DIAGNOSIS OF BACTERIAL DISEASE (BACTERIOLOGICAL EXAMINATION)

1. DIAGNOSIS OF BACTERIAL DISEASE(BACTERIOLOGICAL EXAMINATION) Prof. Dr./ Wafaa Abd El-ghany Professor of Poultry Dis., Fac. Vet. Med., Cairo Univ.

2. Sampling • The sample should be taken either from: • Living birds in ailing condition (exhibit typical signs). • Freshly dead birds (Not exceed 2 hrs in summer and 4 hrs in winter) due to the followings:

3. Sampling • The intestinal and brain barriers of the body are destructed and permit the normal flora to flourish and invade all the body tissues, so misdiagnosis will occur. • Bile impregnation occur around the surrounding tissues with destruction of the bile organisms.

4. Equipments • Bacteriological examination must be undertaken in a closed cabinet under complete aseptic conditions provided by an ultra violet lamp for sterilization of the place. • Sterilized instruments have to be used and flamed instruments dipped in alcohol is sufficient for such purpose.

5. Technique of sampling • Dip the bird in any antiseptic solution to decrease the surface bacterial load. • By sterile scissor, make transverse cuts in the thigh webs. • Reflect the 2 joints. • Make another transverse incision in the keel bone. • Reflect the skin. Fine flaming, not burn the carcass. • Make 2 lateral cuts at the costochondral junction to open the carcass. • Choose the correct or target organ in sterile plastic bag or Petri dish.

6. Sample selection • Each disease has a special sample according to the predilection seat of the organism: • For enterobacterial organisms like Salmonella and E. coli, samples should be taken from bile (gall bladder) as all enterobacteria are bile resistant , liver, heart blood and intestines (for Sgp only as it is not normal inhabitant of intestines as paratyphoid organisms).

7. Sample selection • For Pasturella species, the samples should be taken from heart, heart blood, liver, spleen and bone marrow. NB: Bile slats contain sodium tauroglucolate which destruct Pasteurella spp. (except P. pseudotuberculosis and P. pestis), So Pasteurella species are bile sensitive.

8. Sample selection • For haemophilus paragallinarum, take the samples as swabs from nasal cleft, sinuses and conjunctiva. • For Chlamydia sp., take the samples from airsacs, lungs and pericardial sacs. • For Mycoplasma gallisepticum, the samples are taken from seous membranes (airsacs, pericardiam, perihepatic capsule), lungs and trachae.

9. Sample selection • For Staphyloccocus, take the samples from the joints. NB: in case of autolysed carcasses or frozen imported ones, samples could be taken from the bone marrow for isolation of certain organisms especially enterobacteriacae.

10. Transportation of samples • Carried out quickly as much as possible. • In a portable refrigerator so far minimize autolysis. • In ice bags. • The sample should be labeled (history, clinical exam., and suspected disease).

11. Media • Culture (On agar). • Propagated (In broth). 1- Fluid media (e.g. nutrient broth, selenite F broth,…etc.). 2- Solid media (Nutrient agar, MacConkey agar, Dorest egg media, PPLO agar …etc.) depending upon suspected disease in the examined case.

13. Types of the media • General (ordinary media): It is used for general routine examination as nutrient agar, nutrient broth and peptone water.

14. Types of the media • Enriched media: • Make enhancing and enriching of fastidious organisms like Haemphilus, Pasteurella, Staphylococcus and Streptococcus species. • These media are enriched by blood, serum, vitamins (biotin), etc…..

15. Types of the media • Enriched media: • Blood agar (chocolate blood agar): (Blood of sheep, chickens, horse or bovine) are used for differentiation between haemolytic and non haemolytic organisms. • PPLO media contains inactivated horse or swine serum. • Selenite F broth contains sod. selenite and tetrathionate broth contains iodine.

16. Types of the media • Selective media: Used for isolation of specific organism or group of organisms: • Salmonella Shegilla media (SS media) for Salmonela and Shegilla organisms only. • PPLO media contains penicillin G sodium which prevent the growth of G +ve bacteria and Thallium acetate which prevent the growth of G -ve bacteria except mycoplasma. • MacConkey and EMB media are selective for enterobacteriacae.

