Wastes should be disposed in environmental friendly manner. Items to be discarded should be properly decontaminated and packed using biohazard bags before disposal. Blood samples may be taken for culture, in which case it is usual to use anticoagulants, such as ethylene diamine tetra-acetic acid EDTA or heparin.
They may also be taken for serology, which requires a clotted sample. This can also be accomplished by storing the whole blood sample, in an upright position, overnight at room temperature 22 — oC.
These samples can be used for molecular diagnostic tests. The test can be done on animals suspected to be infected viral diseases and bacterial diseases Using jugular vein puncture technique, collect 5ml to 7ml whole blood using EDTA vacutainer tubes and needles with needle holder. It may be helpful if the swab is first moistened with transport medium.
Note 2: Ensure that swab samples submitted to bacteriology laboratory should not have viral transport media. The antibiotic added in the VTM can hamper the diagnosis. Nasal, , Oropharyngal, lesions and eye. If the shaft is longer than the tube, break or cut it. Microbial replication will be most active at the periphery of the lesion.
Hence, special transport media should be used to keep the viability. OIE manual Chapter 1. Quinn et al. Pages: Title Any change, variation or breech of the procedure within the document will be notified to my line manager immediately.
I understand that it is a disciplinary offence not to follow the procedure documented in this SOP. Anyone using an uncontrolled copy is individually responsible for checking that they have the latest revision of the document prior to use 1.
This virus can persist for long periods in pig products and the environment. It can also become endemic in undomesticated or wild Suidae and in Ornithodoros ticks. The nucleic acid obtained is used as a template for further PCR. Cellular nucleic acids bind selectively to special glass fibers pre-packed in the high pure purification filter tube.
Finally, the nucleic acids are released from the glass fiber using sterile nuclease free water or elution buffer. Briefly centrifuge the 1. Place the high pure filter tube in a collection tube and pipette the sample in the upper reservoir. Centrifuge for one minute at rpm.
Note; for blood samples, repeat the centrifugation step if sample remains in the filter tube. Discard the collection tube and place the filter tube into a clean collection tube.
Discard the collection tube and repeat the washing step. Centrifuge for 10 seconds at rpm to remove residual wash buffer. Discard the collection tube and place the filter tube in a clean 1.
Centrifuge for 1min. Vet Res. It can also become endemic in undomesticated or wild Suidae, and in Ornithodoros ticks. There is no vaccine or treatment.
PCR is a three- step process that is carried out in repeated cycles. The initial step is the denaturation, or separation of the two strands of the DNA molecule, accomplished by heating the starting material to temperatures of about 95oC oF.
Each strand is a template on which a new strand is built. In the second step the temperature is reduced to a predetermined annealing temperature so that the primers can anneal to the template.
At the end of the cycle the temperature is raised and the process begins again. The number of copies doubles after each cycle generating multiple copies of the target DNA. Finally, in the conventional PCR the amplified product will be detected by agarose gel electrophoresis, staining with ethidium bromide that intercalates the double-stranded DNA.
This can be observed under UV light. Store at room temperature. Include all the controls After addition of the template, close the reaction tube and spin down the PCR mix. Place all tubes in an automated thermocycler. Run the incubation program as detailed below. Heat the solution in a microwave oven until the agarose is completely melted. Add the Ethidium bromide BrEt at a final concentration of 0. Shake carefully to homogenate. Prepare the gel tray, seal the ends and place the comb for adequate number of wells.
Pour the melted agarose into the gel tray. Wait until the gel becomes solid approx. Carefully remove the sealing of the tray and place it in the tank. Add the electrophoresis buffer until the gel is covered.
Carefully remove the comb. Connect to power supply and confirm direction of sample s movement DNA samples will move towards the positive electrode Run the gel at a constant voltage of volts for about minutes. Finally, place the gel on an ultraviolet trans-illuminator to visualize the bands. Note: The voltage depends on the percentage and size of the agarose gel.
Compare the size of the PCR products in the test samples and the positive control by reference to the standard molecular weight marker. The contamination could be due to the ASFV itself present in the positive analyzed samples or in the positive controls included in the DNA extraction procedure. It is mandatory that personnel working on PCR follow and carry out strict work-flow rules in order to minimize contamination risk associated to PCR technique.
It is highly recommended to order as dropper solution to minimize its manipulation. Ethidium bromide handling must be performed exclusively in the laboratory assigned to it while observing laboratory safety measures.
