VETERINARY LABORATORY & DIAGNAOSIS

VETERINARY LABORATORY & DIAGNAOSIS 

PRACTICAL NO. 1

MICROBIOLOGICAL INVESTIGATION OF CLINICAL CASES OF THE URINARY TRACT INFECTION

Collection of Urine :

            Urine from most species of animal may be collected for analysis as the animal voids. If voided samples (midstream) are to be collected, the vulva or prepuce should be cleaned of all contamination. It is very necessary that the urine collected should be uncontaminated for this.

a)         Clean containers should be used.

b)        The sample may be collected when animal is urinating.

            However, it is difficult to obtain uncontaminated midstream specimens so catheterization or cystocentosis is preferred.

Catheterization :

            The smallest diameter catheter that will permit urine collection should be selected trauma can be minimized by lubricating the distal end of catheter with a sterilized aqueous lubricant. A glass hypodermic syringe fitted to the catheter with a rubber adopter. It is used to aspirate the urine.

Cytocentosis :

            In smaller domestic animals urine sample can be obtained by cystocentosis. A syringe with a needle attached is inserted through the abdominal wall and into the bladder. Gentle suction is used to remove as much as urine as possible.

Manual Compression of Urinary Bladder

            Urine specimens can sometimes be obtained by manual compression of the urinary bladder.

Isolation :

            A variety of gram positive and gram negative bacteria may be associated with diseases or urinary system, so it is necessary to culture the both groups. This can be done by inoculating both blood agar plate and a plate containing as agar medium selective for gram negative bacteria (MacConkey’s and  Eosin Methylene blue) 2-3 loopfull of well mixed urine are streaked on each plate as follows:

1]        Place a small drop of sterile saline in the center of agar plate.

2]        Transfer one loopful of urine to the drop of saline.

3]        Streak this inoculation over the surface of the plate using a blurt (bert) needle.

4]        Incubate at 35-37 ⁰C for 24 hrs. then count the number of colonies and multiply by 100 to estimate bacterial population.

5]        After counting select typical one and identify organisms.

            The cultures are prepared as soon as possible, after collection, if delay more than 1 hr. then sample may be refrigerated.

DIRECT MICROSCOPIC EXAMINATION OF URINE

            If urine is grossly cloudy or opaque and direct microscopic examination has revealed the presence of large number of microorganisms. A small quantity of uncetralfused urine can be placed directly on the slide allowed to dry heat fixed and stained. However, it is necessary to centrifuge clear urine specimen. The urine should not be spread too thinly on the slide.

MICROORGANISMS

Corynebacterium renale :

            The specific entity pyelonephritis caused by C. renale can often be confirmed by microscopic examination of urine sediment.

Morphology – On Gram’s staining reveal the presence of gram positive rods that tend to form aggregates in palisade like structure.

Leptospira species :

            A laboratory diagnosis of Leptospirosis can not be based safely upon a direct microscopic examination of urine. It is most readily demonstrated by dark field examination dark field illumination for Leptospira species.

            Urine is first centrifuged for a short time at a relatively low speed 1200-1600 rpm to sediment the organisms. The supernatant material is discarded and a small amount of sediment is placed on a slide for dark field examination.

Morphology :

            Spiral rods the ends of which are often bent into the shape of hook demonstration of an increasing titre by use of paired samples is the best method for confirming a diagnosis of leptospirosis.

Fontana method – This method is used for staining of leptospira.

Composition -         

Glacial acetic acid               1 ml

Formaline                              2 ml

Distilled water                      100 ml

Phenol                                    1 gm

Tannic acid                           5 gm

Distilled water                      100 ml

Ammonium silver nitrate

Procedure :

1]        Prepare the smear and treat three times 30 sec. Each time with fixative.

2]        Wash off the fixative with absolute alcohol and allow the alcohol to act for 3 min.

3]        Drain off the excess alcohol and carefully burn off the semender until the field is dry.

