Stability of Genotube® Swabs for African Swine Fever Virus Detection Using Loop-Mediated Isothermal (LAMP) Laboratory Testing on Samples Stored without Refrigeration.

African swine fever (ASF) is a transboundary viral disease which causes high mortality in pigs. In many low- and middle-income countries and in remote areas where diagnostic surveillance for ASF virus (ASFV) is undertaken, access to trained animal health technicians, sample collection, cold chain storage and transport of samples to suitably equipped laboratories can be limiting when traditional sampling and laboratory tests are used. Previously published studies have demonstrated that alternative sampling matrices such as swabs and filter papers can be tested using PCR without refrigeration for up to a week. This study used Genotube® swabs stored in temperate and tropical climates without refrigeration for four weeks after collection to demonstrate there was no change in test performance and results using loop-mediated isothermal amplification (LAMP) ASFV detection on a series of pig serum samples including serum spiked with a synthetic ASFV positive control, naturally acquired ASFV positive serum from Timor-Leste and negative ASFV serum samples. The use of Genotube® swabs for ASFV detection for surveillance purposes, coupled with testing platforms such as LAMP, can provide an alternative to traditional testing methodology where resources are limited and time from collection to testing of samples is prolonged.

Prevalence of Antimicrobial Resistance in Escherichia coli and Salmonella Species Isolates from Chickens in Live Bird Markets and Boot Swabs from Layer Farms in Timor-Leste.

The rapid emergence of antimicrobial resistance is a global concern, and high levels of resistance have been detected in chicken populations worldwide. The purpose of this study was to determine the prevalence of antimicrobial resistance in Escherichia coli and Salmonella spp. isolated from healthy chickens in Timor-Leste. Through a cross-sectional study, cloacal swabs and boot swabs were collected from 25 live bird markets and two layer farms respectively. E. coli and Salmonella spp. from these samples were tested for susceptibility to six antimicrobials using a disk diffusion test, and a subset was tested for susceptibility to 27 antimicrobials using broth-based microdilution. E. coli and Salmonella spp. isolates showed the highest resistance towards either tetracycline or ampicillin on the disk diffusion test. E. coli from layer farms (odds ratio:5.2; 95%CI 2.0-13.1) and broilers (odds ratio:18.1; 95%CI 5.3-61.2) were more likely to be multi-drug resistant than those from local chickens. Based on the broth-based microdilution test, resistance to antimicrobials in the Timor-Leste Antimicrobial Guidelines for humans were low, except for resistance to ciprofloxacin in Salmonella spp. (47.1%). Colistin resistance in E. coli was 6.6%. Although this study shows that antimicrobial resistance in chickens was generally low in Timor-Leste, there should be ongoing monitoring in commercial chickens as industry growth might be accompanied with increased antimicrobial use.

A pilot study using hospital surveillance and a birth cohort to investigate enteric pathogens and malnutrition in children, Dili, Timor-Leste.

In low-to-middle-income countries (LMICs), enteric pathogens contribute to child malnutrition, affecting nutrient absorption, inducing inflammation, and causing diarrhoea. This is a substantial problem in LMICs due to high disease burden, poor sanitation and nutritional status, and the cyclical nature of pathogen infection and malnutrition. This relationship remains understudied in Timor-Leste. In our pilot study of enteric pathogens and malnutrition in Dili, Timor-Leste (July 2019-October 2020), we recruited 60 infants in a birth cohort from Hospital Nacional Guido Valadares (HNGV) with up to four home visits. We collected faecal samples and details of demographics, anthropometrics, diet and food practices, and animal husbandry. Additionally, we collected faecal samples, diagnostics, and anthropometrics from 160 children admitted to HNGV with a clinical diagnosis of severe diarrhoea or severe acute malnutrition (SAM). We tested faeces using the BioFire® FilmArray® Gastrointestinal Panel. We detected high prevalence of enteric pathogens in 68.8% (95%CI 60.4-76.2%) of infants at home, 88.6% of SAM cases (95%CI 81.7-93.3%) and 93.8% of severe diarrhoea cases (95%CI 67.7-99.7%). Diarrhoeagenic Escherichia coli and Campylobacter spp. were most frequently detected. Pathogen presence did not significantly differ in birth cohort diarrhoeal stool, but hospital data indicated associations between Salmonella and Shigella and diarrhoea. We observed wasting in 18.4% (95%CI 9.2-32.5%) to 30.8% (95%CI 17.5-47.7%) of infants across home visits, 57.9% (95%CI 34.0-78.9%) of severe diarrhoea cases, and 92.5% (95%CI 86.4-96.2%) of SAM cases. We associated bottle feeding with increased odds of pathogen detection when compared with exclusive breastfeeding at home (OR 8.3, 95%CI 1.1-62.7). We detected high prevalence of enteric pathogens and signs of malnutrition in children in Dili. Our pilot is proof of concept for a study to fully explore the risk factors and associations between enteric pathogens and malnutrition in Timor-Leste.

