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The influence of growth conditions of Pasteurella multocida on its ability to colonise the nasal mucosa of SPF piglets
Veterinary Microbiology, 24 ( 1990) 81-88 Elsevier Science Publishers B.V., Amsterdam
81
The influence of growth conditions of Pasteurella multocida on its ability to colonise the nasal mucosa of SPF piglets B. l~lifis~, Mfiria Herpay 5, M. Albert 2, Judit Fekete 6, S. Tuboly 3, P. Rafai 4 and E d i t Moln~ir ~ ~Departments of Research Organization, 2pathological Anatomy, 3Epizootiology and 4Animal Hygiene, University of Veterinary Science, H-1400, Budapest, P.O.B 2 (Hungary) SNational lnstitute of Hygiene, H-1966 Budapest, P.O.B. 64 (Hungary) 6public Health Station of Csongrdd County, 1t-6700 Szeged (Hungary) (Accepted 12 December 1989)
ABSTRACT l~lifis, B., Herpay, M., Albert, M., Fekete, J., Tuboly, S., Rafai, P. and Molnfir, E., 1990. The influence of growth conditions of Pasteurella multocida on its ability to colonise the nasal mucosa of SPF piglets. Vet. Microbiol., 24: 81-88. Colonisation of type D Pasteurella multocida was studied in five groups of seven SPF piglets each. Piglets of Group 1 were kept together with seven 5-week-old piglets obtained from a large herd infected with toxigenic P. multocida for 16 weeks (contact infection). These piglets were made free from toxigenic Bordetella bronchiseptica by local immunisation. Piglets of Group 2 were inoculated with 5 X 10 v colony-forming units (cfu) of P. multocida washed from the nasal mucosa of piglets free from toxigenic B. bronehiseptica with fetal calf serum. Piglets of Group 3 were inoculated intranasally with 5 X 10 7 cfu of P. multoeida washed from yeast-extract proteose-peptone cystine (YPC)-blood agar with fetal calf serum. Piglets of Group 4 were inoculated with 5 X 10 7 cfu of P. multocida grown in a YPC-based broth without blood. Piglets of Group 5 served as controls. The piglets of Group 1 did not contract P. multocida infection from infected contact piglets. After a single inoculation one of four, while after three inoculations two of three piglets of Group 2 became infected by P. multocida. After a single inoculation none of four, while after three inoculations one of three piglets of Group 3 were colonised by P. multocida. Both single and repeated inoculation failed in piglets of Group 4.
INTRODUCTION
Progressive atrophic rhinitis (AR) of swine is characterised by necrosis of the nasal respiratory epithelium, atrophy of the nasal turbinates and distortion of the nasal bones (Duncan et al., 1966; Miniats and Johnson, 1980). These lesions develop both in SPF and gnotobiotic piglets intranasally inoculated with toxigenic Bordetella bronchiseptica or repeatedly subjected to lo0378-1135/90/$03.50
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cal treatment of the nasal mucosa with the dermonecrotic toxin (DNT) of B. bronchiseptica (Nakagawa et al., 1974; Hanada et al., 1979 ). Toxigenic Pasteurella multocida also plays a role in producing AR (Pedersen and Barfod, 1981; Pedersen and Elling, 1984), and some consider P. multocida the primary causative agent (Pedersen and Barfod, 1981; Sch6ss, 1987). The interrelationship between P. multocida and B. bronchiseptica in the pathogenesis of AR has not yet been fully elucidated (Pedersen and Barfod, 1982; Rutter and Rojas, 1982; Pedersen and Elling, 1984; Frymus et al., 1986). Authors generally agree that P. multocida shows weak adherence to porcine nasal epithelial cells in vitro (Nakai et al., 1984; Frymus et al., 1986) and to the intact nasal mucosa of pigs in vivo (Oyamada et al., 1986; Nakai et al., 1988 ). Adherence ofP. multocida was generally successful if the nasal mucosa had been exposed to toxigenic B. bronchiseptica a few days before (Rutter and Rojas, 1982; Oyamada et al., 1986 ). In the absence of toxigenic B. bronchiseptica, P. multocida can colonise the nasal mucosa of pigs only if it has been grown on media containing blood (Rutter and Rojas, 1982; Oyamada et al., 1986 ). On the other hand, P. multocida infection of pigs has been reported from herds not infected with toxigenic B. bronchiseptica (Pedersen and Barfod, 1982; Sch/3ss, 1987). The purpose of this work was to study the influence of growth conditions on the ability ofP. multocida to colonise the nasal mucosa. MATERIALS AND METHODS
Experimental animals The SPF piglets were derived by hysterectomy in an SPF laboratory. Up to 2 weeks of age the piglets were kept in isolators, then for 16 weeks, until the end of the experiment, they were reared in climatic chambers in isolation. The large pig herd used in .the trial was infected both with toxigenic B. bronchiseptica and type D P. multocida. At birth, 200 piglets of the herd were inoculated with 5 × 108 colony-forming units (cfu) of an avirulent, non-toxigenic B. bronchiseptica strain (N-104) suspended in PBS via the mucosa of both nostrils (l~li~is et al., 1988 ). The strain had been grown on agar (Bordet and Gengou, 1906 ) (Difco Co., U.S.A. ) containing 10% sterile bovine blood, at 37 °C for 24 h. Bacteriological examination of the piglets was carried out at 4 and 5 weeks of age. Piglets infected with toxigenic type D P. multocida, whose nasal mucosa did not yield B. bronchiseptica, were selected for the experiment. Bacterial isolation and identification B. bronchiseptica was isolated from the nasal mucosa of piglets on BordetGengou and MacConkey agar (Difco) containing 10% sterile bovine blood, at 37°C for 24 h. Isolation of P. multocida was done on YPC agar (cystine agar medium containing yeast-proteose peptone, Difco) containing 10% sterile bovine blood at 37°C for 18 h (Namioka and Murata, 1961 ). The
THE ABILITY OF P. MULTOCIDA TO COLONISE THE NASAL MUCOSA OF SPF PIGLETS
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strains were identified by the criteria of Bergey's Manual (Pittman, 1974; Smith, 1974). The capsular antigens ofP. multocida strains were determined by indirect haemagglutination and by acriflavine and hyaluronidase tests (Carter and Subronto, 1973). The D N T of P. multocida was determined according to Nakai et al. (1984) and that of B. bronehiseptica by the method of l~li~ts et al. ( 1982 ).
