Molecular detection of Francisella tularensis in rodents collected from certain areas of Changbai Mountain / 中国热带医学

@#Abstract: Objective To explore the prevalence and bacterial strains of Francisella tularensis (F. tularensis) in wild rodents in Changbai Mountain area of China, and to further understand the epidemiological characteristics of F. tularensis infections in this area. Methods Wild rodents were captured from forest and forest-edge farmland from Kuandian County and Jianshi Forest District in the Changbai Mountain area, 2012-2014. DNA was extracted from the spleen tissues of the rodents, and the fopA gene of F. tularensis in wild rodents was detected using nested PCR. The infection rates were calculated for different areas and rat species. The bacterial subspecies of positive samples were identified using type-specific primers (C1/C4), and sequencing and comparative analysis were performed. Results A total of 133 wild rodents belonging to 6 rat species were captured. Among them, eight samples from three rat species (Apodemus agrarius, Apodemus peninsulae, Tscherskia triton ) were detected positive, with the overall positive rate of 6.01%. The positive rates of F. tularensis of Ji'an and Kuandian were 7.46% and 4.54%, respectively, and there was no difference in positive rates for different regions (χ2=0.117, P=0.732) and different rat species (χ2=0.641, P=0.986). The subspecies analysis showed that the detected 8 trains of F.tularensisall belonged to F.tularensis type B (F.subspecies subsp. holarctica). Genetic evolution analysis was performed on the fopA gene sequences of three positive samples (JA56, JA33, and JA38), which clustered together with Russia strains(CP009694.1, CP044004.1) and China strains (HM371344.1, HM371343.1) F.tularensis type B, with sequence similarities ranging from 99.21% to 99.47%. Conclusions Infection of F.tularensis subsp. holarctica existed in wild rodents in Changbai Mountain area of China, which suggests the existence of F.tularensis infection risks in this area.

Analyses of tularemia cases and their long-term results

@#Tularemia is a zoonotic disease and endemic in the northern hemisphere. The aim of this study was to evaluate the epidemiological, clinical and laboratory characteristics of tularemia patients, and to re-analyze their lymphadenopathy during the follow-up. The patients who were diagnosed with tularemia were reviewed. They were invited for the long term, physical and radiological evaluations. 69.8% patients had lived in rural areas. 54.7% patients were associated with animal husbandry, the 18.9% had contact with rodents. The most common form was the glandular type (62.3%). The frequency of granulomatous lymphadenitis was significantly higher in patients diagnosed later than 30 days from the onset of symptoms. Lymphadenopathy was undetectable in 61.5% patients, its severity was reduced in 38.4% patients compared to its state at the admission. In rural areas, avoiding contact with wild animals can ensure the protection from the pathogen. Public communities should be made aware of the disease.

Development of dual reporter imaging system for Francisella tularensis to monitor the spatio-temporal pathogenesis and vaccine efficacy

PURPOSE: Study on the pathogen and the pathogen-related disease require the information at both cellular and organism level. However, lack of appropriate high-quality antibodies and the difference between the experimental animal models make it difficult to analyze in vivo mechanism of pathogen-related diseases. For more reliable research on the infection and immune-response of pathogen-related diseases, accurate analysis is essential to provide spatiotemporal information of pathogens and immune activity to avoid false-positive or mis-interpretations. In this regards, we have developed a method for tracking Francisella tularensis in the animal model without using the specific antibodies for the F. tularensis. MATERIALS AND METHODS: A dual reporter plasmid using GFP-Lux with putative bacterioferritin promoter (pBfr) was constructed and transformed to F. tularensis live vaccine strain to generate F. tularensis LVS (FtLVS)-GFP-Lux for both fluorescence and bioluminescence imaging. For vaccination to F. tularensis infection, FtLVS and lipopolysaccharide (LPS) from FtLVS were used. RESULTS: We visualized the bacterial replication of F. tularensis in the cells using fluorescence and bioluminescence imaging, and traced the spatio-temporal process of F. tularensis pathogenesis in mice. Vaccination with LPS purified from FtLVS greatly reduced the bacterial replication of FtLVS in animal model, and the effect of vaccination was also successfully monitored with in vivo imaging. CONCLUSION: We successfully established dual reporter labeled F. tularensis for cellular and whole body imaging. Our simple and integrated imaging analysis system would provide useful information for in vivo analysis of F. tularensis infection as well as in vitro experiments, which have not been fully explained yet with various technical problems.

