Single-step multiplex reverse transcription-polymerase chain reaction for the detection and differentiation of QX-like infectious bronchitis virus from the Thai variant and vaccine strains H120, Ma5, and 4/91.

Background and Aim: QX-like infectious bronchitis virus (IBV) is a highly infectious avian coronavirus that causes respiratory and kidney disease. It is linked to increased mortality and loss of performance in infected chickens worldwide, including Thailand. Thus, a simple and rapid diagnostic method for the diagnosis of QX-like IBV is needed. This study aimed to develop a single-step multiplex reverse transcription-polymerase chain reaction (mRT-PCR) assay to detect and differentiate QX-like IBV from Thai IBV and vaccine strains used in the poultry industry (H120, Ma5, and 4/91). Materials and Methods: Primer sets specific for QX-like and Thai IBV were designed to target the S1 gene. The specificity of the technique was verified using nine isolates of QX-like IBV, four isolates of Thai IBV, and other avian viral respiratory pathogens. The detection limit was evaluated using a serial ten-fold dilution of QX-like and Thai IBV. Results: The results showed that single-step mRT-PCR could detect QX-like IBV and differentiate it from Thai IBV and the vaccine strains H120, Ma5, and 4/91. The limit of detection of the developed assay was 102.2 embryo infectious dose (EID)50/mL for QX-like IBV and 101.8 EID50/mL for Thai IBV. Interestingly, the developed assay could identify mixed infection by both IBVs in a single sample. Conclusion: The single-step mRT-PCR assay developed in this study can potentially discriminate QX-like IBV from Thai IBV and the vaccine strains H120, Ma5, and 4/91 in a single reaction. It is also suitable for use in all laboratories with access to conventional PCR equipment.

Full-length coding sequence analysis of genome segments A and B of infectious bursal disease virus in Thailand: identification of Chinese-like and recombinant virus in the field.

RESEARCH HIGHLIGHTS: For the first time, this work demonstrated a recombinant IBDV strain in Thailand.Two genogroups of IBDV were found in Thailand: including HLJ-504-like and recombinant virus.Analysis of the full coding sequence is essential for monitoring emerging variant IBDV.

Efficacy of common disinfection processes against infective spores (arthroconidia) and mycelia of Microsporum gallinae causing avian dermatophytosis.

Background and Aim: Microsporum gallinae is the major dermatophyte species that causes avian dermatophytosis. Disinfection plays an important role in controlling and preventing dermatophytosis; however, information about the effect of common disinfection processes on M. gallinae is limited. This study aimed to investigate the disinfection efficacy of ultraviolet (UV) irradiation, heat treatment, detergents, and germicides against infective spores (arthroconidia) and vegetative mycelia of M. gallinae. Materials and Methods: The minimum inhibitory and minimum fungicidal concentrations of benzalkonium chloride, chlorhexidine, ethanol, formaldehyde, glutaraldehyde, hydrogen peroxide, phenol, povidone-iodine, and sodium hypochlorite germicides against arthroconidia and mycelia of M. gallinae American type culture collection (ATCC) 90749 were determined by broth microdilution. Time-kill assays were used to determine the fungicidal efficacy of moist heat treatment, UV irradiation, commercially available detergents, and germicides. Results: There were no significant differences between the arthroconidia and mycelia growth stages of M. gallinae ATCC 90749 in the magnitude of the log10 cell reductions in the number of viable fungal cells induced by the disinfection treatments (all p > 0.05). Moist heat treatment at 40°C did not reduce the number of viable fungal cells at any time (1-60 min); however, treatment at 50°C for 25 min and either 60°C or 80°C for 5 min eliminated > 99.999% of viable fungal cells. Irradiation of fungal cultures with UVC and UVB at doses higher than or equal to 0.4 and 0.8 J/cm2, respectively, resulted in a 5-log10 reduction in the number of viable fungal cells, whereas UVA only reduced the number of viable fungal cells by < 2-log10 up to a dose of 1.6 J/cm2. All the tested detergents demonstrated minimal fungicidal effects with < 1-log10 reductions in the number of viable fungal cells at concentrations up to 8% w/v. All of the tested germicides eradicated the fungus after treatment for 1 min at 1-1000× minimum inhibitory concentration (MIC), except for hydrogen peroxide, which was not fungicidal after treatment for 20 min at 100× MIC. Conclusion: Moist heat treatment at temperatures greater than or equal to 50°C, UVC and UVB irradiation at doses higher than or equal to 0.4 and 0.8 J/cm2, respectively, and treatment with all tested germicides except hydrogen peroxide can be considered effective processes for disinfecting the fungus M. gallinae from the equipment employed in poultry farming. In contrast, commercially available detergents are not suitable for use as M. gallinae disinfectants.

Molecular investigation of S2-3a/3b-E-M-4b/4c-5a/5b-N gene of QX-like and variant genotype infectious bronchitis virus isolated in Thailand reveals a distinct E gene.

