Avian influenza H5N1 in viverrids: implications for wildlife health and conservation.

The Asian countries chronically infected with avian influenza A H5N1 are 'global hotspots' for biodiversity conservation in terms of species diversity, endemism and levels of threat. Since 2003, avian influenza A H5N1 viruses have naturally infected and killed a range of wild bird species, four felid species and a mustelid. Here, we report fatal disseminated H5N1 infection in a globally threatened viverrid, the Owston's civet, in Vietnam, highlighting the risk that avian influenza H5N1 poses to mammalian and avian biodiversity across its expanding geographic range.

Potency testing of inactivated erysipelas vaccines by ELISA--influence of the adjuvant on antibody development.

The humoral immune response after vaccination may be greatly influenced by the type of adjuvant used. In this study we investigated the influence of three different adjuvants on the serological response in laboratory mice after immunisation with commercial erysipelas vaccines.

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.

Control of Clostridium perfringens vaccines using an indirect competitive ELISA for the epsilon toxin component - examination of the assay by a collaborative study.

Investigations on the replacement of the mouse neutralisation test for proving vaccine batches of Clostridium (C.) perfringens toxoid vaccines were performed since several years. The European Pharmacopoeia (Ph. Eur.) monograph Clostridium perfringens vaccines for veterinary use (0363) is prescribing a potency test by immunisation of rabbits and checking the induction of specific antibodies against the toxins in a mouse neutralisation test. Since the monograph was revised, immunochemical methods are favoured to detect directly specific antibodies in the rabbit sera. An indirect competitive ELISA using a monoclonal antibody was established at the Paul-Ehrlich-Institut for the detection of antibodies against the epsilon toxin component of C. perfringens. It was revised using the Clostridia rabbit antiserum Ph. Eur. Biological Reference Preparation (BRP) Batch 1 as reference serum. With a defined content of 11 International Units (IU) of C. perfringens epsilon antitoxin this reference serum enables the calculation of the potency of rabbit sera under test. For the collaborative study vaccine products of different composition licensed for the German and European markets were used. Seven international laboratories were included. Aim was to make a prediction on the transferability and precision of the test method. The results showing a satisfactory intermediate precision and transferability of the test confirmed the applicability of the ELISA method for the batch control of C. perfringens vaccines. Therefore a replacement of the mouse neutralisation test is available.

[Ultrasonography of meniscus lesions]. / Die Ultrasonographie von Meniskusläsionen.

Controversy exists about the value of ultrasonography of meniscal tears. We therefore examined 101 knee joints of 99 patients in a prospective study. Prior to the arthroscopy the menisci were scanned from an independent team by using 7.5 and 10.0 MHz ultrasound waves. 81 meniscal tears were seen at arthroscopy; 36% of these tears could not be detected with the scanner (false negative results) while 20% of intact menisci showed positive echogenic structures, which were analysed as meniscal tears. It seems that ultrasonography of the menisci is still of experimental use without any clinical importance.

Effect of three different dihydroxy bile acids on intestinal cholesterol absorption in normal volunteers.

Measurements of cholesterol absorption were performed by an intestinal perfusion technique before and after 4 wk of administration of three different dihydroxy bile acids [ursodeoxycholic acid (1 g/day), chenodeoxycholic acid (1 g/day), and deoxycholic acid (0.4 g/day)] in 6 normal volunteers. Mean hourly absorption of cholesterol declined from 41 mumol/h in the control period to 31 mumol/h (-22%) during feeding of chenodeoxycholic acid (NS). During deoxycholic acid administration, mean cholesterol absorption decreased from 46 to 28 mumol/h (-28%; p less than 0.05). Ursodeoxycholic acid feeding was associated with a reduction of cholesterol absorption from 38 to 13 mumol/h (-70%; p less than 0.05), even though the input of cholesterol from bile was reduced significantly (from 123 to 86 mumol/h). The results suggest that the hydroxy groups at different positions on the bile acid nucleus of these three dihydroxy bile acids differ in their potential for cholesterol absorption.

[Training and conditioning in atopic patients following high altitude climate therapy]. / Training und Abhärtung bei Atopikern nach Hochgebirgsklimatherapie.

The subjective and objective values have changed after a four weeks treatment in high mountain areas under cool climatic conditions. "Terrainkuren" (mountain walking under guidance of a therapist) under cool climatic- and body temperature conditions show besides training results (in sport medicine) also favourable thermoregulatory adaptations. Improvements of the endurance training can be objectivated by means of the reduced heart frequency and the decrease in lactit acid under standardized physical exercises. The simultaneous changes of thermoregulation can be looked upon as part of the reaction of the whole body (also called inurement). Both, the improvement of the training condition and the thermoregulatory change seem to support each other. Thus patients can train more effectively under the same standardized physical training, but cooler conditions. The decrease of comfort temperature of patients is due to the thermoregulatory adaptation. There is a considerable decrease in the sensibility to cold. The training in high mountain areas causes an improvement in the well being and behaviour of the atopic patients which can still be manifested three months after the therapy.

[Ultrasonic diagnosis of meniscus lesions]. / Die Ultrasonographie von Meniskusläsionen.