Tularemia, a re-emerging infectious disease in Iran and neighboring countries / 한국역학회지

OBJECTIVES: Tularemia is a zoonotic disease transmitted by direct contact with infected animals and through arthropod bites, inhalation of contaminated aerosols, ingestion of contaminated meat or water, and skin contact with any infected material. It is widespread throughout the northern hemisphere, including Iran and its neighbors to the north, northeast, and northwest. METHODS: In this paper, the epidemiology of tularemia as a re-emerging infectious disease in the world with a focus on Iran and the neighboring countries is reviewed. RESULTS: In Iran, positive serological tests were first reported in 1973, in wildlife and domestic livestock in the northwestern and southeastern parts of the country. The first human case was reported in 1980 in the southwest of Iran, and recent studies conducted among at-risk populations in the western, southeastern, and southwestern parts of Iran revealed seroprevalences of 14.4, 6.52, and 6%, respectively. CONCLUSIONS: Several factors may explain the absence of reported tularemia cases in Iran since 1980. Tularemia may be underdiagnosed in Iran because Francisella tularensis subspecies holarctica is likely to be the major etiological agent and usually causes mild to moderately severe disease. Furthermore, tularemia is not a disease extensively studied in the medical educational system in Iran, and empirical therapy may be effective in many cases. Finally, it should be noted that laboratories capable of diagnosing tularemia have only been established in the last few years. Since both recent and older studies have consistently found tularemia antibodies in humans and animals, the surveillance of this disease should receive more attention. In particular, it would be worthwhile for clinical researchers to confirm tularemia cases more often by isolating F. tularensis from infected humans and animals.

Genetic diversity of Francisella tularensis subsp.holarctica in China / 中华流行病学杂志

Objective To explore the genetic relationship between the Chinese and the foreign species of Francisella tularensis.Methods Based on our own findings and from the literature,17 SNP,4 INDEL,and 12 VNTR were selected for phylogenetic analysis on 39 strains of F.tularensis,including 10 strains of Chinese F.tularensis and 29 strains of foreign F.tularensis that had been sequenced and published.SNP-INDEL and MLVA were used for the separation and combination.Results Data from the combined analysis indicated that 3 strains of Chinese F.tularensis with Japanese FSC022 were assigned to B5;3 strains,with Swedish FSC200 to B1;3 strains with American OSU18 to B2 and 1 strain with French FTNF002-00,German F92,and American OR96246 to B4,respectively.10 strains of Chinese F.tularensis were assigned to 4 clades and the result demonstrated a wide diversity of F.tularensis subsp.holarctica in China.Conclusion A set of simple and robust typing tools for F.tularensis subsp.holarctica were established in this study.Based on the results,F.tularensis subsp.holarctica might have had its origins in Asia.

The first pediatric case of tularemia in Korea: manifested with pneumonia and possible infective endocarditis / 소아과

Tularemia is a potentially severe zoonotic disease caused by Francisella tularensis. A lack of awareness about tularemia can be embarrassing and could result in delayed treatment because of improper diagnosis. The diagnosis of tularemia is difficult, because the infections are rare and the clinical spectrum is broad. As only 1 adult case has been reported in Korea thus far, pediatricians in Korea may be unfamiliar with tularemia. We report our experience with a 14-year-old male adolescent with tularemia who presented with atypical pneumonia and possible infective endocarditis. Although the infectivity and mortality rates for tularemia are very high if left untreated, we did not suspect tularemia in this case until the incidental isolation of F. tularensis. The present case suggests that clinicians in Korea should be more aware of tularemia. This case also suggests that tularemia should be considered in undetermined cases of atypical pneumonia or acute febrile illness without local signs.

