- Email: [email protected]
Reproduction of proliferative enteritis in hamsters with a pure culture of porcine ileal symbiont intracellularis
E LS EV 1ER
Veterinary Microbiology41 (1994) 1-9
Reproduction of proliferative enteritis in hamsters with a pure culture of porcine ileal symbiont intracellularis S. Jasni, S. McOrist, G.H.K. Lawson Department of Veterinary Pathology, Royal (Dick) School of Veterinary Studies, Universityof Edinburgh, Veterinary FieM Station, Easter Bush, Midlothian EH25 9RG, UK (Received l 1 May 1993; accepted 2 November 1993)
Abstract Hamsters, three weeks old, were inoculated orally with suspensions of intracellular bacteria, grown in tissue culture cells, IEC- 18, rat enterocytes. Cells had been infected with suspensions ofintracellular bacteria derived from the lesions of proliferative haemorrhagic enteropathy occurring naturally in two pigs 916/91 and 1482/89. Infected cell lines containing each separate strain, 916/91 and 1482/89, were passaged one, two or five times and pure cultures of intracellular bacteria, identified as ileal symbiont intracellularis by immunological means, were collected from the cells and used as inocula. Ten of sixteen hamsters dosed with 916/91 passaged one or five times, developed lesions of proliferative enteritis evident at necropsy three weeks after inoculation. Hamsters inoculated with 1482/ 89 passaged twice and stored frozen, or IEC-18 cells alone or those left uninoculated, failed to develop lesions of proliferative enteritis. Campylobacterjejuni infection occurred throughout, in all groups. Marked hyperplasia of ileal enterocytes, associated with numerous intracellular curved bacteria was invariably detected in experimentally affected hamsters. Immunofluorescence reactions with specific antibodies indicated that these intracellular bacteria were also ileal symbiont intracellularis. The results suggested that proliferative enteritis could be reproduced in hamsters with a pure culture of an agent derived from pigs. We concluded that the reproduction of the disease with our inocula containing a single agent clarifies the aetiology of proliferative enteritis in both hamsters and pigs. Keywords: Campylobacterjejuni; Hamster; Proliferativeenteritis; Ileal symbiont intracellularis
1. Introduction Proliferative enteropathy or enteritis presents as a similar clinical and pathological syndrome in pigs and hamsters, with mucosal hyperplasia of immature crypt epithelial cells, primarily in the terminal ileum. Other sites o f the intestines that can be affected include the 0378-1135/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSD10378-1 135 ( 9 3 ) E 0 1 5 9 - F
2
s. Jasni et al. / VeterinaryMicrobiology 41 (1994) 1-9
jejunum, caecum and colon (Rowland and Lawson, 1974; Frisk and Wagner, 1977). Weanling and young adult pigs or hamsters are principally affected with typical clinical manifestation of rapid weight loss and dehydration (Jacoby and Johnson, 1981; Rowland and Lawson, 1992). Natural clinical disease in hamsters has not been reported in the UK, only from the USA, whereas the condition in pigs occurs worldwide. The disease is consistently associated with the presence of intracellular curved bacteria in the cytoplasm of the hyperplastic crypt epithelial cells. The consistent presence of these intracellular bacteria in the early lesions of natural and experimentally induced disease in pigs and hamsters suggests that they are involved in aetiology (Frisk and Wagner, 1977; Jacoby and Johnson, 1981; McOrist et al., 1989). Previous attempts to reproduce the disease in hamsters by oral inoculation of pure cultures of curved organisms such as Campylobacter jejuni have been unsuccessful (Regina and Lonigro, 1982; McOrist and Lawson, 1987). In contrast, hamsters given inocula of diseased hamster ileum free of C. jejuni (Stills and Hook, 1989) or of a cell culture of unidentified intracellular bacteria prepared from diseased hamsters developed the proliferative lesions (Stills, 1991 ). Subsequent work indicated that some of the inocula used in those experiments were contaminated by a Chlamydia sp., not capable of reproducing the disease itself (Stills et al., 1991). The disease can be transmitted from pigs to hamsters, suggesting that the intracellular bacteria involved may be similar in both host species (McOrist and Lawson, 1987). C. jejuni can be regularly isolated from the intestines of healthy hamsters (Fox et al., 1981). Immunological, cultural and DNA studies of the intracellular bacteria in the lesions in pigs have indicated that it is an obligate intracellular bacterium belonging to a new taxonomic group (reviewed in Lawson and McOrist, 1993). Sequencing of the 16S rDNA portion of the genomes of 4 strains of intracellular bacteria derived from lesions in pigs established that this taxonomic group was distinct from all known bacteria - these strains were designated ileal symbiont intracellularis (Gebhart et al., 1993). This study examines the transmissibility of the disease in hamsters with tissue culture bacteria derived from naturally affected pigs.
2. Materials and methods Hamsters Hamsters (Mesocricetus auratus) were obtained from a closed colony naturally colonized with C. jejuni. The hamsters were bred randomly and maintained in a standard laboratory animal facility. Hamsters normally take solid food at ca. 10 days old and are weaned at ca. 21 days old. The solid food given was either Mouse/Hamster diet, Special Diet Service Ltd., Witham, Essex, U.K. or Mouse Chow, Ralston Purina, St Louis, Missouri, USA. These diets are proprietary formulations containing a balanced complete nutrition with 5% and 3% fibre respectively. The hamsters were kept on wood shavings and given feed and water throughout. All hamsters were weaned and inoculated at 3 weeks of age. Control or infected hamsters were placed in separate cages after inoculation.
