Bactericidal effect of normal swine sera on Treponema hyodysenteriae.

Treponema hyodysenteriae was incubated in 20% normal swine sera (NSS) at 37 degrees C for 4 h, and viability was determined by a plate dilution method. NSS was bactericidal for nonpathogenic T. innocens and avirulent T. hyodysenteriae, but not for virulent T. hyodysenteriae isolates. Heat inactivation at 56 degrees C for 30 min, treatment with EDTA or EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N-tetraacetic acid], or removal of immunoglobulin M eliminated the bactericidal activity of NSS. However, removal of the alternate complement pathway with 10 mg of bentonite per ml did not remove bactericidal activity of NSS. Incubation of virulent isolates of T. hyodysenteriae in the presence of specific antisera plus NSS resulted in bactericidal activity. These data suggest that complement and natural antibody may be involved in protecting the host from T. innocens or avirulent T. hyodysenteriae and that T. hyodysenteriae antibody plus complement are involved in protecting convalescent pigs from re-exposure to swine dysentery.

Involvement of lipopolysaccharide in the pathogenicity of Treponema hyodysenteriae.

Treponema hyodysenteriae, the etiologic agent of swine dysentery, caused gross and microscopic lesions in the large intestines of C3HeB/FeJ mice. No gross lesions were observed in the intestines of the closely related, but lipopolysaccharide-resistant, C3H/HeJ strain of mice, and microscopic lesions were mild, if present at all. In the presence of actinomycin D, 1 mg of T. hyodysenteriae lipopolysaccharide (LPS) was lethal for C3HeB/FeJ but not for C3H/HeJ mice. Also, the treponemal LPS was chemotactic for macrophages from C3H/HeJ mice but not for macrophages from C3HeB/FeJ mice. The difference between the two mouse strains in lesion development may be due to the nondestructive nature of LPS in C3H/HeJ mice, which suggests that the treponemal LPS is involved in the pathogenicity of T. hyodysenteriae. T. hyodysenteriae may prove to be a useful bacterium in the study of LPS-resistant C3H/HeJ mice, because resistance to the treponemal LPS and to the treponeme itself appear to correlate.

Serotype-specific opsonization of Treponema hyodysenteriae.

Treponema hyodysenteriae was shown to attach to mouse peritoneal cells in the absence of serum opsonins in vitro. If serotype-specific antiserum from pigs was added to the media and treponemes of that corresponding serotype were employed in the assay, the amount of attachment increased an average of 3.7 times that of the control without pig sera. However, the amount of attachment was increased an average of only 1.5 times that of the control if organisms of any noncorresponding serotype of T. hyodysenteriae were used in the assay. Since the lipopolysaccharide (LPS) extracted from T. hyodysenteriae is the basis for serotyping the treponeme, the ability of these distinct LPS types to block attachment by blocking opsonization of the organisms was tested. Attachment, using corresponding antisera and treponemes, was blocked by LPS extracted from treponemes of that serotype, but not by LPS extracted from treponemes of other serotypes. These results indicate that antibody response to T. hyodysenteriae infection in pigs is serotype-specific. Furthermore, since opsonization and subsequent attachment of bacteria to phagocytes is known to be a protective mechanism, we suggest that the LPS may be an important antigen in the stimulation of host defense against the treponeme.

Biological activity of a lipopolysaccharide extracted from Treponema hyodysenteriae.

A lipopolysaccharide (LPS) was obtained from pathogenic Treponema hyodysenteriae by hot phenol-water extraction. Various effects of the LPS on host cells were examined in vitro. Toxicity for mouse peritoneal macrophages was observed after 10 h of incubation at concentrations as low as 15 micrograms of the LPS per ml. Marked enhancement of both complement (C3) and immunoglobulin G-Fc receptor-mediated internalization was noted in macrophages obtained from mice injected 6 days previously with 75 micrograms of the material. Incorporation of [3H]thymidine into murine splenocytes was elevated approximately fourfold when splenocytes were treated with 5 to 10 micrograms of LPS per ml. Incubation of the LPS with normal porcine serum resulted in the generation of a factor(s) that stimulated the migration of porcine leukocytes. Generation of the chemotactic activity was inhibited by heating the serum at 56 degrees C for 30 min before treatment with LPS. The results suggest that T. hyodysenteriae contains an LPS that is biologically active.

Generation of chemotactic factor for granulocytes and monocytes from serum by fractions of Brucella abortus.

Several fractions isolated from Brucella abortus were examined for their ability to generate chemotactic factor from normal serum. Phenol phase lipopolysaccharides exhibited activity equivalent to that obtained with E. Coli lipopolysaccharide. A carbohydrate-rich aqueous methanol fraction was inhibitory at high concentrations, but a non-dialysable component of this fraction contained a potent stimulator of chemotactic activity. Protein-rich fractions from both strain 19 and strain 2308 were inactive. Preheating the serum at 56 degrees for 30 min prevented generation of chemotactic activity by the various fractions, suggesting a role for serum complement. No chemotactic activity was produced by Brucella fractions in C5-deficient DBA/2J mouse serum.

Detection of cell surface immunoglobulin on leucocytes by agglutination.

The detection of immunoglobulin (Ig) on murine peritoneal cell (PC) surfaces by an agglutination test is described. Addition of anti-mouse IgG (amIgG) to a suspension of PC results in agglutination of the cells. Trypsin or pronase treatment of the cells abrogated agglutination upon addition of amIgG. Incubation of washed murine PC with porcine IgG (pIgG) for 30 minutes resulted in agglutination of the cells upon addition of anti-porcine IgG (apIgG) to the cell suspension. This suggests that the heterologous pIgG is also able to bind to the murine cell surface. The implications of these findings are discussed and compared with respect to the agglutination test described herein and other techniques for detection of cytophilic antibody.

The involvement of sugars and Mg++ in the attachment of phagocytic cells to Ascaris suum juveniles.

In vitro adherence of washed peritoneal exudate cells (PECs) from mice to Ascaris suum juveniles was inhibited by D-glucosamine and D-galactosamine. Other carbohydrates tested had no apparent effect on adherence after incubation for 3 hr at any of the concentrations tested. Furthermore, attachment of PECs to A. suum juveniles was completely inhibited by the addition of ethylenediaminetetraacetate to the medium, but magnesium ethylene glycol bis(trichloroacetate) had no effect on adherence. The results suggest that receptors for glucosamine-like or galactosamine-like molecules in conjunction with Mg++ are involved in the attachment of pECs to A. suum juveniles in the absence of antibody and complement. Adherence mediated by carbohydrated and divalent cations may be involved in early nonspecific mechanisms of resistance against A. suum because the reaction did not depend on the presence of antibody and/or complement for attachment.