Retrospective investigation of the 2019 African swine fever epidemic within smallholder pig farms in Oudomxay province, Lao PDR.

The 2019 African swine fever (ASF) outbreak in the Lao People's Democratic Republic (Lao PDR or Laos) represented a major epidemiologic event where a transitioning lower-middle income nation (LMIC) experienced a viral epidemic in a naïve pig population. The diversity of pig management styles creates challenges for local and regional policymakers when formulating recommendations to control an ASF outbreak. The aim of this study were to investigate the management of pigs in villages of Oudomxay province that were affected by ASF in 2019, as a case study in a smallholder pig-rasing system in northern Laos. The frequencies of well known risk factors were measured in the affected villages and the timelines and household level stock losses due to the outbreak were investigated. These findings were compared to data available from a similar outbreak in the southern province of Savannakhet. Disease control implications of these findings are discussed. Mean losses were 3.0-23.3 pigs per household, with a mean lost herd value of USD 349, 95% CI (294-415). These pig losses reflect those estimated in Savannakhet (6.7 pigs per household). However, the financial loss estimated per household was higher, USD 349 versus USD 215, possibly due to higher pig values and a higher input/output management approach in Oudomxay. The investigation revealed the presence of numerous ASF risk factors, such as swill-feeding and free-ranging. In addition, poor biosecurity practices - such as inappropriate garbage disposal and slaughtering - that could contaminate the environment were present. ASF cases occurred across all villages between June and December 2019, with outbreak periods ranging from 22-103 days. These values are consistent with the outbreak in Savannakhet; however, notable differences in management styles were observed. These findings demonstrate the need for more disease control resources from the village to the Governmental level. Villages need support in enacting context appropriate biosecurity measures, whilst the ongoing surveillance and investigation of ASF require investment in logistical and veterinary resources at the Governmental level.

Evaluating serological tests for foot-and-mouth disease while accounting for different serotypes and uncertain vaccination status.

Controlling foot-and-mouth disease (FMD) by vaccination requires adequate population coverage and high vaccine efficacy under field conditions. To assure veterinary services that animals have acquired sufficient immunity, strategic post-vaccination surveys can be conducted to monitor the coverage and performance of the vaccine. Correct interpretation of these serological data and an ability to derive exact prevalence estimates of antibody responses requires an awareness of the performance of serological tests. Here, we used Bayesian latent class analysis to evaluate the diagnostic sensitivity and specificity of four tests. A non-structural protein (NSP) ELISA determines vaccine independent antibodies from environmental exposure to FMD virus (FMDV), and three assays measuring total antibodies derived from vaccine antigen or environmental exposure to two serotypes (A, O): the virus neutralisation test (VNT), a solid phase competitive ELISA (SPCE), and a liquid phase blocking ELISA (LPBE). Sera (n = 461) were collected by a strategic post-vaccination monitoring survey in two provinces of Southern Lao People's Democratic Republic (PDR) after a vaccination campaign in early 2017. Not all samples were tested by every assay and each serotype: VNT tested for serotype A and O, whereas SPCE and LPBE tested for serotype O, and only NSP-negative samples were tested by VNT, with 90 of them not tested (missing by study design). These data challenges required informed priors (based on expert opinion) for mitigating possible lack of model identifiability. The vaccination status of each animal, its environmental exposure to FMDV, and the indicator of successful vaccination were treated as latent (unobserved) variables. Posterior median for sensitivity and specificity of all tests were in the range of 92-99 %, except for the sensitivity of NSP (∼66%) and the specificity of LPBE (∼71 %). There was strong evidence that SPCE outperformed LPBE. In addition, the proportion of animals recorded as having been vaccinated that showed a serological immune response was estimated to be in the range of 67-86 %. The Bayesian latent class modelling framework can easily and appropriately impute missing data. It is important to use field study data as diagnostic tests are likely to perform differently on field survey samples compared to samples obtained under controlled conditions.

Utilising abattoir sero-surveillance for high-impact and zoonotic pig diseases in Lao PDR.

