Krumholzibacteriota and Deltaproteobacteria contain rare genetic potential to liberate carbon from monoaromatic compounds in subsurface coal seams.

Biogenic methane in subsurface coal seam environments is produced by diverse consortia of microbes. Although this methane is useful for global energy security, it remains unclear which microbes can liberate carbon from the coal. Most of this carbon is relatively resistant to biodegradation, as it is contained within aromatic rings. Thus, to explore for coal-degrading taxa in the subsurface, this study reconstructed relevant metagenome-assembled genomes (MAGs) from coal seams by using a key genomic marker for the anaerobic degradation of monoaromatic compounds as a guide: the benzoyl-CoA reductase gene (bcrABCD). Three MAGs were identified with this genetic potential. The first represented a novel taxon from the Krumholzibacteriota phylum, which this study is the first to describe. This Krumholzibacteriota MAG contained a full set of genes for benzoyl-CoA dearomatization, in addition to other genes for anaerobic catabolism of monoaromatics. Analysis of Krumholzibacteriota MAGs from other environments revealed that this genetic potential may be common, and thus, Krumholzibacteriota may be important organisms for the liberation of recalcitrant carbon in a broad range of environments. Moreover, the assembly and characterization of two Syntrophorhabdus aromaticivorans MAGs from different continents and a Syntrophaceae sp. MAG implicate the Deltaproteobacteria class in coal seam monoaromatic degradation. Each of these taxa are potential rate-limiting organisms for subsurface coal-to-methane biodegradation. Their description here provides some understanding of their function within the coal seam microbiome and will help inform future efforts in coal bed methane stimulation, anoxic bioremediation of organic pollutants, and assessments of anoxic, subsurface carbon cycling and emissions.IMPORTANCESubsurface coal seams are highly anoxic, oligotrophic environments, where the main source of carbon is "locked away" within aromatic rings. Despite these challenges, many coal seams accumulate biogenic methane, implying that the coal seam microbiome is "unlocking" this carbon source in situ. For over two decades, researchers have endeavored to understand which organisms perform these processes. This study provides the first descriptions of organisms with this genetic potential from the coal seam environment. Here, we report metagenomic insights into carbon liberation from aromatic molecules and the degradation pathways involved and describe a Krumholzibacteriota, two Syntrophorhabdus aromaticivorans, and a Syntrophaceae MAG that contain this genetic potential. This is also the first time that the Krumholzibacteriota phylum has been implicated in anaerobic dearomatization of aromatic hydrocarbons. This potential is identified here in numerous MAGs from other terrestrial and marine subsurface habitats, implicating the Krumholzibacteriota in carbon-cycling processes across a broad range of environments.

Draft genome of the bluefin tuna blood fluke, Cardicola forsteri.

The blood fluke Cardicola forsteri (Trematoda: Aporocotylidae) is a pathogen of ranched bluefin tuna in Japan and Australia. Genomics of Cardicola spp. have thus far been limited to molecular phylogenetics of select gene sequences. In this study, sequencing of the C. forsteri genome was performed using Illumina short-read and Oxford Nanopore long-read technologies. The sequences were assembled de novo using a hybrid of short and long reads, which produced a high-quality contig-level assembly (N50 > 430 kb and L50 = 138). The assembly was also relatively complete and unfragmented, comprising 66% and 7.2% complete and fragmented metazoan Benchmarking Universal Single-Copy Orthologs (BUSCOs), respectively. A large portion (> 55%) of the genome was made up of intergenic repetitive elements, primarily long interspersed nuclear elements (LINEs), while protein-coding regions cover > 6%. Gene prediction identified 8,564 hypothetical polypeptides, > 77% of which are homologous to published sequences of other species. The identification of select putative proteins, including cathepsins, calpains, tetraspanins, and glycosyltransferases is discussed. This is the first genome assembly of any aporocotylid, a major step toward understanding of the biology of this family of fish blood flukes and their interactions within hosts.

Enhancement of live vaccines by co-delivery of immune modulating proteins.

