Distribution of Mycobacterium avium subspecies paratuberculosis in clinically asymptomatic bulls and different non-ruminant species

Paratuberculosis is a disease of global distribution, which mainly occurs in ruminants. The disease is triggered by Mycobacterium avium subsp. paratuberculosis (MAP), which causes a chronic, degenerative granulomatous inflammation of the intestinal tract, in turn leading to diarrhoea, weight loss, reduced reproductive performance, and finally death. The economic losses sustained to the agricultural sector, as well as the potential aetiological link between MAP and Crohn's disease in humans, are the main reasons for the indispensability of an efficient control of paratuberculosis. The main challenge in ensuring efficient disease control is that infected animals do not necessarily show clinical symptoms and diagnostic methods are not reliable at this stage of the disease. To examine the unrecognized spread of asymptomatically infected hosts as well as potential transmission pathways, three studies were conducted in this thesis. In an additional study, a recombinase polymerase amplification (RPA) assay was developed to enable the detection of MAP in low-resource settings or directly in the stable. The occurrence of MAP in the faeces, blood, and semen of a naturally infected, but clinically asymptomatic German Fleckvieh bull (related to Simmental: Bos primigenius taurus) was analysed over a period of four years, from the age of seven weeks until necropsy. MAP was intermittently detected by IS-900 based semi-nested PCR, real-time PCR, and bacteriological cultivation, with occurrence of MAP-free intervals of up to nine weeks. A recurring detection pattern was not found. Besides antigen detection, most of the implemented control programmes are based on the detection of an antibody response by ELISA. However, the examined bull was a serologically negative shedder over the entire investigation period. Such animals are one of the main reasons for the reduced success of paratuberculosis control programmes. As well as ruminants, non-ruminants can be infected with MAP, but currently the knowledge concerning potential hosts is incomplete. It has been known for many years that the faeces of infected non-ruminants contains concentrations of up to 106 MAP cells/g and might therefore play a role in the transmission of MAP to ruminants, through shared habitats. In this thesis, the presence of MAP was confirmed in rock hyraxes (Procavia capensis); a common marmoset (Callithrix jacchus); and a cottontop tamarin (Saguinus oedipus). Three of the investigated rock hyraxes were captured in South Africa and their four descendants were subsequently born in a quarantine stable at a German zoological garden. PCR-analysis of a pooled faecal sample of all animals, and subsequent examination of individual faeces, confirmed the excretion of MAP by two rock hyraxes. In contrast, MAP was not detected in the faeces of 20 non-human primates (NHP), belonging to seven different species, although the presence of MAP was confirmed in the ileum of one cottontop tamarin (Saguinus oedipus), and the bone marrow of a common marmoset (Callithrix jacchus). Analogous to the examination of the NHP, the distribution of MAP within the body of the host was also investigated in the bull and the rock hyraxes. MAP manifestation within most body regions of clinically asymptomatic ruminants as well as non-ruminants was observed, including in the tissues of the gastrointestinal tract, urogenital tract, cardiovascular system, and/or respiratory system. Since none of the investigated animals (the bull; the group of rock hyraxes; or either of the non-human primate species) showed typical paratuberculosis clinical symptoms or lesions after necropsy, an asymptomatic infection with MAP was assumed. Therefore, it can be expected that MAP replicates within the asymptomatically infected host without triggering typical pathohistological changes or clinical signs of paratuberculosis. Furthermore, it is reasonable to suspect that non-ruminants excrete the pathogen intermittently and in different concentrations, as clinically asymptomatic ruminants do. In order to control a disease, it is necessary to discover all routes of transmission. Although since 1970 it has been known that MAP is present in the semen and the reproductive organs of bulls with clinical symptoms and/or paratuberculosis-typical pathological changes, only a small number of studies have scrutinised the role of bulls in the transmission of MAP. In this thesis, the presence of MAP was confirmed in the semen and reproductive organs of an asymptomatic infected bull. These results support the hypotheses that MAP might be transmitted to cows via semen under natural mating conditions, or, likely, by artificial insemination; however, the influence of the semen extender on the viability of MAP is unknown. In addition to the investigated bull, MAP was also observed in the reproductive organs of one female and one male rock hyrax. Interestingly, female animals of this species have a haemoendothelial placenta, meaning the foetuses have direct contact with the maternal blood, and that vertical transmission of MAP might also, therefore, be a possibility in non-ruminants. Another interesting finding of this thesis, was the detection of MAP in the bone marrow of the investigated bull and the common marmoset. As it has been demonstrated that a MAP-related pathogen, Mycobacterium tuberculosis, can persist within the mesenchyme cells of the bone marrow in a viable, but not culturable, stage, it was concluded that the bone marrow might be a niche for the establishment of latent tuberculosis infection. Therefore, it might also be assumed that bone marrow plays a key role in the establishment of latent paratuberculosis infection. Currently, available detection methods (PCR, cultivation, ELISA) require sophisticated laboratory equipment; to allow the identification of MAP-infected animals in low-resource settings or directly in the stable, a simple molecular diagnostic tool is needed. In order to fill this gap, an RPA assay targeting the MAP IS900 gene was developed. In contrast to commonly used diagnostic methods (culture and real-time PCR), RPA assays are extremely fast (3-15 minutes), easy to perform, run at a constant temperature of 42°C, and all reagents are cold-chain independent. The developed MAP-RPA assay has a limit of detection of 16 DNA molecules, and does not show any cross-reactivity to other bacterial strains of clinical importance. The clinical specificity and sensitivity is calculated at 100% (n=20) and 90% (n=48), respectively, based on comparison with a well-established real-time PCR. In conclusion, the studies performed for this thesis highlight the potential risk of MAP transmission from asymptomatic animals. Ruminants as well as non-ruminants play an important role in the silent spread of MAP within an animal stock. The obtained results confirm the high occurrence rate of MAP in semen and the male reproductive organs. Thus, bulls might be a part of the transmission cycle of MAP to cows and their descendants or foetuses. Furthermore, non-ruminants seem to be more susceptible to MAP than commonly believed. For the first time, the presence of MAP in rock hyraxes and the common marmoset was confirmed. Further studies should address the pathogenesis of clinically asymptomatic infected ruminants and non-ruminants, and routes of transmission via semen as well as between different species