Salivarian trypanosomosis : a review of parasites involved, their global distribution and their interaction with the innate and adaptive mammalian host immune system

Salivarian trypanosomes are single cell extracellular parasites that cause infections in a wide range of hosts. Most pathogenic infections worldwide are caused by one of four major species of trypanosomes including (i) Trypanosoma brucei and the human infective subspecies T. b. gambiense and T. b. rhodesiense, (ii) Trypanosoma evansi and T. equiperdum, (iii) Trypanosoma congolense and (iv) Trypanosoma vivax. Infections with these parasites are marked by excessive immune dysfunction and immunopathology, both related to prolonged inflammatory host immune responses. Here we review the classification and global distribution of these parasites, highlight the adaptation of human infective trypanosomes that allow them to survive innate defense molecules unique to man, gorilla, and baboon serum and refer to the discovery of sexual reproduction of trypanosomes in the tsetse vector. With respect to the immunology of mammalian host-parasite interactions, the review highlights recent findings with respect to the B cell destruction capacity of trypanosomes and the role of T cells in the governance of infection control. Understanding infection-associated dysfunction and regulation of both these immune compartments is crucial to explain the continued failures of anti-trypanosome vaccine developments as well as the lack of any field-applicable vaccine based anti-trypanosomosis intervention strategy. Finally, the link between infection-associated inflammation and trypanosomosis induced anemia is covered in the context of both livestock and human infections

Virotyping and genetic antimicrobial susceptibility testing of porcine ETEC/STEC strains and associated plasmid types

IntroductionEnterotoxigenic Escherichia coli (ETEC) infections are the most common cause of secretory diarrhea in suckling and post-weaning piglets. For the latter, Shiga toxin-producing Escherichia coli (STEC) also cause edema disease. This pathogen leads to significant economic losses. ETEC/STEC strains can be distinguished from general E. coli by the presence of different host colonization factors (e.g., F4 and F18 fimbriae) and various toxins (e.g., LT, Stx2e, STa, STb, EAST-1). Increased resistance against a wide variety of antimicrobial drugs, such as paromomycin, trimethoprim, and tetracyclines, has been observed. Nowadays, diagnosing an ETEC/STEC infection requires culture-dependent antimicrobial susceptibility testing (AST) and multiplex PCRs, which are costly and time-consuming.MethodsHere, nanopore sequencing was used on 94 field isolates to assess the predictive power, using the meta R package to determine sensitivity and specificity and associated credibility intervals of genotypes associated with virulence and AMR.ResultsGenetic markers associated with resistance for amoxicillin (plasmid-encoded TEM genes), cephalosporins (ampC promoter mutations), colistin (mcr genes), aminoglycosides (aac(3) and aph(3) genes), florfenicol (floR), tetracyclines (tet genes), and trimethoprim-sulfa (dfrA genes) could explain most acquired resistance phenotypes. Most of the genes were plasmid-encoded, of which some collocated on a multi-resistance plasmid (12 genes against 4 antimicrobial classes). For fluoroquinolones, AMR was addressed by point mutations within the ParC and GyrA proteins and the qnrS1 gene. In addition, long-read data allowed to study the genetic landscape of virulence- and AMR-carrying plasmids, highlighting a complex interplay of multi-replicon plasmids with varying host ranges.ConclusionOur results showed promising sensitivity and specificity for the detection of all common virulence factors and most resistance genotypes. The use of the identified genetic hallmarks will contribute to the simultaneous identification, pathotyping, and genetic AST within a single diagnostic test. This will revolutionize future quicker and more cost-efficient (meta)genomics-driven diagnostics in veterinary medicine and contribute to epidemiological studies, monitoring, tailored vaccination, and management

High quality MinION and Flongle long-read nanopore genome assemblies of Mycoplasma bovis using taxon-specific training of the Bonito basecaller

