Prevalence of mycoplasmosis

Mycoplasmosis is quite common in birds kept in zoos. In the United States, prevalence rates of 38.7% and 3.2% have been reported for MS and MM, respectively in peafowl (Pavo cristatus) kept at three Michigan zoos; whereas, the peafowls in all of these zoos were seronegative for MG (Hollamby et al., Reference HOLLAMBY, SIKARSKIE and STUHT2003). Hollamby et al. (Reference HOLLAMBY, SIKARSKIE and STUHT2003) hypothesised that peafowls declared positive by serum plate agglutination (SPA) test were not subjected to culture isolation and, for this reason, the significance of the results was not certain. These agglutination methods usually have low specificity, if results are confirmed by haemagglutination tests and culture characteristics of the causative agent, the reactions are considered as false positives (Charlton, Reference CHARLTON2000).

Marco et al. (Reference MARCO, DELOGU, CATELLI, TERREGINO and GUBERTI2002) conducted a serological study for the detection of MS and MG antibodies in domesticated and wild pheasants. They reported that eight out of 16 inspected farms were seropositive to MS (ranging between 2.6% to 86.8%). While among free living pheasants, only one was positive for MG. Wieliczko et al. (Reference WIELICZKO, TOMANEK and KUCZKOWSKI2003) conducted tests on eleven flocks of ring-necked pheasants from 1997-2000 in Poland and reported 71.4% seroprevalence of MS. In contrast, Racnik et al. (Reference RACNIK, KRAPEZ, DOVC, VOLK, EMERSIC and ROJA2005) recorded 5% and 8% seroprevalence of MG and MS respectively, during health status studies on backyard flocks in Slovenia. Nadeem (Reference NADEEM2010) determined the seroprevalence of MG (29.73%) and MS (16.22%) by using SPA testing in captive peafowl (Pavo cristatus) maintained at five wildlife parks in the Punjab, Pakistan and reported a strong correlation between the prevalence of MG and MS with management practices. This was the first report from Pakistan regarding the seroprevalence of mycoplasmosis in peafowl.

Diagnostic procedures and tools

Diagnosis of mycoplasmosis is necessary for early treatment to improve the bird's health status and to control the further transmission of the disease. Several diagnostic methods/tools are available, including serum plate agglutination (SPA), rapid plate agglutination (RPA), rapid serum plate agglutination (RSPA), haemagglutination inhibition (HI), micro-agglutination (MA), enzyme linked immunosorbant assay (ELISA), polymerase chain reaction (PCR) and culture isolation assays (Avakian et al., Reference AVAKIAN, KLEVEN and GLISSON1988; Ortiz and Kleven, Reference ORTIZ and KLEVEN1992; Cookson and Shivaparasad, 1994; McMartin et al., Reference MCMARTIN, DAMASSA, MCKEEN, READ, DAFT and LAM1996; Bradbury et al., Reference BRADBURY, YAVARI and DARE2001a; Bencina et al., Reference BENCINA, MRZEL, ROJS, BIDOVEC and DOVC2003; Feberwee et al., Reference FEBERWEE, DE-VRIES and LANDMAN2008). The SPA test is the most frequently used diagnostic test for the screening of avian species because of its high sensitivity and easiness to perform (Ahmad et al., Reference AHMAD, KLEVEN, AVAKIAN and GLISSON1988). In the National Poultry Improvement Plan, SPA had been referred to as one of the most reliable tests due to its fairly high sensitivity, inexpensiveness and rapid detection of antibodies for MG, MM and MS (Anonymous, 1985; Ahmad et al., Reference AHMAD, KLEVEN, GLISSON and AVAKIAN1989).