S. Jasni et al. / Veterinary Microbiology 41 (1994) 1-9
3
Pig-derived bacteria
The source material used for infection of cells was derived from the affected intestines of two pigs naturally affected with proliferative haemorrhagic enteropathy designated as 916/91 and 1482/89. The method of preparation of pure suspensions of intracellular bacteria from the lesions has been described previously (McOrist et al., 1987). These bacterial suspensions were re-suspended finally in sucrose potassium glutamate (B ovamick et al., 1950) containing 10% v/v fetal calf serum or in Dulbecco's modification of Eagle's medium (DMEM) with 10% v/v dimethyl sulfoxide (DMSO) in 1 ml vials and stored frozen at - 70°C for 3 to 4 months respectively before they were used. Inocula f o r hamsters
IEC-18 cells, rat enterocytes, European Cell Culture Collection No. 88011801, were grown to a monolayer covering 20 to 30% of 25 cm 2 plastic flasks in 24 h in DMEM supplemented with L-glutamine, fungizone and 10% v/v fetal calf serum. Infection of these cells by intracellular bacteria derived from pigs is described in detail elsewhere (Lawson et al., 1993). Briefly, after rapidly thawing one 1 ml vial of pig-derived bacteria at 37°C, it was added to 14 ml of warm DMEM with supplements as above, but with 7% v/v fetal calf serum. The diluted suspension was then added to the IEC-18 cell monolayers in two 25 cm 2 flasks. Infection of cells was assisted by centrifugation of the flasks at 2000 g for 30 min. Flasks were incubated microaerophilically (8% 02, 8.8% CO2, 82% N2) for 3 h. The cells were then fed with the same media containing neomycin and vancomycin and further incubated as above. At days 2 and 4 post-infection, the infected cell monolayers were re-fed with the same growth media, supplements and antibiotics but with 5% v/v fetal calf serum and finally harvested at day 6 post-inoculation for passage. Passaging of infected ceils was performed by treatment with potassium chloride as described by Lawson et al. (1993), followed by removal of the cells in each flask by a cell scraper. The scraped cells were then ruptured by passage 6 times through a needle before they were used to infect fresh monolayers of IEC- 18 cells in new flasks. The infected new cells were grown for another 6 days before they were prepared either for infecting hamsters or passaged again. The passage number of bacteria grown in cells and used to infect each group of hamsters is given in Table 1. For each flask, the media was removed and replaced with 2 ml of growth medium with 5% v/v fetal calf serum and the cells removed with a cell scraper. Cells from each passage used for each group were then bulked, homogenised briefly for 15 s and hamsters dosed orally with 2 ml each via a 16 G blunt ended needle. The inocula used for hamsters in groups 5, 6 and 7 (see Table 1) were passaged material which had been stored frozen at - 7 0 ° C in tissue culture growth medium with 10% v/v DMSO for approximately 3 months. All remaining test hamsters received freshly harvested bacteria. Control hamsters were either not inoculated or orally inoculated with non-infected IEC18 cells. Confluent monolayers of these cells were detached by trypsinization by standard methods (Paul, 1986), then homogenised for 15 seconds and each hamster dosed with 2 ml.
4
S. Jasni et al. / Veterinary Microbiology41 (1994) 1-9
Table 1 Features of hamsters dosed with tissue culturederivedinocula Group
1 2 3 4 5 6b 7 8 9
Inocula
No. of Hamsters Inoculated
Strain No.
Passage No.
Organisms Present (IIF) ~
916/91 916/91 916/91 916/91 916/91 1492/89 1482/89 IEC-18 ConSols Undosed Controls
1 5 6 7 5 2 2 c
+++ +++ +++ ++ + ++ None
c
c
Histological changes in the intestine Proliferative enteritis
Acute enteritis
No
visible lesions
6 5 5 6 4 6 8 4
4 3 3 0 0 0 0 0
2 2 2 4 4 3 8 3
0 0 0 2 0 3 0 1
28
0
16
12
"Indirectimmunofluorescencestaining (IIF) with monoclonalantibodyIG4. + ++
>5 > 10
+++
>100
<10 ] < I00~ per high power field
J
bSpecialDiet ServiceLtd. diet used. CNotapplicable. Inoculafor groups 5, 6 and 7 had been frozenbefore use. Monitoring o f inocula To monitor the numbers of Ileal symbiont intracellularis in the flasks of each IEC-18 passage, IEC-18 cells on glass coverslips cultured in identical media in small vials were infected in parallel. On day 6 post-infection, the relevant coverslips were collected at the same time as the infected flasks, washed in warm Locke's salt solution (Paul, 1986), fixed in acetone and mounted on glass slides for specific immunoperoxidase staining. Coverslips were stained with monoclonal antibody IG4 as the primary antibody and an anti-mouse peroxidase conjugate used as the secondary antibody in an indirect immunoperoxidase test with haematoxylin counterstaining. Antibody IG4 is known to be specific for ileal symbiont intracellularis (McOrist et al., 1987). In addition, 10/xl of each bulked inoculum used to dose hamsters was smeared onto a glass slide, air-dried, fixed in acetone, and stained with an indirect immunofluorescence assay, incorporating the same primary antibody IG4 followed by fluorescein conjugated sheep anti-mouse antibody. Infected and non-infected IEC- 18 cells stained for the presence of Chlamydia species using Giemsa staining and either anti-Chlamydia lipopolysaccharide (Imogen, Dako Ltd.) or outer membrane protein antibody kits (Microtrak, Syva) were uniformly negative.
S. Jasni et al. / Veterinary Microbiology 41 (1994) 1-9
5
Necropsy procedure A full necropsy was conducted and samples of small and large intestines taken for light microscopy and immunostaining as described previously (McOrist and Lawson, 1987). Briefly, sections of fixed intestines were stained with haematoxylin and eosin or a silver impregnation stain. Further unstained paraffin sections were digested with trypsin and immunofluorescence staining was conducted with monoclonal antibody, IG4. Fluorescein conjugated sheep anti-mouse immunoglobulin was employed as the secondary antibody and sections were examined under a fluorescence microscope. Similar immunofluorescence staining was conducted in all hamsters using an antiserum prepared in rabbits against C. jejuni strain 664/83 isolated from a normal hamster (McOrist and Lawson, 1987).