National disease surveillance systems are essential to a healthy pig industry but can be costly and logistically complex. In 2019, Lao People's Democratic Republic (Lao PDR) piloted an abattoir disease surveillance system to assess for the presence of high impact pig diseases (HIPDs) using serological methods. The Lao Department of Livestock and Fisheries (DLF) identified Classical Swine Fever (CSF), Porcine Respiratory and Reproductive Syndrome (PRRS) and Brucella suis as HIPDs of interest for sero-surveillance purposes. Porcine serum samples (n = 597) were collected from six Lao abattoirs in March to December of 2019. Serological enzyme-linked immunosorbent assay (ELISA) methods were chosen for their high-throughput and relatively low-costs. The true seroprevalence for CSF and PRRS seropositivity were 68.7%, 95% CI (64.8-72.3) and 39.5%, 95% CI (35.7-43.5), respectively. The results demonstrated no evidence of Brucella spp. seroconversion. Lao breed pigs were less likely to be CSF seropositive (P < 0.05), whilst pigs slaughtered at <1 year of age were less likely to be PRRS seropositive (P < 0.01). The testing methods could not differentiate between seropositivity gained from vaccine or natural infection, and investigators were unable to obtain the vaccine status of the slaughtered pigs from the abattoirs. These results demonstrate that adequate sample sizes are possible from abattoir sero-surveillance and lifetime health traceability is necessary to understand HIPDs in Lao PDR.

Abattoir-Based Serological Surveillance and Spatial Risk Analysis of Foot-and-Mouth Disease, Brucellosis, and Q Fever in Lao PDR Large Ruminants.

A national animal disease surveillance network initiated by the Lao PDR government is adopted and reinforced by a joint research project between the National Animal Health Laboratory (NAHL), the Department of Livestock and Fisheries (DLF), and the Mahidol Oxford Tropical Medicine Research Unit (MORU). The network is strengthened by staff training and practical exercises and is utilised to provide zoonotic or high-impact disease information on a national scale. Between January and December 2020, large ruminant samples are collected monthly from 18 abattoirs, one in each province, by provincial and district agriculture and forestry officers. The surveillance network collected a total of 4247 serum samples (1316 buffaloes and 2931 cattle) over this period. Samples are tested for antibodies against Brucella spp., Coxiella burnetii (Q fever) and Foot-and-Mouth Disease Non-Structural Protein (FMD NSP) using commercial ELISA kits and the Rose Bengal test. Seroprevalences of Q fever and brucellosis in large ruminants are low at 1.7% (95% CI: 1.3, 2.1) and 0.7% (95% CI: 0.5, 1.0) respectively, while for FMD NSP it is 50.5% (95% CI: 49.0, 52.0). Univariate analyses show differences in seroprevalences of Q fever between destination (abattoir) province (p-value = 0.005), province of origin (p-value = 0.005), animal type (buffalo or cattle) (p-value = 0.0008), and collection month (p-value = 3.4 × 10−6). Similar to Q fever, seroprevalences of brucellosis were significantly different for destination province (p-value < 0.00001), province of origin (p-value < 0.00001), animal type (p-value = 9.9 × 10−5) and collection month (p-value < 0.00001), plus body condition score (p-value = 0.003), and age (p-value = 0.007). Additionally, risk factors of the FMD NSP dataset include the destination province (p-value < 0.00001), province of origin (p-value < 0.00001), sex (p-value = 7.97 × 10−8), age (p-value = 0.009), collection date (p-value < 0.00001), and collection month (p-value < 0.00001). Spatial analyses revealed that there is no spatial correlation of FMD NSP seropositive animals. High-risk areas for Q fever and brucellosis are identified by spatial analyses. Further investigation of the higher risk areas would provide a better epidemiological understanding of both diseases in Lao PDR. In conclusion, the abattoir serological survey provides useful information about disease exposure and potential risk factors. The network is a good base for field and laboratory staff training in practical technical skills. However, the sustainability of such a surveillance activity is relatively low without an external source of funding, given the operational costs and insufficient government budget. The cost-effectiveness of the abattoir survey could be increased by targeting hotspot areas, reducing fixed costs, and extending the focus to cover more diseases.

Socioeconomic impacts of clinical foot-and-mouth disease and a risk-based partial vaccination campaign for smallholders in Lao People's Democratic Republic.

Foot-and-mouth disease (FMD) is endemic in Lao People's Democratic Republic (PDR). A risk-based partial vaccination campaign was implemented in Champasak, Savannakhet and Xiengkhouang Provinces in Lao PDR in 2016-2020, which had limited effects on reducing the circulation of FMD virus. The objectives of this study were to measure the socioeconomic effects of (i) a clinical FMD occurrence and (ii) the vaccination campaign on livestock production performance of smallholders in Lao PDR in 2016-2020. Two questionnaire surveys were conducted at the beginning (2016) and the end (2020) of the campaign, involving 1609 smallholders in 160 villages comparing areas with and without FMD control. Mixed-effect multivariable regression models were fitted to measure the association between livestock production parameters at the household level (rates of birth, sale, purchase, and death, sale price, costs, gross gains and net gains) and (i) a clinical FMD occurrence and (ii) the vaccination campaign, while adjusting for village, herd size, province, vaccination status and FMD knowledge. We found no significant difference in annual household-level costs, gains and net gains by due absence of FMD. However, smallholders with FMD had a 52% higher cattle death rate, a 43% lower goat sale rate and a 78% lower pig sale price than those without FMD. Smallholders with FMD also had a lower purchase rate of livestock (cattle -15% and pigs -93%) compared with those without FMD. There was a general improvement in livestock production in target villages over the 5 years of the vaccination campaign, whereas there was an overall decline in non-target villages, resulting in USD 218 higher net gains in target villages in 2020 compared with non-target villages, possibly due to reduced severity of disease and improved awareness on FMD and general livestock health. The findings of this study will help improve strategies for sustainable and efficient FMD control in Lao PDR, while the challenges and importance of gaining participation by smallholders are highlighted.

