Rudovick Reuben Kazwala.

An outstanding Tanzanian scientist in the field of veterinary epidemiology and One Health research.

Local and wide-scale livestock movement networks inform disease control strategies in East Africa.

Livestock mobility exacerbates infectious disease risks across sub-Saharan Africa, but enables critical access to grazing and water resources, and trade. Identifying locations of high livestock traffic offers opportunities for targeted control. We focus on Tanzanian agropastoral and pastoral communities that account respectively for over 75% and 15% of livestock husbandry in eastern Africa. We construct networks of livestock connectivity based on participatory mapping data on herd movements reported by village livestock keepers as well as data from trading points to understand how seasonal availability of resources, land-use and trade influence the movements of livestock. In communities that practise agropastoralism, inter- and intra-village connectivity through communal livestock resources (e.g. pasture and water) was 1.9 times higher in the dry compared to the wet season suggesting greater livestock traffic and increased contact probability. In contrast, livestock from pastoral communities were 1.6 times more connected at communal locations during the wet season when they also tended to move farther (by 3 km compared to the dry season). Trade-linked movements were twice more likely from rural to urban locations. Urban locations were central to all networks, particularly those with potentially high onward movements, for example to abattoirs, livestock holding grounds, or other markets, including beyond national boundaries. We demonstrate how livestock movement information can be used to devise strategic interventions that target critical livestock aggregation points (i.e. locations of high centrality values) and times (i.e. prior to and after the wet season in pastoral and agropastoral areas, respectively). Such targeted interventions are a cost-effective approach to limit infection without restricting livestock mobility critical to sustainable livelihoods.

Integrating contact tracing and whole-genome sequencing to track the elimination of dog-mediated rabies: An observational and genomic study.

Background: Dog-mediated rabies is endemic across Africa causing thousands of human deaths annually. A One Health approach to rabies is advocated, comprising emergency post-exposure vaccination of bite victims and mass dog vaccination to break the transmission cycle. However, the impacts and cost-effectiveness of these components are difficult to disentangle. Methods: We combined contact tracing with whole-genome sequencing to track rabies transmission in the animal reservoir and spillover risk to humans from 2010 to 2020, investigating how the components of a One Health approach reduced the disease burden and eliminated rabies from Pemba Island, Tanzania. With the resulting high-resolution spatiotemporal and genomic data, we inferred transmission chains and estimated case detection. Using a decision tree model, we quantified the public health burden and evaluated the impact and cost-effectiveness of interventions over a 10-year time horizon. Results: We resolved five transmission chains co-circulating on Pemba from 2010 that were all eliminated by May 2014. During this period, rabid dogs, human rabies exposures and deaths all progressively declined following initiation and improved implementation of annual islandwide dog vaccination. We identified two introductions to Pemba in late 2016 that seeded re-emergence after dog vaccination had lapsed. The ensuing outbreak was eliminated in October 2018 through reinstated islandwide dog vaccination. While post-exposure vaccines were projected to be highly cost-effective ($256 per death averted), only dog vaccination interrupts transmission. A combined One Health approach of routine annual dog vaccination together with free post-exposure vaccines for bite victims, rapidly eliminates rabies, is highly cost-effective ($1657 per death averted) and by maintaining rabies freedom prevents over 30 families from suffering traumatic rabid dog bites annually on Pemba island. Conclusions: A One Health approach underpinned by dog vaccination is an efficient, cost-effective, equitable, and feasible approach to rabies elimination, but needs scaling up across connected populations to sustain the benefits of elimination, as seen on Pemba, and for similar progress to be achieved elsewhere. Funding: Wellcome [207569/Z/17/Z, 095787/Z/11/Z, 103270/Z/13/Z], the UBS Optimus Foundation, the Department of Health and Human Services of the National Institutes of Health [R01AI141712] and the DELTAS Africa Initiative [Afrique One-ASPIRE/DEL-15-008] comprising a donor consortium of the African Academy of Sciences (AAS), Alliance for Accelerating Excellence in Science in Africa (AESA), the New Partnership for Africa's Development Planning and Coordinating (NEPAD) Agency, Wellcome [107753/A/15/Z], Royal Society of Tropical Medicine and Hygiene Small Grant 2017 [GR000892] and the UK government. The rabies elimination demonstration project from 2010-2015 was supported by the Bill & Melinda Gates Foundation [OPP49679]. Whole-genome sequencing was partially supported from APHA by funding from the UK Department for Environment, Food and Rural Affairs (Defra), Scottish government and Welsh government under projects SEV3500 and SE0421.

