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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 609

Supplemental Assay Method for Potency Assay of Leptospira interrogans

serogroup canicola Bacterins

Date: April 19, 2022

Number: SAM 609.07

Supersedes: SAM 609.06, March 9, 2016

Standard Requirement: 9 CFR 113.103

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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Supplemental Assay Method for Potency Assay of Leptospira interrogans Serogroup 

Canicola Bacterins

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 canicola bacterins as prescribed by title 9, Code of

Federal Regulations (9 CFR), part 113.103.

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

2.1.5 Stomacher® blender and sterile bags (alternatively, Tenbroeck tissue 

grinders, 15 mL, 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-PA semi-solid medium 

2.2.11 0.85% NaCl solution (saline)

2.2.12 L. canicola challenge culture, hamster-virulent 

2.3 Animals

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

tested. Ten hamsters are always required per test session for nonvaccinated 

controls, and an additional twenty hamsters are required for LD50 titration if the 

back-titration exemption is not used. Sufficient hamsters should always be 

available to perform at least three in vivo serial passages of the Leptospira prior to 

challenge.

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

uses golden Syrian hamsters sourced from Envigo. 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, 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 at 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 at 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 six 

months.

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 1/80 of the recommended host-animal dose.

1. For 2-mL-dose products, dilute the bacterin 1:10 in saline. 

2. For 5-mL-dose products, dilute the bacterin 1:4 in saline. 

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 (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 
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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 if required.

4.1.4 Cryopreserved challenge culture should be inoculated into the first 

hamsters for serial passage on the day of vaccination or within sufficient time to 

perform at least three serial passages prior to use in a regulatory test. Leptospira

passage through hamsters is conducted according to the current version of 

CVB-WI-0253, Leptospira Passage through Hamsters.

4.1.5. Challenge all hamsters with a virulent suspension of L. canicola 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. canicola. 

4.2.2 Euthanize the hamster with CO2. 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.
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4.2.5 Using fresh instruments, aseptically remove approximately 1 gram of liver 

tissue. Measure the liver on an analytical scale in a tared sterile container in order 

to obtain 1.0 ± 0.1 gram of infected tissue. Aseptically place liver in a sterile 

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

liver with a Stomacher or tissue grinder, 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, then the 10-6 dilution 

will be the challenge inoculum. (The challenge inoculum is the dose 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 does not contain the appropriate number of 

spirochetes per field, determine which dilution contains between 4–20 

spirochetes/microscopic field. Then utilize the chart below to determine the 

appropriate dilution to use as the challenge inoculum.

Dilution Containing Challenge

4 - 20 spirochetes/field Inoculum Dilution

10-3 10-5

10-4 10-6

10-5 10-7

10-6 10-8

If the 10-3 dilution contains < 4 organisms per field, select another clinically 

ill hamster (Section 4.2.1) and prepare another challenge inoculum.

4.2.9 If the LD50 is required, 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 inoculum may be 

used to infect additional hamsters to serve as a source of inoculum for future 
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potency tests. The dilution and dose volume should be tailored to suit the 

frequency with which serial passage is performed.

4.3 Challenge of test hamsters

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

challenge inoculum 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 if a back-titration is required. These 4 groups of hamsters will be 

used to calculate the LD50 of the challenge inoculum.

4.3.3 The 10-4 dilution should be retained and later tested for serogroup 

specificity through the current version of CVB-TWS-0048, Leptospira ELISA 

Screen.

4.3.4 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 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, 

Dragstedt-Behrens, or Spearman-Kärber method of calculation. CVB typically 

uses the Dragstedt-Behrens method.

5. Interpretation of the Test Results

5.1 Interpret the results as described in 9 CFR 113.103.

5.2 The test is valid if 8 or more of the nonvaccinated hamsters that received the 

challenge inoculum are counted as deaths. A LD50 of 10-10,000 is an additional validity 

requirement without a back-titration exemption.
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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 

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:

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 113.103, U.S. Government Printing Office, 

Washington, DC.

8. Summary of Revisions

Version .07

• 4.2.8 Corrected challenge dilution.

• Minor formatting changes and updated coversheet inserted.

Version .06

• Formatting has been updated according to current practices.

• The LD50 calculation was clarified.

• The use of back-titration hamsters has been updated in agreement with current public 

guidance.

• Serogroup specificity testing and retention of shipping cultures has been updated 

based on current practices.

Version .05

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

current practices.

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
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Version .04

• The Contact information has been updated.

• The title has been changed to be more in agreement with current nomenclature 

practices.

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

• Minor revisions have been made to clarify practices currently in use at the Center for 

Veterinary Biologics. 

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.