Sampling Methods For Different Diseases

Dr. Tariq Mustafa Mohamed AliAl Ain Veterinary labAnimal Health section

Agriculture SectorDepartment of Municipalities and Agriculture

طرق جمع العينات للتشخيص المرضى

د. طارق مصطفى محمد علىالمختبر البيطرى- قسم الثروة

الحيوانيةقطاع الزراعة – دائرة البلديات

والزراعة

APPROACH TO DIAGNOSIS Success of diagnostic veterinary

laboratory depends on submission samples of good quality which will provide optimal opportunity for the diagnosis 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 sample's origin for submission to the laboratory.

Accession or submission forms1. 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 and Blood 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 and Blood 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 and Blood 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.

Fecal swab

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 SUBMISSION STANDARDS 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 NECROPSIED ANIMALS

1. 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 NECROPSIED ANIMALS( cont.)3. Collect observable lesions or

suspected target organs

4. 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 NECROPSIED ANIMALS( 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.

Diarrhea/Enteritis Feces Blood samples Serum samples Food material Affected intestine , Liver, Intestinal

LN

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 AND MICROAEROPHILIC 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.

Approximate sample size table

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 ( Stratified Random sampling)

Prepare a list of camel owner for each clinic , avoid repletion of names .

e.g. Clinic # 1 : 37Clinic # 2 : 101Clinic # 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 95000i.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 TESTAGID , 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 5 Serotype) SPECIMENS

Intestinal content Feces

TYPE OF TEST ELISA

Johne’s Disease (Mycobacterium Para tuberculosis). SPECIMENS

Fecal swabs, mucosal scrapings, intestinal lymph nodes

Serum TYPE OF TEST

ELISA , CFT, PCR

Titers: 1:8 – Negative, 1:16 – Suspicious, 1:32 – Positive

Typical lesions of contagious caprine 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).

Foot And Mouth Disease

Foot And Mouth Disease

Foot And Mouth Disease

PPR in a goatpurulent eye and nose

dischargesDischarges 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 goatInflamed (reddened)

eye membranesReddening 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 cellsEarly pale, grey areas of dead cells on the gums

PPR in a goat later mouth

lesionsThe 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.