Salmonella abortus-ovis Infection in Mice

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1 INFECTION AND IMMUNITY, May 983, p /83/ $02.00/0 Copyright 983, Amerian Soiety for Mirobiology Vol. 40, No. 2 Immunogeniity of a Low-Virulene Vainal Strain Against Salmonella abortus-ovis Infetion in Mie FRtDERIC LANTIER,* PIERRE PARDON, AND JOSE MARLY Institut National de la Reherhe Agronomique, Station de Pathologie de la Reprodution, Nouzilly, Frane Reeived 3 Otober 982/Aepted 7 February 983 Strain Rv6, a streptomyin-independent reverse mutant of Salmonella abortusovis, multiplied and disseminated in vivo to a lesser extent than the virulent parental strain 5/5, as determined by subutaneous inoulation of two outbred strains of mie with different suseptibilities to salmonella infetion. This lower virulene of strain Rv6 was not modified by 0 passages in suseptible mie. Vaination with strain Rv6 indued a dose-dependent apaity of mie to ontrol subsequent infetion with the virulent parental strain. Indued resistane began as early as 3 days after vaination and lasted at least months. Its effiieny was ontrolled by different routes and doses of hallenge. Strain Rv6 indued a greater protetion than that onferred by heat-killed bateria or by tested ommerial killed vaines. Salmonella abortus-ovis is a pathogen speifi for sheep and goats that indues abortion and, to a lesser degree, mortality of young (3). Many European ountries fae diffiulties in ontrolling this disease beause there are no ways to diagnose all infeted animals. Immunization would be one way to redue losses and exretion of the bateria at the time of parturition (3, 23). Beause effetive immunity against salmonellosis is not generally produed by dead vaines (6, 3), researh has been oriented towards a live attenuated strain. Earlier suessful vaination trials against enterobaterial diseases (9, 7, 3) or sheep bruellosis (2, 0) motivated our hoie of mutants for streptomyin. Isolation of suh mutants and sreening of their virulene and immunogeniity with a murine model were reported in a previous paper (6). Subsequent experiments were onduted on sheep. They onfirmed the low virulene of isolated mutants and demonstrated the effiieny of vaination with strain Rv6, a streptomyin-independent reverse mutant (22, 24, 29). Vainal harateristis required of live vaines are well-known: inouousness, effiieny in induing protetion, and harateristis or markers that differentiate vainal mutant strains from wild-type virulent strains (4). The aim of experiments on mie related in this paper was to assess the low virulene, stability, and immunogeniity of strain Rv6. Protetion indued by strain Rv6 against hallenge with virulent S. abortus-ovis was ompared with that indued by killed vaines and by primary infetion with the parental virulent strain. MATERIALS AND METHODS Mie. Conventional female mie from two outbred Swiss strains were bred in a onditioned building (2 C, 60% relative humidity, filtered air) with free aess to water and sterilized food. As already reported (6), CD- mie (Charles River Breeding Laboratories, Elbeuf, Frane) are more resistant to salmonella infetion than OF mie (Iffa-Credo, St-Germain sur l'arbresle, Frane). Bateria. S. abortus-ovis 5/5 and a streptomyin- 60 resistant strain derived from it were used for primary infetion and hallenge, respetively; both strains are of omparable virulene for mie (23). Isolation of strain Rv6 was reported in a previous publiation (6); in brief, strain Rv6 is a streptomyin-suseptible reverse mutant derived from a streptomyin-dependent strain of S. abortus-ovis 5/5. Killed vaine. Heat-killed vaine ( h, 60 C) was prepared from S. abortus-ovis 5/5. Its sterility was heked by plating samples. Commerial killed vaine in oil adjuvant was hosen as the most immunogeni from five ommerial preparations tested with the murine model (data not shown). As indiated by the manufaturer, it ontains 5 x 08 S. abortus-ovis per ml. Preparation of mirobial suspensions, ulture media, and diluent. Organisms were grown on trypti soy agar (Bio Mdrieux, Lyon, Frane). In the ase of the streptomyin-resistant hallenge strain, streptomyin (Speia, Paris, Frane) was added at a onentration of 500,ug/ml of medium. To maintain their onstant virulene, all strains of S.

