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This was a comparative study of the heavy metal levels (Zn, Pb, Cd, Co, Mn, Fe, Cr and Cu) in eggs from free grazing duck, small-scale, and large-scale farms in central and western regions of Thailand. A questionnaire was used to gather demographic data for the analysis of heavy metal contamination in feed, drinking water and wastewater. The correlation between the amounts of heavy metal contamination in eggs was studied against the heavy metals found in feed, drinking water and wastewater. The levels of Pb, Cd, Cr and Cu in eggs from large-scale farms were significantly higher than small farms and free grazing farms at P < 0.001. Zn in eggs from free grazing farms was higher than in the small farms and large-scale farms sampled. The contamination of Pb in eggs from all types of farms exceeded the standard limits of ACFS 6703–2005. The average levels of Pb in the eggs from small-scale farms correlated significantly with the level of Pb found in the feed at P < 0.05, while the average levels of Pb in eggs from free grazing duck farms correlated significantly with the levels of Pb found in the drinking water at P < 0.05. Additionally, the average level of Cu in duck egg from large-scale farms correlated significantly with the level of Cu found in the feeds at P < 0.001. Furthermore, from a calculation of the provisional tolerable daily intake (WHO-FAO) of heavy metals in this study, it was concluded that consumers face health risks from Cd contamination. Thus, heavy metal contamination, especially Pb and Cd in duck egg, must be of concern due to the health risks and the route of crucial heavy metals contamination should be elucidated and long - term monitoring of heavy metals posing health effects in farm systems should be carried out. [ABSTRACT FROM AUTHOR]

High stocking density (HSD) and heat stress (HS) challenge broiler production. While antibiotics can mitigate the adverse effects of HS and HSD, their restricted use underscores the need to explore phytochemicals, particularly their combined effects under such conditions. This study investigated the influence of flavonoids, isoquinoline alkaloids, and their combinations as alternatives to bacitracin on growth performance, inflammatory status, gut morphology, and ceca microbiome in broilers raised under HSD and HS. A total of 2100 one-day-old male Ross 308 broiler chicks were distributed into 70 replicates, randomly assigned to one of seven dietary treatments and raised during the summer for 37 days. The treatments included normal stocking density (NSD, 10 birds/m 2 ); HSD (15 birds/m 2 ); HSD + 50 ppm of bacitracin (BCT); HSD + 300 ppm of flavonoids (FVNs); HSD + 80 ppm of isoquinoline alkaloids (IQAs); HSD + FVNs (1-10 days) and IQAs (11-37 days) (FVN-IQA); and HSD + IQAs (1-10 days) and FVNs (11-37 days) (IQA-FVN). The HS index reached or exceeded 160 during most of the experimental period. From 11 to 24 days of age, the HSD and BCT birds had lower body weight gain. The FVNs, IQAs, and their combinations decreased the corticosterone, IL-6, malondialdehyde, and heterophil-lymphocytes ratio compared to the HSD. Jejunal, ileal, and duodenal villi height/crypt depth ratio was lower in HSD than in other treatments except BCT. The α- and β-diversity, microbiota composition, and metabolic pathways were affected by treatment groups. Overall, FVNs, IQAs, and their combinations improved the growth performance, anti-inflammatory response, and gut health in broilers under HSD and HS, with the combinations exerting synergistic effects.

This study investigated the effect of isoquinoline alkaloids as an alternative to bacitracin on growth performance, oxidative stress, inflammatory status, and ceca microbiome of broilers raised under high stocking density (HSD). A total of 1,500 one-day-old male Ross 308 chicks were randomly assigned to five treatment groups, with 10 replicate pens per group and 30 birds per pen, for 37 days. The treatments included normal stocking density (NSD, 10 birds/m²), HSD (15 birds/m²), HSD with 50 ppm Bacitracin (BCT50), HSD with 80 ppm isoquinoline alkaloids (IQA80), and HSD with 100 ppm isoquinoline alkaloids (IQA100). From days 11 to 24, HSD birds had lower feed efficiency (P < 0.05) compared to those in other treatments. The heterophil-to-lymphocyte ratio and malondialdehyde levels were lower in NSD and IQA80 birds compared to HSD and BCT50 birds (P < 0.05). HSD birds had higher IL-6 and a lower villus height and villus height-to-crypt depth ratio compared to birds in other groups (P < 0.05). Serum TNF-α was lower in NSD and IQA80 birds compared to those in the HSD group. Alpha diversity was not affected by the treatments; however, beta diversity was lower in HSD birds compared to other treatments. HSD birds showed reduced microbial diversity, with a higher prevalence of Enterococcaceae and Peptostreptococcaceae. NSD enhanced the abundance of Lactobacillaceae, Clostridiaceae, and Rikenellaceae. BCT50 increased and decreased the abundance of Enterococcaceae and Rikenellaceae respectively. IQA80 and IQA100 increased the abundance of Lachnospiraceae, Leuconostocaceae, and Coriobacteriaceae. HSD altered metabolic pathways related to carbohydrate and lipid metabolism, and amino acid biosynthesis. BCT50 modulated microbial functions, particularly those related to cell wall synthesis, while isoquinoline alkaloids upregulated pathways involved in energy production, secondary metabolite biosynthesis, and antioxidant production. Both Bacitracin and isoquinoline alkaloids were effective in mitigating the negative effects of HSD on immunity, gut health and microbiota in broilers. Given the concerns about antimicrobial resistance, isoquinoline alkaloids are a potent alternative to bacitracin, with IQA80 being particularly recommended., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Background and Aim: Ducks worldwide are infected with duck circovirus (DuCV), which causes feather abnormality, emaciation, and poor growth performance. DuCV is similar to other circoviruses that induce immunosuppression due to the occurrence of the bursae of Fabricius (BF) and spleen atrophies. In Thailand, retarded ducks with feather losses were submitted for disease investigation. The ducks presented low body weight gain, had small BF and spleens, and were consistent with duck-infected DuCV. Our study investigated the possibility of DuCV infection in duck flocks in Thailand. We also analyzed the genetic characteristics of the virus., Materials and Methods: BF and spleen samples were collected from affected meat and layer ducks from six farms thought to have been infected with DuCV. These tissues were then subjected to histopathological examination and molecular identification using conventional polymerase chain reaction and nucleotide sequencing. To identify DuCV, phylogenetic trees were generated using MEGA version X software. Samples of tissues or swabs were collected to determine whether coinfections with bacteria and viruses existed., Results: Phylogenetic analysis using the entire genome (1995-1996 bp) and cap gene (762 bp) revealed that the DuCV isolates circulating in Thailand belonged to DuCV genotype I, which was further subdivided into two sub-genotypes: sub-genotype I b and an unclassified sub-genotype based on reference sub-genotypes. Thai isolates have variations in 10 amino acid residues in the capsid protein. Ducks infected with Thai DuCV were also coinfected with Riemerella anatipestifer , Escherichia coli , Pasteurella multocida , duck viral enteritis, and duck Tembusu virus, which is consistent with previous DuCV infection studies., Conclusion: Six DuCVs from ducks who were previously found to have feather loss, were underweight, had growth retardation, and had poor body condition were identified in this study as belonging to genotype I and constituting at least two sub-genotypes. Due to the immunosuppressive effects of DuCV, coinfection of bacterial and viral pathogens was typically observed in Thai DuCV-infected ducks., Competing Interests: The authors declare that they have no competing interests., (Copyright: © Kulprasertsri, et al.)

