Francisella tularensis, F. noatunensis subsp. noatunensis y F. noatunensis subsp. orientalis – Culture; Molecular diagnosis (PCR).

Information 12-08-2018.

            Francisella is a genus of gram-negative, aerobic, coccobacillus, non-motile and non-encapsulated bacteria that inhabit diverse ecological niches. Members of the genus Francisella are associated with diverse clinical and environmental sources and include highly virulent human and animal pathogens, opportunistic human pathogens, fish pathogens, tick endosymbionts, and apparently free organisms that inhabit brackish water. Because some species of the genus can cause serious diseases in animals and humans, the detection and identification of species of this genus is fundamental for the treatment of patients and help to prevent or reduce important economic losses in aquaculture.           

            Due to the diversity of environmental niches and the limited genetic diversity among Francisella species, taxonomic relationships between this genus have been difficult to resolve. In recent years, the known diversity within the Francisella genus has expanded significantly. The Francisella species have been isolated from various clinical and environmental sources, and include highly virulent human and animal pathogens (F. tularensis), opportunistic human pathogens (F. novicida, F. hispaniensis, F. philomiragia, and F. opportunistica), pathogens of fish (F. noatunensis) and marine mollusks (F. halioticida), endosymbionts of ticks and ciliates (F. persica and F. endociliophora), and free-living isolates that inhabit seawater (F. salina, F. uliginis, F. novicida TX07-6608) and cooling systems (Francisella spp. W12 -1067, F. frigiditurris and Allofrancisella guangzhouensis).

            The pathogenic species of the genus Francisella are intracellular facultative bacteria, among them Francisella tularensis, a highly virulent bacterium that causes tularemia zoonotic disease. F. tularensis infects rabbits, mice, squirrels, beavers, rats, weasels, foxes, mink, sheep, cats, dogs, horses, pigs, many other wild mammals, more than 25 species of birds, fish species, many blood animals cold and invertebrates. According to epidemiological studies, rodents (mice, rats and squirrels) and hares are the most important reservoirs of tularemia. In these small mammals, the disease usually progresses rapidly, producing distributed necrosis in multiple organs, including the spleen, followed by death, usually occurring in a few days. Alternatively, a type of chronic disease with granulomas in the liver has been described.

            Humans can be infected by direct contact with an infected animal (through cuts or scratches on the skin, tissue damage, conjunctival sac, or oropharyngeal mucosa), through a bite of hematophagous arthropods (e.g., fleas, lice, mosquitoes, and bed bugs), ingesting contaminated water or food, or inhaling contaminated dust or aerosols. The clinical presentation in humans depends on the route of infection and varies in symptoms and severity. F. tularensis is capable of infecting numerous cell types, including dendritic cells, neutrophils, macrophages and hepatocytes or endothelial cells, but it is believed that they replicate in vivo mainly in macrophages. The general effects of tularemia are fever, chills, muscle pain or weakness, and lack of energy. There are six main clinical-pathological types of tularemia with different effects, however, up to 80% of cases are ulceroglandular (skin ulcers and swollen lymph nodes) and are produced by direct contact with infected animals. The different forms of tularemia described are: the ulceroglandular, glandular, oculoglandular, oropharyngeal, pneumonic (respiratory) and septicemic forms. Regardless of the route of entry, F. tularensis can spread from the site of initial infection to the lungs, where it can cause respiratory tularemia, the most severe form of the disease.

Three main subspecies of F. tularensis have been described: tularensis, holarctica, and mediasiatica. These subspecies differ in virulence and geographic origin, but all cause a fulminating disease in mice that is similar to tularemia in humans. Only two subspecies cause the great majority of clinical tularemia in mammals: F. tularensis subsp. tularensis (type A), endemic in North America and associated with increased mortality in humans, and F. tularensis subsp. holarctica (type B) that causes less severe diseases throughout the Northern Hemisphere. F. tularensis subsp. mediasiatica is mainly found in Central Asia and information on its virulence is limited. It is believed that F. tularensis subsp. mediasiatica has a relatively low virulence in humans, and only rare cases of human diseases caused by this subspecies are known.

Due to its low infectious dose, its ability to be transmitted to humans through multiple pathways, and its potential to cause a life-threatening disease, F. tularensis is one of the most infectious bacterial pathogens known and has been classified as a potential agent of biological warfare and bioterrorism, a category A biological weapons agent. Due to this classification, level 3 biosafety facilities (BSL3) are required to study the type A strain.

