Abstract
Classic murine typhus, caused by Rickettsia typhi, is endemic in the continental United States in areas of Texas and southern California. We conducted an environmental investigation in an urban area of Los Angeles identified as the probable exposure site for a case of murine typhus. Four Rattus norvegicus heavily infested with Xenopsylla cheopis (average 32.5 fleas per animal, range 20–42) were trapped, and fleas, blood, and tissues were collected. DNAs from all specimens were tested for R. typhi and Rickettsia felis using a TaqMan assay targeting the rickettsial citrate synthase gene. Although rickettsiemia was not detected, DNA of R. felis was detected in at least one tissue from each rat. Tissues from 3 rats were also positive for R. typhi DNA. R. typhi and R. felis DNAs were detected in fleas collected from each animal with average minimal infection rates of 10% and 32.3%, respectively. Although R. typhi still circulates in urban Los Angeles in the classic Oriental flea–rat cycle, R. felis is more prevalent, even in this association.
Introduction
Infection in the flea is initiated when rickettsiae are ingested with the blood meal and enter the epithelial cells of the midgut. After massive replication within the cytoplasm of the midgut cells, R. typhi are released in the gut lumen and subsequently excreted with the feces and can be transmitted back to a susceptible vertebrate during subsequent feeding (Traub and Wisseman 1978, Arango-Jaramillo et al. 1984, Azad 1990). Transmission to humans presumably results from contamination of broken skin, the respiratory tract, or conjunctivae with infectious flea feces or flea tissues. Murine typhus presents with high fever, headache, generalized pain, weakness, and often other nonspecific symptoms or atypical manifestations (Taylor et al. 1986, Purcell et al. 2007, Civen and Ngo 2008, Gikas et al. 2009).
Since the early 20th century when murine typhus was first described in the United States, it was widely diagnosed and reported (Maxcy 1926, 1928). However, since then, its incidence has sharply declined, mostly because of the widely implemented rodent and flea control programs maintained in urban settings (Hill et al. 1951, PHS Publications 1952). Nowadays, beside sporadic exposures in different parts of the country, particularly in Atlantic and Gulf coastal areas (Betz et al. 1983, Esperanza et al. 1992, Comer et al. 2001, Manea et al. 2001, Purcell et al. 2007), murine typhus is considered a significant public health problem and a reportable disease primarily in California, Texas, and Hawaii (Taylor et al. 1986, Esperanza et al. 1992, Williams et al. 1992, Manea et al. 2001, Civen and Ngo 2008, Adjemian et al. 2010).
In California, Los Angeles and San Diego counties were known as the primary endemic areas for murine typhus with many cases reported from urban localities during 1922–1942 (Meleney and French 1945). The 1950–1960s were characterized by a shift in murine typhus cases in the Los Angeles region from the central and southcentral areas of the city to the foothill areas in the eastern portion of Los Angeles and Orange counties (Adams et al. 1970). Subsequent environmental surveys failed to demonstrate the significant presence of rats in the vicinity of human cases of murine typhus (Beck et al. 1944, Adams et al. 1970, Sorvillo et al. 1993), and the classic flea vector X. cheopis appeared to be absent from the foothill areas (Beck et al. 1944, Sorvillo et al. 1993). Consequently, it was proposed that a suburban cycle involving cats, opossums, and cat fleas was responsible for the human cases of murine typhus reported from the foothill areas (Adams et al. 1970, Williams et al. 1992, Sorvillo et al. 1993). The presence of Rickettsia felis (formerly called ELB agent) was then discovered in cat fleas, Ctenocephilides felis (Williams et al. 1992), and C. felis commonplace association and feeding on both cats and opossums was established (Williams et al. 1992, Karpathy et al. 2009, Reif and Macaluso 2009).
Here, we report the contemporary presence of the classic rat–flea transmission cycle of R. typhi in urban Los Angeles, which was demonstrated during an environmental investigation of a probable, serologically diagnosed, case of murine typhus in a shop owner. Further, we provide the first report, to our knowledge, of R. felis present in rat and fleas in urban Los Angeles, and that coinfection with R. felis and R. typhi may occur in feral R. norvegicus.
Materials and Methods
Sample collection
Four R. norvegicus were trapped using Tomahawk traps (Tomahawk Trap Co., Tomahawk, WI) in September 2008 in an urban area of Los Angeles. Each rat was combed for ectoparasites, and sera and EDTA whole-blood specimens were collected. Archival frozen samples of fleas and sera from 11 R. norvegicus that had been captured in August 2007 within 1.5 km distance of the current investigation site were available. Samples and rat corpses frozen at −70°C were shipped on dry ice to the Centers for Disease Control and Prevention (CDC, Atlanta, GA) for further testing.