"If You Do Not Take the Medicine and Complete the Dose It Could Cause You More Trouble": Bringing Awareness, Local Knowledge and Experience into Antimicrobial Stewardship in Tanzania.

Antimicrobial resistance (AMR) is a global health issue disproportionately affecting low- and middle-income countries. In Tanzania, multi-drug-resistant bacteria (MDR) are highly prevalent in clinical and community settings, inhibiting effective treatment and recovery from infection. The burden of AMR can be alleviated if antimicrobial stewardship (AMS) programs are coordinated and incorporate local knowledge and systemic factors. AMS includes the education of health providers to optimise antimicrobial use to improve patient outcomes while minimising AMR risks. For programmes to succeed, it is essential to understand not just the awareness of and receptiveness to AMR education, but also the opportunities and challenges facing health professionals. We conducted in-depth interviews (n = 44) with animal and human health providers in rural northern Tanzania in order to understand their experiences around AMR. In doing so, we aimed to assess the contextual factors surrounding their practices that might enable or impede the translation of knowledge into action. Specifically, we explored their motivations, training, understanding of infections and AMR, and constraints in daily practice. While providers were motivated in supporting their communities, clear issues emerged regarding training and understanding of AMR. Community health workers and retail drug dispensers exhibited the most variation in training. Inconsistencies in understandings of AMR and its drivers were apparent. Providers cited the actions of patients and other providers as contributing to AMR, perpetuating narratives of blame. Challenges related to AMR included infrastructural constraints, such as a lack of diagnostic testing. While health and AMR-specific training would be beneficial to address awareness, equally important, if not more critical, is tackling the challenges providers face in turning knowledge into action.

Healthcare provider-focused antimicrobial stewardship in sub-Saharan Africa: opportunities and challenges.

To address the antimicrobial resistance (AMR) crisis, governments around the world have created action plans to optimize antimicrobial use (AMU). These plans include antimicrobial stewardship (AMS) that encompasses educational programs for healthcare workers. We discuss these programs in sub-Saharan Africa, including the opportunities and challenges arising from a highly constrained healthcare environment.

Crossover-Use of Human Antibiotics in Livestock in Agricultural Communities: A Qualitative Cross-Country Comparison between Uganda, Tanzania and India.

Antibiotic use in animal agriculture contributes significantly to antibiotic use globally and is a key driver of the rising threat of antibiotic resistance. It is becoming increasingly important to better understand antibiotic use in livestock in low-and-middle income countries where antibiotic use is predicted to increase considerably as a consequence of the growing demand for animal-derived products. Antibiotic crossover-use refers to the practice of using antibiotic formulations licensed for humans in animals and vice versa. This practice has the potential to cause adverse drug reactions and contribute to the development and spread of antibiotic resistance between humans and animals. We performed secondary data analysis of in-depth interview and focus-group discussion transcripts from independent studies investigating antibiotic use in agricultural communities in Uganda, Tanzania and India to understand the practice of antibiotic crossover-use by medicine-providers and livestock-keepers in these settings. Thematic analysis was conducted to explore driving factors of reported antibiotic crossover-use in the three countries. Similarities were found between countries regarding both the accounts of antibiotic crossover-use and its drivers. In all three countries, chickens and goats were treated with human antibiotics, and among the total range of human antibiotics reported, amoxicillin, tetracycline and penicillin were stated as used in animals in all three countries. The key themes identified to be driving crossover-use were: (1) medicine-providers' and livestock-keepers' perceptions of the effectiveness and safety of antibiotics, (2) livestock-keepers' sources of information, (3) differences in availability of human and veterinary services and antibiotics, (4) economic incentives and pressures. Antibiotic crossover-use occurs in low-intensity production agricultural settings in geographically distinct low-and-middle income countries, influenced by a similar set of interconnected contextual drivers. Improving accessibility and affordability of veterinary medicines to both livestock-keepers and medicine-providers is required alongside interventions to address understanding of the differences between human and animal antibiotics, and potential dangers of antibiotic crossover-use in order to reduce the practice. A One Health approach to studying antibiotic use is necessary to understand the implications of antibiotic accessibility and use in one sector upon antibiotic use in other sectors.

