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The molecular clock hypothesis assumes that mutations accumulate on an organism's genome at a constant rate over time, but this assumption does not always hold true. While modelling approaches exist to accommodate deviations from a strict molecular clock, assumptions about rate variation may not fully represent the underlying evolutionary processes. There is considerable variability in rabies virus (RABV) incubation periods, ranging from days to over a year, during which viral replication may be reduced. This prompts the question of whether modelling RABV on a per infection generation basis might be more appropriate. We investigate how variable incubation periods affect root-to-tip divergence under per-unit time and per-generation models of mutation. Additionally, we assess how well these models represent root-to-tip divergence in time-stamped RABV sequences. We find that at low substitution rates (

Abstract A lack of methods to identify individual animals can be a barrier to zoonoses control. We developed and field-tested facial recognition technology for a mobile phone application to identify dogs, which we used to assess vaccination coverage against rabies in rural Tanzania. Dogs were vaccinated, registered using the application, and microchipped. During subsequent household visits to validate vaccination, dogs were registered using the application and their vaccination status determined by operators using the application to classify dogs as vaccinated (matched) or unvaccinated (unmatched), with microchips validating classifications. From 534 classified dogs (251 vaccinated, 283 unvaccinated), the application specificity was 98.9% and sensitivity 76.2%, with positive and negative predictive values of 98.4% and 82.8% respectively. The facial recognition algorithm correctly matched 249 (99.2%) vaccinated and microchipped dogs (true positives) and failed to match two (0.8%) vaccinated dogs (false negatives). Operators correctly identified 186 (74.1%) vaccinated dogs (true positives), and 280 (98.9%) unvaccinated dogs (true negatives), but incorrectly classified 58 (23.1%) vaccinated dogs as unmatched (false negatives). Reduced application sensitivity resulted from poor quality photos and light-associated color distortion. With development and operator training, this technology has potential to be a useful tool to identify dogs and support research and intervention programs.

Abstract: Sustained vaccination coverage of domestic dog populations can interrupt rabies transmission. However, challenges remain including low dog owner participation, high operational costs associated with current (centralized and annually delivered (pulse)) approaches and high dog population turnover. To address these challenges an alternative (community-based continuous mass dog vaccination (CBC-MDV)) approach was designed. We investigated the potential for successful normalization of CBC-MDV into routine practice within the context of local communities and the veterinary system of Tanzania Methods: In a process evaluation of a pilot implementation of CBC-MDV, we conducted in-depth interviews with implementers and community leaders (n = 24), focus group discussion with implementers and community members (n = 12), and non-participant observation (n = 157 h) of delivery of the intervention components. We analyzed these data thematically drawing on the normalization process theory, to assess factors affecting implementation and integration. Main findings: Implementers and community members clearly understood the values and benefits of the CBC-MDV, regarding it as an improvement over the pulse strategy. They had a clear understanding of what was required to enact CBC-MDV and considered their own involvement to be legitimate. The approach fitted well into routine schedules of implementers and the context (infrastructure, skill sets and policy). Implementers and community members positively appraised CBC-MDV in terms of its perceived impact on rabies and recommended its use across the country. Implementers and community members further believed that vaccinating dogs free of charge was critical and made community mobilization easier. However, providing feedback to communities and involving them in evaluating outcomes of vaccination campaigns were reported to have not been done. Local politics was cited as a barrier to collaboration between implementers and community leaders. Conclusion: This work suggests that CBC-MDV has the potential to be integrated and sustained in the context of Tanzania. Involving communities in design, delivery and monitoring of CBC-MDV activities could contribute to improving and sustaining its outcomes.

