Transcript isolation and identification ornithobacterium

Nong lam University, Ho Chi Minh City
Faculty of Animal Science and Veterinary Medicine
ISOLATION AND IDENTIFICATION
ORNITHOBACTERIUM RHINOTRACHEALE
FROM CHICKEN IN VIETNAM
Nguyen Thi Bich Lien1, Vo Thi Tra An1, Tran Thi Ngoc
Han1, Ho Hoang Dung1 and Niwat Chansiripornchai2
Nong Lam University, Vietnam
Chulalongkorn University, Thailand
Content
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ORT infection
ORT - Isolation
ORT - Identification
Further study
Faculty of Animal Science and Veterinary Medicine – NLU - HCMC
INTRODUCTION
Ornithobacterium infection
• Ornithobacterium rhinotracheale (ORT)
• Symptoms: sneezing, moist eyes, swollen
heads, difficult breathing, mortality (2-10%)
• Lesions: airsacculitis, tracheitis, pneumonia
• Risk factors: virus (ND, AI, IBD, IB), ventilation
(NH3, CO2)
• Transmission: horizontal (aerosols, direct
contact, drinkers), vertical (egg shell)
Faculty of Animal Science and Veterinary Medicine – NLU - HCMC
Ornithobacterium rhinotracheale
Gram-negative, rod shaped bacterium,
pleomorphic
Slow growing, easily overgrown by less
fastidious growing bacteria (gentamicin supp.)
For optimal growth
– 5–10% sheep blood agar
– For 48 hours at 37°C
– Microaerophilic conditions (5-10% CO2)
– Small circular grey to grey-white colonies
Faculty of Animal Science and Veterinary Medicine – NLU - HCMC
1981
Turkey
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1987
duck
1991
chicken
1993
Characterized
Isolation
Antibiotic susceptibility
Pathogenic characteristics
Vietnam: no publication on isolation ORT
Faculty of Animal Science and Veterinary Medicine – NLU - HCMC
THE STUDY
Chicken: difficult breathing , sneezing , facial edema
Samples
swab (30), trachea/ lungs (75)
DNA extraction
PCR amplification
16S rRNA ORT
Nucleotide
sequencing
Columbia +
5% sheep blood
+ gentamicin 5 µg/ml
pinpoint small-colony,
grey, greydish white,
non-heamolysis
Gram stain (-), mobility (-),
oxidase (+), catalase (-),
MacConkey (-)
Faculty of Animal Science and Veterinary Medicine – NLU - HCMC
Results
• Generally, positive 4.7%
• No swab samples found
carrying ORT
• 5 isolates cultured from
lung/ tracheal samples
(6.6%)
• 7 samples were positive by
PCR assay (6.7%) (9.3%)
Samples
30
75
Swab
Lung/
trachea
Asadpour et al (2008), 2.3%
Turan và Ak (2002), 11,46%
Faculty of Animal Science and Veterinary Medicine – NLU - HCMC
RESULT: isolation
Samples from
No (swab No.)
Pos.
Rate (%)
Breeding hens
5 (3)
1
20
Layer
8 (5)
0
0
Broiler
21 (2)
2
9,5
Native chicken
40 (17)
0
0
Local breed
18 (3)
1
5,5
Black-Bone Silky Fowl
13 (0)
1
7,7
Total
105 (30)
5
4,7
Seifi, 2002 (2,6%); Turkyilmaz, 2005 (1,2%), Ghanbarpour and Salehi, 2009 (3,5%)
Faculty of Animal Science and Veterinary Medicine – NLU - HCMC
RESULT: PCR
1.
2.
3.
7.
Positive control (ORT, N.
Chansiripornchai, 2007)
Negative control (E.coli)
- 6: positive samples
negative sample
. PCR product was nucleotide-sequenced and compared with
reference gene in GenBank (KC454288).
. The homology of 100% found (several ORT sequences)
Faculty of Animal Science and Veterinary Medicine – NLU - HCMC
RESULT: PCR (direct from samples)
Samples from
No
Pos.
Rate (%)
Breeding hens
5 (3)
1
20
Layer
8 (5)
0
0
Broiler
21 (2)
2
9,5
Native chicken
40 (17)
1
0
Local breed
18 (3)
2
5,5
Black-Bone Silky Fowl
13 (0)
1
7,7
Total
105 (30)
7
4,7
Asadpour et al (2008): ELISA, 62,8%; PCR, 3,1%;
Ozbey et al (2004): ELISA 10,2%; PCR, 2,4%
Faculty of Animal Science and Veterinary Medicine – NLU - HCMC
Conclusion
• First study on isolation and identification of
ORT in Vietnam.
• Further study on antimicrobial susceptibility,
pathogenic characteristics
Faculty of Animal Science and Veterinary Medicine – NLU - HCMC
Thank you for your attention!
Faculty of Animal Science and Veterinary Medicine – NLU - HCMC