Introduction

International Journal of Poultry Science 7 (11): 1058-1064, 2008ISSN 1682-8356 Asian Network for Scientific Information, 2008 Susceptibilities of Mycoplasma gallisepticum and Mycoplasma synoviea
Isolates to Antimicrobial Agents in vitro
Razi Vaccine and Serum Research Institute, Shiraz, Iran Poultry Research Centre, Veterinary School, Shiraz University, Shiraz, Iran Agriculture School, Shiraz University, Shiraz, Iran Mycoplasma Reference Lab. of Razi Vaccine and Serum Research Institute, Karaj, Iran Abstract: The in vitro activities of aivlosin tylosin tiamulin, erythromycin, oxytetracycline, spectinomycin,
lincomycin, lincomycin-spectinomycin (1:2) and enrofloxacin were determined against twenty-eight isolates
of Mycoplasma gallisepticum and 4 isolates of Mycoplasma synoviae using microbroth dilution method. The
isolates showed various susceptibilities to antimicrobial agents. Aivlosin, lincomycin-spectinomycin (1:2),
tylosin, tiamulin, enrofloxacin and also lincomycin were commonly more effective against these field isolates.
However, aivlosin was the most effective drugs against Mycoplasma gallisepticum and Mycoplasma
synoviae as its MIC was the lowest of all. Oxytetracycline, erythromycin and spectinomycin were not effective
against all isolates, as their MICs for some of isolates were extremely high. The MIC of erythromycin and
oxytetracycline were distributed across a broad range. Resistant strains to two mentioned antibiotics were
obtained from the field.
Key words: Mycoplasma sp., antimicrobial agents, poultry
INTRODUCTION
vertical transmissions. Here the most of veterinarians Mycoplasma gallisepticum and Mycoplasma synoviae prescript the consumption of antibiotics on the basis of has long been recognized as common respiratory clinical findings and their experiences in order to pathogens especially in chickens causing lots of treatment affected flocks and improve egg production commercial losses in poultry industries. Mycoplasma rate. It is logical that for a successful and aimed gallisepticum infection commonly induces chronic mycoplasma infection treatment, it is necessary to have respiratory disease in chickens (Ley, 2003).
regular antibiogram tests of Mycoplasma gallisepticum The clinical signs include nasal discharge, sneezing, and or Mycoplasma synoviae in the field for monitoring coughing, tracheal rales and mild conjunctivitis.
susceptibility of Mycoplasma prevalent in the farms.
Mycoplasma synoviae most frequently occurs as asubclinical upper respiratory infection but may result in Antibiotics: Basically Mycoplasma gallisepticum and
airsacculitis and synovitis in chickens and turkeys Mycoplasma synoviae have shown sensitivity in vitro and in vivo to several antimicrobials including macrolides Establishing of the Mycoplasma gallisepticum and (Jordan and Horrocks, 1996) tetracyclines and Mycoplasma synoviae-clean status of breeder flocks and quinolones (Bébéar et al., 1999; Wu et al., 2000), but maintaining that status of can be accomplished by they are resistant to penicillins or other antibiotic participation in control programs (Kleven, 2003; Ley, 2003). Like other countries in Iran chicken primary and On the other hand many antimicrobial agents, such as multiplier breeders and hatcheries generally have oxytetracycline, amino glycosides lincosamides, adopted various mycoplasma control programs. In spite fluoroquinolones and tiamulin have been shown to of these preventive programs, a lot of broiler and layer possess different degrees of in vitro activity against flocks will be affected by those organisms during their various veterinary mycoplasmas (Bradbury et al., 1994; production periods. However , line and parent flocks are Hannan et al., 1997a). However, increasing resistance mycoplasma free, but a large number of breeder flocks of mycoplasmas against tetracyclines (Hannan et al.
