Cryptosporidium is an important water-borne and food-borne parasite with a high burden of disease. This organism has been shown to contaminate various leafy vegetables; however, studies assessing the presence of Cryptosporidium spp in pre-washed and ready-to-eat vegetables are limited. Routine surveillance in the UK revealed a nationwide exceedance of human cases of Cryptosporidium. Therefore, this study aims to assess the presence of this parasite in pre-washed vegetables from supermarkets in the UK. A total of 36 samples were purchased from four different supermarkets. A nested PCR targeting the SSU rRNA was carried out on 24 samples, 58% were PCR-positive for Cryptosporidium. Sanger sequencing confirmed that, of these sequences, 4/24 (17%) produced significant similarities to Cryptosporidium parvum. This study provides evidence for the presence of C. parvum in pre-washed and ready-to-eat vegetables. Future work to identify the point of contamination is required.

A fine-scale phylogenetic and phylogeographic analysis of Peripatopsis lawrencei s.l. was conducted with both mitochondrial and nuclear DNA sequence data, using both external morphology and scanning electron microscopy of taxonomically important characters. A total of 119 sequences were used for the mitochondrial cytochrome c oxidase subunit I (COI ) whereas a single representative specimen from each locality was sequenced for the nuclear 18S rRNA locus. Phylogenetic analyses were conducted on the total COI data set and the combined COI + 18S rRNA data set using a Bayesian analysis and maximum likelihood analyses. For the combined DNA sequence data set, a divergence time estimation was further undertaken in BEAST and specimens placed in a phylogenetic framework including all the described Peripatopsis species from South Africa. In addition, a phylogeographic study was conducted exclusively on P. lawrencei s.s. (clade A) using an analysis of molecular variance and haplotype network. Phylogenetic results indicated that, at the Oubos sample locality, two highly distinct genetic lineages were present (clades A and B), whereas a divergence time estimation suggests a Miocene cladogenesis of the novel Oubos lineage. Marked phylogeographic structure was observed for P. lawrencei s.s. (restricted to clade A) across the distribution range with limited maternal dispersal. Morphologically, the two sympatric lineages at Oubos A and B differed in leg pair number, ventral colour and dorsal scale rank counts, as evident from scanning electron microscopy. Our results support the recognition of a distinct species that occurs in sympatry with P. lawrencei s.s. The new species, P. aereus sp. nov. (clade B) is described and the implication for fine-scale taxonomic studies on saproxylic taxa is discussed. ZooBank: urn:lsid:zoobank.org:pub:AB6E0BDA-7B5F-4FD3-A863-BA7C814E278C.

Over the last six decades, northwest China has undergone a significant climatic shift from \"warm-dry\" to \"warm-wet\", profoundly impacting the structures and functions of lake ecosystem across the region. However, the influences of this climatic transition on the diversity patterns, co-occurrence network, and assembly processes of eukaryotic microbial communities in lake ecosystem, along with the underlying mechanisms, remain largely unexplored. To bridge this knowledge gap, our study focused on Lake Bosten, the largest inland freshwater body in China, conducting a comprehensive analysis. Firstly, we examined the dynamics of key water quality parameters in the lake based on long-term monitoring data (1992-2022). Subsequently, we collected 93 water samples spanning two distinctive periods: low water level (WL) and high total dissolved solids (TDS) (PerWLTDS; 2010-2011; attributed to \"warm-dry\" climate), and high WL and low TDS (PerTDSWL; 2021-2022; associated with \"warm-wet\" climate). Eukaryotic microorganisms were further investigated using 18S rRNA gene sequencing and various statistical methods. Our findings revealed that climatic warming and wetting significantly increased eukaryotic microbial α-diversity (all Wilcox. test: P<0.05), while simultaneously reducing β-diversity (all Wilcox. test: P<0.001) and network complexity. Through the two sampling periods, assembly mechanisms of eukaryotic microorganisms were predominantly influenced by dispersal limitation (DL) and drift (DR) within stochastic processes, alongside homogeneous selection (HoS) within deterministic processes. WL played a mediating role in eukaryotic microbial DL and HoS processes in the PerTDSWL, whereas water quality and α-diversity influenced the DL process in the PerWLTDS. Collectively, these results underscore the direct and indirect impacts of \"warm-wet\" conditions on the eukaryotic microorganisms within Lake Bosten. This study provides valuable insights into the evolutionary dynamics of lake ecosystems under such climatic conditions and aids in predicting the ecological ramifications of global climatic changes.

