Chigger mites of the genus Leptotrombidium: key to species and their distribution in China

Chigger mites of the genus Leptotrombidium (Acari: Trombiculidae) transmit scrub typhus, caused by Rickettsia tsutsugamushi (= R.orientalis) in South-East Asia. In China, eighty-two species of Leptotrombidium have been recorded; these are listed with the names of Provinces where they were found. Five species, L.deliense, L.insularae, L.kaohuense, L.rubellum and L.scutellare, have been implicated as Chinese vectors of scrub typhus. A brief key is given to the larvae of all but three of the Leptotrombidium mites known in China.     

Isolation of Orientia tsutsugamushi from Leptotrombidium fuji and its characterization

In our attempts to isolate Orientia tsutsugamushi from trombiculid mites, a strain was successfully isolated from Leptotrombidium fuji collected in Aichi Prefecture, Japan. This is the first case of isolation of O. tsutsugamushi from L. fuji. A phylogenetic analysis based on the base-sequence homology of the 56-kDa type-specific antigen-gene indicated that the strain is a new type which is not closely related to any strains analyzed previously. Three strains isolated from Leptotrombidium pallidum harvested at the same area were identified as being closely related to the JP-2 type (subtype-2 of Karp type in Japan) by phylogenetic analysis.     

Coincidence between Geographical Distribution of Leptotrombidium scutellare and Scrub Typhus Incidence in South Korea

To clarify the geographical distribution of scrub typhus vectors in Korea, a survey of larval trombiculid mites was conducted from 2005 to 2007 by collecting wild small mammals twice a year (spring and autumn) at 24 sites nationwide. A total of 67,325 mites representing 4 genera and 14 species were collected from 783 trapped rodents, corresponding to a chigger index (number of chigger mites per rodent) of 86.0. The predominant mite species were Leptotrombidium pallidum (52.6%), Leptotrombiduim scutellare (27.1%), Leptotrombidium palpale (8.2%), Leptotrombidium orientale (5.6%), and Neotrombicula tamiyai (1.7%). However, the proportions of L. scutellare in southern areas, including endemic provinces such as Jeollabuk-Do (34.3%), Jeollanam-Do (49.0%), and Gyeongsangnam-Do (88%), were relatively higher than in central Korean regions where L. pallidum was predominant. In autumn, the ratio of L. scutellare increased to 42% while the ratio of L. pallidum decreased. The geographical distribution map of the L. scutellare chigger index was identical to the incidence pattern of scrub typhus, whereas those of overall mites and L. pallidum showed no relationship with case incidence patterns. Distribution mapping analysis shows an identical geographical distribution of L. scutellare and epidemic incidence of scrub typhus in South Korea. L. pallidum could be another vector at all other parts of the Korean peninsula, including the eastern and northern regions that have a low level of scrub typhus incidence.     

Obligate intracellular bacteria diversity in unfed Leptotrombidium scutellare larvae highlights novel bacterial endosymbionts of mites

It is well known that the mite Leptotrombidium scutellare carries the pathogen of scrub typhus, Orientia tsutsugamushi. However, our understanding of other bacterial endosymbionts of mites is limited. This study investigated the diversity of the obligate intracellular bacteria carried by L. scutellare using 16S rRNA gene amplicon analysis with next-generation sequencing. The results showed that the detected bacteria were classified into the genera Rickettsia, Wolbachia, and Rickettsiella and an unknown genus of the order Rickettsiales. For further classification of the detected bacteria, a representative read that was most closely related to the assigned taxonomic classification was subjected to homology search and phylogenic analysis. The results showed that some bacteria of the genus Rickettsia were identical or very close to the human pathogens Rickettsia akari, Rickettsia aeschlimannii, Rickettsia felis, and Rickettsia australis. The genetic distance between the genus Wolbachia bacteria in the present study and in previous reports is highly indicative that the bacteria in the present study can be classified as a new taxon of Wolbachia. This study detected obligate intracellular bacteria from unfed mites; thus, the mites did not acquire bacteria from infected animals or any other infectious sources. Finally, the present study demonstrated that various and novel bacterial endosymbionts of mites, in addition to O. tsutsugamushi, might uniquely evolve with the host mites throughout overlapping generations of the mite life cycle. The roles of the bacteria in mites and their pathogenicity should be further examined in studies based on bacterial isolation.     