In addition to F. tularensis, the genus Francisella also includes opportunistic pathogens. F. novicida, F. hispaniensis and F. philomiragia are rarely pathogenic and only cause disease in immunocompromised humans. There is an ongoing debate about whether F. novicida should be classified as a separate species of the genus Francisella, or as a subspecies of F. tularensis. However, there are clinical, ecological, genomic, virulence and pathogenic differences between these two bacteria that, when considered together with genetic identity, justify maintaining F. novicida and F. tularensis as separate species. F. novicida infections are rare in humans and have only been reported in immunocompromised persons or people with underlying health problems, which reflects that F. novicida is an opportunistic pathogen. However, F. novicida can infect mice, causing a disease similar to tularemia, shares ≥97.7% sequence identity and possesses homologous virulence factors with F. tularensis subsp. tularensis. Therefore, this species is classified by many authors as a fourth subspecies of F. tularensis, and is widely used as a model to study the virulent subspecies F. tularensis subsp. tularensis, with the advantage of its easy genetic manipulation without the requirement of BSL3.

Francisella noatunensis is the causal agent of the Franciselosis of fish. In recent years, bacteria belonging to the species Francisella noatunensis have emerged as serious pathogens of various fish species, both wild and from fish farms, in various geographical regions around the world. Strains of F. noatunensis are highly pathogenic for fish and can cause high mortality and losses, causing epidemics throughout the world, especially in fish farms. Although the species F. tularensis has been associated with infections in fish since 1970, this bacterium has not been associated with fish disease in recent years. In light of the recent description of pathogenic Francisella species of fish, which share several phenotypic traits with F. tularensis, it is thought that these previous descriptions may be due to erroneous identifications. Similarly, it is believed that franciselosis is not a recent disease, but that the usual diagnostic methods have allowed its identification in the epidemics of recent years. Two subspecies of F. noatunensis have been described. F. noatunensis subsp. noatunensis that affects commercially important cold water fish such as Atlantic cod and Atlantic salmon. Meanwhile, F. noatunensis subsp. orientalis is the causative agent of francisellosis in warm-water fish, including tilapia, striped water bass, perch trilineata and ornamental fish.

All reported incidences of francisellosis in fish are manifested in a similar manner, as systemic, chronic, and granulomatous infections, which result in varying degrees of mortality. Externally, diseased fish do not show specific clinical signs. Common manifestations include the observation of a large number of protruding white nodules of various sizes, called granulomas, in the spleen, kidney and liver. However, practically any type of tissue can be affected, and the associated pathological changes in the gills, heart, testicles, muscles, brain and eye have also been described. Factors such as temperature and coinfection with other pathogenic bacteria seem to affect the mortality rate. On the other hand, it should be noted that there are no signs of zoonotic potential for humans, strains of F. noatunensis cannot grow at 37ºC and, therefore, are not virulent to humans.

Due to the high infectivity of F. noatunensis, a high density of susceptible hosts in the aquatic environment of fish farming, a pattern of chronic disease, and largely internal disease, francisellosis has caused great economic losses in aquaculture.

Tests carried out in IVAMI:

  • Detection by isolation of Francisella tularensis in selective culture media.
  • Detection by molecular diagnosis (PCR) of the species F. tularensis, F. noatunensis subsp. noatunensis and F. noatunensis subsp. Orientalis.

Recommended sample:

  • For the analysis of Francisella tularensis in humans or animals, samples of blood extracted with EDTA (2 to 5 mL), exudate from a skin lesion, or exudate obtained by puncture of the affected lymph node are recommended.
  • For the detection of the subspecies of F. noatunensis in fish, the internal organs in which it is mainly located, preferably, spleen, kidney and liver.

Conservation and shipment of the sample:

  • Refrigerated (preferred) for less than 2 days.
  • Frozen: more than 2 days.

      Note: If blood is sent for subsequent plasma separation in the laboratory, the blood sample should not be frozen and should be sent within 48 hours.

Delivery of results:

  • Detection by isolation of Francisella tularensis: 5 days.
  • Molecular detection (PCR) of the species F. tularensis, F. noatunensis subsp. noatunensis and F. noatunensis subsp. orientalis: 24 a48 hours.

Cost of the test:

  • Molecular detection (PCR) of the species F. tulariensis, F. noatunensis subsp. noatunensis and F. noatunensis subsp. orientalis: Consult