Changes in farmer animal health and biosecurity knowledge, attitudes and practices: Insights from Cambodia and Laos.

Livestock agriculture in Cambodia and Laos is severely affected by endemic and exotic transboundary animal diseases, impacting household livelihoods and food and nutritional security. Collaborative animal health and biosecurity projects were conducted in each country between 2015 and 2019 aimed at increasing smallholder livestock production through mainly knowledge-based interventions in large ruminant disease prevention, nutrition, reproduction and marketing access. This study's objectives were to identify baseline animal health and biosecurity knowledge, attitudes and practices (KAP) of farmers, and temporal changes in key attitudes and practices associated with improved knowledge. Data for each country were analysed separately, and a formal between-country comparison was avoided due to major implementation and management differences. Binomial logistic regression without random terms compared the overall knowledge scores between 2015 and 2018 in each country, and binomial logistic regression with random terms was used to investigate possible associations between animal health and biosecurity knowledge scores and attitudes and practices. In Cambodia, the average animal health and biosecurity knowledge score increased from 4.8 (±2.2)/17 in 2015 to 13.0 (±2.8)/17 in 2018 (p < .0001). In Laos, the average scores improved from 7.2 (±2.5)/12 in 2015 to 7.9 (±2.9)/12 in 2018 (p = .0003). Clinical signs of foot-and-mouth disease were accurately reported by 100% of Cambodian and 88% of Lao project farmers, and overall, attitudes and practices improved in both countries over the study period. The belief that vaccination can improve livestock value was associated with increasing knowledge in both countries (Cambodia: OR 1.45, 95% CI 1.24-1.68, p < .0001; Laos: OR 1.43, 95% CI 1.16-1.75, p = .0006) as was the use of the services of village para-veterinarians if they had better training (Cambodia: OR 1.76, 95% CI 1.20-2.58, p = .004; Laos: OR 2.02, 95% CI 1.19-3.41, p = .009). However, the source of livestock information (District veterinary officials) was the only practice associated with knowledge in both Cambodia and Laos. The influence of resource scarcity on farmer decision making and knowledge measures was considered of particular relevance to the findings.

Impact of risk-based partial vaccination on clinical incidence and seroprevalence of foot and mouth disease in Lao PDR.

Foot and mouth disease (FMD) is endemic in the Lao People's Democratic Republic (PDR) and it causes economic loss to smallholder husbandry systems. An intervention programme based on a risk-based partial vaccination strategy was implemented in three provinces of Lao PDR (Champasak, Savannakhet and Xiangkhouang) to immunise domestic cattle and buffalo during 2016-2020. Two cross-sectional surveys were conducted in 2016/17 and 2020 to evaluate the impact of the vaccination programme on the prevalence of FMD virus exposure and clinical incidence of the disease. A total of 212 villages were visited during the two surveys, collecting 1609 household-level questionnaire results and 5931 blood samples of domestic cattle and buffalo. Blood samples were tested for the presence of antibodies to the non-structural proteins of FMD virus, and seroprevalence of 42.5 and 47.5% in 2016/17 and 2020, respectively were found. Multivariable regression analysis indicated that the efficacy of the FMD vaccination programme for reducing FMD virus circulation varied by province. In general, the incidence of clinical FMD increased toward the end of the 5-year intervention period, coinciding with a reduction of vaccine coverage in the last 2 years of the period. The findings suggest that the risk-based vaccination strategy achieved a marginally protective effect against the circulation of FMD virus with the possible limiting factors being operational constraints of public veterinary services, lack of farmers' compliance and unsustainable funding. We conclude that consistent resource availability and higher vaccination coverage is required to successfully control FMD with a risk-based vaccination strategy in Lao PDR.

Foot-and-mouth disease seroprevalence and reporting behaviours in nine northern provinces in Lao PDR: The current situation and challenges for control.

