A comprehensive 8-yr survey of acarine ectoparasites (ticks and mites) of bats was carried out in 18 localities from 2002 to 2009. Most of the surveys were conducted during 14 national biodiversity scientific expeditions throughout Malaysia. The objective was to identify acarines of known public health importance from bats and thus determine whether there is any potential public health risk in Malaysia. Trapping of bats was conducted using Harp traps and Mist nets. In total, 1,579 individuals comprising of 6 families and 52 species of bats were examined alive. In general, 25.6% of the bats were infested with acarines. Infestation rates of ticks, mesostigmatid mites, and chiggers on bats examined were 0.4, 10.4, and 14.7%, respectively. Their prevalence and mean intensity were tabulated. Genera of ticks extracted were Amblyomma, Dermacentor, Ixodes, and Ornithodoros. Of these genera, only two species can be identified to species level and they are Amblyomma cordiferum and Ixodes simplex. In total, 8 genera and 15 species of mesostigmatid mites were found; the species were Ancystropus eonycteris, Ancystropus zeleborii, Echinonysus nasutus, Laelaps aingworthae, Laelaps nuttalli, Laelaps sanguisugus, Laelaps sculpturatus, Longolaelaps longulus, Longolaelaps whartonii, Meristaspis lateralis, Meristaspis macroglossi, Paraperiglischrus rhinolophinus, Spinturnix acuminatus, Spinturnix americanus, and Spinturnix bakeri. Chiggers on bats were represented by 12 genera and 6 species; the species identified were Gahrliepia fletcheri, Riedlinia lipoxena, Trombigastia cadei, Walchiella impar, Walchiella oudemansi, and Whartonia caobangensis. The study produced an up-to-date list of acarine ectoparasites of bats in Malaysia where a total of 38 genera and 47 species of acarines were listed. Findings of the study demonstrated that 5 genera and 1 species of acarines that may pose potential health risks, can be found on bats.
To examine the acaricidal effects of the essential oil of Cymbopogon citratus leaf extract (lemongrass) and ethanolic Azadirachta indica leaf extract (neem) against house dust mites Dermatophagoides farinae (D. farinae) and Dermatophagoides pteronyssinus (D. pteronyssinus).
To identify the presence of acarine ectoparasites and determine whether there is any potential public health risk in Panti Forest Reserve, Johore, Malaysia.
A survey of ticks and other ectoparasites was carried out during a national biodiversity scientific expedition at Ulu Muda Forest Reserve, Kedah, Malaysia from 23-29 March 2003. A total of 161 animals comprising 20 species of birds, 16 species of bats, six species of non-volant small mammals and 12 species of reptiles were examined for ticks and other ectoparasites. From these animals, nine species in five genera of ticks, 10 species in two families of Mesostigmatid mites and five species of chiggers were collected. Three of the ectoparasitic species found, Dermacentor auratus, Ixodes granulatus and Leptotrombidium deliense are of known public health importance. This survey produced the first list of ticks and other ectoparasites in the forest reserve and the third study of ectoparasites in Kedah. Fourteen species of these ectoparasites are new locality records.
A survey of ticks and other ectoparasites was carried out during a national biodiversity scientific expedition at Gunung Stong Forest Reserve, Kelantan, Malaysia from 23-29 May 2003. A total of 272 animals comprised of 12 species of birds, 21 species of bats, 7 species of rodents and 2 species of insects were examined for ticks and other ectoparasites. From these animals, 5 species in 4 genera of ticks; 7 species in 2 families of Mesostigmatid mites and 5 species of chiggers were collected. Among the ectoparasites found were Ixodes granulatus and Leptotrombidium deliense, which are of known medical importance. A tick island consisting of 10 nymphal stages of Dermacentor spp was observed feeding on Rattus tiomanicus.