Bacteriological procedures Samples were taken from all inocula and cultured onto Columbia blood agar, Skirrow's medium and Brucella semi-solid medium and incubated microaerophically for Campylobacter spp. and other bacteria. Mucosal swabs from each hamster's intestine were collected at necropsy and cultured for Campylobacter spp. by standard methods as described previously (McOrist and Lawson, 1987).
3. Results
Bacterial culture of the tissue culture derived inocula revealed no detectable isolates. Immunoperoxidase staining of infected IEC-18 cells set up in parallel to the flasks used to inoculate the hamsters and smears of each inoculum examined by immunofluorescence assay showed numerous curved bacteria reactive with the primary monoclonal antibody (see Table 1). Infected monolayers showed no discernible cytopathic effects during infection and passage of bacteria. C. jejuni was isolated from 33/40 treated and 24/32 control hamsters. All hamsters were clinically healthy throughout. Marked thickening of the intestinal mucosa was not detected in any hamster.
Microscopic findings All hamsters including control animals had varying degrees of localised acute intestinal inflammation. Infiltration of the mucosa with inflammatory cells especially neutrophils, and scanty lymphocytes and macrophages was present. Some hamsters had focal necrosis and infiltration of the crypts with neutrophils. Crypt lumina containing numerous curved bacilli were seen consistently on silver stained sections. Fluorescent curved bacilli were also evident in the lumina of occasional crypts in sections treated with antiserum raised against C.jejuni 664/83. Sections of control hamster intestines appeared normal and sections treated with monoclonal antibody IG4 showed no fluorescence. Microscopic lesions consistent with proliferative enteritis were seen only in hamsters dosed with inocula 916/91 in groups 1,2 and 3 (see Table 1). All affected hamsters showed marked hyperplasia of crypt epithelial cells in the terminal ileum and three hamsters in group 1 also had similar lesions in the jejunum. In these adenomatous lesions, affected crypts were enlarged, had reduced numbers or total absence of goblet cells and many mitotic
6
S. Jasni et al. / Veterinary Microbiology 41 (1994) 1-9
Fig. 1. Ileumof hamster in group 2 affected with proliferativeenteritis.Note enlargedcrypt with proliferationof immatureepithelialcells and reduced numbersof goblet cells. Haematoxylinand eosin stainx 700.
Fig. 2. Ileumof hamsterin group 3 affected with proliferativeenteritis.Note numerousbrightlyfluorescingcurved bacteria in the apical cytoplasm of proliferatingcrypt epithelialcells. Sectionstainedwith monoclonalantibody IG4 in an indirectimmunofluorescenceassayX550. figures were apparent (Fig. 1). Adjacent villi were distorted and reduced to a leaf-like shape. There was a moderate increase in the range of crypt to villus ratio in affected hamsters from the control values of 0.2 to 0.3, to 0.35 to 0.45 in groups 1, 2 and 3. Silver stains of affected intestines showed numerous curved bacterial forms within the apical cytoplasm of crypt cells. Immunofluorescence staining of intestinal sections with monoclonal antibody IG4, showed numerous brightly fluorescing curved bacteria especially in the apical cytoplasm
s. Jasni et al. / VeterinaryMicrobiology 41 (1994) 1-9
7
of cells in the affected crypts and villi (Fig. 2). Curved bacteria in the lumen did not react with this monoclonal antibody. Animals exposed to infected material which did not develop proliferative lesions, failed to show any evidence of luminal or intracellular infection by L intracellularis, either by silver or monoclonal antibody staining.
4. Discussion
Weanling hamsters proved to be susceptible to infection by ileal symbiont intracellularis derived from porcine enteropathy tissue and grown in tissue culture. The severity of the adenomatous lesions which appeared in infected hamsters was relatively mild but apparently identical to the naturally occurring disease in pigs (Rowland and Lawson, 1992) and hamsters (Jacoby and Johnson, 1981 ). Similar disease occurred in hamsters directly dosed with homogenates of other affected porcine mucosas passed through 0.65/zm pore size filters (McOrist and Lawson, 1987). Only hamsters given passaged 916/91 inocula in groups 1, 2 and 3 developed the lesions. The failure of the hamsters given inocula 1482/89 to develop the disease may suggest that only particular strains are capable of infection and initiation of proliferative responses in the intestines of experimentally challenged hamsters. The severity of disease may also be influenced by a number of other factors, including the number of infective bacteria in the inocula. Another possible explanation is that the organism may occur in different infective forms at different stages of development and only the infective form is capable of crypt cell infection. The rickettsial bacterium, Cowdria ruminatum can have three morphologically distinct forms, only two of which are infective (Jongejan et al., 1991). The inability of frozen inocula to initiate the lesions may be attributable to reduction of infective titre by freeze-thawing. A high concentration of the intracellular organism is known to be required to elicit a successful reproduction of proliferative enteritis in hamsters (Frisk and Wagner, 1977). Numerous intracellular curved bacterial forms were observed in the cytoplasm of cells in the hyperplastic crypts and in adjacent crypt lumina. The definite relationship of curved bacterial forms within crypt lumina in both control and infected hamsters and adjacent cells in affected hamsters is still not clear. Although C. jejuni was isolated from many control and infected hamsters and formed colonies in the crypt lumens of the intestines with associated acute intestinal inflammation, there was no indication of any significant relationship of this organism to the proliferative lesions. Similar findings have been reported in previous experimental studies with C. jejuni (Regina and Lonigro, 1982; Stills and Hook, 1989), confirming that the intracellular bacteria are distinct from C.jejuni. This is also clear from the ability of inocula 916/91 to initiate the lesions, as all inocula were grown in cell lines free of C. jejuni at all stages. C. jejuni is a common component of the gut flora of hamsters worldwide (Fox et al., 1981). Colonization is commonly associated with a mild to moderate degree of acute enteritis (Humphrey et al., 1985). We assessed the identity of the intracellular bacteria in the original pig lesions, the infected cell cultures, the inocula and in the hamster lesions by morphological and immunological tests. The positive reactions of all these bacteria with the specific monoclonal antibody IG4 indicates that they are identical to the intracellular organisms present in pig lesions as these
8
S. Jasni et al. / Veterinary Microbiology 41 (1994) 1-9
were purified and used to prepare that antibody (McOrist et al., 1987). Subsequent work established that those organisms had distinctive immunological and DNA profiles, and sequencing of their 16S rDNA confirmed that they belong to a new taxonomic group currently named ileal symbiont intracellularis (McOrist et al., 1990; Gebhart et al., 1993) although further taxonomic study is in progress. The ability to grow these organisms in vitro in tissue culture cell lines has only recently been demonstrated (Lawson et al., 1993). This study represents the first reported reproduction of proliferative enteritis using pure cultures of an agent derived from pigs. Similar results have been reported with tissue culture derived inocula prepared from hamsters (Stills, 1991). Although organisms observed in hamsters in that study appeared similar to ours, their exact identity was not clear and the inocula were probably contaminated with a Chlamydia species (Stills et al., 1991). We found no evidence of viruses or Chlamydia species in the animal cells or tissues examined in this study using specific techniques. We therefore concluded that the reproduction of the disease with our inocula containing a single bacterium clarifies the aetiology of proliferative enteritis in both hamsters and pigs. It is possible that there is some difference in the strains of IS intracellularis present naturally in pigs and hamsters. This would be an explanation for the milder syndrome seen in pig to hamster transmission in this study, compared to hamster to hamster transmission (Stills, 1991). Hamster to pig transmission studies have not been reported. Differences in the strains and disease induced may reflect the number of host animal passages in any one species by a strain, or an innate difference between hamster and pig organisms.