Foot-and-mouth disease (FMD), caused by the FMD virus (FMDV), is one of the most important of global livestock diseases, impacting even-toed ungulates and distributed mostly in less developed countries that are home to 75% of the human population. A progressive control pathway for FMD (PCP-FMD) was developed to assist countries to better manage FMD risks and progress towards control and eradication. This requires evidence of current FMD seroprevalence to enable the informed risk assessment and the disease control planning required to progress along the initial stages of the PCP-FMD. Despite Laos being an active member of the South East Asia and China FMD campaign, these data have been challenging to obtain. To address this gap, a cross-sectional seroprevalence study in nine northern Lao provinces was conducted in early 2019. The study sampled 602 large ruminants and 19 goats from 30 villages. Overall, the large ruminant seroprevalence was 43.7% (95% CI 39.7-47.8), and 90% of sampled villages had at least one seropositive animal. Large ruminants suspected of having had clinical FMD in their lifetime were found to have an increased risk of being seropositive (odds ratio 1.96, 95% CI 1.06-3.65, p = .03). An examination of current reporting behaviours of farmers, village veterinary workers and district and provincial officials found that individuals were using their discretion as to whether disease events required reporting along the chain of command. This, plus the lack of a computerized reporting system, contributed to a loss of historical data, potentially compromising progression on the PCP-FMD. Laos requires ongoing support from donor agencies to improve the current animal disease surveillance system and implement effective FMD control strategies that can enable progress on the PCP-FMD.

Antimicrobial use and resistance data in human and animal sectors in the Lao PDR: evidence to inform policy.

OBJECTIVES: To review the scientific evidence base on antimicrobial use (AMU) and antimicrobial resistance (AMR) in human and animal sectors in the Lao PDR (Laos). METHODS: We reviewed all publications from July 1994 (the first article describing AMR in Laos) to December 2020. Electronic searches were conducted using Google Scholar and PubMed with specific terms relating to AMR and AMU in Lao, French and English languages. FINDINGS: We screened 1,357 peer-reviewed and grey reports by title and abstract and then full articles/reports. Of 80 included, 66 (83%) related to human health, nine (11%) to animal health, four (5%) to both animal and human health and one (1%) to the environment. Sixty-two (78%) were on AMR and 18 (22%) on AMU. Extended spectrum beta lactamase-producing Escherichia coli was the greatest concern identified; the proportion of isolates increased fivefold from 2004 to 2016 (2/28 (7%) to 27/78 (35%)) from blood cultures submitted to the Microbiology Laboratory, Mahosot Hospital, Vientiane. Carbapenem resistant Escherichia coli was first identified in 2015. Methicillin-resistant Staphylococcus aureus (MRSA) was uncommon, with 15 cases of MRSA from blood cultures between its first identification in 2017 and December 2020. AMR patterns of global antimicrobial resistance surveillance system (GLASS) target pathogens from livestock were less well documented. There were few data on AMU in human health and none on AMU in livestock. The first hospital AMU survey in Laos showed that 70% (1,386/1,981) of in-patients in five hospitals from 2017 to 2018 received antimicrobial(s). Antibiotic self-medication was common. CONCLUSION: AMR in Laos is occurring at relatively low proportions for some GLASS pathogens, giving the country a window of opportunity to act quickly to implement strategies to protect the population from a worsening situation. Urgent interventions to roll out new guidelines with enhanced one-health antibiotic stewardship, reduce antibiotic use without prescriptions, enhance surveillance and improve understanding of AMU and AMR are needed.

Detection of Foot-and-Mouth Disease Virus in the Absence of Clinical Disease in Cattle and Buffalo in South East Asia.

Foot-and-mouth disease virus (FMDV) is widespread throughout much of the world, including parts of South East Asia. Surveillance is often limited in endemic areas, relying predominantly on passive outbreak reporting. As part of the World Organisation for Animal Health (OIE)'s South East Asia and China Foot-and-Mouth Disease Project (SEACFMD), field sampling was performed to help understand evidence of widespread virus exposure observed in previous studies. Serum and dry mucosal swabs were collected to evaluate the presence of FMDV RNA on the nasal, oral, and dorsal nasopharyngeal mucosal surfaces of 262 healthy cattle (n = 84 in Laos; n = 125 in Myanmar) and buffalo (n = 48 in Laos; n = 5 in Myanmar) immediately following slaughter in three slaughterhouses. Swabs and serum were tested by the OIE/FAO World Reference Laboratory for foot-and-mouth disease (WRLFMD) using pan-serotypic real-time reverse transcription-PCR (rRT-PCR) and serum was evaluated using the FMD PrioCHECK non-structural protein (NSP) ELISA. In total, 7.3% of animals had detectable FMDV RNA in one or more of the three sites including 5.3% of nasopharyngeal swabs, 2.3% of oral swabs, and 1.5% of nasal swabs. No FMDV RNA was detected in serum. Overall, 37.8% of animals were positive for NSP antibodies, indicating likely past natural exposure to FMDV. Results were comparable for Laos and Myanmar, and for both cattle and buffalo, and were not significantly different between age groups. Detectable FMDV RNA present on the oral and nasal mucosa of clinically-healthy large ruminants in Laos and Myanmar demonstrates the importance of sampling asymptomatic animals as part of surveillance, and may indicate that subclinical infection plays a role in the epidemiology of FMD in these countries.

