Francisella tularensis infection in a stone marten ( Martes foina ) without classic pathological lesions consistent with tularemia

Tularemia is a very complex emerging zoonotic disease caused by the Gram-negative bacterium Francisella tularensis. Depending on the route of infection, involved strains, and hosts, tularemia presents numerous clinical forms.^6,14,15 It is assumed that people become infected via the environment sensu lato, mainly after contact with infected animals, contaminated water, and aerosols or by bites from blood-sucking arthropods. ⁶ However, besides outbreak situations, the source of infection remains essentially unknown and difficult to demonstrate. ¹⁷ Although certain wild animal species are recognized as playing a role in the biological cycle of this bacterium, especially lagomorphs and rodents, the real spectrum of naturally infected species remains hypothetical. ¹¹ This is principally due to the fact that F. tularensis grows only on special culture media^5,18 and is not commonly considered in routine diagnostic investigations. Tularemia may be overlooked in some animal species and more particularly when anamnesis and/or tissue changes are inconsistent. Additionally, similarly to other fastidious organisms, the presence of F. tularensis might be masked by the overgrowth of other microorganisms or go undetected because of poor growth on inadequate isolation media. The current report describes the isolation and genetic characterization of F. tularensis subsp. holarctica belonging to the cluster circulating in Western Europe ⁴ from the spleen of a stone marten (or beach marten, Martes foina), with no pathological observations suggestive of tularemia.

A stone marten was found unresponsive and did not attempt to escape when approached by a game warden in Graenichen, Canton Aargau, Switzerland, in July 2012. The animal was culled and sent to the Centre for Fish and Wildlife Health, University of Bern, Switzerland, for a postmortem examination. A full necropsy was performed on the submitted marten, and representative tissue sections of multiple organs were routinely processed and stained with hematoxylin and eosin according to the standard protocol. ¹⁶ Additionally, special stains were used as appropriate.

Grossly, a 4-mm perforation of the skin just caudal to the left elbow region associated with the presence of subcutaneous light tan creamy material consistent with pus was observed. The suppurative exudate was dissecting the subcutis and extended over the entire left thoracic region and the cranial two-thirds of the left abdominal region. Additionally, a 0.4 cm × 1 cm perforating wound was found on the right front paw. No additional gross findings were observed.

Histologically, the lesions observed were consistent with complication of traumatic wounds, parasite infestation, and kidney disease. None of the lesions were suggestive of tularemia. The most severe tissue change was a chronic-active suppurative cellulitis with intralesional cocci observed in correspondence to the gross skin wounds. A mild myocarditis on a background of mild multifocal fibrosis and focal moderate thickening of the epicardium were seen in the heart. A parasitic pneumonia, with large numbers of nematode parasites, and multifocal parenchymal consolidation with rare small foci of parasite-associated necrosis were seen in the lungs. Grocott and Gram stains did not reveal the presence of detectable organisms. A mild multifocal lymphoplasmacytic interstitial nephritis with segmental tubular necrosis and mineralization along with occasional collection of tubular casts was seen in the kidneys. Multifocal mild mineralization in the lamina propria of the stomach and moderate numbers of cestodes in the lumen of the small intestine were seen in the gastrointestinal tract. Finally, a minute focus of coagulation necrosis was seen in the liver, while a diffuse, minimal lymphoid depletion was observed in the spleen.

Samples from spleen, lung, liver, kidney, and subcutaneous suppurative exudate were submitted for bacteriology analyses. Stone martens are unconventional hosts for tularemia. However, 2 factors were considered: an ongoing project investigating F. tularensis in the environment was being carried out in the laboratory, and an outbreak of tularemia in farmed Mustela sp., another genus belonging to the family Mustelidae, had been previously reported. ¹² Hence, the decision was made to test the stone marten for F. tularensis, despite the absence of typical signs or lesions. Lysates ²⁰ from the organs and a swab of the subcutaneous suppurative exudate were tested by real-time polymerase chain reaction (PCR) for F. tularensis as previously described. ²² The lung, spleen, and subcutaneous pus were positive by this F. tularensis–specific PCR.

All samples were cultivated onto trypticase soy agar (TSA) with 5% sheep blood, ^a chocolate agar with IsoVitaleX ^b (ChocIso), and F. tularensis selective agar (Ftsel; chocolate agar with brain–heart infusion broth ^b as base, 1% soluble hemoglobin powder, ^a 0.1% L-cysteine hydrochloride monohydrate, 0.1% D-glucose and Skirrow Campylobacter selective supplement ^a ) for 3 days at 37°C with 5% CO₂. Mixed flora grew from all samples on TSA, ChocIso, and Ftsel. After colony purification, a strain with colony morphology consistent with F. tularensis could be isolated from Ftsel agar plates inoculated with the spleen.

The strain isolated from the spleen was then genetically characterized. A lysate ²⁰ from the culture was prepared as DNA template. The strain was identified as subspecies holarctica by PCR. ¹ An additional PCR targeting the region of difference 23 was performed as previously described, ⁴ and the amplicon size corresponded to the emerging clone from Western Europe of F. tularensis subsp. holarctica that is circulating in France, Germany, Italy, Spain, and Switzerland.^4,7,10,19 Sequence analysis of the single nucleotide polymorphism markers B.18 (derived state) and B.19 (ancestral state) confirmed the subclade B4.FTNF002-00, ²¹ which is endemic in Switzerland. Multiple-loci variable number of tandem repeats (VNTR) analysis^2,13,19 was performed, and the isolated strain showed a VNTR profile that is circulating in Switzerland. ¹⁹

Awareness of tularemia is emerging in Europe, and increased resources are being invested in order to better understand this disease.^3,8,9,19 The focus on the improvement and refinement of diagnostic methods for F. tularensis identification of the past decade may contribute to an increase in the number of cases reported in human beings and in animals. However, only long-term systematic surveillance programs may answer if the present scenario is the outcome of an actual increment of F. tularensis on the territory or simply an increased attention for this bacterium.

In veterinary diagnostic laboratories, F. tularensis is normally investigated only in cases of suspicion of tularemia (mainly in primates, rodents, and lagomorphs) and typically only when consistent anamnesis is present. In the reported case, the marten was tested in the frame of a study investigating the biological cycle of F. tularensis in the environment sensu lato. It should be emphasized that the bacterium could be isolated only on an appropriate selective medium used specifically for this project and was present as a mixed culture. In other circumstances, it is very likely that this case would have been missed, which raises serious concerns not only for the missed diagnosis of a serious pathogen but also for biosafety, especially for that of the personnel who handled the carcass and samples. The isolation of F. tularensis from an animal species generally not screened for this bacterium and with no consistent pathological lesions may be challenging and dangerous for the personnel of diagnostic laboratories. Awareness of the potential presence of F. tularensis in unconventional hosts should be raised in countries where tularemia is endemic.

Finally, the biological role of martens, if any, in the environmental cycle of F. tularensis is not clear as is the case for many other wild animal species. More investigations are needed because the environmental “reservoir(s)” for tularemia is still unknown.