ObjectivesDog vaccination can eliminate rabies in dogs, but annual delivery strategies do not sustain vaccination coverage between campaigns. We describe the development of a community-based continuous mass dog vaccination (CBC-MDV) approach designed to improve and maintain vaccination coverage in Tanzania and examine the feasibility of delivering this approach as well as lessons for its optimization.MethodsWe developed three delivery strategies of CBC-MDV and tested them against the current annual vaccination strategy following the UK Medical Research Council's guidance: i) developing an evidence-based theoretical framework of intervention pathways and ii) piloting to test feasibility and inform optimization. For our process evaluation of CBC-MDV we collected data using non-participant observations, meeting reports and implementation audits and in-depth interviews, as well as household surveys of vaccination coverage to assess potential effectiveness. We analyzed qualitative data thematically and quantitative data descriptively.ResultsThe final design included delivery by veterinary teams supported by village-level one health champions. In terms of feasibility, we found that less than half of CBC-MDV's components were implemented as planned. Fidelity of delivery was influenced by the strategy design, implementer availability and appreciation of value intervention components, and local environmental and socioeconomic events (e.g. elections, funerals, school cycles). CBC-MDV activities decreased sharply after initial campaigns, partly due to lack of supervision. Community engagement and involvement was not strong. Nonetheless, the CBC-MDV approaches achieved vaccination coverage above the critical threshold (40%) all-year-round. CBC-MDV components such as identifying vaccinated dogs, which village members work as one health champions and how provision of continuous vaccination is implemented need further optimization prior to scale up.InterpretationCBC-MDV is feasible to deliver and can achieve good vaccination coverage. Community involvement in the development of CBC-MDV, to better tailor components to contextual situations, and improved supervision of activities are likely to improve vaccination coverage in future.

Background: Thermostable vaccines greatly improved the reach and impact of large-scale programmes to eliminate infectious diseases such as smallpox, polio, and rinderpest. A study from 2015 demonstrated that the potency of the Nobivac® Rabies vaccine was not impacted following experimental storage at 30°C for 3 months. Whether the vaccine would remain efficacious following storage under more natural, fluctuating temperature conditions remains unknown. We carried out a randomised controlled non-inferiority trial to compare serological responses in dogs following vaccination with doses stored under cold chain conditions with those stored within a locally made Passive Cooling Device (“Zeepot”) under fluctuating temperature conditions.Materials and Methods: Nobivac® Rabies vaccine was stored under either cold-chain conditions or within the Zeepot for 2 months. Daily ambient temperatures and temperatures within the Zeepot were recorded every 3 h. Following storage, 412 domestic dogs were randomly assigned to receive either cold-chain or Zeepot stored Nobivac® Rabies vaccine. Baseline and day 28-post vaccination blood samples were collected. Serological analysis using the Fluorescent Antibody Virus Neutralisation assay was carried out with a threshold of 0.5 IU/ml to determine seroconversion. In addition, the impact of dog Body Condition Score, sex, and age on seroconversion was examined.Results: The serological response of dogs vaccinated using Nobivac® Rabies vaccine stored within the Zeepot was not inferior to the response of dogs vaccinated using cold-chain stored vaccine (z = 1.1, df = 313, p-value = 0.25). Indeed, the 28-day post-vaccination group geometric mean titre was 1.8 and 2.0 IU/ml for cold-chain vs. non-cold-chain storage, respectively. Moreover, the percentage of dogs that seroconverted in each arm was almost identical (85%). There was a positive linear trend between Body Condition Score (O.R. 2.2, 95% CI: 1.1–5.1) and seroconversion, suggesting dogs of poor condition may not respond as expected to vaccination.Conclusions: Our study demonstrated the potency of Nobivac® Rabies vaccine is not impacted following storage under elevated fluctuating temperatures within a Zeepot. These results have potentially exciting applications for scaling up mass dog vaccination programmes in low-and-middle income countries, particularly for hard-to-reach populations with limited access to power and cold-chain vaccine storage.