also involve Mycoplasma gallisepticum and or 1997a) macrolides (Bradbury et al., 1994; Hannan et al., Mycoplasma synoviae infections. However, it is not 1997a; Gautier-Bouchardon et al., 2002) and quinolones widely allowed the use of lived mycoplasma vaccines in (Bébéar et al., 1999; Wu et al., 2000) has been reported the country, control of mycoplasma infections by vaccination is limited. Control of these infections by chemotherapy is MATERIALS AND METHODS
sometimes necessary in complement of biosecurity On this way we carried out a 2 stage research, at first measures to minimize economic losses and lateral and stage isolation and molecular identification of Int, J. Poult. Sci., 7 (11): 1058-1064, 2008 Mycoplasma gallisepticum and Mycoplasma synoviae broader spectrum of activity than the macrolides, being were done. Isolation was carried out by enriched active also against a lot of gram-negative bacteria, which mycoplasma broth and agar. One hundred mycoplasma very often complicate Mycoplasma gallisepticum and isolates were identified genetically by PCR and RFLP Mycoplasma synoviea infections (Ghaleh et al., 2005).
(Garcia et al., 1995; Ghaleh et al., 2005). All of isolates At first 0.2 mL was chosen as final volume for each well gathered in Fars province of Iran and related tests containing 0.05 mL antibiotic solution, 0.05 mL fresh carried out in Shiraz Veterinary Medicin School, Shiraz, broth media solution (and then serially diluted) finally 0.1 mL fresh broth culture of each isolates inoculated toeach well (which they made 0.2 mL of test volume).
Test organisms: In the second stage the antibiogram
However fresh broth culture of each isolates used as test were carried out on 28 field isolates of Mycoplasma primary inoculums, the number of viable mycoplasmas gallisepticum and 4 field isolates Mycoplasma synoviae inoculated in each test of sensitivity to the antibiotics by common antibiotics. The antibiogram test was was determined by micro-broth dilution using designed on the basis of micro dilution method in broth mycoplasma enrichment broth with phenol-red indicator.
medium (Whithear et al., 1983; Hannan et al., 1997b).
Although, considerable data has been obtained on the was needed for the test proper which is added to each in vitro antibiotic susceptibility of well without any serial dilution. The change of color in gallisepticum and Mycoplasma synoviae and other avian the broth medium from red to yellow following mycoplasmas abroad but no information is available inoculation of isolates and incubation at 37°C for 14 about the susceptibility of these organisms in Iran. All days showed growth of the isolates. A micro-broth isolates were between the 20th and 30th medium dilution procedure which is described by Whithear et al. (1983) used in this research except we count thecolonies of 24 fresh broth cultures of isolates before Antibiotics: Antibiotics used to determine the sensitivity
starting the sensitivity tests. The test was carried out in of Mycoplasma gallisepticum and Mycoplasma synoviae 96-well, U bottom micro titration disposable plates with isolates, were Aivlosin tartrate (E.Co.A.H.) Tylosin tartrate cover under sterile conditions using multichannel (Sigma), Tiamulin base (ACS Chemicals), Erythromycin micropipette (Ependorffe Co., Germany). At least six and base (Merck), Oxytetracycline dihydrate (Sigma), sometimes 10-14 replicate doubling dilutions were Spectinomycin dihydrochloride (Sigma), Lincomycin made in experiments to evaluate test variables.
hydrochloride (Sigma), Lincomycin-Spectinomycin (1:2), According to Table 1, 150 µL of mycoplasma enrichment Enrofloxacin base (ACS Chemicals). Erythromycin base broth (pH 7.4) were added to each well containing final were dissolved in 7% ethanol. Each antibiotic solution dilution of antibiotic, containing desired density of was passed through a 450 nm pore size membrane isolates was inoculated in to each well. Positive and filter (Millipore, USA) and then dispensed aseptically.
