COLLECTION PRESERVATION AND DESPATCH OF SPECIMEN FOR LABORATORY DIAGNOSIS

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COLLECTION PRESERVATION AND DESPATCH OF SPECIMEN FOR LABORATORY DIAGNOSIS

Tissue samples are collected from dead or live animal for laboratory examination to confirm the tentative diagnosis.  

PURPOSE:   

  1. Diagnosis of the disease.
  2. Confirmation of tentative diagnosis
  3. Prognosis
  4. To observe the effect of treatment and give direction for future therapy.

GENERAL CONSIDERATIONS  FOR COLLECTION  OF SPECIMENS: 

  1. The diseases most commonly encountered in animals are of bacterial, viral, parasitic, fungal and metabolic origin. Diagnosis based on symptoms and laboratory examination of the relevant materials is essential for initiating treatment at the proper time. In general the following points should be duly considered while collecting materials for laboratory diagnosis.
  2. All materials collected should be accompanied by full history of disease outbreak namely species affected, duration of disease, clinical signs, morbidity and mortality rates, disease suspected etc.
  3. The collected biological specimens should be transported on ice to the nearest laboratory as early as possible.
  4. Materials collected for bacteriological examination should be kept at refrigeration temperature (4 °C) in case of delay of transportation. If a viral etiology is suspected the material can be stored at –20°C to -80 °C.
  5. When sero-diagnosis is required, collect paired serum samples (about 2 ml sera). One serum sample should be collected at the onset of disease and second sera after recovery (3-4 weeks) from disease preferably on 21st day.
  6. If death is reported, the post-mortem examination should be conducted at the earliest as putrefied materials are unfit for laboratory examination & detailed post-mortem report should be attached along with the samples collected during post-mortem.
  7. For Histopathological studies, tissues should be preserved in 10% formalin. The volume of formalin used should be approximately 10 times the volume of material. Specimen bottles with wide mouth should be used for collecting tissues.
  8. All the impression smears before packing, should be fixed in methanol for 1-5 minutes unless otherwise specified.
  9. In case of outbreaks, try to collect materials from as many ailing animals (5-6 or more) as possible at the height of body temperature /clinical signs.

Collection Procedure of different biological material /specimen

Name of specimen         Collection Procedure
Blood Blood: Blood is examined for different purposes as listed below :

 

·     Preparation of blood smears for microscopic examination:                                         The ear vein is the most convenient site for taking blood when required for the preparation of smears or films. In poultry, blood is taken from comb, wattles or wing veins.

·     For cultural examination, transmission experiments and other studies:

In cows, buffaloes, horses, sheep and goats, blood is preferably taken from the jugular vein.

From pig, blood is usually obtained by cutting the end of the tail or tip of the ear, or from anterior vena cava.

In dogs and cats, from the saphenous or radial vein and from fowls brachial vein (on the ventral surface of the wing).From ducks jugular vein is the preferred site.

The site should be prepared by removing hair /feathers and disinfect it with spirit. Blood is collected by sterile syringe. From post-mortem cases, blood is collected from right ventricle.

For serological studies, serum is separated by allowing the blood to clot, keeping the syringe in a slanting position. After complete separation, serum is transferred to a sterile vial.

For haematological studies, blood is not allowed to clot by using anticoagulant (sodium citrate or EDTA). One part of anti-coagulant is added to 9 parts of blood.

Milk: Milk for bacteriological examinations should be collected in sterile container. For isolation purpose; milk should be collected after discarding the first few streams of milk.
Faeces Sample for examination of parasitic eggs or coccidial oocysts are collected from the rectum or at the time of defecation.
Urine: Urine is collected at the time of micturition or obtained with the help of a clean and sterile catheter.
Pus: Smear is made on a clean glass slide. For cultural examination, it should be obtained directly from the abscess in a sterile container, or sterile swab is used.
Nasal/throat/uterine  discharges: Samples should be collected on sterile cotton swabs, then placed in sterile screw cap test tubes.
Exudates/transudates and body fluids Collection has to be made with the help of sterile pipettes or syringes and to be despatched in leak proof sterile screw cap containers.
Tissue, blood and exudates smears: Tissue smears are to be prepared from cut surfaces of organs and fixed over a flame. Blood/exudates smears are drawn as thin film and fixed over a flame or in methyl alcohol.