Differential diagnosis of cervical lymphadenitis mimicking malignancy due to tularemia: Our experiences.

Background: Tularemia is a disease caused by a Gram-negative coccobaci llus Francisella tularensis. This bacterium may cause different types of clinical pictures owing to acquisition route and entrance site, such as ulceroglandular, oropharyngeal, glandular, pneumonic, typhoid and ocular forms. Oropharyngeal tularemia (OPT) is the most common form of tularemia in some regions. OPT may cause tonsillopharyngitis followed by cervical lymphadenopathies (LAPs). Without treatment LAP may p ersist for several months and may mimic other diseases causing cervical LAPs. Materials and Methods: A total of six cases of OPT, fi ve male and one female, between 21 and 31 years old, diagnosed serologically and clinically recorded in GATA Haydarpasa Training Hospital were included in this study. Detailed story including the region they lived for last 6 months, their occupation, family and neighborhood story with similar complaints were obtained. Patient data were also obtained from manually written patients fi les and electronical patient fi le system. Formalin fi xed paraffi n embedded tissue blocks of all biopsy material were submitted for polymerase chain reaction (PCR) study for F. tularensis. Results: A total of six cases with head and neck mass following a story of tonsillopharyngitis admitted to different clinics including infectious diseases, ear-nose-throat and internal medicine in our tertiary care hospital. Physical examination revealed immobile, hard, conglomerated unilateral cervical lymphadenopathy in all cases. Histopathological examination revealed granulomatous infl ammation in four cases. Acute suppurative infl ammatory changes were also seen in two cases. Large necrotic areas mimicking casseifying necrosis were seen in two cases. PCR amplifi cation of F. tularensis genom from isolated deoxyribonucleic acids was successful in fi ve cases. Conclusion: Tularemia should be kept in mind in patients with tonsillopharyngitis not responding to penicillins and beta lactam antibiotics. Furthermore, persisting LAPs mimicking tumor with or without the story of previously experienced sore throat or tonsillopharyngitis in past few days or weeks should be evaluated for glandular or OPT. At this point, easily applicable serological tests such as tularemia micro-agglutination tests will confi rm the diagnosis of OPT. However, if lymph node were already sampled to exclude especially malignancy or T cell lymphoma, tularemia PCR test may be used to make a certain diagnosis.

Familial tularaemia.

Tularaemia is a zoonotic disease caused by Francisella tularensis . In this report, we have presented an early stage case of tularemia with fever and pharyngitis and two cases from the same non-endemic region with typical lymphadenitis. All three patients were treated with non-specific medications in healthcare centres, the treatment being directed towards symptoms resembling those of upper respiratory tract infections. However, there was no regression in their complaints. Because the first case had been treated earlier, his lymphadenopaties regressed and there was no suppuration. The other two cases, which had been suspected to be exposed to the same pathogen based on their histories, were at a mild acute phase and presented to our clinic with typical lymphadenitis. The diagnoses of each of the three patients were made serologically. An early clinical recovery was achieved in the first patient with streptomycin (1 x 1 g/day im) and doxycyline (2 x 100 mg/day peroral) therapy. The therapy was prolonged to 4 weeks in the other two cases according to lymph node response and no complications occurring in their follow-ups. It can be concluded that tularaemia should be considered in the differential diagnosis of patients with fever, pharyngitis, conjunctivitis and cervical lymphadenopathies that do not respond to β -lactam antibiotics.

An Outbreak of Tularemia in Western Black Sea Region of Turkey

The aim of this study was to investigate the source and the size of a tularemia outbreak in a village located in a non-endemic area. Five patients from the same village were admitted to hospital with the same complaints all within one week of September 2001. Tularemia was suspected and a diagnosis was made after physical and anamnesis examinations. The village was visited the same week that the patients were admitted to the hospital, in the January and April 2002. The villagers were examined and screened serologically by microagglutination method and the water sources were investigated bacteriologically. A total of 14 people were found to be infected from the outbreak and the oropharyngeal form was the only clinical presentation. Antibody titers ranged between 1 : 80 and 1 : 640. The patients responded well to the aminoglycoside plus tetracycline therapy. Examination of the pipewater and three springs revealed that all the water sources were contaminated by coliforms, however, Francisella tularensis could not be isolated in glucose-cystine medium. Antibody levels stayed stable or decreased seven months after. Tularemia had not been reported in this area before, so the first patients were misdiagnosed. In conclusion tularemia should be considered in differential diagnosis of patients with fever, sore throat and cervical lymphadenopaties.

