High prevalence of haemosporidian parasites in Eurasian jays.

Avian haemosporidians are vector-borne parasites, infecting a great variety of birds. The order Passeriformes has the highest average infection probability; nevertheless, some common species of Passeriformes have been rather poorly studied. We investigated haemosporidians in one such species, the Eurasian jay Garrulus glandarius (Corvidae), from a forest population in Hesse, Central Germany. All individuals were infected with at least one haemosporidian genus (overall prevalence: 100%). The most common infection pattern was a mixed Haemoproteus and Leucocytozoon infection, whereas no Plasmodium infection was detected. Results on lineage diversity indicate a rather pronounced host-specificity of Haemoproteus and Leucocytozoon lineages infecting birds of the family Corvidae.

Molecular detection of Toxoplasma gondii and Neospora caninum in seabirds collected along the coast of Santa Catarina, Brazil.

Toxoplasma gondii and Neospora caninum are two closely related protozoans that infect a wide range of animals, including birds. However, the occurrence of N. caninum and T. gondii in seabirds is unknown. Therefore, this study aimed to determine the presence of T. gondii and N. caninum DNA in tissue samples of seabirds. Tissue samples of the pectoral muscles, heart, and brain were collected from 47 birds along the coastline of Santa Catarina State, SC, Brazil. The DNA was extracted from the tissues and screened using nested-PCR (nPCR) targeting internal transcribed spacer 1 (ITS1). T. gondii DNA was detected in tissues from seven seabirds (7/47, 14.8%), kelp gull (Larus dominicanus) (5/21), and Manx shearwater (Puffinus puffinus) (2/8). N. caninum DNA was detected in tissues of nine seabirds (9/47, 19.1%), the kelp gull (L. dominicanus) (4/21), Manx shearwater (P. puffinus) (2/8), neotropic cormorant (Phalacrocorax brasilianus) (1/4), brown booby (Sula leucogaster) (1/5), and white-chinned petrel (Procellaria aequinoctialis) (1/1); however, no co-infection was observed. In conclusion, this study showed the circulation of N. caninum and T. gondii in seabirds along the coastline of Santa Catarina State. Further studies are required to clarify the role of these birds in the epidemiology of neosporosis and toxoplasmosis.

First report of Sarcocystis falcatula in naturally infected Razorbill auks (Alca torda) collected in Tunisian Mediterranean Sea shores.

Sarcocystis spp. are apicomplexan cyst-forming parasites that can infect numerous vertebrates, including birds. Sarcosporidiosis infection was investigated in three muscles (breast, right and left thigh muscle) and one organ (heart) of four Razorbill auks (Alca torda) stranded between November and December 2022 on the shores of the Mediterranean Sea in Nabeul and Bizerte governorates, Northern Tunisia. Two of the four tested A. torda were PCR positive for 18S rRNA Sarcocystis spp. gene. Among the examined 16 muscles/organs, only one breast and one right thigh were Sarcocystis spp. PCR-positive (12.5% ± 8.3, 2/16). Our results showed a relatively high molecular prevalence of Sarcocystis spp. in Razorbill auks (A. torda). Sarcocystis spp. sequence described in the present study (GenBank number: OR516818) showed 99.56-100% identity to Sarcocystis falcatula. In conclusion, our results confirmed the infection of Razorbill auks (A. torda) by S. falcatula. Further research is needed on different migratory seabirds' species in order to identify other Sarcocystis species.

New insight into avian malaria vectors in New Zealand.

