LABORATORY DIAGNOSIS OF CANINE AND FELINE DISEASES
S. K. Panda,
Assoc. Professor & Head, Veterinary Pathology, OUAT, Bhubaneswar
Veterinary laboratory diagnosis has become an integral part in modern pet animal practice. Laboratory tests yield useful information and provide definite evidence regarding the alterations during the disease process.
Basic clinical pathology or practitioners laboratory is a small self contained laboratory aiding in the diagnosis of certain diseases in a clinic or dispensary. It deals with routine screening of samples collected by the clinician. Generally examination of wet film of blood, blood smear, blood, urine, faecal sample, skin scrapping, nasal washing and cytological preparations are carried out in such laboratory. Tests could be easily and quickly performed in such laboratory. Complete diagnostic laboratory deals with clinical pathology, biochemical, parasitological and microbiological examination of the clinical samples collected from the diseased pet animals.
Diagnosis of the pet animal diseases mostly depend upon the proper collection of the samples, procedure of the tests and interpretation of the results.
Hematological Examination (Complete Blood Count)
Blood to be used for hematologic tests collected from cephalic vein which is the most commonly used site in dog. Ear vein can be used in the cat, small dog. A marginal ear vein on the dorsal side of the ear is usually selected. Toe or toe nail can be used in small dog and puppies. The capillary bed of the nail is cut into just short of the base of the nail. Femoral, saphenous or tibial vessels used in the dogs and cat. Recurrent tarsal vein can be used for dog (situated at lateral side of hock joint in hind limb). For collection of blood the needle and syringe must be dry since presence of water hemolyses the RBC. The needle should be sterile. Clip the area from where the blood is to be drawn. Apply Tr. Iodine and allow it to dry. Raise the vein by pressure. Use a large (bore) needle that has been sterilized and dried. Insert the needle into the vein; draw into the dry syringe the required quantity of blood. This engages the needle, insert the syringe into the specimen tube containing the anticoagulant to very near its bottom and allow the blood to flow out. Thoroughly mixed with anticoagulant. Never shake the blood vigorously otherwise froth will form, RBC may rupture and erroneous results may be obtained. In collecting blood care should be taken to ensure that needle is of sufficient diameter. A needle of too small gauge may cause disruption of RBC and damage WBC. If it is necessary to transfer the blood from a syringe into a test tube, the needle should be removed, as forcing blood through the needle may damage cells.
A great variety of anticoagulants may be used for hematological examination. Dipotassium and Disodium salt of Ethylene diamine tetracetic acid (EDTA is mostly recommended for routine hematological procedures may be used in either a liquid or in dry form.1mg of powder per ml of blood is required. If liquid is available, 1 drop of a 10% solution is sufficient for 5ml of blood. Care must be taken not to exceed the recommended level of EDTA as it adversely affects the hematological results. Heparin is also commonly used in liquid or dry form (in a test tube). A syringe may be rinsed with a stock solution (1% )of heparin & will contain sufficient anticoagulating activity to preserve 5ml of blood. 1-2 mg (0.2ml of 1% solution) is required for 10 ml of blood.
Taking too much time in obtaining the blood so that the sample has already started to clot before being mixed with the anticoagulants. Failure to agitate the blood sample immediately after placing it in vial and filling the vial to the top cause clotting, as the anticoagulant is not properly mixed with the blood. It is usually safe to take about 0.5 ml blood/Kg body wt. in all spp. Blood should be collected when the animal is at rest and without under excitation.
It is always advisable to start the examination of blood as soon as possible after its collection. But if delay is expected the blood samples must be kept in refrigerator at 40C for 24 hours without much alterations. The refrigerated blood sample should be taken out at least 30 min before the start of exam & the sample should be mixed properly with gentle shaking.
