Sylvatic Typhus Associated with Flying Squirrels ( Glaucomys volans ) in New York State,United States

Abstract

Sylvatic typhus is an infrequent, potentially life-threatening emerging zoonotic disease. In January of 2009, the New York State Department of Health was notified of a familial cluster of two suspected cases. Due to the paucity of typhus cases in New York, epidemiologic and environmental investigations were conducted to establish rickettsial etiology and determine potential sources of infection. Patients presented with symptoms consistent with typhus, and serologic testing of each patient confirmed infection with typhus group rickettsiae. Serologic analysis of blood obtained from southern flying squirrels (Glaucomys volans) captured from the attic crawlspace above an enclosed front porch of the cases' residence indicated evidence of infection with Rickettsia prowazekii, with 100% seroprevalence (n=11). Both patients reported spending significant time on the porch and hearing animal activity above the ceiling prior to onset of illness, implicating these flying squirrels as the likely source of infection.

Introduction

Sylvatic typhus is an uncommon and potentially fatal zoonotic disease caused by the rickettsial organism Rickettsia prowazekii, which is maintained through an enzootic cycle involving southern flying squirrels (Glaucomys volans) and their ectoparasites (McDade et al. 1980, Chapman et al. 2009). Although the exact mode of transmission remains unknown, humans in the United States may be exposed to R. prowazekii through bites from infected lice or fleas associated with flying squirrels and their nesting material, by inhaling dried louse feces, or by inadvertently rubbing rickettsia-laden louse feces in the eyes, mucosal membranes, or insect bite-associated wounds (Bechah et al. 2008, Chapman et al. 2009). Human illness is characterized by high fever (≥38°C), intense headache, myalgia, and rash. The disease can be effectively treated with tetracycline group antibiotics or chloramphenicol.

As a result of decreased prevalence of body lice infestations and associated disease, surveillance of epidemic typhus was discontinued in the 1950s in the United States. However, since 1976, 45 cases of human R. prowazekii infection have been documented in residents of rural or suburban areas of the country with no reported contact with human body lice (Chapman et al. 2009). Approximately one-third of these cases were clearly associated with flying squirrels (Reynolds et al. 2003). The locations of human cases of sylvatic typhus coincide with the geographic distribution of southern flying squirrels, which includes much of the eastern United States (Dolan and Carter 1977, McDade et al. 1980, Duma et al. 1981, Reynolds et al. 2003). In New York, 30 suspected typhus group infections were reported to the New York State Department of Health (NYSDOH) from 1953 to 2008. Previous environmental investigations conducted by the NYSDOH in response to a familial cluster of three individuals with typhus group rickettsial infection in 1986 and 1987 were unable to uncover a source of exposure, although evidence of rodent activity was noted in the attic of the residence (White et al. 1990).

On January 29, 2009, the NYSDOH was notified by the Saratoga County Health Department of a potential typhus case in a 58-year-old resident (patient 1) whose 22-year-old son had also become ill with symptoms consistent with typhus (patient 2). Initial serologic tests conducted at a commercial laboratory for patient 1 demonstrated the presence of antibodies to typhus group rickettsiae, and for patient 2, were positive for presence of antibodies to Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever (RMSF). Subsequent serologic testing by the NYSDOH suggested evidence of current or recent infection with typhus group rickettsiae in both patients. Given the time of year and the infrequency of typhus fever cases in New York, an investigation was conducted to determine potential sources of infection and rickettsial etiology. The results of the epidemiologic investigation are summarized in the following report, and implicate R. prowazekii acquired from flying squirrels as the causative agent of illness.

