Molecular characterisation of virulence in Entamoeba histolytica

Entamoeba histolytica is a parasitic protozoan that infects the human digestive tract. Infection results from ingestion of cyst-contaminated food or water. To date, E. histolytica infection remains a major worldwide public health problem in worldwide endemic areas. The spectrum of clinical manifestations ranges from asymptomatic carrier to mucous and bloody diarrhea or even extraintestinal amoebiasis, usually amoebic liver abscess. Several molecular studies have been carried out to reveal novel aspects of E. histolytica infection. However, the studies focused on genomic-wide analysis comparing between E. histolytica strains are still limited. Thus, the aims of this project are to comprehensively study the comparative analysis of the whole and small RNA transcriptomes amongst nonvirulent and virulent strains of laboratory cultured E. histolytica trophozoites as well as to integrate such transcriptomic findings with the genomic data for advanced understanding of the molecular pathogenesis and virulence in amoebiasis. In this study, genome-wide transcriptome analysis using Illumina RNA-Seq technology can illustrate significant expression differences between nonvirulent and virulent E. histolytica strains. Differential gene expression analysis between nonvirulent Rahman strain and other three virulent strains (i.e. PVBM08B, HM-1:IMSS and IULA:1092:1) reveals that transcripts involved in host cell killing and mucosal invasion, nucleic acid interaction and response to oxidative stress are notably upregulated in the virulent trophozoites. InterProScan results show the upregulation of genes encoding proteolysis-related domains and the co-upregulation of cytoskeleton and actin-modulating domains in the virulent strains. Also, process ontologies related to protein degradation, cellular biosynthesis, DNA metabolism, repair and recombination, mitotic cell division, actin dynamics and response to stress are highly enriched as a core metabolism in the virulent strains, indicating the rapid growing and active metabolic state are the main drivers of virulence. However, the striking underrepresentation of ontologies involved in signaling and regulatory processes was observed in the virulent parasites. It could be inferred that reduced regulation of sensing and correctly responding to the environmental stimuli potentially enable the parasites to become virulent and subsequently cause the invasive infection. Also, NanoString validation reveals the spectrum of virulence-associated gene expression among these four strains, reflecting their different degrees of virulence. Gene copy number variation (CNV) is widespread among the genomes of the E. histolytica strains, reflecting genomic plasticity and variability in gene family content. Herein, this present data show that patterns of CNV contribute to differential expression profiles, therefore it can be extrapolated that differences in gene copy number between genomes could contribute to the variation in phenotypic attributes, including virulence, among E. histolytica strains. Also, genome plasticity can also be seen in Trypanosomes and Leishmania, suggesting that CNV is a potentially important mechanism in generating genetic diversity and regulating gene expression levels in almost exclusively asexual parasite group. For small RNA transcriptomics, the size-fractionated sRNA sequencing data demonstrate the inverse relationship between antisense sRNA abundance and target gene expression levels, strongly suggesting the sRNA-mediated regulation. Differential sRNA regulation in virulence-associated gene expression was found among strains, indicating that sRNA-mediated post-transcriptional regulation may be important in shaping the parasite virulence. In addition, this study identified the novel putative miRNA from the sRNA sequencing data using the biogenesis-based bioinformatic analysis and qPCR validation, implying that miRNA potentially play a regulatory role in E. histolytica. In summary, it can be inferred that genomic plasticity and sRNA-mediated regulation are important mechanisms of virulence modulation in E. histolytica

First Evidence of Co-Circulation of Emerging Leishmania martiniquensis, Leishmania orientalis, and Crithidia sp. in Culicoides Biting Midges (Diptera: Ceratopogonidae), the Putative Vectors for Autochthonous Transmission in Southern Thailand