Vaccines are very effective in providing protection against many infectious diseases. However, it has proven difficult to develop highly efficacious vaccines against some pathogens and so there is a continuing need to improve vaccine technologies. The first successful and widely used vaccines were based on attenuated pathogens (e.g., laboratory passaged Pasteurella multocida to vaccinate against fowl cholera) or closely related non-pathogenic organisms (e.g., cowpox to vaccinate against smallpox). Subsequently, live vaccines, either attenuated pathogens or non-pathogenic microorganisms modified to deliver heterologous antigens, have been successfully used to induce protective immune responses against many pathogens. Unlike conventional killed and subunit vaccines, live vaccines can deliver antigens to mucosal surfaces in a similar manner and context as the natural infection and hence can often produce a more appropriate and protective immune response. Despite these advantages, there is still a need to improve the immunogenicity of some live vaccines. The efficacy of injectable killed and subunit vaccines is usually enhanced using adjuvants such mineral salts, oils, and saponin, but such adjuvants cannot be used with live vaccines. Instead, live vaccines can be engineered to produce immunomodulatory molecules that can stimulate the immune system to induce more robust and long-lasting adaptive immune responses. This review focuses on research that has been undertaken to engineer live vaccines to produce immunomodulatory molecules that act as adjuvants to increase immunogenicity. Adjuvant strategies with varying mechanisms of action (inflammatory, antibody-mediated, cell-mediated) and delivery modes (oral, intramuscular, intranasal) have been investigated, with varying degrees of success. The goal of such research is to define adjuvant strategies that can be adapted to enhance live vaccine efficacy by triggering strong innate and adaptive immune responses and produce vaccines against a wider range of pathogens.

Methanogenic archaea in subsurface coal seams are biogeographically distinct: an analysis of metagenomically-derived mcrA sequences.

The production of methane as an end-product of organic matter degradation in the absence of other terminal electron acceptors is common, and has often been studied in environments such as animal guts, soils and wetlands due to its potency as a greenhouse gas. To date, however, the study of the biogeographic distribution of methanogens across coal seam environments has been minimal. Here, we show that coal seams are host to a diverse range of methanogens, which are distinctive to each geological basin. Based on comparisons to close relatives from other methanogenic environments, the dominant methanogenic pathway in these basins is hydrogenotrophic, with acetoclastic being a second major pathway in the Surat Basin. Finally, mcrA and 16S rRNA gene primer biases were predominantly seen to affect the detection of Methanocellales, Methanomicrobiales and Methanosarcinales taxa in this study. Subsurface coal methanogenic community distributions and pathways presented here provide insights into important metabolites and bacterial partners for in situ coal biodegradation.

In vitro inhibitory activities of sugarcane extract on avian Eimeria sporozoites.

The current in vitro study aimed to investigate the effects of a processed sugarcane extract on the viability of avian Eimeria sporozoites. Treatments were applied to hatched sporozoites: 1) without additives (no-treatment control); 2) with ethanol; 3) with salinomycin; 4) with Polygain™. All treatments were incubated in RPMI media containing live sporozoites at 37 °C for 14 h and then the number of viable sporozoites were counted. Compared to the no-treatment control, Polygain™ decreased (P < 0.001) the counts of E. maxima, E. acervulina, E. bruneti, and E. mitis sporozoites to a level similar to salinomycin (P > 0.05). In conclusion, Polygain™ could be a potential candidate as an anticoccidial agent.

Aromatic compound-degrading taxa in an anoxic coal seam microbiome from the Surat Basin, Australia.

Methane is an important energy resource internationally, and a large proportion of this methane is produced by microbial communities living in coal seams. Despite the value of this resource for human energy security, our understanding of the metabolic roles played by specific taxa during the biodegradation of coal to methane in situ is quite limited. In order to develop a greater understanding of microbial catabolism on coal, a community from a coal seam in the Surat Basin, Australia, was incubated on 10 different aromatic organic compounds: coronene, benzo[a]pyrene, pyrene, phenanthrene, naphthalene, ethylbenzene, phenol, benzoate, vanillate and syringate. Each of these aromatic compounds either occurs in coal or is a possible product of the coal biodegradation process. 16S rRNA sequencing revealed substantial changes to each community in response to each aromatic carbon substrate provided. Abundant taxa from these substrate-specific communities were identified and their probable catabolic roles proposed based on literature searches of related taxa. This study is the first to link specific coal seam taxa to aromatic substrates available in coal seam environments. Two conceptual models of the putative degradation pathways and key taxa responsible are proposed.