Methods

Epidemiologic investigation

Case reports and medical records were reviewed to obtain information pertaining to signs and symptoms of illness, preliminary diagnostic test results, prescribed course of treatment, and potential sources of exposure. Physicians and patients were interviewed. Serologic testing of acute and convalescent sera, when available, was performed as follows at the NYSDOH Wadsworth Center. Immunoglobulin G (IgG) and IgM antibodies to R. rickettsii and R. typhi were detected by indirect immunofluorescence assay (IFA) using commercial antigen slides in a test kit (Focus Diagnostics, Cypress, CA) including positive and negative serum controls provided by the manufacturer, as previously described (Clements et al. 1983). Sera were screened at a 1/64 dilution, and, if reactive, were serially diluted in a two-fold dilution scheme to determine end point titers. Confirmatory IFA testing was performed at the Centers for Disease Control and Prevention (CDC) using R. prowazekii and R. typhi antigens and controls obtained from convalescent and negative donor patient sera (CDC IRB protocol #2526), as described previously (Reynolds et al. 2003, Chapman et al. 2009).

Environmental sampling

On February 10 and 11, 2009, mammal trapping and ectoparasite surveys were conducted on the property surrounding the residence of the cases. Tomahawk traps (5×5×16 in), baited with peanut butter and apple slices, were set along the perimeter of the house, adjacent barn, and in proximity to outdoor equipment stored on the property. Additionally, a licensed pest control specialist contracted by the homeowner (patient 1) set one colony trap (Tomahawk model 202C, 5×5×28 in) baited with crackers in the attic crawl space over the enclosed front porch of the residence on February 11, 2009. Traps were checked at first light and periodically during the day, as snow was present and daytime air temperatures did not exceed 6°C. Captured animals were anesthetized with isoflurane (Isoflo^®, Abbott Laboratories, Abbott Park, IL), field-processed for blood via cardiac puncture, and examined for ectoparasites before being euthanized via isoflurane overdose followed by cervical dislocation. When weather conditions were not conducive, animals were euthanized as described and individually bagged for transport to a NYSDOH laboratory facility for processing in a class 2 biosafety cabinet following Biosafety Level 2 standard safety precautions. Mammal trapping and processing methods were in compliance with current New York State Institutional Animal Care and Use Committee requirements (protocol #08-301).

Squirrel whole blood samples were stored at 4°C, and DNA was extracted using the Magna Pure LC DNA Isolation Kit I (Roche Applied Science, Indianapolis, IN) following the manufacturer's guidelines, with a final elution volume of 100 μL. All ectoparasites were identified to the species level, pooled by animal and type, and stored at −80°C prior to total DNA nucleic acid extraction using the Epicentre^® MasterPure^™ Complete DNA and RNA Purification Kit (Epicentre^® Biotechnologies, Madison, WI). No surface sterilization of ectoparasite samples was performed prior to extraction. Ectoparasite samples were individually dissected (tick) or ground using a disposable tissue grinder (lice, flea, and mite pools) in 200 μL of Epicentre^® MasterPure^™ Tissue and Cell Lysis Solution (Epicentre^® Biotechnologies, Madison, WI). Samples were lysed overnight at 56°C after the addition of 1 μL of Epicentre^® MasterPure^™ Proteinase K (Epicentre^® Biotechnologies, Madison, WI). Lysed samples were subjected to total DNA nucleic acid extraction following the manufacturer's protocol and resuspended with Tris-EDTA buffer in a final volume of 50 μL. Purified genomic DNA from whole blood and ectoparasite samples were tested in duplicate using an experimental TaqMan Rickettsia species real-time PCR assay designed by the NYSDOH Wadsworth Center to amplify a 136-bp region of the nudix hydrolase (invA) gene (Gaywee et al. 2002). For each sample, a 5- μL portion was amplified in a total reaction mixture of 25 μL containing 200 nM Forward Primer (Integrated DNA Technologies, Coralville, IA), 300 nM Reverse Primer (Integrated DNA Technologies, Coralville, IA), 500 nM 5′ROX NH Ester-3′ BHQ labeled probe (Integrated DNA Technologies, Coralville, IA), 4 mM MgCl₂, and Lightcycler FastStart DNA Master Hybridization Probes (Roche Applied Science, Indianapolis, IN). Assay positive control and negative controls were included in each run and consisted of R. prowazekii strain Cairo DNA (BEI Resources, Manassas, VA) and ultrapure water, respectively. A two-step real-time PCR was performed using an Applied Biosystems 7500 Fast Sequence Detection System with the following cycling parameters: Initial 10 min Taq DNA polymerase activation step at 95°C followed by 45 cycles of denaturation at 95°C for 10 sec and extension at 60°C for 30 sec.

