Outbreak Groupings

On August 2, 2002, a total of 163 prairie dogs were found at the exotic animal facility in Texas. These animals were classified into four groups: group A (bin 1, dead), group B (bin 1, live), group C (escapees), and group D (bin 2 and cages, healthy). Group A animals (n = 46) were collected during the last week of July through August 2, 2002. All group A animals had been housed in an uncovered metal tub (bin 1). The live animals remaining in bin 1 were classified as group B (n = 23), with most of the animals being emaciated, dehydrated, and lethargic. Group C (n = 36) comprised escaped prairie dogs that were running free throughout the facility. Group D prairie dogs (n = 58) were physically separated from both group B and C animals, and all group D animals were large, well-nourished, energetic, and noisy. Group D animals were housed in an uncovered metal tub (bin 2) and in several wire cages.

Animals from the Texas facility that had been sent to other locations made up two additional groups. Group E animals comprised seven prairie dogs that originated from the Texas facility, were distributed to pet shops in Texas, and recalled once F. tularensis was identified as the cause of the outbreak. Group F comprised 100 prairie dogs shipped from the Texas facility to the Czech Republic.

Culture Recovery of F. tularensis

All prairie dogs at the Texas facility (n = 163) were necropsied on site, and tissues were surgically removed. Appropriate biosafety measures were adhered to, including the use of closed front gowns, N95 masks, glasses, and gloves. Spleen and liver samples were spread onto cysteine heart agar supplemented with 9% sheep blood (CHAB). Plates were sealed with parafilm and transported in ice coolers (~15°C–20°C) until arrival at the CDC laboratory, Fort Collins, Colorado (~72 hours). Culture plates were then transferred to a biosafety level (BSL) 3 incubator at 37°C for 5 days and checked daily for F. tularensis growth. Some tissues were also spread onto CHAB medium containing antibiotics (10), incubated at 37°C for 7 days, and checked daily for F. tularensis growth. A culture isolate from prairie dogs shipped to the Czech Republic was grown at the State Veterinary Administration, Prague, Czech Republic, and submitted to our laboratory.

Spleen and liver tissues were injected into pathogen-free Swiss-Webster outbred mice for culture recovery of F. tularensis (IACUC Protocol 00-06-018-MUS). Tissues (~1 gm) from individual prairie dogs were ground with mortar and pestle, resuspended in 2 mL of saline and 0.5 mL of the tissue suspension was injected subcutaneously (s.c.) into mouse. All injections were performed in a BSL2 animal facility, and appropriate biosafety measures were followed, including the use of closed front gowns, N95 masks, glasses, and gloves. Animals were euthanized when signs and symptoms of tularemia were evident. After euthanasia was performed, 0.5 to 1.0 mL of whole blood was removed by cardiac puncture with a 1.0 mL tuberculin syringe. Liver and spleen tissues were surgically removed and spread onto CHAB with sterile wooden sticks. All healthy injected mice were euthanized 21 days after injection, and serum was tested for anti-F. tularensis antibody.

Direct Fluorescent Assay (DFA)

Slide touch preparations of tissues were prepared and heat-fixed immediately after necropsy at the Texas animal facility. On arrival at the laboratory, all slides were incubated with FITC-labeled rabbit anti-F. tularensis subsp. tularensis (SchuS4 strain) antibodies (CDC) for 30 minutes at room temperature. Slides were washed twice in phosphate-buffered saline, followed by a final rinse with dH₂O and viewed with a fluorescent microscope using the 40X objective and a 490 nm filter. Direct fluorescent activity was scored independently by two technicians experienced with F. tularensis DFA.

Serologic Findings

For all group B, C, and D animals, blood samples were collected from euthanized animals by cardiac puncture. Blood was collected into Microtainer brand serum separator tubes (Becton Dickinson, Franklin Lakes, NJ) and maintained at 4°C until arrival at the laboratory (~72 h). Serum was separated, heat-inactivated for 30 min at 56°C, and tested for F. tularensis specific antibodies by using a standard microagglutination assay (11). Briefly, serial dilutions of serum were incubated overnight with safranin-stained, formalin-killed F. tularensis subsp. tularensis (SchuS4 strain) cells at room temperature, and a titer was assigned reflecting the last well demonstrated full agglutination. Samples with a titer of 1:128 or greater were reported as positive.

