Small Subunit Ribosomal RNA Gene Research Articles

First report of leaf blight of water chestnut (Trapa natans L.) caused by Sclerotium hydrophilum in central China.

Trapa natans L., or water chestnut, is a currently globally distributed aquatic plant. The Yangtze River basin in China, a site origin of water chestnut (Fan et al., 2022), has extensive cultivation as a vegetable. In June 2021, a survey at the National Aquatic Vegetable Resource Garden in Wuhan, Hubei, China, revealed browning and wilting of water chestnut plants, with abundant white mycelia and brown to black sclerotia on leaves, indicative of southern blight (Sclerotium rolfsii). Field disease incidence a 1 ha area reached 70%, reducing fruit yield by 50%. About 10% of diseased plants showed blackening and rot from petioles to leaves. White sclerotial primordia and small brown to black sclerotia formed on the plant surfaces. By late August, symptoms exceeded 60%. To identify the pathogen, isolations were made from 243 sclerotial samples using 75% alcohol to disinfect, rinsed three times with water, and then incubated on potato dextrose agar (PDA) at 25℃. A total of 129 isolates were obtained, most of which exhibited characteristics of S. rolfsii. However, 21 isolates had sclerotia significantly smaller than those of S. rolfsii (1 to 1.5 mm in diameter). These isolates, cultured on PDA, produced abundant fluffy white aerial hyphae, 3 to 6 μm wide. Optimal mycelium growth was between 25 °C to 30 °C, with an average daily rate of 10 mm. White to light brown sclerotia appeared after 5 days and turned black within 10 to 14 days, averaging 0.34 mm in diameter (n=50). Some isolates produced a light brown pigment. These traits matched the description of S. hydrophilum (Bashyal et al. 2021). Isolates 221 and 238 were selected for molecular identification, with genomic DNA extracted from mycelia using the CTAB method. PCR amplification was conducted using ITS1/ITS4 and NS1/NS6 primers to target the internal transcribed spacer (ITS) and the small subunit ribosomal RNA gene (ssrRNA). Sequence analysis showed that the ITS sequence of isolate 221 (GenBank Acc. No. OR512512) had 99.84% sequence identity with S. hydrophilum Msh6 (GenBank Acc. No. FJ595946), and isolate 238 (GenBank Acc. No. PP035993) had 99.72% identity with S. hydrophilum Whcc-4 (GenBank Acc. No. PP035994). The ssrRNA sequences of both isolates (GenBank Acc. No. PP237261) had 99.69% identity with S. hydrophilum strain Hbq001 (GenBank Acc. No. KY995575), confirming their identification as S. hydrophilum. To assess pathogenicity, 16 water chestnut plants (cultivar Jia-yu Ling) at the rosette stage were placed individually in 32 cm diameter, 10 cm deep containers with fresh water. Eight plants were inoculated with 50 mature sclerotia from PDA cultures of isolates 221 and 238, and incubated at 25 °C for 14 days, with four plants per isolate. The remaining eight plants served as controls. Containers were covered to maintain 100% relative humidity at 25 °C to 32 °C for 3 days. This procedure was repeated twice. After 7 days, inoculated plants developed dark brown lesions on petioles that spread to leaves by 15 days post-inoculation. Fungi isolated from diseased leaves resembled isolates 221 and 238, fulfilling Koch's postulates. S. hydrophilum infecting at least 19 genera of plants, including rice (Zhong et al. 2018), wild rice, water lily (Kernkamp et al. 1977) and watershield (Fu et al. 2024). This is the first report of S. hydrophilum infecting water chestnut (T. natans) in central China, identifying it as a co-pathogen with S. rolfsii. Their combined presence may heighten plant mortality and threaten water chestnut cultivation.

Curcumin is an efficacious therapeutic agent against Chilodonella uncinata via interaction with tubulin alpha chain as protein target