Remaining squirrel blood samples were submitted to the CDC for serologic detection of IgG (heavy plus light chain) polyvalent antibodies against R. prowazekii by IFA, using in-house produced rabbit-derived, anti-flying squirrel, fluorescein isothiocyanate–labeled conjugate, with appropriate positive and negative controls derived from animals whose exposure history to R. prowazekii had been documented during previous CDC investigations. Samples were initially screened at 1/32 dilution, with reactive samples titered to end point. Extracted DNA samples from blood and ectoparasites were also submitted to the CDC for nested PCR testing to amplify the rickettsial genus common 17-kD protein antigen gene, targeting typhus group rickettsiae including R. prowazekii and R. typhi, with appropriate controls, as described previously (Massung et al. 2001, Chapman et al. 2009). All DNA samples were also tested using TaqMan PCR assay targeting rickettsial gltA with a pan Rickettsia probe (5′-HEX-CCTA(T)GGC(T)ATTA(T)GCTTGC-BHQ1-3′; locked nucleic acid bases, shown in parentheses, were incorporated to improve probe binding). The details of the TaqMan assay, reaction conditions, and controls used are described elsewhere (Eremeeva et al. 2008).

Results

Case reports

Patient 1, a 58-year-old female, presented on January 8, 2009, with an 8-day history of fever (39.4°C), chills, sweats, severe headache, malaise, macular/papular rash on her trunk and extremities, and an elevated platelet count (reported onset date of January 1, 2009). Initial commercial laboratory testing of acute and convalescent sera drawn on January 14, 2009, and January 29, 2009, respectively, for typhus group rickettsiae revealed an IgM titer of >1/64 and an IgG titer of >1/256 for the acute specimen, and an IgM-positive RMSF result of 16 PanBio enzyme-linked immunosorbent assay (ELISA) units (reference range: <9 negative; 9–11 equivocal; >11 positive) for the convalescent serum. The patient visited a hospital emergency department on January 16, 2009, with blood in the urine and acute ileitis. She was prescribed oral doxycycline for 14 days (100 mg twice daily) by the attending physician. Her condition improved markedly over the following 10 days; ileitis, rash, fever, and associated symptoms resolved. She returned to work on January 23, 2009, although she continued to experience residual headaches and fatigue for an additional 6 weeks. She had no significant travel history prior to onset of illness. The family did not own any pets. The patient had no history of insect bites and no known animal contact, although eastern gray squirrels (Sciurus carolinensis) are common in the neighborhood, and she suspected mice or squirrels were present in her house.

Patient 2, the 22-year-old son of patient 1, developed fever (39.4°C), chills, sweats, severe headache, malaise, and myalgia on January 1, 2009. His liver enzymes (alanine aminotransferase [ALT] and aspartate aminotransferase [AST]) levels were elevated. Initial testing of acute serum drawn on January 12, 2009, at a commercial laboratory revealed an IgM-positive RMSF result of 14 PanBio ELISA units. The patient was prescribed 10 days of oral doxycycline (100 mg twice daily) by his physician, and his fever resolved within 3 days, although he continued to experience intermittent headaches and fatigue for approximately 1 week following treatment. The patient had no known animal contact except as noted for patient 1, and reported no significant travel history prior to illness.

Repeated requests for additional convalescent serum from both patients were denied. Two other family members, the 27-year-old daughter and 25-year-old son of patient 1, stayed at the residence for a week in late December and remained well following their visit. Family members reported hearing scratching noises above the ceiling of an enclosed front porch used to store holiday decorations and food. The family hosted a gathering of 10 guests in late December, none of whom subsequently became ill. Both patients reportedly spent considerable time on the porch in the weeks prior to onset of illness, while others interviewed did not.

