Bird Sampling Methods: Collection

• Bird sampling methods involve sample collection, transportation, preparation, and preservation.

Sample Collection

• Sample collection focuses on collecting samples from live birds with clinical signs or from dead birds.

Live Bird Sampling: Swabbing Method

• Requirement list: PPE, swabs, and cryovials
• Do not use cotton-tipped or wood stems due to RNAse activity.
• Use Dacron or Rayon tipped swabs, or wire steamed swabs for small birds.
• Use transport tube or cryovials containing viral transport medium (VTM)
• Transport media include Brain-heart infusion broth (BHI) and Viral transport media (VTM) such as PBS.
• BHI contains Penicillin, streptomycin, Nystatin, Polymyxin B, and Amphotericin to minimize bacterial and fungal contamination.
• Nutrient broth (NB) is general and most common
• Peptone broth (PB) is for salmonella.
• 70% alcohol solution
• Optional: Ice and/or liquid nitrogen for storage, markers, and a data form
• Swabbing procedures include cloacal, oropharyngeal, and tracheal swabbing.
• Cloacal swabbing: Put swab into cloacal opening, rotate, and shake to drop fecal residues.
• Oropharyngeal swabbing: Open peak then around the swab into side of bird mouth.
• Tracheal swabbing: Gently pull tongue, expose trachea, wait for breath, and gently touch the trachea.

Live Bird Sampling: Blood Sampling

• Requirement list: PPE, syringe, markers, ice tank, anticoagulant, separator/centrifuge tube, 70% alcohol solution with cotton gauze.
• Serum sample: Collect without anticoagulant at room temperature (22–25 °C) before refrigeration.
• Plasma sample: Collect with anticoagulant and put in refrigerator for 24 hours to avoid hemolysis.
• Whole blood: Collect from wing vein (most common), medial metatarsal vein (duck), or jugular vein (duck).

Dead Bird Sampling: Yolk Sac

• Sampling of the yolk sac is for vertically transmitted diseases.
• Use freshly dead birds (2-4 hours) to avoid putrefaction and migration of organisms.
• Collect the organ which represents lesion.
• Requirement list for sampling collection from freshly dead: Plastic bags, scissors, cleansing agents such as Dettol, ice tank, markers, data form.

Number of Samples

• For serology, 20-30 samples/flock; if infection level is 10% +Ve, flock infected at percent 95%.
• For tissue, 3-5 birds (don’t pool tissue from different birds).
• For swabs, 20-30 (pooled 5/tube).

Sample Transportation

• Transportation depends on the period and distance:
• Within 24-48 hours: use ice blocks.
• More than 48 hours: use liquid nitrogen (-196 °C).

Sample Preservation and Preparation

• Serum preservation: 4 °C for short time & -20 °C for long time.
• Tissue/swab preservation: 4 °C (up to 72-96 hours) & -80 °C for long time.
• Swab sample preparation: prepare as 10-20% suspension in antibiotic solution for 15 minutes, then centrifuge at 3000 rpm for 25 minutes and collect the supernatant.
• Organ sample preparation: thaw frozen tissues, grind in sterile mortar, make 10% suspension of physiological saline with antibiotics, centrifuge at 3000 rpm for 15 minutes, collect supernatant, and culture to check for bacterial presence.

Samples According to Suspected Virus

• AI: Trachea – Lung – Air sac – Sinus exudate – Pancreas – Cloacal swab
• ND: Cloacal swab – Trachea – Lung – Spleen – Brain – Intestine
• ILT: Tracheal exudate – Trachea – Lung
• IB: Tracheal swab – Kidney – Oviduct
• Pox: Wart like nodules – Diphtheritic membrane
• IBD: Bursa – Spleen – Kidney
• DVH: Liver – Spleen – Blood
• MD: Blood - Cellular suspension – tumor – feather follicle.
• AL: Tumor organ
• AE: Brain
• Reo: Synovial fluid – Affected joints – Intestine