BACKGROUND: Mosquitoes (Culicidae) are vectors for most malaria parasites of the Plasmodium species and are required for Plasmodium spp. to complete their life cycle. Despite having 16 species of mosquitoes and the detection of many Plasmodium species in birds, little is known about the role of different mosquito species in the avian malaria life cycle in New Zealand. METHODS: In this study, we used nested polymerase chain reaction (PCR) and real-time PCR to determine Plasmodium spp. prevalence and diversity of mitochondrial cytochrome b gene sequences in wild-caught mosquitoes sampled across ten sites on the North Island of New Zealand during 2012-2014. The mosquitoes were pooled by species and location collected, and the thorax and abdomens were examined separately for Plasmodium spp. DNA. Akaike information criterion (AIC) modeling was used to test whether location, year of sampling, and mosquito species were significant predictors of minimum infection rates (MIR). RESULTS: We collected 788 unengorged mosquitoes of six species, both native and introduced. The most frequently caught mosquito species were the introduced Aedes notoscriptus and the native Culex pervigilans. Plasmodium sp DNA was detected in 37% of matched thorax and abdomen pools. When considered separately, 33% of abdomen and 23% of thorax pools tested positive by nested PCR. The MIR of the positive thorax pools from introduced mosquito species was 1.79% for Ae. notoscriptus and 0% for Cx. quinquefasciatus, while the MIR for the positive thorax pools of native mosquito species was 4.9% for Cx. pervigilans and 0% for Opifex fuscus. For the overall MIR, site and mosquito species were significant predictors of Plasmodium overall MIR. Aedes notoscriptus and Cx. pervigilans were positive for malaria DNA in the thorax samples, indicating that they may play a role as avian malaria vectors. Four different Plasmodium lineages (SYAT05, LINN1, GRW6, and a new lineage of P (Haemamoeba) sp. AENOT11) were identified in the pooled samples. CONCLUSIONS: This is the first detection of avian Plasmodium DNA extracted from thoraxes of native Culex and introduced Aedes mosquito species in New Zealand and therefore the first study providing an indication of potential vectors in this country.

Genetic characteristics of Blastocystis sp. in cattle from Hebei Province, China.

Blastocystis sp. is a protozoan parasite that infects the intestines of humans and animals, causing chronic diseases such as skin rashes, abdominal pain, and irritable bowel syndrome. A survey was conducted to determine the prevalence and genetic diversity of Blastocystis sp. infection in cattle, in Hebei Province, China. 2746 cattle fecal samples were collected from 11 cities in Hebei Province and analyzed using polymerase chain reaction targeting the Blastocystis sp. barcoding gene. MEGA, PhyloSuite, and PopART were used to analyze the subtype, sequence signature, pairwise genetic distance, and genetic diversity indices. The results showed that the Blastocystis sp. detection rate was 12.60% (346/2746). The infection rate in different herds was affected by region, age, breeding mode, and variety; that is, the infection rates in areas of southern Hebei, cattle under one year old, intensive raising, and dairy cattle were higher than the infection rates in northern Hebei, cattle over one year old, scatter feeding, and beef cattle. Seven Blastocystis subtypes were identified, namely, ST1, ST2, ST5, ST10, ST14, ST21, and ST26; ST10 was the dominant subtype, and ST14 was the second most common subtype. A total of 374 polymorphic and conserved sites were obtained, including 273 invariable (monomorphic) sites and 101 variable (polymorphic) sites, accounting for 27.01% of all nucleotides. The nucleotide diversity index (Pi) was 0.07749, and the haplotype (gene) diversity index (Hd) was 0.946. This study provides the first comprehensive information on the epidemiological situation of Blastocystis sp. infection in cattle from Hebei Province, China, and revealed rich genetic diversity of Blastocystis sp.

Multicenter evaluation of the Toxoplasma gondii Real-TM (Sacace) kit performance for the molecular diagnosis of toxoplasmosis.

