sampling exam chapter 2

Sampling method for bacterial examination


Important notes for collecting samples

It should represent the disease suspected as possible.

The specimen should be collected under aseptic technique.

The specimen should be refrigerated at once it’s collected and send to the laboratory as soon as possible.

The chance of bacterial isolation is reduced in case of using antibiotics and chemotherapeutics such as sulpha drugs, so you have to take the specimen before starting treatment.

Type of specimen

1- BloodDisease Produced Mainly in the stage of bacteremia or septicemia e.g. Anthrax, Pasteuellosis, Enterotoxaemia, Acute pneumonia

Suspected microorganisms

e.g. Bacillus anthraces, Pasteurella, Clostridium, Diplococcus

Procedures

Blood for Microbiologic examination must be collected under aseptic conditions.

The area over the vein should be cleaned properly and disinfected by a piece of cotton or gauze soaked in a suitable disinfectant.

The skin is allowed to dry and the blood is collected with a sterile syringe or vacationer tubes (EDTA tube) with sterile needle make sure to mix the collected blood with the anticoagulant (EDTA).

The blood is added to special tubes (according to the type of the microorganism suspected) which contain transport media, (e.g. Stuart’s transport media) this will allow the fastidious microorganism to grow.

Various kinds of tubes, swabs and containers for the collection of different types of the specimens

By dr.khaled fujairah municipality 1


Blood and serum samples Good sample Bad Sample

2- MilkDisease Produced Suspected microorganisms

Mastitis Acute Sub acute Chronic

e.g. Staphylococcus, StreptococcusColiforms,CorynebacteriumPseudomonas

Labeling of the milk specimens RF = Right Fore اليمين األماميالربع .RH = Right Hind الخلفي اليمين .الربعLF = Left Fore اليسار األماميالربع .LH = Left Hind الخلفي اليسار .الربع

Procedures 1. Milk for microbiology must be collected under aseptic Technique and avoid contamination by

other type of bacteria.



2. Clean the udder properly and don’t use disinfectant as it may inhibit the pathogenic microorganism.

3. The first streams usually (3) are discarded as it may contain contaminated bacteria.

4. Collect milk from each quarter in a separated sterile container or tube, and label it as shown in the diagram above.

5. Milk should be refrigerated to ( 4 °C ) at once it’s collected to reduce the overgrowth of normal flora present in the milk.

6. Milk kept more than 24 hours, and / or at ambient temperature, are useless for diagnostic procedure, as the causative agent may be overgrowth by less significant microorganism. So that for longer period the milk may be kept in freeze until it delivery to the laboratory this will have no effect on bacterial diagnosis.

3- OrgansDisease Produced Suspected microorganisms

The disease is either :

1. Involve most of the body organs (heart, liver, spleen, kidneys) in case of Generalized infection (like bacteremia, septicemia).

1. Or it’s involving special parts of the body in case of Localized infection (the ileocecal region in the case of Johne ’s disease).

e.g. - Pasteurella- Coliforms- Clostridium- Pseudomonas- Mycobacterium paratuberculosis

Procedures

1. The knowledge of post mortem skills is necessary for this procedure, e.g., Take specimens from affected organs, Involved lymph nodes should be taken.

2. Aseptic techniques must be adapted to avoid contamination. e.g. Don’t use the instruments which are used to remove skin in obtaining the specimens like heart, liver, spleen as this may lead to contamination.

3. Every piece of tissue or organ must be put separately in a sterile container and labeling, specially the intestine.

4. The specimens should be refrigerated at once or frozen for longer period.5. Avoid using disinfectant with tissues for bacterial isolation, under field condition try to use

boiling water for sterilization of instruments (metal).

4- Serum samplesSerum samples are collected for serological diagnosis.

1. Collect 5-10 ml of blood in plain tubes( with out EDTA ).2. Avoid shaking of the tubes (which contain blood) at transporting to prevent distraction of the

RBCs and hemolysis.3. Try to separate the serum from clotted blood as possible.4. The tubes are placed vertically at room temperature for 1 hour then refrigerated at ( 4 - 8 °C ) for

1- 2 hour. Don’t refrigerate the whole blood immediately after collection.5. Don’t freeze the whole blood.



