Immunohistochemical demonstration of Francisella tularensis in lesions of cats with tularemia.

An immunohistochemical test was developed and validated for detection of Francisella tularensis antigen in tissues of cats with fatal tularemia. Ten cases of naturally occurring tularemia in cats were positive both by isolation of F. tularensis and immunohistochemical identification of F. tularensis antigen. Nine additional cases with lesions typical of tularemia were positive for F. tularensis antigen, although bacterial cultures were not performed. Immunohistochemical identification of F. tularensis in formalin-fixed tissue is valuable for establishing a rapid etiologic diagnosis under circumstances where fresh tissues may not be available for isolation and identification of the organism.     

Tularemia as a cause of fever in a squirrel monkey

CASE DESCRIPTION: A 3-year-old female squirrel monkey (Saimiri sciureus sciureus) was examined because of sudden onset of lethargy and fever. CLINICAL FINDINGS: On initial examination, the monkey was weak and febrile and had petechiae on both thoracic limbs. Following collection, blood samples were slow to clot. During the next week, the monkey developed anemia and thrombocytopenia; Francisella tularensis was isolated from blood samples. TREATMENT AND OUTCOME: Treatment with gentamicin resulted in the monkey's gradual return to health, but inguinal lymphadenopathy developed after drug administration was discontinued. Francisella tularensis was isolated from a fine-needle aspirate of an enlarged lymph node. Treatment with streptomycin resulted in resolution of infection. By use of biochemical and molecular tests, the microbial isolate was characterized as F tularensis subsp holarctica. Results of a microagglutination assay confirmed that the monkey had developed serum antibodies against F tularensis. CLINICAL RELEVANCE: With timely diagnosis, treatment of tularemia in the squirrel monkey was successful. Francisella tularensis is the cause of a highly infectious zoonotic disease, and infection with this microorganism is enzootic in wildlife throughout the Northern Hemisphere. Tularemia should be considered in the differential diagnosis of febrile disease in animals of any species. Even limited or indirect exposure of humans or other animals to outdoor environments in which reservoir hosts and arthropod vectors are present can lead to transmission of F tularensis. Francisella tularensis is a class A agent of bioterrorism, and all cases of tularemia (regardless of species) should be reported to public health officials.     

Characterization of a wild-type strain of Francisella tularensis isolated from a cat.

Francisella tularensis type A is the primary cause of tularemia in animals and humans in North America. The majority of research on F. tularensis has been done with the attenuated live vaccine strain (LVS), which is a type B, but very few wild-type F. tularensis strains have been characterized. A gram-negative coccobacillus that was isolated in pure culture from the lungs of a cat that died after being lost for 5 days was received for identification at the Virginia-Maryland Regional College of Veterinary Medicine Teaching hospital. The isolate (strain TI0902) was not identified (or was misidentified) by commercial identification systems; however, it was identified as F. tularensis subspecies tularensis (type A) by sequencing a portion of the 16S ribosomal RNA gene. Furthermore, repetitive extragenic palindromic sequences-polymerase chain reaction amplified a 4-kb DNA fragment from TI0902 that was characteristic of F. tularensis type A but not type B. The electrophoretic profile of the lipopolysaccharide of strain TI0902 was identical to that of the LVS by Western blotting with antiserum to LVS. The protein-enriched outer membrane of strain TI0902 contained 6-8 proteins, which were similar in molecular size to those from the LVS. Electron microscopy of negatively stained and alcian blue-stained LVS and TI0902 cells showed that both strains were coccobacillary in shape and may be encapsulated. However, after mouse challenge, the TI0902 strain was clearly more virulent than the LVS strain. Results of this study indicate that the genotype and phenotype of wild-type F. tularensis type A strain TI0902 is similar, but not identical, to that of the LVS strain. Further studies will help determine whether pathogenesis and host-pathogen interactions are also similar between the 2 strains.     

Seroprevalence of tularemia in wild bears and hares in Japan

Tularemia is a zoonotic disease caused by Francisella tularensis. The distribution of the pathogen in Japan has not been studied well. In this study, seroprevalence of tularemia among wild black bears and hares in Japan was determined. Blood samples collected from 431 Japanese black bears (Ursus thibetanus japonicus) and 293 Japanese hares (Lepus brachurus) between 1998 and 2009 were examined for antibodies against F. tularensis by micro-agglutination test (MA) or enzyme-linked immunosorbent assay. By subsequent confirmatory tests using western blot (WB) and indirect immunofluorescence assay (IFA), eight sera from Japanese black bears were definitely shown to be seropositive. All of these eight bears were residents of the northeastern part of main-island of Japan, where human tularemia had been reported. On the other hand, no seropositive Japanese hares were found. These results suggest that Japanese black bears can serve as sentinel for tularemia surveillance and may help understand the distribution of F. tularensis throughout the country. This is the first report on detection of antibody to F. tularensis in black bears of Japan.     

