SSU rRNA Gene Research Articles

Long rDNA amplicon sequencing of insect-infecting nephridiophagids reveals their affiliation to the Chytridiomycota and a potential to switch between hosts

Nephridiophagids are unicellular eukaryotes that parasitize the Malpighian tubules of numerous insects. Their life cycle comprises multinucleate vegetative plasmodia that divide into oligonucleate and uninucleate cells, and sporogonial plasmodia that form uninucleate spores. Nephridiophagids are poor in morphological characteristics, and although they have been tentatively identified as early-branching fungi based on the SSU rRNA gene sequences of three species, their exact position within the fungal tree of live remained unclear. In this study, we describe two new species of nephridiophagids (Nephridiophaga postici and Nephridiophaga javanicae) from cockroaches. Using long-read sequencing of the nearly complete rDNA operon of numerous further species obtained from cockroaches and earwigs to improve the resolution of the phylogenetic analysis, we found a robust affiliation of nephridiophagids with the Chytridiomycota—a group of zoosporic fungi that comprises parasites of diverse host taxa, such as microphytes, plants, and amphibians. The presence of the same nephridiophagid species in two only distantly related cockroaches indicates that their host specificity is not as strict as generally assumed.

Evolutionary relationships of Metchnikovella dogieli Paskerova et al., 2016 (Microsporidia: Metchnikovellidae) revealed by multigene phylogenetic analysis.

The species Metchnikovella dogieli (Paskerova et al. Protistology 10:148-157, 2016) belongs to one of the early diverging microsporidian groups, the metchnikovellids (Microsporidia: Metchnikovellidae). In relation to typical ('core') microsporidia, this group is considered primitive. The spores of metchnikovellids have no classical polar sac-anchoring disk complex, no coiled polar tube, no posterior vacuole, and no polaroplast. Instead, they possess a short thick manubrium that expands into a manubrial cistern. These organisms are hyperparasites; they infect gregarines that parasitise marine invertebrates. M. dogieli is a parasite of the archigregarine Selenidium pygospionis (Paskerova et al. Protist 169:826-852, 2018), which parasitises the polychaete Pygospio elegans. This species was discovered in samples collected in the silt littoral zone at the coast of the White Sea, North-West Russia, and was described based on light microscopy. No molecular data are available for this species, and the publicly accessible genomic data for metchnikovellids are limited to two species: M. incurvata Caullery & Mesnil, 1914 and Amphiamblys sp. WSBS2006. In the present study, we applied single-cell genomics methods with whole-genome amplification to perform next-generation sequencing of M. dogieli genomic DNA. We performed a phylogenetic analysis based on the SSU rRNA gene and reconstructed a multigene phylogeny using a concatenated alignment that included 46 conserved single-copy protein domains. The analyses recovered a fully supported clade of metchnikovellids as a basal group to the core microsporidia. Two members of the genus Metchnikovella did not form a clade in our tree. This may indicate that this genus is paraphyletic and requires revision.

First identification and molecular subtyping of Blastocystis sp. in zoo animals in southwestern China

BackgroundBlastocystis sp. is an anaerobic protozoan that parasitizes many animal hosts and the human gastrointestinal tract, and its pathogenicity is controversial. Captive wildlife may be potential reservoirs for human infection with Blastocystis sp. The present study was performed to investigate the prevalence and subtype distribution of Blastocystis sp. in zoo animals in Sichuan Province, southwestern China.MethodsA total of 420 fresh fecal samples were collected from 54 captive wildlife species in four zoos in southwestern China between June 2017 and September 2019. The prevalence and subtype (ST) genetic characteristics of Blastocystis sp. were determined by PCR amplification of the barcode region of the SSU rRNA gene and phylogenetic analysis.ResultsOverall, 15.7% (66/420) of the animal samples and 20.7% (14/54) of the species tested were shown to be infected with Blastocystis sp. The highest prevalence of Blastocystis sp. was found in Panzhihua Zoo (24.3%), which was significantly higher than that in Chengdu Zoo (6.9%), and Xichang Zoo (2.9%) (P < 0.05). There are also significant differences in the prevalence of Blastocystis sp. among different species (P < 0.05), and the highest of Blastocystis sp. prevalence was observed in white-cheeked gibbon, black great squirrel, and red giant flying squirrel (100%). Subtype analysis of Blastocystis sp. revealed nine subtypes, including six zoonotic STs (ST1-5, and ST8) and three animal-specific STs (ST10, ST14, and ST17), with ST17 as the predominant subtype (26/66) in Blastocystis sp.-positive isolates.ConclusionsTo our knowledge, this is the first report on the prevalence and subtype distribution of Blastocystis sp. among captive wildlife in zoos in southwestern China. This study highlights that these animals may serve as reservoirs for human Blastocystis sp. infections.Graphical

