African swine fever is a viral disease with major socioeconomic consequences in countries including Nigeria, where pig farming sustains many livelihoods. Despite the critical role of genomic data in outbreak control, Nigeria has remained underrepresented in global African swine fever virus (ASFV) genomic datasets. Here, we report the first in-country sequencing and genomic analysis of ASFV from recent outbreaks in Nigeria, representing the largest dataset of ASFV genomes generated in Nigeria and Africa to date. Using Oxford Nanopore and Illumina Technology platforms, we assembled 27 whole-genome sequences from field samples collected in Ogun, Osun, and Oyo States. Comparative analyses revealed nearly 100 single-nucleotide polymorphisms (SNPs) in coding regions and more than 250 across coding and non-coding regions, alongside multiple insertions and deletions relative to the Georgia 2007 ASFV reference genome. Notably, we report novel large deletions in variable regions, which may affect genes linked to virulence and host adaptation. Phylogenetic analysis revealed that the Nigerian ASFV genomes form a distinct clade within biotype II, which also diverges considerably from the genome backbone currently utilized for vaccine designs, highlighting the impact of viral divergence on current control measures.

Highly pathogenic avian influenza (HPAI) viruses cross species barriers and have the potential to cause pandemics. In North America, HPAI A(H5N1) viruses related to the goose/Guangdong 2.3.4.4b hemagglutinin phylogenetic clade have infected wild birds, poultry, and mammals. Our genomic analysis and epidemiological investigation showed that a reassortment event in wild bird populations preceded a single wild bird-to-cattle transmission episode. The movement of asymptomatic or presymptomatic cattle has likely played a role in the spread of HPAI within the United States dairy herd. Some molecular markers that may lead to changes in transmission efficiency and phenotype were detected at low frequencies. Continued transmission of H5N1 HPAI within dairy cattle increases the risk for infection and subsequent spread of the virus to human populations.

The Ebolavirus genus contains several of the deadliest zoonotic viruses known. One of these, Bundibugyo virus (BDBV), has been the causative agent of two outbreaks of human disease that have resulted in 211 known cases with a case fatality rate of 33.6%. Although bats are routinely implicated as the possible reservoir species for the ebolaviruses, the source of infection for index cases in almost all outbreaks is unknown with only limited epidemiological evidence directly linking human cases to bats. This lack of evidence leaves open the possibility that maintenance of one or more of these viruses could involve multiple host species or more complex spillover dynamics. Domestic pigs have been found naturally infected with Reston virus (RESTV) and are experimentally susceptible to infection with Ebola virus (EBOV), two other members of the Ebolavirus genus. Infection of pigs resulted in shedding of infectious virus with subsequent transmission to naïve animals being documented, including transmission to humans for RESTV and to nonhuman primates for EBOV. The susceptibility and subsequent viral shedding and pathogenesis of domestic pigs to other ebolaviruses and the potential role this species may play in virus ecology, spillover dynamics, and human public health risk is unknown. For these reasons, we conducted a series of studies aimed at determining the susceptibility of domestic pigs to BDBV thereby demonstrating that pigs are not only susceptible to experimental infection but that the development of productive infection, tissue dissemination, and shedding of infectious virus can also occur while animals remain clinically normal. The role of pigs as a possible interim or amplifying host for ebolaviruses is a concern for both human public health and food security.

African swine fever virus (ASFV) is the causative agent of a severe and highly contagious viral disease affecting domestic and wild swine. The current ASFV pandemic strain has a high mortality rate, severely impacting pig production and, for countries suffering outbreaks, preventing the export of their pig products for international trade. Early detection and diagnosis of ASFV is necessary to control new outbreaks before the disease spreads rapidly. One of the rate-limiting steps to identify ASFV by next-generation sequencing platforms is library preparation. Here, we investigated the capability of the Oxford Nanopore Technologies’ VolTRAX platform for automated DNA library preparation with downstream sequencing on Nanopore sequencing platforms as a proof-of-concept study to rapidly identify the strain of ASFV. Within minutes, DNA libraries prepared using VolTRAX generated near-full genome sequences of ASFV. Thus, our data highlight the use of the VolTRAX as a platform for automated library preparation, coupled with sequencing on the MinION Mk1C for field sequencing or GridION within a laboratory setting. These results suggest a proof-of-concept study that VolTRAX is an effective tool for library preparation that can be used for the rapid and real-time detection of ASFV.