Background and Aim: Marek's disease (MD) is a common lymphoproliferative disease affecting chickens and causing economic losses in commercial poultry. The MD outbreak was noticed in the southern part of Thailand in 2019. The suspected cases were found with an abnormal number of cases of layers dying with clinical signs, for example, weakness and emaciation, with evidence of MD gross lesions. This study aimed to raise awareness of the MD outbreak through value chain analysis (VCA), identifying associated possible risk factors, and estimating the associated economic impact., Materials and Methods: Value chain analysis, including seasonal calendar, value chain diagram, and layer movement mapping of the layer industry, was conducted. High-risk stakeholders were identified on the basis of risk practices and interactions between stakeholders. A case-control study was conducted to determine risk factors associated with the MD outbreak on layer farms, and partial budget analysis was used to estimate economic losses associated with MD., Results: The value chain diagram showed the linkages between stakeholders, including estimation of the percentage of products moved from one stakeholder group to another and the negotiated price. Fourteen out of 35 layer farms were case farms. Farm size and source of birds were significantly associated with the MD outbreak. The MD outbreak caused total economic losses of 295,823 USD. Farms that slaughtered infected birds with additional revenues incurred losses of 140,930 USD, whereas farms that culled infected birds without additional revenue returned incurred losses of 1995 USD., Conclusion: The VCA provided a better understanding of the layer and egg businesses in South Thailand and guided the development of questionnaires for outbreak investigation. The potential risk factor findings suggested the need for further exploration of the source of the MD outbreak., Competing Interests: The authors declare that they have no competing interests., (Copyright: © Dejyong, et al.)

Porcine epidemic diarrhea virus (PEDV) is the causative agent of a highly contagious enteric disease of pigs characterized by diarrhea, vomiting, and severe dehydration. PEDV infects pigs of all ages, but neonatal pigs during the first week of life are highly susceptible; the mortality rates among newborn piglets may reach 80-100%. Thus, PEDV is regarded as one of the most devastating pig viruses that cause huge economic damage to pig industries worldwide. Vaccination of sows and gilts at the pre-fertilization or pre-farrowing stage is a good strategy for the protection of suckling piglets against PEDV through the acquisition of the lactating immunity. However, vaccination of the mother pigs for inducing a high level of virus-neutralizing antibodies is complicated with unstandardized immunization protocol and unreliable outcomes. Besides, the vaccine may also induce enhancing antibodies that promote virus entry and replication, so-called antibody-dependent enhancement (ADE), which aggravates the disease upon new virus exposure. Recognition of the virus epitope that induces the production of the enhancing antibodies is an existential necessity for safe and effective PEDV vaccine design. In this study, the enhancing epitope of the PEDV spike (S) protein was revealed for the first time, by using phage display technology and mouse monoclonal antibody (mAbG3) that bound to the PEDV S1 subunit of the S protein and enhanced PEDV entry into permissive Vero cells that lack Fc receptor. The phages displaying mAbG3-bound peptides derived from the phage library by panning with the mAbG3 matched with several regions in the S1-0 sub-domain of the PEDV S1 subunit, indicating that the epitope is discontinuous (conformational). The mAbG3-bound phage sequence also matched with a linear sequence of the S1-BCD sub-domains. Immunological assays verified the phage mimotope results. Although the molecular mechanism of ADE caused by the mAbG3 via binding to the newly identified S1 enhancing epitope awaits investigation, the data obtained from this study are helpful and useful in designing a safe and effective PEDV protein subunit/DNA vaccine devoid of the enhancing epitope., Competing Interests: KT was employed by the MORENA Solution Company. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Thavorasak, Chulanetra, Glab-ampai, Mahasongkram, Sae-lim, Teeranitayatarn, Songserm, Yodsheewan, Nilubol, Chaicumpa and Sookrung.)