2 602 LANTIER, PARDON, AND MARLY abortus-ovis were passaged one in mie and stored at -70 C in infeted spleens. Overnight ultures from homogenates of thawed spleens were used to prepare suspensions in phosphate-buffered saline (ph 6.9). These were turbidimetrially standardized and tested for purity and number of viable organisms by plating serial dilutions (23). Route of immunization and hallenge. Eah mouse was immunized by a single subutaneous injetion into the nek with 0.2 ml of baterial suspension. For the test of virulene and for the hallenge of immunized mie, the animals were infeted by intravenous inoulation into a tail vein (0.2 ml of baterial suspension), by subutaneous inoulation in the left hind footpad (0.05 ml), or by oral administration in drinking water. Daily onsumption of water was estimated at 4 ml per mouse. Stability of the level of spleni infetion indued by strain Rv6. The stability of Rv6-indued spleen infetion was tested by 0 suessive passages in OF mie. An interval of 3 days between vaination and autopsy was used, orresponding to the maximum of spleni infetion by strain Rv6. For eah passage, bateria from the most infeted spleen (as determined by baterial numeration; see below) of the preeding passage were subultured on trypti soy agar (24 h, 37 C) and reinoulated into five mie (08 bateria per mouse, subutaneously inoulated into the nek). Baterial numeration in organs. Mie were killed by ervial disloation. Spleens, plus popliteal lymph nodes in some experiments, were aseptially removed and weighed. The organs were then homogenized, and the homogenates were diluted and plated as previously desribed (23). Colonies were ounted after 48 h of inubation at 37 C. Contamination of hallenge S. abortus-ovis with any vainal bateria was avoided by using streptomyin-resistant organisms as hallenge and by ultivating infeted organs on trypti soy agar with added streptomyin. Passive transfer of serum in CD- mie. Donor mie were immunized with strain 5/5 (06 bateria per mouse) or Rv6 (08 bateria per mouse) subutaneously inoulated into the nek. They were bled 3 weeks later by setion of axillary vessels. Serum from nonimmunized mie (normal serum) served as a ontrol. Serum samples were sterilized by filtration (pore size, 0.22,um) and stored at -20 C. Reipient mie were intravenously injeted in a tail vein with 0. ml of serum. Subutaneous hallenge with virulent strain 5/5 was arried out 4 h later. Mie were killed on day 3 after infetion. Antibodies in transferred serum samples were titrated by a miroagglutination tehnique with S. abortusovis olored by triphenyltetrazolium and prepared by lassial proedures as somati (0) and flagellar (H) antigens. Presentation of results. Values for subutaneous 50% lethal doses were determined by the method of Reed and Muenh (27). Viable ounts of S. abortus-ovis were expressed as logio units. Means and standard deviations of means were alulated from the loglo values. To simplify the presentation of results, organs whih oasionally (though rarely) gave no positive ultures were regarded as infeted by one baterium, and means were alulated from all mie of the experimental groups. When neessary, the onventional F test was used for statistial analysis of results. RESULTS Low virulene of mutant strain Rv6: omparison with parent strain 5/5. Values for subutaneous 50o lethal doses of strains Rv6 and 5/5 were estimated for CD- and OF mie (Table ). The lethal apaity of strain Rv6 appeared onsiderably dereased (50% lethal dose was 2 09 bateria per mouse). The kinetis of growth in subutaneously inoulated CD- and OF mie were used to ompare in vivo dissemination and multipliation of strains Rv6 and 5/5 (Fig. ). Levels of popliteal lymph node infetion were similar in mie infeted by eah of the baterial strains. Spleni infetion was ontrolled by the host after inoulation with strain Rv6 from day 3 onwards but peaked on day 6 after inoulation with fully virulent strain 5/5, whih killed OF mie. The intensity and duration of spleni infetion were higher in OF than in CD- mie. Spleni infetion did not last longer than month in Rv6-inoulated mie (data not shown). Stability of the low virulene of strain Rv6. Ten passages in OF mie (five mie per passage) did not inrease the level of spleni infetion indued by strain Rv6 on day 3 after inoulation (F409 = 0.3; P > 0.25), despite the use for eah inoulation of bateria subultured from the most infeted spleen of the preeding passage. As alulated from all individual values, the mean was 3.72, with a 5% onfidene interval of 0.39 (extreme values, 3.4 to 4.20). Immunogeniity of strain Rv6: omparisons with killed vaines and first infetion with virulent strain 5/5. Kinetis of growth of the streptomyin-resistant hallenge strain 5/5 were examined in CD- mie immunized month earlier (Fig. 2). Unlike mie infeted for the first time (06 bateria per mouse), those vainated with strain Rv6 (08 bateria per mouse) did not prevent a transient growth of hallenge organisms. This was more apparent after intravenous than after subutaneous inoulation. In these proteted mie, reovery was fast: only a few TABLE. Values for 50% lethal doses of strains Rv6 and 5/5 in miea S. abortus-ovis <50%o lethal dose (no. of bateria per mouse) for mouse strain: strain INFECT. IMMUN. OF CD- Mutant Rv6 Parental 5/5 i09 5 x 04 >09 8 x 07 a Mie were inoulated subutaneously in the left hind footpad. Values were alulated by the method of Reed and Muenh (27) from numbers of mie surviving 8 days after inoulation (9 logarithmi doses were used; eight mie per group).

