Immune defence mechanism against blood-borne Pasteurella multocida in turkeys

Research in Veterinary Science /990, 48, 344-349

Immune defence mechanism against blood-borne Pasteurella multocida in turkeys M. TSUJI, M. MATSUMOTO, College of Veterinary Medicine, Oregon State University, Corvallis, Oregon 97331, USA

Humoral and cellular immune defence factors ments with immune serum or specific IgG antibody involved in controlling blood-borne Pasteurella (Alexander and Soltys 1973, Rebers et al 1975, multocida were investigated in turkeys by the passive Nathanson et al 1980). However, Baba (1984) transfer of immune serum or by the treatment with reported that immunity was adoptively transferable macrophage-activating agents. The treated and into susceptible birds by administration of the spleen untreated birds were intravenously inoculated with a cells from immune birds. He further demonstrated virulent strain of P multocida, and the viable bacteria that the peritoneal macrophages obtained from in the blood, liver and spleen were counted. In immunised birds, but not those from unimmunised untreated birds, the bacteria were rapidly removed birds, were capable of inhibiting the intracellular from the blood, and the majority were recovered from multiplication of P multocida. the liver and spleen 120 minutes after inoculation. Whether hosts permit or prevent the development Neither the transfer of immune serum nor the treat- of fatal septicaemia may depend on the bactericidal ment with macrophage-activating agents significantly capability of the host. A previous study showed that influenced the clearance rate of bacteria from the the bacteria, when inoculated intravenously, were blood. The number of bacteria recovered from the rapidly removed from the bloodstream in both liver 120 minutes after inoculation was slightly lower immune and susceptible birds (Tsuji and Matsumoto in the birds treated with macrophage-activating 1989). Since the majority of bacteria were recovered agents and significantly lower in those given immune from the liver and spleen shortly after the inoculation, serum than in the untreated birds. None of the treat- the bacteria were presumed to be captured by reticuloments, however, significantly changed the number of endothelial phagocytes in these organs. Removal of bacteria recovered from the spleen 120 minutes after bacteria from the blood, however, does not assure inoculation. The results suggest that the phagocytes in their elimination. Specific immunity is a prerequisite the liver, but not in the spleen, play a crucial role in the for the subsequent inactivation of bacteria. The objective of this study was to investigate the intravascular defence against P multocida in the possible factors involved in the intravascular defence presence of specific antibodies. against P multocida at an early stage of intravenous FOWL cholera, a severe septicaemic disease of avian infection in turkeys. Involvement of humoral or species, is caused by Pasteurella multocida. In natural cellular defence factors was tested by the passive infections, the organism probably enters the host transfer of immune serum or treatment with macrofrom the pharynx and, or, upper respiratory tract phage-activating agents, respectively, observing the (Hughes and Pritchett 1930, Maheswaran et al 1973). clearance of bacteria from the bloodstream and the Following the invasion, the bacteria seem to spread recovery of viable bacteria from the liver and spleen via the bloodstream and initially localise in the liver 120 minutes after infection. and spleen (Tsuji and Matsumoto 1989). The bacteria grow primarily in these two organs and are abruptly Materials and methods released into the bloodstream at the terminal stage, Bacterial strain resulting in fatal septicaemia (Pabs-Garnon and Pasteurella multocida strain Pol 059 (serotype 3:A), Soltys 1971). At the time of death, the numbers of bacteria in the liver and spleen usually exceed 109 g - I. a highly virulent, encapsulated strain of turkey origin, Immunity against fowl cholera can be induced by was used. The strain was kindly supplied by Dr K. R. vaccination. Although the nature of the immunity is Rhoades, National Animal Disease Center, Ames, not understood, both humoral and cellular factors Iowa. To maintain the virulence, the strain was passed may play roles. Involvement of humoral factors has six times in susceptible turkeys. Organisms were rebeen demonstrated by passive immunisation experi- isolated from the liver and stored at -70°C. 344

345

Immune defence mechanism against P multocida Immune serum Thirty Nicholas Large White turkeys were subcutaneously immunised twice, at seven and nine weeks old, with an oil-emulsified vaccine consisting of Freund's incomplete adjuvant (Difco) and a protective antigen (designated 2'5S antigen; 250 Jlg per dose) purified from the strain P-1059 as described previously (Syuto and Matsumoto 1982). The birds were intramuscularly challenge-exposed at 11 weeks old with 1·1 x I