Acknowledgements We thank Rebecca Mackie for her kind help with tissue cultures. This work was supported by the Agricultural and Food Research Council of the UK, the Wellcome Trust and the Govemment of Malaysia.
References Bovarnick, M.R., Miller, J.C. and Snyder, J.C., 1950. The influence of certain salts, amino acids, sugars and proteins on the stability of Rickettsia. J. Bacterioi., 59: 509-522. Fox, J.G., Zanotti, S. and Jordan, H.V., 1981. The hamster as a reservoir of Campylobacter fetus subspecies jejuni. J. Infect. Dis., 143: 856. Frisk, C.S. and Wagner, J.E., 1977. Experimental hamster enteritis: an electron microscopic study. Am. J. Vet. Res., 38: 1861-1868. Gebhart, C.J., Barns, S.M., McOrist, S., Lin, G.F. and Lawson, G.H.K., 1993. Ileal symbiont intracellularis, an obligate intracellular bacteria of porcine intestines showing a relationship to Desulfovibrio species. Int. J. Syst. Bacteriol., 43: 533-538. Humphrey, C.D., Montag, D.M. and Pittman, F.E., 1985. Experimental infection of hamsters with Campylobacter jejuni. J. Infect. Dis., 151: 485--493. Jacoby, R.O. and Johnson, E.A., 1981. Transmissible ileal hyperplasia of hamsters. Adv. Exp. Med. Biol. 134: 267-289. Jongejan, F., Zondbergen, T.A., Van de Wiel, R.A., Groot, M.D. and Uilenburg, G., 1991. The tick-borne rickettsia, Cowdria ruminantum has a chlamydia-like developmental cycle. Onderst. J. Vet. Res; 58: 227-237.
S. Jasni et al. / Veterinary Microbiology 41 (1994) 1-9
9
Lawson G.H.K. and McOrist, S., 1993. The enigma of the proliferative enteropathies: a review. J. Comp. Pathol., 108: 41-46. Lawson G.H.K., McOfist, S., Jasni, S. and Mackie, R.A., 1993. The intracellular bacteria of porcine proliferative enteropathy: Cultivation and maintenance in vitro. J. Clin. Microbiol., 31:1136-1142. McOrist, S. and Lawson, G.H.K., 1987. Possible relationship of proliferative enteritis in pigs and hamsters. Vet. Microbiol., 15: 293-302. McOrist S., Boid, R., Lawson, G.H.K. and McConnell, I., 1987. Monoclonal antibodies to the intracellular Campylobacter-like organisms of the porcine proliferative enteropathies. Vet. Rec., 121: 421-422. McOrist S., Lawson, G.H.K., Rowland, A.C. and MacIntyre, N., 1989. Early lesions of proliferative enteritis in pigs and hamsters. Vet. Pathol., 26: 260-264. McOrist, S., Lawson, G.H.K., Roy, D.J. and Boid, R., 1990. DNA analysis of intracellular Campylobacter-like organisms associated with the porcine proliferative enteropathies. FEMS Microbiol. Lett., 69: 189-194. Paul, J., 1975. Cell and Tissue Culture. 5th Ed. Churchill Livingstone, London. p. 484. Regina, M.L. and Lonigro, S., 1982. Isolation of Campylobacter fetus subspecies jejuni from hamsters with proliferative ileitis. Lab. Anim. Sci., 32: 660-662. Rowland, A.C. and Lawson, G.H.K., 1974. Intestinal adenomatosis in the pig: immunofluorescent and electron microscopic studies. Res. Vet. Sci., 17: 323-330. Rowland, A.C. and Lawson, G.H.K., 1992. Porcine proliferative enteropathies. In: Diseases of Swine, 7th Ed. Eds.: A.D. Leman, B.E. Straw, W.L. Mengeling, S.D. Allaire and D.J. Taylor. Iowa State University press, Ames. pp. 560-569. Stills, H.F. and Hook, R.R., 1989. Experimental production of proliferative ileitis in Syrian hamsters by using ileal homogenate free of Campylobacter jejuni. Infect. Immun., 57: 191-195. Stills, H.F., 1991. Isolation of an intracellular bacterium from hamsters with proliferative ileitis and reproduction of the disease with a pure culture. Infect. Immun., 59: 3227-3236. Stills, H.F., Fox, J.G., Paster, B.J. and Dewhirst, F.E., 1991. A new Chlamvdia sp. strain SFPD isolated from transmissible proliferative ileitis in hamsters. Microbial Ecology in Health and Disease. Special Issue, Campylobacter VI, 4: 599.
