High-throughput Real-time PCR Research Articles

Wide spectrum of tick-borne pathogens in juvenile Ixodes ricinus collected from autumn-migrating birds in the Vistula River Valley, Poland

BackgroundMigratory birds serve as potential hosts for ticks and can be reservoirs of tick-borne pathogens (TBPs). The aim of our study was to investigate the prevalence of TBPs in juvenile Ixodes ricinus collected from Erithacus rubecula, Turdus merula, and Turdus philomelos passing through the Vistula River Valley, Poland — one of the most important European north-south routes for migratory birds.MethodsTo detect TBPs in collected ticks we used a high-throughput microfluidic real-time PCR method. In addition, we performed a phylogenetic analysis of Borreliella garinii flaB and Rickettsia helvetica ompB sequences, considering haplotype diversity through a Median Joining Network.ResultsOur results showed a high prevalence and wide spectrum of TBPs in both larvae and nymphs of I. ricinus. Overall, including co-infections, 47.41% of the tested tick specimens were infected with at least one TBP. Borreliaceae spirochetes were detected in ticks collected from all examined bird species. Ticks (larvae and nymphs) collected from T. merula showed the highest prevalence of Bo. garinii (33.33%), Bo. burgdorferi s.s. (7.69%) and Borrelia miyamotoi (2.56%), while the highest number of ticks infected with Bo. valaisiana were collected from T. philomelos (8.11%). In turn, the highest prevalence of R. helvetica (20.00%) was observed in ticks collected from E. rubecula. Additionally, infections with A. phagocytophilum (5.00%), Ehrlichia spp. (2.50%), Ba. divergens (2.50%) and Ba. venatorum (2.50%) were only confirmed in ticks collected from this bird species. The phylogenetic analysis of Bo. garinii revealed that the detected haplotype circulates widely across various hosts and is geographically widespread, while the haplotype of R. helvetica is mainly detected in ticks in Central Europe.ConclusionsTicks carried by T. merula, T. philomelos, and E. rubecula migrating along the Vistula River Valley, Poland are characterized by a high prevalence and a wide spectrum of detected TBPs. Tested ticks carry widespread strains of Bo. garinii, in contrast to R. helvetica, which is mainly found in Central Europe. Therefore, further research on the possible role of birds as reservoirs of TBPs is needed.

Direct comparison of an ultrasensitive real-time PCR assay with droplet digital PCR for the detection of PIK3CA hotspot mutations in primary tumors, plasma cell-free DNA and paired CTC-derived gDNAs

IntroductionDetection of PIK3CA mutations in primary tumors and liquid biopsy samples is of increasing importance for treatment decisions and therapy resistance in many types of cancer. The aim of the present study was to directly compare the efficacy of a relatively inexpensive ultrasensitive real-time PCR with the well-established and highly sensitive technology of ddPCR for the detection of the three most common hotspot mutations of PIK3CA, in exons 9 and 20, that are all of clinical importance in various types of cancer.Patients and methodsWe analyzed 42 gDNAs from primary tumors (FFPEs), 29 plasma-cfDNA samples, and 29 paired CTC-derived gDNAs, all from patients with ER+ metastatic breast cancer, and plasma from 10 healthy donors. The same blood draws were used for CTC isolation using EpCAM beads for positive immunomagnetic enrichment. All FFPEs and plasma-cfDNA samples were analyzed in parallel for PIK3CA mutations by ultrasensitive real-time PCR assay and droplet digital PCR.ResultsIn gDNAs from FFPEs, using ultrasensitive real-time PCR, the p.E545K mutation was detected in 22/42(52.4%), and the p.E542K and p.H1047R mutations were detected in 14/42(33.3%) and 16/42(38.1%), respectively. Using ddPCR, the p.E545K mutation was detected in 22/42(52.4%), p.E542K in 17/42(40.5%), and p.H1047R in 19/42(45.2%) samples, revealing a concordance between the two methodologies of 81%, 78.6% and 78.6% for each mutation respectively. In plasma-cfDNA, using ultrasensitive real-time PCR, the p.E545K mutation was detected in 11/29(38%) and both p.E542K and p.H1047R mutations in 2/29(6.9%).In the same plasma-cfDNA samples using ddPCR, p.E545K was detected in 1/29(3.5%), p.E542K in 2/29(6.9%), and p.H1047R in 3/29(10.5%) samples, revealing a concordance of 65.5%,100% and 93.1% for each mutation respectively. In paired CTC-derived gDNAs p.E545K was detected in 11/29(38%), p.E542K in 3/29(10.3%), and p.H1047R in 7/29(24.1%) samples.ConclusionsThis low-cost, high-throughput and ultrasensitive real-time PCR assay provides accurate and specific detection of PIK3CA hotspot mutations in liquid biopsy samples and gives similar results to ddPCR. This assay can be performed in labs where digital PCR instrumentation is not available. In CTC-derived gDNA and paired plasma-cfDNA, PIK3CA mutations detected were not identical, revealing that CTC and plasma-cfDNA give complementary information.