The molecular detection of Toxoplasma gondii DNA is a key tool for the diagnosis of disseminated and congenital toxoplasmosis. This multicentric study from the Molecular Biology Pole of the French National Reference Center for toxoplasmosis aimed to evaluate Toxoplasma gondii Real-TM PCR kit (Sacace). The study compared the analytical and clinical performances of this PCR assay with the reference PCRs used in proficient laboratories. PCR efficiencies varied from 90% to 112%; linearity zone extended over four log units (R2 > 0.99) and limit of detection varied from 0.01 to ≤1 Tg/mL depending on the center. Determined on 173 cryopreserved DNAs from a large range of clinical specimens, clinical sensitivity was 100% [106/106; 95 confidence interval (CI): 96.5%-100%] and specificity was 100% (67/67; 95 CI: 94.6%-100%). The study revealed two potential limitations of the Sacace PCR assay: the first was the inconsistency of the internal control (IC) when added to the PCR mixture. This point was not found under routine conditions when the IC was added during the extraction step. The second is a lack of practicality, as the mixture is distributed over several vials, requiring numerous pipetting operations. Overall, this study provides useful information for the molecular diagnosis of toxoplasmosis; the analytical and clinical performances of the Sacace PCR kit were satisfactory, the kit having sensitivity and specificity similar to those of expert center methods and being able to detect low parasite loads, at levels where multiplicative analysis gives inconsistently positive results. Finally, the study recommends multiplicative analysis in particular for amniotic fluids, aqueous humor, and other single specimens.

Development of nucleic acid lateral flow immunoassay for molecular detection of Entamoeba moshkovskii and Entamoeba dispar in stool samples.

Entamoeba moshkovskii, recently known as a possible pathogenic amoeba, and the non-pathogenic Entamoeba dispar are morphologically indistinguishable by microscopy. Although PCR was used for differential diagnosis, gel electrophoresis is labor-intensive, time-consuming, and exposed to hazardous elements. In this study, nucleic acid lateral flow immunoassay (NALFIA) was developed to detect E. moshkovskii and E. dispar by post-PCR amplicon analysis. E. moshkovskii primers were labeled with digoxigenin and biotin whereas primers of E. dispar were lebeled with FITC and digoxigenin. The gold nanoparticles were labeled with antibodies corresponding to particular labeling. Based on the established assay, NALFIA could detect as low as 975 fg of E. moshkovskii target DNA (982 parasites or 196 parasites/microliter), and 487.5 fg of E. dispar target DNA (444 parasites or 89 parasites/microliter) without cross-reactivity to other tested intestinal organisms. After testing 91 stool samples, NALFIA was able to detect seven E. moshkovskii (87.5% sensitivity and 100% specificity) and eight E. dispar samples (66.7% sensitivity and 100% specificity) compared to real-time PCR. Interestingly, it detected three mixed infections as real-time PCR. Therefore, it can be a rapid, safe, and effective method for the detection of the emerging pathogens E. moshkovskii and E. dispar in stool samples.

Direct evidence of cheetah (Acinonyx jubatus) as intermediate host of Toxoplasma gondii through isolation of viable strains.

Toxoplasma gondii causes lifelong infection in most definitive and intermediate hosts. Clinical cases of toxoplasmosis in captive cheetahs have been reported. However, there are few reports of viable T. gondii strains isolated from cheetahs. Here, T. gondii infection was investigated using molecular and serological assays in cheetahs from China. Modified agglutination test (MAT) (cut-off: 1:25) indicated that all six examined cheetahs (n = 6) showed T. gondii antibodies. Toxoplasma gondii DNA was detected in three out of five cheetahs. Two viable T. gondii strains were isolated from the striated muscles of two cheetahs using mice bioassay. They were designated as TgCheetahCHn1 and TgCheetahCHn2. Genetic characterization of DNA derived from tachyzoites was performed using RFLP-PCR of 10 markers. Toxoplasma gondii TgCheetahCHn1 is ToxoDB PCR-RFLP genotype #319, and the alleles of ROP18/ROP5 types were 3/7. TgCheetahCHn2 is ToxoDB genotype #9, and the alleles of ROP18/ROP5 were 3/6. The average survival time of TgCheetahCHn1-infected Swiss mice was 22 ± 1 days (n = 23), and the mice did not have detectable T. gondii-specific antibodies until 117 ± 30 days post-inoculation (n = 8), therefore, TgCheetahCHn1 had intermediate virulence. TgCheetahCHn2 was avirulent for Swiss mice. Few brain tissue cysts (0-50) were observed in the mice inoculated with TgCheetahCHn1 or TgCheetahCHn2. The results provide direct evidence of cheetah as intermediate host of T. gondii.

