Direct Detection of Lyme Borrelia: Recent Advancement and Use of Aptamer Technology.

Borrelia burgdorferi sensu lato (B. burgdorferi s.l.), which is predominantly spread by ticks, is the cause of Lyme disease (LD), also known as Lyme borreliosis, one of the zoonotic diseases affecting people. In recent years, LD has become more prevalent worldwide, even in countries with no prior records. Currently, Lyme Borrelia detection is achieved through nucleic acid amplification, antigen detection, microscopy, and in vitro culture. Nevertheless, these methods lack sensitivity in the early phase of the disease and, thus, are unable to confirm active infection. This review briefly discusses the existing direct detection methods of LD. Furthermore, this review also introduces the use of aptamer technology integrated with biosensor platforms to detect the Borrelia antigen. This aptamer technology could be explored using other biosensor platforms targeting whole Borrelia cells or specific molecules to enhance Borrelia detection in the future.

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.

Actinobacteria: An eco-friendly and promising technology for the bioaugmentation of contaminants.

Over the past two decades, various eco-friendly approaches utilizing microbial species to clean up contaminated environments have surfaced. In this aspect, actinobacteria have demonstrated their potential in contaminant degradation. The members of actinobacteria phylum exhibits a cosmopolitan distribution, which means that they can be found widely in both aquatic and terrestrial ecosystems. Actinobacteria play important ecological roles in the environment, such as degrading complex polymers, recycling compounds, and producing bioactive molecules. Hence, using actinobacteria to clean up contaminants is an attractive method in the field of biotechnology. This can be achieved through the green technology of bioaugmentation, whereby the degradative capacity of contaminated areas can be greatly improved through the introduction of specific microorganisms. This review describes actinobacteria as an eco-friendly and a promising technology for the bioaugmentation of contaminants, with focus on pesticides and heavy metals.

Loop-mediated isothermal amplification (LAMP) reaction as viable PCR substitute for diagnostic applications: a comparative analysis study of LAMP, conventional PCR, nested PCR (nPCR) and real-time PCR (qPCR) based on Entamoeba histolytica DNA derived from faecal sample.

BACKGROUND: This study reports the analytical sensitivity and specificity of a Loop-mediated isothermal amplification (LAMP) and compares its amplification performance with conventional PCR, nested PCR (nPCR) and real-time PCR (qPCR). All the assays demonstrated in this study were developed based on Serine-rich Entamoeba histolytica protein (SREHP) gene as study model. RESULTS: A set of SREHP gene specific LAMP primers were designed for the specific detection of Entamoeba histolytica. This set of primers recorded 100% specificity when it was evaluated against 3 medically important Entamoeba species and 75 other pathogenic microorganisms. These primers were later modified for conventional PCR, nPCR and qPCR applications. Besides, 3 different post-LAMP analyses including agarose gel electrophoresis, nucleic acid lateral flow immunoassay and calcein-manganese dye techniques were used to compare their limit of detection (LoD). One E. histolytica trophozoite was recorded as the LoD for all the 3 post-LAMP analysis methods when tested with E. histolytica DNA extracted from spiked stool samples. In contrast, none of the PCR method outperformed LAMP as both qPCR and nPCR recorded LoD of 100 trophozoites while the LoD of conventional PCR was 1000 trophozoites. CONCLUSIONS: The analytical sensitivity comparison among the conventional PCR, nPCR, qPCR and LAMP reveals that the LAMP outperformed the others in terms of LoD and amplification time. Hence, LAMP is a relevant alternative DNA-based amplification platform for sensitive and specific detection of pathogens.

Data on ectoparasites infestation on small mammals from different habitats in east-coast Peninsular Malaysia.

This data article presents on the ectoparasites infestation on small mammals in Peninsular Malaysia. The dataset on ectoparasites infestation is important because it raises a major medical concern regarding the spread of potentially zoonotic disease from wildlife to human. Tick and chigger are the primary ectoparasites as reservoirs of vector-borne diseases found on small mammals in Malaysia. These small mammals that are infested with ectoparasites occupy various types of habitats, including human settlements, could be of community health risks as the carriers of potentially zoonotic diseases. Field samplings were conducted from February 2015 to February 2016 in three different ecological habitats of mixed dipterocarp forest, coastal forest and insular forest, in Terengganu, Malaysia. A total of 35 and 22 species of bats and rodents respectively were captured and examined for ectoparasites. Twenty-three species of bats and 16 species of small mammal were recorded as hosts for at least one species of ectoparasites. These findings show that the highest ectoparasite infestation occurred on bat community.

