Sampling Methods ForDifferent Diseases Dr. Tariq Mustafa Mohamed Ali Al Ain Veterinary lab Animal Health section Agriculture Sector Department of Municipalities and Agriculture
طرق جمع العينات للتشخيص المرضى د. طارق مصطفى محمد علىالمختبر البيطرى- قسم الثروة الحيوانيةقطاع الزراعة – دائرة البلديات والزراعة
APPROACH TODIAGNOSISSuccess of diagnostic veterinarylaboratory depends on submissionsamples of good quality which willprovide optimal opportunity for thediagnosis of disease.
Sampling standards : Provide epidemiological and clinical details with the samples. Always sample several animals in an outbreak. Collect samples from live animals in acute stage of the disease. Keep samples cool during transfer to the laboratory (preferably on melting ice) and reduce the time in transit to the minimum. Mark sample bottles carefully with an indelible pen and record details of each samples origin for submission to the laboratory.
Accession or submissionforms1. Provide the requested information on the Lab form.2. Brief, concise, complete histories are required and aid in providing diagnoses and pertinent advice.3. Please use black ink and write or print legibly.4. List the tissues submitted, also the number of tumors. This will help insure that all submitted specimens are identified
Submission of Serum andBlood samples Blood samples should be collected in sterile tubes containing no anticoagulants. These should be submitted to the laboratory in specially designed Styrofoam holders to avoid breakage. Blood samples should not be frozen or allowed to overheat. If samples cannot be delivered to the laboratory within a reasonable time, serum should be removed and refrigerated or frozen.
Submission of Serum andBlood samples. Blood submitted for culture should be submitted in blood culture bottles or sterile vacutainer. Serum must be fresh, clear, unhemolyzed, and uncontaminated. Submit at least 1.0 ml of serum for each test requested. Refrigerate the serum until shipment. Identify specimen in a way that will avoid confusion when results are reported.
Submission of Serum andBlood samples Be careful that writing will be legible Avoid using animal names to avoid duplication and confusion. Label each tube with tube number and vet code.
Submission of swabs Swab material from the more advanced lesions. Collect swabs from acutely ill animals . Collect swabs from several animals in different stages of the illness . Two swabs should be collected each time
Submission of swabs Swabs for virus isolation should placed in viral transport medium . Most of these virus transport media are balanced salt solutions containing high protein content and antibiotics to prevent bacterial overgrowth. Swab for electron microscopy should be placed in a screw-capped tube containing one or two drops of distilled water.
Feces Feces should be collected from acutely ill animals and placed in leak proof containers. Well-saturated swabs are adequate for many individual examinations. Several milliliters or grams of feces permit a more complete diagnostic work-up including bacteriologic and parasitological examinations. Samples should submitted to the laboratory using cold packs as coolant.
Tears Cotton buds or swabs of absorbent cotton wool are inserted into the conjunctival sac and swirled around to collect tears. The bud/swab is broken off into a container and about 150 microlitres of sterile phosphate-buffered saline (PBS pH 7.2 to 7.6) are added (if available).
Pus Collect samples aseptically and submit in culturette swab. When abscess material is available submit the exudates in a sterile container. Exudates should be collected from non- draining lesion
Tissues It is recommended that the following tissues be collected during post mortem examination: lymph nodes found around the lungs (mediastinal) and alimentary tract (mesenteric); portions of the spleen and the lungs. Two sets of each tissue are required; one set is chilled but not frozen, and the other is put in 10 percent formalin solution to preserve the samples.
Gum debris This material can be collected by a spatula or finger rubbed across the gum and inside the upper and lower lips. The material collected is then scraped into a container and 150 microlitres of PBS are added (if available).
NECROPSY SUBMISSIONSTANDARDS Dead animals should be cooled as soon as possible after death. Large animals should be thoroughly hosed down with cold water. Birds, rabbits, and other fur bearing animals should be soaked in cold, soapy water, placed in a plastic bag, and refrigerated.
