HAM-ART: An optimised culture-free Hi-C metagenomics pipeline for tracking antimicrobial resistance genes in complex microbial communities.

Shotgun metagenomics is a powerful tool to identify antimicrobial resistance (AMR) genes in microbiomes but has the limitation that extrachromosomal DNA, such as plasmids, cannot be linked with the host bacterial chromosome. Here we present a comprehensive laboratory and bioinformatics pipeline HAM-ART (Hi-C Assisted Metagenomics for Antimicrobial Resistance Tracking) optimised for the generation of metagenome-assembled genomes including both chromosomal and extrachromosomal AMR genes. We demonstrate the performance of the pipeline in a study comparing 100 pig faecal microbiomes from low- and high-antimicrobial use pig farms (organic and conventional farms). We found significant differences in the distribution of AMR genes between low- and high-antimicrobial use farms including a plasmid-borne lincosamide resistance gene exclusive to high-antimicrobial use farms in three species of Lactobacilli. The bioinformatics pipeline code is available at https://github.com/lkalmar/HAM-ART.

Ineffective responses to unlikely outbreaks: Hypothesis building in newly-emerging infectious disease outbreaks.

Over the last 30 years, there has been significant investment in research and infrastructure aimed at mitigating the threat of newly emerging infectious diseases (NEID). Core epidemiological processes, such as outbreak investigations, however, have received little attention and have proceeded largely unchecked and unimproved. Using ethnographic material from an investigation into a cryptic encephalitis outbreak in the Brong-Ahafo Region of Ghana in 2010-2013, in this paper we trace processes of hypothesis building and their relationship to the organizational structures of the response. We demonstrate how commonly recurring features of NEID investigations produce selective pressures in hypothesis building that favor iterations of pre-existing "exciting" hypotheses and inhibit the pursuit of alternative hypotheses, regardless of relative likelihood. These findings contribute to the growing anthropological and science and technology studies (STS) literature on the epistemic communities that coalesce around suspected NEID outbreaks and highlight an urgent need for greater scrutiny of core epidemiological processes.

Advancing the quantitative characterization of farm animal welfare.

Animal welfare is usually excluded from life cycle assessments (LCAs) of farming systems because of limited consensus on how to measure it. Here, we constructed several LCA-compatible animal-welfare metrics and applied them to data we collected from 74 diverse breed-to-finish systems responsible for 5% of UK pig production. Some aspects of metric construction will always be subjective, such as how different aspects of welfare are aggregated, and what determines poor versus good welfare. We tested the sensitivity of individual farm rankings, and rankings of those same farms grouped by label type (memberships of quality-assurance schemes or product labelling), to a broad range of approaches to metric construction. We found farms with the same label types clustered together in rankings regardless of metric choice, and there was broad agreement across metrics on the rankings of individual farms. We found woodland and Organic systems typically perform better than those with no labelling and Red tractor labelling, and that outdoor-bred and outdoor-finished systems perform better than indoor-bred and slatted-finished systems, respectively. We conclude that if our goal is to identify relatively better and worse farming systems for animal welfare, exactly how LCA welfare metrics are constructed may be less important than commonly perceived.

Inferring Mycobacterium bovis transmission between cattle and badgers using isolates from the Randomised Badger Culling Trial.

Mycobacterium bovis (M. bovis) is a causative agent of bovine tuberculosis, a significant source of morbidity and mortality in the global cattle industry. The Randomised Badger Culling Trial was a field experiment carried out between 1998 and 2005 in the South West of England. As part of this trial, M. bovis isolates were collected from contemporaneous and overlapping populations of badgers and cattle within ten defined trial areas. We combined whole genome sequences from 1,442 isolates with location and cattle movement data, identifying transmission clusters and inferred rates and routes of transmission of M. bovis. Most trial areas contained a single transmission cluster that had been established shortly before sampling, often contemporaneous with the expansion of bovine tuberculosis in the 1980s. The estimated rate of transmission from badger to cattle was approximately two times higher than from cattle to badger, and the rate of within-species transmission considerably exceeded these for both species. We identified long distance transmission events linked to cattle movement, recurrence of herd breakdown by infection within the same transmission clusters and superspreader events driven by cattle but not badgers. Overall, our data suggests that the transmission clusters in different parts of South West England that are still evident today were established by long-distance seeding events involving cattle movement, not by recrudescence from a long-established wildlife reservoir. Clusters are maintained primarily by within-species transmission, with less frequent spill-over both from badger to cattle and cattle to badger.