Allosteric strand displacement isothermal amplification for the visual detection of Toxoplasma gondii in 30 minutes.

Early detection of Toxoplasma gondii (T. gondii) is critical due to a lack of effective treatment for toxoplasmosis.This study established a simple, cost-effective, and rapid colorimetric detection method for T. gondii. The entire testing process, from sample collection to results, takes only 0.5 h. These characteristics fulfill the demands of researchers seeking rapid target detection with minimal equipment reliance. For genomic extraction, this study evaluated the ability of two filter papers to capture genomes. A rapid genomic extraction device combined with the two filter papers was designed to simplify the genomic extraction process, which was completed within 10 min and increased the detection sensitivity tenfold. The method utilized a simplified primer design for isothermal amplification, namely allosteric strand displacement (ASD), and employed an underutilized commercial color indicator, Bromothymol Blue (BTB), for signal output. Compared with other reported indicators, BTB exhibited a more pronounced color change, shifting from blue to yellow in positive samples, facilitating easier visual differentiation. The reaction was completed in 20 min with a limit of detection (LOD) as low as 0.014 T. gondii per microliter.

Transmission risk evaluation of transfusion blood containing low-density Babesia microti.

Background: Babesia is a unique apicomplexan parasite that specifically invades and proliferates in red blood cells and can be transmitted via blood transfusion, resulting in transfusion-transmitted babesiosis. However, detecting Babesia in blood before transfusion has not received enough attention, and the risk of transfusing blood containing a low density of Babesia microti (B. microti) is unclear, possibly threatening public health and wellness. Purpose: This study aimed to determine the lower detection limit of B. microti in blood and to evaluate the transmission risk of blood transfusion containing low-density B. microti. Methods: Infected BALB/c mouse models were established by transfusing infected whole blood with different infection rates and densities of B. microti. Microscopic examination, nested Polymerase Chain Reaction (nested PCR), and an enzyme-linked immunosorbent assay (ELISA) were used to evaluate the infection status of the mouse models. Meanwhile, the nested PCR detection limit of B. microti was obtained using pure B. microti DNA samples with serial concentrations and whole blood samples with different densities of B. microti-infected red blood cells. Thereafter, whole mouse blood with a B. microti density lower than that of the nested PCR detection limit and human blood samples infected with B. microti were transfused into healthy mice to assess the transmission risk in mouse models. The infection status of these mice was evaluated through microscopic examination, nested PCR tests, and ELISA. Results: The mice inoculated with different densities of B. microti reached the peak infection rate on different days. Overall, the higher the blood B. microti density was, the earlier the peak infection rate was reached. The levels of specific antibodies against B. microti in the blood of the infected mice increased sharply during the first 30 days of infection, reaching a peak level at 60 days post-infection, and maintaining a high level thereafter. The nested PCR detection limits of B. microti DNA and parasite density were 3 fg and 5.48 parasites/µL, respectively. The whole blood containing an extremely low density of B. microti and human blood samples infected with B. microti could infect mice, confirming the transmission risk of transfusing blood with low-density B. microti. Conclusion: Whole blood containing extremely low density of B. microti poses a high transmission risk when transfused between mice and mice or human and mice, suggesting that Babesia detection should be considered by governments, hospitals, and disease prevention and control centers as a mandatory test before blood donation or transfusion.

Epidemiology and genetic diversity of Theileria equi and Babesia caballi in Mongolian horses.

