Epidemiology study and risk assessments of highly pathogenic avian influenza H5N1 in free flying birds in Thailand

The highly pathogenic avian influenza virus H5N1 was the cause of a pandemic of avian influenza in poultry throughout many parts of the world. The role of wild birds in the transmission and cycling of this virus has been uncertain and the current study was designed to collect further data on the role of wild birds in the transmission of H5N1 in Thailand. The study site for the current study was located in Nakorn Pathom province, the central part of Thailand, where both backyard poultry and low biosecurity poultry farms are common and co-exist. The analysis of existing extensive data from the national wild bird surveillance program for HPAI H5N1 virus in Thailand, found that since 2004 the prevalence of infection with H5N1 in wild birds was low (1.0% 95%CI (0.7, 1.2). However, the annual prevalence varied considerably over this period with a peak of 2.7% (95%CI 1.4, 4.1) in 2004, which dropped to 0.5% (95%CI 0.3, 0.8) and 0.6% (95%CI 0.3, 1.0) in 2005 and 2006, respectively, and then rose again to 1.8% (95%CI 1.0, 2.6) in 2007. During this period, sixteen species of wild birds tested positive for H5N1 virus infection. All samples from juvenile birds were negative for H5N1 virus, whereas the virus prevalence in pooled samples from adult birds was 0.6% (95%CI 0.4, 0.9). The positive birds belonged to twelve species which were mainly resident species that are commensal with human activities. Infected wild bird samples were only found in provinces where poultry outbreaks had occurred. A risk factor study conducted in this project using a questionnaire for villagers on farm practices and wild birds observed in the area revealed that factors associated with disease included replacing poultry individually into households/farms, buying native chickens and/or fighting cocks from commercial hatcheries and the presence of lesser whistling ducks (Dendrocygna javanica) on farms. Selecting healthy poultry when purchasing replacement birds was identified as a protective factor in this study. The longitudinal wild bird surveillance programs conducted in this study revealed that the serological and virological prevalence of H5N1 virus were low in the wild bird population. The seroprevalence as tested by the H5N1 serum neutralization test (NT) was 2.1% (95% CI 0.7, 3.5). Species that tested positive to NT were rock pigeon (Columba livia), Asian pied starling (Gracupica contra), spotted dove (Streptopelia chinensis), oriental magpie robin (Copsychus saularis), blue-tailed bee-eater (Merops philippinus), myna (Acridotheres spp.), and pond heron (Ardeola spp.). The prevalence of H5N1 virus detection was 0.5% (95% CI 0.0, 1.1); the two H5N1 virus -positive samples were from Asian pied starling (Gracupica contra) and white vented myna (Acridotheres grandis). Wild birds that tested positive to H5N1 virus were mostly common terrestrial birds, some of which showed no clinical signs of disease. Molecular epidemiology showed that the viruses isolated from the survey were most closely related to poultry viruses isolated in Thailand (A/chicken/Thailand/PC-168/2006, A/chicken/Phichit/NIAH606988 /2006, and A/quail/Thailand /CU-333/06). There was no evidence to support the presence of unique strains in wild birds in Thailand. A wild bird observational study undertaken demonstrated that habitats which contain the potential for a high risk of interspecies transmission of HPAI H5N1 viruses were open system duck farms and household/backyard areas. In these areas wild birds were commonly observed feeding together and in close contact with domestic poultry and pigs. Common terrestrial birds considered as bridge species (e.g. pigeons, sparrows, mynas, starlings, and doves) were likely to be involved in the disease transmission. Moreover, a qualitative risk assessment conducted in this study showed that the risk of wild birds transmitting the disease to poultry was low with an overall risk ranking of ―Medium severity‖. For quantitative risk assessment conducted, the risk of an infected lesser whistling duck defaecating an infectious dose of HPAI H5N1 virus close to a domestic duck in an open system duck farm was 5.8 x 10-6. This risk increased to 2.5 x 10-1 when all ducks visiting an open system duck farm were considered in a year. In conclusion, wild birds can help maintain the virus in wild and domestic bird populations through spill back and spill over. However, risk of wild birds transmitting HPAI H5N1 virus to poultry in the current study was considered to be low. Monitoring of the disease in wild birds and poultry should be performed in Thailand, and the biosecurity of small and backyard poultry farms should be improved

