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Experimental infection of neonatal pigs with Campylobacter sputorum subspecies mucosalis with special reference to the oral cavity
Veterinary Microbiology, 5 (1980) 249---255 Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
249
Short Communication EXPERIMENTAL INFECTION OF NEONATAL PIGS WITH CAMPYLOB A C T E R S P U T O R UM S U B S P E C I E S M U C O S A L I S W I T H S P E C I A L REFERENCE TO THE ORAL CAVITY
L. ROBERTS*, G.H.K. LAWSON** and A.C. ROWLAND** * Veterinary Investigation Centre, Mill of Craibstone, Bucksburn, Aberdeen AB2 9TS (Great Britain) **Department of Veterinary Pathology, University of Edinburgh, Veterinary Field Station, Easter Bush, Roslin, Midlothian (Great Britain) (Accepted 25 March 1980) ABSTRACT Roberts, L., Lawson, G.H.K. and Rowland, A.C., 1980. The experimental infection of neonatal pigs with Campylobacter sputorum subspecies mucosalis with special reference to the oral cavity. Vet. Microbiol., 5: 249--255. After oral exposure Campylobacter sputorum subsp, mucosalis became established in the mouth of piglets for up to eight weeks. C. sputorum subsp, mucosalis was not isolated from the same pigs each week, although some pigs were positive for a number of consecutive weeks. Spread also took place from orally dosed piglets to an undosed litter mate maintalned with them. Other catalase negative, serologically distinct Campylobacters were also demonstrated in the oral cavity. INTRODUCTION T h e t y p e species o f t h e catalase negative C a m p y l o b a c t e r s , Campylobacter sputorum subsp, sputorum is p r e s e n t in the h u m a n m o u t h ( L o e s c h e et al., 1965). In man, m o s t oral m o t i l e organisms, such as spirochaetes and vibrios, are f o u n d in highest c o n c e n t r a t i o n s in t h e gingival crevice area ( G i b b o n s and Van H o u t e , 1 9 7 5 ) . L i t t l e or n o saliva p e n e t r a t e s into this crevice, and it is t h o u g h t t h a t w h e n such m o t i l e organisms are i n t r o d u c e d i n t o t h e m o u t h , active m o v e m e n t enables t h e m t o r e a c h this q u i e s c e n t site ( G i b b o n s and Van H o u t e , 1 9 7 5 ) . H u m a n oral m i c r o b i o l o g y has b e e n a field o f active research i n t o t h e i n v o l v e m e n t o f bacteria in a n u m b e r o f d e n t a l diseases. T h e r e has b e e n little r e p o r t e d v e t e r i n a r y w o r k in this area. An organism resembling C. sputorum subsp, sputorum has b e e n r e c o v e r e d f r o m cases o f p o r c i n e intestinal a d e n o m a t o s i s (PIA) ( L a w s o n and R o w l a n d , 1 9 7 4 ) . This is a c o n d i t i o n o f the p o s t w e a n e d pig characterised b y a n o r e x i a and wasting, t h e a f f e c t e d intestinal e p i t h e l i u m having an a d e n o m a t o u s appearance. T h e b a c t e r i u m has b e e n fully characterised ( L a w s o n e t al., 1 9 7 5 b ) and the n a m e Campylobacter sputorum subsp, mucosalis p r o p o s e d . In PIA, 0378--1135/80/0000--0000/$02.25 © 1980 Elsevier Scientific Publishing Company
250
C. sputorum subsp, mucosalis lies free within the cytoplasm of affected intestinal epithelial cells and undergoes division in that site. C. sputorum subsp. mucosalis has also been recovered from the m o u t h of piglets (Lawson et al., 1975 a). This was a limited study but provided information on the presence of C. sputorum subsp, mucosalis in, and its isolation from, the oral cavity of animals w i t h o u t gross or clinical evidence o f porcine intestinal adenomatosis (PIA); the organism has also been isolated from the m o u t h in cases of PIA (Lawson et al., 1976). The catalase negative Campylobacters of the porcine oral cavity seem to be a heterogenous group, and some isolates are biochemically and serologically typical of C. sputorum subsp, mucosalis, others are biochemically typical but serologically distinct, while a third group differ both biochemically and serologically from C. sputorum subsp, mucosalis (Lawson et al., 1976). In this paper the catalase positive Campylobacters will be referred to as Campylobacter coli. Although not recognised in the eighth edition of Bergey's Manual of Determinative Bacteriology (Smibert, 1974), most veterinary bacteriologists recognise the existence of this organism, and consider it to be distinct from Campylobacter fetus subsp. ]ejuni. The experiment described in the present paper set out to examine the occurrence of Campylobacters in the m o u t h of piglets exposed to C. sputorum subsp, mucosalis in the neonatal period. MATERIALS AND METHODS
Experimental animals and timing o f procedures A Duroc sow was introduced to isolation premises several days before farrowing. Eight of the piglets born were removed from the sow before suckling and seven were dosed orally with C. sputorum subsp, mucosalis approximately 1.5 h after birth of the last piglet. The piglets were returned to the sow 1.5 h later, i.e. 3.0 h after birth of the last piglet. All seven piglets received oral chalk before the C. sputorum subsp, mucosalis suspension. The piglets were weaned. at 51 days of age. They were offered creep (starter creep - Seafield Mill) from 10 days of age and 'ABRO Breeders' meal (Seafield Mill - Roslin) at 3 weeks. Mouth swabs were taken from all of the piglets at 2 days of age and weekly from then onwards until 72 days of age. Mouth and vaginal swabs were taken from the sow on days 30, 37, 44 and 51 of the experiment. The piglet n o t directly exposed to infection was killed on day 37, the remaining seven piglets were killed at 80 days of age and subjected to necropsy. Bacteriological examination was not carried out on these piglets at this time.