Equine piroplasmosis is a tick-borne disease caused by Theileria equi and Babesia caballi in horses. Because of its impact on horse industry, control of this disease is crucial for endemic countries. The control of equine piroplasmosis may be influenced by the genotypic diversity of T. equi and B. caballi. Mongolia, a country with a thriving livestock industry, is endemic for T. equi and B. caballi. However, nationwide epidemiological surveys have not been conducted to determine the current status of infections and genetic diversity of these two parasite species. Therefore, the objective of this research was to investigate the infection rates and genotypes of T. equi and B. caballi in horses across Mongolia. Blood samples were collected from 1353 horses in 15 of Mongolia's 21 provinces, and their DNAs were analyzed with T. equi- and B. caballi-specific PCR assays. Additionally, blood smears were prepared from 251 horses, stained with Giemsa, and examined under a light microscope to identify T. equi and B. caballi. The microscopy revealed that 30 (11.9%) and 4 (1.6%) of the 251 horses were positive for T. equi and B. caballi, respectively. By contrast, PCR assays detected the T. equi and B. caballi in 1058 (78.2%) and 62 (4.6%) horses, respectively. Phylogenetic analysis of 18S rRNA sequences from 42 randomly selected T. equi-positive DNA samples detected the genotypes A and E. On the other hand, the rap-1 sequences from 19 randomly selected B. caballi-positive DNA samples occurred in clades representing the genotypes A and B1, as well as in a distinct clade closely related to the genotype A. Our findings confirm the widespread occurrence of T. equi and B. caballi infections in Mongolian horses, highlighting the need for a comprehensive control approach.

Molecular characteristics of Cryptosporidium spp. in human cases in five Finnish hospital districts during 2021: first findings of Cryptosporidium mortiferum (Cryptosporidium chipmunk genotype I) in Finland.

The aims of the study were to characterise the distribution of Cryptosporidium spp. and subtypes causing infections in Finland during 2021. This was carried out with 60 clinical samples from the hospital districts of Helsinki and Uusimaa, Vaasa, Kymenlaakso, South Karelia, and Central Finland, as well as with Finnish Infectious Diseases Register (FIDR) data. Additionally, the study aimed to explore the potential exposures related to Cryptosporidium mortiferum (Cryptosporidium chipmunk genotype I) infections via interview. Species identification was carried out with quantitative real-time PCR (qPCR) and 18S sequencing. Further typing was performed with gp60 subtyping. Over 70% of the samples were identified as Cryptosporidium parvum and 20% as C. mortiferum, which had not been identified in Finland before. Two cases of Cryptosporidium hominis were identified from patients reported to have travelled outside Europe. The C. parvum subtype IIaA15G2R1 and the C. mortiferum subtype XIVaA20G2T1 were the most common subtypes identified. The interviewed C. mortiferum cases did not report shared exposures such as contact with wild rodents. In conclusion, C. parvum and C. mortiferum were the major causes of cryptosporidiosis in the five studied Finnish hospital districts.

Comparison of Three Methods to Extract Plasmodium falciparum DNA from Whole Blood and Dried Blood Spots.

This study aimed to compare the effectiveness of three DNA extraction methods: the GF-1 Blood DNA Extraction Kit (GF-1 BD Kit), which employs a spin column along with lysing and washing buffers; the tris-ethylenediaminetetraacetic acid and proteinase K (TE-pK) method, which utilizes a combination of TE buffer and proteinase K for cell lysis; and DNAzol® Direct (DN 131), a single reagent combined with heating for the extraction process. Plasmodium falciparum DNA was extracted from both whole blood and dried blook spots (DBSs), with consideration of DNA concentration, purity, cost, time requirement, and limit of parasite detection (LOD) for each method. The target gene in this study was 18S rRNA, resulting in a 395-bp product using specific primers. In the comparative analysis, the DN 131 method yielded significantly higher DNA quantities from whole blood and DBSs than the GF-1 BD Kit and TE-pK methods. In addition, the DNA purity obtained from whole blood and DBSs using the GF-1 BD Kit significantly exceeded that obtained using the TE-pK and DN 131 methods. For LOD, the whole blood extracted using the DN 131, GF-1 BD Kit, and TE-pK methods revealed 0.012, 0.012, and 1.6 parasites/µL, respectively. In the case of DBSs, the LODs for the DN 131, GF-1 BD Kit, and TE-pK methods were 1.6, 8, and 200 parasites/µL, respectively. The results revealed that the TE-pK method was the most cost-effective, whereas the DN 131 method showed the simplest protocol. These findings offer alternative approaches for extracting Plasmodium DNA that are particularly well-suited for large-scale studies conducted in resource-limited settings.

