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Abstract Background Babesia caballi is an intraerythrocytic parasite from the phylum Apicomplexa, capable of infecting equids and causing equine piroplasmosis. However, since there is limited genome information available on B. caballi, molecular mechanisms involved in host specificity and pathogenicity of this species have not been fully elucidated yet. Results Genomic DNA from a B. caballi subclone was purified and sequenced using both Illumina and Nanopore technologies. The resulting assembled sequence consisted of nine contigs with a size of 12.9 Mbp, rendering a total of 5,910 protein-coding genes. The phylogenetic tree of Apicomplexan species was reconstructed using 263 orthologous genes. We identified 481 ves1-like genes and named “ves1c”. In contrast, expansion of the major facilitator superfamily (mfs) observed in closely related B. bigemina and B. ovata species was not found in B. caballi. A set of repetitive units containing an open reading frame with a size of 297 bp was also identified. Conclusions We present a chromosome-level genome assembly of B. caballi. Our genomic data may contribute to estimating gene expansion events involving multigene families and exploring the evolution of species from this genus.

[This corrects the article DOI: 10.1371/journal.pone.0187703.].

Visceral leishmaniasis (VL) is the most severe type of leishmaniasis which is caused by infection of Leishmania donovani complex. In the BALB/c mouse model of VL, multinucleated giant cells (MGCs) with heavy parasite infection consist of the largest population of hemophagocytes in the spleen of L. donovani-infected mice, indicating that MGCs provide the parasites a circumstance beneficial for their survival. Although ATP6V0D2 is a demonstrated factor inducing the formation of hemophagocytic MGCs during L. donovani infection, functions of this protein in shaping the infection outcome in macrophages remain unclear. Here we evaluated the influence of upregulated ATP6V0D2 on intracellular survival of the parasites. L. donovani infection-induced hemophagocytosis of normal erythrocytes by macrophages was suppressed by RNAi-based knockdown of Atp6v0d2. The knockdown of Atp6v0d2 did not improve the survival of amastigotes within macrophages when the cells were cultured in the absence of erythrocytes. On the other hand, reduced intracellular survival of amastigotes in macrophages by the knockdown was observed when macrophages were supplemented with antibody-opsonized erythrocytes before infection. There, increase in cytosolic labile iron pool was observed in the L. donovani-infected knocked-down macrophages. It suggests that ATP6V0D2 plays roles not only in upregulation of hemophagocytosis but also in iron trafficking within L. donovani-infected macrophages. Superior access to iron in macrophages may be how the upregulated expression of the molecule brings benefit to Leishmania for their intracellular survival in the presence of erythrocytes.

Summary: Background: Pulmonary infection with SARS-CoV-2 stimulates host immune responses and can also result in the progression of dysregulated and critical inflammation. Throughout the pandemic, the management and treatment of COVID-19 has been continuously updated with a range of antiviral drugs and immunomodulators. Monotherapy with oral antivirals has proven to be effective in the treatment of COVID-19. However, treatment should be initiated in the early stages of infection to ensure beneficial therapeutic outcomes, and there is still room for further consideration on therapeutic strategies using antivirals. Methods: We studied the therapeutic effects of monotherapy with the oral antiviral ensitrelvir or the anti-inflammatory corticosteroid methylprednisolone and combination therapy with ensitrelvir and methylprednisolone in a delayed dosing model of hamsters infected with SARS-CoV-2. Findings: Combination therapy with ensitrelvir and methylprednisolone improved respiratory conditions and reduced the development of pneumonia in hamsters even when the treatment was started after 2 days post-infection. The combination therapy led to a differential histological and transcriptomic pattern in comparison to either of the monotherapies, with reduced lung damage and down-regulation of expression of genes involved in the inflammatory response. Furthermore, we found that the combination treatment is effective in case of infection with either the highly pathogenic delta or circulating omicron variants. Interpretation: Our results demonstrate the advantage of combination therapy with antiviral and corticosteroid drugs in COVID-19 treatment from the perspective of lung pathology and host inflammatory responses. Funding: Funding bodies are described in the Acknowledgments section.

Visceral leishmaniasis (VL) is an infectious disease caused by parasitic protozoa of the genus Leishmania and manifests clinical symptoms such as splenomegaly, hepatomegaly, anemia, and fever. It has previously been shown that B-cell-activating factor (BAFF) is involved in splenomegaly during VL. Although BAFF is known to be expressed by a variety of cells, the mechanism of elevated BAFF expression in VL is not clear. In this study, we aimed to identify BAFF-producing cells in the spleens of mice infected with Leishmania donovani. Splenocytes of L. donovani-infected mice showed elevated BAFF expression compared to that of naive mice. In the infected spleen, the number of both CD11b+ and F4/80+ cells increased, and the major BAFF-producing cells were CD11b+ cells, which did not serve as host cells of Leishmania. Immunohistochemical/immunofluorescent staining of spleens of infected mice revealed that the increased CD11b+ cells were primarily MRP14+ mononuclear cells. Together, these results suggest the increased BAFF expression in the spleen of L. donovani-infected mice involves a recruitment of inflammatory macrophages distinct from host macrophages for the parasites.

Abstract Zoonotic malaria due to Plasmodium knowlesi infection in Southeast Asia is sometimes life-threatening. Post-mortem examination of human knowlesi malaria cases showed sequestration of P. knowlesi-infected red blood cells (iRBCs) in blood vessels, which has been proposed to be linked to disease severity. This sequestration is likely mediated by the cytoadhesion of parasite-iRBCs to vascular endothelial cells; however, the responsible parasite ligands remain undetermined. This study selected P. knowlesi lines with increased iRBC cytoadhesion activity by repeated panning against human umbilical vein endothelial cells (HUVECs). Transcriptome analysis revealed that the transcript level of one gene, encoding a Schizont Infected Cell Agglutination (SICA) protein, herein termed SICA-HUVEC, was more than 100-fold increased after the panning. Transcripts of other P. knowlesi proteins were also significantly increased, such as PIR proteins exported to the iRBC cytosol, suggesting their potential role in increasing cytoadhesion activity. Transgenic P. knowlesi parasites expressing Myc-fused SICA-HUVEC increased cytoadhesion activity following infection of monkey as well as human RBCs, confirming that SICA-HUVEC conveys activity to bind to HUVECs.

Ovine babesiosis, caused by Babesia ovis, is an acute, lethal, and endemic disease worldwide and causes a huge economic loss to animal industry. Pathogen genome sequences can be utilized for selecting diagnostic markers, drug targets, and antigens for vaccine development; however, those for B. ovis have not been available so far. In this study, we obtained a draft genome sequence for B. ovis isolated from an infected sheep in Turkey. The genome size was 7.81 Mbp with 3,419 protein-coding genes. It consisted of 41 contigs, and the N50 was 526 Kbp. There were 259 orthologs identified among eight Babesia spp., Plasmodium falciparum, and Toxoplasma gondii. A phylogeny was estimated on the basis of the orthologs, which showed B. ovis to be closest to B. bovis. There were 43 ves genes identified using hmm model as well. They formed a discriminating cluster to other ves multigene family of Babesia spp. but showed certain similarities to those of B. bovis, B. caballi, and Babesia sp. Xinjiang, which is consistent with the phylogeny. Comparative genomics among B. ovis and B. bovis elucidated uniquely evolved genes in these species, which may account for the adaptation.