Where Rabies Is Not a Disease. Bridging Healthworlds to Improve Mutual Understanding and Prevention of Rabies.

Deeply embedded in local social, cultural, and religious settings, traditional healing is part of dog bite and rabies management in many rabies endemic countries. Faith healing, which usually encompasses a more holistic approach to health including physical, mental and social dimensions, is rare in the context of rabies. In Gujarat, Western India, the Hindu goddess Hadkai Mata is worshiped by low-caste communities as the Mother of Rabies in the event of a dog bite to a person or their livestock. This belief might influence people's attitudes and behaviors toward rabies prevention but has never been investigated. Through 31 in-depth interviews with healers and staff of Hadkai Mata temples, this paper explores the system of knowledge around dog and human rabies that is built and shared in these places of worship and healing. Qualitative and quantitative data were analyzed looking for convergences and divergences with the recently launched National Action Plan for dog-mediated Rabies Elimination. Results suggest that while the etiology of human rabies as a social illness is usually explained as the goddess's wish to correct misbehaving people and restore positive interpersonal relations, there is some appreciation for the biological processes of infection that lead to rabies as a physical disease. Hadkai Mata is believed to cure rabies if her patients undergo the necessary process of moral growth. Although conventional post-exposure prophylaxis is not opposed per se, it is often delayed by patients who seek traditional treatment first. Some reluctance was expressed toward mass dog vaccination because it is seen as an interference in how the goddess controls dogs, by enraging them-hence infecting them with rabies-and sending them to bite wrongdoers. Addressing these cultural perceptions is likely to be critical in achieving effective control of dog rabies in this region. The study highlights the value of multidisciplinary approaches in the control and elimination of rabies, as well as other zoonoses. This includes the importance of understanding different culturally- and religiously- mediated ways in which humans relate to animals; and looking for points of convergence and mutual understanding, upon which context-tailored, linguistically-accurate, locally acceptable, feasible and effective strategies can be designed.

Participatory mapping identifies risk areas and environmental predictors of endemic anthrax in rural Africa.

Disease mapping reveals geographical variability in incidence, which can help to prioritise control efforts. However, in areas where this is most needed, resources to generate the required data are often lacking. Participatory mapping, which makes use of indigenous knowledge, is a potential approach to identify risk areas for endemic diseases in low- and middle-income countries. Here we combine this method with Geographical Information System-based analyses of environmental variables as a novel approach to study endemic anthrax, caused by the spore-forming bacterium Bacillus anthracis, in rural Africa. Our aims were to: (1) identify high-risk anthrax areas using community knowledge; (2) enhance our understanding of the environmental characteristics associated with these areas; and (3) make spatial predictions of anthrax risk. Community members from the Ngorongoro Conservation Area (NCA), northern Tanzania, where anthrax is highly prevalent in both animals and humans, were asked to draw areas they perceived to pose anthrax risks to their livestock on geo-referenced maps. After digitisation, random points were generated within and outside the defined areas to represent high- and low-risk areas, respectively. Regression analyses were used to identify environmental variables that may predict anthrax risk. Results were combined to predict how the probability of being a high-risk area for anthrax varies across space. Participatory mapping identified fourteen discrete high-risk areas ranging from 0.2 to 212.9 km2 in size and occupying 8.4% of the NCA. Areas that pose a high risk of anthrax were positively associated with factors that increase contact with Bacillus anthracis spores rather than those associated with the pathogen's survival: close proximity to inland water bodies, where wildlife and livestock congregate, and low organic carbon content, which may indicate an increased likelihood of animals grazing close to soil surface and ingesting spores. Predicted high-risk areas were located in the centre of the NCA, which is likely to be encountered by most herds during movements in search for resources. We demonstrate that participatory mapping combined with spatial analyses can provide novel insights into the geography of disease risk. This approach can be used to prioritise areas for control in low-resource settings, especially for diseases with environmental transmission.

