Johne's disease in zoo animals : development of molecular tools for the detection and characterisation of Mycobacterium avium subspecies paratuberculosis /

Johne's disease or paratuberculosis is an incurable chronic enteritis mainly affecting ruminants. It is caused by Mycobacterium avium subspecies paratuberculosis (Map). Paratuberculosis occurs throughout the world and is responsible for enormous financial losses. Map has been associated with Crohn's disease in human but this link has not been proven yet. Up to one third of zoos accredited by the American Zoo and Aquarium Association have reported a minimum of one infected animal since 1995. The existing diagnostic methods are not sufficiently specific, sensitive and rapid to be used in a survey of the occurrence of Map in a zoo environment. The overall objective was the study of the occurrence of Map in zoo animals. To this end new specific and rapid tests had to be developed. Specific objectives included the design of a more specific PCR assay, the development of a faecal DNA extraction method and the development of a PCR based typing method. New specific primers for IS900 en f 57 were designed and evaluated with a panel of mycobacterial and non-mycobacterial isolates, resulting in a detection limit of 1 CFU per PCR. A new faecal DNA extraction method was designed. The best method was a combination of a homogenisation with Tween-20 and a DNA isolation based on a sequence-capture. A detection limit of 100 Map cells per g faeces was obtained. Faecal and tissue samples of zoo ruminants (Antwerp zoo, Belgium) were used in a comparative study between PCR detection and conventional and radiometric culture. For the first time, a commercial non-species dependent ELISA test for the detection of anti-Map antibodies was also used in zoo animals. No Map cells were isolated using culture. However, three tissue samples and one faecal sample were PCR positive. The ELISA results indicated the occurrence of Map in several zoo species.The thesis also describes the attempts to develop a PCR based typing method directly applicable to clinical samples were described. A modified AFLP was used to determine polymorphisms between 74 isolates. It proved difficult to convert the AFLP polymorphism in PCR detectable fragments.Proefschrift voorgelegd tot het verkrijgen van de graad van Doctor in de Farmaceutische WetenschappenJohne's disease or paratuberculosis is an incurable chronic enteritis mainly affecting ruminants. It is caused by Mycobacterium avium subspecies paratuberculosis (Map). Paratuberculosis occurs throughout the world and is responsible for enormous financial losses. Map has been associated with Crohn's disease in human but this link has not been proven yet. Up to one third of zoos accredited by the American Zoo and Aquarium Association have reported a minimum of one infected animal since 1995. The existing diagnostic methods are not sufficiently specific, sensitive and rapid to be used in a survey of the occurrence of Map in a zoo environment. The overall objective was the study of the occurrence of Map in zoo animals. To this end new specific and rapid tests had to be developed. Specific objectives included the design of a more specific PCR assay, the development of a faecal DNA extraction method and the development of a PCR based typing method. New specific primers for IS900 en f 57 were designed and evaluated with a panel of mycobacterial and non-mycobacterial isolates, resulting in a detection limit of 1 CFU per PCR. A new faecal DNA extraction method was designed. The best method was a combination of a homogenisation with Tween-20 and a DNA isolation based on a sequence-capture. A detection limit of 100 Map cells per g faeces was obtained. Faecal and tissue samples of zoo ruminants (Antwerp zoo, Belgium) were used in a comparative study between PCR detection and conventional and radiometric culture. For the first time, a commercial non-species dependent ELISA test for the detection of anti-Map antibodies was also used in zoo animals. No Map cells were isolated using culture. However, three tissue samples and one faecal sample were PCR positive. The ELISA results indicated the occurrence of Map in several zoo species.The thesis also describes the attempts to develop a PCR based typing method directly applicable to clinical samples were described. A modified AFLP was used to determine polymorphisms between 74 isolates. It proved difficult to convert the AFLP polymorphism in PCR detectable fragments