Niwat Chansiripornchai

Detection and molecular characterization of infectious bronchitis virus isolated from recent outbreaks in broiler flocks in Thailand

Thirteen field isolates of infectious bronchitis virus (IBV) were isolated from broiler flocks in Thailand between January and June 2008. The 878-bp of the S1 gene covering a hypervariable region was amplified and sequenced. Phylogenetic analysis based on that region revealed that these viruses were separated into two groups (I and II). IBV isolates in group I were not related to other IBV strains published in the GenBank database. Group 1 nucleotide sequence identities were less than 85% and amino acid sequence identities less than 84% in common with IBVs published in the GenBank database. This group likely represents the strains indigenous to Thailand. The isolates in group II showed a close relationship with Chinese IBVs. They had nucleotide sequence identities of 97-98% and amino acid sequence identities 96-98% in common with Chinese IBVs (strain A2, SH and QXIBV). This finding indicated that the recent Thai IBVs evolved separately and at least two groups of viruses are circulating in Thailand.

Differentiation of avian pathogenic Escherichia coli (APEC) strains by random amplified polymorphic DNA (RAPD) analysis

Here we describe the application of a random amplified polymorphic DNA (RAPD) analysis for molecular genetic typing avian pathogenic Escherichia coli (APEC) strains. The RAPD technique was shown to be highly reproducible. Stable banding patterns with a high discriminatory capacity were obtained using two different primers. Overall, 55 E. coli strains were analyzed with a RAPD technique. The RAPD analysis showed that the E. coli strains isolated from poultry in Thailand and Sweden could be grouped into 50 of RAPD types by using these two different primer sets. Most of these different E. coli RAPD types were not geographically restricted. There was, as expected, a tendency of higher genetic relationship among E. coli strains isolated from the same farm. It is suggested that the RAPD technique may provide a rapid, low cost, simple and powerful tool to study the clonal epidemiology of avian E. coli infections.

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.

Efficacy of live B1 or Ulster 2C Newcastle disease vaccines simultaneously vaccinated with inactivated oil adjuvant vaccine for protection of Newcastle disease virus in broiler chickens

Two hundred, one-day-old broiler chicks were divided into groups 1, 2 and 3 containing 60, 70 and 70 chicks, respectively. The groups were divided into subgroups of 10 chicks that were vaccinated according to the following scheme: group 1 unvaccinated control, group 2 vaccinated subcutaneously at 1 day old with inactivated oil adjuvant vaccine (IOAV) in combination with live B1 vaccine. Group 3 was vaccinated in the same mode as group 2 with IOAV and live Ulster 2C vaccine. All birds were challenged when they were 28 days old. Mortality rate, body weight gain and feed conversion ratio (FCR) were monitored before and after challenge. All the chickens in group 1 died, indicating that there was no disease resistance of this unvaccinated control group of chickens. Conversely, the monitored disease resistance of chickens in groups 2 and 3 was 68.57% ± 18.64 and 88.57% ± 9.00, respectively (P < 0.05). The morbidity of chickens in groups 2 and 3 was 37.89% ± 14.36 and 14.76% ± 12.40, respectively (P < 0.05). The body weight gain, feed intake and FCR of group 3 were significantly better than those of group 2 (P < 0.05) during 1–42 days old. The simultaneous vaccination with B1 or Ulster 2C and IOAV of 1-day-old chicks gave some protection of 28-day-old broilers without a booster vaccination.

Efficacy of sarafloxacin in broilers after experimental infection with Escherichia coli.