Mycoplasma bovis is a major and primary bovine pathogen causing respiratory and reproductive disorders, mastitis and arthritis. Due to its persistent nature it is difficult to combat infections on farms. No effective vaccine is able to prevent M. bovis infection, leaving antimicrobials as the only first-line treatment. Nevertheless, therapy with antimicrobials is rarely efficient since increased resistance to many commercially available antimicrobials has been reported. An accurate diagnosis including antimicrobial resistance profiling is required to combat infections with working antibiotics, but this cannot be achieved fast enough with classical diagnostics. Here we developed a nanopore-based workflow allowing M. bovis species typing and Antimicrobial Resistance (AMR) Profiling applicable in point-of-care settings in control of M. bovis infections. Our new diagnostic tool was verified with 100 field strains of M. bovis for which whole genome sequencing and MIC testing was performed for practice-relevant antibiotics. Besides whole genome species typing, Single Nucleotide Polymorphism (SNP) analysis was performed to associate strain-specific genetic markers with its phenotypic AMR antibiogram. Raw fast5 outputs ranged from 5.4 Gb up to 17.2 Gb, with an average N50 of 5.5 ± 1.3 Kb per run with 11 M. bovis strains. Furthermore, including the M. bovis PG45 type strain within every run as internal control, inter-run accuracy reached up to 99.95% sequence identity. Since computation time presents a new bottleneck in this new workflow, we exploited a GPU-based bioinformatics pipeline speeding up full bioinformatics analysis to be completed within 10 hours for 11 M. bovis genomes. This new M. bovis diagnostics pipeline delivers a high accurate species identification along with an accurate genotypic antibiogram. This will be accelerated even further to facilitate proper antimicrobial therapy selection for the rapid control of bovine mycoplasmosis

Genome sequences of equine herpesvirus 1 strains from a European outbreak of neurological disorders linked to a horse gathering in Valencia, Spain, in 2021

Five equine herpesvirus 1 (EHV-1) genome sequences with links to an EHV-1 outbreak with neurological disorders after a horse gathering in Valencia, Spain, in February 2021, were determined. All strains showed the closest relationships to strains from Belgium and the United Kingdom, indicating a common source of infection

High quality genome assemblies of Mycoplasma bovis using a taxon-specific Bonito basecaller for MinION and Flongle long-read nanopore sequencing

Implementation of Third-Generation Sequencing approaches for Whole Genome Sequencing (WGS) all-in-one diagnostics in human and veterinary medicine, requires the rapid and accurate generation of consensus genomes. Over the last years, Oxford Nanopore Technologies (ONT) released various new devices (e.g. the Flongle R9.4.1 flow cell) and bioinformatics tools (e.g. the in 2019-released Bonito basecaller), allowing cheap and user-friendly cost-efficient introduction in various NGS workflows. While single read, overall consensus accuracies, and completeness of genome sequences has been improved dramatically, further improvements are required when working with non-frequently sequenced organisms like Mycoplasma bovis. As an important primary respiratory pathogen in cattle, rapid M. bovis diagnostics is crucial to allow timely and targeted disease control and prevention. Current complete diagnostics (including identification, strain typing, and antimicrobial resistance (AMR) detection) require combined culture-based and molecular approaches, of which the first can take 1–2 weeks. At present, cheap and quick long read all-in-one WGS approaches can only be implemented if increased accuracies and genome completeness can be obtained

Presence of broad-spectrum beta-lactamase-producing Enterobacteriaceae in zoo mammals

Broad-spectrum beta-lactamase (BSBL)-producing Enterobacteriaceae impose public health threats. With increased popularity of zoos, exotic animals are brought in close proximity of humans, making them important BSBL reservoirs. However, not much is known on the presence of BSBLs in zoos in Western Europe. Fecal carriage of BSBL-producing Enterobacteriaceae was investigated in 38 zoo mammals from two Belgian zoos. Presence of bla-genes was investigated using PCR, followed by whole-genome sequencing and Fourier-transform infrared spectroscopy to cluster acquired resistance encoding genes and clonality of BSBL-producing isolates. Thirty-five putatively ceftiofur-resistant isolates were obtained from 52.6% of the zoo mammals. Most isolates were identified as E. coli (25/35), of which 64.0% showed multidrug resistance (MDR). Most frequently detected bla-genes were CTX-M-1 (17/25) and TEM-1 (4/25). Phylogenetic trees confirmed clustering of almost all E. coli isolates obtained from the same animal species. Clustering of five isolates from an Amur tiger, an Amur leopard, and a spectacled bear was observed in Zoo 1, as well as for five isolates from a spotted hyena and an African lion in Zoo 2. This might indicate clonal expansion of an E. coli strain in both zoos. In conclusion, MDR BSBL-producing bacteria were shown to be present in the fecal microbiota of zoo mammals in two zoos in Belgium. Further research is necessary to investigate if these bacteria pose zoonotic and health risks

