Small Subunit Ribosomal RNA Gene Research Articles

Detailed observations of the life history and phylogenetic placements of the freshwater green alga Oocystaenium elegans (Oocystaceae, Trebouxiophyceae), with an emended description

SUMMARYOocystaenium elegans Gonzalves & Mehra (Oocystaceae, Trebouxiophyceae) was collected from a paddy field in Japan. Its life history was reexamined based on detailed observations of cultured material, and an emended description is given. This species is a large spindle‐shaped green alga that grows in puddles and paddy fields and has been reported in India, Nepal and Cuba. Reproduction occurs asexually via autospore formation and sexually via oogamy. Although this oogamy is considered to involve fertilization by globular egg cells and antherozoids, the results of the present study revealed that the egg cells (female cells) remained spindle‐shaped and became globular zygotes after fertilization. The place on the egg cells where the antherozoid attached swelled to become a receptive papilla, after which the antherozoid invaded. Phylogenetic analysis based on nuclear 18S small subunit ribosomal RNA gene sequences showed that O. elegans is included in the family Oocystaceae.

New Haplotypes of Blastocystis sp. Identified in Faeces from Various Animal Groups in Algeria.

Blastocystis sp. is a single-celled, anaerobic, parasitic protozoan commonly found in the intestinal tract of animals and humans globally. Genetic analysis has revealed significant diversity within its species, leading to the identification of at least 40 subtypes (ST1-ST40). This study aimed to identify and differentiate Blastocystis in faeces samples from various animal hosts in Algeria. A total of 403 fecal samples, collected from both domestic and zoo animals, were subjected to PCR amplification and sequencing of Blastocystis-specific small subunit ribosomal RNA (SSU-RNA) gene. The overall prevalence of Blastocystis in animals was found to be 38.9%. Through comprehensive phylogenetic and phylogeographic analyses, we identified four distinct subtypes (ST1 in both domestic and zoo animals, and ST3, ST4, and ST5 exclusively in zoo animals), encompassing nine different haplotypes, including five that appear original to Algeria. This study represents the first epidemiological molecular investigation of Blastocystis sp. in animals in Algeria.

Genetic diversity and host specificity of Blastocystis in reptiles, Eastern Thailand

Blastocystis inhabits the digestive tracts of a diverse range of hosts. Transmission patterns, including host specificity, and the clinical and public health significance of Blastocystis in humans remain poorly understood. This study aimed to investigate the distribution and genetic diversity of Blastocystis in herbivorous and carnivorous reptiles in Eastern Thailand. A total of 501 faecal samples were collected from 363 iguanas, 79 bearded dragons, 50 tortoises, and nine snakes in an animal breeding farm in Chonburi Province, Eastern Thailand. Detection and differentiation of Blastocystis was based on amplification, sequencing, and phylogenetic analysis of specific small subunit (SSU) ribosomal RNA genes from faecal DNA extracted from the samples. Altogether 101/501 samples (20 %) were polymerase chain reaction (PCR) and sequencing-positive for Blastocystis, 90 (89 %) of which were from iguanas; the remaining positive samples were from African spurred tortoise (n=6), Bearded dragon (n=3), Leopard tortoise (n=1), and Red-footed tortoise (n=1). Phylogenetic analysis revealed that most of the Blastocystis sequences from iguanas were largely similar, and they were distinct from those of the tortoises. Subtype 17 was found in the three bearded dragons and likely reflected Blastocystis from prey animals. This is the largest survey of Blastocystis in reptiles to date. Remarkable differences in Blastocystis colonization rates and genetic diversity were observed between iguanas and other reptile orders, and what was considered Blastocystis colonization was only observed in herbivorous reptiles.

Cryptosporidium occultus in disguise

As data accumulate in GenBank, the difficulties of delineating species of Cryptosporidium based on nuclear small subunit ribosomal RNA (ssu rRNA) gene information alone becomes increasingly evident. Here, we summarize currently available evidence suggesting that several ssu rDNA sequences primarily referred to as Cryptosporidium suis (some of them from non-suid hosts) should be considered Cryptosporidium occultus.

