Chlamydia Infection Remodels Host Cell Mitochondria to Alter Energy Metabolism and Subvert Apoptosis.

Chlamydia infection represents an important cause for concern for public health worldwide. Chlamydial infection of the genital tract in females is mostly asymptomatic at the early stage, often manifesting as mucopurulent cervicitis, urethritis, and salpingitis at the later stage; it has been associated with female infertility, spontaneous abortion, ectopic pregnancy, and cervical cancer. As an obligate intracellular bacterium, Chlamydia depends heavily on host cells for nutrient acquisition, energy production, and cell propagation. The current review discusses various strategies utilized by Chlamydia in manipulating the cell metabolism to benefit bacterial propagation and survival through close interaction with the host cell mitochondrial and apoptotic pathway molecules.

Anesthesia in patients with Brugada syndrome: two case reports.

BACKGROUND: Brugada syndrome is a rare disease. It causes sudden cardiac arrest, which is a serious life-threatening event. Sudden cardiac death mostly results from coronary artery disease. However, patients with Brugada syndrome show normal cardiac anatomy and no evidence of ischemia or electrolyte imbalance. Anesthesia in patients with Brugada syndrome is challenging due to its unpredictable nature, and is worth our attention. CASE PRESENTATION: We report two cases of Brugada syndrome during anesthesia. In case one, a 31-year-old Filipino laborer was scheduled for laparoscopic appendectomy. The patient denied any preexisting cardiac disease. The preoperative vital signs were stable, with mild fever of 37.9 °C. The operation was smooth. During the emergence period, the patient suffered from sudden onset of ventricular tachycardia. After resuscitation, the cardiac rhythm returned to normal. Later, he was confirmed to have a genetic trait of Brugada syndrome. In case two, a young Taiwanese patient with pre-diagnosed Brugada syndrome underwent an operation. The perioperative precautions were taken to prevent the occurrence of ventricular arrhythmia. The surgery was uneventful. CONCLUSIONS: Brugada syndrome, although rare, has the highest incidence in South East Asian healthy young males. It brings attention to possible fatal cardiac arrhythmia in this population. Careful preoperative evaluation and perioperative management can help reduce the harmful outcome of the disease and prevent any untoward events.

Chlamydia trachomatis plasmid-encoding Pgp3 protein induces secretion of distinct inflammatory signatures from HeLa cervical epithelial cells.

BACKGROUND: Genital Chlamydia trachomatis infection is the most common bacterial sexual transmitted disease that causes severe complications including pelvic inflammatory disease, ectopic pregnancy, and infertility in females. The Pgp3 protein encoded by C. trachomatis plasmid has been speculated to be an important player in chlamydial pathogenesis. However, the precise function of this protein is unknown and thus remains to be thoroughly investigated. METHODS: In this study, we synthesized Pgp3 protein for in vitro stimulation in the Hela cervical carcinoma cells. RESULTS AND CONCLUSION: We showed that Pgp3 induced prominent expression of host inflammatory cytokine genes including interleukin-6 (IL-6), IL-8, tumor necrosis factor alpha-induced protein 3 (TNFAIP3), and chemokine C-X-C motif ligand 1 (CXCL1), implying a possible role of Pgp3 in modulating the inflammatory reaction in the host.

Immunomodulatory proteins from hookworms reduce cardiac inflammation and modulate regulatory responses in a mouse model of chronic Trypanosoma cruzi infection.

