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SAM 617.06

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 617

Supplemental Assay Method for Potency Assay Leptospira kirschneri 

serogroup grippotyphosa Bacterins

Date: April 26, 2022

Number: SAM 617.06

Supersedes: SAM 617.05, March 9, 2016

Standard Requirement: 9 CFR 113.104

Contact: Email: Methodsrequest.notification@usda.gov

 Phone: Center for Veterinary Biologics, 515-337-6100

United States Department of Agriculture

Animal and Plant Health Inspection Service

P. O. Box 844

Ames, IA 50010

Mention of trademark or proprietary product does not constitute a guarantee or warranty of the product by USDA 

and does not imply its approval to the exclusion of other products that may be suitable.
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Table of Contents

1. Introduction

2. Materials

2.1 Equipment/instrumentation

2.2 Reagents/supplies

2.3 Animals

3. Preparation for the Test

3.1 Personnel qualifications/training

3.2 Preparation of equipment/instrumentation

3.3 Preparation of reagents

3.4 Preparation of the sample

4. Performance of the Test

4.1 Vaccination of hamsters

4.2 Challenge procedure

4.3 Challenge of test hamsters

4.4 Observation of hamsters after challenge

5. Interpretation of the Test Results

6. Report of Test Results

7. References 

8. Summary of Revisions
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1. Introduction

This Supplemental Assay Method (SAM) describes the hamster vaccination-challenge method 

used to determine potency of Leptospira grippotyphosa bacterins as prescribed by title 9, Code 

of Federal Regulations (9 CFR), part 113.104.

2. Materials

2.1 Equipment/instrumentation

Equivalent equipment or instrumentation may be substituted for any brand name listed 

below.

2.1.1 Microscope with darkfield capability

2.1.2 Forceps, 5 1/2-inch, rat-tooth

2.1.3 Dissecting pins, 1-to 1 1/2-inch

2.1.4 Necropsy board

2.15 Stomacher® blender and sterile bags (alternatively, tissue grinders, 15 mL, 

TenBroeck may be used)

2.1.6 Balance, analytical 

2.2 Reagents/supplies

Equivalent reagents or supplies may be substituted for any brand name listed below.

2.2.1 Syringes, 1-mL tuberculin

2.2.2 Needles, appropriate size

2.2.3 Scalpels

2.2.4 Glass screw-top tubes, 20 x 150-mm (or equivalent container)

2.2.5 Serum bottles with rubber stoppers (or equivalent container for preparing 

dilutions of liver homogenate)

2.2.6 70% (v/v) ethanol
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2.2.7 Microscope slides and coverslips

2.2.8 Pipettes, assorted sizes, cotton-plugged 

2.2.9 1% Bovine Serum Albumin Diluent (BSAD)

2.2.10 P80-BA semi-solid medium 

2.2.11 0.85% NaCl solution (saline) 

2.2.12 L. grippotyphosa challenge culture, hamster-virulent

2.3 Animals

2.3.1 Hamsters, adult, 50-90 g. Ten hamsters are required for each bacterin 

tested. Thirty additional hamsters are required per test session for nonvaccinated 

controls and LD50 titration.

2.3.2 The hamsters must be obtained from the same source and colony. Use 

either all male or all female hamsters for any one test. 

2.3.3 House and feed all hamsters in an identical manner.

3. Preparation for the Test

3.1 Personnel qualifications/training

Technical personnel need a working knowledge of the use of general laboratory 

chemicals, equipment, and glassware and must have specific training and experience in 

the safe handling of live Leptospira spp. Personnel need specific training in the care and 

handling of laboratory hamsters and in the performance of this assay.

3.2 Preparation of equipment/instrumentation

3.2.1 Operate and maintain all equipment according to manufacturers’ 

recommendations and applicable in-house standard operating procedures.

3.2.2 Sterilize all glassware before use.

3.2.3 Use only sterile supplies (pipettes, syringes, needles, rubber stoppers, 

etc.).
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3.3 Preparation of reagents

3.3.1 1% Bovine Serum Albumin Diluent – National Centers for Animal Health 

(NCAH) Media #20133

Sodium phosphate, dibasic 0.664 g

Potassium phosphate, monobasic 0.087 g

Bovine serum albumin, fraction V 10 g

Deionized water q.s. 1.0 L

Mix until dissolved. If necessary, adjust pH to 7.5 ± 0.1. Sterilize by filtration, 

using a 0.22-μm filter. Store 20°- 25°C for no longer than 1 year.

3.3.2 0.85% saline – NCAH Media #30201

Sodium chloride 8.5 g

Deionized water q.s. 1.0 L

Autoclave at 121°- 125°C for 15 to 20 minutes. Store 20°- 25°C for no longer 

than 1 year.

3.3.3 P80-BA semi-solid medium – NCAH Media #10117

Sodium phosphate, dibasic 0.664 g

Potassium phosphate, monobasic 0.087 g

Sodium chloride 1.925 g

Ammonium chloride 0.268 g

Magnesium chloride 0.191 g

Deionized water 790 mL

Stir to dissolve. Add:

Cupric sulfate solution (300 mg/L, pH 5.8) 1 mL

Zinc sulfate solution (0.4g/L, pH 6.3) 10 mL

Ferrous sulfate solution (2.5 g/L) 20 mL

L-cystine 0.2 g

Stir. Do not attempt to dissolve L-cystine completely. Do not heat. 