Genomic surveillance is an important aspect of contemporary disease management but has yet to be used routinely to monitor endemic disease transmission and control in low- and middle-income countries. Rabies is an almost invariably fatal viral disease that causes a large public health and economic burden in Asia and Africa, despite being entirely vaccine preventable. With policy efforts now directed towards achieving a global goal of zero dog-mediated human rabies deaths by 2030, establishing effective surveillance tools is critical. Genomic data can provide important and unique insights into rabies spread and persistence that can direct control efforts. However, capacity for genomic research in low- and middle-income countries is held back by limited laboratory infrastructure, cost, supply chains and other logistical challenges. Here we present and validate an end-to-end workflow to facilitate affordable whole genome sequencing for rabies surveillance utilising nanopore technology. We used this workflow in Kenya, Tanzania and the Philippines to generate rabies virus genomes in two to three days, reducing costs to approximately £60 per genome. This is over half the cost of metagenomic sequencing previously conducted for Tanzanian samples, which involved exporting samples to the UK and a three- to six-month lag time. Ongoing optimization of workflows are likely to reduce these costs further. We also present tools to support routine whole genome sequencing and interpretation for genomic surveillance. Moreover, combined with training workshops to empower scientists in-country, we show that local sequencing capacity can be readily established and sustainable, negating the common misperception that cutting-edge genomic research can only be conducted in high resource laboratories. More generally, we argue that the capacity to harness genomic data is a game-changer for endemic disease surveillance and should precipitate a new wave of researchers from low- and middle-income countries.

Human rabies can be prevented through mass dog vaccination campaigns; however, in rabies endemic countries, pulsed central point campaigns do not always achieve the recommended coverage of 70%. This study describes the development of a novel approach to sustain high coverage based on decentralized and continuous vaccination delivery. A rabies vaccination campaign was conducted across 12 wards in the Mara region, Tanzania to test this approach. Household surveys were used to obtain data on vaccination coverage as well as factors influencing dog vaccination. A total 17,571 dogs were vaccinated, 2654 using routine central point delivery and 14,917 dogs using one of three strategies of decentralized continuous vaccination. One month after the first vaccination campaign, coverage in areas receiving decentralized vaccinations was higher (64.1, 95% Confidence Intervals (CIs) 62.1–66%) than in areas receiving pulsed vaccinations (35.9%, 95% CIs 32.6–39.5%). Follow-up surveys 10 months later showed that vaccination coverage in areas receiving decentralized vaccinations remained on average over 60% (60.7%, 95% CIs 58.5–62.8%) and much higher than in villages receiving pulsed vaccinations where coverage was on average 32.1% (95% CIs 28.8–35.6%). We conclude that decentralized continuous dog vaccination strategies have the potential to improve vaccination coverage and maintain herd immunity against rabies.

Genomic surveillance is an important aspect of contemporary disease management but has yet to be used routinely to monitor endemic disease transmission and control in low- and middle-income countries. Rabies is an almost invariably fatal viral disease that causes a large public health and economic burden in Asia and Africa, despite being entirely vaccine preventable. With policy efforts now directed towards achieving a global goal of zero dog-mediated human rabies deaths by 2030, establishing effective surveillance tools is critical. Genomic data can provide important and unique insights into rabies spread and persistence that can direct control efforts. However, capacity for genomic research in low- and middle-income countries is held back by limited laboratory infrastructure, cost, supply chains and other logistical challenges. Here we present and validate an end-to-end workflow to facilitate affordable whole genome sequencing for rabies surveillance utilising nanopore technology. We used this workflow in Kenya, Tanzania and the Philippines to generate rabies virus genomes in two to three days, reducing costs to approximately £60 per genome. This is over half the cost of metagenomic sequencing previously conducted for Tanzanian samples, which involved exporting samples to the UK and a three- to six-month lag time. Ongoing optimization of workflows are likely to reduce these costs further. We also present tools to support routine whole genome sequencing and interpretation for genomic surveillance. Moreover, combined with training workshops to empower scientists in-country, we show that local sequencing capacity can be readily established and sustainable, negating the common misperception that cutting-edge genomic research can only be conducted in high resource laboratories. More generally, we argue that the capacity to harness genomic data is a game-changer for endemic disease surveillance and should precipitate a new wave of researchers from low- and middle-income countries.

