Molecular characterization and antimicrobial susceptibility profiles of Thai Mycoplasma synoviae isolates.

Mycoplasma synoviae (MS) infection is mainly controlled by pathogen-free flocks' maintenance, medication in infected flocks, and vaccination in high-risk flocks. The effective control strategy requires convenient approach for detecting and differentiating MS strains and reliable drug susceptible evidence for deciding on reasonable antimicrobial usage. This study aimed to characterize the partial vlhA gene of nine Thai MS isolates circulated in chickens in 2020, to verify the PCR-RFLP assay for strain differentiation, and to determine the eight antimicrobial susceptibility profiles using microbroth dilution method. Based on sequence analysis of the partial vlhA gene, Thai MS isolates in 2020 were classified as types E and L with 19 and 35 amino acid lengths, respectively. The developed PCR-RFLP assay could detect and differentiate vaccine and Thai field strains. Most Thai MS isolates in this study were susceptible to tylosin, tylvalosin, tiamulin, doxycycline, oxytetracycline, tilmicosin, and lincomycin-spectinomycin at MIC50 values of 0.0391, 0.0098, 0.0781, 0.1563, 0.1563, 0.625 and 0.625 µg/mL, respectively; and resistance to enrofloxacin at MIC50 value of 10 µg/mL. In conclusion, this study revealed diagnostic assays for differentiating MS strains and the antimicrobial susceptibility profiles of Thai MS, which are necessary to design suitable MS control procedures for poultry flocks.

Genetic characterization of Marek's disease virus in chickens in Thailand reveals a high genetic diversity of circulating strains.

Marek's disease (MD) is a highly contagious lymphoproliferative disease of chickens caused by Gallid alphaherpesvirus 2, commonly known as serotype 1 Marek's disease virus (MDV-1). Despite widespread vaccination, MD-related cases have been frequently observed worldwide, including in Thailand. However, no information is available on the genetic characteristics of MDV-1 field strains circulating in chickens in Thailand. This study investigated the geographic distribution and genetic characteristics of MDV-1 field strains circulating in chickens in Thailand between 2013 and 2021 by analysing the Meq and pp38 genes. Out of a total of the 286 clinical samples obtained from 70 chicken farms located in major chicken raising areas of Thailand, 138 samples (48.25%) from 46 chicken farms (65.71%) tested positive for MDV-1 field strains. Results demonstrated that MDV-1 field strains were extensively distributed in major chicken raising areas. Phylogenetic analyses based on the Meq gene revealed that four clusters of MDV-1 circulated in chickens in Thailand between 2013 and 2021. Among these clusters, cluster 1 was the predominant cluster circulating in chickens in Thailand. Additionally, our findings based on molecular characteristics of Meq and pp38 gene/protein suggested that most of the Thai MDV-1 field strains were potentially highly virulent. In conclusion, our data demonstrated the circulation of different clusters of MDV-1 with virulence characteristics in chickens in Thailand, indicating high genetic diversity of MDV-1 in Thailand. This study highlights the importance of more effective vaccine development and routine MDV-1 surveillance for early detection and control of highly virulent MDV-1.

Live-Attenuated Oral Vaccines to Reduce Campylobacter Colonization in Poultry.

The control of Campylobacter in poultry at the pre-harvest level is critical to reducing foodborne infections with Campylobacter since the consumption of contaminated poultry is the most frequent cause of human campylobacteriosis. Although poultry vaccination is suggested as useful intervention measures, no Campylobacter vaccines are currently available. To develop live-attenuated oral Campylobacter vaccines, in this study, we evaluated the efficacy of pre-colonization by oxidative stress defense mutants, including knockout mutants of ahpC, katA, and sodB, in preventing Campylobacter jejuni from colonizing poultry. Interestingly, when chickens were pre-colonized with ΔahpC and ΔkatA mutants, rather than the ΔsodB mutant, the level of C. jejuni colonization was significantly reduced within 35 days. Further studies demonstrated when chickens were pre-colonized with the ΔahpC mutant by oral challenge with a high dose (ca., 5 × 108 CFU/bird) and a low dose (ca., 5 × 106 CFU/bird), it twice reduced the level of C. jejuni by 3.9 log10CFU/g feces and 3 log10CFU/g feces after 42 days, respectively, compared to the untreated control. Due to a colonization defect, the ΔahpC mutant was removed from chickens within 42 days. After excretion from the host, moreover, the ΔahpC mutant cannot survive in aerobic environments because of compromised aerotolerance. Our findings suggest that the ahpC mutant has a great potential for on-farm application to control C. jejuni at the pre-harvest level.