6. After the serum was separated distribute it equally in to two plastic tubes for serum and keep it in a refrigerator for short periods or in freeze for longer periods.

7. Avoid repeated freezing & thawing as this may affect the protein structure of the serum.

5- SwabsDisease producedSuspected microorganisms

Septicemia (Generalized). Bacteremia (Generalized). Johne’s disease (Localized).

A. Organ swabse.g. Pasteurella, ColiformsClostridium, Pseudomonas,Mycobacterium paratuberculosis

Procedures

1. Organ swab may be taken for culturing or just for making direct smears to determine the guide for working, in both cases careful handling and aseptic techniques should be adapted to prevent contamination.

2. The organs may be containing exudates, pus material, necrotic material, etc. 3. Swabs for culture must be putted in a suitable transport media to prevent dissection of the

material and to maintain and enhance the growth of the microorganisms specially the fastidious once , nowadays swabs with their transport medium it’s available commercially for different kinds of cultures ( aerobic and anaerobic, etc.).

4. Make sure to take enough material by the swab and represent the disease as possible.

Disease producedSuspected microorganisms

e.g.OphthalmiaConjunctivitis

B. Eye swabse.g.Staphylococcus StreptococcusMorexella, Pseudomonas

1. Collect specimens from the infected eye during the period in which the disease is progressing, as at a later time one may obtain only saprophytes.

2. The secretion is collected by using sterile cotton swab, avoid contamination due to contact with margins and angles.

1. It's important to avoid dryness of the specimens as many microorganisms can not survive in dryness.

Disease producedSuspected microorganisms

e.g.Otitis media

C. Ear swabe.g.Staphylococcus, Streptococcus, Pseudomonas, Coliforms, Corynebacterium

• The exudates from external auditory canal must be collected with sterile cotton swab.

• It's important to avoid dryness of the specimens as many microorganisms can not survive in dryness.



( CCPP ) in goat Contagious Caprine Pleuro Pneumonia:Typical lesion, note the yellowish , fibrinous deposit on the surface of the lung adhered to the inner side of the ribs

Pasteurella in sheep: Note the congestion of the lung and presence of the abscesses.

Pasteurella in sheep

Laboratory diagnosis & control of brucellosisIntroduction

Brucellosis is considered as the most wide spread zoonosis in the world and it is considered as True zoonosis ( That mean it is Basically transmitted from animal to human).

The importance of this contagious disease is the economic impact on livestock industry.

Causes sever hazard to human health, through either direct contact with infected animals or the consumption of contaminated milk and dairy products.

Causative bacteria of the diseaseBrucellosis is named after Sir David Bruce, who is in 1886 Isolated the causative agent from a soldier in Malta.

Brucella species are recognized based on the natural Animal host to the following species as shown in table



Table show Brucella species:

1NoneNoneWood rat1Brucella neotomae6.

BenignNoneDog1Brucella canis5.

Sever( except

biovar 2)

Various wild species

Swine1-5Brucella suis4.

None NoneSheep (Ram)

1Brucella ovis3.

SeverWild ruminant cattle, camels

Sheep and Goat

1-3Brucella melitensis2.

Less severWild animals, water buffalo, camels

Cattle1-9Brucella abortus1.

Human disease

Other animal species affected

Naturalhost

BiovarSpeciesNO

Table (1) show Brucella species :

Morphology and staining The bacteria are strictly parasitic and prefer the intracellular habit.

The species of the genus Brucella are small non motile, non spore forming; Gram negative rods and they do not produce true capsules.

They are somewhat resistant to decolonization by weak acids and thus stain red by the modified Ziehl - Neelsen method.

Gram stain of Brucella (Gram – ve) CoccobacilliAntigenic Structure

The designation of the antigens in cultures composed of smooth and rough colonies are shown in the table

The Production of monospecific antisera to A and M antigen can be

used in the identification of the Brucella species.