Angiostrongylus chabaudi in felids: New findings and a review of the literature.

Cardiopulmonary infections by Angiostrongylus chabaudi affect domestic and wild felids but, due to limited information on the biology of this nematode, its pathogenicity remains unclear. This article describes the histopathological alterations associated with Angiostrongylus infection in a wildcat from Bulgaria, and reviews current literature on this feline angiostrongylid. Nematodes were isolated from lung lavage and faecal samples of a road killed wildcat in Southern Bulgaria. The morphological identification of parasite larvae as A. chabaudi was confirmed by molecular analysis of part of the 18S ribosomal RNA gene. Upon histopathological examination, severe granulomatous pneumonia, ranging from multifocal to coalescing, and pulmonary vascular lesions were observed. Extensive alveolar collapse, alveolar emphysematous changes, parenchymal haemorrhages and small artery wall hyperplasia were observed in the parenchyma adjacent to the granulomas. Histopathological examination revealed the presence of cross-sections of adult female parasites within the lumen of the pulmonary artery branches, the intima altered markedly by subendothelial proliferation and oedematous changes. This study compliments current knowledge of the pathogenesis of feline angiostrongylosis by A. chabaudi in wildcats, as well as of the distribution of this little-known parasite.

Paramyosin of canine Onchocerca lupi: usefulness for the diagnosis of a neglected zoonotic disease.

BACKGROUND: Of increasing importance to the medical and veterinary communities is the zoonotic filarioid nematode Onchocerca lupi. Onchocercosis, thus far found in wolves, dogs, cats and humans, is diagnosed via skin snips to detect microfilariae and surgical removal of adults from the eye of the host. These methods are time-consuming, laborious and invasive, highlighting the need for new tools for the diagnosis of O. lupi in susceptible hosts. Symptoms related to the presence of the adults in the eye can range from none apparent to severe, including blindness. No reliable chemotherapeutic protocols are available, as yet, to eliminate the infection. Paramyosin, an invertebrate-specific protein, has been well-studied as an allergen, diagnostic marker and vaccine candidate. The aim of this study, therefore, was to isolate and characterise paramyosin from O. lupi to assess its suitability for the development of a serological diagnostic assay. METHODS: The adult and microfilarial stages of O. lupi were isolated from the eyes and skin of a 3-year-old male dog. Total RNA was extracted and reverse transcribed into single stranded cDNA. Reverse-transcription PCR was used to isolate a full-length paramyosin cDNA from adult worms and to investigate the temporal expression patterns of this gene. All amplicons were sequenced using dideoxy chain termination sequencing. Bioinformatics was used to predict the amino acid sequence of the gene, to compare the DNA and protein sequences with those available in public databases and to investigate the phylogenetic relationship of all molecules. Antibody binding sites were predicted using bioinformatics and mapped along with published antigenic epitopes against the O. lupi paramyosin protein. The native protein, and three smaller recombinantly expressed peptides, were subjected to western blot using serum from dogs both positive and negative for O. lupi. RESULTS: Paramyosin of O. lupi was herein molecularly characterized, encoded by a transcript of 2,643 bp and producing a protein of 881 amino acids (101.24 kDa). The paramyosin transcript was detected, by reverse transcription PCR, in adults and microfilariae, but not in eggs. Phylogenetic analysis indicates that this molecule clusters with paramyosins from other filarioids to the exclusion of those from other taxa. A total of 621 unique antibody binding epitopes were predicted for this protein and another 28 were conserved in other organisms. This information was used to design three peptides, for recombinant expression, to identify the antibody binding epitope(s) and reduce potential cross-reactivity with serum from dogs infected with other filarioid nematodes. Native paramyosin, purified from microfilariae and adults, was detected by antibodies present in serum from dogs with known O. lupi infections. CONCLUSIONS: Data provided herein may assist in the development of a serological diagnostic test, based on antibodies to O. lupi paramyosin, for the diagnosis of this infection, in order to gain more information on the real distribution of this little known filarioid of zoonotic concern.

Crenosoma vulpis in wild and domestic carnivores from Italy: a morphological and molecular study.

