Laboratory evaluation of aromatic essential oils from thirteen plant species as candidate repellents against Leptotrombidium chiggers (Acari: Trombiculidae), the vector of scrub typhus.

Scrub typhus, a rickettsial disease transmitted by several species of Leptotrombidium chiggers (larvae), is endemic in many areas of Asia. The disease is best prevented by the use of personal protective measures, including repellents. In this study commercially produced aromatic, essential oils of 13 plant species and ethanol (control) were tested in the laboratory for repellency against host-seeking chiggers of Leptotrombidium imphalum Vercammen-Grandjean and Langston (Acari: Trombiculidae). A rapid, simple and economic in vitro test method was used by exposing the chigger for up to 5 min. Repellency was based on relative percentages of chiggers attracted to test and control substances. Four of the 13 essential oils showed promise as effective repellent against L. imphalum chiggers. Syzygium aromaticum (clove) oil exhibited 100% repellency at 5% concentration (dilution with absolute ethanol), whereas Melaleuca alternifolia (tea tree) oil exhibited 100% repellency at 40% concentration. Undiluted oils of Zingiber cassamunar (plai) and Eucalyptus globules (blue gum) exhibited 100% repellency. Of the remaining nine essential oils, only 100% Pelargonium graveolens (geranium) exhibited >50% repellency (viz. 57%). Styrax torkinensis (benzoin) oil did not exhibit any repellency. These findings show that several aromatic, essential oils of plants may be useful as chigger repellent for the prevention of scrub typhus. Syzygium aromaticum oil may be safer and more economical to prevent chigger attacks than commercially available synthetic chemicals, such as DEET that may have harmful side effects.

Surveys of rodent-borne disease in Thailand with a focus on scrub typhus assessment.

The epidemiology of many rodent-borne diseases in South-East Asia remains ill-defined. Scrub typhus and lep-tospirosis are common and medically significant, while other zoonotic diseases, such as spotted fever group Rickettsiae have been identified, but their overall medical significance is unknown. Rodent surveillance was conducted from June 2002 to July 2004 in 18 provinces from Thailand. Traps were set up for one to three nights. Blood and serum samples and animal tissue samples (liver, spleen, kidney and urinary bladder) were collected. Chiggermites, ticks and fleas were removed from captured rodents. A total of 4536 wild-caught rodents from 27 species were captured over two years of animal trapping. Rattus rattus was the dominant species, followed by Rattus exulans and Bandicota indica. Almost 43 000 ectoparasites were removed from the captured animals. Approximately 98% of the ectoparasites were chigger-mites, of which 46% belonged to the genus Leptotrombidium (scrub typhus vector). Other genera included Schoengastia and Blankaartia. Tick and flea specimens together comprised less than 1% of the sample. Among the five species of ticks collected, Haemaphysalis bandicota was the predominant species caught, followed by Ixodes granulatus other Haemaphysalis spp., Rhipicephalus spp. and Dermacentor spp. Only two species of fleas were collected and Xenopsylla cheopis (rat flea) was the predominant species. Using both commercial diagnostic kits and in-house molecular assays, animal tissue samples were examined and screened for zoonotic diseases. Seven zoonotic diseases were detected: scrub typhus, leptospirosis, murine typhus, tick typhus, bartonella, babesiosis and trypanosomiasis. Most samples were positive for scrub typhus. Other zoonotic diseases still under investigation include borrelosis, ehrlichiosis, the plague, and other rickettsial diseases. Using geographic information systems, global positioning systems and remote sensing technology, epidemiological and environmental data were combined to assess the relative risk in different biotopes within highly endemic areas of scrub typhus in Thailand.

Comparison of immune response against Orientia tsutsugamushi, a causative agent of scrub typhus, in 4-week-old and 10-week-old scrub typhus-infected laboratory mice using enzyme-linked immunosorbent assay technique.

Using the indirect enzyme-linked immunosorbent assay (ELISA) technique, we compared the immune response of specific antibodies (IgM and IgG) in serum samples of scrub typhus-infected and non-infected mice against Orientia sutsugamushi (a causative agent of scrub typhus). Two different age groups (4-week-old and 10-week-old) of ICR laboratory mice were infected with O. tsutsugamushi by the animal passage procedure. Serum samples were detected for scrub typhus-specific antibodies using ELISA technique. Results from determining the presence of IgM and IgG in the serum samples obtained from scrub typhus-infected mice showed that the IgG was first detected on day-13 after the initial infection in both the 4-week-old and 10-week-old mice. The IgG titer levels of both groups were not significantly different. Although the presence of IgM in the in serum samples obtained from scrub typhus-infected mice was first detected on day 13 in the 4-week-old mice and on day-12 in the 10-week-old mice, the IgM titer in the 4-week-old mice was slightly lower than in the 10-week-old mice. Clinical observations of the scrub typhus-infected mice showed that the older mice become ill on day 9 whereas the younger mice exhibited the symptoms on day 12. Considering the earlier detection and slightly higher level of specific IgM antibody, it could be interpreted that the older mice may appear to have responded against O. tsutsugamushi faster than the younger mice.