Inocula for pigs The inoculum for Group-2 piglets was prepared from the nasal mucosa of 5-week-old piglets rendered free from B. bronchiseptica by local immunisation but infected with toxigenic type D P. multocida. The nasal mucosa collected from killed piglets was washed several times with sterile fetal calf serum (Gibco Laboratories, Grand Island, NY). The number of P. multocida cfu was determined by colony counting. The inoculum for Group-3 piglets was prepared by propagating P. multocida, washed from the nasal mucosa with sterile fetal calf serum, on YPC agar containing 10% sterile bovine blood, at 37°C for 18 h. Agar cultures were washed with sterile fetal calf serum and the bacterial concentration (cfu) was determined by spectrophotometry (SPECOL- 10, Karl Zeiss Jena, G.D.R. ). The inoculum for Group-4 piglets was prepared in a serum-free, YPC-based broth inoculated with Y P C - b l o o d agar culture and incubated at 37 °C for 3 h. The bacterial concentration (cfu) was determined by spectrophotometry. All three inocula were stored in a lyophilized state at - 20 ° C. Experimental design A total of 35 2-week-old SPF piglets were used. The piglets were divided into five groups of seven piglets each, and kept isolated in climatic chambers. The experiment lasted 16 weeks and the piglets were killed at 18 weeks of age. Piglets of Group 1 were kept together for 16 weeks with seven 5-week-old piglets chosen from a large herd (contact infection). The nasal mucosa of the latter piglets was infected with toxigenic type D P. multocida but was free from B. bronchiseptica. Piglets of Group 2 were divided into two subgroups. Four piglets (Group 2a) were inoculated intranasally with 5 X 107 cfu of P. multocida at 2 weeks of age. Three piglets (Group 2b) were inoculated with the same dose of P. multocida repeatedly at 2, 3 and 4 weeks of age. The inoculum was prepared by washing the nasal mucosa of piglets selected from the large herd with sterile fetal calf serum. Piglets of Group 3 were assigned to two subgroups. Four piglets (Group 3a) received a single intranasal dose (5 X 107 cfu) of P. multocida at 2 weeks of age. The other three piglets (Group 3b) were inoculated with the same dose of P. multocida repeatedly at 2, 3 and 4 weeks of age. The inoculum was prepared by propagating on Y P C - b l o o d agar P. multocida washed from the nasal mucosa with sterile calf serum.
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Piglets of Group 4 were also divided into two groups. Four piglets (Group 4a) were inoculated intranasally with 5 X 10 7 cfu of P. multocida at 2 weeks of age. Three piglets (Group 4b) were inoculated intranasally with the same dose ofP. multocida repeatedly, at 2, 3 and 4 weeks of age. The inoculum was prepared in a serum-free YPC broth inoculated with Y P C - b l o o d agar culture. Piglets of Groups 2, 3 and 4 were inoculated with 0.5 ml inoculum into both nostrils, using a syringe. For inoculation, the animals were immobilized with the head upwards. The piglets were examined bacteriologically on the day before inoculation and then at 7-day intervals for 18 weeks. Bacteriological examination included examination of the pharyngeal tonsils of piglets killed at 18 weeks of age.
Ammonium sulphate aggregation test Using phosphate buffer (pH 6.8 ), an a m m o n i a sulphate dilution series was made from 0.2 to 3.2 mol, with a difference of 0.2 tool between the dilutions. This series was used for testing the aggregation of P. multocida strains 3 and 4 (used for inoculating Group 3 and Group 4 respectively) on slides. The cultures were washed twice in saline and centrifuged at 10 000 X g for 30 min at 4 ° C, and the sediment was tested for aggregation. The test was read after 1 min at 20°C (Ljungh and WadstrSm, 1982).
Adsorption to aluminium hydroxide gel Adsorption to aluminium hydroxide gel was tested in five parallel experiments. Using phosphate buffer (pH 7.2 ), twofold dilutions were made from 0.00128 to 0.00002 mol. Two-ml volumes of the dilutions were measured into Wassermann tubes. Into one tube 2 ml Tris-phosphate buffer (pH 7.5 ) was pipetted. To the contents of each tube 1 ml 0.25% Cy quality aluminium hydroxide gel (Behring Werke, F.R.G.) and 1 ml bacterial suspension were added (Czirok et al., 1986 ). P. multocida strain 3 was grown on Y P C - b l o o d agar at 37°C for 8 h, and P. multocida strain 4 was cultured in serum-free YPC broth at 37°C for 3 h. The bacterial suspensions were prepared with saline and contained approximately 5 X 106 cfu m l - ~. The contents of the tubes were mixed thoroughly and incubated at 20 °C for 20 rain. Subsequently the tubes were centrifuged at 2 0 0 0 X g for 10 s, and the germ count (cfu) of the supernatant was determined on Y P C - b l o o d agar. RESULTS
Bacteriological findings P. multocida could be cultured neither from the nasal mucosa of the seven SPF piglets of Group 1 during the 16 weeks of the experiment nor from the surface of the pharyngeal tonsils of the piglets killed at the age of 18 weeks. At the same time, the nasal m u c o s a o f the seven piglets that had come from the
THE ABILITY OF P. MULTOCIDA TO COLONISE THE NASAL MUCOSA OF SPF PIGLETS
85
TABLE1 Isolation of Pasteurella multocida from the nasal turbinate of SPF pigs after contact exposure or experimental inoculation with P. rnultocida Groups
Subgroups
Age (weeks) 1
1 (n=7) 2 (n=7) 3 (n=7) 4 (n=7) 5 (n=7)
2a 2b 3a 3b 4a 4b
(n=4) (n=3) (n=4) (n=3) (n=4) (n=3)
. . . . .