Top-down predators shape soil bacterial community composition while bottom-up nutrients drive bacterial abundance

Although the top-down and bottom-up concept in microbial food-webs has been a primary interest in ecology, less is still known about it in soil ecosystems. Protists are the primary top-down predators of bacterial communities, altering their compositions, while the bottom-up resources are the primary factors limiting bacterial growth. Here, we hypothesized that the top-down predators modulate soil bacterial community composition, while the bottom-up nutrients control the bacterial growth and population. To precisely control nutrient levels, we used an inert soil substitute consisting of a combination of calcined clay and sand. Nutrients equivalent to the reference paddy field soil were added to microcosms as a control treatment. To investigate the effects of C, N, and P, six additional bottom-up treatments in the absence and double amounts of the nutrients were prepared. Four top-down treatments (no protist addition, Acanthamoeba castellanii, Vermamoeba vermiformis, and Heteromita globosa) were set up for each bottom-up treatment. A total of 252 microcosms under 28 treatments were incubated. Bacterial communities were analyzed using high-throughput sequencing and real-time PCR in the 1st, 3rd, and 5th weeks. The results revealed that the top-down predators significantly altered the bacterial community composition, and the bacterial population was predominantly controlled by the bottom-up nutrients. Analysis of absolute abundance data demonstrated that both top-down and bottom-up factors shaped the bacterial community structure (community composition and population). Random forest analysis classified the amplicon sequence variants associated with the treatments, showing that mostly similar families were affected by both bottom-up and top-down factors. In conclusion, the results of this study fully supported our hypothesis that top-down predators alter community composition, while bottom-up factors influence bacterial population dynamics.

Diversity and Distribution of Bacterial and Parasitic Tick-Borne Pathogens in Armenia, Transcaucasia.

Variations in the distribution and prevalence of pathogens in ticks can have significant consequences for human health. Information on these variables in Transcaucasia is scarce, so the aim of our study was to conduct a large-scale study to detect selected tick-borne infectious agents in Armenia. Overall, 209 adult ticks were collected from different hosts including 4 samples from human clothes. We tested ticks using high-throughput microfluidic single-cell real-time PCR to detect 42 genospecies of pathogens. We used GIS to determine biotic and abiotic factors governing the prevalence of pathogens and applied statistical analyses to test the association between prevalence of pathogens depending on hosts, locality and environment. From 209 samples, 134 were positive to targeted pathogens. Anaplasma phagocytophilum Foggie, 1949 was the most prevalent case (44%). The highest overall prevalence was observed in ticks from sheep (74%), followed by cows (67%) and calves (60%). The highest multiple infection rates were also detected in sheep (40%) and calves (40%) followed by cows (28%). One statistically significant association was found among co-infections (P<0.05). The prevalence of pathogens varied according to locality. The abundance of Anaplasma spp. is significantly correlated with "slope" and "vegetation" factors. Similar patterns were detected for other pathogens. This was the first large-scale survey of multiple tick-borne pathogens in Armenia and Transcaucasia. The results of this study shed light on spatial variations in pathogen infection rate among adult ticks found on hosts and underline a number of environmental determinants of pathogen distribution among ticks.

Spatiotemporal Distribution of nirS-Type Denitrifiers in Cascade Reservoir Sediments of the Qinghai Plateau