4]        Poor on the mordant that till steam arises and allow it to act for 30 sec.

5]        Wash well in distilled water and again dry slide.

6]        Treat with ammonium silver nitrate that till stream arises for 30 sec. When the field becomes brown in colour.

7]        Wash well in distilled water dry and mount the specimen under coverslip before the examination to avoid folding of field.

8]        Observe under microscope under oil immersion.

INFERENCE

            Leptospira stain brownish black on a brownish yellow background.

Viral infection in urinary tract :

            Adenovirus infectious canine hepatitis virus cause interstitial nephritis in dogs, pigs and  ferrets.

Isolation of virus :

            Canine hepatitis virus can be cultivated in all cultures derived from the kidney tissue of pig, dog, raevon etc. Cytopathic changes of intranuclear inclusions are observed in infected all cultures.

Direct microscopic examination of urine :

            The white cells are routinely counted in vial sized urine sample by a counting Chakra method. Cast and red cells are best looked for in a gently centrifuge deposit of rush sample of urine from selected patients with suspected renal disease. Numerous granular leucocyte or epithelial cast suggesting renal involvement in infected patient.

PRACTIAL NO. 2

SEROLOGICAL TEST FOR BRUCELLOSIS

Agglutination test :

            It is serological reaction as it involves reaction between particulate antigen and specific antibody.

Principle:

            Antigen (multivalent) and antibodies are bivalent when the antigen and specific antibody are allowed to react at optimum. Concentration antigen and antibody cross each other and form lattice which given agglutination.

            The common serological test i.e. agglutination test can be performed by two techniques or methods namely; plate agglutination and tube agglutination test.

QUICK PLATE METHOD

Use :

            This test is used for quick diagnosis. It is qualitative or  semiqualitative test. The coloured antigen is used is and it can be used for diagnosis of brucellosis.

Materials required :

Brucella abortus, coloured antigen, serum, glass rod, glass slide and inoculation loop.

Procedure :

1]        Place a drop of suspected serum and brucella coloured antigen adjacent to each other on a glass slide.

2]        Mix the reagent and rotate the plate while tilting the slide side slowly water for antigen reaction.

3]        Check the antigen for autoagglutination replacing the serum with normal saline.

STANDARD TUBE AGGLUTINTION

            It is quantitative test i.e. we can note the concentration of specific antibody in serum plain antigen is used in this test.

Material required :

Brucella abortus, plain antigen, serum, normal serological test tube and graduated pipette.

Procedure :

            Arrange the 11 serological tubes in racks and number them 1-11 and add reagent as shown in tables i.e. protocol.

BASIC DIFFERENCE BETWEEN QPAT AND STAT

Sr. No.	Quick plate agglutination test	STAT
1.	It is qualitative test	It is quantitative test
2.	Detects whether the antibody is present or not	Concentration of antibodies is determined
3.	Use of coloured antigen	In this test plain antigen is used.
4.	Source of antibody is serum or whole blood.	As a source of antibody serum is used.
5.	Results are available quickly time required for the test is 1 min.	Delayed type of method it requires 18-24 hr. for result.

Result -  Examine the agglutination control for uniform bacterial suspension and record highest dilution of serum giving agglutination positive reaction.

Titre of serum – Reciprocal of highest dilution of serum gives agglutination test.

Formula – I.U. = 2 × titre of serum

Interpretation :

            Highest dilution of serum showing 50% agglutination is taken as antibody titre.

Cattle, buffalo and horse    1:40                         Positive

                        1:20                         Doubtful

Breeding bull                        1:20                         Positive

                                                1:10                         Doubtful          

Sheep, goat, human              1:20 and above      Positive

PRACTICAL NO. 3

MICROBIAL EXAMINATION IN CLINICAL CASES OF REPRODUCTIVE TRACT INFECTION

Collection of material :

1]        Brucellosis    -           Vaginal swab, uterine swab, placenta foetal lung,
                                 stomach, milk and semen

2]        Vibriosis        -           Prepucial smeal, semen, cervical swab, vaginal
                                 swab, semen, cervical swab

3]        Leptospirosis            -           Urine

4]        IBR and IPV  -           Vaginal swab, liver, spleen, lymphnode, brain,
                                  serum

            Following are the other microorganisms of reproductive tract.