Myxobolus acanthogobii Hoshina, 1952 and Myxobolus selari n. sp. (Myxosporea: Myxobolidae) infecting brain of commercial fishes in Terengganu, Malaysia.

Myxosporean infection in marine water fishes has drawn less attention than in freshwater fishes, which resulted in a higher taxonomic variety in freshwater in Malaysia. This study aimed to address the gap by conducting a myxosporean survey on two commercially significant marine fish species, Nemipterus furcosus (Valenciennes) (Eupercaria incertae sedis: Nemipteridae) and Selar crumenophthalmus (Bloch) (Carangiformes: Carangidae), collected from the northeastern part of peninsular Malaysia. During the examination of the organs, two distinct Myxobolus Bütschli, 1882 species were discovered in the brain tissue of these fishes, despite the absence of any observable pathological signs. The two Myxobolus species were characterized through morphometry, morphology, and analysis of partial small subunit ribosomal RNA (18S rDNA) gene. As a result, Myxobolus acanthogobii Hoshina, 1952, which infects 2.3% of N. furcosus, is synonymous with a myxobolid species commonly found in Japanese waters, based on its morphological traits, tissue tropism, and molecular diagnostics. Furthermore, a novel species, Myxobolus selari n. sp., was described, infecting the brain of one (11%) individual S. crumenophthalmus. This unique species displayed distinctive features, placing it within a well-supported subclade primarily comprising brain-infecting myxobolids. Maximum likelihood analysis further revealed the close relationships among these brain-infecting myxobolids, underscoring the significance of tissue tropism and host taxonomy for myxobolids. This study represents the initial documentation of Myxobolus species within the southern South China Sea, shedding light on the potential diversity of marine myxosporean in this region. This article was registered in the Official Register of Zoological Nomenclature (ZooBank) as urn:lsid:zoobank.org:pub:7C400E35-7CB8-4DEE-92B7-F75FF3926441.

Molecular Identification and Survey of Trichomonad Species in Pigs in Shanxi Province, North China.

Several trichomonad species have already been identified in pigs, and their pathogenic potential may not be ruled out. To date, however, no information is available regarding the prevalence of trichomonads in pigs in Shanxi Province, North China. In the present study, a total of 362 fecal samples collected from pigs in three representative counties (Qi, Jishan, and Shanyin) in this province were examined for Tetratrichomonas buttreyi, Tritrichomonas foetus, and Pentatrichomonas hominis using a nested polymerase chain reaction (PCR) with primers targeting the small subunit ribosomal RNA (SSU rRNA) gene. The overall prevalence of T. buttreyi was 49.72%, and region and age were found to be significantly associated with T. buttreyi infection, respectively. Only one pig fecal sample from Qi County was found to be positive for T. foetus, and all samples were negative for P. hominis. Molecular evolutionary analysis revealed that some T. buttreyi isolates showed complete genetic identity with those reported previously, and some T. buttreyi isolates and one T. foetus isolate showed minor allelic variations compared with those reported previously. This is the report of the molecular epidemiology of T. foetus and T. buttreyi in pigs in Shanxi Province, North China. These findings not only enrich the knowledge on the distribution of these trichomonad species in pigs in China but also provide baseline information for planning future research and control strategies.

Synopsis of the species of OrtholineaShulman, 1962 (Cnidaria: Myxosporea: Ortholineidae)

A synopsis of Ortholinea Shulman, 1962 (Cnidaria: Myxosporea: Ortholineidae) is presented and identifies 26 nominal species presently allocated within this genus. Species morphological and morphometric features, tissue tropism, type-host, and type-locality are provided from original descriptions. Data from subsequent redescriptions and reports is also given. Accession numbers to sequences deposited in GenBank are indicated when available, and the myxospores were redrawn based on original descriptions. The information gathered shows that Ortholinea infect a wide taxonomic variety of freshwater and marine fish. Nonetheless, the broad host specificity reported for several species is not fully supported by morphological descriptions and requires molecular corroboration. The members of this genus are coelozoic and mainly parasitize the urinary system, with few species occurring in the gallbladder. Ortholinea visakhapatnamensis is the only exception, being histozoic in the visceral peritoneum. Molecular data of the small subunit ribosomal RNA gene (SSU rDNA) is available for about one third of Ortholinea species, with genetic interspecific variation ranging between 1.65% and 29.1%. Phylogenetic analyses reveal Ortholinea to be polyphyletic, with available SSU rDNA sequences clustering within the subclades of the highly heterogenous freshwater urinary clade of the oligochaete-infecting lineage. The life cycles of two Ortholinea species have been clarified based on molecular inferences and identify triactinomyxon actinospores as counterparts, and marine oligochaetes of the family Naididae as permissive hosts to this genus.

