Consumer knowledge, attitudes, and behaviour regarding food safety - A perspective from Laos.

Primary research data on food chain safety at consumer level is needed to help policymakers to decrease the risk of foodborne infections in the Lao People's Democratic Republic. Therefore, the objectives of this study are to 1) determine Lao people's food safety knowledge, attitudes, and intentions and identify the most influential factors that shape their food safety behaviour, and 2) to support the government in the development of more efficient risk communication programmes. A paper-based consumer survey was implemented to collect quantitative data on the selected topics about Laotian consumers, as well as on the equipment of households regarding the conditions and tools necessary for safe food preparation and storage. A model was developed based on the extended Theory of Planned Behaviour (TPB), and partial least squares structural equation modelling (PLS-SEM) was applied to analyse the factors that affect food safety behaviour. The elements of Knowledge and Perceived Behavioural Control positively influence behaviours, such as using separate chopping boards for raw and ready-to-eat foods. According to the model, only 10 % of the Intentions would be translated into Behaviours, which gap can be explained by the lack of a hygienic environment. Although the survey indicated that Laotian people intend to follow food safety guidelines, participants felt they lacked sufficient knowledge. This perception was validated by the knowledge test and behavioural part of the study. In the light of these findings, it is essential to raise awareness and promote food safety knowledge and interventions, such as enhancing food safety education in childhood. This would help to facilitate and encourage adopting safe practices to improve overall food safety at the consumer level, delivering positive impacts also to the food business sector.

Social network analysis for the assessment of pig, cattle and buffalo movement in Xayabouli, Lao PDR.

The aim of this study is to understand the role that the movement patterns of pigs, cattle and buffalo play in the spread of foot-and-mouth disease (FMD). A cross-sectional survey consisting of a questionnaire was used in a hotspot area for FMD: Xayabouli Province, Lao People's Democratic Republic. A total of 189 respondents were interviewed. We found that the key players in this network were people who were involved with more than one species of animal or occupation (multipurpose occupational node), which represents the highest number of activities of animals moved off the holding (shown with the highest out-degree centrality) and a high likelihood of being an intermediary between others (shown with the highest betweenness centrality). Moreover, the results show that the animals moved to and away from each node had few connections. Some nodes (such as traders) always received animals from the same group of cattle owners at different times. The subgroup connection within this network has many weak components, which means a connection in this network shows that some people can be reached by others, but most people were not. In this way, the number of connections present in the network was low when we defined the proportion of observed connections with all possible connections (density). These findings indicate that the network might not be busy; only one type of node is dominant which enables increased control of disease spread. We recommend that the relevant authorities implement control measures regarding the key players, which is the best way to effectively control the spread of infectious diseases.

Patterns of Flavivirus Seroprevalence in the Human Population of Northern Laos.

A total of 1,136 samples from 289 households in four provinces in northern Laos were subjected to Japanese encephalitis virus (JEV) and dengue virus hemagglutination inhibition (DENV HI). Overall, antibodies to JEV were detected by HI in 620 (54.6%) of 1,136 people; of which 217 (19.1%) had HI activity against JEV only. Antibodies to DENV4 were detected by HI in 526 (46.3%) of 1,136 people; of which 124 (10.9%) had HI activity against DENV4 only. Antibodies to DENV1-3 were detected by HI in 296 (26.1%), 274 (24.1%), and 283 (24.9) of 1,136 people, respectively; of which 7, 1, and 0, respectively, had HI activity against DENV1-3 only. JEV was the most prevalent Flavivirus in Oudomxay, Luangprabang, and Huaphan provinces and DENV4 was the most prevalent in Xiengkhouang province. Seroprevalence for JEV increased with increasing age and wealth and was higher in villages where rice was cultivated in paddy fields and highest for people of Lao-Tai ethnicity.

Patterns and risks of trichinella infection in humans and pigs in northern Laos.