Introduction: Hookworms are parasitic helminths that secrete a variety of proteins that induce anti-inflammatory immune responses, stimulating increased CD4 + Foxp3+ regulatory T cells and IL-10 production. Hookworm-derived recombinant proteins AIP-1 and AIP-2 have been shown to reduce inflammation in mouse models of inflammatory bowel disease and inflammatory airway disease by inducing CD4+Foxp3+ cells and IL-10 production. In contrast, chronic infection with the protozoal parasite Trypanosoma cruzi, the causative agent of Chagas disease, leads to chronic inflammation in tissues. Persistence of the parasites in tissues drives chronic low-grade inflammation, with increased infiltration of inflammatory cells into the heart, accompanied by increased production of inflammatory cytokines. There are no current antiparasitic drugs that effectively reduce or prevent chronic myocarditis caused by the onset of Chagas disease, thus new therapies are urgently needed. Therefore, the impact of AIP-1 and AIP-2 on myocarditis was investigated in a mouse model of chronic T. cruzi infection. Methods: Female BALB/c mice infected with bioluminescent T. cruzi H1 strain trypomastigotes for 70 days were treated once daily for 7 days with 1mg/kg AIP-1 or AIP-2 protein by intraperitoneal injection. Control mice were left untreated or treated once daily for 14 days with 25mg/kg aspirin in drinking water. At 84 days of infection, splenocytes, cardiac tissue and serum were collected for evaluation. Results: Treatment with both AIP-1 and AIP-2 proteins significantly reduced cardiac cellular infiltration, and reduced cardiac levels of IFNγ, IL-6 and IL-2. AIP-2 treatment reduced cardiac expression of COX-2. Further, while incubation with AIP-1 and AIP-2 proteins did not induce a significant upregulation of an immunoregulatory phenotype in dendritic cells (DC), there was a modest upregulation of CD11c +CD11b+MHCII+SIRPα+ expression, suggesting a regulatory phenotype. Ex-vivo stimulation of splenocytes from the treatment groups with AIP-1 loaded DC induced reduced levels of cytotoxic and pro-inflammatory T cells, stimulation with AIP-2 loaded DC specifically induced enhanced levels of CD4+CD25+Foxp3+ regulatory T cells among treatment groups. Discussion: All in vivo and in vitro results demonstrate that hookworm-derived AIP-1 and AIP-2 proteins reduce T. cruzi induced cardiac inflammation, possibly through multiple anti-inflammatory mechanisms.

The Immunobiology of Nipah Virus.

Nipah virus (NiV) is a highly lethal zoonotic paramyxovirus that emerged in Malaysia in 1998. It is a human pathogen capable of causing severe respiratory infection and encephalitis. The natural reservoir of NiV, Pteropus fruit bats, remains a continuous virus source for future outbreaks, although infection in the bats is largely asymptomatic. NiV provokes serious disease in various mammalian species. In the recent human NiV outbreaks in Bangladesh and India, both bats-to-human and human-to-human transmissions have been observed. NiV has been demonstrated to interfere with the innate immune response via interferon type I signaling, promoting viral dissemination and preventing antiviral response. Studies of humoral immunity in infected NiV patients and animal models have shown that NiV-specific antibodies were produced upon infection and were protective. Studies on cellular immunity response to NiV infection in human and animal models also found that the adaptive immune response, specifically CD4+ and CD8+ T cells, was stimulated upon NiV infection. The experimental vaccines and therapeutic strategies developed have provided insights into the immunological requirements for the development of successful medical countermeasures against NiV. This review summarizes the current understanding of NiV pathogenesis and innate and adaptive immune responses induced upon infection.

Comparative expression of pro-inflammatory and apoptotic biosignatures in chronic HBV-infected patients with and without liver cirrhosis.

The interplay of immune mediators is paramount to optimal host anti-viral immune responses, especially against chronic hepatitis B virus (HBV) infection. Here, we investigated the dynamic changes in host immune responses in chronic HBV-infected individuals with and without liver cirrhosis by examining the signatures of apoptosis and plasma levels of pro-inflammatory cytokines, chemokines, and cytotoxic proteins. A total of 40 chronic HBV patients with and without liver cirrhosis were studied for plasma levels of immune mediators, and signatures of apoptosis in peripheral blood mononuclear cells (PBMCs). The intracellular concentrations of reactive oxygen species (ROS) in patients with chronic HBV with liver cirrhosis was relatively higher as compared to chronic HBV patients. The onset of apoptosis was sustained due to ongoing liver inflammation in concert with plasma TNF-α and IL-6 levels. Plasma VEGF was upregulated among chronic HBV patients with liver cirrhosis, whereas CCL2, CCL5 and granzyme B levels were down-regulated. High levels of ROS, IL-6 and TNF-α correlated with ongoing inflammation among chronic HBV patients with liver cirrhosis, which likely attributed to the expression of biosignatures of apoptosis and activation in immune cells.

Isolation of Streptococcus cuniculi from corneal lesion in laboratory-raised mice.

Corneal lesions appearing as white mass beneath intact epithelium, with ocular discharge in one mouse, was observed in a batch of laboratory-raised BALB/c mice (n=9 of 56). The affected mice remained active, well-groomed and had normal appetite. Isolates recovered from swab cultures of the external and internal contents of the eye had partial 16S rRNA gene sequence of 99.1% similarity to Streptococcus cuniculi. No previous report of S. cuniculi infection in laboratory rodents has been presented. The isolate was susceptible to all antibiotics tested. We suggest S. cuniculi is an opportunistic bacteria in laboratory mice but are uncertain of its source. Our findings revealed that S. cuniculi is able to colonize laboratory mice and should be considered when mice present with eye lesion or ocular discharge.