Infections of chickens with Escherichia coli serotypeO78 can be treated with the antibiotic sarafloxacin. Three experiments were conducted on the administration of this drug to chickens that had been experimentally infected with E. coli. The birds were monitored for 10 days after infection for their average daily gain (ADG) and feed conversion ratio (FCR), and the post-mortem pathology was assessed. In the first experiment, sarafloxacin (20 mg/L, equivalent to 5 mg/kg live weight per day), given in the drinking water for 3 days after infection, led to a reduction in the mortality from 75% to 27%, but the ADG of the treated birds was still less than that of the uninfected controls. In the second experiment, when the sarafloxacin was administered at the same dose in the water but over only 2 h, there was also a considerable reduction in mortality, and the ADG and the FCR also improved significantly. In the third experiment, the dose dependence of the drug was tested. The birds were given 5 and 10 mg/kg per day sarafloxacin in each group, starting within 2 h after infection. This rapid administration of the drug completely prevented mortality, while the ADG and FCR were similar to those of the uninfected controls.

Serovar Identification, Antimicrobial Sensitivity, and Virulence of Avibacterium paragallinarum Isolated from Chickens in Thailand

Summary. 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 µl of bacteria (108 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. Resumen. Identificación de serovariedades, sensibilidad a los antibióticos y virulencia de aislamientos de Avibacterium paragallinarum de pollos en Tailandia. Avibacterium paragallinarum produce coriza infecciosa en los pollos, que es una enfermedad respiratoria aguda que tiene una importancia económica a nivel mundial. Los objetivos de este estudio fueron determinar las serovariedades, la resistencia antimicrobiana y la patogenicidad de A. paragallinarum aislados de pollos en Tailandia. Dieciocho aislamientos de campo de A. paragallinarum fueron confirmados por PCR. Cuando se examinaron mediante serotipificación con una prueba de inhibición de la hemaglutinación, diez aislamientos se clasificaron como serovariedad A, cinco aislamientos como serovariedad B, y tres aislamientos como serovariedad C. La susceptibilidad de los aislamientos a 16 agentes antimicrobianos se puso a prueba mediante un método de difusión en disco. Todos los aislados fueron sensibles a amoxicilina-ácido clavulánico. Se observó un alto nivel de resistencia a la lincomicina y a la eritromicina. Todos los aislados fueron resistentes a la cloxacilina y a la neomicina. Un estudio de fagocitosis y de supervivencia bacteriana en el interior de macrófagos, mostró variación entre los aislamientos, pero no se observó ninguna conexión clara con la serovariedad. Se realizó una prueba de virulencia mediante el desafío de pollas de una estirpe productora de huevo, de cuatro semanas de edad a través de la vía nasal con 400 µl de bacterias (108 unidades formadoras de colonias / ml). Los signos clínicos se observaron diariamente durante siete días, y las aves fueron sometidas a una necropsia a los siete días después del desafío. Todos los 18 aislamientos de campo causaron los signos clínicos típicos de coriza infecciosa y esta pudo ser reaislada a los siete días después del desafío. No hubo diferencias significativas en las puntuaciones clínicas de los aislamientos, excepto que dos cepas (112179 y 102984, clasificados como serovariedades A y B, respectivamente), produjeron puntuaciones significativamente más altas a las observadas con el aislamiento CMU1009 (un aislamiento serovariedad A). No se observó correlación entre serovariedad y la severidad de los signos clínicos.

Seroprevalence and Identification of Ornithobacterium rhinotracheale from Broiler and Broiler Breeder Flocks in Thailand

Abstract Ornithobacteriosis is an infectious disease of avian species that has been reported in almost all countries around the world, except Thailand. The objectives of this study were to determine the seroprevalence of Ornithobacterium rhinotracheale (ORT) and to isolate and identify ORT in broilers and broiler breeders in Thailand. Chicken antibodies had been randomly checked from 17 farms (19 flocks) of broilers and 23 farms (28 flocks) of broiler breeders. The seropositive flocks were 63% and 100% in broilers and broiler breeders, respectively. The sera analysis showed that the individual 280 broiler sera antibody responses were 67.5% negative, 12.9% suspect, and 19.6% positive. The individual antibody responses of 510 broiler breeder sera revealed 12.2% negative, 38.0% suspect, and 49.8% positive samples. The bacteria were isolated and identified by polymerase chain reaction (PCR). Bacterial isolation and identification revealed that nine isolates of the 12 PCR analysis samples showed positive results to PCR analysis. All the positive PCR samples were collected from the broiler breeder farms.

