Diversity Of Pathogens Research Articles

Genetic diversity of vector-borne pathogens in ixodid ticks infesting dogs from Pakistan with notes on Ehrlichia canis, Rickettsia raoultii and Dirofilaria immitis detection

BackgroundVector-/tick-borne pathogens (V/TBPs) pose a potential threat to human and animal health globally. Information regarding canine V/TBPs is scarce and no specific study has been conducted so far to explore the microbial diversity within ticks infesting dogs from Pakistan. Herein, this knowledge gap is addressed by assessing the genetic diversity and prevalence pattern of V/TBPs in ixodid ticks with special implications for public and canine health.MethodsA total of 1150 hard ticks were collected from 300 dogs across central Khyber Pakhtunkhwa (KP), Pakistan. After morpho-molecular identification, 120 tick samples were screened for the presence of V/TBPs by amplifying 16S rRNA/gltA (Rickettsia/Ehrlichia and Wolbachia sp.), 18S rRNA (Theileria sp.) and cox1 (Dirofilaria sp.) genes through PCR followed by sequencing and phylogenetic study.ResultsIn toto, 50 ixodid ticks (50/120, 41.7%) were found positive for V/TBPs DNA. The detected V/TBPs were categorized into five genera and eight species, viz. Ehrlichia (E. canis and Ehrlichia sp.), Rickettsia (R. massiliae, R. raoultii and Rickettsia sp.), Theileria (T. annulata), Dirofilaria (D. immitis) and Wolbachia (Wolbachia sp.). The pathogen prevalence patterns showed that R. massiliae was the most prevalent zoonotic V/TBP (19.5%), followed by E. canis (10.8%), Rickettsia sp. (7.5%), R. raoultii (6.7%), T. annulata (5.8%), D. immitis (5.8%), Wolbachia sp. (4.2%) and Ehrlichia sp. (3.3%), respectively. Among the screened tick species, most Rhipicephalus sanguineus sensu lato samples were found positive for V/TBP DNA (20/20,100%) followed by Rh. turanicus sensu stricto (13/20, 65%), Hyalomma dromedarii (8/20, 40%), Rh. haemaphysaloides (6/20, 30%), Hy. excavatum (2/20, 10%) and Rh. microplus (1/20, 5%). Co-occurrence of V/TBP was also detected in tick specimens (single V/TBP infection: 32 ticks; double and triple: 13 and 5 tick samples). The detected pathogens shared a phylogenetic relationship with similar isolates published in NCBI GenBank from Old and New World countries.ConclusionIxodid ticks infesting dogs harbor a diverse array of V/TBPs including zoonotic agents from Pakistan. Furthermore, the presence of D. immitis in ticks that infest dogs raises the possibility that this parasite has either attained its dead-end host (i.e. the tick) while feeding on dogs or has expanded its range of intermediate/paratenic hosts. Further research work is needed to investigate the epidemiology and confirm the vector competence of screened tick species for these pathogens from Pakistan.Graphical

Hiding in plain sight: Genome-wide recombination and a dynamic accessory genome drive diversity in Fusarium oxysporum f.sp. ciceris

Understanding the origins of variation in agricultural pathogens is of fundamental interest and practical importance, especially for diseases that threaten food security. Fusarium oxysporum is among the most important of soil-borne pathogens, with a global distribution and an extensive host range. The pathogen is considered to be asexual, with horizontal transfer of chromosomes providing an analog of assortment by meiotic recombination. Here, we challenge those assumptions based on the results of population genomic analyses, describing the pathogen's diversity and inferring its origins and functional consequences in the context of a single, long-standing agricultural system. We identify simultaneously low nucleotide distance among strains, and unexpectedly high levels of genetic and genomic variability. We determine that these features arise from a combination of genome-scale recombination, best explained by widespread sexual reproduction, and presence-absence variation consistent with chromosomal rearrangement. Pangenome analyses document an accessory genome more than twice the size of the core genome, with contrasting evolutionary dynamics. The core genome is stable, with low diversity and high genetic differentiation across geographic space, while the accessory genome is paradoxically more diverse and unstable but with lower genetic differentiation and hallmarks of contemporary gene flow at local scales. We suggest a model in which episodic sexual reproduction generates haplotypes that are selected and then maintained through clone-like dynamics, followed by contemporary genomic rearrangements that reassort the accessory genome among sympatric strains. Taken together, these processes contribute unique genome content, including reassortment of virulence determinants that may explain observed variation in pathogenic potential.