High temperatures and low humidity promote the occurrence of microsporidians (Microsporidia) in mosquitoes (Culicidae)

BackgroundIn the context of climate change, a growing concern is that vector-pathogen or host-parasite interactions may be correlated with climatic factors, especially increasing temperatures. In the present study, we used a mosquito-microsporidian model to determine the impact of environmental factors such as temperature, humidity, wind and rainfall on the occurrence rates of opportunistic obligate microparasites (Microsporidia) in hosts from a family that includes important disease vectors (Culicidae).MethodsIn our study, 3000 adult mosquitoes collected from the field over 3 years were analysed. Mosquitoes and microsporidia were identified using PCR and sequencing of the hypervariable V5 region of the small subunit ribosomal RNA gene and a shortened fragment of the cytochrome c oxidase subunit I gene, respectively.ResultsDNA metabarcoding was used to identify nine mosquito species, all of which were hosts of 12 microsporidian species. The prevalence of microsporidian DNA across all mosquito samples was 34.6%. Microsporidian prevalence in mosquitoes was more frequent during warm months (> 19 °C; humidity < 65%), as was the co-occurrence of two or three microsporidian species in a single host individual. During warm months, microsporidian occurrence was noted 1.6-fold more often than during the cold periods. Among the microsporidians found in the mosquitoes, five (representing the genera Enterocytospora, Vairimorpha and Microsporidium) were positively correlated with an increase in temperature, whereas one (Hazardia sp.) was significantly correlated with a decrease in temperature. Threefold more microsporidian co-occurrences were recorded in the warm months than in the cold months.ConclusionsThese results suggest that the susceptibility of mosquitoes to parasite occurrence is primarily determined by environmental conditions, such as, for example, temperatures > 19 °C and humidity not exceeding 62%. Collectively, our data provide a better understanding of the effects of the environment on microsporidian-mosquito interactions.Graphical

First report of Sclerotium hydrophilum causing leaf spot on Brasenia schreberi in central China.