 

Preservation of materials for specific examination

Specific examination Preservatives used
Bacteriological/ Mycological

 

·     Blood and exudates: are to be collected aseptically with sterile Pasteur pipettes/syringes, and then put in sterile tubes or vials without any preservative and to be transported over ice. Especially for Anthrax and HS blood should be collected in sterile container /Cryo vials with (500-1000µl EDTA is sufficient) and without EDTA. After collection cotton soaked with alcohol should be placed on the collection site and burned and transported strictly under cold chain.

·     Tissues: Pieces of affected organs with lesions should be collected in sterile condition and should be transported under cold chain.

 

Virological

 

·     Blood and exudates: to be collected aseptically without any chemical preservative and to be stored and transported on ice. In some viral infections (e.g. Blue Tongue and Classical Swine Fever blood may be collected in vials/vacutainer containing EDTA).

·     TissuesPieces of affected organs are to be collected under aseptic condition and transported either on ice. For FMD 50% buffered glycerine saline is the preferred transport medium

 

Parasitological

 

·     For identification of parasite and helminth ova 10% formalin is the preferred transport medium to preserve the integrity of the ova..

·     For coccidial oocysts, 2.5% Potassium dichromate solution is the preferred transport medium.

Serological tests: ·     After collection, serum samples have to be stored at -20°C and should be transported strictly under cold chain.
Histopathological

 

·     For routine and general histopathological examination tissue pieces are to be collected in 10% formal saline (collection to be done in wide mouth bottles with10 times the volume of tissues)

·     A copy of detailed post-mortem report should be sent.

Toxicological                                ( Forensic laboratory) ·     For chemical analysis fresh tissues and fluids should be sent as soon as possible and on ice. Avoid addition of preservatives to the samples. Use 95% ethanol @ 1ml per gram of sample when necessary.

 

SPECIMENS REQUIRED FOR LABORATORY EXAMINATIONS IN FEW IMPORTANT DISEASES

                                                   Bacterial diseases
Name of Disease Antemortem sample Postmortem sample
Anthrax:
  • Smear from ear vein, discharges or from swelling,
  • Whole blood with and without EDTA.
  • Smear of blood from the ear vein or caudal vein
  • Whole blood  should  be collected from ear vein/caudal vein
  • Exudate or blood mixed soil should be placed in sealed pack
  • Pieces of ear, or muzzle should be placed in sterile container for Ascoli’s test.
Haemorrhagic Septicaemia
  • Smear from ear vein
  • Smear from fluid of swelling of throat.
  • Whole blood from jugular vein with or without EDTA
  • Blood smears or exudates obtained with the help of sterile syringe and fluid from oedematous swelling.
  • Heart blood with and without EDTA
  • Portion of Lungs, spleen and mediastinal lymph nodes, liver on ice
  • Long bones.
  • Lungs & other affected tissue in 10% formalin
Black quarter  /Black leg
  • Smear from affected muscle or swab from the swelling of the affected quarter by making small incision
  • Whole blood from jugular vein with or without EDTA
  • Smear from swelling
  • Affected muscle piece on ice.
Actinomycosis /  Actinobacilosis
  • Pus from mandible and maxilla & Pus smear.
  • Slides from pus for sulfer granules.
  • Tissue from affected parts in 10% formalin
  • Pus in sterile test tube/from edge of lesion.
  • Portion of swelling mass on ice
Brucellosis
  • Serum after 3 weeks of abortion.
  • Vaginal mucus; seminal plasma; uterine discharge
  • Foetal stomach tied off
  • Pieces of stomach, liver, placenta, aborted foetus on ice
Mastitis
  • 10 ml milk in sterile vial on ice                                                         
  • Blood smear, milk and pus from the affected quarter, if available
Tuberculosis
  • Sputum in a sterile swab, sample of milk from infected udder
  • Faeces in a sterile container
  • Smear from lymph glands showing nodular lesions
  • Lungs, lymph glands, intestine, mesenteric lymph glands should be collected if lesions are seen on ice
John’s Disease
  • Rectal pinch, swab or smear
  • Bowel washing in sterile bottle
  • Smear from rectal mucosa
  • Mesenteric lymph node in 10% formalin
  • Smear from thickened areas of intestine, small portion of bowel showing gross lesions
Glanders
  • Nasal discharge; pus from skin lesions
  • Smear from discharge
  • Lung, liver and spleen in 10% formalin
  • Serum
Strangles
  • Smears from pus of abscess; smears from discharge from nasal cavity
  • Smear from pus of affected part.
  • Pieces of affected lymph nodes, lungs, spleen, and trachea on ice
Salmonellosis
  • Blood is collected from wing vein
  • Faeces in sterile container
  • Intestinal content from ileum and large intestine on ice
  • Mesenteric lymph nodes, kidney and gall bladder on ice
Leptospirosis
  • Serum 21 days after abortion.
  • Blood ( at febrile stage ) on ice
  • Milk / urine in Vials( 1 drop of formalin in 20 ml sample
  • Portion of kidney & liver on ice
Listeriosis
  • CSF and whole blood with and without EDTA
  • Half brain in ice.
  • Half brain in 10 % formalin
Colibasilosis
  • Faeces in sterile container
  • Abdominal viscera; heart blood, fresh intestinal tissue in Sterile vial
Tetanus
  • Smear from the exudates/wound
  • Muscles, spinal cord and brain on ice
Enterotoxaemia
  • Whole blood with or without EDTA
  • Small intestinal contents, intestinal smear
Mycoplasmosis / CCPP/CBPP
  • Swabs from sinus/ trachea
  • Nasal and vaginal swabs preferably in Amies transport medium on ice
  • Serum samples (paired serum)
  • Piece of lung preserved in 10% formalin and on ice separately

