Navigating One Health in research-for-development: Reflections on the design and implementation of the CGIAR Initiative on One Health.

Adopting One Health approaches is key for addressing interconnected health challenges. Yet, how to best put One Health into practice in research-for-development initiatives aiming to 'deliver impacts' remains unclear. Drawing on the CGIAR Initiative on One Health - a global initiative to address zoonotic diseases, antimicrobial resistance, and food and water safety - we reflect on challenges during program conception and implementation, prompting us to suggest improvements in multisectoral collaboration, coordination, and communication. Our approach involves conducting a researcher-centered process evaluation, comprising individual interviews that are subsequently thematically analyzed and synthesized. The key takeaway is that limited time for planning processes and short program timelines compared to envisioned development impacts may impede research-for-development efforts. Yet, collaborative work can be successful when adequate time and resources are allocated for planning with minimal disruption throughout implementation. Additionally, due to the multifaceted nature of One Health initiatives, it is important to pay attention to co-benefits and trade-offs, where taking action in one aspect may yield advantages and disadvantages in another, aiding to identify sustainable One Health development pathways. Forming close partnerships with national governments and local stakeholders is essential not only to promote sustainability but also to ensure local relevance, enhancing the potential for meaningful impact. Finally, regularly assessing progress toward development goals is critical as development stands as an overarching objective.

Nanopore sequencing for identification and characterization of antimicrobial-resistant Escherichia coli and Salmonella spp. from tilapia and shrimp sold at wet markets in Dhaka, Bangladesh.

Wet markets in low-and middle-income countries are often reported to have inadequate sanitation resulting in fecal contamination of sold produce. Consumption of contaminated wet market-sourced foods has been linked to individual illness and disease outbreaks. This pilot study, conducted in two major wet markets in Dhaka city, Bangladesh during a 4-month period in 2021 aimed to assess the occurrence and characteristics of Escherichia coli and non-typhoidal Salmonella spp. (NTS) from tilapia (Oreochromis niloticus) and shrimp (Penaeus monodon). Fifty-four individuals of each species were collected. The identity of the bacterial isolates was confirmed by PCR and their susceptibility toward 15 antimicrobials was tested by disk diffusion. The whole genome of 15 E. coli and nine Salmonella spp. were sequenced using Oxford Nanopore Technology. E. coli was present in 60-74% of tilapia muscle tissue and 41-44% of shrimp muscle tissue. Salmonella spp. was found in skin (29%) and gills (26%) of tilapia, and occasionally in muscle and intestinal samples of shrimp. The E. coli had several Multilocus sequence typing and serotypes and limited antimicrobial resistance (AMR) determinants, such as point mutations on glpT and pmrB. One E. coli (BD17) from tilapia carried resistance genes for beta-lactams, quinolones, and tetracycline. All the E. coli belonged to commensal phylogroups B1 and A and showed no Shiga-toxin and other virulence genes, confirming their commensal non-pathogenic status. Among the Salmonella isolates, five belonged to Kentucky serovar and had similar AMR genes and phenotypic resistance patterns. Three strains of this serovar were ST198, often associated with human disease, carried the same resistance genes, and were genetically related to strains from the region. The two undetermined sequence types of S. Kentucky were distantly related and positioned in a separate phylogenetic clade. Two Brunei serovar isolates, one Augustenborg isolate, and one Hartford isolate showed different resistance profiles. This study revealed high fecal contamination levels in tilapia and shrimp sold at two main wet markets in Dhaka. Together with the occurrence of Salmonella spp., including S. Kentucky ST198, a well-known human pathogen, these results stress the need to improve hygienic practices and sanitation standards at markets to improve food safety and protect consumer health.

A multiplexed RT-PCR assay for nanopore whole genome sequencing of Tilapia lake virus (TiLV).

Tilapia lake virus (TiLV) is a highly contagious viral pathogen that affects tilapia, a globally significant and affordable source of fish protein. To prevent the introduction and spread of TiLV and its impact, there is an urgent need for increased surveillance, improved biosecurity measures, and continuous development of effective diagnostic and rapid sequencing methods. In this study, we have developed a multiplexed RT-PCR assay that can amplify all ten complete genomic segments of TiLV from various sources of isolation. The amplicons generated using this approach were immediately subjected to real-time sequencing on the Nanopore system. By using this approach, we have recovered and assembled 10 TiLV genomes from total RNA extracted from naturally TiLV-infected tilapia fish, concentrated tilapia rearing water, and cell culture. Our phylogenetic analysis, consisting of more than 36 TiLV genomes from both newly sequenced and publicly available TiLV genomes, provides new insights into the high genetic diversity of TiLV. This work is an essential steppingstone towards integrating rapid and real-time Nanopore-based amplicon sequencing into routine genomic surveillance of TiLV, as well as future vaccine development.

