**Veterinary Diagnosis of C. difficile Infection**

Clostridium difficile is a Gram positive to Gram variable, anaerobic, spore-forming bacterium that commonly inhabits the intestinal tract of various mammalian species, reptiles and birds, and may also be found in the environment. The organism is an important cause of enteric disease in laboratory rodents and horses. Hamsters, guinea pigs and mice may be affected by pseudomembranous colitis precipitated by antimicrobial therapy (1-4). In neonatal foals, it has been associated with hemorrhagic necrotizing enterocolitis and diarrhea (5,6). Adult horses may develop typhlocolitis and there have been of outbreaks of nosocomially-acquired diarrhea (7-9). C. difficile has recently been described as a cause of typhlocolitis in nursing piglets (10). The organism may also be a cause of chronic diarrhea in dogs and enterotoxemia in ostriches (11,12). Diagnosis involves isolation of the organism and demonstration of toxins A and/or B in clinical materials.

Specimen collection, transport and processing

I. Principle

Proper selection of specimens and prompt transport to the lab for processing are essential for isolation of this organism.

II. Specimen and transport

A.	Large intestinal contents or feces should be submitted in an appropriate anaerobic transport medium unless they can be delivered to the laboratory within 24 hours.
B.	Live, acutely ill, untreated animals should be selected for specimen procurement.
C.	Specimens that cannot be delivered to the lab within 24 hours should be sent refrigerated to be received as soon as possible.

III. Processing specimens (13)

A.	Culture media
	1. Cycloserine-cefoxitin-fructose agar (CCFA)
	2. Clostridium difficile selective agar with 5% horse or bovine blood (CDSA)
	3. Anaerobic blood agar (CDC, Brucella or BHI base)
	4. Plates should be pre-reduced prior to use
B.	Equipment
	1. Anaerobic chamber, jars, pouches
	2. Anaerobic gas generating system, if jars or pouches used
	3. Inoculating loops
	4. Sterile tubes

IV. Media inoculation

A.	Properly obtained clinical specimens are inoculated directly onto selective media (CCFA and CDSA using a 0.1 ml inoculum size
B.	Ethanol shock treatment
	1. Equal amounts of specimen and ethanol (approximately 1 ml of each) are mixed and incubated for 30 minutes at room temperature
	2. One drop (approximately 0.1 ml) of the treated specimen is plated onto anaerobic blood agar
C.	Plates are streaked for isolation and incubated anaerobically at 37oC for 48 hours
D.	Remainder of specimen should be frozen immediately after processing at or below –20oC for use in the toxin detection assay

Examination of primary culture plates and organism identification

I. Principle

The goal of plate examination is to identify any suspicious colonies by their unique odor and colonial morphology. Gram’s stain and fluorescence under long-wave ultraviolet light are also employed. The establishment that the suspect organism is truly anaerobic and a rapid spot test will confirm the identity as C. difficile.

II. Materials

A. Reagents and media

1. Gram stain reagents

2. L-proline aminopeptidase test disks

3. Cinnamaldehyde reagent

4. Routine blood agar plate

B. Supplies

1. Clean microscopic slides

2. Inoculating loops

C. Equipment

1. Light microscope

2. Longwave ultraviolet light

III. Identification procedure (14,15)

A. Plates are initially examined after 48 hours of incubation

B. Colonies have a distinct “horse stable-like” odor

C. Colonial morphology

1. Anaerobic blood agar and CDSA

a. Non-hemolytic, gray, slightly raised, umbonate with a ground glass-like appearance and filamentous edge

2. CCFA

a. Large, slightly flat, circular colonies with a filamentous edge

b. Yellow zone 2-3 mm out from the edge of the colony

c. Inspect suspicious colonies under longwave ultraviolet light for yellow fluorescence

D. Pick single colonies from anaerobe blood agar

1. Gram stain

a. Gram positive to variable long, thin, straight rods

b. Usually many oval subterminal spores

2. Subculture to routine blood agar plate for aerotolerance testing

a. Incubate plate at 37oC in 5-7% CO2 for 48 hours

b. No growth on plate is indicative of an anaerobe

3. L-proline-aminopeptidase disk test

a. Test disk is placed on microscope slide in petri dish

b. Disk is moistened slightly with water

c. Disk is inoculated with a visible amount of test organism using a sterile loop

d. Incubate at room temperature for 2-5 minutes

e. Place one drop of cinnamaldehyde reagent onto disk

f. Wait one minute and observe for a pinkish red color to develop which is a positive test result

g. This test is 99% positive for C. difficile

Toxin detection assay (16)

