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

Opportunities for enhanced surveillance of foot‐and‐mouth disease in endemic settings using milk samples

Under‐reporting of foot‐and‐mouth disease (FMD) masks the true prevalence in parts of the world where the disease is endemic. Laboratory testing for the detection of FMD virus (FMDV) is usually reliant upon the collection of vesicular epithelium and fluid samples that can only be collected from acutely infected animals, and therefore animals with sub‐clinical infection may not be identified. Milk is a non‐invasive sample type routinely collected from dairy farms that has been utilised for surveillance of a number of other diseases. The aim of this study was to examine the application of milk as an alternative sample type for FMDV detection and typing, and to evaluate milk as a novel approach for targeted surveillance of FMD in East Africa. FMDV RNA was detected in 73/190 (38%) individual milk samples collected from naturally infected cattle in northern Tanzania. Further, typing information by lineage‐specific rRT‐PCR assays was obtained for 58% of positive samples, and corresponded with the virus types identified during outbreak investigations in the study area. The VP1‐coding sequence data obtained from milk samples corresponded with the sequence data generated from paired epithelial samples collected from the same animal. This study demonstrates that milk represents a potentially valuable sample type for FMDV surveillance and might be used to overcome some of the existing biases of traditional surveillance methods. However, it is recommended that care is taken during sample collection and testing to minimise the likelihood of cross‐contamination. Such approaches could strengthen FMDV surveillance capabilities in East Africa, both at the individual animal and herd level

Waves of endemic foot-and-mouth disease in eastern Africa suggest feasibility of proactive vaccination approaches

Livestock production in Africa is key to national economies, food security and rural livelihoods, and > 85% of livestock keepers live in extreme poverty. With poverty elimination central to the Sustainable Development Goals, livestock keepers are therefore critically important. Foot-and-mouth disease is a highly contagious livestock disease widespread in Africa that contributes to this poverty. Despite its US$2.3 billion impact, control of the disease is not prioritized: standard vaccination regimens are too costly, its impact on the poorest is underestimated, and its epidemiology is too weakly understood. Our integrated analysis in Tanzania shows that the disease is of high concern, reduces household budgets for human health, and has major impacts on milk production and draft power for crop production. Critically, foot-and-mouth disease outbreaks in cattle are driven by livestock-related factors with a pattern of changing serotype dominance over time. Contrary to findings in southern Africa, we find no evidence of frequent infection from wildlife, with outbreaks in cattle sweeping slowly across the region through a sequence of dominant serotypes. This regularity suggests that timely identification of the epidemic serotype could allow proactive vaccination ahead of the wave of infection, mitigating impacts, and our preliminary matching work has identified potential vaccine candidates. This strategy is more realistic than wildlife-livestock separation or conventional foot-and-mouth disease vaccination approaches. Overall, we provide strong evidence for the feasibility of coordinated foot-and-mouth disease control as part of livestock development policies in eastern Africa, and our integrated socioeconomic, epidemiological, laboratory and modelling approach provides a framework for the study of other disease systems

Development of in vitro assays for detection of anthelmintic resistance in cattle nematodes : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science (Animal Science) at Massey University, Palmerston North, New Zealand

The principle aim of the current research was to modify the larval development assay (LDA) for use with Cooperia from cattle. A series of experiments were conducted in order to modify the LDA protocol to determine the most appropriate culture media and incubation temperature. These initial experiments concluded that, of the protocols examined, a culture medium of 1/8th the concentration of E. coli (EC) + 1/4th the concentration of yeast extract (YE) as generally used to culture sheep nematodes, at a culture temperature of 18ºC, resulted in the optimum number of Cooperia larvae developed to the third larval stage (L3). However, the number of eggs that developed to L3 was still generally low. A comparison was then made using isolates from a farm with a history of resistance in Cooperia to ivermectin (IV) and benzimidazoles (BZ) and two farms with a history of no resistance in this parasite. These experiments were undertaken using 1/8EC + 1/4YE media protocol and 1/2EC + 1/2YE concentration of the standard culture media for sheep nematodes. These three isolates were cultured at temperature of 18ºC and 25 ºC in the commercially available DrenchRite® 96-well microtitre assay plates which contained BZ, levamisole (LV) and IV in doubling dilutions within an agar matrix. The LD50 values were determined from a dose response curve. The resulting LD50 values were very variable, especially for the IV analogues. There was no obvious difference between the resistant and susceptible farms for the LD50 values of BZ or IV. A secondary aim of this research was to investigate the potential usefulness of the larval feeding inhibition assay (LFIA). This was adopted as published and it was determined it could be used to distinguish between susceptible and resistant Teladorsagia circumcincta with a resistance ratio of at least six. This research concluded that further research is required to fully optimise the LDA for Cooperia in cattle but adequate dose response curves were determined to indicate it struggles to distinguish BZ and IV resistance. The LFIA deserves to be further investigated as it offers some scope to detect ivermectin resistance in cattle nematodes as the dose response curves demonstrated a good repeatability for T. circumcincta from sheep. Comparing LDA and LFIA, both assays seemed to be useful but the latter was considered to have greater potential

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

Metadata and sequence quality metrics of Bacillus anthracis isolates included in this study

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