Compared to single damming, the impact of cascade damming on nitrogen-related microorganisms in river ecosystems exhibits greater complexity. However, there is still a lack of research on the response of denitrifiers to the construction of cascade reservoirs. A study was conducted on 10 cascade reservoirs in the upper reaches of the Yellow River to investigate the impact of cascade reservoir construction on nirS-type denitrifying bacteria in sediments. Sediment samples were collected in May (dry season) and August (wet season) of 2023. The spatiotemporal characteristics of the nirS-type denitrifying bacterial community and gene abundance were analyzed using Illumina high-throughput sequencing and real-time fluorescence quantification PCR (qPCR). Redundancy analysis (RDA) and variation partitioning (VP) were utilized to assess the impact of environmental factors on these communities. The results showed the following: (1) Proteobacteria was the predominant phylum of nirS-type denitrifying bacteria in cascade reservoir sediments. At the genus level, unclassified Proteobacteria (69.51–95.64%) showed the highest relative abundance, followed by Paracoccus, Rhodanobacter, and Pseudomonas, indicating that unclassified Proteobacteria may dominate denitrification in these reservoir sediments. (2) The α and β diversity indices of nirS-type denitrifying bacteria were higher in the dry season than in the wet season, and also higher in young reservoirs compared to old reservoirs (p &lt; 0.05). (3) Temporally, the abundance of the nirS gene was significantly higher in the wet season (12.71 × 107 copies/g dry sediment) compared to the dry season (66.35 × 105 copies/g dry sediment). Spatially, the abundance of the nirS gene was higher in the central region, while relatively lower at both ends. (4) RDA and VP analysis indicated that the community structure and abundance of nirS-type denitrifying bacteria were significantly influenced by the total nitrogen in sediments (19.31%) and water temperature (14.13%). Spearman correlation analysis showed that organic carbon significantly affected the diversity of nirS-type denitrifying bacteria (p &lt; 0.05). The results contribute to a better understanding of the nitrogen-related microbial community in cascade reservoir sediments of the Yellow River, providing a scientific basis for reservoir management.

Mosquitoes on a chip-environmental DNA-based detection of invasive mosquito species using high-throughput real-time PCR.

The monitoring of mosquitoes is of great importance due to their vector competence for a variety of pathogens, which have the potential to imperil human and animal health. Until now mosquito occurrence data is mainly obtained with conventional monitoring methods including active and passive approaches, which can be time- and cost-consuming. New monitoring methods based on environmental DNA (eDNA) could serve as a fast and robust complementary detection system for mosquitoes. In this pilot study already existing marker systems targeting the three invasive mosquito species Aedes (Ae.) albopictus, Ae. japonicus and Ae. koreicus were used to detect these species from water samples via microfluidic array technology. We compared the performance of the high-throughput real-time PCR (HT-qPCR) system Biomark HD with real-time PCR (qPCR) and also tested the effect of different filter media (Sterivex® 0.45 µm, Nylon 0.22 µm, PES 1.2 µm) on eDNA detectability. By using a universal qPCR protocol and only 6-FAM-MGB probes we successfully transferred these marker systems on the HT-qPCR platform. All tested marker systems detected the target species at most sites, where their presence was previously confirmed. Filter media properties, the final filtration volume and observed qPCR inhibition did not affect measured Ct values via qPCR or HT-qPCR. The Ct values obtained from HT-qPCR were significantly lower as Ct values measured by qPCR due to the previous preamplification step, still these values were highly correlated. Observed incongruities in eDNA detection probability, as manifested by non-reproducible results and false positive detections, could be the result of methodological aspects, such as sensitivity and specificity issues of the used assays, or ecological factors such as varying eDNA release patterns. In this study, we show the suitability of eDNA-based detection of mosquito species from water samples using a microfluidic HT-qPCR platform. HT-qPCR platforms such as Biomark HD allow for massive upscaling of tested species-specific assays and sampling sites with low time- and cost-effort, thus this methodology could serve as basis for large-scale mosquito monitoring attempts. The main goal in the future is to develop a robust (semi)-quantitative microfluidic-based eDNA mosquito chip targeting all haematophagous culicid species occurring in Western Europe. This chip would enable large-scale eDNA-based screenings to assess mosquito diversity, to monitor species with confirmed or suspected vector competence, to assess the invasion progress of invasive mosquito species and could be used in pathogen surveillance, when disease agents are incorporated.

Distribution and response of electroactive microorganisms to freshwater river pollution

Electroactive microorganisms (EAMs) play a vital role in biogeochemical cycles by facilitating extracellular electron transfer. They demonstrate remarkable adaptability to river sediments that are characterized by pollution and poor water quality, significantly contributing to the sustainability of river ecosystems. However, the distribution and diversity of EAMs remain poorly understood. In this study, 16S rRNA gene high-throughput sequencing and real-time fluorescence quantitative PCR were used to assess EAMs in 160 samples collected from eight rivers within the Pearl River Delta of Southern China. The results indicated that specialized EAMs communities in polluted sediments exhibited variations in response to water quality and sediment depth. Compared to clean sediment, polluted sediments showed a 4.5% increase in the relative abundances of EAMs communities (59 genera), with 45- and 17-times higher abundances of Geobacter and cable bacteria. Additionally, the abundance of cable bacteria decreased with increasing sediment depth in polluted sediments, while the abundance of L. varians GY32 exhibited an opposite trend. Finally, the abundances of Geobacter, cable bacteria, and L. varians GY32 were positively correlated with the abundance of filamentous microorganisms (FMs) across all samples, with stronger interactions in polluted sediments. These findings suggest that EAMs demonstrate heightened sensitivity to polluted environments, particularly at the genus (species) level, and exhibit strong adaptability to conditions characterized by high levels of acid volatile sulfide, low dissolved oxygen, and elevated nitrate nitrogen. Therefore, environmental factors could be manipulated to optimize the growth and efficiency of EAMs for environmental engineering and natural restoration applications.

