Jing Jing Khoo

Toll-like receptors as interferon-regulated genes and their role in disease.

The Toll-like receptors (TLRs) are innate sensors that recognize both microbial and endogenous ligands, initiating the host defense response. TLRs initiate the potent proinflammatory response to infection, are the target for adjuvants, and are essential for the establishment and maturation of adaptive immunity. As such they have been the interest of widespread research and the target of therapeutic intervention on multiple diseases. It has become apparent that expression of a subset of TLRs (TLR1, TLR2, TLR3, TLR5, and TLR7) is induced by Type I interferons (IFN). The role and impact of IFN expression on TLR responses is therefore critical in understanding the role of TLRs in disease, particularly as IFN itself is a downstream gene induced by specific TLRs. In this review we discuss the function and role of IFN-regulated TLRs in disease and how the role of IFN may impact upon TLR induction of the immune response in diseases, particularly in mouse models.

Mitochondrially localised MUL1 is a novel modulator of antiviral signaling

The innate immune response to virus must be balanced to eliminate infection yet limit damaging inflammation. A critical arm of the antiviral response is launched by the retinoic acid‐inducible‐gene I (RIG‐I) protein. RIG‐I is activated by viral RNA then associates with the mitochondrial antiviral signaling (MAVS) protein to subsequently induce potent inflammatory cytokines. Here, we demonstrate the mitochondrial E3 ubiquitin protein ligase 1 (MUL1) is a crucial moderator of RIG‐I signaling. MUL1 is localized to the mitochondria where it interacts with MAVS and catalyzes RIG‐I post‐translational modifications that inhibit RIG‐I‐dependent cell signaling. Accordingly, depletion of MUL1 potentiated RIG‐I mediated nuclear factor‐kappa B (NF‐κB) and interferon (IFN) β reporter activity. Moreover, depletion of MUL1 boosted the antiviral response and increased proinflammatory cytokines following challenge with the RNA mimetic poly I:C and Sendai virus. We therefore submit that MUL1 is a novel regulator of the RIG‐I‐like receptor‐dependent antiviral response, that otherwise functions to limit inflammation.

Bacterial community in Haemaphysalis ticks of domesticated animals from the Orang Asli communities in Malaysia

Ticks are vectors in the transmission of many important infectious diseases in human and animals. Ticks can be readily found in the semi-forested areas such as the settlements of the indigenous people in Malaysia, the Orang Asli. There is still minimal information available on the bacterial agents associated with ticks found in Malaysia. We performed a survey of the bacterial communities associated with ticks collected from domestic animals found in two Orang Asli villages in Malaysia. We collected 62 ticks, microscopically and molecularly identified as related to Haemaphysalis wellingtoni, Haemaphysalis hystricis and Haemaphysalis bispinosa. Bacterial 16s rRNA hypervariable region (V6) amplicon libraries prepared from the tick samples were sequenced on the Ion Torrent PGM platform. We detected a total of 392 possible bacterial genera after pooling and sequencing 20 samples, indicating a diverse bacterial community profile. Dominant taxa include the potential tick endosymbiont, Coxiella. Other dominant taxa include the tick-associated pathogen, Rickettsia, and environmental bacteria such as Bacillus, Mycobacterium, Sphingomonas and Pseudomonas. Other known tick-associated bacteria were also detected, including Anaplasma, Ehrlichia, Rickettsiella and Wolbachia, albeit at very low abundance. Specific PCR was performed on selected samples to identify Rickettsia and Coxiella. Sequence of Rickettsia felis, which causes spotted fever in human and cats, was identified in one sample. Coxiella endosymbionts were detected in three samples. This study provides the baseline knowledge of the microbiome of ticks in Malaysia, focusing on tick-associated bacteria affecting the Orang Asli communities. The role of the herein found Coxiella and Rickettsia in tick physiology or disease transmission merits further investigation.