Population genomics of Bacillus anthracis from an anthrax hyperendemic area reveals transmission processes across spatial scales and unexpected within-host diversity.

Genomic sequencing has revolutionized our understanding of bacterial disease epidemiology, but remains underutilized for zoonotic pathogens in remote endemic settings. Anthrax, caused by the spore-forming bacterium Bacillus anthracis, remains a threat to human and animal health and rural livelihoods in low- and middle-income countries. While the global genomic diversity of B. anthracis has been well-characterized, there is limited information on how its populations are genetically structured at the scale at which transmission occurs, critical for understanding the pathogen's evolution and transmission dynamics. Using a uniquely rich dataset, we quantified genome-wide SNPs among 73 B. anthracis isolates derived from 33 livestock carcasses sampled over 1 year throughout the Ngorongoro Conservation Area, Tanzania, a region hyperendemic for anthrax. Genome-wide SNPs distinguished 22 unique B. anthracis genotypes (i.e. SNP profiles) within the study area. However, phylogeographical structure was lacking, as identical SNP profiles were found throughout the study area, likely the result of the long and variable periods of spore dormancy and long-distance livestock movements. Significantly, divergent genotypes were obtained from spatio-temporally linked cases and even individual carcasses. The high number of SNPs distinguishing isolates from the same host is unlikely to have arisen during infection, as supported by our simulation models. This points to an unexpectedly wide transmission bottleneck for B. anthracis, with an inoculum comprising multiple variants being the norm. Our work highlights that inferring transmission patterns of B. anthracis from genomic data will require analytical approaches that account for extended and variable environmental persistence, as well as co-infection.

How public health crises expose systemic, day-to-day health inequalities in low- and-middle income countries: an example from East Africa.

BACKGROUND: The current Coronavirus disease pandemic reveals political and structural inequities of the world's poorest people who have little or no access to health care and yet the largest burdens of poor health. This is in parallel to a more persistent but silent global health crisis, antimicrobial resistance (AMR). We explore the fundamental challenges of health care in humans and animals in relation to AMR in Tanzania. METHODS: We conducted 57 individual interviews and focus groups with providers and patients in high, middle and lower tier health care facilities and communities across three regions of Tanzania between April 2019 and February 2020. We covered topics from health infrastructure and prescribing practices to health communication and patient experiences. RESULTS: Three interconnected themes emerged about systemic issues impacting health. First, there are challenges around infrastructure and availability of vital resources such as healthcare staff and supplies. Second, health outcomes are predicated on patient and provider access to services as well as social determinants of health. Third, health communication is critical in defining trusted sources of information, and narratives of blame emerge around health outcomes with the onus of responsibility for action falling on individuals. CONCLUSION: Entanglements between infrastructure, access and communication exist while constraints in the health system lead to poor health outcomes even in 'normal' circumstances. These are likely to be relevant across the globe and highly topical for addressing pressing global health challenges. Redressing structural health inequities can better equip countries and their citizens to not only face pandemics but also day-to-day health challenges.

Tackling barriers to collective action for effective vaccination campaigns: rabies in rural Africa as an example.

