Molecular evidence supports that Anopheles anthropophagus from China and Anopheles lesteri from Japan are the same species.

The Hyrcanus group of Anopheles consists of many related species, of which An. sinensis, An. lesteri, and An. anthropophagus are known as malaria vector species. It is not possible to identify these species morphologically in the adult and larval stages. Nucleotide sequence alignment of 2nd internal transcribed spacer (ITS2) regions from 4 specimens of An. lesteri collected in Japan, 2 specimens of An. anthropophagus collected in China, and 1 specimen of An. sinensis collected in Korea were sequenced and compared. Sequences of ITS2 regions varied only at 4 sites between An. lesteri and An. anthropophagus, and individual variations among each An. lesteri and An. anthropophagus were found at 5 and 7 sites, respectively, whereas sequences varied at 161 sites between An. lesteri and An. sinensis. This molecular evidence strongly supports that An. lesteri from Japan and An. anthropophagus from China are the same species.

Allergenicity of recombinant Bla g 7, German cockroach tropomyosin.

BACKGROUND: Cockroach infestation may sensitize and elicit allergic responses to genetically predisposed individuals. Invertebrate tropomyosins are a frequent cause of allergy and highly cross-reactive in nature. In this study, we aimed to produce recombinant German cockroach tropomyosin and investigate its allergenicity. METHODS: German cockroach tropomyosin (Bla g 7) was cloned by reverse transcriptase polymerase chain reaction (RT-PCR). The cloned cDNA was over-expressed in Escherichia coli and purified by affinity chromatography using Ni-nitrilotriacetic (NTA) acid resin. The allergenicity of the recombinant tropomyosin was examined by enzyme-linked immunosorbent assay (ELISA). RESULTS: The cloned Bla g 7 shared up to 91% amino acid sequence identity with other cockroach tropomyosins. ELISA showed a recombinant Bla g 7 sensitization rate of 16.2% to German cockroach allergic sera. Recombinant tropomyosin was able to inhibit 32.4% of the specific IgE binding to cockroach extract. CONCLUSIONS: Tropomyosin represents a minor allergen in cockroach extracts. It is hoped that recombinant tropomyosin will be useful for further studies and clinical applications.

Localization of Der f 2 in the gut and fecal pellets of Dermatophagoides farinae.

BACKGROUND: House dust mite derived materials are known to be the most potent agent inducing allergic diseases. Localization of Der f 2 was attempted to specify the sites and concentrations of Der f 2 within the mite, which may indicate the importance of secreted materials and nonexcreted body components as allergen sources. METHODS: Serial cryostat sections of embedded live mites and the fecal pellets, collected by brush, were immunoprobed using monoclonal antibody (mAb) 2F38 raised against recombinant (r) Der f 2. RESULTS: Highest concentrations were found in the anterior midgut, implying that this is the site of Der f 2 synthesis and secretion. Digestive material and defecated fecal pellets were also labeled with mAb. CONCLUSIONS: Our results show that the major allergen, Der f 2, found in the house dust mite D. farinae is derived from the digestive tract, and is concentrated in the feces.

Monoclonal antibodies to recombinant Der f 2 and development of a two-site ELISA sensitive to major Der f 2 isoallergen in Korea.

BACKGROUND: Der f 2 is a major sensitizing allergen in patients allergic to house dust mites worldwide. Isoforms of Der f 2 have been reported and are known to have different antigenicities. The aim of this study was to facilitate antigenic analysis and to develop an improved method for the detection of Der f 2 isoallergen, which is prevalent in Korea. METHODS: A two-site ELISA was developed with monoclonal antibodies (mAbs) which were produced against recombinant Der f 2 (rDer f 2) and applied to assess Der f 2 in bedding samples. RESULTS: A major isoform of Der f 2, found in Korea, was found to have amino acid variations especially at position 100 from lysine to glutamic acid, which is known to reduce significantly the binding affinity of mAbs when used to assess group 2 allergens. The detection limit of the developed two-site ELISA was determined to be about 8 ng/ml with rDer f 2 and 1 microg/ml with Derntatophagoides farinae crude extract. The average amount of Der f 2 in dust obtained from bedding samples from 89 homes in Seoul was estimated to be 25.61+/-10.70 microg/g dust. CONCLUSIONS: Assays using mAbs for rDer f 2 could be useful for the assessment of environmental allergen exposure and mAbs could be used to further characterize the isoallergens of Der f 2.

Determination and geographical distribution of Orientia tsutsugamushi serotypes in Korea by nested polymerase chain reaction.