Interpretation for Hemoglobin (Hb)
Decreased Hb in anemia (Hypochromic) which is caused by blood loss, hemolytic anemia, auto immuno hemolytic anemia, nutritional, parasitism-anaplasma, piroplasma, bacteria-leptospira, Chemical-lead, phenothiazine, copper, Deficiency-copper,cobalt,vit-B12,folic acid, riboflavin, pyridoxine.
Increased Hb in hemoconcentration, diarrhoea, vomition, excess sweating, burn, polyuria, shock.
Interpretation of Total Leucocyte Count (TLC)
Leucocytosis in generalised infection, localised infection, intoxications including those produced by metabolic disturbances, chemicals, drugs and venoms, rapidly growing neoplasms, acute hemorrhage particularly into one of the body cavities (thoracic peritoneal Joint), sudden haemolysis of erythrocyte, leukemias, trauma, from parenteral administration and excess secretion of adrenal corticosteroids
Leukopenia: The general causes of leukopenia are 4D which are related to alteration in bone marrow. 4D are Degeneration, Depression, Depletion, Destruction. If any of these alterations occurs in the bone marrow, the no. of leukocytes mostly nutrophils in the peripheral circulation is decreased. Viral infections such as canine distemper, infectious canine hepatitis, feline panleukopenia are often accompanied by a decrease in TLC. The decrease is usually observed during the early stage of disease-secondary infection following viral diseases are usually accompanied by a leukocytosis. Overwhelming bacterial infections also result in leukopenia that is commonly accompanied by a diminution of mature neutrophils in the peripheral blood. Early stages of an infectious disease or of a localized severe infection where there is depletion of the pheripheral blood of its leukocytes with concentration in the area of the infection resulting in a leukopenia until the bone marrow catches up with the production. Endotoxins from Gram-ve bacteria produce a significant leukopenia, large doses of endotoxin have severe effects on formed elements of the blood particularly leukocytes & thrombocytes both of which is decreased. Leukopenia is also associated with cachectic and debilitated states and old age that may be caused by lack of certain nutritional factors or by exhaustion of the marrow. In Vitamin B12 and folic acid deficiency there will be mild chronic leukopenia. Physical agents such as x-rays and radioactive substances produce a leukopenia by destroying the cellular elements of the bone marrow. Anaphylactic shock and early stages of a reaction to foreign protein may produce a leukopenia. Bone marrow abnormalities also produce leucopenia. Bone marrow hypoplasia which may be due to metabolic disorders, Ionizing radiation. Chemical agents, cause leucopenia Bone marrow dysplasia caused due to subleukemic or aleukemic Leukemia, Bone marrow displacement (myelophthisic) may cause leucopenia. Rickettsial diseases like ehrlichiosis and protozoan diseases like toxoplasmosis also cause leucopenia. Chemical agent may also produce leuckopenia those are some includes some of the antibiotics–( Chloramphenicol, penicilin streptomycin and oxytetracyline), Analgesics(phenacitin,,Antipyrine and aminopyrine), Antifungal–(griseofulvin), Antihistamines (pyribenzamine), Anticonvulsants (primidone), antithyroid (thiouracil), hemopoetic depressant (cytoxan, 6-mercaptopurine),cortisone products, sulfonamide, inorganic chemicals (lead Benzene, bismuth, mercury, Arsenic, Thallium and others (DDT, Barbiturates, chlorpromazine).
Interpretation for TEC
Decreased TEC occurs in anaemia, esp. in toxic, aplastic, haemolytic and chronic haemorrhagic anaemia.
Increased TEC occurs in polycythemia, haemoconcentration due to loss of fluid, diarrhoea. vomiting. excess sweating, severe burns, polyuria, shock.
Interpretation for PCV
In haematocrit a column of PCV shorter than normal indicates anaemia while one taller than normal indicates hemoconcentration. An anaemic animal with severe hemoconcentration due to loss of fluid from the body (diarrhoea, vomition) may have a PCV falling within normal limits. So while interpreting PCV, the water balance of the animal should be taken into consideration. Approximate leucocyte counts can be made from the buffy coat at the top of the packed erythrocyte layer in the wintrobe tube. The first millimetre equals to 10000 and each 0.1 mm thereafter equals 2000 leucocytes/microliter.(1mm equals 20000) If lower, it is leucocytopenia and if higher, it is leucocytosis. This approximation should never be used as a substitute for TLC since it is possible to be misled by a marked thrombocytosis or the variability in size.
