Porcine ear necrosis: characterization of lesions and associated pathogens.

Porcine ear necrosis (PEN) is characterized by ulcerative lesions of the ear auricle. To investigate that problem, three farms with PEN in nursery pigs were included, and the study aim was to characterize PEN and the potential role of pathogens and mycotoxins. Within each farm, one batch of weaned piglets was included and the prevalence and severity of PEN were monitored for 6-7 weeks. Within each batch, 30 PEN-affected/non-affected animals were randomly selected. Blood samples were taken from these animals, to assess the systemic presence of pathogens and mycotoxins, as well as punch biopsies from the ear auricle for histopathological examination. From 10 animals, scrapings and swabs from the lesions were subjected to nanopore metagenomic sequencing and bacteriological cultivation, respectively. In all three farms, lesions appeared within 3-4 weeks post-weaning. The prevalence at the end of the nursery was 33%, 24%, and 46% for farms A, B, and C, respectively. Most affected pigs had mild to moderate lesions. Blood samples revealed low to very low levels of pathogens and mycotoxins. Different bacteria such as Staphylococcus, Streptococcus, Fusobacterium, Mycoplasma, and Clostridium species were identified by sequencing in the scrapings. The first two pathogens were also most often identified in bacterial cultures. Mycoplasma hyopharyngis was only found in PEN-affected pigs. Histopathological changes were primarily observed in the outer layer of the epidermis. The results suggest that PEN lesions develop by damage to the outer part of the skin e.g. by ear suckling or biting, followed by multiplication of opportunistic pathogens.

Surveillance and Genomic Characterization of Influenza A and D Viruses in Swine, Belgium and the Netherlands, 2019-2021.

During 2019-2021, we isolated 62 swine influenza A viruses in Belgium and the Netherlands. We also detected influenza D in pigs in the Netherlands. The ever-changing diversity of influenza viruses and the identification of influenza D emphasize the need for more virus surveillance.

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

Introduction: Enterotoxigenic 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. Methods: Here, 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. Results: Genetic 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. Conclusion: Our 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.

Prevalence and Molecular Characterization of Methicillin-Resistant Staphylococci (MRS) and Mammaliicocci (MRM) in Dromedary Camels from Algeria: First Detection of SCCmec-mecC Hybrid in Methicillin-Resistant Mammaliicoccus lentus.

Dromedary camels are an important source of food and income in many countries. However, it has been largely overlooked that they can also transmit antibiotic-resistant bacteria. The aim of this study was to identify the Staphylococcaceae bacteria composition of the nasal flora in dromedary camels and evaluate the presence of methicillin-resistant Mammaliicoccus (MRM) and methicillin-resistant Staphylococcus (MRS) in dromedary camels in Algeria. Nasal swabs were collected from 46 camels from seven farms located in two different regions of Algeria (M'sila and Ouargla). We used non-selective media to determine the nasal flora, and antibiotic-supplemented media to isolate MRS and MRM. The staphylococcal isolates were identified using an Autoflex Biotyper Mass Spectrometer (MALDI-TOF MS). The mecA and mecC genes were detected by PCR. Methicillin-resistant strains were further analysed by long-read whole genome sequencing (WGS). Thirteen known Staphylococcus and Mammaliicoccus species were identified in the nasal flora, of which half (49.2%) were coagulase-positive staphylococci. The results showed that four out of seven farms were positive for MRS and/or MRM, with a total of 16 isolates from 13 dromedary camels. The predominant species were M. lentus, S. epidermidis, and S. aureus. Three methicillin-resistant S. aureus (MRSA) were found to be ST6 and spa type t304. Among methicillin-resistant S. epidermidis (MRSE), ST61 was the predominant ST identified. Phylogenetic analysis showed clonal relatedness among M. lentus strains, while S. epidermidis strains were not closely related. Resistance genes were detected, including mecA, mecC, ermB, tet(K), and blaZ. An SCCmec type VIII element was found in a methicillin-resistant S. hominis (MRSH) belonging to the ST1 strain. An SCCmec-mecC hybrid element was detected in M. lentus, similar to what was previously detected in M. sciuri. This study highlights that dromedary camels may be a reservoir for MRS and MRM, and that they contain a specific set of SCCmec elements. This emphasizes the need for further research in this ecological niche from a One Health perspective.

Whole genome sequencing to study antimicrobial resistance and RTX virulence genes in equine Actinobacillus isolates.