Since 1996, autochthonous cases of emerging leishmaniasis caused by Leishmania (Mundinia) martiniquensis and Leishmania (Mundinia) orientalis have been more frequently reported, especially in the northern and southern parts of Thailand. However, the accurate identification of their natural vectors and reservoirs remains unconfirmed. Previous studies have suggested that these emerging parasites might be transmitted by other non-phlebotomine vectors. Herein, we speculated that Culicoides biting midges might act as the competent vectors responsible for autochthonous leishmaniasis in southern Thailand. In this research, 187 non-engorged, parous and gravid Culicoides females and 47 blood-engorged ones were trapped from the residences of two recently diagnosed visceral leishmaniasis patients in Sadao District and the unaffected site in Rattaphum District, Songkhla Province, southern Thailand. Species diversity and abundance of biting midges varied among the trapping sites. Using ITS1-PCR and BLASTn analysis, L. martiniquensis was predominantly detected in several Culicoides species, including C. peregrinus, C. oxystoma, C. mahasarakhamense, and C. huffi from the vicinity of patients’ houses; and in C. fordae and C. fulvus from the unaffected site. L. orientalis was also co-circulated in C. peregrinus and C. oxystoma caught near the second patient’s house. Additionally, Crithidia sp. were also detected using SSU rRNA-PCR across Culicoides spp. Host blood meal analysis of eight different Culicoides species from the unaffected site also revealed that all trapped Culicoides had fed on cows and goats, indicating the possible role of these mammalian species as reservoir hosts. Essentially, this study is the first entomological investigation, revealing the co-circulation of emerging trypanosomatids among several species of Culicoides biting midges and strongly supporting the potential role of this insect group as the main vectors responsible for the epidemiology of autochthonous leishmaniasis in southern Thailand

Molecular Analysis of Canine Filaria and Its Wolbachia Endosymbionts in Domestic Dogs Collected from Two Animal University Hospitals in Bangkok Metropolitan Region, Thailand

Canine filariasis is caused by several nematode species, such as Dirofilaria immitis, Dirofilaria repens, Brugia pahangi, Brugia malayi, and Acanthocheilonema reconditum. Zoonotic filariasis is one of the world’s neglected tropical diseases. Since 2000, the World Health Organization (WHO) has promoted a global filarial eradication program to eliminate filariasis by 2020. Apart from vector control strategies, the infection control of reservoir hosts is necessary for more effective filariasis control. In addition, many studies have reported that Wolbachia is necessary for the development, reproduction, and survival of the filarial nematode. Consequently, the use of antibiotics to kill Wolbachia in nematodes has now become an alternative strategy to control filariasis. Previously, a case of subconjunctival dirofilariasis caused by Dirofilaria spp. has been reported in a woman who resides in the center of Bangkok, Thailand. Therefore, our study aimed to principally demonstrate the presence of filarial nematodes and Wolbachia bacteria in blood collected from domestic dogs from the Bangkok Metropolitan Region, Thailand. A total of 57 blood samples from dogs with suspected dirofilariasis who had visited veterinary clinics in Bangkok were collected. The investigations for the presence of microfilaria were carried out by using both microscopic and molecular examinations. PCR was used as the molecular detection method for the filarial nematodes based on the COI and ITS1 regions. The demonstration of Wolbachia was performed using PCR to amplify the FtsZ gene. All positive samples by PCR were then cloned and sequenced. The results showed that the filarial nematodes were detected in 16 samples (28.07%) using microscopic examinations. The molecular detection of filarial species using COI-PCR revealed that 50 samples (87.72%) were positive; these consisted of 33 (57.89%), 13 (22.81%), and 4 (7.02%) samples for D. immitis, B. pahangi, and B. malayi, respectively. While the ITS1-PCR showed that 41 samples (71.93%) were positive—30 samples (52.63%) were identified as containing D. immitis and 11 samples (19.30%) were identified to have B. pahangi, whereas B. malayi was not detected. Forty-seven samples (82.45%) were positive for Wolbachia DNA and the phylogenetic tree of all positive Wolbachia was classified into the supergroup C clade. This study has established fundamental data on filariasis associated with Wolbachia infection in domestic dogs in the Bangkok Metropolitan Region. An extensive survey of dog blood samples would provide valuable epidemiologic data on potential zoonotic filariasis in Thailand. In addition, this information could be used for the future development of more effective prevention and control strategies for canine filariasis in Thailand

Stimulation of metacyclogenesis in Leishmania ( Mundinia ) orientalis for mass production of metacyclic promastigotes