Independent Emergence of the Cosmopolitan Asian Chikungunya Virus, Philippines 2012

Outbreaks involving the Asian genotype Chikungunya virus (CHIKV) caused over one million infections in the Americas recently. The outbreak was preceded by a major nationwide outbreak in the Philippines. We examined the phylogenetic and phylogeographic relationships of representative CHIKV isolates obtained from the 2012 Philippines outbreak with other CHIKV isolates collected globally. Asian CHIKV isolated from the Philippines, China, Micronesia and Caribbean regions were found closely related, herein denoted as Cosmopolitan Asian CHIKV (CACV). Three adaptive amino acid substitutions in nsP3 (D483N), E1 (P397L) and E3 (Q19R) were identified among CACV. Acquisition of the nsP3-483N mutation in Compostela Valley followed by E1-397L/E3-19R in Laguna preceded the nationwide spread in the Philippines. The China isolates possessed two of the amino acid substitutions, nsP3-D483N and E1-P397L whereas the Micronesian and Caribbean CHIKV inherited all the three amino acid substitutions. The unique amino acid substitutions observed among the isolates suggest multiple independent virus dissemination events. The possible biological importance of the specific genetic signatures associated with the rapid global of the virus is not known and warrant future in-depth study and epidemiological follow-up. Molecular evidence, however, supports the Philippines outbreak as the possible origin of the CACV.

Detection in Malaysia of a Borrelia sp. From Haemaphysalis hystricis (Ixodida: Ixodidae)

Spirochetes from the Borrelia genus are known to cause diseases in humans, namely Lyme disease and relapsing fever. These organisms are commonly transmitted to humans by arthropod vectors including ticks, mite, and lice. Here, we report the molecular detection of a Borrelia sp. from a Haemaphysalis hystricis Supino tick collected from wildlife in an Orang Asli settlement in Selangor, Malaysia. Phylogenetic analyses of partial 16s rRNA and flaB gene sequences revealed that the Borrelia sp. is closely related to the relapsing fever group borreliae, Borrelia lonestari, Borrelia miyamotoi, and Borrelia theileri, as well as a number of uncharacterized Borrelia sp. from ticks in Portugal and Japan. To our knowledge, this is the first report of a Borrelia sp. detected in H. hystricis, and in Malaysia. The zoonotic potential of this Borrelia sp. merits further investigation.

Initiation of primary cell cultures from embryonic Haemaphysalis bispinosa ticks

Abstract Tick cell cultures have been widely used as an important tool for the study of tick-associated microorganisms, specifically for medically important bacteria or viruses that may be difficult to isolate or culture in axenic conditions. In this study, primary embryonic tick cell cultures were initiated separately from each of the egg batches laid by 10 female ticks belonging to the hard tick genus Haemaphysalis. All cultures were maintained at 28°C. After 10 months, 4 healthy cultures were identified with the potential for developing into continuous tick cell lines. These cultures comprise large cells predominantly forming floating cell clumps with multicellular vesicles, which are morphologically similar to cell lines derived from the soft tick Ornithodoros moubata. Subculture has not yet been performed due to the low cell density at the time of writing. Amplification and sequencing of a fragment of the 16S rRNA gene from DNA extracted from the parent ticks showed 99%-100% similarity to published sequences of Haemaphysalis bispinosa. This is the first report of the initiation of embryonic cell cultures from Haemaphysalis ticks found in Malaysia. Such tick cell cultures will be useful for studies of tick-borne pathogens in this region, where recent studies have shown that Haemaphysalis ticks are highly represented and harbor medically important bacteria.

Paenibacillus lautus, an opportunistic bacterial pathogen, isolated from Ixodes granulatus Supino (Acari: Ixodidae) collected from a Müller's giant Sunda rat (Sundamys muelleri)

Abstract Wild rodents are carriers of ectoparasites such as ticks that are vectors of infectious diseases with human and veterinary significances. Due to their close association with human dwellings, synanthropic wild rodents may aid the spread and transmission of tick-borne pathogens. In light of this, an effort was initiated to study the cultivable bacteria within ticks collected from wild rodents trapped in Sungai Congkak Recreational Forest, Selangor. Ticks collected from four different rodent species; Sundamys muelleri, Maxomys whiteheadi, Maxomys rajah and Rattus rattus, were sterilely homogenized and cultured. One unique bacterial isolate originating from an engorged adult female Ixodes granulatus Supino collected off a Sundamys muelleri rat was identified as Paenibacilus lautus via 16S rDNA, ctpA sequencing and biochemical tests. Antimicrobial susceptibility testing revealed that the P. lautus isolate was resistant to ampicillin, penicillin, clindamycin, chloramphenicol, rifampicin and trimethoprim-sulfamethoxazole, mirroring the antimicrobial susceptibility profiles of clinical strains. Recovery of this opportunistic bacterial pathogen from the tick suggests that it may be transmitted to humans or other hosts through tick bites and cause disease once it enters the bloodstream. Therefore, it is important to remain vigilant due to its pathogenic potential and improve our waste disposal practices to avoid attracting rodents.

