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

The dynamics of the microbiome in Ixodidae are shaped by tick ontogeny and pathogens in Sarawak, Malaysian Borneo

Tick-borne diseases have recently been considered a potential emerging public health threat in Malaysia; however, fundamental studies into tick-borne pathogens and microbiome appear limited. In this study, six tick species (Ixodes granulatus, Haemaphysalis hystricis, Haemaphysalis shimoga, Dermacentor compactus, Dermacentor steini and Dermacentor atrosignatus) collected from two primary forests and an oil palm plantation in Sarawak, Malaysian Borneo, were used for microbiome analysis targeting bacterial 16S rDNA using next-generation sequencing (NGS). In addition, bacterial species were further characterized in conventional PCRs to identify potential pathogens. Sequences generated from NGS were first filtered with the Decontam package in R before subsequent microbial diversity analyses. Alpha and beta analyses revealed that the genus Dermacentor had the highest microbial diversity, and H. shimoga significantly differed in microbial composition from other tick species. Alpha and beta diversities were also significantly different between developmental stages of H. shimoga. Furthermore, we observed that some bacterial groups were significantly more abundant in certain tick species and developmental stages of H. shimoga. We tested the relative abundances using pairwise linear discriminant analysis effect size (LEfSe), which also revealed significant microbial composition differences between Borrelia -positive and Borrelia -negative I. granulatus ticks. Finally, pathogenic and potentially pathogenic bacteria circulating in different tick species, such as Rickettsia heilongjiangensis , Ehrlichia sp., Anaplasma sp. and Bartonella spp. were characterized by PCR and sequencing. Moreover, Coxiella and Francisella -like potential symbionts were identified from H. shimoga and D. steini, respectively. More studies are required to unravel the factors associated with the variations observed in this study

First molecular detection of Hemolivia and Hepatozoon parasites in reptile-associated ticks on Iriomote Island, Japan

Hepatozoon and Hemolivia are members of the haemogregarines and are reported in reptiles and reptile-associated ticks. However, no studies have reported on Hepatozoon and Hemolivia in Japanese reptile-associated ticks. This study aimed to molecularly identify and to characterize Hepatozoon and Hemolivia in Japanese reptile-associated ticks, Amblyomma geoemydae (Cantor, 1847) and Amblyomma nitidum (Hirst & Hirst, 1910). A total of 41 and 75 DNA samples from A. geoemydae and A. nitidum ticks, respectively, were used for screening of Hepatozoon and Hemolivia with polymerase chain reaction targeting 18S rDNA. As a result, Hemolivia and Hepatozoon were detected in two A. geoemydae and one A. nitidum, respectively. The sequences of Hemolivia spp. showed a 99.5% (1,050/1,055 bp) identity with Hemolivia parvula (KR069083), and the Hemolivia spp. were located in the same clade as H. parvula in the phylogenetic tree. The sequences of Hepatozoon sp. showed a 98.4% (1,521/1,545 bp) identity with Hepatozoon colubri (MN723844), and the Hepatozoon sp. was distinct from validated Hepatozoon species in the tree. Our findings highlight the first molecular record of Hemolivia in Japan and present the first detection of Hepatozoon in A. nitidum. Further investigations on these tick-borne protozoa are required to understand their life cycle and pathogenicity

Isolation of Rickettsia, Rickettsiella, and Spiroplasma from Questing Ticks in Japan Using Arthropod Cells

Ticks are blood-sucking ectoparasites that transmit zoonotic pathogens to humans and animals. Ticks harbor not only pathogenic microorganisms but also endosymbionts. Although some tick endosymbionts are known to be essential for the survival of ticks, their roles in ticks remain poorly understood. The main aim of this study was to isolate and characterize tick-borne microorganisms from field-collected ticks using two arthropod cell lines derived from Ixodes scapularis embryos (ISE6) and Aedes albopictus larvae (C6/36). A total of 170 tick homogenates originating from 15 different tick species collected in Japan were inoculated into each cell line. Bacterial growth was confirmed by PCR amplification of 16S ribosomal DNA (rDNA) of eubacteria. During the 8-week observation period, bacterial isolation was confirmed in 14 and 4 samples using ISE6 and C6/36 cells, respectively. The sequencing analysis of the 16S rDNA PCR products indicated that they were previously known tick-borne pathogens/endosymbionts in three different genera: Rickettsia, Rickettsiella, and Spiroplasma. These included four previously validated rickettsial species namely Rickettsia asiatica (n = 2), Rickettsia helvetica (n = 3), Rickettsia monacensis (n = 2), and Rickettsia tamurae (n = 3) and one uncharacterized genotype Rickettsia sp. LON (n = 2). Four isolates of Spiroplasma had the highest similarity with previously reported Spiroplasma isolates: Spiroplasma ixodetis obtained from ticks in North America and Spiroplasma sp. Bratislava 1 obtained from Ixodes ricinus in Europe, while two isolates of Rickettsiella showed 100% identity with Rickettsiella sp. detected from Ixodes uriae at Grimsey Island in Iceland. To the best of our knowledge, this is the first report on successful isolation of Rickettsiella from ticks. The isolates obtained in this study can be further analyzed to evaluate their pathogenic potential in animals and their roles as symbionts in ticks