Brasenia schreberi, commonly known as watershield and referred to as 'Chun Cai' in Chinese, is a worldwide aquatic vegetable. It has long been regarded as health- promoting vegetable due to production of mucilage in young shoots, and thus has gained popularity in China. In September 2022, a leaf spot disease was observed at the National Aquatic Vegetable Germplasm Resource Nursery located in Wuhan, Hubei province, China. The disease occurred on watershield leaves. It started with the formation of leaf spots surrounded by halos.These spots ranged in color from yellow to brown and in diameter from 1 to 10 mm. Subsequently, the smaller spots merged, ultimately causing the entire leaves to turn black. Small brown- to black-colored sclerotia were produced on the underside of the diseased leaves. Disease incidence was 30% on average, and yield loss was approximately 15% on average. To isolate the pathogen, leaf tissues at the disease-healthy border area were excised into 5 × 5 mm pieces, these segments were immersed in 75% ethanol for 30 s, followed by immersion in 0.5% sodium hypochlorite for 30 s, and then rinsed twice in sterile water. After air-drying, the leaf pieces were incubated on potato dextrose agar (PDA) in darkness at 28°C for 3 d. Mycelia from the leaf pieces were transferred to new PDA plates for purification, three sclerotia-forming fungal isolates (Whcc-1, Whcc-3, Whcc-4) were finally obtained. They were incubated on PDA at 28°C for 4 to 14 d for observation of colony morphology. At 4 d after incubation (DAI), they grew rapidly with the average growth rate of 2.2 cm/d and formed colonies with whitish substrate mycelia and well-developed aerial mycelia and small white to light brown-colored sclerotia. At 10 to 14 DAI, the sclerotia gradually turned to black, 0.2 to 0.4 mm in diameter (0.26 mm on average, n = 50). These morphological characteristics matched description of Sclerotium hydrophilum (Bashyal et al. 2021). Molecular identification was done to further clarify the species identity of this fungus. Genomic DNA was extracted from isolates Whcc-1, Whcc-3, and Whcc-4. The internal transcribed spacer region of the ribosomal DNA (ITS-5.8S rDNA) and the small subunit ribosomal RNA gene (ssrRNA) were amplified using universal primers ITS1/ITS4 and NS1/NS6, as described by White et al. (1990). Sequence analysis revealed a high degree of similarity between the ITS-5.8S rDNA sequences from Whcc-1, Whcc-3, and Whcc-4 (GenBank Acc. No. OP782030, PP035994, and PP035995, respectively) and those of S. hydrophilum strain CBS201.27 (GenBank Acc. No. FJ231396), with similarities of 99.25%, 99.4%, and 99.25%, respectively. The ssrRNA sequences from Whcc-1, Whcc-3, and Whcc-4 (GenBank Acc. No. PP238401, PP261342 and PP261345) were found to be identical, displaying 100% similarity to the ssrRNA sequences of S. hydrophilum strain ZH11 (GenBank Acc. No. KC354147). Based on both morphological and molecular evidence, it can be inferred that Whcc-1, Whcc-3, and Whcc-4 belong to the species S. hydrophilum. Pathogenicity tests were conducted by inoculation of the mycelial agar plugs of Whcc-1, Whcc-3 or agar plugs of fresh PDA (control) on floating leaves of two watershield plants, 4 leaves (replicates) for each treatment. After inoculation, the treated leaves were sealed in plastic bags to maintain humidity, and grown under natural conditions (18°C to 28°C, with 8 hours of light daily). At 7 DAI, while control leaves remained healthy, the leaves inoculated with Whcc-1 and Whcc-3 leaves formed yellow- to brown-colored spots similar to those observed in the field surveys. S. hydrophilum was re-isolated from the leaf spots, thus verifing Koch's postulates. S. hydrophilum has a wide host range, infecting at least 19 genera of plants, including common rice and wild rice (Johnson et al. 1976), and water lily (Kernkamp et al. 1977). Moreover, it has been reported to infect rice in China (Punter et al. 1984; Zhong et al. 2018). To our knowledge, this is the first report of S. hydrophilum on watershield leaves in central China.

The association of bacterial agents and flagellated protozoans in the etiopathogenesis of subspectacular abscesses in snakes.

To describe the etiology, clinical pathology, and treatment of subspectacular abscesses (SAs) in 16 snakes. Sixteen snakes from private captive collections that were presented at a veterinary teaching hospital and diagnosed with SAs. Exudate was collected from SAs via percutaneous aspiration or by performing a partial spectaculectomy and submitted to direct light microscopic examination and/or microbiological examination. Bacterial isolates were cultured from exudate samples in 12 out of 16 snakes and comprised Pseudomonas aeruginosa isolates that showed identical antimicrobial susceptibility patterns in seven cases. Direct light microscopic examination demonstrated the presence of flagellated protozoa in the exudate in 10 out of 16 snakes and in all of these snakes, concurrent oropharyngeal flagellated protozoan infection could be demonstrated. Combined flagellated protozoan and bacterial SA infection was demonstrated in six snakes. Comparison of small subunit ribosomal RNA gene sequences of flagellated protozoans that were detected in the exudate of SAs from four snakes revealed they represented the parabasalians Monocercomonas colubrorum and Hypotrichomonas acosta in one and three cases, respectively. In all snakes, successful treatment of the SAs was achieved following surgical debridement of the subspectacular space combined with antimicrobial treatment. Bacteria as well as flagellates are considered to reach the subspectacular space through ascending infection from the oral cavity via the lacrimal duct. Although opportunistic bacterial infection presumably constitutes the predominant cause of SAs in snakes, the role of flagellate infection in the pathogenesis of this disorder should be further elucidated.

