Volume 18, Number 1—January 2012
Rickettsia felis Infections, New Zealand
To the Editor: Members of the genus Rickettsia have garnered much attention worldwide in recent years with the emergence of newly recognized rickettsioses. In New Zealand, only Rickettsia typhi and R. felis, belonging to the typhus and spotted fever groups, respectively, have so far been found (1). R. typhi, primarily transmitted by the oriental rat flea (Xenopsylla cheopis), has a worldwide distribution and causes murine typhus in humans (2). At the end of 2009, a total of 47 cases of murine typhus had been recorded in New Zealand. In contrast, although the cat flea (Ctenocephalides felis) can carry R. felis in New Zealand (3), no human infections have been reported. However, because R. felis shares a similar clinical profile to murine typhus, infection can be mistaken for a suspected case of R. typhi (4).
Clinical suspicion of rickettsial infection is widely confirmed by serologic tests with the indirect immunofluorescence assay (IFA) being the standard test. However, antibodies against R. felis in human sera are known to cross-react with R. typhi in IFA (5). Western blot (WB) and cross-adsorption assays, in combination with IFA, can differentiate between several rickettsioses (5,6). We report on the trial in New Zealand of WB and cross-adsorption assays for differentiating retrospectively between past R. typhi and R. felis infections and evidence of R. felis infection in persons living in the country.
Serum samples were obtained from 24 volunteers from the Institute of Environmental Science and Research Limited, Porirua, New Zealand. Samples were tested using R. typhi IFA slides (Australian Rickettsial Reference Laboratory [ARRL], Geelong, Victoria, Australia). After incubation (37°C for 30 min), slides were washed 3 times, incubated with fluorescein-conjugated antihuman IgG, IgM, and IgA (ARRL), and washed again before examination. All samples were then tested by using an IgG IFA kit (Focus Diagnostics, Cypress, CA, USA) against typhus group (TG) R. typhi and spotted fever group (SFG) R. rickettsii.
TG-positive and SFG-negative serum samples may represent R. typhi infections, and SFG-positive and TG-negative serum samples may represent R. felis infections. Because R. typhi can cross-react with SFG rickettsiae (7), and R. felis with R. typhi (5), results that are TG positive and SFG positive may be caused by either rickettsiae. Positive reactivity may also represent overseas-acquired rickettsioses. Thus, WB and cross-adsorption assays using R. typhi (Wilmington) and R. felis (URRWXCal2) antigens (Unité des Rickettsies, Marseilles, France) were used to confirm any R. typhi or R. felis infections (6).
Antigens (2 mg/mL) were solubilized (100°C for 10 min) in 2× Laemmli buffer (6) and subjected to electrophoresis (20 μg/well; 20 mA, 2.5 h) through polyacrylamide gels (12.5% resolving; 4% stacking) (BioRad, Hercules, CA, USA). Resolved antigens were electroblotted (100 V for 1 h) onto 0.45-μm polyvinylidene difluoride membranes, which were blocked by using Tris-buffered saline with 0.1% Tween 20. Each antigen lane was divided into 2 strips before incubation (room temperature for 1 h) with serum (diluted 1:200). After three 10-min washes with 5% milk–Tris-buffered saline with 0.1% Tween 20, strips were incubated (room temperature, 1 h) with horseradish peroxidase–conjugated antihuman IgG (1:150,000; SouthernBiotech, Birmingham, AL, USA) and washed again. Enhanced chemiluminescent detection of bound horseradish peroxidase (ECL Plus; GE Healthcare, Buckinghamshire, UK) enabled identification of reactive band sizes with Precision Plus standards (BioRad). Cross-adsorption was carried out by incubating serum diluted 1:30 in boiled antigen (37°C for 5.5 h, then 4°C overnight) before centrifugation (10,000 × g for 10 min) (6). Supernatants were applied to WB strips and results compared with R. typhi and R. felis antisera.
Of the 24 serum samples, 3 (12.5%) were positive on the ARRL slides, and 11 (45.8%) showed IgG reactivity on Focus slides (Table). Of these 11 serum samples, 8 (33.3%) were SFG positive and TG negative, and 3 (12.5%) were SFG and TG positive. Of the 12 serum samples that showed some IFA reactivity, after cross-adsorption, none had specific reactivity against R. typhi, and 2 were confirmed as R. felis. Both volunteers recorded risk factors associated with R. felis infection. Six serum samples were indeterminate. Detectable antibodies remained after both cross-adsorptions, which may be caused by infection by R. felis and R. typhi, or other rickettsiae or cross-reactive pathogens (5,8).
Although IgG titers decline over time, detectable levels can remain for 4 years and thus exposure to R. felis may have occurred any time during this period (9). Because both R. felis–infected persons had traveled overseas within the past 4 years and R. felis has a wide distribution (4), overseas exposure is possible. R. felis is known to be prevalent in C. felis fleas, including in New Zealand (3,4). This prevalence and the high rate of cat and dog ownership have public health implications and support the recognition of R. felis as an emerging global health threat (4). Infection from R. felis in addition to R. typhi should be considered in the differential diagnosis of fever, headache, myalgia, and rash.
We thank Alice Johnstone, Daniel Kay, Donia Macartney, and Lin Hou for their advice on Western blotting and Pierre-Edouard Fournier for advice on cross-adsorption assays. We also thank John Stenos for assistance and advice in the early stages of our project.
- Kelly P, Roberts S, Fournier PE. A review of emerging flea-borne bacterial pathogens in New Zealand. N Z Med J. 2005;118:1257–65.
- Comer JA, Paddock CD, Childs JE. Urban zoonoses caused by Bartonella, Coxiella, Ehrlichia, and Rickettsia species. Vector Borne Zoonotic Dis. 2001;1:91–118.
- Kelly P, Rolain J-M, Raoult D. Prevalence of human pathogens in cat and dog fleas in New Zealand. N Z Med J. 2005;118:1754–6.
- Pérez-Osorio CE, Zavala-Velázquez JE, Arias León JJ, Zavala-Castro JE. Rickettsia felis as emergent global threat for humans. Emerg Infect Dis. 2008;14:1019–23.
- Pérez-Arellano JL, Fenollar F, Angel-Moreno A, Bolaños M, Hernández M, Santana E, Human Rickettsia felis infection, Canary Islands, Spain. Emerg Infect Dis. 2005;11:1961–4.
- La Scola B, Rydkina L, Ndihokubwayo JB, Vene S, Raoult D. Serological differentiation of murine typhus and epidemic typhus using cross-adsorption and western blotting. Clin Diagn Lab Immunol. 2000;7:612–6.
- Mouffok N, Parola P, Raoult D. Murine typhus, Algeria. Emerg Infect Dis. 2008;14:676–8.
- Kantsø B, Svendsen CB, Jørgensen CS, Krogfelt KA. Evaluation of serological tests for the diagnosis of rickettsiosis in Denmark. J Microbiol Methods. 2009;76:285–8.
- Halle S, Dasch G. Use of a sensitive microplate enzyme-linked immuno-sorbent assay in a retrospective serological analysis of a laboratory population at risk to infection with typhus group rickettsiae. J Clin Microbiol. 1980;12:343–50.