Veterinary Microbiology41 (1994) 1-9
Reproduction of proliferative enteritis in hamsters with a pure culture of porcine ileal symbiont intracellularis S. Jasni, S. McOrist, G.H.K. Lawson Department of Veterinary Pathology, Royal (Dick) School of Veterinary Studies, Universityof Edinburgh, Veterinary FieM Station, Easter Bush, Midlothian EH25 9RG, UK (Received l 1 May 1993; accepted 2 November 1993)
Abstract Hamsters, three weeks old, were inoculated orally with suspensions of intracellular bacteria, grown in tissue culture cells, IEC- 18, rat enterocytes. Cells had been infected with suspensions ofintracellular bacteria derived from the lesions of proliferative haemorrhagic enteropathy occurring naturally in two pigs 916/91 and 1482/89. Infected cell lines containing each separate strain, 916/91 and 1482/89, were passaged one, two or five times and pure cultures of intracellular bacteria, identified as ileal symbiont intracellularis by immunological means, were collected from the cells and used as inocula. Ten of sixteen hamsters dosed with 916/91 passaged one or five times, developed lesions of proliferative enteritis evident at necropsy three weeks after inoculation. Hamsters inoculated with 1482/ 89 passaged twice and stored frozen, or IEC-18 cells alone or those left uninoculated, failed to develop lesions of proliferative enteritis. Campylobacterjejuni infection occurred throughout, in all groups. Marked hyperplasia of ileal enterocytes, associated with numerous intracellular curved bacteria was invariably detected in experimentally affected hamsters. Immunofluorescence reactions with specific antibodies indicated that these intracellular bacteria were also ileal symbiont intracellularis. The results suggested that proliferative enteritis could be reproduced in hamsters with a pure culture of an agent derived from pigs. We concluded that the reproduction of the disease with our inocula containing a single agent clarifies the aetiology of proliferative enteritis in both hamsters and pigs. Keywords: Campylobacterjejuni; Hamster; Proliferativeenteritis; Ileal symbiont intracellularis
1. Introduction Proliferative enteropathy or enteritis presents as a similar clinical and pathological syndrome in pigs and hamsters, with mucosal hyperplasia of immature crypt epithelial cells, primarily in the terminal ileum. Other sites o f the intestines that can be affected include the 0378-1135/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSD10378-1 135 ( 9 3 ) E 0 1 5 9 - F
2
s. Jasni et al. / VeterinaryMicrobiology 41 (1994) 1-9
jejunum, caecum and colon (Rowland and Lawson, 1974; Frisk and Wagner, 1977). Weanling and young adult pigs or hamsters are principally affected with typical clinical manifestation of rapid weight loss and dehydration (Jacoby and Johnson, 1981; Rowland and Lawson, 1992). Natural clinical disease in hamsters has not been reported in the UK, only from the USA, whereas the condition in pigs occurs worldwide. The disease is consistently associated with the presence of intracellular curved bacteria in the cytoplasm of the hyperplastic crypt epithelial cells. The consistent presence of these intracellular bacteria in the early lesions of natural and experimentally induced disease in pigs and hamsters suggests that they are involved in aetiology (Frisk and Wagner, 1977; Jacoby and Johnson, 1981; McOrist et al., 1989). Previous attempts to reproduce the disease in hamsters by oral inoculation of pure cultures of curved organisms such as Campylobacter jejuni have been unsuccessful (Regina and Lonigro, 1982; McOrist and Lawson, 1987). In contrast, hamsters given inocula of diseased hamster ileum free of C. jejuni (Stills and Hook, 1989) or of a cell culture of unidentified intracellular bacteria prepared from diseased hamsters developed the proliferative lesions (Stills, 1991 ). Subsequent work indicated that some of the inocula used in those experiments were contaminated by a Chlamydia sp., not capable of reproducing the disease itself (Stills et al., 1991). The disease can be transmitted from pigs to hamsters, suggesting that the intracellular bacteria involved may be similar in both host species (McOrist and Lawson, 1987). C. jejuni can be regularly isolated from the intestines of healthy hamsters (Fox et al., 1981). Immunological, cultural and DNA studies of the intracellular bacteria in the lesions in pigs have indicated that it is an obligate intracellular bacterium belonging to a new taxonomic group (reviewed in Lawson and McOrist, 1993). Sequencing of the 16S rDNA portion of the genomes of 4 strains of intracellular bacteria derived from lesions in pigs established that this taxonomic group was distinct from all known bacteria - these strains were designated ileal symbiont intracellularis (Gebhart et al., 1993). This study examines the transmissibility of the disease in hamsters with tissue culture bacteria derived from naturally affected pigs.
2. Materials and methods Hamsters Hamsters (Mesocricetus auratus) were obtained from a closed colony naturally colonized with C. jejuni. The hamsters were bred randomly and maintained in a standard laboratory animal facility. Hamsters normally take solid food at ca. 10 days old and are weaned at ca. 21 days old. The solid food given was either Mouse/Hamster diet, Special Diet Service Ltd., Witham, Essex, U.K. or Mouse Chow, Ralston Purina, St Louis, Missouri, USA. These diets are proprietary formulations containing a balanced complete nutrition with 5% and 3% fibre respectively. The hamsters were kept on wood shavings and given feed and water throughout. All hamsters were weaned and inoculated at 3 weeks of age. Control or infected hamsters were placed in separate cages after inoculation.