Acute tularemia in three domestic cats.

Acute Francisella tularensis infection in 3 domestic cats was presumptively diagnosed on the basis of clinical signs and lesions and confirmed by culturing or immunofluorescent demonstration of the organism. Clinical findings include marked signs of depression, oral/lingual ulceration, regional or generalized lymphadenomegaly, hepatosplenomegaly, panleukopenia with severe toxic change of neutrophils, and hyperbilirubinemia with bilirubinuria. Lesions found at necropsy included icterus, oropharyngeal and lingual ulceration, multiple foci of necrosis in lymph nodes, spleen, liver, and lung, and severe segmental or diffuse enterocolitis. Results of serologic testing for F tularensis was positive in only 1 of the 3 cats. The organism was cultured aerobically from several tissues, including aspirated bone marrow obtained before death in 1 cat. Results of an indirect fluorescent antibody test, performed on fresh and formalin-fixed tissues of all cats, were positive. Because of the severe clinical course, opportunity to evaluate therapeutic regimens was not possible. Until now, confirmed diagnosis of feline tularemia only has been made retrospectively, in instances when cats were suspected to have transmitted infection to human beings in whom the primary diagnosis was made. The findings in this report provide a basis for presumptive diagnosis that will help to minimize public health risk associated with this potentially fatal zoonotic disease.     

Francisella tularensis: an arthropod-borne pathogen

Arthropod transmission of tularemia occurs throughout the northern hemisphere. Few pathogens show the adaptability of Francisella tularensis to such a wide array of arthropod vectors. Nonetheless, arthropod transmission of F. tularensis was last actively investigated in the first half of the 20th century. This review will focus on arthropod transmission to humans with respect to vector species, modes of transmission, geographic differences and F. tularensis subspecies and clades.     

Surveillance of Francisella tularensis infection in dogs in Bratislava]

Out of 548 serologically investigated dogs from Bratislava and other regions of Slovakia and Moravia, antibodies to F. tularensis were found in 16.4% (Tabs. I, II). In all the investigated groups of dogs from the region of Bratislava the highest seroprevalence by F. tularensis was recorded in watch dogs kept on farms and in cooperatives--37.5% and in rambling dogs--20.7% (Tab. I). The highest seropositivity was found in one to three year old dogs--22.2% (Tab. III). A similar degree of seroprevalence was also observed in one to three years old police dogs which came from the endemic region of tularemia--West Slovakia (19.3%) and East Slovakia (25.6%)--Tab. IV. These facts indicate the persistence of active natural foci in these regions. Serological investigations of the relatively great number of dogs from different regions of Slovakia showed that the presence of F. tularensis antibodies in this animal species, mainly in the watch dogs group, can be taken as a convenient marker or indicator of the existence of active natural foci of tularemia and as a suitable component for surveillance of this diseases.     

Epizootic of tularemia in an outdoor housed group of cynomolgus monkeys (Macaca fascicularis)

Tularemia is a highly contagious infectious zoonosis, transmissible by inoculation, ingestion, or inhalation of the infectious agent Francisella tularensis. The disease is perpetuated by infected rodents, blood-sucking arthropods, and by contaminated water. Therefore, nonhuman primates housed outdoors may be at risk for exposure. An epizootic of F. tularensis occurred in an indoor/outdoor-housed group of cynomolgus monkeys (Macaca fascicularis) at the German Primate Center. Tularemia was diagnosed in 18 out of 35 animals within a period of 2 years. Six animals died with unspecific clinical symptoms; 12 animals developed seroconversion and were still alive. Pathologic findings were similar in all monkeys that died and resembled the clinical picture of the human disease, including an ulceroglandular syndrome with local lymphadenopathy, gingivostomatitis, and systemic spread, with manifestations such as subacute necrotizing hepatitis, granulomatous splenitis, and pneumonia. Tularemia was diagnosed by culture, real-time polymerase chain reaction, and ELISA techniques. This is the largest outbreak in nonhuman primates and the first report of tularemia in cynomolgus monkeys. An overview of the recent literature about tularemia in nonhuman primates is given.     

Detection of antibodies to Francisella tularensis in cats

Blood samples were obtained from privately owned cats in Connecticut and New York State, USA in 1985-1990, and analyzed for evidence of Francisella tularensis, the etiologic agent of tularemia. Of the 91 sera tested by microagglutination (MA) methods, 11 (12%) contained antibodies to F. tularensis. Analyses of the same sera by indirect fluorescent antibody (IFA) staining methods revealed 22 (24%) positives. There was good agreement in results of both tests (73% concordance). However, we measured higher titers (1:80 to 1:640) with IFA analysis than by MA methods (1:80 to 1:160). Both tests were suitable for general screening purposes. The DNA of F.tularensis was not detected in the 24 antibody-positive sera tested. Cats living in Connecticut and New York State were naturally exposed to F.tularensis or a closely related organism. With exposure to ticks, other biting arthropods, mice, and rabbits, cats are at risk for acquiring F.tularensis infections and can be an important source of information on the presence of this agent in nature.     