The serological evidence of Mycoplasma spp. was first investigated in pheasants suffering from infectious sinusitis by Keymer (Reference KEYMER1958; Reference KEYMER1961). It was carried out using a pleuropneumonia-like organism (PPLO) H-I antigen prepared from D-stain of PPLO isolated from turkeys in the UK. Rapid whole blood plate agglutination tests were performed to detect antibodies against MG and prevalence of MG, and were found in 71.4% of Pavo cristatus maintained in Nandan Khan, State Biological Park, Orissa (Tripathy et al., Reference TRIPATHY, ACHARJYO, SINGH, RAY and MISRA1972). Similarly, MG infection was diagnosed in Chukar partridges, pheasants and peafowl by using SPA and HI tests (Cookson and Shivaparasad, 1994). The antibodies against MG, MS and MM were detected in two backyard operations of game birds, including pheasants and peafowl, in Central and South Slovenia (Bencina et al., Reference BENCINA, MRZEL, ROJS, BIDOVEC and DOVC2003). Welchman et al. (Reference WELCHMAN, BARDBURY, CAVANAGH and AEBISCHER2002) reported serological evidence of MG and MS in pheasants, associated with respiratory disease, by using the RSPA test. Hollamby et al. (Reference HOLLAMBY, SIKARSKIE and STUHT2003) carried out the SPA tests for rapid diagnosis of MS in captive peafowls which were maintained in three zoos in the United States. Failure to detect antibody responses using SPA against Mycoplasma spp. was seen in turkey flocks suffering from severe respiratory disease (Kleven, Reference KLEVEN1990). Consequently, the microagglutination test (MA) was introduced for detection in avian birds (Yamamoto and Ortiz, Reference YAMAMOTO1974; Yoder, Reference YODER1982).

Ortiz and Kleven (Reference ORTIZ and KLEVEN1992) conducted a study to evaluate MA and used a commercial ELISA test to detect antibodies against MS and compared the results of commonly used serological tests such as SPA and HI. They reported that MA had high sensitivity and specificity over ELISA and other serological tests, and could be used as a diagnostic tool in avian flocks. Feberwee et al. (Reference FEBERWEE, MEKKES, DE WIT, HARTMAN and PIJPERS2005) carried out a comparative study between several diagnostic methods including culture isolation, PCR, SPA, HI and ELISA for the detection of MG and MS and reported that high numbers of false positive results were obtained using the SPA and ELISA methods. The specificity of the SPA test proved to be low in avian species for the quick diagnosis and screening of MG infection because the incidence of false positive results occurred more frequently. The administration of oil emulsion vaccines, which are used on a large scale in avian species, proved to be a major factor for the false positive results in the case of the SPA test. Secondly, the vaccines prepared in a medium containing serum, resulted in a higher frequency of false positive SPA results than vaccines containing no serum components, which could minimise this issue (Cullen and Timms, Reference CULLEN and TIMMS1972; Kleven, Reference KLEVEN1975; Glisson et al., Reference GLISSON, DAWE and KLEVEN1984).

Serum proteins present in the medium that were used for the production of MG SPA antigen adsorbed to the surface of the multiplying bacteria, giving more false positive reactions. When serum was brought to the laboratory for MG detection, it contained immunoglobulins that might be involved in binding the serum proteins present on the surface of Mycoplasma spp., thus pertaining to more false positive results (Bradbury and Jordan, Reference BRADBURY and JORDAN1971; Reference BRADBURY and JORDAN1972; Reference BRADBURY and JORDAN1973). A significant reduction of false positive results was observed when MG SPA antigen was prepared in the medium that was free from globulins. However, some non-specific agglutination reaction may occur with the use of such antigens (Windsor and Thornton, Reference WINDSOR and THORNTON1973).

Ahmad et al. (Reference AHMAD, KLEVEN, AVAKIAN and GLISSON1988) suggested that preparation of MG SPA antigen on Frey's modified medium with artificial liposomes as an exogenous source of cholesterol, and by substituting the phospholipids from the serum, could significantly decrease the number of false positive reactions. MS was not isolated or detected by PCR in either tracheal or conjunctival swabs from the birds, but the RSPA test for MS was declared positive (Welchman et al., Reference WELCHMAN, BARDBURY, CAVANAGH and AEBISCHER2002). This was due to their cross-reaction, genetic and antigenic relatedness between MG and MS as reported earlier by Yogev et al. (Reference YOGEV, LEVISOHN and RAZIN1989).