The Effects of Mixed Robinia pseudoacacia and Quercus variabilis Plantation on Soil Bacterial Community Structure and Nitrogen-Cycling Gene Abundance in the Southern Taihang Mountain Foothills.

Mixed forests often increase their stability and species richness in comparison to pure stands. However, a comprehensive understanding of the effects of mixed forests on soil properties, bacterial community diversity, and soil nitrogen cycling remains elusive. This study investigated soil samples from pure Robinia pseudoacacia stands, pure Quercus variabilis stands, and mixed stands of both species in the southern foothills of the Taihang Mountains. Utilizing high-throughput sequencing and real-time fluorescence quantitative PCR, this study analyzed the bacterial community structure and the abundance of nitrogen-cycling functional genes within soils from different stands. The results demonstrated that Proteobacteria, Acidobacteria, and Actinobacteria were the dominant bacterial groups across all three forest soil types. The mixed-forest soil exhibited a higher relative abundance of Firmicutes and Bacteroidetes, while Nitrospirae and Crenarchaeota were most abundant in the pure R. pseudoacacia stand soils. Employing FAPROTAX for predictive bacterial function analysis in various soil layers, this study found that nitrogen-cycling processes such as nitrification and denitrification were most prominent in pure R. pseudoacacia soils. Whether in surface or deeper soil layers, the abundance of AOB amoA, nirS, and nirK genes was typically highest in pure R. pseudoacacia stand soils. In conclusion, the mixed forest of R. pseudoacacia and Q. variabilis can moderate the intensity of nitrification and denitrification processes, consequently reducing soil nitrogen loss.

The Changes of Microbial Diversity and Isolation of Microorganism in Soil for Alleviating the Production Decreasing After Continuous Cultivation of Ganoderma lucidum.

Ganoderma lucidum is a medicinal mushroom usually cultivated in logs and covered with soil. Its production decreases after continuous cultivation. Changes of microbial diversity in soil are suggested to be one of the reasons. This study aims to investigate the changes of microbial diversity and abundance in soil during cultivation, and isolate potential microbial strains that affect the yield of G. lucidum. Soil samples were collected at two different ranges from logs during one complete growth cycle of G. lucidum. The changes in fungi and bacteria were investigated by using high-throughput sequencing and real-time PCR. Results indicated that the relative abundance of Firmicutes in the bacterial community decreased at the short-range site. In the fungal community, the relative abundance of Ganoderma increased to 70% at the long-range site at the end of the cultivation. The abundance of bacteria and fungi decreased significantly at the end of the growth cycle. Recovery of microbial changes in soil should be proceeded separately based on different ranges to logs. The microbial strains in these soil samples were also isolated and identified. Potential strains were assessed in the form of bio-fertilizer. The yield of G. lucidum in the field using bio-fertilizer with isolated bacterial strains from the Firmicutes phylum was about 13% higher than that without using bio-fertilizer, suggesting the possibility of alleviating the production decrease of G. lucidum by this method.

The distribution and key influential factors of antibiotic resistance genes in agricultural soils polluted by multiple heavy metals.

Due to anthropogenic activities such as mining, several agricultural soils are polluted by multiple heavy metals. However, it is still unclear whether multiple heavy metals could affect the distribution of antibiotic resistance genes (ARGs), and how metals affect ARGs. To understand ARGs' distribution in heavy metal-polluted soils, we chose soils contaminated by different types and contents of heavy metals to determine the ARGs' number and abundance through high-throughput quantitative real-time PCR (HT-qPCR) in this study. Additionally, the factors affecting ARGs' distribution, such as soil properties, mobile genetic genes (MGEs), and bacterial communities, were explored. The results demonstrated that the sampled soils were primarily contaminated by Cd, As, Pb, and Zn, and the pollution load index (PLI) values of these metals ranged from 1.3 to 2.7, indicating a low to moderate degree of heavy metal contamination. The number and abundance of ARGs ranged from 44 to 113 and from 2.74 × 107 copies/g to 1.07 × 108 copies/g, respectively. Besides, abundant MGEs in soils, ranging from 1.84 × 106 copies/g to 5.82 × 106 copies/g, were observed. The pathway analysis suggested that MGEs were the most important factor directly affecting ARG abundance (0.89). Notably, heavy metals also affected the ARG abundance. Proteobacteria and Actinobacteria, the main heavy metal tolerant bacteria, were found to be the main hosts of ARGs through network analysis. ARG-carrying pathogens (ACPs) in agricultural soils were found to carry MGEs, indicating a high risk of dissemination. This study provided important information for understanding the ARGs' fate and also the key factors affecting ARGs' spread in multiple heavy metal-contaminated soils.