The impact of African swine fever virus on smallholder village pig production: An outbreak investigation in Lao PDR.

African swine fever virus (ASFV) causes a deadly disease of pigs which spread through southeast Asia in 2019. We investigated one of the first outbreaks of ASFV in Lao People's Democratic Republic amongst smallholder villages of Thapangtong District, Savannakhet Province. In this study, two ASFV affected villages were compared to two unaffected villages. Evidence of ASFV-like clinical signs appeared in pig herds as early as May 2019, with median epidemic days on 1 and 18 June in the two villages, respectively. Using participatory epidemiology mapping techniques, we found statistically significant spatial clustering in both outbreaks (p < 0.001). Villagers reported known risk factors for ASFV transmission - such as free-ranging management systems and wild boar access - in all four villages. The villagers reported increased pig trader activity from Vietnam before the outbreaks; however, the survey did not determine a single outbreak source. The outbreak caused substantial household financial losses with an average of nine pigs lost to the disease, and Monte Carlo analysis estimated this to be USD 215 per household. ASFV poses a significant threat to food and financial security in smallholder communities such as Thapangtong, where 40.6% of the district's population are affected by poverty. This study shows ASFV management in the region will require increased local government resources, knowledge of informal trader activity and wild boar monitoring alongside education and support to address intra-village risk factors such as free-ranging, incorrect waste disposal and swill feeding.

Seroepidemiology of Foot and Mouth Disease using passive surveillance techniques in selected provinces of Lao PDR.

Foot and Mouth Disease (FMD) is a high-impact, contagious transboundary animal disease that is endemic in Southeast Asia. Abattoir samples were routinely collected in six selected provinces between March and December 2019. A total of 1280 samples of abattoir animals were tested for FMD Non-Structural Protein (NSP) antibodies to indicate natural infections. Overall, 22.8% were seropositive for FMD NSP antibodies while seroprevalence of cattle (n = 469), buffalo (n = 214), and pigs (n = 597) were 44.6%, 35.0%, and 1.3%, respectively. The highest seroprevalence destination province was Xiengkhouang (35.3% of 272 samples), followed by Savannakhet (27.0% of 244 samples). Risk factors for evidence of natural infection identified by a multivariate logistic regression model included age groups (p-value = 0.02) and origin provinces (p-value = 2.8 × 10-5) of the animals. There were significant differences of FMD NSP seroprevalence between age groups and origin provinces of the animals. The odds ratio of a seropositive result in the less than 1 year old group was 2.5 (95% CI; 1.4, 4.4) when compared to the 3-4 years old group, while the odds ratios for animals that originated from Khammouane and Xiengkhouang provinces were 4.5 (95% CI; 1.1, 18.7) and 2.4 (95% CI; 1.4, 4.1), respectively, when compared to Champasak province. Serotype-specific antibody ELISA for 44 NSP antibody-positive samples revealed evidence of FMD serotypes O and A virus circulation in some provinces. Despite the passive abattoir survey providing useful information on FMD virus previous exposure and geographic locations of the animals, timely information on FMD virus circulation and distribution is also crucial to an effective control program. Alternative approaches to increase the cost-effectiveness of the surveillance network are also discussed.

The Development of an Abattoir-Based Surveillance System in Lao PDR for the Detection of Zoonoses in Large Ruminants: Q Fever and Brucellosis Seroepidemiology as a Pilot Study.

Although animal health surveillance programmes are useful for gaining information to help improve global health and food security, these programmes can be challenging to establish in developing economies with a low-resource base. This study focused on establishing a national surveillance system initiated by the Lao PDR government using a passive surveillance system of abattoir samples as a pilot model, and to gain information on contagious zoonoses, particularly Q fever and brucellosis, in the large ruminant population. A total of 683 cattle and buffalo samples were collected from six selected provinces of Lao PDR between March-December 2019. Out of 271 samples tested, six samples (2.2%, 95% confidence interval (CI) of 1.0, 4.8) were positive in the Q fever antibody ELISA test. Only one sample (out of 683; 0.2%, 95% CI 0.0, 0.8) tested positive to the Brucella antibody ELISA test. Seroprevalence of these important zoonoses in Lao PDR were relatively low in cattle and buffaloes; however, extensive animal movement within the country was identified which could increase risks of spreading transboundary diseases. The study highlights the importance of ongoing animal health surveillance and the need to find cost-effective approaches for its long-term sustainability.