Haemaphysalis longicornis Neumann, 1901 is one of the most well-known hard ticks because of its medical and veterinary importance. Haemaphysalis longicornis transmit a wide range of pathogens among vertebrates, affecting humans and animals in Asia and Oceania. In Japan, the tick species is a major pest of cattle because it can spread a protozoan parasite Theileria orientalis, which causes theileriosis and produces economic losses to the livestock industry (Yokoyama et al. 2012 [1]). Apart from bovine theileriosis, H. longicornis is a vector of bovine babesiosis caused by Babesia ovata, canine babesiosis caused by Babesia gibsoni, and rickettsiosis and viral diseases in humans. Its habitats are mainly Japan, Australia, New Zealand, New Caledonia, the Fiji Islands, Korea, China, and Russia (Oliver et al. 1973 [2]). In the United States, heavy H. longicornis infestations on cattle and white-tailed deer were reported in 2019, making it now one of the tick species to be an increasing threat to livestock animals and humans globally.Ticks reproduce offspring after mating with female and male ticks, however, interestingly, there are two races of H. longicornis: bisexual (diploid) and parthenogenetic (triploid) races [2]. Parthenogenetic H. longicornis is distributed throughout Japan, while the northern limit of the bisexual race is believed to be Fukushima Prefecture on Honshu Island (Fujita et al. 2013 and Kitaoka et al. 1961 [3,4]). This tick species is also considered to be of great scientific importance, and the parthenogenetic race collected in Okayama prefecture has been reared since 1961, while the bisexual race collected in Oita prefecture has been reared since 2008 under laboratory conditions in Japan (Boldbaatar et al. 2010 and Fujisaki et al. 1976 [5,6]). Namely, the “Okayama strain” and “Oita strain” of H. longicornis have been maintained for more than six decades and 15 years, respectively, stably under laboratory conditions. To obtain reference data of bisexual H. longicornis, we sequenced unfed females with haploid genomes using Illumina and MinION Q20 kit then obtained a draft genome consisting of 2.48 Gbp. The number of the contig was 98,529 and N50 was 46.5 Kb. Genome information derived from our laboratory colony of bisexual H. longicornis ticks would provide fundamental insight into understanding how different reproductive lineages occur within the same species of the tick.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) has been widespread globally in pigs and humans for decades. Nasal colonization of LA-MRSA is regarded as an occupational hazard to people who are regularly involved in livestock production. Our previous study suggested pig-to-human transmission caused by LA-MRSA clonal complex (CC) 398, using traditional molecular typing methods. Instead, this study aimed to investigate the zoonotic transmission of LA-MRSA CC398 using whole genome sequencing (WGS) technologies. A total of 63 LA-MRSA isolates were identified and characterized in Thailand. Further, the 16 representatives of LA-MRSA CC9 and CC398, including porcine and worker isolates, were subjected to WGS on the Illumina Miseq platform. Core-genome single nucleotide polymorphism (SNP)-based analyses verify the zoonotic transmission caused by LA-MRSA CC398 in two farms. WGS-based characterization suggests the emergence of a novel staphylococcal cassette chromosome (SCC) mec type, consisting of multiple cassette chromosome recombinase (ccr) gene complexes via genetic recombination. Additionally, the WGS analyses revealed putative multi-resistant plasmids and several cross-resistance genes, conferring resistance against drugs of last resort used in humans such as quinupristin/dalfopristin and linezolid. Significantly, LA-MRSA isolates, in this study, harbored multiple virulence genes that may become a serious threat to an immunosuppressive population, particularly for persons who are in close contact with LA-MRSA carriers.

Abstract Background The recently discovered Babesia sp. Mymensingh, which causes clinical bovine babesiosis, has a wide geographical distribution. We investigated the phylogenetic position of Babesia sp. Mymensingh using its mitochondrial, plastid, and nuclear genes. Based on morphological and molecular data, Babesia sp. Mymensingh is a unique species and we named it as Babesia naoakii n. sp. Methods A blood DNA sample from a Babesia sp. Mymensingh-infected cow was subjected to genome sequencing to obtain the sequences of mitochondrial, plastid, and nuclear genes. Six phylogenetic trees were then constructed with (1) concatenated amino acid sequences of cytochrome oxidase subunit I, cytochrome oxidase subunit III, and cytochrome b genes of the mitochondrial genome; (2) 16S rRNA of the plastid genome; (3) nucleotide sequences of the elongation factor Tu gene of the plastid genome; (4) ITS1-5.8S rRNA-ITS2; (5) concatenated nucleotide sequences of 89 nuclear genes; and (6) concatenated amino acid sequences translated from the 89 nuclear genes. Results In all six phylogenetic trees, B. naoakii n. sp. formed a sister clade to the common ancestor of Babesia bigemina and B. ovata. The concatenated nuclear genes of B. naoakii n. sp. and their translated amino acid sequences shared lower identity scores with the sequences from B. bigemina (82.7% and 84.7%, respectively) and B. ovata (83.5% and 85.5%, respectively) compared with the identity scores shared between the B. bigemina and B. ovata sequences (86.3% and 87.9%, respectively). Conclusions Our study showed that B. naoakii n. sp. occupies a unique phylogenetic position distinct from existing Babesia species. Our findings, together with morphological differences, identify B. naoakii n. sp. as a distinct parasite species. Graphical Abstract

Molecular assays and capillary electrophoresis sequencing have been used to identify parasites in livestock. The low sample capacity, which increases labor and processing time, is one drawback. Targeted amplicon sequencing (Ampliseq) uses the fast and large sample capacity platform to identify parasites in the target host, overcoming this limitation. DNA was extracted from 162 whole blood samples collected from cattle in three provinces in the Philippines. Using Illumina’s Miseq platform, the V4 hypervariable region of the piroplasma 18S rRNA gene was amplified and sequenced. The AMPtk pipeline was used to obtain distinct amplicon sequence variants (ASVs) and the NCBI BLAST non-redundant database was used to assign taxonomy. In total, 95 (58.64%) samples were positive for piroplasma. Using the AMPTk pipeline, 2179 ASVs were obtained. A total of 79 distinct ASVs were obtained after clustering and filtering, which belonged to genera Babesia (n = 58), Theileria (n = 17), Hepatozoon (n = 2), and Sarcocystis (n = 2). The ASV top hits were composed of 10 species: Babesia bovis, B. bigemina, Theileria orientalis, Babesia sp., Hepatozoon canis, Sarcocystis cruzi, T. annulata, T. equi, T. mutans, and Theileria sp. Thung Song. The results generated in this study demonstrated the applicability of Ampliseq in detecting piroplasmid parasites infecting cattle in the Philippines.

Rotavirus is a major cause of diarrhea globally in animals and young children under 5 years old. Here, molecular detection and genetic characterization of porcine rotavirus in smallholder and commercial pig farms in the Lusaka Province of Zambia were conducted. Screening of 148 stool samples by RT-PCR targeting the VP6 gene revealed a prevalence of 22.9% (34/148). Further testing of VP6-positive samples with VP7-specific primers produced 12 positives, which were then Sanger-sequenced. BLASTn of the VP7 positives showed sequence similarity to porcine and human rotavirus strains with identities ranging from 87.5% to 97.1%. By next-generation sequencing, the full-length genetic constellation of the representative strains RVA/pig-wt/ZMB/LSK0137 and RVA/pig-wt/ZMB/LSK0147 were determined. Genotyping of these strains revealed a known Wa-like genetic backbone, and their genetic constellations were G4-P[6]-I5-R1-C1-M1-A8-N1-T1-E1-H1 and G9-P[13]-I5-R1-C1-M1-A8-N1-T1-E1-H1, respectively. Phylogenetic analysis revealed that these two viruses might have their ancestral origin from pigs, though some of their gene segments were related to human strains. The study shows evidence of reassortment and possible interspecies transmission between pigs and humans in Zambia. Therefore, the “One Health” surveillance approach for rotavirus A in animals and humans is recommended to inform the design of effective control measures.