Crenosoma vulpis is a metastrongyloid nematode primarily associated with respiratory tract infections of red foxes in North America and Europe. Sporadic cases have also been reported in domestic dogs. The present study aimed to provide morphological, molecular, and epidemiological data on the geographical distribution of this nematode throughout Italy. From 2012 to 2014, 12 of the 138 foxes examined, three dogs and one badger scored positive for C. vulpis. Forty adults were isolated from foxes and the badger, whereas first-stage larvae were detected in the three dogs. All specimens were morphologically identified as C. vulpis, and 28 nematodes were also molecularly characterized by sequencing mitochondrial (12S ribosomal DNA (rDNA)) and nuclear (18S rDNA) ribosomal genes. Four haplotypes were identified based on the 12S rDNA target gene, with the most representative (78.5%) designated as haplotype I. No genetic variability was detected for the 18S rDNA gene. The molecular identification was consistent with the distinct separation of species-specific clades inferred by the phylogenetic analyses of both mitochondrial and ribosomal genes. Data herein reported indicates that C. vulpis has a wide distribution in foxes from southern Italy, and it also occurs in dogs from southern and northern regions of the country. Practitioners should consider the occurrence of this nematode in the differential diagnosis of canine respiratory disease, particularly in dogs living close to rural areas where foxes are present.

Occurrence of Ixodiphagus hookeri (Hymenoptera: Encyrtidae) in Ixodes ricinus (Acari: Ixodidae) in southern Italy.

Natural enemies of ticks include the parasitoid wasp Ixodiphagus hookeri (Hymenoptera: Encyrtidae). The aim of this preliminary study was to investigate the occurrence of I. hookeri DNA in a community of ticks (Ixodes ricinus, Dermacentor marginatus, Hyalomma marginatum, Haemaphysalis inermis and Rhipicephalus turanicus). From May 2010 to March 2012, ticks were collected monthly by dragging and flagging, identified, and 481 adults and 305 nymphs screened molecularly for infection with I. hookeri. Of the samples tested (n=786), 3.1% (n=25) were positive for I. hookeri DNA, 7.2% (n=22) in nymphs and 0.6% (n=3) in adults. I. hookeri DNA was only detected in I. ricinus. This study shows that I. hookeri infests I. ricinus in southern Italy, with nymphs being the main developmental stage affected by this wasp.

Impact of next-generation technologies on exploring socioeconomically important parasites and developing new interventions.

High-throughput molecular and computer technologies have become instrumental for systems biological explorations of pathogens, including parasites. For instance, investigations of the transcriptomes of different developmental stages of parasitic nematodes give insights into gene expression, regulation and function in a parasite, which is a significant step to understanding their biology, as well as interactions with their host(s) and disease. This chapter (1) gives a background on some key parasitic nematodes of socioeconomic importance, (2) describes sequencing and bioinformatic technologies for large-scale studies of the transcriptomes and genomes of these parasites, (3) provides some recent examples of applications and (4) emphasizes the prospects of fundamental biological explorations of parasites using these technologies for the development of new interventions to combat parasitic diseases.

Detection of Anaplasma platys in dogs and Rhipicephalus sanguineus group ticks by a quantitative real-time PCR.

Anaplasma platys is an obligate intracellular, tick-borne pathogen of dogs, which causes canine infectious cyclic thrombocytopenia (CICT). The vector role of Rhipicephalus sanguineus group ticks has been only suggested, but definitive evidence is lacking. This study aimed to detect and quantify A. platys DNA in infected dogs and in their respective ticks through a quantitative real-time PCR assay. From March to May 2009, blood and tick samples from dogs residing in a CICT-endemic area were collected and molecularly analysed. Differences (p<0.05) were detected in the bacterial load between tick nymphs (3.5 × 10(-2)± 2.5 × 10(-2)) and adults (female: 1.2 × 10(-1) ± 1.1 × 10(-1); male: 9.3 × 10(-2)± 1.2 × 10(-2)) and between unengorged (1.1 × 10(-1) ± 2.8 × 10(-2)) and partially or fully engorged ticks (1.9 × 10(-1) ± 2.7 × 10(-2)). No difference was found between ticks collected from positive (1.1 × 10(-1)± 0.6 × 10(-1)) and negative (1.2 × 10(-1) ± 0.3 × 10(-1)) dogs (p>0.05). The mean bacterial load detected in positive dogs was lower than that in their respective ticks (p>0.05). This study provides circumstantial evidence of the putative role of Rhipicephalus sp. I as a vector of this pathogen.