Chilodonella, a parasitic ciliate that infects both cold water and warm water fish, can impede the growth of juvenile fish and cause considerable economic losses globally to freshwater aquaculture. In this study, the parasite was collected from both the gills and zygotes of largemouth bass (Micropterus salmoides). Isolated from diseased fish, the parasites were identified as Chilodonella uncinata based on morphological features and genetical diagnostic characterization using the partial small subunit ribosomal RNA gene. To develop an effective approach to treat chilodonellosis caused by C. uncinata in largemouth bass farming, we first developed an in vivo culture model for propagating C. uncinate and thus could use for morphological characterization, molecular analyses and antiparasitic drug screening. Curcumin was successfully identified as an efficacious anti-C. uncinata agent from 26 phytochemical compounds. When administered at a concentration of 6 mg/L, curcumin not only completely cured infected largemouth bass but also shielded uninfected fish from C. uncinata infections. The 24 h median effective concentration (EC50) of curcumin against C. uncinata was 3.098 mg/L. Remarkably, the 96 h median lethal concentration (LC50) of curcumin against largemouth bass was determined to be 17.143 mg/L, approximately 5.533 times higher than EC50. The mechanism of action of curcumin was investigated by the cellular thermal shift assay, demonstrating that tubulin alpha chain was the binding target for curcumin. Moreover, SEM investigations further provided morphological evidence suggesting that curcumin induces parasite demise by disrupting the parasite's body surface and subsequently infiltrating its interior. These findings collectively emphasize the potential of curcumin as a safe and effective therapeutic agent for controlling C. uncinata in aquaculture.

High accuracy meets high throughput for near full-length 16S ribosomal RNA amplicon sequencing on the Nanopore platform.

Small subunit (SSU) ribosomal RNA (rRNA) gene amplicon sequencing is a foundational method in microbial ecology. Currently, short-read platforms are commonly employed for high-throughput applications of SSU rRNA amplicon sequencing, but at the cost of poor taxonomic classification due to limited fragment lengths. The Oxford Nanopore Technologies (ONT) platform can sequence full-length SSU rRNA genes, but its lower raw-read accuracy has so-far limited accurate taxonomic classification and de novo feature generation. Here, we present a sequencing workflow, termed ssUMI, that combines unique molecular identifier (UMI)-based error correction with newer (R10.4+) ONT chemistry and sample barcoding to enable high throughput near full-length SSU rRNA (e.g. 16S rRNA) amplicon sequencing. The ssUMI workflow generated near full-length 16S rRNA consensus sequences with 99.99% mean accuracy using a minimum subread coverage of 3×, surpassing the accuracy of Illumina short reads. The consensus sequences generated with ssUMI were used to produce error-free de novo sequence features with no false positives with two microbial community standards. In contrast, Nanopore raw reads produced erroneous de novo sequence features, indicating that UMI-based error correction is currently necessary for high-accuracy microbial profiling with R10.4+ ONT sequencing chemistries. We showcase the cost-competitive scalability of the ssUMI workflow by sequencing 87 time-series wastewater samples and 27 human gut samples, obtaining quantitative ecological insights that were missed by short-read amplicon sequencing. ssUMI, therefore, enables accurate and low-cost full-length 16S rRNA amplicon sequencing on Nanopore, improving accessibility to high-resolution microbiome science.

Wastewater-based intestinal protozoa monitoring in Shanghai, China.

Intestinal protozoa Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi have been implicated in serious waterborne outbreaks worldwide. Wastewater-based epidemiology (WBE) is a promising approach for evaluating the disease prevalence in a catchment population in that it monitors the contamination level of the intestinal pathogens in wastewater. We collected 48 urban wastewater samples (24 from influents and 24 from effluents) from the Yangpu Wastewater Treatment Plant (YPWTP) in Shanghai, China. We identified Cryptosporidium spp., G. duodenalis, and E. bieneusi by nested polymerase chain reaction (PCR) amplification. Cryptosporidium hominis and subtype IdA14 were identified in two samples by analyzing the sequences of small subunit ribosomal RNA (SSU rRNA) and 60-kDa glycoprotein (gp60) genes, respectively. The G. duodenalis sub-assemblage AII (n = 8) and assemblage C (n = 4) in 12 samples were determined by analyzing triosephosphate isomerase (tpi) gene sequences. The E. bieneusi genotype A was identified in one sample by analyzing the sequence of the internal transcribed spacer (ITS) region of the rRNA gene. These findings suggest that improving wastewater treatment and monitoring the virility of pathogens in effluents is critical. We observed similar prevalence and genotypes/subtypes of the three intestinal protozoa in our wastewater samples as those reported in previous studies, providing evidence that WBE can be used as an effective epidemic management tool.IMPORTANCECryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi are common intestinal protozoa causing diarrhea. The infective oocysts, cysts, and spores released in feces can survive in different environments, including multiple types of water bodies. Humans can acquire these intestinal protozoan infections via the fecal-oral route as in waterborne transmission. Wastewater-based epidemiology can rapidly and reliably detect and monitor the emergence and spread of waterborne diseases. We detected Cryptosporidium spp., G. duodenalis, and E. bieneusi in a wastewater treatment plant in Shanghai, China, reflecting the occurrence and genetic characterizations of the three intestinal pathogens from community members served by the wastewater treatment plant.