Biosurfactants synthesized by endophytic Bacillus strains as control of Moniliophthora perniciosa and Moniliophthora roreri

ABSTRACT: Ecuador is responsible for up to 70 % of the world’s production of fine flavor cocoa. Although fungal diseases have reduced cocoa production, eco–friendly alternatives have been implemented to control the spread of phytopathogens. This study aimed to explore the potential of endophytic strains isolated from fine flavor Nacional cocoa trees to synthetize compounds with antimicrobial and surface active properties. It was determined that under in vitro conditions, seven strains (DS03, DS07, DS18, DS23, DS31, DS34 and DS50) exhibited antifungal activity that inhibited the mycelial growth of Moniliophthora roreri and Moniliophthora perniciosa. The strains were identified based on their morphological (pattern forming Bacilli) and genetic characteristics using the 16S SSU rRNA gene sequences. Furthermore, we determined that the endophytic strains contained genes that encoded the lipopeptides Fengycin (fenA, fenB, fenC, fenE), Iturin (ituB, ituC, ituD), and Bacyllomicin D (bamC, bamD). The production of biosurfactant compounds was determined by a reduction in surface tension in the mineral medium. Additionally, the biosurfactant crude extract (BCE) was tested for its inhibitory activity against M. perniciosa and M. roreri. The minimal inhibitory concentration (MIC) was determined to be 0.07 and 0.035 mg mL−1, respectively, in both cases in a fungistatic action mode. The swelling granulation and fragmentation of both phytopathogens’ hyphae were observed using the BCE treatment. These results suggested the biocontrol capacity of the endophytic strains, which under in vitro conditions, have the ability to synthesize antifungal and surface active compounds that might be used as an ecological alternative in cocoa plants to prevent infection by phytopathogens.

Morphology and phylogeny of a new soil ciliate, Colpodidium zelihayildizi n. sp. (Ciliophora, Nassophorea, Colpodidiidae), from Van, Turkey

new ciliate species Colpodidium zelihayildizae n. sp., which was discovered in a dry irrigation channel in Van (Turkey), was investigated morphologically using standard live and silver impregnation methods, and its SSU rRNA gene phylogeny was provided. This new species was characterized as follows: body size about 40-60 × 15-20 ?m in vivo, elongated ellipsoidal with flat or slightly concave ventral surface; the buccal cavity slightly below equatorial plane; macronucleus spherical or slightly elliptical, usually located in the last third of the cell; cortex distinctly furrowed in the anterior half; 19-22 somatic kineties, 4 postoral kineties; paroral membrane has 20 dikinetids; 3 nassulid organelles (NOs), with NO1 very close to the left edge of the paroral membrane, which consists of 1 dikinetid pair. Phylogenetic analyses conducted on the SSU rDNA sequences determined that Colpodidium zelihayildizae was clustered with Colpodidium caudatum and formed a clade with Apocolpodidium etoschense and Colpodididae sp.

Genetic characterization and zoonotic potential of Blastocystis from wild animals in Sichuan Wolong National Natural Reserve, Southwest China.