Potent antibacterial activity in surgical wounds with local administration of D-PLEX100

Despite significant advances in infection control guidelines and practices, surgical site infections remain a substantial cause of morbidity, prolonged hospitalization, and mortality. The most effective component of SSI reduction strategies is the preoperative administration of intravenous antibiotics; however, systemic antibiotics drug exposure diminishes rapidly and may result in insufficient prophylactic activity against susceptible and resistant SSI pathogens at the wound. D-PLEX100 (D-PLEX) is an antibiotic-releasing drug (doxycycline) that is supplied as a sterile powder for paste reconstitution with sterile saline. D-PLEX paste is administered locally into the incision site along the entire length of soft tissue and sternal bone wound surfaces prior to skin closure. A single D-PLEX administration is intended for 30 days of constant antimicrobial prophylaxis in the prevention of incisional SSIs. We evaluated D-PLEX minimal bactericidal concentration (MBC) against a panel of bacteria that is prevalent in the abdominal wall and sternal surgical procedures including doxycycline susceptible and resistant strains. D-PLEX in vivo efficacy was assessed in incisional infection rabbit models (abdominal wall and sternal) challenged with a similar bacterial panel. The D-PLEX drug exposure profile was determined by in vitro release assay, and in vivo by quantitative pharmacokinetic parameters of local and systemic doxycycline concentrations released from D-PLEX after local administration in incisional rabbit models. Analyses of pathogens and variations in antibiotic resistance from wound isolates were determined from patients who participated in a previously reported prospective randomized trial that assessed the SSI rate in D-PLEX plus standard of care (SOC) versus SOC alone in colorectal resection surgery. The D-PLEX MBC values demonstrated >3- Log10 reduction in all the organisms tested relative to untreated controls, including doxycycline-resistant bacteria (i.e., Methicillin-resistant Staphylococcus aureus (MRSA), K. pneumoniae, and P. aeruginosa). In vivo, D-PLEX significantly reduced the bacterial loads in all the bacteria tested in both animal models (p=0.0001) with a marked impact observed in E. Coli (>6.5 Log10 reduction). D-PLEX exhibited a zero-order release kinetics profile in vitro for 30 days (R2 = 0.971) and the matched in vivo release profile indicated a constant local release of protein-unbound doxycycline for 30 days at 3-5 mcg/mL with significantly lower (>3 orders of magnitudes) systemic levels. In colorectal surgery patients, where significant SSI reduction was observed, analysis of the positive cultures in the overall population indicated similar pathogen diversity and antibiotic resistance rates in both treatment arms. However, almost all the patients with positive culture in the SOC arm were adjudicated as SSI (94%) compared to only 28% in the D-PLEX arm. The SSI-adjudicated D-PLEX patients also exhibited lower resistance rates to the SOC antibiotics and to MDRs compared to patients in the SOC arm. Thus, D-PLEX provides safe and effective prophylaxis activity against the most prevalent SSI pathogens including doxycycline-susceptible and resistant bacteria. Our findings suggest that D-PLEX is a promising addition to SSI prophylactic bundles and may address the gaps in current SSI prophylaxis. D-PLEX is now evaluated in Phase 3 clinical trial.

ReporTree: a surveillance-oriented tool to strengthen the linkage between pathogen genetic clusters and epidemiological data

BackgroundGenomics-informed pathogen surveillance strengthens public health decision-making, playing an important role in infectious diseases’ prevention and control. A pivotal outcome of genomics surveillance is the identification of pathogen genetic clusters and their characterization in terms of geotemporal spread or linkage to clinical and demographic data. This task often consists of the visual exploration of (large) phylogenetic trees and associated metadata, being time-consuming and difficult to reproduce.ResultsWe developed ReporTree, a flexible bioinformatics pipeline that allows diving into the complexity of pathogen diversity to rapidly identify genetic clusters at any (or all) distance threshold(s) or cluster stability regions and to generate surveillance-oriented reports based on the available metadata, such as timespan, geography, or vaccination/clinical status. ReporTree is able to maintain cluster nomenclature in subsequent analyses and to generate a nomenclature code combining cluster information at different hierarchical levels, thus facilitating the active surveillance of clusters of interest. By handling several input formats and clustering methods, ReporTree is applicable to multiple pathogens, constituting a flexible resource that can be smoothly deployed in routine surveillance bioinformatics workflows with negligible computational and time costs. This is demonstrated through a comprehensive benchmarking of (i) the cg/wgMLST workflow with large datasets of four foodborne bacterial pathogens and (ii) the alignment-based SNP workflow with a large dataset of Mycobacterium tuberculosis. To further validate this tool, we reproduced a previous large-scale study on Neisseria gonorrhoeae, demonstrating how ReporTree is able to rapidly identify the main species genogroups and characterize them with key surveillance metadata, such as antibiotic resistance data. By providing examples for SARS-CoV-2 and the foodborne bacterial pathogen Listeria monocytogenes, we show how this tool is currently a useful asset in genomics-informed routine surveillance and outbreak detection of a wide variety of species.ConclusionsIn summary, ReporTree is a pan-pathogen tool for automated and reproducible identification and characterization of genetic clusters that contributes to a sustainable and efficient public health genomics-informed pathogen surveillance. ReporTree is implemented in python 3.8 and is freely available at https://github.com/insapathogenomics/ReporTree.