2
3 .
. . . .
. +(1) l +(1) . . . .
4 .
5 .
+(1) +(2) .
. +(1)
. . .
. . .
. +(1) +(2) . +(1) . . .
6-17
182
+(1) +(2)
+(1) +(2)
+(1)
+(1)
.
. . . .
qsolation of P. m ultocida (number of piglets in parentheses). Group 1 was in contact with infected pigs; Group 2 was inoculated with organisms washed from the nasal mucosa; Group 3 was inoculated with organisms grown in media containing blood; Group 4 was inoculated with organisms grown in media without blood; Group 5 was an untreated control. Subgroups 2a, 3a and 4a were inoculated at the age of 2 weeks; subgroups 2b, 3b and 4b were inoculated at 2, 3 and 4 weeks of age. /Isolation from the pharyngeal tonsils of piglets at 18 weeks of age.
large herd and were kept together with the SPF piglets consistently yielded toxigenic type D P. multocida. The nasal mucosa in one of the four Group-2 piglets that received a single inoculation yielded P. multocida. Isolation ofP. multocida was successful from the nasal mucosa of two of the three repeatedly inoculated piglets. The four Group-3 piglets inoculated once proved negative throughout. The nasal mucosa of one of the three piglets inoculated three times yielded P. multocida. Bacteriological examination of the killed piglets gave the same results. No P. multocida was isolated from the nasal mucosa and from the surface of the pharyngeal tonsils of Group-4 piglets. The piglets of Group 5 proved free from P. multocida throughout the experiment (Table 1 ). Ammonium sulphate aggregation test P. multocida strains 3 and 4 gave dissimilar results in the ammonium sulphate aggregation test. Strain C, grown on YPC-blood agar, was aggregated by ammonium sulphate solutions of > 2.4 mol, whereas solutions of at least 2.6 mol were needed to aggregate strain D, a strain grown in YPC broth without blood. Adsorption to aluminium hydroxide gel The adsorption ofP. multocida strains 3 and 4 to aluminium hydroxide gel was complete in tubes containing Tris buffer. Colony counts indicated that
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B. I~LI,~SETAL.
TABLE 2 Adsorption to aluminium hydroxide gel of Pasteurella multocida strains used for inoculating SPE piglets of Group 3 and Group 4 in the presence of phosphate buffer of different molarity Strain
Tris buffer
Phosphate buffer (mol) 0.00002 0.00004 0.00008-0.00128 Colony forming units
Pasteurella multocida strain 3 Pasteurella multocida strain 4 n.c.
=
-
149 _+8 136 _+5
186 _+ 10 144 +_ 13
n.c. n.c.
not countable.
Pasteurella multocida strain 3 was grown on YPC-blood agar (Group 3 ). Pasteurella multocida strain 4 was grown in YPC-based broth culture (Group 4).
the media were sterile. In the phosphate buffer dilution range from 0.00128 to 0.00008 mol, the colonies of both strains were uncountable. The supernarant from tubes containing 0.00004 mol phosphate buffer yielded 186+ 10 and 144+ 13 colonies ( m e a n + S D ) o f P . multocida strains 3 and 4 respectively. In the supernatant of tubes containing 0.00002 mol phosphate buffer the colony counts of P. multocida strain 3 and 4 were 149+ 8 and 136_+ 5 (mean _+SD ) respectively (Table 2 ).
DISCUSSION
The purpose of this study was to determine whether prolonged contact can result in colonisation of the nasal mucosa of toxigenic B. bronchiseptica-free piglets by P. multocida. The study was justified by results obtained by Pedersen and Barfod (1982) and Sch6ss (1987), who reported the isolation oftoxigenic P. multocida from the nasal mucosa of pigs suffering from progressive AR but free from toxigenic B. bronchiseptica. Another objective was to monitor changes in the hydrophobicity of P. multocida during cultures on media with or without blood. Knowledge of these changes would permit interpretation of the results obtained in infection experiments with P. multocida (Oyamada et al., 1986; Rutter and Rojas, 1982). Over a period of 16 weeks, the SPF piglets in Group 1 did not contract infection from the P. multocida-infected contact animals, even at an age when they were most susceptible. This observation indicates that, by direct contact, fewer P. multocida bacteria are transmitted than are needed to colonise the nasal mucosa. This study has failed to confirm the transmissibility ofP. mul-
THE ABILITY OF P. MULTOCIDA TO COLONISE THE NASAL MUCOSA OF SPF PIGLETS
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tocida infection from live pigs to B. bronchiseptica-free pigs having intact nasal mucosa. For the successful infection of three piglets of Group 2, 5 × 107 cfu of P. multocida originating from the nasal mucosa, transmitted together with uninfected surface structures in fetal calf serum, were needed. Even three inoculations with rather high P. multocida counts failed to result in colonisation of the nasal mucosa in all piglets, indicating that P. multocida living on the nasal mucosa had poor colonising ability. The results provide a basis for comparing changes in the colonising ability of P. multocida living on the nasal mucosa and that grown on artificial media. In Group 3, even repeated inoculation with 5 × 107 cfu ofP. multocida grown on YPC agar containing 10% blood was successful in only one piglet. In agreement with the results o f O y a m a d a et al. ( 1986 ) and Rutter and Rojas ( 1982 ), this allows us to conclude that propagation on blood-containing m e d i u m does not cause damage to the surface structures of P. multocida that would hinder its colonising ability. None of the Group 4 piglets was colonised by P. multocida grown in serumfree YPC broth. The results of aluminium hydroxide gel adsorption tests in five parallel experiments indicated significant differences. In addition to the poor colonising capacity ofP. rnultocida strains, these tests revealed the weak adherence of P. multocida strain 4 to the gel. On the analogy of studies on Escherichia coli (Czirok et al., 1986; Ljungh and WadstrSm, 1982 ), the combined results of the two in vitro experiments indicate strong hydrophobicity of P. multocida strains in general, and particularly strong hydrophobicity of P. multocida strain 3. The results of the in vivo experiments are insufficient to establish a close correlation between colonising and hydrophobicity of P. multocida. However, together with the in vitro results they suggest that such a relationship probably exists. ACKNOWLEDGEMENTS
We thank the National Scientific Research F u n d (OTKA) Granting Board for encouragement and financial support which enabled us to carry out this work.