Evaluation and comparison of hemagglutination inhibition and indirect immunofluorescence tests for the detection of antibodies against duck Tembusu virus.

Currently, duck Tembusu virus (DTMUV), an emerging avian pathogenic flavivirus, is widely spread and becomes endemic in duck populations in Asia, causing significant economic losses in the duck producing industry. To early detection and control of DTMUV, the well-validated diagnostic tests for efficient detection of DTMUV infection in ducks are needed. In this study, we validated and compared hemagglutination inhibition (HI) and indirect immunofluorescence (IFA) tests for identifying antibodies against DTMUV in duck serum samples. Our results demonstrated that HI and IFA tests can both be used to detect antibodies against DTMUV in duck serum samples with high sensitivity (100%), specificity (>87%) and overall agreement with the gold standard serum neutralization (SN) test (>90%). Additionally, DTMUV-specific antibody titres determined by HI and IFA tests correlated well with the neutralizing antibody titres obtained by SN test. No cross-reactivity against common duck viruses and other flaviviruses was observed in both tests. It is interesting to note that HI test had higher diagnostic specificity and exhibited a stronger positive correlation with SN test than IFA test. Evaluating the performance of HI and IFA tests with experimental and field serum samples revealed that both tests showed comparable performance with SN test in terms of antibody kinetic and detection rate. Collectively, these findings support the use of both tests, particularly HI test, as the alternative to SN test for measuring the antibody responses against DTMUV in ducks. These tests could be the suitable choices for DTMUV diagnosis, epidemiological study and vaccine efficacy evaluation.

Patterns of duck Tembusu virus infection in ducks, Thailand: a serological study.

Duck Tembusu virus (DTMUV), a mosquito-borne flavivirus, has been identified as a causative agent of an emerging viral disease in ducks, causing significant economic losses to the duck-producing industry. In Thailand, DTMUV has been detected sporadically in ducks since the first report in 2013. However, information on the patterns of DTMUV infection in ducks in Thailand is limited. In this study, a serological survey of DTMUV on ducks raised in farming and free-grazing systems was conducted during 2015-2016. Blood samples of farm ducks (n = 160) and free-grazing ducks (n = 240) were collected in the summer, rainy, and winter seasons during 2015-2016 and tested for DTMUV infection. Our results showed that DTMUV infection in ducks in Thailand occurred all year-round; however, the patterns of DTMUV infection varied between 2 duck-raising systems. Significant seasonal pattern was found in free-grazing ducks, whereas no seasonality was observed in farm ducks. Notably, DTMUV infection in ducks in Thailand was highest in the winter season. In conclusion, our data indicate distinct patterns of DTMUV infection between farm and free-grazing ducks, and the year-round circulation of DTMUV in ducks in Thailand, with peaks in the winter season. This information will help reduce the risk of DTMUV transmission through prevention and control strategies focusing on the peak period. Routine surveillance of DTMUV in ducks is essential for early detection of DTMUV allowing the implementation of control measures in a timely manner.

Genetic characterization of infectious bronchitis viruses in Thailand, 2014-2016: identification of a novel recombinant variant.