Filter through triple thickness #1 Whatman paper. If filtrate is not clear, filter 

again.
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Combine filtered media with:

Vitamin B12 solution (10 mg/L) 20 mL

Thiamine HCl solution (2 g/L, pH 3.8) 0.1 mL

Tween 80 1.2 mL

Deionized water q.s. 1 L

Place 800 mL of this mixture in a large container and add 1.3 g purified agar. 

Autoclave at 121°- 125°C for 20 to 25 minutes. Cool to 56°± 1°C. 

Combine the following:

Bovine serum albumin, fraction V 20 g

Sodium phosphate, dibasic 0.133 g

Potassium phosphate, monobasic 0.017 g

Deionized water q.s. 200 mL

Adjust pH to 7.4 to 7.6 and sterilize by filtration (0.2 μm).

Add filtered albumin solution to the cooled (56°C) solution prepared previously.

Adjust to pH 7.2 to 7.8 with sterile 10% NaOH. Dispense in 9-mL aliquots into 

screw-capped tubes. Store, tightly capped at 20°- 25°C for no longer than 1 year.

3.4 Preparation of the sample

3.4.1 Shake each bacterin to mix contents thoroughly.

3.4.2 Disinfect the top of the bacterin container with 70% ethanol.

3.4.3 Dilute each bacterin with saline so that 1 hamster dose (0.25 mL) is 

equivalent to 1/800 of the recommended host-animal dose.

1. For 2-mL-dose products, dilute the bacterin 1:100 in saline. 

2. For 5-mL-dose products, dilute the bacterin 1:40 with saline. 
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4. Performance of the Test

4.1 Vaccination of hamsters

4.1.1 For each bacterin to be tested, vaccinate 10 hamsters with 0.25 mL of 

appropriately diluted bacterin (see Section 3.4.3) using the route recommended by 

the manufacturer. If the recommended vaccination route is intramuscular, or if the 

product is labeled for either intramuscular or subcutaneous use, vaccinate the 

hamsters intramuscularly in the hind leg. If the label limits administration of that 

product to the subcutaneous route, vaccinate the hamsters subcutaneously in the 

abdominal area. For all vaccinations, use a 1.0-mL syringe fitted with an 

appropriate size needle. 

4.1.2 Retain 10 nonvaccinated hamsters as controls. 

4.1.3 Retain 20 nonvaccinated hamsters to determine the LD50 of the challenge 

inoculum.

4.1.4 Challenge all hamsters with a virulent suspension of L. grippotyphosa 14 

to 18 days after vaccination.

4.2 Challenge procedure

The challenge inoculum is a liver homogenate from a clinically ill hamster. The Center 

for Veterinary Biologics (CVB) maintains virulent challenge organisms by serial passage 

through hamsters on a routine basis.

4.2.1 Select a clinically ill (preferably moribund) hamster from a group of 

hamsters that were infected 3 to 4 days previously with L. grippotyphosa.

4.2.2 Euthanize the hamster with CO2. Rapid administration from a compressed 

gas cylinder is recommended because the inflow to the chamber can be regulated 

precisely. Follow the euthanasia procedure approved by the Animal Care and Use 

Committee.

4.2.3 Pin the dead hamster to a posting board (ventral aspect up) and disinfect 

the skin with 70% ethanol.

4.2.4 Using aseptic technique, reflect the abdominal skin. Discard the 

instruments used to expose the abdominal musculature. Using fresh instruments, 

reflect the abdominal musculature to expose the abdominal viscera. Discard the 

instruments used to open the abdominal cavity.

4.2.5 Using fresh instruments, aseptically remove approximately 1 gram of liver 

tissue and place in a sterile previously weighed container. Weigh the container 
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again with liver tissue and add or subtract additional tissue to obtain a mass as 

close to 1.0 gram as possible ± 0.1 gram. Aseptically place liver in a sterile 

blender bag and add 9 mL of sterile BSAD to the bag. Thoroughly homogenize 

the liver, taking care to avoid foam formation. This suspension is considered the 

1:10 dilution.

4.2.6 Prepare 5 additional serial tenfold dilutions (10-2 through 10-6) of the 

tissue suspension in BSAD (1.0 mL suspension + 9.0 mL diluent). Hold the 

dilutions at room temperature (20°- 25°C) and complete challenge inoculations 

within 1 hour after preparation of dilutions.

4.2.7 Place 2 drops of the 10-4 dilution on a microscope slide, cover with a 

coverslip, and examine under a 200X magnification with a darkfield microscope. 

The 10-4 dilution should have approximately 4 to 20 organisms per field.

4.2.8 If the 10-4 dilution has 4 to 20 organisms per field, use the 10-6 dilution for 

the challenge inoculum. (This usually is sufficient to deliver the required 

challenge of 10-10,000 LD50.) The challenge inoculum should be prepared in an 

appropriate sterile container.