Sampling A sterile c o t t o n wool swab was rotated along the gingival margin of the piglet. In the laboratory the swab was broken off into a McCartney bottle con-
251 talning 5 ml sterile saline and shaken for five minutes. Each sample was then treated as previously described (Lawson et al., 1975 a}.
Infecting inoculum The suspension of C. sputorum subsp, mucosalis used to orally dose four of the piglets (N 1--4) contained 3.5 × l 0 s viable C. sputorum subsp, mucosalis per ml. The other three orally dosed piglets (D 1--3) received a ten-fold dilution in tryptose phosphate broth of that suspension. Each dosed piglet received 15 ml of one of the C. sputorum subsp, mucosalis suspensions.
Differentiation of oral Campylobacters The criteria for the differentiation of the oral Campylobacters were those used in the initial separation of C. sputorum subsp, mucosalis and C. coli (Lawson and Rowland, 1974) dealing with intestinal isolates. Selected isolates were subsequently examined biochemically and found to conform to the original description (Lawson et al., 1975 b). Catalase negative, slide agglutination negative Campylobacters were those organisms which proved negative in slide catalase tests and did n o t react in slide agglutination tests with C. sputorum subsp, mucosalis anti-serum (serotype A, NCTC 11,000). These isolates were n o t subjected to extensive biochemical investigation, although a number of m o u t h isolates have been examined (Lawson et al., 1976) and found to be characteristic of the genus Campylobacter. These organisms are therefore referred to as Campylobacters, although the possibility that some of them may n o t belong to this genus cannot be definitely excluded. In the tables and text they are referred to as non-agglutinating Campylobacters (NACS). RESULTS From 21 days of age, the piglet n o t directly exposed to C. sputorum subsp. mucosalis was noticeably smaller in size and less active compared to its litter mates. This piglet was killed on day 37 as it had become progressively weaker although on no occasion was diarrhoea seen. At necropsy this piglet was in very poor bodily condition. Histopathologically there was no evidence of PIA and C. sputorum subsp, mucosalis was n o t recovered from the alimentary tract. When the remaining pigs were killed at the end of the experiment there was no evidence of PIA. The recovery of C. sputorum subsp, mucosalis from the m o u t h of these piglets is summarised in Table I. With the exception of week 2, C. sputorum subsp. mucosalis was isolated from a variable number of piglets each week, up to 8 weeks post-infection. The number of piglets from which C. sputorum subsp. rnucosalis was isolated increased to a maximum at week 5, and then decreased until no recoveries were made in weeks 9--11. C. sputorum subsp, rnucosalis was n o t recovered from the same piglets each week, except for piglet D3 which
252 TABLE
I
R e c o v e r y o f C a m p y l o b a c t e r s o n w e e k l y s a m p l i n g in t h e litter in w h i c h all p i g l e t s w e r e s a m p l e d w e e k l y Piglet
Week 1
2
3
4
5
6
7
8
9
10
11
NI
M
C
O
O
M
C
C
O
O
NACS
N2
O
C
O
C
M
C
C
O
O
M
C
C
C NACS C
O
N3
C NACS O
C NACS C
O
C
N4
O
O
NACS
O
C
M
O
D1
O
O
C
M
O M
O O
M M
M M
M C C M
C
D2 D3
M C O M
Control
C
O
C
M
M
C
C M NACS . .
C NACS C NACS C NACS O O
2/8 + 118 0/8
0/8 1/8 0/8
3/8 2/8 1/8
3/8 3/8 0/8
6/8 2/8 0/8
3/8 5/8 1/8
1/7 5/7 1/7
C
M C C NACS C NACS .
.
O
O
O
O
C O
C C
0/7 1 /7 0/7
0/7 4/7 1/7
.
O v e r a l l recovery
from swabs C. s p u t o r u m subsp. mucosalis C. c o l i
NACS M ffi C .