3 VOL. 40, 983 CL 4,2-4 5, n o 0 (2) days after inoulation FIG.. Kinetis of growth of virulent parental strain 5/5 (A) and vainal strain Rv6 (0) in mie inoulated by the subutaneous route in the left hind footpad with 06 bateria per mouse. Exept where the number of dead mie is shown (in parentheses), eah point represents the mean of values for 0 mie; standard errors are also shown. salmonellae (around log0) ould be reovered from their spleens and popliteal lymph nodes on day 0 after hallenge (Fig. 2). Compared with nonvainated ontrols, mie immunized with hallenge : slubutaneous spleen 5 ; (:\ + 3 / \ - ut spleen (4) 5 intravenous VACCINAL S. ABORTUS-OVIS MUTANT 603 heat-killed bateria (08 bateria per mouse) did not aquire an inreased apaity to eliminate hallenge organisms from infeted tissues, although heat-killed vaine did indue a redution of log0 in the level of maximal spleni infetion on day 6 after hallenge. This time interval of 6 days between hallenge and autopsy was used in subsequent experiments. The result of inreasing the vainal dose of strain Rv6 in CD- and OF mie was to inrease protetion against subutaneous inoulation with the fully virulent strain 5/5 (Fig. 3). Vaination with 08 Rv6 bateria gave the greatest differene between ontrols and vainated mie; among the latter, no salmoneilae ould be reovered from the spleens of 6 out of 0 OF mie and 8 out of 0 CD- mie. Unlike live vaine, heat-killed S. abortusovis indued non-dose-dependent protetion against subutaneous hallenge. There was a differene of around log0 between the levels of spleni infetion in nonvainated ontrols and in mie immunized with different doses varying from 05 to 09 killed bateria per mouse (Table 2). The effet of hanging the time interval between vaination and hallenge was tested in CD- mie immunized with a redued dose of bateria of strain Rv6 (07 bateria per mouse) to avoid the noninfetion of spleens by hallenge organisms. As measured by the level of spleni infetion on day 6 after subutaneous hallenge, mie were proteted as early as 3 days after EA. 8 n -'4 a 0 C lymph node 3f a-c_ I days after hallenge o unvainated ontrols heat-killed vaine o Llive vaine strain Rv A primary infetion days after hallenge strain 5i5 FIG. 2. Kinetis of growth of hallenge strain S. abortus-ovis 5/5 in mie subutaneously immunized month earlier with 08 bateria of the indiated strain per mouse. Eah point represents the mean of values for five mie. 40 CX 0 4 L og 0 6 i (vaiidl uusess) FIG. 3. Evidene that protetion indued by strain Rv6 inreases with vainal dose in CD- mie (treated, 0; ontrol, 0) and OFI mie (treated, *; ontrol, O). Three weeks after subutaneous immunization, mie were subutaneously hallenged in the left hind footpads with 5 x 05 S. abortus-ovis 5/5 apiee. Exept where the number of dead mie is shown (in parentheses), eah point represents the mean of values for 0 mie; standard errors are also shown.