MG and MS synthesise haemagglutinins that are immunogenic and variably expressed surface proteins. The haemagglutinins of MG (pMGA) and MS (VlhA) are lipoproteins encoded by related multigene families and appear to have arisen by horizontal gene transfer. Therefore, the variant haemagglutinins of MG (pMGA1.7) and MS (diverse VIhA forms) share a common sequence that could be responsible for antigenic cross-reactions between these two avian Mycoplasma spp. (Bencina, Reference BENCINA2002). Isolation of MG and MS in pheasants and peafowls on culture media is necessary to overcome these problems. MG and MS have been confirmed through culture isolation in a number of avian species in the world, including sparrows (Passer montanus) in Japan, guinea fowl (Numida meleagris) in Italy, racing pigeons (Columba livia) in Australia, red legged partridges (Alectoris rufa) in Spain, Japanese quail (Coturnix coturnix japonica), geese (Anser albiforns), turkeys (Meleagris gallopavo), ducks (A. platyrhynchos domesticus), chickens (Gallus domesticus) in Slovenia, house finches (Carpodacus mexicanus) in North America, pheasants (Phasianus colchicus) in the UK and peafowl (Pavo cristatus) in Slovenia (Pascucci et al., Reference PASCUCCI, MAESTRINI, GOVONI and PRATI1976; Shimizu et al., Reference SHIMIZU, NUMANO and UCHIDA1979; Poveda et al., Reference POVEDA, FERNANDEZ, CARRANZA, HERMOSO and PEREDA1986; Recce et al., 1986a; Bencina et al., Reference BENCINA, DORRER and TADINA1987b, Reference BENCINA, TADINA and DORRER1988b; Ley et al., Reference LEY, BERKHOFF and MCLAREN1996; Bradbury et al., Reference BRADBURY, YAVARI and DARE2001a; Reference BRADBURY, YAVARI and DARE2001b; Bencina et al., Reference BENCINA, MRZEL, ROJS, BIDOVEC and DOVC2003).

During a survey reviewing the causes of mortality in British birds, infectious sinusitis was first described by Keymer (Reference KEYMER1958) in the family Phasianidae. Later on, pathogenic and non pathogenic Mycoplasma organisms were isolated from the tracheal and mucous obtained from both sinuses on special media (Keymer, Reference KEYMER1961). MG and MS were successfully isolated from chickens, turkeys, pigeons and Japanese quail, but not from geese and ducks by Bencina et al. (Reference BENCINA, MRZEL, TADINA and DORRER1987a; Reference BENCINA, DORRER and TADINA1987b; Reference BENCINA, TADINA and DORRER1988a). Racnik et al. (Reference RACNIK, KRAPEZ, DOVC, VOLK, EMERSIC and ROJA2005) reported similar observations when duck and geese were declared seronegative for MG and MS out of twelve avian species from a backyard flock in Slovenia.

Tripathy et al. (Reference TRIPATHY, ACHARJYO, SINGH, RAY and MISRA1972) isolated MG from the tracheal swabs of peafowl showing swelling of the infraorbital sinuses. After collection, the swabs were immediately dipped in PPLO broth and, through serial passage techniques, typical Mycoplasma colonies were obtained on PPLO agar plates. The growth characteristics, teterazolium blue reduction test and clinical symptoms manifested by the affected peafowl indicated that the isolated MG was pathogenic. Cookson and Shivaparasad (1994) isolated MG through serology and culturing method from tracheal, sinual and choanal samples of chukar partridges, pheasants and peafowl on Frey's broth and agar media by adding 12% equal volumes of equine and swine sera. Mostly non-pathogenic Mycoplasma spp. were isolated during this study. It was difficult to isolate MG from cultures taken from birds suffering from sinusitis, because of the overgrowth of other rapidly growing avian Mycoplasama spp. (Cookson and Shivaparasad, 1994; Kempf, Reference KEMPF1998; Bradbury et al., Reference BRADBURY, YAVARI and DARE2001b). This was confirmed from birds that were declared positive for MG by PCR testing, but could not be isolated on culture media (Bradbury et al., Reference BRADBURY, YAVARI and DARE1996). Bradbury et al. (Reference BRADBURY, YAVARI and DARE2001b) demonstrated that culturing other species including M. glycophilum, M. gallinaceum, M. pullorum, M. iners, M. imitans and M. lipofaciens from pheasants suffering from respiratory diseases raised queries regarding the role of MG and MS as disease pathogens in the family Phesanidae.

Welchman et al. (Reference WELCHMAN, BARDBURY, CAVANAGH and AEBISCHER2002) was not able to detect MS from the tracheal samples of pheasants with respiratory disease by using RSAT. However, tracheal samples showed positive results for MS when subjected to culturing and PCR tests. Bradbury et al. (Reference BRADBURY, YAVARI and DARE2001a) showed that MS could not be isolated on culture media from pheasants suffering from respiratory diseases, but were deemed positive by PCR. Pourbakhsh et al. (Reference POURBAKHSH, SHOKRI, BANANI, ELHAMNIA and ASHTARI2010) suggested that these false negative results from culturing might be due to the deficiency of several substances (NAD, serum, CO₂ and certain other materials) in the medium and during transportation, over-chilling of the specimen and destruction of viable MS at room temperature.