17. Fried egg shaped colonies of Mycoplasma species

18. Types of the media • Differential media: • MacConkey medium differentiates between lactose fermenter (E. coli) and non-lactose fermenter organisms (Salmonellae) due to it contains neutral red indicator that transformed into lactic acid (pink colour) in case of fermentation. • Coagulase medium for differentiation between pathogenic and non pathogenic Staphylococcus.

19. Types of the media • Sensitivity test media: • Mollar Hinkton agar which contains Nacl in certain concentration that allows maximum diffusion of the antibiotics. • Media used for certain tests: • Indol, Citrate, etc…..

20. Procedure for isolation • Direct plating on the medium from the sample. • Insertion of a sterilized wire loop into a burned surface of the organ (hot spatula). • Cutting a part of the burned surface with a cold scissor. • Immersion of the swab or parts of the organs into the medium.

21. Procedure for isolation • Enrichment of the organism before plating: • As in case of Salomonella organisms: 1. By small sterile scissor make mincing of the organ in a tube containing Selenite F broth or tetrathionate broth. 2. After mincing, the organism will exposed in the broth and it increases in NO. after incubation at 42C for 14-16 hours.

22. Procedure for isolation • Enrichment of the organism before plating: • As in case of Salomonella organisms: The incubation of Salmonella organisms should not exceed 16 hours to avoid the growth of E. coli which shift the pH of the media toward the acidic site which kill Salmonellae. Positive growth appears as sediment.

23. Procedure for isolation • Enrichment of the organism before plating: • As in case of Salomonella organisms: Make plating on specific media for salmonella like SS, MacConkey, EMB, XLD. Incubate at 37-42C for 24-48 hours. NB: Salmonella organism is the only one which tolerate this high level of temperature.

24. Procedure for isolation • Enrichment of the organism before plating: • As in case of Haemophilus paragallinarum: It’s growth needs 2 factors (X Haemin) and (V NAD by plating of Staph epidermidis), incubate under microaerophilic condition (10% CO2, 85% N2 and 5% O2). • Clostridial group needs strict anaerobic condition for growth.

26. Identification • Colonial morphology: • In liquid medium preparation of sediment or homogeneous turbidity. • On solid media the appearance is including; size (diameter in mm.) outline (circular, rhizoid, crecented…etc.) elevation (flat, raised, low convex…etc.) transparency (clean transparent, or opaque), colour (colourless, white, pigmented) production of haemolysis in blood containing medium or not. • Characteristic Fried egg shape appearance.

27. Identification • Morphology and staining reaction: • Unstained wet film examined with dark ground microscope for observation of exact morphology specially used for Spirochaetes and Campylobacters. • A stained smears (e.g. Gram stain, Ziehl Neelson stain, Giemsa, Leishmans stain …etc.) to determine Gram reaction, size, shape and grouping of bacteria. • Acute P. mulocida give a characteristic bioplarity between nucleated RBCs in blood stained films by Giemsa stain.

28. Salmonella, E. coli species are gram negative Clostridium Acid fast bacilli Acid­alcohol­fast bacilli (AFB).

29. This impression smear, made from the exudate of infected tissues, shows intracytoplasmic inclusions associated with Chlamydophila psittaci infection.

30. Bipolar P.multocida in between nucleated RBCS in acute infection

31. Identification • Biochemical reactions: Sugar fermentation: Growth of bacteria in liquid medium will ferment particular sugars with production of acid or acid and gas. Production of a particular end producte.g. Indol, H2S or nitrate when the organism grows in a suitable medium. Possessing of a certain enzymatic activities e.g. oxidase, catalase, urease depends upon the fact that the serum of an animal immunized against a microorganism contain specific antibodies for the homologous species) which react in a characteristic manner with the particular microorganism.

32. Identification • Animal pathogeniciy: • Laboratory animal inoculation (Biological properties). • Virulent pathogenic organisms in laboratory animals produce characteristic lesions. e.g. • P. multocida in mice or rabbits induced deaths within 24 hr, isolate the organism from heart Blood (bipolarity). • Chlamydia organism in mice or G.pig death with lesions, stain to see inclusion bodies. • Avian tuberculosis in guinea pigs.