Vaccine-based protection in populations that are vulnerable to infectious diseases represents a public good, whose successful attainment requires collective action. We investigated participation in mass domestic dog vaccination against dog-mediated human rabies endemic in Tanzania as a prototypical example of these issues. We employed advertising interventions, text messaging and/or engagement through community leaders, as well as operational adjustments to increase the saliency of rabies risks and reduce barriers to participation in vaccination campaigns. Neither advertising strategies were effective on their own, however, when taken together, the two advertising strategies substantially improved vaccination coverage. Operational interventions, such as increasing vaccination stations and extending time windows of delivery, greatly enhanced participation. Our experimental and theoretical findings highlight the importance of both salience and context: sparking successful collective action requires decision-making bodies to understand and respond to the challenges encountered by intended beneficiaries in their local contexts.

The Power of Music to Prevent and Control Emerging Infectious Diseases.

Music is a powerful approach to engage communities and disseminate information. Specifically, health campaigns employing music have been used to promote behaviors that can prevent emerging infectious diseases (EIDs). For example, hip hop artists supported campaigns to prevent acquired immunodeficiency syndrome in the 70s in the United States, while Brazilian funk promoted vaccination to mitigate the ongoing COVID-19 pandemic. Similarly, we broadcast musical messages in local languages to increase community awareness and support prevention measures in Guinea and Liberia in response to the recent Ebola outbreak in 2021. Given the potential of music to promote both individual and population-level behavioral changes to prevent transmission, there is a need to consolidate information on music-based health interventions, and on how we can measure their effectiveness. In this perspective, we provide examples of relevant initiatives, discussing challenges and solutions associated with implementing interventions based on our experience with the 2021 Ebola outbreak. We recommend four steps for a successful music-based health intervention including (1) establishing a task force, (2) compose a "catchy" song including critical preventive measures, (3) deliver the song to the target audience, and (4) evaluate the campaign effectiveness. We argue that close interactions between scientists and musicians can produce rapid musical content for disease prevention. We also identify and discuss several methodological frameworks for testing the effectiveness of such interventions. We conclude that support from public health authorities, government media departments, and international agencies, is necessary to deliver wide outreach and long-term sustainability of musical messaging toward effective EID prevention.

Antigenic Diversity in Theileria parva Populations From Sympatric Cattle and African Buffalo Analyzed Using Long Read Sequencing.

East Coast fever (ECF) in cattle is caused by the Apicomplexan protozoan parasite Theileria parva, transmitted by the three-host tick Rhipicephalus appendiculatus. The African buffalo (Syncerus caffer) is the natural host for T. parva but does not suffer disease, whereas ECF is often fatal in cattle. The genetic relationship between T. parva populations circulating in cattle and buffalo is poorly understood, and has not been studied in sympatric buffalo and cattle. This study aimed to determine the genetic diversity of T. parva populations in cattle and buffalo, in an area where livestock co-exist with buffalo adjacent to the Serengeti National Park, Tanzania. Three T. parva antigens (Tp1, Tp4, and Tp16), known to be recognized by CD8+ and CD4+ T cells in immunized cattle, were used to characterize genetic diversity of T. parva in cattle (n = 126) and buffalo samples (n = 22). Long read (PacBio) sequencing was used to generate full or near-full length allelic sequences. Patterns of diversity were similar across all three antigens, with allelic diversity being significantly greater in buffalo-derived parasites compared to cattle-derived (e.g., for Tp1 median cattle allele count was 9, and 81.5 for buffalo), with very few alleles shared between species (8 of 651 alleles were shared for Tp1). Most alleles were unique to buffalo with a smaller proportion unique to cattle (412 buffalo unique vs. 231 cattle-unique for Tp1). There were indications of population substructuring, with one allelic cluster of Tp1 representing alleles found in both cattle and buffalo (including the TpM reference genome allele), and another containing predominantly only alleles deriving from buffalo. These data illustrate the complex interplay between T. parva populations in buffalo and cattle, revealing the significant genetic diversity in the buffalo T. parva population, the limited sharing of parasite genotypes between the host species, and highlight that a subpopulation of T. parva is maintained by transmission within cattle. The data indicate that fuller understanding of buffalo T. parva population dynamics is needed, as only a comprehensive appreciation of the population genetics of T. parva populations will enable assessment of buffalo-derived infection risk in cattle, and how this may impact upon control measures such as vaccination.

