Ladawan Sariya

COMPARISON OF OUTBREAKS OF H5N1 HIGHLY PATHOGENIC AVIAN INFLUENZA IN WILD BIRDS AND POULTRY IN THAILAND

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.

Indigenous sources of 2007–2008 H5N1 avian influenza outbreaks in Thailand

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.

Detection and phylogenetic characterization of hepatitis E virus genotype 3 in a captive wild boar in Thailand.

Hepatitis E virus (HEV) was studied in different types of wild boar captive settings in Thailand, including a wildlife breeding research station, zoo, and commercial wild boar farm, which were located in different locations of Thailand. Fifty-one fecal samples were collected and screened for HEV RNA and then analyzed. One sample obtained from a wildlife breeding research station in Ratchaburi province was HEV positive. Phylogenetic characterization revealed that the virus was HEV genotype 3 and belongs to subgroup 3e, which is closely related to HEV recently isolated from domestic pigs and humans in the country. It was hypothesized that HEV is shared among wild boars, domestic pigs, and humans in Thailand.

Virological and molecular epidemiological investigations into the role of wild birds in the epidemiology of influenza A/H5N1 in central Thailand

A serological and virological surveillance program to investigate the HPAI H5N1 virus in wild bird populations was undertaken from February 2007 to October 2008. The purpose of the survey was to investigate the infection status in free ranging wild birds in Banglane district, Nakhon Pathom province, central Thailand. Samples from wild birds were collected every two months. Choanal and cloacal swabs, serum and tissue samples were collected from 421 birds comprising 44 species. Sero-prevalence of the virus tested by H5N1 serum neutralization test (using a H5N1 virus clade 1; A/chicken/Thailand/vsmu-3-BKK/2004) was 2.1% (8 out of 385 samples; 95% CI 0.7, 3.5). Species that were antibody positive included rock pigeons (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.). Prevalence by H5N1 virus isolation was 0.5% (2 out of 421 samples; 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). Positive virological samples were collected in June 2007 while all positive serology samples were collected between May and August except for one sample collected in December 2007. No positive samples were collected in 2008. Molecular studies revealed that the wild bird H5N1 viruses were closely related to poultry viruses isolated in other parts of Thailand. However, there was no poultry H5N1 prevalence study performed in the study site during the time of this wild bird survey. Interpretation of source of virus isolates would include spill-over of H5N1 viruses from contaminated sources due to movement of domestic poultry and/or fomites from other areas; or infection of wild birds within the outbreak locations and then translocation by wild bird movement and interaction with wild birds inhabiting distant locations.

Expression of foot and mouth disease virus nonstructural polyprotein 3ABC with inactive 3Cpro in Escherichia coli

Nonstructural 3ABC protein of foot and mouth disease virus (FMDV) was widely used to differentiate vaccinated from natural FMDV-infected animals. 3ABC is a polyprotein which is auto-processed to 3A, three copies of 3B and 3C(pro) by 3C(pro) protease. The 3ABC gene was cloned and expressed in Escherichia coli as native or mutated 3ABC (mu3ABC) forms. Cysteine residues 142 and 163 of the catalytic triad within the 3C(pro) of mu3ABC were changed to serine and glycine, respectively, to inhibit its protease activity. Both native and mutated 3ABC ORFs were cloned into BamHI and HindIII restriction sites of an expression vector, pQE80L. The expression of the recombinant native 3ABC and mu3ABC genes in E. coli BL21 was induced with 0.2mM isopropyl-beta-d-thiogalactopyranoside at 37 °C for 5h. SDS-PAGE and Western blot analysis revealed that the full length 3ABC was present in the lysate from mu3ABC but not native 3ABC transformed cells. The recombinant mu3ABC was expressed mainly in the inclusion body and presented as monomer and dimer. In addition, the mu3ABC reacted strongly with a convalescent serum from a natural FMDV-infected cattle but very weakly with a serum from vaccinated cattle. This study clearly demonstrates that successful expression of the full length 3ABC occurs only when the protease active sites within the 3C(pro) were completely abolished. This information would accelerate in house development of the 3ABC-based diagnostic test that can distinguish between vaccinated and FMDV-infected animals.

