Biosafety and Biosecurity Challenges Facing Veterinary Diagnostic Laboratories in Lower-Middle Income Countries in Southeast Asia: A Case Study of Thailand.

INTRODUCTION: Global concerns over emerging and transboundary infectious zoonotic diseases have increased disease diagnostic demands, especially in the veterinary sector. In developing or newly developed countries where the sector often works under limited capacity, biosafety and biosecurity are unlikely to be high-priority issues. A recent development program supported by the Biological Threat Reduction Program of the Defense Threat Reduction Agency funded by the US government aimed to increase biosafety and biosecurity measures of government veterinary diagnostic and research laboratories in Thailand. OBJECTIVE: The purpose of this article is to identify biosafety and biosecurity challenges, opportunities, and recommendations. METHODS: Eleven government laboratory centers were assessed against the Biosafety in Microbiological and Biomedical Laboratories (BMBL) biosafety level 2 (BSL-2) requirements checklist. The BMBL assessment outcomes were then combined with the outcomes of discussion sessions, and the results of pre- and post-test questionnaires conducted during biosafety assessment workshops and self-evaluation reports using the Food and Agriculture Organization Biosafety Laboratory Mapping Tool of each laboratory center were reviewed and summarized. RESULTS: Despite established national policies on laboratory biosafety and biosecurity, major challenges included (1) harmonization and enforcement of these policies, especially at the regional level, and (2) engagement of personnel in implementations of biosafety and biosecurity measures. CONCLUSION: Consistent biosafety policy and allocated resources together with regular training are required to develop sustainable biosafety and biosecurity at the national level. Collaboration between regional countries, international organizations, and donors is essential for improving biosafety and biosecurity on a global scale through setting regional priorities, enacting regulatory standards, and providing technical and financial support.

Pathogenicity of Genetically Similar, H5N1 Highly Pathogenic Avian Influenza Virus Strains in Chicken and the Differences in Sensitivity among Different Chicken Breeds.

Differences in the pathogenicity of genetically closely related H5N1 highly pathogenic avian influenza viruses (HPAIVs) were evaluated in White Leghorn chickens. These viruses varied in the clinical symptoms they induced, including lethality, virus shedding, and replication in host tissues. A comparison of the host responses in the lung, brain, and spleen suggested that the differences in viral replication efficiency were related to the host cytokine response at the early phase of infection, especially variations in the proinflammatory cytokine IL-6. Based on these findings, we inoculated the virus that showed the mildest pathogenicity among the five tested, A/pigeon/Thailand/VSMU-7-NPT/2004, into four breeds of Thai indigenous chicken, Phadu-Hung-Dang (PHD), Chee, Dang, and Luang-Hung-Khao (LHK), to explore effects of genetic background on host response. Among these breeds, Chee, Dang, and LHK showed significantly longer survival times than White Leghorns. Virus shedding from dead Thai indigenous chickens was significantly lower than that from White Leghorns. Although polymorphisms were observed in the Mx and MHC class I genes, there was no significant association between the polymorphisms in these loci and resistance to HPAIV.

Host cytokine responses of pigeons infected with highly pathogenic Thai avian influenza viruses of subtype H5N1 isolated from wild birds.

Highly pathogenic avian influenza virus (HPAIV) of the H5N1 subtype has been reported to infect pigeons asymptomatically or induce mild symptoms. However, host immune responses of pigeons inoculated with HPAIVs have not been well documented. To assess host responses of pigeons against HPAIV infection, we compared lethality, viral distribution and mRNA expression of immune related genes of pigeons infected with two HPAIVs (A/Pigeon/Thailand/VSMU-7-NPT/2004; Pigeon04 and A/Tree sparrow/Ratchaburi/VSMU-16-RBR/2005; T.sparrow05) isolated from wild birds in Thailand. The survival experiment showed that 25% of pigeons died within 2 weeks after the inoculation of two HPAIVs or medium only, suggesting that these viruses did not cause lethal infection in pigeons. Pigeon04 replicated in the lungs more efficiently than T.sparrow05 and spread to multiple extrapulmonary organs such as the brain, spleen, liver, kidney and rectum on days 2, 5 and 9 post infection. No severe lesion was observed in the lungs infected with Pigeon04 as well as T.sparrow05 throughout the collection periods. Encephalitis was occasionally observed in Pigeon04- or T.sparrow05-infected brain, the severity, however was mostly mild. To analyze the expression of immune-related genes in the infected pigeons, we established a quantitative real-time PCR analysis for 14 genes of pigeons. On day 2 post infection, Pigeon04 induced mRNA expression of Mx1, PKR and OAS to a greater extent than T.sparrow05 in the lungs, however their expressions were not up-regulated concomitantly on day 5 post infection when the peak viral replication was observed. Expressions of TLR3, IFNα, IL6, IL8 and CCL5 in the lungs following infection with the two HPAIVs were low. In sum, Pigeon04 exhibited efficient replication in the lungs compared to T.sparrow05, but did not induce excessive host cytokine expressions. Our study has provided the first insight into host immune responses of pigeons against HPAIV infection.

