Diagnostic Tests for Brucellosis

Diagnostic Tests for Brucellosis

1.      Milk Ring Test

It can be perform at herd level, milk tank, or individual level.
Principle:
It is based on detecting specific agglutinin in the milk which cause agglutination in the milk.
Specific for detecting agglutinant agent which agglutinate stain antigen adding to the milk sample of the cow.
After incubation the presence of agglutination is determine by agglutination of stained antigen which rises with the cream layer.
In this test antigen is comprise of killed brucella abortis with dye (Hematoxin blue or Tetrazolium red)
Result:

·         If ring form than test +ve

·         If no ring form than test -ve

2.      Rose Bangal test

It is a serological test.
It is modification of serological test or serum agglutination test.
These are routinely perform for screening of brucella in dairy herd.
Material;
2-things important for it
1-Serum
2-Rose Bangal Dye (Antigen of brucella)
So this test is Antigen-Antibody reaction between Rose Bangal Dye and Brucella.
p.H is adjusted at 3.6 to prevent non-specific growth of other bacteria.
Procedure:
0.3ml serum and 0.3ml Rose Bangal dye mix and wait for 4-mint to check agglutination.

·         if agglutination than test +

·         if no-agglutination than -

Advantage:

·         Easy to perform

·         Cheaper          

·         Immediate result

·         It is better than SAT

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Milk Tests for Mastitis Diagnosis in Animals

Milk Tests for Mastitis Diagnosis in Animals

1.      California Mastitis Test

This is rapid mastitis test.
Take 5 ml milk in shallow and take equal amount of NaOH.
NaOH is California mastitis test (CMT) reagent.
Rotate and mix plate.
Any thickening, gel formation, or change in pH of milk.
Gel formation is due to this material react with bacteria.
1mg indicator in the regent bromcresol purple indicates the pH of the milk by changing the color of milk.

2.      NAGase Test

This based on the measurement of cell associated enzymes Sodium Acetyl beta D- glucosaminidase in milk.  
This enzyme in milk actually come from neutrophils and damage epithelial cells.
Highly active this enzyme high level of somatic cells and this indicate high level of mastitis because somatic cell count (SCC) increase in mastitis by inflammation of udder.

3.      Pour Plate Method

Principle:
In this method we will be able to understand the pour plate method in which the bacteria are mixed evenly distributed and separated evenly in the throughout the liquid.
The method agar is then poured into an empty palate and allowed to solidify
Material:

·         Nutrient agar liquefied

·         Sterile petri dish

·         Test tubes

·         Microscope

Procedure:
Take a test tube in which milk sample and molten broth is present.
Place that sample on petri dish.
Swirl the petri dish in this way both sample and nutrient agar mix up.
Agar at that temp would killed the bacteria when are introduced so the agar is cooled to 60C and held in the water bath to maintain temp.
Inoculate the plate at 37C for 24hrs to 48 hrs.
After 48hr examine the petri dish and count the CFU
Result:
Colonies were obtained from surface of petri dish.

4.      Somatic Cell Count

It is number of somatic cells in milk.
Poor quality milk has a high number of SC.
Most herd milk contain b/w 200000-500000 SC/ml of milk.
This test is used to evaluate the udder health of all individual cows in herd.
To determine the level of mastitis in herd.
To tell us the quality of milk.
SC count increase with the severity of udder infection. However the corresponding milk loss does not increase at the same rate.
In healthy cows SCC up to 100000 / ml.
SC also depends on lactation number:
*1st Lactation cow have up to 35000 /ml
*2nd Lactation cow have up to 50000 /ml
*3rd Lactation cow have up to 60000 /ml
If SCC/ml < 200000 than no mastitis. Test is negative. Mixture remains liquid with no evidence of thickening or formation of precipitate. Test score negative.
If SCC/ml 150000-500000 than animal is suspicious. Than test is Trace. Slightly thickness. Tests score 0.
If SCC/ml 400000-1500000 than weak mastitis present. Tests score 1. Distinct thickening. But no gel formation. Thickening may disappear after prolonged rotation of paddle.
If SCC/ml 800000-5000000 than distinct mastitis present. Its score 2. Mixture thicken immediately. If paddle the milk thick material move in center.
If SCC/ml > 5000000 than sever mastitis present. Distinct mass. Abrupt thickness and also gel form.