In case of any unintended contact, wash immediately with abundant water and contact the biosafety officer. This control should be sourced from the OIE. All the equipment must be put under planned maintenance in accordance with the Quality Manual regulations.
Pages: Any change, variation or breach of the procedure within the document will be notified to my line manager immediately. The condition is caused by bacteria of the genus Brucella, which occur in different variants in different animal species. For example, Brucella abortus is mostly associated with cattle and B. In animals it is characterized by abortion, retained placenta, orchitis and epididymitis and in human it is associated with undulating fever, fatigue, malaise, headache, backache, and arthralgia.
It is the responsibility of the head of laboratory to ensure that all the staff performing the test receive copies of the SOP. The sample together with monoclonal antibody mAb specific to an epitope on the o-polysaccharide portion of the S-LPS antigen, are exposed to Brucella abortus smooth o-polysaccharide S-LPS coated wells on micro titre plates.
If Brucella antibodies are present in the test sample, they will bind to the antigen in the well and block these antigen sites. If Brucella antibodies are absent in the sample, these sites will remain free and the mAb which was added together with the sample will bind to these antigenic sites.
After an incubation period the unbound material are removed by rinsing and conjugated IgG is added to the plate. The conjugate will bind to the specific mAb in the absence of Brucella antibodies in the sample.
Unbound materials are removed by rinsing prior to the addition of the substrate. Subsequently a blue colour develops which is due to the conversion of the substrate by the conjugate. A negative result is indicated by the development of a blue colour.
The reaction is stopped by addition of stop solution, the colour changes to yellow. In the presence of antibodies, no coloration appears. The test plate is read at nm. The pH of the buffer must be between 7. All reagents should equilibrate to room temperature 0c before use. For confirmation purpose it is recommended to run the control sera in duplicate. The sample can be tested in singlicates or in duplicates.
However for confirmation purposes it is recommended to run the samples in duplicates. Seal the plates and mix the reagents thoroughly for 5 minute, either by using a plate shaker or by tapping the sides of the plate. Incubate the plates at room temperature c for 30 minutes. Seal the plates and incubate at room temperature c for 30 minutes.
Repeat step no. Begin timing after the first plate is filled. Remember to add the stop solution in the same order as the substrate solution was added in step no. Measure the optical density OD of the controls and samples at nm in a micro plate photometer use air as a blank. Measure the OD within 15 minutes after addition of stop solution to prevent fluctuation in OD values. Chapter 2. Mantur B. G, Amarnath S.
K, Shinde R. Review of clinical and laboratory features of human brucellosis. Indian J Med Microbiol ; — Pages: 9. Fresh serum can also be used directly after centrifugation. In short, the microplate wells are coated with purified Brucella abortus lipopolysaccharide, LPS.
Specific antibodies present in the test sera bind to the coated antigen on the microwells, the anti Brucella antibodies if present form an antibody — antigen complex. After washing, a multi species horseradish peroxidase conjugate HRP is added to the microwells.
This fixes to the anti Brucella antibodies, forming an antigen — antibody conjugate peroxidase complex. The complex is revealed when HRP substrate TMB is added to form a blue compound that will turn yellow when the reaction is stopped. Thus the intensity of the colour formed is directly proportional to the amount of bound sample antibody. If it is not to be used immediately then prepare for 1 plate i. Top up to 1 litre with distilled water to make the PBS solution. Therefore 0. Guideline of conjugate dilution depending on the number of plates No of plates Conjugate ml Diluent buffer 24 ml 1 1 9 2 2 18 3 3 27 4 4 36 7.
Gently shake the plate until the coloured solution is homogenised; Wipe carefully the bottom of the plate; Read and record the OD at nm; Interpret and record the results. The kit contains a strong positive control, and negative control. Not applicable 7. The disease in animals is characterized by abortions or reproductive failure.
Animals typically recover, and are able to have live offspring following the initial abortion but they may continue to shed the bacteria. Brucellosis in cattle is caused by B.
Brucellosis is a notifiable disease and is listed as one of the diseases that must be reported to the World Organisation for Animal Health OIE. In man, Brucellosis manifests as an undulant fever commonly known as Malta fever. The vials are then labelled and assigned a laboratory number. Agglutination indicates presence of antibody in the serum sample. Bring the test serum, controls and reagents to room temperature C before use; 2. Using a marker pen, label each square on the white tile s with the sample ID; 3.