 

Rabies
  • Saliva on ice if possible
  • Brain on ice for demonstration of viral antigen, viral inclusions and isolation of virus.
F.M.D
  • Vesicular fluid
  • Tongue epithelium from areas of ruptured vesicles in sterile vials containing 50% buffered glycerine
  • Oesophageal and pharyngeal fluid, collected by probang with mouth gag.
  • .Lymph nodes, kidney, adrenal gland, heart &Thyroid gland on ice
PPR
  • Ocular, buccal, rectal, nasal swabs on ice, no preservative should be added.
  • Paired serum samples

 

  • Pieces of spleen, lymph nodes, lungs, liver on ice.
  • Lungs, liver, spleen, tonsil in 10% formalin for HP
Pox
  • .Pox scab on ice
  • Pieces of skin and other organ with lesion in 10% formal saline
Classical Swine Fever
  • Whole blood in EDTA on ice is the most preferred sample
  • Pieces of spleen, mesenteric lymph glands, intestine especially ileocaecal region in 50% glycerol saline for isolation.
  • Pieces of brain, lung, intestines, ileocaecal region & kidney for HP
Ehrlichiosis
  • Blood smear, whole blood in EDTA
  • Spleen and lungs on ice
Ring worm
  • Skin scrapings from the lesions, including some hair roots, unpreserved in a tight container.
  • Skin scrapings from the lesions, including some hair roots, unpreserved in a tight container
Mange
  • Scabs and deep skin scrapings
  • Portion of skin showing lesion
Poisoning Cases
  •  Blood samples on ice
  • Stool, vomitus & urine sample on ice.
  • Stomach and intestinal contents should be sent after proper ligation at both the end sent it in ice to avoid putrefaction
  • Liver spleen, kidneys should be sent in separate container on ice.
  • Feed sample should be sent in separate container on ice

 

Despatch of Samples to Animal Health Laboratories:

Following points must be kept in mind while despatching the material to laboratory for diagnosis:

  1. Describe the clinical signs, lesions, tentative diagnosis and treatment given to animal in your letter; also mention the type of test you want with your tentative diagnosis.
  2. Write correct address on letter as well as on the parcel preferably with pin code, if the material is sent through post.
  3. Mark the parcel “Biological Material” “Handel with Care” “Glass Material” etc .in order to avoid damage in parcel, also mark the side to be kept on upper side with arrows.
  4. Seal the container so that it should not leak in transit.
  5. A separate specimen box should preferably be used for each caseso that they do not contaminate each other, or leak in transit..
  6. Try to send the material as soon as after collection from animal.
  7. Keep one copy of cover letter inside the parcel and another copy by hand or post in a separate  cover
  8. Put a frozen coolant pad into each tin when packing the specimens for despatch.
  9. In poisoning cases the address of Forensic Laboratory should be clearly written.
  10. The bottle or container should be sealed and labelled properly indicating the name of owner, identification of animal (tag no /mark/colour/sex/ breed /age/species) type of tissue collected and preservative used (if any).The examination requested and disease or poisoning suspected should also be written.
  11. A copy with details of post-mortem report and containing above information should be sent separately under separate cover
  12. All the container should be packed with cloth and sealed with sealing wax and should be sent through a person in order to avoid any breakage in transit.