Occurrence rate and species and subtypes of Cryptosporidium spp. in pet dogs in Yunnan Province, China

BackgroundCryptosporidium spp. is a ubiquitous, globally distributed intestinal protozoan infecting humans and at least 260 animal hosts. Due to close human contact with pet dogs and identification of zoonotic Cryptosporidium species and subtypes in these animals, dog health is not only a veterinarian issue but also a public health issue. This study aimed to understand occurrence and genetic characterization at both genotype and subtype levels in pet dogs in Yunnan Province, China.ResultsA total of 589 fresh fecal specimens were collected from adult pet dogs in the rural areas of eight cities/autonomous prefectures of Yunnan Province, China. 16 fecal specimens were positive for Cryptosporidium spp. by polymerase chain reaction (PCR) amplification and sequence analysis of the small subunit ribosomal RNA (SSU rRNA) gene, with an average occurrence rate of 2.7% (16/589) being observed. Three zoonotic Cryptosporidium species were identified: C. parvum (n = 7), C. suis (n = 5) and C. canis (n = 4). At the 60-kDa glycoprotein (gp60) locus, only three C. parvum and two C. canis specimens were successfully amplified and sequenced, with subtype IIaA17G2R1 (n = 3) and subtypes XXa4 (n = 1) and XXa5 (n = 1) being identified, respectively.ConclusionsThe present finding of three zoonotic Cryptosporidium species in dogs implied that dogs infected with Cryptosporidium spp. may pose a threat to human health. C. suis was identified in dogs in this study for the first time, expanding the host range of this species. Identification of C. parvum subtype IIaA17G2R1 and C. canis subtypes XXa4 and XXa5 will be helpful to explore the source attribution of infection/contamination and assess the transmission dynamics of C. parvum and C. canis in the investigated areas in the future.

First Molecular Detection and Genetic Characterization of Tetratrichomonas buttreyi and Pentatrichomonas hominis in Donkeys in Shanxi Province, China.

Two species of trichomonads, Tetratrichomonas buttreyi and Pentatrichomonas hominis, are common intestinal parasites that can impact animal health and productivity. Severe infection by these parasites can lead to diarrhea and wasting in affected animals. Notably, P. hominis is known to cause diarrhea and has the potential to be transmitted between animals and humans. Donkeys hold significant economic importance in China's agricultural sector. However, whether donkeys are infected with T. buttreyi and P. hominis remains unknown globally. To address this gap in knowledge, 815 fecal samples were collected from donkeys in three representative regions in Shanxi Province, North China. Then, the presence and genetic characteristics of T. buttreyi and P. hominis were examined using species-specific PCR primers amplifying the small subunit ribosomal RNA genes. The overall prevalence was detected to be 25.4% (207/815) for T. buttreyi and 0.7% (6/815) for P. hominis in donkeys in Shanxi Province. All obtained P. hominis sequences were identified as genotype CC1. Genetic analysis revealed that all P. hominis isolates from donkeys were clustered into the same branch with isolates detected in humans, suggesting possible zoonotic transmission. This study is the first to report the occurrence and prevalence of T. buttreyi and P. hominis in donkeys globally. These findings expand the host range of trichomonads and improve our understanding of their genetic diversity and zoonotic potential, providing essential baseline data for the prevention and control of these parasites in donkeys in the region.

Coral Reef Water Microbial Communities of Jardines de la Reina, Cuba.

Globally, coral reef ecosystems are undergoing significant change related to climate change and anthropogenic activities. Yet, the Cuban archipelago of Jardines de la Reina (JR) has experienced fewer stressors due to its geographical remoteness and high level of conservation. This study examines the surface and benthic reef water microbial communities associated with 32 reef sites along the JR archipelago and explores the relationship between the community composition of reef microorganisms examined using bacterial and archaeal small subunit ribosomal RNA gene (16S rRNA gene) sequencing compared to geographic, conservation/protection level, environmental, physicochemical, and reef benthic and pelagic community features. Reef nutrient concentrations were low and microbial communities dominated by picocyanobacteria and SAR11 and SAR86 clade bacteria, characteristic of an oligotrophic system. Reef water microbial community alpha and beta diversity both varied throughout the archipelago and were strongly related to geography. Three sites in the western archipelago showed unique microbial communities, which may be related to the hydrogeography and influences of the channels linking the Ana Maria gulf with the Caribbean Sea. Overall, this work provides the first extensive description of the reef microbial ecology of the Caribbean's 'Crown Jewel' reef system and a framework to evaluate the influence of ongoing stressors on the reef microorganisms.

