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 (). R. typhi, primarily transmitted by the oriental rat flea (Xenopsylla cheopis), has a worldwide distribution and causes murine typhus in humans (). 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 (), 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 ().

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 (). Western blot (WB) and cross-adsorption assays, in combination with IFA, can differentiate between several rickettsioses (,). 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 (), and R. felis with R. typhi (), 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 ().

Antigens (2 mg/mL) were solubilized (100°C for 10 min) in 2× Laemmli buffer () 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) (). 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 (,).

Table

Serologic data and risk factors of volunteers that showed positive reactivity in rickettsial IFA, New Zealand*

Volunteer no.	IFA serologic titers				WB and cross-adsorption results	Risk factors in the past 4 years
	ARRL kit†		Focus kit‡			Flea or animal contact	Traveled overseas
	Rickettsia typhi		R. typhi	R. rickettsii
4	Neg		64	64	Indeterminate§	Rat, cat, dog	Yes
5	Neg		Neg	64	Indeterminate	Cat, dog	Yes
8	Neg		Neg	128	Neg¶	Flea, rat, cat, dog	Yes
9	Neg		Neg	64	Indeterminate	Flea, cat, dog	Yes
16	Neg		Neg	128	Indeterminate	Rat, cat, dog	Yes
17	Neg		Neg	128	R. felis	Rat, cat, dog	Yes
18	128		64	64	Neg	Cat, cow, hen	Yes
19	128		Neg	64	Indeterminate	Cat, dog	Yes
21	Neg		64	256	R. felis	Flea, cat, dog, possum	Yes
22	Neg		Neg	64	Neg	Cat, dog	Yes
23	256		Neg	Neg	Neg	Rat, cat, dog	No
25	Neg		Neg	128	Indeterminate	None	Yes

*IFA, indirect immunofluorescence assay; ARRL, Australian Rickettsial Reference Laboratory; WB, Western blot; neg, negative.
†The cutoff titer for seropositivity was 128 as recommended by the manufacturer.
‡According to kit instructions, endpoint titers ≥64 and <256 indicate either past infection or early response to a recent infection, and ≥256 are considered presumptive evidence of recent or current infection.
§Serum samples that still reacted with R. typhi and R. felis after cross-adsorption were classified as indeterminate responses.
¶Serum samples that showed no specific reactions to R. typhi and R. felis in the WB assay were classified as negative for R. typhi and R. felis..

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 (). Because both R. felis–infected persons had traveled overseas within the past 4 years and R. felis has a wide distribution (), overseas exposure is possible. R. felis is known to be prevalent in C. felis fleas, including in New Zealand (,). 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 (). Infection from R. felis in addition to R. typhi should be considered in the differential diagnosis of fever, headache, myalgia, and rash.