I00006Evaluation of the virulence of rhea S. hyodysenteriae strains for swine.
Thad B. Stanton (1), Neil S. Jensen (1), Brad T. Bosworth (1), Robert A. Kunkle (2)
(1) Enteric Diseases and Food Safety Research Unit and (2) Avian and Swine Respiratory Disease Research Unit, National Animal Disease Center, USDA, Agricultural Research Service, Ames, IA 50010
Abstract:
Serpulina hyodysenteriae (S. hyo.) is the etiologic agent of swine dysentery. Although the natural host range of this spirochete is generally considered limited to swine, recent findings suggest that S. hyo. has the potential to cause intestinal disease in other animals. S. hyo. strains were isolated from rheas with a severe necrotizing typhlocolitis (Vet Micro 52:259). In these experiments, we evaluated whether S. hyo. strains R1 and R358, isolated from dead rheas in Ohio and Iowa, respectively, were virulent for swine. The rheas had died due to severe typhlocolitis. In two experiments, postweaning piglets, 5-8 weeks old, were given on two consecutive days, 100 ml of BHIS broth culture containing 10e10 to 6 x 10e10 cfu of S. hyo. cells intra-gastrically. Animals in control groups received either 100 ml of sterile BHIS broth or cultures of S. hyo. B204, a strain virulent for swine. Swine given S. hyo. B204 cells first appeared ill within 24-36 hours after the second inoculation and all (12/12) of the B204-infected swine eventually showed clincal signs of swine dysentery, including positive tests for fecal occult blood and shedding of detectable levels of viable S. hyo. cells in feces. of 11 B204-infected animals had significant weight loss and, in (blind) histopathological analyses, had large intestine mucosal lesions consistent with swine dysentery, including multifocal, superficial erosions partially covered by loosely-adherent fibrinosuppurative and/or catarrhal exudate. By contrast, none of the swine given rhea strain R1 (14 animals), strain R358 (6 animals) or sterile culture broth developed dysentery. Only two animals (R1 group) had detectable S. hyo. cells in its feces and only at 24 h after the challenge inoculation. Histopathological examinations did not detect differences in the appearance of intestinal tissues between control animals and animals challenged with the rhea S. hyo. strains. These results indicate that S. hyodysenteriae strains R1 and R358, isolated from diarrheic rheas, are avirulent for swine. Comparisons of the biochemical and genetic differences between these S. hyo. rhea strains and swine strains could lead to the identification of characteristics essential for the virulence of S. hyo. in swine.
Introduction:
S. hyodysenteriae is an anaerobic spirochete and the causative agent of swine dysentery. Recently, anaerobic spirochetes were isolated from rheas suffering from severe typhlo-colitis and identified as strains of Serpulina hyodysenteriae (1,3). This discovery extends the known animal host range for S. hyodysenteriae and suggests the possibility that rheas (or other avian species) might be natural reservoirs for S. hyodysenteriae and transmit the pathogenic spirochete to swine or other mammals. The objective of the research described in this poster was to determine whether or not S. hyodysenteriae strains isolated from rheas are virulent for swine.
Materials and Methods:
Bacterial strains and culture conditions: S. hyodysenteriae B204 was isolated from a dysenteric swine and is virulent in experimental infections of swine (2). S. hyodysenteriae strains R-1 and R358 were independently isolated from intestinal contents of dead rheas in Ohio and Iowa. Pre- and postmortem examination indicated the birds had died from severe diarrhea. They exhibited a severe, fibrinous, and hemorrhagic enterocolitis and a colonic mucosa populated with large numbers of spirochetes. Strains R-1 and R358 were identified as S. hyodysenteriae strains by the following characteristics: form strongly beta-hemolytic colonies; cultures positive in tests for indole production; 16S rDNA restriction patterns and protein immunoblots nearly identical to those of porcine S. hyodysenteriae strains (1). Strain R-1 has also been identified as a strain of S. hyodysenteriae by multilocus enzyme electrophoresis (MEE) analysis and by DNA homology studies (5, unpublished observations). All S. hyodysenteriae strains were cultured in pre-reduced BHIS (brain heart infusion broth containing 10% heat treated calf serum) broth beneath an atmosphere of O2:N2 (1:99). Cultures (700 ml) of cells in the logarithmic phase of growth (3-5 x 10e8 cfu/ml) were used to inoculate animals. Viable cell counts (cfu/ml) were determined after the cultures had been used to inoculate animals.
Animals and inoculation conditions: Weaned piglets, mixed sexes, outbred, between 6-8 wks old at the time of inoculation were used. Animals were held for an acclimation period of two weeks before the experiment. During the acclimation period they were tested and determined to be free of Salmonella and S. hyodysenteriae. Animal groups:
I. Animals received sterile BHIS broth (negative control group).
II. Animals inoculated with S. hyodysenteriae B204 (positive control group).
III. Animals inoculated with S. hyodysenteriae R-1.
IV. Animals inoculated with S. hyodysenteriae R358.
In the first experiment, groups I, II, and III were used and in the second experiment, all groups were used. Each group was housed in a pen in a separate room. Animals were given food and water ad lib, except during the inoculation period when food was removed (2). Each animal was given, by intragastric gavage, 100 ml of either sterile broth or S. hyodysenteriae culture on two consecutive days.
Monitoring animals for dysentery: Throughout the experiments, animals were monitored two or three times daily for clinical signs of disease (stool consistency, bloody diarrhea, lethargy).
