I), specific-pthogen-free (SPF) chicken serum (medium II), nd het-inctivted SPF chicken serum (medium III). Ser used in broth medi. The nti-221 chicken serum ws prepred by intrvenously injecting thimerosl-inctivted orgnisms of strin 221 into 7-weekold chickens. The ntiserum contined gglutinin ginst hyluronidse (HU) (Sigm Chemicl Co., St. Louis, Mo.)-treted ntigen, prepred from encpsulted orgnisms of strin 221 t titer of 1:10, nd hemgglutintion inhibition (HI) ntibody ginst HU ntigen t titer of 1:80. SPF chicken serum ws collected from H&N (Pfizer Tito Co., Shizuok, Jpn) nd line S (Nisseiken Co., Ymnshi, Jpn) 20-week-old chickens. Colony selection. Freeze-dried cultures were reconstituted in phosphte-buffered sline (PBS; ph 7.0) in n mpule nd inoculted onto S gr. After 15 h of incubtion t 37 C in 5% C02, highly iridescent (ir+) colonies (hi vrint) were trnsferred to S gr. The culture grown under the conditions described bove ws hrvested in PBS nd djusted to 109 cells per ml by spectrophotometry (1010 cells per ml = opticl density t 650 nm of 0.386) with model 6/20 spectrophotometer (Colemn Instruments Division, Perkin- Elmer Co., Ok Brook, Ill.). This suspension contined pproximtely 1 x 108 to 5 x 108 CFU/ml. Then, 0.2 ml of the suspension ws dministered vi the nsl cvity to 7-week-old chickens. At 1 or 2 dys postinocultion, orgnisms were recovered from chickens showing typicl signs of coryz, i.e., runny nose nd fcil swelling, nd the orgnisms were serilly trnsferred to the other chickens until the ir+ colonies were predominnt (.99% of the totl colonies) on S gr, stored under lyophilized conditions, nd used s strting mterils to obtin the vrint. Subculture in S broth. Vrints were obtined by serilly subculturing in the three kinds of S broth (medi I, li, nd III) t 3- to 4-dy intervls for 80 subcultivtions. The colonies grown on S gr could be divided into seven vrints. Ech vrint in ech subcultivtion ws considered positive when 25 or more of 500 colonies (.o5%) were identicl. The most predominnt type in ech subcultivtion ws lso recorded. Ech vrint considered to be stble on S gr ws stored in lyophilized stte for exmintion of its serologicl properties. Microscopy. After 13 to 15 h of incubtion t 37 C in 5% C02, colonies grown on S gr were exmined with model SMZ-10 stereomicroscope (Nikon Co., Tokyo, Jpn) under trnsmitted nd obliquely reflected light. The colonil morphologies observed under trnsmitted light could be divided into three types: S, R, nd n intermedite type between S nd R (SR). The sizes of the colonies were clculted by comprison with ir+ colonies. Colony iridescence ws determined by using two ngles between the source of illumintion nd mirror (pproximtely 30 nd 600) under obliquely reflected light. The grdes of iridescence were recorded s follows: ir+, positive t both ngles; ir' (low iridescence), positive t only 30 ; nd ir- (noniridescence), negtive t both ngles. The colony color observed under obliquely reflected light ws lso recorded. The cellulr morphologies of Grm- nd cpsule-stined (10) orgnisms were ob- J. CLIN. MICROBIOL. served with model XF-31 light microscope (Nikon). Antigens. The cultures incubted in S broth supplemented with nonheted SPF chicken serum t 37 C for 10 h were centrifuged t 10,000 x g for 20 min t 4 C nd wshed once in PBS. The cells were then suspended in PBS contining 0.01% (wt/vol) thimerosl nd djusted to concentrtion of 10l1 cells per ml by spectrophotometry. This