Abattoir-based serological surveillance for transboundary and zoonotic diseases in cattle and swine in Cambodia: a pilot study in Phnom Penh province during 2019 and 2020

A pilot animal disease surveillance program was implemented at four abattoirs in Phnom Penh, Cambodia, between October 2019 and January 2020. A total of 1141 samples were collected from 477 cattle and 664 swine. Serological testing was performed using commercial antibody ELISA kits for zoonotic and high-impact animal diseases, namely brucellosis, Q fever, classical swine fever (CSF), porcine reproductive and respiratory syndrome (PRRS) and African swine fever (ASF). Only two samples tested positive for Brucella antibodies (0.2%, 95% CI 0.4–0.6, n = 1141). The seroprevalence of Q fever was 0.8% (95% CI 0.3–2.1, n = 477) in the cattle samples, while CSF, PRRS and ASF in pigs were 55.4% (95% CI 51.6–59.2, n = 655), 81.2% (95% CI 78.1–84.0, n = 655) and 2.6% (95% CI 1.6–4.1, n = 664), respectively. All 38 doubtful and 17 positive ASF antibody ELISA samples were negative when tested by real-time PCR. Univariate analyses demonstrated that the factor significantly associated with positive results of ASF was the abattoir location (p-value = 0.002). Based on logistic regression models, significant risk factors for CSF were province of origin (p-value = 1.7 × 10−6), abattoir (p-value = 3.6 × 10−11) and PRRS positivity (p-value = 0.004), and for PRRS were province of origin (p-value = 0.0004) and CSF positivity (p-value = 0.001). In conclusion, the seroprevalences of zoonotic diseases in this study were very low. The high prevalence of CSF and PRRS antibodies were most likely the result of vaccination. All ASF seropositive pigs, including those that gave equivocal results, originated from large-scale Cambodian-based commercial farms, as well as Thailand, which raises questions about possible illegal vaccination or low-pathogenicity ASF variants. The pilot abattoir serological surveillance program described here has the potential to provide a sentinel for incursions of novel and endemic pathogens, although further work is required to demonstrate its capacity to provide information on the longitudinal disease trends

Risk mapping using serologic surveillance for selected One Health and transboundary diseases in Cambodian goats

In Cambodia, goat production and meat consumption are customary among Muslim communities. Recently, goat meat has gained popularity among Cambodians. Goat farmers use a traditional management system, including grazing, requiring minimal labour. The close proximity between humans and animals could increase the risk of zoonotic disease transmission. A serological survey was undertaken to estimate the prevalence of some priority zoonoses and high-impact animal diseases in the Cambodian goat population. A total of 540 samples were collected from goats in six provinces and analysed with commercially available enzyme-linked immunosorbent assays for Brucella species, Q fever (Coxiella burnetii), Foot and Mouth Disease virus non-structural protein (FMDV NSP) and Peste des Petits Ruminants virus (PPRV). True seroprevalences with a 95% Confidence Interval (CI), taking into account imperfect tests, risk factors and odds ratios (ORs), were calculated to better understand the disease distribution and epidemiology. Independent variables used in statistical modellings included sex, body condition score, age, vaccination history, province and commune, while dependent variables were ELISA test results. The overall true prevalence of antibodies to Brucella spp., C. burnetii, FMDV and PPRV, were 0.1% (95% CI 0.0, 1.0), 7.2% (95% CI 5.3, 9.7), 57.7% (95% CI 53.1, 62.3) and 0.0% (95% CI 0.0, 0.0), respectively. There was no identified risk factor for brucellosis and PPR. The two risk factors for C. burnetii seropositivity were sex (p-value = 0.0005) and commune (p-value <0.0001). However, only the OR of C. burnetii seropositive female goat was significant at 9.7 (95% CI 2.7, 35.5) times higher than male. The risk factors of FMD NSP seropositivity were age (p-value = 0.001) and commune (p-value <0.0001). Only the age ’more than two-year-old’ group with a significant OR of 6.2 (95% CI 2.1, 18.4) using the ’up to one-year-old’ group as the reference. In summary, Brucella spp. seroprevalence was low, while no evidence of PPRV antibodies was detected in the goat populations. C. burnetii seroprevalence in female goats was significantly higher than for males, and there were significant differences in C. burnetii seroprevalence between communes. The overall FMDV NSP seroprevalence was high, especially in older animals. Vaccination should be advocated to protect animals from FMDV and improve productivity. As the impacts of these zoonoses on human and animal health were still unknown, further investigation of these zoonotic diseases’ epidemiology is recommended