S. Jasni et al. / Veterinary Microbiology 41 (1994) 1-9
3
Pig-derived bacteria
The source material used for infection of cells was derived from the affected intestines of two pigs naturally affected with proliferative haemorrhagic enteropathy designated as 916/91 and 1482/89. The method of preparation of pure suspensions of intracellular bacteria from the lesions has been described previously (McOrist et al., 1987). These bacterial suspensions were re-suspended finally in sucrose potassium glutamate (B ovamick et al., 1950) containing 10% v/v fetal calf serum or in Dulbecco's modification of Eagle's medium (DMEM) with 10% v/v dimethyl sulfoxide (DMSO) in 1 ml vials and stored frozen at - 70°C for 3 to 4 months respectively before they were used. Inocula f o r hamsters
IEC-18 cells, rat enterocytes, European Cell Culture Collection No. 88011801, were grown to a monolayer covering 20 to 30% of 25 cm 2 plastic flasks in 24 h in DMEM supplemented with L-glutamine, fungizone and 10% v/v fetal calf serum. Infection of these cells by intracellular bacteria derived from pigs is described in detail elsewhere (Lawson et al., 1993). Briefly, after rapidly thawing one 1 ml vial of pig-derived bacteria at 37°C, it was added to 14 ml of warm DMEM with supplements as above, but with 7% v/v fetal calf serum. The diluted suspension was then added to the IEC-18 cell monolayers in two 25 cm 2 flasks. Infection of cells was assisted by centrifugation of the flasks at 2000 g for 30 min. Flasks were incubated microaerophilically (8% 02, 8.8% CO2, 82% N2) for 3 h. The cells were then fed with the same media containing neomycin and vancomycin and further incubated as above. At days 2 and 4 post-infection, the infected cell monolayers were re-fed with the same growth media, supplements and antibiotics but with 5% v/v fetal calf serum and finally harvested at day 6 post-inoculation for passage. Passaging of infected ceils was performed by treatment with potassium chloride as described by Lawson et al. (1993), followed by removal of the cells in each flask by a cell scraper. The scraped cells were then ruptured by passage 6 times through a needle before they were used to infect fresh monolayers of IEC- 18 cells in new flasks. The infected new cells were grown for another 6 days before they were prepared either for infecting hamsters or passaged again. The passage number of bacteria grown in cells and used to infect each group of hamsters is given in Table 1. For each flask, the media was removed and replaced with 2 ml of growth medium with 5% v/v fetal calf serum and the cells removed with a cell scraper. Cells from each passage used for each group were then bulked, homogenised briefly for 15 s and hamsters dosed orally with 2 ml each via a 16 G blunt ended needle. The inocula used for hamsters in groups 5, 6 and 7 (see Table 1) were passaged material which had been stored frozen at - 7 0 ° C in tissue culture growth medium with 10% v/v DMSO for approximately 3 months. All remaining test hamsters received freshly harvested bacteria. Control hamsters were either not inoculated or orally inoculated with non-infected IEC18 cells. Confluent monolayers of these cells were detached by trypsinization by standard methods (Paul, 1986), then homogenised for 15 seconds and each hamster dosed with 2 ml.
4
S. Jasni et al. / Veterinary Microbiology41 (1994) 1-9
Table 1 Features of hamsters dosed with tissue culturederivedinocula Group
1 2 3 4 5 6b 7 8 9
Inocula
No. of Hamsters Inoculated
Strain No.
Passage No.
Organisms Present (IIF) ~
916/91 916/91 916/91 916/91 916/91 1492/89 1482/89 IEC-18 ConSols Undosed Controls
1 5 6 7 5 2 2 c
+++ +++ +++ ++ + ++ None
c
c
Histological changes in the intestine Proliferative enteritis
Acute enteritis
No
visible lesions
6 5 5 6 4 6 8 4
4 3 3 0 0 0 0 0
2 2 2 4 4 3 8 3
0 0 0 2 0 3 0 1
28
0
16
12
"Indirectimmunofluorescencestaining (IIF) with monoclonalantibodyIG4. + ++
>5 > 10
+++
>100
<10 ] < I00~ per high power field
J
bSpecialDiet ServiceLtd. diet used. CNotapplicable. Inoculafor groups 5, 6 and 7 had been frozenbefore use. Monitoring o f inocula To monitor the numbers of Ileal symbiont intracellularis in the flasks of each IEC-18 passage, IEC-18 cells on glass coverslips cultured in identical media in small vials were infected in parallel. On day 6 post-infection, the relevant coverslips were collected at the same time as the infected flasks, washed in warm Locke's salt solution (Paul, 1986), fixed in acetone and mounted on glass slides for specific immunoperoxidase staining. Coverslips were stained with monoclonal antibody IG4 as the primary antibody and an anti-mouse peroxidase conjugate used as the secondary antibody in an indirect immunoperoxidase test with haematoxylin counterstaining. Antibody IG4 is known to be specific for ileal symbiont intracellularis (McOrist et al., 1987). In addition, 10/xl of each bulked inoculum used to dose hamsters was smeared onto a glass slide, air-dried, fixed in acetone, and stained with an indirect immunofluorescence assay, incorporating the same primary antibody IG4 followed by fluorescein conjugated sheep anti-mouse antibody. Infected and non-infected IEC- 18 cells stained for the presence of Chlamydia species using Giemsa staining and either anti-Chlamydia lipopolysaccharide (Imogen, Dako Ltd.) or outer membrane protein antibody kits (Microtrak, Syva) were uniformly negative.
S. Jasni et al. / Veterinary Microbiology 41 (1994) 1-9
5
Necropsy procedure A full necropsy was conducted and samples of small and large intestines taken for light microscopy and immunostaining as described previously (McOrist and Lawson, 1987). Briefly, sections of fixed intestines were stained with haematoxylin and eosin or a silver impregnation stain. Further unstained paraffin sections were digested with trypsin and immunofluorescence staining was conducted with monoclonal antibody, IG4. Fluorescein conjugated sheep anti-mouse immunoglobulin was employed as the secondary antibody and sections were examined under a fluorescence microscope. Similar immunofluorescence staining was conducted in all hamsters using an antiserum prepared in rabbits against C. jejuni strain 664/83 isolated from a normal hamster (McOrist and Lawson, 1987).
Bacteriological procedures Samples were taken from all inocula and cultured onto Columbia blood agar, Skirrow's medium and Brucella semi-solid medium and incubated microaerophically for Campylobacter spp. and other bacteria. Mucosal swabs from each hamster's intestine were collected at necropsy and cultured for Campylobacter spp. by standard methods as described previously (McOrist and Lawson, 1987).