Characterization of Skoliomonas gen. nov., a haloalkaliphilic anaerobe related to barthelonids (Metamonada).

Metamonads are a large and exclusively anaerobic group of protists. Additionally, they are one of the three clades proposed to ancestrally possess an "excavate" cell morphology, with a conspicuous ventral groove accompanied by a posterior flagellum with a vane. Here, we cultivate and characterize four anaerobic bacterivorous flagellates from hypersaline and alkaline soda lake environments, which represent a novel clade. Small subunit ribosomal RNA (SSU rRNA) gene phylogenies support recent phylogenomic analyses in placing them as the sister of barthelonids, a group that is itself sister to or deeply branching within Fornicata (Metamonada). The new isolates have a distinctive morphology: the hunchbacked cell body is traversed by a narrow ventral groove ending in a large opening to a conspicuous recurrent cytopharynx. The right margin of the groove is defined by a thin "lip." The posterior flagellum bears a wide ventral-facing vane. The narrow ventral groove and elongate cytopharynx are shared with barthelonids. We describe one isolate as Skoliomonas litria, gen. et sp. nov. Further investigation of their mitochondrial-related organelles (MROs) and detailed ultrastructural studies would be important to understanding the adaptation to anaerobic conditions in Metamonads-especially fornicates-as well as the evolution of the "excavate" cell architecture.

Veterinary perspectives on the urbanization of leishmaniosis in Morocco

BackgroundLeishmaniosis caused by Leishmania infantum, L. major and L. tropica is endemic in Morocco. Growing evidence of both human and canine Leishmania infections in urban centres has been reported. Since many forms of the disease are zoonotic, veterinarians play an important role in leishmaniosis control by intervening at the parasite host level. This study aimed to bring together One Health principles to connect canine and feline leishmaniosis epidemiology within urban centres of Morocco (Rabat and Fez) and assess the level of awareness of Moroccan veterinarians about facing this threat.MethodsA molecular survey was conducted for Leishmania DNA detection in canine (n = 155) and feline (n = 32) whole-blood samples. Three conventional polymerase chain reaction (PCR) protocols were implemented. The first PCR aimed at identifying infected animals by targeting Leishmania spp. kinetoplast minicircle DNA (kDNA). The second and third PCR targeted the Leishmania internal transcribed spacer region (ITS-1) and the Leishmania small subunit ribosomal RNA (SSUrRNA) gene, respectively, aiming at identification of the infecting species after Sanger sequencing-positive amplicons. Total immunoglobulin G (IgG) against Leishmania spp. was evaluated in 125 dogs by enzyme-linked immunosorbent assays (ELISA) using an in-house protocol, including three Leishmania-specific antigens (SPLA, rKDDR and LicTXNPx). Sera from 25 cats were screened for total IgG to Leishmania spp. by an indirect immunofluorescence antibody test (IFAT). An online questionnaire was presented to Moroccan veterinarians addressing their knowledge and practices towards animal leishmaniosis.ResultsOverall, 19.4% of the dogs tested positive for Leishmania kDNA and ITS-1 and sequencing revealed infection with L. infantum among PCR-positive dogs. These animals presented a wide range of ELISA seropositivity results (16.7%, 34.9% and 51.6%) according to the tested antigens (rKDDR, SPLA and LicTXNPx, respectively). Use of kDNA-PCR revealed 12.5% cats positive to Leishmania spp. otherwise found to be seronegative by IFAT.ConclusionsA considerable prevalence of infection was identified in dogs from urban centres of Morocco. Additionally, this is the first report of feline infection with Leishmania spp. in this country and in urban settings. Moroccan veterinarians are aware that animal leishmaniosis is endemic in Morocco, representing a public health threat, and are knowledgeable about canine leishmaniosis diagnosis and treatment.Graphical