Abstract Objective To disseminate the portable sequencer MinION in developing countries for the main purpose of battling infectious diseases, we found a consortium called Global Research Alliance in Infectious Diseases (GRAID). By holding and inviting researchers both from developed and developing countries, we aim to train the participants with MinION’s operations and foster a collaboration in infectious diseases researches. As a real-life example in which resources are limited, we describe here a result from a training course, a metagenomics analysis from two blood samples collected from a routine cattle surveillance in Kulan Progo District, Yogyakarta Province, Indonesia in 2019. Results One of the samples was successfully sequenced with enough sequencing yield for further analysis. After depleting the reads mapped to host DNA, the remaining reads were shown to map to Theileria orientalis using BLAST and OneCodex. Although the reads were also mapped to Clostridium botulinum, those were found to be artifacts derived from the cow genome. An effort to construct a consensus sequence was successful using a reference-based approach with Pomoxis. Hence, we concluded that the asymptomatic cow might be infected with T. orientalis and showed the usefulness of sequencing technology, specifically the MinION platform, in a developing country.

Abstract Background Postviral olfactory dysfunction (PVOD) following a viral upper respiratory tract infection (URI) is one of the most common causes of olfactory disorders, often lasting for over a year. To date, the molecular pathology of PVOD has not been elucidated. Methods A murine model of Toll-like receptor 3 (TLR3)-mediated upper respiratory tract inflammation was used to investigate the impact of URIs on the olfactory system. Inflammation was induced via the intranasal administration of polyinosinic–polycytidylic acid (poly(I:C), a TLR3 ligand) to the right nostril for 3 days. Peripheral olfactory sensory neurons (OSNs), immune cells in the olfactory mucosa, and glial cells in the olfactory bulb (OB) were analyzed histologically. Proinflammatory cytokines in the nasal tissue and OB were evaluated using the quantitative real-time polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay (ELISA). Results In the treated mice, OSNs were markedly reduced in the olfactory mucosa, and T cell and neutrophil infiltration therein was observed 1 day after the end of poly(I:C) administration. Moreover, there was a considerable increase in microglial cells and slight increase in activated astrocytes in the OB. In addition, qPCR and ELISA revealed the elevated expression of interleukin-1 beta, interleukin-6, tumor necrosis factor-alpha, and interferon-gamma both in the OB and nasal tissue. Conclusions Taken together, the decreased peripheral OSNs, OB microgliosis, and elevated proinflammatory cytokines suggest that immunological changes in the OB may be involved in the pathogenesis of PVOD.

A novel multiplex loop-mediated isothermal amplification (LAMP) method combined with DNA chromatography was developed for the simultaneous detection of three important respiratory disease-causing viruses: severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus, and influenza B virus. Amplification was performed at a constant temperature, and a positive result was confirmed by a visible colored band. An in-house drying protocol with trehalose was used to prepare the dried format multiplex LAMP test. Using this dried multiplex LAMP test, the analytical sensitivity was determined to be 100 copies for each viral target and 100-1000 copies for the simultaneous detection of mixed targets. The multiplex LAMP system was validated using clinical COVID-19 specimens and compared with the real-time qRT-PCR method as a reference test. The determined sensitivity of the multiplex LAMP system for SARS-CoV-2 was 71% (95% CI: 0.62-0.79) for cycle threshold (Ct) ≤ 35 samples and 61% (95% CI: 0.53-0.69) for Ct ≤40 samples. The specificity was 99% (95%CI: 0.92-1.00) for Ct ≤35 samples and 100% (95%CI: 0.92-1.00) for the Ct ≤40 samples. The developed simple, rapid, low-cost, and laboratory-free multiplex LAMP system for the two major important respiratory viral diseases, COVID-19 and influenza, is a promising field-deployable diagnosis tool for the possible future 'twindemic, ' especially in resource-limited settings.

Neuro-Behçet’s disease (NBD) involves the nervous system and has a poorer prognosis. Since the typical delay in the onset of neurological symptoms is several years, difficulties are associated with diagnosing NBD when neurological disorders occur almost simultaneously with other major symptoms. We herein report a rare case of NBD that presented with the almost simultaneous manifestation of principal symptoms and neurological disorders. A 32-year-old Japanese woman presented with severe pharyngitis with a white coating and ulcers. On the third day of admission, she suddenly fainted while walking with urinary incontinence. T2-weighted MRI showed multiple high-intensity signals in the brainstem. Genital ulcers and pseudofolliculitis on the back were also detected. Stabilometry showed principal involvement of the peripheral vestibular system. A cerebrospinal fluid examination revealed an increase in the number of cells with lymphocyte dominance. The interleukin-6 level was markedly elevated. Pulse corticosteroid therapy led to the attenuation of symptoms.

Abstract Nucleic acid test (NAT), most typically quantitative PCR, is one of the standard methods for species specific flavivirus diagnosis. Semi-comprehensive NATs such as pan-flavivirus PCR which covers genus Flavivirus are also available; however, further specification by sequencing is required for species level differentiation. In this study, a semi-comprehensive detection system that allows species differentiation of flaviviruses was developed by integration of the pan-flavivirus PCR and Nanopore sequencing. In addition, a multiplexing method was established by adding index sequences through the PCR with a streamlined bioinformatics pipeline. This enables defining cut-off values for observed read counts. In the laboratory setting, this approach allowed the detection of up to nine different flaviviruses. Using clinical samples collected in Vietnam and Brazil, seven different flaviviruses were also detected. When compared to a commercial NAT, the sensitivity and specificity of our system were 66.7% and 95.4%, respectively. Conversely, when compared to our system, the sensitivity and specificity of the commercial NAT were 57.1% and 96.9%, respectively. In addition, Nanopore sequencing detected more positive samples (n = 8) compared to the commercial NAT (n = 6). Collectively, our study has established a semi-comprehensive sequencing-based diagnostic system for the detection of flaviviruses at extremely affordable costs, considerable sensitivity, and only requires simple experimental methods.

ABSTRACT Metagenomic next-generation sequencing (mNGS) offers a hypothesis-free approach for pathogen detection, but its applicability in clinical diagnosis, in addition to other factors, remains limited due to complicated library construction. The present study describes a PCR-free isothermal workflow for mNGS targeting RNA, based on a multiple displacement amplification, termed circular whole-transcriptome amplification (cWTA), as the template is circularized before amplification. The cWTA approach was validated with clinical samples and nanopore sequencing. Reads homologous to dengue virus 2 and chikungunya virus were detected in clinical samples from Bangladesh and Brazil, respectively. In addition, the practicality of a high-throughput detection system that combines mNGS and a group testing algorithm termed mNGS screening enhanced by a group testing algorithm (mEGA) was established. This approach enabled significant library size reduction while permitting trackability between samples and diagnostic results. Serum samples of patients with undifferentiated febrile illnesses from Vietnam (n = 43) were also amplified with cWTA, divided into 11 pools, processed for library construction, and sequenced. Dengue virus 2, hepatitis B virus, and parvovirus B19 were successfully detected without prior knowledge of their existence. Collectively, cWTA with the nanopore platform opens the possibility of hypothesis-free on-site comprehensive pathogen diagnosis, while mEGA contributes to the scaling up of sample throughput. IMPORTANCE Given the breadth of pathogens that cause infections, a single approach that can detect a wide range of pathogens is ideal but is impractical due to the available tests being highly specific to a certain pathogen. Recent developments in sequencing technology have introduced mNGS as an alternative that provides detection of a wide-range of pathogens by detecting the presence of their nucleic acids in the sample. However, sequencing library preparation is still a bottleneck, as it is complicated, costly, and time-consuming. In our studies, alternative approaches to optimize library construction for mNGS were developed. This included isothermal nucleic acid amplification and expansion of sample throughput with a group testing algorithm. These methods can improve the utilization of mNGS as a diagnostic tool and can serve as a high-throughput screening system aiding infectious disease surveillance.