Field rodents and chigger mites were collected at 30 locations in Korea in October and November 1997-1999 to determine the serotypes of Orientia tsutsugamushi and their geographical distribution. A nested polymerase chain reaction was performed with the spleen tissues from 546 field-striped mice (Apodemus agrarius) and 104 pools of chigger mites. The positivity rate of O. tsutsugamushi was 45.6% in A. agrarius and 39.4% in the chigger mite pools. Two serotypes, Boryong and Karp, were found in these samples; the former was predominant (78.3% in the mice and 82.9% in the chigger mite pools), with wide distribution throughout the country, including Cheju-do. The latter was confined to the middle of the Korean peninsula, with positivity rates of 15.7% in the mice and 12.2% in the chigger mite pools. The double infection of Karp and Boryong serotypes was found in 15 (6.0%) A. agrarius mice. Gilliam serotype was not detected at any of the study locations. The Boryong and Kuroki serotypes were identical in amino acid sequence of the 56-kDa protein, although they differed in virulence to BALB/c mice.

Comparison of allergenic components between German cockroach whole body and fecal extracts.

BACKGROUND: Cockroaches have been demonstrated to be an etiologic factor in allergic diseases. Further, sensitivity to cockroach places patients with asthma at risk for exacerbations that require emergency medical care. OBJECTIVE: This study compared the differences in allergenic components between German cockroach whole body and German cockroach fecal extracts (GWBE and GFE). METHODS: Patients with asthma and/or allergic rhinitis were skin prick tested with German cockroach extract (Bayer Corporation, West Haven, CT). Serum specimens from these patients, 25 with positive skin tests and 8 with negative tests, were used for the ELISA and immunoblot experiments. RESULTS: By ELISA, 72% (18 of 25) and 60% (15 of 25) of positive responders' sera showed IgE antibodies to GWBE and GFE, respectively, and the IgE levels to GWBE were highly correlated with those to GFE (r = .84, P < .01). In inhibition ELISA experiments, extensive cross-reactivity was observed between GWBE and GFE, slight cross-reactivity between GWBE and Dermatophagoides farinae, and no cross-reactivity between GFE and D. farinae. The two-site monoclonal antibody ELISA detected more of the German cockroach major allergens in GFE compared with GWBE; 6.2 times (2420 vs 390 U/mL) for Bla g 1 and 3 times (15.32 vs 5.07 microg/mL) for Bla g 2. In the immunoblot comparison of patients' sera, the IgE antibodies binding to GWBE were apparently different from those binding to GFE in all the positive responders' sera; eg, 50% or more of the 25 positive responders' sera reacted to 43- to 67-kDa proteins in GWBE and to 28- to 30-kDa proteins in GFE, respectively. No IgE antibodies bound to components in GWBE and GFE in the 8 negative responders' sera. CONCLUSIONS: There are major differences between the allergenic components of GWBE and GFE. Based on the amounts of major allergens (Bla g 1, Bla g 2), German cockroach feces are a more important source of allergen than the whole body in respiratory allergic diseases.

Daily survival and human blood index of Anopheles sinensis, the vector species of malaria in Korea.

To evaluate the vector efficiency of Anopheles sinensis in transmitting vivax malaria in the northern part of Gyonggi-do, South Korea, daily survival and feeding host preferences were studied during the period of June-October 1999. Ovaries of unfed and freshly fed An. sinensis females were dissected and parity or nulliparity were observed. The parous rates were 75.2% in July, 56.5% in August, 78.5% in September, and 60.0% in October at Gusan-dong, Goyang-si, Gyonggi-do. The average probability of daily survival was 0.890. To determine the host feeding patterns of An. sinensis, outdoor-resting bloodfed mosquitoes were collected, and the sources of the blood meals were analyzed by enzyme-linked immunosorbent assay, using 6 different animal immunoglobulin G antibodies. Out of 305 blood meals tested, 0.7% were positive from humans, 89.8% from bovines, 3.3% from swine, 0.7% from dogs, 1.6% from chickens, and 0.7% from bovines and swine mixed. No blood meals were positive from mice. Though the vector efficiency of An. sinensis was poor because of a low human blood index and a moderate rate of daily survival, vectorial capacity would be high because of high density of the population.

Localization of a major allergen, Der p 2, in the gut and faecal pellets of Dermatophagoides pteronyssinus.