Prevalence and diversity of Bartonella in rodents of northern Thailand: a comparison with Bartonella in rodents from southern China.

We report results of the first study to investigate the distribution and diversity of Bartonella in rodents from Thailand. Whole blood from 195 rodents, representing six species, was tested for the presence of Bartonella species using standard culture techniques. Isolates were obtained from 17 (8.7%) of the samples, and 14 of those isolates represented distinct strains, based upon partial sequencing of the citrate synthase (gltA) gene. Phylogenetic analysis of the isolates and other Bartonella species indicated that five unique isolates from Bandicota indica form a cluster that may represent a new Bartonella species. Two additional isolates from B. indica clustered together, and were nearly identical to an isolate from Apodemus draco collected in southern China. Importantly, a number of the isolates from Thailand rodents are closely related to B. grahamii and B. elizabethae, species which have been associated with human illness.

Occurrence of Orientia tsutsugamushi in small mammals from Thailand.

Extensive sampling of small mammals was conducted in eight provinces of Thailand between September 9, 1992 and April 29, 2001. A total of 3,498 specimens representing 22 species were collected. Eighty-eight percent (3,089 of 3,498) of the animals were collected from a region in Chiangrai Province, which is commonly recognized as endemic for human scrub typhus. Blood and tissue samples from each animal were tested for the presence of Orientia tsutsugamushi, the etiologic agent of scrub typhus. The predominant species collected were Rattus rattus (53%, n = 1,863), R. losea (18%, n = 638), Bandicota indica (16%, n = 564), and R. exulans (4%, n = 146). Orientia tsutsugamushi was detected in 10 of the 22 species of mammals that included R. bukit (25% infected, 1 of 4), R. rattus (23%, 419 of 1,855), R. argentiventer (22%, 5 of 23), R. berdmorei (22%, 2 of 9), R. losea (13%, 82 of 638), B. indica (9%, 52 of 564), R. koratensis (8%, 1 of 12), B. savilei (3%, 1 of 30), R. exulans (1%, 2 of 146), and Tupaia glis (2%, 1 of 49). Infected animals were found in Chiangrai (18% infected, 563 of 3,084), Bangkok (11%, 1 of 9), Sukothai (3%, 1 of 30), and Nonthaburi (1%, 1 of 69) Provinces. The implications towards scrub typhus maintenance and transmission are discussed.

Development of an in vitro method for the evaluation of candidate repellents against Leptotrombidium (Acari: Trombiculidae) chiggers.

We developed a rapid and economical in vitro procedure with which to evaluate the efficacy of candidate repellents against chiggers. The procedure requires only 5 min and a small number of chiggers to obtain a valid estimate of the median effective dose. We used this procedure to evaluate the repellent activity of 11 compounds against the chigger, Leptotrombidium imphalum Vercammen-Grandjean and Langston. Median effective doses were determined for 10 of the 11 compounds. DM-165-2 (N,N-diethyl-3-flurobenzamide) was the only compound that was significantly more effective than deet.

Efficiency of Leptotrombidium chiggers (Acari: Trombiculidae) at transmitting Orientia tsutsugamushi to laboratory mice.

Thirteen different laboratory colonies of Leptotrombidim chiggers [L. chiangraiensis Tanskul & Linthicum, L. deliense Walch and L. imphalum (Vercammen-Grandjean &Langston)] were evaluated for their ability to transmit Orientia tsutsugamushi (Hyashi) to mice. Of 4,372 transmission attempts using individual chiggers from all 13 colonies, 75% (n = 3,275) successfully infected mice. Transmission rates for the individual chigger colonies ranged from 7 to 80%. Increasing the number of chiggers that fed on a given mouse generally increased transmission rates. Transmission of O. tsutsugamushi to mice by different generations (F1-F11) of certain chigger colonies was stable; however, transmission rates varied greatly in other colonies. Transmission rates (both vertical and horizontal) of several L. changraiensis colonies and the L. deliense colony were the highest, suggesting that these colonies may be useful for the development of a chigger-challenge model that can be used to evaluate the efficacy of candidate scrub typhus vaccines or therapeutic agents in laboratory mice.