Cattle cryptosporidiosis is noted worldwide with varied frequency of infection prevalence depending on geographical, environmental and husbandry factors. In this study, the prevalence of Cryptosporidium infections in cattle was determined on the basis of molecular results obtained by testing 1601 faecal samples collected from calves up to 4 months of age housed in all Polish provinces from 2014 to 2018. Detection and identification of Cryptosporidium species was performed at the 18 small subunit ribosomal RNA (18S rRNA) locus by conducting PCR-RFLP analysis of the amplified DNA fragments. The prevalence of Cryptosporidium infections in the cattle population was 45.3% (CI 95%: 42.8-47.7; 725/1601). The infected animals were housed on 233/267 (87.3%) of monitored farms with regional prevalence ranging from 27.8 to 62%. The restriction pattern of 18S rRNA amplicons for positive samples was characteristic of C. parvum, C. bovis, C. ryanae, C. andersoni, and unexpectedly also of C. baileyi and C. suis. Infections of C. bovis and C. ryanae prevailed in the studied cattle population relegating C. parvum to third in prevalence. Likewise, mixed infections caused by C. bovis and C. ryanae as well as C. parvum and C. bovis were observed. A relationship between the infecting parasite species and animal breed was found. For instance, C. parvum prevailed in Black and White lowland breed, C. ryanae in Limousine cattle and C. andersoni in dairy animals of mixed dairy breeds. Furthermore, differences in prevalence of particular parasite species between cattle breeds were also shown.

The vast diversity of microalgae imposes the challenge of identifying them through the most common and economical identification method, morphological identification, or through using the more recent molecular-level identification tools. Here we report an approach combining enrichment and metagenomic molecular techniques to enhance microalgae identification and identify microalgae diversity from environmental water samples. From this perspective, we aimed to identify the most suitable culturing media and molecular approach (using different primer sets and reference databases) for detecting microalgae diversity. Using this approach, we have analyzed three water samples collected from the River Nile on several enrichment media. A total of 37 microalgae were identified morphologically to the genus level. While sequencing the three-primer sets (16S rRNA V1-V3 and V4-V5 and 18S rRNA V4 region) and aligning them to three reference databases (GG, SILVA, and PR2), a total of 87 microalgae were identified to the genus level. The highest eukaryotic microalgae diversity was identified using the 18S rRNA V4 region and alignment to the SILVA database (43 genera). The two 16S rRNA regions sequenced added to the eukaryotic microalgae identification, 26 eukaryotic microalgae. Cyanobacteria were identified through the two sequenced 16S rRNA regions. Alignment to the SILVA database served to identify 14 cyanobacteria to the genera level, followed by Greengenes, 11 cyanobacteria genera. Our multiple-media, primer, and reference database approach revealed a high microalgae diversity that would have been overlooked if a single approach had been used over the other.