Interpretation for ESR
Increased ESR in acute generalised infection, acute localized infection of serosal membranes like peritonitis, pleuritis, pericarditis. chronic localised infection like suppurative conditions, skin alterations like inflammation, hypothyroidism and hyperadrenocorticism, tissue injury, malignant tumors where considerable reaction, vascularity and tissue breakdown, pregnancy.
Specific conditions characterised by an increased ESR are dog-distemper, leptospirosis, pyometra, chronic interstitial nephritis, radiation injury, filariasis, acute and subacute bacterial endocarditis, myocarditis, myocardial degeneration, pneumonia, pleuritis, peritonitis, ICH, salmon diseases, hypothyroidism, hyper adrenocorticism, bone fracture, trypanosomiasis.
Blood smear
Examination of a smear of peripheral blood is one of the most informative laboratory procedures. It should be made within 15 min. after the collection, but the best smear are made immediately from fresh blood. Inspect the blood smear under low power to note distribution of cells & select a portion of the smear near the thin end where the erythrocytes do not overlap. Switch to the oil immersion objective for making the rest of the examination.
Erythrocytes should be inspected in regard to
- Size – Variation(Anisocytosis)- Normocytic, Macrocytic or Microcytic)
- Shape – Variation(Poikilocytosis)-target cells,sperocytes, acanthocytes, leptocyte)
[Toxic disease may cause change in size or shape of RBC]
- Colour – Normochromic, Hyperchromic, hypochromic
- Erythrocytic inclusions(Polychromasia)- Howell-Jolleybodies,Basophilic stippling as in Lead poisoning, hinz bodies
- Polychromatophilia, Nucleated erythrocytes, reticulocytes in brilliant crysyl blue stain or new methylene [N.B- Reticulocytes are immature RBC & larger in size & non nucleated. Since this cell represents a more immature form of erythrocyte, its presence in periphery blood is of importance in assessing bone morrow response.
Bacteria & parasites : Parasites likeTryps, Anaplasma, Piroplasma, Micro filaria, and bacteria in septicemia.
Thrombocytes or platelets :- Estimate the number present as normal, increased or decreased or none. [Four or more per oil immersion field is normal]. Note variation in size and morphology. Mostly seen in clusters but may occur singly or in small clumps. Giant forms are occasionally seen with a distinct cell membrane surrounding light colored cytoplasm and deep reddish purple staining granules. Greatly increased no. would suggest increased activity of bone marrow.
Leukocytes :- Interpretation of DC
Neutrophilia in Non – infectious conditions like neoplasia, foreign body, trauma, sterile abscess, tissue manipulation uremia (toxemia), stress and Infectious conditions like bacterial (acute infection), mycotic, protozoan or parasitic.
Neutropenia in overwhelming bacterial infection, viral infections, rickettsia, chronic infections, malignancy, deficiency of Vit-B-12 & Folic acid, per – acute bacterial infection, bone marrow depression
Eosinophilia in skin allergies Eosinophilic pneumonia, enteritis, parasitism, and leukemia
Eosinopenia in any stress (release of corticosteroids), administration of ACTH or corticoids
Basophilia in basophilic granulocytic leukemia. But it is rare.
Lymphocytosis in neutropenia may have relative lymphocytosis, Lymphocytic leukemia, recovery stage of certain infections, adrenocortical insufficiency, following vaccination, chronic infection, hyperthyroidism, lymphadenitis, blood parasites (Babesia, trypanosoma), excitement
Lymphocytopenia in some viral disease like canine distemper, ICH, parvo, pan leucopenia etc., stress – due to glucocorticoids, Injection of ACTH and hyperadrenocortcism, Ionizing radiation and mmunosuppressive drugs.