suspension is referred to herein s nontreted (NT) ntigen. The HU ntigen ws prepred from the cell suspension in PBS by incubtion with 100 U of HU (Sigm) per ml in wter bth t 37 C for 2 h, followed by wshing once in PBS, nd ws djusted to concentrtion of 1011 cells per ml by spectrophotometry. Finlly, 0.01% thimerosl ws dded for gglutintion, hemgglutintion (HA), nd bsorption tests. Antiser. Antiser were prepred by injecting rbbits with NT ntigen prepred from hi vrints dissocited from strin 221 nd with NT ntigens prepred from the remining six vrints dissocited from strin 221. About 109 cells per ml of ntigen were injected subcutneously into rbbits (ech weighing bout 2 kg) twice week, beginning with 0.5 ml of the ntigen given with 0.5 ml of Freund complete djuvnt (Difco). Subsequent injections were given intrvenously six times in progressively lrger doses, from 0.5 to 4 ml, t 3-dy intervls. At 1 week fter the finl injections were given, blood smples were withdrwn, nd the ser obtined were stored t -20 C (16). HA test. The NT nd HU ntigens (1011 cells per ml) prepred from ech vrint were tested for HA bility ginst freshly collected erythrocytes (RBCs) from line S (Niiseiken) 60-dy-old chickens by procedures reported previously (18). The hemgglutinins showing vrious HA titers were treted by trypsin (P-L Biochemicls Inc., Milwukee, Wis.) or heting t 65C for 30 min to determine their trypsin or het sensitivities, respectively. The procedures used were s reported previously (16). Serologicl tests. The gglutintion test ws performed by rpid plte gglutintion (RPA) method, described in n erlier report (16), with ntiserum diluted 1:12.5 to 1:3,200 with PBS nd NT nd HU ntigens. The HI test ws performed by method described previously (16) with ntiser which hd isohemgglutinin removed by dding 5% (vol/vol) chicken RBC suspension to finl dilution of 1:5. Agglutinin nd HI ntibody bsorption tests were performed by dding 1.15 ml of HU ntigen from hi vrints dissocited from strin 221 (221-hi-HU), concentrted to 1012 cells per ml in PBS, to 0.1 ml of undiluted ntiserum. The mixture ws llowed to stnd t 370C for 3 h nd then t 40C for t lest 12 h. After centrifugtion, gglutinin or HI ntibody remining in the superntnt ws titrted by RPA nd HI tests. Chickens. Eggs from stock known to be free from Hemophilus orgnisms nd certin bcteril nd virl infections obtined from the H&N poultry frm (Phizer Tito) were incubted in the lbortory, nd the chickens htched were housed in seprte room. Seven-week-old chicks were used for the pthogenicity test. Pthogenicity test. The chickens were intrnslly given 0.2 ml of PBS suspension of the culture incubted on S gr for 10 h. The vible cells were djusted to bout 5 x 108 cells per 0.2 ml per chicken.

3 VOL. 18, 1983 TABLE 1. Reltionships between virulence nd morphologicl or serologicl properties of vrints dissocited from H. prgllinrum serotype 1 Vrint- Recov- Vri- Colony Irides- Cp- specific Viru- ery of nt morphol- cence sule ntigen lence orgogy L A- nisms hi S ir' + +b +b + + li-a S ir' + + +C +C li-b S ir' -- ni-a S ir ni-b SR ir R-A R ir _ + R-B R ir Het-lbile, trypsin-sensitive, hyluronidse-resistnt gglutinogen (L) nd hemgglutinin (HA-L) locted under the cpsule. Antigens L nd HA-L re vrint-specific ntigens mong the seven serotype 1 vrints. b Lck of gglutinbility by cpsule, but ctivity ppered soon fter tretment with hyluronidse. c Vrition from li-a to hi vrint ws observed in