16S rRNA amplicon sequencing and antimicrobial resistance profile of intensive care units environment in 41 Brazilian hospitals.

Infections acquired during healthcare setting stay pose significant public health threats. These infections are known as Healthcare-Associated Infections (HAI), mostly caused by pathogenic bacteria, which exhibit a wide range of antimicrobial resistance. Currently, there is no knowledge about the global cleaning process of hospitals and the bacterial diversity found in ICUs of Brazilian hospitals contributing to HAI. Characterize the microbiome and common antimicrobial resistance genes present in high-touch Intensive Care Unit (ICU) surfaces, and to identify the potential contamination of the sanitizers/processes used to clean hospital surfaces. In this national, multicenter, observational, and prospective cohort, bacterial profiles and several antimicrobial resistance genes from 41 hospitals across 16 Brazilian states were evaluated. Using high-throughput 16S rRNA amplicon sequencing and real-time PCR, the bacterial abundance and resistance genes presence were analyzed in both ICU environments and cleaning products. We identified a wide diversity of microbial populations with a recurring presence of HAI-related bacteria among most of the hospitals. The median bacterial positivity rate in surface samples was high (88.24%), varying from 21.62 to 100% in different hospitals. Hospitals with the highest bacterial load in samples were also the ones with highest HAI-related abundances. Streptococcus spp., Corynebacterium spp., Staphylococcus spp., Bacillus spp., Acinetobacter spp., and bacteria from the Flavobacteriaceae family were the microorganisms most found across all hospitals. Despite each hospital particularities in bacterial composition, clustering profiles were found for surfaces and locations in the ICU. Antimicrobial resistance genes mecA, bla KPC-like, bla NDM-like, and bla OXA-23-like were the most frequently detected in surface samples. A wide variety of sanitizers were collected, with 19 different active principles in-use, and 21% of the solutions collected showed viable bacterial growth with antimicrobial resistance genes detected. This study demonstrated a diverse and spread pattern of bacteria and antimicrobial resistance genes covering a large part of the national territory in ICU surface samples and in sanitizers solutions. This data should contribute to the adoption of surveillance programs to improve HAI control strategies and demonstrate that large-scale epidemiology studies must be performed to further understand the implications of bacterial contamination in hospital surfaces and sanitizer solutions.

Comparing Occurrence of Bovine Respiratory Pathogens Detected by High-Throughput Real-Time PCR in Nasal Swabs and Non-Endoscopic Bronchoalveolar Lavage Samples from Dairy and Veal Calves.

This study aimed to enhance our understanding of the agreement between two sampling methods for the detection of bovine respiratory disease (BRD) pathogens in calves using high-throughput real-time qPCR (ht-RT-qPCR). In total, 233 paired nasal swab (NS) and non-endoscopic bronchoalveolar lavage (nBAL) samples were collected from 152 calves from 12 Danish cattle herds. In 202 of the observations, the calves were examined using a standardized clinical protocol. Samples were tested for three viruses (bovine respiratory syncytial virus, bovine corona virus, and influenza D virus) and six bacteria (Histophilus somni, Mannheimia haemolytica, Mycoplasma bovis, Mycoplasma species, Pasteurella multocida, and Truepurella pyogenes). The results showed age-related differences in disease and pathogen occurrence, with the highest detection rates in calves aged 35 days or older. Poor to moderate agreement was found between the NS and nBAL results. The presence of Mannheimia haemolytica in both NS and nBAL in younger calves and in nBAL in older calves was associated with clinical BRD. There was a potential link between BRD and influenza D virus in older calves, although it was only found in one herd in a small sample size. Overall, NS was a relatively poor predictor of pathogens in the lower respiratory tract. The present study confirms the complexity of pathogen detection in BRD, with marked influences of age and the sampling method on pathogen detection and disease associations.