Mites are known causes of allergic diseases. Currently, identification of mites based on morphology is difficult if only one mite is isolated from a (dust) sample, or when only one gender is found, or when the specimen is not intact especially with the loss of the legs. The purpose of this study was to use polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the ITS2 gene, to complement the morphological data for the identification of mites to the species level. For this, six species were cultured: Dermatophagoides pteronyssinus, D. farinae, Blomia tropicalis, Tyrophagus putrescentiae, Aleuroglyphus ovatus and Glycycometus malaysiensis. Genomic DNA of the mites was extracted, quantified, amplified and digested individually with restriction enzymes. Hinf I and Ple I differentiated the restriction patterns of D. pteronyssinus and D. farinae. Bfa I and Alu I enzymes differentiated B. tropicalis and G. malaysiensis. Ple I enzyme was useful for the differentiation between T. putrescentiae and A. ovatus. Bfa I was useful for the differentiation of G. malaysiensis from the rest of the species. In conclusion, different species of mites can be differentiated using PCR-RFLP of ITS2 region. With the established PCR-RFLP method in this study, identification of these mites to the species level is possible even if complete and intact adult specimens of both sexes are not available. As no study to date has reported PCR-RFLP method for the identification of domestic mites, the established method should be validated for the identification of other species of mites that were not included in this study.
Allergens of Dermatophagoides and Blomia species are well-characterized but not for other species. This study was conducted to determine the prevalence of allergic sensitization to house dust (HDM) and storage mites (SM). One hundred adult subjects (aged ≥ 18) were recruited. The mite specific IgE of all allergic subjects were higher compared with healthy subjetcs despite being not statistically significant except for D. farinae and G. malaysiensis. The mean serum IgE levels against HDM and SM for allergic subjects were significantly higher compared with those in healthy subjects. They were mainly sensitized to Dermatophagoides farinae (35%) and Glycycometus malaysiensis (37%). Immunoblots revealed not all allergic subjects showed positive immuno-reactivity against the mites tested. Single or multiple bands were observed for different species. The subjects were commonly sensitized to Group 2 (9-12 kDa), 10 (38 kDa) and 18 (40-48 kDa) allergens. Twenty-one out of 60 allergic subjects were sensitized to either one or more species. The majority of them (71%) were sensitized to single species. The allergic subjects were mainly sensitized to D. pteronyssinus, followed by Tyrophagus putrecentiae and Aleuroglyphus ovatus. Seven were solely sensitized to HDM while 10 were solely sensitized to SM. Four subjects were sensitized to both. Pre-adsorption study revealed no cross-reactivity. There was difference between the prevalence and reactivity to allergens of HDM and SM in these subjects. Both ELISA and immunoblot did not correlate well but can complement each other in improving the detection of mite allergens to the species level.
To examined the immediate and 24 hours post- irradiation germicidal effects of UV-C lamp on eggs and adults of house dust mites Dermatophagoides pteronyssinus (D. pteronyssinus) and Dermatophagoides farinae (D. farinae).
Four commercial repellents were evaluated in the laboratory against Leptotrombidium deliense chiggers. Both in vitro and in vivo methods were used to determine repellency of the compounds. The repellents were Kellis (containing citronella oil, jojoba oil and tea tree oil), Kaps (containing citronella oil), BioZ (containing citronella oil, geranium oil and lemon grass oil) and Off (containing DEET). The combination of three active ingredients: citronella oil, geranium oil, lemon grass oil gave the highest repellency (87%) followed by DEET (84%). In vitro repellencies ranged from 73% to 87%. There was no significant difference between the four products. All the repellents had 100% in vivo repellency compared to 41-57% for the controls.
The DNA sequences encompassing two hypervariable regions, VD II and III of the 56 kDa immunodominant protein gene of 21 Malaysian strains of Orientia tsutsugamushi were determined. Two strains demonstrated a 100% DNA homology with the Gilliam prototype strain, and one with TH1817 strain and TA678 strain respectively. High percentages of DNA similarity (95-99%) were observed with Karp (4 strains), Gilliam (2 strains), TH1817 (4 strains), TC586 (3 strains) and TA763 (1 strain). The remaining strains demonstrated the highest DNA similarity with TA763 (1 strain, 89%), TA678 (1 strain, 86%) and TA686 (1 strain, 87%). Our study provides additional evidence on the existence and the genetic heterogeneity of TA strains of the Southeast Asia and their closely related strains in Malaysia.
