AVIAN MYCOPLASMOSIS. Prof. Dr./ Wafaa Abd El-ghany. Professor of Poultry Dis., Fac. Vet. Med., Cairo Univ.

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1 AVIAN MYCOPLASMOSIS Prof. Dr./ Wafaa Abd El-ghany Professor of Poultry Dis., Fac. Vet. Med., Cairo Univ.

2 Economic importance Increase condemnation rate due to serositis. Poor feed conversion rate. Growth retardation. Reduction in egg production. Decrease in the fertility and hatchability. Increase the cost of prevention and medication.

3 The shared properties of viruses and mycoplasmas 1. Very small in size (smallest free living m.o.) 2. Pass through 0.45um filters. 3. Haemagglutinating. 4. Morphology under electron-microscopy. 5. Ether and chloroform sensitive. 6. Penicillins resistant. 7. In vitro cytopathic effect. 8. Neutralization by homologus antisera.

4 The causative agent Mycoplasmas are very small prokaryotes. Lack of a cell wall: 1. Resistant to penicillin. 2. Formation of fried egg shaped colonies. 3. Sensitivity to detergents.

5 The causative agent Mycoplasmas is polymorphic organism, when stained with Giemsa appeared as weekly Gram negative, cocoid or filamentous in old culture. There are at least 25 sp., the most common pathogenic ones are: M. gallisepticum (MG) M. synoviae (MS) M. meleagridis (MM) M. iowae

6 The causative agent Haeagglutinating organism to avian (chickens and turkeys) RBCS. Infection with mycoplasmas alone rarely induces a disease condition, but it should be presence of other concomitant complicating factors as the followings:

7 The causative agent Complicating factors Infectious Non-Infectious Bacteria (E.coli, H.paragallinarum, P.multocida) Virus (NDV, IBV, ILTV). Fungi (Aspergillus spp.). Living virus vaccines. Overcrowdness Bad ventilation Dustness High ammonia Immunsupression

8 Susceptibility All avian species (domestic and wild birds) are susceptible. Gallinaceous birds particularly chickens, turkeys, pheasants and quails are highly susceptible. Mycoplasmas infect the chickens and turkeys at any age, while young birds (4-10 weeks old) are more susceptible.

9 Mode of infection Vertical infection: From infected hens to the embryos, causing embryonic mortalities or infection of day old chicks. Horizontal infection: Through aerosol (air born) droplet infection.

10 Mode of transmission 1. Vertical (in-ovo) transmission: Only 2% of the laid eggs from mycoplasma infected carriers breeders carry the organism. Oviduct infection may result from extension from infected abdominal air sacs.

11 Mode of transmission 2. Horizontal transmission: Direct or indirect contact of susceptible birds to clinical or subclinical infected birds. 3. Mechanical (Fomites): workers, free living birds, utensils, visitors, cars, feed bags, etc.

12 Mode of transmission 4. Venereal transmission: In case of M. melaegridis. This organism is present in the phallus and cloacae of the males. The transmission from males to females through mating or artificial insemination with the infected semen.

13 MYCOPLASMA GALLISEPTICUM (MG) [Chronic Respiratory Disease (CRD) of chickens] (Infectious Sinusitis of turkeys)

14 Definition MG infections are respiratory affections of slow development and long course characterized by; Respiratory rales, coughing, nasal discharge and conjunctivitis in chickens; Swollen infra orbital sinus is frequently occurred in turkeys.

15 Clinical signs of MG In chickens (CRD): 1) Respiratory signs (Tracheal rals, nasal discharge, coughing, swelling of eye lids with increased lacrimation). 2) Loss of body weight. 3) High morbidity, while mortality occurred sequentially due to concurrent infections. 4) Drop in egg production, fertility and hatchability.

