8. Examine a small aliquot of the contents from each DMC for
the presence of leptospires under dark-field microscopy.
9. Proceed to downstream applications (e.g., RNA and/or DNA
isolation, analysis by one- and two-dimensional SDS-PAGE).
4 Notes
1. The quality of the BSA used for the cultivation of leptospires is
critical. Please verify that the albumin used for DMC implantation supports the growth of virulent leptospires under standard
in vitro growth conditions (e.g., when used as a component of
EMJH medium).
2. This procedure is performed most often with Sprague-Dawley
rats, but Wistar and Fisher rats also have been used successfully.
While sex and age of rats do not appear to affect the experimental outcome (i.e., host adaptation), a minimal size/weight
of 175–200 g is recommended. Smaller rats require a reduction
in DMC volume. Larger DMCs are not recommended due to
difficulty with handling when inserting into the peritoneal
cavity.
3. Buprenorphine (0.05–0.1 mg per kg), administered subcutaneously every 6–12 h, also may be used for postoperative pain
relief. However, animals may develop pica after receiving this
drug. For this reason, carprofen is preferred.
4. Two percent chlorhexidine (w/v) in 70% isopropyl alcohol is
preferred for preparation of the surgical site. Iodophor (e.g.,
Betadine) activity may be reduced in the presence of organic
matter.
Fig. 4 Recovery of host-adapted leptospires. After 7–10 days postimplantation, the animal is euthanized and
placed in supine position on a sterile workspace inside a biosafety cabinet (BSL-2). By this point, the incision
site should be completely healed (a). The skin and abdominal wall are opened using scalpel blade and/or
scissors, and the DMC is located. Using forceps to hold up the DMC, collect contents using an 18 G needle
(1.5 in.) attached to 10 ml syringe (b) and transfer to a sterile tube for processing
Cultivation of Leptospira interrogans within DMCs
239
the presence of leptospires under dark-field microscopy.
9. Proceed to downstream applications (e.g., RNA and/or DNA
isolation, analysis by one- and two-dimensional SDS-PAGE).
4 Notes
1. The quality of the BSA used for the cultivation of leptospires is
critical. Please verify that the albumin used for DMC implantation supports the growth of virulent leptospires under standard
in vitro growth conditions (e.g., when used as a component of
EMJH medium).
2. This procedure is performed most often with Sprague-Dawley
rats, but Wistar and Fisher rats also have been used successfully.
While sex and age of rats do not appear to affect the experimental outcome (i.e., host adaptation), a minimal size/weight
of 175–200 g is recommended. Smaller rats require a reduction
in DMC volume. Larger DMCs are not recommended due to
difficulty with handling when inserting into the peritoneal
cavity.
3. Buprenorphine (0.05–0.1 mg per kg), administered subcutaneously every 6–12 h, also may be used for postoperative pain
relief. However, animals may develop pica after receiving this
drug. For this reason, carprofen is preferred.
4. Two percent chlorhexidine (w/v) in 70% isopropyl alcohol is
preferred for preparation of the surgical site. Iodophor (e.g.,
Betadine) activity may be reduced in the presence of organic
matter.
Fig. 4 Recovery of host-adapted leptospires. After 7–10 days postimplantation, the animal is euthanized and
placed in supine position on a sterile workspace inside a biosafety cabinet (BSL-2). By this point, the incision
site should be completely healed (a). The skin and abdominal wall are opened using scalpel blade and/or
scissors, and the DMC is located. Using forceps to hold up the DMC, collect contents using an 18 G needle
(1.5 in.) attached to 10 ml syringe (b) and transfer to a sterile tube for processing
Cultivation of Leptospira interrogans within DMCs
239