REFERENCES Bordet, J. and Gengou, O., 1906. Le microbe de la Congueluch. Ann. Inst. Pasteur (Paris), 20: 731-741. Carter, G.R. and Subronto, P., 1973. Identifications of type D strains ofPasteurella multocida with acriflavine. Am. J. Vet. Res., 34: 293-294. Czirok, 1~., Pethe6, G., SzSll6sy, E., Milch, H., Herpay, M. and Csik, M., 1986. Adsorption to
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AL(OH)3 gel ofEscherichia coli is correlated with O and K antigens and with type of extraintestinal infection. Acta Microbiol. Hung., 34:219-224. Duncan, J.R., Ross, R.F., Switzer, W.P. and Ramsey, F.K., 1966. Pathology of experimental Bordetella bronchiseptica infection in swine: atrophic rhinitis. Am. J. Vet. Res., 27: 457-466. 121i4s, B., Krfiger, M. and R4tz, F., 1982. Epizootiologische Untersuchungen zur Rhinitis atrophicans des Schweines. II. Biologische Eigenschaften der von Schweinen isolierten Bordetella bronchiseptica-StSmme. Zbl. Vet. Med. B, 29:619-635. t~lifis, B., Gergely, P. and Boros, G., 1988. Epizootiological studies on porcine atrophic rhinitis. XV. Results of local immunization with a live Bordetella bronchiseptica strain in a large-scale pig herd affected by atrophic rhinitis. Acta Vet. Hung., 36:195-204. Frymus, T., Wittenbrink, M.-M. and Petzold, K., 1986. Failure to demonstrate adherence of Pasteurella multocida involved in atrophic rhinitis to swine nasal epithelial cells. J. Vet. Med. B, 33: 140-144. Hanada, M., Shimoda, K., Tomita, S., Nakase, Y. and Nishiyama, Y., 1979. Production of lesions similar to naturally occurring swine atrophic rhinitis by cell-free sonicated extract of Bordetella bronchiseptica. Jap. J. Vet. Sci., 41: 1-8. Ljungh, A. and Wadstr6m, T., 1982. Salt aggregation test for measuring cell surface hydrophobicity of urinary Escherichia coli. Eur. J. Clin. Microbiol., 1: 388-393. Miniats, O.P. and Johnson, J.A., 1980. Experimental atrophic rhinitis in gnotobiotic pigs. Can. J. Comp. Med., 44: 358-365. Nakagawa, M., Shimizu, T. and Motoi, Y., 1974. Pathology of experimental atrophic rhinitis in swine infected with Alcaligenes bronchiseptica or Pasteurella multocida. Natl. Inst. Anita. Health Q. (Yatabe), 14: 61-71. Nakai, T., Sawata, A., Tsuji, M., Samejima, Y. and Kume, K., 1984. Purification of dermonecrotic toxin from a sonic extract ofPasteurella multocida SP-72 serotype D. Infect. Immun. 46: 429-434. Nakai, T., Kume, K., Yoshikawa, H., Oyamada, T. and Yoshikawa, T., 1988. Adherence of Pasteurella multocida or Bordetella bronchiseptica to the swine nasal epithelial cell in vitro. Infect. Immun., 56: 234-240. Namioka, S. and Murata, M., 1961. Serological studies on Pasteurella multocida. 1. A simplified method for capsular typing of the organisms. Cornell Vet., 51: 498-507. Oyamada, T., Yoshikawa, T., Yoshikawa, H., Shimizu, M., Nakai, T. and Kume, K., 1986. Lesions induced in the nasal turbinates of neonatal pigs by Pasteurella multocida or Bordetella bronchiseptica. Jap. J. Vet. Sci., 48: 377-387. Pedersen, K.B. and Barfod, K., 1981. The aetiological significance of Bordetella bronchiseptica and Pasteurella multocida in atrophic rhinitis of swine. Nord. Vet. Med., 33:513-522. Pedersen, K.B. and Barfod, K., 1982. Effect of the incidence of atrophic rhinitis on vaccination of sows with a vaccine containing Pasteurella multocida toxin. Nord. Vet. Med., 34: 293302. Pedersen, K.B. and Elling, F., 1984. The pathogenicity of atrophic rhinitis in pigs induced by toxigenic Pasteurella multocida. J. Comp. Pathol., 94:203-214. Pittmann, M., 1974. Genus Bordetella Moreno-L6pes 1952. In: R.E. Buchanan and N.E. Gibbons (Editors), Bergey's Manual of Determinative Bacteriology, 8th edn. Williams and Wilkins, Baltimore, pp. 282-283. Rutter, J.M. and Rojas, X., 1982. Atrophic rhinitis in gnotobiotic piglets: differences in pathogenicity ofPasteurella multocida in combined infections with Bordetella bronchiseptica. Vet. Rec., 110: 531-535. Sch6ss, P., 1987. Neues fiber die Rhinitis atrophicans des Schweines. Wien. tier~irztl. Mschr., 74: 301-305. Smith, J.E., 1974. Genus Pasteurella. In: R.E. Buchanan and N.E. Gibbons (Editors), Bergey's Manual of Determinative Bacteriology, 8th edn. Williams and Wilkins, Baltimore, pp. 370373.
