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Background: Asian Openbills, Anastomus oscitans, have long been known to migrate from South to Southeast Asia for breeding and nesting. In Thailand, the first outbreak of H5N1 highly pathogenic avian influenza (HPAI) infection in the Openbills coincided with the outbreak in the poultry. Therefore, the flyways of Asian Openbills was determined to study their role in the spread of H5N1 HPAI virus to poultry and wild birds, and also within their flocks., Results: Flyways of 5 Openbills from 3 colonies were monitored using Argos satellite transmitters with positioning by Google Earth Programme between 2007 and 2013. None of the Openbills tagged with satellite telemeters moved outside of Thailand. Their home ranges or movement areas varied from 1.6 to 23,608 km 2 per month (95% utility distribution). There was no positive result of the viral infection from oral and cloacal swabs of the Openbills and wild birds living in the vicinity by viral isolation and genome detection during 2007 to 2010 whereas the specific antibody was not detected on both Openbills and wild birds by using microneutralization assay after 2008. The movement of these Openbills did not correlate with H5N1 HPAI outbreaks in domestic poultry but correlated with rice crop rotation and populations of the apple snails which are their preferred food. Viral spread within the flocks of Openbills was not detected., Conclusions: This study showed that Openbills played no role in the spread of H5N1 HPAI virus, which was probably due to the very low prevalence of the virus during the monitoring period. This study revealed the ecological factors that control the life cycle of Asian Openbills.

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.

Brown-headed gulls (Larus brunnicephalus), winter visitors of Thailand, were tracked by satellite telemetry during 2008-2011 for investigating their roles in the highly pathogenic avian influenza (HPAI) H5N1 virus spread. Eight gulls negative for influenza virus infection were marked with solar-powered satellite platform transmitters at Bang Poo study site in Samut Prakarn province, Thailand; their movements were monitored by the Argos satellite tracking system, and locations were mapped. Five gulls completed their migratory cycles, which spanned 7 countries (China, Bangladesh, India, Myanmar, Thailand, Cambodia, and Vietnam) affected by the HPAI H5N1 virus. Gulls migrated from their breeding grounds in China to stay overwinter in Thailand and Cambodia; while Bangladesh, India, Myanmar, and Vietnam were the places of stopovers during migration. Gulls traveled an average distance of about 2400 km between Thailand and China and spent 1-2 weeks on migration. Although AI surveillance among gulls was conducted at the study site, no AI virus was isolated and no H5N1 viral genome or specific antibody was detected in the 75 gulls tested, but 6.6% of blood samples were positive for pan-influenza A antibody. No AI outbreaks were reported in areas along flyways of gulls in Thailand during the study period. Distance and duration of migration, tolerability of the captive gulls to survive the HPAI H5N1 virus challenge and days at viral shedding after the virus challenging suggested that the Brown-headed gull could be a potential species for AI spread, especially among Southeast Asian countries, the epicenter of H5N1 AI outbreak.

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., (Copyright © 2010 Elsevier Inc. All rights reserved.)

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.

All 16 subtypes of avian influenza viruses of low pathogenicity (LPAIV) as well as their hemagglutinin (H) antigens, and four 2009 pandemic influenza A (H1N1) virus isolates were assayed for hemagglutinating activity against 5 erythrocyte species: goose, guinea pig, human group O, chicken and horse. Of all viruses and antigens assayed, the highest hemagglutination (HA) titers were obtained with goose and guinea pig erythrocytes. Hemagglutinating activity of replicating LPAIV and LPAIV antigens decreased, in order, with chicken and human group O; meanwhile, horse erythrocytes yielded lowest or no HA titer. Moreover, the 2009 pandemic viruses did not agglutinate both horse and chicken erythrocytes. Our study concluded that goose and guinea pig erythrocytes are the best in HA assay for all subtypes of influenza viruses., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Wild bird surveillance for highly pathogenic avian influenza (HPAI) H5N1 virus from 2004 to 2007 in Thailand indicated that the prevalence of infection with avian influenza H5N1 virus in wild birds was low (1.0%, 95% confidence interval [CI]: 0.7-1.2, 60/6,263 pooled samples). However, the annual prevalence varied considerably over this period, with a peak of 2.7% (95% CI: 1.4, 4.1) in 2004. Prevalence 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 increased to 1.8% (95% CI: 1.0, 2.6) in 2007. During this period, 16 species from 12 families of wild birds tested positive for H5N1 virus infection. All samples from juvenile birds were negative for H5N1 virus, whereas 0.6% (95% CI: 0.4, 0.9) of pooled samples from adult birds were positive. Most positive samples originated from peridomestic resident species. Infected wild bird samples were only found in provinces where poultry outbreaks had occurred. Detection of H5N1 virus infection in wild birds was reported up to 3 yr after eradication of the poultry outbreaks in those provinces. As observed with outbreaks in poultry, the frequencies of H5N1 outbreaks in wild birds were significantly higher in winter. Further understanding of the mechanisms of persistence and ongoing HPAI H5N1 transmission between wild birds and domestic poultry is needed.