Design and evaluation of loop-mediated isothermal amplification for rapid detection of Enterocytozoon bieneusi

Enterocytozoon bieneusi is one of the most prevalent microsporidia species, responsible for more than 90% of human and animal microsporidiosis. Microsporidia species, particularly E. bieneusi, are frequently reported from waterborne and foodborne outbreaks. Therefore, early detection is crucial in clinics and outbreak investigations. This study aimed to design a loop-mediated isothermal amplification (LAMP) for rapid detection of E. bieneusi. Total DNA was extracted from 30 E. bieneusi –positive samples, which had been confirmed with nested PCR. LAMP primers were designed based on the identical fragment of small subunit ribosomal RNA (SSU rRNA) gene. LAMP reactions were performed at 63 °C for 60 min. The sensitivity and specificity of the assay were analyzed and the results of amplification were compared to real-time PCR. Our results showed that the LAMP assay successfully amplified 25/30 (83.3%) samples. The specificity results indicated no false positive with other microorganisms. Furthermore, the LAMP method exhibited a sensitivity (limit of detection, LoD) as low as 34 ag/μL of total DNA. Compared to the LAMP assay, real-time PCR was able to detect all 30 nested PCR-positive samples. Our findings showed that the LAMP assay was able to detect 83.3% of E. bieneusi-positive samples. Although the current assay was not able to detect all nested PCR-positive samples, the lack of need for specific instruments, rapid processes, and high specificity makes LAMP assay a suitable tool for screening.

The first molecular characterization of Sarcocystis cameli in the Indian dromedary camels (Camelus dromedarius).

In the present study, tissue samples (tongue, esophagus and heart) were investigated from dromedary camels of India for identification and characterization of Sarcocystis spp. using histopathology, PCR and gene sequencing. Genomic DNA extracted from these tissue samples was used for PCR amplification of the cytochrome c oxidase subunit I gene (cox1) of Sarcocystis spp. and the partial sequence of small subunit ribosomal RNA (18S rRNA) gene of the S. cameli. The PCR products were purified, sequenced and analyzed using bioinformatics tools. Based on phylogenetic analysis of the cox1 gene, the sequences of the present study clustered with those of S. cameli, hosted by dromedary camels of Iraq and a close association was observed with S. masoni hosted by dogs and alpacas of China. Until now, there are no 18S rRNA sequences of S. cameli available in GenBank and this is the first study recording 18S rRNA sequences of S. cameli which were grouped with S. masoni from alpaca of China and guanaco and llama of Argentina in phylogenetic analysis. These findings could be useful for further studies on the characterization through molecular epidemiology, genetic diversity and host specificity of S. cameli.

The Use of the Internal Transcribed Spacer Region for Phylogenetic Analysis of the Microsporidian Parasite Enterocytozoon hepatopenaei Infecting Whiteleg Shrimp (Penaeus vannamei) and for the Development of a Nested PCR as Its Diagnostic Tool.

The increasing economic losses associated with growth retardation caused by Enterocytozoon hepatopenaei (EHP), a microsporidian parasite infecting penaeid shrimp, require effective monitoring. The internal transcribed spacer (ITS)-1 region, the non-coding region of ribosomal clusters between 18S and 5.8S rRNA genes, is widely used in phylogenetic studies due to its high variability. In this study, the ITS-1 region sequence (~600-bp) of EHP was first identified, and primers for a polymerase chain reaction (PCR) assay targeting that sequence were designed. A newly developed nested-PCR method successfully detected the EHP in various shrimp (Penaeus vannamei and P. monodon) and related samples, including water and feces collected from Indonesia, Thailand, South Korea, India, and Malaysia. The primers did not cross-react with other hosts and pathogens, and this PCR assay is more sensitive than existing PCR detection methods targeting the small subunit ribosomal RNA (SSU rRNA) and spore wall protein (SWP) genes. Phylogenetic analysis based on the ITS-1 sequences indicated that the Indonesian strain was distinct (86.2% nucleotide sequence identity) from other strains collected from Thailand and South Korea, and also showed the internal diversity among Thailand (N = 7, divided into four branches) and South Korean (N = 5, divided into two branches) samples. The results revealed the ability of the ITS-1 region to determine the genetic diversity of EHP from different geographical origins.