Actinobacillus equuli is mostly associated with disease in horses and is most widely known as the causative agent of sleepy foal disease. Even though existing phenotypic tools such as biochemical tests, 16S rRNA gene sequencing, and Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) can be used to identify members of the Actinobacillus genus, these methods struggle to differentiate between certain species and do not allow strain, virulence, and antimicrobial susceptibility typing. Hence, we performed in-depth analysis of 24 equine Actinobacillus isolates using phenotypic identification and susceptibility testing on the one hand, and long-read nanopore whole genome sequencing on the other hand. This allowed to address strain divergence down to the whole genome single nucleotide polymorphism (SNP) level. While lowest resolution was observed for 16S rRNA gene classification, a new multi-locus sequence typing (MLST) scheme allowed proper classification up to the species level. Nevertheless, a SNP-level analysis was required to distinguish A. equuli subspecies equuli and haemolyticus. Our data provided first WGS data on Actinobacillus genomospecies 1, Actinobacillus genomospecies 2, and A. arthritidis, which allowed the identification of a new Actinobacillus genomospecies 1 field isolate. Also, in-depth characterization of RTX virulence genes provided information on the distribution, completeness, and potential complementary nature of the RTX gene operons within the Actinobacillus genus. Even though overall low prevalence of acquired resistance was observed, two plasmids were identified conferring resistance to penicillin-ampicillin-amoxicillin and chloramphenicol in one A. equuli strain. In conclusion our data delivered new insights in the use of long-read WGS in high resolution identification, virulence gene typing, and antimicrobial resistance (AMR) of equine Actinobacillus species.

Successful Whole Genome Nanopore Sequencing of Swine Influenza A Virus (swIAV) Directly from Oral Fluids Collected in Polish Pig Herds.

Influenza A virus (IAV) is a single-stranded, negative-sense RNA virus and a common cause of seasonal flu in humans. Its genome comprises eight RNA segments that facilitate reassortment, resulting in a great variety of IAV strains. To study these processes, the genetic code of each segment should be unraveled. Fortunately, new third-generation sequencing approaches allow for cost-efficient sequencing of IAV segments. Sequencing success depends on various factors, including proper sample storage and processing. Hence, this work focused on the effect of storage of oral fluids and swIAV sequencing. Oral fluids (n = 13) from 2017 were stored at -22 °C and later transferred to -80 °C. Other samples (n = 21) were immediately stored at -80 °C. A reverse transcription quantitative PCR (RT-qPCR) pre- and post-storage was conducted to assess IAV viral loads. Next, samples were subjected to two IAV long-read nanopore sequencing methods to evaluate success in this complex matrix. A significant storage-associated loss of swIAV loads was observed. Still, a total of 17 complete and 6 near-complete Polish swIAV genomes were obtained. Genotype T, (H1avN2, seven herds), P (H1N1pdm09, two herds), U (H1avN1, three herds), and A (H1avN1, 1 herd) were circulated on Polish farms. In conclusion, oral fluids can be used for long-read swIAV sequencing when considering appropriate storage and segment amplification protocols, which allows us to monitor swIAV in an animal-friendly and cost-efficient manner.

Viral and Bacterial Profiles in Endemic Influenza A Virus Infected Swine Herds Using Nanopore Metagenomic Sequencing on Tracheobronchial Swabs.