Br.canis and Br.ovis grow as rough colonies that do not possess either of the surface antigens A and M, but instead of that they have R antigen.

Cultural characteristics1. Culture media



There are two major types of media for cultivation of Brucella.

A. Basal MediumDirect isolation and culture of Brucella are usually performed on solid media.This enables the developing colonies to be isolated and limit the development of contaminants. There are many Kinds of commercial media, e.g. Brucella medium base, Trypticase soy agar, Columbia agar, Serum- dextrose agar or Glycerol-dextrose agar.The addition of 2-5 % Bovine or Equine serum is necessary for the growth of strains such as B. abortus biovar 2.

B. Selective MediaAppropriate antibiotics are added in order to suppress the growth of organisms other than Brucella. The most widely used medium is Farrell’s medium, which is prepared by the addition of six antibiotics : Polymyxin B sulphate,Bacitracin, Cycloheximide, Nalidixic acid, Nystatin, Vancomycin

•2. Colony Morphology

Brucella colonies are visible after 3-5 days incubation period at 37 ºC on suitable solid media, and they are aerobic or microaerophilic.

Cultures should not be discarded as negative until 8-10 days have elapsed.

Brucella colonies are 1-2 mm in diameter, round, entire, smooth, glistening, translucent, and a pale honey color when plates are viewed in the daylight through a transparent medium.

Brucella colonies on blood agar

Epidemiology of the disease 1. Transmission of the disease

• Animal to animal Transmission The oral route, Contamination of the udder during milking and contact with aborted fetuses and infected newborn lambs are considered to be common methods of spread, also the venereal

transmission of the disease is occur due to infected male or contaminated semen.

• Animal to human Transmission Infected tissues and contaminated materials must be handled under (biosafty 3) conditions. Transmission could be either by contaminated food, invasion by intact skin, inhalation of aerosols containing the bacteria and aerosol contamination of the conjunctiva.



One of the most important route of animal to human transmission of Brucella

• Brucella melitensis (biovar 1, 2 or 3) is the main causative agent of caprine and ovine brucellosis. Sporadic cases caused by B.abortus have been observed. The infection is widespread world-wide.

• Brucella abortus is usually causes bovine brucellosis, less frequently by brucella melitensis.

2. The economic losses of brucellosis1. Losses due to abortion in the affected animal population.2. Diminished milk production, mastitis and contamination of milk.3. Cull and condemnation of infected animals due to breeding failure.4. Human brucellosis causing reduced work capacity of the affected people.5. Government costs on research and eradication schemes.6. Losses of financial investments.

Collection of the samplesA. For serological examinations:

1. Serum samples are collected for serological diagnosis.2. Collect 5-10 ml of blood in plain tubes (with out EDTA).3. Avoid shaking of the tubes (which contain blood) at transporting to prevent distraction of the RBCs and hemolysis.4. Try to separate the serum from clotted blood as possible as you can.5. The tubes are placed vertically at room temperature for 1 hour then refrigerated at (4 - 8 °C) for

1- 2 hour. Don’t refrigerate the whole blood immediately after collection.6. Don’t freeze the whole blood.7. After the serum was separated distribute it equally in to two plastic tubes for serum and keep it in a refrigerator for short periods or in freeze for longer periods. 8. Avoid repeated freezing & thawing as this may affect the protein structure of the serum.

B. For bacterial isolation

1. the most valuable samples from live animals are semen, vaginal swabs, and milk.2.After necropsy, the preferred organs are epididymas, seminal vesicles & inguinal lymph nodes in rams, and the uterus, iliac and supra mammary lymph nodes in ewes.3. In aborted and stillborn lambs the preferred culture sites are abomasal content and lung. 4. Samples for culture should be transported to the laboratory on ice as soon as possible after collection.



Classes of Immunoglobulin

There are five major classes of antibodies which have diverse functions.

1. IgM (Immunoglobulin M) The class of serum antibody first produced during an infection. It is a large pentameric molecule that is active in agglutination pathogens and activating complement.