Babesia are tick-borne protozoan parasites that can infect livestock, pets, wildlife animals, and humans. In the mammalian host, they invade and multiply within red blood cells (RBCs). To support their development as obligate intracellular parasites, Babesia export numerous proteins to modify the RBC during invasion and development. Such exported proteins are likely important for parasite survival and pathogenicity and thus represent candidate drug or vaccine targets. The availability of complete genome sequences and the establishment of transfection systems for several Babesia species have aided the identification and functional characterization of exported proteins. Here, we review exported Babesia proteins; discuss their functions in the context of immune evasion, cytoadhesion, and nutrient uptake; and highlight possible future topics for research and application in this field.

Leishmaniasis is caused by infection with protozoan parasites of the genus Leishmania. In both clinical and experimental visceral leishmaniasis, macrophage multinucleation is observed in parasitized tissues. However, the feature and the mechanism of macrophage multinucleation remained unclear. Here, we report that infection of Leishmania donovani, a causative agent of visceral leishmaniasis, induces multinucleation of bone marrow-derived macrophages (BMDMs) in vitro. When these infection-induced multinucleated macrophages were compared with cytokine-induced multinucleated giant cells, the former had higher phagocytic activity on red blood cells but no apparent changes on phagocytosis of latex beads. BMDMs infected with L. donovani had increased expression of ATP6V0D2, one of the components of V-ATPase, which was also upregulated in the spleen of infected mice. Infection-induced ATP6V0D2 localized in a cytoplasmic compartment, which did not overlap with the mitochondria, endoplasmic reticulum, or lysosomes. When ATP6V0D2 expression was recombinantly induced in BMDMs, the formation of multinucleated macrophages was induced as seen in the infected macrophages. Taken together, L. donovani infection induces multinucleation of macrophages via ATP6V0D2 upregulation leading to a unique metamorphosis of the macrophages toward hemophagocytes.

Leishmaniases are neglected tropical diseases of humans and animals. We detected Leishmania infantum in 3 mixed-breed dogs in Zambia that had no travel history outside the country. Our findings suggest presence of and probable emergence of leishmaniasis in Zambia, indicating the need for physicians and veterinarians to consider the disease during diagnosis.

Bats are of significant interest as reservoirs for various zoonotic viruses with high diversity. During the past two decades, many herpesviruses have been identified in various bats worldwide by genetic approaches, whereas there have been few reports on the isolation of infectious herpesviruses. Herein, we report the prevalence of herpesvirus infection of bats captured in Zambia and genetic characterization of novel gammaherpesviruses isolated from striped leaf-nosed bats (Macronycteris vittatus). By our PCR screening, herpesvirus DNA polymerase (DPOL) genes were detected in 29.2% (7/24) of Egyptian fruit bats (Rousettus aegyptiacus), 78.1% (82/105) of Macronycteris vittatus, and one Sundevall’s roundleaf bat (Hipposideros caffer) in Zambia. Phylogenetic analyses of the detected partial DPOL genes revealed that the Zambian bat herpesviruses were divided into seven betaherpesvirus groups and five gammaherpesvirus groups. Two infectious strains of a novel gammaherpesvirus, tentatively named Macronycteris gammaherpesvirus 1 (MaGHV1), were successfully isolated from Macronycteris vittatus bats, and their complete genomes were sequenced. The genome of MaGHV1 encoded 79 open reading frames, and phylogenic analyses of the DNA polymerase and glycoprotein B demonstrated that MaGHV1 formed an independent lineage sharing a common origin with other bat-derived gammaherpesviruses. Our findings provide new information regarding the genetic diversity of herpesviruses maintained in African bats.

Abstract DNA methylation plays important functions in gene expression regulation that is involved in individual development and various diseases. DNA methylation has been well studied in human and model organisms, but only limited data exist in companion animals like dog. Using methylation-sensitive restriction enzyme-based next generation sequencing (Canine DREAM), we obtained canine DNA methylation maps of 16 somatic tissues from two dogs. In total, we evaluated 130,861 CpG sites. The majority of CpG sites were either highly methylated (> 70%, 52.5–64.6% of all CpG sites analyzed) or unmethylated (

Toxoplasma gondii bradyzoites establish chronic infections within their host cells. Recent studies have demonstrated that several parasite effector proteins are translocated to host cells during the bradyzoite stage of chronic infection. To understand the interaction between host cells and bradyzoites at the transcriptomic landscape level, we utilized single-cell RNA-sequencing (scRNA-Seq) to characterize the bradyzoite-induced host cell response. Distinct gene expression profiles were observed in infected host, cells with low parasite mapped reads, and mock (non-exposed) control cells. Gene set enrichment analysis showed that c-Myc and NF-κB signaling and energy metabolic pathways were upregulated by infection. Type I and II interferon response pathways were upregulated in cells with low parasite mapped reads compared to the non-exposed host control cells, and this upregulation effect was reversed in infected cells. Differences were observed in the host cells depending on the differentiation status of the parasites, as determined by BAG1 and SAG1 expression. NF-κB, inflammatory response pathways, and IFN-γ response pathways were downregulated in host cells containing T. gondiiBAG1+/SAG1−, whereas this downregulation effect was reversed in case of T. gondiiBAG1−/SAG1+. We also identified two distinct host cell subsets that contained T. gondiiBAG1+/SAG1−, one of which displayed distinct transcriptomes with upregulated c-Myc expression. Overall, these data clearly demonstrate that host cell transcriptional alteration by bradyzoite infection is different from that of tachyzoite infection, indicating fine-tuning of the host immune response.

Rickettsia asembonensis is a flea-related Rickettsia with unknown pathogenicity to humans. We detected R. asembonensis DNA in 2 of 1,153 human blood samples in Zambia. Our findings suggest the possibility of R. asembonensis infection in humans despite its unknown pathogenicity.

Abstract Background Piroplasms are vector-borne intracellular hemoprotozoan parasites that infect wildlife and livestock. Wildlife species are reservoir hosts to a diversity of piroplasms and play an important role in the circulation, maintenance and evolution of these parasites. The potential for likely spillover of both pathogenic and non-pathogenic piroplasm parasites from wildlife to livestock is underlined when a common ecological niche is shared in the presence of a competent vector. Method To investigate piroplasm diversity in wildlife and the cattle population of the greater Kafue ecosystem, we utilized PCR to amplify the 18S rRNA V4 hyper-variable region and meta-barcoding strategy using the Illumina MiSeq sequencing platform and amplicon sequence variant (ASV)-based bioinformatics pipeline to generate high-resolution data that discriminate sequences down to a single nucleotide difference. Results A parasite community of 45 ASVs corresponding to 23 species consisting of 4 genera of Babesia, Theileria, Hepatozoon and Colpodella, were identified in wildlife and the cattle population from the study area. Theileria species were detected in buffalo, impala, hartebeest, sable antelope, sitatunga, wild dog and cattle. In contrast, Babesia species were only observed in cattle and wild dog. Our results demonstrate possible spillover of these hemoprotozoan parasites from wildlife, especially buffalo, to the cattle population in the wildlife-livestock interface. Conclusion We demonstrated that the deep amplicon sequencing of the 18S rRNA V4 hyper-variable region for wildlife was informative. Our results illustrated the diversity of piroplasma and the specificity of their hosts. They led us to speculate a possible ecological cycle including transmission from wildlife to domestic animals in the greater Kafue ecosystem. Thus, this approach may contribute to the establishment of appropriate disease control strategies in wildlife-livestock interface areas.