Seroprevalence of Nipah Virus Infection in Peninsular Malaysia.

Nipah virus (NiV) outbreak occurred in Malaysia in 1998. The natural host reservoir for NiV is Pteropus bats, which are commonly found throughout Malaysia. Humans become infected when NiV spills over from the reservoir species. In this study, NiV serosurveillance in Peninsular Malaysia, particularly among the indigenous population, was performed. The collected samples were tested for presence of NiV antibodies using a comparative indirect enzyme-linked immunosorbent assay based on the recombinant NiV nucleocapsid (rNiV-N) protein. We found that 10.73% of the participants recruited in this study had antibodies against rNiV-N, suggesting possible exposure to NiV.

Copro-molecular study of Entamoeba infection among the indigenous community in Malaysia: A first report on the species-specific prevalence of Entamoeba in dogs.

BACKGROUND: Entamoeba is a free-living protozoan parasitic species that infect a variety of hosts. In humans, Entamoeba histolytica is the causative agent of amoebiasis. Entamoeba species has also been reported in dogs. However, little is known about the molecular epidemiology and the specific species of this parasite in dogs globally, including Malaysia. As dogs are important companion animals for the indigenous community, and close contact with dogs is part of the natural living conditions for this community, this study aims to determine the prevalence and molecular epidemiology of Entamoeba species in human and dogs in Malaysia. METHOD: The presence of Entamoeba species was examined in 504 fresh fecal samples, collected randomly from 411 humans and 93 dogs using microscopy and polymerase chain reaction (PCR) amplifying 16 s ribosomal RNA (rRNA). Data was analyzed using appropriate statistical analysis. RESULTS: The microscopy data showed an overall occurrence of Entamoeba species of 26.3% (108/411) and 36.6% (34/93) in humans and dogs respectively. In humans, the most common species was a single infection of E. dispar (26.5%; 13/49), followed by E. histolytica and E. moshkovskii, (20.4% for each species respectively). Double infection of E. dispar + E. moshkovskii was detected at 10.2%, followed by E. dispar + E. histolytica (8.2%) and E. moshkovskii and E. histolytica (6.1%). 8.2% of the samples had triple infection with all three species. In animals, E. moshkovskii (46.7%) was the most common species detected, followed by E. histolytica, and E. dispar, at 20.0% and 13.3% respectively. Double infection with E. moshkovskii + E. histolytica and a triple infection were found in 2 samples (13.3%) and 1 (6.7%) sample respectively. Risk factor analysis showed that members of the community who used untreated water were more prone to be infected with Entamoeba. CONCLUSION: This study provides information on the species-specific occurrence of Entamoeba infection, the potential risk factors and their zoonotic potential to humans. This is the first report to describe the molecular occurrence of Entamoeba species in dogs in Malaysia. The presence of pathogenic Entamoeba species implies that dogs could be a reservoir or mechanical host for human amoebiasis. Further studies need to be conducted to better understand the transmission dynamics and public health significance of Entamoeba species in human and animal hosts.

Diversity of endocervical microbiota associated with genital Chlamydia trachomatis infection and infertility among women visiting obstetrics and gynecology clinics in Malaysia.

The cervical microbiota constitutes an important protective barrier against the invasion of pathogenic microorganisms. A disruption of microbiota within the cervical milieu has been suggested to be a driving factor of sexually transmitted infections. These include Chlamydia trachomatis which frequently causes serious reproductive sequelae such as infertility in women. In this study, we profiled the cervical microbial composition of a population of 70 reproductive-age Malaysian women; among which 40 (57.1%) were diagnosed with genital C. trachomatis infection, and 30 (42.8%) without C. trachomatis infection. Our findings showed a distinct compositional difference between the cervical microbiota of C. trachomatis-infected subjects and subjects without C. trachomatis infection. Specifically, significant elevations of mostly strict and facultative anaerobes such as Streptococcus, Megasphaera, Prevotella, and Veillonella in the cervical microbiota of C. trachomatis-positive women were detected. The results from the current study highlights an interaction of C. trachomatis with the environmental microbiome in the endocervical region.