The use of the internal transcribed spacer region for phylogenetic analysis of the microsporidian parasite Enterocytozoon hepatopenaei infecting whiteleg shrimp (Penaeus vannamei) and for the development of a nested PCR as its diagnostic tool.

The increasing economic losses associated with growth retardation caused by Enterocytozoon hepatopenaei (EHP), a microsporidian parasite infecting penaeid shrimp, require effective monitoring. The internal transcribed spacer (ITS)-1 region, the non-coding region of ribosomal clusters between 18S and 5.8S rRNA genes, is widely used in phylogenetic studies due to its high variability. In this study, the ITS-1 region sequence (~600-bp) of EHP was first identified, and primers for a polymerase chain reaction (PCR) assay targeting that sequence were designed. A newly developed nested-PCR method successfully detected the EHP in various shrimp (Penaeus vannamei and P. monodon) and related samples, including water and feces collected from Indonesia, Thailand, South Korea, India, and Malaysia. The primers did not cross-react with other hosts and pathogens, and this PCR assay is more sensitive than existing PCR detection methods targeting the small subunit ribosomal RNA (SSU rRNA) and spore wall protein (SWP) genes. Phylogenetic analysis based on the ITS-1 sequences indicated that the Indonesian strain was distinct (86.2%) from other strains collected from Thailand and South Korea, and also showed the internal diversity among Thailand (N = 7, divided into four branches) and South Korean (N = 5, divided into two branches) samples. The results revealed the ability of the ITS-1 region to determine the genetic diversity of EHP from different geographical origins.

A NEW SPECIES OF THELOHANELLUS (CNIDARIA: MYXOSPOREA: MYXOBOLIDAE) FROM THE GILL OF QUILLBACK, CARPIODES CYPRINUS (CYPRINIFORMES: CATOSTOMIDAE), FROM THE ARKANSAS RIVER DRAINAGE OF OKLAHOMA.

During May 2022 and again in March 2023, 5 quillbacks, Carpiodes cyprinus, were collected from the Verdigris River, Wagoner County, Oklahoma (n = 1), and the Black River, Lawrence County, Arkansas (n = 4), and their gill, gallbladder, fins, integument, musculature, and other major organs were macroscopically examined for myxozoans. Gill lamellae from the single quillback from the Verdigris River was infected with a new myxozoan, Thelohanellus oklahomaensis n. sp. Qualitative and quantitative morphological data were obtained from fresh and formalin-fixed preserved myxospores, and molecular data consisted of a 1,767 base pair sequence of the partial small subunit (SSU) ribosomal RNA gene. Phylogenetic analysis grouped T. oklahomaensis n. sp. with myxozoans known to infect North American catostomids and Eurasian cyprinids. Histological examination localized plasmodia to an intralamellar developmental site and revealed a possible vestige of a second polar capsule. Although plasmodia markedly expanded lamellae, there were no associated epithelial or inflammatory changes. Thelohanellus oklahomaensis n. sp. is the only member of the genus known to infect the gills of C. cyprinus.

Acanthamoeba Keratitis in China: Genotypic and Clinical Correlations.