Inoculum preparations for clearance test For each clearance experiment, the strain P-I059 was streaked on a dextrose starch agar (Difco) plate and incubated overnight at 37°C. Three to five colonies were picked and spread out on another plate. The plate was incubated at 37°C for three to four hours and harvested in Dulbecco's phosphate buffered saline at pH 7·4 (PBS). For groups 1,2,3,5, 6 and 9, bacterial suspensions were diluted with PBS to give an optical density value of O' 30 at 600 nm, corresponding to approximately 6 x 108 cfu ml : I.

For groups 4, 7 and 8, bacterial concentrations were adjusted at the optical density value of O·60 and the suspensions were mixed with an equal volume of immune serum (diluted 1:3 with PBS). The mixtures were incubated at 37°C for 20 minutes. This treatment did not cause any agglutination of bacteria. Each inoculum was kept on ice and used within two hours. Actual inoculum doses, determined by plate colony-counting with heart infusion agar (Difco), are listed in Table I.

Treatment of turkeys for clearance tests Clearance experiments were carried out in nine groups. Groups 1 to 8 consisted of four or five Wrolstad Medium White turkeys. In group 9 Nicholas Large White turkeys were used. The plan of inoculation of birds with immune serum and bacteria is indicated in Table 1. Two macrophage-activating agents were used; Mycobacterium butyricum (killed dry cells; Difco) in group 2, and Propionibacterium acnes (previously termed Corynebacterium parvum; heat killed cells in Iyophilised form; Ribi ImmunoChern Research) in groups 3 and 8. Both M butyricum and P acnes were suspended at 10 mg ml : 1 (by dry weight) in a 0·85 per cent sodium chloride solution containing 1 per cent gelatin and O·1 per cent Tween 80. A dose of 25 mg (in 2' 5 ml) was inoculated intravenously five days before the clearance experiments (groups 2, 3 and 8). This dose was determined in a preliminary experiment, where doses over 50 mg caused some toxic effects. Immune serum was intravenously administered at 3 ml kg- 1 of bodyweight either 30 minutes before (in''groups 5, 7 and 8) or 30 minutes

TABLE 1: Design for clearance experiments with P multocida strain P-1 059 in turkeys

Group

Treatment of turkeys

1 2 3 4 5 6 7 8

None M butvricumP ecnes) None IS ( - 30 minutes) II IS ( + 30 minutes)" IS (- 30 minuteslll P series- and IS (- 30 minutes) II

9

lrnrnunet"

tt

Inoculum of bacteria Sensitised Dose with (log 10)

Number used

Turkeys Age SIB (weeks I ratio"

Antibody

titre"

None None None IS§ None None IS§

8·7 8'8 8·4 9·1 8·5 8·7 9·1

5 5 4 5 4 4 4

11 14 15 12 10 10 15

0·9 1 ·3 1 ·8 1·0 1·0 0·9 0'9

1·01 1·67 1·86 1 ·14 0·88 1 ·19 0·98

IS§ None

8·8 9·0

4 5

9 13

1·5 0·9

1·76±0·25 4·31 ± 0'30

± ± ± ± ± ± ±

0·16 0·11 0·21 0·17 0·16 0·08 0·40

Ratio of the mean spleen weight relative to the mean bodyweight (g kg - ,) Serum samples were obtained about one hour before the clearance tests, and the antibody tit res (mean ± standard deviation in log,o) against the 2' 5S antigen of P multocida P-1059 were determined by ELISA. Titres below 1 . 50 were considered to be negative Inoculated intravenously, at 25 mg per dose, five days before the clearance tests P multocida were incubated with immune serum (IS) before inoculation Immune serum was administered intravenously, at 3 ml kg - , of bodyweight, 30 minutes before inoculation of P multocida Immune serum was administered 30 minutes after inoculation of P multocida in the identical manner Survivors on day 16 after vaccination and challenge with P multocida P-1 059