Blastocystis is a prevalent eukaryotic parasite that has been identified in a wide range of hosts. Several species are considered potential sources of Blastocystis infection in humans, but little is known about the prevalence of Blastocystis in wild animals. In this study, the prevalence and subtypes of Blastocystis were investigated to assess the zoonotic potential of wild animals in Sichuan Wolong National Natural Reserve. A total of 300 fecal samples were collected from 27 wildlife species in three areas of the Reserve. The subtype (ST), genetic characteristics, and prevalence of Blastocystis were determined by PCR amplification of part (~600 bp) of the SSU rRNA gene. Thirty fecal samples (10.0%) were Blastocystis-positive. The highest prevalence of Blastocystis was found in Yinchanggou (18.3%), with significantly less found in Niutoushan (7.5%) and Genda (5.5%) (p < 0.05). No significant differences were associated with different orders of animals in prevalence, which may be because of the small number of positive samples obtained. Sequence analysis showed five subtypes (ST1, ST3, ST5, ST13, and ST14), with ST13 and ST14 being predominant (33% each), followed by ST1 (20%). This is the first molecular investigation of Blastocystis infection in the wild animals of southwestern China. Subtypes ST1, ST3, ST5, and ST14 have previously been identified in humans, suggesting that wild animals may be potential reservoirs of Blastocystis for humans.

An Illumina MiSeq-Based Amplicon Sequencing Method for the Detection of Mixed Parasite Infections Using the Blastocystis SSU rRNA Gene as an Example.

Parasite mixed infections remain a relatively unexplored field in part due to the difficulties of unraveling complex mixtures of parasite DNA using classical methods of sequencing. Next-generation amplicon sequencing (NGS) is a powerful tool for exploring mixed infections of multiple genetic variants of the same parasite in clinical, environmental (water or soil), or food samples. Here, we provide a method for NGS-based detection of mixed parasite infections which uses the Blastocystis SSU rRNA gene as an example and includes steps for parasite concentration, DNA extraction, sequencing library preparation, and bioinformatic analysis.

Blastocystis infection and subtype distribution in domestic animals in the Qinghai-Tibetan Plateau Area (QTPA) in China: A preliminary study.

Blastocystis is a common intestinal protozoan parasite of humans and a variety of animal species. To date, the prevalence of Blastocystis and major subtypes distribution in the domestic animals inhabiting in the Qinghai-Tibetan Plateau Area (QTPA) of China is yet poorly studied. In this study, we investigated the distribution and genetic diversity of Blastocystis in seven animal species in QTPA in China. Four hundred and five fresh fecal samples were collected from domestic animals in Qinghai, Yunnan, and Tibet of China and analyzed using nested PCR and SSU rRNA gene sequencing. It was found that the overall prevalence of Blastocystis infection was 40.2% (163/405) in the animals studied. The most predominant subtype of Blastocystis was ST10 (57.7%) followed by ST14 (28.8%) and ST2 (13.5%). These results reveal the epidemiological features of Blastocytis infection in animals in the high altitude plateau area. The finding of presence of ST2 in a number of animal species suggests a zoonotic nature of Blastocystis and might be of importance of public health.

Morphology, ultrastructure and molecular phylogeny of Johsia chumphonensis gen. et sp. nov. and Parvodinium parvulum comb. nov. (Peridiniopsidaceae, Dinophyceae)

ABSTRACT The family Peridiniopsidaceae encompasses mainly freshwater species of the genera Peridiniopsis, Palatinus and Parvodinium. Only one benthic, marine species ‘Scrippsiella’ hexapraecingula has been attributed to this family. Here we established five strains by isolating single Parvodinium-like cells from the marine Gulf of Thailand, Hainan Island waters (China), off Manado (Indonesia) and from a freshwater reservoir in Fuzhou (China). All strains were examined with light, scanning and transmission electron microscopy, and their SSU, ITS-5.8S and partial LSU rRNA regions were sequenced. Four marine strains share a plate formula of Po, cp, X, 4′, 2a, 6′′, 6C, 4S, 5′′′, 2′′′′ and are herein attributed to a new genus Johsia as J. chumphonensis gen. et sp. nov. Its theca is characterized by an epitheca 1.5 times as long as the hypotheca in dorsal view and a 2a plate about half the size of 1a. A type B eyespot was observed in J. chumphonensis comprising two rows of lipid globules within a chloroplast with a single row of crystals overlying the eyespot. Production of spherical cysts was observed in culture. The freshwater strain shows a plate pattern of Po, cp, X, 4′, 2a, 7′′, 6C, 5S, 5′′′, 2′′′′, characterized by two unequal antapical plates and a lack of antapical spines, fitting the description of Peridinium parvulum, which was transferred to Parvodinium as P. parvulum comb. nov. In this species, a type A eyespot was observed comprising four rows of lipid globules within a chloroplast. A molecular phylogeny was inferred based on concatenated data from SSU, ITS-5.8S and partial LSU rRNA gene sequences using maximum likelihood and Bayesian inference. Our results show that Johsia is nested within the Peridiniopsidaceae and is a sister clade to Peridiniopsis borgei and the strain UTEX1948 identified as ‘Scrippsiella’ hexapraecingula with three anterior intercalary plates.