16 Clinical signs of MG In turkeys (Infectious sinusitis): 1) Unilateral or bilateral swelling of the infraorbital sinuses with facial swelling. 2) Partial or complete closure of eye. 3) Nasal discharge with foamy eye secretion. 4) Tracheal rales, coughing and labored breathing.

17 Clinical signs of MG In turkeys (Infectious sinusitis): 5) Decreased feed intake and weight loss. 6) MG can induce encephalitic form in weeks old commercial meat turkey with torticollis and opithotenous. 7) Breeder flocks show drop in egg production.

18 Post-mortem lesions of MG In case of CRD (MG without complications): 1) Catarrhal rhinitis, sinusitis (turkeys), conjunctivitis, trachitis and bronchitis. 2) Lung congestion and pneumonia. 3) Mild pericarditis, perihepatitis and airsacculitis.

19 Post-mortem lesions of MG In case of CCRD (MG complications): 1) Serofibrinous or fibrinous (casous or purulent) pericarditis, perihepatitis and airsacculitis. 2) Peritonitis. 3) Salpingitis. 4) Lung congestion and pneumonia.

20 Severe swelling of the infraorbital sinuses

21 Severe swelling of the infraorbital sinuses and conjunctivitis

22 Severe swelling of the infraorbital sinuses and conjunctivitis

23 Severe swelling of the infraorbital sinuses and conjunctivitis

24 Keratoconjunctivitis and occasionally corneal opacities

25 Mild airsacculitis with light presence of foam. Airsacculitis with caseous exudate Severe airsacculitis with abundant foam and aggregates of caseous exudate.

26 Airsacculitis with caseous exudate

27 Severe perihepatitis and pericarditis.

28 Severe perihepatitis and pericarditis.

29 pericarditis and fibrinous pneumonia Fibrinous pericarditis

30 Differential diagnosis ND IB AI IC FC Chlamydiosis Asprigellosis

31 MYCOPLASMA SYNOVIAE (MS) INFECTION Infectious synovitis

32 Definition MS induces acute or chronic infectious synovitis in chickens and turkeys characterized by exudative synovitis and tenovaginitis or sternal bursitis. MS can also induce respiratory infection (air sac disease).

33 Clinical signs of MS In chickens and turkeys (Infectious synovitis): 1) Swellings around joints especially hocks, foot pad and the sternal bursa is enlarged (turkeys). 2) Lameness. 3) Retardation in growth. 4) Mild respiratory signs. 5) Slight drop egg production and egg quality.

34 Post-mortem lesions of MS In chickens and turkeys (Infectious synovitis): 1) The synovial membranes of tendon sheaths of affected joints and keel bursa have a viscous creamy to caseous exudates. 2) Articular surfaces become eroded. 3) Sternal bursitis (breast blister) in turkeys. 4) Caseous exudates may be found in air sacs.

35 SUSP.DIS. : Viral Arthritis (REO) Arthritis

36 Arthritis with caseous exudates in the hock joint

37 Sternal bursitis or breast blister

38 Differential diagnosis Staph.aureus E. coli FC Salmonellae viral tenosynovitis (REO)

39 MYCOPLASMA MELEAGRIDIS (MM) INFECTION

40 Definition MG is a specific pathogen of turkeys, causing egg transmitted disease with primary lesions in air sacs with decrease hatchability, skeletal abnormalities and poor performance.

41 Clinical signs of MM Airsaculitis deficiency syndrome (TS-65): 1) Hock joint swelling. 2) Bowing. 3) Twisting and shortening tarsometatarsal bone. of the 4) Deformity of the cervical vertebrate. 5) Lameness. 6) Stunting and abnormal feathering. 7) Retardation in growth. 8) Severe late embryonic mortality. 9) Respiratory signs.

42 Post-mortem lesions of MM 1) Airsaculitis deficiency syndrome (TS-65): 2) Airsacculitis. 3) Skelatal deformaties (tibial dyschondroplasia and chondrodystrophy). 4) Synovitis and sternal bursitis. 5) Sinusitis.