Three rabbit hemorrhagic disease virus type 2 (RHDV2) coding-complete genome sequences were obtained from domestic and wild rabbits in Washington State in June and July 2023. These three RHDV2 sequences are <82% identical to previous RHDV2 sequences in North America and likely indicate a discrete incursion.

In September 2022, deaths of pigs manifesting pox-like lesions caused by swinepox virus were reported in Tshuapa Province, Democratic Republic of the Congo. Two human mpox cases were found concurrently in the surrounding community. Specific diagnostics and robust sequencing are needed to characterize multiple poxviruses and prevent potential poxvirus transmission.

Domestic pigs are a critical component of the food supply and one of the most commonly raised production animals. Pork consumption has driven the intensification of pig production expanding into environments conducive to increased emergence and spread of infectious diseases, including the spillover of pathogens into human populations. One of these emerging viruses, Reston virus (RESTV), is an enigma among the Orthoebolavirus genus in that its lack of human pathogenicity is in stark contrast to the high virulence associated with most other ebolaviruses. RESTV is, however, associated with outbreaks of highly lethal hemorrhagic disease in non-human primates (NHP), as well as poorly understood clinical manifestations of mixed virulence and lethality in naturally and experimentally infected domestic pigs. Our results show it is possible for RESTV derived from an NHP to infect domestic pigs resulting in a spectrum of disease, from asymptomatic to severe respiratory distress. Further, we report on the first experimental transmission of RESTV between infected pigs and a co-housed, naïve animal, as well as the first report of the successful use of group oral fluids for the detection of RESTV RNA and virus-specific IgA antibodies.

Foot-and-mouth disease (FMD) is a severe and extremely contagious viral disease of cloven-hoofed domestic and wild animals, which leads to serious economic losses to the livestock industry globally. FMD is caused by the FMD virus (FMDV), a positive -strand RNA virus that belongs to the genus Aphthovirus , within the family Picornaviridae . Early detection and characterization of FMDV strains are key factors to control new outbreaks and prevent the spread of the disease. Here, we describe a direct RNA sequencing method using Oxford Nanopore Technology (ONT) Flongle flow cells on MinION Mk1C (or GridION) to characterize FMDV. This is a rapid, low cost, and easily deployed point of care (POC) method for a near real-time characterization of FMDV in endemic areas or outbreak investigation sites.

Blood samples were collected from pigs at six abattoirs in the Kampala, Uganda metropolitan area from May 2021 through June 2022, and tested for African swine fever virus. Thirty-one samples with cycle threshold values < 26 from pigs with different geographic origins, clinical and pathologic signs, and Ornithodoros moubata exposure underwent whole genome sequencing. The p72 gene was used to genotype the isolates, and all were found to be genotype IX; whole genome sequences to previous genotype IX isolates confirmed their similarity. Six of the isolates had enough coverage to evaluate single nucleotide polymorphisms (SNPs). Five of the isolates differed from historic regional isolates, but had similar SNPs to one another, and the sixth isolate also differed from historic regional isolates, but also differed from the other five isolates, even though they are all genotype IX. Whole genome sequencing data provide additional detail on viral evolution that can be useful for molecular epidemiology, and understanding the impact of changes in genes to disease phenotypes, and may be needed for vaccine targeting should a commercial vaccine become available. More sequencing of African swine fever virus isolates is needed in Uganda to understand how and when the virus is changing.

Lakin, S. M., O'Donnell, V. K., Xu, L., Barrette, R. W., Barnabei, J., Nunez, R., Holland, R., Faburay, B., & Robbe-Austerman, S. (2022). Whole genome sequencing and molecular epidemiology of the 2021 African swine fever virus outbreak in the Dominican Republic. Transboundary and Emerging Diseases, 00, 00-00. The above article, published online on October 31, 2022 in Wiley Online Library (wileyonlinelibrary.com), has been withdrawn by agreement between the authors, Steven Lakin and Suelee Robbe Austerman, journal Editor in Chief, Michael Ward, and Wiley-VCH GmbH. The withdrawal has been agreed following no response from author Rafael Nunez to requests to sign the Journal's publishing license.