The Genetic Diversity and Antimicrobial Resistance of Pyogenic Pathogens Isolated from Porcine Lymph Nodes

According to the Food and Agriculture Organization of the United Nations, pork remains the most consumed meat in the world. Consequently, it is very important to ensure that it is of the highest microbiological quality. Many of the pathogens that cause lymph node lesions in pigs are zoonotic agents, and the most commonly isolated bacteria are Mycobacterium spp., Streptococcus spp., Staphylococcus aureus and Rhodococcus equi (synonymous with Prescottella equi). The prevention and treatment of zoonotic infections caused by these bacteria are mainly based on antimicrobials. However, an overuse of antimicrobials contributes to the emergence and high prevalence of antimicrobial-resistant strains, which are becoming a serious challenge in many countries. The aim of this study was to evaluate the genetic diversity and antimicrobial resistance of the Streptococcus spp. (n = 48), S. aureus (n = 5) and R. equi (n = 17) strains isolated from swine lymph nodes with and without lesions. All isolates of S. dysgalactiae, S. aureus and R. equi were subjected to PFGE analysis, which showed the genetic relatedness of the tested bacteria in the studied pig populations. Additionally, selected tetracycline and macrolide resistance genes in the streptococcal strains were also studied. The results obtained in the present study provide valuable data on the prevalence, diversity, and antimicrobial resistance of the studied bacteria. Numerous isolated bacterial Streptococcus spp. strains presented resistance to doxycycline, and almost half of them carried tetracycline resistance genes. In addition, R. equi and S. aureus bacteria presented a high level of resistance to beta-lactam antibiotics and to cefotaxime, respectively.

Real-time detection and analysis of foodborne pathogens via machine learning based fiber-optic Raman sensor

Food safety is a critical concern worldwide, and in recent years, it has become a growing public concern in the United States due to mass production and multi-channel distribution of high-nutrient and fresh-cut foods that have increased the prevalence and diversity of foodborne pathogens (e.g., Escherichia coli, Salmonella, Listeria, etc.). Traditional methods for the detection of these pathogens rely heavily on low-efficiency techniques, which may expose the public to contaminated foods that have not been tested or identified. Therefore, rapid, simple, sensitive, and inexpensive detection methods are urgently needed for food safety investigations with higher efficiency and accuracy. One of the promising methods for the detection of foodborne pathogens is Raman spectroscopy, which is a molecular-level analytical tool with the advantages of high selectivity and sensitivity and simple operation at a relatively low cost. In this study, a novel fiber-optic-based portable Raman probe was developed and used for real-time detection of a panel of pathogen-specific molecular fingerprint volatile organic compounds (VOCs). Furthermore, machine learning (ML) algorithms were applied to assist in the extraction of molecular information from the raw Raman spectra, resulting in high accuracy prediction of complex VOC mixtures, even at high dilution folds (100x). The developed ML-assisted Raman probe has immense potential for rapid on-site detection of complex chemical mixtures in food safety and beyond. This innovative approach achieves high accuracy in comprehensively sorting mixed chemicals with varying concentrations and provides several advantages over previous studies, including speed, portability, non-contact operation, and precise classification of foodborne pathogen VOCs.