Livestock movement informs the risk of disease spread in traditional production systems in East Africa.

In Africa, livestock are important to local and national economies, but their productivity is constrained by infectious diseases. Comprehensive information on livestock movements and contacts is required to devise appropriate disease control strategies; yet, understanding contact risk in systems where herds mix extensively, and where different pathogens can be transmitted at different spatial and temporal scales, remains a major challenge. We deployed Global Positioning System collars on cattle in 52 herds in a traditional agropastoral system in western Serengeti, Tanzania, to understand fine-scale movements and between-herd contacts, and to identify locations of greatest interaction between herds. We examined contact across spatiotemporal scales relevant to different disease transmission scenarios. Daily cattle movements increased with herd size and rainfall. Generally, contact between herds was greatest away from households, during periods with low rainfall and in locations close to dipping points. We demonstrate how movements and contacts affect the risk of disease spread. For example, transmission risk is relatively sensitive to the survival time of different pathogens in the environment, and less sensitive to transmission distance, at least over the range of the spatiotemporal definitions of contacts that we explored. We identify times and locations of greatest disease transmission potential and that could be targeted through tailored control strategies.

Combining Multiple Assays Improves Detection and Serotyping of Foot-and-Mouth Disease Virus. A Practical Example with Field Samples from East Africa.

Multiple serotypes and topotypes of foot-and-mouth disease virus (FMDV) circulate in endemic areas, posing considerable impacts locally. In addition, introductions into new areas are of great concern. Indeed, in recent years, multiple FMDV outbreaks, caused by topotypes that have escaped from their original areas, have been recorded in various parts of the world. In both cases, rapid and accurate diagnosis, including the identification of the serotype and topotype causing the given outbreaks, plays an important role in the implementation of the most effective and appropriate measures to control the spread of the disease. In the present study, we describe the performance of a range of diagnostic and typing tools for FMDV on a panel of vesicular samples collected in northern Tanzania (East Africa, EA) during 2012-2018. Specifically, we tested these samples with a real-time RT-PCR targeting 3D sequence for pan-FMDV detection; an FMDV monoclonal antibody-based antigen (Ag) detection and serotyping ELISA kit; virus isolation (VI) on LFBKαVß6 cell line; and a panel of four topotype-specific real-time RT-PCRs, specifically tailored for circulating strains in EA. The 3D real-time RT-PCR showed the highest diagnostic sensitivity, but it lacked typing capacity. Ag-ELISA detected and typed FMDV in 71% of sample homogenates, while VI combined with Ag-ELISA for typing showed an efficiency of 82%. The panel of topotype-specific real-time RT-PCRs identified and typed FMDV in 93% of samples. However, the SAT1 real-time RT-PCR had the highest (20%) failure rate. Briefly, topotype-specific real-time RT-PCRs had the highest serotyping capacity for EA FMDVs, although four assays were required, while the Ag-ELISA, which was less sensitive, was the most user-friendly, hence suitable for any laboratory level. In conclusion, when the four compared tests were used in combination, both the diagnostic and serotyping performances approached 100%.

Addressing antimicrobial resistance by improving access and quality of care-A review of the literature from East Africa.