I. Principle

A commercial assay kit uses antibodies against C. difficile toxins A and B that are coated into microtiter wells. An aliquot of specimen is diluted and placed into the test wells. If toxins A and B are present, they will bind to the antibodies in the wells. Substrate added to the wells, will produce a color change due to the enzyme-antibody-antigen complex that will form in the presence of toxins. Also see the Techlab website for more information.

For information on detection of toxin in tissue culture using CHO cells, click here.

II. Materials

A. Test kit

B. Wash bottle

C. Test tubes

D. Timer

E. Paper towels

F. Biohazard container

G. Distilled water

H. Incubator

III. Procedure

A. Kit is removed from storage in the refrigerator and allowed to warm to room temperature

B. Specimens are removed from freezer and thawed

C. Specimens are diluted in 0.2 ml kit diluent

D. The number of test wells to be used is determined

E. One drop of kit conjugate is added to each well

F. One well per specimen to be tested, one for the positive control and one for the negative control are to be used

G. Two drops of diluted specimen is added to a well

H. Add one drop positive control reagent to the appropriate well

I. Add one drop of diluent to the negative control well

J. Cover wells with plastic adhesive

K. Incubate plates at 37oC for 50 minutes in aerobic incubator

L. Remove plastic and shake out well contents into biohazard container

M. Wash each well with buffer four times

N. Add one drop of Substrate A to each well, followed by one drop substrate B

O. Gently tap wells to mix

P. Incubate at room temperature for 10 minutes

Q. Add one drop of stop solution to each well

R. Visual readings are made against a white background in good light

IV. Test interpretation

A. Wells that visibly have a yellow color are considered positive for toxins

B. Wells that are colorless are negative

C. If controls fail, test is invalid and should be repeated

References

1. Chang J, Rohwer RG. 1991. Clostridium difficile infection in adult hamsters. Lab Anim Sci 41:548-552.

2. Rehg JE, Lu Y-S. 1982. Clostridium difficile typhilitis in hamsters not associated with antibiotic therapy. J Am Vet Med Assoc 181:1422-1423.

3. Lowe BR, Fox JG, Bartlett JG. 1980. Clostridium difficile associated cecitis in guinea pigs exposed to penicillin. Am J Vet Res 41:1277-1279.

4. Rehg JE, Pakes SP. 1982. Implications of Clostridium difficile and Clostridium perfringens iota toxins in experimental lincomycin-associated colitis in rabbits. Lab Anim Sci 32:253-257.

5. Jones RL, Adney WS, Alexander AF, Shideler RK, Traub-Dargatz JL. 1988. Hemorrhagic necrotizing enterocolitis associated with Clostridium difficile infection in four foals. J Am Vet Med Assoc 193:76-79.

6. Weese JS, Parsons DA, Staempfli HR. 1999. Association of Clostridium difficile with enterocolitis and lactose intolerance in a foal. J Am Vet Med Assoc 214:229-232.

7. Perrin J, Cosmetatos I, Gallusser A, Lobsiger L, Straub R, Nicolet J. 1993. Clostridium difficile associated with typhlocolitis in an adult horse. J Vet Diagn Invest 5:99-101.

8. Madewell BR, Tang YJ, Jang S, Madigan JE, Hirsh DC, Gumerlock PH, Silva J. 1995. Apparent outbreaks of Clostridium difficile-associated diarrhea in horses in a veterinary medical teaching hospital. J Vet Diagn Invest 7:343-346.

9. Donaldson MT, Palmer JE. 1999. Prevalence of Clostridium perfringens enterotoxin and Clostridium difficile toxin A in feces of horses with diarrhea and colic. J Am Vet Med Assoc 215:358-361.

10. Waters EH, Orr JP, Clark EG, Schaufele CM. 1998. Typhlocolitis caused by Clostridium difficile in suckling piglets. J Vet Diagn Invest 10:104-108.

