OCCURRENCE, MOLECULAR IDENTIFICATION AND ANTIBIOTIC RESISTANCE PROFILING OF Mycoplasma gallisepticum AND Mycoplasma synoviae FROM CHRONIC RESPIRATORY DISEASE CASES IN POULTRY BIRDS AND FARM ENVIRONMENT

Abu Baker Siddique, Sajjad Ur Rahman, Mazhar Ulhaq, Rasheeha Naveed

Abstract


Avian mycoplasmosis is an important risk for commercial poultry production leading to enormous losses in terms of disease and productivity. The main causative agents are Mycoplasma gallisepticum and Mycoplasma synoviae. To study the variable degree of resistance to commonly prescribed and used antibiotics in mycoplasmosis, a total of 115 samples including tissue specimen and swabs were collected from chronic respiratory disease (CRD) cases of broiler and layer birds and their contaminated farm environment. The samples were directly passaged into the Brain Heart Infusion broth (supplemented with 10 % horse serum, NAD, cysteine, penicillin and thallium acetate). Positive samples were transferred to Brain Heart Infusion agar (Difco) for the isolation of Mycoplasma spp. while negative samples were declared after the third passage. Of the samples, 61.5% were found positive for Mycoplasma spp., which were recovered mostly after second passage. Out of total culture positive cases, Mycoplasma gallisepticum (MG) was identified in 62% cases and Mycoplasma synoviae (MS) in 38%, as confirmed through Polymerase Chain Reaction (PCR) using specific primers. The MG and MS isolates showed variable degrees of sensitivity against the commercially available drug of choice, tylosin. The highest Minimum Inhibitory Concentration (MIC) of enrofloxacin (112.38±4.34 µg/ml) was recorded against MG, followed by tetracyclin (91.58±4.66µl/ml), gentamicin (54.33±2.98 µg/ml), spiromicin (52.23±3.99 µg/ml) and tylosin (52.58±2.69 µg/ml). The highest MIC for enrofloxacin (168.24 ±3.82 µg/ml) was recorded against MS followed by tetracyclin (115.48±2.62 µg/ml), spiromicin (95.96 ±2.17 µg/ml), tylosin (84.84±2.56 µg/ml) and gentamicin (46.4±2.18 µg/ml). Multiplex PCR is a time tested tool for the molecular diagnosis and confirmation of Mycoplasma species.

Key words: avian mycoplasmosis; chronic respiratory distress; minimum inhibitory concertation; multiplex polymerase chain reaction

 

POJAVNOST, MOLEKULARNA IDENTIFIKACIJA IN UGOTAVLJANJE ODPORNOSTI NA ANTIBIOTIKE MIKOBAKTERIJ Mycoplasma gallisepticum in Mycoplasma synoviae IZOLIRANIH IZ KOKOÅ I S KRONIÄŒNIMI DIHALNIMI OBOLENJI IN IZ NJIHOVEGA BIVALNEGA OKOLJA
 
