Genomic epidemiology of Escherichia coli: antimicrobial resistance through a One Health lens in sympatric humans, livestock and peri-domestic wildlife in Nairobi, Kenya.

BACKGROUND: Livestock systems have been proposed as a reservoir for antimicrobial-resistant (AMR) bacteria and AMR genetic determinants that may infect or colonise humans, yet quantitative evidence regarding their epidemiological role remains lacking. Here, we used a combination of genomics, epidemiology and ecology to investigate patterns of AMR gene carriage in Escherichia coli, regarded as a sentinel organism. METHODS: We conducted a structured epidemiological survey of 99 households across Nairobi, Kenya, and whole genome sequenced E. coli isolates from 311 human, 606 livestock and 399 wildlife faecal samples. We used statistical models to investigate the prevalence of AMR carriage and characterise AMR gene diversity and structure of AMR genes in different host populations across the city. We also investigated household-level risk factors for the exchange of AMR genes between sympatric humans and livestock. RESULTS: We detected 56 unique acquired genes along with 13 point mutations present in variable proportions in human and animal isolates, known to confer resistance to nine antibiotic classes. We find that AMR gene community composition is not associated with host species, but AMR genes were frequently co-located, potentially enabling the acquisition and dispersal of multi-drug resistance in a single step. We find that whilst keeping livestock had no influence on human AMR gene carriage, the potential for AMR transmission across human-livestock interfaces is greatest when manure is poorly disposed of and in larger households. CONCLUSIONS: Findings of widespread carriage of AMR bacteria in human and animal populations, including in long-distance wildlife species, in community settings highlight the value of evidence-based surveillance to address antimicrobial resistance on a global scale. Our genomic analysis provided an in-depth understanding of AMR determinants at the interfaces of One Health sectors that will inform AMR prevention and control.

Population genomics of Escherichia coli in livestock-keeping households across a rapidly developing urban landscape.

Quantitative evidence for the risk of zoonoses and the spread of antimicrobial resistance remains lacking. Here, as part of the UrbanZoo project, we sampled Escherichia coli from humans, livestock and peri-domestic wildlife in 99 households across Nairobi, Kenya, to investigate its distribution among host species in this rapidly developing urban landscape. We performed whole-genome sequencing of 1,338 E. coli isolates and found that the diversity and sharing patterns of E. coli were heavily structured by household and strongly shaped by host type. We also found evidence for inter-household and inter-host sharing and, importantly, between humans and animals, although this occurs much less frequently. Resistome similarity was differently distributed across host and household, consistent with being driven by shared exposure to antimicrobials. Our results indicate that a large, epidemiologically structured sampling framework combined with WGS is needed to uncover strain-sharing events among different host populations in complex environments and the major contributing pathways that could ultimately drive the emergence of zoonoses and the spread of antimicrobial resistance.

Mycotoxins in Poultry Feed and Feed Ingredients from Sub-Saharan Africa and Their Impact on the Production of Broiler and Layer Chickens: A Review.

The poultry industry in sub-Saharan Africa (SSA) is faced with feed insecurity, associated with high cost of feeds, and feed safety, associated with locally produced feeds often contaminated with mycotoxins. Mycotoxins, including aflatoxins (AFs), fumonisins (FBs), trichothecenes, and zearalenone (ZEN), are common contaminants of poultry feeds and feed ingredients from SSA. These mycotoxins cause deleterious effects on the health and productivity of chickens and can also be present in poultry food products, thereby posing a health hazard to human consumers of these products. This review summarizes studies of major mycotoxins in poultry feeds, feed ingredients, and poultry food products from SSA as well as aflatoxicosis outbreaks. Additionally reviewed are the worldwide regulation of mycotoxins in poultry feeds, the impact of major mycotoxins in the production of chickens, and the postharvest use of mycotoxin detoxifiers. In most studies, AFs are most commonly quantified, and levels above the European Union regulatory limits of 20 µg/kg are reported. Trichothecenes, FBs, ZEN, and OTA are also reported but are less frequently analyzed. Co-occurrences of mycotoxins, especially AFs and FBs, are reported in some studies. The effects of AFs on chickens' health and productivity, carryover to their products, as well as use of mycotoxin binders are reported in few studies conducted in SSA. More research should therefore be conducted in SSA to evaluate occurrences, toxicological effects, and mitigation strategies to prevent the toxic effects of mycotoxins.