Mycorrhizal symbiosis modulates the rhizosphere microbiota to promote rhizobia–legume symbiosis

Plants establish symbioses with mutualistic fungi, such as arbuscular mycorrhizal (AM) fungi, and bacteria, such as rhizobia, to exchange key nutrients and thrive. Plants and symbionts have coevolved and represent vital components of terrestrial ecosystems. Plants employ an ancestral AM signaling pathway to establish intracellular symbioses, including the legume–rhizobia symbiosis, in their roots. Nevertheless, the relationship between the AM and rhizobial symbioses in native soil is poorly understood. Here, we examined how these distinct symbioses affect root-associated bacterial communities in Medicago truncatula by performing quantitative microbiota profiling (QMP) of 16S rRNA genes. We found that M. truncatula mutants that cannot establish AM or rhizobia symbiosis have an altered microbial load (quantitative abundance) in the rhizosphere and roots, and in particular that AM symbiosis is required to assemble a normal quantitative root-associated microbiota in native soil. Moreover, quantitative microbial co-abundance network analyses revealed that AM symbiosis affects Rhizobiales hubs among plant microbiota and benefits the plant holobiont. Through QMP of rhizobial rpoB and AM fungal SSU rRNA genes, we revealed a new layer of interaction whereby AM symbiosis promotes rhizobia accumulation in the rhizosphere of M. truncatula. We further showed that AM symbiosis-conditioned microbial communities within the M. truncatula rhizosphere could promote nodulation in different legume plants in native soil. Given that the AM and rhizobial symbioses are critical for crop growth, our findings might inform strategies to improve agricultural management. Moreover, our work sheds light on the co-evolution of these intracellular symbioses during plant adaptation to native soil conditions.

Rapid and sensitive colorimetric detection of microsporidian Enterocytozoon hepatopenaei (EHP) based on spore wall protein (SWP) gene using loop-mediated isothermal amplification combined with DNA functionalized gold nanoparticles as probes

Enterocytozoon hepatopenaei (EHP) is a newly emerging microsporidian parasite that causes growth retardation in cultured shrimp. Currently, EHP infection can be detected by standard polymerase chain reaction (PCR)-based methods, as well as loop-mediated isothermal amplification (LAMP) methods that target EHP's small subunit (SSU) ribosomal RNA (rRNA) gene (LAMP-SSU). However, this gene target has a propensity for generating false positive test results due to cross-reactivity of LAMP primers with SSU-rRNA genes from closely related microsporidia that infect other aquatic organisms. Herein, we describe the development of a simple, specific and sensitive method for detection of EHP by the naked eye. The method is a LAMP technique targeting the spore wall protein (SWP) gene combined with DNA-functionalized gold nanoparticle probes (LAMP-SWP). It has analytical sensitivity comparable to that of nested-PCR (10 copies of plasmid DNA; 1 pg of total DNA) and other reported LAMP methods. Furthermore, no cross-reactivity was observed with other pathogens known to infect shrimp. LAMP-SWP had a sensitivity of 97% (95% CI: 88.2–99.9%) and specificity of 100% (95% CI: 88.4–100%)for EHP detection, while the corresponding sensitivity and specificity of the LAMP-SSU we used for comparison were 100% (95% CI: 91.9–100%) and 56% (95% CI: 37.4–74.5%), respectively. Hence, our novel colorimetric LAMP-SWP combines high specificity for EHP (due to the SWP target), high sensitivity for 10 target copies (similar to conventional nested-PCR), confirmation of PCR amplicons by DNA hybridization and colorimetric visualization of PCR amplicons by the naked-eye (i.e., no electrophoresis or fluorometric equipment needed). It is a rapid method that could allow for real-time management decisions at shrimp hatcheries and farms without the need for sophisticated and expensive laboratory equipment.