Mycobacterium tuberculosis

Actinomyces bovis

Pseudomonas spp.

Listeria spp.

Chlamydia spp.

Epivag

F.M.D. viruses

Sr. No.	Bacteria	Morphology	Media colony character		Confirmation
1.	Brucella	Coccoid rod, non-motile, non-sporulated thin capsule gram – ve	Serum dextrose agar, serum potato infusion agar	Small pin point mucoid or rough yellow brown pigmentation	ABR/Milk ring test QPAT, STAT, CFT Agglutination test HA
2.	Vibriosis Compylobacter foetus	Comma shaped, non-sporulated, non-capsulated	Blood agar Thiol medium broth	Fine pin point broth and protease growth of organisms in smooth and rough mucoid colony	Agglutination test HA

                                                                                                                        

3.	Leptospira	Spiral with bend hook or end.	----	Media used for isolation of microorganism	Dark field examination in G. pig, HAI.
4.	IBR/IPV	Alpha virus 130-180 nm in diameter thermoliable half life at 37 0C for 1016 stable at pH 6.9	Bovine kidney testicle cell culture	Infected cell become rounded refractive form synction forms large nuclear inclusion bodies cell found in infected cell	CPE characterized by rounding bunch of grapes and high gloss lost cloudy take inclusion body formation STAT, FAT

PRACTICAL NO. 4

MICROBIOLOGICAL INVESTIGATION IN CLINICAL CASES OF MASTITIS

Collection of sample :

            Since absolute diagnosis of mastitis and identification of its causative agent are based on the isolation and identification of bacteria all specimen for laboratory examination should be collected with as little contamination as possible. Samples should consist of foremilk taken at least following a regular milking samples should be collected as follows:

1]        Clean the udder well by brushing

2]        Wash the udder thoroughly with clean cloth soaked in a disinfectant solution

3]        Allow the udder to dry and tract the teat orifice with tincture of iodine solution and allow it to dry.

4]        Label the tubes as to cow and quarter from which the sample will be taken eg. RH, LH, LF and RF

5]        The cap of sterile tube is carefully removed and help between the finger in such a manner that the inside of the cap is facing downward. The tubes should be hold at a slight angle to prevent contamination of sample by falling particles.

6]        Immediately following collection refrigeration of the sample for transportation to the laboratory.

            In herd survey, it may not be necessary to collect individual quarter samples and composite samples may be used.

PROCESSING OF MILK SAMPLES FOR ISOLATION

            A nutrient medium containing blood is recommended for isolation of bacteria from milk samples and requires only the addition of distilled water for preparation. The agar should be sterilized by autoclaving. Following sterilization agar is cooled to 45-48 ⁰C and sufficient sterile citrated as defibrinated blood is added to provide a concentration of 5% most commonly used blood is from sheep. Agar is then pored into sterile petridish and allowed to harder before the material is streaked. Sufficient medium should be placed to provide an even ¼ to 3/8 thick layer of agar.

            In streaking milk into agar a wire inoculating loop is preferred. Milk should be streaked into one quarter of blood plate and incubate overnight at 37 ⁰C. The plate is examined for bacterial colonies. Any colony found should be examined and a gram staining is performed to assist in positive identification.

ISOLATION AND IDENTIFICATION OF ETIOLOGICAL AGENT

1]        Milk sample is inoculated

2]        On blood agar – Staphylococci, Streptococci and other organisms

3]        Edward medium containing blood agar, crystal violet and esculine as crystal violet inhibit the growth of staph.

