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2 nd International Conference on Animal & Dairy Sciences Prevalence of Mycoplasma gallisepticum Infection 2 nd International Conference on Animal & Dairy Sciences Prevalence of Mycoplasma gallisepticum Infection in Indian Poultry Farms M R Reddy Principal Scientist Directorate of Poultry Research (ISO 9001: 2008 Certified Institution) Rajendranagar, Hyderabad-500 030

Background • Mycoplasmas are highly versatile and successful pathogen • Chronic Respiratory Disease: Once Background • Mycoplasmas are highly versatile and successful pathogen • Chronic Respiratory Disease: Once infected, infection remains for life • Mycoplasma lack a cell wall: resistant to penicillin group of antibiotics • Antimicoplasmal drugs are bacteriostatic • Antibodies can not eliminate infection • Field infection (MG/MS) level is high due to vertical transmission and low level of biosecurity • Raising mycoplasma clean flocks is not practicable

Major pathogenic species M. gallisepticum M. synoviae M. meliagridis M. iowae - Chickens, Turkeys Major pathogenic species M. gallisepticum M. synoviae M. meliagridis M. iowae - Chickens, Turkeys - Turkeys Smallest self replicating prokaryotes Lack of cell wall, bounded by cell membrane Fragile – easily killed out side its host by disinfectants

Economic significance • Costliest Disease in Poultry • • Mortality Reduced feed conversion Loss Economic significance • Costliest Disease in Poultry • • Mortality Reduced feed conversion Loss of weight Complications with – IB, ND, E coli Condemnations Drop in egg production Reduced hatchability & chick viability Cost of eradication and control programs

MG related losses Excluding medication costs Effect Body weight gain FCR Egg production Embryo MG related losses Excluding medication costs Effect Body weight gain FCR Egg production Embryo mortality Mortality Carcass down grading Per cent 20 -30 10 -20 5 -10 10 -20 Stipkovits and Kempf (1996)

Transmission • Horizontal – Within a flock - contact or aerosols – Between flocks Transmission • Horizontal – Within a flock - contact or aerosols – Between flocks - windborne • Vertical – Parent to the chick through egg • Venereal

Target organs: – Respiratory system – Synovial membranes – Reproductive system Incubation period: – Target organs: – Respiratory system – Synovial membranes – Reproductive system Incubation period: – Chronic slow spreading – Varies from 6 -21 days

Clinical Signs • • • Coughing, sneezing, Nasal discharge Foamy secretions in the eye Clinical Signs • • • Coughing, sneezing, Nasal discharge Foamy secretions in the eye Open mouthed breathing Tracheal rales Reduced feed consumption Loss of weight – more stunted chicks Drop in egg production – layer, breeder Reduced hatchability, chick viability Lameness Morbidity – up to 100% Mortality – up to 30% in young, negligible in adults

Gross Lesions • • Sinusitis and conjunctivitis Tracheitis with excessive mucus Airsacculitis Pneumonia Synovitis Gross Lesions • • Sinusitis and conjunctivitis Tracheitis with excessive mucus Airsacculitis Pneumonia Synovitis Osteomyelitis Salpingitis

Accumulation of Cheesy mass in thoracic air sac Frothy exudate in Air sac Accumulation of Cheesy mass in thoracic air sac Frothy exudate in Air sac

Complicated Chronic Respiratory Disease Air-saculitis, Pericarditis, perihepatitis Complicated Chronic Respiratory Disease Air-saculitis, Pericarditis, perihepatitis

STUDY OBJECTIVE To determine the prevalence of M. gallisepticum from different regions in Indian STUDY OBJECTIVE To determine the prevalence of M. gallisepticum from different regions in Indian poultry industry

Methodology • Sampling • Antibody detection by ELISA • Isolation and Identification of MG Methodology • Sampling • Antibody detection by ELISA • Isolation and Identification of MG

Poultry map of India Breeder Population – 15. 6 m Broiler Population – 2000 Poultry map of India Breeder Population – 15. 6 m Broiler Population – 2000 m Layer Population - 165 m High population Moderate population Low population Zone South N W West/ Central E S North East State Andhra Pradesh, Tamil Nadu, Karnataka, Kerala Maharastra, Gujarat, MP, Chattishgarh Punjab, Haryana , Rajasthan, HP, UP, J&K West Bengal, Orissa, Jharkhant, North East Share 40% 25% 20% 15%

Flow Chart for Serology, isolation and identification of Mycoplasma gallisepticum Sample collection Layers, Br Flow Chart for Serology, isolation and identification of Mycoplasma gallisepticum Sample collection Layers, Br Parents, Broilers Blood Choanal swabs Tracheal Swabs Synovial fluid swabs Serum Mycoplasma broth Mycoplasma agar MG ELISA DNA PCR for MG MG Positive Analysis of MG incidence Sequencing

Target Number of Samples and Geographic Regions Geographic Location North Samples Serum Swabs 250 Target Number of Samples and Geographic Regions Geographic Location North Samples Serum Swabs 250 South 500 Central East TOTAL 300 100 1150

Serum separation for ELISA Collection of choanal swab Serum separation for ELISA Collection of choanal swab

Filling Sample Submission Form Filling Sample Submission Form

Number of Samples processed from different Geographic Regions Region Central East North South Total Number of Samples processed from different Geographic Regions Region Central East North South Total Choanal Swabs Serum Samples Number of birds flocks Birds flocks sampled 440 22 441 22 200 10 284 15 368 26 791 27 818 28 1715 64 1827 86