Duck tembusu virus (DTMUV) is an emerging duck pathogen in China and other Asian countries. It is unclear whether this emerging zoonotic infection poses a threat to humans. A previous study in 2012 showed surprisingly high rates of seropositivity and positive viral detection by RT-PCR in duck farm workers in China. To understand the nature of the threat of this emerging virus, we studied the neutralizing antibody response to a local isolate of DTMUV in an at-risk population, who were workers in duck farms and residents around farming areas in Central Thailand where DTMUV had been previously detected, and in a not-at-risk population, who were people living in the same or neighbouring province, but at a distance from the farms and who had no contact with ducks. The sera from the at-risk population showed higher anti-DTMUV neutralizing antibody titres as compared with those of the not-at-risk population. However, within the at-risk population, workers with direct contact with ducks did not show higher neutralizing titres than those without direct contact. Interestingly, some people in the not-at-risk group also displayed high neutralizing antibody titres to DTMUV. These sera were tested against other endemic Flaviviruses and showed no or low cross-reactivity suggesting the specificity of the neutralizing activity against DTMUV. These data raise a possibility of DTMUV as a potential zoonotic pathogen but the mode of transmission of the virus from ducks or other possible hosts to humans should be explored further., (© 2021 Wiley-VCH GmbH.)

Porcine epidemic diarrhea virus (PEDV) causes devastating enteric disease that inflicts huge economic damage on the swine industry worldwide. A safe and highly effective PEDV vaccine that contains only the virus-neutralizing epitopes (not enhancing epitope), as well as a ready-to-use PEDV neutralizing antibody for the passive immunization of PEDV vulnerable piglets (during the first week of life) are needed, particularly for PEDV-endemic farms. In this study, we generated monoclonal antibodies (mAbs) to the recombinant S1 domain of PEDV spike (S) protein and tested their PEDV neutralizing activity by CPE-reduction assay. The mAb secreted by one hybrodoma clone (A3), that also bound to the native S1 counterpart from PEDV-infected cells (tested by combined co-immunoprecipitation and Western blotting), neutralized PEDV infectivity. Epitope of the neutralizing mAb (mAbA3) locates in the S1A subdomain of the spike protein, as identified by phage mimotope search and multiple sequence alignment, and peptide binding-ELISA. The newly identified epitope is shared by PEDV G1 and G2 strains and other alphacoronaviruses. In summary, mAbA3 may be useful as a ready-to-use antibody for passive immunization of PEDV-susceptible piglets, while the novel neutralizing epitope, together with other, previously known protective epitopes, have potential as an immunogenic cocktail for a safe, next-generation PEDV vaccine.

Salmonella is an important foodborne bacterium that has become increasingly resistant to critical antimicrobial and disinfectant agents. The aim of this study was to characterize antimicrobial and disinfectant resistance of Salmonella spp. isolated from ducks raised for meat in Nakhon Pathom province, Thailand. A total of 694 fecal samples from ducks were collected in 2018. Of which, 85 samples were positive for Salmonella (12.2%), and 12 Salmonella serovars were identified from 125 Salmonella isolates. The Altona serovar was the predominant serotype found in this study (36.5%). All isolates showed resistance to at least one class of antimicrobial, and 23.2% displayed multidrug resistance (MDR) phenotype. The bla TEM , aadA2 , strA , and dfrA12 genes were detected in antibiotic-resistant strains of Salmonella , whereas the genes within a plasmid-borne qnr family that presented in fluoroquinolone-susceptible Salmonella strains were qnrB (3.8%) and qnrS (1.5%). The minimum inhibitory concentrations of benzalkonium chloride (BKC), cetylpyridium chloride (CPC), and hexadecyltrimethyl ammonium bromide (CTAB) ranged between 128 and 512 μg/mL, while that of didecyldimethylammonium chloride (DDAC) was between 32 and 128 μg/mL. The presences of qacEΔ1 , mdfA , sugE(c) , sugE(p) , and ydgE genes were less prevalent (0.8-1.6%). Taken together, our results indicate that duck is an important source of Salmonella with antimicrobial resistance in food-producing animals. Active surveillance programs for antimicrobial and disinfectant resistance in duck production are needed for an early detection of resistance strains of public health importance.

Zinc, Pb, Cd, Mn, Fe, Cr, and Cu levels in duck meat from large-scale farms have been found to be significantly higher than those from free-grazing duck farms. Zinc, Co, Mn, Cr, and Cu contamination levels in duck liver from large-scale farms were significantly higher than those from free-grazing farms; only Cd in duck liver from free-grazing farms was higher than in liver samples from large-scale farms at P < 0.05. Lead, Cd, Fe, and Cr levels in duck intestine samples from free-grazing farms were higher than large-scale farms at P < 0.001. Moreover, the average concentrations of Pb in duck meat and liver samples from large-scale farms and Cd levels in duck liver samples from free-grazing farm also exceeded the FAO/WHO and Codex Alimentarius limits by 100% (55/55), 100% (54/54), and 67.6% (23/34), respectively. PCA analysis showed a strong positive relationship between the eight metals in meat, liver, and intestine was > 0.69, > 0.69, and > 0.72, in order. The relationship of the liver combined with the intestine was > 0.65. This study indicated that consumers may incur health risks from long-term consumption of duck due to high Pb and Cd concentrations from both types of farms, particularly from large-scale duck farms.