Proposed vector candidate: Leptotrombidium palpale for Shimokoshi type Orientia tsutsugamushi

To identify the vector species for Shimokoshi type Orientia tsutsugamushi, a survey of larval trombiculid mites was conducted in Yamagata Prefecture, Japan from April to May 2012. In all, 2889 larval trombiculid mites were obtained from 21 Apodemus speciosus rodent hosts, 2600 of which were morphologically classified into eight species in three genera. After screening of O. tsutsugamushi DNA in individual larval trombiculid mites using real‐time PCR targeting the 16S ribosomal RNA gene, serotype‐specific nested PCRs targeting the 56 kDa protein gene were performed, followed by sequencing analysis. As a result, Shimokoshi type O. tsutsugamushi DNA was identified from 3 (1.9%) of 157 Leptotrombidium palpale. This is the first study to identify Shimokoshi type O. tsutsugamushi DNA in L. palpale. The results indicate that L. palpale is a possible vector for Shimokoshi type O. tsutsugamushi.     

Transovarial Transmission of Orientia tsutsugamushi in Leptotrombidium palpale (Acari: Trombiculidae)

Transovarial transmission of Orientia tsutsugamushi in colonies of Leptotrombidium palpale was studied in the parent and F₁ and F2 generations. Both transovarial transmission and filial infection rates were 100% in the parent and F₁ generations of Leptotrombidium palpale. The filial infection rate in the F₁ generation was 100%, but it declined to 94.3% in the F₂ progeny. The sex ratio of the F₁ generation from infected L. palpale was 1∶0.8 (male:female) and the proportion of males was relatively high. This study is the first to report on the transovarial transmission of O. tsutsugamushi in L. palpale. High transovarial transmission rates in L. palpale suggest that this species might be one of the major vectors of tsutsugamushi disease in Korea.     

Variable clinical responses of a scrub typhus outbred mouse model to feeding by Orientia tsutsugamushi infected mites

Rodents are the natural hosts for Leptotrombidium mites that transmit Orientia tsutsugamushi, the causative agent of scrub typhus, a potentially fatal febrile human disease. Utilizing mite lines that included O. tsutsugamushi infected and non-infected Leptotrombidium species we investigated the varied infection response of outbred mice (ICR) exposed to L. chiangraiensis (Lc), L. imphalum (Li) and L. deliense (Ld). Each of six mite lines (Lc1, Lc5, Li3, Li4, Li7 and Ld) was separately placed in the inner ears of ICR mice either as a single individual (individual feeding, IF) or as a group of 2-4 individuals (pool feeding, PF). The species of infected chigger feeding on mice significantly affected mortality rates of the mice, with mite lines of Lc causing higher mean (±SE) mortality (90.7 ± 3.6 %) than mite lines of Li (62.9 ± 5.6 %) or Ld (53.6 ± 5.8 %). Mouse responses which included time to death, food consumption and total mice weight change depended on mite species and their O. tsutsugamushi genotype, more than on feeding procedure (IF vs. PF) except for mite lines within the Lc. Infected mite lines of Lc were the most virulent infected mites assessed whereas the infected Ld species was the least virulent for the ICR. Mice killed by various mite lines showed enlarged spleens and produced ascites. The results of this investigation of the clinical responses of ICR mice to feeding by various infected mite lines indicated that the different species of infected mites and their O. tsutsugamushi genotype produced different clinical presentations in ICR mice, a scrub typhus mouse model which mimics the natural transmission of O. tsutsugamushi that is critical for understanding scrub typhus disease in terms of natural transmission, host-pathogen-vector interaction and vaccine development.     

Multiplex PCR assay for identifying five species of chigger mites in the Republic of Korea

Chigger mites (Trombiculidae), which are known vectors of Orientia tsutsugamushi, have significantly fewer molecular data available than other known species, with a focus on morphological species identification rather than molecular species identification. However, chigger mites are easily damaged because of small size. In addition, natural variation and adult similarity would interrupt accurate classification and identification. Therefore, in this study, a multiple polymerase chain reaction (PCR) method using the internal transcribed spacer (ITS) region of five frequently occurring species (Euschoengastia koreaensis, Leptotrombidium orientale, Leptotrombidium pallidum, Leptotrombidium palpale, and Leptotrombidium scutellare) among eight species known to mediate the spread of is presented. This is the first report using molecular species identification for the classification method of chigger mites, which was only possible with morphological species identification, and is expected to be used as basic data for vector surveillance.     