Differential host gene responses in mice infected with two highly pathogenic avian influenza viruses of subtype H5N1 isolated from wild birds in Thailand.

In Thailand, highly pathogenic avian influenza (HPAI) viruses of subtype H5N1 had been isolated from various wild birds during the HPAI outbreak in poultries. In this study, we examined the pathogenicity of two wild bird isolates (A/Pigeon/Thailand/VSMU-7-NPT/2004; Pigeon04 and A/Tree sparrow/Ratchaburi/VSMU-16-RBR/2005; T.sparrow05) in mice. They showed similar replication in several organs and lethal outcome. However, on day 3 post-infection, Pigeon04 induced mRNA expression of proinflammatory cytokines (IL6 and TNFα) and MIP-2, neutrophil chemoattractant, in the lungs, resulting in severe pneumonia that was accompanied by neutrophil infiltration. In contrast, on day 7 post-infection, T.sparrow05 induced the expression of several cytokines to a greater extent than Pigeon04; it also potently induced mRNA expression of several cytokines in brains of the infected mice that triggered frequent inflammatory events. In sum, our study demonstrated that two HPAI viruses induced different host responses, despite having similar replications, resulting in lethal outcome in mice.

Ecologic risk factor investigation of clusters of avian influenza A (H5N1) virus infection in Thailand.

This study was conducted to investigate space and time clusters of highly pathogenic avian influenza A (H5N1) virus infection and to determine risk factors at the subdistrict level in Thailand. Highly pathogenic avian influenza A (H5N1) was diagnosed in 1890 poultry flocks located in 953 subdistricts during 2004-2007. The ecologic risk for H5N1 virus infection was assessed on the basis of a spatial-based case-control study involving 824 case subdistricts and 3296 control subdistricts from 6 study periods. Risk factors investigated in clustered areas of H5N1 included human and animal demographic characteristics, poultry production systems, and wild birds and their habitats. Six variables remained statistically significant in the final model: flock density of backyard chickens (odds ratio [OR], 0.98), flock density of fighting cocks (OR, 1.02), low and high human density (OR, 0.60), presence of quail flocks (OR, 1.21), free-grazing duck flocks (OR, 2.17), and a poultry slaughterhouse (OR, 1.33). We observed a strong association between subdistricts with H5N1 virus-infected poultry flocks and evidence of prior and concomitant H5N1 infection in wild birds in the same subdistrict.

Molecular epidemiological analysis of highly pathogenic avian influenza H5N1 subtype isolated from poultry and wild bird in Thailand.

A comprehensive molecular epidemiological analysis was performed on highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype derived from poultry and wild bird during 2004-2007 in Thailand. Sequence analysis followed by phylogenetic analysis was applied to all eight segments of the viruses. Viruses belonging to clades 1 and 2.3.4 in the HA phylogenetic tree have been shown to circulate in Thailand. Our analysis revealed differential evolution of the HPAI viruses among clade 1 strains. Isolates from Phichit province in 2006 resided in two distinct branches, designated 1.p1 and 1.p2. A hemagglutination inhibition test with a panel of monoclonal antibodies demonstrated a possible antigenic drift between the Phichit isolates. Involvement of free-grazing duck practice in the area was discussed as a cause of the differential evolution among the Phichit isolates. A branch, designated 1-TGWB and consisting exclusively of isolates from zoological tigers and wild birds, was evident in all phylogenetic trees constructed in the study. The branch's existence indicated that the HPAI viruses could have been maintained in the wild bird population for a certain period, although no involvement of wild birds in HPAI transmission to poultry was evident in this study.