Epidemiology of antimicrobial-resistant Escherichia coli carriage in sympatric humans and livestock in a rapidly urbanizing city.

There are substantial limitations in understanding of the distribution of antimicrobial resistance (AMR) in humans and livestock in developing countries. This papers present the results of an epidemiological study examining patterns of AMR in Escherichia coli isolates circulating in sympatric human (n = 321) and livestock (n = 633) samples from 99 households across Nairobi, Kenya. E. coli isolates were tested for susceptibility to 13 antimicrobial drugs representing nine antibiotic classes. High rates of AMR were detected, with 47.6% and 21.1% of isolates displaying resistance to three or more and five or more antibiotic classes, respectively. Human isolates showed higher levels of resistance to sulfonamides, trimethoprim, aminoglycosides and penicillins compared with livestock (P<0.01), while poultry isolates were more resistant to tetracyclines (P = 0.01) compared with humans. The most common co-resistant phenotype observed was to tetracyclines, streptomycin and trimethoprim (30.5%). At the household level, AMR carriage in humans was associated with human density (P<0.01) and the presence of livestock manure (P = 0.03), but keeping livestock had no influence on human AMR carriage (P>0.05). These findings revealed a high prevalence of AMR E. coli circulating in healthy humans and livestock in Nairobi, with no evidence to suggest that keeping livestock, when treated as a single risk factor, contributed significantly to the burden of AMR in humans, although the presence of livestock waste was significant. These results provide an understanding of the broader epidemiology of AMR in complex and interconnected urban environments.

Clinically relevant antimicrobial resistance at the wildlife-livestock-human interface in Nairobi: an epidemiological study.

BACKGROUND: Antimicrobial resistance is one of the great challenges facing global health security in the modern era. Wildlife, particularly those that use urban environments, are an important but understudied component of epidemiology of antimicrobial resistance. We investigated antimicrobial resistance overlap between sympatric wildlife, humans, livestock, and their shared environment across the developing city of Nairobi, Kenya. We use these data to examine the role of urban wildlife in the spread of clinically relevant antimicrobial resistance. METHODS: 99 households across Nairobi were randomly selected on the basis of socioeconomic stratification. A detailed survey was administered to household occupants, and samples (n=2102) were collected from the faeces of 75 wildlife species inhabiting household compounds (ie, the household and its perimeter; n=849), 13 livestock species (n=656), and humans (n=333), and from the external environment (n=288). Escherichia coli, our sentinel organism, was cultured and a single isolate from each sample tested for sensitivity to 13 antibiotics. Diversity of antimicrobial resistant phenotypes was compared between urban wildlife, humans, livestock, and the environment, to investigate whether wildlife are a net source for antimicrobial resistance in Nairobi. Generalised linear mixed models were used to determine whether the prevalence of antimicrobial resistant phenotypes and multidrug-resistant E coli carriage in urban wildlife is linked to variation in ecological traits, such as foraging behaviour, and to determine household-level risk factors for sharing of antimicrobial resistance between humans, wildlife, and livestock. FINDINGS: E coli were isolated from 485 samples collected from wildlife between Sept 6,2015, and Sept 28, 2016. Wildlife carried a low prevalence of E coli isolates susceptible to all antibiotics tested (45 [9%] of 485 samples) and a high prevalence of clinically relevant multidrug resistance (252 [52%] of 485 samples), which varied between taxa and by foraging traits. Multiple isolates were resistant to one agent from at least seven antimicrobial classes tested for, and a single isolate was resistant to all antibiotics tested for in the study. The phenotypic diversity of antimicrobial-resistant E coli in wildlife was lower than in livestock, humans, and the environment. Within household compounds, statistical models identified two interfaces for exchange of antimicrobial resistance: between both rodents, humans and their rubbish, and seed-eating birds, humans and their rubbish; and between seed-eating birds, cattle, and bovine manure. INTERPRETATION: Urban wildlife carry a high burden of clinically relevant antimicrobial-resistant E coli in Nairobi, exhibiting resistance to drugs considered crucial for human medicine by WHO. Identifiable traits of the wildlife contribute to this exposure; however, compared with humans, livestock, and the environment, low phenotypic diversity in wildlife is consistent with the hypothesis that wildlife are a net sink rather than source of clinically relevant resistance. Wildlife that interact closely with humans, livestock, and both human and livestock waste within households, are exposed to more antimicrobial resistant phenotypes, and could therefore act as conduits for the dissemination of clinically relevant antimicrobial resistance to the wider environment. These results provide novel insight into the broader epidemiology of antimicrobial resistance in complex urban environments, characteristic of lower-middle-income countries. FUNDING: UK Medical Research Council and CGIAR Research Program on Agriculture for Nutrition and Health.