Spatial Distribution and Pathogen Profile of Dermacentor reticulatus Ticks in Southeastern Poland: A Genetic and Environmental Analysis

In recent years, significant changes have been observed in the distribution and abundance of local Dermacentor reticulatus populations. However, changes in D. reticulatus dynamics have not been studied in southeastern Poland. Our objective was to enhance our understanding of the environmental factors influencing the occurrence and density of D. reticulatus in this area. Additionally, we sought to investigate the genetic diversity of the tick population and the prevalence of tick-borne pathogens (TBPs). To this end, we established 45 study sites in the Subcarpathian province. Ticks were collected during their peak activity in both spring and autumn. A subset of randomly selected specimens underwent molecular analysis for TBPs screening, using high-throughput microfluidic real-time PCR. Positive amplicons were then sequenced, and phylogenetic analyses were conducted. Our findings confirmed the presence of D. reticulatus ticks in 24 surveyed sites, primarily concentrated in the northern and eastern parts of the region. The mean density of D. reticulatus ticks in their compact range was 5.8 ± 6.4 specimens/100 m2. Notably, air temperature and altitude emerged as significant factors influencing the species’ activity. We also identified a high prevalence of Rickettsia raoultii infections in adult D. reticulatus, reaching up to 84.21%. Additionally, 9.52% of ticks were found to be infected with R. helvetica and 4.76% with Anaplasma phagocytophilum. Furthermore, our genetic analyses confirmed the identity of D. reticulatus in the Subcarpathian region, aligning with haplotypes found in other regions of Poland, Czechia, Croatia, and Portugal. In conclusion, our study suggests that the surveyed region represents the current boundary of the compact range of D. reticulatus in Poland in which this tick species exhibits low genetic diversity and a narrow spectrum of detected TBPs.

Discrepant soil microbial community and C cycling function responses to conventional and biodegradable microplastics

Biodegradable microplastics (MPs) are promising alternatives to conventional MPs and are of high global concern. However, their discrepant effects on soil microorganisms and functions are poorly understood. In this study, polyethylene (PE) and polylactic acid (PLA) MPs were selected to investigate the different effects on soil microbiome and C-cycling genes using high-throughput sequencing and real-time quantitative PCR, as well as the morphology and functional group changes of MPs, using scanning electron microscopy and Fourier transform infrared spectroscopy, and the driving factors were identified. The results showed that distinct taxa with potential for MP degradation and nitrogen cycling were enriched in soils with PLA and PE, respectively. PLA, smaller size (150-180 μm), and 5% (w/w) of MPs enhanced the network complexity compared with PE, larger size (250-300 μm), and 1% (w/w) of MPs, respectively. PLA increased β-glucosidase by up to 2.53 times, while PE (150–180 μm) reduced by 38.26%–44.01% and PE (250–300 μm) increased by 19.00%–22.51% at 30 days. Amylase was increased by up to 5.83 times by PLA (150–180 μm) but reduced by 40.26%–62.96% by PLA (250–300 μm) and 16.11%–43.92% by PE. The genes cbbL, cbhI, abfA, and Lac were enhanced by 37.16%-1.99 times, 46.35%-26.46 times, 8.41%-69.04%, and 90.81%-5.85 times by PLA except for PLA1B/5B at 30 days. These effects were associated with soil pH, NO3-–N, and MP biodegradability. These findings systematically provide an understanding of the impact of biodegradable MPs on the potential for global climate change.

Arbuscular mycorrhizal fungal communities in soils where astragalus had grown for 2 years were similar to those in the abandoned farmland.

Astragalus-cultivated soils are enriched in arbuscular mycorrhizal fungi (AMF); however, the community changes of AMF between years in stragalus-cultivated soils are still unclear. To illustrate this, using high-throughput amplicon sequencing and quantitative real-time PCR, we analyzed the AMF communities of the abandoned farmlands and interannual astragalus-cultivated soils for 1-, 2-, 3-, and 4-years, including community composition, dominant, core, specific and significantly fluctuating AMF, co-occurrence network, alpha diversity, and beta diversity. A total of 74 OTUs were classified into one phylum, Glomeromycota; one class, Glomeromycetes; four orders; four families; and six genera. The 2-year soil had the highest number of reads among the interannual soils. Only one OTU was shared among all interannual soils. The treatments significantly affected the Ace, Shannoneven, and Shannon estimators of the communities. The 2-year soil had the highest richness, evenness, and diversity among all interannual soils and was the closest to the abandoned farmland in terms of alpha diversity. Glomus of the family Glomeraceae was the dominant genus present in all treatments, and the composition of the dominant genus in interannual soils was different. Both Glomus and Diversispora were the core AMF in interannual soils, and specific AMF existed in different interannual soils. Glomus is a genus that exhibits significant interannual variation. The interannual time significantly affected the network connectivity. The results of the principal coordinate analysis showed that the community composition of the interannual soils was close to each other and separated from the abandoned farmland, and that the interannual time significantly affected the community composition. Among the interannual soils, the 2-year soil may be more suitable for A. sinensis seedling rotation.