Genetic characterization and antimicrobial profiling of bacterial isolates collected from Nile tilapia (Oreochromis niloticus) affected by summer mortality syndrome.

In recent years, Egyptian tilapia aquaculture has experienced mortality episodes during the summer months. The causative agents responsible for such mortalities have not been clearly identified. A total of 400 fish specimens were collected from affected tilapia farms within five Egyptian governorates. A total of 344 bacterial isolates were identified from the examined fish specimens. Bacterial isolates were grouped into seven genera based on API 20E results. The most prevalent pathogens were Aeromonas spp. (42%), Vibrio spp. (21%), and Streptococcus agalactiae (14.5%). Other emerging infections like, Plesiomonas shigelloides (10%), Staphyloccocus spp. (8%), Pseudomonas oryzihabitans, and Acinetobacter lwoffii (2.3%) were also detected. Sequence analysis of the 16S ribosomal RNA bacterial gene of some isolates, confirmed the phenotypic identification results. The analysis of antibiotic resistance genes revealed the presence of aac(6')-Ib-cr (35.7%), blaCTX gene (23.8%), qnrS (19%), ampC (16.7%), floR (14.3%), sul1, tetA, and van.C1 (2.4%) genes in some isolates. The antimicrobia resistance gene, qac was reported in 46% of screened isolates. Bacterial strains showed variable virulence genes profiles. Aeromonas spp. harboured (act, gcat, aerA, lip, fla, and ser) genes. All Vibrio spp. possessed the hlyA gene, while cylE, hylB, and lmb genes, were detected in S. agalactiae strains. Our findings point to the possible role of the identified bacterial pathogens in tilapia summer mortality syndrome and highlight the risk of the irresponsible use of antibiotics on antimicrobial resistance in aquaculture.

Concentration and quantification of Tilapia tilapinevirus from water using a simple iron flocculation coupled with probe-based RT-qPCR.

Background: Tilapia tilapinevirus, also known as tilapia lake virus (TiLV), is a significant virus that is responsible for the die-off of farmed tilapia across the globe. The detection and quantification of the virus using environmental RNA (eRNA) from pond water samples represents a potentially non-invasive and routine strategy for monitoring pathogens and early disease forecasting in aquaculture systems. Methods: Here, we report a simple iron flocculation method for concentrating viruses in water, together with a newly-developed hydrolysis probe quantitative RT-qPCR method for the detection and quantification of TiLV. Results: The RT-qPCR method designed to target a conserved region of the TiLV genome segment 9 has a detection limit of 10 viral copies per µL of template. The method had a 100% analytical specificity and sensitivity for TiLV. The optimized iron flocculation method was able to recover 16.11 ± 3.3% of the virus from water samples spiked with viral cultures. Tilapia and water samples were collected for use in the detection and quantification of TiLV disease during outbreaks in an open-caged river farming system and two earthen fish farms. TiLV was detected from both clinically sick and asymptomatic fish. Most importantly, the virus was successfully detected from water samples collected from different locations in the affected farms (i.e., river water samples from affected cages (8.50 × 103 to 2.79 × 105 copies/L) and fish-rearing water samples, sewage, and reservoir (4.29 × 103 to 3.53 × 104 copies/L)). By contrast, TiLV was not detected in fish or water samples collected from two farms that had previously experienced TiLV outbreaks and from one farm that had never experienced a TiLV outbreak. In summary, this study suggests that the eRNA detection system using iron flocculation, coupled with probe based-RT-qPCR, is feasible for use in the concentration and quantification of TiLV from water. This approach may be useful for the non-invasive monitoring of TiLV in tilapia aquaculture systems and may support evidence-based decisions on biosecurity interventions needed.

Systems-thinking approach to identify and assess feasibility of potential interventions to reduce antibiotic use in tilapia farming in Egypt.