43 Bowing or skelatal deformity Bowing, poor feathering and stunted growth

44 Isolation on the culture media Samples are taken from the nasal clefts, trachea, lungs, airsacs, joint exudate, phallus, vagina, semen or fertile eggs. It is preferable to isolate mycoplasma organism in acute condition, difficult isolation in chronic cases. Mycoplasma species are fastidious organisms due to that, they difficult to grow and need long time for growth. Mycoplasma species need enriched media like PPLO (pleuro-pneumonia like organism) media or modified Frey s FM media.

45 Isolation on the culture media Pencillin G-sodium (1000 IU/ml) to prevent the growth of Gram positive bacteria and thallus acetate (1:4000) to prevent the growth of Gram negative bacteria in addition to inactivated horse or swine sera (10-15%) as growth promoters and yeast extract or yeast autolysate 1%. Cystien hydrochloride is added as reducing agent. Polymixin B (100 unit/ml) and mycostatin (50 IU/ml) inhibit fungal growth. Phenol red or 2,3,5 phenyl tetrazolium as indicators.

46 Isolation on the culture media For isolation of MS, NAD (0.01%) should be added to the media. The culture is grown in PPLO broth, and filtrated 2 or 3 ten fold serial dilution then plating on PPLO solid media. At the same time inoculate the culture directly on PPLO solid agar media. The broth is incubated at ph 7 under microaerobic conditions (10%) Co2 in Co2 incubator or candle jar at 37C for 24 hr.

47 Isolation on the culture media Positive culture appears as turbidity and it should be re-inculated on PPLO agar and incubated at the previously mentioned conditions. Positive agar culture appears as a characteristic Fried egg shaped appearance colonies under dissecting or steromicroscope. Negative broth culture should be re-inoculted into PPLO broth again or 14 days till obtained positive reaction. No turbidity of the broth after 14 days is considered as negative sample for avian mycoplasmas.

48 Fried egg shape colonies of Mycoplasmas

49 Isolation in ECE Inoculation of the suspected mycoplasma material or broth culture in the yolk sac (5-7 days) of ECE induce death of the embryos within 5-8 days with curling and dwarfing of the embryo, generalized oedema, liver necrosis and enlarged spleen. Serial passages of harvested yolk may be required to induce the deaths in the embryo.

50 Serum plate agglutination (SPA) test SPA test is considered as the first step to screen flocks for antibody response. SPA test is in-expensive, very sensitive, but poor specific test as false positive results are common by this test. If the test is positive, another more specific tests as HI and ELISA tests should be done as a confirmative tests.

51 Serum plate agglutination (SPA) test On the clean porcelain or glass plate, add one drop of MG or MS specific coloured (stained) antigen, then add on drop of the serum of the bird. Mix well, and rotate the plate in a circular manner. Positive reaction appears with in 2 minutes as the form of agglutination (flocculation or granulation), suspected cases should be confirmed using HI test.

52 Serum plate agglutination (SPA) test SPA test is considered as a flock test, that is mean presence of one positive bird indicate that all the birds in the flock are positive due to mycoplasma organism is transmit rapidly and mainly through the respiratory rout. We don t use the blood in this test due to mycoplasma organism is haemagglutinating organism to the avian RBCS (natural phenomonon).

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55 Serological and molecular diagnosis 1) Tube agglutination test. 2) HI test. 3) ELISA test. 4) Micro-immunofluoresence or peroxidase test. 5) AGPT. 6) DNA probs. 7) PCR.

56 Prevention of avian mycoplasmosis in breeders I. Serological monitoring program of breeder flocks: serum plate agglutination test, HI or ELISA. Positive mycoplasma birds should be : 1. Treated with medication. 2. Egg treatment. 3. Vaccination. 4. Hygienic measures. 5. Egg and hatchery sanitation.