Knowledge, attitudes and practices of government animal health workers on antibiotic use and antibiotic resistance in Timor-Leste.

Introduction: Antibiotic resistance is a global health threat, and there is growing concern on the inappropriate use of antibiotics in the livestock sector especially in low and middle income countries. The purpose of the study was to understand the knowledge, attitudes and practices on antibiotic use and antibiotic resistance of government animal health workers in Timor-Leste. Method: A cross-sectional survey using a census approach was conducted between August 2021 and January 2022 focusing on government animal health workers involved in field work and access to antibiotics. Interviews were face-to-face in the local Tetun language. Descriptive and regression analysis informed by causal diagrams were performed. Result: The study found poor knowledge of antibiotics among participants, with only 8.0% (13/162) able to correctly answer questions on how antibiotics worked. Knowledge of antibiotic resistance was poor as only 29.0% (47/162) of participants had heard of antibiotic resistance and were able to accurately identify that it made antibiotics less effective. Knowledge of antibiotics and knowledge of antibiotic resistance were crudely associated with being a veterinary technician and having university education. Attitude scores were positively influenced by knowledge of antibiotics and antibiotic resistance. Antibiotics were most commonly used in pigs, cattle and buffalo, with oxytetracycline being the most commonly used antibiotics in pigs and chicken. However, most participants reported a lack in supply of this antibiotic (137/162, 78.4%) and other antibiotics. Empiric use of antibiotics in sick animals was common, and some participants used antibiotics for parasitic diseases. Less than a fifth of participants reported ever using human antibiotics, and use of antibiotics for growth promotion was uncommon. Conclusion: There is a need to develop Timor-Leste specific treatment guidelines, strengthen veterinary diagnostic support, improve antibiotic procurement, and develop training programs to address knowledge gaps and poor practices found in this study.

Community engagement strengthens pig disease knowledge and passive surveillance in Timor-Leste.

Smallholder pig production in Timor-Leste is culturally and economically important for most households. However, regular and ongoing disease surveillance and pig husbandry training for farmers are limited. This article describes collaborative social and diagnostic research followed by a pilot community engagement program to improve farmer and technician knowledge, skills, and working relationships. There were three phases: (1) A qualitative study in 2020 to explore the experiences and knowledge of 133 pig farmers, 6 village leaders, and 16 district veterinary technicians on pig diseases and reporting, treatment methods, and access to information or assistance. (2) A pilot community engagement program in 3 villages in 2021 with the diagnostic investigation with samples analyzed from 27 dead pigs, and (3) Evaluation of community engagement and training outcomes. Results of the qualitative study revealed limited reporting of sick or dead pigs by farmers to veterinary technicians due to a lack of trust in the veterinary diagnostic system. Most technicians lacked experience with sampling or post-mortems so diagnostic training was undertaken for the pilot disease investigation. Evaluation results showed improved knowledge, motivation, and confidence of government staff and farmers. The credibility of veterinary technicians improved and gave them more confidence to work with communities. Farmers felt supported because all aspects of pig husbandry were addressed, and they were more willing to report dead or sick pigs. The project indicates that improved passive disease surveillance can be achieved by engaging communities in smallholder pig farming in Timor-Leste. Further research and testing of the approach in other districts and countries is recommended.

Investigating the Socio-Economic and Livelihoods Impacts of African Swine Fever in Timor-Leste: An Application of Spatial Group Model Building.