Phylogenomic analysis of Mycoplasma bovis from Belgian veal, dairy and beef herds

M. bovis is one of the leading causes of respiratory disease and antimicrobial use in cattle. The pathogen is widespread in different cattle industries worldwide, but highest prevalence is found in the veal industry. Knowledge on M. bovis strain distribution over the dairy, beef and veal industries is crucial for the design of effective control and prevention programs, but currently undocumented. Therefore, the present study evaluated the molecular epidemiology and genetic relatedness of M. bovis isolates obtained from Belgian beef, dairy and veal farms, and how these relate to M. bovis strains obtained worldwide. Full genomes of one hundred Belgian M. bovis isolates collected over a 5-year period (2014–2019), obtained from 27 dairy, 38 beef and 29 veal farms, were sequenced by long-read nanopore sequencing. Consensus sequences were used to generate a phylogenetic tree in order to associate genetic clusters with cattle sector, geographical area and year of isolation. The phylogenetic analysis of the Belgian M. bovis isolates resulted in 5 major clusters and 1 outlier. No sector-specific M. bovis clustering was identified. On a world scale, Belgian isolates clustered with Israeli, European and American strains. Different M. bovis clusters circulated for at least 1.5 consecutive years throughout the country, affecting all observed industries. Therefore, the high prevalence in the veal industry is more likely the consequence of frequent purchase from the dairy and beef industry, than that a reservoir of veal specific strains on farm would exist. These results emphasize the importance of biosecurity in M. bovis control and prevention

Comparison of primary virus isolation in pulmonary alveolar macrophages and four different continuous cell lines for type 1 and type 2 porcine reproductive and respiratory syndrome virus

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) has a highly restricted cellular tropism. In vivo, the virus primarily infects tissue-specific macrophages in the nose, lungs, tonsils, and pharyngeal lymphoid tissues. In vitro however, the MARC-145 cell line is one of the few PRRSV susceptible cell lines that are routinely used for in vitro propagation. Previously, several PRRSV non-permissive cell lines were shown to become susceptible to PRRSV infection upon expression of recombinant entry receptors (e.g., PK15Sn-CD163, PK15S10-CD163). In the present study, we examined the suitability of different cell lines as a possible replacement of primary pulmonary alveolar macrophages (PAM) cells for isolation and growth of PRRSV. The susceptibility of four different cell lines (PK15Sn-CD163, PK15S10-CD163, MARC-145, and MARC-145Sn) for the primary isolation of PRRSV from PCR positive sera (both PRRSV1 and PRRSV2) was compared with that of PAM. To find possible correlations between the cell tropism and the viral genotype, 54 field samples were sequenced, and amino acid residues potentially associated with the cell tropism were identified. Regarding the virus titers obtained with the five different cell types, PAM gave the highest mean virus titers followed by PK15Sn-CD163, PK15S10-CD163, MARC-145Sn, and MARC-145. The titers in PK15Sn-CD163 and PK15S10-CD163 cells were significantly correlated with virus titers in PAM for both PRRSV1 (p < 0.001) and PRRSV2 (p < 0.001) compared with MARC-145Sn (PRRSV1: p = 0.22 and PRRSV2: p = 0.03) and MARC-145 (PRRSV1: p = 0.04 and PRRSV2: p = 0.12). Further, a possible correlation between cell tropism and viral genotype was assessed using PRRSV whole genome sequences in a Genome-Wide-Association Study (GWAS). The structural protein residues GP2:187L and N:28R within PRRSV2 sequences were associated with their growth in MARC-145. The GP5:78I residue for PRRSV2 and the Nsp11:155F residue for PRRSV1 was linked to a higher replication on PAM. In conclusion, PK15Sn-CD163 and PK15S10-CD163 cells are phenotypically closely related to the in vivo target macrophages and are more suitable for virus isolation and titration than MARC-145/MARC-145Sn cells. The residues of PRRSV proteins that are potentially related with cell tropism will be further investigated in the future