BACKGROUND: Tsetse flies can transmit various Trypanosoma spp. that cause trypanosomiasis in humans, wild animals, and domestic animals. Amplicon deep sequencing of the 12S ribosomal RNA (rRNA) gene can be used to detect mammalian tsetse hosts, and the 18S rRNA gene can be used to detect all associated eukaryotic pathogens, including Trypanosoma spp.
METHODS: Tsetse flies were collected from the Serengeti National Park (n = 48), Maswa Game Reserve (n = 42), and Tarangire National Park (n = 49) in Tanzania in 2012-13. Amplicon deep sequencing targeting mammal-specific 12S rRNA and 18S rRNA genes was performed to screen the blood-feeding sources of tsetse flies and eukaryotic parasites in tsetse flies, respectively.
RESULTS: 12S rRNA gene deep sequencing revealed that various mammals were blood-feeding sources of the tsetse flies, including humans, common warthogs, African buffalos, mice, giraffes, African elephants, waterbucks, and lions. Genes of humans were less frequently detected in Serengeti (P = 0.0024), whereas African buffaloes were detected more frequently as a blood-feeding source (P = 0.0010). 18S rRNA gene deep sequencing showed that six tsetse samples harbored the Trypanosoma gene, which was identified as Trypanosoma godfreyi and Trypanosoma simiae in subsequent ITS1 gene sequencing.
CONCLUSIONS: Through amplicon deep sequencing targeting the 12S rRNA and 18S rRNA genes, various mammalian animals were identified as blood-meal sources, and two Trypanosoma species were detected in tsetse flies collected from the Maswa Game Reserve, Serengeti National Park, and Tarangire National Park in Tanzania. This study illustrates the patterns of parasitism of tsetse fly, wild animals targeted by the fly, and Trypanosoma spp. carried by the fly in Tanzania. It may provide essential data for formulating better strategies to control African trypanosomes.

Myzus persicae is a globally important pest with the ability to adjust to a wide range of environmental situations, and many molecular technologies have been developed and applied to understand the biology and/or control this pest insect directly. Reverse-transcription quantitative real-time PCR (RT-qPCR) is a primary molecular technology that is used to quantify gene expression. Choosing a stable reference gene is significantly important for precisely clarifying the expression level of the target gene. Actin and 18S have been recommended as stable compounds for real-time RT-qPCR in M. persicae under the tested biotic and abiotic conditions. In this study, we checked the stability of Actin and 18S by analyzing the relative expression levels of the cytochrome 450 monooxygenase family member genes CYP6CY3 and CYP6-1, carboxylesterase gene E4 and vacuolar protein sorting gene VPS11 via RT-qPCR under various conditions. The expression levels of these four target genes were normalized using both Actin and 18S individually and the combination of these two genes. Our results confirmed that Actin and 18S can be used as reference genes to normalize the expression of target genes under insecticide treatment and starvation in M. persicae. However, at the developmental stages of M. persicae, the expression of the four tested target genes was normalized stably by Actin but not 18S, with the latter presenting a problematic change with the developmental stages. Thus, the stability of reference genes in response to diverse biotic and abiotic factors should be evaluated before each RT-qPCR experiment.

Although culture-based methods remain a staple element of microbiology analysis, advanced molecular methods increasingly supplement the testing repertoire. Since the advent of 16s and 18s ribosomal RNA PCR in the 2000s, there has been interest in its utility for pathogen detection. Nonetheless, studies assessing the impact on antimicrobial prescribing are limited. We report a single-centre experience of the influence of 16s and 18s PCR testing on antimicrobial treatment, including a cost-analysis.
Data were collected retrospectively for all samples sent for 16s and 18s PCR testing between January 2014 and December 2020. Results were compared to any culture-based result. Assessment focused on any change of antimicrobial treatment based on PCR result, or use of the result as supportive evidence for microbiological diagnosis.
310 samples relevant to 268 patients were referred for 16s/18s rRNA PCR testing during the period. Culture was performed for 234 samples. Enrichment culture was performed for 83 samples. 82 of 300 samples sent for 16s PCR had positive results (20.8%). When culture was performed, enrichment reduced the outcome of 16s PCR only positive results (4/36 [11.1%] versus 14/35 [40.0%], p = 0.030 where a pathogen found). 18s PCR yielded 9 positive results from 67 samples. The 16s PCR result influenced antimicrobial change for 6 patients (2.2%). We estimated the cost for 16s PCR testing to result in one significant change in antimicrobial therapy to be €3,340. 18s PCR did not alter antimicrobial treatment.
There was limited impact of 16s PCR results on antimicrobial treatments. Relevance to practice was affected by relatively long turn-around-time for results. Utility may be increased in specialised surgical centres, or by reducing turn-around-time. Enrichment culture should be considered on samples where 16s PCR is requested. There remains limited evidence for use of 18s PCR in clinical management, and further studies in this area are likely warranted.