3. Results
Bacterial culture of the tissue culture derived inocula revealed no detectable isolates. Immunoperoxidase staining of infected IEC-18 cells set up in parallel to the flasks used to inoculate the hamsters and smears of each inoculum examined by immunofluorescence assay showed numerous curved bacteria reactive with the primary monoclonal antibody (see Table 1). Infected monolayers showed no discernible cytopathic effects during infection and passage of bacteria. C. jejuni was isolated from 33/40 treated and 24/32 control hamsters. All hamsters were clinically healthy throughout. Marked thickening of the intestinal mucosa was not detected in any hamster.
Microscopic findings All hamsters including control animals had varying degrees of localised acute intestinal inflammation. Infiltration of the mucosa with inflammatory cells especially neutrophils, and scanty lymphocytes and macrophages was present. Some hamsters had focal necrosis and infiltration of the crypts with neutrophils. Crypt lumina containing numerous curved bacilli were seen consistently on silver stained sections. Fluorescent curved bacilli were also evident in the lumina of occasional crypts in sections treated with antiserum raised against C.jejuni 664/83. Sections of control hamster intestines appeared normal and sections treated with monoclonal antibody IG4 showed no fluorescence. Microscopic lesions consistent with proliferative enteritis were seen only in hamsters dosed with inocula 916/91 in groups 1,2 and 3 (see Table 1). All affected hamsters showed marked hyperplasia of crypt epithelial cells in the terminal ileum and three hamsters in group 1 also had similar lesions in the jejunum. In these adenomatous lesions, affected crypts were enlarged, had reduced numbers or total absence of goblet cells and many mitotic
6
S. Jasni et al. / Veterinary Microbiology 41 (1994) 1-9
Fig. 1. Ileumof hamster in group 2 affected with proliferativeenteritis.Note enlargedcrypt with proliferationof immatureepithelialcells and reduced numbersof goblet cells. Haematoxylinand eosin stainx 700.
Fig. 2. Ileumof hamsterin group 3 affected with proliferativeenteritis.Note numerousbrightlyfluorescingcurved bacteria in the apical cytoplasm of proliferatingcrypt epithelialcells. Sectionstainedwith monoclonalantibody IG4 in an indirectimmunofluorescenceassayX550. figures were apparent (Fig. 1). Adjacent villi were distorted and reduced to a leaf-like shape. There was a moderate increase in the range of crypt to villus ratio in affected hamsters from the control values of 0.2 to 0.3, to 0.35 to 0.45 in groups 1, 2 and 3. Silver stains of affected intestines showed numerous curved bacterial forms within the apical cytoplasm of crypt cells. Immunofluorescence staining of intestinal sections with monoclonal antibody IG4, showed numerous brightly fluorescing curved bacteria especially in the apical cytoplasm
s. Jasni et al. / VeterinaryMicrobiology 41 (1994) 1-9
7
of cells in the affected crypts and villi (Fig. 2). Curved bacteria in the lumen did not react with this monoclonal antibody. Animals exposed to infected material which did not develop proliferative lesions, failed to show any evidence of luminal or intracellular infection by L intracellularis, either by silver or monoclonal antibody staining.
4. Discussion
Weanling hamsters proved to be susceptible to infection by ileal symbiont intracellularis derived from porcine enteropathy tissue and grown in tissue culture. The severity of the adenomatous lesions which appeared in infected hamsters was relatively mild but apparently identical to the naturally occurring disease in pigs (Rowland and Lawson, 1992) and hamsters (Jacoby and Johnson, 1981 ). Similar disease occurred in hamsters directly dosed with homogenates of other affected porcine mucosas passed through 0.65/zm pore size filters (McOrist and Lawson, 1987). Only hamsters given passaged 916/91 inocula in groups 1, 2 and 3 developed the lesions. The failure of the hamsters given inocula 1482/89 to develop the disease may suggest that only particular strains are capable of infection and initiation of proliferative responses in the intestines of experimentally challenged hamsters. The severity of disease may also be influenced by a number of other factors, including the number of infective bacteria in the inocula. Another possible explanation is that the organism may occur in different infective forms at different stages of development and only the infective form is capable of crypt cell infection. The rickettsial bacterium, Cowdria ruminatum can have three morphologically distinct forms, only two of which are infective (Jongejan et al., 1991). The inability of frozen inocula to initiate the lesions may be attributable to reduction of infective titre by freeze-thawing. A high concentration of the intracellular organism is known to be required to elicit a successful reproduction of proliferative enteritis in hamsters (Frisk and Wagner, 1977). Numerous intracellular curved bacterial forms were observed in the cytoplasm of cells in the hyperplastic crypts and in adjacent crypt lumina. The definite relationship of curved bacterial forms within crypt lumina in both control and infected hamsters and adjacent cells in affected hamsters is still not clear. Although C. jejuni was isolated from many control and infected hamsters and formed colonies in the crypt lumens of the intestines with associated acute intestinal inflammation, there was no indication of any significant relationship of this organism to the proliferative lesions. Similar findings have been reported in previous experimental studies with C. jejuni (Regina and Lonigro, 1982; Stills and Hook, 1989), confirming that the intracellular bacteria are distinct from C.jejuni. This is also clear from the ability of inocula 916/91 to initiate the lesions, as all inocula were grown in cell lines free of C. jejuni at all stages. C. jejuni is a common component of the gut flora of hamsters worldwide (Fox et al., 1981). Colonization is commonly associated with a mild to moderate degree of acute enteritis (Humphrey et al., 1985). We assessed the identity of the intracellular bacteria in the original pig lesions, the infected cell cultures, the inocula and in the hamster lesions by morphological and immunological tests. The positive reactions of all these bacteria with the specific monoclonal antibody IG4 indicates that they are identical to the intracellular organisms present in pig lesions as these
8
S. Jasni et al. / Veterinary Microbiology 41 (1994) 1-9
were purified and used to prepare that antibody (McOrist et al., 1987). Subsequent work established that those organisms had distinctive immunological and DNA profiles, and sequencing of their 16S rDNA confirmed that they belong to a new taxonomic group currently named ileal symbiont intracellularis (McOrist et al., 1990; Gebhart et al., 1993) although further taxonomic study is in progress. The ability to grow these organisms in vitro in tissue culture cell lines has only recently been demonstrated (Lawson et al., 1993). This study represents the first reported reproduction of proliferative enteritis using pure cultures of an agent derived from pigs. Similar results have been reported with tissue culture derived inocula prepared from hamsters (Stills, 1991). Although organisms observed in hamsters in that study appeared similar to ours, their exact identity was not clear and the inocula were probably contaminated with a Chlamydia species (Stills et al., 1991). We found no evidence of viruses or Chlamydia species in the animal cells or tissues examined in this study using specific techniques. We therefore concluded that the reproduction of the disease with our inocula containing a single bacterium clarifies the aetiology of proliferative enteritis in both hamsters and pigs. It is possible that there is some difference in the strains of IS intracellularis present naturally in pigs and hamsters. This would be an explanation for the milder syndrome seen in pig to hamster transmission in this study, compared to hamster to hamster transmission (Stills, 1991). Hamster to pig transmission studies have not been reported. Differences in the strains and disease induced may reflect the number of host animal passages in any one species by a strain, or an innate difference between hamster and pig organisms.