negative culture controls containing mycoplasma FOR every antibiogram test the solutions prepared enrichment broth plus different isolates without under sterile conditions and in WFI water as stock antibiotics and mycoplasma broth plus different isolates solutions. The stock solutions were diluted in and antibiotics and zero culture controls included only mycoplasma broth to dilution two times that required mycoplasma enrichment broth (without any isolates and and were dispensed in 1 mL aliquots and then frozen at antibiotics), were included in all test plates. Plates were covered with their covers to prevent the evaporation ofthe broth medium during incubation period at 37°C. As Media: The media used were the mycoplasma broth and
it mentioned above the standardized test microbroth or mycoplasma agar. Mycoplasma broth medium was dilution procedure which is described by Whithear et al. formulated and described by Frey et al. (1968). Added (1983) was applied for Mycoplasma gallisepticum and glucose (dextrose) to broth medium mycoplasma Mycoplasma synoviae isolates. The standardized test fermentative growth was enhanced and provided an was used to determine the sensitivity of 28 strains of indicator of growth when glucose fermentation produced Mycoplasma gallisepticum, four strains of Mycoplasma acids in the media containing phenol red. The color of synoviae to the antibiotics. Mycoplasma broth prepared medium gradually changed from red to orange or yellow by the described formula. Erythromycin base were if the growing fermentative species of mycoplasmas dissolved in 7% ethanol. Each antibiotic solution was passed through a 450 nm membrane filter (Millipore, Sometimes the effectiveness of various antibiotics USA) and then dispensed aseptically. For every towards Mycoplasma gallisepticum and Mycoplasma antibiogram test antibiotic solutions were prepared synoviea in the field is not very different but some freshly and immediately used. Solutions were diluted in antibiotics show better advantages. Enrofloxacin still mycoplasma broth in two fold dilution started in 100 retains the acceptable advantage because it has µg/mL for first time but for second and third time the Int, J. Poult. Sci., 7 (11): 1058-1064, 2008 Table 1: In vitro activities of, oxytetracycline, aivlosin, tylosin, tiamulin, erythromycin, spectinomycin, lincomycin, L/S and enrofloxacin against *Isolate numbers 23, 24, 31 and 32 are Mycoplasma synoviae (MS) see Table 2 narrower rage of titration was required started in 25, 6.25 and 0.195 µg/mL. The ranges of concentrations of active The in vitro activities of lincomycin-spectinomycin compounds used in the final tests to survey the combination (1:2), erythromycin, oxytetracycline, sensitivity of avian mycoplasmas to the antibiotics were lincomycin, spectinomycin, enrofloxacin, tylosin, aivlosin as follows: Tiamulin 25 µg/mL to 1.56 µg/mL, and tiamulin against the 28 isolates of Mycoplasma spectinomycin, oxytetracycline, erythromycin, lincomycin, gallisepticum and 4 isolates of Mycoplasma synoviae, tylosin and enrofloxacin from 6.25 µg/mL to 0.195 µg/mL as determined by the micro-broth technique, are shown to 0.012 µg/mL (Hannan et al., 1997a,b). All antibiotic in Table 1. Of the eight antimicrobials, oxytetracycline solution pH were adjusted to the pH of the mycoplsma had the highest and aivlosin had the lowest MIC values.
The results for Mycoplasma synoviae are given in Table2 and again demonstrate the greater sensitivity of the Antibiogram tests were carried out in 96 well sterilized isolates to aivlosin and lincomycin-spectinomycin than micro plate U bottoms. When growth occurred in the to oxytetracycline. Of the other antimicrobials well, a definite change was observed of the phenol red enrofloxacin, tylosin erythromycin, tiamulin, spectino- to yellow (usually after 140 h of incubation) and the mycin and lincomycin showed the lowest MICs with minimum inhibitory concentration (MIC) was determined.