IncobotulinumtoxinA for the Treatment of Glabellar Frown Lines: A Prospective, Multicenter, Single-arm Study in Taiwan.

Objective: We assessed clinical effectiveness, longevity of treatment effects, and patient satisfaction with incobotulinumtoxinA for glabellar frown lines (GFL) treatment in Asian patients. Design, Setting, and Participants: This was a prospective, multicenter, single-arm, open-label study at six sites in Taiwan. Patients aged 20 to 65 years with mild to very severe GFLs (Merz scale: 1-4 points) were eligible; 45 patients [including 23 BoNT/A-naïve and 22 previously-treated ("switch") patients were enrolled. Patients received intramuscular incobotulinumtoxinA injection at up to five injection points. Total doses ranged from 12 to 20U. Measurements: Investigators assessed improvements in dynamic GFLs at Days 14 and 120 using the validated five-point Merz scale (0=no lines; 4=very severe lines). Treatment satisfaction was self-reported by patients via questionnaire. Results: All patients showed excellent response to treatment in that Merz scores at Day 14 were 0 or 1 point(s). Both groups showed a mean improvement of 2.9 points; the response rate (1-point improvement or more from baseline) was 100 percent. GFL improvement was maintained over at least four months in both groups (mean improvements at Day 120: 1.5 points, naïve; 1.7 points, switch). Patient satisfaction ratings remained high (almost 100% in both groups) throughout the study. There were no statistically significant differences between groups regarding treatment satisfaction or GFL improvement (Merz score) at Days 14 and 120. No adverse events occurred. Conclusion: In Asian patients, incobotulinumtoxinA treatment for dynamic GFLs is effective and long lasting, with no expected differences between BoNT/A-naïve patients and those switching from other BoNT/As. IncobotulinumtoxinA yields consistent and natural-looking results for first and subsequent treatments.

Nipah Virus Infection of Immature Dendritic Cells Increases Its Transendothelial Migration Across Human Brain Microvascular Endothelial Cells.

Nipah virus (NiV) can infect multiple organs in humans with the central nervous system (CNS) being the most severely affected. Currently, it is not fully understood how NiV spreads throughout the body. NiV has been shown to infect certain leukocyte populations and we hypothesized that these infected cells could cross the blood-brain barrier (BBB), facilitating NiV entry into the CNS. Here, three leukocyte types, primary immature dendritic cells (iDC), primary monocytes (pMO), and monocytic cell line (THP-1), were evaluated for permissiveness to NiV. We found only iDC and THP-1 were permissive to NiV. Transendothelial migration of mock-infected and NiV-infected leukocytes was then evaluated using an in vitro BBB model established with human brain microvascular endothelial cells (HBMEC). There was approximately a threefold increase in migration of NiV-infected iDC across endothelial monolayer when compared to mock-infected iDC. In contrast, migration rates for pMO and THP-1 did not change upon NiV infection. Across TNF-α-treated endothelial monolayer, there was significant increase of almost twofold in migration of NiV-infected iDC and THP-1 over mock-infected cells. Immunofluorescence analysis showed the migrated NiV-infected leukocytes retained their ability to infect other cells. This study demonstrates for the first time that active NiV infection of iDC and THP-1 increased their transendothelial migration activity across HBMEC and activation of HBMEC by TNF-α further promoted migration. The findings suggest that NiV infection of leukocytes to disseminate the virus via the "Trojan horse" mechanism is a viable route of entry into the CNS.

Oral immunization of a non-recombinant Lactococcus lactis surface displaying influenza hemagglutinin 1 (HA1) induces mucosal immunity in mice.

Mucosal immunization of influenza vaccine is potentially an effective approach for the prevention and control of influenza. The objective of the present study was to evaluate the ability of oral immunization with a non-recombinant Lactococcus lactis displaying HA1/L/AcmA recombinant protein, LL-HA1/L/AcmA, to induce mucosal immune responses and to accord protection against influenza virus infection in mice. The LL-HA1/L/AcmA was orally administered into mice and the immune response was evaluated. Mice immunized with LL-HA1/L/AcmA developed detectable specific sIgA in faecal extract, small intestine wash, BAL fluid and nasal fluid. The results obtained demonstrated that oral immunization of mice with LL-HA1/L/AcmA elicited mucosal immunity in both the gastrointestinal tract and the respiratory tract. The protective efficacy of LL-HA1/L/AcmA in immunized mice against a lethal dose challenge with influenza virus was also assessed. Upon challenge, the non-immunized group of mice showed high susceptibility to influenza virus infection. In contrast, 7/8 of mice orally immunized with LL-HA1/L/AcmA survived. In conclusion, oral administration of LL-HA1/L/AcmA in mice induced mucosal immunity and most importantly, provided protection against lethal influenza virus challenge. These results highlight the potential application of L. lactis as a platform for delivery of influenza virus vaccine.