Triplex qPCR assay for Campylobacter jejuni and Campylobacter coli monitoring in wastewater

Campylobacter spp. is one of the most frequent pathogens of bacterial gastroenteritis recorded worldwide. Campylobacter jejuni (C. jejuni) and Campylobacter coli (C. coli) are the two major disease-associated species, accounting for >95 % of infections, and thus have been selected for disease surveillance. Monitoring temporal variations in pathogen concentration and diversity excreted from community wastewater allows the early detection of outbreaks. Multiplex real-time/quantitative PCR (qPCR) enables multi-target quantification of pathogens in various types of samples including wastewater. Also, an internal amplification control (IAC) is required for each sample when adopting PCR-based methods for pathogen detection and quantification in wastewater to exclude the inhibition of the wastewater matrix. To achieve reliable quantification of C. jejuni and C. coli towards wastewater samples, this study developed and optimized a triplex qPCR assay by combining three qPCR primer-probe sets targeting Campylobacter jejuni subsp. jejuni, Campylobacter coli, and Campylobacter sputorum biovar sputorum (C. sputorum), respectively. This triplex qPCR assay not only can directly and simultaneously detect the concentration of C. jejuni and C. coli in wastewater but also can achieve the PCR inhibition control using C. sputorum primer-probe set. This is the first developed triplex qPCR assay with IAC for C. jejuni and C. coli, to be used in the wastewater-based epidemiology (WBE) applications. The optimized triplex qPCR assay enables the detection limit of the assay (ALOD100%) and wastewater (PLOD80%) as 10 gene copy/μL and 2 log10 cells/mL (2 gene copies/μL of extracted DNA), respectively. The application of this triplex qPCR to 52 real raw wastewater samples from 13 wastewater treatment plants demonstrated its potential as a high-throughput and economically viable tool for the long-term monitoring of C. jejuni and C. coli prevalence in communities and the surrounding environments. This study provided an accessible methodology and a solid foundation for WBE-based monitoring of Campylobacter spp. relevant diseases and paved the road for future WBE back-estimation of C. jejuni and C. coli prevalence.

Clostridium perfringens-Opportunistic Foodborne Pathogen, Its Diversity and Epidemiological Significance.

The C. perfringens species is associated with various environments, such as soils, sewage, and food. However, it is also a component of the gastrointestinal (GI) microflora (i.e., microbiota) of sick and healthy humans and animals. C. perfringens is linked with different systemic and enteric diseases in livestock and humans, such as gas gangrene, food poisoning, non-foodborne diarrhoea, and enterocolitis. The strains of this opportunistic pathogen are known to secrete over 20 identified toxins that are considered its principal virulence factors. C. perfringens belongs to the anaerobic bacteria community but can also survive in the presence of oxygen. The short time between generations, the multi-production capability of toxins and heat-resistant spores, the location of many virulence genes on mobile genetic elements, and the inhabitance of this opportunistic pathogen in different ecological niches make C. perfringens a very important microorganism for public health protection. The epidemiological evidence for the association of these strains with C. perfringens-meditated food poisoning and some cases of non-foodborne diseases is very clear and well-documented. However, the genetic diversity and physiology of C. perfringens should still be studied in order to confirm the importance of suspected novel virulence traits. A very significant problem is the growing antibiotic resistance of C. perfringens strains. The aim of this review is to show the current basic information about the toxins, epidemiology, and genetic and molecular diversity of this opportunistic pathogen.

Population genomics and pathotypic evaluation of the bacterial leaf blight pathogen of rice reveals rapid evolutionary dynamics of a plant pathogen.

The Xanthomonas oryzae pv. oryzae (Xoo) is a bacterial pathogen causing bacterial blight disease in rice, resulting in significant yield reductions of up to 50% in rice production. Despite its serious threat to food production globally, knowledge of its population structure and virulence evolution is relatively limited. In this study, we employed whole-genome sequencing to explore the diversity and evolution of Xoo in the main rice-growing areas of China over the past 30 years. Using phylogenomic analysis, we revealed six lineages. CX-1 and CX-2 primarily contained Xoo isolates from South China, while CX-3 represented Xoo isolates from North China. Xoo isolates belonging to CX-5 and CX-6 were the most prevalent across all studied areas, persisting as dominant lineages for several decades. Recent sporadic disease outbreaks were primarily caused by Xoo isolates derived from the two major lineages, CX-5 and CX-6, although Xoo isolates from other lineages also contributed to these outbreaks. The lineage and sub-lineage distributions of Xoo isolates were strongly correlated with their geographical origin, which was found to be mainly determined by the planting of the two major rice subspecies, indica and japonica. Moreover, large-scale virulence testing was conducted to evaluate the diversity of pathogenicity for Xoo. We found rapid virulence evolution against rice, and its determinant factors included the genetic background of Xoo, rice resistance genes, and planting environment of rice. This study provides an excellent model for understanding the evolution and dynamics of plant pathogens in the context of their interactions with their hosts, which are shaped by a combination of geographical conditions and farming practices. The findings of this study may have important implications for the development of effective strategies for disease management and crop protection in rice production systems.