Uncovering the endemic circulation of rabies in Cambodia.

In epidemiology, endemicity characterizes sustained pathogen circulation in a geographical area, which involves a circulation that is not being maintained by external introductions. Because it could potentially shape the design of public health interventions, there is an interest in fully uncovering the endemic pattern of a disease. Here, we use a phylogeographic approach to investigate the endemic signature of rabies virus (RABV) circulation in Cambodia. Cambodia is located in one of the most affected regions by rabies in the world, but RABV circulation between and within Southeast Asian countries remains understudied. Our analyses are based on a new comprehensive data set of 199 RABV genomes collected between 2014 and 2017 as well as previously published Southeast Asian RABV sequences. We show that most Cambodian sequences belong to a distinct clade that has been circulating almost exclusively in Cambodia. Our results thus point towards rabies circulation in Cambodia that does not rely on external introductions. We further characterize within-Cambodia RABV circulation by estimating lineage dispersal metrics that appear to be similar to other settings, and by performing landscape phylogeographic analyses to investigate environmental factors impacting the dispersal dynamic of viral lineages. The latter analyses do not lead to the identification of environmental variables that would be associated with the heterogeneity of viral lineage dispersal velocities, which calls for a better understanding of local dog ecology and further investigations of the potential drivers of RABV spread in the region. Overall, our study illustrates how phylogeographic investigations can be performed to assess and characterize viral endemicity in a context of relatively limited data.

From farm to table: exploring food handling and hygiene practices of meat and milk value chain actors in Ethiopia.

Livestock value chains constitute a source of livelihood for meat and milk value chain actors in Ethiopia, from dairy farmers to other associated value chain actors such as milk traders, abattoir workers, public health officials, veterinarians, butcheries selling meats, milk cooperatives, artisanal milk processors, and transporters. The development of these livestock value chains, however, is constrained by poor food safety and quality, while consumers are also exposed to public health risks due to milk and meat value chain actors' food handling and hygiene practices.This study used Photovoice and participant observation to explore meat and milk value chain actors' food handling and hygiene practices in suburban areas of Addis Ababa and neighbouring Oromia in Ethiopia. The results of this study reveal that milk and meat value chain actors' food handling practices are not aligned with the recommended Ethiopian food safety and quality standards. Low compliance with food safety and quality standards reflected a combination of factors such as lack of incentives, poor road infrastructure and low enforcement of food safety standards.Participatory and visual research methods enable a researcher to collect context-aware data that can lead to the development of policies and intervention strategies that reflect local needs and priorities. The results of this study affirm the need to identify socially acceptable and economically viable policies and intervention strategies that are acceptable to all chain actors; and suggest there is an imperative to train milk and meat value chain actors on good hygiene handling practices, improve road infrastructure, and facilitate access equipment such as fridges and freezers that can contribute to maintaining food safety and quality.

Milk and meat consumption patterns and the potential risk of zoonotic disease transmission among urban and peri-urban dairy farmers in Ethiopia.