11. Songer JG. 1996. Clostridial enteric disease of domestic animals. Clin Microbiol Rev 9:216-234.

12. Frazier KS, Herron AJ, Hines ME, Gaskin JM, Altman NH. 1993. Diagnosis and enterotoxemia due to Clostridium difficile in captive ostriches (Struthio camelus). J Vet Diagn Invest 5:623-625.

13. Marler LM, Siders JA, Wolters LC, Pettigrew Y, Skitt BL, Allen SD. 1992. Comparison of five cultural procedures for isolation of Clostridium difficile from stools. J Clin Microbiol 30:514-516.

14. Fedorko DP and Williams EC. 1997. Use of cycloserine-cefoxitin-fructose agar and L-proline-aminopeptidase (PRO discs) in the rapid identification of Clostridium difficile. J Clin Microbiol 35:1258-1259.

15. Knoop FC, Owens M, Crocker C. 1993. Clostridium difficile: clinical disease and diagnosis. Clin Microbiol Rev 6:251-265.

16. Techlab Product Insert #92-007-03. 1997. C. difficile TOX A/B test. TechLab Inc., Blacksburg, Va.

Appendix 1 Sources of anaerobic transport supplies

Becton Dickinson Microbiology Systems, Cockeysville, MD 21030

Transport for anaerobe swabs Catalog number

Anaerobic culturette

62218

Port-a-cul tube

21606

Anaerobic pouches

Biobag type A

4361214

Scott Laboratories Inc., Fisherville, RI or Carson, CA

Transport for anaerobic swabs

Anaswab Set

3200-1000

Anaerobe Systems

Transport for anaerobic swabs

Anaerobic Transport Medium Sterile Pack with tube and swabs

AS-912

Appendix 2 Partial list of suppliers for anaerobic plated media

    Anaerobe Systems
    15906 Concord Circle
    Morgan Hill, CA 95037
    408-782-7557

    BBL
    PO Box 243
    Cockeysville, MD
    800-638-8663

    Remel, Inc.
    12076 Santa Fe Dr.
    Lenexa, KS 66215
    800-255-6730

Appendix 3 Media recipes for anaerobic plates

General anaerobic blood agar

A. CDC anaerobe agar

trypticase soy agar base (BBL)	40 gm
agar	5 gm
yeast extract	5 gm
hemin	5mg
L-cysteine	400 mg
vitamin K1 stock solution	1 ml
distilled water	1000 ml

Suspend above ingredients in water and heat with frequent agitation until boiling. Autoclave at 121oC at 15 psi for 15 min. Cool in water bath to 45oC and aseptically add 5% defibrinated animal blood. Mix well and dispense into sterile petri dishes. Store in plastic bags at 2-8oC. Shelf-life is up to 1 month.

B. Brucella formulation (BBL or Difco)

Polypeptone	23 gm
glucose	1 gm
yeast extract	2 gm
sodium chloride	5 gm
hemin (1% solution)	10 ml
vitamin K1 (1% solution)	1 ml
L-cysteine	0.4 gm
agar	15 gm
distilled water	1000 ml

Suspend the above ingredients in water and heat with frequent agitation until boiling. Autoclave at 121oC at 15 psi for 15 min. Cool in water bath to 45oC and aseptically add 5% defibrinated animal blood. Mix well and aseptically dispense into sterile petri dishes. Store in plastic bags at 2-8oC. Shelf-life is up to 1 month.

C. BHI blood agar

BHI base (BBL, Difco)	26 gm
agar	2.5 gm
distilled water	1000 ml

D. CCFA (Clostridium difficile agar by BBL) makes 1 liter

casein peptone	18 gm
glucose	1 gm
BHI meat peptone	8 gm
dipotassium phosphate	2.5 gm
sodium chloride	5.5 gm
yeast extract	2 gm
fructose	6 gm
neutral red	30 mg
cycloserine	500 mg
cefoxitin	16 mg
agar	15 gm
distilled water	1000 ml

Suspend all the above ingredients in water except the cycloserine and cefoxitin. Heat with frequent agitation until boiling. Autoclave at 121oC at 15 psi for 15 min. Cool in water bath to 45oC and aseptically add the cycloserine and cefoxitin. Mix well. Aseptically dispense into sterile petri plates. Store in plastic bags at 2-8oC. Shelf-life is up to 2 weeks.