1/7 5/7 3/7
0/7 5/7 4/7
s p u t o r u m subsp, m u c o s a l i s r e c o v e r e d : C ffi C . c o l i r e c o v e r e d .
N A C S = Catalase n e g a t i v e , slide a g g l u t i n a t i o n negative C a m p y l o b a c t e r s r e c o v e r e d . O = N o C a m p y l o b a c t e r i s o l a t e d : C o n t r o l piglet ffi t h e piglet n o t d i r e c t l y e x p o s e d t o C. s p u t o r u m s u b s p .
mucosalis. + N u m e r a t o r = n u m b e r o f s w a b s p o s i t i v e : d e n o m i n a t o r ffi t o t a l a n i m a l s s a m p l e d .
proved to be infected in 6 of the 7 weeks in which this organism was isolated from some members of the litter. With the exception of the single isolation of NACS made from piglet N4 in week 3, NACS were not recovered until week 6, after which time they were isolated from a considerable proportion of the piglets (Table I). Piglet D3 is of interest since C. sputorurn subsp, rnucosalis was isolated from the oral cavity of this piglet each week, except for week 2, up to week 7. In week 7, both C. sputorum subsp, mucosalis and NACS were isolated from the same swab. In week 8, only NACS were recovered. C. sputorum subsp, rnucosalis was not isolated from either the oral cavity or vagina of the sow on any of four occasions when sampled between days 30 and 61. Catalase positive Campylobacters were isolated from both sites and on one occasion NACS were also recovered from the mouth. DISCUSSION The establishment of irregular oral infection with C. sputorum subsp, mucosalis in a litter of neonatal piglets for eight weeks after oral exposure is described. The method of sampling was relatively crude and it cannot be said that C. sputorum subsp, mucosalis was absent from the mouth of those piglets from
253 whose m o u t h swabs it was not isolated. It seems reasonable, however, that taken as a whole, these results provide an indication of the presence of C. sputorum subsp, mucosal/s within the oral cavity of some members of the litter. The duration of infection of eight weeks is comparable to the period of clinical signs in PIA (Rowland and Rowntree, 1972), the experimental disease (Roberts et al., 1977a; Roberts, 1978), and the persistence of infection in the intestinal tract of orally dosed neonatal piglets (Roberts, 1978; Roberts et al., 1980). There was spread from dosed piglets to the undosed piglet. With Streptococcus mutans in rats, transmission from m o u t h to m o u t h occurs mainly b y coprophagy (Van H o u t e et al., 1976). These workers suggest that the faecal count of S. mutans in their experiments was directly related to the m o u t h population of the organism. It is possible that spread b y coporphagy occurred within this litter of piglets, or alternatively spread could have occurred by suckling contaminated teats. During the period of the experiment, there was no clinical evidence of PIA and when piglets were necropsied at the termination of the experiment, 11 weeks after oral dosing with C. sputorum subsp, mucosalis, no gross changes of the disease were found. Little information is available a b o u t the establishment of micro-organisms in the oral cavity (Van Houte, 1976), although it is recognised that the host's age is important in oral colonisation b y bacteria. In rats, younger animals are more susceptible to infection with S. mutans (Van H o u t e and Upeslacis, 1976), whereas in the same t y p e of rats, susceptibility to oral infection with Actinomyces viscosus increases after weaning (Brecher and Van Houte, 1976). It is interesting that a number of isolates of C. sputorum subsp, mucosalis were made from the m o u t h of orally dosed neonatal piglets, b u t only one isolation from the post-weaned group (Roberts, 1978; Roberts et al., 1980). The susceptibility of the neonatal piglet to oral infection with C. sputorum subsp. mucosalis is confirmed in this paper. In a manner analogous to the rapid clearance of bacteria from the intestine, Bloomfield (1919, 1920 a,b) demonstrated that various bacteria when introduced into the m o u t h and nasopharynx of man were rapidly cleared. There is a great variability in the bacterial composition of the oral cavity in the first few days of life ( B u m e t t et al., 1976). Possibly during this period, establishment of introduced organisms is easier and this may be true of the establishment of C. sputorum subsp, mucosalis in the m o u t h of the neonatal pig. In the experiment described in this paper, in addition to the recovery of C. sputorum subsp, mucosalis from the oral cavity, NACS were also isolated. The indication was that these appeared, and certainly increased in number, after C. sputorum subsp, rnucosalis started to disappear. The relationship of these organisms to serotype B C. sputorum subsp, rnucosalis (Roberts et al., 1977 b) is not known at the present time. Antigenic variation has been described in S. mutans colonising the intestine of gnotobiotic rats (Bratthall and