2. IgG (Immunoglobulin G) The predominant immunoglobulin class in serum. Has function such as neutralizing toxins, opsonizing bacteria, activating complement and crossing the placenta to protect the fetus and neonate.3. IgA (Immunoglobulin A)

The class of immunoglobulin that is present in dimeric form in many body secretions (e.g., saliva, tears, bronchial and intestinal secretions) and protects body surfaces.

4. IgE (Immunoglobulin E) The class of immunoglobulin that binds to mast cell and basophils, and is responsible for type I or anaphylactic hypersensitivity. It is also involved in resistance to helminth parasites.

5. IgD (Immunoglobulin D) The class of immunoglobulin found on the surface of many B lymphocytes; thought to serve as antigen receptor in the stimulation of antibodies synthesis.

Types of antibody responseA. Primary antibody response.

It is stimulated during immunization procedures (and also in naturally acquired immunity) the type of immunoglobulin is (IgM) and it is characterized by low antibodies titer.B. Secondary antibody response. It is characterized by producing the (IgG) type of immunoglobulin with high level and affinity for the antigen. It depends on the formation of the memory B cells.

By dr.khaled fujairah municipality

AntibodyTiter

Days

7 14 21 28 35 42

IgG

IgM

Primary antibody response

Secondary antibody response

9


Diagram show the primary and secondary antibody response and the types of immunoglobulin

The most common serological test

1. Rose Bengal Test (RBT).2. Complement Fixation Test (CFT).3. Enzyme Linked Immuno Sorbent Assay (ELISA).4. Milk Ring Test (MRT).5. Standard Agglutination Test (SAT).

Rose Bengal Test (RBT)A. Principle The buffered – acid antigen stained with Rose Bengal is used for the early detection of Brucella specific agglutinins. B. Reagents

Rose Bengal Antigen. Positive and Negative controls.

Important notes: Shake before use. Do not freeze.

Store at 4 °C in a dark place.C. Equipments

Precision pipettes calibrated to 30 μl. Enamel plate or disposable agglutination cards. Disposable stirring sticks.

D. Procedure Allow reagents and serum samples to reach room temperature (18-25 °C). Gently shake the reagents. Check the reagent against positive and negative controls (as follows). Place a drop (30 μl) of undiluted serum onto a circle of the slide. Add a drop of the reagent (Rose Bengal Brucella antigen) next to the drop of the serum. Mix both drops by the disposable stirring stick, spreading them over the full surface of the circle. Rotate the plate (card) manually or with mechanical rotator at (80- 100) R.P.M (Round per

minute) for 4 minutes.E. Reading

The reading must be carried out exactly 4 minutes from the beginning of shacking. Beyond this time nonspecific reaction may occurs (False Positive). No agglutination = (- Ve) Negative result. Any visible agglutination (even slight) = (+Ve) Positive results (Presence of specific antibodies).

Positive and negative results for RBT (Rose Bengal test)


Positive Negative


F. Important Notes

The test is very sensitive especially in vaccinated animals.

Positive samples should be retested by confirmatory test such as CFT or ELISA.

False Negative reaction may occur and can be detected by retesting animals at intervals over a period of at least 3 months.

Complement Fixation Test (CFT)• The complement fixation test (CFT) is widely used for the diagnosis of brucellosis in cattle, sheep

and goat.• This test is relatively insensitive to antibody produced in response to vaccination with the living

attenuated vaccine (for Br. Abortus, Br. Melitensis).• Whilst being highly sensitive and specific in animals naturally infected with brucellosis.• The test is some what complex and in mass testing a screen test such as Rose Bengal test, is

often used to reduce the number of samples that need to be tested by (CFT).

A.Principle

• In the complement fixation reactin an antigen-antibody reaction is demonstrated by binding of complement.

• The reaction can be visualized with the aid of an indicator – reactin (hemolytic system).

• The hemolytic system consists of sheep erythrocytes and antiserum to sheep erythrocytes (amboceptor).

• If no antigen-antibody reaction develops with the consumption of complement, the erythrocyte will be lysis completely and the hemoglobin will be released.