Several outbreaks of trichinellosis associated with the consumption of raw pork have occurred in Laos since 2004. This cross-sectional study was conducted in four provinces of northern Laos to investigate the seroepidemiology of trichinellosis in the human population and determine the prevalence and species of Trichinella infection in the domestic pig population. Serum samples and questionnaire data were obtained from 1419 individuals. Serum samples were tested for Trichinella antibodies by ELISA using larval excretory-secretory (ES) antigens and a subset of 68 positive samples were tested by western blot. The seroprevalence of Trichinella antibodies was 19.1% (95% confidence interval (CI) = 17.1-21.1%). The risk of having antibodies detected by ELISA using ES antigens increased with age, being of Lao-Tai ethnicity, living in Oudomxay province and being male. Tongue and diaphragm muscle samples were collected from 728 pigs and tested for Trichinella larvae by the artificial digestion method. Trichinella larvae were isolated from 15 pigs (2.1%) of which 13 were identified as T. spiralis by molecular typing; the species of the two remaining isolates could not be determined due to DNA degradation. Trichinella spp. are endemic in the domestic environment of northern Laos and targeted preventative health measures should be initiated to reduce the risk of further outbreaks occurring.

The economic impact of pig-associated parasitic zoonosis in Northern Lao PDR.

The parasitic zoonoses human cysticercosis (Taenia solium), taeniasis (other Taenia species) and trichinellosis (Trichinella species) are endemic in the Lao People's Democratic Republic (Lao PDR). This study was designed to quantify the economic burden pig-associated zoonotic disease pose in Lao PDR. In particular, the analysis included estimation of the losses in the pork industry as well as losses due to human illness and lost productivity. A Markov-probability based decision-tree model was chosen to form the basis of the calculations to estimate the economic and public health impacts of taeniasis, trichinellosis and cysticercosis. Two different decision trees were run simultaneously on the model's human cohort. A third decision tree simulated the potential impacts on pig production. The human capital method was used to estimate productivity loss. The results found varied significantly depending on the rate of hospitalisation due to neurocysticerosis. This study is the first systematic estimate of the economic impact of pig-associated zoonotic diseases in Lao PDR that demonstrates the significance of the diseases in that country.

A cross-sectional study of Taenia solium in a multiple taeniid-endemic region reveals competition may be protective.

We conducted cross-sectional surveys for taeniasis and cysticercosis in humans, pigs, and dogs in four northern provinces of Laos. Human cysticercosis and taeniasis prevalence was 2.2% (95% confidence interval [CI] = 1.4-3.0%) and 8.4% (95% CI = 6.9-9.9%), respectively. Eating uncooked beef, being male, province of residence, age, and ethnicity were significant risk factors for taeniasis and only province of residence was a significant risk factor for cystiercosis. Thirty-five human tapeworms were recovered during the survey and 33 (94.3%) and 2 (5.7%) were identified as Taenia saginata and T. solium, respectively. Maximum-likelihood adjusted prevalence of T. solium and T. hydatigena in pigs was 4.2% (95% CI = 0.5-7.9%) and 55.9% (95% CI = 47.5-64.3%), respectively, and T. hydatigena taeniasis in dogs was 4.8% (95% CI = 0.0-11.3%). Taenia hydatigena and T. saginata were the most prevalent taeniids in the respective pig and human populations and together may suppress T. solium transmission.

Seroprevalence of major bovine-associated zoonotic infectious diseases in the Lao People's Democratic Republic.

