DIAGNOSIS
Detailed description of laboratory diagnostics and protocols are found in the World Organisation for Animal Health (OIE) Manual of Diagnostic Tests and Vaccines for Terrestrial AnimaU10 and the World Health Organization (WHO) GGuidelines on TularaemicΓv,.
Field examination of carcasses is not recommended when tularaemia is suspected, because of the real potential for human exposure and the risk of contaminating the environment. There is a high risk of infection of humans by direct contact with F. tularensis- infected tissues. Special precautions, including the wearing of gloves, masks and eyeshields are recommended when handling infective materials. Procedures should be performed within secure biosafety containment facilities (biosafety level 2 or 3). Diagnosis is based on the combined results of necropsy findings and the demonstration of F tularensis from the samples or tissues.F. tularensis appear as numerous small, Gram- negative bacteria in impression smears or in histological sections of spleen, liver, lung, kidney, bone marrow and lymph node as well as in blood smears. Francisella tularensis can be demonstrated specifically by direct or indirect fluorescent antibody tests. Immunohistochemical assay is a very useful and sensitive method for the detection of F tularensis in domestic and wild animals(6). Abundant bacterial antigen can be observed, often extracellularly, in foci of necrosis. Intracellular F tularensis is found in macrophages and giant cells and less frequently in other cell types.
Francisella tularensis can be identified by culture. The preferred samples for culture are, in acute cases, heart blood, spleen, liver or bone marrow and in subacute/ chronic cases, the granulomatous lesions. Owing to the highly fastidious culture requirements of F. tularensis, isolation can be difficult, as it grows poorly on ordinary culture media.
Francis medium, McCoy and Chapin medium or modified Thayer-Martin agar are recommended. The colonies are small, round and do not appear within the first 48 hours of incubation at 37°C. Isolation of F. tularensis from carcasses may be difficult because of overgrowth of other bacteria. Penicillin, polymyxin B and cycloheximide can be added to prepare selective culture media. If it is difficult to isolate F tularensis on primary culture, it may be isolated following inoculation of tissue suspension from suspect cases into laboratory animals, such as mice or guinea pigs.A variety of polymerase chain reaction (PCR) methods have been described for the detection of F. tularensis DNA in both clinical and environmental specimens. The gelbased PCR assays target the genes encoding the outer membrane proteins, fopA or tul4, show good specificity and allow for the rapid detection of F tularensis in specimens. However, Francisella-Yike endosymbionts of ticks (bacteria closely related to F. tularensis found in ticks) can produce non-specific positive results in these assays. Realtime PCR methods show no evidence of cross- reactivity with non-F tularensis bacteria (environmental bacteria and vector- borne organisms) and the detection limit of very low numbers of organisms increases the likelihood of detecting F. tularensis in environmental samples in which the number of organisms is low(11). Molecular methods are also applied to cultured F tularensis to provide resolution among the Francisella species, subspecies and within- subspecies strains(5).
Serology can be carried out for investigating exposure to F. tularensis in species that are relatively resistant to the disease, such as sheep, cattle, pigs, moose (Alces alces), dogs, cats or birds(10). A slide agglutination test, using one drop of stained bacteria and one drop of whole blood, is a widely used field method for screening the European brown hare populations in Central Europe. The standard serological test is the tube agglutination. Commercially available antigen for slide and tube agglutination tests is available (Bioveta Inc., Ivanovice na Hane, Czech Republic). Possible cross-reaction with Brucella abortus, B. melitensis, B. suis, Legionella spp. and Yersinia spp. may occur. The enzyme-linked immunosorbent assay (ELISA) allows an early diagnosis of tularaemia(10), and a commercial kit is also available (Serion Immundiagnostica GmbH, Wurzburg, Germany).