The QX-like infectious bronchitis virus (IBV) and variant genotype have been discovered worldwide including Thailand. In order to know the origin of QX-like and variant genotype IBV in Thailand, the genetic analysis on multiple genes was investigated. Seven IBVs including four QX-like and three variant genotype were randomly selected from IBVs isolated in Thailand during 2008 and 2010. Phylogenetic analysis of the S2-3a/3b-E-M-4b/4c-5a/5b-N gene showed that Thai QX-like and variant genotype IBV were grouped together in a separate branch from other IBV strains. The isolated IBVs shared nucleotide identities of 96-99.9% with each other. They exhibited a high level of similarity (93.8%) with KM91 strain in South Korea. Phylogenetic analysis of the S2 and 3a/3b gene showed a relationship to KM91 strain. The E gene was distinct from other IBV strains. The M, 4a/4b and 5a/5b gene were closely related to Massachusetts type. The N gene was classified into two groups which were a group of unique to Thailand (variant genotype) and a relationship with Massachusetts type (QX-like). Recombination analysis identified the occurrence of recombination events in the genome of viruses. These findings demonstrated that the QX-like IBV and variant genotype isolates in Thailand were the recombinant viruses. Thai QX-like IBV had a genetic relationship with KM91 strain, Massachusetts type and unknown IBV whereas variant genotype had a genetic relationship with Thai QX-like IBV and Connecticut strain.

Pathology and molecular characterization of Leucocytozoon caulleryi from backyard chickens in Khon Kaen Province, Thailand.

AIM: The aim of this study was to characterize Leucocytozoon caulleryi from backyard chickens in Khon Kaen Province, Thailand. MATERIALS AND METHODS: Tissue samples were collected from backyard chickens suspected to have leucocytozoonosis and subjected to histopathology examination. The BLAST Tool at NCBI GenBank (Basic Local Alignment Research Tools) (http://www.ncbi.nlm.nih.gov/BLAST) was used to identify the nucleotide sequence of the partial cytochrome c oxidase subunit I (cox I) gene. A Phylogenetic tree for analysis of L. caulleryi was constructed by using MEGA 7.0 software (https://www.megasoftware.net/). RESULTS: The necropsy results revealed the subcutaneous hemorrhages of pectoral muscles, multifocal hemorrhages of the thymus and pectoral muscles, hemorrhage of the proventriculus and peritoneal cavity, and megaloschizonts of the pancreas and intestine, including subcapsular hemorrhages of the liver. Microscopic examination revealed numerous megaloschizonts of Leucocytozoon spp. in the pectoral muscles, intestine, pancreas, and thymus. Molecular analysis of the partial cox I gene showed that the causal agent was closely related to L. caulleryi reported in Japan. CONCLUSION: From these results, L. caulleryi was determined to be the causal agent of leucocytozoonosis and was closely associated with L. caulleryi reported in Japan.

In Vivo Efficacy of Clove Essential Oil Ointment for Microsporum gallinae Avian Dermatophytosis-A Randomized Controlled Trial.

Avian favus (dermatophytosis) is a superficial mycosis caused by Microsporum gallinae in poultry. This disease is an important problem in poultry husbandry, but the standard antifungal treatment can leave drug residues in farm products. The aim of this study was to compare the efficacy of a clove essential oil ointment (3%, w/w) with commercially available ketoconazole cream (2%, w/w) for the treatment of M. gallinae infection in chickens. An in vitro time-kill assay showed that clove essential oil ointment reduced the number of viable M. gallinae ATCC 90749 by 99.99% within 1 hr. A randomized controlled trial showed that the therapeutic efficacy of clove essential oil ointment (3%, w/w) was noninferior to ketoconazole cream (2%, w/w) in M. gallinae-infected chickens. The percentage of completely recovered (culture-negative) animals in both treatment groups was 90% in day 35 after initial treatment. This study indicates that clove essential oil is suitable for preparation as an alternative topical treatment for avian dermatophytosis.

Eficacia in vivo de una pomada de aceite esencial de clavo para la dermatofitosis aviar por Microsporum gallinae: Ensayo controlado y aleatorio. El favus aviar (dermatofitosis) es una micosis superficial causada por Microsporum gallinae en la avicultura. Esta enfermedad es un problema importante en la avicultura, pero el tratamiento antifúngico estándar puede dejar residuos de medicamentos en los productos agrícolas. El objetivo de este estudio fue comparar la eficacia de una pomada de aceite esencial de clavo (3%, peso a peso; P/P) con la crema de ketoconazol disponible comercialmente (2%, P/P) para el tratamiento de la infección por M. gallinae en pollos. Un ensayo in vitro para determinar el tiempo de eliminación mostró que la pomada de aceite esencial de clavo redujo el número de M. gallinae ATCC 90749 viables en un 99.99% en una hora. Un ensayo controlado y aleatorio mostró que la eficacia terapéutica de la pomada de aceite esencial de clavo (3%, P/P) no fue inferior a la crema de ketoconazol (2%, P/P) en pollos infectados con M. gallinae. El porcentaje de animales completamente recuperados (con cultivo negativo) en ambos grupos de tratamiento fue del 90% en el día 35 después del tratamiento inicial. Este estudio indica que el aceite esencial de clavo es adecuado para su preparación como tratamiento tópico alternativo para la dermatofitosis aviar.

Developing an indirect ELISA based on recombinant hexon protein for serological detection of inclusion body hepatitis in chickens.

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.