Biochemical responses and oxidative stress in Francisella tularensis infection: a European brown hare model

Background

The aim of the present study was to investigate biochemical and oxidative stress responses to experimental F. tularensis infection in European brown hares, an important source of human tularemia infections.

Methods

For these purposes we compared the development of an array of biochemical parameters measured in blood plasma using standard procedures of dry chemistry as well as electrochemical devices following a subcutaneous infection with a wild Francisella tularensis subsp. holarctica strain (a single dose of 2.6 × 10⁹ CFU pro toto).

Results

Subcutaneous inoculation of a single dose with 2.6 × 10⁹ colony forming units of a wild F. tularensis strain pro toto resulted in the death of two out of five hares. Plasma chemistry profiles were examined on days 2 to 35 post-infection. When compared to controls, the total protein, urea, lactate dehydrogenase, aspartate aminotransferase and alanine aminotransferase were increased, while albumin, glucose and amylase were decreased. Both uric and ascorbic acids and glutathione dropped on day 2 and then increased significantly on days 6 to 12 and 6 to 14 post-inoculation, respectively. There was a two-fold increase in lipid peroxidation on days 4 to 8 post-inoculation.

Conclusions

Contrary to all expectations, the present study demonstrates that the European brown hare shows relatively low susceptibility to tularemia. Therefore, the circumstances of tularemia in hares under natural conditions should be further studied.     

Tularemia in range sheep: an overlooked syndrome?

Abortion and death caused by Francisella tularensis were well recognized in range flocks of domestic sheep in Idaho, Montana, and Wyoming in the first 6 decades of the 20th century. The current report describes 4 episodes of tularemia in 3 range flocks in Wyoming and South Dakota in 1997 and 2007 (1 flock was affected twice). Flock owners reported that ticks were unusually numerous and commonly present on sheep during outbreaks. Tularemia presented as late-term abortions (3 episodes) or listlessness and death in lambs and, to a lesser extent, ewes (1 episode). Lesions were multifocal pinpoint necrotic foci in tissues, particularly spleen, liver, and lung. An immunohistochemical procedure demonstrated F. tularensis, particularly in necrotic foci. The diagnosis was corroborated by bacterial isolation and, in individual cases, by serology, fluorescent antibody assay, and/or polymerase chain reaction detection of F. tularensis. Diagnosticians in endemic areas should include tularemia as a differential diagnosis when investigating late-term abortions or outbreaks of fatal illness in young lambs, particularly in years of high tick activity and when characteristic necrotic foci occur in spleen, liver, and lung.     

Tularemia

Tularemia is a rare but potentially fatal disease that develops in numerous wild and domestic animals, including lagomorphs, rodents, cats, and humans. The disease occurs throughout much of the United States and should be considered in the differential diagnosis of acute febrile illness, particularly when risk factors such as contact with wild mammals or tick exposure are present. Veterinarians may be at increased risk of acquiring tularemia from contact with infected animals, but standard precautions should greatly reduce this risk. Outbreaks of tularemia warrant investigation, especially given the possibility of the use of F tularensis as an agent of bioterrorism.     

Tularemia in Alaska, 1938 - 2010

Tularemia is a serious, potentially life threatening zoonotic disease. The causative agent, Francisella tularensis, is ubiquitous in the Northern hemisphere, including Alaska, where it was first isolated from a rabbit tick (Haemophysalis leporis-palustris) in 1938. Since then, F. tularensis has been isolated from wildlife and humans throughout the state. Serologic surveys have found measurable antibodies with prevalence ranging from < 1% to 50% and 4% to 18% for selected populations of wildlife species and humans, respectively. We reviewed and summarized known literature on tularemia surveillance in Alaska and summarized the epidemiological information on human cases reported to public health officials. Additionally, available F. tularensis isolates from Alaska were analyzed using canonical SNPs and a multi-locus variable-number tandem repeats (VNTR) analysis (MLVA) system. The results show that both F. t. tularensis and F. t. holarctica are present in Alaska and that subtype A.I, the most virulent type, is responsible for most recently reported human clinical cases in the state.     