Monocytosis in chronic diseases, granulomatous reaction, fungal infection, T.B, brucellosis, protozoan infection, erysipelas, listeriosis, monocytic leukemia, acute stress, hyper adenocorticism, recovery / late phase of diseases.
Wet Blood Smear can be employed for microfilaria
Urine Examination
Interpretation of urine examination
When there will be pathophysiological change of the body there may be alteration in the quantity and quality of the urine excreted. Hence examination of urine helps in assessing the health of the body. A critical examination of urine is an important diagnostic procedure.
The urine must be collected in a clean container from the mid stream of the morning sample. Urine can be collected by catheterization, manual compression of urinary bladder. Urine analysis should be completed as quickly as possible after collection as chemical and cytological changes occur rapidly in urine. Bacteria multiply rapidly. If urine to be preserved then kept in refrigerator and added with the preservatives like toluene, thymol etc.4-6 drops of formaline(40%) can be added but gives false reaction for sugar. Thymol(0.1gm/100ml)may be used but false reaction for albumin. A pinch of camphor can preserve 30 ml of urine. In leptospire either directly examine the slides under dark ground illumination. In case of parasite in urine 2 drops of 40% formalin is added to 30-40 ml of urine and dispatch.
Interpretation of physical changes:
Volume: urine volume is dependant upon several physiological factors including water and fluid intake, environment, diet, activity of animals. Increases in urine volume (polyuria) may be present transiently owing to diuretic therapy or increased fluid intake and followimg parental administration of fluid or administration of corticosteroids. Pathologic polyuria associated with acute and chronic generalized nephritis, diabetes mellitus, insipidus, nephrogenic diabetes insipidus, diduretic phase of toxic nephrosis, primary renal glucosuria, pyometra. renal amyloidosis, pyelonephritis, compulsive polydypsia and some liver diseases. Urine volume will decrease (oliguria) with decreased fluid intake, high environmental temperature, hyper ventilation. Oliguria seen in dehydration, decreased BP, acute nephritis, prolonged fever, circulatory dysfunction.
Colour and Transparency: Normal urine is pale yellow and clear. Colour becomes red in hematuria amd hemoglobinuria, greenish yellow in jaundice, green when given methylene blue(urinary antiseptic)brown or coffee in hemoglobinuria, dark yellow in acute nephritis, fever, dehydration etc. Turbid(thick) urine indicates pyogenic process in urinary tract and epithelial cells(protein). Pathologically cloudy urine may be observed when any of the following like leucocytes, erythrocytes, epithelial cells, bacteria. Transparency is tested by viewing against light in a test tube. In congenital porphyria, a faint pink. Pink colour also in medication of phenothiazine.
Specific gravity: Increased specific gravity seen in acute interstitial nephritis, cystitis, diabetes mellitus, dehydration, vomition, diarrhoea, fever, reduced fluid intake, hypovolumeic shock, burns. Decreased specific gravity seen in increased fluid intake, interstitial nephritis, advanced state of uremia, diabetes inspidus, hyperadrenocortisim, pyometra.
Odour: It is not diagnostic although the urine of males of feline and has an especially strong odour. An odour of ammonia may appear if urea is being converted to ammonia by bacterial action. Strong ammonia smell suggest cystitis due to proteus organism. Ketone bodies impart a sweetish and fruity odour and may be detected in urine and associated with pregnancy disease, acetonemia, diabetes mellitus. Rotten grapes like smell of urine suggest diabetes mellitus.
Foam:if shaken after collection, normal urine produes a white foam that is limited in quantity. if there is proteinuria excess foam produced and slowly disappear. if bile-foam is green,yellow,yellow brown, if hemoglobin-foam is red to brown.