Mapping Nairobi's dairy food system: An essential analysis for policy, industry and research.

Demand for dairy products in sub-Saharan Africa, is expected to triple by 2050, while limited increase in supply is predicted. This poses significant food security risk to low income households. Understanding how the dairy food system operates is essential to identify mitigation measures to food insecurity impact. This study aims to determine the structure and functionality of Nairobi's dairy system using a value chain mapping approach. Primary data were gathered through focus group discussions and key informant interviews with dairy value chain stakeholders in Nairobi to obtain qualitative information on people and products in the chains while describing their interactions and flows. Qualitative thematic analysis combined with flowcharts created by participants enabled identification of key food system segments and the development of chain profiles (or flow-diagrams) which together form Nairobi's dairy system. Seven chain profiles forming Nairobi's dairy value chain were identified. These were found to be dominated by small-scale individuals who operate largely independently. Our profiles for the urban and peri-urban farming systems were structurally similar in their downstream networks, obtaining inputs from similar sources. Upstream, the urban systems were shorter, supplying mostly to immediate neighbours or based on own consumption, while the peri urban systems supplied to a wider network and showed some affiliations to producers' associations. Two distinct profiles characterize the milk flow from traders belonging either to a Dairy Traders Association (DTA) or those not belonging to this association (non-DTA). DTA traders sell mainly to fixed retailers and non-DTA traders to mobile retailers (hawkers or roadside vendors). Profiles associated with medium and large cooperatives were driven by networks of collection centres, but with medium-sized cooperatives selling half of their production to large processing companies, and large cooperatives only to fixed retailers. Large processing companies' profiles indicated distribution of high volumes and value addition processing. They reported strategic milk collection arrangements with suppliers on long, medium - or short - term contracts and with well-established product distribution channels. We have identified numerous inter-linkages across dairy chain profiles in Nairobi's complex system, demonstrating significant interdependency among the stakeholders. Therefore, enhancing the system's efficiency requires a holistic, system-wide approach and any policy interventions should consider every segment of the value chain. This study provides a methodological approach for organizations and policy makers to understand and address structural and functional vulnerabilities within food systems more broadly. The insights from this study are relevant to other rapidly growing cities in the region.

Social and gender determinants of risk of cryptosporidiosis, an emerging zoonosis, in Dagoretti, Nairobi, Kenya.

The aim of the study was to investigate the social and gender determinants of the risk of exposure to Cryptosporidium from urban dairying in Dagoretti, Nairobi. Focus group discussions were held in six locations to obtain qualitative information on risk of exposure. A repeated cross-sectional descriptive study included participatory assessment and household questionnaires (300 randomly selected urban dairy farming households and 100 non-dairying neighbours). One-hundred dairy households randomly selected from the 300 dairy households participated in an additional economic survey along with 40 neighbouring non-dairy households. We found that exposure to Cryptosporidium was influenced by gender, age and role in the household. Farm workers and people aged 50 to 65 years had most contact with cattle, and women had greater contact with raw milk. However, children had relatively higher consumption of raw milk than other age groups. Adult women had more daily contact with cattle faeces than adult men, and older women had more contact than older men. Employees had greater contact with cattle than other groups and cattle faeces, and most (77 %) were male. Women took more care of sick people and were more at risk from exposure by this route. Poverty did not affect the level of exposure to cattle but did decrease consumption of milk. There was no significant difference between men and women as regards levels of knowledge on symptoms of cryptosporidiosis infections or other zoonotic diseases associated with dairy farming. Awareness of cryptosporidiosis and its transmission increased significantly with rising levels of education. Members of non-dairy households and children under the age of 12 years had significantly higher odds of reporting diarrhoea: gender, season and contact with cattle or cattle dung were not significantly linked with diarrhoea. In conclusion, social and gender factors are important determinants of exposure to zoonotic disease in Nairobi.

