Complete mitochondrial genome analyses confirm that bat Polychromophilus and ungulate Plasmodium constitute a distinct clade independent of other Plasmodium species.

In recent phylogenetic studies, bat Polychromophilus and ungulate Plasmodium, two relatively understudied haemosporidian parasites within the Apicomplexa phylum, have often been overlooked. Instead, the focus has been primarily on haemosporidian parasites in primates, rodents, and birds. Several phylogenetic analyses of bat Polychromophilus have relied on limited datasets and short informative DNA sequences. As a result of these inherent limitations, the substantiation of their evolutionary stance has encountered a diminished degree of robust validation. This study successfully obtained complete mitochondrial genome sequences from 11 Polychromophilus parasites originating from Hipposideros gentilis and Myotis siligoensis bats for the first time. Additionally, the authors have sequenced the apicoplast caseinolytic protease C genes from Polychromophilus murinus and a potentially new Polychromophilus species. These mitochondrial genomes range in length from 5994 to 6001 bp and consist of three protein-coding genes (PCGs), seven small subunit ribosomal RNA genes (SSU rRNA), 12 large subunit ribosomal RNA genes (LSU rRNA), and seven miscellaneous RNA genes. Phylogenetic analyses using Bayesian Inference and Maximum Likelihood methods indicated robust support for the grouping of ungulate Plasmodium and bat Polychromophilus in a single clade separate from other Plasmodium spp., confirming previous reports, albeit with stronger evidence in this study. The divergence between Polychromophilus in bats and Plasmodium in ungulates occurred approximately 29.61 to 55.77 million years ago (Mya), with a node age estimated at 40.63 Mya. These findings highlight that the genus Plasmodium, which includes species found in ungulates, birds, reptiles, and other mammals, does not form a monophyletic group. By incorporating Polychromophilus in bats and Plasmodium in ungulates, this study contributes significantly to understanding the phylogenetic relationships within the Haemosporida order. It provides valuable insights into the evolutionary history and interconnections among these diverse parasites, thereby expanding knowledge in this field.

Genetic characterization of genes encoding the major surface proteins of Anaplasma marginale from cattle isolates in Thailand reveals multiple novel variants.

Bovine anaplasmosis is a serious tick-borne disease that is responsible for economic loss worldwide. The major surface proteins (MSPs), encoded by msp1 to msp5 genes of Anaplasma marginale, play an important role in host-pathogen and tick-pathogen interactions. These markers have been used for genetic characterization and phylogenetic studies. Despite domestic reports concerning suspected outbreaks of anaplasmosis in Thailand, genetic analysis of A. marginale in the country remains largely limited. Therefore, we aim to investigate the infection rate of the rickettsia organism in the Anaplasmataceae family throughout five regions of Thailand and to further characterize the key genetic markers: msp1a, msp2, and msp5 of A. marginale. From 2016 to 2021, we collected a total of 384 cattle blood samples across 18 provinces. Overall, the infection rate of the rickettsia organism in the Anaplasmataceae family was 46.1%. Over 65% of the positive samples were confirmed as A. marginale. We successfully obtained a total of 138 A. marginale msp1a (38), msp2 (79), and msp5 (21) sequences from all regions of the country. The msp1a and msp2 genes exhibit a high degree of genetic diversity, while the msp5 gene is highly conserved among the Thai isolates. Our findings regarding msp1a corroborated the genetic heterogeneity of A. marginale strains in endemic regions worldwide. Additionally, we found multiple novel variants for the first time in the current nationwide survey. We found 45 tandem repeat characters of the msp1a sequence. Among them, 24 characters were not shared with other countries. Collectively, we expanded the extent of genetic diversity in key markers; msp1a and msp2 genes, and further confirmed the previous finding that msp5 was highly conserved. The msp1a and msp2 genes could be useful for the surveillance of newly introduced strains. The current data may also be useful in designing a vaccine containing potential epitopes of different antigens in the future.

Myzorhynchus series of Anopheles mosquitoes as potential vectors of Plasmodium bubalis in Thailand.