Image diagnosis of zoonotic onchocercosis by Onchocerca lupi.

Onchocerca lupi, a zoonotic nematode infecting the eyes of carnivores, has been increasingly reported in dogs from Europe and the USA. In order to improve the current status of knowledge on this neglected filarioid, diagnostic imaging tools (i.e., ultrasound scan, computed tomography and magnetic resonance imaging) are herein used to diagnose canine onchocercosis in two dogs, which scored positive for O. lupi microfilariae at the skin snip test and to assess the anatomical location of the nematode within the ocular apparatus. Results indicate that ultrasound tools are useful to address the diagnosis of O. lupi in dogs and to evaluate the localization of nodules or cysts containing the adult nematode.

A preliminary investigation of serological tools for the detection of Onchocerca lupi infection in dogs.

Onchocerca lupi is a neglected filarioid causing nodular lesions associated with acute or chronic ocular disease in dogs. Despite the recent appraisal of its zoonotic potential, human cases are increasingly reported in the Old and New Worlds. Therefore, the development of accurate tools for the rapid diagnosis of O. lupi infections in dogs is becoming a priority. In this study, we conducted a preliminary investigation aimed at evaluating the usefulness of a commercially available ELISA test for the detection of O. lupi antigens in canine sera. The potential use of this tool for larger epidemiological studies of canine onchocerciasis is discussed.

Simultaneous detection of the feline lungworms Troglostrongylus brevior and Aelurostrongylus abstrusus by a newly developed duplex-PCR.

In addition to Aelurostrongylus abstrusus (Strongylida: Angiostrongylidae), referred to as the feline lungworm, Troglostrongylus brevior (Strongylida: Crenosomatidae) has recently been identified as an agent of bronco-pulmonary infestations in cats. These two parasites have a similar biology, share ecological niches, potentially co-infesting cats, but are difficult to be differentiated due to the morphological similarities of their first-stage larvae (L1). This paper describes a molecular tool, based on single-step duplex polymerase chain reaction (duplex-PCR) on the ribosomal internal transcribed spacer 2 region (ITS-2) for the simultaneous detection and differentiation of T. brevior and A. abstrusus. L1 of both species were collected from faecal samples, morphologically identified, and single larval specimens isolated. An aliquot of faeces was used as a test sample for a case of mixed natural infestation. The duplex-PCR was performed using species-specific forward primer sets for the ITS-2 region (i.e., A. abstrusus: 220bp; T. brevior: 370bp). The detection limit of the molecular assay was also assessed by serial dilutions of DNA from single larvae of both species (from ≈ 4.0 to 4.0 × 10(-5) µg/µl). The duplex-PCR carried out on individual DNA samples was able to detect as low as 5.2 × 10(-3) µg/µl of DNA for A. abstrusus, 4.9 × 10(-3)µg/µl for T. brevior, and as low as 4.0 × 10(-3) µg/µl for samples containing both species. Species-specific bands of the expected sizes and two bands were simultaneously amplified from the faecal sample containing both species. The phylogenetic analyses of the ITS-2 sequences here examined and those available for other metastrongyloids were concordant in clustering them with those of other Troglostrongylus brevior and A. abstrusus sequences available in GenBank database. This molecular approach proved to be effective and highly sensitive for the simultaneous detection of the two lungworms species and it might be used for molecular epidemiological studies and for monitoring therapeutic protocols.

The genome and developmental transcriptome of the strongylid nematode Haemonchus contortus.