Lakin, S. M., O'Donnell, V. K., Xu, L., Barrette, R. W., Barnabei, J., Núñez, R., Holland, R., Faburay, B., & Robbe-Austerman, S. (2022). Whole genome sequencing and molecular epidemiology of the 2021 African swine fever virus outbreak in the Dominican Republic. Transboundary and Emerging Diseases, 00, 00-00. https://doi.org/10.1111/tbed.14751 The above article, published online on October 31, 2022 in Wiley Online Library (wileyonlinelibrary.com), has been withdrawn by agreement between the authors, Steven Lakin and Suelee Robbe Austerman, journal Editor in Chief, Michael Ward, and Wiley-VCH GmbH. The withdrawal has been agreed following no response from author Rafael Núñez to requests to sign the Journal's publishing license.

AbstractBackgroundCopper associated hepatopathy (CAH) has become an important and prevalent disease since the 1990's, coincidental with changes in copper (Cu) content in commercial dog foods. Knowing the demographic and histopathologic features related to hepatic Cu concentrations might aid in diagnosing CAH in dogs.Hypothesis/ObjectivesThe primary aim was to identify demographic and histopathologic features associated with abnormally high hepatic Cu concentrations.AnimalsDogs that underwent liver histopathology and Cu quantification at a veterinary diagnostic laboratory between July 2010 and February 2020.MethodsData was retrospectively collected from an electronic database. A Gaussian multiple regression model on the log scale was used to evaluate associations between hepatic Cu and a set of demographic and histologic features selected with machine learning methods.ResultsOf 4559 cases meeting criteria, 50% had hepatic Cu > 400 and 19% had Cu > 1000 ppm (parts per million) dry weight (reference range 120‐400). Median hepatic Cu was 391 ppm, range 4.5 to 31500. Age was negatively associated (P < .02), but specific breeds (Doberman pinscher, Labrador retriever, and West Highland white terrier) were positively associated with abnormally high hepatic Cu (P < .001). Severity of inflammation (mild, moderate, and severe) and necrosis/apoptosis were associated with abnormally high hepatic Cu (P < .01).Conclusion and Clinical ImportanceAbnormally high hepatic Cu is prevalent in hepatic biopsies from dogs. Machine learning modeling showed that necroinflammation, not cholestasis or cirrhosis, on hepatic histopathology, is predictive of higher hepatic Cu and might be a reliable histologic predictor of CAH.

African swine fever (ASF) is a high-consequence transboundary disease of domestic and wild swine often characterized by high case mortality rates. On July 29, 2021, the Dominican Republic announced the African swine fever virus (ASFV) had been detected in samples collected in early- to mid-July 2021. Retrospective testing of samples collected as part of a collaborative surveillance project between the United States and the Dominican Republic identified ASFV in samples collected as early as May 13, 2021. These detections represent a new outbreak of ASF in the Dominican Republic, which had been declared free of the disease since 1981. Overall, 73 whole genomes of ASFV were sequenced from clinical samples received during the outbreak across 18 provinces. The genomic sequence data have been deposited in public databases to support and expand global data sharing on this impactful disease. While the sequences show a high degree of nucleotide identity to publicly available ASFV genomes from Europe and Asia, they are genetically distant from genomes in the public repositories by at least 8 previously undescribed single nucleotide polymorphisms.

Antimicrobial resistance spread is a worldwide health challenge, stemming in large part from the ability of microorganisms to share their genetic material through horizontal gene transfer. To address this issue, many countries and international organizations have adopted a One Health approach to curtail the proliferation of antimicrobial-resistant bacteria.