Molecular detection of Babesia and Hepatozoon species and morphological characteristics of Babesia species in Japanese wild boars

We investigated intraerythrocytic Babesia parasites in 21 Japanese wild boars, Sus scrofa leucomystax, captured in Wakayama Prefecture on the mainland from 2008 to 2009 and in 31 Japanese wild boars from 2011 to 2013 in Kochi Prefecture on Shikoku Island, Japan. We detected small subunit ribosomal RNA (18S rRNA) gene (SSUrDNA) fragments of a Babesia species in 17 boars from Wakayama and 18 boars from Kochi. The nearly full SSUrDNA sequence (1669 bps) of this species was determined. A FASTA search revealed that the SSUrDNA sequence of the Babesia sp. in Japanese wild boars was the most homologous to those of several Babesia isolates reported as Babesia gibsoni. Phylogenetic analysis showed that the Babesia sp. found in Japanese wild boars was the closest relative to B. gibsoni but made a different clade from B. gibsoni. The Babesia sp. in Japanese wild boars was completely different from Babesia sp. Suis found in a European domestic pig, Sus scrofa domesticus. By microscopic examination, ring-shaped, oval and pear-shaped small sized intraerythrocytic parasites were observed on blood smears of 12 of 18 Japanese wild boars whose blood smears could be examined in Wakayama. We also detected SSUrDNA fragments of a Hepatozoon species in 6 of the 21 wild boars from Wakayama. The nearly full SSUrDNA sequence (1774 bps) of the Hepatozoon sp. was shown to be identical to that of Hepatozoon apri.

Molecular prevalence and subtype characteristics of Blastocystis among school children in Hainan, the tropical island province of China

Blastocystis is one of the most common zoonotic intestinal protozoa with global distribution and can cause gastrointestinal syndrome mainly characterized by diarrhea. School children are the main susceptible population. No epidemiological data on Blastocystis among school children in Hainan, the only tropical island province in China. Between March 2021 and June 2023, 1973 fecal samples were collected from school children across three regions in Hainan province. Blastocystis was examined by amplifying the small subunit ribosomal RNA (SSU rRNA) gene via polymerase chain reaction (PCR), and subtypes were identified through DNA sequencing and phylogenetic analysis. The overall prevalence of Blastocystis was 7.3 % (144/1973). The differences in infection rates across different regions, nationalities, and educational stages are statistically significant (P < 0.001). Five subtypes were identified, of which ST3 was the dominant subtype (60.4 %; 87/144), followed by ST1 (27.8 %; 40/144), ST7 (10.4 %; 15/144), ST6 (0.7 %; 1/144), and ST2 (0.7 %; 1/144). 42 known sequences and 15 novel sequences were identified including eight new variations of the ST1 (ST1–16∼ST1–23) with similarities ranging from 98.3 % to 99.78 % and seven new variations of the ST7 (ST7–7∼ST7–13) with similarities ranging from 97.7 % to 99.79 % by intra-subtype genetic polymorphisms analysis. The results evaluate the public health risks of Blastocystis among school children in Hainan and the sources of infection were discussed, providing important basic data for the effective prevention and control of intestinal parasitic diseases in Hainan.

Trypanosoma infection and bloodmeal analysis in post-feeding sand flies across Thailand

Phlebotomine sand flies are recognized as a primary vector of Leishmania and are also suspected vectors of Trypanosoma. The transmission cycle of these parasites relies on the distribution of sand fly vectors, parasites, and reservoir animals. This study aimed to detect Leishmania and Trypanosoma DNA and identify the sources of bloodmeals in post-feeding sand flies captured across Thailand. A total of 42,911 field female sand flies were collected from 11 provinces across Thailand using CDC light traps. Among these, 253 post-feeding sand flies were selected for analysis. The predominant species in this study was Sergentomyia khawi (33.60 %). The DNA was extracted from individual female sand flies. Polymerase chain reaction (PCR), specific to the internal transcribed spacer 1 (ITS1) and the small subunit ribosomal RNA (SSU rRNA) gene regions were used to detect the presence of Leishmania and Trypanosoma DNA, respectively. Additionally, cytochrome c oxidase subunit I (COI) gene region was utilized to identify the sources of host bloodmeals. Leishmania DNA was not detected in any specimens. The analysis of SSU rRNA sequences revealed the presence of Trypanosoma DNA (11.46 %, 29/253) in sand fly samples. Among these samples, T. noyesi (1.58 %, 4/253) was identified in Idiophlebotomus longiforceps and Phlebotomus asperulus, Trypanosoma Anura01+02/Frog2 (1.18 %, 3/253) in Se. khawi, and Trypanosoma Anura04/Frog1 (8.70 %, 22/253) in Se. khawi, Se. hivernus and Grossomyia indica. Bloodmeal analysis utilizing the COI gene revealed a diverse range of vertebrate hosts’ blood, including bird, bat, frog and sun skink. Our findings confirm the presence of Trypanosoma DNA and identify the sources of bloodmeals from vertebrate hosts in various sand fly species, suggesting their potential as possible vectors for Trypanosoma in Thailand. Furthermore, our study is the first to provide molecular evidence using the COI gene to identify frogs as a host blood source for sand flies in Thailand. Further studies focusing on the isolation of live parasites in sand flies to confirm vector potential and examining the role of animal reservoirs will enhance our understanding of the host-parasite relationship and enable more efficient control for disease transmission.