5.      Standard Plate Count

Standard plate count is a common microbial test to check the quality of milk.
SPC indicate the number of bacterial colonies growing on a non-specific solid nutrient agar (medium) after a given period of incubation.
Materials:

·         Milk sample

·         Test tube

·         Sterile petri plate

·         Microscope

·         Nutrient agar

·         Register

Procedure:
Setup a dilution scheme and raw milk is normally diluted to 1:10, 1: 100, 1: 1000 , 1: 10000 , 1: 100000.
Before starting the dilution procedure, shake the milk sample 25 times in the customary manner.
After dilution we pour the samples on petri dishes in which already solidify nutrient agar is present.
Incubate the plate at 35C for 24hrs then count the colonies on plate that has been between 30 and 300 colonies.
Calculation:
Numbre of colonies on plate × Reciprocal of dilution of sample = Number of bacteria/ml
e.g. 32 × 10000 = 320000 /ml.

6.      Strip Cup Test

Use for the physical examination of the milk.
This test is only use for clinical mastitis.
Shiny black plate with 4 shallow should be used.
Take milk sample its 1st 2, 3 stream.
Take 5-10 ml of milk sample from every teat.
Check for any discoloration, clot, or flakes formation or any purulent material.
Discoloration may be blood stain or any watery appearance shows positive case of mastitis.

7.      Surf Field Mastitis Test

Quick method for diagnosis of sub-clinical mastitis.
These all test directly related to somatic cell count.
Take a black plate and make 3% solution of surf.  
Take milk 5ml from 4teat&take in 4 shallow and mix in each 5ml surf solution and mix for 1 mint.
After that we examine the mixture for any thicking or clot or flake or color or any abnormality.
Quality of thickness indicate the condition of mastitis
Surf contains Sodium alkyl aryl sulphate which break down the cell wall of bacteria and react with their DNA.

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Blood Sampling in Animals

Blood Sampling in Animals

Blood

Blood is composed of blood cells (erythrocytes, platelets, 5 types of leukocytes) and plasma.

Blood withdrawn from a vessel must immediately be mixed with an anticoagulant to prevent initiation of clot formation and to keep cells and other components in suspension.

Analysis of whole blood must be rapid because the cells die within few hours and the sample become unacceptable for analysis. 

COMMON DISEASES AND CONDITIONS IN WHICH BLOOD SAMPLE IS REQUIRED

Bacterial Diseases.

1. Chronic strangles
2. Strptococcal infections
3. Anthrax
4. Botulism
5. HS
6. CCPP
7. Brucellosis
8. Leptospirosis
9. Bacillary heamoglobinurea
10. Borelliosis
11. Jhones disease
12. Tuberculosis

Viral Diseases

1. EVA
2. Equine viral rhinopneuminitis
3. Equine influenza
4. Equine adenovirus
5. FMD
6. Vesicular stomatitis
7. RP
8. PPR
9. MCF BVD 
10. BT 
11. Equine herpes virus infection
12. IBR
13. Maedi-visna disease
14. Venzuelan viral encephalomyelitis of horses
15. Pseudo rabies
16. Louping ill
17. Border disease
18. Bovine ulcerative mammalitis

Parasitic diseases

1. Babesiosis
2. Anaplasmosis
3. Eperythrozoonosis
4. Q- fever
5. Theleriosis
6. Trypanosomiasis

Metabolic/ Nutritional Disorders

1. PPH
2. Carbohydrate engorgment
3. Milk fever
4. Downer cow
5. Acute hypokalemia of cow
6. Lactational tetany of mares
7. Hypomagnesemic tetany
8. Ketosis
9. Pregnancy toxemia
10. Fatty liver of cattle 
11. Equine hyperlipidemia
12. Disorders of thyroid
13. White muscle disease
14. Deficiency/ poisoning of minerals

DISEASES & CONDITIONS THAT CAUSE NEUTROPHILIA

1. Inflammatory

Infections- bacterial, fungal, viralImmune heamolytic anemiaNecrosis- heamolysis,, heamorrhage, infarcts, burns, neoplasia

2. Glucocorticoid-associated

      a)      Stress

      b)      Glucocorticoid therapy

DISEASES & CONDITIONS THAT CAUSE NEUTROPENIA

1. Inflammatory- Equine salmonellosis, canine/ feline parvo virus, equine influenza, mastitis, pneumonia
2. Immune neutropenia
3. Toxic- Estrogen, chemotherapeutic drugs, chlorumphenicol (cats), bracken fern
4. Marrow necrosis
5. Myelofibrosis