Povzetek: PtiÄja mikoplazmoza je resno težava v perutninski proizvodnji, ki vodi v velike izgube zaradi obolevanja perutnine in poslediÄno povzroÄa ekonomske izgube. Glavni povzroÄitelji mikoplazmoz so Mycoplasma gallisepticum in Mycoplasma synoviae. Za preuÄevanje spremenljive stopnje odpornosti na obiÄajno predpisane in uporabljene antibiotike pri mikoplazmozi je bilo odvzetih skupno 115 vzorcev, vkljuÄno z vzorci tkiva in brisom, pitovnih piÅ¡Äancev, nesnic s kroniÄnimi boleznimi dihal (CRD) in iz njihovega bivalnega okolja. Vzorci so bili preneseni v tekoÄe gojiÅ¡Äe BHI (iz angl. Brain Heart Infusion), z dodatkom 10 % konjskega seruma, NAD, cisteina, penicilina in talijevega acetata. Pozitivne vzorce smo prenesli v agar BHI (Difco) za izolacijo Mycoplasma spp. Vzorci so bili doloÄeni kot negativni po tretji pasaži. Med vzorci je bilo 61,5 % pozitivnih na prisotnost Mycoplasma spp., ki smo jih veÄinoma ugotovili po drugi pasaži. Od vseh pozitivnih primerov je bila ugotovljena Mycoplasma gallisepticum (MG) v 62 % primerov, Mycoplasma synoviae (MS) pa v 38 %, kar je bilo potrjeno z verižno reakcijo s polimerazo (PCR) z uporabo specifiÄnih primerjev. Izolati MG in MS so pokazali spremenljivo stopnjo obÄutljivosti na komercialno dostopno zdravilo tilozin. Minimalna zaviralna koncentracija (MIC) pri MG  je bila najviÅ¡ja pri enrofloksacinu (112,38 ± 4,34 µg/ml), sledili pa so tetraciklin (91,58 ± 4,66 µl/ ml), gentamicin (54,33 ± 2,98 µg/ml), spiromicin (52,23 ± 3,99 µg/ml) in tilozin (52,58 ± 2,69 µg/ml). NajviÅ¡jo MIC proti MS smo ravno takougotovili pri enrofloksacinu (168,24 ± 3,82 µg/ml), ki so mu sledili tetraciklin (115,48 ± 2,62 µg/ml), spiromicin (95,96 ± 2,17 µg/ml), tilozin (84,84 ± 2,56 µg/ml) in gentamicin (46,4 ± 2,18 µg/ml).

KljuÄne besede: ptiÄja mikoplazmoza; kroniÄna bolezen dihal; minimalna zaviralna koncentracija; mnogokranta PCR reakcija


Full Text:

PDF

References


(1.) OIE. Avian mycoplasmosis (Mycoplasma gallisepticum, M. synoviae). In: Manual of diagnostic tests and vaccines for terrestrial animals. Paris : Office International des Epizooties, 2008: 482–96.

(2.) Kleven SH, Ferguson-Noel N. Mycoplasma synoviae infection. In: Saif YM, Barnes HJ, Glisson JR, et al., eds. Diseases of poultry. London : Blackwell Publishers, 2008: 845–56.

(3.) Kleven SH. Control of avian mycoplasma infections in commercial poultry. Avian Dis 2008; 52: 367–74.

(4.) Luciano RL, Cardoso ALSP, Stoppa GFZ, et al. Comparative study of serological tests for Mycoplasma synoviae diagnosis in commercial poultry breeders. Vet Med Int 2011; 2011: Art 304349. doi: 10.4061/2011/304349

(5.) Mohana MA, Haider M, Kadhim AH, et al. Molecular diagnosis of avian respiratory diseases in commercial broiler chicken flocks in province of Najaf, Iraq. Sci Res Essays 2013; 8: 1191–5.

(6.) Yashpal SM, Devi PP, Sagar MG. Detection of three avian respiratory viruses by single-tube multiplex reverse transcription-polymerase chain reaction assay. J Vet Diagn Invest 2004; 16: 244–8.

(7.) Gabriel SC, Shivaprasad HL, Chin RP. Systemic Mycoplasma synoviae infection in broiler chickens. Avian Pathol 2005; 34: 137–42.

(8.) Ley DH. Mycoplasma gallisepticum infection. In: Saif, YM, et al., eds. Diseases of poultry. Ames : Iowa State University Press, 2008: 805–33.

(9.) Jeon EO, Kim JN, Lee HR, et al. Eggshell apex abnormalities associated with Mycoplasma synoviae infection in layers. J Vet Sci 2014; 15: 579–82.

(10.) Levisohn S, Kleven SH. Avian mycoplasmosis (Mycoplasma gallisepticum). Rev Sci Tech 2000; 19: 425–42.

(11.) Gautier-Bouchardon AV, Reinhardt AK, Kobisch M, Kempf I. In vitro development of resistance to enrofloxacin, erythromycin, tylosin, tiamulin and oxytetracycline in Mycoplasma gallisepticum, Mycoplasma iowae and Mycoplasma synoviae. Vet Microbiol 2002; 88: 47–58.