Case serologic studies

Comparative results of serologic tests performed by the NYSDOH and CDC for patient 1 and patient 2 are shown in Table 1.

Table 1.

IFA Serologic Results and Clinical Symptoms of Patients Diagnosed with Typhus Group Infection, New York State, February, 2009

				Reciprocal serologic titer ^a				Clinical symptoms ^b
			R. prowazekii	R. typhi		R. rickettsii
Patient no. (Age, years)	Gender	Specimen collection date	IgG	IgG	IgM	IgG	IgM	Rash	Myalgia	Elevated platelet count	Elevated ALT/AST
1 (58)	Female	16-Jan-09	4096	2048 (512)	256	NS^c	NS^c	Yes	No	Yes	No
		29-Jan-09	4096	2048 (256)	256	NS^c	NS^c
2 (22)	Male	12-Jan-09	4096	512 (512)	≥1024	128	NS^c	No	Yes	No	Yes

Initial NYSDOH Wadsworth Center serologic results for R. typhi (CDC confirmatory results).

Both patients had fever of 39.4°C, chills, sweats, headache, and malaise.

NS, nonspecific binding at 1/64 dilution.

ALT, alanine aminotransferase; AST, aspartate aminotransferase.

Environmental investigation

The residence of the patients was constructed in 1930 and is situated on 0.6 wooded acres adjacent to a river, in a semirural neighborhood in Saratoga County, New York. The exterior is sheathed in wooden siding with an asphalt shingle roof. Interior walls are plaster, consistent with the age of the house. Squirrel nesting material and droppings were noted during inspection of the porch attic space, and an entry hole was visible in the wooden exterior fascia of the porch roof.

Live animal trapping conducted by the NYSDOH for 2 consecutive days in February 2009 (48 trap nights) yielded three eastern gray squirrels. An additional 11 southern flying squirrels were captured by the pest control specialist in the porch attic (one trap night) in mid-February. Whole blood samples obtained from each animal varied from approximately 100 μL to 1 mL in volume. A total of 26 pools of ectoparasites obtained from the animals included fleas (Orchopeas howardi), lice (Neohaematopinus sciuropteri and N. sciuri), mites (Euhaemogamasus ambulans and Androlaelaps fahrenholzi), and ticks (Ixodes marxi). The presence of Rickettsia species DNA was undetectable in squirrel blood, individual ticks, and pooled ectoparasites (mites, fleas, or lice) by real-time or nested PCR assays; however, all 11 flying squirrels were seropositive for R. prowazekii in CDC serologic tests, with titers ranging from 1/64 to 1/512 (geometric mean titer of 1/256). All eastern gray squirrels were seronegative at the 1/32 screening dilution.

Discussion

Our investigation provides supporting evidence for occurrence of sylvatic typhus in suburban New York State. Both patients had spent considerable time on the enclosed front porch of their home, where flying squirrels found harborage from the cold winter weather. Although the presence of R. prowazekii could not be confirmed in squirrel blood or ectoparasites by PCR, the high seroprevalence (100%) found in the flying squirrels trapped from the crawl space above the porch confirms past infection and suggests enzootic maintenance of R. prowazekii in the local animal reservoir population. All eastern gray squirrels were seronegative at the 1/32 screening dilution and their ectoparasites were PCR negative, providing suggestive evidence that eastern gray squirrels were not involved in the maintenance of this pathogen.