We compared the antibody responses and persistence of the reduced-dose, 9 µg hemagglutinin (HA)/strain intradermal (ID) injection via the Mantoux technique and the 15 μg HA/strain intramuscular (IM) injection of the repeated annual identical trivalent, inactivated, split-virion vaccine 2011-2012 in chronic obstructive pulmonary disease (COPD) patients. Eighty patients were randomized to ID (n = 41) and IM (n = 39) groups. Four weeks post-vaccination, the antibody responses of the two groups were similar; those for influenza A(H1N1)pdm09 and influenza A(H3N2)-but not influenza B-met the criteria of the Committee for Proprietary Medicinal Products (CPMP). The antibody responses for influenza A(H1N1)pdm09 rapidly declined in both groups, especially with the ID injection, whereas those for influenza A(H3N2) maintained above the CPMP criteria throughout 12 months post-vaccination. The geometric mean titres for influenza A(H1N1)pdm09 persisted above the protective threshold (≥ 40) until 6 months post-vaccination in both the ID and IM groups. The seroprotection rates of the ID and IM groups were above 60% until 3 months and 6 months post-vaccination, respectively. In conclusion, the 9 μg HA/strain ID injection of vaccine 2011-2012 elicited antibody responses similar to the standard dose of 15 μg of the HA/strain IM injection at 4 weeks post-vaccination. However, the antibody responses for influenza A(H1N1)pdm09 rapidly declined, especially in the case of the ID injection, whereas they were comparable for influenza A(H3N2). Additional strategies for increasing vaccine durability should be considered, especially for new pandemic strains affecting elderly COPD patients.

Bovine coronavirus (BCoV) is involved mainly in enteric infections in cattle. This study reports the first molecular detection of BCoV in a diarrhea outbreak in dairy cows in the Central Region, Thailand. BCoV was molecularly detected from bloody diarrheic cattle feces by using nested PCR. Agarose gel electrophoresis of three diarrheic fecal samples yielded from the 25 samples desired amplicons that were 488 base pairs and sequencing substantiated that have BCoV. The sequence alignment indicated that nucleotide and amino acid sequences, the three TWD isolated in Thailand, were more quite homologous to each other (amino acid at position 39 of TWD1, TWD3 was proline, but TWD2 was serine) and closely related to OK-0514-3strain (virulent respiratory strain; RBCoV).The amino acid sequencing identities among TWD1, TWD2,TWD3, and OK-0514-3 strain were 96.0 to 96.6%, those at which T3I, H65N, D87G, H127Y, andQ136R were changed. In addition, the phylogenetic tree of the hypervariable region S1subunit spike glycoprotein BCoV gene was composed of three major clades by using the 54 sequences generated and showed that the evolutionally distance, TWD1, TWD2, and TWD3 were the isolated group together and most similar to OK-0514-3 strain (98.2 to 98.5% similarity). Further study will develop ELISA assay for serologic detection of winter dysentery disease.

The role of gender in the progression of fatty liver due to chronic high-fat high-fructose diet (HFFD) has not been studied. The present investigation assessed whether HFFD induced hepatic perturbations differently between the sexes and examined the potential mechanisms. Male, female, and ovariectomized (OVX) Sprague-Dawley rats were fed either a control diet or HFFD for 12 wk. Indexes of liver damage and hepatic steatosis were analyzed biochemically and histologically together with monitoring changes in hepatic gene and protein expression. HFFD induced a higher degree of hepatic steatosis in females, with significant increases in proteins involved in hepatic lipogenesis, whereas HFFD significantly induced liver injury, inflammation, and oxidative stress only in males. Interestingly, a significant increase in hepatic fibroblast growth factor 21 (FGF21) protein expression was observed in HFFD-fed males but not in HFFD-fed females. Ovarian hormone deprivation by itself led to a significant reduction in FGF21 with hepatic steatosis, and HFFD further aggravated hepatic fat accumulation in OVX rats. Importantly, estrogen replacement restored hepatic FGF21 levels and reduced hepatic steatosis in HFFD-fed OVX rats. Collectively, our results indicate that male rats are more susceptible to HFFD-induced hepatic inflammation and that the mechanism underlying this sex dimorphism is mediated through hepatic FGF21 expression. Our findings reveal sex differences in the development of HFFD-induced fatty liver and indicate the protective role of estrogen against HFFD-induced hepatic steatosis., (Copyright © 2017 the American Physiological Society.)