DISEASE & CONDITIONS THAT CAUSE LYMPHOCYTOSIS

1. Chronic inflammation- bacterial, viral, fungal. Protozoan infections
2. Neoplasia- lymphoma, lymphocytic leukemia

DISEASES AND CONDITIONS THAT CAUSE LYMPHOPENIA

1. Acute inflammation- acute bacterial & viral infections, endotoxemia 
2. Glucocorticoid associated- stress, glucocorticoid therapy 
3. Loss of afferent lymph- alimentary lymphoma, enteric neoplasm, granulomatous enteritis, paratuberculosis 
4. Immunosupressive drugs

DISEASES & CONDITIONS THAT CAUSE MONOCYTOSIS

      1.       Inflammation-

           a)      Infections-baterial, viral, fungal. Protozoa 

           b)      Necros

      2.       Glucocorticoid associated- stress, glucocorticoid therapy

      3.       Monocytic leukemia

DISEASES & CONDITIONS THAT CAUSE EOSINOPHILIA

1.  Allergic disorder

·         Flea bite dermatitis 

·         Hypersensetivity to streptococcal, staphlococcal proteins 

·         Milk allergy in ruminants 

·         Asthma & eosinophillic respiratory disorders 

2.  Parasitism

3.  Idiopathic eosinophillic condition

4.  Mast cell neoplasia

DISEASES & CONDITIONS THAT CAUSE EOSINOPHENIA

1. Glucocorticoid associated- stress, glucocorticoid therapy 
2. Acute inflammation

DISEASES & CONDITIONS THAT CAUSE BASOPHILIA

1. Allergic reactions
2. Parasitism
3. Neoplasia- basophilic leukemia, mast cell neoplasia, polycythemia vera etc. 

MORPHOLOGIC FEATURES OF ERYTHROCYTES- CLINICAL SIGNIFICANCE AND IMPORTANCE

1. Discocytes- mature erythrocyte of each domestic mammal are discs with different degrees of bioconcavity that creats central pallor
2. Rouleau- aggregate of RBCs in stalk like manner
3. Agglutination
4. Rubricytosis
5. Ghost cell
6. Hypochromis erythrocyte
7. Polychromatophil
8. Reticulocy

ERYTHROCYTE PARASITE

Organism - Anaplasma marginale 

Identifying features - Marginal body is a small, dark staininng coccus about 0.5μm diameter on internal margin of erythrocyte typicall one organism per cell but may be multiple

Organism - Anaplasma centrale

Identifying Features - Small dark staining coccus about 0.5μm diameter with in erythrocyres typically one organism per cell but may be multiple

Organism - Babesia spp 

Identifying Features - Intracellular oval to tear drop or pearl shaped trophozoites; size varries with species typically pale blue with darker outter membrane and reddish purple eccentric nucleus

Organism - Cytauxzoon felis 

Identifying Features - Intracellular oval structure (0.1 – 2 μm) with outter thin rim and eccentric nucles; may resemble signet ring or saftey pin; one to several piroplasm per cell

Organism - Eperythrozoon spp 

Identifying Features - Rings, rods or cocci on surface of RBCs 0.3 – 1 μm diameter

Organism - Heamobartonella canis 

Identifying Features - Typically thin chain of cocci on membrane that form pleomorphic pattrens; occasionaly seen as individual cocci or rods

Organism - Heamobartonela felis 

Identifying Features - Typically cocci (individual or in short chains), may form small ring or doughnuts (1μm) on erythrocyte surface, stain blue-grey to pale purple

Organism - Theileria spp

Identifying Features - Highly pleomorphic piroplasm including cocci, rings, rods, pears and maltese crosses

INCLUSIONS OTHER THAN PARASITES

Inclusion - Basophillic stippling 

Identifying Features - Fine to coarse, blue to dark purple dots or specks that represent aggregated ribosome disperesed in an erythrocytes cytoplasm 

Inclusion - Distemper inclusion in dogs 

Identifying Features - Round or variable shaped, pale blue or auzrophillic homogeneous inclusion, variable sizes

Inclusion - Heinz body 

Identifying Features - Slightly pale round structure that creats a membrane defect, protrudes from erthrocyte, or occurs as free body, stains blue with NMB stain.