Confirming F. tularensis

Prairie dogs were confirmed positive on recovery of an isolate with characteristic growth on CHAB and positive results by DFA or ISFtu2 polymerase chain reaction (PCR). Animals were considered presumptive positive if tissues tested positive by DFA or PCR, but no isolate was obtained. Prairie dogs were considered negative if all three diagnostic tests (culture, DFA, serologic testing) failed to detect any evidence of F. tularensis infection. For negative samples, recovery of culture included passage of the spleen and liver tissues through mice injected with F. tularensis.

F. tularensis Subtyping

For molecular subtyping, DNA was prepared after injection of a 1 μL loop of culture into 200 μL TE buffer. Cells were lysed by boiling at 95°C for 10 min. A differential PCR, based on the presence or absence of the ISFtu2 element (GenBank accession no. AY06), was performed by using 1 μL of the lysed bacterial supernatant and the primers TuF1705 (5′-GATAGATACACGCCTTGCTCACA-3′) and TuBR431(5′-ACCCAGCCAATGCCTAAATA-3′) (Y. Zhou, unpub. data). The amplification program included a denaturation cycle at 95°C for 2 min, followed by 35 amplification cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 1 min, and a final elongation cycle of 72°C for 5 min. PCR products were analyzed by agarose gel electrophoresis, followed by staining with EtBr and visualization with a Bio-Rad Gel Doc UV system (Bio-Rad Laboratories, Hercules, CA). For biochemical subtyping, the 96-well automated MicroLog MicroStation System with GN2 Microplates (Biolog Inc, Hayward, CA) was used. Microplates were set up and analyzed per the manufacturer’s instructions.

Statistical Analysis

McNemar’s test was used for statistical analysis. Sensitivities of different diagnostic tests were evaluated for their ability to detect F. tularensis in a given population of animals (either live or dead animals).

Laboratory Findings

F. tularensis–infected prairie dogs from the Texas animal facility were traced to Texas pet shops and animals shipped to the Czech Republic. From these three sources, 177 prairie dogs (1 animal whose illness initiated the investigation [12], 163 animals that remained onsite at the Texas facility, 7 animals recalled from Texas pet shops, and 6 animals shipped to the Czech Republic) were tested. Of these animals, 63 were confirmed positive, 13 were identified as presumptive positives, and 101 were confirmed negative for F. tularensis infection (Table 1).

F. tularensis Isolates from Infected Prairie Dogs

Because prairie dogs were sold commercially as pets and the risk for pet-to-human transmission was unknown, determining which groups (A–D) of animals were potentially infectious was important. Subtyping the F. tularensis isolates was also important, since this outbreak carried the threat of international dissemination. Therefore, our laboratory efforts focused on recovery of viable organisms. In total, 63 isolates were recovered (Table 1): 61 from prairie dogs at the Texas facility (groups A–C), 1 isolate from prairie dogs recalled from Texas pet shops (group E), and 1 isolate from prairie dogs distributed to the Czech Republic (group F).

Biochemical and Molecular Typing of F. tularensis Isolates

For subtyping of the 63 F. tularensis isolates (63 isolates described here, including 1 isolate that initiated the investigation, TX021935 [12]), a combination of biochemical and molecular typing was used. Biochemical characterization was performed on 15 isolates representative of all five groups of F. tularensis–positive animals (groups A, B, C, E, F). All 15 F. tularensis isolates were unable to use glycerol as a carbon source and thus were classified as Type B (data not shown). In addition, six representative isolates were tested for antimicrobial susceptibilities and demonstrated MICs consistent with those published previously for Type B (data not shown, 13).

Figure 1

Figure 1. Molecular subtyping of representative Francisella tularensis isolates from Groups A, B, C, E, and F prairie dogs. The expected size PCR fragments for F. tularensis subsp. tularensis (Type A) and holarctica...

To distinguish molecularly between Type A and Type B, a differential PCR based on the presence or the absence of the ISFtu2 element was performed (Y. Zhou, unpub. data). For Type A, a PCR product of 390 bp was amplified, whereas for Type B, a product of 1,249 bp was amplified. When ISFtu2 PCR subtyping was performed on all 63 isolates, all were shown to be Type B, including the single isolate received from the Czech Republic. Representative ISFtu2 PCR subtyping for the five groups (A, B, C, E, and F) of F. tularensis–positive animals is shown in Figure 1. Additional analysis with ISFtu2 restriction fragment length polymorphisms southern blotting demonstrated that the F. tularensis isolates were molecularly indistinguishable (data not shown).