The MIC was the lowest concentration of antibiotic Against 4 isolates of MS by the micro-broth method inhibiting growth (no color change of phenol red).
aivlosin showed the best in vitro activity giving MICs of When the MIC was determined, approximately 0.01 mL 0.042 µg/mL for most isolates. Also, lincomycin- of broth from each tube was placed on an agar plate.
spectinomycin (1:2) combination had MICs of The plates were incubated at 37ºC in a moist container 0.0485/0.097 µg/mL for most isolates. Tiamulin and and examined microscopically after 5 days for erythromycin had similar MICs of 0.39-0.78 µg/mL for the mycoplasma colonies (Gautier-Bouchardon et al., 2002, isolates. Spectinomycin and lincomycin had MICs Bradbury et al., 1994). All MIC values were expressed in ranging from 0.78-1.56 µg/mL and oxytetracycline had µg/mL of each active compound. Also all sensitivity tests repeated second times to determine the accuracy of The combination of lincomycin-spectinomycin (1:2) appeared to have a synergistic effect for all isolates.
Int, J. Poult. Sci., 7 (11): 1058-1064, 2008 In vitro activities of oxytetracycline, aivlosin, tylosin, tiamulin, erythromycin, spectinomycin, lincomycin, lincomycin-spectinomycin andenrofloxacin against Mycoplasma synoviae isolates According to the results all MS strains are resistant to Review of the published literatures describing sensitivity oxytetracycline and sensitive to aivlosin, tylosin, tiamulin, testing of mycoplasmas reveals that there is lincomycin, lincomycin - spectinomycin combination considerable various workers on this title. There (1:2) and enrofloxacin. These strains were sensitive to spectinomycin except isolate No.32 and semi-sensitive mycoplasmologists about standard procedures for or resistant to erythromycin except isolate No. 23.
testing of mycoplasmas for sensitivity to antibiotics, forexample, Lin had been used agar dilution method to DISCUSSION
determine susceptibility of avian mycoplasmas to The purpose of the study was to determine the in vitro antibiotics (Lin, 1987) Boughton also demonstrated susceptibilities of Mycoplasma gellisepticum and tylosin susceptibility in 10 isolates of Mycoplasma Mycoplasma synoviae, which isolated from broiler gallisepticum tested by disc diffusion techniques chicken farms, Fars, Iran. in vitro susceptibility testing of mycoplasmas presents several problems that make In this research the MICs were determined by a glucose standardization of methods difficult. Because no single metabolic inhibition method. Therefore, a micro-broth medium is suitable for in vitro testing of all species, we dilution procedure was chosen as the basic test design, had to use a different medium for each of the two because it was relatively simple and quick to perform species. It was recommended to add NAD to mediums and required only small volumes of media and other for cultivating Mycoplasma synoviea. A pH indicator such necessary materials such as different antibiotics. The as phenol red visualizes the pH shift due to biochemical test was also easy to read, since inhibition of activities of multiplying mycoplasmas. One of the major mycoplasma growth by antibiotics could be deduced by ways for Mycoplasma gallisepticum and Mycoplasma the inability of the organism to ferment glucose and thus synoviae control is antibiotic therapy of affected flocks to produce a color change in a phenol red indicator in and infected hatching eggs (Alls et al., 1963). In Iran the medium from red to orange-yellow and generally antibiotic administration has been accepted and more acceptable between mycoplasma scientists. As different antibiotics are available and administered mentioned before, replicate doubling dilutions of against mycoplasma infection for several decades. This antibiotics were made in 150 microliter volumes of is important to know that administration of antibiotics mycoplasma broth in sterile 96-well U-bottomed micro despite of dosage and duration of administration could titration plates (Hirose et al., 2003; Gautier-Bouchardon not eradicate these organisms from the infected flocks.
et al., 2002; Hannan et al., 1997a; Whithear et al., 1983) But antibiotics are able to reduce the severity of clinical using 8-channel micropipette (Ependorff, Germany). The signs and lesions, decrease egg production losses and test was read when the phenol red indicator in the other economic losses such as downgrading medium of a culture control (without antibiotic) had carcasses. Proper antibiotic therapy could significantly changed from deep red (pH 7.8) to orange-yellow (pH 7).
reduce population of Mycoplasma gallisepticum and It is believed that the number of viable mycoplasmas Mycoplasma synoviea in the poultry respiratory tract.