Prevalence and antimicrobial resistance profile of bacterial foodborne pathogens in Nile tilapia fish (Oreochromis niloticus) at points of retail sale in Nairobi, Kenya

Proteus spp., Staphylococcus spp., Pseudeomonas spp., and pathogenic Vibrios are among the major foodborne pathogens associated with the consumption of contaminated fish. The increasing occurrence of antimicrobial resistance in these pathogens is a serious public health concern globally and therefore continuous monitoring of antimicrobial resistance of these bacteria along the food chain is crucial for for control of foodborne illnesses. The aim of this study was to assess the prevalence, antimicrobial resistance patterns, antibiotic resistance genes, and genetic diversity of bacterial foodborne pathogens recovered from fresh Nile tilapia (Oreochromis niloticus) obtained from retail markets in Nairobi, Kenya. A total of 68 O. niloticus fish with an average weight of 300.12 ± 25.66 g and body length of 23.00 ± 0.82 cm were randomly sampled from retail markets and tested for the presence of Proteus, Staphylococcus aureus, Pseudomonas aeruginosa, Vibrio cholerae, and Vibrio parahaemolyticus. Standard culture-based microbiological and Kirby–Bauer agar disk diffusion methods were used to isolate and determine the antimicrobial resistance patterns of the isolates to 11 selected antibiotics. Statistical analysis was performed using Minitab v17.1, with p < 0.05 considered significant. The genetic diversity of the multidrug-resistant (MDR) and extensively drug-resistant (XDR) bacteria was determined using 16S rRNA sequencing and phylogenetic analysis, and polymerase chain reaction (PCR) was used for detection of antibiotic resistance genes in MDR bacterial isolates. High levels of bacterial contamination were detected in fresh O. niloticus fish (44/68, 64.71%). The most prevalent bacteria were Proteus spp. (44.12%), with the rest of the bacterial species registering a prevalence of 10.29%, 4.41%, 2.94%, and 2.94% (for S. aureus, P. aeruginosa, V. cholerae, and V. parahaemolyticus, respectively). Antimicrobial resistance was detected in all the bacteria species and all the isolates were resistant to at least one antibiotic except cefepime (30 µg). Additionally, 86.36% of the isolates exhibited multidrug resistance, with higher multiple antibiotic resistance indices (MAR index >0.3) indicating that fresh O. niloticus fish were highly contaminated with MDR bacteria. Results of 16S rRNA sequences, BLASTn analysis, and phylogenetic trees confirmed the identified MDR bacterial isolates as Proteus mirabilis and other Proteus spp., S. aureus, P. aeruginosa, V. cholerae, and V. parahaemolyticus. PCR analysis confirmed the presence of multiple antibiotic resistance genes blaTEM-1, blaCMY-2, tetA, tetC, Sul2, dfrA7, strA, and aadA belonging to β-lactamases, tetracycline, sulfonamide, trimethoprim, and aminoglycosides in all the MDR bacterial isolates. There was strong correlation between antibiotic- resistant genes and phenotypic resistance to antibiotics of MDR bacteria. This study showed high prevalence of multidrug resistance among foodborne bacterial isolates from fresh O. niloticus fish obtained from retail markets. From this study, we conclude that fresh O. niloticus fish are a potential source of MDR bacteria, which could be a major risk to public health as a consequence of their dissemination along the human food chain. These results highlight the prevalence of antimicrobial-resistant foodborne pathogens in fish purchased from retail markets and underscore the risk associated with improper handling of fish.

PPE Barcoding Identifies Biclonal Mycobacterium ulcerans Buruli Ulcer, Côte d'Ivoire.