BACKGROUND: In the Ethiopian dairy farming system, prevalence of zoonotic diseases such as bovine tuberculosis (bTB) is high in the cattle population. This, combined with some risky milk and meat consumption habits, such as raw milk and uninspected raw meat consumption, poses a considerable risk of zoonotic disease transmission. A survey was conducted to investigate milk and meat consumption patterns, and the level of exposure to urban and peri-urban dairy-keeping households for risks of zoonotic disease transmission. METHODS: Data on milk and meat consumption behaviours and other socioeconomic and demographic variables were collected from 480 urban and peri-urban dairy farms randomly surveyed in major towns in Ethiopia (Mekele, Hawassa, and Gondar towns, Addis Ababa city, as well as five Oromia towns around Addis Ababa). Determinants of raw milk consumption associated with a number of demographic and socio-economic factors were analysed using a generalised ordered logistic model. RESULTS: The results indicated that about 20% the population consumed raw milk and their awareness about pasteurisation and its benefits were low. Location, gender of the household head, previous bTB testing of cattle on the farm, knowledge of zoonotic risks associated with raw milk consumption, household size, and per-capita milk consumption were found to be important determinants of the frequency of raw milk consumption. About 60% of the respondents were exposed to the risk of zoonotic diseases through their habit of frequently consuming raw meat. This was despite that over 90% of the respondents were aware of possible zoonotic risks of raw meat consumption. The determinants of raw meat consumption behaviours were associated with location, gender and age of the household head, household size, meat type preference, per-capita meat consumption, knowledge about disease transmission risks, and training on zoonoses. CONCLUSION: Creating awareness about the risk factors for zoonotic transmission of diseases through training and media campaigns, improving meat hygiene through better abattoir services, and inducing behavioural change around meat sourcing, raw meat and raw milk consumption, are all crucial to the successful prevention and control of the spread of zoonotic diseases, including bTB.

Viral CpG Deficiency Provides No Evidence That Dogs Were Intermediate Hosts for SARS-CoV-2.

Due to the scope and impact of the COVID-19 pandemic there exists a strong desire to understand where the SARS-CoV-2 virus came from and how it jumped species boundaries to humans. Molecular evolutionary analyses can trace viral origins by establishing relatedness and divergence times of viruses and identifying past selective pressures. However, we must uphold rigorous standards of inference and interpretation on this topic because of the ramifications of being wrong. Here, we dispute the conclusions of Xia (2020. Extreme genomic CpG deficiency in SARS-CoV-2 and evasion of host antiviral defense. Mol Biol Evol. doi:10.1093/molbev/masa095) that dogs are a likely intermediate host of a SARS-CoV-2 ancestor. We highlight major flaws in Xia's inference process and his analysis of CpG deficiencies, and conclude that there is no direct evidence for the role of dogs as intermediate hosts. Bats and pangolins currently have the greatest support as ancestral hosts of SARS-CoV-2, with the strong caveat that sampling of wildlife species for coronaviruses has been limited.

Environmental limits of Rift Valley fever revealed using ecoepidemiological mechanistic models.

Vector-borne diseases (VBDs) of humans and domestic animals are a significant component of the global burden of disease and a key driver of poverty. The transmission cycles of VBDs are often strongly mediated by the ecological requirements of the vectors, resulting in complex transmission dynamics, including intermittent epidemics and an unclear link between environmental conditions and disease persistence. An important broader concern is the extent to which theoretical models are reliable at forecasting VBDs; infection dynamics can be complex, and the resulting systems are highly unstable. Here, we examine these problems in detail using a case study of Rift Valley fever (RVF), a high-burden disease endemic to Africa. We develop an ecoepidemiological, compartmental, mathematical model coupled to the dynamics of ambient temperature and water availability and apply it to a realistic setting using empirical environmental data from Kenya. Importantly, we identify the range of seasonally varying ambient temperatures and water-body availability that leads to either the extinction of mosquito populations and/or RVF (nonpersistent regimens) or the establishment of long-term mosquito populations and consequently, the endemicity of the RVF infection (persistent regimens). Instabilities arise when the range of the environmental variables overlaps with the threshold of persistence. The model captures the intermittent nature of RVF occurrence, which is explained as low-level circulation under the threshold of detection, with intermittent emergence sometimes after long periods. Using the approach developed here opens up the ability to improve predictions of the emergence and behaviors of epidemics of many other important VBDs.

Disentangling serology to elucidate henipa- and filovirus transmission in Madagascar fruit bats.

