Surveillance of ticks on a tropical island with introduced ungulates.

The abundance of hard ticks can be determined by abiotic factors and the presence of suitable hosts. Since deer represent important hosts for many tick species, it is imperative to investigate whether deer introduction will increase the number of ticks, which can transmit a wide variety of pathogens. The sika deer (Cervus nippon) was introduced to Green Island, Taiwan, to supply velvet antlers, a traditional Chinese medicine. However, they were later released into the wild after a steep decline in antler prices. We conducted surveys for questing ticks, ticks on rodents and shrews, and the fecal pellet groups of ungulates (deer and goats) in 31 transects within 3 habitat types (forest, grassland, and roadside) on Green Island every 2 months in 2019. A total of 5,321 questing ticks were collected. All collected ticks were Haemaphysalis mageshimaensis Saito and Hoogstraal, a species first identified on an island with an abundance of sika deer. Additionally, 48 ticks collected from 126 trapped rodents and shrews were almost invariably Ixodes granulatus (except for 1 larval Haemaphysalis sp.). We did not find a positive correlation between the number of questing ticks and the number of fecal pellet groups, suggesting that factors other than ungulate abundance affect tick abundance. Nevertheless, large populations of questing ticks along some roadsides suggest a high risk of tick-borne diseases to tourists on this tropical island.

Metagenomic profile of the bacterial communities associated with Ixodes granulatus (Acari: Ixodidae): a potential vector of tick-borne diseases.

Ixodes granulatus Supino, 1897 (Acari: Ixodida) is one of Malaysia's most common hard ticks and is a potential vector for tick-borne diseases (TBDs). Despite its great public health importance, research on I. granulatus microbial communities remains largely unexplored. Therefore, this study aimed to investigate the bacterial communities of on-host I. granulatus collected from three different recreational areas on the East Coast of Peninsular Malaysia using high throughput Next Generation Sequencing (NGS). A total of 9 females on-host I. granulatus were subjected to metabarcoding analysis targeting V3-V4 regions of 16S ribosomal RNA (rRNA) using the Illumina MiSeq platform. This study identified 15 bacterial phyla corresponding to 19 classes, 54 orders, and 90 families from 435 amplicon sequence variants (ASVs), revealing a diverse bacterial community profile. Together with 130 genera assigned, local I. granulatus harbored 4 genera of pathogens, i.e., Rickettsia da Rocha Lima, 1916 (Rickettsiales: Rickettsiaceae) (58.6%), Borrelia Swellengrebel 1907 (Spirochaetales: Borreliaceae) (31.6%), Borreliella Adeolu and Gupta 2015 (Spirochaetales: Borreliaceae) (0.6%), and Ehrlichia Cowdria Moshkovski 1947 (Rickettsiales: Ehrlichiaceae) (39.9%). Some endosymbiont bacteria, such as Coxiella (Philip, 1943) (Legionellales: Coxiellaceae), Wolbachia Hertig 1936 (Rickettsiales: Ehrlichiaceae), and Rickettsiella Philip, 1956 (Legionellales: Coxiellaceae), were also detected at very low abundance. Interestingly, this study reported the co-infection of Borrelia and Ehrlichia for the first time, instilling potential health concerns in the context of co-transmission to humans, especially in areas with a high population of I. granulatus. This study successfully characterized the tick microbiome and provided the first baseline data of I. granulatus bacterial communities in Malaysia. These results support the need for way-forward research on tick-associated bacteria using NGS, focusing on medically important species toward TBD prevention.

Characterization of the complete mitochondrial genome of Ixodes granulatus (Ixodidae) and its phylogenetic implications.

Ticks are deemed to be second only to mosquitoes as the most common vector of human infectious diseases worldwide that give rise to human and animal diseases and economic losses to livestock production. Our understanding of the phylogenetic analysis between tick lineages has been restricted by the phylogenetic markers of individual genes. Genomic data research could help advance our understanding of phylogenetic analysis and molecular evolution. Mitochondrial genomic DNA facilitated the phylogenetic analysis of eukaryotes containing ticks. In this study, we sequenced and assembled the circular complete mitogenome information of Ixodes granulatus. The 14,540-bp mitogenome consists of 37 genes, including 13 protein-coding genes (PCGs), two genes for ribosomal RNA (rRNAs), and 22 genes for transfer RNA (tRNAs), and the origin of the L-strand replication region. The directions of the coding strand and component genes in the non-Australasian Ixodes mitochondrial genome were similar to those found in most other Australasian Ixodes, except for the loss of a lengthy control region. The phylogenetic tree based on maximum likelihood (ML) and Bayesian inference (BI) computational algorithms showed that I. granulatus exhibits a close relationship with I. hexagonus and I. ricinus. To our knowledge, this is the first study exploring the complete mitogenome for the species I. granulatus. Our results provide new insights for further research on the evolution, population genetics, systematics, and molecular ecology of ticks.