Universal access to healthcare, including quality medicines, is a fundamental human right but is still out of reach for many in low- and middle-income countries (LMICs). An existing framework capturing variability of access to healthcare in low-resource settings includes the 5 dimensions: availability, accessibility, affordability, adequacy, and acceptability. This framework encompasses key components, including health infrastructure and means to access it as well as service organisation, costs, and factors that influence users' satisfaction. However, in reality, the effectiveness of accessed healthcare is measured by the likelihood of a positive outcome. We therefore propose an expansion of this framework to include an additional dimension, "aspects of quality," incorporating quality, which critically influences the ability of the accessed services to generate optimal health outcomes. Within this framework, we explore literature from East Africa likely relevant to a range of LMIC contexts, mainly focusing on the provision of widely used antimicrobials such as antimalarials and antibiotics. We argue that major inadequacies exist across all 6 dimensions of access and quality of drugs and their provision. While the global focus is on curbing excessive antimicrobial use to tackle the antimicrobial resistance (AMR) crisis, major constraints around access shape patients' health-seeking decisions leading to potentially problematic practices that might exacerbate the AMR problem. We advocate for a holistic approach to tackling these inadequacies, encompassing all dimensions of access and quality of healthcare in order to improve health outcomes while simultaneously counteracting the AMR crisis.

Dog ownership practices and responsibilities for children's health in terms of rabies control and prevention in rural communities in Tanzania.

Interventions tackling zoonoses require an understanding of healthcare patterns related to both human and animal hosts. The control of dog-mediated rabies is a good example. Despite the availability of effective control measures, 59,000 people die of rabies every year worldwide. In Tanzania, children are most at risk, contributing ~40% of deaths. Mass dog vaccination can break the transmission cycle, but reaching the recommended 70% coverage is challenging where vaccination depends on willingness to vaccinate dogs. Awareness campaigns in communities often target children, but do not consider other key individuals in the prevention chain. Understanding factors related to dog ownership and household-level responsibility for dog vaccination and child health is critical to the design of vaccination strategies. We investigated who makes household decisions about dogs and on health care for children in rural Tanzania. In the Kilosa district, in-depth interviews with 10 key informants were conducted to inform analysis of data from a household survey of 799 households and a survey on Knowledge Attitudes and Practices of 417 households. The in-depth interviews were analysed using framework analysis. Descriptive analysis showed responsibilities for household decisions on dogs' and children's health. Multivariate analysis determined factors associated with the probability of dogs being owned and the number of dogs owned, as well as factors associated with the responsibility for child health. Dog ownership varied considerably between villages and even households. The number of dogs per household was associated with the size of a household and the presence of livestock. Children are not directly involved in the decision to vaccinate a dog, which is largely made by the father, while responsibility for seeking health care if a child is bitten lies with the mother. These novel results are relevant for the design and implementation of rabies interventions. Specifically, awareness campaigns should focus on decision-makers in households to improve rabies prevention practices and on the understanding of processes critical to the control of zoonoses more broadly.

Practical and effective diagnosis of animal anthrax in endemic low-resource settings.

Anthrax threatens human and animal health, and people's livelihoods in many rural communities in Africa and Asia. In these areas, anthrax surveillance is challenged by a lack of tools for on-site detection. Furthermore, cultural practices and infrastructure may affect sample availability and quality. Practical yet accurate diagnostic solutions are greatly needed to quantify anthrax impacts. We validated microscopic and molecular methods for the detection of Bacillus anthracis in field-collected blood smears and identified alternative samples suitable for anthrax confirmation in the absence of blood smears. We investigated livestock mortalities suspected to be caused by anthrax in northern Tanzania. Field-prepared blood smears (n = 152) were tested by microscopy using four staining techniques as well as polymerase chain reaction (PCR) followed by Bayesian latent class analysis. Median sensitivity (91%, CI 95% [84-96%]) and specificity (99%, CI 95% [96-100%]) of microscopy using azure B were comparable to those of the recommended standard, polychrome methylene blue, PMB (92%, CI 95% [84-97%] and 98%, CI 95% [95-100%], respectively), but azure B is more available and convenient. Other commonly-used stains performed poorly. Blood smears could be obtained for <50% of suspected anthrax cases due to local customs and conditions. However, PCR on DNA extracts from skin, which was almost always available, had high sensitivity and specificity (95%, CI 95% [90-98%] and 95%, CI 95% [87-99%], respectively), even after extended storage at ambient temperature. Azure B microscopy represents an accurate diagnostic test for animal anthrax that can be performed with basic laboratory infrastructure and in the field. When blood smears are unavailable, PCR using skin tissues provides a valuable alternative for confirmation. Our findings lead to a practical diagnostic approach for anthrax in low-resource settings that can support surveillance and control efforts for anthrax-endemic countries globally.