254
Gibbons, 1975b), and the suggestion has been made that variation is due to the selection pressure of antibodies acting on the bacteria. The evidence presented for this is the changing agglutination activities of salivary immunoglobulin A against oral streptococci over a period o f time (Bratthall and Gibbons, 1975 a). It remains to be established if the NACS are the result of such selection pressure mediated b y antibodies against C. sputorum subsp, mucosalis. ACKNOWLEDGEMENTS
The work described here was supported financially by the WeUcome Trust and one of us (L. Roberts) was in receipt of a WeUcome Trust Scholarship for the duration of the work. The authors wish to express thanks to Miss P. Wooding for excellent technical assistance and help in management of experimental animals, and to Mrs. M. Buchan for typing this paper. REFERENCES Bloomfield, A.L., 1919. The fate of bacteria introduced into the upper air passages. Bull. Johns Hopkins Hosp., 30: 317--322. Bloomfield, A.L., 1920a. The fate of bacteria introduced into the upper air passages. II. B. coli and Staphylococcus albus. Bull. Johns Hopkins Hosp., 31: 14--19. Bloomfield, A.L., 1920b. The fate of bacteria introduced into the upper air passages. V. The Friedlander bacilli. Bull. Johns Hopkins Hosp., 31: 203--206. Bratthall, D. and Gibbons, R.J., 1975 a. Changing agglutination activities of salivary immunoglobulin A preparation against oral streptococci. Infect. Immun., 11: 603--606. Bratthall, D. and Gibbons, R.J., 1975 b. Antigenic variation of Streptococcus mutans colonizing gnotobiotic rats. Infect. Immun., 12: 1231--1236. Brecher, S. and Van Houte, J., 1976. Oral establishment of Actinomyces viscosus in SpragueDawley rats of various age. J. Dent. Res., 55: Special Issue B, B177, abstr. 462. Burnett, G.W., Scherp, H.W. and Schuster, G.S., 1976. Oral Microbiology and Infectious Disease. 4th edition. Williams and Williams Company Limited, Baltimore, p. 219. Gibbons, R.J. and Van Houte, J., 1975. Bacterial adherence in oral microbial ecology. Annu. Rev. Microbiol., 29: 19--44. Lawson, G.H.K. and Rowland, A.C., 1974. Intestinal adenomatosis in the pig: a bacteriological study. Res. Vet. Sci., 17: 331--336. Lawson, G.H.K., Rowland, A.C. and Roberts, L., 1975 a. Isolation of Campylobacter sputorum subspecies mucosalis from oral cavity of pigs. Vet. Rec., 97: 308. Lawson, G.H.K., Rowland, A.C. and Wooding, P., 1975 b. The characterisation of Campylobacter sputorum subspecies mucosalis isolated from pigs. Res. Vet. Sci., 18: 121--126. Lawson, G.H.K., Rowland, A.C. and Roberts, L., 1976. Studies on Campylobacter sputorum subspecies mucosalis. J. Med. Microbiol., 9: 163--171. Loesche, W.J., Gibbons, R.J. and Socransky, S.S., 1965. Biochemical characteristics of Vibrio sputorum and relationship to Vibrio bubulus and Vibrio fetus. J. Bacteriol., 89: 1109--1116. Roberts, L., 1978. A study of porcine intestinal adenomatosis. PhD. thesis, The University of Edinburgh, 439 pp. Roberts, L., Rowland, A.C. and Lawson, G.H.K., 1977 a. Experimental reproduction of porcine intestinal adenomatosis and necrotic enteritis. Vet. Rec., 100: 12--13.
255 Roberts, L., Lawson, G.H.K. and Rowland, A.C., 1977 b. Porcine intestinal adenomatosis associated with serologically distinct Campylobacter sputorum subspecies mucosalis. Res. Vet. Sci., 23: 257--258. Roberts, L., Lawson, G.H.K. and Rowland, A.C., 1980. The experimental infection of neonatal pigs with Campylobacter sputorum subspecies mucosalis. Res. Vet. Sci., 28: 145--147. Rowland, A.C. and Rowntree, P.G.M., 1972. A haemorragic bowel syndrome associated with intestinal adenomatosis in the pig. Vet. Rec., 91: 235--241. Smibert, R.M., 1974. Genus Campylobacter. In: R.E. Buchanan and N.E. Gibbons (coEditors), Bergey's Manual of Determinative Bacteriology, Eight Edition, Williams and Wilkins,Company, Baltimore, pp. 207--212. Van Houte, J., 1976. Oral bacterial colonization: mechanisms and implications. In: M. Stiles, W. Loesche and T. O'Brien (Editors), Proceedings, Microbiol. Aspects of Dental Caries. Special Supplement to Microbiol. Abstr., 1 : 3--32. Van Houte, J. and Upeslacis, V.N., 1976. Decreased establishment of Streptococcus mutans on the teeth of conventional Sprague-Dawley rats during their aging. J. Dent. Res., 55: Special Issue B, B178, abstr. 467. Van Houte, J., Upeslacis, V.N., Jordan, H.V., Skobe, Z. and Green, D.B., 1976. Role of sucrose in colonization of Streptococcus mutans in conventional Sprague-Dawley rats. J. Dent. Res., 55: 202--215.