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The influence of growth conditions of Pasteurella multocida on its ability to colonise the nasal mucosa of SPF piglets B. l~lifis~, Mfiria Herpay 5, M. Albert 2, Judit Fekete 6, S. Tuboly 3, P. Rafai 4 and E d i t Moln~ir ~ ~Departments of Research Organization, 2pathological Anatomy, 3Epizootiology and 4Animal Hygiene, University of Veterinary Science, H-1400, Budapest, P.O.B 2 (Hungary) SNational lnstitute of Hygiene, H-1966 Budapest, P.O.B. 64 (Hungary) 6public Health Station of Csongrdd County, 1t-6700 Szeged (Hungary) (Accepted 12 December 1989)
ABSTRACT l~lifis, B., Herpay, M., Albert, M., Fekete, J., Tuboly, S., Rafai, P. and Molnfir, E., 1990. The influence of growth conditions of Pasteurella multocida on its ability to colonise the nasal mucosa of SPF piglets. Vet. Microbiol., 24: 81-88. Colonisation of type D Pasteurella multocida was studied in five groups of seven SPF piglets each. Piglets of Group 1 were kept together with seven 5-week-old piglets obtained from a large herd infected with toxigenic P. multocida for 16 weeks (contact infection). These piglets were made free from toxigenic Bordetella bronchiseptica by local immunisation. Piglets of Group 2 were inoculated with 5 X 10 v colony-forming units (cfu) of P. multocida washed from the nasal mucosa of piglets free from toxigenic B. bronehiseptica with fetal calf serum. Piglets of Group 3 were inoculated intranasally with 5 X 10 7 cfu of P. multoeida washed from yeast-extract proteose-peptone cystine (YPC)-blood agar with fetal calf serum. Piglets of Group 4 were inoculated with 5 X 10 7 cfu of P. multocida grown in a YPC-based broth without blood. Piglets of Group 5 served as controls. The piglets of Group 1 did not contract P. multocida infection from infected contact piglets. After a single inoculation one of four, while after three inoculations two of three piglets of Group 2 became infected by P. multocida. After a single inoculation none of four, while after three inoculations one of three piglets of Group 3 were colonised by P. multocida. Both single and repeated inoculation failed in piglets of Group 4.
INTRODUCTION
Progressive atrophic rhinitis (AR) of swine is characterised by necrosis of the nasal respiratory epithelium, atrophy of the nasal turbinates and distortion of the nasal bones (Duncan et al., 1966; Miniats and Johnson, 1980). These lesions develop both in SPF and gnotobiotic piglets intranasally inoculated with toxigenic Bordetella bronchiseptica or repeatedly subjected to lo0378-1135/90/$03.50
© 1990 Elsevier Science Publishers B.V.
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B. I~LIASET AL.
cal treatment of the nasal mucosa with the dermonecrotic toxin (DNT) of B. bronchiseptica (Nakagawa et al., 1974; Hanada et al., 1979 ). Toxigenic Pasteurella multocida also plays a role in producing AR (Pedersen and Barfod, 1981; Pedersen and Elling, 1984), and some consider P. multocida the primary causative agent (Pedersen and Barfod, 1981; Sch6ss, 1987). The interrelationship between P. multocida and B. bronchiseptica in the pathogenesis of AR has not yet been fully elucidated (Pedersen and Barfod, 1982; Rutter and Rojas, 1982; Pedersen and Elling, 1984; Frymus et al., 1986). Authors generally agree that P. multocida shows weak adherence to porcine nasal epithelial cells in vitro (Nakai et al., 1984; Frymus et al., 1986) and to the intact nasal mucosa of pigs in vivo (Oyamada et al., 1986; Nakai et al., 1988 ). Adherence ofP. multocida was generally successful if the nasal mucosa had been exposed to toxigenic B. bronchiseptica a few days before (Rutter and Rojas, 1982; Oyamada et al., 1986 ). In the absence of toxigenic B. bronchiseptica, P. multocida can colonise the nasal mucosa of pigs only if it has been grown on media containing blood (Rutter and Rojas, 1982; Oyamada et al., 1986 ). On the other hand, P. multocida infection of pigs has been reported from herds not infected with toxigenic B. bronchiseptica (Pedersen and Barfod, 1982; Sch/3ss, 1987). The purpose of this work was to study the influence of growth conditions on the ability ofP. multocida to colonise the nasal mucosa. MATERIALS AND METHODS
Experimental animals The SPF piglets were derived by hysterectomy in an SPF laboratory. Up to 2 weeks of age the piglets were kept in isolators, then for 16 weeks, until the end of the experiment, they were reared in climatic chambers in isolation. The large pig herd used in .the trial was infected both with toxigenic B. bronchiseptica and type D P. multocida. At birth, 200 piglets of the herd were inoculated with 5 × 108 colony-forming units (cfu) of an avirulent, non-toxigenic B. bronchiseptica strain (N-104) suspended in PBS via the mucosa of both nostrils (l~li~is et al., 1988 ). The strain had been grown on agar (Bordet and Gengou, 1906 ) (Difco Co., U.S.A. ) containing 10% sterile bovine blood, at 37 °C for 24 h. Bacteriological examination of the piglets was carried out at 4 and 5 weeks of age. Piglets infected with toxigenic type D P. multocida, whose nasal mucosa did not yield B. bronchiseptica, were selected for the experiment. Bacterial isolation and identification B. bronchiseptica was isolated from the nasal mucosa of piglets on BordetGengou and MacConkey agar (Difco) containing 10% sterile bovine blood, at 37°C for 24 h. Isolation of P. multocida was done on YPC agar (cystine agar medium containing yeast-proteose peptone, Difco) containing 10% sterile bovine blood at 37°C for 18 h (Namioka and Murata, 1961 ). The
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83
strains were identified by the criteria of Bergey's Manual (Pittman, 1974; Smith, 1974). The capsular antigens ofP. multocida strains were determined by indirect haemagglutination and by acriflavine and hyaluronidase tests (Carter and Subronto, 1973). The D N T of P. multocida was determined according to Nakai et al. (1984) and that of B. bronehiseptica by the method of l~li~ts et al. ( 1982 ).