Small-scale pig farming is highly important to the economic and social status of households in Timor-Leste. The presence of an African Swine Fever (ASF) outbreak in Timor-Leste was confirmed in 2019, a major concern given that around 70% of agricultural households practice pig farming. This research used a virtual spatial group model building process to construct a concept model to better understand the main feedback loops that determine the socio-economic and livelihood impacts of the ASF outbreak. After discussing the interaction of reinforcing and balancing feedback loops in the concept model, potential leverage points for intervention are suggested that could reduce the impacts of ASF within socio-economic spheres. These include building trust between small-scale farmers and veterinary technicians, strengthening government veterinary services, and the provision of credit conditional on biosecurity investments to help restock the industry. This conceptual model serves as a starting point for further research and the future development of a quantitative system dynamics (SD) model which would allow ex-ante scenario-testing of various policy and technical mitigation strategies of ASF outbreaks in Timor-Leste and beyond. Lessons learned from the blended offline/online approach to training and workshop facilitation are also explored in the paper.

Complete Genome Sequence of African Swine Fever Virus Isolated from a Domestic Pig in Timor-Leste, 2019.

Here, we report the complete genome sequence of the African swine fever virus (ASFV) isolate ASFV/Timor-Leste/2019/1, isolated from a domestic pig during the first outbreak of ASF in Timor-Leste in 2019. Using target enrichment short-read Illumina data combined with long-read Oxford Nanopore data, we assembled a full-length genome sequence of 192,237 bp.

Use of Field Based Loop Mediated Isothermal Amplification (LAMP) Technology for a Prevalence Survey and Proof of Freedom Survey for African Swine Fever in Timor-Leste in 2019.

African Swine Fever (ASF) has been spreading in numerous southeast Asian countries since a major incursion in mainland China in 2018. Timor-Leste confirmed an outbreak of ASF in September 2019 which resulted in high mortalities in affected pigs. Pigs in Timor-Leste are the second most common type of livestock kept by villagers and represent a traditionally important source of income and prestige for householders. In order to understand the extent of ASF infected villages in Timor-Leste a prevalence survey was designed and conducted in November-December 2019. Timor-Leste has limited laboratory facilities and access to qPCR diagnostic tests. Therefore, a loop mediated isothermal amplification (LAMP) assay was used to detect ASF positive blood samples collected during the prevalence survey. The LAMP assay was proven to be a robust, highly specific and sensitive laboratory test for ASF suitable for use in the field and where there are limited laboratory facilities. The results of the prevalence survey allowed the extent of the ASF incursion to be delineated and the introduction of a disease response strategy to limit the spread of ASF and assist in the recovery of the pig population in Timor-Leste.

Antimicrobial Use in Animals in Timor-Leste Based on Veterinary Antimicrobial Imports between 2016 and 2019.

Monitoring veterinary antimicrobial use is part of the global strategy to tackle antimicrobial resistance. The purpose of this study was to quantify veterinary antimicrobials imported into Timor-Leste between 2016 and 2019 and describe the antimicrobial import profile of importers. Data were obtained from import applications received by the Ministry of Agriculture and Fisheries (MAF) of Timor-Leste. Import quantities were analysed by antimicrobial class, importance for human medicine, recommended route of administration and type of importer. An average of 57.4 kg (s.d. 31.0 kg) and 0.55 mg/kg (s.d. 0.27 mg/kg) animal biomass of antimicrobials was imported per year. Tetracyclines (35.5%), penicillins (23.7%), and macrolides (15.9%) were the commonly imported antimicrobial classes. Antimicrobials imported for parenteral administration were most common (60.1%). MAF was the largest importer (52.4%). Most of the critically important antimicrobials for human medicine were imported by poultry farms for oral administration and use for growth promotion could not be ruled out. In conclusion, the use of antimicrobials in animals in Timor-Leste is very low, in keeping with its predominantly subsistence agriculture system. Farmer education, development of treatment guidelines, and strengthening of the veterinary service is important for addressing the potential future misuse of antimicrobials especially in the commercial poultry industry.

Knowledge and Practices on Antibiotic Use and Antibiotic Resistance Among Smallholder Pig Farmers in Timor-Leste.