Label two empty squares with control IDs Positive and Negative ; 4. Gently shake the Rose Bengal antigen bottle to ensure a uniform suspension; 7. Start the timer immediately after mixing the first well contents and agitate the tile on the shaker for up to 4 minutes; Observe the mixtures for any agglutination in the various squares; Record the results obtained. In cattle, it causes heavy losses to farmers such as weight loss, low milk production and death. The disease is characterized by anorexia, fever and respiratory signs, such as laboured breathing, grunting when breathing, dyspnoea, polypnoea, coughing and nasal discharges.
The head and neck of the infected cattle are extended when standing with front legs apart. Lameness is observed in calves up to six months of age due to painful limb joints. Transmission occurs by direct contact between infected and healthy animals. CBPP also affects buffaloes. Antibodies against CBPP present in the test sera will bind to the MmmSC antigens coated on the micro-titre plate competing with the monoclonal antibodies for the specific epitopes.
The conjugate composed of anti-mouse IgG serum conjugated to horseradish peroxidase HRP added binds to any monoclonal antibodies fixed on the wells. If antibodies specific to MmmSC antigen are present in the test sera, they will outcompete and displace the monoclonal antibodies. Hence, the conjugate will not be able to bind. The intensity of the colour is an inverse measure of the proportion of MmmSC antibodies present in the test sera.
If the buffer is not for immediate use, prepare the amount required for washing 1 plate i. OR dissolve one PBS tablet in 1 litre of distilled water as recommended by the manufacturer, add 2. Transfer this to washing trough.
Dilute the buffer further by adding 4 litres of distilled water, mix well, label and store at room temperature oC for not more than one week. OR dissolve one PBS sachet in one litre of distilled water to give a buffer of 0. Transfer this to wash fluid container with a tap to which tubing may be attached.
Dilute the buffer further by adding 4 litres of distilled water, mix well, label and store at room temperature for not more than one week. NOTE: Prepare wash buffer according to the workload.
Wash 3 times with wash solution; empty plate completely by turning it upside down and fill it with ul with wash solution. Repeat this 3 times. Empty the plate and tap it upside down on absorbent towel to remove the remaining wash buffer; 4. Wash 3 times as in 10 above; 7. Cover plate and incubate for 30minutes at 37oC under gentle agitation; 9. Gently shake the plate until the coloured solution is homogenous; Wipe carefully the bottom of the plate.
Read the ODs at nm. The usefulness of these reagents as controls for the test is assured when the positive and negative controls produce results indicated in the cELISA kit. OD of Cm must be between 0. OD of Cc must be below 0. Le Goff, F. Thiaucourt Veterinary Microbiology 60 This organism is closely related to three other Mycoplasma: M.
After washing away unbound material, an anti-mouse antibody enzyme conjugate is added. In a negative reaction, unbound conjugate is washed away and enzyme substrate is oxidized TMB if added. In the presence of enzyme, the Substrate is oxidized and develops a blue compound becoming yellow after stopping. Subsequent color development is inversely proportional to the amount of anti-MmmLc antibodies in the test sample 7.
If the buffer is not for immediate use, prepare the amount required for cleaning 1 plate i. Guideline of wash buffer dilution depending on the number of plates No of plates Wash concentrate ml Distilled Water ml 1 20 2 40 3 60 4 80 7. Note: This test procedure is based on Idexx test kit. Pre-plate dilution 1. Dispense ul of dilution detection solution into each wells of the pre-plate except in Conjugate control CC and Mab control wells already ul detection solution ; Testing 1.
Measure and record the absorbance value of samples and control at nm. The usefulness of these reagents as controls for the test is assured when the positive and negative controls produce results as indicated in the cELISA kit. Diagnosis and control of contagious caprine pleuropneumonia F. Pages: 8. Exotic breeds are more susceptible to the disease than traditional breeds.
While the disease is rarely fatal in adult animals, there is often high mortality in the young ones due to myocarditis or lack of milk when the dam is infected by the disease. FMD is characterized by fever and blister-like sores on the tongue and lips, in the mouth, on the teats and between the hooves.
The disease causes severe production losses and while majority of affected animals recover, the disease often leaves them weakened and debilitated. The serum is collected in sterile vials, labeled and assigned a laboratory number and entered into the registry book.