Helminth parasites occurrence in wild proboscis monkeys (Nasalis larvatus), endemic primates to Borneo Island.

BACKGROUND: Nasalis larvatus are well-known attraction for tourists in the mangrove forest of Bako National Park (BNP). Little is known regarding the infestation of helminth parasites in proboscis monkeys. Therefore, the objectives of this study are to determine the prevalence species of major helminth parasites of public health importance in proboscis monkeys in BNP. METHODS: A total of 65 faecal samples screened for helminth parasites via sodium nitrate floatation and faecal sedimentation techniques. RESULTS: A total of 14 helminth parasite species comprising of eight genera of nematodes, two species of cestodes and two trematodes were identified. Eggs of Trichuris trichiura were the most frequently encountered in proboscis monkeys. CONCLUSION: This is the first survey on the fauna of helminth parasites of proboscis monkeys living in mangrove forests, and therefore, it implies the important baseline information that increases our current knowledge for future research regarding parasite-host ecology in primates.

Ticks (Argasidae, Ixodidae) and tick-borne diseases of continental Southeast Asia.

Knowledge of the tick fauna of continental Southeast Asia is either patchy or, in some cases, for example Cambodia and Myanmar, poor. Nevertheless, 97 species have been recorded from this region, making it one of the most diverse for ticks worldwide. Throughout Southeast Asia, work on tick-borne diseases of stock and companion animals, as well as of humans, is in its infancy, and the medical, veterinary and socio-economic importance of these diseases is largely unknown. Here we review current knowledge of Southeast Asian ticks and tick-borne diseases, with the aim of stimulating further research on this subject.

Gastrointestinal parasites of zoonotic importance observed in the wild, urban, and captive populations of non-human primates in Malaysia.

BACKGROUND: A study was undertaken to determine gastrointestinal (GI) parasites commonly found in Malaysia's non-human primates (NHP) living in three different types of populations (wild, urban, and captive) and the basis of major GI parasites of zoonotic importance. METHODS: A total of 308 samples was collected and microscopically screened from the NHP in the wild (n = 163), urban (n = 76), and captive (n = 69) populations. The samples were taken from 12 species of local NHPs. RESULTS: At least, 44 species of GI parasites comprising of protozoans (seven species), nematodes (26 species), cestodes (five species), trematodes (five species), and pentastomida (one species) were detected. There were no significant differences for the overall prevalence and no great differences in GI parasite species among the wild, urban, and captive NHP populations. CONCLUSION: The most common GI parasite was Ascaris spp. (49.7%), followed by Oesophagostomum spp. (26.9%), and 31 species discovered in this study are of known public health importance.

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.

Molecular identification of blood meal sources of ticks (Acari, Ixodidae) using cytochrome b gene as a genetic marker.

Blood meal analysis (BMA) from ticks allows for the identification of natural hosts of ticks (Acari: Ixodidae). The aim of this study is to identify the blood meal sources of field collected on-host ticks using PCR analysis. DNA of four genera of ticks was isolated and their cytochrome b (Cyt b) gene was amplified to identify host blood meals. A phylogenetic tree was constructed based on data of Cyt b sequences using Neighbor Joining (NJ) and Maximum Parsimony (MP) analysis using MEGA 5.05 for the clustering of hosts of tick species. Twenty out of 27 samples showed maximum similarity (99%) with GenBank sequences through a Basic Local Alignment Search Tool (BLAST) while 7 samples only showed a similarity range of between 91-98%. The phylogenetic trees showed that the blood meal samples were derived from small rodents (Leopoldamyssabanus, Rattustiomanicus and Sundamysmuelleri), shrews (Tupaiaglis) and mammals (Tapirusindicus and Prionailurusbengalensis), supported by 82-88% bootstrap values. In this study, Cyt b gene as a molecular target produced reliable results and was very significant for the effective identification of ticks' blood meal. The assay can be used as a tool for identifying unknown blood meals of field collected on-host ticks.

A survey of acarine ectoparasites of bats (Chiroptera) in Malaysia.