The Kafue ecosystem is a vast conservation protected area comprising the Kafue National Park (KNP) and the Game Management Areas (GMA) that act as a buffer around the national park. The KNP has been neglected as a potential foci for rhodesiense sleeping sickness despite the widespread presence of the tsetse vector and abundant wildlife reservoirs. The aim of this study was to generate information on circulating trypanosomes and their eminent threat/risk to public health and livestock production of a steadily growing human and livestock population surrounding the park. We detected various trypanosomes circulating in different mammalian wildlife species in KNP in Zambia by applying a high throughput ITS1-polymerase chain reaction (PCR)/nanopore sequencing method in combination with serum resistant associated-PCR/Sanger sequencing method. The prevalence rates of trypanosomes in hartebeest, sable antelope, buffalo, warthog, impala and lechwe were 6.4%, 37.2%, 13.2%, 11.8%, 2.8% and 11.1%, respectively. A total of six trypanosomes species or subspecies were detected in the wildlife examined, including Trypanosoma brucei brucei, T. godfreyi, T. congolense, T. simiae and T. theileri. Importantly we detected human infective T. b. rhodesiense in buffalo and sable antelope with a prevalence of 9.4% and 12.5%, respectively. In addition, T. b. rhodesiense was found in the only vervet monkey analyzed. The study thus reaffirmed that the Kafue ecosystem is a genuine neglected and re-emerging foci for human African trypanosomiasis. This is the first assessment of the trypanosome diversity circulating in free-ranging wildlife of the KNP.

With an increasing number of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) sequences gathered worldwide, we recognize that deletion mutants and nucleotide substitutions that may affect whole-genome sequencing are accumulating. Here, we propose an additional strategy for tiling PCR for whole-genome resequencing, which can make the pipeline robust for mutations at the primer annealing site by a redundant amplicon scheme. We further demonstrated that subtracting overrepresented amplicons from the multiplex PCR products reduced the bias of the next-generation sequencing (NGS) library, resulting in decreasing required sequencing reads per sample. We applied this sequencing strategy to clinical specimens collected in Bangladesh. More than 80% out of the 304 samples were successfully sequenced. Less than 5% were ambiguous nucleotides, and several known variants were detected. With the additional strategies presented here, we believe that whole-genome resequencing of SARS-CoV-2 from clinical samples can be optimized.

Schistosoma mekongi, a blood fluke that causes Asian zoonotic schistosomiasis, is distributed in communities along the Mekong River in Cambodia and Lao People’s Democratic Republic. Decades of employing numerous control measures including mass drug administration using praziquantel have resulted in a decline in the prevalence of schistosomiasis mekongi. This, however, led to a decrease in sensitivity of Kato–Katz stool microscopy considered as the gold standard in diagnosis. In order to develop a serological assay with high sensitivity and specificity which can replace Kato–Katz, recombinant S. mekongi thioredoxin peroxidase-1 protein (rSmekTPx-1) was expressed and produced. Diagnostic performance of the rSmekTPx-1 antigen through ELISA for detecting human schistosomiasis was compared with that of recombinant protein of S. japonicum TPx-1 (rSjTPx-1) using serum samples collected from endemic foci in Cambodia. The sensitivity and specificity of rSmekTPx-1 in ELISA were 89.3% and 93.3%, respectively, while those of rSjTPx-1 were 71.4% and 66.7%, respectively. In addition, a higher Kappa value of 0.82 calculated between rSmekTPx-1 antigen ELISA and Kato–Katz confirmed better agreement than between rSjTPx-1 antigen ELISA and Kato–Katz (Kappa value 0.38). These results suggest that ELISA with rSmekTPx-1 antigen can be a potential diagnostic method for detecting active human S. mekongi infection.

Abstract Background Infection with Toxoplasma gondii is thought to damage the brain and be a risk factor for neurological and psychotic disorders. The immune response-participating chemokine system has recently been considered vital for brain cell signaling and neural functioning. Here, we investigated the effect of the deficiency of C-C chemokine receptor 5 (CCR5), which is previously reported to be associated with T. gondii infection, on gene expression in the brain during T. gondii infection and the relationship between CCR5 and the inflammatory response against T. gondii infection in the brain. Results We performed a genome-wide comprehensive analysis of brain cells from wild-type and CCR5-deficient mice. Mouse primary brain cells infected with T. gondii were subjected to RNA sequencing. The expression levels of some genes, especially in astrocytes and microglia, were altered by CCR5-deficiency during T. gondii infection, and the gene ontology and Kyoto Encyclopedia of Genes and Genomes analysis revealed an enhanced immune response in the brain cells. The expression levels of genes which were highly differentially expressed in vitro were also investigated in the mouse brains during the T. gondii infections. Among the genes tested, only Saa3 (serum amyloid A3) showed partly CCR5-dependent upregulation during the acute infection phase. However, analysis of the subacute phase showed that in addition to Saa3, Hmox1 may also contribute to the protection and/or pathology partly via the CCR5 pathway. Conclusions Our results indicate that CCR5 is involved in T. gondii infection in the brain where it contributes to inflammatory responses and parasite elimination. We suggest that the inflammatory response by glial cells through CCR5 might be associated with neurological injury during T. gondii infection to some extent.

Abstract Background Flea-borne spotted fever is a zoonosis caused by Rickettsia felis, a Gram-negative obligate intracellular bacterium. The disease has a worldwide distribution including western and eastern sub-Saharan Africa where it is associated with febrile illness in humans. However, epidemiology and the public health risks it poses remain neglected especially in developing countries including Zambia. While Ctenocephalides felis (cat fleas) has been suggested to be the main vector, other arthropods including mosquitoes have been implicated in transmission and maintenance of the pathogen; however, their role in the epidemiological cycle remains to be elucidated. Thus, the aim of this study was to detect and characterize R. felis from animal hosts and blood-sucking arthropod vectors in Zambia. Methods Dog blood and rodent tissue samples as well as cat fleas and mosquitoes were collected from various areas in Zambia. DNA was extracted and screened by polymerase chain reaction (PCR) targeting genus Rickettsia and amplicons subjected to sequence analysis. Positive samples were further subjected to R. felis-specific real-time quantitative polymerase chain reactions. Results Rickettsia felis was detected in 4.7% (7/150) of dog blood samples and in 11.3% (12/106) of rodent tissue samples tested by PCR; this species was also detected in 3.7% (2/53) of cat fleas infesting dogs, co-infected with Rickettsia asembonensis. Furthermore, 37.7% (20/53) of cat flea samples tested positive for R. asembonensis, a member of spotted fever group rickettsiae of unknown pathogenicity. All the mosquitoes tested (n = 190 pools) were negative for Rickettsia spp. Conclusions These observations suggest that R. felis is circulating among domestic dogs and cat fleas as well as rodents in Zambia, posing a potential public health risk to humans. This is because R. felis, a known human pathogen is present in hosts and vectors sharing habitat with humans.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) have significantly impacted the global epidemiology of the pandemic. From December 2020 to April 2022, we conducted genomic surveillance of SARS-CoV-2 in the Southern Province of Zambia, a region that shares international borders with Botswana, Namibia, and Zimbabwe and is a major tourist destination. Genetic analysis of 40 SARS-CoV-2 whole genomes revealed the circulation of Alpha (B.1.1.7), Beta (B.1.351), Delta (AY.116), and multiple Omicron subvariants with the BA.1 subvariant being predominant. Whereas Beta, Delta, and Omicron variants were associated with the second, third, and fourth pandemic waves, respectively, the Alpha variant was not associated with any wave in the country. Phylogenetic analysis showed evidence of local transmission and possible multiple introductions of SARS-CoV-2 VOCs in Zambia from different European and African countries. Across the 40 genomes analysed, a total of 292 mutations were observed, including 182 missense mutations, 66 synonymous mutations, 23 deletions, 9 insertions, 1 stop codon, and 11 mutations in the non-coding region. This study stresses the need for the continued monitoring of SARS-CoV-2 circulation in Zambia, particularly in strategically positioned regions such as the Southern Province which could be at increased risk of introduction of novel VOCs.