Antimicrobial resistance (AR) spread is a worldwide health challenge, stemming in large part, from the ability of microbes to share their genetic material through horizontal gene transfer (HGT). Overuse and misuse of antibiotics in clinical settings and in food production have been linked to this increased prevalence and spread of AR. Consequently, public health and consumer concerns have resulted in a remarkable recent reduction in antibiotics used for food animal production. This is driven by the assumption that removing this selective pressure will favor the recovery of antibiotic susceptible taxa and will limit AR sharing through HGT, allowing the currently available antibiotic arsenal to be effective for a longer period. In this study we used broiler chicks raised antibiotic-free and Salmonella enterica serovar Heidelberg (SH), as a model food pathogen, to test this hypothesis. Our results show that neonatal broiler chicks challenged with an antibiotic susceptible SH strain and raised without antibiotics carried susceptible and multidrug resistance SH strains 14 days after challenge. SH infection perturbed the microbiota of broiler chicks and gavaged chicks acquired antibiotic resistant SH at a higher rate. We determined that the acquisition of a plasmid from commensal Escherichia coli population conferred multidrug resistance phenotype to SH recipients and carriage of this plasmid increased the fitness of SH under acidic selection pressure. These results suggest that HGT of AR shaped the evolution of SH and that antibiotic use reduction alone is insufficient to limit antibiotic resistance transfer from commensal bacteria to Salmonella . Importance The reported increase in antibiotic resistant bacteria in humans have resulted in a major shift away from antibiotics use in food animal production. This has been driven by the assumption that removing antibiotics will select for antibiotic susceptible bacterial taxa, and this in turn will allow the currently available antibiotic arsenal to be more effective. This shift in practice has highlighted new questions that need to be answered to assess the effectiveness of antibiotic removal in reducing the spread of antibiotic resistance bacteria. This research demonstrates that antibiotic susceptible Salmonella Heidelberg strains can acquire multidrug resistance from commensal bacteria present in the gut of neonatal broiler chicks, even in the absence of antibiotic selection. We demonstrate that exposure to acidic pH drove the horizontal transfer of antimicrobial resistance plasmids and suggests that simply removing antibiotics from food-animal production might not be sufficient to limit the spread of antimicrobial resistance.

The overuse and misuse of antibiotics in clinical settings and in food production have been linked to the increased prevalence and spread of antimicro-bial resistance (AR). Consequently, public health and consumer concerns have resulted in a remarkable reduction in antibiotics used for food animal production. However, there are no data on the effectiveness of antibiotic removal in reducing AR shared through horizontal gene transfer (HGT). In this study, we used neonatal broiler chicks and Salmonella enterica serovar Heidelberg, a model food pathogen, to test if chicks raised antibiotic free harbor transferable AR. We challenged chicks with an antibiotic-susceptible S. Heidelberg strain using various routes of inoculation and determined if S. Heidelberg isolates recovered carried plasmids conferring AR. We used antimicrobial susceptibility testing and whole-genome sequencing (WGS) to show that chicks grown without antibiotics harbored an antimicrobial resistant S. Heidelberg population at 14 days after challenge and chicks challenged orally acquired AR at a higher rate than chicks inoculated via the cloaca. Using 16S rRNA gene sequencing, we found that S. Heidelberg infection perturbed the microbiota of broiler chicks, and we used metagenomics and WGS to confirm that a commensal Escherichia coli population was the main reservoir of an IncI1 plasmid acquired by S. Heidelberg. The carriage of this IncI1 plasmid posed no fitness cost to S. Heidelberg but increased its fitness when exposed to acidic pH in vitro. These results suggest that HGT of plasmids carrying AR shaped the evolution of S. Heidelberg and that an-tibiotic use reduction alone is insufficient to limit antibiotic resistance transfer from commensal bacteria to Salmonella enterica. IMPORTANCE The reported increase in antibiotic-resistant bacteria in humans has resulted in a major shift away from antibiotic use in food animal production. This shift has been driven by the assumption that removing antibiotics will select for anti-biotic susceptible bacterial taxa, which in turn will allow the currently available anti-biotic arsenal to be more effective. This change in practice has highlighted new questions that need to be answered to assess the effectiveness of antibiotic removal in reducing the spread of antibiotic resistance bacteria. This research demonstrates that antibiotic-susceptible Salmonella enterica serovar Heidelberg strains can acquire multidrug resistance from commensal bacteria present in the gut of neonatal broiler chicks, even in the absence of antibiotic selection. We demonstrate that exposure to acidic pH drove the horizontal transfer of antimicrobial resistance plasmids and suggest that simply removing antibiotics from food animal production might not be sufficient to limit the spread of antimicrobial resistance.