vivo. After inocultion, clinicl chnges, especilly runny nose nd fcil swelling, were recorded once dy in ech chick for 2 weeks. The coryz signs were divided into four ctegories: severe (severe runny nose nd fcil swelling), moderte (severe runny nose nd slight fcil swelling); mild (slight nsl dischrge only when pushed on the nsl cvity); nd none (no signs). Isoltion of Hemophilus orgnisms from the nsl cvity of ech chicken ws performed t 16 h nd 2, 3, 7, nd 14 dys fter inocultion. Smples obtined on cotton swbs were directly streked on S gr, nd morphologicl nd serologicl properties of the recovered orgnisms were determined. VIRULENCE OF H. PARAGALLINARUM VARIANT 51 RESULTS Vrints dissocited from serotype 1 strins. Seven vrints with different morphologicl or serologicl properties (vrints hi, li-a, li-b, ni- A, ni-b, R-A, nd R-B) were obtined from H. prgllinrum serotype 1 strins during seril trnsfer in medium I, II, or III (Tble 1). Colonil ppernce of vrints. Vrints could be divided into three forms by stereomicroscopic observtions under trnsmitted light: S (hi, li-a, li-b, nd ni-a vrints), R (R-A nd R-B vrints), nd SR (ni-b vrint). S colonies were highly or modertely convex nd smooth, with regulr edges; R colonies were flt nd rough, with modertely or mrkedly irregulr edges; nd SR colonies were slightly convex nd intermedite in ppernce between S nd R, with modertely irregulr edges. Under obliquely reflected light, hi vrints showed reddish or reddish-green, high iridescence (ir+); li-a nd li- B vrints hd light bluish-green, low iridescence (ir+); nd ni-a, ni-b, R-A, nd R-B vrints showed no iridescence (ir-). All except R-A vrints produced trnsprent colonies on S gr when observed under trnsmitted or obliquely reflected light. Cellulr morphology of vrints. hi vrints hd uniform, short rods or coccobcilli, wheres li-a vrints hd rods of vrious lengths nd slight pleomorphism. li-b nd ni-a vrints hd rods of vrious sizes with filments; nd R-B vrints hd uniform, medium-sized rods with clumps. Cpsules surrounding the cells were observed in most of the bcilli from hi vrints by the method of Moller. The cpsules in li-a nd li-b vrints were not s evident s those in hi vrint, nd ni-a, ni-b, R-A, nd R-B vrints hd no cpsules. Dissocition pttern in S broth. hi vrints of strins 221, 083, W, 7411, nd 7682 were serilly trnsferred in medium I, II, or III for up to 80 subcultivtions. li-a vrints were dissocited to vrious degrees from the originl hi vrints in 3 to 5 subcultivtions. Between 21 nd 30 subcultivtions, hi, li-a, li-b, nd ni-a vrints were observed; hi vrints were still predominnt in medium I, wheres li-a vrints were predominnt in medi II nd III. Between 41 nd TABLE 2. Agglutintion rections involving NT nd HU ntigens prepred from H. prgllinrum serotype 1 vrints Vrint Anti- Hemgglutinting Agglutintion titerb gen ctivity' 221-hi-NT 221-hi-HU hi NT <2 <12.5 <12.5 HU ,600 1,600 li-a NT HU ,600 1,600 li-b NT <2 <12.5 <12.5 HU < ni-a NT HU ,600 ni-b NT <2 <12.5 <12.5 HU <2 <12.5 <12.5 R-A NT HU R-B NT <2 <12.5 <12.5 HU <2 <12.5 <12.5 Hemgglutinting ctivity ginst fresh chicken RBCs expressed s reciprocl of the titers (strins 221, 083, nd 7411). The initil ntigen dilution ws 1:2. b Expressed s reciprocl of the titers (strin 221). The initil serum dilution ws 1:12.5. All the NT nd HU ntigens prepred from vrints hi, li-a, ni-a, nd R-A lcked gglutinting ctivities fter tretment with trypsin or by heting t 65 C for 30 min.