Microfluidic PCR and network analysis reveals complex tick-borne pathogen interactions in the tropics

BackgroundIxodid ticks, particularly Rhipicephalus sanguineus s.l., are important vectors of various disease-causing agents in dogs and humans in Cuba. However, our understading of interactions among tick-borne pathogens (TBPs) in infected dogs or the vector R. sanguineus s.l. remains limited. This study integrates microfluidic-based high-throughput real-time PCR data, Yule's Q statistic, and network analysis to elucidate pathogen-pathogen interactions in dogs and ticks in tropical western Cuba.MethodsA cross-sectional study involving 46 client-owned dogs was conducted. Blood samples were collected from these dogs, and ticks infesting the same dogs were morphologically and molecularly identified. Nucleic acids were extracted from both canine blood and tick samples. Microfluidic-based high-throughput real-time PCR was employed to detect 25 bacterial species, 10 parasite species, 6 bacterial genera, and 4 parasite taxa, as well as to confirm the identity of the collected ticks. Validation was performed through end-point PCR assays and DNA sequencing analysis. Yule's Q statistic and network analysis were used to analyse the associations between different TBP species based on binary presence-absence data.ResultsThe study revealed a high prevalence of TBPs in both dogs and R. sanguineus s.l., the only tick species found on the dogs. Hepatozoon canis and Ehrlichia canis were among the most common pathogens detected. Co-infections were observed, notably between E. canis and H. canis. Significant correlations were found between the presence of Anaplasma platys and H. canis in both dogs and ticks. A complex co-occurrence network among haemoparasite species was identified, highlighting potential facilitative and inhibitory roles. Notably, H. canis was found as a highly interconnected node, exhibiting significant positive associations with various taxa, including A. platys, and E. canis, suggesting facilitative interactions among these pathogens. Phylogenetic analysis showed genetic diversity in the detected TBPs.ConclusionsOverall, this research enhances our understanding of TBPs in Cuba, providing insights into their prevalence, associations, and genetic diversity, with implications for disease surveillance and management.Graphical abstract

Beach sand plastispheres are hotspots for antibiotic resistance genes and potentially pathogenic bacteria even in beaches with good water quality

Massive amounts of microplastics are transported daily from the oceans and rivers onto beaches. The ocean plastisphere is a hotspot and a vector for antibiotic resistance genes (ARGs) and potentially pathogenic bacteria. However, very little is known about the plastisphere in beach sand. Thus, to describe whether the microplastics from beach sand represent a risk to human health, we evaluated the bacteriome and abundance of ARGs on microplastic and sand sampled at the drift line and supralittoral zones of four beaches of poor and good water quality. The bacteriome was evaluated by sequencing of 16S rRNA gene, and the ARGs and bacterial abundances were evaluated by high-throughput real-time PCR. The results revealed that the microplastic harbored a bacterial community that is more abundant and distinct from that of beach sand, as well as a greater abundance of potential human and marine pathogens, especially the microplastics deposited closer to seawater. Microplastics also harbored a greater number and abundance of ARGs. All antibiotic classes evaluated were found in the microplastic samples, but not in the beach sand ones. Additionally, 16 ARGs were found on the microplastic alone, including genes related to multidrug resistance (blaKPC, blaCTX-M, tetM, mdtE and acrB_1), genes that have the potential to rapidly and horizontally spread (blaKPC, blaCTX-M, and tetM), and the gene that confers resistance to antibiotics that are typically regarded as the ultimate line of defense against severe multi-resistant bacterial infections (blaKPC). Lastly, microplastic harbored a similar bacterial community and ARGs regardless of beach water quality. Our findings suggest that the accumulation of microplastics in beach sand worldwide may constitute a potential threat to human health, even in beaches where the water quality is deemed satisfactory. This phenomenon may facilitate the emergence and dissemination of bacteria that are resistant to multiple drugs.