Serologic testing
Indirect immunofluorescence assay (IFA) was performed using antigen of R. typhi grown in chicken yolk sacs as described previously (Adjemian et al. 2010). Suspended antigen was dotted onto 24-well glass slides (Erie Scientific Corp., Portsmouth, NH), air-dried, fixed in acetone, and stored at −70°C. Sera were tested by IFA at consecutive twofold serum dilutions starting at 1:64 to end-point using fluorescein-labeled affinity-purified goat anti-rat IgG(H + L) antibody (KPL, Gaithersburg, MD). IgG titers ≥1:64 were considered positive for this study.
Sample processing and preparation of DNA
Animals were thawed, necropsy was performed, and 25–50 mg fragments of brain, lung, liver, spleen, kidney, testes, and ear tissues were collected. The QIAamp DNA Mini Kit (Qiagen, Valencia, CA) was used to extract DNA from the tissue samples and 200 μL of blood as described previously (Eremeeva et al. 2008). DNA was eluted with 200 μL of Qiagen AE buffer and stored at 4°C.
Fleas were identified as X. cheopis using standard taxonomic keys (PHS Publications 1969) and pairs from the same animal were placed in Eppendorf vials (22-281; Axygene, Union City, CA). Fleas were surface disinfected by washing in 10% bleach, 70% ethanol, and three washes of sterile distilled water, frozen in liquid nitrogen, and pulverized using Kontes pestles (Kimble-Kontes, Vineland, NJ). Flea powder was re-suspended in 220 μL of lysis buffer consisting of 160 μL of nuclei lysis solution (Promega, Madison, WI), 40 μL of 0.5 M EDTA, and 20 μL of 20 mg/mL Proteinase K (Qiagen), and incubated overnight at 56°C. DNA was extracted using the Wizard SV 96 Genomic DNA purification system (Promega) and a Biomek 2000 Robotic Workstation (Beckman Coulter, Fullerton, CA); it was eluted with 120 μL of sterile nuclease-free water and stored at 4°C, as reported previously (Moriarity et al. 2005, Eremeeva et al. 2008).
Polymerase chain reaction, cloning, and sequencing
Rat and flea DNAs were tested for the presence of R. typhi and R. felis DNAs using the Brilliant QPCR reagent kit (Stratagene, Cedar Creek, TX) and a TaqMan assay for the citrate synthase (gltA) gene of Rickettsia. Species-specific probes, primers, and amplification conditions were used as described previously (Eremeeva et al. 2008, Karpathy et al. 2009). Data were analyzed using an iCycler real-time Polymerase chain reaction (PCR) thermocycler and software (BioRad, Richmond, CA).
PCR fragments were cloned using pCR2.1 plasmid vector and INVαF-competent E. coli cells according to the manufacturer's instructions (Invitrogen, Carlsbad, CA). Plasmid DNA was prepared from selected individual colonies and inserts were sequenced using M13 and T7 primers (Invitrogen). All sequence reactions were done in both directions using the Big Dye Terminator Sequencing Kit and purified using ABI BigDye XTerminator kit (Applied Biosystems, Carlsbad, CA). Assembly and sequence analysis was performed using Sequencher 4.8 software (Gene Codes, Ann Arbor, MI).
Results
Serologic testing on rats
Three of 4 R. norvegicus trapped during the 2008 investigation tested positive for IgG antibodies with R. typhi antigen (Table 1) with titers 1:64, 1:512, and 1:1024 (geometric mean titer 1:323). The IgG antibody titer of the subadult rat (rat 08-346) was negative (<1:64). Three of 11 R. norvegicus trapped in 2007 also had R. typhi IgG antibodies (geometric mean titer 1:203).
Testing of rat tissue and blood for the presence of rickettsial DNA
Tissue samples from 4 R. norvegicus rats trapped in 2008 were tested for R. typhi and R. felis DNAs (Table 2). DNA of R. typhi was detected in tissues from 3 rats. The brain tissues of two adult rats (rat 08-343 and rat 08-345) were positive for R. typhi DNA. Every organ tested, including the brain, from the subadult rat (rat 08-346) was positive for R. typhi DNA. DNA of R. felis was detected in tissues of all four rats tested, including spleen, kidney, brain, heart, and ear. Ear tissue of the subadult female rat contained both R. typhi and R. felis DNA. The presence of both DNAs was confirmed by cloning and sequencing of 12 randomly selected clones: 2 clones contained R. typhi DNA and 10 clones contained R. felis DNA. No R. typhi or R. felis DNA was detected in the blood of any of the four rats.