Stylostome organization in feeding Leptotrombidium larvae (Acariformes: Trombiculidae)

The stylostome of larvae of the trombiculids Leptotrombidium scutellare (Nagayo et al.), Leptotrombidium fletcheri (Womersley et Heaslip) and Leptotrombidium deliense (Walch) was studied experimentally at different time intervals after larval attachment using the histological method. The stylostome of these species has the same organization and belongs to the epidermal combined with the mixed type, developing more in width than in length. Neither transverse nor conspicuous longitudinal layers are present within the stylostome walls, which stain predominantly in red with Azan, also showing longitudinal portions with blue staining. Larvae tend to attach closely to each other and scabs, consisting of the hyperkeratotic epidermal layers fusing with migrating inflammatory cells, develop around the attachment sites. The dermis shows inflammatory foci with dilated capillaries and inflammatory cells inserting in the connective tissue layer underneath the stylostome. The feeding cavity, which is moderately expressed, may be found either in the epidermis or in the dermis. It contains inflammatory cells and their debris in the liquefied host tissues. The stylostome length depends on the character of the attachment site (the thicker epidermis or scab the longer the stylostome), and does not directly correspond to the stages of larval feeding. Nevertheless, at the 48-h time interval, nearly all attached larvae are found to be fully fed and their midgut cells are filled with nutritional globules.     

Larval Chigger Mites Collected from Small Mammals in 3 Provinces,Korea

A total of 9,281 larval chigger mites were collected from small mammals captured at Hwaseong-gun, Gyeonggi-do (Province) (2,754 mites from 30 small mammals), Asan city, Chungcheongnam-do (3,358 mites from 48 mammals), and Jangseong-gun, Jeollanam-do (3,169 for 62 mammals) from April-November 2009 in the Republic of Korea (= Korea) and were identified to species. Leptotrombidium pallidum was the predominant species in Hwaseong (95.8%) and Asan (61.2%), while Leptotrombidium scutellare was the predominant species collected from Jangseong (80.1%). Overall, larval chigger mite indices decreased from April (27.3) to June (4.9), then increased in September (95.2) and to a high level in November (169.3). These data suggest that L. pallidum and L. scutellare are the primary vectors of scrub typhus throughout their range in Korea. While other species of larval chigger mites were also collected with some implications in the transmission of Orientia tsutsugamushi, they only accounted for 11.2% of all larval chigger mites collected from small mammals.     

Identification of potential hosts and vectors of scrub typhus and tick-borne spotted fever group rickettsiae in eastern Taiwan

Scrub typhus and tick-borne spotted fever group (SFG) rickettsioses are transmitted by chiggers (larval trombiculid mites) and hard ticks, respectively. We assessed exposure to these disease vectors by extensively sampling both chiggers and ticks and their small mammal hosts in eastern Taiwan during 2007 and 2008. The striped field mouse Apodemus agrarius Pallas (Rodentia: Muridae) was the most common of the small mammals (36.1% of 1393 captures) and presented the highest rate of infestation with both chiggers (47.8% of 110 760) and ticks (78.1% of 1431). Leptotrombidium imphalum Vercammen-Grandjean & Langston (Trombidiformes: Trombiculidae) and immature Rhipicephalus haemaphysaloides Supino (Ixodida: Ixodidae) were the most abundant chiggers (84.5%) and ticks (>99%) identified, respectively. Immunofluorescent antibody assay revealed high seropositive rates of rodents against Orientia tsutsugamushi Hyashi (Rickettsiales: Rickettsiaceae), the aetiological agent of scrub typhus (70.0% of 437 rodents), and tick-borne SFG rickettsiae (91.9% of 418 rodents). The current study represents a first step towards elucidating the potential hosts and vectors in the enzootic transmission of O. tsutsugamushi and tick-borne SFG rickettsiae in Taiwan. Further studies should focus on characterizing pathogens in L. imphalum and R. haemaphysaloides, as well as the proclivity of both vectors to humans. Uncovering the main hosts of adult ticks is also critical for the prevention of SFG rickettsial infections.     