We clarified the genetic diversity of Trypanosoma spp. within the Kafue ecosystem, using PCR targeting the internal transcribed spacer 1 and the cathepsin L-like cysteine protease (CatL) sequences. The overall prevalence of Trypanosoma spp. in cattle and tsetse flies was 12.65 and 26.85%, respectively. Cattle positive for Trypanosoma vivax had a significantly lower packed cell volume, suggesting that T. vivax is the dominant Trypanosoma spp. causing anemia in this area. Among the 12 operational taxonomic units (OTUs) of T. vivax CatL sequences detected, one was from a known T. vivax lineage, two OTUs were from known T. vivax-like lineages, and nine OTUs were considered novel T. vivax-like lineages. These findings support previous reports that indicated the extensive diversity of T. vivax-like lineages. The findings also indicate that combining CatL PCR with next generation sequencing is useful in assessing Trypanosoma spp. diversity, especially for T. vivax and T. vivax-like lineages. In addition, the 5.42% prevalence of Trypanosoma brucei rhodesiense found in cattle raises concern in the community and requires careful monitoring of human African trypanosomiasis.

Babesia bovis causes a pathogenic form of babesiosis in cattle. Following invasion of red blood cells (RBCs) the parasite extensively modifies host cell structural and mechanical properties via the export of numerous proteins. Despite their crucial role in virulence and pathogenesis, such proteins have not been comprehensively characterized in B. bovis. Here we describe the surface biotinylation of infected RBCs (iRBCs), followed by proteomic analysis. We describe a multigene family (mtm) that encodes predicted multi-transmembrane integral membrane proteins which are exported and expressed on the surface of iRBCs. One mtm gene was downregulated in blasticidin-S (BS) resistant parasites, suggesting an association with BS uptake. Induced knockdown of a novel exported protein encoded by BBOV_III004280, named VESA export-associated protein (BbVEAP), resulted in a decreased growth rate, reduced RBC surface ridge numbers, mis-localized VESA1, and abrogated cytoadhesion to endothelial cells, suggesting that BbVEAP is a novel virulence factor for B. bovis.

Human African trypanosomiasis (HAT) is one of the neglected tropical diseases in sub-Saharan Africa. Early diagnosis and treatment prior to disease progression are crucial for the survival of HAT patients. We had previously established a loop-mediated isothermal amplification (LAMP) method for HAT diagnosis in which the reagents were dried for field-use purposes. In this study, we used a semi-automated process to produce the test tubes using a bio-inkjet printer to achieve an accurate production. The performance of the inkjet printer-produced dried LAMP test (CZC-LAMP) was found to be stable after storage for up to 180 days at 30 °C. The diagnostic accuracy of CZC-LAMP HAT was evaluated using DNA samples that were extracted from 116 Trypanosoma brucei gambiense patients and 66 T. b. rhodesiense patients. The sensitivity was 72% for T. b. gambiense (95%CI: 63%-80%) and 80% for T. b. rhodesiense (95%CI: 69%-89%). The specificity determined using DNA from 116 endemic control DNA samples was 95% (95%CI: 89%-98%). The performance of the CZC-LAMP HAT and CZC-LAMP rHAT were also evaluated using 14 crude blood lysate samples obtained from T. b. rhodesiense patients and endemic control samples collected from Rumphi District in Malawi. The sensitivity and specificity were both 100% (95%CI: 77%-100%). As the developed CZC-LAMP test does not require a cold chain or a sophisticated laboratory, it holds promise for use as a routine simple molecular tool for point-of-care HAT diagnosis in endemic areas.

Legionella pneumophila is an environmental bacterium that has evolved to survive predation by soil and water amoebae such as Acanthamoeba castellanii, and this has inadvertently led to the ability of L. pneumophila to survive and replicate in human cells. L. pneumophila causes Legionnaire's Disease, with human exposure occurring via the inhalation of water aerosols containing both amoebae and the bacteria. These aerosols originate from aquatic biofilms found in artifical water sources, such as air-conditioning cooling towers and humidifiers. In these man-made environments, A. castellanii supports L. pneumophila intracellular replication, thereby promoting persistence and dissemination of the bacteria and providing protection from external stress. Despite this close evolutionary relationship, very little is known about how A. castellanii responds to L. pneumophila infection. In this study, we examined the global transcriptional response of A. castellanii to L. pneumophila infection. We compared A. castellanii infected with wild type L. pneumophila to A. castellanii infected with an isogenic ΔdotA mutant strain, which is unable to replicate intracellularly. We showed that A. castellanii underwent clear morphological and transcriptional rewiring over the course of L. pneumophila infection. Through improved annotation of the A. castellanii genome, we determined that these transcriptional changes primarily involved biological processes utilizing small GTPases, including cellular transport, signaling, metabolism and replication. In addition, a number of sirtuin-encoding genes in A. castellanii were found to be conserved and upregulated during L. pneumophila infection. Silencing of sirtuin gene, sir6f (ACA1_153540) resulted in the inhibition of A. castellanii cell proliferation during infection and reduced L. pneumophila replication. Overall our findings identified several biological pathways in amoebae that may support L. pneumophila replication and A. castellanii proliferation in environmental conditions.

Abstract Background The recent spread of artemisinin (ART)-resistant Plasmodium falciparum represents an emerging global threat to public health. In Southeast Asia, the C580Y mutation of kelch13 (k13) is the dominant mutation of ART-resistant P. falciparum. Therefore, a simple method for the detection of C580Y mutation is urgently needed to enable widespread routine surveillance in the field. The aim of this study is to develop a new diagnostic procedure for the C580Y mutation using loop-mediated isothermal amplification (LAMP) combined with the MinION nanopore sequencer. Results A LAMP assay for the k13 gene of P. falciparum to detect the C580Y mutation was successfully developed. The detection limit of this procedure was 10 copies of the reference plasmid harboring the k13 gene within 60 min. Thereafter, amplicon sequencing of the LAMP products using the MinION nanopore sequencer was performed to clarify the nucleotide sequences of the gene. The C580Y mutation was identified based on the sequence data collected from MinION reads 30 min after the start of sequencing. Further, clinical evaluation of the LAMP assay in 34 human blood samples collected from patients with P. falciparum malaria in Indonesia revealed a positive detection rate of 100%. All LAMP amplicons of up to 12 specimens were simultaneously sequenced using MinION. The results of sequencing were consistent with those of the conventional PCR and Sanger sequencing protocol. All procedures from DNA extraction to variant calling were completed within 3 h. The C580Y mutation was not found among these 34 P. falciparum isolates in Indonesia. Conclusions An innovative method combining LAMP and MinION will enable simple, rapid, and high-sensitivity detection of the C580Y mutation of P. falciparum, even in resource-limited situations in developing countries.