Revolutionizing viral and bacterial diagnostics in veterinary medicine using nanopore sequencing

Diagnostic tools in the field of medicine are required to support examination procedures as performed by practitioners. In Belgium, serological tests still represent the highest number (66%) of all tests performed in pig and cattle industry, followed by bacterial cultures (7%) and molecular assays (3%). The most important reason for this trend is the costs associated with most diagnostic tests as they do not deliver sufficient information within a single test. Even though multiplex quantitative PCRs have been designed to support detection of respiratory disease complexes in cattle and pigs or virotyping of enterotoxigenic Escherichia coli (ETEC), its cost is considered high (approximately 125 EUR) in relation to the delivered information. Over the last decades, various sequencing technologies have been released, allowing a significant reduction in sequencing cost. The availability of real-time single molecule (SMRT) nanopore sequencing (Oxford Nanopore Technologies) initiated a new era in the field of diagnostics (reviewed in Chapter 1). The use of this technology in a metagenomic workflow, allows the co-identification of viral and bacterial pathogens without prior pathogen selection. The added value of this was shown for known respiratory disease complexes in swine and cattle (PRDC and BRDC, respectively), highlighting the co-circulation of primary, secondary, and lesser-known pathogens (e.g., porcine hemagglutinating encephalomyelitis virus (PHEV) and Mycoplasmopsis arginini in PRDC and BRDC, respectively) (Chapter 3). Furthermore, it allowed (semi-)quantitative evaluation of all microbes (both viral and bacterial) to assess their relevance within the disease complex along with sequence information for viruses. The latter is an important asset as it allows viral (sub)typing which is often required to distinguish vaccines from wild type infections (e.g., porcine reproductive and respiratory syndrome virus (PRRSV)) and the subtyping of swine influenza A virus (swIAV). Obtaining these viral genome sequences from a single test also showed to be of great value in surveillance, epidemiology, and tailoring of vaccination as exemplified for porcine parvovirus type 1 (PPV1). Also, in the context of outbreaks it quickly provides sequencing information (e.g., equine herpes virus 1 (EHV-1)). While (m)any host species and sample types (including fecal, serum, respiratory, tissue, and urine) can be subjected to metagenomics, some samples require an additional targeted enrichment to deliver complete viral genomes as shown for swIAV from “dirty” oral fluids (Chapter 4). Costs associated with current tests lie within the range of multiplex PCRs, allowing its use by progressive veterinarians in livestock, companion animals, and exotics, along with various applications for academic institutions and pharmaceutical companies to date. Though, feedback from the field highlighted the need of bacterial virotyping and antimicrobial susceptibility testing (AST) to allow its extended application in routine veterinary diagnostics. This represents a tougher task since existing ad-random metagenomic workflows are not able to deliver sufficient depth on bacterial genomic information yet. Here, a spike-in approach to enrich for specific targets, while maintaining metagenomic sensitivity, is thought to guarantee its wider implementation in routine diagnostic laboratories. Therefore, fundamental knowledge on genetic virulence- and antimicrobial resistance-associated markers should be obtained. To do so, accurate and complete bacterial genomes should be available. Current databases (e.g., NCBI) do not provide enough genomic and phenotypic metadata on veterinary bacteria, thus extensive efforts were made to provide new information for relevant bacterial species. The use of long-read only data for genome assemblies was examined and performed excellent in delivering accurate and complete genomes for non-fastidiously growing bacteria such as E. coli and Actinobacillus species (Chapter 5). This is majorly due to significant improvements in raw read accuracies via new pore chemistries and base calling models over the last years. For fastidiously growing bacterial species, such as Mycoplasmopsis bovis and Brachyspira hyodysenteriae, the generation of a taxon-specific base calling model was shown of great importance to deliver