Swine influenza A virus (swIAV) plays an important role in porcine respiratory infections. In addition to its ability to cause severe disease by itself, it is important in the multietiological porcine respiratory disease complex. Still, to date, no comprehensive diagnostics with which to study polymicrobial infections in detail have been offered. Hence, veterinary practitioners rely on monospecific and costly diagnostics, such as Reverse Transcription quantitative PCR (RT-qPCR), antigen detection, and serology. This prevents the proper understanding of the entire disease context, thereby hampering effective preventive and therapeutic actions. A new, nanopore-based, metagenomic diagnostic platform was applied to study viral and bacterial profiles across 4 age groups on 25 endemic swIAV-infected German farms with respiratory distress in the nursery. Farms were screened for swIAV using RT-qPCR on nasal and tracheobronchial swabs (TBS). TBS samples were pooled per age, prior to metagenomic characterization. The resulting data showed a correlation between the swIAV loads and the normalized reads, supporting a (semi-)quantitative interpretation of the metagenomic data. Interestingly, an in-depth characterization using beta diversity and PERMANOVA analyses allowed for the observation of an age-dependent interplay of known microbial agents. Also, lesser-known microbes, such as porcine polyoma, parainfluenza, and hemagglutinating encephalomyelitis viruses, were observed. Analyses of swIAV incidence and clinical signs showed differing microbial communities, highlighting age-specific observations of various microbes in porcine respiratory disease. In conclusion, nanopore metagenomics were shown to enable a panoramic view on viral and bacterial profiles as well as putative pathogen dynamics in endemic swIAV-infected herds. The results also highlighted the need for better insights into lesser studied agents that are potentially associated with porcine respiratory disease. IMPORTANCE To date, no comprehensive diagnostics for the study of polymicrobial infections that are associated with porcine respiratory disease have been offered. This precludes the proper understanding of the entire disease landscape, thereby hampering effective preventive and therapeutic actions. Compared to the often-costly diagnostic procedures that are applied for the diagnostics of porcine respiratory disease nowadays, a third-generation nanopore sequencing diagnostics workflow presents a cost-efficient and informative tool. This approach offers a panoramic view of microbial agents and contributes to the in-depth observation and characterization of viral and bacterial profiles within the respiratory disease context. While these data allow for the study of age-associated, swIAV-associated, and clinical symptom-associated observations, it also suggests that more effort should be put toward the investigation of coinfections and lesser-known pathogens (e.g., PHEV and PPIV), along with their potential roles in porcine respiratory disease. Overall, this approach will allow veterinary practitioners to tailor treatment and/or management changes on farms in a quicker, more complete, and cost-efficient way.

Predictive Power of Long-Read Whole-Genome Sequencing for Rapid Diagnostics of Multidrug-Resistant Brachyspira hyodysenteriae Strains.

Infections with Brachyspira hyodysenteriae, the etiological agent of swine dysentery, result in major economic losses in the pig industry worldwide. Even though microbial differentiation of various Brachyspira species can be obtained via PCR, no quick diagnostics for antimicrobial susceptibility testing are in place, which is mainly due to the time-consuming (4 to 7 days) anaerobic growth requirements of these organisms. Veterinarians often rely on a clinical diagnosis for initiating antimicrobial treatment. These treatments are not always effective, which may be due to high levels of acquired resistance in B. hyodysenteriae field isolates. By using long-read-only whole-genome sequencing and a custom-trained Bonito base-calling model, 81 complete B. hyodysenteriae genomes with median Q51 scores and 99% completeness were obtained from 86 field strains. This allowed the assessment of the predictive potential of genetic markers in relation to the observed acquired resistance phenotypes obtained via agar dilution susceptibility testing. Multidrug resistance was observed in 77% and 21% of the tested strains based on epidemiological cutoff and clinical breakpoint values, respectively. The predictive power of genetic hallmarks (genes and/or gene mutations) for antimicrobial susceptibility testing was promising. Sensitivity and specificity for tiamulin [tva(A) and 50SL3N148S, 99% and 67%], valnemulin [tva(A), 97% and 92%), lincomycin (23SA2153T/G and lnuC, 94% and 100%), tylvalosin (23SA2153T/G, 99% and 93%), and doxycycline (16SG1026C, 93% and 87%) were determined. The predictive power of these genetic hallmarks is promising for use in sequencing-based workflows to speed up swine dysentery diagnostics in veterinary medicine and determine proper antimicrobial use. IMPORTANCE Diagnostics for swine dysentery rely on the identification of Brachyspira species using molecular techniques. Nevertheless, no quick diagnostic tools are available for antimicrobial susceptibility testing due to extended growth requirements (7 to 14 days). To enable practitioners to tailor antimicrobial treatment to specific strains, long-read sequencing-based methods are expected to lead to rapid methods in the future. Nevertheless, their potential implementation should be validated extensively. This mainly implies assessing sequencing accuracy and the predictive power of genetic hallmarks in relation to their observed (multi)resistance phenotypes.

IABS/DCVMN webinar on next generation sequencing.

Next Generation Sequencing (NGS) is a new technology that could overcome some of the limitations of the current viral testing methods for demonstrating the absence of adventitious agents in biologics. This report is for the webinar that was organized by the International Alliance for Biological Standardization (IABS) and the Developing Countries Vaccine Manufacturers Network (DCVMN), held on July 20, 2022, as an introduction to the technical and bioinformatics concepts of NGS and to some of the strengths and limitations of using the technology for those working in vaccine production or development. The current state of scientific knowledge and readiness of NGS to replace or supplement the current viral tests was further discussed in the 3rd Conference on NGS for Adventitious Virus Detection in Biologics for Humans and Animals that was held in Rockville, Maryland, USA, on September 27-28, 2022. The application of NGS to supplement or replace current in vivo and in vitro assays in adventitious virus testing during vaccine production is promising; however, assay performance (sensitivity, specificity, and reproducibility) needs to be demonstrated, which may include laboratory and bioinformatics work. Efforts from regulatory authorities, industry, and researchers are ongoing to facilitate validation and establishment of NGS as a new method for virus detection.

