INFLUENCE OF GUINEA PIG PLASMA FACTORS ON PHAGOCYTOSIS

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1 INFLUENCE OF GUINEA PIG PLASMA FACTORS ON PHAGOCYTOSIS OF PASTEURELLA PESTIS II. PLASMA FROM PLAGUE-INFECTED GUINEA PIGS W. G. STANZIALE1 AND J. D. WHITE U. S. Army Chemical Corps Biological Laboratories, Fort Detrick, Frederick, Maryland Received for publication August 1, 1961 ABSTRACT STANZIALE, W. G. (Fort Detrick, Frederick, Md.) AND J. D. WHITE. Influence of guinea pig plasma factors on phagocytosis of Pasteurella pestis. II. Plasma from plague-infected guinea pigs. J. Bacteriol. 83: The phagocytosis enhancing property of normal guinea pig plasma was altered during experimental plague infection. The most notable changes occurred in the plasma from afebrile, moribund animals and from those convalescing from acute infection. The plasma of the moribund guinea pigs, which was obtained 7 to 8 days after exposure to Pasteurella pestis, inhibited phagoeytosis to a considerable degree. Plasma from convalescent guinea pigs enhanced phagocytosis to a higher degree than the plasma of normal animals. In contrast, plasma from recovered guinea pigs in which cultural or serological evidence of infection was not demonstrated enhanced phagocytosis to a degree equivalent to that of normal plasma. Plasma taken from guinea pigs during the febrile phase of plague infection enhanced phagocytosis to a lower degree than did normal plasma. MATERIALS AND METHODS Methods and preparation of materials for assaying in vitro phagocytosis were essentially the same as in the previous investigation. Experimental infection of guinea pigs. A total of 85 guinea pigs weighing 300 to 350 g were each administered 0.2 ml of a suspension of P. pestis strain Alexander (virulent). This volume was injected intraperitoneally, and contained approximately 1,000 viable bacteria by plate count estimation. After incubation at 37 C for 24 hr, blood agar base cultures were emulsified with brain heart infusion broth. The resulting suspension was adjusted to desired density with a Coleman spectrophotometer at 610 m,u. Concentration of bacteria was determined from a standard curve. The desired dilution for injection was also prepared with brain heart infusion broth. Surface plate counts of appropriate dilutions were made on blood agar base plates incubated at 28 C for 48 hr. Blood cultures. Evidence of plague infection in exposed animals was obtained during the febrile phase of infection by culturing 1-ml samples of cardiac blood in heart infusion broth. The cultures were incubated at 28 C for 7 days before being considered negative. Species identification was made serologically and by the fluores- The enhancing effect of blood plasma from normal guinea pigs on the in vitro phagocytosis cent antibody technique. of Pasteurella pestis was reported in the preceding paper (Stanziale and WVhite, 1962). The 18 guinea pigs underwent streptomycin therapy Streptomycin therapy. An additional group of present study is concerned with the influence for 5 consecutive days (Meyer and Quan, 1949). of plasma from plague-infected guinea pigs. The Treatment consisted of daily intramuscular enhancing or inhibiting effects of plasma during injections of 25 mg of streptomycin sulfate various phases of infection were compared with (Eli Lilly & Co., Indianapolis, Ind.) per pound of those of normal plasma. body weight. This was initiated as soon as the I Present address: Department of Biology, St. infected animals were febrile and blood cultures Joseph College, West Hartford, Conn. had been taken. 182