Illumina-level accuracy genomes (>99.999% accuracy) (Chapter 6). Applying genome-wide association studies (GWAS) on these bacterial datasets allowed the identification of various genetic markers (genes, point mutations, and plasmids) with high diagnostic predictive power for virotyping and genetic AST (Chapter 5 and 6). Even though not all resistance phenotypes could be fully explained by these markers, epigenetics (e.g., methylation) along with transcriptomic profiling could still be addressed in the future. For the former, available raw nanopore data could be re-analyzed when more widely applicable methylation software and models are available. This would allow to provide an even broader view on the complex landscape of AMR but will also represent a (very) big challenge to get implemented in a metagenomic context. Prior to the use of these genetic markers in a “virulence and genetic AST enriched” metagenomic workflow, it is recommended to still perform extensive validation via targeted mutagenesis to deliver proof-of-concept for their actual link with virulence and AMR phenotypes (as discussed in Chapter 7). In conclusion, existing metagenomic diagnostic workflows can be enriched to detect genes associated with virulence and AST in a relatively short time frame. First fundaments for its broader application and integration in routine veterinary diagnostic laboratories can be delivered when genetic markers as described in this work will be implemented. A similar approach can be exploited to augment existing metagenomic tools for a wider variety of relevant viruses and bacteria depending on the needs within the field. In the end, this will deliver accurate all-in-one diagnostic tests which will speed-up identification and AST for relevant bacteria in acute infections and support antimicrobial drug tailorship.Diagnostische testen zijn in de diergeneeskunde noodzakelijk om dierenartsen bij het examineren te ondersteunen. In België worden serologische testen nog steeds het vaakst (66%) toegepast in de varkens- en runderindustrie, gevolgd door bacteriële culturen (7%) en moleculaire testen (3%). De belangrijkste reden voor deze trends is de kosten die gepaard gaan met laatstgenoemde testen omdat deze vaak onvoldoende informatie leveren in één test. Ook al werden multiplex en kwantitatieve PCR’s al ontworpen om de detectie van respiratoire ziektecomplexen in runderen en varkens of pathotypering van enterotoxigene Escherichia coli (ETEC) te ondersteunen, de geassocieerde kost is nog steeds te hoog (ongeveer 125 euro per staal). De laatste jaren werden verschillende technologieën voor sequentieanalyse geïntroduceerd, wat toeliet om de kost voor het sequeneren significant te verlagen. Het beschikbaar hebben van de realtime single molecule (SMRT) nanopore technologie (Oxford Nanopore Technologies) opende een nieuw tijdperk in de diagnostiek (gereviewd in Hoofdstuk 1). Het gebruik van deze technologie in een metagenomische workflow laat toe om zowel virale als bacteriële ziekteverwekkers in een enkele test te detecteren zonder vooraf pathogenen te selecteren. De toegevoegde waarde van deze toepassing werd aangetoond voor gekende respiratoire ziektecomplexen bij varken en rund. Dit toonde aan dat er cocirculatie van primaire, secundaire, en minder gekende pathogenen (zoals porcien hemagglutinating encephalomyelitis virus (PHEV) en Mycoplasmopsis arginini respectievelijk bij varken en rund (Hoofdstuk 3)). Daarnaast laat de technologie ook toe om alle microben (zowel viraal als bacterieel) op een (semi-)kwantitatieve manier te evalueren om zo hun relevantie binnen het ziektecomplex te bepalen. Dit samen met sequentieanalyses voor virale genomen. Dat laatste is een belangrijk voordeel omdat dit het (sub)typeren van virussen toelaat, wat vaak noodzakelijk is om een onderscheid te kunnen maken tussen vaccinstammen en wild type infecties (vb. porcien reproductief en respiratoir syndroom virus (PRRSV)) en het subtyperen van influenza A-virus bij varkens (swIAV)). De beschikbaarheid van deze genoomsequenties uit één enkele test laat toe om een bijdrage te leveren bij surveillance, epidemiologie, en het eventueel op punt stellen van vaccinaties zoals voorgesteld voor het porcien parvovirus type 1 (PPV1). Ook in de context van een virale uitbraak kan het gebruikt worden om snel sequentie-informatie te bekomen (vb. equien herpes virus 1 (EHV1)). Zo goed als alle dieren en staaltypes (inclusief