A trans-disciplinary study on the health risks of cryptosporidiosis from dairy systems in Dagoretti, Nairobi, Kenya: study background and farming system characteristics.

This paper characterises the dairy farming system in Dagoretti, Nairobi. Characterisation was part of a broader ecohealth project to estimate the prevalence and risk of cryptosporidiosis and develop risk mitigation strategies. In the project a trans-disciplinary team addressed epidemiological, socioeconomic, environmental and policy aspects of cryptosporidiosis, an emerging zoonosis. This paper also provides background and describes sampling methods for the wider project. Three hundred dairy households were probabilistically sampled from a sampling frame of all dairy households in five of the six locations of Dagoretti, one of the eight districts of Nairobi Province. Randomly selected households identified 100 non-dairy-keeping households who also took part in the study. A household questionnaire was developed, pre-tested and administered in the dry and wet seasons of 2006. An additional study on livelihood and economic benefits of dairying took place with 100 dairy farmers randomly selected from the 300 farms (as well as 40 non-dairy neighbours as a control group), and a risk-targeted survey of environmental contamination with Cryptosporidium was conducted with 20 farmers randomly selected from the 29 farmers in the wider survey who were considered at high risk because of farming system. We found that around 1 in 80 urban households kept dairy cattle with an average of three cattle per household. Cross-breeds of exotic and local cattle predominate. Heads of dairy-keeping households were significantly less educated than the heads of non-dairy neighbours, had lived in Dagoretti for significantly longer and had significantly larger households. There was a high turnover of 10 % of the cattle population in the 3-month period of the study. Cattle were zero grazed, but productivity parameters were sub-optimal as were hygiene and husbandry practices. In conclusion, dairy keeping is a minor activity in urban Nairobi but important to households involved and their community. Ecohealth approaches are well suited to tackling the complex problem of assessing and managing emerging zoonoses in urban settings.

Participatory probabilistic assessment of the risk to human health associated with cryptosporidiosis from urban dairying in Dagoretti, Nairobi, Kenya.

We carried out a participatory risk assessment to estimate the risk (negative consequences and their likelihood) from zoonotic Cryptosporidium originating in dairy farms in urban Dagoretti, Nairobi to dairy farm households and their neighbours. We selected 20 households at high risk for Cryptosporidium from a larger sample of 300 dairy households in Dagoretti based on risk factors present. We then conducted a participatory mapping of the flow of the hazard from its origin (cattle) to human potential victims. This showed three main exposure pathways (food and water borne, occupational and recreational). This was used to develop a fault tree model which we parameterised using information from the study and literature. A stochastic simulation was used to estimate the probability of exposure to zoonotic cryptosporidiosis originating from urban dairying. Around 6 % of environmental samples were positive for Cryptosporidium. Probability of exposure to Cryptosporidium from dairy cattle ranged from 0.0055 for people with clinical acquired immunodeficiency syndrome in non-dairy households to 0.0102 for children under 5 years from dairy households. Most of the estimated health burden was born by children. Although dairy cattle are the source of Cryptosporidium, the model suggests consumption of vegetables is a greater source of risk than consumption of milk. In conclusion, by combining participatory methods with quantitative microbial risk assessment, we were able to rapidly, and with appropriate 'imprecision', investigate health risk to communities from Cryptosporidium and identify the most vulnerable groups and the most risky practices.

Outcome mapping for fostering and measuring change in risk management behaviour among urban dairy farmers in Nairobi, Kenya.

A study was undertaken to investigate and mitigate the risk from zoonotic Cryptosporidium associated with dairy farming in Dagoretti division, Nairobi, Kenya. Outcome mapping (OM), a relatively new tool for planning and evaluation, was used to foster and then monitor changes in farmer management of health risks. Elements of the OM framework, including the vision, mission and expected progress markers, were developed in participatory sessions and a set of progress markers was used for monitoring behaviour change in farmers participating in the project (the boundary partners). Behaviour change (the outcome challenge) was supported by a range of awareness and educational campaigns, working with strategic partners (extension agents and administrative leaders). The farmers the project worked with made considerable progress according to the markers; they demonstrated an understanding of cryptosporidiosis, established or maintained clean and well drained cattle sheds, and took conscious effort to reduce possible infection. Farmers who did not participate in the project (non-contact farmers) were found to be less advanced on the progress marker indicators. Non-contact farmers who carried out risk-reducing practices had done so independently of the project team. The administration leaders, as strategic partners, had a positive attitude towards the project and confidence in their ability to support project objectives. The study demonstrates the utility of OM in helping to identify and support behavioural change.