SPECIMENS FROM NECROPSIEDANIMALS1. Collect all specimens as aseptically as possible. Liberal portions of each organ should be collected. If the outside of the specimen is accidentally contaminated, wash the specimen with clean tap water.2. Refrigerate (wet ice packs) all specimens to prevent saprophytic growth.
SPECIMENS FROM NECROPSIEDANIMALS ( cont.)3. Collect observable lesions or suspected target organs4. For neonatal diarrhea, submit a tied off 4-5 cm segment of jejunum, ileum, and colon with the accompanying lymph nodes for culture of pathogenic bacteria.
SPECIMENS FROM NECROPSIEDANIMALS ( cont.)5. Tissue specimens should be placed in individual leak-proof plastic bags and identified (use water-proof ink on bags)
MASTITIS MILK SPECIMENS1. Wash udder to remove dirt and allow to dry.2. Scrub teat end with alcohol soaked cotton and let dry.3. Samples should be collected in a sterile container immediately prior to regular milking without discarding any streams of milk (since the foremilk usually contains the greatest number of the infecting micro-organisms.
ABORTIONS Diagnosis cause of abortion is difficult and complex. Fetus, placenta, fetal stomach contents, uterine contents and serum are the favorite specimens. Submit multiple specimens to increase the probability of diagnosis.
ABORTIONS (cont.) Rinse the fetus and placenta with clean tap water and place them in a plastic bag. Force the air out of the bag before sealing it. All specimens should be refrigerated . If a toxic condition is suspected, submit samples of the aborting animal’s feed and water. If you suspect nitrate toxicity send Eye or aqueous humor.
ABORTIONS (cont.) Collect and submit the first of paired serum samples from the suspected aborting animal. The second serum sample should be collected and submitted in 2-3 weeks.
SPECIMENS for ANAEROBIC ANDMICROAEROPHILIC culture. The success of culture for anaerobic and microaerophilic organisms is heavily dependent on sample selection and shipment. Sample should be taken from a living animal or a fresh carcass. Specimens for Campylobacter isolation should be submitted in a transport media that limits or excludes air from the sample such as Amies media, containing Cary-Blair or thioglycolate broth.
MYCOLOGY1. Submit skin scrapings from the outer edges of a lesion and submit plucked (not cut) hairs.2. Skin, hair, and nails should by shipped to the laboratory without refrigeration.3. Submit internal organs or internal lesions suspected of fungal infection.4. Internal specimens should be sent refrigerated (wet ice packs) and not frozen.
Surveillance Continuous investigation of a given population to detect the occurrence of disease for control purpose
Active surveillance Advantage of Active surveillance Better information quality Reflect the true situation Faster Cheaper
Passive surveillance Compulsory notification Laboratory submission data Disadvantages of Passive surveillance Under reporting system Expense Non representative report
Monitoring Constitutes on going programmes directed at the detection of changes in the prevalence of a disease in a given population What you are looking for?????? Estimate disease prevalence Estimate disease incidence Detect disease or demonstrate freedom from disease
Prevalence The proportion of number of sick animals at a single point in time to the total population at risk at the same point of time In the previous example, Prevalence is 50%
Incidence rate It is a measure of average speed at which the disease is spreading Incidence rate = Total new cases during a period of time av. No. of animals at risk X time period
Example :A small animal farm consists of 2000 goat suffer from an outbreak of PPR. The first animal start to get sick on the 3rd of March. By 5th of march many animal are dying. The owner contact the veterinarian on the 6th of march . The veterinarian count 56 sick animals and the owner said that 143 animal have already died and 28 animals had been sick but recovered. A number of 1801 apparently healthy .