Bencina et al. (Reference BENCINA, MRZEL, ROJS, BIDOVEC and DOVC2003) characterised the MG strains obtained from backyard game birds, including pheasants and peafowl, during two outbreaks in 1997 and 1999 in Central and South Slovenia. They isolated one MG strain (ULB 992) from the infra-orbital sinuses of peafowl and two MG strains (ULB 971 (trachea) and ULB 972) in vitro from pheasants. One strain of MG was isolated from pheasants which had a unique PGMA gene sequence containing a repetition of 12 nucleotides, and which was different from other MG strains isolated in Slovenia. In addition, this strain contained a small amount of cytadhesins (MGC1 and MGC3) and no PvpA protein (Milosevic-Berlic et al., Reference MILOSEVIC-BERLIC, BENCINA and DOVC2000). Bencina et al. (Reference BENCINA, MRZEL, ROJS, BIDOVEC and DOVC2003) documented that the pathogenicity of strain ULB 972, which was isolated from the sinus of a pheasant, was very high for chicken embryos, because the entire embryo died within nine days. The second strain ULB 971 was associated with respiratory disease and caused death in peafowl, but affected the chicken flocks more frequently. The strain ULB 992 lacked PvpA protein, but synthesised small quantity of MGC3, a truncated form of MGC1. However, it expressed many proteins which were immunogenic for both game birds infected with MG.

Nadeem (Reference NADEEM2010) successfully isolated MG and MS from captive peafowl on Frey's modified broth and agar media. The only modification made to both of the media was the replacement of swine serum with horse serum. MG was isolated by removing nicotinamide adenine dinucleotide (NAD) and cysteine hydrochloride from modified Frey's broth during the serial passage technique which eliminated the growth of MS (Boettger and Dohmsa, Reference BOETTGER and DOHMSA2006). Kleven (Reference KLEVEN1998) reported that NAD and reduced cysteine hydrochloride were required for the growth of MS. Ewing et al. (Reference EWING, COOKSON, PHILLIPS, TURNER and KLEVEN1998) suggested that culture isolation of avian Mycoplasma spp. could be a costly and time consuming method with inconclusive results. It required approximately 3-4 weeks for the growth of avian Mycoplasma spp. on culture media and, even then, results could be negative or hampered by mixed infection or growth of other Mycoplasma species (Bradbury and McLenaghan, Reference BRADBURY and MCLENAGHAN1982; Zain and Bradbury, Reference ZAIN and BRADBURY1995; Bradbury et al., Reference BRADBURY, YAVARI and DARE2001b).

Bayatzadeh et al. (Reference BAYATZADEH, POURBAKHSH, HOMAYOUNIMEHR, ASHTARI and ABTIN2011) reported that PCR took only one day for the detection of Mycoplasmas and was not dependent upon the viability of the organisms. It was suggested that molecular techniques like PCR are one of the most sensitive and specific methods for the detection of avian Mycoplasma spp. (Kempf et al., Reference KEMPF, BLANCHARD, GESBERT, GUITTET and BENNEJEAN1993; Lauerman et al., Reference LAUERMAN, HOERR, SHARPTON, SHAH and SANTEN1993). Bradbury et al. (Reference BRADBURY, YAVARI and DARE2001a; Reference BRADBURY, YAVARI and DARE2001b). In addition, Bencina et al. (Reference BENCINA, MRZEL, ROJS, BIDOVEC and DOVC2003) used PCR for the detection of MG and MS in pheasants, partridges and peafowl successfully. Other researchers have reported that sometimes MS was not detected by PCR in infected birds because of one or more mutations in the genomes of the highly passaged strain at the target sequence of the PCR primer (Feberwee et al., Reference FEBERWEE, MEKKES, DE WIT, HARTMAN and PIJPERS2005). These results were consistent with the findings of Bayatzadeh et al. (Reference BAYATZADEH, POURBAKHSH, HOMAYOUNIMEHR, ASHTARI and ABTIN2011), where samples were PCR negative but yielded positive results on Frey's modified culture media. Amores et al. (Reference AMORES, CORRALES, GOMEZ MARTI, SANCHEZ, CONTRERAS and DE LA FE2010) reported that the gene sequence variations and presence of Taq DNA polymerase inhibitor led to false negative PCR results.