A comprehensive 8-yr survey of acarine ectoparasites (ticks and mites) of bats was carried out in 18 localities from 2002 to 2009. Most of the surveys were conducted during 14 national biodiversity scientific expeditions throughout Malaysia. The objective was to identify acarines of known public health importance from bats and thus determine whether there is any potential public health risk in Malaysia. Trapping of bats was conducted using Harp traps and Mist nets. In total, 1,579 individuals comprising of 6 families and 52 species of bats were examined alive. In general, 25.6% of the bats were infested with acarines. Infestation rates of ticks, mesostigmatid mites, and chiggers on bats examined were 0.4, 10.4, and 14.7%, respectively. Their prevalence and mean intensity were tabulated. Genera of ticks extracted were Amblyomma, Dermacentor, Ixodes, and Ornithodoros. Of these genera, only two species can be identified to species level and they are Amblyomma cordiferum and Ixodes simplex. In total, 8 genera and 15 species of mesostigmatid mites were found; the species were Ancystropus eonycteris, Ancystropus zeleborii, Echinonysus nasutus, Laelaps aingworthae, Laelaps nuttalli, Laelaps sanguisugus, Laelaps sculpturatus, Longolaelaps longulus, Longolaelaps whartonii, Meristaspis lateralis, Meristaspis macroglossi, Paraperiglischrus rhinolophinus, Spinturnix acuminatus, Spinturnix americanus, and Spinturnix bakeri. Chiggers on bats were represented by 12 genera and 6 species; the species identified were Gahrliepia fletcheri, Riedlinia lipoxena, Trombigastia cadei, Walchiella impar, Walchiella oudemansi, and Whartonia caobangensis. The study produced an up-to-date list of acarine ectoparasites of bats in Malaysia where a total of 38 genera and 47 species of acarines were listed. Findings of the study demonstrated that 5 genera and 1 species of acarines that may pose potential health risks, can be found on bats.

Establishment of a molecular tool for blood meal identification in Malaysia.

OBJECTIVE: To establish a polymerase chain reaction (PCR) technique based on cytochrome b (cytb) gene of mitochondria DNA (mtDNA) for blood meal identification. METHODS: The PCR technique was established based on published information and validated using blood sample of laboratory animals of which their whole gene sequences are available in GenBank. PCR was next performed to compile gene sequences of different species of wild rodents. The primers used were complementary to the conserved region of the cytb gene of vertebrate's mtDNA. A total of 100 blood samples, both from laboratory animals and wild rodents were collected and analyzed. The obtained unknown sequences were compared with those in the GenBank database using BLAST program to identify the vertebrate animal species. RESULTS: Gene sequences of 11 species of wild animals caught in 9 localities of Peninsular Malaysia were compiled using the established PCR. The animals involved were Rattus (rattus) tanezumi, Rattus tiomanicus, Leopoldamys sabanus, Tupaia glis, Tupaia minor, Niviventor cremoriventor, Rhinosciurus laticaudatus, Callosciurus caniseps, Sundamys muelleri, Rattus rajah and Maxomys whiteheadi. The BLAST results confirmed the host with exact or nearly exact matches (>89% identity). Ten new gene sequences have been deposited in GenBank database since September 2010. CONCLUSIONS: This study indicates that the PCR direct sequencing system using universal primer sets for vertebrate cytb gene is a promising technique for blood meal identification.

Establishment of a molecular tool for blood meal identification in Malaysia

Objective: To establish a polymerase chain reaction (PCR) technique based on cytochrome b (cytb) gene of mitochondria DNA (mtDNA) for blood meal identification. Methods: The PCR technique was established based on published information and validated using blood sample of laboratory animals of which their whole gene sequences are available in GenBank. PCR was next performed to compile gene sequences of different species of wild rodents. The primers used were complementary to the conserved region of the cytb gene of vertebrate’s mtDNA. A total of 100 blood samples, both from laboratory animals and wild rodents were collected and analyzed. The obtained unknown sequences were compared with those in the GenBank database using BLAST program to identify the vertebrate animal species. Results: Gene sequences of 11 species of wild animals caught in 9 localities of Peninsular Malaysia were compiled using the established PCR. The animals involved were Rattus (rattus) tanezumi, Rattus tiomanicus, Leopoldamys sabanus,Tupaia glis, Tupaia minor, Niviventor cremoriventor, Rhinosciurus laticaudatus, Callosciurus caniseps, Sundamys muelleri, Rattus rajah and Maxomys whiteheadi. The BLAST results confirmed the host with exact or nearly exact matches (>89% identity). Ten new gene sequences have been deposited in GenBank database since September 2010. Conclusions: This study indicates that the PCR direct sequencing system using universal primer sets for vertebrate cytb gene is a promising technique for blood meal identification.