Rapid diagnosis and quantification of Francisella tularensis in organs of naturally infected common squirrel monkeys (Saimiri sciureus)

Francisella tularensis, a small Gram-negative facultative intracellular bacterium, is the causative agent of tularaemia, a severe zoonotic disease transmitted to humans mostly by vectors such as ticks, flies and mosquitoes. The disease is endemic in many parts of the northern hemisphere. Among animals, the most affected species belong to rodents and lagomorphs, in particular hares. However, in the recent years, many cases of tularaemia among small monkeys in zoos were reported. We have developed a real-time PCR that allows to quantify F. tularensis in tissue samples. Using this method, we identified the spleen and the kidney as the most heavily infected organ containing up to 400 F. tularensis bacteria per simian host cell in two common squirrel monkeys (Saimiri sciureus) from a zoo that died of tularaemia. In other organs such as the brain, F. tularensis was detected at much lower titres. The strain that caused the infection was identified as F. tularensis subsp. holarctica biovar I, which is susceptible to erythromycin. The high number of F. tularensis present in soft organs such as spleen, liver and kidney represents a high risk for persons handling such carcasses and explains the transmission of the disease to a pathologist during post-mortem analysis. Herein, we show that real-time PCR allows a reliable and rapid diagnosis of F. tularensis directly from tissue samples of infected animals, which is crucial in order to attempt accurate prophylactic measures, especially in cases where humans or other animals have been exposed to this highly contagious pathogen.     

Tularemia: emergence/re-emergence

Francisella tularensis is a gram-negative coccobacillus and the etiologic agent of the zoonotic disease tularemia. First described in 1911 in Tulare County, California, it has since been reported throughout the Northern Hemisphere, with natural infections reported among an unusually wide range of vertebrates and invertebrates. In recent years, tularemia has emerged in new geographic locations, populations, and settings. This review will serve to highlight mechanisms contributing to the recent emergence of tularemia as well as a repertoire of diagnostic tools useful for detecting and diagnosing disease.     

An outbreak of tularemia in mink

An outbreak of tularemia in farm raised mink is reported. Twenty-six of approximately 5000 mink succumbed within a 10 day period. Prodromal signs were minimal. Necropsy revealed necrotic nodules scattered in the parenchyma of the lungs, liver, spleen, and mesenteric lymph nodes. Francisella tularensis was isolated from spleens, livers and lungs.     

An endemic case of tularemia in the mountain hare (Lepus timidus) on the island of Stora Karls?

In September 1983, an epizootic of unknown cause broke out among the mountain hares on the island of Stora Karls?, which is located outside the coast of Gotland. One hare was sent in the National Veterinary Institute for post-mortem examination. The hare showed lesions in liver, spleen, and bone-marrow typical for tularemia. The specimens were positive for Francisella tularensis with fluorescent-antibody test. Tularemia has not been observed earlier in this part of Sweden. It is supposed that the disease might have been spread to Stora Karls? with migrating birds from Finland, where there was an epizootic of tularemia during the summer 1983.     

Tularemia in a group of nonhuman primates.

In an episode of tularemia in a Canadian zoologic garden, three black and red tamarins (Sanguinus nigricollis) and one talapoin (Cercopithecus talapoin) died. A second talapoin developed abscesses in the tongue and submandibular area; this animal recovered with treatment. Francisella tularensis was isolated from lung, liver, and spleen from each dead monkey and from pus collected from the tongue abscess of the sick talapoin. The attending veterinarian contracted the disease from a tamarin bite. The source of the disease was identified as wild ground squirrels, and the causative organism was recovered from the liver and spleen of one squirrel and from fleas found on it.     

An isolated cryptococcal urinary tract infection in a cat

A male domestic shorthaired cat was presented for evaluation of stranguria and pollakiuria. A cryptococcal urinary tract infection (UTI) was diagnosed cytologically and via fungal culture. No evidence of systemic involvement was found. Chronic renal failure was a concurrent disease in this cat. Treatment consisted of oral fluconazole. Clinical signs resolved after 2 weeks of therapy, and fluconazole was discontinued after 6 months when negative urine culture results indicated resolution of the infection. This case demonstrates that correct identification of cryptococcal UTI allows for administration of therapy that can be associated with resolution of clinical signs.     

An outbreak of Francisella tularensis in captive prairie dogs: an immunohistochemical analysis.

An immunohistochemical assay was developed and tested for detection of Francisella tularensis lipopolysaccaride antigen in tissues of captive prairie dogs (Cynomys ludovicianus). Tissues from 59 cases of F. tularensis were examined by this technique, which was corroborated by direct fluorescent antibody assay and direct isolation of the organism. In infected prairie dogs, studies indicated multiple, severe, necroprurulent foci occurring in the liver, lung, spleen, terminal ileum, and mandibular lymph node. Immunohistochemical analysis of the same formalin-fixed tissues indicated the presence of F. tularensis antigen in neutrophils and macrophages of these lesions and occurring extracellularly in areas of necrosis. This report demonstrates that immunohistochemical analysis is a rapid procedure that can be used to determine the pathogenesis of F. tularensis in rodent populations.