Isolation of Rickettsia, Rickettsiella, and Spiroplasma from Questing Ticks in Japan Using Arthropod Cells

Ticks are blood-sucking ectoparasites that transmit zoonotic pathogens to humans and animals. Ticks harbor not only pathogenic microorganisms but also endosymbionts. Although some tick endosymbionts are known to be essential for the survival of ticks, their roles in ticks remain poorly understood. The main aim of this study was to isolate and characterize tick-borne microorganisms from field-collected ticks using two arthropod cell lines derived from Ixodes scapularis embryos (ISE6) and Aedes albopictus larvae (C6/36). A total of 170 tick homogenates originating from 15 different tick species collected in Japan were inoculated into each cell line. Bacterial growth was confirmed by PCR amplification of 16S ribosomal DNA (rDNA) of eubacteria. During the 8-week observation period, bacterial isolation was confirmed in 14 and 4 samples using ISE6 and C6/36 cells, respectively. The sequencing analysis of the 16S rDNA PCR products indicated that they were previously known tick-borne pathogens/endosymbionts in three different genera: Rickettsia, Rickettsiella, and Spiroplasma. These included four previously validated rickettsial species namely Rickettsia asiatica (n = 2), Rickettsia helvetica (n = 3), Rickettsia monacensis (n = 2), and Rickettsia tamurae (n = 3) and one uncharacterized genotype Rickettsia sp. LON (n = 2). Four isolates of Spiroplasma had the highest similarity with previously reported Spiroplasma isolates: Spiroplasma ixodetis obtained from ticks in North America and Spiroplasma sp. Bratislava 1 obtained from Ixodes ricinus in Europe, while two isolates of Rickettsiella showed 100% identity with Rickettsiella sp. detected from Ixodes uriae at Grimsey Island in Iceland. To the best of our knowledge, this is the first report on successful isolation of Rickettsiella from ticks. The isolates obtained in this study can be further analyzed to evaluate their pathogenic potential in animals and their roles as symbionts in ticks

The effects of administering lactic acid bacteria sealed in a capsule on the intestinal bacterial flora of cattle

We examined the effects of encapsulated lactic acid bacteria administrated orally to lactating cattle on the intestinal flora. A dose of 3 × 1011 colony forming unit (cfu) of freeze-dried Lactobacillus coryniformis subsp. torquens (JCM1099) encapsulated in an enteric capsule capable of bypassing the rumen was administered for seven days. DNA was extracted from feces 0 and 24 hr after daily administration. Metagenomic analysis showed an increasing trend of the alpha diversity, an index of the species diversity. Furthermore, principal component analysis of intestinal flora revealed that cattle could be differentiated by JCM1099 capsule and suspension administration via principal components 1, 2, and 3. We conclude that administration of encapsulated JCM1099 can alter the intestinal bacterial flora of cattle

Prevalence and Association of Trypanosomes and Sodalis glossinidius in Tsetse Flies from the Kafue National Park in Zambia

Tsetse flies are obligate hematophagous vectors of animal and human African trypanosomosis. They cyclically transmit pathogenic Trypanosoma species. The endosymbiont Sodalis glossinidius is suggested to play a role in facilitating the susceptibility of tsetse flies to trypanosome infections. Therefore, this study was aimed at determining the prevalence of S. glossinidius and trypanosomes circulating in tsetse flies and checking whether an association exists between trypanosomes and Sodalis infections in tsetse flies from Kafue National Park in Zambia. A total of 326 tsetse flies were sampled from the Chunga and Ngoma areas of the national park. After DNA extraction was conducted, the presence of S. glossinidius and trypanosome DNA was checked using PCR. The Chi-square test was carried out to determine whether there was an association between the presence of S. glossinidius and trypanosome infections. Out of the total tsetse flies collected, the prevalence of S. glossinidius and trypanosomes was 21.8% and 19.3%, respectively. The prevalence of S. glossinidius was 22.2% in Glossina morsitans and 19.6% in Glossina pallidipes. In relation to sampling sites, the prevalence of S. glossinidius was 26.0% in Chunga and 21.0% in Ngoma. DNA of trypanosomes was detected in 18.9% of G. morsitans and 21.4% of G. pallidipes. The prevalence of trypanosomes was 21.7% and 6.0% for Ngoma and Chunga, respectively. The prevalences of trypanosome species detected in this study were 6.4%, 4.6%, 4.0%, 3.7%, 3.1%, and 2.5% for T. vivax, T. simiae, T. congolense, T. godfreyi, T. simiae Tsavo, and T. b. brucei, respectively. Out of 63 trypanosome infected tsetse flies, 47.6% of the flies also carried S. glossinidius, and the remaining flies were devoid of S. glossinidius. A statistically significant association was found between S. glossinidius and trypanosomes (p < 0.001) infections in tsetse flies. Our findings indicated that presence of S. glossinidius increases the susceptibility of tsetse flies to trypanosome infections and S. glossinidius could be a potential candidate for symbiont-mediated vector control in these tsetse species