Bats are reservoirs for emerging human pathogens, including Hendra and Nipah henipaviruses and Ebola and Marburg filoviruses. These viruses demonstrate predictable patterns in seasonality and age structure across multiple systems; previous work suggests that they may circulate in Madagascar's endemic fruit bats, which are widely consumed as human food. We aimed to (a) document the extent of henipa- and filovirus exposure among Malagasy fruit bats, (b) explore seasonality in seroprevalence and serostatus in these bat populations and (c) compare mechanistic hypotheses for possible transmission dynamics underlying these data. To this end, we amassed and analysed a unique dataset documenting longitudinal serological henipa- and filovirus dynamics in three Madagascar fruit bat species. We uncovered serological evidence of exposure to Hendra-/Nipah-related henipaviruses in Eidolon dupreanum, Pteropus rufus and Rousettus madagascariensis, to Cedar-related henipaviruses in E. dupreanum and R. madagascariensis and to Ebola-related filoviruses in P. rufus and R. madagascariensis. We demonstrated significant seasonality in population-level seroprevalence and individual serostatus for multiple viruses across these species, linked to the female reproductive calendar. An age-structured subset of the data highlighted evidence of waning maternal antibodies in neonates, increasing seroprevalence in young and decreasing seroprevalence late in life. Comparison of mechanistic epidemiological models fit to these data offered support for transmission hypotheses permitting waning antibodies but retained immunity in adult-age bats. Our findings suggest that bats may seasonally modulate mechanisms of pathogen control, with consequences for population-level transmission. Additionally, we narrow the field of candidate transmission hypotheses by which bats are presumed to host and transmit potentially zoonotic viruses globally.

Network analysis of dairy cattle movement and associations with bovine tuberculosis spread and control in emerging dairy belts of Ethiopia.

BACKGROUND: Dairy cattle movement could be a major risk factor for the spread of bovine tuberculosis (BTB) in emerging dairy belts of Ethiopia. Dairy cattle may be moved between farms over long distances, and hence understanding the route and frequency of the movements is essential to establish the pattern of spread of BTB between farms, which could ultimately help to inform policy makers to design cost effective control strategies. The objective of this study was, therefore, to investigate the network structure of dairy cattle movement and its influence on the transmission and prevalence of BTB in three emerging areas among the Ethiopian dairy belts, namely the cities of Hawassa, Gondar and Mekelle. METHODS: A questionnaire survey was conducted in 278 farms to collect data on the pattern of dairy cattle movement for the last 5 years (September 2013 to August 2018). Visualization of the network structure and analysis of the relationship between the network patterns and the prevalence of BTB in these regions were made using social network analysis. RESULTS: The cattle movement network structure display both scale free and small world properties implying local clustering with fewer farms being highly connected, at higher risk of infection, with the potential to act as super spreaders of BTB if infected. Farms having a history of cattle movements onto the herds were more likely to be affected by BTB (OR: 2.2) compared to farms not having a link history. Euclidean distance between farms and the batch size of animals moved on were positively correlated with prevalence of BTB. On the other hand, farms having one or more outgoing cattle showed a decrease on the likelihood of BTB infection (OR = 0.57) compared to farms which maintained their cattle. CONCLUSION: This study showed that the patterns of cattle movement and size of animal moved between farms contributed to the potential for BTB transmission. The few farms with the bulk of transmission potential could be efficiently targeted by control measures aimed at reducing the spread of BTB. The network structure described can also provide the starting point to build and estimate dynamic transmission models for BTB, and other infectious diseases.

Demodicosis in a captive African straw-coloured fruit bat (Eidolon helvum).

