Search

Lawsonia (L.) intracellularis, an obligately intracellular bacterium, causes proliferative enteropathy (PE) in swine and, occasionally, in other animals. To determine the spread of the agent among German pig herds pooled fecal samples of five animals each of clinically normal Hessian pig herds collected between november 1998 and february 1999 as well as feces (n = 1684) from individual animals representing 648 herds, sent to our laboratory by veterinarians from all parts of Germany, were tested for L. intracellularis using the polymerase chain reaction (PCR). In addition, fecal samples from diarrhoic foals (n = 46), dogs (n = 57), cats (n = 50), calves (n = 37), hedge hogs (n = 9), seals (n = 8) and one giraffe were also studied. DNA was extracted from feces using high concentrations of chaotropic salt and diatomaceous earth. For PCR, primers flanking a 279 bp fragment of L. intracellularis DNA were used (JONES, G. F., WARD, G. E., MURTAUGH, M. P., LINN, G. (1993), J. Clin. Microbiol. 31, 2611-2615). Amplificates were separated by agarose gel electrophoresis and visualized under UV-light. L. intracellularis was found in 26 (12.8%) samples from 21 (30.0%) of the Hessian pig herds without symptoms of diarrhoea. In feces of pigs with diarrhoea (n = 1684) the agent was present in 431 (25.6%) samples originating from 224 (34.6%) herds. Of the other animal species studied, L. intracellularis was detected in feces of 4 (7.0%) dogs, 2 (5.4%) calves, 3 (33.3%) hedge hogs and in the sample of the giraffe. The remaining species were all tested negative.

Bacterial lysates of different bacterial strains (E. coli, B. bronchiseptica, P. haemolytica) were prepared by heating, acid- and alkaline-hydrolysis. Lysates were tested for their immunostimulating effect in bacterial infection models and with chromium 51 test demonstrating spontaneous (natural) cytotoxicity. Lysate production was standardized by protein- and Lps-determination. The alkaline-hydrolysis reduced toxicity of Lps and increased the content of soluble bacterial protein. Heating and acid-hydrolysis did not alter bacterial suspensions with respect to Lps-toxicity and protein-content. Mice infected with P. aeruginosa, P. multocida, E. coli and L. monocytogenes (5-10 LD50) had a significantly longer survival time after prophylactic immunostimulation with bacterial lysates than control animals. No protection was observed in immunostimulated mice infected with Erysipelothrix rhusiopathiae. In the Pseudomonas infection model, bacterial lysates prepared by alkaline-hydrolysis had a 10 times higher immunostimulating effect than lysates prepared by acid-hydrolysis or heating. Bacterial lysates stimulated spontaneous cytotoxicity of natural mouse peritoneal killer cells after intraperitoneal application. Whole bacterial lysates had a higher NK-activity as their corresponding purified lipopolysaccharide portion.

The risk of a bacterial invasion by pseudomonas, e. coli, proteus, citrobacter and gaffkya via an open skin wound was investigated in combined injured mice (infliction of a skin wound and irradiations). Suspensions of different bacterial strains were applied on the wound and then the lethality was determined. Besides of apathogenic (citrobacter, proteus, gaffkya) and high pathogenic bacteria (e. coli and pseudomonas) an e. coli strain of the 0-Group 81 was found that seems to induce a decrease of lethality even after sublethal whole body irradiation.

Mandatory vaccination against smallpox was abolished on the account of smallpox-eradication proclaimed by the WHO and the postvaccinal complications detected after smallpox vaccination. At the same time vaccine banks with the vaccinia virus strain "Elstree" were organized. Should mass vaccinations with this vaccinia virus strain be carried out in a case of emergency, severe postvaccinal diseases and complications can arise in overaged and immunosuppressed vaccinees after primovaccination. Therefore attenuated vaccinia virus strains should be used for vaccine banks, which cannot be activated, or increase in virulence in impaired vaccinees after primovaccination. For these individuals the vaccinia virus strain "MVA", among other attenuated vaccinia strains, is recommended. The MVA virus strain can be applied parenterally without complications. From the scientific and field-relevant point of view it was tried to combine the vaccinia virus strain "MVA" with tetanus toxoid and to develop a combination vaccine "tetanus-smallpox". In immunization experiments using mice, piglets and monkeys, safety and efficacy of the vaccine were investigated. Efficacy was demonstrated by means of postvaccinal antibody determination and by the mouse protection test. Tetanus antitoxin was measured by ELISA and indirect hemagglutination test, antibody levels to vaccinia virus were investigated employing the neutralization test and hemagglutination inhibition test. No significant differences in potency could be demonstrated between the combination vaccine and the corresponding monovalent vaccines in mice, piglets and monkeys. The combination vaccine consisted of 12 Lf tetanus toxoid and 10 TCID50 vaccinia virus "MVA" preserved with gelatine and glucosamine. The double intramuscular immunization of monkeys stimulated average tetanus antitoxin titers of 1:310 and average vaccinia virus titers of 1:195 2 weeks p. revacc. Similar results were obtained in mice and piglets. Side reactions were not observed in mice and piglets. Except for occasional local reactions of short duration at the injection site of the monkeys, similarly no adverse reactions were observed after intramuscular vaccination with the combination vaccine.