Inocula for pigs The inoculum for Group-2 piglets was prepared from the nasal mucosa of 5-week-old piglets rendered free from B. bronchiseptica by local immunisation but infected with toxigenic type D P. multocida. The nasal mucosa collected from killed piglets was washed several times with sterile fetal calf serum (Gibco Laboratories, Grand Island, NY). The number of P. multocida cfu was determined by colony counting. The inoculum for Group-3 piglets was prepared by propagating P. multocida, washed from the nasal mucosa with sterile fetal calf serum, on YPC agar containing 10% sterile bovine blood, at 37°C for 18 h. Agar cultures were washed with sterile fetal calf serum and the bacterial concentration (cfu) was determined by spectrophotometry (SPECOL- 10, Karl Zeiss Jena, G.D.R. ). The inoculum for Group-4 piglets was prepared in a serum-free, YPC-based broth inoculated with Y P C - b l o o d agar culture and incubated at 37 °C for 3 h. The bacterial concentration (cfu) was determined by spectrophotometry. All three inocula were stored in a lyophilized state at - 20 ° C. Experimental design A total of 35 2-week-old SPF piglets were used. The piglets were divided into five groups of seven piglets each, and kept isolated in climatic chambers. The experiment lasted 16 weeks and the piglets were killed at 18 weeks of age. Piglets of Group 1 were kept together for 16 weeks with seven 5-week-old piglets chosen from a large herd (contact infection). The nasal mucosa of the latter piglets was infected with toxigenic type D P. multocida but was free from B. bronchiseptica. Piglets of Group 2 were divided into two subgroups. Four piglets (Group 2a) were inoculated intranasally with 5 X 107 cfu of P. multocida at 2 weeks of age. Three piglets (Group 2b) were inoculated with the same dose of P. multocida repeatedly at 2, 3 and 4 weeks of age. The inoculum was prepared by washing the nasal mucosa of piglets selected from the large herd with sterile fetal calf serum. Piglets of Group 3 were assigned to two subgroups. Four piglets (Group 3a) received a single intranasal dose (5 X 107 cfu) of P. multocida at 2 weeks of age. The other three piglets (Group 3b) were inoculated with the same dose of P. multocida repeatedly at 2, 3 and 4 weeks of age. The inoculum was prepared by propagating on Y P C - b l o o d agar P. multocida washed from the nasal mucosa with sterile calf serum.
84
B. I~LI,g,S ET AL.
Piglets of Group 4 were also divided into two groups. Four piglets (Group 4a) were inoculated intranasally with 5 X 10 7 cfu of P. multocida at 2 weeks of age. Three piglets (Group 4b) were inoculated intranasally with the same dose ofP. multocida repeatedly, at 2, 3 and 4 weeks of age. The inoculum was prepared in a serum-free YPC broth inoculated with Y P C - b l o o d agar culture. Piglets of Groups 2, 3 and 4 were inoculated with 0.5 ml inoculum into both nostrils, using a syringe. For inoculation, the animals were immobilized with the head upwards. The piglets were examined bacteriologically on the day before inoculation and then at 7-day intervals for 18 weeks. Bacteriological examination included examination of the pharyngeal tonsils of piglets killed at 18 weeks of age.
Ammonium sulphate aggregation test Using phosphate buffer (pH 6.8 ), an a m m o n i a sulphate dilution series was made from 0.2 to 3.2 mol, with a difference of 0.2 tool between the dilutions. This series was used for testing the aggregation of P. multocida strains 3 and 4 (used for inoculating Group 3 and Group 4 respectively) on slides. The cultures were washed twice in saline and centrifuged at 10 000 X g for 30 min at 4 ° C, and the sediment was tested for aggregation. The test was read after 1 min at 20°C (Ljungh and WadstrSm, 1982).
Adsorption to aluminium hydroxide gel Adsorption to aluminium hydroxide gel was tested in five parallel experiments. Using phosphate buffer (pH 7.2 ), twofold dilutions were made from 0.00128 to 0.00002 mol. Two-ml volumes of the dilutions were measured into Wassermann tubes. Into one tube 2 ml Tris-phosphate buffer (pH 7.5 ) was pipetted. To the contents of each tube 1 ml 0.25% Cy quality aluminium hydroxide gel (Behring Werke, F.R.G.) and 1 ml bacterial suspension were added (Czirok et al., 1986 ). P. multocida strain 3 was grown on Y P C - b l o o d agar at 37°C for 8 h, and P. multocida strain 4 was cultured in serum-free YPC broth at 37°C for 3 h. The bacterial suspensions were prepared with saline and contained approximately 5 X 106 cfu m l - ~. The contents of the tubes were mixed thoroughly and incubated at 20 °C for 20 rain. Subsequently the tubes were centrifuged at 2 0 0 0 X g for 10 s, and the germ count (cfu) of the supernatant was determined on Y P C - b l o o d agar. RESULTS
Bacteriological findings P. multocida could be cultured neither from the nasal mucosa of the seven SPF piglets of Group 1 during the 16 weeks of the experiment nor from the surface of the pharyngeal tonsils of the piglets killed at the age of 18 weeks. At the same time, the nasal m u c o s a o f the seven piglets that had come from the
THE ABILITY OF P. MULTOCIDA TO COLONISE THE NASAL MUCOSA OF SPF PIGLETS
85
TABLE1 Isolation of Pasteurella multocida from the nasal turbinate of SPF pigs after contact exposure or experimental inoculation with P. rnultocida Groups
Subgroups
Age (weeks) 1
1 (n=7) 2 (n=7) 3 (n=7) 4 (n=7) 5 (n=7)
2a 2b 3a 3b 4a 4b
(n=4) (n=3) (n=4) (n=3) (n=4) (n=3)
. . . . .