(12.) Hannan PC, Windsor GD, De Jong A, Schmeer N, Stegemann M. Comparative susceptibilities of various animal-pathogenic mycoplasmas to fluoroquinolones. Antimicrob Agents Chemother 1997; 41: 2037–40.

(13.) Ter Laak EA, Noordergraaf JH, Verschure MH. Susceptibilities of Mycoplasma bovis, Mycoplasma dispar, and Ureaplasma diversum strains to antimicrobial agents in vitro. Antimicrob Agents Chemother 1993; 37: 317–21.

(14.) Bradbury JM, Yavari CA, Giles CJ. In vitro evaluation of various antimicrobials against Mycoplasma gallisepticum and Mycoplasma synoviae by the microâ€broth method, and comparison with a commerciallyâ€prepared test system. Avian Pathol 1994; 23: 105–15.

(15.) Hannan PC. Guidelines and recommendations for antimicrobial minimum inhibitory concentration (MIC) testing against veterinary mycoplasma species. Vet Res 2000; 31(4): 373-–95.

(16.) Wu CM, Wu H, Ning Y, Wang J, Du X, Shen J. Induction of macrolide resistance in Mycoplasma gallisepticum in vitro and its resistance-related mutations with domain V of 23S rRNA. FEMS Microbiol Lett 2005; 247: 199–205.

(17.) Nicholas RAJ, Ayling RD. Mycoplasma bovis: disease, diagnosis, and control. Res Vet Sci 2003; 74: 105–12.

(18.) Ahmad A, Khan TA, Kanwal B, et al. Molecular identification of agents causing respiratory infections in chickens from southern region of Pakistan from October 2007 to February 2008. Int J Agr Biol 2009; 11: 325–8.

(19.) Stipkovits L. Kempf I. Mycoplasmosis in poultry. Rev Sci Tech 1996; 15: 1495–525.

(20.) Bencina D, Bradbury JM, Stripkovits L, et al. Isolation of Mycoplasma capriculum-like strains from chickens. Vet Microbiol 2006; 112(1): 23–31.

(21.) Jackwood MW, Hilt DA, Williams SM, et al. Molecular and serologic characterization, pathogenicity, and protection studies with infectious bronchitis virus field isolates from California. Avian Dis 2007; 51: 527–33.

(22.) Siddique AB, Rahman SU, Hussain I, et al. Frequency distribution of opportunistic avian pathogens in respiratory distress cases of poultry. Pak Vet J 2012; 32: 386–9.

(23.) Marois C, Gesbert FD, Kempf I. Polymerase chain reaction for the detection of Mycoplasma gallisepticum from environmental samples. Avian Pathol 2002; 31: 163–8.

(24.) Wang C, Ewing M, A'arabi SY. In vitro susceptibility of avian mycoplasmas to enrofloxacin, sarafloxacin, tylosin, and oxytetracycline. Avian Dis 2001; 45: 456–60.

(25.) Cerda RO, Giacoboni GI, Xavier JA, Sansalone PI, Landoni MF. In vitro antibiotic susceptibility of field isolates of Mycoplasma synoviae in Argentina. Avian Dis 2002; 46: 215–8.

(26.) Gerchman I, Lysnyansky I, Perk S, Levisohn S. In vitro susceptibilities to fluoroquinolones in current and archived Mycoplasma gallisepticum and Mycoplasma synoviae isolates from meat-type turkeys. Vet Microbiol 2008; 131: 266–76.

(27.) Ehtisham-ul-Haque S, Rahman SU, Siddique M, Qureshi AS. Involvement of Mycoplasma synoviae in respiratory distress cases of broilers. Pak Vet J 2011; 31: 117–9.

(28.) Seifi S, Asasi K, Mohammadi A. Natural co-infection caused by avian influenza H9 subtype and infectious bronchitis viruses in broiler chicken farms. Vet Arh 2010; 80: 269–81.