4 604 LANTIER, PARDON, AND MARLY TABLE 2. Evidene that immunization with heatkilled S. abortus-ovis indues a non-dose-dependent redution in the level of spleni infetion by hallenge salmonellae Dose (no. of heat-killed Salmonellae per spleen (loglo, bateria per mouse)a mean ± SE)b ± 0.33 i ± ± ± 0.33 a Mie were immunized by subutaneous inoulation in the nek with heat-killed bateria ( h, 60 C) suspended in saline. They were hallenged 3 weeks later with 5 x 05 S. abortus-ovis 5/5 apiee subutaneously injeted in the left hind footpads. b Counted on day 6 after hallenge; 0 mie per group. vaination. Protetion remained stable from day 7 after immunization until the end of the experiment months later (Fig. 4). Different doses of subutaneous hallenge were used to ompare a ommerial killed vaine in oil adjuvant (08 bateria per mouse) with the live strain Rv6 vaine (08 bateria per mouse) in their abilities to protet CD- and OF mie (Table 3). Live vaine redued the level of spleni infetion more than killed vaine did, but both types of immunization indued protetion against death. The transfer to reipient mie of immune sera from CD- mie immunized with strains Rv6 or 5/5 redued the level of spleni infetion by hallenge bateria in a similar manner (Table 4). Titers of these two immune sera in agglutinins against S. abortus-ovis did not differ. In normal serum transferred as a ontrol, antibodies speifi for salmonellae ould not be deteted. When hallenged by the oral route with a high dose of virulent S. abortus-ovis, OF mie immunized with heat-killed bateria from strains Rv6 or 5/5 showed no aquired resistane against infetion (Table 5). Immunization with living Rv6 bateria signifiantly redued the number of hallenge organisms olonizing the spleen 6 days after inoulation. DISCUSSION The results reported in this paper provide evidene for the stability of the low virulene of strain Rv6 and its immunogeniity for CD- and OF mie. The low virulene of strain Rv6 was onfirmed by the kinetis of infetion in subutaneously inoulated mie. As in intravenously inoulated OF mie (6), ontrol by the host of the vainal spleni infetion ourred as early as 3 days after immunization. The stability of the low virulene of strain Rv6 was tested by using 0 suessive passages in mie with an interval of 3 days between vaination and autopsy. Inoulations of organ homogenates would have provided a better demonstration than intermediate in vitro ultures, but in vivo multipliation of strain Rv6 was too low to obtain inoula of suffiiently high dosage. This problem might be solved by an abortive model involving diret inoulations with infeted produts of abortion (3) or strains of mie that are very suseptible to salmonellosis (25). However, our model seemed sensitive enough to detet any virulent form. Suh reversion was not observed. In addition, the virulene of strain Rv6 for mie was not modified when it was reisolated from the lymph nodes of vainated ewes, nor was it modified after seven suessive ultures in a bath-type fermentor (unpublished data). Strain Rv6 persisted 2 to 3 weeks in CD- mie and no longer than month in OF mie, whih are more suseptible to salmonella infetion. Persistene was suffiient to onfer on these strains of mie an early and long-lasting immunity, lasting muh longer than the persistene of vainal bateria in the host. This fat indiates the indution of an immunologial memory (7). Resistane to hallenge as aquired by immunization with live or killed vaine was assessed by omparing the rates of growth or inativation of hallenge organisms in spleens of different groups (6). The results obtained by Germanier (2), whih were similar to those presented here and in a previous publiation (6), suggest that to produe good protetion a vainal strain must indue a proess that resembles baterial olonization by wild-type strains. In mie vainated with strain Rv6 or infeted for the first time with virulent strain 5/5, the mehanisms of s U 3 E - E!3 (U INFECT. IMMUN days * 4 months time between vaination and hallenge FIG. 4. Preoity and duration of protetion indued by strain Rv6 in CD- mie. Mie subutaneously immunized with 07 bateria per mouse (O) and unvainated ontrols (0) were subutaneously hallenged in the left hind footpads with around 5 x 05 S. abortus-ovis 5/5 per mouse. They were killed 6 days after hallenge. Eah point represents the mean of values for eight mie; standard errors are also shown.