Milk lactose protects against porcine group A rotavirus infection.

Rotavirus A (RVA) is an important pathogen causing acute gastroenteritis in animals and humans. Attachment to the host receptor is a crucial step for virus replication. The VP8* domain is the distal terminal region of the RVA spike protein VP4 (expressed by the P gene) and is important for rotavirus binding and infectivity. Recent studies have indicated a role for non-sialylated glycans, including mucin core 2 and histo-blood group antigens (HBGAs), in the infectivity of human and animal group A rotaviruses. In the present study, we determined if porcine rotavirus-derived recombinant VP8* of the endemic strains 14R103 G5P[6], 13R054 G5P[7], 12R010 G4P[13], 12R046 G9P[23], and 12R022 G2P[27] interact with hitherto uncharacterized glycans. We successfully produced five recombinant GST-VP8* proteins of genotype P[6], P[7], P[13], P[23], and P[27]. The hemagglutination assay showed genotypes P[7] and P[23] hemagglutinate porcine and human red blood cells. In an array screen of > 300 glycans, recombinant VP8* of rotavirus genotype P[6], P[7], and P[13] showed specific binding to glycans with a Gal-ß-1,4-Glc (ß-lactose) motif, which forms the core structure of HBGAs. The specificity of glycan-binding was confirmed through an ELISA-based oligosaccharide binding assay. Further, 13R054 G5P[7] and 12R046 G9P[23] infectivity was significantly reduced by ß-lactose in MA104 cells and primary porcine enterocytes. These data suggest that lactose, the main natural sugar in milk, plays an important role in protecting piglets from enteric viral replication and diarrhea.

Molecular epidemiology of Porcine Parvovirus Type 1 (PPV1) and the reactivity of vaccine-induced antisera against historical and current PPV1 strains.

Porcine Parvovirus Type 1 (PPV1) contributes to important losses in the swine industry worldwide. During a PPV1 infection, embryos and fetuses are targeted, resulting in stillbirth, mummification, embryonic death, and infertility (SMEDI syndrome). Even though vaccination is common in gilts and sows, strains mainly belonging to the 27a-like group have been spreading in Europe since early 2000s, resulting in SMEDI problems and requiring in-depth studies into the molecular epidemiology and vaccination efficacy of commercial vaccines. Here, we show that PPV1 has evolved since 1855 [1737, 1933] at a rate of 4.71 × 10-5 nucleotide substitutions per site per year. Extensive sequencing allowed evaluating and reassessing the current PPV1 VP1-based classifications, providing evidence for the existence of four relevant phylogenetic groups. While most European strains belong to the PPV1a (G1) or PPV1b (G2 or 27a-like) group, most Asian and American G2 strains and some European strains were divided into virulent PPV1c (e.g. NADL-8) and attenuated PPV1d (e.g. NADL-2) groups. The increase in the swine population, vaccination degree, and health management (vaccination and biosafety) influenced the spread of PPV1. The reactivity of anti-PPV1 antibodies from sows vaccinated with Porcilis© Parvo, Eryseng© Parvo, or ReproCyc© ParvoFLEX against different PPV1 field strains was the highest upon vaccination with ReproCyc© ParvoFLEX, followed by Eryseng© Parvo, and Porcilis© Parvo. Our findings contribute to the evaluation of the immunogenicity of existing vaccines and support the development of new vaccine candidates. Finally, the potential roles of cluster-specific hallmark amino acids in elevated pathogenicity and viral entry are discussed.

The Attenuated Pseudorabies Virus Vaccine Strain Bartha Hyperactivates Plasmacytoid Dendritic Cells by Generating Large Amounts of Cell-Free Virus in Infected Epithelial Cells.

