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      Molecular Detection of Rickettsia typhi in Cats and Fleas

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          Abstract

          Background

          Rickettsia typhi is the etiological agent of murine typhus (MT), a disease transmitted by two cycles: rat-flea-rat, and peridomestic cycle. Murine typhus is often misdiagnosed and underreported. A correct diagnosis is important because MT can cause severe illness and death. Our previous seroprevalence results pointed to presence of human R . typhi infection in our region; however, no clinical case has been reported. Although cats have been related to MT, no naturally infected cat has been described. The aim of the study is to confirm the existence of R . typhi in our location analyzing its presence in cats and fleas.

          Methodology/Principal Findings

          221 cats and 80 fleas were collected from Veterinary clinics, shelters, and the street (2001-2009). Variables surveyed were: date of collection, age, sex, municipality, living place, outdoor activities, demographic area, healthy status, contact with animals, and ectoparasite infestation. IgG against R . typhi were evaluated by indirect immunofluorescence assay. Molecular detection in cats and fleas was performed by real-time PCR. Cultures were performed in those cats with positive molecular detection. Statistical analysis was carried out using SPSS. A p < 0.05 was considered significant.

          Thirty-five (15.8%) cats were seropositive. There were no significant associations among seropositivity and any variables. R . typhi was detected in 5 blood and 2 cultures. High titres and molecular detection were observed in stray cats and pets, as well as in spring and winter. All fleas were Ctenocephalides felis . R . typhi was detected in 44 fleas (55%), from shelters and pets. Co-infection with R . felis was observed.

          Conclusions

          Although no clinical case has been described in this area, the presence of R . typhi in cats and fleas is demonstrated. Moreover, a considerable percentage of those animals lived in households. To our knowledge, this is the first time R . typhi is detected in naturally infected cats.

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          Most cited references27

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          A highly sensitive and specific real-time PCR assay for the detection of spotted fever and typhus group Rickettsiae.

          A highly specific real-time polymerase chain reaction (PCR) assay was developed to detect spotted fever and typhus group rickettsiae using the citrate synthase gene as the target. The assay amplified rickettsial members of the spotted fever and typhus group including Rickettsia akari, R. australis, R. conorii, R. honei, "R. marmionii," R. sibirica, R. rickettsii, R. typhi, and R. prowazekii. The ancestral group rickettsia, R. bellii, did not produce a positive reaction, nor did other members of the order Rickettsiales or any non-rickettsial bacteria. The assay had a sensitivity of one target copy number per reaction as determined by serial dilutions of a plasmid containing a spotted fever group target sequence. This quantitative assay is useful for the enumeration of rickettsiae in clinical specimens and the diagnosis of rickettsial illnesses, when rickettsial numbers are very low.
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            Development of quantitative real-time PCR assays to detect Rickettsia typhi and Rickettsia felis, the causative agents of murine typhus and flea-borne spotted fever.

            Rickettsia typhi and Rickettsia felis are the etiologic agents of murine typhus and flea-borne spotted fever, respectively. We have constructed two quantitative real-time polymerase chain reaction (qPCR) assays to detect these pathogenic rickettsiae. The qPCR assays were developed utilizing unique sequences of the R. typhi and R. felis outer membrane protein B genes (ompB) to design the specific primers and molecular beacon probes. The assays were found to be species-specific and did not yield false-positive reactions with nucleic acid from other rickettsiae, orientiae, neorickettsiae or unrelated bacteria. In addition, the assays were sensitive enough to detect three target sequence copies per reaction and were capable of detecting R. typhi and R. felis nucleic acid in the cat flea, Ctenocephalides felis. These results demonstrate that two sensitive and specific qPCR assays have been successfully developed to detect and enumerate R. typhi and R. felis.
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              Typhus and typhuslike rickettsiae associated with opossums and their fleas in Los Angeles County, California.

              The recent discovery of cat fleas (Ctenocephalides felis) infected with a typhuslike rickettsia (designated the ELB agent) raises the question of whether similar rickettsial infections exist in wild cat flea populations. We verified the presence of the ELB agent and Rickettsia typhi in urban and suburban areas of Los Angeles, Calif. Opossums trapped in close proximity to the residences of human murine typhus cases in Los Angeles county and other areas within the city of Los Angeles were tested for the presence of typhus group rickettsiae by the polymerase chain reaction (PCR). The presence of rickettsiae in the spleen tissues of three opossums (n = 9) and in 66 opossum fleas (n = 205) was determined by PCR and was verified by dot blot and Southern transfer hybridization. Further analysis of the amplified PCR products generated by a series of primer pairs derived from either the 17-kDa antigen gene or the citrate synthase gene revealed that both R. typhi and the ELB agent were present in the tested samples. Dual infection was not noted in the samples; however, the fleas were infected with either R. typhi or the ELB agent. The presence of the ELB agent in the cat flea population may have implications for public health. Whether this agent is responsible for the mild cases of human murine typhus in urban and suburban areas of Los Angeles or in other endemic foci remains to be determined.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2013
                6 August 2013
                : 8
                : 8
                : e71386
                Affiliations
                [1 ]Department of Infectious Diseases. Corporació Sanitària i Universitària Parc Taulí - Institut Universitari Parc Taulí – Autonomous University of Barcelona, Sabadell, Barcelona, Spain
                [2 ]Department of Animal Health, Autonomous University of Barcelona, Bellaterra, Barcelona, Spain
                [3 ]Animal Shelter Company (Vallès Oriental), Granollers, Barcelona, Spain
                [4 ]Clinical Veterinary, Sabadell, Barcelona, Spain
                [5 ]Department of Medicine, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain
                University of Minnesota, United States of America
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: MMN FS. Performed the experiments: MMN IP. Analyzed the data: MMN IP FS. Contributed reagents/materials/analysis tools: MMN IP JP AO JM JC. Wrote the manuscript: MMN.

                Article
                PONE-D-13-20078
                10.1371/journal.pone.0071386
                3735526
                23940746
                67162f16-4229-4d43-9a3c-1db76752e83f
                Copyright @ 2013

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 14 May 2013
                : 2 July 2013
                Funding
                This study was supported by Ministerio de Sanidad y Consumo (Grant number FIS06/0433). Supported partially by Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III – FEDER, Spanish Network for the Research in Infectious Diseases -REIPI (Grant number RD06/0008); and Catalan Society of Infectious Diseases and Microbiology. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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