4 52 SAWATA AND KUME J. CLIN. MICROBIOL. TABLE 3. Agglutintion nd HI ntibody titers of ntiser prepred from H. prgllinrum serotype 1 vrints before nd fter bsorption with 221-hi-HU ntigen Agglutintion titer" HI ntibody titer' Antiserum Before bsorption After bsorption Before After bsorption bsorption 221-hi-NT 221-hi-HU 221-hi-NT 221-hi-HU (221-hi-HU)b (221-hi-HU) 221-hi-NT < <12.5 < < li-A-NT <12.5 1,600 <12.5 < < li-B-NT < <12.5 <12.5 <5 < ni-A-NT < <12.5 < < ni-B-NT <12.5 <12.5 <12.5 <12.5 <5 < R-A-NT < <12.5 < < R-B-NT <12.5 <12.5 <12.5 <12.5 <5 <12.5 Expressed s reciprocl of the titers. The initil serum dilution ws 1:12.5. b Expressed s reciprocl of the titers. The initil serum dilution ws 1:5. 50 subcultivtions, hi, li-a, li-b, ni-a, ni-b, nd R-A vrints were observed; li-b vrints were predominnt in medium I, wheres li-a nd ni-a vrints were predominnt in medi II nd III, respectively. At 80 subcultivtions, ni-b vrints were predominnt in medium I, wheres li- A nd ni-a vrints were still predominnt in medi II nd III, respectively. R-B vrints were found only in medium I. Smll numbers of hi vrints were still found in medi I nd II even t 80 subcultivtions, but never in medium III. HA ctivity of NT nd HU ntigens from ech vrint. HA tests were performed with NT nd HU ntigens prepred from ech vrint of strins 221, 083, nd 7411 nd with the RBC suspension collected from line S chickens (Tble 2). All the hi-nt ntigens lcked the bility to gglutinte chicken RBCs, but hi-hu ntigen rendered them ble to do so t dilutions of 1:32 to 1:128. However, trypsiniztion or heting t 65 C for 30 min completely eliminted this bility, suggesting tht the hemgglutinin is het lbile, trypsin sensitive, nd HU resistnt. This hemgglutinin, termed HA-L hemgglutinin, ws found in both NT nd HU ntigens from li- A, ni-a, nd R-A vrints, but not in those from li-b, ni-b, or R-B vrints. The HA-L hemgglutinin ws found to be vrint-specific hemgglutinin mong the seven serotype 1 vrints. Agglutinbility of NT nd HU ntigens from ech vrint. Cross-gglutintion tests were performed with NT nd HU ntigens prepred from ech vrint of strin 221 nd with their corresponding ntiser, 221-hi-NT nd 221-hi-HU (Tble 2). hi-nt ntigen filed to rect with either ntiser, but hi-hu ntigen rected with both ntiser. Both NT nd HU ntigens of li-a, ni-a, nd R-A vrints lso rected with the ntiser, but those of li-b, ni-b, or R-B vrints did not. The gglutinin ws found to be hetlbile, trypsin-sensitive, nd HU-resistnt L gglutinogen, which ws found to be vrintspecific gglutinogen mong the seven serotype 1 vrints. Absorption tests with 221-hi-HU ntigen. Before bsorption with 221-hi-HU ntigen, gglutinin nd HI ntibody ginst 221-hi-HU ntigen were detected in NT ntiser prepred from vrints hi, li-a, ni-a, nd R-B, but these were bsorbed completely with 221-hi-HU ntigen (Tble 3). HI ntibody ginst 221-hi-HU ntigen ws not found, even in unbsorbed ntiser of li-b-nt, ni-b-nt, nd R-B-NT vrints. Virulence in chickens. At 1 or 2 dys postinocultion, ll the chickens given hi vrints showed moderte to severe coryz signs which persisted throughout the experiment (Tble 4). At 2 to 5 dys post-inocultion, 21 of 25 chickens given li-a vrints showed moderte to severe coryz signs, but the remining 4 chickens showed only mild coryz signs during the experimentl periods. In contrst, none of the chickens given li-b, ni-a, ni-b, R-A, or R-B vrints showed coryz signs. Recovery of orgnisms from inoculted chickens. During the experimentl periods, hi vrints were predominntly recovered from ll the chickens given hi vrints, lthough minor mount of li-a vrint ws recovered (Tble 5). In 21 of 25 chickens inoculted with li-a vrints, in vivo vritions from li-a to hi vrints were observed, except for the isoltion done t 16 h post-inocultion, when predominntly hi vrints were obtined. These chickens showed moderte to severe coryz signs. li-a vrints were predominntly recovered from the remining four chickens showing mild coryz signs throughout the experimentl periods. Despite the lck of virulence, ni-a nd R-A vrints were minly recovered from ll the chickens given these vrints during the first 7 dys postinocultion, but bcteril isoltion from these birds ws negtive t 14 dys post-inocultion. No orgnisms were recovered from ny of the