Managing Welfare and Antimicrobial-Resistance Issues in Treating Foot-and-Mouth Disease Lesions: A New Therapeutic Approach.

INTRODUCTION: Foot-and-mouth disease (FMD) causes negative impacts on global food security, the livestock trade, national economies, and farming-family livelihoods, particularly in resource-poor developing countries with inadequate biosecurity and low levels of vaccination from inadequate veterinary services. As smallholder farmers have limited understanding of disease-risk management, their focus in FMD outbreaks is on accessing clinically effective therapies. However, most are provided inappropriate traditional treatments and/or topical or parenteral antibiotics, often delivered by paraveterinarians inadequately trained in antimicrobial custodianship. This results in negative financial impacts on livelihoods plus risks of food safety and development of antimicrobial resistance. We report the use of a novel pain-relief therapy for FMD. METHODS: Clinical examinations in an outbreak of suspected FMD in April 2019 in Muang Khay village in Luang Prabang province, Laos confirmed signs and lesions of severe, subacute, ulcerative glossitis and interdigital dermatitis, typical of FMD. All affected buffalo (n=99) and cattle (n=37) presented for treatment in a population of 238 large ruminants, from 15 of 136 households, were administered a topical anesthetic pain-relief product (PRP) wound gel by spray-on (10-30 mL per animal) formulation developed for aversive husbandry procedures (Tri-Solfen, Animal Ethics, Australia). RESULTS: Treatment with PRP resulted in immediate improvement in demeanor and locomotion, and no adverse events were observed. On follow-up interview, all owners confirmed that their animals were eating within 2 days and lesions had healed within 5 days. Having experienced the positive clinical impacts of PRP on affected animals, these and surrounding farmers were keen to purchase the PRP for future use. The veterinary authorities rapidly registered the PRP for FMD therapy in Laos due to the observed efficacy. DISCUSSION: These findings suggest a potential paradigm shift from treating FMD with expensive antimicrobials, which risks antimicrobial resistance, to a new, less expensive therapeutic approach that reduces animal suffering and may motivate farmers to report disease to access treatment. Use of the PRP is suggested as an innovation that may improve future FMD management, particularly in developing countries.

Serological Evidence of Foot-and-Mouth Disease Infection in Goats in Lao PDR.

Foot and Mouth Disease (FMD) causes significant economic loss in Lao PDR (Laos) and perpetuates the cycle of smallholder poverty mainly through large ruminant productivity losses, increased costs of production and potential limitations to market access for trade in livestock and their products. Goats are emerging as an important livestock species in Laos, and there is an increasing trend in the number of households with goats, often farmed alongside cattle and buffalo. Although an FMD susceptible species, very little is known about the role of goats in the epidemiology of the disease in Laos. A cross-sectional seroprevalence study was conducted by detecting antibodies to the non-structural proteins (NSP), an indication of a previous infection, and serotype-specific structural proteins (SP) that could be due to vaccination or infection. The study commenced in late 2017 and sera were collected from 591 goats in 26 villages of northern, central and southern Laos. For a subset of sera samples, paired oral swab samples were also collected by a simple random sampling method to detect the prevalence of FMD virus infection at the time of collection. The NSP seroprevalence in the provinces of Borkeo and Xayabouli in the north was 42 and 8%, respectively and in Khammoune in the center, it was 20%. In the other five provinces, Luang Namtha and Luang Prabang (northern Laos), Xieng Khouang and Savannaket (central Laos), and Champasak (southern Laos), the seroprevalence was close to zero. The multivariable analysis indicated that age (p < 0.001) was positively associated with animal-level seropositivity and males were less likely to be seropositive than females (OR: 0.29; 95%CI: 0.10-0.83; p = 0.017). Continued sero-surveillance for FMD in goats is recommended to improve our understanding of their role in the epidemiology of FMD in the region and to extend support to FMD control decisions, particularly regarding vaccination.

Evaluation of the diagnostic accuracy of an affordable rapid diagnostic test for African Swine Fever antigen detection in Lao People's Democratic Republic.