fecaal, serum, respiratoir, weefsel en urine) kunnen gebruikt worden voor het sequeneren in een metagenomische context. Sommige staaltypes hebben echter nood aan een extra doelgerichte verrijking om complete virale genomen te bekomen. Dit werd aangetoond voor swIAV uit “vuile” orale vloeistoffen (Hoofdstuk 4). Kosten die geassocieerd worden met huidige testen liggen in de orde van multiplex PCR’s die toelaten gebruikt te worden door progressieve dierenartsen van vee, gezelschapsdieren en exotische dieren, samen met toepassingen voor academische instituten en farmaceutische bedrijven. Feedback uit het veld suggereert dat er nood is aan verdere typering van bacteriën (vb. virotypering) en het bepalen van antimicrobiële gevoeligheid om de huidige test breder bij routinediagnostiek te kunnen toepassen. Dit houdt een grotere opgave in omdat bestaande ad random metagenomische workflows nog niet in staat zijn om voldoende diepgang te leveren voor bacteriële genomische informatie. Hierbij zou een doelgerichte spike-in benadering toelaten om een verrijking uit te voeren voor specifieke genen, met het behoud van metagenomische gevoeligheid. Dit zou een uitgebreidere toepassing bieden voor routinediagnostiek in diagnostische laboratoria. Om dit toe te laten is fundamentele kennis noodzakelijk rond genen die geassocieerd worden met virulentie en antibioticaresistentie, waarvoor accurate en complete bacteriële genomen voorhanden moeten zijn. Huidige databases, zoals NCBI, hebben vaak onvoldoende genomische en fenotypische metadata voor veterinaire bacteriën beschikbaar. Dus werd moeite gedaan om deze informatie te voorzien voor relevante bacteriële species. Het gebruik van lange reads voor genoomassemblage werd uitgebreid getest en kon voor “standaard” bacteriën zoals E. coli en Actinobacillus species excellente genomen met voldoende accuraatheid en compleetheid afleveren (Hoofdstuk 5). Dit is vooral mogelijk door de significante verbetering in accuraatheid van ruwe reads via nieuwe technologie-updates waaronder nanopore chemie en base calling algortimes over de laatste jaren heen. Voor “niet-standaard” bacteriële species, zoals Mycoplasmopsis bovis en Brachyspira hyodysenteriae, was een taxon-specifiek base calling model noodzakelijk om gouden standaardniveau genomen (Illumina; >99.999% accuraatheid) af te leveren (Hoofdstuk 6). Het toepassen van genoom-wijde associatiestudies (GWAS) op deze bacteriële datasets liet toe om verscheidene genetische merkers (genen, puntmutaties en plasmides) te identificeren met hoge diagnostische voorspellende kracht voor zowel virotypering en bepaling van antibioticagevoeligheid (Hoofdstuk 5 en 6). Ook al konden niet alle resistentie fenotypes compleet voorspeld worden, in de toekomst kunnen deze via epigenetica (vb. methylatie) of het profileren van het bacterieel transcriptoom verder onderzocht worden. Voor die eerste kan de beschikbare ruwe nanopore data opnieuw geanalyseerd worden als er meer wijder toepasbare methylatie software en modellen voorhanden zijn. Dit zou toelaten om het complexe landschap van antibioticaresistentie breder te bestuderen. Het zal een (zeer) grote uitdaging zijn om deze data in een metagenomische context en workflow te implementeren. Vooraleer deze genetische merkers in een “virulentie en genetische gevoeligheidsbepaling aangereikte” metagenomische workflow kunnen gebruikt worden, is het aangeraden om nog uitgebreide validaties uit te voeren via doelgerichte mutagenese om zo proof-of-concept te bieden voor de bijdrage van deze merkers tot het virulentie en/of resistentie fenotype (zoals besproken in Hoofdstuk 7). Conclusie: in een relatief korte tijd kunnen metagenomische diagnostische workflows aangereikt worden met genetische markers, die geassocieerd worden met virulentie en antibioticaresistentie. Deze eerste fundamenten kunnen worden getest door de genetische informatie – zoals beschreven in dit werk – te implementeren om zo een breder toepassingsveld en integratie in veterinaire diagnostische laboratoria te bekomen. Een vergelijkbare benadering kan gebruikt worden om bestaande metagenomische workflows op punt te stellen voor een wijdere variëteit aan relevante virussen en bacteriën in het veld. Finaal zal dit ertoe leiden dat bij acute infecties accurate all-in-one diagnostische tests identificatie, typering en genetische antibioticaresistentiebepaling voor relevante bacteriën kunnen versnellen om zo het gebruik van antibiotica beter te sturen