BACKGROUND: The house dust mite Dermatophagoides ptronyssinus is one of the most significant indoor sensitizing agents of allergy. Allergen localization may indicate the importance of secreted materials, faeces, and nonexcreted mite body components as allergen sources. OBJECTIVE: This study attempted to localize the sites and concentrations of Der p 2 in the cryostat sections of D. pteronyssinus using antirecombinant Der p 2 monoclonal antibody. METHODS: Male and female mites and mite faeces collected separately from both sexes were used. Live mites were embedded and serial cryostat sections for light microscopy were performed. Anti-recombinant Der p 2 monoclonal antibody previously produced by the authors was used. For immunoprobing, mite cryostat sections were incubated in the following antibody-containing solutions: monoclonal antibody against Der p 2 was initially applied to the sections and fluorescent isothiocyanate conjugated antimouse immunoglobulin G was reacted as the secondary antibody. The faecal pellets were treated the same as described above. RESULTS: Immunofluorescent probing of cryostat sections with the monoclonal antibody showed labelling of the gut lining, gut contents and defecated faecal pellets. No other internal organs were identified as positively labelled. CONCLUSION: This study suggested that a major allergen, Der p 2, found in the house dust mite D. pteronyssinus is derived from the digestive tract and concentrated in the faeces.

Unstable vivax malaria in Korea.

Korean vivax malaria had been prevalent for longtime throughout the country with low endemicity. As a result of the Korean war (1950-1953), malaria became epidemic. In 1959-1969 when the National Malaria Eradication Service (NMES) was implemented, malaria rates declined, with low endemicity in the south-west and south plain areas and high endemic foci in north Kyongsangbuk-do (province) and north and east Kyonggi-do. NMES activities greatly contributed in accelerating the control and later eradication of malaria. The Republic of Korea (South Korea) was designated malaria free in 1979. However, malaria re-emerged in 1993 and an outbreak occurred in north Kyonggi-do and north-west Kangwon-do (in and/or near the Demilitarized Zone, DMZ), bordering North Korea. It has been postulated that most of the malaria cases resulted from bites of sporozoite-infected females of An. sinensis dispersed from North Korea across the DMZ. Judging from epidemiological and socio-ecological factors, vivax malaria would not be possible to be endemic in South Korea. Historical data show that vivax malaria in Korea is a typical unstable malaria. Epidemics may occur when environmental, socio-economical, and/or political factors change in favor to malaria transmission, and when such factors change to normal conditions malaria rates become low and may disappear. Passive case detection is a most feasible and recommendable control measure against the unstable vivax malaria in Korea in cost-effect point of view.

Comparative study on longevity of Anopheles sinensis in malarious and non-malarious areas in Korea.

An outbreak of vivax malaria has been occurring in northern part of Kyonggi-do and north-western part of Kangwon-do, where are located near the demilitarized zone, since 1993. For understanding of epidemiological features of malaria, the probability of daily survival of Anopheles sinensis, the vector species of malaria was compared in malarious and non-malarious areas in July-August, 2000. Total 915 females collected at three locations in malarious areas were dissected for ovaries, and 64.6% of the parous rate was found. Total 758 females collected at three locations in non-malarious areas were dissected, and 57.8% of the parous rate was observed. It was estimated from the parous rates that the probability of daily survival of An. sinensis females was 0.864 in malarious areas and 0.850 in non-malarious areas, which was not significantly different.

Evolution of Hypervariable Regions, V4 and V7, of Insect 18S rRNA and Their Phylogenetic Implications.

We compared primary and secondary structures of V4 (helices E23-2 to E23-5) and V7 (helix 43) regions of 18S rRNAs in insects and the other three major arthropod groups (crustaceans, myriapods, and chelicerates) known so far. We found that the lengths of primary sequences and the shapes of secondary structures of these two hypervariable regions of insect 18S rRNA even at infraclass levels are phylogenetically informative and reflect major steps in insect evolution. The long sequence insertion and bifurcated shape of helices E23-2 to E23-5 in the V4 region are unique synapomorphic characters for winged insects (Pterygota). The long sequence insertion and expanded stem length of helix 43 in the V7 region are synapomorphic characters for holometabolous insects which conduct complete metamorphosis. The strongly conserved secondary structures suggest the possibility that these hypervariable regions may be related with certain important cellular functions unknown thus far. The comparison with insect fossil records revealed that the pterygote synapomorphy (V4) and the holometabolous synapomorphy (V7) were established prior to the acquisition of insect wings (flight system) and prior to the development of complete metamorphosis, respectively. These synapomorphies have been also relatively stable over at least 300 Myr and 280 Myr, respectively as well. It implies that the expansion events of the V4 and V7 regions have not occurred simultaneously but independently at different periods during the insect evolution. Then this suggests that V4 and V7 regions are not functionally correlated as recently suggested by Crease and Coulbourn.