Initial antibody testing of sera from both patients demonstrated high titers of IgG and IgM antibodies to R. typhi, as well as nonspecific binding to R. rickettsii antigen using a commercial test kit. This kit did not screen for antibodies specific to R. prowazekii, and a high degree of human antibody cross-reactivity with rickettsial antigens has been extensively documented (White et al. 1990, La Scola et al. 2000). The significantly higher titer to R. typhi, particularly in patient 2, strongly suggested the agent of infection for both patients was unlikely to be R. rickettsii. Furthermore, case exposure occurred during the winter months, when the primary vector of RMSF in New York, the American Dog tick (Dermacentor variabilis), is not active. In confirmatory testing conducted by the CDC, sera from both patients demonstrated higher titers to R. prowazekii antigen than to R. typhi. Although there was no observed change in titer between acute and convalescent specimens of patient 1, only 13 days intervened between collections. Typically, a 3-week period is necessary between acute sera and convalescent phase sera draws, if detection of a four-fold rise in titer is expected.

This is the second reported familial cluster of sylvatic typhus in New York associated with squirrel infestation of the residence during winter months. In 1986–1987, three family members residing in a suburban neighborhood approximately 70 km north of New York City removed holiday decorations from their attic approximately 2 weeks before the onset of acute febrile illness, which was subsequently diagnosed as typhus group infection by serologic and immunoblot tests with R. typhi and R. prowazekii antigens (White et al. 1990). During the course of that investigation, rodent droppings were found in the attic, although an eastern gray squirrel and white-footed mouse (Peromyscus leucopus) trapped and tested were negative for the presence of rickettsiae. Previous studies have demonstrated that R. prowazekii seroprevalence in flying squirrels is highest in autumn and early winter months, when squirrels congregate (Sonenshine et al. 1978). The seasonality of sylvatic typhus in New York, and across the northeastern United States, is likely the result of human exposure to infectious materials from flying squirrels overwintering in residences or camps, during the time of the year when a large proportion of the animals may be infected with R. prowazekii and potential arthropod vector burdens are greatest (Sonenshine et al. 1978, Comer et al. 2001).

Our investigation is subject to the following limitations. We were unable to elucidate the exact mode of transmission of R. prowazekii to humans from flying squirrels. Both patients could have potentially been exposed to R. prowazekii through inhalation or mucosal contact with louse feces or microscopic contaminated nesting material rendered airborne by squirrel activity. Alternatively, it is possible that transmission occurred via inapparent insect bite; however, the species of lice found during our investigation, particularly N. sciuropteri, are highly host specific and not known to feed on humans. These lice likely play a role in transmission of R. prowazekii within squirrel reservoir populations, but they are not a probable source of human typhus infection via parasitism (Sonenshine et al. 1978, McDade et al. 1980). The flea O. howardi presents a much more feasible alternative because it has been documented to feed on humans and may be of public health importance with respect to sylvatic typhus (Bozeman et al. 1981, Comer et al. 2001). Second, the scope of our environmental investigation was limited to the area immediately surrounding the residence where the cases occurred, during the weeks following diagnosis. Future studies are warranted to identify the primary transmission mechanism(s) involved in sylvatic typhus and accurately assess the prevalence, seasonality, and geographic distribution of R. prowazekii in reservoir mammals, and thus to determine risk at the statewide or regional scale.

Our investigation further supports the notion that prolonged or repeated exposure to flying squirrels and their associated nesting materials and ectoparasites presents a significant risk of transmission of sylvatic typhus to humans. Homeowners, pest control specialists, and renovation contractors in endemic areas should be educated on the symptoms of sylvatic typhus and take appropriate precautions to reduce risk of exposure (Chapman et al. 2009). Clinicians should consider a diagnosis of sylvatic typhus when patients present with acute febrile illness and history of contact with flying squirrels or putative rodent infestation of their residence or worksite.