The serum is brought to 40C before use. The kit contains a microtitre plate, which is pre- coated with recombinant 3ABC antigen on the well. For testing, pre- coated plates are incubated with an equal mixture of serum and mAb HRP dilutions in the conjugate diluent for 90 minutes at 37oC. During first incubation, if present in the test sample, the antibodies against 3ABC in the test serum and HRP conjugated monoclonal antibodies against 3ABC competitively bind to the antigens in the well.
Following this incubation, all unbound material is removed by aspiration and washing before the addition of a substrate solution. The residual enzyme activity found in the well will thus be directly inverse proportional to the anti-3ABC antibodies in serum or plasma, and evidenced by incubating the solid-phase with a substrate solution. The reaction is stopped by addition of the stop solution and colorimetric reading will be performed by using a spectrophotometer at nm and nm.
The specially selected 3ABC antigens are used as capture material in the test. Preparation of wash solution 10X concentrated : The washing solution must be diluted 1 to 9 with distilled or de-ionised water before use, e. Use the solution after dissolving crystals by placing the vials at 37oC for few minutes.
Dilutions of Wash Concentrate depending on the number of plates No of plates Wash concentrate ml Distilled Water ml 1 Allow all the reagents and samples to equilibrate at room temperature oC for 30 min and shake them gently before use; 2.
Prepare the strip wells for negative control, positive control and each of the samples; 3. Shake the plate s gently and cover the plate s with an adhesive plate sealer, shaking is very important to get the reproducible results; 8.
Blank the spectrophotometer with air. Measure and record the absorbance of the samples and controls at nm in a bichromatic spectrophotometer with reference wavelength at nm immediately after the end of assay, within 30min. Calculate the results. Use fresh samples. Hemolyzed or contaminated samples might cause false results. Remove the blood corpuscles in sample before use for they may cause non specific reaction. Use disposable gloves while handling potentially infectious materials and performing the assay, after assay, wash hands with sanitizers.
Store all reagents at oC in the dark, bring to oC before use, and return to oC after use. Unused micro plate wells should be stored while sealed in plastic bags at oC. Do not inter-mix components from kits with different batch numbers. Substrate and stopping solutions can cause irritation or burns to the skin and eyes; in case of an accident, rinse immediately with fresh cold water.
Do not expose substrate solution directly to light or to any oxidizing agents, handle all substrate solutions with new clean glass or plastic ware. Do not use reagents after expire dates. Careful pipetting, timing and washing throughout this procedure are necessary to maintain precision and accuracy. Dispose containers and residues safely in accordance with national regulations. Working dilution of the washing solution once prepared must be stored at 25oC for one week Optimal results will be obtained by strict adherence to this SOP.
If these specifications are not met, the test is to be repeated. NB: repeat the test for ambiguous or doubtful results. It is also the most important economic threat to the livestock industry.
It is therefore vital to have guidelines on an activity that facilitates proper diagnosis. The SOP is applicable to all staff involved in the testing, analysis and reporting of results. The Head of FMD Laboratory is also responsible for ensuring that the staff using the SOP are appropriately qualified and trained to carry out the procedures. Follow universal safety practices Good Laboratory Practices when in the laboratory and when handling diagnostic specimens, reagents and chemicals.
All the work with live virus must be carried out in a room that is fitted with a LAF- cab. Materials that were in contact with virus must be deposited in a container with 0. Sterile techniques must be observed in all steps. Serial dilutions of heat inactivated test serum are incubated with known viral suspension of infectious virus.
If antibodies to the virus are present, it binds to the virus, preventing its attachment to and subsequent infection of cells. After this incubation period the test is read by examining each well of the plate for the presence of viral infection by direct microscopy or staining of the test wells of the plate for evidence of viral cytopathic effect CPE.
Sectioning Plastic Blocks. The " Bone Histomorphometry Booklet " contains all the protocols relative to plastic thin sectioning and histomorphometric analyses used by the P30 Histology Assessment Core. In this booklet, you will find information on preparing and submitting specimens to the Core, embedding the specimens and sectioning, staining the sections, acquiring microscope images of the sections, and histomorphometric analysis of the images.
Karl Jepsen, PhD kjepsen med. Carol Whitinger whitinge med. Search this site. Histology Workflows and SOPs. Report abuse. Page details. Page updated. This site uses cookies from Google to deliver its services and to analyze traffic. Information about your use of this site is shared with Google. By using this site, you agree to its use of cookies.
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