Infectious bronchitis (IB) causes severe economic losses to the poultry industry worldwide owing to frequent emergence of novel infectious bronchitis virus (IBV) variants, which potentially affect the effectiveness of the currently used IBV vaccine. Therefore, continuous monitoring of IBV genotypes and lineages recently circulating in chickens worldwide is essential. In this study, we characterized the complete S1 gene from 120 IBVs circulating in chickens in Thailand from 2014 to 2016. Phylogenetic analysis of the complete S1 gene of 120 Thai IBVs revealed that the 2014-2016 Thai IBVs were divided into 3 lineages (GI-1, GI-13, and GI-19) and a novel IBV variant. Our results also showed that GI-19 lineage has become the predominant lineage of IBV circulating in chicken flocks in Thailand from 2014 to 2016. It is interesting to note that a novel IBV variant, which was genetically different from the established IBV lineages, was identified in this study. The recombination analysis demonstrated that this novel IBV variant was a recombinant virus, which was originated from the GI-19 and GI-13 lineage viruses. In conclusion, our data demonstrate the circulation of different lineages of IBV and the presence of a novel recombinant IBV variant in chicken flocks in Thailand. This study highlights the high genetic diversity and continued evolution of IBVs in chickens in Thailand, and the importance of continued IBV surveillance for effective control and prevention of IB.

Genetic characterization of reticuloendotheliosis virus in chickens in Thailand.

Reticuloendotheliosis virus (REV) causes an immunosuppressive, runting, and oncogenic disease in poultry, posing a significant threat to the poultry industry. In Thailand, an unidentified disease associated with runting-stunting syndrome and neoplasia, resembling REV infection, has been continuously observed in several chicken farms. However, REV infection in Thailand has never been reported. In this study, we investigated the occurrence and genetic characteristics of REVs in chickens in Thailand from 2013 to 2016. Of the 130 clinical samples obtained from 29 chicken farms from 9 provinces located in the major chicken-raising regions of Thailand, including the central, eastern, northern, and northeastern parts of Thailand, 51 samples (39.23%) and 21 farms (72.41%) were REV-positive. REV-positive samples were detected in all 9 provinces tested. Our results demonstrated that REV was extensively distributed in the major chicken-raising regions of Thailand. Phylogenetic analysis of the whole genome sequence showed that Thai REV was most closely related to Chinese, Taiwanese, and the US REV strains isolated from different avian species and clustered into REV subtype III. This finding indicates that REV subtype III was predominantly circulated in Thai chicken flocks. This study is the first report on REV infection in chickens in Thailand. Our findings raise the awareness of REV as another causative agent of runting and oncogenic disease in chickens in Thailand and highlight the wide distribution of REV infection among chickens worldwide.

Serological evidence of duck Tembusu virus infection in free-grazing ducks, Thailand.

Duck Tembusu virus (DTMUV) has been reported in ducks raised in farming system since its emergence in 2010. No information is available on DTMUV infection in free-grazing ducks, which are commonly raised and widespread in several Asian countries. To determine the presence of DTMUV infection in free-grazing ducks in Thailand, retrospective serum samples collected from 1,000 free-grazing ducks during 2008-2015 were tested for DTMUV infection. Our result showed that 91 (9.10%) were positive for DTMUV neutralizing antibodies and DTMUV seropositive ducks have been detected in Thailand since 2008. To further investigate the seroprevalence and geographic distribution of DTMUV infection in free-grazing ducks in Thailand, a cross-sectional serological survey of DTMUV was conducted in 2016. Of 1,200 free-grazing ducks in the 60 flocks from 20 provinces located in the major free-grazing duck raising areas of Thailand, 365 (30.42%) were positive for DTMUV neutralizing antibodies and 56 flocks (93.33%) had at least one DTMUV seropositive duck. Additionally, DTMUV seropositive ducks were observed in all provinces tested. In conclusion, our data demonstrated the presence of DTMUV infection in free-grazing ducks since 2008 and widespread DTMUV infection in free-grazing ducks in Thailand with a relatively high seroprevalence. These findings suggest the potential role of free-grazing ducks in the dissemination of DTMUV and highlight the necessity of systemic DTMUV surveillance in free-grazing ducks in addition to farm ducks for early detection, prevention, and control of this emerging disease.

The Efficacy of Chitosan-Adjuvanted, Mycoplasma gallisepticum Bacterin in Chickens.