Identification and characterization of three microsporidian genera concurrently infecting a silkworm, Bombyx mori, in Brazil

Microsporidia are important entomopathogens known for infecting insects such as the silkworm (Bombyx mori) thus impairing global silk production. This study aimed to identify and characterize the microsporidia isolated from a diseased larva of silkworm, collected from a sericulture farm in southern Brazil. Identification was performed by phylogenetic analysis of the nucleotide sequences of the SSU rRNA genes. Characterization was performed by analyzing spore sizes, tissue tropism, internal and external symptoms, and pathogenicity against B. mori. Microsporidia belonging to three different genera were identified, namely, Endoreticulatus, Nosema and Tubulinosema. After inoculation of the mixed spores of the microsporidian isolates into B. mori larvae, a high prevalence of Tubulinosema spp. was observed. This isolate showed high prevalence on the silk glands and a late mortality, initially of around 10% until the 20th day post-inoculation but reaching 91.5% upon pupation. Therefore, we demonstrated that Tubulinosema spp. causes chronic infection with slow pathogenicity. We identified for the first time three different microsporidians concurrently infecting B. mori in Brazil. Tubulinosema is of particular interest because of its potential threat to silk production; it affects the formation of silk glands in B. mori while not presenting distinguishable external symptoms or causing the immediate death of these insects. Further studies focusing on this species, mainly regarding its life cycle within the host and the sublethal effects of surviving individuals, demonstrate the importance of describing it as a new species and improving the characterization of the disease in order to prevent its spread.

PhyloFlash: Rapid Small-Subunit rRNA Profiling and Targeted Assembly from Metagenomes.

The small-subunit rRNA (SSU rRNA) gene is the key marker in molecular ecology for all domains of life, but it is largely absent from metagenome-assembled genomes that often are the only resource available for environmental microbes. Here, we present phyloFlash, a pipeline to overcome this gap with rapid, SSU rRNA-centered taxonomic classification, targeted assembly, and graph-based binning of full metagenomic assemblies. We show that a cleanup of artifacts is pivotal even with a curated reference database. With such a filtered database, the general-purpose mapper BBmap extracts SSU rRNA reads five times faster than the rRNA-specialized tool SortMeRNA with similar sensitivity and higher selectivity on simulated metagenomes. Reference-based targeted assemblers yielded either highly fragmented assemblies or high levels of chimerism, so we employ the general-purpose genomic assembler SPAdes. Our optimized implementation is independent of reference database composition and has satisfactory levels of chimera formation. phyloFlash quickly processes Illumina (meta)genomic data, is straightforward to use, even as part of high-throughput quality control, and has user-friendly output reports. The software is available at https://github.com/HRGV/phyloFlash (GPL3 license) and is documented with an online manual.IMPORTANCE To track organisms across all domains of life, the SSU rRNA gene is the gold standard. Many environmental microbes are known only from high-throughput sequence data, but the SSU rRNA gene, the key to visualization by molecular probes and link to existing literature, is often missing from metagenome-assembled genomes (MAGs). The easy-to-use phyloFlash software suite tackles this gap with rapid, SSU rRNA-centered taxonomic classification, targeted assembly, and graph-based linking to MAGs. Starting from a cleaned reference database, phyloFlash profiles the taxonomic diversity and assembles the sorted SSU rRNA reads. The phyloFlash design is domain agnostic and covers eukaryotes, archaea, and bacteria alike. phyloFlash also provides utilities to visualize multisample comparisons and to integrate the recovered SSU rRNAs in a metagenomics workflow by linking them to MAGs using assembly graph parsing.