Pseudorabies virus (PRV) is a porcine alphaherpesvirus and the causative agent of Aujeszky's disease. Successful eradication campaigns against PRV have largely relied on the use of potent PRV vaccines. The live attenuated Bartha strain, which was produced by serial passaging in cell culture, represents one of the hallmark PRV vaccines. Despite the robust protection elicited by Bartha vaccination, very little is known about the immunogenicity of the Bartha strain. Previously, we showed that Bartha-infected epithelial cells trigger plasmacytoid dendritic cells (pDC) to produce much higher levels of type I interferons than cells infected with wild-type PRV. Here, we show that this Bartha-induced pDC hyperactivation extends to other important cytokines, including interleukin-12/23 (IL-12/23) and tumor necrosis factor alpha (TNF-α) but not IL-6. Moreover, Bartha-induced pDC hyperactivation was found to be due to the strongly increased production of extracellular infectious virus (heavy particles [H-particles]) early in infection of epithelial cells, which correlated with a reduced production of noninfectious light particles (L-particles). The Bartha genome is marked by a large deletion in the US region affecting the genes encoding US7 (gI), US8 (gE), US9, and US2. The deletion of the US2 and gE/gI genes was found to be responsible for the observed increase in extracellular virus production by infected epithelial cells and the resulting increased pDC activation. The deletion of gE/gI also suppressed L-particle production. In conclusion, the deletion of US2 and gE/gI in the genome of the PRV vaccine strain Bartha results in the enhanced production of extracellular infectious virus in infected epithelial cells and concomitantly leads to the hyperactivation of pDC. IMPORTANCE The pseudorabies virus (PRV) vaccine strain Bartha has been and still is critical in the eradication of PRV in numerous countries. However, little is known about how this vaccine strain interacts with host cells and the host immune system. Here, we report the surprising observation that Bartha-infected epithelial porcine cells rapidly produce increased amounts of extracellular infectious virus compared to wild-type PRV-infected cells, which in turn potently stimulate porcine plasmacytoid dendritic cells (pDC). We found that this phenotype depends on the deletion of the genes encoding US2 and gE/gI. We also found that Bartha-infected cells secrete fewer pDC-inhibiting light particles (L-particles), which appears to be caused mainly by the deletion of the genes encoding gE/gI. These data generate novel insights into the interaction of the successful Bartha vaccine with epithelial cells and pDC and may therefore contribute to the development of vaccines against other (alphaherpes)viruses.

Organ-specific genome diversity of replication-competent SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is not always confined to the respiratory system, as it impacts people on a broad clinical spectrum from asymptomatic to severe systemic manifestations resulting in death. Further, accumulation of intra-host single nucleotide variants during prolonged SARS-CoV-2 infection may lead to emergence of variants of concern (VOCs). Still, information on virus infectivity and intra-host evolution across organs is sparse. We report a detailed virological analysis of thirteen postmortem coronavirus disease 2019 (COVID-19) cases that provides proof of viremia and presence of replication-competent SARS-CoV-2 in extrapulmonary organs of immunocompromised patients, including heart, kidney, liver, and spleen (NCT04366882). In parallel, we identify organ-specific SARS-CoV-2 genome diversity and mutations of concern N501Y, T1027I, and Y453F, while the patient had died long before reported emergence of VOCs. These mutations appear in multiple organs and replicate in Vero E6 cells, highlighting their infectivity. Finally, we show two stages of fatal disease evolution based on disease duration and viral loads in lungs and plasma. Our results provide insights about the pathogenesis and intra-host evolution of SARS-CoV-2 and show that COVID-19 treatment and hygiene measures need to be tailored to specific needs of immunocompromised patients, even when respiratory symptoms cease.

Genome-Wide Association Study Reveals Genetic Markers for Antimicrobial Resistance in Mycoplasma bovis.