Mycobacterium ulcerans, an environmental opportunistic pathogen, causes necrotic cutaneous and subcutaneous lesions, named Buruli ulcers, in tropical countries. PCR-derived tests used to detect M. ulcerans in environmental and clinical samples do not allow one-shot detection, identification, and typing of M. ulcerans among closely related Mycobacterium marinum complex mycobacteria. We established a 385-member M. marinum/M. ulcerans complex whole-genome sequence database by assembling and annotating 341 M. marinum/M. ulcerans complex genomes and added 44 M. marinum/M. ulcerans complex whole-genome sequences already deposited in the NCBI database. Pangenome, core genome, and single-nucleotide polymorphism (SNP) distance-based comparisons sorted the 385 strains into 10 M. ulcerans taxa and 13 M. marinum taxa, correlating with the geographic origin of strains. Aligning conserved genes identified one PPE (proline-proline-glutamate) gene sequence to be species and intraspecies specific, thereby genotyping the 23 M. marinum/M. ulcerans complex taxa. PCR sequencing of the PPE gene correctly genotyped nine M. marinum/M. ulcerans complex isolates among one M. marinum taxon and three M. ulcerans taxa in the African taxon (T2.4). Further, successful PPE gene PCR sequencing in 15/21 (71.4%) swabs collected from suspected Buruli ulcer lesions in Côte d'Ivoire exhibited positive M. ulcerans IS2404 real-time PCR and identified the M. ulcerans T2.4.1 genotype in eight swabs and M. ulcerans T2.4.1/T2.4.2 mixed genotypes in seven swabs. PPE gene sequencing could be used as a proxy for whole-genome sequencing for the one-shot detection, identification, and typing of clinical M. ulcerans strains, offering an unprecedented tool for identifying M. ulcerans mixed infections. IMPORTANCE We describe a new targeted sequencing approach that characterizes the PPE gene to disclose the simultaneous presence of different variants of a single pathogenic microorganism. This approach has direct implications on the understanding of pathogen diversity and natural history and potential therapeutic implications when dealing with obligate and opportunistic pathogens, such as Mycobacterium ulcerans presented here as a prototype.

A 5-Year Review of Coinfections in Acanthamoeba keratitis From South India.

To ascertain the frequency of coinfections in Acanthamoeba keratitis, the nature of copathogens involved, and to analyze the implications in the context of current research on amoebic interactions. A retrospective case review from a Tertiary Care Eye Hospital in South India. Smear and culture data for coinfections in Acanthamoeba corneal ulcers were collected from records over a 5-year period. The significance and relevance of our findings in the light of current research on Acanthamoeba interactions were analyzed. Eighty-five cases of culture-positive Acanthamoeba keratitis were identified over a 5-year period (43 of them being coinfections). Fusarium was most commonly identified species, followed by Aspergillus and the dematiaceous fungi. Pseudomonas spp was the commonest bacterial isolate. Coinfections with Acanthamoeba are common at our centre, and account for 50% of Acanthamoeba keratitis. The diverse nature of the organisms involved in coinfections suggest that such amoebic interactions with other organisms are probably more widespread than recognized. To the best of our knowledge, this is the first documentation from a long-term study of pathogen diversity in Acanthamoeba coinfections. It is possible that Acanthamoeba itself may be virulence enhanced and secondary to the co-organism, breaching the ocular surface defenses in an already compromised cornea. However, observations from the existing literature on Acanthamoeba interactions with bacteria and certain fungi are based mainly on nonocular or nonclinical isolates. It would be illuminating if such studies are performed on Acanthamoeba and coinfectors from corneal ulcers-to ascertain whether interactions are endosymbiotic or virulence enhanced through amoebic passage.

Net cleaning impacts Atlantic salmon gill health through microbiome dysbiosis

IntroductionNet biofouling has a significant impact for the global salmon industry in the seawater grow-out stage in terms of its management. Current mitigation strategies occur primarily through the regular removal of biofouling using in situ cleaning. While in situ net cleaning is effective there is uncertainty as to whether the equipment or dispersed material has an impact upon the fish in the cages. Through direct contact with the environment, the significant surface area of the gill including its microbiome is directly exposed to the acute environmental changes generated by net cleaning. This study aimed to provide a detailed understanding of the impact of in situ net cleaning on Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) gill health.MethodsThree field trials were conducted on commercial fish farms in western Norway. Fouling organisms on net pens and flushed particles during in situ cleaning were identified and screened for major fish pathogens. Hydrographic profile measurements were performed to measure the impact on water quality. Gill samples were examined for histopathological changes, immune gene expression, and the prevalence of major pathogens. 16S rRNA amplicon sequencing was employed to explore the impact of net cleaning on gill microbiome.Results and discussionData obtained from these trials identified a diversity of fouling species including hydroids, algae, skeleton shrimps, and filter feeders on net pens, a direct impact on measured water quality indicators, a moderate change in gill inflammatory and antigen presentation activity at the level of mRNA, and a large significant change in gill microbiome. Observed changes in gill microbial community involved a decrease in bacterial richness coupled to an increase in identified bacterial genera related to negative health consequences. Parallel analyses for pathogens load in biofouling organisms and flushed particles highlighted the presence of several fish bacteria and parasites. However, minor changes were detected in salmon gill pathogen diversity and loading. Our results suggest that biofouling organisms may act as transient reservoirs for some fish pathogens but not viruses and that gill microbial dysbiosis could be related to the host stress response during and post net cleaning.