5 VOL. 18, 1983 Vrint VIRULENCE OF H. PARAGALLINARUM VARIANT 53 TABLE 4. Virulence of H. prgllinrum serotype 1 vrints in chickens No. of subcultivtions in S broth of following No. of strin: chickens No. with coryz signs inocu W lted None Mild Moderte Severe hi li-a li-b ni-a ni-b R-A R-B chickens given li-b, ni-b, or R-B vrints, even t s erly s 16 h post-inocultion. Reltionships between virulence nd morphologicl or serologicl properties of vrints. Correltions between virulence nd morphologicl or serologicl properties of H. prgllinrum serotype 1 vrints re summrized in Tble 1. Encpsulted hi vrints with vrint-specific L nd HA-L ntigens showed strong virulence in chickens. li-a vrints with L nd HA-L ntigens showed reltively strong virulence, but encpsulted orgnisms were not s clerly visible s those of hi vrint; however, in vivo vrition of li-a to hi vrints ws observed. Regrdless of the ntigen structure nd colonil or cellulr morphology, li-b, ni-a, ni-b, R-A, nd R-B vrints cused no coryz signs in chickens upon inocultion. hi, li-a, ni-a, nd R- A vrints with ntigens L nd HA-L were recovered from inoculted chickens, wheres li- B, ni-b, nd R-B vrints, which lck these ntigens, were not recovered. DISCUSSION There re few reports concerning the virulence of H. prgllinrum. Using strin 2403 of Pge's serotype B (11), Hinz (2) demonstrted correltion between his colony types, M nd R, nd virulence in chickens. Employing strins 221 nd H-18 (6) of serotypes 1 nd 2 (15), Swt et l. (16) reported the presence of two colony types, iridescent nd noniridescent, of S colonies. The former, encpsulted strin, possessing L gglutinogen; het-lbile, trypsin-resistnt gglutinogen HL; nd het-stble, trypsin-resistnt gglutinogen HS, ws virulent in chickens, where s the ltter nonencpsulted strin, lcking L gglutinogen, ws virulent (7, 16). L ntigen seems to be cpsulr ntigen becuse its dissocition is lwys ccompnied by loss of cpsule (16). In ddition, NT ntigens prepred from encpsulted orgnisms of both serotypes filed to rect in ntiser prepred from NT ntigens of nonencpsulted orgnisms. Therefore, we postulted tht cpsulr substnce of H. prgllinrum is relted to virulence, s hs been demonstrted in Hemophilus influenze (1, 12). In the present study, we ttempted to chrcterize the morphologicl nd serologicl properties of the vrints dissocited from five serotype 1 strins in reltion to their virulence in chickens. The vrints could be divided into seven colony types on the bsis of different morphologicl or serologicl properties. The results showed tht close correltion existed between virulence nd the mount of the cpsule substnce. The encpsulted orgnisms pprently cuse coryz in chickens, wheres nonen- TABLE 5. Recovery of Hemophilus orgnisms with cotton swbs from nsl cvities of chickens given H. prgllinrum serotype 1 vrints intrnslly No. of Recovery of orgnisms (no. of chickens with positive recovery; most predominnt vrint) Vrint chickens t: inoculted' 16 h 2 dys 3 dys 7 dys 14 dys hi 25 + (25; hi) + (25; hi) + (25; hi) + (25; hi) + (25; hi) li-a 25 + (25; li-a) + (25; hi) + (25; hi) + (25; hi) + (25; hi) li-b ni-a 25 + (10; ni-a) + (25; ni-a) + (25; ni-a) + (22; ni-a) ni-b R-A 15 + (14; R-A) + (15; R-A) + (15; R-A) + (15; R-A) - R-B Sme birds noted in Tble 4.