High-throughput screening of pathogens in Ixodes ricinus removed from hosts in Lombardy, northern Italy

Ticks are important vectors of many pathogens in Europe, where the most impactful species is Ixodes ricinus. Recently, the geographical distribution of this tick species has been expanding, resulting in an increased risk of human exposure to tick bites. With the present study, we aimed to screen 350 I. ricinus specimens collected from humans and wild animals (mainly ungulates), to have a broader understanding of the tick-borne pathogens circulating in the Lombardy region, in northern Italy. To do so, we took advantage of a high-throughput real-time microfluidic PCR approach to screen ticks in a cost-effective and time-saving manner. Molecular analysis of the dataset revealed the presence of four genera of bacteria and two genera of protozoa: in ungulates, 77 % of collected ticks carried Anaplasma phagocytophilum, while the most common pathogen species in ticks removed from humans were those belonging to Borrelia burgdorferi sensu lato group (7.6 %). We also detected other pathogenic microorganisms, such as Rickettisa monacensis, Rickettsia helvetica, Neoehrlichia mikurensis, Babesia venatorum, and Hepatozoon martis. Besides, we also reported the presence of the pathogenic agent Borrelia miyamotoi in the area (1.4 % overall). The most common dual co-infection detected in the same tick individual involved A. phagocytophilum and Rickettsia spp. Our study provided evidence of the circulation of different tick-borne pathogens in a densely populated region in Italy.

Detection of bacterial and protozoan pathogens in individual bats and their ectoparasites using high-throughput microfluidic real-time PCR.

Among the most studied mammals in terms of their role in the spread of various pathogens with possible zoonotic effects are bats. These are animals with a very complex lifestyle, diet, and behavior. They are able to fly long distances, thus maintaining and spreading the pathogens they may be carrying. These pathogens also include vector-borne parasites and bacteria that can be spread by ectoparasites such as ticks and bat flies. In the present study, high-throughput screening was performed and we detected three bacterial pathogens: Bartonella spp., Neoehrlichia mikurensis and Mycoplasma spp., and a protozoan parasite: Theileria spp. in paired samples from bats (blood and ectoparasites). In the samples from the bat-arthropod pairs, we were able to detect Bartonella spp. and Mycoplasma spp. which also showed a high phylogenetic diversity, demonstrating the importance of these mammals and the arthropods associated with them in maintaining the spread of pathogens. Previous studies have also reported the presence of these pathogens, with one exception, Neoehrlichia mikurensis, for which phylogenetic analysis revealed less genetic divergence. High-throughput screening can detect more bacteria and parasites at once, reduce screening costs, and improve knowledge of bats as reservoirs of vector-borne pathogens. IMPORTANCE The increasing number of zoonotic pathogens is evident through extensive studies and expanded animal research. Bats, known for their role as reservoirs for various viruses, continue to be significant. However, new findings highlight the emergence of Bartonella spp., such as the human-infecting B. mayotimonensis from bats. Other pathogens like N. mikurensis, Mycoplasma spp., and Theileria spp. found in bat blood and ectoparasites raise concerns, as their impact remains uncertain. These discoveries underscore the urgency for heightened vigilance and proactive measures to understand and monitor zoonotic pathogens. By deepening our knowledge and collaboration, we can mitigate these risks, safeguarding human and animal well-being.

Monitoring bovine dairy calf health and related risk factors in the first three months of rearing

BackgroundRearing replacement heifers is pivotal for the dairy industry and is associated with high input costs for the preweaned calves, due to their higher susceptibility to diseases. Ensuring calf health and viability calls for systematic approaches in order to mitigate the costs induced by managing sick calves and to ensure animal welfare. The objective of this study was to develop a systematic and feasible health-monitoring tool for bovine dairy calves based on repeated clinical observations and diagnostic results of calves at three time points; the 1st (T0), the 3rd (T1) and the 12th (T3) week of age. The study included observations from 77 dairy heifer calves in nine Danish commercial dairy herds. Immunisation status was assessed by serum Brix% at T0. Clinical scoring included gastrointestinal disease (GD) and respiratory disease (RD). The average daily weight gain (ADWG) was estimated from heart-girth measurements. Pathogen detection from nasal swabs and faecal samples were analysed for 16 respiratory and enteric pathogens by means of high-throughput real time-PCR. All measures obtained in each herd were visualised in a panel to follow the health status of each calf over time.ResultsThe individual clinical observations combined with diagnostic information from immunisation and pathogen detection form each enrolled calf are presented in a herd dashboard illustrating the health status over the study period. This monitoring revealed failure of passive transfer (Brix% < 8.1) in 31% of the 77 enrolled calves, signs of severe GD peaked at T0 with 20% affected calves, while signs of severe RD peaked at T2 with 42% affected calves. ADWG over the first eight weeks was estimated to be 760 g (± 190 g). Pathogen profiles varied between herds.ConclusionsThe large variation in both clinical disease and pathogen occurrence across herds emphasizes the need for herd specific monitoring. Combining the results of the present study from measures of immunisation, health and growth from individual calves in one visualisation panel allowed for the detection of patterns across age groups in the specific herds, showing promising potential for early detection and interventions that can lead to enhanced calf health and welfare.