Brain abscesses are often polymicrobial and of unclear primary origin. Here, we compare the use of next-generation sequencing (NGS) technology with classical microbiological diagnostics for identification of clinically relevant microorganisms and describe the microbiome profiling with respect to the primary source of brain abscess. Thirty-six samples from 36 patients, with primary brain abscesses, were subjected to both culture- and 16S/18S rRNA Sanger sequencing-based diagnostics (\"standard methods\") and compared to a 16S/18S amplicon-based NGS, which were also subjected to a microbiome diversity analyses. Forty-seven species were identified with \"standard methods\" compared to 96 species with NGS, both confirming and adding to the number of species identified (p < 0.05). The variation of the brain abscess microbiome diversity was not continuous but could be stratified comparing the presumable origin of infection (\"dental,\" \"sinus,\" \"disseminated,\" or \"unknown\"). Alpha diversity did not differ (p > 0.05) between groups while beta diversity differed significantly (p = 0.003) comparing disseminated vs the other presumable origin of infection. Interesting, clustering was also detected between \"dental\" and \"sinusitis,\" although not significantly (p = 0.07). Microbiome-based diagnostics can increase sensitivity without losing specificity. The bacterial beta diversity differed between the presumably origin of the brain abscess and might help to clarify the primary source of infection.

BACKGROUND: Epidemiological surveys in Oman have revealed a high prevalence of the co-occurrence of the pathogenic Theileria lestoquardi and the non-pathogenic Theileria ovis among sheep in the Barka region, Oman. Our most recent data illustrated an interaction and reduced mortality risk in animals co-infected with T. lestoquardi and T. ovis, suggesting that the latter confers protection against pathogenicity of T. lestoquardi. The present study extends the above findings and examines disease outcomes; clinical markers, hematological parameters, and parasite density in mixed and single T. lestoquardi infections.
METHODS: A total of 390 blood samples were collected from 16 sheep pens located in Barka, Oman between July and November 2019. Theileria spp. were detected and quantified using qPCR assay targeting 18S rRNA, and the extent of genetic diversity was estimated by a panel of T. lestoquardi specific micro- and mini-satellites. The association of some disease markers with the presence of Theileria spp. and genetic diversity was tested.
RESULTS: Theileria spp. were detected in 75 (19.2%) sheep; of these 65 (86.7%) had mixed infections (T. lestoquardi plus T. ovis), 8 (10.6%) were infected with T. lestoquardi alone, and 2 (2.7%) with only T. ovis. Exotic breeds had a higher risk for Theileria spp. infection. The density (18S rRNA gene copies) of both parasites was higher in single infection against mixed infection, and there was a relatively lower density of T. lestoquardi in mixed infections. However, there was no difference in hematological indices between single T. lestoquardi and mixed infections. High genetic diversity was observed among T. lestoquardi in Barka, with no differences of T. lestoquardi in single and mixed infections. The extent of diversity seen in Barka was higher (He = 0.772) than that reported in Oman in 2019 (He = 0.582), with distinct T. lestoquardi genotypes.
CONCLUSIONS: The lower density of T. lestoquardi as mixed infection with T. ovis compared to single infection supports the hypothesis that T. ovis confers protection against lethal T. lestoquardi infection. However, there were no differences in disease correlations (clinical markers, hematological parameters, and density of parasites) or the extent of diversity of T. lestoquardi between the two types of infection. The presence of distinct T. lestoquardi genotypes in Barka, compared to that reported earlier in Oman, likely reflects movement of carrier animals and highlights the need for further analysis of the parasite populations to inform novel approaches for controlling malignant ovine theileriosis.