Maintaining Warp Speed: Policy Requirements for a Just-in-Time, Capability-Based, Scalable Medical Countermeasure Research and Development Enterprise.

Maintaining Warp Speed: Policy Requirements for a Just-in-Time, Capability-Based, Scalable Medical Countermeasure Research and Development Enterprise.

Anthropogenic and natural disturbances along a river and its estuary alter the diversity of pathogens and antibiotic resistance mechanisms

Antibiotic resistance (AMR) in pathogens threatens human health worldwide, and antibiotic-resistant bacteria (ARB) are widespread in the environment. In particular, anthropogenically-disturbed rivers became reservoirs of ARBs and hotspots of antibiotic resistance gene (ARG) transmission. However, the diversity and sources of ARB, and the mechanisms of ARG transmission are not fully known. Here, we used deep metagenomic sequencing to study the dynamics of pathogens and their antibiotic resistance mechanisms along the Alexander River (Israel), affected by sewage and animal farm runoffs. Putative pathogens such as Aeromicrobium marinum and Mycobacterium massilipolynesiensis were enriched in western stations, following the inputs of polluted Nablus River. Aeromonas veronii was dominant in eastern stations in Spring. Several AMR mechanisms showed distinct patterns in Summer-Spring (dry season) and Winter (rainy season). We found low abundance beta-lactamases conferring carbapenem resistance: e.g., OXA-912 was linked to A. veronii in Spring; OXA-119 and OXA-205 to Xanthomonadaceae in Winter. We classified 33 % of ARG-containing contigs as putative plasmid sequences, indicating the high potential for resistome transmission. A limited number of ARGs were linked to putative phages. Our results suggest that this model river is a hotspot for AMR activity and transmission, and highlight the merit of deep sequencing for AMR discovery.

Integrative population genetics and metagenomics reveals urbanization increases pathogen loads and decreases connectivity in a wild bee.

As urbanization continues to increase, it is expected that two-thirds of the human population will reside in cities by 2050. Urbanization fragments and degrades natural landscapes, threatening wildlife including economically important species such as bees. In this study, we employ whole genome sequencing to characterize the population genetics, metagenome and microbiome, and environmental stressors of a common wild bee, Ceratina calcarata. Population genomic analyses revealed the presence of low genetic diversity and elevated levels of inbreeding. Through analyses of isolation by distance, resistance, and environment across urban landscapes, we found that green spaces including shrubs and scrub were the most optimal pathways for bee dispersal, and conservation efforts should focus on preserving these land traits to maintain high connectivity across sites for wild bees. Metagenomic analyses revealed landscape sites exhibiting urban heat island effects, such as high temperatures and development but low precipitation and green space, had the highest taxa alpha diversity across all domains even when isolating for potential pathogens. Notably, the integration of population and metagenomic data showed that reduced connectivity in urban areas is not only correlated with lower relatedness among individuals but is also associated with increased pathogen diversity, exposing vulnerable urban bees to more pathogens. Overall, our combined population and metagenomic approach found significant environmental variation in bee microbiomes and nutritional resources even in the absence of genetic differentiation, as well as enabled the potential early detection of stressors to bee health.

Large‐scale eDNA monitoring of multiple aquatic pathogens as a tool to provide risk maps for wildlife diseases

AbstractMultiple parasites and pathogens cause disease in aquatic wildlife and in aquaculture species, generating a need for monitoring and management. Conventional disease monitoring methods involve laborious, costly, and invasive capture and examination of host species, and require specialized expertise for every host and pathogen of interest. Environmental DNA could provide simultaneous occurrence data for multiple pathogens across different host taxa, valuable for using parasite diversity as, for example, a bioindicator of ecosystem disturbance. Here, we tested the potential for simultaneous detection of four wildlife pathogens in water samples from 280, mainly riverine, sites across Switzerland. We targeted the crayfish pathogen Aphanomyces astaci, the amphibian pathogen Batrachochytrium dendrobatidis, and the fish pathogens Saprolegnia parasitica and Tetracapsuloides bryosalmonae. The eDNA detection showed a widespread distribution of A. astaci, S. parasitica, and T. bryosalmonae, although A. astaci and T. bryosalmonae were not detected in some alpine river catchments. B. dendrobatidis was detected only rarely, which was expected since the sampling did not target amphibian breeding sites. Co‐detection rates were higher in rivers than in lakes, likely reflecting the habitat preferences and distributions of the host species. We discuss the advantages and limitations of eDNA‐based pathogen monitoring and list a set of recommendations for managers. Our study illustrates how eDNA‐based techniques can monitor several pathogen species concurrently, thus facilitating more comprehensive disease monitoring schemes. Combined with metabarcoding approaches in the future, eDNA‐based sampling and detection can facilitate the incorporation of parasite and pathogen occurrence and diversity as an indicator for aquatic ecosystem health, and for revealing the hidden biodiversity and structure of parasite communities.