2
3 .
. . . .
. +(1) l +(1) . . . .
4 .
5 .
+(1) +(2) .
. +(1)
. . .
. . .
. +(1) +(2) . +(1) . . .
6-17
182
+(1) +(2)
+(1) +(2)
+(1)
+(1)
.
. . . .
qsolation of P. m ultocida (number of piglets in parentheses). Group 1 was in contact with infected pigs; Group 2 was inoculated with organisms washed from the nasal mucosa; Group 3 was inoculated with organisms grown in media containing blood; Group 4 was inoculated with organisms grown in media without blood; Group 5 was an untreated control. Subgroups 2a, 3a and 4a were inoculated at the age of 2 weeks; subgroups 2b, 3b and 4b were inoculated at 2, 3 and 4 weeks of age. /Isolation from the pharyngeal tonsils of piglets at 18 weeks of age.
large herd and were kept together with the SPF piglets consistently yielded toxigenic type D P. multocida. The nasal mucosa in one of the four Group-2 piglets that received a single inoculation yielded P. multocida. Isolation ofP. multocida was successful from the nasal mucosa of two of the three repeatedly inoculated piglets. The four Group-3 piglets inoculated once proved negative throughout. The nasal mucosa of one of the three piglets inoculated three times yielded P. multocida. Bacteriological examination of the killed piglets gave the same results. No P. multocida was isolated from the nasal mucosa and from the surface of the pharyngeal tonsils of Group-4 piglets. The piglets of Group 5 proved free from P. multocida throughout the experiment (Table 1 ). Ammonium sulphate aggregation test P. multocida strains 3 and 4 gave dissimilar results in the ammonium sulphate aggregation test. Strain C, grown on YPC-blood agar, was aggregated by ammonium sulphate solutions of > 2.4 mol, whereas solutions of at least 2.6 mol were needed to aggregate strain D, a strain grown in YPC broth without blood. Adsorption to aluminium hydroxide gel The adsorption ofP. multocida strains 3 and 4 to aluminium hydroxide gel was complete in tubes containing Tris buffer. Colony counts indicated that
86
B. I~LI,~SETAL.
TABLE 2 Adsorption to aluminium hydroxide gel of Pasteurella multocida strains used for inoculating SPE piglets of Group 3 and Group 4 in the presence of phosphate buffer of different molarity Strain
Tris buffer
Phosphate buffer (mol) 0.00002 0.00004 0.00008-0.00128 Colony forming units
Pasteurella multocida strain 3 Pasteurella multocida strain 4 n.c.
=
-
149 _+8 136 _+5
186 _+ 10 144 +_ 13
n.c. n.c.
not countable.
Pasteurella multocida strain 3 was grown on YPC-blood agar (Group 3 ). Pasteurella multocida strain 4 was grown in YPC-based broth culture (Group 4).
the media were sterile. In the phosphate buffer dilution range from 0.00128 to 0.00008 mol, the colonies of both strains were uncountable. The supernarant from tubes containing 0.00004 mol phosphate buffer yielded 186+ 10 and 144+ 13 colonies ( m e a n + S D ) o f P . multocida strains 3 and 4 respectively. In the supernatant of tubes containing 0.00002 mol phosphate buffer the colony counts of P. multocida strain 3 and 4 were 149+ 8 and 136_+ 5 (mean _+SD ) respectively (Table 2 ).
DISCUSSION
The purpose of this study was to determine whether prolonged contact can result in colonisation of the nasal mucosa of toxigenic B. bronchiseptica-free piglets by P. multocida. The study was justified by results obtained by Pedersen and Barfod (1982) and Sch6ss (1987), who reported the isolation oftoxigenic P. multocida from the nasal mucosa of pigs suffering from progressive AR but free from toxigenic B. bronchiseptica. Another objective was to monitor changes in the hydrophobicity of P. multocida during cultures on media with or without blood. Knowledge of these changes would permit interpretation of the results obtained in infection experiments with P. multocida (Oyamada et al., 1986; Rutter and Rojas, 1982). Over a period of 16 weeks, the SPF piglets in Group 1 did not contract infection from the P. multocida-infected contact animals, even at an age when they were most susceptible. This observation indicates that, by direct contact, fewer P. multocida bacteria are transmitted than are needed to colonise the nasal mucosa. This study has failed to confirm the transmissibility ofP. mul-
THE ABILITY OF P. MULTOCIDA TO COLONISE THE NASAL MUCOSA OF SPF PIGLETS
87
tocida infection from live pigs to B. bronchiseptica-free pigs having intact nasal mucosa. For the successful infection of three piglets of Group 2, 5 × 107 cfu of P. multocida originating from the nasal mucosa, transmitted together with uninfected surface structures in fetal calf serum, were needed. Even three inoculations with rather high P. multocida counts failed to result in colonisation of the nasal mucosa in all piglets, indicating that P. multocida living on the nasal mucosa had poor colonising ability. The results provide a basis for comparing changes in the colonising ability of P. multocida living on the nasal mucosa and that grown on artificial media. In Group 3, even repeated inoculation with 5 × 107 cfu ofP. multocida grown on YPC agar containing 10% blood was successful in only one piglet. In agreement with the results o f O y a m a d a et al. ( 1986 ) and Rutter and Rojas ( 1982 ), this allows us to conclude that propagation on blood-containing m e d i u m does not cause damage to the surface structures of P. multocida that would hinder its colonising ability. None of the Group 4 piglets was colonised by P. multocida grown in serumfree YPC broth. The results of aluminium hydroxide gel adsorption tests in five parallel experiments indicated significant differences. In addition to the poor colonising capacity ofP. rnultocida strains, these tests revealed the weak adherence of P. multocida strain 4 to the gel. On the analogy of studies on Escherichia coli (Czirok et al., 1986; Ljungh and WadstrSm, 1982 ), the combined results of the two in vitro experiments indicate strong hydrophobicity of P. multocida strains in general, and particularly strong hydrophobicity of P. multocida strain 3. The results of the in vivo experiments are insufficient to establish a close correlation between colonising and hydrophobicity of P. multocida. However, together with the in vitro results they suggest that such a relationship probably exists. ACKNOWLEDGEMENTS
We thank the National Scientific Research F u n d (OTKA) Granting Board for encouragement and financial support which enabled us to carry out this work.