For identification of MG and MS in pheasants and peafowl, many biochemical methods have been used, such as the indirect immunofluorescent antibodies technique, direct immunoperoxidase technique, indirect fluorescent antibody test, glucose fermentation, arginine hydrolysis, film and the spot assay (Shimizu et al., Reference SHIMIZU, ERNO and NAGATOMO1978; Cookson and Shivaparasad, 1994; Bradbury et al., Reference BRADBURY, YAVARI and DARE2001a; Reference BRADBURY, YAVARI and DARE2001b; Welchman et al., Reference WELCHMAN, BARDBURY, CAVANAGH and AEBISCHER2002; Bencina et al., Reference BENCINA, MRZEL, ROJS, BIDOVEC and DOVC2003; Nadeem, Reference NADEEM2010).

Bayatzadeh et al. (Reference BAYATZADEH, POURBAKHSH, HOMAYOUNIMEHR, ASHTARI and ABTIN2011) suggested PCR as a molecular detection method that successfully eliminated or replaced the need for biochemical tests, which were required for the identification of avian Mycoplasma spp.. PCR could provide an alternative method for accurate identification of MS infection in breeder flocks (Pourbakhsh et al., Reference POURBAKHSH, SHOKRI, BANANI, ELHAMNIA and ASHTARI2010). Hong et al. (Reference HONG, GARCÍA, LEITING, BENČINA, DUFOUR-ZAVALA, ZAVALA and KLEVEN2004) and Harada et al. (Reference HARADA, KIJIMA-TANAKA, UCHIYAMA, YAMAMOTO, OISHI, ARAO and TAKAHASHI2009) reported that the PCR assay proved to be useful in the detection of the variable haemagglutinin gene (vlhA gene) and for typing the different strains of MG and MS. Various researchers have reported that PCR is a rapid, effective, sensitive, specific, inexpensive, less laborious and useful method than the traditional standard cultural techniques for the detection of MG and MS in pheasants, peafowl and other avian species (Lauerman et al., Reference LAUERMAN, HOERR, SHARPTON, SHAH and SANTEN1993; Salisch et al., Reference SALISCH, HINZ and GRAACK1998; Bradbury et al., Reference BRADBURY, YAVARI and DARE2001a; Reference BRADBURY, YAVARI and DARE2001b; Bencina et al., Reference BENCINA, MRZEL, ROJS, BIDOVEC and DOVC2003; Feberwee et al., Reference FEBERWEE, MEKKES, DE WIT, HARTMAN and PIJPERS2005; Pourbakhsh et al., Reference POURBAKHSH, SHOKRI, BANANI, ELHAMNIA and ASHTARI2010; Bayatzadeh et al., Reference BAYATZADEH, POURBAKHSH, HOMAYOUNIMEHR, ASHTARI and ABTIN2011).

Treatment of mycoplasmosis

In the treatment of avian mycoplasmosis, antibiotics are frequently used in naturally infected birds (Hamdy et al., Reference HAMDY, SAIF and KASSON1982; Glisson et al., Reference GLISSON, CHENG, BROWN and STEWARD1989; Charleston et al., Reference CHARLESTON, GATE, AITKEN and REEVE-JOHNSON1998; Hannan, Reference HANNAN2000) but the development of resistance against various antibiotics has been reported by various scientists across the globe (Bradbury et al., Reference BRADBURY, YAVARI and GILES1994; Gautier-Bouchardon et al., Reference GAUTIER-BOUCHARDON, REINHART, KOBISCH and KEMPF2002; Pakpinyo and Sasipreeyajan, Reference PAKPINYO and SASIPREEYAJAN2007).

Tripathy et al. (Reference TRIPATHY, ACHARJYO, SINGH, RAY and MISRA1972) treated the infraorbital swelling of peafowl with vitamin A and terramycin (intramuscular; I/M) and terramycin (0.5ml) infused into the swelling after removal of exudate. Clinical signs disappeared within ten days and birds resumed their normal feeding. The signs did not reappear until after the six months of therapy. Reece et al. (Reference REECE, IRELAND and BARR1986b) reported that treatment with sulphonamide did not reduce the number of new cases within the flock, and so tylosin was recommended in infectious sinusitis associated with MG in peafowl, turkeys and other game birds. Reece et al. (Reference REECE, IRELAND and SCOTT1986a) successfully treated mycoplasmosis in racing pigeons with tylosin followed by oxytetracycline or chlortetracycline at a dose rate of 0.2-0.5 g per litre of drinking water for five days. Bird health improved gradually. Wissman and Parsons (Reference WISSMAN and PARSONS1996) treated MS infection in the common rhea (Rhea americana) by injecting long acting doxycycline at a dose rate of 20 mg/kg of body weight (I/M) and tylosin at the dose rate of 250 g/8 ounce in drinking water, with the addition of vitamin A, D₃ and B-complex (thiamine) as supportive therapy. All of the birds rapidly responded to this therapy and complete resolution of ocular and nasal lesions was observed.