Note: If the 10-4 dilution contains > 20 organisms per field, dilute the 

challenge with BSAD until numbers of organisms are between 4 and 20 per 

field. If < 4 organisms are present per field in the 10-4 dilution examine the 

10-3 dilution; if the 10-3 dilution contains the required number of organisms, 

challenge with the 10-5 dilution. If the 10-3 dilution contains < 4 organisms 

per field, select another clinically ill hamster (Section 4.2.1) and prepare 

another challenge inoculum that more closely matches the desired organism 

density.

4.2.9 Prepare 4 additional tenfold dilutions beyond the dilution selected for the 

challenge inoculum. Retain these dilutions to determine the LD50 of the challenge 

inoculum. 

4.2.10 The remaining liver homogenate from the challenge preparation may be 

used to infect additional hamsters to serve as a source of inoculum for future 

potency tests. The dilution and dose volume should be tailored to suit the 

frequency with which serial passage is performed.

4.2.11 Transfer 1 mL of the challenge inoculum into 9 mL of P80-BA semisolid 

medium. Incubate semisolid cultures at 25°- 30° C for 2 to 4 weeks. Cultures may 

be maintained at 20°- 25°C for up to 6 months. These cultures serve as an archival 

source of the challenge inoculum if additional analysis is warranted. The CVB 

prepares additional cultures (i.e., duplicate cultures of inoculum from each of the 

10-4 through 10-10 dilutions) for internal use.
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4.3 Challenge of test hamsters

4.3.1 Within 1 hour after preparation, inject intraperitoneally (IP) 0.2 mL of the 

dilution selected in Section 4.2.8 into each of the vaccinated hamsters and 10 

nonvaccinated control hamsters. Use a 1.0-mL syringe fitted with an appropriate 

size needle.

4.3.2 Inject 5 hamsters (0.2 mL, IP) with each of the dilutions prepared in 

Section 4.2.9. These 4 groups of hamsters will be used to calculate the LD50 of 

the challenge. 

4.3.3 Disinfect all work surfaces with 70% ethanol. Sterilize all contaminated 

equipment and supplies in the autoclave. 

4.4 Observation of hamsters after challenge

4.4.1 Observe all hamsters daily for 14 days following challenge. Record 

deaths.

Note: Moribund animals exhibiting clinical signs consistent with the 

expected disease pathogenesis that are unable to rise or move under their 

own power may be humanely euthanized and considered as deaths as 

outlined in 9 CFR 117.4.

4.4.2 At the end of the 14-day observation period, count the remaining hamsters 

and record results. 

4.4.3 Calculate the LD50 of the challenge inoculum using the Reed-Muench or 

Spearman-Kärber method of calculation. 

5. Interpretation of the Test Results

5.1 Interpret the results as described in 9 CFR 113.104.

5.2 If 8 or more nonvaccinated controls die and the hamsters received a challenge of 

10-10,000 LD50, the test is valid. 

5.3 If 3 or 4 vaccinates die in the first stage test, conduct a second stage test in a 

manner identical to the first stage. If the second stage is used, evaluate each serial 
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according to the second part of the table. Serials pass or fail on the basis of cumulative 

results. Evaluate the results according to the following table:

Stage

Number of

Vaccinates

Cumulative

Number of

Vaccinates

Cumulative

Total Dead

Hamsters for

Satisfactory

Serial

Cumulative

Total Dead

Hamsters for 

Unsatisfactory 

Serial

1 10 10 2 or less 5 or more

2 10 20 5 or less 6 or more

6. Report of Test Results

Report the results of the test(s) as described by standard operating procedures.

7. References

Title 9, Code of Federal Regulations, part 113.104, U.S. Government Printing Office, 

Washington, DC.

8. Summary of Revisions

Version .06

• The coversheet has been updated

Version .05

• The Contact information has been updated, and minor edits to accurately reflect 

current practices.

Version .04

• Leptospira interrogans Serovar grippotyphosa has been changed to Leptospira

kirschneri serogroup grippotyphosa to reflect the most current molecular 

identification results.

• The Contact information has been updated.

• 3.3.1: The pH for the BSA diluent has been altered to allow ± 0.1 deviation from 7.5.

• References to NVSL have been changed to NCAH throughout the document.
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Version .03

• Minor changes have been made to update the document to current formatting 

practices.

Version .02

This document was revised to clarify practices currently in use at the Center for Veterinary 

Biologics and to provide additional detail. While no significant changes were made that impact 

the outcome of the test, the following changes were made to the document:

• 2.1 A Stomacher® blender and analytical balance have been added to the list of 

equipment and instrumentation.

• 2.2 The list of reagents and supplies has been updated to more accurately reflect what 

is used for this assay method.

• 3.3 The formulas have been updated to reflect that the solution may be stored at 

room temperature for up to one year.

• 3.3.3 P80-BA semi-solid medium has been added to the Preparation of Reagents 

section.

• References to internal CVB documents have been replaced with summary 

information.

• The contact person has been changed to Mary C. Rasmusson.