Cryptosporidium species detected in calves and cattle in Dagoretti, Nairobi, Kenya.

A total of 1,734 cattle faecal samples from 296 dairy-keeping households were collected from urban settings in Nairobi, Kenya. Modified Ziehl-Neelsen staining method and an immunofluorescence assay were used to identify those samples with Cryptosporidium oocyst infection. Oocysts from positive faecal samples were isolated by Sheather's sucrose flotation method and picked from the concentrate using cover slips. Genomic DNA was extracted from 124 of the faecal samples that were positive for Cryptosporidium and was used as template for nested PCR of the 18S rRNA gene. Twenty-five samples (20 %) were PCR-positive for Cryptosporidium, and 24 of the PCR products were successfully cloned and sequenced. Sequence and phylogenetic analysis identified 17 samples (68 %) as Cryptosporidium parvum-like, four samples (16 %) as Cryptosporidium ryanae, three samples (12 %) as Cryptosporidium andersoni and one sample (4 %) as Cryptosporidium hominis. To the best of our knowledge, this is the first genotyping study to report C. parvum-like, C. andersoni and C. hominis in cattle from Kenya. The results of this study show Cryptosporidium infections in calves and cattle may be potential zoonotic reservoirs of the parasite that infects humans.

Aflatoxin B1 and M1 contamination of animal feeds and milk from urban centers in Kenya.

BACKGROUND: Aflatoxin M1 (AFM1) is the principal hydroxylated AFB1 metabolite present in milk of cows fed with a diet contaminated with AFB1and excreted within 12 hours of administration of contaminated feeds. OBJECTIVE: This study was initiated to assess the knowledge and practices of urban dairy farmers and feed millers about aflatoxin in feeds and milk, determine the prevalence and quantify the levels of AFB1 and AFM1 in animal feeds and milk respectively from urban environs in Kenya. METHODS: This work was carried out in the Department of Public Health Pharmacology and Toxicology, University of Nairobi, Kenya, between February 2006 and March 2007. RESULTS: A total of 830 animal feed and 613 milk samples from four urban centers were analyzed for aflatoxin B1 and M1 respectively using competitive enzyme immunoassay. Eighty six percent (353/412) of the feed samples from farmers were positive for aflatoxin B1 and 67% (235/353) of these exceeded the FAO/WHO level of 5µ gKg-1. Eighty one percent (197/243) of the feed samples from feed millers and 87% (153/175) from agrochemical shops were positive, while 58% (115/197) and 66% (92/153) of the positive samples exceeded the FAO/WHO limits respectively. Seventy two percent (315/439) of the milk from dairy farmers, 84% (71/85) from large and medium scale farmers and 99% (88/89) of the pasteurized marketed milk were positive for aflatoxin M1, and 20%, 35% an 31% of positive milk from dairy farmers, medium and large scale farmers and market outlets respectively, exceeded the WHO/FAO levels of 0.05µ g/Kg-1. Sixty seven percent of the urban smallholder dairy farmers had no knowledge that milk could be contaminated with aflatoxin M1 and neither knew how they could mitigate against this exposure. Feed millers knew about aflatoxin B1 in grains and excretion of aflatoxin M1 in milk, but were not alleviating exposure to animals. CONCLUSION: There is need to create awareness and establish routine monitoring of animal feeds and milk to reduce animal and consequently human response.

First report of Cryptosporidium deer-like genotype in Kenyan cattle.

The objective of the study was to identify Cryptosporidium genotypes from feces collected from urban and peri-urban dairy cattle in Nairobi, Kenya, in order to determine their zoonotic potential. DNA was extracted from 34 samples that were diagnosed positive by the modified Ziehl-Neelsen technique. Two Cryptosporidium isolates examined at the 18S rRNA locus were identified as the deer-like genotype by DNA sequencing. As public health officials are facing the difficult decision whether to allow urban livestock production because of its economic benefits and a livelihood asset to the urban communities, or to ban it for its public health risks, the finding of non-zoonotic genotypes in a smallholder dairy system has significant public health as well as economic implications that merit further investigation.