Collection of Parasitological Samples from Livestock and Birds

Materials to be collected for Parasitological Analysis

  1. Faecal Sample                                     2. Blood Slide
  2. Lymph node smears                            4. Whole Blood ,  5. Serum
  3. Impression Smear                               7. Adult Helminths
  4. Skin scrapings                                     9. Ticks/fleas/lice/any other arthropod

Faecal Sample

Collection

  1. a) Fresh faecal material
  2. b) Collect immediately after defecation
  3. c) Collect directly from the rectum of large animals
  4. d) Collect at least several gram of faeces (the size of human’s thumb) for small animals

Preservation

  1. a)  If collected faecal sample cannot be examined within a few hours, the sample should be refrigerated until it is tested.
  2. b) Faeces should not be frozen
  3. c) Faeces should be examined immediately for suspected case of protozoan infection             (Trophozoites)
  4. d) Faecal sample containing helminth eggs, can be preserved with an equal volume of 10% formalin
  5. e) Adult Nematode can be preserved in 70% alcohol/ 10% formalin
  6. f) Adult trematodes –first flattened in between two slides and tied up with tags, then preserved in 10% formalin
  7. g) Adult Cestodes- Preserved in 10% formalin or 70% alcohol, precaution should be taken during preservation of scolices.
  8. h) Faecal Sample for Poultry coccidiosis -The dropping may be kept in 2.5% Potassium–dichromate (KMnO4) solution.

Despatch

  1. a) Faeces should be dispatched/ submitted in a sealed glass or plastic container, clearly marked with the time & date of collection, species of animal, animal’s name, owner’s name, age of the animal and any other information (History) relevant to the case
  2. b) The physical condition of the stool should be noted for color, consistency, and presence of parasites
  3. c) When material is to be sent another laboratory, it should be packaged with ice packs.

QUALITATIVE METHODS OF FAECAL EXAMINATION FOR PARASITES

Macroscopic Examination

  1. Intact worms or proglottids may be identified on the surface of faecal material
  2. Consistency of faecal material(Liquid/Soft/Formed/Semisolid), soft or liquid faecal material should be examined within one hour of passage to ensure the motility of the parasites
  3. Colour of the faecal material: if muddy colour, it may be due to ascarid worm infestation, greenish color may be due to strongyloidiosis, whitish due to cryptosporidiosis.

Microscopic examination

Direct smear method

  1. Thin smear of  emulsified faeces (to demononstrate the presence of parasite stages-eggs/larvae of helminthes, oocyst of Eimeriasp.)
  2. Small quantity of faeces is placed on a slide
  3. Mixed / dilute with N.S. or ordinary water
  4. A drop of the mixed faecal material spread into a thin layer
  5. Put coverslip on the faecal smear
  6. Observed under the microscope at low (X100) and high (X400) power objective lens to identify the specific parasitic egg
  7. At least 2-3 slides to be examined before giving conclusion.

Parasitic ova found with this method: Coccidian oocyst , helminthic eggs, cestode & trematode eggs (mainly birds)

Concentration Method

Sedimentation by gravitation     

  • Take approximately 3 g of faeces, mix it with water in a pastle morter
  • Filter the faecal suspension through a tea strainer or double layer of cheesecloth into urin glass/beaker
  • Add water to wash strainer until the urine glass is almost full
  • Keep it standstill for 10-15 min to sediment
  • Remove (pipette, decant) the supernatant very carefully
  • Repeat the washing step for 4-5 times till the supernatant becomes clear
  • Take a drop of sediment to examine under microscope

Sedimentation by centrifugation

      1. Emulsify small amount of faeces in water
      1. Strain it into centreifuge tube
      1. Centrifuge at 2000 rpm for 3-4 min
      1. Discard the supernatant and mix the sediment with water again
      1. Repeat washing by centrifugation followed by discard of supernartant for 2-3 times
      1. Examine the drop of sediment as before

Application: The procedure can be used to assess the presence of trematode infections like liver fluke (Fasciola) and amphistome eggs.

