YERSINIA PESTIS: ISOLATION and IDENTIFICATION Introduction: Yersinia Pestis Is a Major Animal Pathogen and Can Cause Fatal Disease in Mammalian Species

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YERSINIA PESTIS: ISOLATION AND IDENTIFICATION Introduction: Yersinia pestis is a major animal pathogen and can cause fatal disease in mammalian species. The main route of infection is through flea bites, although contact of organism with mucus membranes or inhalation are other routes. Disease is manifested as lymphadenomegaly, fever, shock and death, most often in cats and people. Principle: This procedure describes methods for isolation and identification of Yersinia pestis from animal tissue. Each laboratory will need to select tests appropriate to their facility/expertise. Specimens: The preferred specimens for culture are needle aspirates from lymph nodes, blood, or tissues. It is important to collect samples from animals that are freshly dead and to avoid contact with purulent material during collection. Specimens should be refrigerated but not frozen, packaged to conform with federal regulations for transport of hazardous agents and sent to a reference laboratory for culture. Materials: Biohazard safety cabinet (Class IIA/B III) Microscope (oil objective) Gloves Disposable lab coats or disposable arm covers Disposable forceps Disposable scalpels Absorbent bench covers Swabs Masks Saline Disposable loops (10 ul) Biohazard bags (8” X 12” and 19” X 23”) 37C and 28C incubator 10% bleach solution gram’s stain Wright-Giemsa or Wayson stain Staining rack for slides Heat source for fixing slides Sheep blood agar or equivalent Eosin methylene blue agar or MacConkey agar Brain heart infusion broth or equivalent Blood culture system Glass slides Oxidase reagents Catalase reagent (3% hydrogen peroxide) Urease test (eg Christensen agar, biochemical kit) Procedure: ALL WORK SHOULD BE DONE IN A BIOLOGICAL SAFETY CABINET. GLOVES, A MASK, AND A DISPOSABLE LAB COAT OR ARM COVERS SHOULD BE WORN AT ALL TIMES. Gram stain: 1. Perform Gram stain procedure according to standard laboratory protocol. Smears of specimens for staining may be prepared in order of likely positive results (i.e., cultures, lymph node aspirates, tissue, blood) 2. Examine slides at 100X (oil immersion). 3. Gram reaction and size: Cells appear as plump, gram-negative rods, 1-2 µm X 0.5 µm, seen mostly as single cells or pairs and short chains in liquid media.

Wright-Giemsa or Wayson stain: The Wright-Giemsa stain often reveals the bipolar staiing characteristic of Y. pestis, whereas the Gram stain may not. Wayson stain is another polychromatic stain that can be used instead of Wright-Giemsa. Culture: 1. Use established inoculation and plating procedure. Then, tape plates shut in 2 places (or use alternative method) to prevent inadvertent opening. 2. Incubation of cultures: a. Temperature: 28 C (optimal); 35-37 C (grows more slowly).

b. Atmosphere: Ambient, 5% CO2 is acceptable. c. Length of incubation: Hold primary plates for 5 days (7 days if the patient has been treated with antibiotics). 3. Growth characteristics: a. Agar plates: Y. pestis grows as gray-white, translucent colonies, usually too small to see as individual colonies at 24 h. After 48 h of incubation, colonies are 1-2 mm in diameter, gray-white to slightly yellow, and opaque. Under 4X magnification, after 48-72 h of incubation, colonies have a raised, irregular “fried egg” appearance, which becomes more prominent as the culture ages. Colonies also are described as having a “hammered copper”, shiny surface. There is little or no hemolysis of sheep red blood cells. It grows as small, non-lactose fermenting colonies on MacConkey or EMB agar. b. Broth tubes: Y. pestis grows in clumps that are typically described as flocculant or “stalactite” in appearance when the culture is not shaken or mixed. At 24 h, growth is seen as clumps that hang along the side of the tube. Subsequently the growth settles to the bottom of the tube and is described as “cotton fluff”.

Biochemical Reactions/Tests Use established laboratory procedures for performing and interpreting catalase, oxidase, and urease tests. Note that commercial biochemical identification systems are not recommended at this stage. Any isolate with the following major characteristics should be suspected as being Y. pestis: a. Bipolar-staining rod (Wright-Giemsa) on direct smear b. Pinpoint colony at 24 h on Sheep blood agar c. Non-lactose fermenter, may not be visible on MacConkey or EMB at 24 h d. Oxidase and urease negative e. Catalase positive f. Growth often better at 28 C

Keep in mind the following limitations: a. Since the growth rate of Y. pestis is slower than that of most other bacteria, its presence may be masked by organisms that replicate faster. b. Bipolar staining of cells is not an exclusive feature of Y. pestis. Other gram-negative organisms, particularly Pasteurella spp., but also other Yersinia spp. and enteric bacteria can exhibit the same staining characteristic. c. Clumped growth in unshaken broth culture is not an exclusive feature of Y. pestis. Some Y. pseudotuberculosis and Streptococcus pneumoniae can exhibit the same growth feature. d. Some of the automated identification systems do not identify Y. pestis adequately. It has been falsely identified as Y. pseudotuberculosis,

Shigella, H2S-negative Salmonella, or Acinetobacter. Y. pestis is alkaline slant/acid butt in triple sugar iron agar slants. In most conventional biochemical or commercial identification systems, the organism appears relatively inert, making further biochemical testing of little value.

Confirmatory Tests: If Y. pestis is suspected, several options exist for confirming the identity of the isolate: 1. Referral to a reference laboratory, such as NVSL or the CDC’s Division of Vectorborne Infectious Disease, PO Box 2087, Ft. Collins, Colorado 80522, (970) 221-6444. 2. Fluorescent antibody procedure as follows:

a. Make impressions of tissues on FA slide; let air dry 15 minutes b. Heat fix impressions on the slides c. Place a drop or two of Yersinia pestis FA conjugate on slide (enclosed in a wax pencil circle). Treat a positive-control slide in the same manner. d. Place slides in a moisture chamber and incubate at 37 C for 30 minutes e. Wash gently with tap water f. Rinse in fresh FA buffer 3 times, rinsing 3 minutes each time in separate staining racks g. Blot slides dry; coverslip using FA mounting fluid (90% glycerol and 10% PBS) h. Bacteria will show a brilliant apple-green fluorescence when read on FA scope.

Plague FA conjugate is available (free) from CDC in Fort Collins (address above). 3. Polymerase Chain Reaction protocols have been developed (Hinnebusch, J. and Schwan, T.G. 1993. New method for plague surveillance using polymerase chain reaction to detect Yersinia pestis in fleas. J Clin Microbiol 31:1511-1514.) 4. DNA sequence analysis of 16S rDNA gene, comparing isolate to GenBank database.