Serological evidence for Japanese encephalitis and West Nile virus infections in domestic birds in Cambodia

Mosquito-borne flaviviruses with an enzootic transmission cycle like Japanese encephalitis virus (JEV) and West Nile virus (WNV) are a major public health concern. The circulation of JEV in Southeast Asia is well-documented, and the important role of pigs as amplification hosts for the virus is long known. The influence of other domestic animals especially poultry that lives in high abundance and close proximity to humans is not intensively analyzed. Another understudied field in Asia is the presence of the closely related WNV. Such analyses are difficult to perform due to the intense antigenic cross-reactivity between these viruses and the lack of suitable standardized serological assays. The main objective of this study was to assess the prevalence of JEV and WNV flaviviruses in domestic birds, detailed in chickens and ducks, in three different Cambodian provinces. We determined the flavivirus seroprevalence using an hemagglutination inhibition assay (HIA). Additionally, we investigated in positive samples the presence of JEV and WNV neutralizing antibodies (nAb) using foci reduction neutralization test (FRNT). We found 29% (180/620) of the investigated birds positive for flavivirus antibodies with an age-depended increase of the seroprevalence (OR = 1.04) and a higher prevalence in ducks compared to chicken (OR = 3.01). Within the flavivirus-positive birds, we found 43% (28/65) with nAb against JEV. We also observed the expected cross-reactivity between JEV and WNV, by identifying 18.5% double-positive birds that had higher titers of nAb than single-positive birds. Additionally, seven domestic birds (10.7%) showed only nAb against WNV and no nAb against JEV. Our study provides evidence for an intense JEV circulation in domestic birds in Cambodia, and the first serological evidence for WNV presence in Southeast Asia since decades. These findings mark the need for a re-definition of areas at risk for JEV and WNV transmission, and the need for further and intensified surveillance of mosquito-transmitted diseases in domestic animals

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

Japanese Encephalitis in Small-Scale Pig Farming in Rural Cambodia: Pig Seroprevalence and Farmer Awareness

Japanese encephalitis (JE) is endemic in Cambodia, but circulation of JE virus (JEV) among domestic pigs has previously only been studied in the southern part of the country. The main purpose of this study was to determine the seroprevalence of JEV antibodies in smallholder pigs held in rural areas of Kampong Thom, Preah Vihear, Ratanakiri, and Stung Treng provinces, northeastern Cambodia. Another purpose was to identify possible associations between serologic status and other factors, such as reproductive disorders, and to investigate the farmers' knowledge of mosquito-borne diseases and use of preventive measures. In October 2019, 139 households were visited throughout the study area, and 242 pigs were sampled for blood. The sera were analysed with ELISA for JEV antibodies. Household representatives were interviewed, and data were recorded for each sampled pig. The apparent seroprevalence was 89.1% in pigs between 3 and 6 months of age, and 100% in pigs over 6 months of age. In total, 93.0% of the pigs tested positive. Province appeared to be the only factor significantly associated with serologic status (p < 0.001). Almost all (97.8%) respondents knew that mosquitos could transmit diseases, and 70.5% had heard of JE. However, only one respondent knew that JEV is transmitted to people through mosquito bites. Very few respondents knew that pigs can become infected with JEV, and no one knew that mosquitos transmit the virus. All families used some sort of mosquito protection for themselves, but only 15.1% protected their pigs from mosquito bites. The children were vaccinated against JE in 93 households, while adults only were vaccinated in eight households. The results suggest that JEV transmission is intense in northeastern Cambodia, and that people's knowledge about the transmission route of JEV and the role of pigs in the transmission cycle is low. Fortunately, people are well aware of mosquito-borne diseases in general and use mosquito protection, and many children are vaccinated against JE. Nonetheless, it is important that national vaccination is continued, and that people-especially in rural areas where pigs are commonly kept-are educated on the ecology and transmission of JEV

Quantitative risk assessment of salmonellosis in Cambodian consumers through chicken and pork salad consumption

Salmonella is a globally important foodborne bacterial pathogen that poses a high risk to human health. This study aimed to estimate the risk to Cambodian consumers from acquiring salmonellosis after consuming chicken and pork salad, using a quantitative microbial risk assessment (QMRA). Chicken and pork salads are typical Cambodian dishes containing raw vegetables and boiled chicken meat or pork. As previously described, chicken meat and pork samples (n = 204 of each) were collected from traditional markets in 25 Cambodian provinces to generate data on Salmonella contamination. Salad preparation and consumption practices were surveyed in 93 Cambodian households and this information was used to design an experiment to assess Salmonella cross-contamination from raw meat to ready-to-eat salad. In the part of the study reported here, data on consumption, Salmonella in salad, dose-response, and predicted salmonellosis were modeled using Monte Carlo simulations at 10,000 iterations. The prevalence of Salmonella in chicken meat and pork were set to 42.6 and 45.1%, respectively, with average most probable number (MPN) per gram of Salmonella in chicken meat was 10.6 and in pork 11.1 MPN/g, based on an earlier study. Half of the interviewed households cooked meat for the salad directly after purchase. The QMRA model showed that the modeled annual risk of salmonellosis from consuming chicken salad, pork salad and both chicken and pork salad were 11.1% probability of illness per person per year (90% CI 0.0–35.1), 4.0% (90% CI 0.0–21.3), and 14.5% (90% CI 0.0–33.5), respectively. The factors most influencing the estimate were cross-contamination while preparing the salad, followed by the prevalence of Salmonella in chicken meat and pork at the market. The wide confidence interval for the incidence was mainly due to the variability in reducing bacteria concentration by cooking and salad consumption. The predicted risk of salmonellosis due to chicken and pork salad consumption is high, and the study provides evidence supporting control measures of improving the safety of retailed chicken and pork obtained from markets to households and improving food preparation methods in the household

