Distribution ofMycobacterium aviumsubsp.paratuberculosisin a Subclinical Naturally Infected German Fleckvieh Bull

Abstract

Although it has been known for years that Mycobacterium avium subsp. paratuberculosis (MAP) is detectable in the reproductive organs and semen of infected bulls, only few studies have been conducted on this topic worldwide. This study surveyed the MAP status of a bull, naturally infected due to close contact with its subclinically infected parents over a period of 4 years. From the age of 7 weeks to necropsy, faecal, blood and, after sexual maturity, semen samples were drawn repeatedly. Already at the first sampling day, MAP-DNA was detected in faeces by semi-nested PCR. True infection was confirmed by the detection of MAP-DNA in blood at the age of 40 weeks. In total, MAP-DNA was present in 25% faecal (34/139), 16% blood (23/140) and 5% semen (4/89) samples, including MAP-free intervals of up to 9 weeks. MAP genome equivalents (MAP-GE) of up to 6.3 × 10 /g faeces and 1.8 × 10 /ml blood were determined. Cultivation of MAP occurred only in three of 137 faecal and two of 109 blood, but never in semen samples. Over the whole period, the bull was a serological negative MAP shedder. During necropsy, 42 tissue samples were collected. Neither macroscopic nor histological lesions characteristic of a MAP infection were observed. Cultivation of MAP in tissue sections failed. However, MAP-DNA was spread widely in the host, including in tissues of the lymphatic system (7/15), digestive tract (5/14) and the urogenital tract (5/9) with concentrations of up to 3.9 × 10 MAP-GE/g tissue. The study highlighted the detection of MAP in male reproductive organs and semen. It supports the hypothesis that bulls may probably transmit MAP, at least under natural mating conditions. In artificial insemination, this might not be relevant, due to antibiotics included currently in semen extenders. However, the survivability of MAP in this microenvironment should be investigated in detail.

Development of a Recombinase Polymerase Amplification Assay for Rapid Detection of the Mycobacterium avium subsp. paratuberculosis

“…The limit of detection using a molecular standard was 16 DNA molecules and 500 fg by employing the genomic DNA. The latter corresponds to approximately 95 MAP genomes as one MAP genome has a weight of 5.29 fg [16]. …”

“…Both the molecular plasmid and genomic DNA standard were tested using a published quantitative real-time PCR protocol ([16], patent number: EP2841596A1) as described below.…”

“…DNA extraction was performed by QIAamp DNA Blood Mini Kit (QIAgen GmbH, Hilden, Germany) using the recommended modifications as described previously [16]. …”

“…The clinical performance of the MAP RPA assay was evaluated by 48 MAP-positive and 20 MAP-negative blood, sperm, faecal and tissue samples. All results were compared by a well-established real-time PCR [16]. …”

Detection of Mycobacterium avium subsp. paratuberculosis in non‐human primates

“…Homogenized samples were incubated in a thermomixer at 37°C and at 900 rpm for 30 minutes. Further purification of the tissue samples was performed, as described elsewhere . To the homogenized fecal samples, 400 μL lysis buffer (AL buffer) and 40 μL ready‐to‐use proteinase K solution (20 mg/mL) were added.…”