2 1962] PHAGOCYTOSIS OF P. PESTIS. II. 183 RESULTS Phases of experimental plague infection. Normal base line temperatures were established for each guinea pig during 2 days preceding challenge. Average base line temperatures were within a range of to F. After exposure to P. pestis, temperatures were recorded every 6 to 8 hr for 15 days; 72 of 85 animals exhibited a temperature increase approximately TABLE 1. Comparison of untreated and streptomycintreated guinea pigs infected with plague Number of guinea pigs* Untreated Streptomycintreated Number of animals exposed Febrile Convalescent 1 6 Survivor Dead 67 1 * Each guinea pig infected cells, intraperitoneally so - A 0Vl X 9 C8 1\ Z50 t\i z. Il < 40 - E with 1000 bacterial - NORMAL PLASMA FEBRILE PLASMA *- AFEBRILE PLASMA 4 to 5 days after exposure (Table 1). Average maximum temperatures ranged from to F. Plasma obtained from animals at this time was designated "febrile" plasma. Guinea pigs succumbing to plague became afebrile and moribund 6 to 10 days after exposure (average 7 to 8 days). These animals had subnormal temperatures ranging from 94.0 to 96.5 F. Plasma from seven of these animals was designated "afebrile" plasma. Plasma was obtained from surviving guinea pigs 27 days after exposure to P. pestis. This plasma was designated "convalescent" llasma if a precipitin titer was demonstrated or if positive blood cultures were demonstrated during the febrile phase. If serological or cultural evidence for infection was not obtained, the plasma was denoted "survivor" plasma. Effect of febrile plasma. The phagocytic response in the presence of plasma from plagueinfected guinea pigs was compared with data previously established with normal plasma. Phagocytosis was measured with plasma concentrations ranging from 1.25 to 50% PLASMA CONCENTRATION (PER CENT) FIG. 1. Comparison of in vitro phagocytosis of Pasteurella pestis in plasma from normal, febrile, and afebrile guinea pigs. 40

3 184 STANZIALE AND WHITE [VOL. 83 Phagocytosis of P. pestis was enhanced in the presence of 2.5 to 25% febrile plasma. Maximal enhancement (85% phagoeytosis) was observed with 6.25% plasma, but there was no difference between the effect of 6.25, 12.5, and 25% concentrations. Phagocytosis in the presence of 50% plasma was the same as in control mixtures devoid of plasma (Fig. 1). Although the curves for normal and febrile plasmas appear to follow a similar trend, the degree of phagoeytosis promoted by 5, 12.5, and 50% febrile plasma was statistically lower than that promoted by the same concentrations of normal plasma. The activity of "febrile" plasma was statistically lower than "convalescent" plasma in all concentrations from 5 to 50%; 95% confidence limits for these plasma concentrations are presented in Table 2. The determination of statistically significant differences between plasmas at points of overlap was made by application of the t-test. Effect of afebrile plasma. Phagoeytosis of P. pestis was inhibited to a profound degree by afebrile plasma (Fig. 1). Inhibition of phagocytosis was noted with all plasma concentrations tested. This is in contrast to the enhancing influence of plasma from normal noninfected guinea pigs. Cultural evidence of plague had been obtained in all animals during the febrile phase of infection. Additional evidence of infection was obtained at death from liver imprints, which were positive for P. pestis when stained bv the fluorescent antibody technique. Effect of convalescent plasma. Phagocytosis of P. pestis was greatly enhanced in convalescent plasma concentrations ranging from 5 to 50%. The plasma samples assaved were obtained from plague-recovered guinea pigs in which a cultural diagnosis of infection had been made during the febrile phase, and which had received streptomycin therapy. From one untreated animal, plasma that was culturally negative, but had an agar-gel precipitin titer of 1:128, was also assayed. No differences were observed between the two types of convalescent plasma described above. Maximal phagoeytic response (94 to 98% phagoeytosis) occurred consistently within 6.25 to 50% l)lasma concentrations (Fig. 2). Convalescent plasma promotes a uniformly higher range of phagoeytic response than either normal or febrile plasmas. There is no overlap in 95% confidence limits between convalescent and normal or convalescent and febrile plasmas, within the range of 5 to 50%G plasma concentrations (Table 2). Effect of survivor plasma. The plasma of guinea pigs which survived exposure to P. pestis and were culturally, serologically, and clinically negative enhanced phagoeytosis within a plasma concentration range of 5% to 50%. The curve established with "survivor" plasma was similar to that of normal plasma (Fig. 2), except that the enhancing effect of survivor plasma was lower in 12.5% concentration than was that of normal plasma. The over-all phagocytie response in survivor plasma was somewhat lower than in convalescent plasma (Fig. 2). A consideration of the 95 % confidence limits of survivor and convalescent plasma at 5, 12.5, and 50% concentrations indicates that survivor plasma promoted a statistically lower degree of phagoeytosis (probability less than 0.05). At a 25%7 concentration, however, a statistical difference was not detected at this probability level. TABLE 2. Statistical evaluation of phagocytosis-enhancing influence of guinea pig blood plasma 95% Confidence limits* Concentration (%) Plasma type Normal Febrile Convalescent Survivor (79)75-83t (68) (87)83-91 (79)74-84 (84)82-86 (85)80-90 (95)93-97 * t-test applied where overlapping occurred. t Figures in parentheses represent the arithmetic means. (93)92-94 (85)80-90 (98)97-99 (75)68-82 (85)83-87 (81)76-86 (94)93-95 (86)73-99 (83)81-85 (54)49-59 (92)89-95 (86)83-89