Variability in Leptotrombidium europaeum and two new related chigger mite species (Acari: Trombiculidae) from Caucasus

Two new chigger mite species closely related to Leptotrombidium europaeum (Daniel et Brelih, 1959) are described from small mammals collected in Caucasus and Transcaucasia. L. alanicum sp. n. differs from L. europaeum in having shorter legs (TaIII = 61-81, Ip = 734-927 versus 72-90, and 855-1017), shorter scutal and idiosomal setae (D(min) = = 30-45, D(max) = 48-67, H = 59, PL = 58 versus 40-52, 54-69, 64, 63), slightly smaller scutum (AP = 25, SD = 47, PW = 89 versus 28, 50, 91), and more numerous idiosomal setae (87 versus 81). L. montanum sp. n. differs from L. europaeum in having more numerous idiosomal setae (102 versus 81), longer scutal and idiosomal setae (AM = 61, AL = 44, H = 68, D(max) = 66 versus 56, 41, 64, 62), thicker legs (TaW = 19 versus 18), and broader scutum (PW = 95 versus 91). Exact identification of both new species is possible only using classification functions constructed by means of discriminant analysis. These three Leptotrombidium species expose sympatric distribution in Daghestan (Eastern Caucasus). L. alanicum and L. montanum also occurred together in Krasnodar Territory (Western Caucasus). Each of these species includes a number of local geographical forms precisely distinguished from each other. Morphometric differences between L. alanicum and L. montanum agree with eco-geographical rules being previously found in chigger mites from other genera. However, differences between local forms of these species show other tendencies directed controversially in part. Therefore it is probable that interspecific differences in this case correspond to the variability which took place in the process of speciation.     

Stylostome formation by Leptotrombidium mites (Acari: Trombiculidae).

The modes of stylostome formation by larvae (chiggers) of Leptotrombidium intermedium, L. fletcheri, L. arenicola, and L. deliense in parasitized mouse skin were studied histologically in relation to their capacity to transmit Rickettsia tsutsugamushi. Three types of stylostome formation were recognized among the different species: the epidermal stylostome formed by the larva of L. intermedium; the mesenchymal stylostome formed by the larva of L. fletcheri; and, the mixed stylostome formed by the larva of both L. arenicola and L. deliense. Dermal inflammations related to the three types of stylostomes were histologically defined. The possible importance of stylostome characteristics to the transmission of rickettsial organisms is discussed.     

Trombiculid mites (Acarina: Trombiculidae) from Apodemus agrarius coreae caught at Pochun-gun, Gyeonggi Province and Chinhae-City, Kyongnam Province

During October-December 1987, chigger mites infested on the striped field mice (Apodemus agrarius coreae) collected at Pochun-gun, Gyeonggi Province and Chinhae city, Kyongnam Province were identified. 1. Of 171 back-striped mice collected, chigger mites were found from 58 mice, showing 33.9% of infestation rate. 2. Total 865 chigger mites collected were classified into seven species; Leptotrombidium pallidum (4.3%), L. palpale(23.0%), L. orientalis(20.2), L. zetum(19.0%). Neotrombicula tamiyai(32.9), N. japonica(0.2%) and Euschongastia koreaensis(0.3%). Though the most dominant species in number was N. tamiyai(32.9% of the total), this species was collected at very limited locality and for a short period. Therefore, most common species in Korea seems to be L. palpale which was found at all localities throughout the whole survey period. L. pallidum which is known as the vector species of tsutsugamushi disease in Korea was collected in a very low number(4.3% of the total chiggers collected). 3. The number of chigger mites infested on a host animal showed great variations from one chigger up to 207 chiggers. The present study has shown that there may be other Leptotrombidium species mite(s) for the vector and host of tsutsugamushi disease in Korea.     