M. Tsuji, M. Matsumoto

346

after (in group 6) inoculation of P multocida. The immune birds used in group 9 have been described earlier. Serum samples were obtained from every bird before each clearance experiment, and the antibody tit res against the 2·5 S antigen were determined by the ELISA. The treated or untreated birds were intravenously inoculated with immune-serum-sensitised or unsensitised bacteria. Blood samples were collected from each bird at 10, 20, 30, 60, 90 and 120 minutes after infection with P multocida. Immediately after the last bleeding, the birds were killed by electrocution (120 V AC) and a portion of the livers (about 4 g) and the whole spleens were quickly removed. Bodyweights, as well as whole liver and spleen weights, were obtained from individual birds. The number of viable bacteria in each specimen was determined by plate colony-counting with heart infusion agar. Details of specimen sampling, tissue homogenisation and bacterial enumeration methods have been described (Tsuji and Matsumoto 1989). Bacterial concentration of each blood sample (cfu ml- 1) was converted to the number per total blood volume, and, in turn, to the recovery rate relative to the inoculum dose using the following formula: Recovery rate (per cent) = cfu ml- 1 x bod~weight x 0·08 x 100 cfu of the Inoculum The total blood volume was estimated at 8 per cent of the bodyweight (Sturkie and Griminger 1986). Recovery rates from the liver and spleen were calculated as follows: Recovery rate (per cent) = cfu g -I X organ weight ~~~~-'='-----,"-:::'x 100 cfu of the inoculum

Passive protection test Protective potency of the immune serum was tested in 31-day-old Nicholas Large White turkeys. The birds were intramuscularly inoculated with various doses of the immune serum. After 24 hours, the birds were intravenously challenge-exposed with 1·2 X 103 cfu of P multocida P-1059. Mortality was recorded daily, and survivors at day 14after exposure were subjected to euthanasia. All birds were examined at necropsy, and isolation of P multocida was attempted from the liver using dextrose starch agar plates.

Statistical analysis The recovery rates were transformed into log., values and the mean and standard deviation were calculated in each group. Differences among the mean recovery rates were examined in one-way tables of analysis of variance using Fisher's protected LSD

test. Differences in mortality rates were tested by Fisher's exact test. Results

Clearance ofP multocidafrom the bloodstream Nine experiments were conducted with untreated or treated turkeys (Table 1). The results are shown in Fig I. In all the treated and untreated birds, more than 99·9 per cent of inoculum disappeared from the bloodstream within 30 minutes after infection. Clearance of bacteria, however, was slower in the M butyricum-treated or in the P acnes-treated birds (groups 2 and 3, respectively) than in the untreated ones (group I). Clearance rate was slightly enhanced when P multocida had been sensitised with immune serum (group 1 versus 4). The passive transfer of immune serum did not enhance the rate of clearance within 30 minutes after infection (group 1 versus 5), although the numbers of bacteria in the blood at 90 and 120 minutes after infection were significantly lower in group 5 than in group 1 (P
Bacterial recovery at 120 minutes after infection Fig 2 shows the recovery rates (per cent of inoculum) of viable bacteria from the blood, liver and spleen at 120 minutes after infection. Major differences among the nine groups were observed in the recovery rate from the liver. The mean recovery rate from the livers of immune birds was 800-fold less than that from untreated birds (group 1 versus 9). On the concentration basis (cfu g-l), the numbers of bacteria in the liver and in the spleen of the untreated birds were about 1000 and 3000 times higher, respectively, than those in the blood. In the immune birds, by contrast, the concentrations in the liver and in the blood were 1000 and 30,000 times less, respectively, than those in the spleen. The recovery rate from the liver of M butyricum- or P aenes-treated birds was slightly lower than that from untreated birds (group I versus 2 or 3); the diffference between groups I and 3 was significant (P < 0 ·01), but the difference between groups 1 and 2 was not (P > 0·05). Sensitisation of P multocida with immune serum significantly reduced the bacterial recovery from the liver (group 1versus 4,

Immune defence mechanism against P multocida 100

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347

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Group 1

_

Group 4

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Group 2

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Group 5

. "D·"

Group 8

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Group 6

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-Group7

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0·1

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if'. 0·01

0·001

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30

I

60

o

120

90

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30 60 90 Time after injection (mini