Abstract Background Genetic manipulation techniques, such as transfection, have been previously reported in many protozoan parasites. In Babesia, stable transfection systems have only been established for bovine Babesia parasites. We recently reported a transient transfection system and the selection of promoter candidates for Babesia gibsoni. The establishment of a stable transfection system for B. gibsoni is considered to be urgent to improve our understanding of the basic biology of canine Babesia parasites for a better control of babesiosis. Results GFP-expressing parasites were observed by fluorescence microscopy as early as two weeks after drug selection, and consistently expressed GFP for more than 3 months without drug pressure. Genome integration was confirmed by PCR, sequencing and Southern blot analysis. Conclusions We present the first successful establishment of a stable transfection system for B. gibsoni. This finding will facilitate functional analysis of Babesia genomes using genetic manipulation and will serve as a foundation for the development of tick-Babesia and host-Babesia infection models.

In 2017, an unprecedented increase in febrile illness was observed in Dhaka, Bangladesh. Real-time reverse transcription PCR confirmed that 603 (40.2%) of 1,500 cases were chikungunya fever. Phylogenetic analysis revealed circulation of the non-A226V East/Central/South African genotype of chikungunya virus in Bangladesh.

Highly pathogenic avian influenza viruses (HPAIVs) with H5 and H7 hemagglutinin (HA) subtypes are derived from their low pathogenic counterparts following the acquisition of multiple basic amino acids in their HA cleavage site. It has been suggested that consecutive adenine residues and a stem-loop structure in the viral RNA region that encodes the cleavage site are essential for the acquisition of the polybasic cleavage site. By using a reporter assay to detect non-templated nucleotide insertions, we found that insertions more frequently occurred in the RNA region (29 nucleotide-length) encoding the cleavage site of an H5 HA gene that was predicted to have a stem-loop structure containing consecutive adenines than in a mutated corresponding RNA region that had a disrupted loop structure with fewer adenines. In virus particles generated by using reverse genetics, nucleotide insertions that created additional codons for basic amino acids were found in the RNA region encoding the cleavage site of an H5 HA gene but not in the mutated RNA region. We confirmed the presence of virus clones with the ability to replicate without trypsin in a plaque assay and to cause lethal infection in chicks. These results demonstrate that the stem-loop structure containing consecutive adenines in HA genes is a key molecular determinant for the emergence of H5 HPAIVs.

Members of the genus Borrelia are arthropod-borne spirochetes that are human and animal pathogens. Vertebrate hosts, including wild animals, are pivotal to the circulation and maintenance of Borrelia spirochetes. However, information on Borrelia spirochetes in vertebrate hosts in Zambia is limited. Thus, we aimed to investigate the presence of Borrelia spirochetes in wild animals and cattle in Zambia. A total of 140 wild animals of four species and 488 cattle DNA samples from /near the Kafue National Park were collected for real-time PCR screening, followed by characterization using three different genes with positive samples. Five impalas and 20 cattle tested positive using real-time PCR, and sequence analysis revealed that the detected Borrelia were identified to be Borrelia theileri, a causative agent of bovine borreliosis. This is the first evidence of Borrelia theileri in African wildlife and cattle in Zambia. Our results suggest that clinical differentiation between bovine borreliosis and other bovine diseases endemic in Zambia is required for better treatment and control measures. As this study only included wild and domestic animals in the Kafue ecosystem, further investigations in other areas and with more wildlife and livestock species are needed to clarify a comprehensive epidemiological status of Borrelia theileri in Zambia.

Visceral leishmaniasis (VL) is the most severe form of leishmaniasis caused by protozoan parasites of the genus Leishmania. Hepatomegaly is one of the most frequent clinical manifestations of VL, whereas immunopathology of the symptom has not been well investigated. Using our chronic model of experimental VL, we examined the influence of Leishmania donovani infection on the liver by clinical, histological, and biochemical analyses. The infected mice showed increased liver weight 24 weeks post-infection. Although an increase in serum ALT and inflammatory cell accumulation were observed in the livers of infected mice, no apparent parenchymal necrosis or fibrosis was observed. Tissue water content analyses demonstrated that increased liver weight was predominantly due to an increase in water weight. Together with the finding of hepatic sinusoidal dilation, these results suggested that edema associated with sinusoidal dilation causes hepatomegaly in L. donovani infection. Immunostaining of platelets and erythrocytes showed no thrombus formation or damage to the sinusoidal endothelium in the liver of infected mice. Taken together, these results suggest that hepatomegaly during experimental VL is caused by non-obstructive sinusoidal dilation.

Rickettsial pathogens are amongst the emerging and re-emerging vector-borne zoonoses of public health importance. Though traditionally considered to be transmitted by ixodid ticks, the role of argasid ticks as vectors of these pathogens is increasingly being recognized. While bat-feeding (Ornithodoros faini) and chicken-feeding (Argas walkerae) argasid ticks have been shown to harbor Rickettsia pathogens in Zambia, there are currently no reports of Rickettsia infection in southern Africa from warthog-feeding (Phacochoerus africanus) soft ticks, particularly Ornithodoros moubata and Ornithodoros porcinus. Our study sought to expand on the existing knowledge on the role of soft ticks in the epidemiology of Rickettsia species through screening for Rickettsia pathogens in warthog burrow-dwelling soft ticks from two national parks in Zambia. The tick species from which Rickettsia were detected in this study were identified as Ornithodoros porcinus, and an overall minimal Rickettsia infection rate of 19.8% (32/162) was observed. All of the sequenced Rickettsia were identified as Rickettsia lusitaniae based on nucleotide sequence similarity and phylogenetic analysis of the citrate synthase (gltA) and 17kDa common antigen (htrA) genes. Utilizing all of the gltA (n = 10) and htrA (n = 12) nucleotide sequences obtained in this study, BLAST analysis showed 100% nucleotide similarity to Rickettsia lusitaniae. Phylogenetic analysis revealed that all of the Zambian gltA and htrA gene sequences could be grouped with those of Rickettsia lusitaniae obtained in various parts of the world. Our data suggest that Rickettsia lusitaniae has a wider geographic and vector range, enhancing to our understanding of Rickettsia lusitaniae epidemiology in sub-Saharan Africa.

Abstract Background Babesia ovata, belonging to the phylum Apicomplexa, is an infectious parasite of bovids. It is not associated with the manifestation of severe symptoms, in contrast to other types of bovine babesiosis caused by B. bovis and B. bigemina; however, upon co-infection with Theileria orientalis, it occasionally induces exacerbated symptoms. Asymptomatic chronic infection in bovines is usually observed only for B. ovata. Comparative genomic analysis could potentially reveal factors involved in these distinguishing characteristics; however, the genomic and molecular basis of these phenotypes remains elusive, especially in B. ovata. From a technical perspective, the current development of a very long read sequencer, MinION, will facilitate the obtainment of highly integrated genome sequences. Therefore, we applied next-generation sequencing to acquire a high-quality genome of the parasite, which provides fundamental information for understanding apicomplexans. Results The genome was assembled into 14,453,397 bp in size with 5031 protein-coding sequences (91 contigs and N50 = 2,090,503 bp). Gene family analysis revealed that ves1 alpha and beta, which belong to multigene families in B. bovis, were absent from B. ovata, the same as in B. bigemina. Instead, ves1a and ves1b, which were originally specified in B. bigemina, were present. The B. ovata and B. bigemina ves1a configure one cluster together even though they divided into two sub-clusters according to the spp. In contrast, the ves1b cluster was more dispersed and the overlap among B. ovata and B. bigemina was limited. The observed redundancy and rapid evolution in sequence might reflect the adaptive history of these parasites. Moreover, same candidate genes which potentially involved in the distinct phenotypes were specified by functional analysis. An anamorsin homolog is one of them. The human anamorsin is involved in hematopoiesis and the homolog was present in B. ovata but absent in B. bigemina which causes severe anemia. Conclusions Taking these findings together, the differences demonstrated by comparative genomics potentially explain the evolutionary history of these parasites and the differences in their phenotypes. Besides, the draft genome provides fundamental information for further characterization and understanding of these parasites.