Antibiotics are used in aquaculture to maintain the health and welfare of stocks; however, the emergence and selection of antibiotic resistance in bacteria poses threats to humans, animals and the environment. Mitigation of antibiotic resistance relies on understanding the flow of antibiotics, residues, resistant bacteria and resistance genes through interconnecting systems, so that potential solutions can be identified and issues around their implementation evaluated. Participatory systems-thinking can capture the deep complexity of a system while integrating stakeholder perspectives. In this present study, such an approach was applied to Nile tilapia (Oreochromis niloticus) production in the Nile Delta of Egypt, where disease events caused by antibiotic-resistant pathogens have been reported. A system map was co-produced with aquaculture stakeholders at a workshop in May 2018 and used to identify hotspots of antibiotic use, exposure and fate and to describe approaches that would promote fish health and thus reduce antibiotic use. Antibiotics are introduced into the aquaculture system via direct application for example in medicated feed, but residues may also be introduced into the system through agricultural drainage water, which is the primary source of water for most fish farms in Egypt. A follow-up survey of stakeholders assessed the perceived feasibility, advantages and disadvantages of potential interventions. Interventions that respondents felt could be implemented in the short-term to reduce antibiotic usage effectively included: more frequent water exchanges, regular monitoring of culture water quality parameters, improved storage conditions for feed, use of probiotics and greater access to farmer and service providers training programmes. Other potential interventions included greater access to suitable and rapid diagnostics, high quality feeds, improved biosecurity measures and genetically-improved fish, but these solutions were expected to be achieved as long-term goals, with cost being of one of the noted barriers to implementation. Identifying feasible and sustainable interventions that can be taken to reduce antibiotic use, and understanding implementation barriers, are important for addressing antibiotic resistance and ensuring the continued efficacy of antibiotics. This is vital to ensuring the productivity of the tilapia sector in Egypt. The approach taken in the present study provides a means to identify points in the system where the effectiveness of interventions can be evaluated and thus it may be applied to other food production systems to combat the problem of antibiotic resistance.

Transcriptome analysis of liver elucidates key immune-related pathways in Nile tilapia Oreochromis niloticus following infection with tilapia lake virus.

Nile tilapia (Oreochromis niloticus) is one of the most important aquaculture species farmed worldwide. However, the recent emergence of tilapia lake virus (TiLV) disease, also known as syncytial hepatitis of tilapia, has threatened the global tilapia industry. To gain more insight regarding the host response against the disease, the transcriptional profiles of liver in experimentally-infected and control tilapia were compared. Analysis of RNA-Seq data identified 4640 differentially expressed genes (DEGs), which were involved among others in antigen processing and presentation, MAPK, apoptosis, necroptosis, chemokine signaling, interferon, NF-kB, acute phase response and JAK-STAT pathways. Enhanced expression of most of the DEGs in the above pathways suggests an attempt by tilapia to resist TiLV infection. However, upregulation of some of the key genes such as BCL2L1 in apoptosis pathway; NFKBIA in NF-kB pathway; TRFC in acute phase response; and SOCS, EPOR, PI3K and AKT in JAK-STAT pathway and downregulation of the genes, namely MAP3K7 in MAPK pathway; IFIT1 in interferon; and TRIM25 in NF-kB pathway suggested that TiLV was able to subvert the host immune response to successfully establish the infection. The study offers novel insights into the cellular functions that are affected following TiLV infection and will serve as a valuable genomic resource towards our understanding of susceptibility of tilapia to TiLV infection.

Two-year surveillance of tilapia lake virus (TiLV) reveals its wide circulation in tilapia farms and hatcheries from multiple districts of Bangladesh.

Tilapia lake virus (TiLV) is an emerging pathogen in aquaculture, reportedly affecting farmed tilapia in 16 countries across multiple continents. Following an early warning in 2017 that TiLV might be widespread, we executed a surveillance programme on tilapia grow-out farms and hatcheries from 10 districts of Bangladesh in 2017 and 2019. Among farms experiencing unusual mortality, eight out of 11 farms tested positive for TiLV in 2017, and two out of seven tested positive in 2019. Investigation of asymptomatic broodstock collected from 16 tilapia hatcheries revealed that six hatcheries tested positive for TiLV. Representative samples subjected to histopathology confirmed pathognomonic lesions of syncytial hepatitis. We recovered three complete genomes of TiLV from infected fish, one from 2017 and two from 2019. Phylogenetic analyses based on both the concatenated coding sequences of 10 segments and only segment 1 consistently revealed that Bangladeshi TiLV isolates formed a unique cluster within Thai clade, suggesting a close genetic relation. In summary, this study revealed the circulation of TiLV in 10 farms and six hatcheries located in eight districts of Bangladesh. We recommend continuing TiLV-targeted surveillance efforts to identify contaminated sources to minimize the countrywide spread and severity of TiLV infection.

Identifying hotspots for antibiotic resistance emergence and selection, and elucidating pathways to human exposure: Application of a systems-thinking approach to aquaculture systems.