Mycoplasma gallisepticum (MG) is one of the major pathogens that cause respiratory signs in the poultry industry. To control MG infection, vaccination is the useful procedure. In this study, MG vaccine was developed by using the local Thai MG isolate (AHRL 20/52). Chitosan, a polysaccharide adjuvant derived from crustaceans, has been successfully used in various vaccines. The objectives of this study were to prepare MG bacterins by using chitosan, serving as an adjuvant, to determine protection against the field Thai MG isolate and to evaluate tissue reaction at the injection site. Six groups of 6-wk-old layers (20 birds/group) were intramuscularly vaccinated with bacterin containing various concentrations of chitosan (0.25, 0.5, and 1%), a commercially available MG bacterin, respectively. Sham-negative and sham-positive controls were included in the experiment. Six weeks postvaccination, all groups excluding the negative control were intratracheally challenged with 100 µl of 108 colony-forming units of Thai MG isolate (AHRL 58/46). At 1, 2, 3, and 4 wk postchallenge, five birds from each group were euthanatized and necropsied to determine the gross and histopathologic lesions. For a tissue reaction study, three groups of 24 birds each including sham negative control, 0.5% chitosan bacterin and commercial vaccine were given as previously described. At 1, 2, and 3 wk postvaccination, 8 birds from each group were randomly selected to euthanatize, necropsy, and determine the gross lesions, and 3 out of 8 birds were randomly selected to determine the histopathologic lesions. The results demonstrated that prepared bacterins induced lower numbers of positive antibody birds compared to the commercial vaccine, but groups receiving bacterin containing 0.5 and 1% chitosan exhibited significantly lower tracheal lesions than the positive control and commercial vaccine groups (P < 0.05). Chitosan formulation caused less tissue reaction than the commercial vaccine. These results demonstrated that the prepared MG bacterins could effectively reduce MG-induced pathologic lesions and that chitosan could be used as adjuvant in MG bacterins.

Characterization of Thai Mycoplasma synoviae Isolates by Sequence Analysis of Partial vlhA Gene.

Mycoplasma synoviae (MS), a remarkable pathogen in poultry, causes subclinical infection of the upper respiratory tract and an infectious synovitis, especially in the tendon sheaths and synovial membranes of joints. Because the specific detection of MS 16S rRNA gene-based PCR was unsuitable for strain differentiation, vlhA gene-based PCR was designed to differentiate the MS strains. The vlhA gene of MS encodes for hemagglutinin and other immunodominant membrane proteins involved in colonization, antigenic variations, and virulence. Sequence analysis of the vlhA gene based on the nucleotide insertion/deletion of the proline-rich repeat (PRR) region and the nucleotide polymorphisms of the RIII region in vlhA gene fragments was useful for typing and subtyping of MS strains. This study aimed to characterize the Thai MS field isolates and to differentiate the field and vaccine strains in Thailand by using sequence analysis of the partial vlhA gene. In total, 20 MS field isolates submitted from registered chicken farms in Thailand during 2015 were identified as Type C1 (n = 1), C2 (n = 4), E1 (n = 9), E2 (n = 1), and L (n = 5). The results revealed that six of the nine isolates resulting in respiratory signs were Type E1. In addition, four isolates from lame chickens showing joint swelling were identified as Type L, with a length of 105 nucleotides. This study provides the first molecular data of Thai MS isolates and the first evidence of Type L for being an arthropathic strain that differs from a previous study demonstrating that only MS Type B, with a longer PRR of 135 nucleotides, could be highly invasive strains and associated with infectious synovitis in chickens. Furthermore, one farm showed coinfection of MS Types E and L, but most of the farms were affected by only one type of MS. The results indicated that sequence analysis of the partial vlhA gene can be used as a tool for tracing MS characterization.

The efficacy of artesunate, chloroquine, doxycycline, primaquine and a combination of artesunate and primaquine against avian malaria in broilers.

The efficacy of 5 antimalarial drugs was evaluated on P. gallinaceum infected broilers. One hundred and forty-seven 19-day-old broilers were divided into 7 groups of 21 chicks each. Group 1 was the unmedicated, uninfected control. Groups 2-6 were infected and medicated with artesunate, chloroquine, doxycycline, primaquine and an artesunate-primaquine combination, respectively. Group 7 was the unmedicated, infected control. Infectivity, mortality, parasitemia, schizonts in tissues and body weight gain were monitored. The results revealed that the two most effective drugs for treating P. gallinaceum at the asexual erythrocyte stage were chloroquine and doxycycline. Tissue schizonts of P. gallinaceum in all the medicated groups were significantly fewer than the unmedicated, infected control (P<0.05). The mortality rate of all the medicated groups was significantly lower than the unmedicated, infected control (P<0.05).