E. CDSA (Clostridium difficile selective agar) makes 500 ml

Bacto Brain Heart Infusion Agar	18.5 gm
Clostridium difficile antimicrobic supplement CC	5ml
sodium taurocholate	0.5 gm
defibrinated bovine blood	25 ml
distilled water	500 ml

Suspend the BHI agar and sodium taurocholate in the distilled water. Heat with frequent agitation until boiling. Autoclave at 121oC for 15 minutes. Cool to 45oC in a water bath. Immediately prior to pouring aseptically add the CC supplement (containing cycloserine and cefoxitin) and the bovine blood. Mix well. Dispense into sterile petri dishes. Cool and store in plastic bags at 2-8oC. Shelf-life is up to 2 weeks.

Appendix 4 Partial list of suppliers of disks, toxin assay detection systems, and anaerobic chambers

    PRO discs
    Remel PRO kit (contains discs and cinnamaldehyde reagent) catalog # 21-1357
    Remel, Inc.
    12076 Santa Fe Dr.
    Lenexa, KS 66215
    800-255-6730

    C. difficile TOX A/B TEST Kit
    Tech Lab., Inc.
    Corporate Research Park
    1861 Pratt Dr.
    Blacksburg, VA 24060
    800-TECHLAB

    Bactron I-IV Anaerobe Chambers
    Sheldon Manufacturing
    300 N. 26th Ave.
    Cornelius, OR 97113
    800-322-4897

This document is also available in pdf.
To view pdf files, download Acrobat Reader free at the Adobe web site.

Last modified October 1, 2005

A.	Reagents and media
	1. Gram stain reagents
	2. L-proline aminopeptidase test disks
	3. Cinnamaldehyde reagent
	4. Routine blood agar plate
B.	Supplies
	1. Clean microscopic slides
	2. Inoculating loops
C.	Equipment
	1. Light microscope
	2. Longwave ultraviolet light

A.	Plates are initially examined after 48 hours of incubation
B.	Colonies have a distinct “horse stable-like” odor
C.	Colonial morphology
	1. Anaerobic blood agar and CDSA
	a. Non-hemolytic, gray, slightly raised, umbonate with a ground glass-like appearance and filamentous edge
	2. CCFA
	a. Large, slightly flat, circular colonies with a filamentous edge
	b. Yellow zone 2-3 mm out from the edge of the colony
	c. Inspect suspicious colonies under longwave ultraviolet light for yellow fluorescence
D.	Pick single colonies from anaerobe blood agar
	1. Gram stain
	a. Gram positive to variable long, thin, straight rods
	b. Usually many oval subterminal spores
	2. Subculture to routine blood agar plate for aerotolerance testing
	a. Incubate plate at 37oC in 5-7% CO2 for 48 hours
	b. No growth on plate is indicative of an anaerobe
	3. L-proline-aminopeptidase disk test
	a. Test disk is placed on microscope slide in petri dish
	b. Disk is moistened slightly with water
	c. Disk is inoculated with a visible amount of test organism using a sterile loop
	d. Incubate at room temperature for 2-5 minutes
	e. Place one drop of cinnamaldehyde reagent onto disk
	f. Wait one minute and observe for a pinkish red color to develop which is a positive test result
	g. This test is 99% positive for C. difficile

A.	Test kit
B.	Wash bottle
C.	Test tubes
D.	Timer
E.	Paper towels
F.	Biohazard container
G.	Distilled water
H.	Incubator

A.	Kit is removed from storage in the refrigerator and allowed to warm to room temperature
B.	Specimens are removed from freezer and thawed
C.	Specimens are diluted in 0.2 ml kit diluent
D.	The number of test wells to be used is determined
E.	One drop of kit conjugate is added to each well
F.	One well per specimen to be tested, one for the positive control and one for the negative control are to be used
G.	Two drops of diluted specimen is added to a well
H.	Add one drop positive control reagent to the appropriate well
I.	Add one drop of diluent to the negative control well
J.	Cover wells with plastic adhesive
K.	Incubate plates at 37oC for 50 minutes in aerobic incubator
L.	Remove plastic and shake out well contents into biohazard container
M.	Wash each well with buffer four times
N.	Add one drop of Substrate A to each well, followed by one drop substrate B
O.	Gently tap wells to mix
P.	Incubate at room temperature for 10 minutes
Q.	Add one drop of stop solution to each well
R.	Visual readings are made against a white background in good light