Comparative molecular epidemiology, subtype distribution, and zoonotic potential of Blastocystis sp. in Equus animals (horses, donkeys, and mules) in northwestern Iran.

A total of 500 fecal samples were collected from Equus animals in six different cities (Ardabil, Namin, Nir, Meshginshahr, Germi, and Khalkhal) of Ardabil Province, northwestern Iran, with 200 samples from horses, 200 from donkeys, and 100 from mules. Of the horse samples, 100 were from racing horses under special monitoring and care, while the remaining 100 were from non-racing horses, including those used for herding or in rural areas. All fecal samples were examined for the presence of Blastocystis sp. using PCR amplification of the SSU rRNA gene's barcode region after DNA extraction. The molecular prevalence of Blastocystis infection in Equus animals was 7.6% (38/500). Blastocystis was more common in horses [11.5% (23/200)] than in donkeys [5.5% (11/200)] and mules [4% (4/100)] (P > 0.05). Compared to racing horses [3% (3/100)], non-racing/rural horses [20% (20/100)] exhibited a substantially higher prevalence of Blastocystis (P < 0.05). The prevalence of Blastocystis in diarrheal samples and younger animals was remarkably higher than in formed samples and older animals, respectively (P < 0.05). No significant difference in Blastocystis infection prevalence was found between the genders of examined animals (P > 0.05). In Equus animals, 38 Blastocystis isolates included eight STs: ST10 [31.6% (12/38)], ST1 [21.1% (8/38)], ST2 [15.8% (6/38)], ST3 [10.5% (4/38)], ST4 [7.9% (3/38)], ST7 [5.2% (2/38)], ST14 [5.2% (2/38)], and ST6 [2.6% (1/38)]. These results suggest that Equus animals act as a proper reservoir for numerous Blastocystis STs, consequently playing a crucial part in the spread of this protozoan infection to humans, animals, and water reservoirs.

A SUMO E3 ligase promotes long non-coding RNA transcription to regulate small RNA-directed DNA elimination.

Small RNAs target their complementary chromatin regions for gene silencing through nascent long non-coding RNAs (lncRNAs). In the ciliated protozoan Tetrahymena, the interaction between Piwi-associated small RNAs (scnRNAs) and the nascent lncRNA transcripts from the somatic genome has been proposed to induce target-directed small RNA degradation (TDSD), and scnRNAs not targeted for TDSD later target the germline-limited sequences for programmed DNA elimination. In this study, we show that the SUMO E3 ligase Ema2 is required for the accumulation of lncRNAs from the somatic genome and thus for TDSD and completing DNA elimination to make viable sexual progeny. Ema2 interacts with the SUMO E2 conjugating enzyme Ubc9 and enhances SUMOylation of the transcription regulator Spt6. We further show that Ema2 promotes the association of Spt6 and RNA polymerase II with chromatin. These results suggest that Ema2-directed SUMOylation actively promotes lncRNA transcription, which is a prerequisite for communication between the genome and small RNAs.

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

BACKGROUND: The 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. METHODS: Cryptosporidium-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. RESULTS: Between 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. CONCLUSION: Infection 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.

Selective whole-genome sequencing of Plasmodium parasites directly from blood samples by nanopore adaptive sampling.