The effect of a catastrophic flood disaster on livestock farming in Nakhon Sawan province, Thailand.

In 2011, a catastrophic flood disaster in Thailand affected not only humans but also took animal lives. Data on livestock losses, including death, loss, and decreased production, were collected in Nakhon Sawan province. The time-series map of the flooded area from August to December 2011 was available online from the Geo-informatics and Space Technology Development Agency. To evaluate the high-density areas of livestock loss, a spatial hot spot analysis was performed. The Getis-Ord Gi statistic with weighted zone of indifference and the Euclidean distance measurement were employed to identify spatial clusters of species that were affected by the flood. The results indicated that the majority of livestock losses were from poultry and swine farms. The density of poultry and swine loss was significantly different between sub-districts with clusters of high-density loss alongside the river, particularly in Chum Saeng and Kao Liew. Using spatial hot spot analysis as a tool to classify and rank the areas with high flood risks provides an informative outline for farmers to be aware of potential flood damage. To avoid unexpected loss from flooding, poultry and swine farms in risk areas should be properly managed, particularly during the flooding season between August and December.

Comparative study of pandemic (H1N1) 2009, swine H1N1, and avian H3N2 influenza viral infections in quails.

Quail has been proposed to be an intermediate host of influenza A viruses. However, information on the susceptibility and pathogenicity of pandemic H1N1 2009 (pH1N1) and swine influenza viruses in quails is limited. In this study, the pathogenicity, virus shedding, and transmission characteristics of pH1N1, swine H1N1 (swH1N1), and avian H3N2 (dkH3N2) influenza viruses in quails was examined. Three groups of 15 quails were inoculated with each virus and evaluated for clinical signs, virus shedding and transmission, pathological changes, and serological responses. None of the 75 inoculated (n = 45), contact exposed (n = 15), or negative control (n = 15) quails developed any clinical signs. In contrast to the low virus shedding titers observed from the swH1N1-inoculated quails, birds inoculated with dkH3N2 and pH1N1 shed relatively high titers of virus predominantly from the respiratory tract until 5 and 7 DPI, respectively, that were rarely transmitted to the contact quails. Gross and histopathological lesions were observed in the respiratory and intestinal tracts of quail inoculated with either pH1N1 or dkH3N2, indicating that these viruses were more pathogenic than swH1N1. Sero-conversions were detected 7 DPI in two out of five pH1N1-inoculated quails, three out of five quails inoculated with swH1N1, and four out of five swH1N1-infected contact birds. Taken together, this study demonstrated that quails were more susceptible to infection with pH1N1 and dkH3N2 than swH1N1.

Serovar identification, antimicrobial sensitivity, and virulence of Avibacterium paragallinarum isolated from chickens in Thailand.

Avibacterium paragallinarum causes infectious coryza in chickens, an acute respiratory disease that has worldwide economic significance. The objectives of this study were to determine the serovars, antimicrobial resistance, and pathogenicity of A. paragallinarum isolated from chickens in Thailand. Eighteen field isolates of A. paragallinarum were confirmed by PCR. When examined by serotyping in a hemagglutination inhibition test, 10 isolates were serovar A, five isolates were serovar B, and three isolates were serovar C. The susceptibility of the isolates to 16 antimicrobial agents was tested by a disk diffusion method. All isolates were susceptible to amoxicillin-clavulanic acid. There was a high level of resistance to lincomycin and erythromycin. All isolates were resistant to cloxacillin and neomycin. A study of bacterial entry into, and survival within, chicken macrophages showed variation between isolates but no clear connection to serovar. A virulence test was performed by challenging 4-wk-old layers via the nasal route with 400 dl of bacteria (10(8) colony-forming units/ml). Clinical signs were observed daily for 7 days, and the birds were subjected to a postmortem necropsy at 7 days postchallenge. All 18 field isolates caused the typical clinical signs of infectious coryza and could be re-isolated at 7 days after challenge. There was no significant difference in the clinical scores of the isolates except that two isolates (112179 and 102984, serovars A and B, respectively) gave a significantly higher score than did isolate CMU1009 (a serovar A isolate). No correlation between serovar and severity of clinical signs was found.