Ungulate malaria parasites and their vectors are among the least studied when compared to other medically important species. As a result, a thorough understanding of ungulate malaria parasites, hosts, and mosquito vectors has been lacking, necessitating additional research efforts. This study aimed to identify the vector(s) of Plasmodium bubalis. A total of 187 female mosquitoes (133 Anopheles spp., 24 Culex spp., 24 Aedes spp., and 6 Mansonia spp. collected from a buffalo farm in Thailand where concurrently collected water buffalo samples were examined and we found only Anopheles spp. samples were P. bubalis positive. Molecular identification of anopheline mosquito species was conducted by sequencing of the PCR products targeting cytochrome c oxidase subunit 1 (cox1), cytochrome c oxidase subunit 2 (cox2), and internal transcribed spacer 2 (ITS2) markers. We observed 5 distinct groups of anopheline mosquitoes: Barbirostris, Hyrcanus, Ludlowae, Funestus, and Jamesii groups. The Barbirostris group (Anopheles wejchoochotei or Anopheles campestris) and the Hyrcanus group (Anopheles peditaeniatus) were positive for P. bubalis. Thus, for the first time, our study implicated these anopheline mosquito species as probable vectors of P. bubalis in Thailand.

Molecular detection and characterization of tick-borne parasites in goats and ticks from Thailand.

Ticks and tick-borne pathogens (TTBPs) pose a serious economic threat to ruminant production worldwide. Despite this, investigations focused on goats remain limited compared to those for pathogens infecting cattle. We carried out PCR-based surveys and phylogenetic analyses to examine TTBPs from 6 provinces in Thailand between January 2016 and June 2020. A total of 93 tick samples were collected as well as 969 blood samples from goats. All ticks were morphologically identified as Rhipicephalus microplus and confirmed for species based on 16S rRNA and cox1 gene sequences. The mitochondrial cox1 sequences in the present study were clustered into clades A and C. The overall infection rates of Anaplasma spp., piroplasmids, and co-infections of both parasites in goats were 13.5% (131/969), 2.7% (24/880), and 0.7% (7/969), respectively. We observed no statistically significant association between TTBP infections and age or sex. However, TTBP infections and the rainy season were linked (p < 0.05). Anaplasma bovis, Anaplasma marginale, and Anaplasma ovis were detected for the first time in goats in the country using primers targeting the chaperonin GroEL (groEL), major surface protein 2 (msp2), and major surface protein 4 (msp4) genes, while Anaplasma capra and Anaplasma phagocytophilum were not detected. Anaplasma bovis, A. marginale, and A. ovis isolates were clustered in a subclade that differed from the strains found in other countries. Among piroplasmids, only Theileria luwenshuni was detected in the current investigation. This work will add to the current understanding regarding the prevalence, genetic diversity, and genetic relationships of A. bovis, A. marginale, A. ovis, and T. luwenshuni among global isolates and those in Thailand.

Molecular identification and genetic diversity of Bartonella spp. in 24 bat species from Thailand.

The study of bacterial zoonoses has been under-pursued despite the fact that bacteria cause the majority of zoonotic diseases, of which 70% have a wildlife origin. More Bartonella species are being identified as the cause of human diseases, and several of them have been linked to domestic and wild animals. Bats are outstanding reservoirs for Bartonella species because of their wide distribution, mobility, roosting behaviour, and long life span. Here, we carried out a PCR-based survey on bats that were collected from 19 sampling sites in eight provinces of Thailand from February 2018 to April 2021. Bartonella infection was investigated in a total of 459 bats that belong to 24 different bat species (21 species of which had never been previously studied in Thailand). PCR diagnostics revealed that 115 out of 459 (25.5%) blood samples tested positive for Bartonella. The nucleotide identities of the Bartonella 16S rRNA sequences in this study were between 95.78-99.66% identical to those of known zoonotic species (Bartonella ancashensis, Bartonella henselae, Bartonella bacilliformis and Bartonella australis) as well as to an unidentified Bartonella spp. In addition, the citrate synthase (gltA) and RNA polymerase-beta subunit (rpoB) genes of Bartonella were sequenced and analyzed in positive samples. The gltA and rpoB gene sequences from Hipposideros gentilis and Rhinolophus coelophyllus bat samples showed low nucleotide identity (<95%) compared to those of the currently deposited sequences in the GenBank database, indicating the possibility of new Bartonella species. The phylogenetic inference and genetic diversity were generated and indicated a close relationship with other Bartonella species previously discovered in Asian bats. Overall, the current study demonstrates the primary evidence pointing to a potential novel Bartonella species in bats. This discovery also contributes to our current understanding of the geographical distribution, genetic diversity, and host ranges of bat-related Bartonella.

Development and application of a novel multiplex PCR assay for the differentiation of four haemosporidian parasites in the chicken Gallus gallus domesticus.