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AL(OH)3 gel ofEscherichia coli is correlated with O and K antigens and with type of extraintestinal infection. Acta Microbiol. Hung., 34:219-224. Duncan, J.R., Ross, R.F., Switzer, W.P. and Ramsey, F.K., 1966. Pathology of experimental Bordetella bronchiseptica infection in swine: atrophic rhinitis. Am. J. Vet. Res., 27: 457-466. 121i4s, B., Krfiger, M. and R4tz, F., 1982. Epizootiologische Untersuchungen zur Rhinitis atrophicans des Schweines. II. Biologische Eigenschaften der von Schweinen isolierten Bordetella bronchiseptica-StSmme. Zbl. Vet. Med. B, 29:619-635. t~lifis, B., Gergely, P. and Boros, G., 1988. Epizootiological studies on porcine atrophic rhinitis. XV. Results of local immunization with a live Bordetella bronchiseptica strain in a large-scale pig herd affected by atrophic rhinitis. Acta Vet. Hung., 36:195-204. Frymus, T., Wittenbrink, M.-M. and Petzold, K., 1986. Failure to demonstrate adherence of Pasteurella multocida involved in atrophic rhinitis to swine nasal epithelial cells. J. Vet. Med. B, 33: 140-144. Hanada, M., Shimoda, K., Tomita, S., Nakase, Y. and Nishiyama, Y., 1979. Production of lesions similar to naturally occurring swine atrophic rhinitis by cell-free sonicated extract of Bordetella bronchiseptica. Jap. J. Vet. Sci., 41: 1-8. Ljungh, A. and Wadstr6m, T., 1982. Salt aggregation test for measuring cell surface hydrophobicity of urinary Escherichia coli. Eur. J. Clin. Microbiol., 1: 388-393. Miniats, O.P. and Johnson, J.A., 1980. Experimental atrophic rhinitis in gnotobiotic pigs. Can. J. Comp. Med., 44: 358-365. Nakagawa, M., Shimizu, T. and Motoi, Y., 1974. Pathology of experimental atrophic rhinitis in swine infected with Alcaligenes bronchiseptica or Pasteurella multocida. Natl. Inst. Anita. Health Q. (Yatabe), 14: 61-71. Nakai, T., Sawata, A., Tsuji, M., Samejima, Y. and Kume, K., 1984. Purification of dermonecrotic toxin from a sonic extract ofPasteurella multocida SP-72 serotype D. Infect. Immun. 46: 429-434. Nakai, T., Kume, K., Yoshikawa, H., Oyamada, T. and Yoshikawa, T., 1988. Adherence of Pasteurella multocida or Bordetella bronchiseptica to the swine nasal epithelial cell in vitro. Infect. Immun., 56: 234-240. Namioka, S. and Murata, M., 1961. Serological studies on Pasteurella multocida. 1. A simplified method for capsular typing of the organisms. Cornell Vet., 51: 498-507. Oyamada, T., Yoshikawa, T., Yoshikawa, H., Shimizu, M., Nakai, T. and Kume, K., 1986. Lesions induced in the nasal turbinates of neonatal pigs by Pasteurella multocida or Bordetella bronchiseptica. Jap. J. Vet. Sci., 48: 377-387. Pedersen, K.B. and Barfod, K., 1981. The aetiological significance of Bordetella bronchiseptica and Pasteurella multocida in atrophic rhinitis of swine. Nord. Vet. Med., 33:513-522. Pedersen, K.B. and Barfod, K., 1982. Effect of the incidence of atrophic rhinitis on vaccination of sows with a vaccine containing Pasteurella multocida toxin. Nord. Vet. Med., 34: 293302. Pedersen, K.B. and Elling, F., 1984. The pathogenicity of atrophic rhinitis in pigs induced by toxigenic Pasteurella multocida. J. Comp. Pathol., 94:203-214. Pittmann, M., 1974. Genus Bordetella Moreno-L6pes 1952. In: R.E. Buchanan and N.E. Gibbons (Editors), Bergey's Manual of Determinative Bacteriology, 8th edn. Williams and Wilkins, Baltimore, pp. 282-283. Rutter, J.M. and Rojas, X., 1982. Atrophic rhinitis in gnotobiotic piglets: differences in pathogenicity ofPasteurella multocida in combined infections with Bordetella bronchiseptica. Vet. Rec., 110: 531-535. Sch6ss, P., 1987. Neues fiber die Rhinitis atrophicans des Schweines. Wien. tier~irztl. Mschr., 74: 301-305. Smith, J.E., 1974. Genus Pasteurella. In: R.E. Buchanan and N.E. Gibbons (Editors), Bergey's Manual of Determinative Bacteriology, 8th edn. Williams and Wilkins, Baltimore, pp. 370373.