6 54 SAWATA AND KUME cpsulted orgnisms do not. Regrdless of the virulence, orgnisms with both vrint-specific L nd HA-L ntigens could be recovered from inoculted chickens, wheres vrints lcking such ntigens did not persist in the host. Severity of gross lesions in chickens ws closely correlted with the mount of cpsule substnce when the orgnisms were dministered vi the prnsl sinus route. The encpsulted orgnisms of hi vrints were highly virulent; slightly encpsulted li-a vrints were reltively virulent; nd nonencpsulted ni-a, ni-b, R-A, nd R-B vrints were virulent. Slightly encpsulted li-b vrints were virulent, but the orgnisms lcked L nd HA-L ntigens. The presence of L nd HA-L ntigens hd significnt (P < 0.05) reltionship with the persistnce of the orgnisms in vivo. In ddition, in vivo trnsformtion of li-a to hi vrints ws frequently observed in li-a vrint-inoculted chickens, nd the trnsformtion ccompnied typicl coryz signs. The results suggested tht the ppernce of virulence in chickens is relted to substnce present in the cpsule or substnce specificlly produced from the encpsulted orgnisms. Hinz (2) reported tht the presence of cpsule is lwys ccompnied by colony iridescence. The sme phenomenon ws lso reported by Kume et l. (7). In the present investigtion, iridescence ws found to be divided into two types, ir+ nd ir+, ccording to our ctegories, nd the grde of iridescence ws significntly correlted not only to in vivo virulence but lso to the mount of cpsule substnce(s). Selection of colony ccording to our ctegories of iridescence, therefore, would be useful tool in estimting the virulence of orgnisms in vitro. The presence of hyluronic cid in some H. prgllinrum strins hs been reported previously (3, 13, 14). Rimler hs stted tht, lthough it is not present in ll pthogenic strins, hyluronic cid, when present, cn msk both gglutinogen (14) nd hemgglutinin (13). Using strin 221, Iritni et l. (3) lso reported tht cpsultion might interfere with HA ctivtion, lthough these workers (3, 13, 14) hve not definitely proven this. More recently, Swt et l. found similr phenomenon in H. prgllinrum serotype 2 orgnisms which did not gglutinte fresh chicken RBCs (15), lthough tretment with either potssium thiocynte or soniction ctivted their hemgglutinin (18). The present dt showed tht the cpsule in hi vrints inhibited the gglutinbility of both homologous ntiserum nd freshly collected chicken RBCs, nd this inhibition ws esily eliminted by tretment with HU. It is pprent tht ll the encpsulted orgnisms of serotype 1, which formed hi vrints, contined hyluronic cid- J. CLIN. MICROBIOL. like substnce in this cpsule, suggesting tht both NT nd HU ntigens prepred from the sme strin should be used in serologicl nlyses Ḣi vrints completely lcked HA bility but, when inoculted into chickens, produced typicl coryz signs ws predominntly recovered even 2 weeks fter inocultion. In nother experiment, we observed tht hi vrints were predominntly recovered from the host for t lest 5 weeks post-inocultion, nd bcteril clernce ws observed fter the detection of serotype 1- specific L gglutinin (16) nd HI ntibody (18) in chickens. Despite the lck of the cpsule nd virulence, ni-a nd R-A vrints with L nd HA-L ntigens were lso recovered from inoculted chickens during week 1 post-inocultion, but no orgnisms were recovered 2 weeks fter inocultion. The difference in the bcteril recovery period observed between encpsulted nd non-encpsulted orgnisms with L nd HA-L ntigens suggested tht the cpsule my be relted to resistnce ginst bctericidl ctivity. Iritni et l. (4) hve reported tht the ntigens before nd fter tretment with trypsin hd the sme ntigenicity nd recommended the use of trypsin-treted ntigen for the RPA test to detect the gglutinin. The previous dt showed tht serotype specificity resided in trypsin-sensitive L ntigen, which ws detectble by the RPA test, but not in trypsin-resistnt HL or HS ntigens (16, 17), nd lso in trypsin-sensitive HA-L hemgglutinin (18). In the present investigtion, loss of L ntigen ws lwys ccompnied by loss of HA-L hemgglutinin. No ntigens which hd the sme ntigenicity before nd fter tretment with trypsin (4) hve been confirmed by us. Hinz reported the presence of R to M vrition in vivo (2). The present results strongly suggested tht his M vrint corresponds to our li-a vrint becuse his vrint ws virulent in chickens nd NT ntigen prepred from his M vrint rected in the ntiserum prepred from M vrint without tretment with HU. The identity of his R vrint, however, remins uncler. On the other hnd, encpsulted orgnisms reported by Swt et l. (16) lso correspond to li-a vrint, wheres nonencpsulted orgnisms (16) should be clssified s n intermedite colony type between li-b nd ni-b vrints. In the present investigtion, we clrified morphologicl nd serologicl properties of the seven vrints dissocited from H. prgllinrum serotype 1 in reltion to virulence in chickens. The vrints should be useful tool in nlyzing the mechnisms of infection with nd immunity to H. prgllinrum serotype 1.

