[The importance of allergic skin test with Johnin, antibody ELISA, cultural fecal test as well as vaccination for the sanitation of three chronically paratuberculosis-infected dairy herds in Rhineland-Palatinate]. / Zur Bedeutung von allergischem Hauttest mit Johnin, Antikörper-ELISA, kultureller Kotuntersuchung sowie der Impfung für die Sanierung dreier chronisch Paratuberkulose-infizierter Milchviehherden in Rheinland-Pfalz.

Three chronically paratuberculosis infected herds were tested for six years twice a year (intradermal Johnin test, antibody ELISA (IDEXX Corp.), microbial culture) according to a sanitary program. Culling of shedding animals and vaccination of calves with NEOPARASEC (Merial Corp.) were part of the program. In course of experiment, 1015 samples of 228 non vaccinated cows and 1502 samples of 293 vaccinated cattle have been tested. 3.8% of the vaccinated animals proved positive in microbial culture. Nearly all vaccinated calves developed granulomas sized from hazelnut to loaf at the injection site. Positive reactions in intradermal test as well as in antibody ELISA were found in very young calves. 24.3%, 33.7%, 25.9%, respectively of the non vaccinated animals were identified as shedders of M. avium subsp. paratuberculosis (MAP) by microbial culture. In the first and in the second herd most shedders of MAP were found in the first herd examination (66.7%, 42.9%, respectively), whereas in the third herd they were detected in the fifth examination (31.0%). At the beginning, 17.9% of non vaccinated animals proved positive in intradermal test, 14.4% in antibody ELISA. Afterwards, the number of positive test results decreased but increased again towards the end of the experiment. 48.5% of the 66 shedders showed positive reactions in intradermal test, 57.6% in antibody ELISA, 77.3% in at least one of these both tests. Antibodies in ELISA were found in rising frequency from two years before the time of shedding. 50.0% of the shedders reacted positive in ELISA at the time of shedding. In selected shedders first positive results were found at the age of about two years. Unfortunately, only incomplete hygienic measures were realized by the farmers. Under field conditions the realisation of attending sanitary programs is difficult. MAP is spread mainly by buying of animals, therefore a certification program for paratuberculosis free herds is urgently necessary as well as an improvement of diagnostic methods.

Batch potency testing of inactivated erysipelas vaccines by ELISA--development, validation and implementation.

Inactivated erysipelas vaccines are widely used to protect pigs against erysipelas disease caused by the bacterium Erysipelothrix (E.) rhusiopathiae. Quality control tests for this vaccine are laid down in the European Pharmacopoeia (Ph.Eur.) Monograph No. 64. A laboratory animal model using a vaccination-challenge procedure is currently required as batch potency test. More than 10 years ago we initiated the first studies to develop an alternative ELISA potency model to replace this regulatory challenge test in mice. A short retrospective outline of the various steps from the development of the method until implementation into the regulatory requirements is described.

Immune reactions in cattle after immunization with a Mycobacterium paratuberculosis vaccine and implications for the diagnosis of M. paratuberculosis and M. bovis infections.

After immunization of four calves with a live modified Mycobacterium paratuberculosis vaccine the course of the humoral and cell mediated immune reactions was studied during a 2-year clinical investigation. Furthermore, the possibility of shedding of the vaccine strain and the influence of the vaccination on the tuberculin skin test was determined. In addition to standard procedures recently developed diagnostic methods (antibody enzyme-linked immunosorbent assay, interferon-gamma test, polymerase chain reaction) were used. A cell-mediated immune reaction, reflected in an increased, specifically induced, interferon-gamma production developed much earlier (1-2 weeks post-immunization) than humoral immunity (8-16 weeks post-gamma immunization). While the increase in antibody titres was transient, declining to extremely low levels 48-60 weeks post-immunization, cell-mediated immunity remained detectable until the end of the investigation. Spread of the vaccine strain into the body and shedding were never detected during the whole course of the study except for one colon site in one calf. As late as 2 years after vaccine application positive or doubtful skin reactions against M. bovis purified protein derivative were measured, reflecting possible interference of the immunization with the diagnosis of bovine tuberculosis. At the end of the investigation, a positive cell-mediated immune reaction was detected the control animal although clinical, pathological and bacteriological examinations gave no indication for a mycobacterial infection.

[Comparative investigations of a combined vaccine against parvovirus and erysipelas and corresponding monovaccines in different vaccination schedules. 1: Field trial]. / Vergleichende Untersuchungen über den Einsatz eines Parvovirose-Rotlauf-Kombinationsimpfstoffes sowie entsprechender Monovakzinen bei unterschiedlichen Revakzinationszeitpunkten. Teil 1: Feldversuch

In a field trial, the development of antibodies of a combined vaccine against the porcine parvovirus (PPV) as well as against swine erysipelas was compared with corresponding mono vaccines. Furthermore, these vaccines were used in different vaccination schedules. The tests were carried out on 109 gilts in three closed farms. In all gilts, a basic immunization repeated twice was carried out at the age of six months and at intervals of three weeks. The revaccination was carried out four months after the basic immunization with half of the animals, and six months after the basic immunization with the remaining gilts. Between the combined vaccine and the mono vaccine no significant differences in the development of antibodies against PPV could be found according to different vaccination schedules. The gilts having been vaccinated with the mono vaccine and boostered six months later showed significantly higher antibody titers against Erysipelothrix rhusiopathiae. Between the remaining vaccination groups no significant difference in the development of the antibodies against swine erysipelas could be found. On only one farm, a continuous decrease of antibody titers against PPV in case of altogether 238 non-vaccinated piglets until the sixth month of life could be observed. On the two other farms, an increase of antibody titers against PPV could be found at different points of time, which indicates an infection of the piglets. Between the individual vaccination groups no significant antibody titers against PPV could be measured in milk tests. With regard to the number of piglets born alive per litter, the number of piglets born dead per litter and the number of mummies, a significant difference could neither be found between the vaccination groups 1-4.

Potency Testing of Swine Erysipelas Vaccines by Serology Results of a Pre-validation Study.

The European Pharmacopoeia (Ph. Eur.) monograph on Swine Erysipelas Vaccine (inactivated) (Ph. Eur., 1997) requires the potency of each batch to be demonstrated in a mouse protection test. In this parallel-line bioassay, mice are challenged with a virulent strain of Erysipelothrix (E.) rhusiopathiae after immunisation with different doses of either the standard preparation or of the test vaccine. More than one hundred animals are necessary for the routine testing of a single batch. In previous studies (Beckmann und Cubetaler, 1994; Rosskopf-Streicher et al., 1998), we have shown that an indirect enzyme-linked immunosorbent assay (ELISA) may be used to quantify the humoral response of mice. This method can replace the challenge model for the purposes of potency testing in the following manner: Ten mice are immunised subcutaneously with 1/10 of the vaccine dose required for pig vaccination. After three weeks, the mice are bled under anaesthesia. Serum samples are pooled and the antibody content is compared to that of a reference serum. In view of animal welfare the advantages of the alternative model are obvious: A highly reduced number of animals and the replacement of challenge exposure. This includes the discontinuation of the control group with a mortality rate of 100%. A pre-validation study was initiated to evaluate the performance of the serological method. Eight laboratories used the test kits to evaluate pooled serum samples from vaccinated mice. Both the reagents and the test protocol were shown to be satisfactory. The intra- and inter-laboratory reproducibility that was achieved indicates that the method is a strong candidate for validation.