5. After the third month, start monitoring the animals: look for
anti-PGL-1 serum antibodies [41], WBC, hematocrit, and liver
enzymes (LDH, ALT, and AST). Anti-PGL-1 serum antibodies are expected after 9 months post-inoculation. Beginning
3 months postinfection, the armadillos are monitored for production of anti-PGL-1 antibodies using a sandwich ELISA
[41]. Monitoring continues every 3 months until seroconversion occurs (>0.700 OD at 490 nm), at which time they are
then monitored on a monthly basis until disease fully disseminates. Animals are sacrificed when anti-PGL-1 antibody titers
are >1.00 OD at 490 nm. Hematocrit and blood chemistry,
including liver enzymes, are also monitored every 3 months
postinfection using an HT5 veterinary hematology analyzer
and Element DC veterinary chemistry analyzer (HESKA),
respectively. At anti-PGL-1 seroconversion, hematocrit and
blood chemistry monitoring continues on a monthly basis.
Normal hematocrit values are >30, whereas at full dissemination, the hematocrit decreases to <20 due to hemolysis of
RBC. Regarding liver enzymes, LDH values >1500 indicate
RBC lysis. These, along with low hematocrit, are additional
indicators of fully disseminated disease.
6. After 18–24 months, it is expected to observe generalized
dissemination, and the recovery is expected to be for up to
10
12 bacilli from a single armadillo.
3.13 Harvesting Liver
and Spleen
of M. leprae-Infected
Armadillos
Once dissemination is attained, the armadillo is humanely sacrificed
and liver and spleen are harvested aseptically.
1. At least 4 h prior to sacrifice, the armadillo is given Gentamicin
(1 mL) and Penicillin (1 mL) intramuscularly (IM) in order to
avoid contamination of internal organs.
2. The armadillo is anesthetized using a combination of Ketamine
(0.6 mL) and Dexdomitor (0.4 mL) given IM, and the skin of
the abdomen is shaved and thoroughly cleaned with iodine,
70% ethanol, and water.
3. The armadillo is then euthanized by exsanguination and placed
in a BSC where the tissues are aseptically removed and placed
into sterile jars on ice.
4. To check for contaminants, 1 g piece from each organ is
removed and processed for acid-fast staining, microscopic
counting, and sterility testing. Mycobacterial contamination is
ascertained by inoculating two of each 7H11 and L-J slants;
one of each is incubated at 33
C and one of each is incubated
at 37
C.
5. Slants are examined weekly for 8 weeks.
42
Elizabeth Wallace et al.
anti-PGL-1 serum antibodies [41], WBC, hematocrit, and liver
enzymes (LDH, ALT, and AST). Anti-PGL-1 serum antibodies are expected after 9 months post-inoculation. Beginning
3 months postinfection, the armadillos are monitored for production of anti-PGL-1 antibodies using a sandwich ELISA
[41]. Monitoring continues every 3 months until seroconversion occurs (>0.700 OD at 490 nm), at which time they are
then monitored on a monthly basis until disease fully disseminates. Animals are sacrificed when anti-PGL-1 antibody titers
are >1.00 OD at 490 nm. Hematocrit and blood chemistry,
including liver enzymes, are also monitored every 3 months
postinfection using an HT5 veterinary hematology analyzer
and Element DC veterinary chemistry analyzer (HESKA),
respectively. At anti-PGL-1 seroconversion, hematocrit and
blood chemistry monitoring continues on a monthly basis.
Normal hematocrit values are >30, whereas at full dissemination, the hematocrit decreases to <20 due to hemolysis of
RBC. Regarding liver enzymes, LDH values >1500 indicate
RBC lysis. These, along with low hematocrit, are additional
indicators of fully disseminated disease.
6. After 18–24 months, it is expected to observe generalized
dissemination, and the recovery is expected to be for up to
10
12 bacilli from a single armadillo.
3.13 Harvesting Liver
and Spleen
of M. leprae-Infected
Armadillos
Once dissemination is attained, the armadillo is humanely sacrificed
and liver and spleen are harvested aseptically.
1. At least 4 h prior to sacrifice, the armadillo is given Gentamicin
(1 mL) and Penicillin (1 mL) intramuscularly (IM) in order to
avoid contamination of internal organs.
2. The armadillo is anesthetized using a combination of Ketamine
(0.6 mL) and Dexdomitor (0.4 mL) given IM, and the skin of
the abdomen is shaved and thoroughly cleaned with iodine,
70% ethanol, and water.
3. The armadillo is then euthanized by exsanguination and placed
in a BSC where the tissues are aseptically removed and placed
into sterile jars on ice.
4. To check for contaminants, 1 g piece from each organ is
removed and processed for acid-fast staining, microscopic
counting, and sterility testing. Mycobacterial contamination is
ascertained by inoculating two of each 7H11 and L-J slants;
one of each is incubated at 33
C and one of each is incubated
at 37
C.
5. Slants are examined weekly for 8 weeks.
42
Elizabeth Wallace et al.