Morphological and Molecular Characterization of Three Myxosporean Species of the Genera Myxobolus, Henneguya, and Myxidium (Cnidaria: Myxozoa) Infecting Freshwater Fish, Isolated for the First Time in Japan.

The majority of myxosporean species (Cnidaria: Myxozoa) of the genera Myxobolus (35 species), Henneguya (8 species), and Myxidium (9 species) from freshwater or brackish fish in Japan were recorded more than 30 years ago (accumulatively 81.1% [43/53]). The re-discovery and molecular-genetic characterization of these species is a current research priority. During our myxosporean survey in Japanese freshwater fish, we detected three species that had never been recorded in Japan, but in the Russian Far East (Sakhalin Island, and Maritime Province): Myxobolus tribolodonus sp. n., forming cysts in the gills of Tribolodon sachalinensis (syn. M. marinus sensu Aseeva, 2000; M. marinus sensu Sokolov et Frolova, 2015, recorded from the gills of Pseudaspius (syn. Tribolodon) spp.); Henneguya pungitii Achmerov, 1953, forming cysts in the subcutis of external skin and buccal submucosa of Pungitius sinensis; and Myxidium salvelini Konovalov et Shulman, 1966, in the urinary bladder of Oncorhynchus masou ishikawae. These new isolates were characterized by integrated taxonomic approaches, i.e., myxospore morphology and molecular-genetic characterization of the small subunit ribosomal RNA gene (SSU rDNA). These new isolates were phylogenetically differentiated from any species whose SSU rDNA sequences were deposited in the DNA databases, and concurrently compared with recorded species based on classical morphological criteria. All three species were differentiated from myxosporeans previously recorded in Japan, indicating new distribution records out of the Russian Far East. For reliable species identification, accumulation of at least SSU rDNA sequences of known species worldwide is critically important.

New species of Myxobolus in potamodromous catfish from the eastern Amazon, Brazil

The mapará (Hypophthalmus marginatus) is a commercially important fish in the Brazilian Amazon and has been described as a host for numerous myxosporid species. The integrated taxonomy of a new species, Myxobolus mickeyii n. sp., discovered in the urinary bladder of H. marginatus, is undertaken in this study. In 105 specimens of H. marginatus, plasmodia and myxospores were observed in the urinary bladder fluid, the myxospores measuring 20.5 (19.6–21.3) μm in length and 14.0 (13.2–14.9) μm in width. The posterior valves of the spore body were thick, with valvulogenic nuclei, endoplasmic reticulum, and the presence of secretory vesicles. Two elliptical, rounded appendages attached to the valve, containing tubular filaments. The two polar capsules, symmetry, measuring 6.1 (5.9–6.3) μm in length and 4.4 (3.6–6.2) μm in width, with polar tubules of 3 to 5 turns. Phylogenetic analyses of the small subunit ribosomal RNA gene (SSU rDNA) sequencing revealed that M. mickeyii n. sp. is part of a Myxobolidae family clade with freshwater fish of the Siluriformes order, with a genetic distance of 19% to the nearest species. This work contributes to the wide diversity of myxozoans in this host, as other taxa have previously been reported infecting different tissues.

An insight into the cryptic diversity of Fragilariaceae (Bacillariophyta), with the description of a new Antarctic species, Gedaniella antarctica sp. nov.