Antibiotic resistance is an emerging global health threat which is linked to the overuse and misuse of antibiotics. This study was conducted to understand the knowledge and practices of smallholder pig farmers on antibiotic use and resistance in Timor-Leste. A cross-sectional study using a structured face-to-face interview was conducted in three municipalities. The interview was piloted and implemented in the local Tetun language. This study found that knowledge of antibiotics was very poor as only 12.7% (95% CI: 6.3-23.9) of farmers reported knowing what antibiotics were, and of these only one was able to correctly explain how an antibiotic worked. None of the farmers knew about antibiotic resistance and were able to explain the concept correctly. After the definition of antibiotic was explained to the farmer, only 3.6% (95% CI: 0.8-14.9) reported that their pigs had ever received antibiotics, and the majority of farmers whose pigs had not received antibiotics reported the lack of access to veterinary services. When used, antibiotics were only used for treatment with no reported use for disease prevention or growth promotion. None of the commonly used antibiotics were critically important antimicrobials. Compliance with withdrawal periods was not routinely followed. There is a need to improve access to government veterinary services for farmers in Timor-Leste, while addressing identified knowledge gaps on antibiotics and promoting prudent use practices. The findings from this study serve as baseline information to inform future interventions.

Field Verification of an African Swine Fever Virus Loop-Mediated Isothermal Amplification (LAMP) Assay During an Outbreak in Timor-Leste.

Recent outbreaks of African swine fever virus (ASFV) have seen the movement of this virus into multiple new regions with devastating impact. Many of these outbreaks are occurring in remote, or resource-limited areas, that do not have access to molecular laboratories. Loop-mediated isothermal amplification (LAMP) is a rapid point of care test that can overcome a range of inhibitors. We outline further development of a real-time ASFV LAMP, including field verification during an outbreak in Timor-Leste. To increase field applicability, the extraction step was removed and an internal amplification control (IAC) was implemented. Assay performance was assessed in six different sample matrices and verified for a range of clinical samples. A LAMP detection limit of 400 copies/rxn was determined based on synthetic positive control spikes. A colourmetric LAMP assay was also assessed on serum samples. Comparison of the LAMP assay to a quantitative polymerase chain reaction (qPCR) was performed on clinical ASFV samples, using both serum and oral/rectal swabs, with a substantial level of agreement observed. The further verification of the ASFV LAMP assay, removal of extraction step, implementation of an IAC and the assessment of a range of sample matrix, further support the use of this assay for rapid in-field detection of ASFV.

Counting the cost: The potential impact of African Swine Fever on smallholders in Timor-Leste.

In September 2019, Timor-Leste, where pigs are kept by more than 70 percent of households, became the eleventh Asian country to report African Swine Fever (ASF). Drawing on our recent, as-yet unpublished research we show that while national pork consumption is low, pigs hold tremendous monetary value for smallholders within the economy of ceremonies. Given the sums of money paid for live pigs, the value of the national pig herd is around USD160 million - more than USD1000 per pig keeping household. Accordingly, pigs serve to buffer families against shocks and pressures, especially for health and education expenses. While not a zoonosis, the potential for ASF to lead to significant, negative impacts for smallholder farmers in Timor-Leste - some of the world's most vulnerable people - cannot be underestimated. We argue that Timor-Leste faces significant challenges in responding to ASF and there is a strong case for international support.

Family poultry: Multiple roles, systems, challenges, and options for sustainable contributions to household nutrition security through a planetary health lens.

Achieving sustainable production of eggs by family poultry production systems that meet both environmental health and welfare standards is a complex endeavour. Humans have been raising different species of poultry for thousands of years across many different agroecological zones. The Food and Agriculture Organization of the United Nations has identified four different family poultry production systems: small extensive, extensive, semi-intensive, and intensive. Each of these systems varies in terms of inputs, outputs, gender dimensions, poultry health and welfare, and environmental impacts. This paper addresses key issues associated with the production of family poultry eggs in support of both improved maternal and child nutrition and sustainable, nutrition-sensitive agricultural practices. It provides an overview of the history of poultry raising; characteristics of the different family poultry production systems; challenges and solutions to poultry production in low- and middle-income countries; poultry husbandry (including breeds, nutrition, and shelter); infectious disease prevention and control in line with national and international animal health regulations; and food safety (microbial pathogens, toxins, and egg storage). To ensure that bird, human, and environmental health can flourish, it is essential for interdisciplinary research and development teams to work in collaboration with communities to ensure the long-term environmental and economic sustainability of family poultry production enterprises that are a good fit with local circumstances.