Abstract Background A simple and accurate molecular diagnostic method for malaria is urgently needed due to the limitations of conventional microscopic examination. In this study, we demonstrate a new diagnostic procedure for human malaria using loop mediated isothermal amplification (LAMP) and the MinION™ nanopore sequencer. Methods We generated specific LAMP primers targeting the 18S–rRNA gene of all five human Plasmodium species including two P. ovale subspecies (P. falciparum, P. vivax, P. ovale wallikeri, P. ovale curtisi, P. knowlesi and P. malariae) and examined human blood samples collected from 63 malaria patients in Indonesia. Additionally, we performed amplicon sequencing of our LAMP products using MinION™ nanopore sequencer to identify each Plasmodium species. Results Our LAMP method allowed amplification of all targeted 18S–rRNA genes of the reference plasmids with detection limits of 10–100 copies per reaction. Among the 63 clinical samples, 54 and 55 samples were positive by nested PCR and our LAMP method, respectively. Identification of the Plasmodium species by LAMP amplicon sequencing analysis using the MinION™ was consistent with the reference plasmid sequences and the results of nested PCR. Conclusions Our diagnostic method combined with LAMP and MinION™ could become a simple and accurate tool for the identification of human Plasmodium species, even in resource-limited situations.

Abstract The recent development of a nanopore-type portable DNA sequencer has changed the way we think about DNA sequencing. We can perform sequencing directly in the field, where we collect the samples. Here, we report the development of a novel method to detect and genotype tropical disease pathogens, using dengue fever as a model. By combining the sequencer with isothermal amplification that only requires a water bath, we were able to amplify and sequence target viral genomes with ease. Starting from a serum sample, the entire procedure could be finished in a single day. The analysis of blood samples collected from 141 Indonesian patients demonstrated that this method enables the clinical identification and serotyping of the dengue virus with high sensitivity and specificity. The overall successful detection rate was 79%, and a total of 58 SNVs were detected. Similar analyses were conducted on 80 Vietnamese and 12 Thai samples with similar performance. Based on the obtained sequence information, we demonstrated that this approach is able to produce indispensable information for etiologically analyzing annual or regional diversifications of the pathogens.

Cancer patients having anti-programmed cell death-1 (PD-1)/PD ligand 1 (L1)-unresponsive tumors may benefit from advanced immunotherapy. Double-stranded RNA triggers dendritic cell (DC) maturation to cross-prime antigen-specific cytotoxic T lymphocytes (CTLs) via Toll-like receptor 3 (TLR3). The TLR3-specific RNA agonist, ARNAX, can induce anti-tumor CTLs without systemic cytokine/interferon (IFN) production. Here, we have developed a safe vaccine adjuvant for cancer that effectively implements anti-PD-L1 therapy. Co-administration of ARNAX with a tumor-associated antigen facilitated tumor regression in mouse models, and in combination with anti-PD-L1 antibody, activated tumor-specific CTLs in lymphoid tissues, enhanced CTL infiltration, and overcame anti-PD-1 resistance without cytokinemia. The TLR3-TICAM-1-interferon regulatory factor (IRF)3-IFN-β axis in DCs exclusively participated in CD8+ T cell cross-priming. ARNAX therapy established Th1 immunity in the tumor microenvironment, upregulating genes involved in DC/T cell/natural killer (NK) cell recruitment and functionality. Human ex vivo studies disclosed that ARNAX+antigen induced antigen-specific CTL priming and proliferation in peripheral blood mononuclear cells (PBMCs), supporting the feasibility of ARNAX for potentiating anti-PD-1/PD-L1 therapy in human vaccine immunotherapy.

Abstract Background Malaria still poses one of the major threats to human health. Development of effective antimalarial drugs has decreased this threat; however, the emergence of drug-resistant Plasmodium falciparum, a cause of Malaria, is disconcerting. The antimalarial drug chloroquine has been effectively used, but resistant parasites have spread worldwide. Interestingly, the withdrawal of the drug reportedly leads to an increased population of susceptible parasites in some cases. We examined the prevalence of genomic polymorphisms in a malaria parasite P. falciparum, associated with resistance to an antimalarial drug chloroquine, after the withdrawal of the drug from Indonesia. Results Blood samples were collected from 95 malaria patients in North Sulawesi, Indonesia, in 2010. Parasite DNA was extracted and analyzed by polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) for pfcrt and pfmdr1. In parallel, multiplex amplicon sequencing for the same genes was carried out with Illumina MiSeq. Of the 59 cases diagnosed as P. falciparum infection by microscopy, PCR–RFLP analysis clearly identified the genotype 76T in pfcrt in 44 cases. Sequencing analysis validated the identified genotypes in the 44 cases and demonstrated that the haplotype in the surrounding genomic region was exclusively SVMNT. Results of pfmdr1 were successfully obtained for 51 samples, where the genotyping results obtained by the two methods were completely consistent. In pfmdr1, the 86Y mutant genotype was observed in 45 cases (88.2%). Conclusions Our results suggest that the prevalence of the mutated genotypes remained dominant even 6 years after the withdrawal of chloroquine from this region. Diversified haplotype of the resistance-related locus, potentially involved in fitness costs, unauthorized usage of chloroquine, and/or a short post-withdrawal period may account for the observed high persistence of prevalence.

Visceral leishmaniasis (VL) is caused by parasitic protozoa of the genus Leishmania and is characterized by clinical manifestations such as fever, hepatosplenomegaly and anemia. Hemophagocytosis, the phenomenon of phagocytosis of blood cells by macrophages, is found in VL patients. In a previous study we established an experimental model of VL, reproducing anemia in mice for the first time, and identified hemophagocytosis by heavily infected macrophages in the spleen as a possible cause of anemia. However, the mechanism for parasite-induced hemophagocytosis or its role in parasite survival remained unclear. Here, we established an in vitro model of Leishmania-induced hemophagocytosis to explore the molecules involved in this process. In contrast to naïve RAW264.7 cells (mouse macrophage cell line) which did not uptake freshly isolated erythrocytes, RAW264.7 cells infected with L. donovani showed enhanced phagocytosis of erythrocytes. Additionally, for hemophagocytes found both in vitro and in vivo, the expression of signal regulatory protein α (SIRPα), one of the receptors responsible for the 'don't-eat-me' signal was suppressed by post-transcriptional control. Furthermore, the overlapped phagocytosis of erythrocytes and Leishmania parasites within a given macrophage appeared to be beneficial to the parasites; the in vitro experiments showed a higher number of parasites within macrophages that had been induced to engulf erythrocytes. Together, these results suggest that Leishmania parasites may actively induce hemophagocytosis by manipulating the expression of SIRPα in macrophages/hemophagocytes, in order to secure their parasitism.