inoculated in each test of sensitivity to antibiotic was very Mycoplasma gallisepticum and Mycoplasma synoviea important to reliability of the test. In this way to determine have shown sensitivity in vitro to several antimicrobials the concentration of each isolates in the mycoplasma broth and for making more accuracy the colony counting fluoro-quinolones and others, but also are resistant to procedure was done (Hirose et al., 2003; Zanella et al., other antibiotics such as cephalosporins and penicillins 1998). Some of other scientists preferred to determine which act by inhibiting cell wall biosynthesis.
the number of viable organisms by color changing unit Mycoplasma gallisepticum may develop resistance and (CCU) in the original culture by consulting the tables even demonstrate cross-resistance, to commonly used which were published by Meynell and Meynell (1970), antibiotics (Bradbury et al., 1994; Zanella et al., 1998).
(Hannan et al., 1997a,b; Whithear et al., 1983). There appears to be no general agreement among There is a wide range of bacteria and mycoplasma mycoplasmologist about standard procedures for susceptibility to antibiotics in different area and testing of mycoplasmas for sensitivity to antibiotics.
countries. On the other hand another problem with Int, J. Poult. Sci., 7 (11): 1058-1064, 2008 antibiogram test of mycoplasma is the different growth less sensitive to tylosin. However, in our research the Mycoplasma gallisepticum and Mycoplasma synoviea According to the results of our research, 28 isolates of isolates were more sensitive to tylosin than Mycoplasma gallisepticum and four isolates of Mycoplasma synoviea showed various susceptibilities Aivlosin, lincomycin-spectinomycin (1:2), tylosin, to the nine different antimicrobial agents which used in tiamulin, enrofloxacin and also lincomycin were commonly very effective in the field isolates. These The breakpoints of MICs of each antimicrobial agent findings are similar to the reports of Hannan (2000) and group: oxytetracycline, macrolides (aivlosin, tylosin and Zanella (1998). Although, macrolides and oxytetracycline erythromycin), aminoglycoside (including spectino- were considered to be effective against mycoplasmas, mycin), lincosamides (lincomycin), fluoroquinolone these drugs (erythromycin and oxytetracycline) did not (enrofloxacin) and tiamulin were 1.0, 0.5-2.0, 2.0-4.0 and prove to be effective against Mycoplasma synoviea 1-2 µg/mL, 0.5-2.0 and 0.1-0.05 µg/mL, respectively isolates. These MICs are higher than breakpoints of (Hirose et al., 2003). When the MIC of the oxytetracycline macrolides (0.5-0.2 µg/mL) or oxytetracycline (1 µg/mL).
was =1.0 µg/mL the isolate was considered susceptible, According to the commercial costs of used antibiotics when the MIC was >1.0 µg/mL, the isolate was against Mycoplasma gallisepticum and Mycoplasma synoviea tetracyclines such as oxytetracyline is not In contrast to Mycoplasma gallisepticum isolates, all of expensive and have been previously used in large Mycoplasma synoviea isolates have been shown scales to control and treat suspected broiler flocks to resistance to oxytetracycline and almost erythromycin.
mycoplasma and E. coli infections and now observed This finding is in complete agreement with research that mycoplasma showed highly resistance to these results of Bradbury et al. (1994) and Whithear et al. kinds of antibiotics. On the other hand, aivlosin are more (1983). Also, they used micro-broth dilution procedure expensive then its use has limited. Also, aivlosin hasbeen more recently introduced to producer. Also, our for MIC detection in their research. The results also in field experiences support these findings.
agreement with reports of Kleven and Anderson (1971) However, aivlosin was one of the most effective drugs and also Bradbury (1994) in the case of MICs of against Mycoplasma gallisepticum and Mycoplasma lincomycin-spectinomycin in comparison with synoviea as its MIC was the lowest of all oxytetracycline, oxytetracycline. Results of the present study agreed with erythromycin and spectinomycin were not effective those of Tanner and Wu (1992) who used broth micro- against all isolates, as its MIC for some of isolates was dilution test and showed Mycoplasma gallisepticum extremely high. The MIC of erythromycin and isolates were susceptible to lincomycin-spectinomycin oxytetracycline were distributed across a broad range.