Calculation of Prevalence percent. Prevalence at 6th March = 56 / (2000-143 ”DEAD”) = 56 / 1857 = 3%
Calculation of Incidence rate New cases =(143 + 28 +56) =227 Av.Population at risk in the 4 Days =(2000 – 227) + 2000/2=1886.5 Incidence rate : = 227/1886.5 X 4 “Period of time” = 0.03 animal /day = 21 animal /100 animals/ week
Sample size The sample size is independent of the total number of animals in the population It depends on 3 factors : 1. Expected prevalence. 2. Level of confidence wanted (90 0r 95 or 99%). 3. Desired absolute precision.
Sample size in infinite population Suppose the true prevalence is thought to be about 40% and the desired estimate at precision of 5% at 95% level of confidence. From the table, the sample size will be 369 animals.
Sample size in infinite population Suppose we have 900 animals at the same prevalence ,precision of 5% and confidence level . The sample size (1/n)=1/n∞ + 1/N =1/369 +1/900 =1/262
Sampling frame ( StratifiedRandom sampling)Prepare a list of camel owner for each clinic , avoid repletion of names . e.g. Clinic # 1 : 37 Clinic # 2 : 101 Clinic # 3 : 76 .Calculate the total No of owners e.g. 214
Sampling frame (Cont.)Determine the proportion allocation of owners belonging to each clinic ( Considering that the number of owners reflect the animal population density in each clinic ) i.e. Clinic # 1 37 / 214 = 17% Clinic # 2 101/214 = 47% Clinic # 3 76/214 = 36%As far as we determine the sample size as 15000 animal from total true animal population 95000 i.e. we select 15000/95000 = 1/6 i.e. one owner from every 6 owner randomly .
Sampling frame (Cont.)Calculate the total no of owners that will be sampled , in our example it will be as follows: 214 / 6 = 36 ownersCalculate how many owners will be sampled from each clinic by multiplying the obtained proportion allocation of each clinic by the total No of owners that should be sampling
Sampling frame (Cont.) Clinic # 1: 17% X 36 = 6 owners will be selected on random basis Clinic # 2 : 47 % X 36= 47 owners will be selected on random basis Clinic # 3 : 36% X 76 = 13 owners will be selected on random basis
Sampling frame (Cont.) If we get less number of animals than that required we should go back to re-select randomly another ? This will depend mainly on the animal density exists for each clinic .
Avian Influenza SPECIMEN Serum , cloacal, tracheal, oropharyngeal Swabs TYPE OF TEST AGID , Imunochromatography, PCR ,HI
Avian Chlamydia infection SPECIMEN Spleen, liver, lung, Air sac, conjunctival swab TYPE OF TEST FA ELISA
Avian Mycoplasma Spp. SPECIMEN Serum TYPE OF TEST HI Plate agglutination test
Salmonella pullorum SPECIMEN Serum TYPE OF TEST Micro agglutination
Canine Corona virus SPECIMEN Serum Feces / small intestine TYPE OF TEST Imunochromatography ELISA IF
Canine Distemper Virus SPECIMEN Lung, kidney, spleen, urinary bladder, brain, stomach, liver, blood smear Serum TYPE OF TEST IF, IIF ,chromatography
Canine Parvovirus (CPV) SPECIMEN Intestine (jejunum, ileum), spleen, mesenteric lymph node Serum TYPE OF TEST IF , IIF , chromatography
Bluetongue disease in a sheep Note the bluish discoloration of the coronary bands of the hoof. The lips will usually be found to be swollen and discolored blue at the same time
Blue tongue SPECIMENS Serum TYPE OF TEST AGID , ELISA
Bovine Leucosis (BLV) SPECIMEN Serum TYPE OF TEST AGID
Bovine Respiratory Syncytial Virus(BRSV) SPECIMEN Lung, bronchial lymph node Serum TYPE OF TEST IF ,IIF , ELISA Serum Samples are tested at 1:50 dilution
Bovine Viral Diarrhea (BVD) SPECIMEN Lung, intestine, turbinate, trachea, swabs from lesions, fetal organs Ear notches are the samples of choice in case of persistent infection Serum TYPE OF TEST IF , SNT , ELISA