Texas Facility Investigation (Groups A–D)

The animals remaining at the Texas facility (groups A–D) provided insight into how tularemia was transmitted among the prairie dogs. When necropsies were performed on animals in groups A–D, cannibalization, as indicated by partially eaten prairie dog carcasses, was noted among group A animals. In addition, all group A and most of group B animals displayed swollen submandibular lymph nodes, suggesting that all animals ingested the bacteria.

Because all prairie dogs from the Texas facility (groups A–D) were tested and classified as confirmed positive, presumptive positive, or negative (Table 1), diagnostic test sensitivities could be determined. From the 68 prairie dogs at the Texas facility that tested positive for F. tularensis by one or more diagnostic methods (culture, DFA, serologic testing), 61 isolates were recovered, yielding an overall culture recovery rate of 89.7%.

Detecting F. tularensis in Live, Infected Animals

For determining F. tularensis infection in live, infected animals, the sensitivities of culture versus DFA and serologic testing were compared (Table 2). Testing all 59 animals in groups B and C, confirmed 21 animals as F. tularensis–positive and 38 animals as F. tularensis–negative (Table 1). For the 20 animals confirmed positive by analysis of spleen and liver tissues, culture detected F. tularensis in 100% of cases. In contrast, both DFA and serologic testing detected F. tularensis in 10 of 20 animals, yielding a sensitivity of only 50% (Table 2). These differences were significant (p < 0.05) and demonstrate that culture of spleen and liver tissues is more sensitive than DFA or serologic testing for detecting F. tularensis in live, infected prairie dogs.

Since the outbreak was consistent with oropharyngeal tularemia, submandibular lymph nodes of group B animals were also analyzed. When lymph nodes were cultured, an additional case was confirmed by isolation of F. tularensis from animal B17 (Table 3). The bacterium was not cultured from the spleen and liver of this prairie dog even on passage of tissues through Swiss-Webster mice. This finding suggested that prairie dog B17 had recently ingested F. tularensis and that the infection was localized to the submandibular lymph nodes.

To determine if lymph node tissues were a better tissue source than either spleen or liver tissues for detection of F. tularensis, DFA was used for direct comparison of tissues from culture-positive group B animals (Table 2). When spleen and liver tissues were analyzed, the sensitivity of DFA was 50%, whereas for analysis of submandibular lymph node tissues the sensitivity of DFA was 89.5% (Table 2). This difference in sensitivities was significant (p < 0.05) and demonstrates that for cases of oropharyngeal tularemia, submandibular lymph node tissues are the most appropriate source for detecting infection by DFA.

Detecting F. tularensis in Fatal Cases of Tularemia

For fatal cases of tularemia, the sensitivity of culture and DFA was also compared (Table 2). Of the 47 animals in group A, 40 were confirmed positive, and 7 were presumptive positive for F. tularensis (Table 1). Direct fluorescence analysis of spleen and liver tissues identified all 47 animals as F. tularensis positive, yielding a sensitivity of 100%. In contrast, 40 F. tularensis isolates were obtained, yielding a sensitivity of 85.1%. These results were significant and demonstrated that DFA was more sensitive than culture for detection of F. tularensis in carcasses (p < 0.05).

Seropositivity and Decreased F. tularensis Levels in Live, Infected Animals

To test for evidence of seroconversion in live, infected animals, serum samples from group B prairie dogs were checked for anti-F. tularensis antibodies. Ten animals showed evidence of seroconversion, displaying titers against F. tularensis as great as 1:4,096 (Table 3). To our knowledge, this evidence is the first that prairie dogs can develop specific antibodies on infection with F. tularensis. In addition, F. tularensis was successfully recovered from the spleen of all 10 seropositive animals, suggesting that prairie dogs may become persistently infected.

Figure 2

Figure 2. Direct fluorescent assay (DFA) results on spleen tissues from a seronegative (panel a) and seropositive (panel b) prairie dog.

Comparison of DFA results for seropositive and seronegative prairie dogs indicated that the levels of F. tularensis in liver and spleen were greatly decreased in seropositive prairie dogs. In 7 of 10 seropositive prairie dogs, F. tularensis was not detectable by DFA analysis of spleen and liver tissues (Table 3, Figure 2, panel b). Conversely, 7 of 10 seronegative animals were positive by DFA analysis of spleen and liver tissues (Table 3, Figure 2, panel a). These findings demonstrate that seropositivity in prairie dogs leads to decreased levels of F. tularensis and may suggest that seropositive prairie dogs can survive an acute infection of oropharyngeal tularemia.