Complement fixation test



B. Reagents

1

B. Reagents

0.1% NaCl + (0.1% MgCl2+CaCl2)pH =7.3

Complement fixation buffer4.

3% suspension of sheep red blood cells (SRBC) sensitized with rabbit anti-Sheep RBC (amboceptor).

Hemolytic system(Sensitized RBC)3.

Guinea pig complementComplement 2.

Brucella abortus strain 99 orBrucella abortus strain 1119-3

Standardized antigen1.

C. ResultsThe test is read by the eye :

Sedimentation of the sensitized RBC = Positive result.Complete lysis of the sensitized RBC = Negative result.

C. ResultsThe test is read by the eye:Sedimentation of the sensitized RBC = Positive result.Complete lysis of the sensitized RBC = Negative result. Milk Ring Test (MRT)The Ring test method is used for the detection of brucella antibody in milk samples. This technique is very easy to perform it especially in dairy herds.



A. Principle It is uses the principle of agglutination between antibodies contained in milk and dyed colored bacterial antigen of brucella to form antigen-antibody complexes that are progressively carried by the fat towards the surface of the milk and formed a blue violet ring

B. ReagentsInactivated bacterial culture of Brucella abortus S 99 inactivated by heat and phenol and stained with hematoxyline.Positive control for milk ring test.C. ProcedureKeep the antigen for 1 hour at room temperature, and shake it gently before the beginning of the test.Carefully mix the tested milk, and put 1 ml in a test tube.Add 50 µl of the antigen and mix carefully.Place in incubator for 1 hour at 37 OC, after that for (18-20 hours) at 4 OC, and then read the result.

D. ResultsRing of cream equal or more colored than the underlying milk = Positive result.Ring of cream less colored than the underlying milk = Negative result.

Types of Brucella vaccines and vaccination Vaccination increases animal resistance to systemic infection and in infected animals decreases

the probability of placental infection, abortion and massive shedding of infections organisms.

Live vaccines induce a long lasting immunity and, are normally administered to the young animals. Adults should also be vaccinated with reduced doses or by the conjunctival route in order to restrict the serological response.

The following are the recommended vaccination procedures of the 3 live vaccines for B. melitensis and B. abortus.

Brucella vaccines

A. Brucella melitensis (Rev-1 Vaccine):

1. Freeze-dried of live B. melitensis Rev-1 strain.2. For sheep and goats. 3. At 3-6 months as a single S/C dose.4. Conjunctival route (reduced dose)

Disadvantages: – Persistent serological response. – Abortion if given to pregnant animals. – Human risk.

B. Brucella abortus (S19):1. Freeze-dried of live B. abortus strain 19.2. For cattle.3. At 3-6 months as a single S/C dose.4. Conjunctival route (reduced dose)

Disadvantages: – Persistent serological response.– Abortion if given to pregnant animals.– Human risk.

C. Brucella abortus (RB51):1. Freeze-dried of live B. abortus strain RB51.2. Lesser abortafecient effect than S19.3. At 4-12 months as a single S/C dose.



4. Does not produce persistent Ab titers.

Specifications of the Ideal Brucella Vaccine

– Effective (solid and durable immunity) but without inducing a long lasting vaccine infection.

– Not interfering with diagnostic tests (Compatible with eradication plans).– With no limitation of its use, e.g. in pregnant animals.– Safe for man when performing the vaccination.– Not expensive and readily available world wide.

Brucella vaccine quality control testing

– Identity test for the bacterial and colony morphology.– Absence of contaminating organisms.– Number of viable organisms.– Dissociation test.– Stability test.– Virulence in guinea pigs and/ or mice.– Safety.

Diseases to be considered in the Differential diagnosis

– Vibriosis.– Salmonellosis.– Toxoplasmosis.– Leptospirosis.– Enzootic abortion of ewes.– Rift Valley fever.

Laboratory identifications of the causative organisms a requirement for the diagnosis.


sampling exam chapter 2

Documents

khaled fujairah municipality

secondary antibody response

persistent serological response

rose bengal test

complement fixation test

primary antibody response

sterile cotton swab

milk ring test