恙螨子代幼虫群体形态在分类上的应用

采自湖南吉首和福建永定的两群形态相似的纤恙螨在实验室中饲养。将它们1雌1雄进行配对。当幼虫孵出后测量并比较其体各部的测量数,同时将其成虫与地里纤恙螨成虫酯酶同工酶酶谱进行比较。结果表明此两群形态相似的纤恙螨系英帕纤恙螨(Leptotrombidium imphalum)的不同地理亚种,它们形态与福建东部地区恙虫病媒介微红纤恙螨十分相似。恙螨子代幼虫群体形态的测量数系恙螨分类中的主要参数。     

青海省寄生螨类的区系分布

根据建国以来的大量研究调查资料,对青海地区已记载的寄生螨类（主要包括革螨、恙螨）计10科33属100余种进行了初步的区系分布、数量与宿主关系的探讨。（1）河湟谷地小区[1]：计有45种,主要有：赛氏血革螨、秉氏血革螨、旱獭青厉螨、毒厉螨、鼠兔赫刺螨、褪色巨螯螨、似田鼠新恙螨、旱獭新恙螨等;（2）柴达木盆地小区：计有15种,具代表性种类为乌兰纤恙螨;（3）祁连青南小区：计有85种,具代表性种类为久治真棒恙螨、玉树羽叶恙螨和朱雀禽棒恙螨;（4）羌塘高原小区：计有3种,皆为血厉螨属种类。     

山东部分地区小兽类寄生恙螨的群落结构

用鼠笼捕捉活鼠,收集体外恙螨,分类计数,定量分析小兽类体外寄生恙螨的群落结构。探讨山东小兽类体外寄生恙螨群落结构及其与疾病的关系。结果表明,当地5种小兽类体外共有6种寄生恙螨。黑线姬鼠、大仓鼠体外寄生螨种较丰富,各有5种寄生螨。小盾纤恙螨宿主范围广,在4种小兽的体外均有发现。大仓鼠体外太平洋无前恙螨的带螨率和带螨指数最高(72．73％、52．00),临淮岗纤恙螨、泰山纤恙螨、须纤恙螨普遍寄生于黑线姬鼠、大仓鼠体外。黑线姬鼠与大仓鼠寄生螨群落多样性指数相对较高。黑线姬鼠寄生螨群落与大仓鼠的相似系数最大(39．2)。黑线姬鼠体外恙螨群落结构随季节变化而发生变化,恙螨群落的丰富度和多样性指数以冬季(12～2月)最低,春、秋季较高。     

Abundances and host relationships of chigger mites in Yunnan Province,China

This paper reports on ectoparasitic chigger mites found on small mammals in Yunnan Province, southwest China. Data were accumulated from 19 investigation sites (counties) between 2001 and 2009. A total of 10 222 small mammal hosts were captured and identified; these represented 62 species, 34 genera and 11 families in five orders. From the body surfaces of these 10 222 hosts, a total of 92 990 chigger mites were collected and identified microscopically. These represented 224 species, 22 genera and three subfamilies in the family Trombiculidae (Trombidiformes). Small mammals were commonly found to be infested by chigger mites and most host species harboured several species of mite. The species diversity of chigger mites in Yunnan was much higher than diversities reported previously in other provinces of China and in other countries. A single species of rodent, Eothenomys miletus (Rodentia: Cricetidae), carried 111 species of chigger mite, thus demonstrating the highest species diversity and heaviest mite infestation of all recorded hosts. This diversity is exceptional compared with that of other ectoparasites. Of the total 224 mite species, 21 species accounted for 82.2% of all mites counted. Two species acting as major vectors for scrub typhus (tsutsugamushi disease), Leptotrombidium scutellare and Leptotrombidium deliense, were identified as the dominant mite species in this sample. In addition to these two major vectors, 12 potential or suspected vector species were found. Most species of chigger mite had a wide range of hosts and low host specificity. For example, L. scutellare parasitized 30 species of host. The low host specificity of chigger mites may increase their probability of encountering humans, as well as their transmission of scrub typhus among different hosts. Hierarchical clustering analysis showed that similarities between different chigger mite communities on the 18 main species of small mammal host did not accord with the taxonomic affinity of the hosts. This suggests that the distribution of chigger mites may be strongly influenced by the environment in which hosts live.     

Ultrastructural study on Japanese isolates of spotted fever group rickettsiae

Japanese isolates of spotted fever group rickettsiae were observed under a transmission electron microscope. In Vero cells persistently infected with Japanese isolates, small numbers of intracytoplasmic rickettsiae were seen. On the other hand, moderate numbers of rickettsiae were found in the cytoplasm of productively infected BHK cells. The electron-lucent, halo-like zone was found to surround organisms in the cytoplasm of their host cells, which is a prominent characteristic of spotted fever group rickettsiae. Fine structural features of the cell wall revealed thin outer and thick inner leaflets like those observed in other spotted fever group rickettsiae.