Identification and characterization of Corynebacterium lactis isolated from Amblyomma testudinarium of Sus scrofa in Malaysia

Abstract Ticks are vectors for a number of important human and animal pathogens. In this study, Corynebacterium lactis was isolated from Amblyomma testudinarium Koch tick sampled from wild boar in Malaysia. Imaging with transmission electron microscope and complete genome sequencing were performed for C. lactis which shared similar morphology to other Corynebacterium species and was susceptible to most of the commonly used antibiotics. The draft genome revealed a total length of 2,568,615 bp with G+C content of 64.3%. This is the first description of C. lactis isolation from ticks, raising the possibility that ticks could be a vector for this emerging pathogen of companion animals.

Prevalence of on-host ticks (Acari: Ixodidae) in small mammals collected from forest near to human vicinity in Selangor, Malaysia

Abstract Ticks are important vectors that transmit a variety of pathogenic microorganisms known to be medically important worldwide. Many vertebrate groups have become host to this organism, and their presence and abundance are an indicator of the condition of both host and its habitat. This study was conducted to determine ticks infestation and its prevalence on small mammals residing in the recreational forests (RF) and semi-urban (SU) residential areas which have encountered Leptospirosis outbreak and cases in Hulu Langat, Selangor Malaysia. Trapping of the small mammals involved deploying two hundred cage traps in a systematic one-hectare plot (100 m × 100 m), as well as along the stream and forest trails at random. Ticks were extracted from the captured individual hosts. Identification of the tick species was performed based on morphological features and molecular approach using 16S rDNA and COI (cytochrome oxidase subunit I) genes. A total of 278 individuals of small mammals belonging to 15 species (13 Rodentia, 1 Scandentia and 1 Insectivora) were captured in the study areas. From these, 34 individuals from eight small mammal species were infested with ticks. The most infested host species was Mullers giant Sunda rat (Sundamys muelleri) with 5.80% (n=16). Ticks prevalence was slightly higher in RF with 6.40% (n=18) compared to SU with 5.80% (n=16). A total of 107 adult ticks (103 female and 4 male) were collected from the infested host. Ixodes granulatus was the most dominant tick species encountered (70.40%, n=85), followed by Dermacentor sp. (18.60%, n=20), while Amblyomma sp. was the least abundant (2%, n=2). This study provides information on tick species present and tick burden on small mammal hosts within the study areas. Our findings suggest that the visitors to the recreational forests and the residents of the semi-urban area were not only exposed to Leptospirosis bacteria but also tick bites and potentially tick-borne disease, therefore, precaution should be taken to avoid contact with small mammal hosts.

Detection of a Borrelia sp. From Ixodes granulatus Ticks Collected From Rodents in Malaysia

Abstract The Borrelia genus consists of spirochete bacteria known to cause Lyme disease (LD) and relapsing fever in humans. Borrelia pathogens are commonly transmitted via arthropod vectors such as ticks, mites, or lice. Here, we report the molecular screening of LD group Borrelia sp. from ticks (Acari: Ixodidae) collected from rodents trapped in recreational forests and a semiurban residential area in the Selangor state in Malaysia. Of 156 adult ticks surveyed, 72 ticks were determined as positive for Borrelia sp. by polymerase chain reaction (PCR). All Borrelia PCR-positive ticks belonged to the Ixodes granulatus Supino species. Borrelia sp. was not detected in other tick species examined, including Dermacentor sp. and Amblyomma sp. ticks. Thirteen Borrelia PCR-positive tick samples were selected for further sequence analyses. Phylogenetic analyses of partial flaB gene sequences revealed that the Borrelia sp. were closely related to the LD group borreliae, Borrelia yangtzensis; a novel Borrelia genospecies reported in East Asian countries including Japan, Taiwan, and China. To our knowledge, this is the first report of Borrelia sp. related to Borrelia yangtzensis detected in Malaysia and Southeast Asia. The zoonotic potential of the Borrelia sp. reported here merits further investigation, as it may explain the previously reported serological evidence for borrelial infections in Malaysia.