and in the case of erythromycin and tylosin who found Resistant isolates to two mentioned antibiotics were the susceptibility varied among their isolates that is similar to our findings. But they reported their isolates Results showed that the sensitivity of isolates to were sensitive to oxytetracycline. On the other hand spectinomycin and licomycin lonely was less than their Bradbury (1994) reported that tylosin had the highest sensitivity to lincomycin-spectinomycin in 1:2 activity followed closely by lincomycin, oxytetracycline, spectinomycin. Erythromycin was less effective, but like Sometimes the efficacy of various antibiotics towards our findings lincomycin-spectinomycin had good activity Mycoplasma gallisepticum and Mycoplasma synoviea in against Mycoplasma gallisepticum. the field is equal but some antibiotics show better According to our research aivlosin had greater activity advantages. Enrofloxacin still retains the acceptable during 9 antibiotics used with a range 0.012-0.042 advantage because it has broader spectrum of activity µg/mL. The reason may be aivlosin is a newer antibiotic than the macrolides, being active also against a lot of which is recently used in Iran against Mycoplasma gram-negative bacteria, which very often complicate gallisepticum and Mycoplasma synoviea. However, Mycoplasma gallisepticum and Mycoplasma synoviea according to our knowledge the use of aivlosin in poultry is relatively limited in Europe and not at all in the USA.
In some cases when an appropriate antibiotic, on the Comparison of MIC values with tylosin and tilmicosin basis of antibiogram test, administrates the results may would be interesting. Zanella et al. (1998) investigating be out of our prediction and treatment results are not development antibiotics resistance in Mycoplasma satisfy. It seems that some of avian respiratory viruses gallisepticum, also reported that cross-sensitivity tests such as infectious bronchitis virus newcastle disease using strains with induced resistance to the different virus and pneumoviruses and also E. coli may interfere antibiotics demonstrated that those which were resistant with Mycoplasma gallisepticum and Mycoplasma to tylosin were also resistant to other macrolides, where synoviea antibacterial treatments (Kleven, 1998a, b).
as strains made resistant to erythromycin appeared only The treatment of these cases may be difficult because of Int, J. Poult. Sci., 7 (11): 1058-1064, 2008 some viral coinfections such as infectious bronchitis, Conclusion: Whichever method of control is chosen, the
avian influenza or infectious bursal disease and then need for effective antibiotics is apparent. According to infection may remain for a long period. This finding is in our findings drug resistance must be taken into agreement with other reports (Kleven and Anderson, consideration in mycoplasma and this fact stresses the 1971; Bradbury et al., 1994). Therefore, some of desirability for a choice between several antibiotics. The countries prefer to eradicate mycoplasma infections in vitro sensitivity of 28 different isolates of Mycoplasma from their flocks by test and slaughter strategy. gallisepticum and 4 isolate Mycoplasma synoviea havebeen tested using 9 different antibiotics includingaivlosin and lincomycin-spectinomycin (1:2). These 2 Specimens in Iran: Ever since the establishment of the
antibiotics showed the highest effect followed by tylosin, sensitivity to broad spectrum antibiotics (such as enrofloxacin, tiamulin and lincomycin.
tetracyclines, macrolides and etc.) of Mycoplasmagallisepticum and Mycoplasma synoviea these and ACKNOWLEDGEMENT
more recently even other antibiotics like tylosin and I wish to thank Dr. Rajaeeyan for his cooperation and erythromycin have been widely used in both preventive preparing the different antibiotics at Pharmacology Dept.
and curative treatment of mycoplasma infection in of Shiraz Veterinary School. This research was poultry. This is true even when programs for the creation financially supported by Shiraz University.
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