The antibody responses of a reduced-dose intradermal seasonal influenza vaccination have never been studied in COPD patients soon after a pandemic. A total of 149 COPD patients (60 y of age or older) were randomized to receive trivalent influenza vaccine (Sanofi-Pasteur, France) either 9 µg of hemagglutinin (HA) per strain split into 2-site intradermal (ID) injections via the Mantoux technique or one intramuscular (IM) injection of 15 µg of HA per strain. The geometric mean titers, seroconversion factors, seroconversion rates and seroprotection rates for influenza A(H3N2) and B administered through the ID injection (n = 75) were similar to those obtained with the IM injection (n = 74) 4 weeks post-vaccination. The antibody responses for influenza A(H1N1)pdm09 administered through the ID injection were lower than those obtained with the IM injection, but all of these responses met the 3 criteria proposed by the Committee for Proprietary Medicinal Products (CPMP) for annual re-licensure. The seroprotection rates 4 weeks post-vaccination for influenza A(H1N1)pdm09 were 64.0% (95%CI 52.7-74.0%) in the ID group vs. 78.4% (95% CI 67.6-86.3%) in the IM group (p = 0.053). Influenza-related acute respiratory illness (ARI), diagnosed as a 4-fold rise in HI titers with a convalescent titer > 1:40, and/or the RT-PCR between the ID group (5.3%) and the IM group (8.1%) were not significantly different. The reduced-dose intradermal influenza vaccine may expand vaccine coverage in cases of vaccine shortage.

A new anti-influenza remedy that can tolerate the virus antigenic variation is needed. Influenza virus matrix protein-1 (M1) is highly conserved and pivotal for the virus replication cycle: virus uncoating, assembly and budding. An agent that blocks the M1 functions should be an effective anti-influenza agent. In this study, human scFv that bound to recombinant M1 middle domain (MD) and native M1 of A/H5N1 was produced. Phage mimotope search and computerized molecular docking revealed that the scFv bound to the MD conformational epitope formed by juxtaposed helices 7 and 9 of the M1. The scFv was linked molecularly to a cell penetrable peptide, penetratin (PEN). The PEN-scFv (transbody), when used to treat the cells pre-infected with the heterologous clade/subclade A/H5N1 reduced the viral mRNA intracellularly and in the cell culture fluids. The transbody mitigated symptom severity and lung histopathology of the H5N1 infected mice and caused reduction of virus antigen in the tissues as well as extricated the animals from the lethal challenge in a dose dependent manner. The transbody specific to the M1 MD, either alone or in combination with the cognate human scFvs specific to other influenza virus proteins, should be an effective, safe and mutation tolerable anti-influenza agent.

Fowl adenovirus (FAdv) serotype 2 causes inclusion body hepatitis (IBH) disease which adversely affects the broiler industry in Thailand. We developed an indirect ELISA based on the recombinant hexon protein produced by E. coli. The recombinant hexon protein was tested with sera, in both infected and noninfected chickens. The recombinant hexon protein was standardized with an antigen concentration of 3.75 µg/ml and test sera. The intra- and inter-assays were repeatable. The cutoff value from TG-ROC curve analysis was 0.106. The specificity and sensitivity were 80 and 80%, respectively. The correlation coefficient (r) of absorbance values from this ELISA compared with the serum neutralization test was 0.76. This ELISA might be helpful for IBH diagnosis and surveillance.

Currently, there is a need of new anti-influenza agents that target influenza virus proteins other than ion channel M2 and neuraminidase. Non-structural protein-1 (NS1) is a highly conserved multifunctional protein which is indispensable for the virus replication cycle. In this study, fully human single chain antibody fragments (HuScFv) that bound specifically to recombinant and native NS1 were produced from three huscfv-phagemid transformed Escherichia coli clones (nos. 3, 10 and 11) selected from a human ScFv phage display library. Western blot analysis, mimotope searching/epitope identification, homology modeling/molecular docking and phage mimotope ELISA inhibition indicated that HuScFv of clone no. 3 reacted with NS1 R domain important for host innate immunity suppression; HuScFv of clone nos. 10 and 11 bound to E domain sites necessary for NS1 binding to the host eIF4GI and CPSF30, respectively. The HuScFv of all clones could enter the influenza virus infected cells and interfered with the NS1 activities leading to replication inhibition of viruses belonging to various heterologous A subtypes and type B by 2-64-fold as semi-quantified by hemagglutination assay. Influenza virus infected cells treated with representative HuScFv (clone 10) had up-expression of IRF3 and IFN-β genes by 14.75 and 4.95-fold, respectively, in comparison with the controls, indicating that the antibodies could restore the host innate immune response. The fully human single chain antibodies have high potential for developing further as a safe (adjunctive) therapeutic agent for mitigating, if not abrogating, severe symptoms of influenza., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: Novel effective anti-influenza agent that tolerates influenza virus antigenic variation is needed. Highly conserved influenza virus M2 protein has multiple pivotal functions including ion channel activity for vRNP uncoating, anti-autophagy and virus assembly, morphogenesis and release. Thus, M2 is an attractive target of anti-influenza agents including small molecular drugs and specific antibodies., Methods: Fully human monoclonal single chain antibodies (HuScFv) specific to recombinant and native M2 proteins of A/H5N1 virus were produced from huscfv-phagemid transformed E. coli clones selected from a HuScFv phage display library using recombinant M2 of clade 1 A/H5N1 as panning antigen. The HuScFv were tested for their ability to inhibit replication of A/H5N1 of both homologous and heterologous clades. M2 domains bound by HuScFv of individual E. coli clones were identified by phage mimotope searching and computerized molecular docking., Results: HuScFv derived from four huscfv-phagemid transformed E. coli clones (no. 2, 19, 23 and 27) showed different amino acid sequences particularly at the CDRs. Cells infected with A/H5N1 influenza viruses (both adamantane sensitive and resistant) that had been exposed to the HuScFv had reduced virus release and intracellular virus. Phage peptide mimotope search and multiple alignments revealed that conformational epitopes of HuScFv2 located at the residues important for ion channel activity, anti-autophagy and M1 binding; epitopic residues of HuScFv19 located at the M2 amphipathic helix and cytoplasmic tail important for anti-autophagy, virus assembly, morphogenesis and release; epitope of HuScFv23 involved residues important for the M2 activities similar to HuScFv2 and also amphipathic helix residues for viral budding and release while HuScFv27 epitope spanned ectodomain, ion channel and anti-autophagy residues. Results of computerized homology modelling and molecular docking conformed to the epitope identification by phages., Conclusions: HuScFv that bound to highly conserved epitopes across influenza A subtypes and human pathogenic H5N1clades located on different functional domains of M2 were produced. The HuScFv reduced viral release and intracellular virus of infected cells. While the molecular mechanisms of the HuScFv await experimental validation, the small human antibody fragments have high potential for developing further as a safe, novel and mutation tolerable anti-influenza agent especially against drug resistant variants.