Sample Distribution Region Sample (%) Central East North South 24. 1 10. 8 20. Sample Distribution Region Sample (%) Central East North South 24. 1 10. 8 20. 0 45. 1 Flock Type Sample (%) Br Parent Broilers Layers 41. 5 13. 1 45. 5

Antibody ELISA • Serum Dilution: 1: 500 • Addition of diluted sera onto a Antibody ELISA • Serum Dilution: 1: 500 • Addition of diluted sera onto a MS/MG antigencoated plate, • Incubation 30 min, then wash • Addition of HRP labeled, anti-chicken antibody (conjugated antibody) • Incubation 30 min, then wash • Addition of substrate • Incubation 15 min, addition of Stop solution • Reading absorbance at 650 nm • Calculation of titer (≥ 1076 positive)

Isolation and Identification Swab in Frey’s Media DNA Frey’s broth Subculture Frey’s Agar DNA Isolation and Identification Swab in Frey’s Media DNA Frey’s broth Subculture Frey’s Agar DNA PCR Confirmation by Sequencing

MG PCR Primers Primer name Sequence MGC 2 2 F 5’-CGCAATTTGGTCCTAATCCCCAACA-3’ MGC 2 2 MG PCR Primers Primer name Sequence MGC 2 2 F 5’-CGCAATTTGGTCCTAATCCCCAACA-3’ MGC 2 2 R 5’-TAA ACCCACCTCCAGCTTTATTTCC-3’ MG 14 F 5’-GAGCTAATCTGTAAAGTTGGTC-3’ MG 13 R 5’-GCTTCCTTGCGGTTAGCAAC-3’ Product size (bp) 300 185

Frey’s Media (1000 ml) • • Frey’s broth base Glucose (10%) Thallous Acetate (5%) Frey’s Media (1000 ml) • • Frey’s broth base Glucose (10%) Thallous Acetate (5%) NAD (1%) Cysteine HCL (1%) Phenol red (0. 1%) Pig serum Penicillin 2 lakh IU/ml 22. 3 g 10 ml 20 ml 120 ml 5 ml

RESULTS RESULTS

Seroprevalance of Mycoplasma gallisepticum by ELISA (Based on Flocks) Region No. of Flocks Tested Seroprevalance of Mycoplasma gallisepticum by ELISA (Based on Flocks) Region No. of Flocks Tested No. of Flocks Positive % Positive Central 22 10 26 28 86 17 07 22 21 67 77. 3 70. 0 84. 6 75. 0 77. 9 East North South Total

Seroprevalance of M. gallisepticum by ELISA (Based on Flocks) 100 91. 7 90 Seroprevalence Seroprevalance of M. gallisepticum by ELISA (Based on Flocks) 100 91. 7 90 Seroprevalence 77. 5 80 70 60 50 40 30 30 20 10 0 Layers Br Parents Broilers

Seroprevalance of M. gallisepticum by ELISA (Based on Samples tested) Region Central East North Seroprevalance of M. gallisepticum by ELISA (Based on Samples tested) Region Central East North South Total No. of Flocks Tested No. of Flocks Positive % Positive 441 200 368 818 1827 220 113 212 258 803 49. 9 56. 5 57. 6 31. 5 43. 9

Seroprevalance of M. gallisepticum by ELISA (Based on samples tested) 60 54. 4 50 Seroprevalance of M. gallisepticum by ELISA (Based on samples tested) 60 54. 4 50 39. 6 40 30 20. 8 20 10 0 Layer Br parents Broilers

Prevalance of M. gallisepticum by Isolation and Identification (Based on Flocks) Region No. of Prevalance of M. gallisepticum by Isolation and Identification (Based on Flocks) Region No. of Flocks Tested No. of Flocks Positive % Positive Central 22 10 15 27 64 7 1 2 7 17 31. 8 10. 0 13. 3 25. 9 26. 6 East North South Total

Prevalance of M. gallisepticum by Isolation and Identification (Based on Flocks tested) 35 31 Prevalance of M. gallisepticum by Isolation and Identification (Based on Flocks tested) 35 31 30 25 MG prevelance 20 20 15 10 10 5 0 Layer Br parents Broilers

Prevalance of M. gallisepticum by Isolation and Identification (Based on Samples tested) Region Central Prevalance of M. gallisepticum by Isolation and Identification (Based on Samples tested) Region Central East North South Total No. of Samples Tested Positive 440 200 284 791 1715 82 2 5 89 178 % Positive 18. 6 1. 0 1. 8 11. 3 10. 4

Prevalance of M. gallisepticum by Isolation and Identification (Based on samples tested) 14 12. Prevalance of M. gallisepticum by Isolation and Identification (Based on samples tested) 14 12. 5 MG prevelance 12 9. 2 10 7. 9 8 6 4 2 0 Layer Br parents Broilers

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Conclusions • This study demonstrated the high prevalence of M. gallisepticum infection in Commercial Conclusions • This study demonstrated the high prevalence of M. gallisepticum infection in Commercial Layers, Broiler Parents and Commercial Broilers in all major poultry growing areas of the country. • Because of high value of individual parent breeder hen and their ability to infect progeny by vertical transmission, economic losses are potentially more severe when MG infection occurs in breeder flocks. • The high prevalence and wide distribution of MG infection warrants development and adaptation of strategies to prevent or minimize economic impact of MG infection.

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