Leishmania (Mundinia) orientalis is a human pathogen causing leishmaniasis and studies on the properties of metacyclic promastigotes, the parasite’s infective stage, are required for a better understanding of its transmission and infection. However, information on cultivation for mass production of L. orientalis metacyclic promastigotes and factors that stimulate their metacyclogenesis is limited. Therefore, the objective of this study was to develop a suitable methodology for generating promastigote cultures containing a high proportion and number of L. orientalis metacyclic promastigotes. Various media, i.e., Schneider’s insect medium, Medium 199 and Grace’s insect medium, supplemented with various quantities of dithiothreitol, Basal Medium Eagle vitamins, pooled human urine, and fetal bovine serum, were optimized for metacyclogenesis. The results revealed that the optimum culture medium and conditions of those tested were Schneider’s insect medium supplemented with 100 μM dithiothreitol, 1% (v/v) Basal Medium Eagle vitamins, 2% (v/v) pooled human urine, and 10% (v/v) fetal bovine serum, pH 5.0 at 26°C. We also demonstrated that L. orientalis metacyclic promastigotes could be purified and enriched by negative selection using peanut lectin. Under these culture conditions, the highest yield of metacyclic promastigotes was obtained with a significantly higher percentage of parasite survival, resistance to complement-mediated lysis, and infection index in THP-1 macrophage cells compared to parasites cultured without media supplements at neutral pH. This is the first report providing a reliable method for mass production of L. orientalis metacyclic promastigotes for in vivo infections and other experimental studies of this emerging parasite in the future

First Report of Anuran <i>Trypanosoma</i> DNA in Flat-Tailed House Geckos (Reptilia: Gekkonidae) Collected from Southern Thailand: No Evidence as a Reservoir for Human Trypanosomatids

Over the years, cases of autochthonous leishmaniasis have been dramatically increasing in Thailand. Recently, several publications have claimed certain species of the phlebotomine sand flies and biting midges potentially serve as natural vectors of Leishmania and Trypanosoma species in this country. However, more information regarding the vector–parasite relationships, as well as their natural reservoirs in the country, still needs to be explored. Herein, we hypothesized that synanthropic reptiles in the leishmaniasis-affected area might be a natural reservoir for these parasites. In this present study, a total of nineteen flat-tailed house geckos were collected from the house of a leishmaniasis patient in Songkhla province, southern Thailand, and then dissected for their visceral organs for parasite detection. Small subunit ribosomal RNA (SSU rRNA) gene and internal transcribed spacer 1 (ITS-1)-specific amplifications were conducted to verify the presence of Trypanosoma and Leishmania parasites, respectively. Only Trypanosoma DNA was screened positive in eight gecko individuals by SSU rRNA-PCR in at least one visceral organ (4, 4, and 6 of the heart, liver, and spleen, respectively) and phylogenetically related to the anuran Trypanosoma spp. (An04/Frog1 clade) previously detected in three Asian sand fly species (Phlebotomus kazeruni, Sergentomyia indica, and Se. khawi). Hence, our data indicate the first detection of anuran Trypanosoma sp. in the flat-tailed house geckos from southern Thailand. Essentially, it can be inferred that there is no evidence for the flat-tailed house gecko (Hemidactylus platyurus) as a natural reservoir of human pathogenic trypanosomatids in the leishmaniasis-affected area of southern Thailand

The Prevalence of <i>Bartonella</i> Bacteria in Cattle Lice Collected from Three Provinces of Thailand