120

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120

FIG 1: Clearance of P multocida from the bloodstream in the nine groups of turkeys. Vertical bars on each mean indicate standard deviation. The group numbers correspond to those shown in Table 1

P O' 90). The birds in groups 2, 3 and 8 had enlarged spleens, and their antibody titres "0

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FIG 2: The percentage bacterial recovery from the blood (BI. liver (LI and spleen (51 120 minutes after the intravenous inoculation of P multocida. Vertical bars on each mean indicate standard deviation. The group numbers correspond to those shown in Table 1

in ELISA were slightly higher than those of untreated birds (Table 1). The immune birds had high antibody titres (Table 1).

Passive protection The birds which had received the immune serum were protected against the intravenous challenge infection with P multocida strain P-1059 (Table 2). Protection rates were roughly proportional to the dose of immune serum. A 50 per cent protective dose ofthe immune serum was estimated at approximately I ml kg - I 0 f bodyweigh t. Discussion Rapid removal of P multocida from the bloodstream was observed not only in the immune but also in the untreated birds. The clearance process, therefore, appears to be essentially independent of specific immunity. The clearance rate in the birds which had been treated with macrophage-activating agents was slower than that in the untreated birds. In the treated birds, some bacteria may have been associated with the monocytes in the bloodstream, because intravenous inoculation of P acnes has been shown to induce activation of reticuloendothelial phagocytes as well as monocytes in the peripheral blood (Ghaffar 1981). Whether specific antibody serves as an effective opsonin for the phagocytes in the blood vascular system is not clear. The passive transfer of immune serum did not enhance the clearance rate within 30 minutes after infection. The organisms may have been removed too rapidly to be sufficiently opsonised by the antibody. In fact, clearance rate was slightly

M. Tsuji, M. Matsumoto

348

TABLE 2: Protective potency of the immune serum tested by administration into 31-day-old turkeys followed by intravenous challenge infection with P muitocide strain P-1059 Group

Serum sample"

1

Immune Immune Immune Immune

2

3

4 5

serum serum serum serum

Normal serum

Dose (mil 3·0 1 ·0 0·3 0·1 3·0

Mortalityt

1/1011 3/1011 7/10 8/10 10/10

MDT:

Isolation of P muJrocida §

9·0 9·3 5·6 5·0 1·6

1/10 4/10 7/10 8/10 10/10

Intramuscularly administered into the birds. which weighed 691 ± 80 g (mean ± standard deviation), 24 hours before the intravenous challenge with 1 . 2 x 10 3 cfu of P muJtocida strain P-1059 Number of birds deadltotal infected Median death time (days) Number of P multocida positiveltotal examined Significantly different from group 5 (P < 0·01 )

accelerated when the bacteria had been incubated with immune serum before the inoculation (in group 4 but not in group 7). However, even if specific antibody acts as an effective opsonin, such a role has only a secondary importance because the clearance capability in the absence of specific antibody was effective enough to remove more than 99· 9 per cent of the inoculum from the bloodstream within 30 minutes. Effects of various treatments were most evident on the recovery rate of bacteria from the liver 120 minutes after infection. This sampling period was determined based on the results of the previous study (Tsuji and Matsumoto 1989), which demonstrated that the numbers of bacteria in the liver of untreated birds were relatively constant between 10 and 120 minutes after infection. In immune birds, by contrast, the bacterial number in the liver decreased about 300-fold during this period. It has also been shown that the presence or absence of immunity did not significantly change the distribution of bacteria among the liver, spleen and blood 10 minutes after infection. Therefore, the difference between the untreated and each treated group reflects primarily the extent of bactericidal activity in the liver in each group. The bacterial recovery from the liver was reduced to a greater extent by the transfer of immune serum than by the treatment with macrophage-activating agents. Thus, specific antibodies may be more important than activation of macrophages for the bactericidal activity in the liver. In the immune birds, however, both factors appeared to work synergistically. The birds that had been treated with both immune serum and P aenes (group 8) had low numbers of bacteria in the livers, which was comparable with those of the immune birds. The mode of action by which the specific antibody facilitates the bactericidal activity in the liver is not clear. The authors' working hypothesis was that the hepatic phagocytes are capable of killing P multocida