Development of an inactivated 3Cpro-3ABC (mu3ABC) ELISA to differentiate cattle infected with foot and mouth disease virus from vaccinated cattle

Foot and mouth disease, a highly contagious disease of cloven-hoofed animals, is still endemic in Asia, Africa, and a few countries in South America. Subclinical and persistent infections usually occur in vaccinated cattle exposed to FMDV. Successful control and eradication measures need a diagnostic assay that can distinguish between immune responses to infection and vaccination. The non-structural 3ABC ELISA is the most reliable differential diagnostic assay. However, expression of the native 3ABC gene in insect cells yielded truncated versions of the proteins; thus, a monoclonal antibody to capture digested proteins is needed to develop the assay. The purpose of this study was to develop a simple indirect 3ABC ELISA using complete 3ABC protein. The full-length mutated 3ABC protein with inactive 3C(pro) (mu3ABC) gene was constructed. The histidine-tagged mu3ABC protein was produced in insect cells for easy purification and measuring. This permits simple assay design and reproducible assay development. mu3ABC ELISA had diagnostic specificity and sensitivity of 96.6% and 84%, respectively, compared to Ceditest(®) FMDV-NS. Agreement of both assays was excellent with κ value of 0.823 (p<0.05). The mu3ABC ELISA could distinguish infected from vaccinated animals. These factors are necessary for the successful development of an in-house NSP-based ELISA. Availability of a reliable assay with acceptable costs would facilitate successful disease control and the establishment of disease-free zones. Expansion of such zones may ultimately decrease the risk of introducing FMDV into disease-free countries, thus accelerating global FMD control.

Development of a SYBR Green I-based real-time PCR for detection of elephant endotheliotropic herpesvirus 1 infection in Asian elephants (Elephas maximus).

Elephant endotheliotropic herpesvirus 1 (EEHV1) can cause fatal hemorrhagic disease in Asian elephants (Elephas maximus). Several studies have described this virus as a major threat to young Asian elephants. A SYBR Green I-based real-time polymerase chain reaction (PCR) was developed to identify EEHV1 on trunk swabs and necropsied tissues. Two of 29 (6.9%) trunk swab samples from healthy Asian elephants were positive for EEHV1. The viruses were analyzed and classified as EEHV1A based on 231 nucleotides of the terminase gene. Necropsied spleen and heart tissue showed the highest level and second highest levels of DNA virus copy accumulation, respectively. The detection limit of the test was 276 copies/μl of DNA. There was no cross-reaction with other mammalian herpesviruses, such as herpes simplex virus 1 and equine herpesvirus 2. Inter- and intra-assay showed low coefficients of variation values indicating the reproducibility of the test. The results indicated that the test can be practically used for epidemiological study, clinical diagnosis, and management and control of EEHV1.

Molecular characterization of Hepatozoon felis in Rhipicephalus sanguineus ticks infested on captive lions (Panthera leo)

Hepatozoon spp. are protozoan parasites that infect a wide range of domestic and wild animals. The infection occurs by ingestion of an infected tick. This study was carried out to detect and characterize Hepatozoon spp. in ticks collected from captive lions (Panthera leo) in Thailand based on the partial 18S rRNA gene sequence. A total of 30 ticks were collected and identified as Rhipicephalus sanguineus. The collected ticks were separated into 10 tick pools by sex and life stages. Of the 10 tick pools examined, only one (10%) was found to be infected with the Hepatozoon species. Sequencing and phylogenetic analysis showed a clustering of the partial 18S rRNA gene sequence like that of H. felis from the GenBank database. This is the first report of H. felis in R. sanguineus ticks collected from captive lions in Thailand. Our results indicated that R. sanguineus may be a possible vector of feline Hepatozoon in Thailand.

Detection and characterization of Chlamydophila psittaci in asymptomatic feral pigeons (Columba livia domestica) in central Thailand.

OBJECTIVE To detect and characterize Chlamydophila psittaci (C. psittaci) in asymptomatic feral pigeons in central Thailand. METHODS A total 814 swabs from the trachea and cloacae of 407 non-clinical feral pigeons in central Thailand were collected and tested for the presence of C. psittaci. RESULTS A 10.8% of feral pigeons in the sample group were positive as determined by nested PCR primer specific to C. psittaci. The outer membrane protein A (ompA) gene of positive samples exhibited amino acid identity of C. psittaci ranging from 71 to 100% and were grouped in genotype B. Exceptionally, BF1676-56 isolate was closely related to Chlamydia avium with 99% identification of the 16S ribosomal (r) RNA gene. CONCLUSIONS This is the first report on C. psittaci isolated from asymptomatic feral pigeons in Thailand, which provides knowledge for the disease status in pigeon populations in Thailand.

Generation of a pig induced pluripotent stem cell (piPSC) line from embryonic fibroblasts by incorporating LIN28 to the four transcriptional factor-mediated reprogramming: VSMUi001-D

Pig induced pluripotent stem cell (piPSC) line was generated from embryonic fibroblast cells using retroviral transduction approaches carrying human transcriptional factors: OCT4, SOX2, KLF4, c-MYC and LIN28. The generated piPSC line, VSMUi001-D, was positive for alkaline phosphatase activity and expressed the pluripotency associated transcription factors including OCT4, SOX2, NANOG and surface markers SSEA-1, all iPSC hallmarks of authenticity. Furthermore, VSMUi001-D exhibited a normal karyotype and formed embryoid bodies in vitro and teratomas in vivo. Upon cardiac differentiation, VSMUi001-D displayed spontaneous beating and expressed cardiomyocyte markers, like cardiac Troponin T.