African Swine Fever (ASF) is a transboundary animal disease of pigs and wild suids that appeared in Lao People's Democratic Republic (Lao PDR) in mid-2019, having spread across China and Vietnam in the months prior. Despite the scale of the Asian ASF pandemic and the availability of pen-side rapid diagnostic tests (RDT) on the market, few locally produced and easily available ASF RDTs have been evaluated for diagnostic accuracy. In this study, an ASF antigen detection RDT from Shenzhen Lvshiyuan Biotechnology Co. Ltd was evaluated using clinical field samples submitted to the National Animal Health Laboratory (NAHL) from ASF suspect cases between June and December 2019 in Lao PDR. Positive (n = 57) and negative (n = 50) samples of whole blood, serum and haemolysed serum were assessed by RDT and PCR, with the latter used as the gold standard reference comparator. Overall the RDT had a diagnostic sensitivity (DSe) of 65 %, 95 % CI [51-77] and diagnostic specificity (DSp) of 76 %, 95 % CI [62-87]. The RDT demonstrated improved performance on samples with lower PCR cycle threshold (ct) values with each additional cycle reducing the odds of the RDT returning a positive by 17 % relative to the previous cycle, 95 % CI [8 %-28 %] (P < 0.01). While this test shows promise for field application, complete validation of diagnostic accuracy requires a larger sample size.

Which species is in the faeces at a time of global livestock movements: single nucleotide polymorphism genotyping assays for the differentiation of Fasciola spp.

Fasciolosis, caused by Fasciola hepatica and Fasciola gigantica, is a globally distributed zoonotic disease of livestock. While F. hepatica and F. gigantica have temperate and tropical distributions, respectively, parasite sympatry occurs in parts of Asia and Africa. A growing protein demand has the potential to facilitate the translocation of parasites from endemic to non-endemic areas, via associated international livestock movements. Such is the case in Southeast Asia, where livestock trade from F. hepatica-endemic countries into China and Vietnam may account for detection of F. hepatica hybrid/introgressed forms. Of particular importance is Lao People's Democratic Republic, which acts as a major livestock thoroughfare for the region. Our ability to understand the impacts of livestock-associated Fasciola spp. movements on local animal and human health is hindered by a lack of ante-mortem diagnostic tools allowing species differentiation. Molecular tools have been developed for Fasciola spp. differentiation, however those rely on access to pure DNA from adult specimens, limiting their application to post-mortem use. Our aim was to detect and differentiate F. hepatica from the endemic F. gigantica in local smallholder cattle in a region of Southeast Asia with frequent livestock trafficking. To do this we designed and validated ante-mortem molecular assays for Fasciola spp. differentiation targeting single-nucleotide polymorphisms (SNPs) within ITS1 and lsrRNA. We then deployed these SNP genotyping assays to diagnose Fasciola spp. infection in 153 local cattle from 27 villages in Northern Laos. We demonstrate the presence of F. hepatica DNA, confirmed by qualitative Sanger and quantitative Illumina amplicon sequencing of ITS1 and lsrRNA, and highlight the shortfalls of Sanger sequencing for Fasciola spp. identification due to the preferential amplification of F. gigantica nucleotides in mixed DNA samples. The outlined protocol enables rapid surveillance of faecal samples for the presence of Fasciola species eggs, their co-infection and/or infection with F. hepatica/F. gigantica hybrids.

Risk factors for Neospora caninum, bovine viral diarrhoea virus, and Leptospira interrogans serovar Hardjo infection in smallholder cattle and buffalo in Lao PDR.

Smallholder large ruminant production in Lao People's Democratic Republic (Laos) is characterised by low reproductive efficiency. To determine if common abortifacient bovid infectious diseases are involved, a serological investigation was conducted. Sera was collected from stored and fresh cattle (n = 390) and buffalo (n = 130) samples from 2016-18 from, and then examined for associations in a retrospective risk factor study of 71 herds. The sera were assayed for antibodies to Neospora caninum, bovine viral diarrhoea virus (BVDV), Leptospira interrogans serovar Hardjo and Brucella abortus using commercially available enzyme-linked immunosorbent assay kits. These pathogens were detected in buffalo samples at 78.5% (95% CI 71.4-85.6), 0%, 2.3% (95% CI 0-4.9) and 0%, respectively, and in cattle at 4.4% (95% CI 2.4-6.4), 7.7% (95% CI 3.1-12.3), 12.8% (95% CI 9.5-16.1) and 0.26% (95% CI 0-0.8), respectively. Exposure of buffalo to N. caninum was positively associated with buffalo age, with a predicted seropositivity at birth of 52.8%, increasing to 97.2% by 12 years of age (p = 0.037). Exposure of cattle to L. interrogans serovar Hardjo was more prevalent in females compared to males, was associated with higher titres of BVDV, and was more prevalent in the wet season compared to the dry season. Exposure of cattle to BVDV was more prevalent in males compared to females, the wet and dry seasons were comparable, and was associated with rising antibody titres against N. caninum and L. interrogans serovar Hardjo. The risk factor survey identified that the probability of herds being N. caninum positive increased with farmer age, if farmers believed there were rodents on farm, and if farmers weren't aware that canids or rodents could contaminate bovid feed on their farm. The probability of a herd being positive to L. interrogans serovar Hardjo increased on farms where multiple cows shared the same bull, where farmers had lower husbandry knowledge, and on farms that used water troughs. The probability of a herd being BVDV seropositive increased with increasing herd size and increasing titres to N. caninum. The benchmarking of bovid exposure to emerging abortifacient pathogens and identification of their risk factors potentially informs disease prevention strategies, supporting efforts to establish a biosecure beef supply for enhanced smallholder livestock productivity, public health and food security in Laos and surrounding countries.