IMPORTANCE: Malaria is caused by parasites of the genus Plasmodium, and reached a global disease burden of 247 million cases in 2021. To study drug resistance mutations and parasite population dynamics, whole-genome sequencing of patient blood samples is commonly performed. However, the predominance of human DNA in these samples imposes the need for time-consuming laboratory procedures to enrich Plasmodium DNA. We used the Oxford Nanopore Technologies' adaptive sampling feature to circumvent this problem and enrich Plasmodium reads directly during the sequencing run. We demonstrate that adaptive nanopore sequencing efficiently enriches Plasmodium reads, which simplifies and shortens the timeline from blood collection to parasite sequencing. In addition, we show that the obtained data can be used for monitoring genetic markers, or to generate nearly complete genomes. Finally, owing to its inherent mobility, this technology can be easily applied on-site in endemic areas where patients would benefit the most from genomic surveillance.

Molecular detection and internal transcribed spacer-1 sequence diversity of Trypanosoma evansi in goats from Cavite, Philippines.

Goat production is an important source of livelihood and food. Goats may serve as reservoir of surra affecting livestock production. Here, forty-two free-roaming goats from Cavite, Philippines were screened using two primer sets, Trypanosoma brucei minisatellite chromosome for initial detection and the internal transcribed spacer 1 (ITS-1) to determine phylogeny. Initial PCR detection showed that 19/42 (45%) goats were positive, much higher than the rate previously reported in goats from Cebu (34%). The infectivity rate was higher in male (56%) than in female (42%) and the rate was higher in young ≤1 year old (100%) than in adult >1 year old (43%). Phylogenetic analysis of the ITS-1 sequences between T. evansi goat samples and other isolates indicate potential interspecies transmission.

Pseudovampyrella gen. nov.: A genus of Vampyrella-like protoplast extractors finds its place in the Leptophryidae.

Vampyrellid amoebae are predatory protists, which consume a variety of eukaryotic prey and inhabit freshwater, marine and terrestrial ecosystems. Although they have been known for almost 150 years, much of their diversity lacks an in-depth characterization. To date, environmental sequencing data hint at several uncharacterized lineages, to which no phenotype is associated. Furthermore, there are numerous historically described species without any molecular information. This study reports on two new vampyrellid strains from moorlands, which extract the protoplasts of Closterium species (Zygnematophyceae). Our data on morphology, prey range specificity and feeding strategy reveal that the studied vampyrellids are very similar to the historically described Vampyrella closterii. However, phylogenetic analyses demonstrate that the two strains do not belong to the genus Vampyrella and, instead, form a distinct clade in the family Leptophryidae. Hence, we introduce a new genus of algivorous protoplast extractors, Pseudovampyrella gen. nov., with the species P. closterii (= V. closterii) and P. minor. Our findings indicate that the genetic diversity of morphologically described vampyrellid species might be hugely underrated.

Division of Blastocystis ST10 into three new subtypes: ST42-ST44.

The Blastocystis subtype ST10 has been recognized to contain a great deal of diversity at the sequence level, potentially indicating the presence of multiple new STs within the clade. However, the data needed to validate these new STs were not available. To help resolve this diversity, full-length small subunit (SSU) rRNA gene reference sequences were generated using Oxford Nanopore MinION long-read sequencing from 21 samples representing multiple domestic and wild hosts and geographic regions and covering the sequence diversity previously described using fragments of the SSU rRNA gene. Phylogenetic and pairwise distance analyses were used to compare full-length sequences of the SSU rRNA gene generated in this study with all other valid STs of Blastocystis. We present data supporting the division of ST10/ST23 cluster into five subtypes, ST10, ST23, and three new subtypes with the proposed ST designations of ST42, ST43, and ST44. As the host range of Blastocystis continues to expand with new subtypes and new hosts being frequently identified, the reference sequences provided in this study will assist in accurate sequence classification and help to clarify the epidemiology of this common intestinal microeukaryote.