Colony characters :

Streptococcus agalactiae :

            Small colonies transparent bluish gray in colour haemolytic or non-haemolytic Streptococcus disagalactiae.

            Non-haemolytic discolouration of medium Streptococcus uberis.

            Dark colored colonies surrounded by black or brown zone of coloration due to hydrolysis of esculin.

Antibiotic Sensitivity Test:

Material required :

            Nutrient agar, milk sample, antibiotic discs, sterile forceps.

Procedure :

            In a sterile enrichment with burns burner as spirit lamp open the petridish of nutrient agar from one corner, poor around 4 ml of broth culture on the surface with the help of pipette and close the dish.

1]        Slowly rotate the plate for ever spread

2]        After about 10 min. with the help of sterile Pasteur pipette remove excess broth

3]        At an equal distance with the help of a sterile forceps carefully place antibiotic discs.

4]        Incubate the plate at 37⁰C overnight

5]        Read in front of light source and record the zone of inhibition around the disc.

PRACTICAL NO. 5

MICROBIOLOGICAL INVESTIGATION OF INFECTIONS OF EYE, EAR AND SKIN

Diseases of skin :

Bacterial diseases    :           Staphylococcal infection, scaled skin syndrome,
           erysipelpyoderma, pyote female impetigo done,
           bursa infection.

Viral diseases           :           Rubella, measles, chicken pox, small pox,
           cow pox, warts.

Fungal diseases        :           Dermatophytes, ringworm etc.

Parasitic diseases     :           Scabies, demodecosis and other dermatological
           conditions.

Diseases of eye :

Bacterial diseases    :           Opthalmia, neonatum traitoma, bacterial
            conjunctivitis, pink eye.

Viral diseases           :           Epidermia, keratoconjunctivitis, acute
            haemorrhagic conjunctivitis

Disease of Ear           :           Otitis externa, otitis media, otitis interna

METHODS OF SAMPLE COLLECTION

Skin scrapping :

            The hairs of the affected part of the body of animal are timed. Then that part is cleaned washed with soap and water. Then tincture iodine is painted on that part and skin scrapping is collected in a sterile bottle using sterile blade. The scrapping be taken until some blood losses out from that part.

ISOLATION AND IDENTIFICATION

            Collected skin scrapping is taken in the test tube. It should be mixed with 5-10 ml KOH or NaOH. Then boil it on the flame so that keratinised layer of the mites or ticks should be dissolved in the solution. Then get it cooled and keep it for sedimentation. After sometime discard the supernatant and take a drop of sediment on a clean glass slide and put a cover slip on it and observe under microscope.

            The mites can be observed under microscope for fungal infection hair, plucking should be used. After that keep a hair root in the clean slide and stain it with lactophenol blue. The fungal hyphae or spore should be observed under microscope.

Ear swab/Eye swab :

Method of collection :

            Ear swab should be collected for culture of Streptococci before the start of antibiotic therapy care should be taken to sample. The inflamed site and the swab do not contaminate by touching the other parts of ear. The swab should be put into stuarts or other suitable transport medium. If a day or more than 1 hr. is expected before the swab could be plated out on blood agar, it should be refrigerated as soon as possible. If not contaminated frequently encounter under field condition may once grow the agents and make its isolation difficult as impossible.

            For Streprococci infection in ear/eye these should be isolated after overnight. Incubation of the culture plate likewise the isolation is done for bacterial recovery. Incubation of freshly prepared solid media in the field has the advantage of providing information or the number and type of bacteria present such preparation should be incubated by practitioner and those with no growth in colonies readily recognized need not be sent to the laboratory. Transport media of specimen used are leibouty media source solution or soly etc. The peptides amino acids as sugar in these solution and in the presented of viruses and bacteria.

Identification :

            It is done by performing gram staining identify the bacteria either with gram negative gram stain of ear/eye swabs may reveal numerous organism.