To investigate the relationship between Acanthamoeba genotypes, clinical manifestations, and outcomes in Acanthamoeba keratitis (AK) patients. This retrospective study included 159 culture-confirmed AK patients. Patients' data were collected, including demographics, initial diagnosis, treatments, and clinical features. The genotype of Acanthamoeba was identified through sequencing the Diagnostic Fragment 3 (DF3) region in the small ribosomal subunit RNA genes. The phylogenetic tree was constructed using the ClustalW model and maximum likelihood method. Cases with "poor outcome" were defined based on specific clinical criteria, including corneal perforation, keratoplasty, other eye surgery, duration of anti-amoebic therapy ≥8.0months, and final visual acuity ≤20/80. "Better outcome" cases were the remainder. The correlation between T4 subtypes, clinical phenotypes, and clinical prognosis were further analyzed. In this study, AK was primarily attributed to the T4A genotype, with a positive correlation between geographical and genetic distances. The primary clinical associated with T4 subtypes was deep stromal infiltration. Results was also showed a significant association between T4 subtypes and clinical outcomes (P = 0.021). Further analysis revealed that T4C was closely associated with a better prognosis (P = 0.040) and T4D with worse outcomes (P = 0.013). In China, AK was predominantly caused by the T4A subtype. Geographical distance positively correlated with genetic distance. Clinical prognosis varied among different subtypes, notably in T4C and T4D. This study demonstrated the association between T4 subtypes and clinical phenotypes, as well as the effects of T4 subtypes on clinical prognosis.

Genetic diversity of the S-type small subunit ribosomal RNA gene of Plasmodium knowlesi isolates from Sabah, Malaysian Borneo and Peninsular Malaysia

Objective: To determine the genetic diversity of Plasmodium (P.) knowlesi isolates from Sabah, Malaysian Borneo and Peninsular Malaysia, targeting the S-type SSU rRNA gene and including aspects of natural selection and haplotype. Methods: Thirty-nine blood samples infected with P. knowlesi were collected in Sabah, Malaysian Borneo and Peninsular Malaysia. The S-type SSU rRNA gene was amplified using polymerase chain reaction, cloned into a vector, and sequenced. The natural selection and haplotype of the S-type SSU rRNA gene sequences were determined using DnaSP v6 and illustrated using NETWORK v10. This study's 39 S-type SSU rRNA sequences and eight sequences from the Genbank database were subjected to phylogenetic analysis using MEGA 11. Results: Overall, the phylogenetic analysis showed no evidence of a geographical cluster of P. knowlesi isolates from different areas in Malaysia based on the S-type SSU rRNA gene sequences. The S-type SSU rRNA gene sequences were relatively conserved and with a purifying effect. Haplotype sharing of the S-type SSU rRNA gene was observed between the P. knowlesi isolates in Sabah, Malaysian Borneo, but not between Sabah, Malaysian Borneo and Peninsular Malaysia. Conclusions: This study suggests that the S-type SSU rRNA gene of P. knowlesi isolates in Sabah, Malaysian Borneo, and Peninsular Malaysia has fewer polymorphic sites, representing the conservation of the gene. These features make the S-type SSU rRNA gene suitable for comparative studies, such as determining the evolutionary relationships and common ancestry among P. knowlesi species.

Cryptosporidium species and subtypes identified in human domestic cases through the national microbiological surveillance programme in Sweden from 2018 to 2022

BackgroundThe intestinal protozoan parasite Cryptosporidium is an important cause of diarrheal disease worldwide. A national microbiological surveillance programme was implemented in Sweden in 2018 in order to increase knowledge of the molecular epidemiology of human cryptosporidiosis to better understand transmission patterns and potential zoonotic sources. This article summarises the results of the first five years of the surveillance programme.MethodsCryptosporidium-positive faecal and DNA samples from domestically acquired infections were collected from clinical microbiological laboratories in Sweden. Species and subtype determination was performed using 60 kDa glycoprotein and/or small subunit ribosomal RNA gene analysis.ResultsBetween 2018 and 2022, 1654 samples were analysed and 11 different species were identified: C. parvum (n = 1412), C. mortiferum (n = 59), C. hominis (n = 56), C. erinacei (n = 11), C. cuniculus (n = 5), C. meleagridis (n = 3), C. equi (n = 2), C. ubiquitum (n = 2), and one each of C. canis, C. ditrichi and C. felis. Subtyping revealed seven subtype families of C. parvum (new subtype families IIy and IIz) and 69 different subtypes (11 new subtypes). The most common C. parvum subtypes were IIdA22G1c, IIdA24G1, IIdA15G2R1 and IIaA16G1R1b. For C. hominis, four different subtype families and nine different subtypes (two new subtypes) were identified. For additional species, two new subtype families (IIIk and VId) and nine new subtypes were identified. All successfully subtyped C. mortiferum cases were subtype XIVaA20G2T1, confirming previous findings in Sweden. Several outbreaks were identified of which the majority were foodborne and a few were due to direct contact with infected animals.ConclusionInfection with C. parvum is the leading cause of human cryptosporidiosis acquired in Sweden, where more than 90% of domestic cases are caused by this zoonotic species and only a small proportion of cases are due to infection with other species. The rodent-associated C. mortiferum is considered an emerging zoonotic species in Sweden and the number of domestically acquired human cases has surpassed that of infection with C. hominis. A high diversity of species and subtypes, as well as diversity within the same subtype, was detected. Also, cryptosporidiosis appears to affect adults to a great extent in Sweden.