ABSTRACT Fragilariaceae is a paraphyletic family of araphid diatoms commonly used as bio-indicators, in environmental assessments and in paleoenvironmental reconstructions, and with various potential industrial applications. Recent molecular taxonomic research has highlighted significant limitations in traditional morphology-based investigations of these diatom species. Most descriptions of species and genera of the Fragilariaceae present broad morphological character definitions and many diagnostic characters are indistinguishable by light microscopy. In this sense, taxon misidentification is common and could have serious implications for environmental surveys and laboratory experiments. To better understand the diversity of the Fragilariaceae, we (1) performed phylogenetic analyses of the small subunit ribosomal RNA (18S rRNA), rbcL and psbC gene sequences, (2) inferred a cladogram from key morphological features used in the traditional identification of Fragilariaceae, (3) tested if the topologies of the tree recovered from the molecular phylogeny, of the tree based on morphology and of the trees constraining to monophyly the genera Sarcophagodes, Pseudostaurosira and Nanofrustulum (together and separately) were significantly different and (4) mapped morphological character states on the ML tree and inferred their evolution based on maximum parsimony. Our results supported the monophyly of a group of Fragilariaceae within small araphid diatoms including the genera Cratericulifera, Plagiostriata, Castoridens, Opephora, Staurosira, Staurosirella, Punctastriata, Psammotaenia, Hendeyella, Stauroforma, Pseudostaurosira sensu Li, Nanofrustulum sensu Li, Serratifera sensu Li and Gedaniella sensu Li. Molecular phylogeny and topology tests suggested that the latest circumscriptions of the genera Sarcophagodes, Pseudostaurosira and Nanofrustulum sensu Morales were not monophyletic. Analyses of the Antarctic strain IMA070A collected during the XXXIV Italian Antarctic Expedition using fine structural features of the frustule and molecular data revealed that this diatom belongs to a distinct lineage within Gedaniella, which we describe here as Gedaniella antarctica sp. nov.

Molecular detection and identification of Enteromonas species in human and animal hosts using polymerase chain reaction and DNA sequencing

Enteromonas hominis, a human intestinal protozoan parasite of the diplomonad group, has been overlooked because of its commensal features; therefore, molecular studies on this parasite are limited. To address this gap, we designed a molecular screening protocol using polymerase chain reaction (PCR) and DNA sequencing targeting the 18S small subunit ribosomal RNA gene and applied this screening method to the molecular epidemiological analysis of Enteromonas spp. in humans and various livestock. We validated our methodology using stool samples collected from 215 humans and 270 animal hosts (buffaloes, pigs, dogs, goats, horses, rodents, chickens, and ducks) during an annual epidemiological investigation conducted from 2013 to 2016 on Sumba Island, Indonesia. The overall prevalences of Enteromonas spp. were 33.9 % (n = 73/215) in humans and 25.2 % (n = 68/270) in mammals and avians. The positive predictive value of this PCR method for Enteromonas spp., as evaluated through sequencing, was 90.1 % in human samples and 58.1 % in non-human samples (particularly low, 11.4 % in rodents). Although the specificity of the PCR approach may not be perfect, in combination with DNA sequencing, it was effective in detecting and identifying a partial sequence (1458 bp) of the target gene region in Enteromonas species.

Molecular detection and characterization of Anaplasma marginale and Babesia canis vogeli infecting dogs in Luxor, Egypt

Tick-borne diseases in animals are increasing rapidly worldwide, but there is insufficient information about tick-borne diseases infecting dogs in southern Egypt. Thus, in the current study, we detected the presence of Anaplasma marginale (A. marginale) and Babesia canis vogeli (B. canis vogeli) in the blood of dogs. The results revealed that 4/100 (4%) were positive, and a higher infection rate was found in males (75%), than females (25%). The phylogenetic analysis for the major surface protein 4 (msp4) gene in this study was compared with amplicons separate from other reported isolates with alignment by identity 100% with cattle and camels from Egypt, and the phylogenetic analysis for the B. canis vogeli small subunit ribosomal RNA (SSU rRNA) gene in this study identified identity by 99.89% with dogs from Egypt. This report is considered the first report in southern Egypt about A. marginale in dogs based on the sequence analysis of the msp4 gene, providing new data for the classification and identification of A. marginale in dogs compared to A. marginale isolated from other animals in southern Egypt.