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

Experimental cross-contamination of chicken salad with Salmonella enterica serovars Typhimurium and London during food preparation in Cambodian households

Non-typhoidal Salmonellae are common foodborne pathogens that can cause gastroenteritis and other illnesses in people. This is the first study to assess the transfer of Salmonella enterica from raw chicken carcasses to ready-to-eat chicken salad in Cambodia. Twelve focus group discussions in four Cambodian provinces collected information on typical household ways of preparing salad. The results informed four laboratory experiments that mimicked household practices, using chicken carcasses inoculated with Salmonella. We developed four scenarios encompassing the range of practices, varying by order of washing (chicken or vegetables first) and change of chopping utensils (same utensils or different). Even though raw carcasses were washed twice, Salmonella was isolated from 32 out of 36 chicken samples (88.9%, 95% CI: 73.0–96.4) and two out of 18 vegetable samples (11.1%, 95% CI: 1.9–36.1). Salmonella was detected on cutting boards (66.7%), knives (50.0%) and hands (22.2%) after one wash; cross-contamination was significantly higher on cutting boards than on knives or hands (p-valu

Aerosol exposure of live bird market workers to viable influenza A/H5N1 and A/H9N2 viruses, Cambodia

Live bird markets (LBMs) have been identified as key factors in the spread, persistence and evolution of avian influenza viruses (AIVs). In addition, these settings have been associated with human infections with AIVs of pandemic concern. Exposure to aerosolised AIVs by workers in a Cambodian LBM was assessed using aerosol impact samplers. LBM vendors were asked to wear an air sampler for 30 min per day for 1 week while continuing their usual activities in the LBM during a period of high AIV circulation (February) and a period of low circulation (May). During the period of high circulation, AIV RNA was detected from 100% of the air samplers using molecular methods and viable AIV (A/H5N1 and/or A/H9N2) was isolated from 50% of air samplers following inoculation into embryonated chicken eggs. In contrast, AIV was not detected by molecular methods or successfully isolated during the period of low circulation. This study demonstrates the increased risk of aerosol exposure of LBM workers to AIVs during periods of high circulation and highlights the need for interventions during these high-risk periods. Novel approaches, such as environmental sampling, should be further explored at key high-risk interfaces as a potentially cost-effective alternative for monitoring pandemic threats

Diversity of A(H5N1) clade 2.3.2.1c avian Influenza viruses with evidence of reassortment in Cambodia, 2014-2016

In Cambodia, highly pathogenic avian influenza A(H5N1) subtype viruses circulate endemically causing poultry outbreaks and zoonotic human cases. To investigate the genomic diversity and development of endemicity of the predominantly circulating clade 2.3.2.1c A(H5N1) viruses, we characterised 68 AIVs detected in poultry, the environment and from a single human A(H5N1) case from January 2014 to December 2016. Full genomes were generated for 42 A(H5N1) viruses. Phylogenetic analysis shows that five clade 2.3.2.1c genotypes, designated KH1 to KH5, were circulating in Cambodia during this period. The genotypes arose through multiple reassortment events with the neuraminidase (NA) and internal genes belonging to H5N1 clade 2.3.2.1a, clade 2.3.2.1b or A(H9N2) lineages. Phylogenies suggest that the Cambodian AIVs were derived from viruses circulating between Cambodian and Vietnamese poultry. Molecular analyses show that these viruses contained the hemagglutinin (HA) gene substitutions D94N, S133A, S155N, T156A, T188I and K189R known to increase binding to the human-type α2,6-linked sialic acid receptors. Two A(H5N1) viruses displayed the M2 gene S31N or A30T substitutions indicative of adamantane resistance, however, susceptibility testing towards neuraminidase inhibitors (oseltamivir, zanamivir, lananmivir and peramivir) of a subset of thirty clade 2.3.2.1c viruses showed susceptibility to all four drugs. This study shows that A(H5N1) viruses continue to reassort with other A(H5N1) and A(H9N2) viruses that are endemic in the region, highlighting the risk of introduction and emergence of novel A(H5N1) genotypes in Cambodia