Background: Validation of hemagglutination inhibition (HI) assays is important for evaluating antibody responses to influenza virus, and selection of erythrocytes for use in these assays is important. This study aimed to determine the correlation between receptor binding specificity and effectiveness of the HI assay for detecting antibody response to pandemic influenza H1N1 (pH1N1) virus., Methods: Hemagglutination (HA) tests were performed using erythrocytes from 6 species. Subsequently, 8 hemagglutinating units of pH1N1 from each species were titrated by real-time reverse transcription-PCR. To investigate the effect of erythrocyte binding preference on HI antibody titers, comparisons of HI with microneutralization (MN) assays were performed., Results: Goose erythrocytes showed most specific binding with pH1N1, while HA titers using human erythrocytes were comparable to those using turkey erythrocytes. The erythrocyte binding efficiency was shown to have an impact on antibody detection. Comparing MN titers, HI titers using turkey erythrocytes yielded the most accurate results, while those using goose erythrocytes produced the highest geometric mean titer. Human blood group O erythrocytes lacking a specific antibody yielded results most comparable to those obtained using turkey erythrocytes. Further, pre-existing antibody to pH1N1 and different erythrocyte species can distort HI assay results., Conclusions: HI assay, using turkey and human erythrocytes, yielded the most comparable and applicable results for pH1N1 than those by MN assay, and using goose erythrocytes may lead to overestimated titers. Selection of appropriate erythrocyte species for HI assay allows construction of a more reliable database, which is essential for further investigations and control of virus epidemics.

Background: Exposure to inadequate chemotherapy may alter cancer cell behavior including their metastatic potential. Because the molecular basis of such a phenomenon is largely unclear, we investigated the possible impact of cisplatin on anoikis response on human lung carcinoma cells., Materials and Methods: Using molecular and pharmacological tools, Caveolin-1 (CAV1) overexpressing and knock-down H460 cells were generated by stable transfection. The levels of CAV1 were determined by western blotting and reactive oxygen species (ROS) were detected by specific probes., Results: Sub-toxic concentrations of cisplatin suppressed anoikis response in H460 cells. The anoikis attenuation observed, was found to be caused by CAV1 up-regulation. Exposure to cisplatin induced superoxide anion and hydrogen peroxide generation; however, only hydrogen peroxide was found to be responsible for the CAV1 elevation., Conclusion: Exposure to cisplatin at sub-toxic concentrations induced hydrogen peroxide generation and the subsequent increase of ROS further regulated CAV1 levels and anoikis resistance. Our findings demonstrate a novel effect of cisplatin treatment on cancer cells which may lead to a better understanding of cancer biology and in the improvement of chemotherapy.

Background: Influenza A viruses are capable of crossing the specific barrier between human beings and animals resulting in interspecies transmission. The important factor of potential infectivity of influenza A viruses is the suitability of the receptor binding site of the host and viruses. The affinities of avian and human influenza virus to bind with the receptors and the distributions of receptors in animals are different., Objective: This study aims to investigate the anatomical distribution of avian and human influenza virus receptors using the double staining lectin histochemistry method., Methods: Double staining of lectin histochemistry was performed to identify both SA alpha2,3 Gal and SA alpha2,6 Gal receptors in trachea and lung tissue of dogs, cats, tigers, ferret, pigs, ducks and chickens., Results: We have demonstrated that avian and human influenza virus receptors were abundantly present in trachea, bronchus and bronchiole, but in alveoli of dogs, cats and tigers showed SA alpha2,6 Gal only. Furthermore, endothelial cells in lung tissues showed presence of SA alpha2,3 Gal., Conclusion: The positive sites of both receptors in respiratory tract, especially in the trachea, suggest that all mammalian species studied can be infected with avian influenza virus. These findings suggested that dogs and cats in close contact with humans should be of greater concern as an intermediate host for avian influenza A in which there is the potential for viral adaptation and reassortment.