Testes from 08-343 male rat contained no detectable Rickettsia typhi or R. felis DNA.
RT, Rickettsia typhi; RF, Rickettsia felis.
Testing of fleas for R. typhi and R. felis DNA
Only X. cheopis were obtained from the trapped rats. Twenty to 42 X. cheopis per rat (flea index 32.5) were collected from four rats trapped during the 2008 investigation (Table 3). Of 65 pools of two fleas, 42 contained R. felis DNA and 13 were positive for R. typhi DNA. Six to 14 flea pools per rat were positive for R. felis (median = 10.5 R. felis-positive pools per rat) with corresponding minimum flea infection rates for each rat ranging from 30% to 33.3% (32.4% median). DNA of R. typhi was found in 1 to 6 positive pools per rat (median = 3.25) with minimum flea infection rates ranging from 2.4% to 17.6% (9.85% median). Six flea pools from rats 08-343, 08-345, and 08-346 had both R. felis and R. typhi DNAs (median = 2 pools per rat).
Numerator indicates number of positive pools detected by TaqMan; denominator indicates number of pools tested.
Min, minimum infection rate is calculated based on the assumption that only one of two fleas in a positive pool was positive; Max, maximum infection rate calculated based on the assumption that both fleas in a pool were positive.
Rats collected in 2007 were trapped from 0.4 to 1.5 km from the 2008 site with a median distance of 0.8 km. One to 25 X. cheopis per rat (n = 11) were collected (flea index 9.4). Of 54 pools of fleas tested, 1 pool was positive for R. felis DNA and 17 pools were positive for R. typhi DNA. One pool from rat 07-190 was positive for R. felis with a minimum infection rate of 8.3%. The presence of R. typhi DNA ranged from 1 to 4 positive flea pools with a median of 3 among positive rats and minimum infection rates ranging from 8.3% to 27.3% (21.4% median). The pool of fleas with R. felis DNA also tested positive for R. typhi DNA.
Discussion
We conducted an environmental investigation of an urban area that was identified as the probable exposure site for a serologically diagnosed case of murine typhus that occurred in the fall of 2008. Four R. norvegicus were trapped at the patient's place of employment, a stall within a multiunit retail and wholesale building in urban Los Angeles, California. That area of the city hosts a persistent rat infestation because of poor sanitation, aging infrastructure, and a dense human population that provide an ideal environment for maintaining a significant rat population. Beside sewers, rats populate alleyways and residential buildings in areas with good access to food sources from unsecured garbage containers.
Immediately after this case occurred, measures had been taken in this area to reduce the rat population by distribution of bait stations by a professional pest control company. It is possible that a decrease in the rat population caused by the control measures may explain the unusually high numbers of Oriental rat fleas collected from each rat in 2008, with a flea index of 32.5 compared to 9.4 collected from rats in 2007 (p = 0.008, Student's t-test). It is a common concern that rodent extermination without accompanying control of ectoparasites can result in migration of the ectoparasites to new hosts, either rodent or human (Brettman et al. 1981, Krinsky 1983). The 2008 rats were all trapped in the specific stall used by the case patient. Laboratory testing of the 2008 rats confirmed that R. typhi commonly infected both the rats and their fleas, and demonstrated the presence in 2007 of R. typhi in rat fleas and R. typhi antibodies in rats up to 1.5 km from the primary investigation site. Although the 2007 rat tissues were not available for PCR testing for rickettsial DNA, we established the seropositivity of 27% of those rats with R. typhi antigen by IFA. Interestingly, R. felis was also detected in the same populations of rats and ectoparasites, suggesting cocirculation of both flea-borne rickettsiae in this zoonotic focus. No cats were reported inside the building, but cats were reported outside the building, and are commonly observed in other areas of downtown Los Angeles. The presence of opossums is not known in that part of Los Angeles (by visual observation, road kill, or detection by actual trapping).