33. Identification • Serological identification: • The most known field test is agglutination test by using specific known mono-valent or poly valent hyperimmune antisera. • On a glass slide, put one drop of saline emulsed purified colonies and one drop of antisera and mix well. Appearance of floccules indicates positive reactions (Salmonella, E.coli). • Another tests as FAT and ELISA are used with known antigens to detect specific antibodies.

34. Identification • Bacteriophage typing. • PCR.

35. Sensitivity test • Commonly there are marked differences in antibiotic sensitivity between different strains of a species. • It is required as a guide to the choice of drug for therapy.

36. Sensitivity test • Subculture 24 hrs colonies to regain its normal enzymatic and biochemical characters. • Pick up some identified colonies and suspend in broth (peptone water). • Incubate the bacteria with broth at 37C for 4 hrs to initiate a Log phase and obtained maximum metabolic activity).

37. Sensitivity test • Adjustment of the bacterial concentration by Maccferland tube (opacity tube) to NO. ½ (0.1 ml of barium chloride with 9.9 ml of sulphoric acid, decant 5 ml and add 5 ml dist. water). • Shack the bacterial tube till obtain bacterial concentration similar to tube NO. ½. • Sensitivity agar plate (should be dried in incubator for ½ hr).

38. Sensitivity test • Pour the broth containing bacteria on the plate and leave it for 15 minutes. • Discard any excess broth on the plate surface and leave it in incubator for 15 minutes in reversed position to evaporate excess fluid and leave it to dry. • Dispense discs, the distance between the center disc and the peripheries ones 3 Cm. • Incubate at 37C for 24 hrs and read inhibitory zone.

40. Y= X-0.6 2 0.6= Disc diameter. Y= distance bet. Disc and the last zone. X= Zone diameter

41. Whole blood agglutination test (WBAT) (Pullorum test) (Rapid test) (Field test) (Qualitative test)

42. Whole blood agglutination test Aim Used for detection of salmonella gallinarum pullorum chronic carriers in the breeder flocks, when the birds reach to 50% of egg production. As the sexual hormones reach to balance point after 50% egg production which leads to formation of antibodies to the infection).

43. Whole blood agglutination test Requirements • Standard stained full antigen: • The antigen must contain the full antigenic structure of S.g.p (1,9,12 1,2,3). • It is a dead bacterial suspension in phenol with adjustment to the NO. which is specified to the reaction. This suspension contain crystal violet die to make the reading of the test is more easier.

44. Whole blood agglutination test Requirements • Standard stained full antigen should be : • Stored in the refrigerator (2-4C), don’t freeze or kept at the room temperature. • Kept few minutes at room temperature before using. • Re-suspended (slowly shacked) before using to avoid false negative result. • Not exposed to heat. • Not expired (valid).

45. Whole blood agglutination test Requirements • Porcelain plate should be: • Completely dry. • Dust free (use a piece of cotton and water only to clean the plate without using detergent that block the reaction). • Grease free (not touch it with your fingers). • Divided into 8-10 wells.

46. Whole blood agglutination test Requirements • Lope and needle: • It should be immersed in water after each test. • The place should be: • Good lighted. • Closed. • Dust free. • Proceed at warm conditions (not less than 20C).

47. Whole blood agglutination test Requirements • The bird should be: • Numbered by leg or wing bands. • Represent 50% of egg production.

48. Whole blood agglutination test Procedure • Put one drop of the coloured antigen in the identified numbered square by dropper. • Add to it drop of blood from the wing vain at the elbow joint by the needle. • Make rapid through and circular mixing of the antigen and the blood by the loop (before blood clotting). • Measure the time.

49. Whole blood agglutination test Interpretation • Positive agglutination reaction appears within one minute as the form of granules or floccules. These birds must be discarded from the flock and used for human consumption and their eggs also not used in hatching and used as table eggs. (This test is individual test as each bird is express itself as the organism is localized in the ovary).

50. Whole blood agglutination test Interpretation • Suspected case which give agglutination with in 1-2 minutes should be handled as the following: • If low NO. (15-20), they considered as positive. • If large NO. they must be re-tested until obtaining 2 successive negative results 2-3 weeks interval (due to intermittent shedding of the organism).