Demodicosis is most frequently observed in the domestic dog (Canis familiaris), but it has rarely been reported in bats (Chiroptera). The overpopulation of Demodex spp. that causes dermatological changes is generally associated with a compromised immune system. We describe the gross and histological features of generalized demodicosis in an adult female African straw-coloured fruit bat (Eidolon helvum) drawn from a captive research colony. The histology of the lesions revealed comedones and follicular infundubular cysts harbouring numerous Demodex spp. mites, eliciting a minimal inflammatory response in the adjacent dermis. The histological examination of a full set of tissues did not reveal clear evidence of immunosuppression, although a clinical history of recent abortion and possible stressors due to captivity could be considered risk factors for the demodicosis. Attempts to determine the Demodex species using PCR on DNA extracted from the formalin fixed paraffin embedded tissue failed. This is the first clinical and histological description of demodicosis in Eidolon helvum.

Brucellosis in the Addis Ababa dairy cattle: the myths and the realities.

BACKGROUND: Bovine brucellosis is considered as an important disease among livestock and people in sub-Saharan African countries including Ethiopia. A cross-sectional study was conducted from November 2016 to May 2017 to estimate the prevalence and associated risk factors, and to assess knowledge-attitude and practices (KAP) of farm workers about bovine brucellosis in Addis Ababa dairy farms. RESULTS: A total of 1550 cattle from 127 dairy farms were serially tested using the Rose Bengal Plate Test (RBPT), Competitive Enzyme-Linked Immunosorbant Assay (c-ELISA) and Complement Fixation Test (CFT). Forty-three (2.77%) of the collected sera were positive by the RBPT and only one of these was positive by c-ELISA (0.06%) and none was positive by CFT. The knowledge of farm workers towards the disease was very low and risk factors associated with Brucella infection were apparent in the study area. CONCLUSION: Seropositivity for Brucella spp. was found in only a very small percentage by c-ELISA test, although risk factors for transmitting Brucella infection were present. The results suggest that bovine brucellosis is currently not a generalized problem in dairy cattle of Addis Ababa. Since this favorable disease situation is not the result of informed policy, there is no guarantee that it will continue unchanged. Setting clear policy in control of the disease and implementing "One Health" are the most constructive approaches recommended.

Evidence for several waves of global transmission in the seventh cholera pandemic.

Vibrio cholerae is a globally important pathogen that is endemic in many areas of the world and causes 3-5 million reported cases of cholera every year. Historically, there have been seven acknowledged cholera pandemics; recent outbreaks in Zimbabwe and Haiti are included in the seventh and ongoing pandemic. Only isolates in serogroup O1 (consisting of two biotypes known as 'classical' and 'El Tor') and the derivative O139 can cause epidemic cholera. It is believed that the first six cholera pandemics were caused by the classical biotype, but El Tor has subsequently spread globally and replaced the classical biotype in the current pandemic. Detailed molecular epidemiological mapping of cholera has been compromised by a reliance on sub-genomic regions such as mobile elements to infer relationships, making El Tor isolates associated with the seventh pandemic seem superficially diverse. To understand the underlying phylogeny of the lineage responsible for the current pandemic, we identified high-resolution markers (single nucleotide polymorphisms; SNPs) in 154 whole-genome sequences of globally and temporally representative V. cholerae isolates. Using this phylogeny, we show here that the seventh pandemic has spread from the Bay of Bengal in at least three independent but overlapping waves with a common ancestor in the 1950s, and identify several transcontinental transmission events. Additionally, we show how the acquisition of the SXT family of antibiotic resistance elements has shaped pandemic spread, and show that this family was first acquired at least ten years before its discovery in V. cholerae.

Potential benefits of cattle vaccination as a supplementary control for bovine tuberculosis.