249
Short Communication EXPERIMENTAL INFECTION OF NEONATAL PIGS WITH CAMPYLOB A C T E R S P U T O R UM S U B S P E C I E S M U C O S A L I S W I T H S P E C I A L REFERENCE TO THE ORAL CAVITY
L. ROBERTS*, G.H.K. LAWSON** and A.C. ROWLAND** * Veterinary Investigation Centre, Mill of Craibstone, Bucksburn, Aberdeen AB2 9TS (Great Britain) **Department of Veterinary Pathology, University of Edinburgh, Veterinary Field Station, Easter Bush, Roslin, Midlothian (Great Britain) (Accepted 25 March 1980) ABSTRACT Roberts, L., Lawson, G.H.K. and Rowland, A.C., 1980. The experimental infection of neonatal pigs with Campylobacter sputorum subspecies mucosalis with special reference to the oral cavity. Vet. Microbiol., 5: 249--255. After oral exposure Campylobacter sputorum subsp, mucosalis became established in the mouth of piglets for up to eight weeks. C. sputorum subsp, mucosalis was not isolated from the same pigs each week, although some pigs were positive for a number of consecutive weeks. Spread also took place from orally dosed piglets to an undosed litter mate maintalned with them. Other catalase negative, serologically distinct Campylobacters were also demonstrated in the oral cavity. INTRODUCTION T h e t y p e species o f t h e catalase negative C a m p y l o b a c t e r s , Campylobacter sputorum subsp, sputorum is p r e s e n t in the h u m a n m o u t h ( L o e s c h e et al., 1965). In man, m o s t oral m o t i l e organisms, such as spirochaetes and vibrios, are f o u n d in highest c o n c e n t r a t i o n s in t h e gingival crevice area ( G i b b o n s and Van H o u t e , 1 9 7 5 ) . L i t t l e or n o saliva p e n e t r a t e s into this crevice, and it is t h o u g h t t h a t w h e n such m o t i l e organisms are i n t r o d u c e d i n t o t h e m o u t h , active m o v e m e n t enables t h e m t o r e a c h this q u i e s c e n t site ( G i b b o n s and Van H o u t e , 1 9 7 5 ) . H u m a n oral m i c r o b i o l o g y has b e e n a field o f active research i n t o t h e i n v o l v e m e n t o f bacteria in a n u m b e r o f d e n t a l diseases. T h e r e has b e e n little r e p o r t e d v e t e r i n a r y w o r k in this area. An organism resembling C. sputorum subsp, sputorum has b e e n r e c o v e r e d f r o m cases o f p o r c i n e intestinal a d e n o m a t o s i s (PIA) ( L a w s o n and R o w l a n d , 1 9 7 4 ) . This is a c o n d i t i o n o f the p o s t w e a n e d pig characterised b y a n o r e x i a and wasting, t h e a f f e c t e d intestinal e p i t h e l i u m having an a d e n o m a t o u s appearance. T h e b a c t e r i u m has b e e n fully characterised ( L a w s o n e t al., 1 9 7 5 b ) and the n a m e Campylobacter sputorum subsp, mucosalis p r o p o s e d . In PIA, 0378--1135/80/0000--0000/$02.25 © 1980 Elsevier Scientific Publishing Company
250
C. sputorum subsp, mucosalis lies free within the cytoplasm of affected intestinal epithelial cells and undergoes division in that site. C. sputorum subsp. mucosalis has also been recovered from the m o u t h of piglets (Lawson et al., 1975 a). This was a limited study but provided information on the presence of C. sputorum subsp, mucosalis in, and its isolation from, the oral cavity of animals w i t h o u t gross or clinical evidence o f porcine intestinal adenomatosis (PIA); the organism has also been isolated from the m o u t h in cases of PIA (Lawson et al., 1976). The catalase negative Campylobacters of the porcine oral cavity seem to be a heterogenous group, and some isolates are biochemically and serologically typical of C. sputorum subsp, mucosalis, others are biochemically typical but serologically distinct, while a third group differ both biochemically and serologically from C. sputorum subsp, mucosalis (Lawson et al., 1976). In this paper the catalase positive Campylobacters will be referred to as Campylobacter coli. Although not recognised in the eighth edition of Bergey's Manual of Determinative Bacteriology (Smibert, 1974), most veterinary bacteriologists recognise the existence of this organism, and consider it to be distinct from Campylobacter fetus subsp. ]ejuni. The experiment described in the present paper set out to examine the occurrence of Campylobacters in the m o u t h of piglets exposed to C. sputorum subsp, mucosalis in the neonatal period. MATERIALS AND METHODS
Experimental animals and timing o f procedures A Duroc sow was introduced to isolation premises several days before farrowing. Eight of the piglets born were removed from the sow before suckling and seven were dosed orally with C. sputorum subsp, mucosalis approximately 1.5 h after birth of the last piglet. The piglets were returned to the sow 1.5 h later, i.e. 3.0 h after birth of the last piglet. All seven piglets received oral chalk before the C. sputorum subsp, mucosalis suspension. The piglets were weaned. at 51 days of age. They were offered creep (starter creep - Seafield Mill) from 10 days of age and 'ABRO Breeders' meal (Seafield Mill - Roslin) at 3 weeks. Mouth swabs were taken from all of the piglets at 2 days of age and weekly from then onwards until 72 days of age. Mouth and vaginal swabs were taken from the sow on days 30, 37, 44 and 51 of the experiment. The piglet n o t directly exposed to infection was killed on day 37, the remaining seven piglets were killed at 80 days of age and subjected to necropsy. Bacteriological examination was not carried out on these piglets at this time.