The protective effect of a combined rotavirus/enterotoxigenic E. coli K99 vaccine against naturally occurring neonatal diarrhoea was evaluated in two dairy and two beef herds. Before vaccination, frequency of diarrhoea in these herds was 46% up to 60% with losses of 7% to 20%. After vaccination of pregnant cows and heifers mild diarrhoea occurred in 14% to 20% of their calves, one out of 144 calves died with enteric symptoms. In 13 out of 26 calves of nonvaccinated controls where diarrhoea was noted, one of them died. K99-positive enterotoxigenic E. coli were not detected in any of the faeces tested. Rotavirus was found in one of the beef herds.

Histopathologic and electron microscopic examination of intestines of three calves and two cats revealed attaching effacing bacteria characteristic of enteropathogenic Escherichia coli (EPEC) in ileum, cecum, and colon. The attaching effacing bacteria in one of the calves contained bacteriophages, and an E. coli isolate from that calf was shown to produce Shiga-like toxin. These findings contribute to emerging evidence that attaching effacing intestinal bacteria are globally distributed pathogens in a variety of host species and that bacteriophage-mediated production of Shiga-like toxin is related to the virulence of such bacteria.

Model experiments in mice were used for the investigation of oral immunization against tetanus. The efficacy of oral tetanus toxoid application was tested by subcutaneous challenge (10 LD50 tetanus toxin) and antitoxin determination by L+-method. Reasonable protection could only be achieved when toxoid was in intensive contact with the mucous membranes of the oral cavity, whereas immunization by way of the gastrointestinal tract failed completely. After a single oral vaccination with 200 Lf fluid tetanus toxoid onto mucous membranes of the oral cavity the efficiency index was 75. A 100% protection was obtained in challenge after 3 applications of 200 Lf toxoid. In comparison, a single subcutaneous dose of 2 Lf tetanus toxoid resulted in an efficiency index of 100 and a single intranasal immunization with 100 Lf in an efficiency index of 93. Development of immunity after oral immunization was much accelerated if compared with parenteral immunization. 20% of the orally treated animals were immune 5 days after vaccination. After the same period subcutaneously treated animals had failed to develop any protection at all. Antitoxin titers after one oral vaccination (200 Lf) were similar to the ones after one subcutaneous dose (2 Lf). A distinct increase in antitoxin titers could only be observed when revaccination was done subcutaneously. The results obtained with mice apply analogically to guinea pigs.

Model-experiments in mice were used for the investigation of oral immunization with tetanus-toxoid. The efficacy of oral toxoid application was tested by challenge and anti-toxin determination. The oral immunization with tetanus-toxoid induced only a sufficient protection, when toxoid was in intensive contact with the mucous membranes of the oral cavity. Local immunization via gastro-intestinal-tract failed. After a single vaccination with 200 Lf tetanus-toxoid onto mucous membranes of the oral cavity the efficiency index was 75. In comparison, a single subcutaneous application of 2 Lf tetanus-toxoid resulted in an efficiency index of 100 and a single intranasal immunization of 100 Lf in an efficiency index of 93. So the application of toxoid subcutaneously or onto nasal mucous membranes induced higher protection than oral application. Temporary development of immunity after immunization via the mucous membranes of the oral cavity was much better than parenteral immunization. 20 per cent of orally immunized animals were immune 5 days p. vacc. At the same time subcutaneously immunized animals failed to develop any protection. The efficiency rates obtained in challenge correlate with antitoxin content in the serum.