Haemosporidian infections in domestic chickens (Gallus gallus domesticus) are not only widely prevalent but also cause economic loss. Diagnosis is usually made by microscopic examination; however, the method has several drawbacks such as requiring an experienced microscopist, being unreliable when parasitemia is low and being unable to accurately differentiate between co-infections from multiple parasite species. Therefore, the current extent of haemosporidian infections might be underestimated and neglected. We have developed a novel multiplex PCR assay to simultaneously detect and differentiate between four haemosporidian parasites: Leucocytozoon caulleryi, Leucocytozoon sabrazesi, Plasmodium juxtanucleare and Plasmodium gallinaceum. Primers in the present study specifically amplified the corresponding targets with no cross-species amplification detected. The multiplex PCR exhibited a significantly greater detection rate when compared with microscopic examination (p = 0.0001). The results demonstrate that the detection rate of multiplex PCR for L. sabrazesi, P. juxtanucleare, and P. gallinaceum are all greater than that of microscopic examination with p = 0.002, 0.0001 and 0.004, respectively. Co-infections were also detected more effectively by multiplex PCR. We applied the current method to field samples originating from Nan, Prachinburi, and Chachoengsao Provinces. The current study revealed that positive rates of haemosporidian parasites in chickens in the three study sites ranging from 39.5%-93.8%. The present assay offers a timesaving option for molecular diagnosis instead of using singleplex PCRs for detecting the parasites individually. Within a single reaction, this assay would be a useful tool for the detection of avian haemosporidian parasites either single or under co-infection conditions and for large-scale epidemiology studies.

Three-Dimensional RAW264.7 Cell Model on Electrohydrodynamic Printed Poly(ε-Caprolactone) Scaffolds for In Vitro Study of Anti-Inflammatory Compounds.

Inflammation plays an essential role in the human immune system, and anti-inflammatory compounds are important to promote health. However, the in vitro screening of these compounds is largely dependent on flat biology. Herein, we report our efforts in establishing a 3D inflammation murine macrophage model. Murine macrophage RAW 264.7 cells were cultured on poly(ε-caprolactone) (PCL) scaffolds fabricated through an electrohydrodynamic jetting 3D printer and their behavior were examined. Cells on PCL scaffolds showed a 3D shape and morphology with multilayers and a lower proliferation rate. Moreover, macrophages were not activated by scaffold material PCL and 3D microenvironment. The 3D cells showed greater sensitivity to lipopolysaccharide stimulation with higher production activity of nitric oxide (NO), nitric oxide synthases (iNOS), and cyclooxygenase-2 (COX-2). Additionally, the 3D macrophage model showed lower drug sensitivity to commercial anti-inflammatory drugs including aspirin, ibuprofen, and dexamethasone, and natural flavones apigenin and luteolin with higher IC50 for NO production and lower iNOS and COX-2 inhibition efficacy. Overall, the 3D macrophage model showed promise for higher accurate screening of anti-inflammatory compounds. We developed, for the first time, a 3D macrophage model based on a 3D-printed PCL scaffold that provides an extracellular matrix environment for cells to grow in the 3D dimension. 3D-grown RAW 264.7 cells showed different sensitivities and responses to anti-inflammatory compounds from its 2D model. The 3D cells have lower sensitivity to both commercial and natural anti-inflammatory compounds. Consequently, our 3D macrophage model could be applied to screen anti-inflammatory compounds more accurately and thus holds great potential in next-generation drug screening applications.

Antitumor Efficacy of the Herbal Recipe Benja Amarit against Highly Invasive Cholangiocarcinoma by Inducing Apoptosis both In Vitro and In Vivo.

Thailand is the country with highest incidence and prevalence of cholangiocarcinoma (CCA) in the world. Due to the frequently late diagnosis that is associated with this disease, most CCA patients are prescribed chemotherapy as a form of treatment. However, CCA is able to resist the presently available chemotherapy, so to the prognosis of this disease is still very poor. In this study, we investigated the anticancer potential of a Thai herbal recipe, Benja Amarit (BJA) against CCA and the relevant mechanisms of action that are involved. We found that BJA inhibited CCA cell viability in a dose-dependent manner, especially in highly invasive KKU-213 cells. The extract induced mitochondrial- and caspase-dependent apoptosis in CCA cells by regulating the nuclear factor-κB (NF-κB) signaling pathway. BJA also triggered autophagy in CCA cells. Nonetheless, the inhibition of autophagy enhanced BJA-induced CCA cell death via apoptosis. An in vivo xenograft model revealed the growth-inhibiting and death-inducing effects of BJA against CCA by targeting apoptosis. However, general toxicity to blood cells, kidneys and the liver, as well as changes in body weight, did not appear. Our findings suggest that the herbal recipe BJA might be used as a potentially new and effective treatment for cholangiocarcinoma patients.