only when the bacteria have been sensitised (or opsonised) with specific antibody before or during the phagocytosis. The results obtained in groups 4, 5 and 7 support this hypothesis, but the result in group 6 does not. The birds in group 6 received the immune serum 30 minutes after the inoculation of P muftocida. Judging from the clearance profile, there seemed to be enough time for the hepatic phagocytes to engulf the bacteria by the time of immune serum administration. If so, the antibodies should not have access to the intraceIlular bacteria. Nonetheless, the bacterial recovery from the liver in this group was significantly lower than that in the untreated group. The immune serum may have possibly contained some unknown factors, besides the specific antibodies, which externaIly enhance the bactericidal activity of hepatic phagocytes. Further trials should be done with the purified immunoglobulin fraction at various periods after inoculation. None of the treatments facilitated the inactivation of bacteria in spleen within 120 minutes after infection. In contrast to the hepatic phagocytes, the splenic macrophages appear to be poorly capable of inactivating P muftocida at an early period of infection. Similar findings have previously been reported (Tsuji and Matsumoto 1989). However, since the passively immunised birds were protected against intravenous chaIlenge infection (Table 2), the inactivation of bacteria in the spleen may have been observed at later periods. The results of the passive protection test clearly indicate the importance of humoral factors for protection against blood-borne P muftocida, but they do not necessarily exclude the importance of ceIlular factors. It is possible that the specific antibody, in association with complement, directly inactivated the bacteria without the aid of phagocytes. However, if solely the humoral factors were responsible for the killing of bacteria, the decrease in bacterial recovery should have been observed not only in the liver but also in the spleen.

Immune defence mechanism against P multocida The evidence presented here suggests that the phagocytes in the liver, but not in the spleen, playa crucial role in controlling the blood-borne P multocida at an early phase of infection. Neither specific antibody nor macrophage activation is required for the initial, rapid removal of the bacteria from the blood stream. The presence of specific antibody, however, appears to be essential for the hepatic phagocytes to be capable of inactivating the bacteria. Activation of macrophages may also contribute to enhancing the bactericidal activity in the liver. Acknowledgements

This study was supported by the Oregon Agricultural Experiment Station fund 00168 and by a special grant from USDA, Science and Education, 87-CRSR-23123. The study was included in a thesis submitted by the senior author to the graduate school of Oregon State University as partial fulfillment of the requirements for the PhD degree in microbiology. A portion of the results was presented at the 37th Western Poultry Disease Conference at Davis, California, March 1988.

349

References ALEXANDER, A. M. & SOLTYS, M. A. (1973) Journal of Comparative Pathology 83, 191-198 BABA, T. (1984) Research in Veterinary Science 36,225-230 GHAFFAR, A. (1981) Manual of Macrophage Methodology. Eds H. B. Herscowitz, H. T. Holden, J. A. Bellanti and A. Ghaffar. New York, Marcel Dekker. pp 441-446 HUGHES, T. P. & PRITCHETT, I. D. (1930) Journal of Experimental Medicine 51, 236-248 MAHESWARAN, S. K., McDOWELL, J. R. & POMEROY, B. S. (1973) Avian Diseases 17,396-405 NATHANSON, R. M., HOFSTAD, M. S. & JESKA, E. L. (1980) American Journal of Veterinary Research 41, 1285-1287 PABS-GARNON, L. F. & SOLTYS, M. A. (1971) Canadian Journal of Comparative Medicine 35, 147-149 REBERS, P. A., HEDDLESTON, K. L., WRIGHT, B. & GILLETTE, K. (1975) Carbohydrate Research 40, 99-110 STURKIE, P. D. & GR1MINGER, P. (1986) Avian Physiology. Ed P. D. Sturkie. 4th edn. New York, Springer-Verlag. pp 102-130 SYUTO, B. & MATSUMOTO, M. (1982) Infection and Immunity 37,1218-1226 TSUJI, M. & MATSUMOTO, M. (1988) American Journal of Veterinary Research 49,1516-1521 TSUJI, M. & MATSUMOTO, M. (1989) Avian Diseases 33, 238-247

Received April 18, 1989 Accepted September 18, 1989