Identification of the cell binding domain in Nipah virus G glycoprotein using a phage display system.

Nipah virus (NiV) is a highly pathogenic zoonotic paramyxovirus with unusual broad host tropism and is designated as a Category C pathogen by the U.S. National Institute of Allergy and Infectious Diseases. NiV infection is initiated after binding of the viral G glycoprotein to the host cell receptor. The aim of this study was to map the NiV G glycoprotein cell binding domain using a phage display system. The NiV G extracellular domain was truncated and displayed as attachment proteins on M13 phage g3p minor coat protein. The binding efficiency of recombinant phages displaying different regions of NiV G to mammalian cells was evaluated. Results showed that regions of NiV G consisting of amino acids 396-602 (recombinant phage G4) and 498-602 (recombinant phage G5) demonstrated the highest binding to both Vero (5.5×103 cfu/ml and 5.6×103 cfu/ml) and THP-1 cells (3.5×103 cfu/ml and 2.9×103 cfu/ml). However, the binding of both of these recombinant phages to THP-1 cells was significantly lower than to Vero cells, and this could be due to the lack of primary host cell receptor expression on THP-1 cells. Furthermore, the binding between these two recombinant phages was competitive suggesting that there was a common host cell attachment site. This study employed an approach that is suitable for use in a biosafety level 2 containment laboratory without the need to use live virus to show that NiV G amino acids 498-602 play an important role for attachment to host cells.

CD8+ T cells of chronic HCV-infected patients express multiple negative immune checkpoints following stimulation with HCV peptides.

Hepatitis C virus (HCV)-specific CD4+ and CD8+ T cells are key to successful viral clearance in HCV disease. Accumulation of exhausted HCV-specific T cells during chronic infection results in considerable loss of protective functional immune responses. The role of T-cell exhaustion in chronic HCV disease remains poorly understood. Here, we studied the frequency of HCV peptide-stimulated T cells expressing negative immune checkpoints (PD-1, CTLA-4, TRAIL, TIM-3 and BTLA) by flow cytometry, and measured the levels of Th1/Th2/Th17 cytokines secreted by T cells by a commercial Multi-Analyte ELISArray™ following in vitro stimulation of T cells using HCV peptides and phytohemagglutinin (PHA). HCV peptide-stimulated CD4+ and CD8+ T cells of chronic HCV (CHC) patients showed significant increase of CTLA-4. Furthermore, HCV peptide-stimulated CD4+ T cells of CHC patients also displayed relatively higher levels of PD-1 and TRAIL, whereas TIM-3 was up-regulated on HCV peptide-stimulated CD8+ T cells. Whereas the levels of IL-10 and TGF-ß1 were significantly increased, the levels of pro-inflammatory cytokines IL-2, TNF-α, IL-17A and IL-6 were markedly decreased in the T cell cultures of CHC patients. Chronic HCV infection results in functional exhaustion of CD4+ and CD8+ T cells likely contributing to viral persistence.

Serum from Nipah Virus Patients Recognises Recombinant Viral Proteins Produced in Escherichia coli.

The genes for Nipah virus (NiV) proteins were amplified from viral RNA, cloned into the plasmid pTriEx-3 Hygro, expressed, and purified using immobilized metal affinity chromatography. The recombinant N, F, and G NiV proteins (rNiV-N, rNiV-F, and rNiV-G), were successfully expressed in Escherichia coli and purified with a yield of 4, 16, and 4 mg/L, respectively. All 3 recombinant viral proteins reacted with all 19 samples of NiV-positive human sera. The rNiV-N and rNiV-G proteins were the most immunogenic. The recombinant viral proteins did not react with any of the 12 NiV-negative sera. However, serum from a patient with a late-onset relapsing NiV infection complication was found to be primarily reactive to rNiV-G only. Additionally, there is a distinctive variation in the profile of antigen-reactive bands between the sample from a case of relapsing NiV encephalitis and that of acute NiV infection. The overall findings of this study suggest that the recombinant viral proteins have the potential to be developed further for use in the detection of NiV infection, and continuous biosurveillance of NiV infection in resource-limited settings.