A non-egg, non-culture based influenza vaccine that intervenes large influenza outbreaks and protects against heterosubtypic infections is needed. Candidates of such vaccine are likely to be conserved influenza virus proteins or their coding DNA. The vaccine must be conveniently produced at reasonable cost, safe, highly immunogenic and should be able to recall rapidly the immunological memory upon the antigenic re-exposure. In this study vaccines made of full length recombinant NP and M2 of the H5N1 influenza A virus were entrapped either alone or together into liposome (L) made of phosphatidylcholine and cholesterol. The vaccines (L-NP, L-M2 or L-NP+M2) and mocks (L or PBS) were safe without causing any adverse reaction in the intramuscularly injected mice. They were readily immunogenic at a single dose and a recalled response could be detected within one day post booster. Cytokine and antibody data indicated that the vaccines induced a Th1 bias immune response. NP containing vaccines stimulated a marked increase of cytotoxic lymphocytes, i.e., CD8(+), intracellular IFNγ(+) cells, while M2 containing vaccines elicited good antibody response which neutralized infectivity of heterologous influenza viruses. Although the three vaccines elicited different immunological defense factors; nevertheless, they similarly and readily abrogated lung histopathology mediated by viruses belonging to different H5N1 clade/subclade and heterosubtypes including swine H1N1 and human H1N1/2009 viruses. They protected the vaccinated mice against lethal challenges with mouse adapted avian H5N1 virus. The liposome adjuvanted vaccines which demonstrated high protective efficacy in mice warrant testing further in a non-rodent model as well as in humans., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The highly pathogenic avian influenza virus H5N1 is known to induce high level of tumor necrosis factor alpha (TNF-alpha) from primary macrophages. However, it is still unclear whether current H5N1 strains also induce high TNF-alpha production, as most of the data were derived from extinct clade 0 H5N1 strain. Here, we show that current clade 1 and 2 H5N1 strains induce variable levels of TNF-alpha that are not necessarily higher than those induced by seasonal influenza viruses. The result suggests that hyper-induction of TNF-alpha in human macrophages is not always associated with a highly pathogenic phenotype. We further tested the contribution of the NS gene segment from H5N1 isolates to TNF-alpha induction by using reverse genetics. While NS conferred some variation in TNF-alpha induction when incorporated into an H1N1 virus genetic background, it did not affect TNF-alpha induction in an H5N1 virus genetic background, suggesting that other viral genes are involved.

A cell penetrating format of human single chain antibody (HuScFv) specific to matrix protein (M1) of influenza A virus was produced by molecular linking of the gene sequence encoding the HuScFv (huscfv) to a protein transduction domain, i.e., penetratin (PEN) of the Drosophila homeodomain. DNA of a recombinant phagemid vector carrying the huscfv was used as a platform template in a three-step PCR for generating a nucleotide sequence encoding a 16 amino acid PEN peptide. The PEN-HuScFv had negligible cytotoxicity on living MDCK cells. They were readily translocated across the cell membrane and bound to native M1 in the A/H5N1-infected cells as revealed by immunofluorescent confocal microscopy. The PEN-HuScFv, when used to treat the influenza virus infected cells, reduced the number of viruses released from the cells. In conclusion, the cell penetrating M1-specific HuScFv, a transbody, produced in this study affected the influenza A virus life cycle in living mammalian cells. While the molecular mechanisms of the PEN-HuScFv need more investigation, the reagent warrants further testing in animals before developing it into a human immunotherapeutic anti-influenza formula.

A double-antigen sandwich ELISA was developed for the detection of antibodies to influenza A viruses. A recombinant nucleoprotein (rNP) of influenza A virus was used as a capture antigen and an HRP-conjugate for detecting the antibodies. A total of 125 serum samples from birds of different species including chickens, geese, open-billed storks, Khaki Campbell ducks, lesser whistling ducks, and pigeons with known antibodies were tested by ELISA. The sensitivity and the specificity of ELISA were found to be 98% and 97.3%, respectively. The assay was able to detect the presence of influenza A antibodies as early as the fourth day post-inoculation in ducks infected experimentally with influenza A (H5N1) virus. Excellent agreement (97.6%) was obtained between this sandwich ELISA and the hemagglutination inhibition (HI) tests (kappa=0.95). The double-antigen sandwich ELISA correlated well with a commercial avian influenza (AI) multispecies ELISA and was slightly more sensitive than the AI multispecies ELISA. These findings indicate that the double-antigen sandwich ELISA based on rNP may offer an effective screening method for serodiagnosis of influenza A virus. The double-antigen sandwich ELISA also enables the detection of antibodies to influenza A viruses in different species without the need for species-specific secondary antibodies., (2009 Elsevier B.V. All rights reserved.)

Highly pathogenic avian influenza (HPAI) H5N1 viruses have seriously affected the Asian poultry industry since their occurrence in 2004. Thailand has been one of those countries exposed to HPAI H5N1 outbreaks. This project was designed to compare the molecular evolution of HPAI H5N1 in Thailand between 2004 and 2008. Viruses with clade 1 hemagglutinin (HA) were first observed in early 2004 and persisted until 2008. Viruses with clade 2.3.4 HA were first observed in the northeastern region of Thailand between 2006 and 2007. Phylogenetic analysis among Thai isolates indicated that clade 1 viruses in Thailand consist of three distinct lineages: CUK2-like, PC168-like, and PC170-like viruses. The CUK2-like virus represents the predominant lineage and has been circulating throughout the course of the 4-year outbreaks. Analysis of recently isolated viruses has shown that the genetic distance was slightly different from viruses of the early outbreak and that CUK2-like viruses comprise the native strain. Between 2005 and 2007, PC168-like and PC170-like viruses were first observed in several areas around central and lower northern Thailand. In 2008, viruses reassorted from these two lineages, PC168-like and PC170-like viruses, were initially isolated in the lower northern provinces of Thailand and subsequently spread to the upper central part of Thailand. On the other hand, CUK2-like viruses were still detected around the lower northern and the upper central part of Thailand. Furthermore, upon emergence of the reassorted viruses, the PC168-like and PC170-like lineages could not be detected, suggesting that the only predominant strains still circulating in Thailand were CUK2-like and reassorted viruses. The substitution rate among clade 1 viruses in Thailand was lower. The virus being limited to the same area might explain the lower nucleotide substitution rate. This study has demonstrated that nationwide attempts to monitor the virus may help curb access and propagation of new HPAI viral genes.

