Leptospira Licerasiae a Possible Contaminant of Cell Culture Processes

Leptospira licerasiae

A possible contaminant of cell culture processes

Leptospirosis is a disease that can cross between species e.g. animal to human. The strain Leptospira Licerasiae is associated with animal urine contamination and mainly effects town and rural populations in tropical regions. When contracted it is known to cause Weils disease, severe pulmonary haemorrhage syndrome, and renal problems.
      The bacteria’s morphology is spiral in shape (as in Fig.1) and only 0.1um in width making them hard to detect.
Fig.1 Leptospira bacteria image using SEM
They are undetectable using normal gram stain and endotoxin tests (Ahmed et al, 2009). Conventional filtration of 0.1 is not sufficient and the bacteria can pass through. This is especially challenging for the Bio-medical industry as it is very difficult to identify contaminated cultures, products, facilities etc. The live bacteria can be observed using darkfield microscopy.
Leptospires are obligate aerobes and have an optimum growth temperature of 29°C. This is similar to most culture conditions enabling the bacteria to form biofilms and toxins that can kill other cells in a culture.
The most common method used to detect Leptospira licerasiae is real time PCR technique. DNA is first ‘extracted, purified and eluted in 0.1xTE buffer pH 8.0 by using the QIAamp DNA extraction kit (Qiagen, GmbH, D-40724 Hilden, Germany)’ (Ahmed et al 2009)
This is a process of SDS cell wall degradation and several centrifuge steps. The resulting pellet of DNA is added to a PCR solution.
The PCR solution has the Primers needed (also includes nucleotides, buffer, taq polymerase) to mark the sequence to be amplified. In this case it is ‘SecYIVF (5′-GCGATTCAGTTTAATCCTGC-3′) and SecYIV (5′-GAGTTAGAGCTCAAATCTA- AG-3′)’

The PCR process is followed ; denaturation for 30 seconds at 95 oC, annealing for 30 seconds at 50°C, elongation for 30 seconds at 72°C and Extension step for 7 minutes at 72°C.

A DNA-binding...