Insights into the Biology of Leucocytozoon Species (Haemosporida, Leucocytozoidae): Why Is There Slow Research Progress on Agents of Leucocytozoonosis?

Blood parasites of the genus Leucocytozoon (Leucocytozoidae) only inhabit birds and represent a readily distinct evolutionary branch of the haemosporidians (Haemosporida, Apicomplexa). Some species cause pathology and even severe leucocytozoonosis in avian hosts, including poultry. The diversity of Leucocytozoon pathogens is remarkable, with over 1400 genetic lineages detected, most of which, however, have not been identified to the species level. At most, approximately 45 morphologically distinct species of Leucocytozoon have been described, but only a few have associated molecular data. This is unfortunate because basic information about named and morphologically recognized Leucocytozoon species is essential for a better understanding of phylogenetically closely related leucocytozoids that are known only by DNA sequence. Despite much research on haemosporidian parasites during the past 30 years, there has not been much progress in taxonomy, vectors, patterns of transmission, pathogenicity, and other aspects of the biology of these cosmopolitan bird pathogens. This study reviewed the available basic information on avian Leucocytozoon species, with particular attention to some obstacles that prevent progress to better understanding the biology of leucocytozoids. Major gaps in current Leucocytozoon species research are discussed, and possible approaches are suggested to resolve some issues that have limited practical parasitological studies of these pathogens.

Comparative Genomics of Barley-Infecting Xanthomonas translucens Shows Overall Genetic Similarity but Globally Distributed Virulence Factor Diversity.

Xanthomonas translucens pv. translucens (Xtt) is a global barley patho-gen and a concern for resistance breeding and regulation. Long-read whole genome sequences allow in-depth understanding of pathogen diversity. We have completed long-read PacBio sequencing of two Minnesotan Xtt strains and an in-depth analysis of available Xtt genomes. We found that average nucleotide identity (ANI)-based approaches organize Xtt strains different from the previous standard multilocus sequencing analysis approach. According to ANI, Xtt forms a separate clade from X. translucens pv. undulosa and consists of three main groups which are represented on multiple continents. Some virulence factors, such as 17 Type III-secreted effectors, are highly conserved and offer potential targets for the elicitation of broad resistance. However, there is a high degree of variation in virulence factors, meaning that germplasm should be screened for resistance with a diverse panel of Xtt.

Identification of Fungal Species Associated with Apple Canker in Tarim Basin, China.

Apple canker has decreased yields of the economically important apple (Malus domestica) crop in China in recent years. Pathogen identity is highly challenging and the disease is poorly understood. Specimens of 339 fungi were isolated from apple trees in the primary apple-producing region in the Tarim Basin during the current study. In total, nine species of Cytospora and five species of the family Botryosphaeriaceae were identified by morphological observation and multilocus phylogenetic analyses: internal transcribed spacer (ITS), actin, translation elongation factor (TEF), and β-tubulin (TUB) gene regions for Cytospora and ITS, TEF, and TUB for Botryosphaeriaceae. Cytospora pyri from the Cytospora genus was the dominant species causing apple canker in the Tarim Basin. C. melnikii, C. tritici, C. euonymina, Diplodia seriata, and Botryosphaeria dothidea have been described as the cause of apple canker in China. Apple (Red Fuji) branches were utilized to assess the pathogenicity of 24 representative fungal isolates from the 14 species, and branches from seven distinct woody plants (Korla pear, walnut, Chinese date, Xinjiang poplar, sand jujube, Populus euphratica, and willow) were utilized to analyze the host range. The main pathogenic fungal species of apple canker around the Tarim Basin were identified and biological characteristics explored. Pathogen diversity and regional source diversity were assessed with host range and pathogenicity. The aim was to provide a theoretical foundation for the prevention and treatment of apple canker.