Mycoplasma bovis causes many health and welfare problems in cattle. Due to the absence of clear insights regarding transmission dynamics and the lack of a registered vaccine in Europe, control of an outbreak depends mainly on antimicrobial therapy. Unfortunately, antimicrobial susceptibility testing (AST) is usually not performed, because it is time-consuming and no standard protocol or clinical breakpoints are available. Fast identification of genetic markers associated with acquired resistance may at least partly resolve former issues. Therefore, the aims of this study were to implement a first genome-wide association study (GWAS) approach to identify genetic markers linked to antimicrobial resistance (AMR) in M. bovis using rapid long-read sequencing and to evaluate different epidemiological cutoff (ECOFF) thresholds. High-quality genomes of 100 M. bovis isolates were generated by Nanopore sequencing, and isolates were categorized as wild-type or non-wild-type isolates based on MIC testing results. Subsequently, a k-mer-based GWAS analysis was performed to link genotypes with phenotypes based on different ECOFF thresholds. This resulted in potential genetic markers for macrolides (gamithromycin and tylosin) (23S rRNA gene and 50S ribosomal unit) and enrofloxacin (GyrA and ParC). Also, for tilmicosin and the tetracyclines, previously described mutations in both 23S rRNA alleles and in one or both 16S rRNA alleles were observed. In addition, two new 16S rRNA mutations were possibly associated with gentamicin resistance. In conclusion, this study shows the potential of quick high-quality Nanopore sequencing and GWAS analysis in the evaluation of phenotypic ECOFF thresholds and the rapid identification of M. bovis strains with acquired resistance. IMPORTANCE Mycoplasma bovis is a leading cause of pneumonia but also causes other clinical signs in cattle. Since no effective vaccine is available, current M. bovis outbreak treatment relies primarily on the use of antimicrobials. However, M. bovis is naturally resistant to different antimicrobials, and acquired resistance against macrolides and fluoroquinolones is frequently described. Therefore, AST is important to provide appropriate and rapid antimicrobial treatment in the framework of AMR and to prevent the disease from spreading and/or becoming chronic. Unfortunately, phenotypic AST is time-consuming and, due to the lack of clinical breakpoints, the interpretation of AST in M. bovis is limited to the use of ECOFF values. Therefore, the objective of this study was to identify known and potentially new genetic markers linked to AMR phenotypes of M. bovis isolates, exploiting the power of a GWAS approach. For this, we used high-quality and complete Nanopore-sequenced M. bovis genomes of 100 isolates.

Evaluation of Nanopore Sequencing as a Diagnostic Tool for the Rapid Identification of Mycoplasma bovis from Individual and Pooled Respiratory Tract Samples.

Rapid identification of Mycoplasma bovis infections in cattle is a key factor to guide antimicrobial therapy and biosecurity measures. Recently, Nanopore sequencing became an affordable diagnostic tool for both clinically relevant viruses and bacteria, but the diagnostic accuracy for M. bovis identification is undocumented. Therefore, in this study Nanopore sequencing was compared to rapid identification of M. bovis with matrix-assisted laser desorption ionization-time of flight mass spectrometry (RIMM) and a triplex real-time PCR assay in a Bayesian latent class model (BLCM) for M. bovis in bronchoalveolar lavage fluid (BALf) samples obtained from calves. In practice, pooling of samples is often used to save money, but the influence on diagnostic accuracy has not been described for M. bovis. Therefore, a convenience sample of 17 pooled samples containing 5 individual BALf samples per farm was analyzed as well. The results for the pooled samples were compared with those for the individual samples to determine sensitivity and specificity. The BLCM showed good sensitivity (77.3% [95% credible interval, 57.8 to 92.8%]) and high specificity (97.4% [91.5 to 99.7%]) for Nanopore sequencing, compared to RIMM (sensitivity, 93.0% [76.8 to 99.5%]; specificity, 91.3% [82.5 to 97.0%]) and real-time PCR (sensitivity, 94.6% [89.7 to 97.7%]; specificity, 86.0% [76.1 to 93.6%]). Sensitivity and specificity of pooled analysis for M. bovis were 85.7% (95% confidence interval, 59.8 to 111.6%) and 90.0% (71.4 to 108.6%%), respectively, for Nanopore sequencing and 100% (100% to 100%) and 88.9% (68.4 to 109.4%) for RIMM. In conclusion, Nanopore sequencing is a rapid, reliable tool for the identification of M. bovis. To reduce costs and increase the chance of M. bovis identification, pooling of 5 samples for Nanopore sequencing and RIMM is possible.

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.

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.

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.

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

BACKGROUND: 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. RESULTS: Here, a taxon-specific custom-trained Bonito v.0.1.3 basecalling model (custom-pg45) was implemented in various WGS assembly bioinformatics pipelines. Using MinION sequencing data, we showed improved consensus accuracies up to Q45.2 and Q46.7 for reference-based and Canu de novo assembled M. bovis genomes, respectively. Furthermore, the custom-pg45 model resulted in mean consensus accuracies of Q45.0 and genome completeness of 94.6% for nine M. bovis field strains. Improvements were also observed for the single-use Flongle sequencer (mean Q36.0 accuracies and 80.3% genome completeness). CONCLUSIONS: These results implicate that taxon-specific basecalling of MinION and single-use Flongle Nanopore long reads are of great value to be implemented in rapid all-in-one WGS tools as evidenced for Mycoplasma bovis as an example.