Presented to……..Dr Ashar Mahfooz

Presented by………Fakhar Un Nisa(2009 ag 3369)

SCREENING TESTS

WHAT IS EQUINE ANHYDROSIS…….

Equine anhidrosis, dry coat, and nonsweating are all terms used to

describe the disease in horses characterized by the inability to sweat effec-tively in response to appropriate stimuli

HOW IT CAN BE DIAGNOSED:

Diagnosis of anhidrosis can be tentatively made based on

clinical signs and performance.

It should be emphasized that, based on the development of

increased respiratory effort

hyperthermia,

anhidrotic horses are com-monly presented to veterinary practitioners for diagnosis and treatment of respiratory disease.

A MORE SPECIFIC TEST FOR ANHYDROSIS IS….

a semiquantitative test to evaluate sweating by induction of the sweat response, using dilute concentrations of epineph-rine injected intradermally, has been developed, and will provide a more definitive diagnosis.

DISADVANTAGE OF THIS TEST………..

epinephrine is not completely suitable for such a diagnostic test because it has bothb- anda-adrenergic receptor ago-nist properties, and therefore, may cause leukotrichia at the injection sites.

ANOTHER ALTERNATIVE……..

Salbutamol sulfate, a specificb2-agonist, has been described for use in a semiquantitative test for anhidrosis in Thoroughbred horses. A

control of sterile water and six dilutions of salbutamol, from 103w\v to 108 w\v, were injected intradermally. The results were read 20

minutes later.

RESULTS…….

Horses with long-standing anhidrosis did not sweat at any of the dilutions.

Normal horses sweated in response to the 10 8 w\v dilution,

partially anhidrotic horses responded with dilutions between 10 4

w\v and 10 6 w\v.

A SECOND ALTERNATIVE………

semiquantitative test using terbutaline sulfate, another specific b2-adrenergic receptor agonist, is also diagnostic . The protocol and results are similar to that of the test using salbutamol, except a physiol-ogic saline solution control is used.

BRUCILOSIS

• Major test widely used for its detection is RBT which is in detailed describe here.

INTENDED USE

Brucella antigens are bacterial suspensions for use in slide agglutination tests to detect the presence of bacterial agglutinins associated with bacterial infection or previous exposure to a related organism.

This slide test is recommended as a screening procedure only to establish the presence or absence of homologous antibody.

PRINCIPLE OF THE TEST

In the course of any infection with any pathogenic microbiological agent, a variety of antibodies are formed. Among these antibodies are the agglutinins.

An agglutinin when combined with homologous antigen (agglutinogen) under the properly controlled conditions is capable of causing agglutination.

The Rose Bengal stained Brucella antigen is used for the early detection of Brucella agglutinins.

REAGENTS

• 1- Rose Bengal Brucella Antigen (0.5% phenol)

• 2- Positive Control (0.01% sodium azide)

• 3- Negative Control (0.01% sodium azide)

WARNING AND STORAGE

WARNING

Brucella Antigens and control antisera are to be used for In Vitro Diagnostic Use Only.

STORAGE

Brucella antigen and control antisera must be stored at 2-8°C when not in use.

SPECIMEN COLLECTION & PREPARATION

. Collect 5ml whole blood samples aseptically from the patient.

2. Allow blood to clot and remove serum as soon as possible to prevent excess haemolysis.

3. Store serum at 2-8°C until testing can be performed.

PROCEDURE

Allow reagents and serum samples to reach room temperature for testing.

2- Shake the antigen bottle gently to insure a uniform suspension.

3- Place 40ul sample serum onto the selected ring of the slide.

4- Place one drop of the Rose Bengal antigen onto serum sample.

CONTINUE…..

Mix serum sample with Rose Bengal antigen using Stirring stick.

6- Repeat these steps using the positive and negative controls instead of serum sample.

7- Gently rock the slide for 2 minutes (automatic rotator can also be used).

8- Observe for agglutination after 2 minutes from beginning of shaking (this is the

optimum time limit).

RESULTS

Negative: No agglutination

Positive: (Presence of Specific Antibodies): Agglutination

LIMITATIONS OF THE PROCEDURE

detectable agglutinins are not always produced in bacterial infection. In some instances, non-specific agglutinins may appear which will react with Brucella antigens.

MILK RING TEST

The antigen is prepared from a concentrated cell suspension of Co2 independent smooth strain of Br. abortus (Strain 99). The cells are stained with haematoxylin and suspended in buffered diluent (pH 4.0) .

INDICATIONS :

It is used to determine the possible presence of Brucellosis in a herd of cows by the milk ring test (MRT). It has not proved effective with pasteurized or treated milk, also with milk of sheep and goats.

DIRECT OF USE :

Collection of milk samples : Milk contained in cans or holding tanks is thooughly mixed. Samples of milk is obtained from each can of milk, but from no more than 3 cans should be pooled in each collection tube (14 x 100 mm.). When collecting milk from holding tanks, milk from no more than one holding tanks should be put in each collection tube

METHOD OF PERFORMING THE TEST :

1. Mix the milk thoroughly and transfer on ml to a narrow test (11 x 100 mm.) . 2. Add one drop (0.03 ml) of the antigen and mix.3. Incubate at 37o C for one hour and read using a uniform light source.4. If the intensity of blue colour in the cream layer is equal to or deeper than in the skim portion, the test is considered negative.5. If the intensity of the colour in the cream layer is less than in the skim potion, the test is considered negative.

. False positive may occur at periods where a high proportion of the animals in the herd are drying off, or where mastitis is

prevalent.

PRECAUTIONS :

1. Milk samples should be mixed thoroughly before applying the test.2. Shake the antigen very well before use.3. Known positive and negative samples should be used in each test.