            Other confirmatory diagnosis should be done by colony character and other biochemical reaction. For identification of viral infection various serological reactions should be used confirmatory diagnosis should be by chicken embryo inoculation or animal inoculation.

PRACTIAL NO. 6

MICROBIOLOGICAL EXAMINATION OF NERVOUS SYSTEM INFECTIONS: ISOLATION AND IDENTIFICATION OF MICROORGANISMS

I.         Collection of C.F.S. :

Sr. No.	Animal	Site of collection of C.S.F.
1.	Cattle and buffalo	i) Lumbosacral region ii) 1st and 2nd coccygeal space
2.	Goat and sheep	i) Suboccipital ii) Lumbosacral region
3.	Horse	i) Suboccipital ii) Lumbosacral region
4.	Dog	i) Suboccipital ii) Lumbosacral region

II.        Isolation of identification of virus :

Sr. No.	Disease	Virus	Material collected	Identification
1.	Rabies	Rabies virus (Rhabdovirus)	Brain, hippocampus, saliva of milk	Bullet shape, demonstration of negribodies
2.	C.D.	Herpes virus	Saliva, brain, spinal cord	It produces thickening of CAM, CPE induces granular demonstration and vacillation of giant cells and synctia formation
3.	Aujesk’s disease	Porcine, Herpes virus	Saliva, nasal discharge, piece of lung, pharyme, vaginal swab	CAM → Pock formation and inclusion body production
4.	Equine encephalitis	Alpha virus	Serum piece of muscle pharyme, LN, edematous fluid	Neutralization test, IPAT, HI virus causes noem. Within 24-48 hrs.
5.	Japanese encephalomyelitis	Japanese B, encephalomyelitis virus	Serum blood, edematous fluid	CPE production, SNT, HI, CFT, FAT

PRACTICAL NO. 7

ISOLATION AND IDENTIFICATION OF ORGANISM FROM CLINICAL CASES OF POULTRY DISEASES

Isolation and identification of bacteria

Collection of material :

            The site for collection of specimen is related to clinical sign and knowledge of pathogenesis of suspected disease. The liver, heart, blood, spleen, intestinal, cloacal contents egg, shell, air sac, yolk sac, serum, synovial fluid etc.

Clinical cases of poultry diseases

Sr. No.	Morphology and staining	Biochemical test	Confirmatory test
			Biological	Serological
1.	Rods capsulated motile	IMViC   : + + - - Utilizes glucose mannitol with A and G production	Rabbit illeal loop test	----
2.	Rod non-capsulated, non-motile non-sporulated accusingly Gram negative	IMViC: + - + Glucose Fructose       +ve with A Maltose        & G                     production	----	----
3.	Acid fast organism occurring singly or pairs	Not imp.	G. pig, rabbit inoculation	ELISA tuberculin test
4.	Gram – ve	Bipolar capsulated	Indole, glucose, Hb, mannitol (+ ve)	-----
5.	Gram – ve cell wall absent	Urea + ve	----	AGPT, HA
6.	Comma or S shaped motile Gram – ve	Sugar      –ve Indole     – ve Nitrate   + ve Catalase + ve Hb          – ve	----	Indirect HA test
7.	Large bacillus, motile, sporu-spore terminal, racket appear Gram + ve	Gelatin liquifaction + ve Glucose Maltose (+ ve with A & G production	G. Pig inoculation test	Neutralization test
8.	Oval capsule, motile, gram + ve arranged in like clusters	Catalase     – ve Glucose      + ve Mannitol     + ve Maltose       + ve	Kitten test	ELISA
9.	In a chain of variable gram + ve	Not imp.	Dick test	ELISA
10.	Motile, non-capsulated forming, straight or curve	Maltose and glucose with acid production	Lab animal inoculation diagnosis and septicemia	ELISA

Isolation and identification of fungi in poultry :

            The most common fungi occurring in poultry animals is Aspergillus, fumigatus, Aspergillus flavus, causes Aspergillosis, thrush, morilla, albicans, M. krusei.