Simple flotation method

  1. Put approximately 3 g of faeces with floatation fluid (saturated sodium chloride solution) in a pestle morter
  2. Mix the contents thoroughly
  3. Pour the faecal suspension through strainer into centrifuge tube erect in stand
  4. Add floatation fluid upto the brim of the tube
  5. put a cover slip on the top of the tube
  6. Allow to stand for 10-20 min.
  7. Gently mount the cover slip on micro slide for microscopic examination of egg /larvae

Flotation by centrifugation

This method provides higher concentration of parasite objects, practically free from detritus by combining the principle of gravitation and floatation.

    1. Follow the step of sedimentation by centrifugation method
      1. After last centrifugation, the sediment resuspended in floatation fluid
      1. Centrifuge again  at 2000 rpm for 1-2 min
  1. Keep the tube in erect stand
  2. Add floatation fluid  with a pipette upto the brim of the tube
  3. put a cover slip on the top of the tube
  4. Allow to stand for 5-10 min.
  5. Gently lift the cover slip and place it on clean slide to examine under microscope

 Application: This is simple technique for use in initial surveys & to detect low numbers of helminth eggs especially nematode ova.

Formalin-Ether technique

This method is very useful in the circumstances which require preservation of faecal sample and floatation method is not so effective due to presence of fat and fatty acids.

  1. Emulsify 1-2 gm of faeces in 15 ml water
  2. Strain it and transfer the filtrate into 15 ml centrifuge tube
  3. Centrifuge at 2000 rpm for 2 min and decant the supernatant
  4. Repeat the washing steps until supernatant is clear
  5. Mix 10 ml of formalin (10%) to the sediment and allow to stand for 10 min or longer for fixation
  6. Add 4 ml of ether and shake vigorously using stopper
  7. Centrifuge at 2000 rpm for 2 min
  8. Carefully loosen the plug at ether-formalin interface with a pipette
  9. Pour off the entire supernatant along with the plug
  10. Take a drop of sediment, mix with a drop of 2% iodine
  11. Put a cover slip and examine under microscope

QUANTITATIVE METHODS OF FAECAL EXAMINATION FOR PARASITES

Stoll’s method

  1. Weigh 1 gm of faecal sample in a balance
  2. Mix with 15 ml of water ( 1 gm in 15 ml)
  3. Strain it, then shake the filtrate to form uniform mixture
  4. Take out 0.15 ml (1/100) on a slide and put coverslip over it
  5. Count the eggs of whole content (0.15 ml)
  6. Multiply the number with 100 to get E.P.G (eggs per gram of faeces) value

McMaster’s method

  1. Weight 1 gm of faecal sample in a balance
  2. Mix with 15 ml of floatation fluid ( 1 gm in 15 ml)
  3. Strain it, then shake the filtrate to form uniform mixture
  4. Charge on special (McMaster slide) where exactly 0.15 ml can be charged into each of the 2 chamber
  5. Count the eggs within ruled areas of both the chamber under low power (X100) microscope
  6. Multiply the number with 100 to get EPG value

FAECAL CULTURE FOR COCCIDIAN OOCYST IN ANIMALS AND BIRDS

Coccidian infections are very much common and important for poultry and ruminant especially in young animals. Many species are responsible for causing coccidiosis in animals and birds and are strictly host specific. When passed in faeces the coccidian oocyst are unsporulated and can not be differentiated. Therefore, culture of faecal sample for sporulation of coccidian oocysts is very much essential for diagnosis as well as for epidemiological study.

Procedure

  1. Take a small amount (3-5 gm) of faeces in pestle-mortar
  2. Add 2.5% potassium dichromate (KMnO4) to emulsify it
  3. Filter through a tea strainer
  4. Pour the filtrate into petridish to the depth of ½ cm only (if necessary, add KMnOsolution)
  5. Keep at room temperataure
  6. Examine under microscope at every 6-12 hrs for  complete sporulation
  7. Record shape, size my micrometry and the sporulation time for identification of different species.