Cattle lice are obligatory blood-sucking parasites, which is the cause of animal health problems worldwide. Recently, several studies have revealed that pathogenic bacteria could be found in cattle lice, and it can act as a potential vector for transmitting louse-borne diseases. However, the cattle lice and their pathogenic bacteria in Thailand have never been evaluated. In the present study, we aim to determine the presence of bacterial pathogens in cattle lice collected from three localities of Thailand. Total genomic DNA was extracted from 109 cattle louse samples and the Polymerase Chain Reaction (PCR) of 18S rRNA was developed to identify the cattle louse. Moreover, PCR was used for screening Bartonella spp., Acinetobacter spp., and Rickettsia spp. in cattle louse samples. The positive PCR products were cloned and sequenced. The phylogenetic tree based on the partial 18S rRNA sequences demonstrated that cattle lice species in this study are classified into two groups according to reference sequences; Haematopinus quadripertusus and Haematopinus spp. closely related to H. tuberculatus. The pathogen detection revealed that Bartonella spp. DNA of gltA and rpoB were detected in 25 of 109 samples (22.93%) both egg and adult stages, whereas Acinetobacter spp. and Rickettsia spp. were not detected in all cattle lice DNA samples. The gltA and rpoB sequences showed that the Bartonella spp. DNA was found in both H. quadripertusus and Haematopinus spp. closely related to H. tuberculatus. This study is the first report of the Bartonella spp. detected in cattle lice from Thailand. The finding obtained from this study could be used to determine whether the cattle lice can serve as a potential vector to transmit these pathogenic bacteria among cattle and may affect animal to human health

Molecular Analysis of Medically and Veterinary Important Muscid Flies (Diptera: Muscidae) in Thailand

We demonstrated the using of the internal transcribed spacer (ITS2) of ribosomal DNA as a tool for identification of medically and veterinary important Muscidae flies in Thailand. A total of 27 fly samples were collected from various regions of Thailand. Six fly species in three subfamilies including Azeliinae (Hydrotaea spinigera), Muscinae (Musca domestica, M. sorbens) and Stomoxyinae (Stomoxys calcitrans, S. indicus and S. sitiens) were identified base on morphological taxonomy. PCR amplicons of the ITS2 gene of these flies varied between 312-377 bp with A+T content of 76.6%. ITS2 sequences of the flies in this study were 93-100% identity to sequences in database and 21 samples were compatible with morphological identification, while sequences of 6 samples did not match any sequences in the database. The intra- and inter-specific divergence analysis results showed that the maximum of intra-specific (within species) variation (6.9%) was found in M. domestica while the minimum inter-specific (between species) variation (11.9%) was found in the sister grouped couple of S. sitiens and S. indicus. No overlapping between intra- and inter-specific divergences was found in all species of this study. The bootstrapped NJ tree constructed showed ability to classify each subfamily in to monophyletic clades. PCR-RFLP using XapI restriction enzyme digestion was able to differentiate between the three Stomoxys species. Data obtained from this study would be valuable for both medical and veterinary entomologists for more accurate identification of important fly species. Therefore, it could be used for population dynamics studies and enrolled in integrated pest management control program

Relationship of serum IL-6, C-reactive protein, erythrocyte sedimentation rate, and knee skin temperature after total knee arthroplasty: a prospective study

Knee osteoarthritis is a common cause of severe pain and functional limitation. Total knee arthroplasty is an effective procedure to relieve pain, restore knee function, and improve quality of life for patients with end stage knee arthritis. The aim of this study was to investigate the inflammatory process in patients with primary knee osteoarthritis before surgery and in subsequent periods following total knee arthroplasty. A prospective study of 49 patients undergoing primary total knee replacements was conducted. The patients were evaluated by monitoring serum interleukin-6 (IL-6), C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), knee skin temperature, and clinical status. Measurements were carried out preoperatively and postoperatively on day one and at two, six, 14, and 26 weeks during follow-up review in the knee clinic. The serum IL-6 and CRP were elevated on the first postoperative day but fell to preoperative values at two weeks postoperatively. Both returned to within the normal range by six weeks postoperatively. In addition, the postoperative ESR showed a slow rise with a peak two weeks after surgery and returned to the preoperative level at 26 weeks postoperatively. The difference in skin temperature between operated and contralateral knees had a mean value of +4.5°C at two weeks. The mean value decreased to +3.5°C at six weeks, +2.5°C at 14 weeks, and +1.0°C at 26 weeks. The difference in skin temperature decreased gradually and eventually there was no statistically significant difference at 26 weeks after surgery. A sustained elevation in serum IL-6, CRP, ESR, and skin temperature must raise the concern of early complication and may suggest the development of postoperative complication such as haematoma and/or infection