BACKGROUND: The barber's pole worm, Haemonchus contortus, is one of the most economically important parasites of small ruminants worldwide. Although this parasite can be controlled using anthelmintic drugs, resistance against most drugs in common use has become a widespread problem. We provide a draft of the genome and the transcriptomes of all key developmental stages of H. contortus to support biological and biotechnological research areas of this and related parasites. RESULTS: The draft genome of H. contortus is 320 Mb in size and encodes 23,610 protein-coding genes. On a fundamental level, we elucidate transcriptional alterations taking place throughout the life cycle, characterize the parasite's gene silencing machinery, and explore molecules involved in development, reproduction, host-parasite interactions, immunity, and disease. The secretome of H. contortus is particularly rich in peptidases linked to blood-feeding activity and interactions with host tissues, and a diverse array of molecules is involved in complex immune responses. On an applied level, we predict drug targets and identify vaccine molecules. CONCLUSIONS: The draft genome and developmental transcriptome of H. contortus provide a major resource to the scientific community for a wide range of genomic, genetic, proteomic, metabolomic, evolutionary, biological, ecological, and epidemiological investigations, and a solid foundation for biotechnological outcomes, including new anthelmintics, vaccines and diagnostic tests. This first draft genome of any strongylid nematode paves the way for a rapid acceleration in our understanding of a wide range of socioeconomically important parasites of one of the largest nematode orders.

Whole-genome sequence of Schistosoma haematobium.

Schistosomiasis is a neglected tropical disease caused by blood flukes (genus Schistosoma; schistosomes) and affecting 200 million people worldwide. No vaccines are available, and treatment relies on one drug, praziquantel. Schistosoma haematobium has come into the spotlight as a major cause of urogenital disease, as an agent linked to bladder cancer and as a predisposing factor for HIV/AIDS. The parasite is transmitted to humans from freshwater snails. Worms dwell in blood vessels and release eggs that become embedded in the bladder wall to elicit chronic immune-mediated disease and induce squamous cell carcinoma. Here we sequenced the 385-Mb genome of S. haematobium using Illumina-based technology at 74-fold coverage and compared it to sequences from related parasites. We included genome annotation based on function, gene ontology, networking and pathway mapping. This genome now provides an unprecedented resource for many fundamental research areas and shows great promise for the design of new disease interventions.

Key strongylid nematodes of animals - Impact of next-generation transcriptomics on systems biology and biotechnology.

The advent and integration of high-throughput 'omic technologies (e.g., genomics, transcriptomics, proteomics and metabolomics) are becoming instrumental to assist fundamental explorations of the systems biology of organisms. In particular, these technologies now provide unique opportunities for global, molecular investigations of parasites. For example, studies of the transcriptomes (all transcripts in an organism, tissue or cell) of different species and/or developmental stages of parasitic nematodes provide insights into aspects of gene expression, regulation and function, which is a major step to understanding their biology. The purpose of this article was to review salient aspects of the systematics and biology of selected species of parasitic nematodes (particularly key species of the order Strongylida) of socio-economic importance, to describe conventional and advanced sequencing technologies and bioinformatic tools for large-scale investigations of the transcriptomes of these parasites and to highlight the prospects and implications of these explorations for developing novel methods of parasite intervention.

Detection of diarrhoeal pathogens in human faeces using an automated, robotic platform.

Infectious diarrhoeal diseases represent a major socio-economic burden to humans, and are linked to a range of pathogens, including viruses, bacteria and protists. The accurate detection of such pathogens is central to control. However, detection often relies on methods that have limited diagnostic sensitivity and specificity. Here, we assessed an automated, robotic platform for the simultaneous detection of eight major pathogens associated with infectious diarrhoea. Genomic DNA samples (n = 167) from faeces from humans with diarrhoea and diagnosed as cryptosporidiosis, and 100 uninfected control subjects, were tested for adenovirus 40/41, norovirus, Clostridium difficile, Campylobacter, Salmonella, Shigella, Cryptosporidium and Giardia by multiplexed-tandem PCR, and also characterized by single-strand conformation polymorphism analysis (SSCP) and selective sequencing. All 167 samples tested positive for Cryptosporidium, five for adenovirus 40/41, four for Campylobacter, three for C. difficile and seven for Shigella spp., with no false positive results for any assay. The automated PCR exhibited a high sensitivity, with <10 individual pathogens being readily detected. The robotic detection platform assessed here represents a sensitive, high-throughput tool for key pathogens linked to infectious diarrhoea in humans. This platform requires little molecular biological expertise and is well suited to various diagnostic facilities and settings.