7 VOL. 18, 1983 VIRULENCE OF H. PARAGALLINARUM VARIANT 55 ACKNOWLEDGMENTS We thnk A. Ghod, Kitsto University, Sgmihr, Kngw, Jpn, nd M. Mtsumoto, School of Veterinry Medicine, Oregon Stte University, Corvllis, Oreg., for reviewing the mnuscript nd N. Kneko, Kitsto Institute, for technicl ssistnce. LITERATURE CITED 1. Chndler, C. A., L. D. Fothergill, nd J. H. Dingle The pttern of dissocition in Hemophilus influenze. J. Bcteriol. 37: Hinz, K. H Beitrg zur Differenzierung von Hemophilus-Stimmen us Huhnern. 4. Mitteilung: Untersuchungen Ober die Dissozition von Hemophilus prgllinrum. Avin Pthol. 5: Iritni, Y., S. Hidk, nd K. Ktgri Production nd properties of hemgglutinin of Hemophilus gllinrum. Avin Dis. 21: Iritni, Y., K. Ktgri, nd K. Tsuji Slide-gglutintion test of Hemophilus gllinrum ntigen treted by trypsin. Avin Dis. 22: Kto, K., nd H. Tsubhr Infectious coryz of chickens. II. Identifiction of isoltes. Bull. Ntl. Inst. Anim. Helth 45: Kume, K., A. Swt, nd Y. Nkse Hemophilus infections in chickens. Chrcteriztion of Hemophilus prgllinrum isolted from chickens ffected with coryz. Jpn. J. Vet. Sci. 40: Kume, K., A. Swt, nd Y. Nkse Reltionship between protective ctivity nd ntigen structure of Hemophilus prgllinrum serotypes 1 nd 2. Am. J. Vet. Res. 41: Kume, K., A. Swt, nd Y. Nkse Immunologic reltionships between Pge's nd Swt's serotype strins of Hemophilus prgllinrum. Am. J. Vet. Res. 41: Mky, K. A., nd R. B. Truscott Reversion of vin pleuropneumonilike orgnisms to bcteri. Ann. N.Y. Acd. Sci. 79: Moller, A new method for stining bcteril cpsules. Act Pthol. Microbiol. Scnd. 28: Pge, L. A Hemophilus infections in chickens. 1. Chrcteristics of 12 Hemophilus isoltes recovered from disesed chickens. Am. J. Vet. Res. 23: Pittmn, M Vrition nd type specificity in the bcteril species Hemophilus influenze. J. Exp. Med. 58: Rimler, R. B Studies of pthogenic vin hemophili. Avin Dis. 24: Rimler, R. B., R. B. Dvis, nd R. K. Pge Infectious coryz: cross-protection studies, using seven strins of Hemophilus gllinrum. Am. J. Vet. Res. 38: Swt, A., K. Kume, nd Y. Nkse Hemophilus infections in chickens. II. Types of Hemophilus prgllinrum isoltes from chickens with infectious coryz, in reltion of Hemophilus gllinrum strin no Jpn. J. Vet. Sci. 40: Swt, A., K. Kume, nd Y. Nkse Antigenic structure nd reltionship between serotypes 1 nd 2 of Hemophilus prgllinrum. Am. J. Vet. Res. 40: Swt, A., K. Kunie, nd Y. Nkse Biologic nd serologic reltionships between Pge's nd Swt's serotypes of Hemophilus prgllinrum. Am. J. Vet. Res. 41: Swt, A., K. Kune, nd Y. Nkse Hemgglutinin of Hemophilus prgllinrum serotype 2 orgnisms: occurrence nd immunologic properties of hemgglutinin. Am. J. Vet. Res. 43: Schlm, 0. W., nd J. R. Bech Studies of infectious coryz of chickens with specil reference to its etiology. Poultry Sci. 15: Schlm, 0. W., nd J. R. Bech Culturl requirements of the fowl-coryz bcillus. J. Bcteriol. 31: Downloded from on April 8, 2018 by guest