Interpretation of chemical examination:
Reaction of urine PH: Acid urine or aciduria seen in normally carnivore’s urine, nursing calves if diets with excess protein but pathologically seen in starvation, fever, acidosis, administration of acidic salt like NaCl, ammonium chloride,CaCl2, proteineuos diet. Alkaline urine or alkalanurea seen normally in carnivoress with veg diet., but pathologically seen in cystitis, retention of urine, administration of alkaline salts like acetate, bicarbonate, citrate or nitrite of sodium or potassium, rapid adsorption of transudates.
Glucose(sugar): Glycosuria is seen in emotional states-fear, excitement, where there is sudden release of adrenaline which causes hyperglycemia with resultant glycosuria, heavy meal of carbohydrate, diabetes mellitus, after general anaesthesia, hyperthyroidism due to rapid absorption of glucose from the bowel, chronic pancreatitis, acute pancreatic necrosis, hyperpituitarism, overactivity of adrenal cortex, shock, intravenous glucose administration, chronic liver diseases.
Ketone Bodies(acetone): Ketonurea seen in diabetes mellitus, starvation, high fever, cachetic condition.
Protein: Proteinuria found in physiological conditions like excess proteinous diet intake, excess muscular excretion, emotional stress, in convulsion and pathological conditions like nephritis by increased permeability of the glomerular filter. In acute interstitial nephritis—protein with cast in urine. In chronic interstitial nephritis—slight protein with casts. In pyelonephritis –marked proteinuria with leucocytes & RBC. In nephrosis due to poisioning by phenol, arsenic, lead, phosphrous, mercury, sulphonamide,turpentine,ether,bismuth, salicylic acid -excess proteinuria. In amyloidosis, renal infraction & neoplasms—proteinurea. In renal congestion due to cardiac congestion, pressure on abdominal veins due to ascites, tumor—slight protenuria. In postrenal condition when protein enters urine after it leaves the kidney tubules like cystitis, vaginal or preputial discharges,prostitis,pyelitis,uretheritis,ureteritis,urolithiasis,trauma with haemorrages –proteinurea observed.
Blood: Hematurea is differentiated from hemoglobinurea by centrifuging the urine. In hematuria sediment examined for presence of RBC and supernetent is colourless. In hemoglobinuria the fluid is red or coffee colour and erythrocytes are lacked. Hematurea seen in pyleonephritis, acute nephritis, ureteritis, cystitis, pyelitis, urolithiasis, passive congestion of kidney, infarction of kidney, neoplasm of kidney, bladder, or prostrate, abscess of kidney, during estrus, at postpartum in females, prostitis, severe infection in leptospirosis, Rubarth’s disease, trauma to urethra during improper catheterization, toxic chemical agent-copper, phenol, sulfonamide, mercury, arsenic and thalium poisoning, sweet clover poisoning, shock-capillary hemorrhage, parasite- Dictyophyma renale,Dirofilaria,Capillaria plica. If blood found in last drops of urine then source is bladder, if urine is red throughout source of blood is kidney and if first portion of urine is red then source of blood is some urethrallesions. Hemoglobinuria found in leptospirosis, postparturient hemoglobinuria, babesiosis, photosensitization, bacillary hemoglobinuria-clostridium hemolyticum infection, Chemical hemolytic agents like copper,mercury and sulfonamides, severe burns, hemolytic disease of the new born, incompatible blood transfusion, drinking large volume of water, plant poisons
Myoglobinurea (azoturea) when urine contains myoglobin and absence of erythrocytes in the sediment. When muscle pigment myoglobin is found in urine, the condition is called as myoglobinurea. it is suggestive of severe destruction of muscle fibres. Myoglobinurea also occurs in electric shock and snake venum.
Calcium: normally very small amount present. Decreased in Bovine Hypocalcemia, tetany, hypothy- roidism, osteomalacia. Increased in renal osteodystrophy,hyperthyroidism,hypervitaminosis.
