Sampling and Sample Preparation PDF

Sampling and sample preparation Characteristics of ideal virological samples: Specimens collected for isolation of viruses must have certain criteria like: 1- Aseptically as possible (of minimum contamination). 2- As fresh as possible (living animals or freshly dead animals within 2 hours...

Sampling and sample preparation Characteristics of ideal virological samples: Specimens collected for isolation of viruses must have certain criteria like: 1- Aseptically as possible (of minimum contamination). 2- As fresh as possible (living animals or freshly dead animals within 2 hours after death). 3- Containing the virus (From the anatomical sites of viral tropism). 4- Representative sample (0.1%) – Minimum of five samples. General consideration should be taken during sampling: 1- The nature of clinical sings: (Respiratory, intestinal, nervous…. etc)  Respiratory sings in poultry (suspect: Newcastle disease virus "NDV", Infectious bronchitis virus "IBV", Infectious laryngotracheitis virus "ILT", avian influenza "AI").  Diarrhea in cattle (suspect: Bovine Rotavirus, Bovine Coronavirus…). 2- Age of herd or flock: Each virus has a susceptible age as:  Viruses causing tumor in poultry (Marek's disease virus "MDV" infect the poultry at any age and Avian leucosis virus "ALV" infect the poultry over 14 weeks of age). 3- Morbidity and mortality rate: Viruses have different constant patterns of morbidity and mortality.  Morbidity rate = No. of infected animals / total No. of susceptible animals.  Mortality rate = No. of dead animals / total No. of susceptible animals.  Viruses causing respiratory sings in poultry: A) NDV and Avian influenza (high morbidity and high mortality). B) IBV and ILT (moderate to low morbidity and mortality). 6  4- History of vaccination: Proper vaccination limits the chance of infection with the vaccinated viruses. 5- History of new entries: The appearance of a new viral disease or new viral strain may be sometime related to entrance of new animals to the flock or to the country.  The entrance of Foot and Mouth disease virus "FMD" A and SAT-2 serotypes to Egypt through the importation of live animals from Africa. 6- Season: The spread of some viruses is sometimes related to the season.  Arthropod born viruses (ARBO): spread in summer season.  Influenza viruses: spread in winter season. 7- Geographical distribution: Some viruses are endemic in a geographical region and not in other(s).  Bovine ephemeral fever (Absent in Europe and the Americas and endemic in Africa and Asia).  Rabies virus (absent in Japan, Australia and most European countries). Sample collection: Sampling requires proper selection of both animals and organs. A) Selection of animals: 1- Live animals (actual infected, apparently health or convalescent). 2- Dead animals (moribund, freshly dead "within 2 hours after death"). B) Selection of organs: The proper sample must be collected from the anatomical sites of the viral tropism. Viral tropism: It is the affinity "ability" of certain virus to invade and replicate in a specific host tissues or cells. 7 Factors affecting the viral tropism: 1- Presence of specific cellular receptors. 2- Temperature: MDV replicates in feather follicles as it needs low temperature. 3- pH: Rotavirus and Coronavirus replicates in alkaline pH "intestine” while the enteroviruses replicates in acidic pH "stomach". 4- Presence of certain metabolites: Rabies virus replicates in nervous tissues due to the presence of catecholamine. Classification of viruses according to tropism: 1- Dermotropic: Pox, Herpes, Papplioma. 2- Viscerotropic: Rota, Corona, Entero 3- Neurotropic: Rabies, Avian encephalomyelitis, Equine encephalomyelitis. 4- Pneumotropic: Influenza, IBV, ILT. 5- Lymphotropic: Avian and bovine leucosis, MDV, Gumboro Virus. 6- Endotheliotropic: Bluetongue, African horse sickness. 7- Pantropic: NDV, RPV. Sample transportation: The specimen(s) should be collected in sterile, tightly-closed, properly labeled vessels and sent to the laboratory with complete history (type of sample, date, animal data "age, sex, number…" and suspected diagnosis). Also, the specimen(s) collected by swabs should be dipped in transport medium (phosphate buffered saline "PBS", minimal essential medium "MEM" Hank's balanced salt solution "HBSS" and glycerol in PBS 1:1 "not used in RP sample"). The specimen(s) must be transported to the laboratory in ice box if the ambient temperatures are moderate and transit time to the laboratory is less than 1 day or on dry ice (-70oC) if the environmental temperature is high and transit time is longer than a day. Sample preservation: Storage temperature of specimens depends on the properties of viral agent to be tested and duration of preservation. 8 1- Refrigerator (4oC): used for sample preservation up to 24 hours. If the viral agent to be isolated can be inactivated during this period, a portion of the specimen should be frozen at -70oC or lower. 2- Deep freezer (-20 and -40oC): used for sample preservation for weeks to few months. 3- Deep freezer (-70oC): used for sample preservation for months to few years. 4- Liquid nitrogen (-196oC): used for sample preservation for several years. 5- Lyophilization (Freeze-drying): (For long-term storage of virus suspensions). Dehydration of freeze viral suspension under low pressure and low temperature using a lyophilizer. The lyophilized material can be stored at 4oC. Types of samples: Types of samples Liquid Solid Viscous Fluid Blood Soft Tissue Hard Tissue Sputum Urine Whole blood Liver Trachea Nasal discharge Milk Serum Spleen Joint capsule Vaginal discharge Vesicular fluid Hart Tendon Semen Intestine Saliva Brain Diarrhea Kidney Cerebrospinal fluid Stool Preparation of samples: 1- Processing of fluid samples: 1- Clarify the sample by centrifugation at 3000 rpm/30minutes (differs according to the rotor diameter of the centrifuge). 2- Collect the supernatant. 3- Add antimicrobial agent to supernatant and allow standing for 1 hour at room temperature. N.B. Antimicrobial includes: -Antibiotic as gentamycin (40 µg/ml). 9  -Antimycoplasma as lincospectin -Antimycotic as Fungizone (2.5 µg/ml). 2- Processing of Viscous samples: 1- Dilute the sample with PBS. 2- Complete as steps 1, 2 and 3 in fluid sample. 3- Processing of Blood samples: A- Whole blood: 1- Collect the blood with anticoagulant. 2- Centrifugation at 1500rpm/7-10 minutes. 3- Collect the different blood fractions separately. B- Serum: 1- Collect the blood without anticoagulant. 2- Leave the tube in an oblique position at 37ºC for 1 hour. 3- Transfer the tube to refrigerator and leave it overnight. 4- Centrifugation at 5000rpm/10 minutes. 4- Processing of soft tissue samples: 1- In a mortar, cut the sample into small pieces using forceps and scissor. 2- Add equal amount of sterile sand. 3- Grind the sample with pistil till complete homogenization. 4- Dilute the sample 1/5 or 1/10 with PBS. 5- Collect the tissue homogenate in a centrifuge tube. 6- Apply 3 cycles of freezing and thawing for rupture of cells and release of virus outside the cells. 7- Clarify the sample by centrifugation at 3000rpm/30minutes. 8- Collect the supernatant. 9-Add antimicrobial agent to supernatant and allow standing for 1 hour at room temperature. 10 5- Processing of hard tissue samples: 1- Homogenization of hard tissue using tissue homogenizer/stomacher. 2- Complete as soft tissue sample from step 4. Questions: 1- What is the purpose of freezing and thawing during the processing of virological samples? 2- What is the purpose for addition of antimicrobial agent during preparation of samples? 3- Why the degenerated samples not used in virus isolation? 4- Why RP suspected samples not transported in (1:1) glycerol: PBS transport medium? 11