Sample collection
A total of 128 samples were collected from infectious coryza suspected 73 farms of 85 poultry flocks as well as samples available with Poultry Disease Diagnosis and Surveillance Laboratory, Namakkal, Tamil Nadu. Information regarding farm/flock strength, age, method of rearing, production performance, clinical symptoms manifested and mortality patterns from various organized poultry farms were recorded. The collected samples comprising of heart blood swabs, tracheal, eye and infra orbital swabs. The collected specimens were transported in Ames transport medium and stored in refrigerator till further processing.
Sampling from live birds
Ailing birds with history of respiratory disease and showing signs of infectious coryza
viz., swollen infra-orbital sinuses, serous to mucoid oculo-nasal discharge, eye and facial swelling, were selected and gentle pressure was exerted on each infraorbital sinus to flush out mucus through nostrils and sampled using sterile disposable cotton swabs.
Sampling from autopsied birds
Samples were taken from birds either already dead or after scarifying by decapitating the birds showing clinical signs of coryza. The head was swabbed with 70% alcohol and allowed to dry. The mucoid fluid from the infra orbital sinus and nasal or conchal cavities was collected with a nichrome loop.
Primary isolation of NAD-dependent Av. paragallinarum
Blood agar (5-7% v/v sheep blood) with haemolytic
Staphylococcus sp.as feeder culture was used as culture media for preliminary isolation of
Av. paragallinarum from suspected samples. Chocolate agar with 0.01% (w/v) NADH and Brain Heart Infusion broth were used for further sub culturing and studying growth characteristics as well as purification of genomic DNA. Collected samples were streaked directly onto the chocolate and blood agar plates. The plates were incubated at 37°C for 48 hrs in a traditional candle jar supplying with 5-10% CO
2.The colonies grown on Blood agar and Chocolate agar at 37°C after 14 hrs of incubation in 5% CO
2 were examined by Gram’s staining. The organism was identified as per Cowan and Steel’s Manual for Identification of Medical Bacteria (
Cowan, 1974). All the isolates were subjected to catalase, oxidase, nitrate reduction, indole production and urea hydrolysis tests.
All the isolates, were inoculated into 5-7 days old embryonated chicken eggs via yolk sac route and inoculated eggs were kept at 37°C for 48 hrs. Embryos dying after 24 hrs of inoculation were considered as infected. After 48 hrs the yolk fluid was harvested, dispensed in screw capped 10 ml glass bottles and stored at -20°C for further use.
Molecular detection
DNA extraction
Genomic DNA was isolated from the overnight grown bacterial culture by using Genomic DNA purification Kit (Promega, USA) and stored at -20°C until use.
Polymerase chain reaction
Initial PCR screening was carried out by using the primer pair targeting the
hpg-2 gene of
Av. paragallinarum N1 (5'-TGAGGGTAGTCTTGCACGCGAAT-3') and R1 (5'-CAAGGTATCGATCGTCTCTCTACT-3') to amplify specific 500bp fragment (
Chen et al., 1998). The amplification was carried out using PCR master mix (2X) (Thermo scientific, USA), with an initial denaturation at 94°C for 4 min, followed by 35 cycles at 94°C for 1 min, 63°C for 1 min, 72°C for 1 min and a final extension at 72°C for 10 min. For sequencing further, the
hagA gene was amplified using primer pairs HA1 (TGT AGC TCAAGC AGC TCC ACA AG) and HA2 (TCA AGC GAT AAG TGC TTT ACG ACC) to amplify 900-bp fragment
(Rhonda et al., 2002). The amplification was carried out using PCR Master Mix (2X) (Thermo scientific, USA), with an initial denaturation at 94°C for 5 min, followed by 35 cycles at 94°C for 1 min, 46°C for 1 min, 72°C for 1.5 min and a final extension at 72°C for 10 min. PCR products were purified employing ExoSAP-IT ® (Affymetrix, USA) and sequenced commercially.
Phylogenetic analysis
The nucleotide sequence data generated were edited and aligned using Sequencing Analyses Software v5.3 (Applied Biosystems, USA) and MEGA 6 software. Reference sequences were downloaded from GenBank database. Phylogenetic analyses and evolutionary associations were inferred in MEGA 6.0 using the Maximum Likelihood algorithm with Kimura-2P correction and 1000 bootstrap replications.
Experimental pathological studies of Av. paragallinarum
Pathogenicity of field isolates of
Av. paragallinarum was studied in experimentally infected 7 weeks old chicken. The birds were examined daily for 7 days for clinical symptoms of Infectious coryza, mortality and progression of diseases. The experimentation was performed after obtaining IAEC and IBSC permission (IAEC approval no. 4/2015, dt. 07.07.2015, VCRI, Namakkal and Lr. No. 1675/DFBS/IBSC/IBSC/2015, dt. 04.09.2015, the Dean, faculty of Basic Sciences, MVC, Chennai).
Preparation of inoculum
The inoculum for the experimental infection of birds was prepared according to
Reid and Blackall (1984) and
Sobti et al., (2001) with slight modifications. Pure bacterial colonies grown on chocolate agar plates were picked and inoculated into Brain Heart infusion broth in test tubes. The test tubes were then incubated for 48 hr under reduced O
2 tension. Cultural suspension of 1011 CFU of each isolate was used as the inoculum for pathogenicity tests.
Infection of birds
A total 90 birds were randomly divided into 6 groups (15 birds each group). Group1-5 was infected with one isolate each (Isolates 1 to 5) of
Av. paragallinarum. Group-6 were kept as unchallenged control.
Method of inoculation
An inoculum of 0.2 ml of each isolate with viable organisms was inoculated to susceptible seven-week-old chicks through intranasal and infraorbital sinus route for three days.
Post infection observation and Re-isolation of Av. paragallinarum
All the experimentally infected birds were daily observed for clinical symptoms and progression of diseases for 7 days. After the development of clinical signs of infectious coryza, the birds were scarified and decapitated for observation of post-mortem lesions. Tissue samples were collected and the organisms were re-isolated.