Urobilinogen: Normally small amounts are excreted in urine. Decreased amount or absence of urine urobilinogen in obstruction of biliary passages, decreased RBC destruction, impaired intestinal absorption, diarrohea, nephritis and some times due to polyuria causing dilution. Increased urine urobilinogen seen in hepatitis, cirrhosis, hemolytic jaundice.
Bile:Presence of excess bilirubin in urine is bilirubinurea. Found in hepatocellular disease, Infectious canine hepatitis, cirrhosis, neoplasm, toxicity, obstruction of bile duct, jaundice, acute enteritis and intestinal obstruction.
Interpretation of microscopical examination:
For microscopical examination sediments of centrifuged urine always taken.
Epithelial cells– Squamous epithelial cells are laregest and having small round nucleus. Transitionalbepithelial cells are smaller,granular and having small round nucleus. Renal epithelial cells are smallest with a nucleus. Pathologically large number of renal epithelial cells found in acute interstitial nephritis. Transtitional epithelial cells found in cystitis and polynephritis.
Erythrocytes- Indicates hemorrhage in some part of genitourinary tract and in faulty catheterization.
Leucocytes (pus cells)– larger than erythrocytes and smaller than epithelial cells and granular cytoplasm. Increased leucocytes in urine is pyuria seen in nephritis, pyelonephritis, pyelitis, ureteritis, cystitis, vulvitis, vaginitis, balanitis
Casts- Hyaline cast indicates a mild form of renal irritation. These are composed of protein as homogenous, semitransparent, colourless, cylindrical structure having rounded ends. Granular casts indicates severe type or renal disease in tubular epithelium. Granular casts are hyaline casts contain granules. Epithelial casts derived from desquamated epithelial cells. It indicates desquamation of epithelial cells. Waxy Casts similar to hyaline being homogenous but appear more opaque than hyaline. it indicates advanced or severe nephritis. Fatty casts contain small droplets of fat as refractile bodies.It indicates deposits of fatty material in tubules. Blood casts are homogenous cylindrical masses having a deep yellow to orange colour. Erythrocytes within these casts degenerate. This indicates glomerulitis, haemorrhges. Leucocyte casts means presence of many pus cells. it indicates nephritis, pyelonephritis, kidney abscess etc. Mucus thread indicates presence of contamination from the genital secretion. Parasites:ova seen in urine like Dictyophyma ranale-giant kidney worm of dog, Capillaria plica-bladder worm of dog,cat,fox. Crystals:if acid urine-amorphous urates, uric acid, calcium oxalate and hippuric acid. If alkaline –triple phosphate, amorphous phosphate, calcium carbonate, ammonium urate.
Amorphous phosphate-Powder, Calcium oxalate-diamond/Envelope, Triple phosphate- Prism, Sulfa crystal-Long prism, Uric acid-Radiating needle are some of the morphological pictures. Bacteria-seen in cystitis, pyelonephritis, genital tract infection. Yeast and fungi-only contaminatants. Fat seen in diabetes mellitus, high fatty diet, obesity, hypothyroidism, contamination.
Biochemical examination:
Blood serum or plasma can be tested for various enzyme levels which are elevated and depressed in various disease conditions. The results can be correlated with diseases.
Parasitological Examination:
The presence of ova or larvae of endo and ecto parasites can be easily demonstrated by direct examination or examining centrifuged faecal samples, skin scrapings etc. Faecal examination requires centrifugation after mixing with water, saline etc. and digestion of skin scrapping in 10% KOH or NaOH.
Microbiological Examination:
Bacteriology: Smears prepared from clinical samples can be stained for identifying Gm+ve or Gm –ve bacteria, acid fast bacilli etc. Cultural examination and antibiotic sensitivity test can be utilized for identification of the bacteria and to determine the susceptibility of bacteria to antibiotics.
Virological methods: Some commonly used virological methods employed in disease diagnosis are virus isolation, HI, ELISA, immunofluorescence, virus neutrasilation, electron microscopy, molecular methods like PCR etc.