Geographical distribution of Brucella melitensis inferred from rpoB gene variation.

INTRODUCTION: Currently available tests have limitations for the identification of Brucella species and strains, and their genetic lineage. The genome sequence of the rpoB gene encoding the ß-subunit of DNA-dependent RNA polymerase was investigated for its use in genotyping Brucella melitensis. METHODOLOGY: Complete rpoB gene sequences of globally distributed Brucella melitensis strains were analyzed. Single nucleotides polymorphisms (SNPs) of the rpoB gene sequences were identified and used to type Brucella melitensis strains. RESULTS: Six DNA polymorphisms were identified, of which two (nucleotides 3201 and 558) were novel. Analysis of the geographical distribution of the strains revealed a spatial clustering pattern with rpoB type 1 representing European and American strains, rpoB type 2 representing European, African, and Asian strains, rpoB type 3 representing Mediterranean strains, and rpoB type 4 representing African (C3201T) and European (C3201T/T558A) strains. CONCLUSIONS: We report the discovery of two novel SNPs of rpoB gene that can serve as useful markers for epidemiology and geographical tracking of B. melitensis.

Insertion of single-chain variable fragment (scFv) peptide linker improves surface display of influenza hemagglutinin (HA1) on non-recombinant Lactococcus lactis.

Heterologous protein displayed on the surface of Lactococcus lactis using the binding domain of N-acetylmuramidase (AcmA) has a potential application in vaccine delivery. In this study, we developed a non-recombinant L. lactis surface displaying the influenza A (H1N1) 2009 hemagglutinin (HA1). Three recombinant proteins, HA1/L/AcmA, HA1/AcmA, and HA1 were overexpressed in Escherichia coli, and purified. In the binding study using flow cytometry, the HA1/L/AcmA, which contained the single-chain variable fragment (scFv) peptide linker showed significantly higher percentage of binding counts and mean fluorescence binding intensity (MFI) (51.7 ± 1.4% and 3,594.0 ± 675.9, respectively) in comparison to the HA1/AcmA without the scFv peptide linker (41.1 ± 1.5% and 1,652.0 ± 34.1, respectively). Higher amount of HA1/L/AcmA (∼2.9 × 104 molecules per cell) was displayed on L. lactis when compared to HA1/AcmA (∼1.1 × 104 molecules per cell) in the immunoblotting analysis. The HA1/L/AcmA completely agglutinated RBCs at comparable amount of protein to that of HA1/AcmA and HA1. Computational modeling of protein structures suggested that scFv peptide linker in HA1/L/AcmA kept the HA1 and the AcmA domain separated at a much longer distance in comparison to HA1/AcmA. These findings suggest that insertion of the scFv peptide linker between HA1 and AcmA improved binding of recombinant proteins to L. lactis. Hence, insertion of scFv peptide linker can be further investigated as a potential approach for improvement of heterologous proteins displayed on the surface of L. lactis using the AcmA binding domain. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:154-162, 2017.

Coxiella Detection in Ticks from Wildlife and Livestock in Malaysia.

Recent studies have shown that ticks harbor Coxiella-like bacteria, which are potentially tick-specific endosymbionts. We recently described the detection of Coxiella-like bacteria and possibly Coxiella burnetii in ticks found from rural areas in Malaysia. In the present study, we collected ticks, including Haemaphysalis bispinosa, Haemaphysalis hystricis, Dermacentor compactus, Dermacentor steini, and Amblyomma sp. from wildlife and domesticated goats from four different locations in Malaysia. Coxiella 16s rRNA genomic sequences were detected by PCR in 89% of ticks tested. Similarity analysis and phylogenetic analyses of the 16s rRNA and rpoB partial sequences were performed for 10 representative samples selected based on the tick species, sex, and location. The findings here suggested the presence of C. burnetii in two samples, each from D. steini and H. hystricis. The sequences of both samples clustered with published C. burnetii sequences. The remaining eight tick samples were shown to harbor 16s rRNA sequences of Coxiella-like bacteria, which clustered phylogenetically according to the respective tick host species. The findings presented here added to the growing evidence of the association between Coxiella-like bacteria and ticks across species and geographical boundaries. The importance of C. burnetii found in ticks in Malaysia warrants further investigation.

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.