Sampling A sterile c o t t o n wool swab was rotated along the gingival margin of the piglet. In the laboratory the swab was broken off into a McCartney bottle con-
251 talning 5 ml sterile saline and shaken for five minutes. Each sample was then treated as previously described (Lawson et al., 1975 a}.
Infecting inoculum The suspension of C. sputorum subsp, mucosalis used to orally dose four of the piglets (N 1--4) contained 3.5 × l 0 s viable C. sputorum subsp, mucosalis per ml. The other three orally dosed piglets (D 1--3) received a ten-fold dilution in tryptose phosphate broth of that suspension. Each dosed piglet received 15 ml of one of the C. sputorum subsp, mucosalis suspensions.
Differentiation of oral Campylobacters The criteria for the differentiation of the oral Campylobacters were those used in the initial separation of C. sputorum subsp, mucosalis and C. coli (Lawson and Rowland, 1974) dealing with intestinal isolates. Selected isolates were subsequently examined biochemically and found to conform to the original description (Lawson et al., 1975 b). Catalase negative, slide agglutination negative Campylobacters were those organisms which proved negative in slide catalase tests and did n o t react in slide agglutination tests with C. sputorum subsp, mucosalis anti-serum (serotype A, NCTC 11,000). These isolates were n o t subjected to extensive biochemical investigation, although a number of m o u t h isolates have been examined (Lawson et al., 1976) and found to be characteristic of the genus Campylobacter. These organisms are therefore referred to as Campylobacters, although the possibility that some of them may n o t belong to this genus cannot be definitely excluded. In the tables and text they are referred to as non-agglutinating Campylobacters (NACS). RESULTS From 21 days of age, the piglet n o t directly exposed to C. sputorum subsp. mucosalis was noticeably smaller in size and less active compared to its litter mates. This piglet was killed on day 37 as it had become progressively weaker although on no occasion was diarrhoea seen. At necropsy this piglet was in very poor bodily condition. Histopathologically there was no evidence of PIA and C. sputorum subsp, mucosalis was n o t recovered from the alimentary tract. When the remaining pigs were killed at the end of the experiment there was no evidence of PIA. The recovery of C. sputorum subsp, mucosalis from the m o u t h of these piglets is summarised in Table I. With the exception of week 2, C. sputorum subsp. mucosalis was isolated from a variable number of piglets each week, up to 8 weeks post-infection. The number of piglets from which C. sputorum subsp. rnucosalis was isolated increased to a maximum at week 5, and then decreased until no recoveries were made in weeks 9--11. C. sputorum subsp, rnucosalis was n o t recovered from the same piglets each week, except for piglet D3 which
252 TABLE
I
R e c o v e r y o f C a m p y l o b a c t e r s o n w e e k l y s a m p l i n g in t h e litter in w h i c h all p i g l e t s w e r e s a m p l e d w e e k l y Piglet
Week 1
2
3
4
5
6
7
8
9
10
11
NI
M
C
O
O
M
C
C
O
O
NACS
N2
O
C
O
C
M
C
C
O
O
M
C
C
C NACS C
O
N3
C NACS O
C NACS C
O
C
N4
O
O
NACS
O
C
M
O
D1
O
O
C
M
O M
O O
M M
M M
M C C M
C
D2 D3
M C O M
Control
C
O
C
M
M
C
C M NACS . .
C NACS C NACS C NACS O O
2/8 + 118 0/8
0/8 1/8 0/8
3/8 2/8 1/8
3/8 3/8 0/8
6/8 2/8 0/8
3/8 5/8 1/8
1/7 5/7 1/7
C
M C C NACS C NACS .
.
O
O
O
O
C O
C C
0/7 1 /7 0/7
0/7 4/7 1/7
.
O v e r a l l recovery
from swabs C. s p u t o r u m subsp. mucosalis C. c o l i
NACS M ffi C .
1/7 5/7 3/7
0/7 5/7 4/7
s p u t o r u m subsp, m u c o s a l i s r e c o v e r e d : C ffi C . c o l i r e c o v e r e d .