PREPARATION AND EXAMINATION OF BLOOD AND TISSUE SMEARS FOR THE PRESENCE OF HAEMOPARASITES & MICROFILARIAE

  1. Blood Slide
  2. Thin /Thick Smear must be prepared from a single drop of blood
  3. Use Clean grease free slides
  4. Smear can also be prepared from anti-coagulated (EDTA) blood sample submitted in a vacutainer
  5. Prepare film should be smooth & even, it should be 3-4 cm. long
  6. Labeled the film/ mark the slide
  7. Air dry the film immediately by waving in the air (this avoids cremation of the erythrocytes)
  8. Blood Slide may be sent through slide mailer.

METHODS:

Preparation of Thin smears

  1. Place a small drop of blood (1.5 –2 cm. from one end of a clean slide).
  2. Hold the spreader at one angle of 45°in front of the drop of blood and bring back to touch the blood allowing it to spread along the edge.
  3. Make the film by pushing the spreader forward a direct and even movement.
  4. The film should be 3-4 cm. long.
  5. Air dry rapidly by waving the film in the air
  6. Labeled the film either by writing with a pencil in the film itself or, if in the laboratory, marks the slide using a diamond tipped pen.
  7. Fix the film in methanol for 2 minutes as soon as the film detoriates rapidly and so it is advisable to carry a small bottle of methanol for this purpose when out doing fieldwork.

Preparation of Thick smears

  1. Place a drop of blood at one end of a slide.
  2. Touch the drop of blood with a spreader & make thin film starting at 3 cm. from the end of the slide.
  3. Spread the remaining blood in the shape of a cube (3 x 20 mm). If the film is too thick, it will peel off the slide.
  4. Air dry and draw a line between two films.
  5. The haemoglobin can be removed from the thick film by inverting the slide and placing it an angle in distilled water (2 min).

Examination of a wet-smear

  1. Take a drop of freshly collected blood on a clean glass slide and put a cover slip on it
  2. And examine under light microscope (first 10X and then 40X objective lens)
  3. By this method, diagnosis of Trypanosomasp. and microfilaria is very easy, because the viable and motile organisms can easily be seen.
  4. Protozoa in faecal smears and Trichomonas sp.in vaginal smear can also be identified in fresh smear.

Lymph node Smear

  1. Locate the position of lymph node (sub-scapular region and other superficial lymph node) and hold it firmly
  2. By means of syringe and needle pierce the gland & move the needle  up to medulla
  3. Pull the plunger of the syringe to aspirate the sample of the contents of the gland & withdraw the needle
  4. Expel the contents to a clean slide and make the smear
  5. Lymph node smear is required for identification of Koch’s blue body and for diagnosis of bovine tropical theileriosis.

Whole Blood/Serum

  1. Blood may be collected with EDTA and send it through ice-packs
  2. Serum (Blood without EDTA- Serum) may be collected & send in vial packaged with ice packs
  3. Blood should be collected at the time of pyrexia.
  4. Proper identification no. and history of the case must be included during sending of samples

STAINING OF BLOOD/LYMPH NODE SMEARS

Giemsa’s staining  

This technique varies according to individual preference. The stain usually used in Giemsa diluted 1:9 (timing, dilution and pH may vary slightly with different batches of stain)

  1. At first, the smear is fixed with conc. Methanol and air dried
  2. Measure 1 ml of concentrated (stock) Giemsa stain  into a  clear empty well ( the stock satin may need to be passed through filter to remove particles)
  3. Add 9 ml of water pH 7.2 ( the ideal pH vary with batches of stain)
  4. Mix well and pour the smear and assure that the whole smear should be covered with stain.
  5. Allow staining for 45 minutes.
  6. Wash the smear in distilled water or tap water
  7. Dry in air and examine under oil immersion objective.

Leishman’s Staining:

  1. Fix air-dried smears in Leishman stain for 2 minutes.
  2. Add an equal volume of buffer distilled water.
  3. Mix by gently rocking the slide to allow even distribution.
  4. Leave staining for 10-15 mints.
  5. Rinse in buffer and immerse until the smear appears pink (1-2 mints) and air dry.
  6. Observe under oil-immersion.

EXAMINATION OF BLOOD SMEARS FOR THE PRESENCE OF MICROFILARIAE

Microfilariae are the larval stage of helminthic parasites, which may occur in the blood, e.g. the microfilariae of canine heart worm, i.e. Dirofilaria immitis. Following methods are applied for diagnosis of microfilaria.

Direct smear

It is very simplest and rapid method sensitivity of the this method is very poor. However, this technique may be used to evaluate the pattern of movement of  microfilariae.