Under field conditions, the comparative effectiveness of oral, nasal and intramuscular vaccination of humans with tetanus toxoid was investigated. The effectiveness was tested by the determination of antitoxin titers with the L+-method. Oral booster vaccination produced only very low and irregular increases in antitoxin titer even at the highest dose of 6,000 Lf toxoid. In the most favourable case following triple oral immunization, a rise from 6.2 to 6.6 I.U. was achieved. Primary oral vaccination failed to stimulate measurable antitoxin production. After nasal booster vaccination with 1,000 Lf toxoid, a ten-fold rise in antitoxin titer was detectable. Following primary nasal vaccination, an average neutralization titer of 1 I.U. was achieved. The results of intramuscular and nasal booster vaccinations were similar although the antitoxin titers were significantly higher after primary intramuscular vaccination than after primary nasal vaccination. The average antitoxin titer amounted to 11.6 I.U. These results indicate that primary and booster vaccination against tetanus by the nasal route is a safe and effective procedure.

Using the "tetanus-mouse" model the differences in immunity development between animals exposed to irradiation (300 R and 400 R) and animals not exposed to irradiation after nasal and subcutaneous vaccination was investigated. Immunization was carried out 1, 3, 6 and 10 days before and after exposure to irradiation. Efficacy of immunization was tested by challenge with 10 X LD50 tetanus-toxin and by antitoxin determination with L+-method. The degree of the immune response was dependent on 1. the irradiation dose, 2. the interval between active immunization and the ensuing or preceding irradiation, 3. the mode of vaccination (local or parenteral) and 4. the vaccination dose.

Hitherto the term "non-parenteral" (local) vaccination meant all those modes of vaccination where the vaccine is applied on the skin or the mucous membranes. The most common types of local immunization are oral and nasal vaccination. Using model studies with the mouse immunization per wound was investigated as an alternative mode of vaccination. Immunization was done with tetanus-toxoid, efficiency was tested by challenge with 10 LD50 tetanus toxin. For immunization per wound only slightly higher antigen doses are necessary compared with subcutaneous immunization. A single subcutaneous vaccination with 2 Lf tetanus-toxoid and a single application per wound of 10 Lf resulted in the same efficiency index of 100. Immunization per wound has to be carried out immediately after injury. It seems to be optimal within the first 3 to 5 hours. At a later time the same efficiency index can only be achieved by use of a higher antigen doses. Immunization per wound is possible with all kinds of wound, even with burns. Size of wound and wound-supply do not influence the efficacy of wound-immunization. Immunization per wound does not disturb wound healing. The experiments reported here showed, for the first time, that immunization per wound is as effective as parenteral immunization. The most important advantage of wound-immunization is the speed and ease with which it can be administered, a fact which predestines it for vaccination in emergency cases. In practice it will be useful to combine wound-vaccination with wound-sprays.

The effectiveness of paramunization as a antigen nonspecific method to activate mechanisms against wound-infections due to Pseudomonas aeruginosa was studied using a model of direct infection of mice with a "mice-pathogenic" Ps. aeruginosa strain on artificially set wounds. Active paramunization by means of a biological inducer "PIND-AVI" (M-HP 438) significantly reduced the mortality rate between treated and placebo animals. The best results were obtained by parenteral prophylactic application. A four times repeated injection of PIND-AVI before the wound-infection reduced the mortality rate from 80% (placebo animals) to 26.6%. Almost equally good results were obtained by clinically useful therapeutic application of the preparation. A four times repeated treatment of the mice after wound infection lead to a decrease of mortality rates from 86.6% to 36.6%. The paramunization inducer PIND-AVI caused no side effect in any of the experiments. The mode of inducer action in Pseudomonas aeruginosa wound infections appears to be complex. Increased phagocytosis by nonspecific opsonisation, increased macrophage activity and concurrent stimulation of the lymphopoetic system could possible occur. On the other hand the nonspecific action of mediators could also play a role due to the inducer stimulated T-cells and cellular antigens of Pseudomonas aeruginosa. However both mechanisms in cooperation with specific and nonspecific humoral factors are probably interacting together. To what extent a simultaneous synthesis resp. release of endogeneous interferon plays a role is not known.