Skin microbiome of beluga whales: spatial, temporal, and health-related dynamics

BackgroundHost-specific microbiomes play an important role in individual health and ecology; in marine mammals, epidermal microbiomes may be a protective barrier between the host and its aqueous environment. Understanding these epidermal-associated microbial communities, and their ecological- or health-driven variability, is the first step toward developing health indices for rapid assessment of individual or population health. In Cook Inlet, Alaska, an endangered population of beluga whales (Delphinapterus leucas) numbers fewer than 300 animals and continues to decline, despite more than a decade of conservation effort. Characterizing the epidermal microbiome of this species could provide insight into the ecology and health of this endangered population and allow the development of minimally invasive health indicators based on tissue samples.ResultsWe sequenced the hypervariable IV region of bacterial and archaeal SSU rRNA genes from epidermal tissue samples collected from endangered Cook Inlet beluga whales (n = 33) and the nearest neighboring population in Bristol Bay (n = 39) between 2012 and 2018. We examined the sequences using amplicon sequence variant (ASV)-based analyses, and no ASVs were associated with all individuals, indicating a greater degree of epidermal microbiome variability among beluga whales than in previously studied cetacean species and suggesting the absence of a species-specific core microbiome. Epidermal microbiome composition differed significantly between populations and across sampling years. Comparing the microbiomes of Bristol Bay individuals of known health status revealed 11 ASVs associated with potential pathogens that differed in abundance between healthy individuals and those with skin lesions or dermatitis. Molting and non-molting individuals also differed significantly in microbial diversity and the abundance of potential pathogen-associated ASVs, indicating the importance of molting in maintaining skin health.ConclusionsWe provide novel insights into the dynamics of Alaskan beluga whale epidermal microbial communities. A core epidermal microbiome was not identified across all animals. We characterize microbial dynamics related to population, sampling year and health state including level of skin molting. The results of this study provide a basis for future work to understand the role of the skin microbiome in beluga whale health and to develop health indices for management of the endangered Cook Inlet beluga whales, and cetaceans more broadly.

Occurrence and genotypes of Cryptosporidium spp., Giardia duodenalis, and Blastocystis sp. in household, shelter, breeding, and pet market dogs in Guangzhou, southern China

Cryptosporidium spp., Giardia duodenalis, and Blastocystis sp. are common intestinal protozoans that infect humans and animals worldwide. A survey that assessed the prevalence, molecular characteristics, and zoonotic potential of these pathogens was conducted on a variety of dogs in Guangzhou, southern China. A total of 651 canine stool samples from household (n = 199), shelter (n = 149), breeding (n = 237), and pet market dogs (n = 66) were collected from eight districts in Guangzhou. Cryptosporidium spp., Giardia duodenalis, and Blastocystis sp. were detected by PCR amplification of the SSU rRNA gene. Giardia duodenalis-positive specimens were further assigned into assemblages using the glutamate dehydrogenase gene. Cryptosporidium spp., G. duodenalis, and Blastocystis sp. were found in 21 (3.2%), 20 (3.1%), and 35 (5.4%) samples, respectively. The overall prevalence of shelter dogs (40.28%, 60/149) was significantly higher than that of household (3.0%, 6/199), breeding (2.1%, 5/237), and pet market dogs (7.5%, 5/66) (χ2 = 154.72, df = 3, P < 0.001). Deworming in the past 12 months had a strong protective effect on the risk of contracting parasite infections (P < 0.001). No significant differences were detected between age or sex groups (P > 0.05). Dog-specific C. canis (n = 19) and zoonotic C. parvum (n = 2) were the only two Cryptosporidium species. Sequence analysis revealed the presence of three G. duodenalis assemblages: dog-specific assemblages D (n = 14) and C (n = 5), and cat-specific F (n = 1). Zoonotic Blastocystis ST3 (n = 28) was the dominant subtype, followed by ST1 (n = 6) and ST10 (n = 1). To our knowledge, this is the first large-scale investigation on the occurrence and molecular characteristics of Blastocystis sp. in dogs in China. Our results indicated that the dogs seemed to play a negligible role as reservoirs for Cryptosporidium spp. and G. duodenalis transmission to humans, but they are potential novel suitable hosts of Blastocystis sp. A strict sentinel surveillance system of dogs should be established to minimise the zoonotic risk of spreading blastocystosis among humans and dogs.