A cross-sectional survey of Blastocystis sp. and Dientamoeba fragilis in non-human primates and their caregivers in Czech zoos

Intestinal protists in the gut microbiome are increasingly studied, but their basic epidemiology is not well understood. We explored the prevalence, genetic diversity, and potential zoonotic transmission of two protists colonizing the large intestine - Blastocystis sp. and Dientamoeba fragilis - in 37 species of non-human primates (NHPs) and their caregivers in six zoos in the Czech Republic. We analyzed 179 fecal samples (159 from NHPs, 20 from humans) by qPCR. Blastocystis sp. was detected in 54.7% (98/179) of samples, in 24 NHP species and in 57.2% of NHP samples (prevalence ranged between 36 and 80%), and in 35% of human samples (prevalence ranged between 0 and 67%). Using next generation amplicon sequencing, nine Blastocystis subtypes (ST1-ST5, ST7, ST8, and two novel subtypes) were identified. The two new Blastocystis subtypes (named ST47 and ST48) were described using Nanopore sequencing to produce full-length reference sequences of the small subunit ribosomal RNA gene. Some subtypes were shared between NHPs and their caregivers, suggesting potential zoonotic transmission. Mixed subtype colonization was frequently observed, with 52% of sequenced samples containing two or more subtypes. Dientamoeba was found only in NHPs with a prevalence of 6%. This study emphasizes the critical role of molecular diagnostics in epidemiological and transmission studies of these protists and calls for further research to better understand their impact on public health.

Molecular Identification and Survey of Cyclospora spp. in Cattle in Shanxi Province, North China.

To date, more than 20 species in the genus Cyclospora have been reported. Among them, Cyclospora cayetanensis has been recognized as the causative agent of human cyclosporiasis, which is characterized by severe intestinal injury and prolonged diarrhea in patients with immune dysfunction. The presence of C. cayetanensis in cattle has been confirmed. To date, however, no surveillance data are available on the occurrence and prevalence of Cyclospora spp. in cattle in Shanxi Province, North China. In the present study, a total of 761 fecal samples collected from cattle in three representative counties (Qi, Jishan, and Shanyin) in this Province were examined for Cyclospora spp. by using a polymerase-chain-reaction-restriction-fragment-length polymorphism (PCR-RFLP) test based on the nuclear small subunit ribosomal RNA (SSU rRNA) gene. The prevalence of Cyclospora spp. in cattle was 2.1%, and region, age, sex, and breed were not identified to be risk factors. Molecular evolutionary analysis based on the SSU rRNA sequences revealed that all 12 of the isolates were relatively distant from the human pathogen C. cayetanensis; seven isolates were grouped with Cyclospora colobi, whereas the others were grouped with cattle Cyclospora spp. reported previously. Though C. cayetanensis was not detected in cattle in the present study, more investigations should be performed in human populations, other animal species, or cattle from other regions of Shanxi Province and other environmental sources from the One Health perspective.

Prevalence and subtypes of Blastocystis in wild rodents from three provinces in China.

Blastocystis is one of the most critical intestinal protozoans in various hosts, including humans and mice. To determine the status of Blastocystis infection in wild rodents in China. A total of 344 faecal samples were collected from seven wild rodent species from three provinces, and the small subunit ribosomal RNA (SSU rRNA) genes of Blastocystis were amplified to determine their prevalence and subtypes. Of the 344 samples, 54 (15.70%) were detected as Blastocystis-positive. The prevalence of Blastocystis was 26.14% (40/153), 7.95% (7/88), and 6.80% (7/103) in wild rodents from Hunan Province, Yunnan Province, and Guangxi Province, respectively. The prevalence of Blastocystis in different wild rodent species varied from 0.00% (0/13) in Mus musculus to 40.00% (2/5) in Rattus rattus sladeni. The prevalence of Blastocystis in samples from the lake beach area (27.40%, 40/146) was significantly higher than in those from the mountain (6.80%, 7/103) and field regions (7.37%, 7/95). The prevalence in different seasons was 26.14% in summer (40/153), 7.95% in autumn (7/88), and 6.80% in winter (7/103). Moreover, a total of two Blastocystis subtypes were identified in the investigated wild rodents, including ST4 and ST5. The present study discovered the existence of Blastocystis infection in Rattus favipectus, Microtus fortis, Apodemus agrarius, Bandicota indica, Rattus rattus sladeni, and Rattus losea, expanding the host range of this parasite. The findings also demonstrate that wild rodents may be an important potential infection source for Blastocystis infection in humans and other animals.