57 Prevention of avian mycoplasmosis in breeders I. Serological monitoring program of breeder flocks: By using of serological tests (serum plate agglutination test, HI or ELISA): Negative mycoplasma birds: should be maintained under strict isolation facilities to avoid disease introduction. Strict sanitary measures. Vaccination of replacement flocks obtained from mycoplasma free flocks.

58 Prevention of avian mycoplasmosis in breeders II. In case of MM: Avoid the cycle of venereal transmission through: Examination of the phallus and cloacae of adult male turkeys for MM and confirm the results serologically, bacteriologically and molecularly before the breeding season. Using non-infected (non genetically carriers) birds for breeding (mating).

59 Prevention of avian mycoplasmosis in breeders II. In case of MM: Avoid the cycle of venereal transmission through: 3. Examination of the female turkeys (oviduct swabs) or eggs for MM and confirm the results serologically, bacteriologically or molecularly before the breeding season. 4. Good hygienic methods and special care of artificial insemination and sexing of day old pullets.

60 Control of avian mycoplasmosis in breeders 1. Serological monitoring. 2. Breeder medication. 3. Egg treatment. 4. Hatchery hygiene. 5. Biosecurity measures.

61 Control of avian mycoplasmosis in broilers 1.Biosecurity measures (strict hygienic measures). 2.Broilers medication.

62 Biosecurity and management practice 1. Proper housing and feeding. 2. Good air, litter and water quality. 3. All in all out system. 4. Optimal stocking density. 5. Optimal hatchery sanitation. 6. Avoid predisposing and stress factors. 7. Avoid immunosuppression. 8. Avoid mechanical routs of transmission.

63 Medication Mycoplasmas are resistant to antibiotics which inhibit the cell wall synthesis (pencillins), or that inhibit the membrane synthesis (polymyxin B). However, Mycoplasmas are sensitive to antibiotics that inhibiting the protein or nucleic acid synthesis.

64 Medication Effective antibiotics to avian mycoplasmas include: 1. Teteracyclines: Oxy, chloro, deoxy. 2. Macrolides: Erythromycin, tylosin, spiramycin, lincomycin and kitasamycin. 3. Aminoglycosides: Spectinamycin. 4. Pleuromutilin: Tiamulin. 5. Quinolones: Norfloxacin,enrofloxacin and danofloxacin.

65 Medication Medication helps only to control clinical signs and lesions and slow the spread of infection, but it doesn't eliminate the infection. Complete elimination of mycoplasmas from all birds in the infected flocks using medication is not expected, as the treatment is regarded as a method of short-term amelioration of the disease, rather than as long term solution of the problem.

66 Egg treatment A) Egg inoculation: Any antimycoplasmal antibiotics is injected into the egg cell, this method may kill the embryo. B) Egg dipping: The eggs are warmed to 37-38C then immersed in cold antibiotic solution (1-4C) for 20 minutes. Due to the temperature differences, cooling of the egg contents and the antibiotic is pulled through the egg shell.

67 Egg treatment C) Pressure differential vacuum (system) Decreasing the pressure above the egg 25 Cm Hg by vacuum pump hen the pressure return slowly to the atmospheric one. This method is not completely eliminate the organism but not affect on the hatchability. D) Heat forced incubator: the eggs are heated in forced air incubator for 40C then putted at room temperature for cooling at 25.6C, it is the most effective method but it reduces the hatchability to 8-12% with high embryonic mortalities.

68 Vaccination Types of MG and MS vaccines: 1. Inactivated vaccines (bacterins). 2. Live vaccines. 3. Subunit vaccine or Recombinant vaccines. Both living and inactivated vaccines are used commercially while Subunit and Recombinant Vaccines are still under development.

69 1. F strain. Types of MG living 2. 6/85 strain. 3. Ts-11 strain. vaccines 4. New K 5054 strain. Chickens are vaccinated with live MG vaccine between 12 and 16 weeks of age. Vaccination can be carried out a 2-4 weeks of age.

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