Catabolic Machinery of the Human Gut Microbes Bestow Resilience Against Vanillin Antimicrobial Nature.

Vanillin is a phenolic food additive commonly used for flavor, antimicrobial, and antioxidant properties. Though it is one of the widely used food additives, strategies of the human gut microbes to evade its antimicrobial activity await extensive elucidation. The current study explores the human gut microbiome with a multi-omics approach to elucidate its composition and metabolic machinery to counter vanillin bioactivity. A combination of SSU rRNA gene diversity, metagenomic RNA features diversity, phylogenetic affiliation of metagenome encoded proteins, uniformly (R = 0.99) indicates the abundance of Bacteroidetes followed by Firmicutes and Proteobacteria. Manual curation of metagenomic dataset identified gene clusters specific for the vanillin metabolism (ligV, ligK, and vanK) and intermediary metabolic pathways (pca and cat operon). Metagenomic dataset comparison identified the omnipresence of vanillin catabolic features across diverse populations. The metabolomic analysis brings forth the functionality of the vanillin catabolic pathway through the Protocatechuate branch of the beta-ketoadipate pathway. These results highlight the human gut microbial features and metabolic bioprocess involved in vanillin catabolism to overcome its antimicrobial activity. The current study advances our understanding of the human gut microbiome adaption toward changing dietary habits.

A Trypanosoma species detected in Rhipicephalus (Boophilus) microplus ticks from Argentina

Specimens of a Trypanosoma sp. were found in a haemolymph sample of Rhipicephalus microplus from Argentina. Polymerase chain reaction (PCR) was done targeting the SSU rRNA gene of Trypanosoma spp. and a fragment of 2300 base pairs (bp) was amplified, subsequently a phylogenetic analysis was conducted, based on an alignment of 905 bp, containing the sequence of the Argentina isolate and sequences of different Trypanosoma species retrieved from GenBank. Phylogenetic analysis revealed that this trypanosome is not related to Trypanosoma theileri as was previously thought, instead the strain of Trypanosoma detected in this study can be provisionally determined as belonging to the recently described organism Trypanosoma rhipicephalis. Furthermore, phylogenetic analysis performed in this work revealed that T. rhipicephalis belongs to a novel clade of tick-related trypanosomes, most with limited genetic data, for which essential aspects of both the vertebrate and invertebrate life cycles are lacking. The lack of basic information restricts the inferences that can be done from the present finding and, in addition, points out a clear knowledge gap in the biology of this group of trypanosomes.

New vectors in northern Sarawak, Malaysian Borneo, for the zoonotic malaria parasite, Plasmodium knowlesi