Inclusion - Heamoglobin crstals 

Identifying Features - Intensily stained, crstallized heamoglobin that forms a pencil, parallelogram,cube or other polyhedron with in the erythrocyte

Inclusion - Howell- jolley body 

Identifying Features - Usually a homogeneous, dark pruple staining, round structure in erythrocytes, not assocated with membrane can be ring forms

Inclusion - Siderotic granule 

Identifying Features - Loose aggregate of granular basophillic inclusions; stained blue with Fe stains

ABNORMAL ERYTHROCYTE SIZE

Acanthocyte (acantho= spur)

Other name - Spur cell, burr cell 

Identifying Feature - 2-20 irregulary spaced membrane projections of variable lengths, projections may be blunt spurs or club 

Codocyte (codo=hat)

Other name - Target cell, mexican hat cell 

Identifying Feature - Cenral focus of hgb that is surrounded by  a ring of pallor that separates it from peripheral Hbg; one form leptocyte 

Darkocyte (darko= tear)

Identifying Feature - Tear drop shape 

Eccentrocyte (eccentro= eccentric)

Other name - Bite cell, cross bonded cell, hemighost cell 

Identifying Feature - Eccentric dense staining Hgb and adjacent clear space or cresent

Echinocyte (echino= spiny)

Other name - Burr cell 

Identifying Feature - Varry from irregulary shaped cell to regularly shaped cell

Keratocyte (kerato= horn)

Other name - Helmet cell 

Identifying Feature - Noteched, flattened margins between two membrane projections, varient has one horn

Leptocyte  (lepto= thin)

Identifying Feature - Thin cell that appear as hypochromic cell with increased central pallor 

Ovalocyte (ovalo= egg)

Other name - Elliptocyte 

Identifying Feature - Elliptical or oval 

Pincered cell

Identifying Feature - Button or knob joined to rest of cell by apinched area 

Pyknocyte (pykno = condensed)

Other name - Irregularly contracted cell 

Identifying Feature - Spheroid erythrocyte with condensed or contracted Hgb and perhaps small tag of condensed membranes

Schizocyte (schizo= cut)

Other name - RBC fragment, schizocyte 

Identifying Feature - Triangular, comma-shaped, small round or irregularly shaped piece of an erythrocyte 

Spherocyte (sphero= round)

Identifying Feature - Decreased central pallor, decreased cell diameter, increased Hgb staining intensity, and smooth margins

Stomatocyte (stomato= mouth)

Identifying Feature Elongated area of cytoplasmic pallor

Torocyte (toro = donut shape)

Identifying Feature - Punched out, central clear space that creata donut-shaped cell. 

VENOUS BLOOD COLLECTION

      1.       Occlude the vein with a tourniquet or digital pressure. 

      2.       Wipe the skin and hair on top of the vein with an alcohol soaked cotton ball to help identify the vein. 

      3.       Insert the needle in to vein through skin at angle of 25 degree. 

      4.       Slowly retract the syringe plunger, and collect a blood sample. 

      5.       Release the pressure on vein, & release the syringe plunger when sufficient volume of bolo has been collected. 

      6.       Remove the needle from vein. 

      7.       Apply digital pressure to venipuncture site for heamostasis. 

BLOOD COLLECTION SITES IN DIFFERENT ANIMALS

Lab Animals BLOOD COLLECTION SITES

1.       Tail vein puncture

2.       Saphenous vein

3.       Jugular vein

4.       Cardiac

5.       Caudal vena cava

6.       Ear vein

Rabbit BLOOD COLLECTION SITES

      1.       Cardiac 

      2.       Marginal ear vein 

      3.       Central ear artery  

      4.       Jugular vein 

Chicken BLOOD COLLECTION SITES

1.       Wing vein

2.       Anterior vena cava

3.       Jugular vein

4.       Wattles/ comb

5.       cardiac

Cat/ Dog BLOOD COLLECTION SITES

      1.       Cephalic vein

      2.       Sephanous vein

      3.       Cardiac

      4.       Jugular vein

      5.       Femoral vein

Sheep/ Goat/ Cattle BLOOD COLLECTION SITES

     1.       jugular vein

ANTICOAGULANTS

The best anticoagulant is the one which prevent coagulation with least cellular damage.

The choice of anticoagulant depends on the type of examination to be carried out.

The most important anticoagulants are:

   1.   Ethylene Diamine Tetra-acetic acid (EDTA).

   2.   Heparin.

   3.   Ammonium and potassium oxalate mixture.

   4.   Sodium citrate.

   5.   Sodium fluoride and potassium oxalate mixture.

Ethylene Diamine Tetra-Acetic Acid (EDTA)

1.       Disodium or dipotassium salt of EDTA, also known as Sequestrene, Disodium versenate or Versene.

2.       The disodium salt has maximum solubility in water of 10 % concentration. While, the dipotassium salt has the advantage of being more soluble than the disodium salt, but it is much more costly.

Mode of action: Precipitation of calcium ions.