Four novel Pleurocordyceps (Polycephalomycetaceae) species from China.

Entomopathogenic fungi comprise an ecologically important group of specialized pathogens infecting other fungi, invertebrates, and plants. These fungi are species-rich with high diversity and broad distribution worldwide. The majority of entomopathogenic fungi belong to clavicipitoids, which consist of the hypocrealean families, Clavicipitaceae, Cordycipitaceae, Ophiocordycipitaceae, and Polycephalomycetaceae. The latter is a newly established entomopathogenic family that recently separated from the family Ophiocordycipitaceae to accommodate the genera, Perennicordyceps, Pleurocordyceps, and Polycephalomyces. In recent years, Polycephalomycetaceae has been enriched with parasitic and hyperparasitic fungi. With 16 species spread across China, Ecuador, Japan, and Thailand, Pleurocordyceps is the most speciose genus in the family. In this study, we expand the number of taxa in the genus by introducing four new Pleurocordyceps species from China, namely, P. clavisynnema, P. multisynnema, P. neoagarica, and P. sanduensis. We provide detailed descriptions and illustrations and infer genus-level phylogenies based on a combined 6-loci gene sequence dataset comprising the internal transcribed spacer gene region (ITS), small subunit ribosomal RNA gene region (SSU), large subunit rRNA gene region (LSU), translation elongation factor 1-alpha gene region (TEF-1α), RNA polymerase II largest subunit gene region (RPB1), and RNA polymerase II second largest subunit (RPB2). This study contributes to knowledge with regard to the diversity of Pleurocordyceps specifically and entomopathogenic Hypocreales more broadly.

Kudoa tanakai n. sp. (Myxozoa: Myxosporea: Multivalvulida), a new kudoid species with spheroid myxospores from the scalpel sawtail (Actinopterygii: Prionurus scalparum) from western Japan.

We describe a new kudoid species, Kudoa tanakai n. sp., in the scalpel sawfish, Prionurus scalprum (Actinopterygii: Acanthuriformes: Acanthuridae), from the natural water around western Japan. The plasmodia were filamentous, localized in pseudocysts in the myofibers of the trunk muscles. The occurrence of plasmodia in the trunk muscle showed no site preference. Its myxospores were spheroid, measuring 6.6-7.6 (7.0) µm by 5.8-6.9 (6.3) µm in apical view (width) and 5.7-6.6 (6.2) in length (n = 30), with four shell valves and a corresponding number of spheroid polar capsules. Shell valves lacked apical protrusions, but scanning electron microscopy revealed that one of the four shell valves had two semi-lunar flaps at its apical terminus. Nucleotide sequencing of the small and large subunit ribosomal RNA genes of the present isolate showed phylogenetic affinities to kudoid species characterized by spheroid myxospores, such as K. musculoliquefaciens, K. hemiscylli, and K. carcharhini, but was molecularly and morphometrically distinct from these and other kudoid species. For direct comparison, Kudoa hemiscylli was collected from the Pacific spadenose shark, Scoliodon macrorhynchos (Elasmobranchii: Carcharhiniformes: Carcharhinidae), in the South China Sea off Guangdong Province, China, and the myxospore surface of the species was observed using scanning electron microscopy. Our study describes the new host and distribution record of this kudoid species originally described from a variety of elasmobranchs in the Australian Coral Sea.