BackgroundPlasmodium knowlesi is a significant cause of human malaria in Sarawak, Malaysian Borneo. Only one study has been previously undertaken in Sarawak to identify vectors of P. knowlesi, where Anopheles latens was incriminated as the vector in Kapit, central Sarawak. A study was therefore undertaken to identify malaria vectors in a different location in Sarawak.MethodsMosquitoes found landing on humans and resting on leaves over a 5-day period at two sites in the Lawas District of northern Sarawak were collected and identified. DNA samples extracted from salivary glands of Anopheles mosquitoes were subjected to nested PCR malaria-detection assays. The small subunit ribosomal RNA (SSU rRNA) gene of Plasmodium was sequenced, and the internal transcribed spacer 2 (ITS2) and mitochondrial cytochrome c oxidase subunit 1 (cox1) gene of the mosquitoes were sequenced from the Plasmodium-positive samples for phylogenetic analysis.ResultsTotals of 65 anophelines and 127 culicines were collected. By PCR, 6 An. balabacensis and 5 An. donaldi were found to have single P. knowlesi infections while 3 other An. balabacensis had either single, double or triple infections with P. inui, P. fieldi, P. cynomolgi and P. knowlesi. Phylogenetic analysis of the Plasmodium SSU rRNA gene confirmed 3 An. donaldi and 3 An. balabacensis with single P. knowlesi infections, while 3 other An. balabacensis had two or more Plasmodium species of P. inui, P. knowlesi, P. cynomolgi and some species of Plasmodium that could not be conclusively identified. Phylogenies inferred from the ITS2 and/or cox1 sequences of An. balabacensis and An. donaldi indicate that they are genetically indistinguishable from An. balabacensis and An. donaldi, respectively, found in Sabah, Malaysian Borneo.ConclusionsPreviously An. latens was identified as the vector for P. knowlesi in Kapit, central Sarawak, Malaysian Borneo, and now An. balabacensis and An. donaldi have been incriminated as vectors for zoonotic malaria in Lawas, northern Sarawak.

Detection of Giardia lamblia by Microscopic Examination, Rapid Chromatographic Immunoassay Test, and Molecular Technique.

BackgroundGiardia lamblia is a pathogenic intestinal flagellate transmitted by the ingestion of contaminated water or food with the cyst stage of the parasite. Giardiasis can cause severe acute diarrhea and malabsorption or may persist as a chronic infection. Effective treatment and control measures depend on proper laboratory diagnosis using diagnostic methods with high sensitivity and specificity.ObjectiveTo compare the sensitivity and specificity of direct smear, Ritchie sedimentation technique, two brands of rapid chromatographic immunoassay test, and real-time polymerase chain reaction (PCR) for the detection of G. lamblia in clinical human fecal samples.Materials and methodsUnpreserved 100 stool specimens were collected in clean plastic containers and labeled with the patient’s information and examined through light microscopy, immunochromatographic test (ICTs), and real-time PCR.ResultsOut of 100 fresh stool samples obtained from workers analyzed, real-time PCR targeting the SSU rRNA gene was able to detect Giardia deoxyribonucleic acid (DNA) in (42) samples followed by ImmunoCard STAT! (31) samples (Meridian Bioscience, Germany), direct smear (23) samples, CerTest (19) samples (Biotec, Zaragoza, Spain), and Ritchie technique (17) samples. Real-time PCR was the most sensitive for the diagnosis of G. lamblia in comparison to the other techniques.ConclusionsAll the techniques investigated were sensitive for the detection of G. lamblia in stool samples. Further studies are recommended using multiplex real-time PCR assay in order to increase the possibility of the presence or absence of the infection.

Molecular Characterization of the Myoliquefactive Fish Parasite Kudoa mirabilis (Cnidaria, Kudoidae) from SE Indian Ocean and Its Phylogenetic Relationship with the Kudoa thyrsites Species Complex.

Myxosporean parasites of the genus Kudoa are fish parasites of great economic importance, as some species can affect the fish fillet quality by producing macroscopic cysts or generating post mortem myoliquefaction, commonly referred to as ‘soft flesh’. Kudoa mirabilis is a ‘soft flesh’-inducing species originally described based on morphology in the musculature of Trichiurus lepturus from the Indian Ocean. An integrative morphological and genetic characterization of K. mirabilis from the type host caught off the coast of Tanzania is here provided. The spores were stellate with four unequal polar capsules, showing similarities to Kudoa thyrsites. For comparative and validation purpose, K. mirabilis was compared morphologically and genetically with K. thyrsites reference isolates, including new obtained samples from the type host Thyrsites atun caught in the SE Atlantic Ocean. Morphological analyses of spores revealed key diagnostic characters clearly distinguishing the two Kudoa species. Phylogenetic analyses based on SSU and LSU rRNA genes demonstrated that K. mirabilis is a distinct and valid species, representing a sister group to a K. thyrsites subclade that comprises several isolates from Japan and one single isolate from South Africa. This finding raises questions about the true diversity likely hidden in the K. thyrsites complex.