Matrix protein (M1) is predominant and has pivotal role in the influenza A virus replication and assembly. It is therefore an attractive target for antiviral drugs, siRNA studies, and therapeutic antibodies. Nevertheless, therapeutic antibody that interferes with the M1 multiplex function has never been developed. In this study, human single monoclonal antibody fragments (HuScFvs) to M1 were generated. Full length recombinant M1 (rM1) was produced from cDNA prepared from genome of highly pathogenic avian influenza virus, A/H5N1. The rM1 was used as an antigen in phage bio-panning to select phage clones displaying HuScFv from a human antibody phage display library. Several phage clones displaying HuScFv bound to the rM1 and harboring the respective huscfv gene inserts were isolated. RFLP experiments revealed multiple DNA banding patterns which indicated epitope/affinity diversity of the HuScFv. The HuScFv were tested for their binding to native M1 of homologous and heterologous influenza A viruses using ELISA as well as incorporating immunostaining and immunofluorescence studies with infected MDCK cells. One such protein produced from a selected phage clone blocked binding of M1 to viral RNA. The HuScFv in their in vivo functional format, e.g. cell-penetrating molecules, should be developed and tested as a broad spectrum anti-A/influenza.

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.

Background: Human antibodies that interfere with the biological activity of haemagglutinins (HAs) of influenza viruses have high potential as an antiviral agent., Methods: Human single-chain antibody fragments (HuScFv) to recombinant and native HAs of the influenza virus H5N1 subtype were produced using a human antibody phage display library with the intention to increase the therapeutic arsenal against this highly pathogenic virus., Results: The HuScFv inhibited HA activity and neutralized infectivity of both homologous and heterologous strains and clades of the H5N1 subtype in Madin-Darby canine kidney cell cultures. Intraperitoneally injected HuScFv also mediated immunotherapeutic protection in mice that were intranasally challenged with highly pathogenic H5N1 viruses belonging to different strains and clades., Conclusions: Our data indicate that it might be worth pursuing these HuScFv further for future consideration as candidates for influenza intervention and treatment.

Native Thai chicken, an equatorial species breeds throughout the year, whereas turkeys are seasonal temperate zone breeder whose reproductive cycle is terminated by the onset of photorefractoriness. This study investigated VIPergic activity throughout a reproductive cycle in both species, hypothesizing that the differential expression of vasoactive intestinal peptide (VIP) would provide an insight into the differing reproductive strategies of the two species. Distribution of VIP neurons in the native Thai chicken and a comparison of VIPergic activity in the nucleus inferioris hypothalami (IH) and nucleus infundibuli hypothalami (IN) were investigated. VIP immunoreactivity was found throughout the native Thai chicken brain, predominantly located within the IH-IN. The pattern of VIP distribution in the native Thai chicken supports the findings reported in temperate zone species. Unlike the turkey, where there is a dissociation between VIPergic activity and prolactin levels during photorefractoriness, in the native Thai chicken, which do not express photorefractoriness, changes in VIP immunoreactive (VIP-ir) neurons within the IH-IN were directly correlated with prolactin throughout the reproductive cycle. VIPergic activity reached its lowest level after hatching of the chicks in the native Thai chicken, while in the turkey VIPergic activity was lowest only after exposure to a short day photoperiod and the acquisition of photosensitivity. This suggests that VIP neurons in the IH-IN may play a pivotal role in regulating the reproductive cycle and its differential expression following hatching of the young may, in part, account for the difference in reproductive mode between equatorial, continually breeding, non-photoperiodic birds and seasonally breeding, photoperiodic birds.

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.

Dopamine (DA) has a pivotal role in avian prolactin (PRL) secretion, acting centrally through D(1) DA receptors to stimulate PRL secretion by operating through vasoactive intestinal peptide (VIP). DA also inhibits PRL secretion by activating D(2) DA receptors at the pituitary level. This study was designed to investigate the distribution of DA neurons in the native Thai chicken, utilizing tyrosine hydroxylase (TH) as a marker for dopaminergic neurons. The differential expression of hypothalamic TH immunoreactive (TH-ir) neurons was also compared across the reproductive cycle. The results revealed that TH-ir neurons and fibers were found throughout the brain of the laying hen and were predominantly located within the diencephalon and mesencephalon. The observed distribution pattern of TH immunoreactivity was consistent with that reported previously in several avian species. However, changes in the number of TH-ir neurons in the nucleus intramedialis (nI) were observed across the reproductive cycle and correlated directly with variations in PRL levels. The population of TH-ir neurons in the nI increased significantly during the egg incubation period, where circulating PRL levels were the greatest. This study indicates, for the first time, that an association exists between DA neurons and the regulation of the reproductive system in the native Thai chicken. There is a paucity of information about the reproductive neuroendocrine regulation of tropical non-seasonally breeding avian species and it is suggested that the differential expression of DA neurons in the nI might play a role in the control of VIP secretion and subsequent PRL release in such birds.