4 1962] PHAGOCYTOSIS OF P. PESTIS. II O- p u I z U z NORMAL PLASMA -----CONVALESCENT PLASMA -*- SURVIVOR PLASMA 3.;25 PLASMA CONCENTRATION (PER CENT) FIG. 2. Comparison of in vitro phagocytosis of Pasteurella pestis in plasma from normal, surviving, and convalescent guinea pigs. DISCUSSION As with normal plasma, relatively low concentrations of febrile, convalescent, and survivor plasmas enhance in vitro phagocytosis of P. pestis. A host defense mechanism, consisting of a thermolabile and a thermostable plasma component, was described in the previous paper (Stanziale and White, 1962). Although the mechanism can be demonstrated during or after infection, the present studies indicate that the potential of this system to enhance phagocytosis in vitro is affected by the phase of plague infection. Antibody titers greater than 1:2 were observed only rarely. A generally lower enhancing effect was promoted by febrile lplasma than by convalescent plasma. With the latter, an optimal condition was achieved in vitro in which practically 100% of the neutrophils were actively ingesting P. pestis cells. When the effect of convalescent plasma is compared with the effect of plasma from normal animals and survivors (those in which infection was not demonstrated), it may be concluded that infection and4 recovery from plague resulted in an increase in the ability of guinea pig plasma to enhance in vitro phagocytosis. In contrast, the plasma of guinea pigs succumbing to plague elicited a considerable phagocytosis-inhibiting effect. A thermostable phagocytosis-inhibiting factor, detectable in normal serum but not in plasma, was also described in the previous paper. The presence of this factor, in addition to the enhancing factors in plasma and serum, offers a basis for reconsidering the role of phagocytosis in the pathogenesis of plague in the guinea pig. It may be speculated that in the initial phases of plague the enhancing factors in normal plasma are in relatively high concentration, and the phagoeytic mechanism is very active. As infection proceeds, the concentration of these factors may decrease below that of the serum inhibiting factor, so that phagocytosis decreases in intensity. The inhibiting factor becomes predominant and the animals become moribund as a

5 186 STANZIALE AND:WHITE [VOL. 83 result, at least in part, of phagocytosis inhibition. If the concentration of enhancing factors does not decrease below that of the serum inhibiting factor, sufficient phagocytosis is promoted to control the multiplication of P. pestis, and the infected animal recovers. A consideration of the role of complement as one of the phagocytosis-enhancing factors is suggested by the work of Ecker, Seifter, and Dozois (1945), who reported that complement titers often decrease to near zero during the course of infection with other pathogenic bacteria. In 14 of 300 cases, this coincided with a serious phase in the patient's condition. In addition, Zhukov-Verezhnikov and Faddeva (1937) observed an increase in the percentage of guinea pigs which survived plague infection when they treated infected animals with antiplague serums modified by addition of fresh complement, as compared with animals treated only with antiplague serum. LITERATURE CITED ECKER, E. E., S. SEIFTER, AND T. F. Dozois Human complement. J. Lab. Clin. Med. 30: MEYER, K. F., AND S. F. QUAN Plague, p In S. A. Waksman [ed.], Streptomycin. Williams & Wilkins Co., Baltimore. STANZIALE, W. G., AND J. D. WHITE Influence of guinea pig plasma factors on phagocytosis of Pasteurella pestis. I. Plasma from normal guinea pigs. J. Bacteriol. 83: ZHUKOV-VEREZHNIKOV, N., AND T. FADDEVA Immunology in plague. VI. Bacteriolysis. Vestnik Mikrobiol. Parazitol. Epidemiol. 16: Downloaded from on August 21, 2018 by guest