Outbreaks of H5N1 avian influenza show strong seasonality. It is not clear where the source of virus originates from in each new outbreak season. This study sought to understand the nature of viral resurgence in recent outbreak seasons in Thailand, where the epidemic is relatively well controlled. In such a situation, indigenous viruses surviving the inter-outbreak season would have to pass through a bottleneck. In order to look for evidence of the bottleneck effect, viral genome sequences from recent outbreaks in the country were analysed. H5N1 avian influenza viruses were isolated from six outbreaks in the rainy season and winter of 2007 through to early 2008. Most of the outbreaks were in the Yom-Nan River basin in the southern part of the northern region of the country. Sequences of these viral isolates were identified as clade 1, genotype Z, similar to viruses from previous years in the central region of the country. The sequences clustered into two groups, one of which was closely related to viruses isolated from the same area in July 2006. These analyses indicated that there was a strong bottleneck effect on the virus population and that only a few lineages remained in the area. In addition, evidence of reassortment among these viruses was found. These indicated re-emergence of viruses from a small pool of indigenous sources that had been silently perpetuated over the dry summer months. Therefore, an approach to eradicate H5N1 avian influenza from the area by eliminating these local reservoirs may be feasible and should be seriously considered.

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.

The aim of this study was to determine the performance of real-time amplification based methods - NASBA, TaqMan, RT-FRET, and RT-PCR LUXtrade mark formats - for the detection of influenza A (H5N1) virus RNA. In an analysis of 54 samples obtained from a range of animal species in Thailand during the period 2003-2006, results showed that the NASBA (H5=98.2%, N1=96.3%), TaqMan (H5=98.2%, N1=96.3%) and FRET (H5=98.2%, N1=96.3%) had significantly higher rates of positive detection than LUX (H5=94.4%, N1=50.0%; P<0.001) for influenza A, H5 and N1 isolates. There were no false-positive results from any methods used in the negative-control group of samples. The limits of analytical detection were at least 10copies/reaction in real-time NASBA and LUX assays, while FRET and TaqMan assay appeared to be less sensitive at > or =100copies/reaction. The assays were relatively specific without cross-reactivity to a number of other influenza strains or viral pathogens. In conclusion, our study demonstrated that real-time NASBA, TaqMan and FRET assays can be used to detect influenza A (H5N1) from a wide range of hosts, and be specific for H5N1 samples obtained during different outbreaks (2003-2006). All assays provided the benefit of rapid influenza H5N1 identification for early diagnosis, in the range of hours, and they are well suited to high throughput analyses.

Toxoplasma gondii can infect all species of warm-blooded animals, including humans, and causes serious diseases in immunocompromized hosts. Live tachyzoites derived from serial passage in HeLa culture were used in the Sabin-Feldman dye test for detection of Toxoplasma gondii antibody in serum samples of 21 captive wild felids including one fishing cat (Prion nailurus viverrina), one leopard (Panthera pardus), two flat-headed cats (Prion nailurus planiceps), 6 tigers (Panthera tigris), two leopard cats (Felis bengalensis), two clouded leopards (Felis nebulosa), 3 pumas (Puma concolor), and 4 jungle cats (Felis chaus). Antibodies to Toxoplasma gondii were founded in 9 of 21 felids (42.8%). This study revealed that cell culture-derived tachyzoites can be used successfully as a source of live organisms in a gold standard Sabin-Feldman dye test, which is simpler, cheaper and less ethically sensitive than in vivo inoculation.

Normal chicken fecal manure (pH 8.23 and 13.7% moisture) was investigated for infectivity of the avian influenza virus (AIV; H5N1). The manure was divided into three groups; each group was inoculated with AIV at 2.38 x 10(5.25) ELD50. After viral inoculation, the first group was incubated at 25 degrees C. The second group was kept at 40 degrees C, and the last group was exposed to ultraviolet light at 4-5 microw/cm2 at room temperature. After incubation, a 20% suspension of manure was filtered and the filtrates were inoculated into 9-11 day-old embryonated chicken eggs per WHO protocol (2002). The results showed that at 25 degrees C the virus lost its infectivity within 24 hours, and at 40 degrees C within 15 minutes. UV light, however, could not destroy the infectivity of the virus even after exposure for 4 hours.