Aquaculture systems are highly complex, dynamic and interconnected systems influenced by environmental, biological, cultural, socio-economic and human behavioural factors. Intensification of aquaculture production is likely to drive indiscriminate use of antibiotics to treat or prevent disease and increase productivity, often to compensate for management and husbandry deficiencies. Surveillance or monitoring of antibiotic usage (ABU) and antibiotic resistance (ABR) is often lacking or absent. Consequently, there are knowledge gaps for the risk of ABR emergence and human exposure to ABR in these systems and the wider environment. The aim of this study was to use a systems-thinking approach to map two aquaculture systems in Vietnam - striped catfish and white-leg shrimp - to identify hotspots for emergence and selection of resistance, and human exposure to antibiotics and antibiotic-resistant bacteria. System mapping was conducted by stakeholders at an interdisciplinary workshop in Hanoi, Vietnam during January 2018, and the maps generated were refined until consensus. Thereafter, literature was reviewed to complement and cross-reference information and to validate the final maps. The maps and component interactions with the environment revealed the grow-out phase, where juveniles are cultured to harvest size, to be a key hotspot for emergence of ABR in both systems due to direct and indirect ABU, exposure to water contaminated with antibiotics and antibiotic-resistant bacteria, and duration of this stage. The pathways for human exposure to antibiotics and ABR were characterised as: occupational (on-farm and at different handling points along the value chain), through consumption (bacterial contamination and residues) and by environmental routes. By using systems thinking and mapping by stakeholders to identify hotspots we demonstrate the applicability of an integrated, interdisciplinary approach to characterising ABU in aquaculture. This work provides a foundation to quantify risks at different points, understand interactions between components, and identify stakeholders who can lead and implement change.

The role of infectious disease impact in informing decision-making for animal health management in aquaculture systems in Bangladesh.

The aquaculture sector in Bangladesh is an important employer and a significant source of foreign exchange. In addition, it contributes significantly to food security due to the role of fish in peoples' diets, the most important source of protein and micronutrients. However, infectious diseases represent an important barrier to sector development due to economic losses and vulnerability of smallholders. The aim of this study was to gain an overview of the impact of infectious diseases in the aquaculture sector, and to assess the usefulness and use of impact studies in decision making for animal health management and biosecurity governance in Bangladesh. A review of scientific and grey literature on infectious disease impact in different aquaculture systems was conducted and their methodologies and findings summarised. Subsequently, interviews with 28 stakeholders from the private and public sector were conducted to enquire about decision-making structures in animal health management. The data were analysed using the framework method to allow the development of themes, by using the information, experiences and opinions inductively obtained from interviewees, deductively through the reviewed literature. Results showed a substantial socio-economic impact of infectious diseases. The numerous stakeholders involved in the decision-making process explained that key barriers to effective aquaculture health management were insufficient resources to investigate and tackle infectious aquatic animal diseases, a dearth of legislation and capacity for disease surveillance, a reliance on reactive response, and a lack of impact and evidence-based approaches for prioritising problem-solving, commonly based on anecdotal evidence. Furthermore, communication among the multiple stakeholders involved was reported to be weak. This complex situation requires a multi-level response, which should span from strengthening the knowledge of farmers and professionals in the field to the improvement of surveillance and diagnostic systems. Improved systems along with evidence on disease impact could inform the prioritisation of diseases and resource allocation for disease control in Bangladesh. Further, this evidence needs to be used to advise decisions to have a true value, for which establishing and strengthening communication pathways and processes is critical to make systematic use of the information and improve animal health management. In the light of future threats to Bangladesh such as climate change, increasing population density and demand for animal source foods, it is crucial to strengthen animal health management systems to reduce livelihoods vulnerability, food insecurity and the likelihood of disease emergence.

An assessment of health management practices and occupational health hazards in tiger shrimp (Penaeus monodon) and freshwater prawn (Macrobrachium rosenbergii) aquaculture in Bangladesh.

Diseases have been recognized as the major obstacle to the shrimp (Penaeus monodon) and prawn (Macrobrachium rosenbergii) aquaculture production in Bangladesh. This study provides an assessment of shrimp and prawn diseases/syndromes, health management practices, and occupational health hazards associated with the handling of chemical and biological products to prevent and treat shrimp and prawn diseases. A survey was conducted using a semi-structured questionnaire with 380 shrimp and prawn farmers in the southwest of Bangladesh during February and June of 2016. The farms were categorized on the basis of the three cropping patterns: shrimp polyculture, prawn polyculture, and shrimp and prawn polyculture. Eight different diseases and/or symptoms were reported by the surveyed farmers. The white spot disease and the broken antenna and rostrum symptom were the most common in shrimp and prawn species, respectively. In total, 35 chemical and biological products (4 antibiotics, 15 disinfectants, 13 pesticides, 2 feed additives and probiotics) were used to treat and/or prevent diseases in the all farm categories. The major constraints for disease management were limited access to disease diagnostic service, inadequate product application information and lack of knowledge on better management practices. Handling chemicals and preparation of medicated feed with bare hands was identified as a potential occupational health hazard. This study suggests improvements in farmers' knowledge and skill in disease diagnostics and health management practices, and appropriate handling of potentially hazardous chemicals.