Molecular Detection and Genetic Identification of Rickettsia Infection in Ixodes granulatus Ticks, an Incriminated Vector for Geographical Transmission in Taiwan.

Tick-borne Rickettsia pathogens have become an emerging source of zoonotic infections and have a major impact on human health worldwide. In this study, the prevalence and genetic identity of Rickettsia infections in Ixodes granulatus ticks was firstly determined in Kinmen Island of Taiwan. A total of 247 I. granulatus ticks were examined for Rickettsia infection by nested-PCR assay targeting the citrate synthase (gltA) gene of Rickettsia. The Rickettsia infection was detected with a general infection rate of 4.86%, and was detected in nymph, male and female stages with an infection rate of 3.81%, 0% and 6.84%, respectively. Phylogenetic relationships were analyzed by comparing the gltA sequences obtained from four Taiwan strains and 19 other strains representing 13 genospecies of Rickettsia. Phylogenetic analyses reveal that all Taiwan strains were genetically affiliated to the genospecies of spotted fever (R. parkeri) and transitional (R. felis) groups of Rickettsia. Our findings reveal the first detection of R. parkeri-like and R. felis in I. granulatus ticks from Kinmen Island. As a tourist island between Taiwan and mainland China, these results demonstrate the epidemiological significance of diverse Rickettsia species existed in I. granulatus ticks and highlight the potential threat of geographical transmission among humans in the Taiwan area.

Detection of Borrelia burgdorferi Sensu Lato and Relapsing Fever Borrelia in Feeding Ixodes Ticks and Rodents in Sarawak, Malaysia: New Geographical Records of Borrelia yangtzensis and Borrelia miyamotoi.

Members of the Borrelia burgdorferi sensu lato (Bbsl) complex are etiological agents of Lyme disease (LD), and Borrelia miyamotoi is one of the relapsing fever Borrelia (RFB). Despite the serological evidence of LD in Malaysia, there has been no report from Sarawak, Malaysian Borneo. Thus, this study aimed to detect and characterize Borrelia in rodents and Ixodes ticks from primary forests and an oil palm (OP) plantation in Sarawak. Borrelia yangtzensis (a member of the Bbsl complex) was detected in 43.8% (14/32) of Ixodes granulatus; most of the positive ticks were from the OP plantation (13/14). Out of 56 rodents, B. yangtzensis was detected in four Rattus spp. from the OP plantation and B. miyamotoi was detected in one rodent, Sundamys muelleri, from the primary forest. Further, the positive samples of B. yangtzensis were randomly selected for multilocus sequence analysis (MLSA). The MLSA results of successfully amplified tick samples revealed a clustering with the sequences isolated from Japan and China. This study is the first evidence of B. miyamotoi, a known human pathogen in Malaysia, and B. yangtzensis, which is circulating in ticks and rodents in Sarawak, Malaysian Borneo, and presenting a new geographical record of the Borrelia spp.

A checklist and key to the tick fauna (Acari: Ixodidae, Argasidae) of Pulau Tioman, Malaysia.

Pulau Tioman is a popular tourist island off the east coast of Peninsula Malaysia, known for its vast areas of intact vegetation. Six tick species have been recorded on the island but little is known about their local ecology or public health importance. To facilitate studies of the tick fauna of Pulau Tioman, a checklist of all species and host associations is presented with the first host records, from Pulau Tioman, of Amblyomma helvolum from the Asian water monitor (Varanus salvator) and Amblyomma varanense from the keeled rat snake (Ptyas carinata). A pictorial key is also presented for all tick species known from the island.