Controversy exists about the value of ultrasonography of meniscal tears. We therefore examined 101 knee joints of 99 patients in a prospective study. Prior to the arthroscopy the menisci were scanned from an independent team by using 7.5 and 10.0 MHz ultrasound waves. 81 meniscal tears were seen at arthroscopy; 36% of these tears could not be detected with the scanner (? false negative results) while 20% of intact menisci showed positive echogenic structures, which were analysed as meniscal tears. It seems that ultrasonography of the menisci is still of experimental use without any clinical importance.

Serum-cholesterol-lowering effect of metronidazole and possible mechanisms of action.

In five patients with Crohn's disease long-term therapy with metronidazole (400 mg b.i.d.) was followed by a significant reduction of total serum cholesterol from 179 mg/dl to 156 mg/dl, 134 mg/dl, and 143 mg/dl, after 2-4 months, 6 months, and 9-12 months, respectively. Lipoprotein analysis before and after 3 weeks of administration of metronidazole (400 mg/day) to five normolipemic volunteers revealed that LDL-cholesterol was reduced by 21% (P less than 0.05), whereas HDL-cholesterol remained unchanged. Biliary secretion of cholesterol and bile acids were reduced by 13% and 20% (P less than 0.05), respectively, which might suggest a decreased sterol synthesis. The amount and percentage of intestinal cholesterol absorption were decreased by 33% and 22% (P less than 0.05). Thus, a possible decrease in sterol synthesis and a reduction of cholesterol absorption might be responsible for the serum-cholesterol-lowering effect of metronidazole. However, caution should be taken when considering metronidazole for long-term treatment of patients with hypercholesterolemia due to possible side effects.

Quality control of inactivated erysipelas vaccines: results of an international collaborative study to establish a new regulatory test.

According to the specifications of the European Pharmacopoeia (Ph. Eur.) monograph (Swine Erysipelas Vaccine (Inactivated), Monograph no. 64, European Pharmacopoeia, 3rd edn., 1997) on erysipelas vaccines for veterinary use, batch potency is estimated in a multi-dilution assay after immunisation and infection of mice. Recently, we described a serological assay system (ELISA) which has the potential to replace this challenge-based model (Beckmann R, Cussler K. Wirksamkeitsprüfung von Rotlaufimpfstoffen an der Labormaus. ELISA kontra Infektionsversuch. ALTEX 1994;Suppl. 1:39-45; Rosskopf-Streicher U, Johannes S, Hausleithner D, Gyra H, Cussler K. Suitability of an ELISA for the batch potency test in laboratory mice. Pharmeuropa BIO 1998;1:65-70). The humoral immune response is quantified in pooled sera of ten mice three weeks after immunisation. The results are expressed as relative potency (RP) in comparison to a reference serum. After a pre-validation study had been performed with success (Rosskopf-Streicher U, Johannes S, Wilhelm M, Gyra H, Cussler K. Potency testing of swine erysipelas vaccines by serology - results of a pre-validation study. ALTEX 1999;16:123-8), we initiated an international collaborative study with five European manufacturers and seven regulatory authorities to validate the assay and model. All participants were provided with blind-coded erysipelas vaccines of different potencies, the ELISA kit and test instructions. The participants had to immunise mice, to prepare serum samples and to perform the ELISA. Inter-laboratory reproducibility was reported by the pass/fail criteria of the vaccines under test. Intra-laboratory precision was assessed by comparing repeated measurements on three consecutive days. Day-to-day variation within the laboratories was statistically analysed by comparing pairs of RPs using Lin's concordance correlation coefficient. The results show that the ELISA is indeed a suitable alternative to replace the vaccination-challenge test. Furthermore, this new model reduces the number of animals required for the potency test by approximately 80%.

Collaborative study for the establishment of erysipelas ELISA coating antigen. European Biological Reference Preparation batch no. 1.

The development and validation of suitable alternatives for the replacement of in vivo challenge testing in the evaluation of vaccines is an important goal for national authorities and manufacturers involved in the assessment of quality, safety and efficacy of such products. To that end, 13 laboratories from 9 European countries, including 5 manufacturers, 7 authorities and EDQM, have taken part in a collaborative study to evaluate the suitability of a candidate reference preparation of erysipelas coating antigen for ELISA as a European Pharmacopoeia Biological Reference Preparation (Ph. Eur. BRP No. 1). The new Ph. Eur. BRP is intended for use in a serological assay, which would significantly reduce the suffering of animals in the potency assays of inactivated erysipelas vaccines. Participants were provided with sufficient study material, including the candidate coating antigen, and a panel of test sera from mice which had been immunised with vaccines representative of products on the European market, in order to evaluate the performance of the coating antigen in an enzyme-linked immunosorbent assay (ELISA) which had previously performed successfully in a prevalidation study [1] and in an international validation study [2]. Results of the collaborative study indicate that the candidate batch of erysipelas ELISA coating antigen is suitable to act as a Ph. Eur. biological reference preparation. The final study report was presented at the 110th session of the Ph. Eur. Commission (June 19-21, 2001) and the material was duly adopted as Erysipelas ELISA Coating Antigen Ph. Eur. BRP No. 1 for use in the enzyme-linked immunosorbent assay in the context of the serological potency assay for inactivated erysipelas vaccines.

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.