Fiorentin et al. (Reference FIORENTIN, SONCINI, DACOSTA, MORES, TREVISOL, TODA and VIEIRA2003) reported the eradication of mycoplasmosis by oral administration of oxytetracycline, enrofloxacin and norfloxacin in feed and drinking water of broiler breeding flocks. Roussan et al. (Reference ROUSSAN, EHAB, BASHA and HADDAD2006) demonstrated that administering tilmicosin (Provitil^® powder) at a dose rate of 30 mg/kg of body weight for three successive days and repeated every five weeks for four months in Galliformes successfully controlled the infection of MG in eight flocks. Charleston et al. (Reference CHARLESTON, GATE, AITKEN and REEVE-JOHNSON1998) reported the effectiveness of tilmicosin towards air sacculitis caused by MG in avian species at the dose rate of 50 mg/l in drinking water for three to five days. In a therapeutic trial, Nadeem (Reference NADEEM2010) reported a 75% recovery rate in captive peafowl by using Tylax (tylosin) oral powder (Routhas^®UK) at the dose rate of 200 mg, and a 100% recovery rate by the use of Oxytet (oxytetracycline; The Russell^® USA) at the dose rate of 1 gm in two litres of drinking water for five days every two weeks. Clinical signs disappeared after seven to nine days, and birds improved their feed consumption. Forrester et al. (Reference FORRESTER, BRADBURY, DARE, DOMANGUE, WINDSOR, TASKER and MOCKETT2011) treated infectious sinusitis associated with MG in pheasants with tylvalosin (TVN; aivlosin, a macrolide) at a dose rate of 25 mg/kg of body weight for three consecutive days. They reported that TVN had better efficacy over other macrolides (tylosin and tilmicosin) because of its high absorption from the alimentary tract from where it entered the lungs, especially the epithelial lining of the respiratory tract.

Association of mycoplasmosis with management practices

Most of the commonly occurring infections caused by MG and MS in captive peafowl and pheasants are associated with respiratory diseases and are characterised by foamy eyes, swollen infraorbital sinuses, respiratory distress and death, but in peafowl its mechanism of transmission is unknown (Cookson and Shivaprasad, Reference COOKSON and SHIVAPRASAD1994; Hollamby et al., Reference HOLLAMBY, SIKARSKIE and STUHT2003). Transmission may be associated with infected hosts at shared feed stations or shelter areas in the winter season (Hollamby et al., Reference HOLLAMBY, SIKARSKIE and STUHT2003).

Other avian species, including turkeys, chickens and bantams, may be the cause of transmission of MG in peafowl maintained in cages, and further transmission of the disease may occur by farm to farm movement of workers, visitors and other personnel on farm (Mason and Maiers, Reference MASON and MAIERS1984). Christensen et al. (Reference CHRISTENSEN, YAVARI, MCBAIN and BRADBURY1994) reported that MG remained alive in human hair for up to three days and MS up to eight hours, and on the nose and clothes MG and MS survived 12-24 hours and two to four days, respectively.

Avian mycoplasmosis may be transmitted vertically through the eggs, or horizontally by direct contact between sick or unaffected carriers and susceptible animals. Indirect transmission via people, wild birds, drinking water, litter or breeding material may play a major role in the initiation of MS outbreaks because of the possible persistence of Mycoplasma spp. in the environment (Marois et al., Reference MAROIS, OUFOUR-GEBERT and KEMPF2000). MG and MS infections were frequently found in game birds where multiple housing of different avian species was practiced (Reece et al., Reference REECE, IRELAND and BARR1986b; Cooksoon and Shivaparasad, 1994; Hollamby et al., Reference HOLLAMBY, SIKARSKIE and STUHT2003; Nadeem, Reference NADEEM2010). Bencina et al. (Reference BENCINA, MRZEL, ROJS, BIDOVEC and DOVC2003) reported that addition of new birds within the flocks, without serological screening, could be a possible cause of infection in pheasants and peafowl. Christensen et al. (Reference CHRISTENSEN, YAVARI, MCBAIN and BRADBURY1994) reported that MS and MG survived in the feathers of birds up to three or four days, respectively, which showed that newly introduced birds may act as carrier for MG and MS infection within flocks.