Host microbiome homeostasis ensures that gut conditions are unfavorable to an invading pathogen such as Salmonella enterica. Consequently, fostering a “balanced” gut microbiome through the administration of microbes that can competitively exclude pathogens has gained a lot of attention and use in human and animal medicine. However, little is known on how competitive exclusion affects the transfer of antibiotic resistance. To shed more light on this question, we challenged neonatal broiler chicks raised on reused broiler chicken litter – a complex environment comprising of decomposing pine shavings, feces, uric acid, feathers, and feed, with Salmonella Heidelberg (S. Heidelberg), a model pathogen. We show that chicks raised on reused litter carried lower abundance of Salmonella and harbored a more uniform and diverse microbiome comprising of bacterial species that are known to provide colonization resistance towards Salmonella compared to chicks raised on fresh bedding composed of pine shavings. Additionally, these bacterial species were associated with a lower horizontal transfer of multidrug resistance genes to S. Heidelberg. Using in vitro competition experiments, we confirmed that conjugation between S. Heidelberg and E. coli strains from chicks raised on fresh litter resulted in the acquisition of multidrug resistant plasmids. Contrastingly, bacteriophage-mediated recombination between S. Heidelberg and E. coli strains made the acquisition of plasmid-mediated β-lactamase gene (blaCMY-2) possible. Collectively, this study demonstrates that competitive exclusion can reduce the transfer of antibiotic resistance and provides information on the bacterial species that can be explored for their benefits to reduce antibiotic resistance transfer. Importance/Significance Antimicrobial resistance spread is a worldwide health challenge, stemming in large part, from the ability of microorganisms to share their genetic material through horizontal gene transfer. To address this issue, many countries and international organization have adopted a one health approach to curtail the proliferation of antibiotic resistant bacteria. This includes the removal and reduction of antibiotics used in food animal production and the development of alternatives to antibiotics. However, there is still a significant knowledge gap in our understanding of how antimicrobial resistance spreads in the absence of antibiotic selection and the role commensal bacteria play in reducing antibiotic resistance transfer. In this study, we demonstrate that commensal bacteria play a key role in reducing the horizontal transfer of antibiotic resistance to Salmonella and provide the identity and characteristics of the bacterial species that performs this function in broiler chickens.

Eight isolates of Streptococcus equi subsp. zooepidemicus were isolated from mares with clinical cases of endometritis. S. equi subsp. zooepidemicus strains were chosen for sequencing based on differing levels of biofilm production in vitro . Using Illumina short-read sequencing in conjunction with MinION sequencing, we report the genomes of eight isolates.

The multinomial and related distributions have long been used to model categorical, count-based data in fields ranging from bioinformatics to natural language processing. Commonly utilized variants include the standard multinomial and the Dirichlet multinomial distributions due to their computational efficiency and straightforward parameter estimation process. However, these distributions make strict assumptions about the mean, variance, and covariance between the categorical features being modeled. If these assumptions are not met by the data, it may result in poor parameter estimates and loss in accuracy for downstream applications like classification. Here, we explore efficient parameter estimation and supervised classification methods using an alternative distribution, called the Beta-Liouville multinomial, which relaxes some of the multinomial assumptions. We show that the Beta-Liouville multinomial is comparable in efficiency to the Dirichlet multinomial for Newton-Raphson maximum likelihood estimation, and that its performance on simulated data matches or exceeds that of the multinomial and Dirichlet multinomial distributions. Finally, we demonstrate that the Beta-Liouville multinomial outperforms the multinomial and Dirichlet multinomial on two out of four gold standard datasets, supporting its use in modeling data with low to medium class overlap in a supervised classification context.

Gold standard datasets used in the publication Fast Maximum Likelihood Estimation and Supervised Classification for the Beta-Liouville Multinomial. Datasets were prepared by Cardoso-Cachopo (2007).