Bovine-associated zoonotic infectious diseases pose a significant threat to human health in the Lao People's Democratic Republic (Lao PDR). In all, 905 cattle and buffalo serum samples collected in northern Lao PDR in 2006 were used to determine seroprevalence of five major bovine zoonotic infectious diseases that included Taenia saginata cysticercosis, bovine tuberculosis, Q-fever, bovine brucellosis, and bovine leptospirosis. Five enzyme-linked immunosorbent assays (ELISAs) were used to test for the presence of antibodies to the diseases, except Taenia saginata, for which we tested for the presence of Taenia metacestode circulating antigens. The overall highest prevalence was for T. saginata (46.4%), with lower prevalence for Q-fever (4%), leptospirosis (3%), tuberculosis (1%), and brucellosis (0.2%). Although there were no significant differences in the proportion of seroprevalence between sex and age of the animals sampled, there were significant differences between the provincial distributions. Further studies are required to determine the seroprevalence of these infections in other locations in Lao PDR, as well as other animal species including humans, in order to develop effective prevention and control strategies. This is the first study to investigate the prevalence of bovine zoonotic infectious agents in the Lao PDR. Positivity was demonstrated for all diseases investigated, with the highest prevalence for T. saginata antigen and Coxiella burnetti antibodies. For T. saginata, there were significant differences in the provincial distribution. Approximately 16% seroprevalence of Coxiella burnetti was noted in Xayabuly Province; however, there are no clear reasons why this was the case, and further studies are required to determine risk factors associated with this observation.

Serologic study of pig-associated viral zoonoses in Laos.

We conducted a serologic survey of four high-priority pig-associated viral zoonoses, Japanese encephalitis virus (JEV), hepatitis E virus (HEV), Nipah virus (NiV), and swine influenza virus (SIV), in Laos. We collected blood from pigs at slaughter during May 2008-January 2009 in four northern provinces. Japanese encephalitis virus hemagglutination inhibition seroprevalence was 74.7% (95% confidence interval [CI] = 71.5-77.9%), JEV IgM seroprevalence was 2.3% (95% CI = 1.2-3.2%), and HEV seroprevalence was 21.1% (95% CI = 18.1-24.0%). Antibodies to SIV were detected in 1.8% (95% CI = 0.8-2.8%) of pigs by screening enzyme-linked immunosorbent assay, and only subtype H3N2 was detected by hemagglutination inhibition in two animals with an inconclusive enzyme-linked immunosorbent assay result. No NiV antibody-positive pigs were detected. Our evidence indicates that peak JEV and HEV transmission coincides with the start of the monsoonal wet season and poses the greatest risk for human infection.

Soil-transmitted helminthiasis in Laos: a community-wide cross-sectional study of humans and dogs in a mass drug administration environment.

We conducted a community cross-sectional survey of soil-transmitted helminthiasis in humans and dogs in four provinces in northern Laos. We collected and tested human and dog fecal samples and analyzed results against sociodemographic data. The prevalence of Ascaris lumbricoides, Trichuris trichiura, hookworm, and Strongyloides stercoralis was 26.1% (95% confidence interval [CI] = 23.7-28.4%), 41.5% (95% CI = 38.8-44.1%), 46.3% (95% CI = 43.3-49.0%), and 8.9% (95% CI = 7.4-10.4%), respectively. We observed strong heterogeneity for helminthiasis by ethnicity, province, and wealth status, which coincided with a risk profile demonstrating that Mon-Khmer persons and the poorest households are highly vulnerable. Necator americanus was the dominant hookworm species infecting humans and Ancylostoma ceylanicum was the only Ancylostoma species detected. Hookworm prevalence in village dogs was 94%, and the dominant species was A. ceylanicum. Necator americanus was also detected in dogs. It appears that dogs have a role in human hookworm transmission and warrant further investigation.

Hepatitis E virus is prevalent in the pig population of Lao People's Democratic Republic and evidence exists for homogeneity with Chinese Genotype 4 human isolates.