Infectious Bovine Rhinotracheitis(IBR) . SPECIMENS Lung, trachea, turbinate, aborted fetal tissues Serum TYPE OF TEST IF , SNT , ELISA
Caprine Arthritis-Encephalitis(CAE) / Ovine Progressive Pneumonia(OPP) SPECIMENS Serum TYPE OF TEST AGID
Chlamydia SPECIMENS Lymph node, tissues of aborted fetus, joint fluid , TYPE OF TEST IF , ELISA
Clostridium SPECIMENS Intestinal content Affected lesions (Liver , Int., Muscles) Serum TYPE OF TEST IF , ELISA
Bovine Corona virus , Rotavirus,Cryptosporidium Infection SPECIMENS Intestinal content , Feces TYPE OF TEST IF , ELISA , Immuno chromatography
Salmonellosis SPECIMENS Feces, Feed, Water, Environmental samples TYPE OF TEST Culturing Agglutination test
E.coli Pilus (K 99 ,F 5Serotype) SPECIMENS Intestinal content Feces TYPE OF TEST ELISA
Typical lesions of contagiouscaprine pleuropneumonia (CCPP)in a goat Note the yellowish, fibrinous deposit on the surface of the lungs and adhesions to the inside of the rib cage.
Contagious caprine pleuropneumonia(CCPP) SPECIMENS Lung Serum TYPE OF TEST IF , L Agglutination
Leptospirosis SPECIMENS Kidney Serum TYPE OF TEST IF , Micro agglutination Test for 6 serovars - canicola, grippotyphosa, hardjo, icterohemorrhagiae, pomona, and bratislava. Samples are tested at an initial dilution 1:100.
Listeria SPECIMENS Cerebellum, pons, medulla, fetus, uterine secretions Serum TYPE OF TEST Card Agglutination Test for Type 1 and Type 4 serotypes. Serum is screened at an initial dilution 1:20.
Ruminant Anaplasmosis SPECIMENS Serum TYPE OF TEST Card Agglutination , CFT , ELISA
Toxoplasmosis SPECIMENS Serum TYPE OF TEST Latex agglutination , IHA A titer > 1:64 is considered positive.
Specimens for FMD The preferred sample for virus isolation is the epithelium (at least 1-2 cm square) from unruptured or freshly ruptured vesicles. Vesicular fluid should be added if available. Samples should be collected into a transport medium consisting of equal amounts of phosphate buffer and glycerol at pH 7.2-7.6 (with added antibiotics).
PPR in a goat purulent eye and nose discharges Discharges from the nose and eyes in advanced PPR infection; the hair below the eyes is wet and there is matting together of the eyelids as well as partial blockage of the nostrils by dried-up purulent discharges
PPR in a goat Inflamed (reddened) eye membranes Reddening of the mucous membranes of the eye (the conjunctiva) in the early stages of infection. Note the purulent eye discharges
PPR in a goat Early mouth lesions showing areas of dead cells Early pale, grey areas of dead cells on the gums
PPR in a goat later mouth lesions The membrane lining the mouth is completely obscured by a thick cheesy material; shallow erosions are found underneath the dead surface cells.
Specimens for RFV Blood in anticoagulant from any animals with a fever of 40.5-42°C Liver and spleen from any freshly dead animals, on ice, in glycerol buffered saline and/or in buffered formalin Liver, spleen and brain from fresh fetuses
RFV Sheep, fetus. Both the pleural and peritoneal cavities contain excessive clear, straw-colored fluid.
RFV Sheep, fetus, kidney. There is severe perirenal edema.
RFV Sheep, liver. The cut surface of the swollen liver is pale and contains many petechiae.
RVF Sheep, colon. Severe hemorrhagic colitis.
RVF Sheep, colon. There is severe locally extensive mucosal hemorrhage.
RVF Sheep, liver. Section reveals that the liver is pale, swollen and contains multiple foci of hemorrhage.
RFV Sheep, liver. Liver is pale and swollen and contains many areas of severe congestion.