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 objective of the present study was to determine the potential for house flies (Musca domestica L.) (Diptera: Muscidae) to harbour the avian influenza (AI) H5N1 virus. Laboratory-reared flies were experimentally fed with a mixture containing the AI virus. Exposed flies were washed with brain-heart infusion broth and followed by 70% alcohol before preparation of whole fly homogenate. The homogenate was inoculated into six 10-day-old embryonated chicken eggs (ECEs). Allantoic fluids were collected to determine the virus using the haemagglutination (HA) test, reverse transcription-polymerase chain reaction (RT-PCR) or quantitative real-time RT-PCR (RRT-PCR). In the first experiment, ECEs that were inoculated with the 50 AI virus exposed fly homogenates died within 48 h and HA and RT-PCR were positive for AI virus. In the second experiment, ECEs that were inoculated with only one fly died with positive HA test and RT-PCR. In the last experiment, a group of exposed flies was collected at 0, 6, 12, 24, 36, 48, 72 and 96 h post-exposure. Fly homogenates of each time point were tested by virus titration in ECEs and RRT-PCR. Virus titres declined in relation to exposure time. Furthermore, RRT-PCR results were positive at any time point. The present study shows that the flies may harbour the AI virus and could act as a mechanical vector of the AI virus.

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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.

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A competitive enzyme-linked immunosorbent assay (cELISA) was developed for detection of turkey coronavirus (TCV) antibodies. The cELISA utilized a recombinant baculovirus (Autographa californica nuclear polyhedrosis virus)-expressed TCV nucleocapsid (N) protein and biotin-labeled TCV N protein-specific monoclonal antibody. Sensitivity and specificity of the cELISA for detection of TCV antibodies were determined by comparison with the indirect fluorescent antibody test (IFAT) with 1269 reference, experimentally derived, and field-origin sera. Sera with discordant cELISA and IFAT results were further evaluated by western immunoblot analyses. The cELISA detected antibodies specific for TCV and infectious bronchitis virus, a closely related coronavirus, but did not detect antibodies specific for other avian viruses. A high degree of concordance was observed between the cELISA and IFAT; sensitivity and specificity of the cELISA relative to IFAT were 92.9% and 96.2%, respectively. Western immunoblot analyses provided additional evidence of cELISA specificity. The findings indicate that the cELISA is a rapid, sensitive, and specific serologic test for detection of TCV antibodies in turkeys.

Enteropathogenic Escherichia coli (EPEC) previously were identified in poult enteritis-mortality syndrome (PEMS)-affected turkeys and associated as a cause of this disease. In the present study, the prevalence of EPEC in PEMS-affected turkeys was examined retrospectively with archived tissues and intestinal contents collected from 12 PEMS-affected turkey flocks in 1998. Formalin-fixed intestinal tissues were examined by light and electron microscopy for attaching and effacing (AE) lesions characteristic of EPEC, and frozen (-75 C) intestinal contents were examined for presence of EPEC. Escherichia coli isolates were characterized on the basis of epithelial cell attachment, fluorescent actin staining (FAS) test, and presence of E. coli attaching/effacing (EAE), shigalike toxin (SLT) type I, SLT II, and bundle-forming pilus (BFP) genes by polymerase chain reaction procedures. EPEC isolates were examined for pathogenicity and ability to induce AE lesions in experimentally inoculated young turkeys. AE lesions were identified by light microscopy in Giemsa-stained intestines from 7 of 12 PEMS-affected turkey flocks. Lesions consisted of bacterial microcolonies attached to epithelial surfaces with epithelial degeneration at sites of attachment and inflammatory infiltration of the lamina propria. Electron microscopy confirmed the identity of AE lesions in six of seven flocks determined to have AE lesions by light microscopy. EPEC were identified in 4 of 12 flocks on the basis of the presence of EAE genes a nd absence of SLT I and SLT II genes; all isolates lacked BFP genes. EPEC isolates produced AE lesions and variable mortality in turkeys coinfected with turkey coronavirus. In total, EPEC were associated with 10 of 12 (83%) naturally occurring PEMS cases on the basis of identification of AE lesions and/or EPEC isolates. These findings provide additional evidence suggesting a possible role for EPEC in the pathogenesis of PEMS.