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

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 KOKOŠI S KRONIČNIMI DIHALNIMI OBOLENJI IN IZ NJIHOVEGA BIVALNEGA OKOLJA Povzetek: Ptičja mikoplazmoza je resno težava v perutninski proizvodnji, ki vodi v velike izgube zaradi obolevanja perutnine in posledično povzroča ekonomske izgube. Glavni povzročitelji mikoplazmoz so Mycoplasma gallisepticum in Mycoplasma synoviae. Za preučevanje spremenljive stopnje odpornosti na običajno predpisane in uporabljene antibiotike pri mikoplazmozi je bilo odvzetih skupno 115 vzorcev, vključno z vzorci tkiva in brisom, pitovnih piščancev, nesnic s kroničnimi boleznimi dihal (CRD) in iz njihovega bivalnega okolja. Vzorci so bili preneseni v tekoče gojišče 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 določeni kot negativni po tretji pasaži. Med vzorci je bilo 61,5 % pozitivnih na prisotnost Mycoplasma spp., ki smo jih večinoma 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 specifičnih primerjev. Izolati MG in MS so pokazali spremenljivo stopnjo občutljivosti na komercialno dostopno zdravilo tilozin. Minimalna zaviralna koncentracija (MIC) pri MG  je bila najvišja 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). Najvišjo 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).Ključne besede: ptičja mikoplazmoza; kronična bolezen dihal; minimalna zaviralna koncentracija; mnogokranta PCR reakcija