Efficacy of Autogenous Killed Vaccine of Avibacterium paragallinarum

Abstract 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 1010 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 108 CFU/ml in 400 µl 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.

Characterization of plasmid pOR1 from Ornithobacterium rhinotracheale and construction of a shuttle plasmid

ABSTRACT The bacterium Ornithobacterium rhinotracheale has been recognized as an emerging pathogen in poultry since about 10 years ago. Knowledge of this bacterium and its mechanisms of virulence is still very limited. Here we report the development of a transformation system that enables genetic modification of O. rhinotracheale. The system is based on a cryptic plasmid, pOR1, that was derived from an O. rhinotracheale strain of serotype K. Sequencing indicated that the plasmid consisted of 14,787 nucleotides. Sequence analysis revealed one replication origin and several rep genes that control plasmid replication and copy number, respectively. In addition, pOR1 contains genes with similarity to a heavy-metal-transporting ATPase, a TonB-linked siderophore receptor, and a laccase. Reverse transcription-PCR demonstrated that these genes were transcribed. Other putative open reading frames exhibited similarities with a virulence-associated protein in Actinobacillus actinomycetemcomitans and a number of genes coding for proteins with unknown function. An Escherichia coli-O. rhinotracheale shuttle plasmid (pOREC1) was constructed by cloning the replication origin and rep genes from pOR1 and the cfxA gene from Bacteroides vulgatus, which codes for resistance to the antibiotic cefoxitin, into plasmid pGEM7 by using E. coli as a host. pOREC1 was electroporated into O. rhinotracheale and yielded cefoxitin-resistant transformants. The pOREC1 isolated from these transformants was reintroduced into E. coli, demonstrating that pOREC1 acts as an independent replicon in both E. coli and O. rhinotracheale, fulfilling the criteria for a shuttle plasmid that can be used for transformation, targeted mutagenesis, and the construction of defined attenuated vaccine strains.

Antimicrobial Resistance in Bacterial Poultry Pathogens: A Review

Antimicrobial resistance (AMR) is a global health threat, and antimicrobial usage and AMR in animal production is one of its contributing sources. Poultry is one of the most widespread types of meat consumed worldwide. Poultry flocks are often raised under intensive conditions using large amounts of antimicrobials to prevent and to treat disease, as well as for growth promotion. Antimicrobial resistant poultry pathogens may result in treatment failure, leading to economic losses, but also be a source of resistant bacteria/genes (including zoonotic bacteria) that may represent a risk to human health. Here we reviewed data on AMR in 12 poultry pathogens, including avian pathogenic Escherichia coli (APEC), Salmonella Pullorum/Gallinarum, Pasteurella multocida, Avibacterium paragallinarum, Gallibacterium anatis, Ornitobacterium rhinotracheale (ORT), Bordetella avium, Clostridium perfringens, Mycoplasma spp., Erysipelothrix rhusiopathiae, and Riemerella anatipestifer. A number of studies have demonstrated increases in resistance over time for S. Pullorum/Gallinarum, M. gallisepticum, and G. anatis. Among Enterobacteriaceae, APEC isolates displayed considerably higher levels of AMR compared with S. Pullorum/Gallinarum, with prevalence of resistance over >80% for ampicillin, amoxicillin, tetracycline across studies. Among the Gram-negative, non-Enterobacteriaceae pathogens, ORT had the highest levels of phenotypic resistance with median levels of AMR against co-trimoxazole, enrofloxacin, gentamicin, amoxicillin, and ceftiofur all exceeding 50%. In contrast, levels of resistance among P. multocida isolates were less than 20% for all antimicrobials. The study highlights considerable disparities in methodologies, as well as in criteria for phenotypic antimicrobial susceptibility testing and result interpretation. It is necessary to increase efforts to harmonize testing practices, and to promote free access to data on AMR in order to improve treatment guidelines as well as to monitor the evolution of AMR in poultry bacterial pathogens.