Isolation of fungi :

            For isolation of fungi (SDA) medium is used i.e. inoculate the material on SDA and incubate at 37⁰C and 28⁰C for 7 – 21 days up to 1 month after I.P. observe the characters.

Identification of fungi :

Lactophenol blue staining :

1]        Place a drop of 95% alcohol on slide or 4% KOH

2]        Gently tease the fragments of culture in alcohol

3]        Let most of alcohol to evaporate then add a drop of stain

4]        Restore excess stain around the coverslip by blotting paper

5]        Let the stain penetrate

6]        Observe the morphology under low and high power objective

7]        Confirm the moulds on the microscope examination.

Aspergillus species :

            On SDA Aspergillus spp. Develop a white filamentous growth, which increase in size up to 2-3 cm and become dark green or green in colour.

            Microscopically hyphae are septate numerous conidiophores, each conidiophore expands into large vesicle at end caused columnella, which is covered by finger like structure sterigmata.

Cellophane tape method :

            They are also used for identification of fungi.

Black technique :

            Isolation and Identification of virus in poultry :

Collection of Material :

1]        Nasal or nasopharyngeal swab for ortho, paramyxo herpes and adenovirus

2]        Vesicle fluid – pox virus

3]        Faecal and cloacal swab – for entire virus entero parvovirus

4]        Lymphnode – RP mucosal disease

5]        Liver – IH, adenovirus, enterovirus

6]        Spleen – ND, IBD, RP

7]        CNS – Fluid

Cultivation of virus :

Embryonated chicken eggs :

            The presence of virus can be detected by mortality or changes in embryo like deformities, harm pox and edema of CAM, presence of specific antigen in fluid, live HA, CFT, antigen detection etc.

Route of inoculation :

            Yolk sac, CAM, Allantoic, Sac, amniotic cavity, I/V.

Cell culture :

1]        Monolayer cell culture

2]        Suspension culture

3]        Organ culture are used for cultivation of virus

Identification of virus :

a)        Cytopathic effect :

                        Most virus produce degenerative changes, the changes are rounding, refraction, synctia formation and detachment.

b)        Immunofluorescence test :

                        Specific antisera labelled with fluorescence can be used in direct or indirect test to detect presence of cytopathic or non-cytopathic virus in cell culture.

c)         H.A. :

                        Virus acquire property to damp the RBC of certain species of birds, eg. Myxo, paramyxo virus.

d)        Haemodespin (Maemadsorption) :

                        Orthomyxo, paramyxo, togavirus, in bird form cytoplasmic membrane, acquire ability to absorb RBC.

Animal inoculation :

            Rabbit, guinea pig, adult and suckling mice, ferret are used for virus detection.

Other serological methods are –

1]        Virus neutralization test

2]        Immunofluoroscence and immunoperoxidase

3]        Radioimmune assay

4]        ELISA

5]        Immuno electromicroscope

6]        Agar gel double diffusion test

7]        HA and HI

8]        CFT

PRACTICAL NO. 8

MICROBIOLOGICAL INVESTIGATION OF SURGICAL CASES

Abscess

Fistula

Arthritis

Material to be collected :

a)                 Abscess : Straw colour fluid from mature abscess should be collected immature abscess should not opened with the help of sterilized cotton swab.

b)                 Fistula : Discharge from it should be collected

c)                 Arthritis : Sinovial fluid is collected

d)                 Wound : Pus discharge from wound should be collected in sterile cotton swab.

Isolation and identification of microorganisms :

1]  Swab from affected wound, fistula, arthritis, abscess should be collected in sterile glass bottle.

2]  Culture the suspected material in broth with in 6 hrs. maximum growth of bacteria occurs.

3]  Make the smear over the glass slide heat and stain it with gram’s staining and observe under high power microscope with oil immersion.