  1. Place one drop of venous blood into a clean microscopic slide.
  2. Place a cover slip over the drop of blood.
  3. Examine the cover slip area under low magnification (x100) of microscope.
  4. Notice for undulating movement of the larvae, which may retain their motility for as long as 24 hrs.

Haematocrit test

This method is slightly more sensitive than direct smear

  1. This technique is not used extensively
  2. Draw fresh whole blood into a micro-haematocrit tube, as for routine packed-cell volume test.
  3. Spin for three minutes in a haematocrit tube centrifuge.
      1. Examine the plasma portion of the separated blood under low magnification (x100). Swimming microfilariae may be present in the plasma above the buffy coat.

Knotts method

Concentration method for detection of microfilariae in blood. It is generally considered preferred technique for heartworm screening   because it   is standard, quick, and inexpensive.

    1. Blood is collected into a syringe containing anticoagulant such as heparin or EDTA
      1. Mix 1 ml of the blood with 9 ml of a 2 % formalin solution. If not well mixed, the red cells will not thoroughly lysed, making the test much more difficult to read. Microfilariae, but not red cells, will be fixed by 2% formalin. If 10% formalin is used (the concentration used for fixation of tissues) red cells will also be fixed.
      1. Centrifuge the mixture at 1200 rpm for 5 minutes and discard the supernatant fluid.
      1. Take the sediment on a microscopic slide and add 1 drop of 0.1% of methylene blue using a Pasteur pipette
      1. Examine under low magnification (x10) under microscope. Microfilariae will be fixed an extended position with nuclei stained blue.

DIAGNOSIS OF MANGE INFESTATION IN DOMESTIC ANIMALS

Mange is a disease condition of skin caused by wide range of obligatory external parasites, commonly known as mites and classified under the order Acarina, which inhabit different layer of the skin. Mange is a pretty common skin affection encountered in almost all domestic and wild animals, birds and humans. The clinical manifestations included redness, thickening of skin, scaliness and itchiness. Various mange mites encountered in domestic animals and birds are Dedodex spp., Sarcoptes spp., Psoroptes spp., Otodectis spp., Cnemidocoptes spp., Notoedres spp.

Skin Scraping examination

  1. Scraping are taken from the affected area of the body by a scalpel blade or a sharp-edge spoon. The area selected for scraping should be at the edge of a visible lesion and their hair over the area should be clipped away. It is useful to moisten the skin with liquid paraffin so that scrapings adhere to the scalpel. Scraping should be continued until the blood oozes from the surface.
  2. The scraping is transferred on a  clean glass slide and a drop of any mineral oil is added and is mixed properly with a stick.
  3. A cover slip should then be applied and examined under a low objective (10X)
  4. Demonstration of adult mite or any of the life stages including eggs of mites confirms the diagnosis.
  5. Direct examination of the skin scraping is not very sensitive  for the diagnosis of mange especially, if it is a case of sarcoptic mange in canine.
  6. The following method should be applied to increase the sensitivity.
  7. The skin scrapping after collection as described above is taken in a centrifuge tube and mixed it with 5-7 ml of 10% KOH and boil the preparation ( while boiling, precautions should be taken to prevent the content of the tube to bump off).
  8. The material is then centrifuged at 1000 rpm for 3-5 min. The supernatant is discarded and the whole sediment is examined microscopically.

COLLECTION & TRANSPORTATION OF ARTHROPODS/SNAILS

Collection

  1. Adult ticks and their developmental stages (larva and nymph) remain attached by keeping their mouthparts embedded in skin. Care should be taken during collection, so that no mouthpart may break which is having identification mark.
  2. Mites remain embedded either superficially or deep into the skin of these hosts and can be collected by taking the skin scrapings.
  3. Lice & Fleas pick up with the help of forceps into the container.
  4. Snails are to be collected form water body/pond in a clean air tight container and send it to the laboratory within 24 hr.

Transportation& preservation

  1. Arthropods are usually transported as living specimens in proper containers like plastic /stopper vials
  2. For shorter distances, the arthropods can be transported simply in plastic vials congaing paper labeling and moistened filter paper.
  3. For temporary storage , the arthropods can best be stored at cool temperatures
  4. If stored for a longer period, then it can be stored in wet preservatives like 10% formalin or 70% ethanol.
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