GoPrime: Development of an In Silico Framework to Predict the Performance of Real-Time PCR Primers and Probes Using Foot-and-Mouth Disease Virus as a Model.

Real-time PCR (rPCR) is a widely accepted diagnostic tool for the detection and quantification of nucleic acid targets. In order for these assays to achieve high sensitivity and specificity, primer and probe-template complementarity is essential; however, mismatches are often unavoidable and can result in false-negative results and errors in quantifying target sequences. Primer and probe sequences therefore require continual evaluation to ensure they remain fit for purpose. This paper describes the development of a linear model and associated computational tool (GoPrime) designed to predict the performance of rPCR primers and probes across multiple sequence data. Empirical data were generated using DNA oligonucleotides (n = 90) that systematically introduced variation in the primer and probe target regions of a diagnostic assay routinely used to detect foot-and-mouth disease virus (FMDV); an animal virus that exhibits a high degree of sequence variability. These assays revealed consistent impacts of patterns of substitutions in primer and probe-sites on rPCR cycle threshold (CT) and limit of detection (LOD). These data were used to populate GoPrime, which was subsequently used to predict rPCR results for DNA templates (n = 7) representing the natural sequence variability within FMDV. GoPrime was also applicable to other areas of the FMDV genome, with predictions for the likely targets of a FMDV-typing assay consistent with published experimental data. Although further work is required to improve these tools, including assessing the impact of primer-template mismatches in the reverse transcription step and the broader impact of mismatches for other assays, these data support the use of mathematical models for rapidly predicting the performance of rPCR primers and probes in silico.

Carnivore Parvovirus Ecology in the Serengeti Ecosystem: Vaccine Strains Circulating and New Host Species Identified.

Carnivore parvoviruses infect wild and domestic carnivores, and cross-species transmission is believed to occur. However, viral dynamics are not well understood, nor are the consequences for wild carnivore populations of the introduction of new strains into wild ecosystems. To clarify the ecology of these viruses in a multihost system such as the Serengeti ecosystem and identify potential threats for wildlife conservation, we analyzed, through real-time PCR, 152 samples belonging to 14 wild carnivore species and 62 samples from healthy domestic dogs. We detected parvovirus DNA in several wildlife tissues. Of the wild carnivore and domestic dog samples tested, 13% and 43%, respectively, were positive for carnivore parvovirus infection, but little evidence of transmission between the wild and domestic carnivores was detected. Instead, we describe two different epidemiological scenarios with separate routes of transmission: first, an endemic feline parvovirus (FPV) route of transmission maintained by wild carnivores inside the Serengeti National Park (SNP) and, second, a canine parvovirus (CPV) route of transmission among domestic dogs living around the periphery of the SNP. Twelve FPV sequences were characterized; new host-virus associations involving wild dogs, jackals, and hyenas were discovered; and our results suggest that mutations in the fragment of the vp2 gene were not required for infection of different carnivore species. In domestic dogs, 6 sequences belonged to the CPV-2a strain, while 11 belonged to the CPV-2 vaccine-derived strain. This is the first description of a vaccine-derived parvovirus strain being transmitted naturally.IMPORTANCE Carnivore parvoviruses are widespread among wild and domestic carnivores, which are vulnerable to severe disease under certain circumstances. This study furthers the understanding of carnivore parvovirus epidemiology, suggesting that feline parvoviruses are endemic in wild carnivores in the Serengeti National Park (SNP), with new host species identified, and that canine parvoviruses are present in the dog population living around the SNP. Little evidence of transmission of canine parvoviruses into wild carnivore species was found; however, the detection of vaccine-derived virus (described here for the first time to be circulating naturally in domestic dogs) highlights the importance of performing epidemiological research in the region.