Quail as a potential mixing vessel for the generation of new reassortant influenza A viruses.

Quail has been proposed as one of the intermediate hosts supporting the generation of newly reassortant influenza A viruses (IAVs) with the potential to infect humans. To evaluate the role of quail as an intermediate host of IAVs, co-infections of quail with swine-origin pandemic H1N1 2009 (pH1N1) and low pathogenic avian influenza (LPAI) duck H3N2 (dkH3N2) viruses (n=10) or endemic Thai swine H1N1 (swH1N1) and dkH3N2 viruses (n=10) were conducted. Three additional groups of five quail were each inoculated with pH1N1, swH1N1 and dkH3N2 as control groups to verify that each virus can infect quail. Our result showed that co-infected quail shed higher viral titers from the respiratory tract than single virus infected quail. This study confirmed that reassortant viruses could be readily generated in the respiratory tract of quail from both the pH1N1/dkH3N2 co-infected group (100% of quail generating reassortant viruses) and the swH1N1/dkH3N2 (33% of quail generating reassortant viruses) co-infected group without discernible clinical signs. The reassortment efficacy between the two combination of viruses was different in that the frequency of reassortant viruses was significantly higher in pH1N1/dkH3N2 co-infected quail (21.4%) compared to swH1N1/dkH3N2 co-infected quail (0.8%), indicating that gene combinations in pH1N1 have a higher potential to reassort with dkH3N2 compared to swH1N1. In summary, our result confirmed that quail could be an intermediate host of IAVs for generating new reassortant viruses. Our finding highlights the importance of monitoring IAVs especially pH1N1 in quail.

Sequence analysis of S1 genes of infectious bronchitis virus isolated in Thailand during 2008-2009: identification of natural recombination in the field isolates.

During 2008-2009, fifteen field infectious bronchitis viruses (IBVs) were isolated from commercial chicken farms in Thailand. After sequencing of the complete S1 gene, phylogenetic analysis was performed and this found that the Thai IBV isolates were divided into three distinct groups, unique to Thailand (group I), QX-like IBV (group II), and Massachusetts type (group III). This finding indicated that the recent Thai IBVs evolved separately and that at least three groups of viruses are circulating in Thailand. The recombination analysis of the S1 gene demonstrated that the 5'-terminus of the group I was similar to isolate THA001 which was unique to Thailand, isolated in 1998 whereas the 3'-terminus was similar to the group II. Moreover, the analysis of the S1 gene of the group II showed that the 5'-terminus was similar to QXIBV, isolated in China whereas the remaining region at the 3'-terminus was similar to the Chinese strain JX/99/01. The results indicated that the recombination events occurred in the S1 gene between the field strains. Based on these facts, the field IBV in Thailand has undergone genetic recombination.

Genetic characterization of 2008 reassortant influenza A virus (H5N1), Thailand.

In January and November 2008, outbreaks of avian influenza have been reported in 4 provinces of Thailand. Eight Influenza A H5N1 viruses were recovered from these 2008 AI outbreaks and comprehensively characterized and analyzed for nucleotide identity, genetic relatedness, virulence determinants, and possible sites of reassortment. The results show that the 2008 H5N1 viruses displayed genetic drift characteristics (less than 3% genetic differences), as commonly found in influenza A viruses. Based on phylogenetic analysis, clade 1 viruses in Thailand were divided into 3 distinct branches (subclades 1, 1.1 and 1.2). Six out of 8 H5N1 isolates have been identified as reassorted H5N1 viruses, while other isolates belong to an original H5N1 clade. These viruses have undergone inter-lineage reassortment between subclades 1.1 and 1.2 and thus represent new reassorted 2008 H5N1 viruses. The reassorted viruses have acquired gene segments from H5N1, subclade 1.1 (PA, HA, NP and M) and subclade 1.2 (PB2, PB1, NA and NS) in Thailand. Bootscan analysis of concatenated whole genome sequences of the 2008 H5N1 viruses supported the reassortment sites between subclade 1.1 and 1.2 viruses. Based on estimating of the time of the most recent common ancestors of the 2008 H5N1 viruses, the potential point of genetic reassortment of the viruses could be traced back to 2006. Genetic analysis of the 2008 H5N1 viruses has shown that most virulence determinants in all 8 genes of the viruses have remained unchanged. In summary, two predominant H5N1 lineages were circulating in 2008. The original CUK2-like lineage mainly circulated in central Thailand and the reassorted lineage (subclades 1.1 and 1.2) predominantly circulated in lower-north Thailand. To prevent new reassortment, emphasis should be put on prevention of H5N1 viruses circulating in high risk areas. In addition, surveillance and whole genome sequencing of H5N1 viruses should be routinely performed for monitoring the genetic drift of the virus and new reassorted strains, especially in light of potential reassortment between avian and mammalian H5N1 viruses.