The objective of this study was to determine the prevalence and genotypic range of Hepatitis E virus (HEV) in the pig population of northern Lao People's Democratic Republic (PDR). We collected 181 faecal samples from indigenous-breed pigs ≤ 6 months of age and the faeces was stored in RNA stabilisation buffer due to cold-chain and transport limitations. Twenty-one (11.6%) pigs had detectable HEV RNA and 43.5% of village pig herds were infected. Based on a 240 base pair-nucleotide sequence flanking the junction of open reading frames 1, 2 and 3 (ORF1, ORF2 and ORF3) the isolates were phylogenetically classified within genotype 4. Phylogenetic analyses revealed distinct genetic groupings of the Lao HEV isolates and two groups clustered with human and pig HEV isolates from China. This was the first study to demonstrate genotype 4 HEV in Lao PDR and indicates pigs are a potential reservoir for human HEV infection.

Does interspecific competition have a moderating effect on Taenia solium transmission dynamics in Southeast Asia?

It is well understood that sociocultural practices strongly influence Taenia solium transmission; however, the extent to which interspecific parasite competition moderates Taenia transmission has yet to be determined. This is certainly the case in Southeast Asia where T. solium faces competition in both the definitive host (people) and the intermediate host (pigs). In people, adult worms of T. solium, T. saginata and T. asiatica compete through density-dependent crowding mechanisms. In pigs, metacestodes of T. solium, T. hydatigena and T. asiatica compete through density-dependent immune-mediated interactions. Here, we describe the biological and epidemiological implications of Taenia competition and propose that interspecific competition has a moderating effect on the transmission dynamics of T. solium in the region. Furthermore, we argue that this competitive ecological scenario should be considered in future research and surveillance activities examining T. solium cysticercosis and taeniasis in Southeast Asia.

[Analysis of tdh gene and its adjacent loci of Vibrio parahaemolyticus isolates from seafoods].

OBJECTIVE: To analyze the structural characteristics of tdh gene and its adjacent loci of Vibrio parahaemolyticus isolates from seafoods. METHODS: Long distance PCR and genome walking were used to amplify the DNA sequences flanking the tdh gene, and the sequences were analyzed by blastn against the NCBI database. RESULTS: The genetic structure of tdh-adjacent loci (VPA1312-VPA1327) from isolate ZS34 was similar to that of the reference strain RIMD2210633, with the nucleotide identity of 98.3%. The tdh gene of isolates FJ14 and WZ64 was located in the loci different from the reference strain and ZS34, and showed high nucleotide similarity to tdh3 gene. In the genome of isolate FJ14, tdh was 15kb away from the trh- ure cluster, with IS-like elements and transposase genes inserted therein. Isolate WZ64 lacked trh gene, but also harbored IS-like elements upstream of tdh. CONCLUSION: The tdh-adjacent loci of V. parahaemolyticus seafood isolates exhibit high diversity, an additional evidence of lateral gene transfer in this particular species.

Pathogenetic characterization of Vibrio parahaemolyticus isolates from clinical and seafood sources.

A total of 216 Vibrio parahaemolyticus isolates from seafood and clinical samples in eastern China were investigated for their hemolytic and urea-producing phenotypes, presence of putative virulence genes tdh and trh. Twenty-one clinical isolates (84%, 21/25) and 3 seafood isolates (1.57%, 3/191) were tdh-positive while only 3 clinical isolates (12%) and 7 seafood isolates (3.66%) were positive for trh gene. We further examined the pathogenicity of selected V. parahaemolyticus isolates in in vitro and in vivo systems. The clinical isolates were apparently more enteropathogenic (74.26 per thousand vs 62.07 per thousand expressed as intestine/body weight ratio, P<0.01) and more virulent than their seafood counterparts to mice (log LD(50) 6.86 vs 7.40 via orogastric route, P<0.05). They were also more adherent to in vitro cultured cells and of higher cytotoxicity as measured by LDH release of the HeLa cells although there were no statistical differences. The tdh-positive V. parahaemolyticus isolates were of higher enteropathogenicity (P<0.05, 74.24 per thousand vs 60.55 per thousand) and more virulent (log LD(50) 6.55 vs 7.21 via intraperitoneal route, P<0.05) than tdh-negative isolates. The tdh-positive isolates were generally more cytotoxic and adhesive to the cultured cell lines as well. From the in vitro and in vivo pathogenicity profiles, trh-positive isolates seemed to line between tdh-positive isolates and those without tdh and trh. There were two isolates H8 and H10 from clinical cases having moderate enteropathogenicity and virulence to mice, but were tdh-negative yet trh-positive. These results seem to suggest that hemolysins TDH and/or TRH may not be necessarily the only virulence factors of pathogenic V. parahaemolyticus isolates.

