A Longitudinal Study to Characterize the Distribution Patterns ofMycobacterium aviumssp.paratuberculosisin Semen, Blood and Faeces of a Naturally Infected Bull by IS 900 Semi-Nested and Quantitative Real-Time PCR

Abstract

Johne's disease is caused by Mycobacterium avium ssp. paratuberculosis (MAP) and has been recognized as an important bacterial infection in ruminants. Although MAP has been detected in semen and within the reproductive organs of bulls, the bacterial distribution and shedding patterns are currently not well characterized. Our investigation was performed to detect and quantify MAP in faeces, semen and blood samples repeatedly drawn from a naturally infected but asymptomatic 18-month-old German Simmental breeding bull candidate over a period of 3 years (June 2007-November 2010). Qualitative and quantitative polymerase chain reaction (PCR) techniques were used to correlate the presence and matrix-specific amounts of MAP. In total, 65 sampling dates were selected. Mycobacterium avium ssp. paratuberculosis was detected intermittently in all matrices with MAP-free intervals of up to 18 weeks by an IS900 semi-nested PCR. The number of MAP-positive results from semen and blood samples was higher than from faecal samples. A quantitative polymerase chain reaction detected the highest MAP contents in faeces (10(3) -10(6) MAP/g), while lower amounts were found in semen and blood samples (10(2) -10(5) MAP/ml). Although no significant agreement was calculated between the presence of MAP in faeces and blood, a statistically significant positive correlation between its occurrence in semen and blood was determined (r = 0.38, P < 0.05, n = 29). The present study contributes to a more detailed understanding of MAP distribution patterns in faeces, semen and blood of a subclinically infected breeding bull candidate. It highlights the possible role of breeding bulls as a source of MAP transmission and indicates the need for further monitoring and hygienic measures to prevent the spread of the infection via semen.

Fecal shedding and tissue infections demonstrate transmission of Mycobacterium avium subsp. paratuberculosis in group-housed dairy calves

“…Others have reported intermittent shedding; however, the interval between positive samples largely depended on the interval between samplings [15, 43]. In the current study, positive fecal samples were followed by negative fecal samples for anywhere from 2 days to 5 weeks before another positive sample was detected.…”

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

“…Nevertheless, the possible distribution of MAP via feces of non‐human primates was not measurable as part of this study, even though it is known from the literature that MAP can be excreted in concentrations of 10 6 MAP genome equivalent/g feces from asymptomatic non‐human primates . Similar MAP concentrations were also measurable in feces of other animals asymptomatically infected, but it is known that the excretion of MAP appears usually intermittently . Hence, it is admitted that diagnostic tests may lead temporarily to negative results during shedding‐free intervals of the animals.…”

“…The sensitivity of MAP cultivation from the feces of subclinically infected animals is considered to be 23%‐49%, whereas cultivation of MAP from animals with clinical signs of paratuberculosis is thought to reach a sensitivity of 98% . Also in other studies, a discrepancy between the results of PCR and cultivation was observed . In one of these previous studies, 875 of 1906 (45.91%) investigated fecal samples from dairy cattle were tested positively for the presences of MAP DNA by an IS900‐based real‐time PCR.…”

“…The number of MAP‐GE in fecal or tissue sample per g was calculated by the following formula: Concentration of DNA (fg/μL) × volume of eluted DNA (50 μL)/ 5.29 fg × 20 (50 mg fecal or tissue sample)=MAP‐GE per extracted sample aliquot, per mL or g sample, respectively. The detection limit of the snPCR was 2.5 fg MAP DNA/reaction (approximately 1 genome equivalents/reaction), whereas the qPCR had a detection limit of 50 fg/reaction (approximately 10 MAP genome equivalents/reaction) and a quantification limit of 5 pg MAP DNA/reaction (approximately 950 MAP genome equivalents/reaction) . Due to the lower sensitivity of the qPCR, only aliquots that tested positive for MAP by snPCR were further analyzed by qPCR in three independent qPCR runs.…”

A Study on Prevalence of Bovine Paratuberculosis in Semen Station in India

“…The common source of infection may be faeco-oral route, contaminated feed, water, milk or colostrums for the calves (Sweeney, 1996). Spreading of the organism through vertical infection to the fetus (Larson and Kopecky, 1970) and infection in semen (Munster et al, 2012) has also been reported. Trangadia et al (2012) found apparent prevalence of JD in Gujarat and Andhra Pradesh 13.39% (true prevalence, 15.68%) and 16.26% (true prevalence, 19.31%) respectively in serological diagnosis.…”