The inactivation of avian influenza virus subtype H5N1 isolated from chickens in Thailand by chemical and physical treatments.

The objectives of this study were to determine the survival of avian influenza virus (AIV) subtype H5N1 under various physical and chemical treatments, including disinfectants, temperature and pH. The highly pathogenic AIVs subtype H5N1 were isolated from internal organs of suspected chickens and were characterized by the inoculation into chicken embryonated eggs (CEEs), hemagglutination (HA) test, hemagglutination inhibition (HI) test, reverse transcriptase polymerase chain reaction (RT-PCR) and nucleotide sequencing of hemagglutinin (H) and neuraminidase (N) genes. Three H5N1 isolates, at the concentration of 10(9) 50% embryo lethal dose (ELD(50))/ml, were used for the determination of the survival of the virus under different chemical and physical treatments. The chemical treatments were performed by incubating the viruses with various types of disinfectants including glutaraldehyde (Glu), hydrogen peroxide, quaternary ammonium compounds (QAC), Glu+QAC, iodine, chlorine, formalin and phenol, at 25 and 37 degrees C, for 0, 5, 7, and 14 days. The physical treatments included incubation of the viruses at 55, 60, 65, 70 and 75 degrees C for 10, 15, 30, 45 and 60 min or pH 3, 5, 7, 9 and 12. The results revealed that AIV H5N1 reference viruses, 2004.1, CUK-2/04 and 2004.2, showed low or no resistance against Glu+QAC, chlorine and phenol at both tested temperatures. Incubations at 70 degrees C for 60 min or at least 75 degrees C for at least 45 min could effectively inactivate all of the isolates, whereas all ranges of pH could not inactivate any of them. In this study, CUK-2/04 was more resistant to the disinfectants, temperatures, and pH compared to the other isolates.

Efficacy of autogenous killed vaccine of Avibacterium paragallinarum.

The efficacy of killed vaccine of Avibacterium paragallinarum with mineral oil adjuvant and aluminum hydroxide gel adjuvant was tested for antibody titers and protection. The autogenous vaccines at a concentration of 10(10) colony-forming units (CFU)/ml were administered to 5-wk-old male layers by subcutaneous injection in the neck twice at a 3-wk interval. Each chicken was challenged with 10(8) CFU/ml in 400 microl of an homologous isolate of A. paragallinarum serotype A, IR1, at 4 wk after the second vaccination via the nasal route. Sera were collected and the antibodies were tested by the hemagglutination inhibition test. The results revealed that the autogenous mineral oil adjuvant vaccine provided the antibody titer significantly faster than the other groups (P < 0.05). The average antibody titers of the group vaccinated with autogenous mineral oil adjuvant vaccine were higher than those of the group vaccinated with autogenous aluminum hydroxide gel adjuvant vaccine. The protective ability of vaccines was assessed by infraorbital sinus swab after 5 days postchallenge. The autogenous vaccines prepared with mineral oil adjuvant and aluminum hydroxide gel adjuvant protected all the chickens after challenge. No bacteria were isolated from the infraorbital sinuses of chickens in either autogenous vaccine group with either high or low antibody titers. The commercial vaccines prepared from mineral oil or aluminum hydroxide gel adjuvant revealed some protection. This is in contrast to the unvaccinated control group, in which facial edema and serous nasal discharge was found, and bacteria could be isolated from all chickens in the group.