N A C S = Catalase n e g a t i v e , slide a g g l u t i n a t i o n negative C a m p y l o b a c t e r s r e c o v e r e d . O = N o C a m p y l o b a c t e r i s o l a t e d : C o n t r o l piglet ffi t h e piglet n o t d i r e c t l y e x p o s e d t o C. s p u t o r u m s u b s p .
mucosalis. + N u m e r a t o r = n u m b e r o f s w a b s p o s i t i v e : d e n o m i n a t o r ffi t o t a l a n i m a l s s a m p l e d .
proved to be infected in 6 of the 7 weeks in which this organism was isolated from some members of the litter. With the exception of the single isolation of NACS made from piglet N4 in week 3, NACS were not recovered until week 6, after which time they were isolated from a considerable proportion of the piglets (Table I). Piglet D3 is of interest since C. sputorurn subsp, rnucosalis was isolated from the oral cavity of this piglet each week, except for week 2, up to week 7. In week 7, both C. sputorum subsp, mucosalis and NACS were isolated from the same swab. In week 8, only NACS were recovered. C. sputorum subsp, rnucosalis was not isolated from either the oral cavity or vagina of the sow on any of four occasions when sampled between days 30 and 61. Catalase positive Campylobacters were isolated from both sites and on one occasion NACS were also recovered from the mouth. DISCUSSION The establishment of irregular oral infection with C. sputorum subsp, mucosalis in a litter of neonatal piglets for eight weeks after oral exposure is described. The method of sampling was relatively crude and it cannot be said that C. sputorum subsp, mucosalis was absent from the mouth of those piglets from
253 whose m o u t h swabs it was not isolated. It seems reasonable, however, that taken as a whole, these results provide an indication of the presence of C. sputorum subsp, mucosal/s within the oral cavity of some members of the litter. The duration of infection of eight weeks is comparable to the period of clinical signs in PIA (Rowland and Rowntree, 1972), the experimental disease (Roberts et al., 1977a; Roberts, 1978), and the persistence of infection in the intestinal tract of orally dosed neonatal piglets (Roberts, 1978; Roberts et al., 1980). There was spread from dosed piglets to the undosed piglet. With Streptococcus mutans in rats, transmission from m o u t h to m o u t h occurs mainly b y coprophagy (Van H o u t e et al., 1976). These workers suggest that the faecal count of S. mutans in their experiments was directly related to the m o u t h population of the organism. It is possible that spread b y coporphagy occurred within this litter of piglets, or alternatively spread could have occurred by suckling contaminated teats. During the period of the experiment, there was no clinical evidence of PIA and when piglets were necropsied at the termination of the experiment, 11 weeks after oral dosing with C. sputorum subsp, mucosalis, no gross changes of the disease were found. Little information is available a b o u t the establishment of micro-organisms in the oral cavity (Van Houte, 1976), although it is recognised that the host's age is important in oral colonisation b y bacteria. In rats, younger animals are more susceptible to infection with S. mutans (Van H o u t e and Upeslacis, 1976), whereas in the same t y p e of rats, susceptibility to oral infection with Actinomyces viscosus increases after weaning (Brecher and Van Houte, 1976). It is interesting that a number of isolates of C. sputorum subsp, mucosalis were made from the m o u t h of orally dosed neonatal piglets, b u t only one isolation from the post-weaned group (Roberts, 1978; Roberts et al., 1980). The susceptibility of the neonatal piglet to oral infection with C. sputorum subsp. mucosalis is confirmed in this paper. In a manner analogous to the rapid clearance of bacteria from the intestine, Bloomfield (1919, 1920 a,b) demonstrated that various bacteria when introduced into the m o u t h and nasopharynx of man were rapidly cleared. There is a great variability in the bacterial composition of the oral cavity in the first few days of life ( B u m e t t et al., 1976). Possibly during this period, establishment of introduced organisms is easier and this may be true of the establishment of C. sputorum subsp, mucosalis in the m o u t h of the neonatal pig. In the experiment described in this paper, in addition to the recovery of C. sputorum subsp, mucosalis from the oral cavity, NACS were also isolated. The indication was that these appeared, and certainly increased in number, after C. sputorum subsp, rnucosalis started to disappear. The relationship of these organisms to serotype B C. sputorum subsp, rnucosalis (Roberts et al., 1977 b) is not known at the present time. Antigenic variation has been described in S. mutans colonising the intestine of gnotobiotic rats (Bratthall and