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

Spatiotemporal drivers of the African swine fever epidemic in Lao PDR

African swine fever (ASF) is a devastating transboundary disease of swine. Following the frst report of ASF in China in August 2018, ASF spread through South-East Asian nations in 2019. Without control and containment measures, ASF can decimate smallholder pig holdings and disrupt value chains. Tis study aimed to describe the spatiotemporal spread of the 2019 Lao PDR ASF epidemic in domestic pigs, identify environmental (protected areas and forests), production (pig ownership), transportation (roads), and social (poverty and ethnicity) risk factors, and recommend measures that could reduce ASF spread. A retrospective spatiotemporal study was conducted at the village level. Information on the date that ASF was frst reported from each case location was collected, and the outcome variable of interest “epidemic day” was created. Risk factor information from diferent sources was extracted for each case location. Te association or correlation between epidemic day and risk factors for the spread of ASF was investigated using Kruskal–Wallis tests and Spearman rank correlation statistics. Te epidemic started on June 16 and lasted for 190 days, displaying a right-skewed epidemic curve. Te directional distribution was rotated approximately 305°, from Southeast to Northwest Laos. Signifcant risk factors for ASF associated with epidemic day were location in terms of distance from the closest protected natural area (P = 0.020), pig ownership (P = 0.005), road networks (P = 0.003), and poverty indices (P < 0.001). Cases were reported earlier in this epidemic at locations that were closer to protected natural areas, of higher pig ownership, more connected via the national road network, and which experienced elevated poverty. Te spatiotemporal pattern described suggests that ASF was introduced via infected pigs from Vietnam. Based on study fndings, recommendations for smallholder pig production in Southeast Asia include improving knowledge of swine value chains to inform disease risk and control, monitoring pig transportation, implementing stricter biosecurity measures on the domestic pig population, and providing biosecurity support and education to smallholder pig farmers in poverty

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

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

The importance and challenges of implementing and maintaining biorepositories for high-consequence veterinary and one health pathogens in South-East Asia

Introduction: Emerging infectious diseases pose a threat to public health and the economy, especially in developing countries. Southeast Asian veterinary laboratories handle numerous high-risk pathogens, making pathogen accountability crucial for safe handling and storage. Methods: Thirteen veterinary laboratories in Cambodia (n = 1), Lao People's Democratic Republic (n = 1), and Thailand (n = 11) participated in a study conducted between 2019 and 2020. Data were collected using a questionnaire, group discussions, and interviews. Conclusion: Significant gaps in biosecurity and biorepository management were recognized and discussed in the context of regional biosafety and biosecurity. Laboratories could use the findings and recommendations of the study to develop or improve their pathogen inventory and biosecurity systems. Governments play a significant role in setting standards and regulations and providing necessary support for laboratories to maintain inventory controls sustainably and have a very important role to play in ensuring biosafety and biosecurity compliance

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