Monoclonal antibodies to recombinant Der p 2, a major house dust mite allergen: specificity, epitope analysis and development of two-site capture ELISA.

House dust mite allergens have been well established as sensitizing agents that are important in the induction of allergic diseases. In order to analyze epitopes of the allergen and to develop a quantitative method of the allergen exposure, monoclonal antibodies against a recombinant Der p 2 (rDer p 2), one of the major allergens of Dermatophagoides pteronyssinus, were produced. Four monoclonal antibodies produced were species-specific and did not cross-react to the D. farinae crude extract. Two of the monoclonal antibodies were found to be IgG1 and the others were IgM. For the analysis of epitopes, a Der p 2 cDNA encoding 126 amino acids (aa) was dissected into three fragments with several overlapping peptides. A (aa residues 1-49), B (44-93), and C fragment (84-126). Three monoclonal antibodies showed reactivities to the recombinant B fragment and to the full-length rDer p 2, but one monoclonal antibody reacted only with the full-length rDer p 2. Two-site capture ELISA was developed using two different monoclonal antibodies for quantitating Der p 2 in house dust. The sensitivity limit was 4 ng/ml with rDer p 2 and 8 micrograms/ml with the D. pteronyssinus crude extract. The result suggested that the assay using monoclonal antibodies against rDer p 2 could be useful for the environmental studies and for the standardization of mite allergen extracts.

Identification of Chironomus kiiensis allergens, a dominant species of non-biting midges in Korea.

Non-biting midges are known to contain potent inhalant allergens. IgE antibody responses to the crude extract of Chironomus kiiensis adults, a dominant chironomid species in Korea, were examined. With the IgE-ELISA or passive cutaneous anaphylaxis reactions, increased levels of chironomid-specific IgE were detected in the skin test positive human sera, or immunized BALB/c mouse sera with the crude extract adsorbed to alum. IgE-immunoblot analysis showed major IgE-reacting protein band patterns, which reacted with more than 50% of the skin test positive human sera, at 110, 80, 73, 46, 40, 37, 34, and 31 kDa. The reactive band patterns were largely similar between skin test positive humans and immune BALB/c mice. However, the bands of 55, 31, 27, 26, 24, and 23 kDa were found only in sensitized humans, but not in immunized mice.

Identification and purification of IgE-reactive proteins in German cockroach extract.

Cockroaches have been implicated as a cause of respiratory allergy in urban areas worldwide. IgE-reactive German cockroach proteins were identified with molecular weights (MWs) of 90, 66, 50, 43 and 36 KD by immunoblot analysis in both immune BALB/c mice and sensitized humans. Prominent IgE-reactive proteins were purified using FPLC by ion-exchange chromatography, gel filtration and hydrophobic chromatography. The N-terminal amino acid sequence of a purified protein with a MW of 66 KD on SDS-PAGE was Val-Thr-Leu-Lys-Lys(Val)-Met-Ile-Lys-Thr-Phe-Tyr. No homologous protein was found through a search of GenBank that indicated a novel IgE-reactive protein in German cockroach extract. Another purified protein with a MW of 36 KD reacted strongly with a monoclonal antibody against Bla g 2.

An infestation of the mite Sancassania berlesei (Acari: Acaridae) in the external auditory canal of a Korean man.

We here report the case of a storage mite, Sancassania berlesei, infestation in the external auditory canal of a 46-yr-old male. He complained of feeling a foreign body and itching in the left external auditory canal for 1 mo, with accompanying otalgia for 3 days. Considering the duration of the patient's complaint and the 8-9-day life cycle of the mite, the mites are believed to have lived in the patient's ear for more than 3 generations.

An aural myiasis case in a 54-year-old male farmer in Korea.

A 54-year-old male farmer residing in Chunchon, Korea, complaining of blood tinged discharge and tinnitus in the left ear for two days, was examined in August 16, 1996. Otoscopic examination revealed live maggots from the ear canal. The patient did not complain of any symptoms after removal of maggots. Five maggots recovered were identified as the third stage larvae of Lucilia sericata (Diptera: Calliphoridae). This is the first record of aural myiasis in Korea.

Cross-reactivity of Tyrophagus putrescentiae with Dermatophagoides farinae and Dermatophagoides pteronyssinus in urban areas.