Isolation of rickettsiae from patients' blood samples and organ samples of wild rodents from areas with high seroprevalence of rickettsial infections was attempted using cell culture assay and animal passages. L929 mouse fibroblast cells grown in 24 well tissue culture plate were inoculated with buffy coat of febrile patients and examined for the growth of rickettsiae by Giemsa, Gimenez staining and direct immunofluorescence assay. No rickettsiae were isolated from 48 patients' blood samples. No symptomatic infections were noted in mice or guinea pigs infected with 50 organ samples of wild rodents. There was no rickettsial DNA amplified from these samples using various PCR detection systems for Orientia tsutsugamushi, typhus and spotted fever group rickettsiae.
The pathogenicity of Malaysian isolates of Orientia tsutsugamushi was investigated by a mouse virulence assay. The isolates could be differentiated as low (4 isolates), moderately (3 isolates) and highly virulent (2 isolates) based on the different responses in infected mice. No direct correlation between severity of human scrub typhus infections and virulence of the O. tsutsugamushi in mice was observed. Mice infected with virulent strains of O. tsutsugamushi showed splenomegaly, ascitis accumulation and enlargement of kidneys and livers whereas avirulent O. tsutsugamushi strains were asymptomatic and exhibited ruffled fur for a short period after infection. There was low antibody response in mice infected with isolates of low pathogenicity as compared with those of highly virulent isolates. Upon dissection of the infected mice, enlargement of mouse organs such as spleen, kidney and liver was noted. Presence of rickettsemia in mice was confirmed by the growth of O. tsutsugamushi in the L929 cells when inoculated with blood from infected mice. O. tsutsugamushi was also cultured from the peritoneal exudates of the infected mice. However, DNA of O. tsutsugamushi was only detected in the peritoneal exudates (by PCR) and blood (by cell culture) and not from other tissue samples.
The seroprevalence of various Orientia tsutsugamushi (OT) strains among Malaysian patients with suspected scrub typhus infections was determined using an indirect immunoperoxidase (IIP) assay. IgG against a single OT strain were detected in six sera (3 Karp, 1 Gilliam and 2 TC586), whereas IgM antibodies against a single OT strain (Gilliam) were noted in 3 sera (Gilliam). IgG reactive to all OT strains were present in 33 (47.1%) of the 70 sera and IgM reactive to all OT strains were present in 22 (78.6%) of the 28 sera. The fact that most sera were reactive to multiple OT strains suggests that group-specific antigens are involved in scrub typhus infections, whereas very few were due to strain-specific epitopes present on these strains. Peak IgG and IgM titers were noted more frequently against Gilliam, Karp, and TA763 strains: this suggests that these strains may be the commonest infecting strains among Malaysian patients. Two predominant OT polypeptides consistently reacted with patients' sera were the 70 kDa and 56 kDa proteins.
In this study, recombinant proteins that encompassed the AD I-AD III regions of 56 kDa immunodominant gene of 2 Orientia tsutsugamushi (OT) serotypes; Gilliam and TA763 were expressed in Escherichia coli. Both recombinant proteins exhibited serologic cross-reactivity with the rabbit antisera against various OT serotypes, as evaluated by enzyme-linked immunosorbent assay (ELISA), but not against other rickettsial species, including Rickettsia typhi, R. prowazekii and TT118 SFG rickettsiae. The feasibility of using the recombinant proteins as a diagnostic reagent was further evaluated by ELISA using sera from blood donors and scrub typhus patients. The results suggested a higher affinity of the antihuman IgM than IgG with both recombinant proteins. The IgM ELISA findings were agreeable with the results of indirect immunoperoxidase (IIP) assay especially with sera of high antibody (1:1600). However, more than one antigen are probably needed for development of an effective assay for serodiagnosis of scrub typhus in endemic areas.