A.	Wells that visibly have a yellow color are considered positive for toxins
B.	Wells that are colorless are negative
C.	If controls fail, test is invalid and should be repeated

1.	Chang J, Rohwer RG. 1991. Clostridium difficile infection in adult hamsters. Lab Anim Sci 41:548-552.
2.	Rehg JE, Lu Y-S. 1982. Clostridium difficile typhilitis in hamsters not associated with antibiotic therapy. J Am Vet Med Assoc 181:1422-1423.
3.	Lowe BR, Fox JG, Bartlett JG. 1980. Clostridium difficile associated cecitis in guinea pigs exposed to penicillin. Am J Vet Res 41:1277-1279.
4.	Rehg JE, Pakes SP. 1982. Implications of Clostridium difficile and Clostridium perfringens iota toxins in experimental lincomycin-associated colitis in rabbits. Lab Anim Sci 32:253-257.
5.	Jones RL, Adney WS, Alexander AF, Shideler RK, Traub-Dargatz JL. 1988. Hemorrhagic necrotizing enterocolitis associated with Clostridium difficile infection in four foals. J Am Vet Med Assoc 193:76-79.
6.	Weese JS, Parsons DA, Staempfli HR. 1999. Association of Clostridium difficile with enterocolitis and lactose intolerance in a foal. J Am Vet Med Assoc 214:229-232.
7.	Perrin J, Cosmetatos I, Gallusser A, Lobsiger L, Straub R, Nicolet J. 1993. Clostridium difficile associated with typhlocolitis in an adult horse. J Vet Diagn Invest 5:99-101.
8.	Madewell BR, Tang YJ, Jang S, Madigan JE, Hirsh DC, Gumerlock PH, Silva J. 1995. Apparent outbreaks of Clostridium difficile-associated diarrhea in horses in a veterinary medical teaching hospital. J Vet Diagn Invest 7:343-346.
9.	Donaldson MT, Palmer JE. 1999. Prevalence of Clostridium perfringens enterotoxin and Clostridium difficile toxin A in feces of horses with diarrhea and colic. J Am Vet Med Assoc 215:358-361.
10.	Waters EH, Orr JP, Clark EG, Schaufele CM. 1998. Typhlocolitis caused by Clostridium difficile in suckling piglets. J Vet Diagn Invest 10:104-108.
11.	Songer JG. 1996. Clostridial enteric disease of domestic animals. Clin Microbiol Rev 9:216-234.
12.	Frazier KS, Herron AJ, Hines ME, Gaskin JM, Altman NH. 1993. Diagnosis and enterotoxemia due to Clostridium difficile in captive ostriches (Struthio camelus). J Vet Diagn Invest 5:623-625.
13.	Marler LM, Siders JA, Wolters LC, Pettigrew Y, Skitt BL, Allen SD. 1992. Comparison of five cultural procedures for isolation of Clostridium difficile from stools. J Clin Microbiol 30:514-516.
14.	Fedorko DP and Williams EC. 1997. Use of cycloserine-cefoxitin-fructose agar and L-proline-aminopeptidase (PRO discs) in the rapid identification of Clostridium difficile. J Clin Microbiol 35:1258-1259.
15.	Knoop FC, Owens M, Crocker C. 1993. Clostridium difficile: clinical disease and diagnosis. Clin Microbiol Rev 6:251-265.
16.	Techlab Product Insert #92-007-03. 1997. C. difficile TOX A/B test. TechLab Inc., Blacksburg, Va.

Transport for anaerobe swabs	Catalog number
Anaerobic culturette	62218
Port-a-cul tube	21606
Anaerobic pouches
Biobag type A	4361214

Transport for anaerobic swabs
Anaerobic Transport Medium Sterile Pack with tube and swabs	AS-912