Precise identification of different stages of a tick, Ixodes granulatus Supino, 1897 (Acari:Ixodidae)

Objective: To identify different stages of Ixodes granulatus (I. granulatus) based on morphological characters prior to molecular identification which is significant for con-firming and identifying the nymphal stages of I. granulatus. Methods: A total of 14 individuals of adult, engorged and nymphal ticks collected from three different localities were examined morphologically using taxonomic keys, followed by PCR using cytochrome oxidase subunit I (COI). Clustering analysis based on COI sequences was carried out by constructing neighbor-joining and maximum parsimony tree to clarify the genetic variation and diversity of local I. granulatus. Results: Based on external morphological characterizations, nine individuals (64.3%) were successfully identified as I. granulatus, while five individuals were recognized only as Ixodes sp. due to lack of morphological characters visible and development during that stage. Molecular analysis of local I. granulatus using COI gene revealed 93%–94%sequence homology from available sequence in GenBank and was in concordance with the morphological identification. Furthermore, a low intraspecific variation was observed among the species of I. granulatus collected from different localities (0%–3.7%). Conclusions: These findings demonstrated for the first time the establishment of COI gene for identifying I. granulatus nymphal tick which is of paramount importance to the control of potential tick-borne infections in Malaysia. Moreover, this study provides evidence that a combination of morphology and molecular data was corroborated as an accurate tool for tick identification.

Precise identification of different stages of a tick, Ixodes granulatus Supino, 1897 (Acari: Ixodidae)

Objective To identify different stages of Ixodes granulatus (I. granulatus) based on morphological characters prior to molecular identification which is significant for confirming and identifying the nymphal stages of I. granulatus. Methods A total of 14 individuals of adult, engorged and nymphal ticks collected from three different localities were examined morphologically using taxonomic keys, followed by PCR using cytochrome oxidase subunit I (COI). Clustering analysis based on COI sequences was carried out by constructing neighbor-joining and maximum parsimony tree to clarify the genetic variation and diversity of local I. granulatus. Results Based on external morphological characterizations, nine individuals (64.3%) were successfully identified as I. granulatus, while five individuals were recognized only as Ixodes sp. due to lack of morphological characters visible and development during that stage. Molecular analysis of local I. granulatus using COI gene revealed 93%–94% sequence homology from available sequence in GenBank and was in concordance with the morphological identification. Furthermore, a low intraspecific variation was observed among the species of I. granulatus collected from different localities (0%–3.7%). Conclusions These findings demonstrated for the first time the establishment of COI gene for identifying I. granulatus nymphal tick which is of paramount importance to the control of potential tick-borne infections in Malaysia. Moreover, this study provides evidence that a combination of morphology and molecular data was corroborated as an accurate tool for tick identification.

Acarine ectoparasites of Panti Forest Reserve in Johore, Malaysia.

OBJECTIVE: To identify the presence of acarine ectoparasites and determine whether there is any potential public health risk in Panti Forest Reserve, Johore, Malaysia. METHODS: Trapping of animals and avifauna was conducted simultaneously along 5 expedition trails using 150 wire traps, 10 harp traps and 30 mist nets for 6 consecutive nights. A total of 140 animals consisting of 7 species of birds, 19 species of bats, 6 species of rodents and 1 species of tree-shrew as well as 8 myriapods were examined. RESULTS: Infestation rates of ticks, mesostigmatid mites and chiggers on animals examined were 24.3%, 28.6% and 27.9%, respectively. Infestation on bats was low (1.5%) and none occurred on birds. Majority of ticks extracted were at immature stages (78.9%). Genera of ticks on animals were Amblyomma, Dermacentor, Haemaphysalis and Ixodes. Ixodes granulatus was the only species of ticks identified from the animals. Examination of ticks under vegetation revealed 54% adults leading to identification of 3 species of ticks. A total of 7 species of mesostigmatid mites were found. 6 species were on rodent, Maxomys surifer and another one species, Laelaps nuttalli was found only on Leopoldamys sabanus. Laelaps sanguisugus was the only mesostigmatid found infesting tree-shrews. Seven genera of chiggers were identified. From this, 5 genera were on rodents, 4 genera on tree-shrews and 1 genus on a bat. CONCLUSIONS: A total of 16 genera, 2 sub-genus and 14 species of acarine ectoparasites were found in this area. Findings of the survey demonstrate the presence of three spesies of acarine ectoparasites which have potential health risk i.e. Ixodes granulatus, Laelaps nuttalli and Leptotrombidium deliense.