Somsak Pakpinyo

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.

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.

Experimental Assessment of Houseflies as Vectors in Avian Influenza Subtype H5N1 Transmission in Chickens

SUMMARY In this study, laboratory-reared houseflies were experimentally exposed to the high pathogenicity avian influenza virus (HPAI) subtype H5N1 virus to evaluate the houseflies as vectors in HPAI-H5N1 virus transmission in chickens. One hundred and fifty houseflies (Musca domestica L.) were equally allocated into three groups. Groups 2 and 3 were exposed to the HPAI-H5N1 virus by allowing the flies to consume food containing the virus for 15 min, while the flies in group 1 were allowed to consume H5N1-free food and would serve as a negative control group. Group 2 flies were euthanatized immediately after H5N1 exposure, while group 3 were held at room temperature for 24 hr and euthanatized. The houseflies in the transmission of the HPAI-H5N1 virus were examined by challenging three groups of housefly homogenates into layer chickens via the oral drop. Morbidity and mortality were observed for 14 days, and virus shedding monitored via oropharyngeal swabs (OS) and cloacal swabs (CS), which were collected daily and determined by real-time reverse transcription-PCR and virus titration. Experimental challenge showed that all the chickens of groups 2 and 3 died within 7 days of inoculation. The OS had higher concentrations of virus than CS. Moreover, the chickens of group 2 had higher concentrations of virus shedding than the chickens of group 3. Immunohistochemistry detected the nucleoprotein of the type A influenza virus in all tissue samples collected, including the trachea, duodenum, pancreas, and brain. In summary, this study demonstrates that houseflies could serve as vectors in HPAI-H5N1 virus transmission in chickens under experimental conditions. RESUMEN Evaluación experimental de las moscas domésticas como vectores para la transmisión a los pollos del virus de la influenza aviar subtipo H5N1. En este estudio, moscas domésticas criadas en laboratorio se expusieron experimentalmente al virus de la influenza aviar de alta patogenicidad subtipo H5N1 para evaluar a estos insectos como vectores para la transmisión de este virus a los pollos. Ciento cincuenta moscas domésticas (Musca domestica L.) se asignaron de manera igual en tres grupos. Los grupos dos y tres fueron expuestos al virus de la influenza aviar alimentándolas por 15 minutos con alimento que contenía el virus, mientras que a las moscas en el grupo uno se les permitió consimir alimento libre del virus y servir como control negativo. A las moscas del grupo dos se les practicó la eutanasia inmediatamente después de la exposición con el virus de influenza aviar H5N, mientras que el grupo tres se mantuvo a temperatura ambiente por 24 horas antes de ser sometidas a la eutanasia. Las moscas domésticas se examinaron en la transmisión del virus de influenza aviar de alta patogenicidad H5N1 mediante el desafío de aves de postura con macerados de moscas por vía oral. Se observaron la morbilidad y la mortalidad por 14 días, y la eliminación viral se determinó mediante hisopos orofaríngeos y cloacales, los cuales fueron recolectados diariamente se analizaron por transcripción reversa y PCR en tiempo real además por titulación viral. El desafío experimental mostró que todos los pollos de los grupos dos y tres murieron dentro de los siete días después de la inoculación. Los hisopos orofaríngeos mostraron las concentraciones virales más altas en comparación con los hisopos cloacales. Además, los pollos del grupo 2 mostraron concentraciones más altas de eliminación viral en comparación con el grupo tres. Mediante inmunohistoquímica, se detectó a la nucleoproteína del virus de la influenza A en todas muestras de tejidos recolectadas, incluyendo la tráquea duodeno, páncreas y cerebro. En resumen, este estudio demuestra que las moscas domésticas pueden servir como vectores en la transmisión del virus de la influenza aviar de alta patogenicidad subtipo H5N1 a los pollos bajo condiciones experimentales.

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.

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

SUMMARY 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.

Chitosan-adjuvanted Mycoplasma gallisepticum bacterin via intraocular administration enhances Mycoplasma gallisepticum protection in commercial layers

ABSTRACT Mycoplasma gallisepticum (MG) causes respiratory signs and economic losses in the poultry industry. MG vaccination is one of the effective prevention and control measures that have been used around the world. Our previous study demonstrated that chitosan‐adjuvanted MG bacterin could effectively reduce pathological lesions induced by MG and that chitosan could be used as an adjuvant in MG bacterin. The present study determining the efficacy of MG bacterins against the Thai MG strain was based on vaccine programs. Seven groups (25 layers/group) were received MG bacterins containing 0.5% chitosan or a commercial bacterin via intramuscular (IM) or intraocular (IO) route at 6 and 10 wk of age. Sham‐negative and sham‐positive controls were groups 1 and 2, respectively. Group 3: IM route of chitosan bacterin followed by IM route of chitosan bacterin; group 4: commercial bacterin via IM route followed by chitosan bacterin via IO route; group 5: commercial bacterin via IM route followed by commercial bacterin via IM route; group 6: chitosan bacterin via IM followed by chitosan bacterin via IO route; and group 7: chitosan bacterin via IO route followed by chitosan bacterin via IO route were determined. At 16 wk of age, all groups, excluding group 1, were challenged intratracheally with 0.1 mL containing Thai MG strain 107 colony‐forming unit. At 17, 18, and 20 wk of age, 5 birds in each group were bled for serological testing and swabbed at the choanal cleft for the quantitative real‐time PCR assay, the euthanized and necropsied. The results showed that birds vaccinated with a commercial intramuscular bacterin followed by an intraocularly chitosan adjuvant bacterin showed the best protection against the MG challenge. The study indicated that chitosan could be the effective mucosal adjuvant and increased the effectiveness of MG bacterin.

Persistence of Chlamydia psittaci in Various Temperatures and Times

SUMMARY Chlamydia psittaci, an obligate intracellular gram-negative bacteria, causes an important zoonotic disease in humans, namely, psittacosis. The objective of this study was to determine the persistent viability of C. psittaci at various temperature conditions. The cloacal swab samples were collected from feral and racing pigeons to find a C. psittaci field strain. The bacterial isolation showed that 1.3% of feral pigeons were PCR positive, while all samples of racing pigeons were PCR negative. Also, bacterial characterization suggested that it belonged to genotype B, which had bacterial titers 3.2 and 3.89 log 50% lethal dose/ml, respectively. A bacterial persistence test was performed, and the results showed that C. psittaci could survive at 56 C for up to 72 hr. In conclusion, C. psittaci could be found in feral pigeons in central Thailand. The bacteria can survive in equatorial temperature areas. This study was the first to report that C. psittaci could survive and has infectivity at 56 C for 72 hr. Therefore, awareness of C. psittaci infection in humans is necessary and should be a public health concern.

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

SUMMARY 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.