BACKGROUND: Tyrophagus putrescentiae (TP) have cohabited with D. pteronys-sinus (DP) and D. farinae (DF) in more than 25% of houses in urban areas of Korea, and many atopic subjects have also been cosensitized to TP and Dermatophagoides species. OBJECTIVE: We evaluated the cross-reactivity of TP with DF and DP in atopic subjects of urban inhabitants. METHODS: The cross-reactivity was evaluated with inhibition ELISA and immunoblotting. Allergenic components of TP were evaluated with IgE immunoblotting of the sera from 25 individual atopics. All enrolled subjects lived in urban areas. RESULTS: In ELISA inhibition with pooled sera, all TP, DP, and DF extract inhibited TP-specific IgE by more than 90%, and the 50% inhibitory concentrations of TP, DP, and DF extract were 0.4 microg/mL, 0.8 microg/mL and 0.8 microg/mL, respectively. The maximum inhibition, however, of DP-specific and DF-specific IgE by TP extracts was 32% and 29%, respectively. With six individual sera, the TP-specific IgE was also inhibited by more than 88% with DF extract in all cases. In inhibition immunoblotting, all of the TP, DP, and DF extracts completely inhibited the TP-specific IgE bands at a concentration of 2.0 microg/mL. Fifteen allergenic components in TP were found. Among them, the 16-kD allergen was most prevalent (52%) and its IgE binding was completely inhibited by 0.1 microg/mL of purified Der f2 and it also bound with 2 different monoclonal antibodies to the group 2 allergen of Dermatophagoides species. CONCLUSIONS: Our results suggested considerable cross-reactivity between TP and the two Dermatophagoides species in urban areas where TP and Dermatophagoides species cohabit. The 16-kD allergen, which shared common epitopes with the group 2 allergen of Dermatophagoides, is one of the most prevalent allergens of TP.

Occurrence of Culex (Barraudius) inatomii Kamimura and Wada (Diptera: Culicidae) in a large marsh adjacent to the coast of Ulsan, Korea.

The occurrence of Culex (Barraudius) inatomii Kamimura and Wada was observed from a large reedy marsh with fairly polluted brackish water near the coast of Ulsan, Korea, at an industrial complex. On average, 263.6 larvae per dip (450-cc dipper) were collected, and 408 females were caught by human biting collection in an hour. A CDC light trap set up outdoors collected 511 Cx. inatomii (96.8% of the total mosquitoes). The occurrence of this rarely reported mosquito raises questions about the effects of habitat modification and the need for further study.

Identification of Tyrophagus putrescentiae allergens and evaluation of cross-reactivity with Dermatophagoides pteronyssinus.

House dust mites are the most common cause of allergic sensitization in respiratory allergic patients in the world. Tyrophagus putrescentiae (TP), which was followed by Dermatophagoides farinae (DF) and Dermatophagoides pteronyssinus (DP), has been reported as the third most common house mite in Korea. We previously reported that many respiratory allergic patients had become concomitantly sensitized to DP, DF and TP. The aims of this study were to identify the allergic components of TP and to evaluate the cross-reactivity between TP and DP. The allergenic components of TP and DP extracts were determined with SDS-PAGE and IgE immunoblotting analysis. The cross-reactivity as evaluated by ELISA inhibition and inhibitory immunoblotting experiments. According the SDS-PAGE, the protein components of the two extracts were somewhat different, although a few components displayed identical molecular weights. The 18 kD protein of TP was the most prevalent allergen in the sera of patients sensitized to TP and DP. Both of the maximum inhibition percentages of optic densities of TP-specific IgE in ELISA with TP and DP extract were 100%, respectively and the 50% inhibitory dose (ID50) of TP extract and DP extract were 0.01 micrograms/ml and 0.02 micrograms/ml, respectively. Maximum inhibition of optic densities of DP-specific IgE in ELISA with TP and DP extracts were 29% and 100%, respectively and the ID50 of DP extract was 0.007 micrograms/ml. On inhibitory immunoblotting of DP specific IgE, 5 micrograms/ml of TP extract completely inhibited 16 kD without inhibiting the other allergic component. Whereas, on inhibitory immunoblotting of TP-specific IgE, 5 micrograms/ml of DP extract completely inhibited all the IgE binding components of TP. These results suggested that the major allergen of TP may be the 18 kD component and we also concluded that TP allergens have a strong cross-reactivity with DP extracts, but that DP allergens only have partial cross-reactivity with TP extracts.

A human case of tick bite by Ixodes nipponensis.

A human case of the tick bite on the back of 36-year-old man was found in September 1995. On admission he complained of itching sensation and pain at the site. The removed tick was identified morphologically as Ixodes nipponensis.