(29.) Yousaf AM, Aradaib IE, Khairalla KMS, Sbdalla MA, Karrar ARE, ElHussein ARM. Evaluation of RT-PCR for rapid detection of Sudanese isolates and vaccine strains of Newcastle disease. Pak J Biol Sci 2005; 8: 418–20.

(30.) Wang H, Fadl AA, Khan MI. Multiplex PCR for avian pathogenic mycoplasmas. Mol Cell Probes 1997; 11: 211–6.

(31.) Hong Y, García M, Leiting V, et al. Specific detection and typing of Mycoplasma synoviae strains in poultry with PCR and DNA sequence analysis targeting the hemagglutinin encoding gene vlhA. Avian Dis 2004; 48: 606–16.

(32.) Harada K, Tanaka MK, Uchiyama M, et al. Molecular typing of Japanese field isolates and live commercial vaccine strain of Mycoplasma synoviae using improved pulsed-field gel electrophoresis and vlhA gene sequencing. Avian Dis 2009; 53(4): 538–43.

(33.) Behbahan NGG, Asasi K, Afsharifar AR, Pourbakhsh SA. Susceptibilities of Mycoplasma gallisepticum and Mycoplasma synoviea isolates to antimicrobial agents in vitro. Int J Poult Sci 2008; 7: 1058–64.

(34.) Reinhardt AK, Gautier-Bouchardon AV, Gicquel-Bruneau M, Kobisch M, Kempf I. Persistence of Mycoplasma gallisepticum in chickens after treatment with enrofloxacin without development of resistance. Vet Microbiol 2005; 106(1/2): 129–37.

(35.) Gharaibeh S, Al-Rashdan M. Change in antimicrobial susceptibility of Mycoplasma gallisepticum field isolates. Vet Microbiol 2011; 150(3/4): 379–83.

(36.) Jordan FT, Horrocks BK. The minimum inhibitory concentration of tilmicosin and tylosin for Mycoplasma gallisepticum and Mycoplasma synoviae and a comparison of their efficacy in the control on Mycoplasma gallisepticum infection in broiler chicks. Avian Dis 1996; 40: 326–34.

(37.) Jordan FT. Forrester CA. Ripley PH, Burch DG. In vitro and in vivo comparison of valnemulin, tiamulin, tylosin, enrofloxacin and lincomycin/spectinomycin against Mycoplasma gallisepticum. Avian Dis 1998; 42(4): 738–45.

(38.) Le Carrou J, Reinhardt AK, Kemf I, Gautier-Bouchardon AV. Persistence of Mycoplasma synoviae in hens after two enrofloxacin treatments and detection of mutations in the parC gene. Vet Res 2006; 37(1): 145–54.

(39.) Senties CG, Shivaprasad HL, Chin RP. Systemic Mycoplasma synoviae infection in broiler chickens. Avian Pathol 2005; 34: 137–42.

(40.) Nascimento ER, Pereira VL, Nascimento MG, Barreto ML. Avian mycoplasmosis update. Braz J Poult Sci 2005; 7(1): e1. doi: 10.1590/S1516-635X2005000100001

(41.) Kursa, O, Pakuła,A, Tomczyk G, et al. Eggshell apex abnormalities caused by two different Mycoplasma synoviae genotypes and evaluation of eggshell anomalies by full-field optical coherence tomography. BMC Vet Res 2019; 15(1): e1. doi: 10.1186/s12917-018-1758-8.




DOI: https://doi.org/10.26873/SVR-598-2020

Refbacks

  • There are currently no refbacks.


SLOVENIAN VETERINARY RESEARCH, Veterinary Faculty
Our journal is indexed in:
Science Citation Index Expanded, Journal Citation Reports/Science Edition, Agris, Biomedicina Slovenica, CAB Abstracts, IVSI Urlich’s International Periodicals Directory
Gerbičeva 60, SI-1000 Ljubljana, Slovenia, T: +386 (0)1 47 79 100, F: +386 (0)1 28 32 243, E: slovetres@vf.uni-lj.si
Published by computing.si