Occurrence of pandemic clones of Vibrio parahaemolyticus isolates from seafood and clinical samples in a Chinese coastal province.

Fifty-four isolates of Vibrio parahaemolyticus were examined for hemolytic and urease-producing phenotypes as well as presence of virulence markers by polymerase chain reaction (PCR). All clinical isolates (11/11, 100%) and one out of 42 isolates from seafood (2.4%) possessed the tdh gene and showed hemolysis. This tdh-positive seafood isolate as well as four clinical isolates belonged to the new pandemic clone O3:K6 according to serotyping and sequencing of the toxRS locus. The new O3:K6 clone, O1:KUT, and O4:K68 shared the same characteristic variations from the old O3:K6 clone at six base positions from 576 to 1244 of the toxRS locus. Seven clinical isolates (63.6%) were positive on both toxRS- and orf8-based PCRs. Our results indicate that the new pandemic O3:K6 clonal group of V. parahaemolyticus isolates from clinical and seafood sources are present in the southeastern coastal Chinese province Zhejiang and the pandemic clones of V. parahaemolyticus should be targeted for control of seafood-related transmission to humans.

Virulence characterization and genotypic analyses of Listeria monocytogenes isolates from food and processing environments in eastern China.

In this study, twenty L. monocytogenes food-related isolates collected from eastern China Zhejiang province were compared by in vivo LD50 assays as well as in vitro cytopathic plaque forming assay. Nineteen L. monocytogenes isolates (19/20) were as virulent as reference strain 10403S, while the isolate M4 had low pathogenicity. The unique isolate M4 fell into lineage III based on the partial nucleotide variations of actA, while the other isolates belonged to the more common lineages I and II. L. monocytogenes isolates were grouped in 17 to 19 subtypes using pulsed-field gel electrophoresis (PFGE) with SmaI digestion, and multilocus sequence typing (MLST) based on three virulence genes (actA, inlA and inlB) and four housekeeping genes (betL, dat, recA and sigB). The virulence genes based MLST had better discriminatory power than that targeting the housekeeping genes (0.990 vs 0.895), similar to PFGE (0.976). An isolate from the processing desk was found having the same pulsotype as the two isolates from final shrimp products in the same plant, indicating that process contamination could be the source of Listeria contamination.

[Analysis of major phenotypes and hemolysin-related genotypes of Vibrio parahaemolyticus isolates from clinical and seafood samples].

Vibrio parahaemolyticus is a gram-negative, halophilic bacterium that inhabits the marine and estuarine environments. It is an important human pathogen causing gastroenteritis when raw or partially-cooked seafoods are consumed. Its pathogenicity is believed to be related to hemolysins such as thermostable direct hemolysin (TDH), TDH-related hemolysin (TRH) and thermolabile hemolysin (TLH). PCR method was used to examine three different hemolysin genes in isolates from clinical and seafood samples in Zhejiang province. The tlh gene was found in all isolates. The tdh gene was positive in all eleven clinical strains but only in one out of a total of 42 seafood isolates. The Kanagawa phenomenon was positive for all tdh-positive isolates. None of the isolates was positive for the trh gene. The urease test was negative for all isolates. Thus, it was assumed that the urease gene could be linked with trh gene. Further research is required to examine the relationship between low prevalence of the major virulence factor TDH and the high incidence of foodborne V. parahaemolyticus infections,and its pathogenesis.