Advantages: 

      1.       It has an excellent preserving power and it is recommended for use in routine blood examination.

      2.       Doesn’t alter the erythrocyte size when employed in the recommended concentration.

      3.       Leucocytes staining are excellent.

      4.       EDTA blood was used in determination of creatinine, urea nitrogen, glucose, phosphorus and uric acid.

Disadvantages: 

      1.       Higher concentration of salt withdraws water from red cells and reduces PCV values.

      2.       Not suitable for estimation of the level of alkaline phosphatase activity because it combine with magnesium ion which are needed for activation of ALP.

      3.       Chloride estimation in EDTA blood was always higher than results with other anticoagulants.

Amount required: 

1mg / 1 ml blood.

Precaution

1.       Excess of EDTA will produce shrinkage of erythrocytes and leucocytes (above 2 mg / ml blood).

2.       ESR does not change during the 1st 6 hr. with the blood samples at room temperature and may be unchanged for up to 24 hr. in samples held in refrigerator.

3.       In fact, there is a difficulty in solubility of disodium salt in blood when 1stly dissolved in water and then evaporated to dryness.

Heparin 

    It is a natural anticoagulant occurring in various tissues and it is found abundantly in the liver from which its name is derived.

Mode of action: Heparin prevents blood coagulation by interfering with conversion of prothrombin to thrombin.

Advantages 

      1.       Does not alter the erythrocyte volume, so it is suitable for haematocrit determination.

      2.       It has the least effect on erythrocyte haemolysis.

      3.       It is especially recommended for blood of cats.

      4.       Lithium salt does not interfere with estimation of the level of calcium, sodium or urea.

Disadvantages 

1.       More expensive (about 150 times than oxalate).

2.       Unsuitable for smears for its poor stain affinity.

3.       Will not prevent clotting of blood for more than 8 hr.

4.       Cause the cells to stain bluish with wrights stain.

Amount required 

0.1 ml of 0.75 % solution and evaporate to dryness at room temperature, this is sufficient for 5 ml blood.

It can be used in liquid form as Heparine injection to coat the inside of the syringe.

Precaution  

Heparin not used for blood of rabbit, this because it need special co-factor which is absent in this species.

Ammonium and Potassium Oxalate Mixture (Heller And Paul Mixture) 

Advantages 

      1.       It is cheaper than EDTA. 

      2.       Easy to prepare and use.

      3.       Little cellular distortion occurs if the sample is examined within the 1st hour of collection.

      4.       Cause very little haemolysis or changes in the volume of RBCs.

Disadvantages 

1.       It doesn’t prevent clumping of platelets.

2.       It is poisonous.

3.       It can’t be used in estimation of the blood levels of potassium or serum ALP or urea by reason of ammonium salt.

Mode of action: Binding Ionized calcium.

Amount required 

      1.       Ammonium oxalate   1.2 gm.

      2.       Potassium oxalate     0.8 gm.

      3.       D.W.                       100 ml.

1ml of the solution in a tube, then dry at 60C. This is sufficient for 10 ml blood or, 2 mg / ml blood.

Precaution  

      1.       The inorganic salts have the effect of altering the volume of the erythrocytes so that cell plasma ratio varies with the type and concentration of anticoagulants

      2.       Ammonium salts increase the cell volume whereas sodium, potassium and lithium salts produces an opposite effect, so that the combination of potassium and ammonium oxalate in ration of 2 : 3 keep the morphology of the RBCs.

Sodium Citrate

    Sodium citrate is employed in blood transfusion because the citrate is metabolized and excreted rapidly.

Mode of action: Binding ionized calcium.

Advantages 

    The best anticoagulant that preserve cellular constituent, biochemical constituent and keep cell morphology.

Disadvantages 

      1.       Interfere with many biochemical tests.  

      2.       Prevent the clotting for only few hr.

      3.       Increased concentration may cause shrinkage of cells.

Amount required

The strength of the stock solution is 3.8 %.

Nine volumes of blood are added to one volume of the sodium citrate solution and mixed immediately.

Sodium citrate is also the anticoagulant most widely used in the estimation of the ESR: for this, four volumes of venous blood are diluted with 1 volume of the sodium citrate solution.

Sodium Fuoride and Potassium Oxalate Mixture

      1.       It is better to use the mixture than sodium fluoride alone as it increase the anticoagulant effect.

      2.       The mixture consists of 4 parts sodium fluoride to 5 parts of potassium oxalate.

      3.       To prepare tubes add 0.5 ml of 2.25 % solution of the mixture and evaporate off the water, this is sufficient for 5 ml blood.

Mode of action: Binding ionized calcium.

Advantages

    Used only for determination of glucose level, since it is effective in inhibiting the glycolytic enzyme which breakdown glucose in blood.

Disadvantages

      1.       It is poisonous.

      2.       Unsuitable for determination the level of ALP or urea determination.

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