Vaccination for the control of bovine tuberculosis (bTB) in cattle is not currently used within any international control program, and is illegal within the EU. Candidate vaccines, based upon Mycobacterium bovis bacillus Calmette-Guérin (BCG) all interfere with the action of the tuberculin skin test, which is used to determine if animals, herds and countries are officially bTB-free. New diagnostic tests that Differentiate Infected from Vaccinated Animals (DIVA) offer the potential to introduce vaccination within existing eradication programs. We use within-herd transmission models estimated from historical data from Great Britain (GB) to explore the feasibility of such supplemental use of vaccination. The economic impact of bovine Tuberculosis for farmers is dominated by the costs associated with testing, and associated restrictions on animal movements. Farmers' willingness to adopt vaccination will require vaccination to not only reduce the burden of infection, but also the risk of restrictions being imposed. We find that, under the intensive sequence of testing in GB, it is the specificity of the DIVA test, rather than the sensitivity, that is the greatest barrier to see a herd level benefit of vaccination. The potential negative effects of vaccination could be mitigated through relaxation of testing. However, this could potentially increase the hidden burden of infection within Officially TB Free herds. Using our models, we explore the range of the DIVA test characteristics necessary to see a protective herd level benefit of vaccination. We estimate that a DIVA specificity of at least 99.85% and sensitivity of >40% is required to see a protective benefit of vaccination with no increase in the risk of missed infection. Data from experimentally infected animals suggest that this target specificity could be achieved in vaccinates using a cocktail of three DIVA antigens while maintaining a sensitivity of 73.3% (95%CI: 61.9, 82.9%) relative to post-mortem detection.

Network analysis of host-virus communities in bats and rodents reveals determinants of cross-species transmission.

Bats are natural reservoirs of several important emerging viruses. Cross-species transmission appears to be quite common among bats, which may contribute to their unique reservoir potential. Therefore, understanding the importance of bats as reservoirs requires examining them in a community context rather than concentrating on individual species. Here, we use a network approach to identify ecological and biological correlates of cross-species virus transmission in bats and rodents, another important host group. We show that given our current knowledge the bat viral sharing network is more connected than the rodent network, suggesting viruses may pass more easily between bat species. We identify host traits associated with important reservoir species: gregarious bats are more likely to share more viruses and bats which migrate regionally are important for spreading viruses through the network. We identify multiple communities of viral sharing within bats and rodents and highlight potential species traits that can help guide studies of novel pathogen emergence.

Eliminating bovine tuberculosis in cattle and badgers: insight from a dynamic model.

Bovine tuberculosis (BTB) is a multi-species infection that commonly affects cattle and badgers in Great Britain. Despite years of study, the impact of badgers on BTB incidence in cattle is poorly understood. Using a two-host transmission model of BTB in cattle and badgers, we find that published data and parameter estimates are most consistent with a system at the threshold of control. The most consistent explanation for data obtained from cattle and badger populations includes within-host reproduction numbers close to 1 and between-host reproduction numbers of approximately 0.05. In terms of controlling infection in cattle, reducing cattle-to-cattle transmission is essential. In some regions, even large reductions in badger prevalence can have a modest impact on cattle infection and a multi-stranded approach is necessary that also targets badger-to-cattle transmission directly. The new perspective highlighted by this two-host approach provides insight into the control of BTB in Great Britain.

Bat flight and zoonotic viruses.

Bats are sources of high viral diversity and high-profile zoonotic viruses worldwide. Although apparently not pathogenic in their reservoir hosts, some viruses from bats severely affect other mammals, including humans. Examples include severe acute respiratory syndrome coronaviruses, Ebola and Marburg viruses, and Nipah and Hendra viruses. Factors underlying high viral diversity in bats are the subject of speculation. We hypothesize that flight, a factor common to all bats but to no other mammals, provides an intensive selective force for coexistence with viral parasites through a daily cycle that elevates metabolism and body temperature analogous to the febrile response in other mammals. On an evolutionary scale, this host-virus interaction might have resulted in the large diversity of zoonotic viruses in bats, possibly through bat viruses adapting to be more tolerant of the fever response and less virulent to their natural hosts.

Evolution of equine influenza virus in vaccinated horses.

Influenza A viruses are characterized by their ability to evade host immunity, even in vaccinated individuals. To determine how prior immunity shapes viral diversity in vivo, we studied the intra- and interhost evolution of equine influenza virus in vaccinated horses. Although the level and structure of genetic diversity were similar to those in naïve horses, intrahost bottlenecks may be more stringent in vaccinated animals, and mutations shared among horses often fall close to putative antigenic sites.