At scheduled intervals (i.e. 0, 1, 2, 5, 7, 9, 14, 21, 28 days after the second inoculation) rectal swabs were taken and animals were weighed. Rectal swab samples were analyzed for occult blood and for viable S. hyodysenteiae cells (4). Animals judged severely ill during the experiments were euthanized with an overdose of sodium pentobarbital administered intravenously. All remaining animals were euthanized in the same manner at the end of the experiment.
Histopathology: During necropsies, tissue samples from the colon, cecum, and small intestine were fixed in formalin. Sections (5 micrometer thick) of paraffin-embedded tissues were collected on glass slides, and stained with either hematoxylin and eosin or modified Dieterle's spirochete stain. Damage to tissues due to infection was assessed during light microscopy examinations of the sections by two microscopists who had no prior knowledge of the tissue origin.
(The experimental design, handling, and welfare of the animals in these studies were approved and monitored independently of the principal investigators by the Animal Care and Use Committee and the Veterinary Services Group of the NADC.)
Results:
There were no significant differences in viable cell counts among the
S. hyodysenteriae cultures used to inoculate animals. Each animal given bacterial
cultures received between 1 x 10e10 and 6 x 10e10 cfu on each of two
consecutive days.
Group I swine given sterile culture medium appeared
healthy throughout these studies. The animals were negative in tests for swine dysentery
and consistently gained weight (Fig. 1). From histopathological observations,
eight of the animals had healthy, normal appearing tissues (Fig. 2) and two had minimal
alterations of a non-specific etiology (Fig 3).
Group II swine infected with
S. hyodysenteriae strain B204 showed signs of swine dysentery within 24-48 hours
after the second inoculation. Within a two week period every animal had viable
S. hyodysenteriae cells in its feces and was positive for fecal occult blood (Fig. 1).
Nine of 11 animals lost weight. Colonic and cecal tissues of most animals had lesions
typical of swine dysentery (Fig. 3; Fig. 4 & 5).
Group III and IV swine
inoculated with either rhea S. hyodysenteriae strain R-1 or R358 exhibited no
signs of swine dysentery (Fig. 1, 3) and remained healthy for the duration of these
studies (4 weeks after inoculation). S. hyodysenteriae cells were recovered
from two rectal swab samples taken within 48 h after the second inoculation.
Histopathological observations of colonic and cecal tissues of the animals did not detect
dysenteric lesions and the appearance of the tissues was similar to that of control
animals in Group I (Fig. 2; Fig. 3).
Discussion and Conclusions:
Our experimental findings indicate that S. hyodysenteriae strains R-1 and R358,
isolated from rheas, are avirulent for swine. Our inability to recover
S. hyodysenteriae R-1 and R358 from rectal swab samples, except in two samples
early after inoculation (Fig. 1) suggests that these strains are unable to colonize the
swine intestinal tract. Comparisons of the biochemical and genetic properties of these
avirulent strains with those of the virulent S. hyodysenteriae strain B204 could
lead to the identification of virulence factors which permit B204 cells to colonize and
damage swine tissues.
Strains R-1 and R358 and other strains of
S. hyodysenteriae have been isolated from rheas suffering from severe dysentery
and displaying colonic lesions resembling those seen in cases of swine
dysentery. S. hyodysenteriae strains isolated from rheas with typhlo-colitis
induce intestinal mucosal lesions typical of typhlitis when inoculated into one day old
rhea chicks (6). Our finding that rhea strains R-1 and R358 do not infect swine suggests
the strains are host specific for virulence and that rheas are not a significant source
for transmitting S. hyodysenteriae strains that cause swine dysentery. It should
be noted, however, that this hypothesis of host specific virulence would receive additional
support if further experiments demonstrated that S. hyodysenteriae strains R-1 and
R358 induced typhlo-colitis when inoculated into adult rheas.
References:
1. Jensen, N.S., T.B. Stanton, and D.E. Swayne. 1996. Identification of the swine pathogen Serpulina hyodysenteriae in rheas (Rhea americana). Vet. Microbiol. 52:259-269.
2. Kinyon, J.M., D.L. Harris, and R.D. Glock. 1977. Enteropathogenicity of various isolates of Treponema hyodysenteriae. Infect. Immun. 15:638-646.
3. Sagartz, J.E., D.E. Swayne, K.A. Eaton, J. Hayes, K.D. Amass, R. Wack, and L. Kramer. 1992. Necrotizing typhlocolitis associated with a spirochete in rheas (Rhea americana). Avian Dis. 36:282-289.
4. Stanton, T.B. and N.S. Jensen. 1993. Monitoring experimental swine dysentery: rectal swab
blood test and Serpulina (Treponema) hyodysenteriae detection. Vet. Microbiol. 34:389-396.
5. Stanton, T.B., D.J. Trott, J.I. Lee, A.J. McLaren, D.J. Hampson, B.J. Paster, N.S. Jensen. 1996. Differentiation of intestinal spirochaetes by multilocus enzyme electrophoresis analysis and 16S rRNA sequence comparisons. FEMS Micro. Lett. 136:181-186.
6. Swayne, D.E., and A.J. McLaren. 1997. Avian intestinal spirochaetes and avian intestinal spirochaetosis. pp. 267-300. In: Intestinal Spirochaetes in Domestic Animals and Humans.
D.J. Hampson and T.B. Stanton (ed.) CAB International, Wallingford and New York.
Comments:
Address questions and comments about this abstract to Thad Stanton ( tstanton@iastate.edu).