254
Gibbons, 1975b), and the suggestion has been made that variation is due to the selection pressure of antibodies acting on the bacteria. The evidence presented for this is the changing agglutination activities of salivary immunoglobulin A against oral streptococci over a period o f time (Bratthall and Gibbons, 1975 a). It remains to be established if the NACS are the result of such selection pressure mediated b y antibodies against C. sputorum subsp, mucosalis. ACKNOWLEDGEMENTS
The work described here was supported financially by the WeUcome Trust and one of us (L. Roberts) was in receipt of a WeUcome Trust Scholarship for the duration of the work. The authors wish to express thanks to Miss P. Wooding for excellent technical assistance and help in management of experimental animals, and to Mrs. M. Buchan for typing this paper. REFERENCES Bloomfield, A.L., 1919. The fate of bacteria introduced into the upper air passages. Bull. Johns Hopkins Hosp., 30: 317--322. Bloomfield, A.L., 1920a. The fate of bacteria introduced into the upper air passages. II. B. coli and Staphylococcus albus. Bull. Johns Hopkins Hosp., 31: 14--19. Bloomfield, A.L., 1920b. The fate of bacteria introduced into the upper air passages. V. The Friedlander bacilli. Bull. Johns Hopkins Hosp., 31: 203--206. Bratthall, D. and Gibbons, R.J., 1975 a. Changing agglutination activities of salivary immunoglobulin A preparation against oral streptococci. Infect. Immun., 11: 603--606. Bratthall, D. and Gibbons, R.J., 1975 b. Antigenic variation of Streptococcus mutans colonizing gnotobiotic rats. Infect. Immun., 12: 1231--1236. Brecher, S. and Van Houte, J., 1976. Oral establishment of Actinomyces viscosus in SpragueDawley rats of various age. J. Dent. Res., 55: Special Issue B, B177, abstr. 462. Burnett, G.W., Scherp, H.W. and Schuster, G.S., 1976. Oral Microbiology and Infectious Disease. 4th edition. Williams and Williams Company Limited, Baltimore, p. 219. Gibbons, R.J. and Van Houte, J., 1975. Bacterial adherence in oral microbial ecology. Annu. Rev. Microbiol., 29: 19--44. Lawson, G.H.K. and Rowland, A.C., 1974. Intestinal adenomatosis in the pig: a bacteriological study. Res. Vet. Sci., 17: 331--336. Lawson, G.H.K., Rowland, A.C. and Roberts, L., 1975 a. Isolation of Campylobacter sputorum subspecies mucosalis from oral cavity of pigs. Vet. Rec., 97: 308. Lawson, G.H.K., Rowland, A.C. and Wooding, P., 1975 b. The characterisation of Campylobacter sputorum subspecies mucosalis isolated from pigs. Res. Vet. Sci., 18: 121--126. Lawson, G.H.K., Rowland, A.C. and Roberts, L., 1976. Studies on Campylobacter sputorum subspecies mucosalis. J. Med. Microbiol., 9: 163--171. Loesche, W.J., Gibbons, R.J. and Socransky, S.S., 1965. Biochemical characteristics of Vibrio sputorum and relationship to Vibrio bubulus and Vibrio fetus. J. Bacteriol., 89: 1109--1116. Roberts, L., 1978. A study of porcine intestinal adenomatosis. PhD. thesis, The University of Edinburgh, 439 pp. Roberts, L., Rowland, A.C. and Lawson, G.H.K., 1977 a. Experimental reproduction of porcine intestinal adenomatosis and necrotic enteritis. Vet. Rec., 100: 12--13.
255 Roberts, L., Lawson, G.H.K. and Rowland, A.C., 1977 b. Porcine intestinal adenomatosis associated with serologically distinct Campylobacter sputorum subspecies mucosalis. Res. Vet. Sci., 23: 257--258. Roberts, L., Lawson, G.H.K. and Rowland, A.C., 1980. The experimental infection of neonatal pigs with Campylobacter sputorum subspecies mucosalis. Res. Vet. Sci., 28: 145--147. Rowland, A.C. and Rowntree, P.G.M., 1972. A haemorragic bowel syndrome associated with intestinal adenomatosis in the pig. Vet. Rec., 91: 235--241. Smibert, R.M., 1974. Genus Campylobacter. In: R.E. Buchanan and N.E. Gibbons (coEditors), Bergey's Manual of Determinative Bacteriology, Eight Edition, Williams and Wilkins,Company, Baltimore, pp. 207--212. Van Houte, J., 1976. Oral bacterial colonization: mechanisms and implications. In: M. Stiles, W. Loesche and T. O'Brien (Editors), Proceedings, Microbiol. Aspects of Dental Caries. Special Supplement to Microbiol. Abstr., 1 : 3--32. Van Houte, J. and Upeslacis, V.N., 1976. Decreased establishment of Streptococcus mutans on the teeth of conventional Sprague-Dawley rats during their aging. J. Dent. Res., 55: Special Issue B, B178, abstr. 467. Van Houte, J., Upeslacis, V.N., Jordan, H.V., Skobe, Z. and Green, D.B., 1976. Role of sucrose in colonization of Streptococcus mutans in conventional Sprague-Dawley rats. J. Dent. Res., 55: 202--215.