A species of house dust mite, Suidasia pontifica, was recently shown to produce allergens affecting man. The species may be as important as other allergen producing mite in sensitization and causing allergic symptoms in Malaysians. Surveys conducted demonstrated that 80% of the houses surveyed were positive for this mite with densities ranged from 2 to 50 mites per gram of dust. Colonies of the species has been successfully established and materials from those colonies have been used to produce extracts for studies on sensitization to the mites. A total of 85 suspected allergic rhinitis patients were tested and 74.1% demonstrated positive reactions. Extract of this mite should be considered for routine diagnostic testing and possible immunotherapy.
A serosurvey was conducted in 1995-97 among 1596 febrile patients from 8 health centres in Malaysia for antibodies against Orientia tsutsugamushi (OT), Rickettsia typhi (RT) and TT118 spotted fever group rickettsiae (SFGR) by using an indirect immunoperoxidase assay. A total of 51.4% patients had antibody against at least 1 of those rickettsiae. Antibody to SFGR was most prevalent (42.5%), followed by RT (28.1%) and OT (24.9%). The seroprevalences of antibodies to SFGR, RT or OT alone were 12.4, 3.6 and 4.3%, respectively. Antibodies against more than 1 species of rickettsiae were presence in 31.1% of the patients, suggesting the possibility of co-infection, previous exposures or serological cross-reactivities. Seroprevalence of the various rickettsiae varied according to locality, with SFGR antibodies being the most prevalent in most areas. There was no significant association of prevalence of rickettsial antibody with gender. The seroprevalence of OT, SFGR and RT increased with patient age but an increase of antibody titre with age was not significant. Those working in the agricultural sectors had significantly higher seroprevalence of OT, SFGR and RT than those not related with agricultural activities. Scrub typhus remains a public health problem with an estimated annual attack rate of 18.5%. Tick typhus and murine typhus as shown in this serosurvey appear much more widespread than scrub typhus in this country.
Using cultured mouse fibroblast L929 cells, this study demonstrated the hemolytic and cytotoxic activities and induction of apoptosis in cells infected with Orientia tsutsugamushi. Low levels of hemolytic activity were detected using heavily infected cells. No hemolysin or cytotoxin were detected in the infected culture fluid regardless of the pathogenicity of the O. tsutsugamushi strains in mice. Using propidium iodide uptake assay, acridine orange/ethidium bromide staining and terminal deoxynucleotide transferase-mediated dUTP-digoxigenin nick-end labeling assay, apoptosis was observed in L929 cells infected with Karp and Gilliam strains.
Aerosampling using Rotorod samplers was conducted in the Institute for Medical Research, Kuala Lumpur, Malaysia, from December 1991 to November 1993. Samples were collected twice a week between 10.00 hours to 12.00 hours. Rods were stained and examined microscopically. A total of 8 and 20 types of pollens and mold spores were collected, respectively. More mold spores were collected than pollens. Grass pollen constituted more than 40 percent of total pollen counts. Gramineae pollen counts peaked in March and September. The most abundant mold spore was Cladosporium followed by Rust, Nigrospora, Curvularia and Smut. Cladosporium counts peaked in February and August. Rust counts peaked in June and December whereas counts for Nigrospora peaked in February and October. Highest counts of Smut were recorded in March and October. Curvularia counts peaked in January, June and September.
A dot-immunobinding assay (DIBA) was compared with a direct fluorescent antibody technique (DFAT) for the detection of Rickettsia tsutsugamushi infection in Leptotrombidium fletcheri (Womersley & Heaslip). Laboratory colonies of infected and noninfected chiggers were examined. The relative proportions of positive, negative, and indeterminate results were significantly different between DIBA and DFAT for infected but not for noninfected chiggers. DIBA was more sensitive and had a better negative predictive value and a lower false negative percentage than DFAT. It was concluded that DIBA is a suitable alternative to DFAT for detecting scrub typhus infection in chiggers.