5 VOL. 40, 983 TABLE 3. VACCINAL S. ABORTUS-OVIS MUTANT 605 Evidene that live vaine strain Rv6 indues higher protetion than ommerial killed vaine against lethal or nonlethal dose of hallengea Salmonella per spleen (loglo, mean ± SE)' Immunization proedure OF mie hallenged with (no. of CD- mie hallenged with (no. of bateria): bateria): 2 x 04 2 x 06 2 x 06 2 x 08 None (ontrols) 3.92 ± 0.73 () 6.84 ± 0.35 (7) 4.79 ± ± 0.29 (4) Commerial killed vaine.53 ± ± ± ± 0.26 Live vaine Rv ± ± ± 0.6 a Mie were immunized by subutaneous inoulation in their neks with 08 bateria per mouse and were hallenged month later with the indiated dose of S. abortus-ovis 5/5 subutaneously injeted in the left hind footpad. b Values for surviving mie on day 6 after hallenge (0 mie per group); when mortality ourred, number of dead mie is shown in parentheses. resistane of hallenge appeared to be similar in the following ways. (i) Immunization with either strain redued the dissemination and multipliation of hallenge organisms and inreased the rate of their elimination from infeted tissues. (ii) Protetion indued by strain Rv6 inreased with the dose of immunization, as it does in the ase of primary infetion (23). (iii) Antibody responses on similar levels were provoked by the administration of either strain; furthermore, sera from mie immunized with strain Rv6 and with 5/5 produed a similar level of protetion against S. abortus-ovis when passively transferred to reipient mie. Therefore, the slightly higher immunogeniity of virulent strain 5/5 might be due more to quantitative than to qualitative fators, i.e., to the in vivo apaity of multipliation, dissemination, and persistene rather than to modifiations of the ell wall (whih may involve proteting antigens). But this hypothesis has not yet been properly investigated. Our results, however, obtained with a partiular serotype of S. abortus-ovis, onfirm the TABLE 4. Immunization proedurea superior effiay of live over heat-killed salmonella vaine (9, 2, 7). The effiay of live vaine has been explained by residual multipliation in the host, in vivo development of a omplete array of antigeni strutures, or both, depending upon the importane attributed to humoral or ellular immunity in salmonellosis (6, 29). In fat, ontrol of baterial infetion involves at least three types of mehanisms, eah essential for the survival of the host (28). First, baterial implantation is restrited by early nonspeifi ativities of marophages (5, 20) and soluble fators of serum (). Seond, salmonella dissemination and multipliation is delayed by antibodies speifi for antigens from the ell wall, i.e., to a great extent for the lipopolysaharides (8, 5, 9, 30). In addition, it should be noted that an early synthesis of antibodies has been shown with various models (, 4, 2). Third, the development of a ell-mediated immunity, whih oinides with ontrol of infetion by a normal nonimmunized host, enables the latter to eliminate bateria from infeted tissues (6, 8). In the absene of omplete Protetion indued by passive transfer of immune serum in CD- mie Agglutinin titers for donor mieb Salmonellae per spleen for reipient mie (logl0, Anti-O Anti-H mean ± SE)C None <0 < ± 0.25 Live S. abortus-ovis 5/5 (06 per mouse) /60 / ± 0.33 Rv6 (08 per mouse) /60 / ± 0.29 a Donor mie were immunized by subutaneous inoulation in the neks. They were bled 22 days later. I Agglutinins were titrated by a miroagglutination tehnique with olored S. abortus-ovis as antigens. Values are the highest dilutions at whih signifiant agglutination ould be observed. Reipient mie were immunized with 0. ml of immune serum apiee intravenously inoulated in a tail vein. They were hallenged 4 h later with 5 x 0' S. abortus-ovis 5/5 subutaneously injeted in the left hind footpad. Spleen ounts were made on day 3 after hallenge, eight mie per group.

6 606 LANTIER, PARDON, AND MARLY TABLE 5. Protetion indued by live vaine strain Rv6 in OFI mie hallenged by the oral route' *No. dead Salmonellae per Immunization proedure (total, No ed 0) mean spleen + (logl0, SE)b None ± 0.26 Heat-killed S. abortus-ovis 5/5 Rv ± ± 0.20 Live S. abortus-ovis Rv ± 0.36 a Mie were immunized by subutaneous injetion of 08 viable or heat-killed ( h, 60 C) bateria suspended in saline. They were hallenged month later by daily administration for 3 days of drinking water ontaining 08 S. abortus-ovis 5/5 per ml, i.e., a total of around.2 x 09 bateria per mouse. b Day 5 after the end of hallenge. Freund adjuvant, urrently available killed vaine, unlike live vaines, seems unable to indue T-ell-mediated mehanisms of resistane (7) Ṫhe results of our experiments on mie have been onfirmed to a ertain extent by experimental infetion of pregnant ewes. When vainated with strain Rv6, ewes were proteted against a subutaneous hallenge that provoked abortion in 90% of nonimmunized ontrols and in 60%o of the animals vainated with ommerial killed vaines (29). These results onfirm the effiieny of vaination with a streptomyin-independent reverse mutant, whih was first demonstrated fgainst bruellosis (2, 0). They make possible the development of a murine model of ontrol (26) for the eventual ommerial prodution of vaine Rv6, the effiay of whih has now to be demonstrated in field experiments. ACKNOWLEDGMENTS We are grateful to C. Le Louede, S. Russell, and Alison Tait for their ritial review and valuable help in preparing the manusript, and to M. Salbreux for seretarial assistane. M. Plommet and R. Fensterbank are aknowledged for their ontinuing interest and support. Thanks are also due to E. Rabouan, L. Maurer, and G. Bourgy for are of mie. 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