Extracts of Bridelia ovata and Croton oblongifolius induce apoptosis in human MDA­MB­231 breast cancer cells via oxidative stress and mitochondrial pathways.

Breast cancer is the most common type of cancer and is also the second leading cause of cancer­associated death in women worldwide. Thus, there is an urgent requirement for the development of effective treatments for this disease. Bridelia ovata and Croton oblongifolius are herbs used in Thai traditional medicine that have been used to treat various health problems; B. ovata has traditionally been used as a purgative, an antipyretic, a leukorrhea treatment and as a birth control herb. C. oblongifolius has been used to increase breast milk production, for post­partum care (where it is used as a hot bath herb), and as a treatment for flat worms and dysmenorrhea. However, there is little research investigating the anticancer properties of these herbs. The present study aimed to investigate the anticancer properties of crude ethyl acetate extracts of B. ovata (BEA) and C. oblongifolius (CEA) in order to explore their underlying mechanisms in breast cancer cell death. The phytoconstituents of the crude extracts of BEA and CEA were studied using gas chromatography­mass spectrometry (GC­MS). GC­MS analysis showed that the primary compound in BEA is friedelan­3­one, and kaur­16­en­18­oic acid in CEA. Cytotoxicity was investigated using an MTT assay, both BEA and CEA showed greater toxicity against MDA­MB­231 breast cancer cells compared with their effect on MCF10A normal epithelial mammary cells. BEA and CEA exerted various effects, including inducing apoptotic cell death, reducing mitochondrial transmembrane potential, increasing the levels of intracellular ROS, activating caspases, upregulating pro­apoptotic and downregulating anti­apoptotic genes and proteins. BEA and CEA were shown to have anticancer activity against breast cancer cells and induce apoptosis in these cells via a mitochondrial pathway and oxidative stress.

Potential of Thai Herbal Extracts on Lung Cancer Treatment by Inducing Apoptosis and Synergizing Chemotherapy.

The incidence of lung cancer has increased while the mortality rate has continued to remain high. Effective treatment of this disease is the key to survival. Therefore, this study is a necessity in continuing research into new effective treatments. In this study we determined the effects of three different Thai herbs on lung cancer. Bridelia ovata, Croton oblongifolius, and Erythrophleum succirubrum were extracted by ethyl acetate and 50% ethanol. The cytotoxicity was tested with A549 lung cancer cell line. We found four effective extracts that exhibited toxic effects on A549 cells. These extracts included ethyl acetate extracts of B. ovata (BEA), C. oblongifolius (CEA), and E. succirubrum (EEA), and an ethanolic extract of E. succirubrum (EE). Moreover, these effective extracts were tested in combination with chemotherapeutic drugs. An effective synergism of these treatments was found specifically through a combination of BEA with methotrexate, EE with methotrexate, and EE with etoposide. Apoptotic cell death was induced in A549 cells by these effective extracts via the mitochondria-mediated pathway. Additionally, we established primary lung cancer and normal epithelial cells from lung tissue of lung cancer patients. The cytotoxicity results showed that EE had significant potential to be used for lung cancer treatment. In conclusion, the four effective extracts possessed anticancer effects on lung cancer. The most effective extract was found to be E. succirubrum (EE).

The Effect of Transfusion-Dependent Thalassemia Patient's Serum on Peripheral Blood Mononuclear Cell Viability.

Iron overload is a major complication in transfusion-dependent thalassemia (TDT) patients. Chronic oxidative stress from iron overload may lead to cellular damage and viability. This is a cross-sectional study. Transfusion-dependent thalassemia patients aged ⩾18 years old were enrolled. Transfusion-dependent thalassemia patient's serum and normal volunteer's serum were separately incubated with healthy peripheral blood mononuclear cells (PBMCs). The cell viability was measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay at 24, 48, and 72 hours. Sixty-nine TDT patients and 22 healthy controls were enrolled. The mean of PBMCs viability after incubation with serum from TDT patients was lower than that with the controls (88.65% vs 103.56% at 24 hours, 78.77% vs 112.04%% at 48 hours, and 71.18% vs 132.16%% at 72 hours, respectively). High serum ferritin level (correlation -0.29, P < .05) and white blood cell (WBC) count negatively affected cell viability (correlation -2.86, P = .05). From multivariate analysis, serum ferritin level is the only significant risk factor that is independently associated with cell viability (correlation -11.42, P < .001). Our findings showed that TDT patient's serum causes decreased cell viability. Serum ferritin level was a significant independent factor influencing cell viability.