Effect of the expression of transferrin receptor 1 on the invasion of Francisella into macrophages / 解放军医学杂志

Objective To evaluate the influential factors of iron acquisition during Francisella tularensis LVS infection of mouse macrophages.Methods F.tularensis LVS expressing green fluorescent protein was used to infect murine macrophage J774A.1 cells.Transferrin receptor 1(Tfr1)was detected with mono-antibody and visualized with a goat-anti mouse IgG conjugated to Alexa 594.The expression profile of 5 iron metabolism related genes of J774A.1 murine macrophages uninfected or infected with F.tularensis LVS was determined with real-time PCR.Immunoblot analysis was used to compare the Tfr1 expression of live Francisella infected macrophage with dead bacteria.Tfr1 knock-off in J774A.1 cells was performed with siRNA.The transfected cells were infected with Francisella for immunoblotting and microscopy and infection assay.Results It was revealed that the live vaccine strain of F.tularensis induced the expression of Tfr1 in host macrophages.Gene expression analysis indicated that F.tularensis LVS drove an active iron acquisition program with induction of Tfr1 and iron regulatory proteins(Irp1 and Irp2).It was shown by Western-blotting that the siRNA-Tfrc-1 could knock off about 75% of Tfr1 in J774A.1 cells.It was determined by infection assay that,Tfr1 was knocked off,the bacteria number at 1h infection with Francisella was not different from that of control(F=1.06,P=0.326 5),while it was decreased significantly after 24h of infection(F=24.12,P=0.000 6).Conclusions It is demonstrated that upregulation of the Tfr1 may be mediated by post-transcriptional regulation during early infection,but sustained later through increased expression of Irp 1 and Irp 2.Increased expression of Tfr1 expands the intracellular iron pool through transferrin-mediated delivery and may thus be readily available for uptaking by Francisella.Knocking off the expression of Tfr1 does not affect bacterial invasion.Francisella,however,may fail to proliferate in macrophages in which the expression of transferrin receptor has been suppressed.

A Case of Tularemia Caused by Francisella Tularensis / 대한임상병리학회지

Tularemia is a major laboratory acquired zoonoses caused by Francisella tularensis that have high virulence, and usually transmitted to humans from direct contact with infected wild animals like rabbits or insect vectors like ticks. Clinical tularemia can be divided with 6 major syndromes that are delineated by the mode of organism aquisition, in which ulceroglandular type is the most common. F. tularensis have 3 different biogroups which have homogeneous antigenecity, type A (biogroup tularensis), type B (biogroup palearctica) and biogroup novicida, and can be confirmed by serology most frequently. In the domestic area, there was no reports of tularemia in humans or presence of bacteria in the reservoirs. Authers experienced a case of tularemia which is suspected as F. tularensis type B, ulceroglandular type. A healthy 40-year-old man admitted the hospital for lymph node swelling in both axillary and upper arm area and for furuncles in both forearm and palm. He contacted with dead rabbit and eated it after cooking before 20 days from admission day. In laboratory cultures, F. tularensis did not grow in any of the routine or anaerobic culture media except for one blood agar plate at 5 days. After subculturing that to cystine containing chocolate agar plate at 37C degree, 5% CO2 incubator, we could see the accelerating growth of colony. In microbiological test, it was oxidase and urease negative. In acid production in cystine trypticase agar base, it was glucose positive and sucrose, maltose, glycerol negative. In agglutinating test, F. tularensis antiserum titer (Difco, USA) with isolates was 1:160 or over and antibody titer to F. tularensis antigen (Difco, USA) was 1:320 or over. Anti-F. tularensis-IF assay and Anti-F. tularensis-indirect-EIA with isolates were positive.