Acknowledgements We thank Rebecca Mackie for her kind help with tissue cultures. This work was supported by the Agricultural and Food Research Council of the UK, the Wellcome Trust and the Govemment of Malaysia.
References Bovarnick, M.R., Miller, J.C. and Snyder, J.C., 1950. The influence of certain salts, amino acids, sugars and proteins on the stability of Rickettsia. J. Bacterioi., 59: 509-522. Fox, J.G., Zanotti, S. and Jordan, H.V., 1981. The hamster as a reservoir of Campylobacter fetus subspecies jejuni. J. Infect. Dis., 143: 856. Frisk, C.S. and Wagner, J.E., 1977. Experimental hamster enteritis: an electron microscopic study. Am. J. Vet. Res., 38: 1861-1868. Gebhart, C.J., Barns, S.M., McOrist, S., Lin, G.F. and Lawson, G.H.K., 1993. Ileal symbiont intracellularis, an obligate intracellular bacteria of porcine intestines showing a relationship to Desulfovibrio species. Int. J. Syst. Bacteriol., 43: 533-538. Humphrey, C.D., Montag, D.M. and Pittman, F.E., 1985. Experimental infection of hamsters with Campylobacter jejuni. J. Infect. Dis., 151: 485--493. Jacoby, R.O. and Johnson, E.A., 1981. Transmissible ileal hyperplasia of hamsters. Adv. Exp. Med. Biol. 134: 267-289. Jongejan, F., Zondbergen, T.A., Van de Wiel, R.A., Groot, M.D. and Uilenburg, G., 1991. The tick-borne rickettsia, Cowdria ruminantum has a chlamydia-like developmental cycle. Onderst. J. Vet. Res; 58: 227-237.
S. Jasni et al. / Veterinary Microbiology 41 (1994) 1-9
9
Lawson G.H.K. and McOrist, S., 1993. The enigma of the proliferative enteropathies: a review. J. Comp. Pathol., 108: 41-46. Lawson G.H.K., McOfist, S., Jasni, S. and Mackie, R.A., 1993. The intracellular bacteria of porcine proliferative enteropathy: Cultivation and maintenance in vitro. J. Clin. Microbiol., 31:1136-1142. McOrist, S. and Lawson, G.H.K., 1987. Possible relationship of proliferative enteritis in pigs and hamsters. Vet. Microbiol., 15: 293-302. McOrist S., Boid, R., Lawson, G.H.K. and McConnell, I., 1987. Monoclonal antibodies to the intracellular Campylobacter-like organisms of the porcine proliferative enteropathies. Vet. Rec., 121: 421-422. McOrist S., Lawson, G.H.K., Rowland, A.C. and MacIntyre, N., 1989. Early lesions of proliferative enteritis in pigs and hamsters. Vet. Pathol., 26: 260-264. McOrist, S., Lawson, G.H.K., Roy, D.J. and Boid, R., 1990. DNA analysis of intracellular Campylobacter-like organisms associated with the porcine proliferative enteropathies. FEMS Microbiol. Lett., 69: 189-194. Paul, J., 1975. Cell and Tissue Culture. 5th Ed. Churchill Livingstone, London. p. 484. Regina, M.L. and Lonigro, S., 1982. Isolation of Campylobacter fetus subspecies jejuni from hamsters with proliferative ileitis. Lab. Anim. Sci., 32: 660-662. Rowland, A.C. and Lawson, G.H.K., 1974. Intestinal adenomatosis in the pig: immunofluorescent and electron microscopic studies. Res. Vet. Sci., 17: 323-330. Rowland, A.C. and Lawson, G.H.K., 1992. Porcine proliferative enteropathies. In: Diseases of Swine, 7th Ed. Eds.: A.D. Leman, B.E. Straw, W.L. Mengeling, S.D. Allaire and D.J. Taylor. Iowa State University press, Ames. pp. 560-569. Stills, H.F. and Hook, R.R., 1989. Experimental production of proliferative ileitis in Syrian hamsters by using ileal homogenate free of Campylobacter jejuni. Infect. Immun., 57: 191-195. Stills, H.F., 1991. Isolation of an intracellular bacterium from hamsters with proliferative ileitis and reproduction of the disease with a pure culture. Infect. Immun., 59: 3227-3236. Stills, H.F., Fox, J.G., Paster, B.J. and Dewhirst, F.E., 1991. A new Chlamvdia sp. strain SFPD isolated from transmissible proliferative ileitis in hamsters. Microbial Ecology in Health and Disease. Special Issue, Campylobacter VI, 4: 599.