Financial impact of an outbreak of clinically diagnosed blackleg - a case study from Lao PDR.

Blackleg (BL) is an acute to peracute highly fatal infectious disease of mainly large ruminants characterised by lesions of myonecrosis caused by Clostridium chauvoei, usually presenting as a sudden onset of sporadic mortalities. In Southeast Asia, 'BL' is considered a cause of occasional outbreaks of a subacute febrile illness, although there are few published reports available. Investigation of a major outbreak of clinically diagnosed BL occurring in large ruminants (cattle and buffalo) in three neighbouring villages in central Laos in mid-2017, was conducted to determine the financial impacts of BL on smallholder livelihoods. Owners of BL-infected large ruminants in the three affected villages were interviewed (n = 27) and financial losses including losses due to mortality, morbidity and costs of treatments, were determined. The reports of clinical signs of subcutaneous swelling with palpable crepitus in febrile animals were considered consistent with a diagnosis of BL. The outbreak occurred in 47 of a total 449 households with large ruminants across the three villages, affecting 147 of a total population of 3505 'at risk' large ruminants with 71 deaths reported. At the household level, the mean morbidity and mortality rates were 5 ± 4(95% CI: 3-6) and 3 ± 2(95% CI: 1-4) heads per household, respectively. The estimated financial losses due to BL per affected household was USD 822 ± 692(95% CI: 518-1125), being 122% of their annual household income from large ruminant sales. The comparison between the estimated losses due to BL per village and cost of annual BL vaccination programmes, indicated a potential economic benefit of USD3.09 and USD12.37 for every dollar invested in the vaccination programme, if BL outbreaks occur every 20 and 5 years, respectively. This study indicates that clinically diagnosed BL can cause significant losses to smallholder households, and requires Lao animal health authorities to consider vaccination interventions to prevent losses from re-emergence of BL in the known endemically affected areas.

Risk factors for emergence of exotic foot-and-mouth disease O/ME-SA/Ind-2001d on smallholder farms in the Greater Mekong Subregion.

Foot-and-mouth disease (FMD) is a significant endemic transboundary animal disease in Lao People's Democratic Republic (Lao PDR) and throughout the Greater Mekong Subregion (GMS). The disease has been shown to perpetuate the cycle of smallholder poverty through reduced animal production, plus limitations on market access for trading in livestock and their products. Despite significant national and multilateral efforts to control FMD over the past two decades, endemic FMD viruses (FMDVs) continue to circulate in Lao PDR. Further, the threat from new and emerging FMDVs is increasing as transboundary movements in the region intensify in response to increasing regional demand for meat. Although the economic impacts of FMD on smallholder farmers in Lao PDR are significant, studies investigating household-level risk factors for FMD are lacking. Following an outbreak of a novel FMDV (O/ME-SA/Ind2001d) in Lao PDR in 2015, a questionnaire and serological study were conducted in Naxaythong District to identify household-level risk factors associated with this outbreak, as well as endemic circulating viruses in the outbreak area. Data were analysed using a multivariable generalised estimating equation (GEE) model with a logit link function and associations were calculated as odds ratios (OR) with 95% confidence intervals (CI95%). After adjusting for other variables, the practice of quarantining new livestock for a minimum of two weeks prior to introduction to a herd was found to be a significant protective factor during the 2015 outbreak (OR 0.225, CI95% [0.06, 0.88], p-value 0.003). In addition, households owning one or more animals with titres to the non-structural proteins of FMDV, indicating prior infection, had 5.5 times the odds (CI95% [6.16, 49.11], p-value <0.001) of sharing communal grazing land with neighbouring villages. These findings indicate that implementing basic on-farm biosecurity and improved husbandry measures to minimise FMDV circulation at the household level are important and reinforce the need to enhance the education of smallholder farmers in infectious disease control.