Cocirculation of R. typhi and R. felis has been previously reported for X. cheopis collected from mice in Hawaii and from Rattus spp. in Indonesia (Jiang et al. 2006, Eremeeva et al. 2008). The prevalence of R. felis ranges from 5% to 45.8% for large collections of cat fleas (Ctenocephalides felis Bouché), sometimes up to 100% when small collections are tested (Bitam et al. 2006, De Sousa et al. 2006, Horta et al. 2007), and is often much higher than the prevalence of R. typhi, as in our study samples in 2008 but not 2007. Coinfection with R. felis and R. typhi has also been reported in experimentally infected cat fleas (Noden et al. 1998). It is not known if either pathogen has any relative advantage for acquisition, life-long persistence, and transmission by fleas, or benefit to the fleas. However, it is striking that high 2008 flea burdens were associated with the presence of R. felis in the tissues of all of the rats collected in 2008. Reports of R. felis DNA in animal tissues are relatively rare in other studies with lower flea infestation rates (Williams et al. 1992, Higgins et al. 1994b, Schriefer et al. 1994, Boostrom et al. 2002). X. cheopis collected in 2007 from rats with low infestation rates had only 1 of 54 pools positive for R. felis DNA, from a rat trapped only 0.4 km from the 2008 patient case site. This suggests that in this area of Los Angeles R. felis may have been present only in low levels among the ectoparasite population in 2007. In contrast, 17 of 54 pools were positive for R. typhi in 2007, so the probability of R. typhi transmission by exposure to a flea was quite comparable to that in 2008 (13 of 65, p = 0.15 chi-square test).
Previous studies conducted in suburban areas of California did not find any evidence of seropositivity of R. norvegicus (n = 35) and R. rattus (n = 105) by complement fixation with R. typhi antigen performed during the period of low case incidence during the 1960s (Adams et al. 1970). Similarly, Sorvillo et al. (1993) reported only 5.6% (2 out of 36) of seropositive R. rattus by complement fixation and indirect fluorescent antibody assay with R. typhi antigen, and 35 seronegative R. norvegicus that had been collected during investigation of 33 cases of murine typhus in residents of the northcentral suburban foothills of Los Angeles in 1984–1988. These findings are in marked contrast to the present investigation where 75% of rats were seropositive in 2008 and 27.3% were seropositive in 2007. X. cheopis was not found on any rats in these two previous studies, so it cannot be determined whether this urban focus was recently established or represents an expansion of a persistent low level endemic focus known in Los Angeles.
Consistent with the idea that this may be a re-emerging urban focus of R. typhi and emerging site for R. felis within the city limits, serosurveillance conducted in 2002 among 200 residents of a Los Angeles area serviced by a free clinic did not detect exposure to R. typhi, in contrast to a 3.5% to 12.5% seroprevalence to Bartonella that is also often found in association with rats and both rat and cat fleas (Smith et al. 2002). Surveys conducted from 1996 through 1998 of Norway rats in downtown Los Angeles revealed a 25.9% seroprevalence (n = 259) to R. typhi (Smith et al. 2002), indicating at least episodic exposure of rat populations to flea-borne rickettsiae despite aggressive vector and rodent management control program (LACPH 2010).
Serology is not routinely performed for R. felis infections because antigen is not yet widely available and the extent to which serum from confirmed cases cross-reacts with both spotted fever and typhus group antigens has been controversial (Perez-Arellano et al. 2005, Renvois et al. 2009). Indeed, only a single case of R. felis infection has been confirmed by molecular methods in the United States, and that was from Texas (Schriefer et al. 1994a). Consequently, suitable patient samples from California for R. felis infection are not available yet to define its serological specificity. Indeed, it is possible that a spectrum of serological responses may occur given that R. felis is known to vary genetically (Bauer et al. 2006, Fournier et al. 2008). Although most attention has been paid to R. felis in the cat flea, C. felis, it is clear from the present study and our previous study in Hawaii (Eremeeva et al. 2008) that R. typhi and R. felis can both attain sufficient prevalence in X. cheopis that human exposure to both of these agents is likely. Since both agents were detected in rat tissues, R. felis from this source has the capacity to infect vertebrates. However, the importance of rats and, potentially, mice as reservoirs for R. felis will need additional investigation.
Footnotes
Acknowledgments
Kyle F. Abramowicz was supported by a fellowship with the Oak Ridge Institute for Science and Education through Oak Ridge Associated Universities and the U.S. Department of Energy. The authors thank Kathryn Dirks and Sandor Karpathy for laboratory and protocol assistance, and Gregory A. Dasch for reviewing the article and helpful suggestions.
Disclosure Statement
All authors declared that no competing financial interests exist.