Footnotes

Acknowledgments

This study was a joint investigation by the NYSDOH and CDC using state and federally appropriated funds. The authors express our gratitude to Robert Massung, Jennifer McQuiston, and John Krebs, at the CDC, for guidance. The authors thank Debra Simmerly, NYSDOH Capital District Regional Office, and Gayle Zimmerman, Saratoga County Public Health Nursing Service, Kathleen Downey, and David Rockwell, for their involvement with human case investigation. We also thank Catherine Turcotte, New York State Department of Health, and John Berghammer, Ace Pest Control, for assistance with mammal trapping and/or processing. The authors had full access to all study data and take responsibility for the integrity of the data and accuracy of analysis. The findings and conclusions of this study are those of the authors and do not necessarily reflect the views of the NYSDOH or U.S. Department of Health and Human Services.

Author Disclosure Statement

No competing financial interests exist.

References

Bechah

, Capo

, Mege

, Raoult

. Epidemic typhus. Lancet Infect Dis, 2008; 8:417–426.

Bozeman

, Sonenshine

, Williams

, Chadwick

, et al. Experimental infection of ectoparasitic arthropods with Rickettsia prowazekii (GvF-16 strain) and transmission to flying squirrels. Am J Trop Med Hyg, 1981; 30:253–263.

Chapman

, Swerdlow

, Dato

, Anderson

, et al. Cluster of sylvatic epidemic typhus cases associated with flying squirrels, 2004–2006. Emerg Infect Dis, 2009; 15:1005–1011.

Clements

, Dumier

, Fiset

, Wisseman

Jr. , et al. Serodiagnosis of Rocky Mountain Spotted fever: Comparison of IgM and IgG enzyme-linked immunosorbent assays and indirect fluorescent antibody test. J Infect Dis, 1983; 148:876–880.

Comer

, Paddock

, Childs

. Urban zoonoses caused by Bartonella, Coxiella, Ehrlichia, and Rickettsia species. Vector Borne Zoonotic Dis, 2001; 1:91–118.

Dolan

, Carter

. Mammalian Species: Glaucomys volans . Am Soc Mammal, 1977; 78:1–6.

Duma

, Sonenshine

, Bozeman

, Veazey

, et al. Epidemic typhus in the United States associated with flying squirrels. JAMA, 1981; 245:2318–2323.

Eremeeva

, Warashina

, Sturgeon

, Buchholz

, et al. Rickettsia typhi and R. felis in rat fleas (Xenopsylla cheopis), Oahu, Hawaii. Emerg Infect Dis, 2008; 14:1613–1615.

Gaywee

, Xu

, Radulovic

, Bessman

, et al. The Rickettsia prowazekii invasion gene homolog (invA) encodes a nudix hydrolase active on adenosine (5′)-pentaphospho-(5′)-adenosine. Mol Cell Proteomics, 2002; 1:179–185.

10.

La Scola

, Rydkina

, Ndihokubwayo

, Vene

, et al. Serological differentiation of murine typhus and epidemic typhus using cross-adsorption and western blotting. Clin Diag Lab Immun, 2000; 7:612–616.

11.

Massung

, Davis

, Slater

, McKechnie

, et al. Epidemic typhus meningitis in the southwestern United States. Clin Infect Dis, 2001; 32:979–982.

12.

McDade

, Shepard

, Redus

, Newhouse

, et al. Evidence of Rickettsia prowazekii infections in the United States. Am J Trop Med Hyg, 1980; 29:277–284.

13.

Pan American Health Organization. Scientific pub. no. 503. In: Acha

, Szyfres

, ed. Zoonoses and Communicable Diseases Common to Man and Animals. 2nd Edition. Washington DC: Pan American Health Organization, 1987:525–528.

14.

Reynolds

, Krebs

, Comer

, Sumner

, et al. Flying squirrel-associated typhus, United States. Emerg Infect Dis, 2003; 9:1341–1343.

15.

Sonenshine

, Bozeman

, Williams

, Masiello

, et al. Epizootiology of epidemic typhus (Rickettsia prowazekii) in flying squirrels. Am J Trop Med Hyg, 1978; 27:339–349.

16.

White

, Smith

, Hechemy

, Veltman

, et al. Possible typhus-group infection in New York State: Presentation of four suspect cases. Ann NY Acad Sci, 1990; 590:256–265.