Thermotolerant vaccines greatly improved the reach and impact of large-scale vaccination programs to eliminate diseases such as smallpox, polio and rinderpest. A recent study demonstrated that the potency of the Nobivac® Canine Rabies vaccine was not impacted following experimental storage at 30 °C for three months. We conducted a study to develop a passive cooling device (PCD) that could store thermotolerant vaccines under fluctuating subambient temperatures. Through a participatory process with local communities in Northern Tanzania, we developed innovative PCD designs for local manufacture. A series of field experiments were then carried out to evaluate the effectiveness of five PCDs for vaccine storage under varying climatic conditions. Following iterative improvement, a final prototype “Zeepot Clay” was developed at the cost of US$11 per unit. During a further field-testing phase over a 12-month period, the internal temperature of the device remained below 26 °C, despite ambient temperatures exceeding 42 °C. Our study thus demonstrated that locally designed PCDs have utility for storing thermotolerant rabies vaccines at subambient temperatures. These results have application for the scaling up of mass dog vaccination programs in low-and-middle income countries, particularly for hard-to-reach populations with limited access to power and cold-chain vaccine storage.

[This corrects the article DOI: 10.1371/journal.pone.0116059.].

Alcelaphine herpesvirus-1 (AlHV-1), a causative agent of malignant catarrhal fever in cattle, was detected in wildebeest (Connochaetes taurinus) placenta tissue for the first time. Although viral load was low, the finding of viral DNA in over 50% of 94 samples tested lends support to the possibility that placental tissue could play a role in disease transmission and that wildebeest calves are infected in utero. Two viral loci were sequenced to examine variation among virus samples obtained from wildebeest and cattle: the ORF50 gene, encoding the lytic cycle transactivator protein, and the A9.5 gene, encoding a novel polymorphic viral glycoprotein. ORF50 was well conserved with six newly discovered alleles differing at only one or two base positions. In contrast, while only three new A9.5 alleles were discovered, these differed by up to 13% at the nucleotide level and up to 20% at the amino acid level. Structural homology searching performed with the additional A9.5 sequences determined in this study adds power to recent analysis identifying the four-helix bundle cytokine interleukin-4 (IL4) as the major homologue. The majority of MCF virus samples obtained from Tanzanian cattle and wildebeest encoded A9.5 polypeptides identical to the previously characterized A9.5 allele present in the laboratory maintained AlHV-1 C500 strain. This supports the view that AlHV-1 C500 is suitable for the development of a vaccine for wildebeest-associated MCF.

This study is the first to partially quantify the potential economic benefits that a vaccine, effective at protecting cattle against malignant catarrhal fever (MCF), could accrue to pastoralists living in East Africa. The benefits would result from the removal of household resource and management costs that are traditionally incurred avoiding the disease. MCF, a fatal disease of cattle caused by a virus transmitted from wildebeest calves, has plagued Maasai communities in East Africa for generations. The threat of the disease forces the Maasai to move cattle to less productive grazing areas to avoid wildebeest during calving season when forage quality is critical. To assess the management and resource costs associated with moving, we used household survey data. To estimate the costs associated with changes in livestock body condition that result from being herded away from wildebeest calving grounds, we exploited an ongoing MCF vaccine field trial and we used a hedonic price regression, a statistical model that allows estimation of the marginal contribution of a good's attributes to its market price. We found that 90 percent of households move, on average, 82 percent of all cattle away from home to avoid MCF. In doing so, a herd's productive contributions to the household was reduced, with 64 percent of milk being unavailable for sale or consumption by the family members remaining at the boma (the children, women, and the elderly). In contrast cattle that remained on the wildebeest calving grounds during the calving season (and survived MCF) remained fully productive to the family and gained body condition compared to cattle that moved away. This gain was, however, short-lived. We estimated the market value of these condition gains and losses using hedonic regression. The value of a vaccine for MCF is the removal of the costs incurred in avoiding the disease.