Sampling for Virus Isolation in Animals

Sampling and Sample Preparation

• Specimens for virus isolation must meet specific criteria:

  • Aseptic collection (minimal contamination)
  • Fresh specimens (living or freshly dead within 2 hours)
  • Contain the virus in anatomical sites of viral tropism
  • Representative sample (minimum 5 samples)

• General considerations during sampling:

  • Clinical signs: Observe respiratory, intestinal, and nervous symptoms (e.g., suspect Newcastle disease virus (NDV), Infectious bronchitis virus (IBV), Infectious laryngotracheitis virus (ILT), avian influenza (AI) in poultry; Bovine Rotavirus and Bovine Coronavirus in cattle).
  • Age of herd/flock: Susceptibility varies by age (e.g., Marek's disease virus (MDV) affects all ages, Avian leucosis virus (ALV) affects poultry over 14 weeks).
  • Morbidity and mortality rates: Calculate morbidity (infected animals/total susceptible) and mortality (dead animals/total susceptible). -Different viruses have different morbidity and mortality rates. -Newcastle Disease Virus (NDV) and Avian influenza (high morbidity and mortality) -Infectious bronchitis virus (IBV) and Infectious laryngotracheitis virus (ILT) (moderate to low morbidity and mortality)

Sample Collection

• Selecting animals:
  • Live, infected, apparently healthy, or convalescing animals
  • Dead/moribund animals (within 2 hours of death)
• Selecting organs: Collect samples from the anatomical sites where the virus is most likely to be found (viral tropism).

Viral Tropism

• Viral tropism is the ability of a virus to infect and replicate in a specific type of host cells or tissues.
• Factors affecting viral tropism:
  • Presence of specific cellular receptors
  • Temperature (e.g., MDV replicates in feather follicles at low temperatures)
  • pH (e.g., Rotavirus and Coronaviruses replicate in alkaline pH, enteroviruses replicate in acidic pH)
  • Presence of certain metabolites (e.g., rabies virus replicates in tissues with catecholamines)

Classification of Viruses by Tropism

• Dermotropic: Pox, Herpes, Papilloma
• Viscerotropic: Rota, Corona, Entero
• Neurotropic: Rabies, Avian encephalomyelitis, Equine encephalomyelitis
• Pneumotropic: Influenza, IBV, ILT
• Lymphotropic: Avian/bovine leucosis, MDV, Gumboro Virus
• Endotheliotropic: Bluetongue, African horse sickness
• Pantropic: NDV, RPV

Sample Transportation

• Specimens should be collected in sterile, labelled containers with complete history of the animal and suspected diagnosis.
• Use appropriate transport media (e.g., phosphate-buffered saline (PBS), minimal essential medium (MEM), Hank's balanced salt solution (HBSS), glycerol in PBS, etc).
• Transport specimens on ice or dry ice (-70°C) based on duration of transit.

Sample Storage

• Preservation temperatures depend on the virus. -Refrigerator (4°C) up to 24 hours. -Deep freezer (-20°C to -40°C) or lower, for weeks to months -Deep freezer (-70°C), for months to years -Liquid nitrogen (-196°C), for years -Lyophilization (freeze-drying).

Types of Samples

• Liquid: Sputum, urine, nasal discharge, vaginal discharge, semen, saliva, diarrhea, cerebrospinal fluid.
• Solid: Liver, spleen, heart, intestine, brain, kidney, stool, trachea, joint capsule, tendon etc. -Viscous samples (e.g., viscous fluids) should be diluted with PBS.

Sample Processing

• Liquid samples: Clarify by centrifugation, collect supernatant, add antimicrobial agent (e.g., gentamycin) and incubate.

• Blood samples: Anticoagulant (e.g., EDTA) required, centrifuge blood to separate the components.

• Soft tissue samples: Cut sample into small pieces using forceps and scissor, add sterile sand, homogenize, dilute with PBS, apply freeze-thaw cycles, centrifuge, collect supernatant, add antimicrobial agent (gentamycin) and incubate.

• Hard tissue samples: Homogenize, complete as soft tissue samples.

Questions

• Freezing and thawing: Breaks cell membranes, releasing viral particles for isolation.
• Antimicrobial agents: Inhibit bacterial or fungal contamination.
• Degenerated samples: Cell/tissue damage may prevent virus isolation.
• RP suspected samples: Glycerol-PBS not used, as glycerol/PBS prevents the inactivation of the virus and makes it possible to transport the sample for investigation.