Incidence and risk factors of Mycoplasma synoviae infection in broiler breeder farms of Iran

Mycoplasma synoviae (MS) is an important pathogen of poultry worldwide, causing respiratory tract infection and infectious synovitis in chickens and turkeys. The aim of this study was to determine the incidence and risk factors (age, size of flock, locale, sex and strain) of Mycoplasma synoviae in broiler breeder farms in Iran. The study was based on Rapid Serum Plate Agglutination (SPA) and enzyme linked immunosorbent assay (ELISA) tests. The prevalence was highest (44%) in winter and lowest (34%) in summer. Ross, Cobb, Arian, Hubbard and Arbor Acres strains had 40%, 44%, 32%, 27% and 45% infection, respectively. The prevalence was recorded highest in above 60 weeks of age (47.8%), but at 10-20 weeks it was lowest (14.2%). No significant difference was seen in flocks up to 30,000 population (41.9%), 30,000-40,000 (52%) and upper 40,000 (57%). The prevalence of Mycoplasmosis in foothills was significantly (p<0.05) higher (41%) than coastal area (34.5%). The results showed that occurrence of MS have a significant relationship with the age and zone of sampling.


Introduction
Provisional diagnosis and monitoring of M. synoviae infection is usually made using serological Mycoplasmas are very small prokaryotes devoid assays, while definitive diagnosis is made by isolation of cell walls, bounded by a plasma membrane only and identification of the organism.The most used (Kleven, 2008).Mycoplasma synoviae (MS) is one of serological tests are serum plate agglutination (SPA), the most important pathogenic chickens Mycoplasmas.hemagglutination inhibition (HI), and enzymelinked MS infection occurs as a subclinical upper respiratory immunosorbent assay (ELISA) (Kleven, 1998).SPA infection.It may cause air sac lesions when combined reactors must generally be confirmed by the HI or with Newcastle disease (ND), infectious bronchitis ELISA tests.The HI test is less sensitive but more (IB), or both.Less frequently, MS becomes systemic specific than the SPA test.It is however, a longer and results in infectious synovitis, an acute to chronic procedure and the reagents are not commercially infectious disease of chickens and turkeys, involving available.In general the ELISA test is more sensitive primarily the synovial membranes of joints and tendon than the HI test and more specific than the SPA test sheaths, and bursitis (Kleven and Ferguson-Noel, (Kempf and Gesbert, 1998;Kempf et al., 1994Kempf et al., ). 2008).Lameness and respiratory disorder caused by Culturing of MS could be costly, time-consuming and this infection results in decreased growth rate and loss inconclusive (Ewing et al., 1998).In Iran, serum plate of egg production and causing economic loss in agglutination (SPA) is used for flocks monitoring as intensive production.As the vertical transmission screening test.SPA is used as the screening test plays a major role in spread of MS in chickens the most because it's rapid, has high sensitivity, and low specificity, as well as being inexpensive (Kleven, effective method of control is regular flocks monitoring and eliminating of positive breeder flocks.1998).ELISA has been proved to have good sensitivity and more specificity compared to SPA.Reliable and rapid diagnosis is needed to prevent Control of MS is very dependent on serologic dissemination of infection (Lockaby et al., 1998; monitoring.Some factors such as: age, size of flock, Kleven and Ferguson-Noel, 2008).locale, etc., may effect on severity of this disease.
was placed in each well (Goat antichicken: horse The aim of the present study was to determine radish peroxidase conjugate HRPO).Plates were the risk factors (age, size of flock, locale, sex and incubated for about 30 minutes and washed again.strain) and seroprevalence of Mycoplasma synoviae Finally, 0.1mL of the substrate solution (tetramethyl-(MS) in broiler breeders in Iran.benzidine or TMB) was dispensed into each well and incubated for 15 minutes at room temperature.The reaction was blocked with 0.1mL of stop solution.

Ethical approval:
Absorbance was measured at 650 nm.Results were Use of birds in this study was expressed as serum-to-positive ratios (S/P ratios) approved by the Institutional Animal Ethical Committee.
relative to a standard positive control.Serum samples, Samples: The study was conducted at the commercial with S/P ratios greater than 0.5 (titers greater than farms of Iran, from May 2002 to October 2008.350 1,076) were considered positive.broiler breeder farms were followed for this study and Statistical analysis: Results were analyzed using three samples for every thousand were obtained randomly.
SPSS 13 (chi square test and Pearson correlation).Blood collection and serum preparation: Blood samples were aseptically collected from the wing veins using 5-mL sterile disposable syringes and The choice of the SPA test to study the needles.Blood was allowed to clot in the syringe and seroprevalence of M. synoviae was based on previous was kept for about 1 hour at room temperature.After research in which the specificity and sensitivity of this this, serum of each sample was separated, centrifuged, laboratory test was compared with that of culture, and transferred to sterile microtubes kept at -20?C polymerase chain reaction (PCR) and various until used.commercial enzyme-linked immunosorbent assay kits.The SPA test was found to perform equally as well Serum Plate Agglutination (SPA) Test: All serum as the tested enzyme-linked immunosorbent assay kits samples were inactivated by heating at 56?C/30 (Feberwee et al., 2005).Sera samples were collected minutes to destroy nonspecific inhibitory substances during seven years.The highest (45.7%) and lowest and tested for MS by SPA, using a commercial antigen (0%) Prevalence of MS infection was found in 2003 for the diagnosis of Mycoplasma synoviae by serum and 2008 respectively (Table 1).plate agglutination (Synovitest-Laboratorio BioVet-Brazil), according to the manufacturer's instructions.In short, 0.02mL of the serum to be tested was mixed synoviae infection and various risk factors with 0.02mL of the commercial antigen (1: 1) in a glass plate.After that, the plate was placed under a light source, and samples that showed agglutination (presence of clots) were considered positive.Positive sera were diluted 1: 5 and 1: 10 with 0.5M phosphatebuffered saline (PBS), pH 7.2.Both dilutions were tested again by SPA as described above.Sera were considered positive when clots were observed in dilutions up to 1: 10.

Enzyme-Linked Immunosorbent Assay (ELISA):
Sera that were positive in SPA were analyzed for antibodies against MS using a commercially available ELISA antibody test kit (Mycoplasma synoviae antibody Test Kit-IDEEX Laboratories, Inc., Maine, USA) according to the manufacturer's instructions.Briefly, samples were diluted five-hundredfold (1: 500) with the diluent, and 0.1mL of each sample was dispensed in a well of a plate previously coated with MS antigen.Plates were incubated for about 30 minutes at room temperature.After that, plates were The prevalence was highest (44%) in winter and washed with deionized water, and 0.1mL of the conjugate lowest (36.1%) in summer.Ross, Cobb, Arian,

Risk factor
Level Hubbard and Arbor Acres had 40%, 44%, 32%, 27% flock do not have an impressive effect in appearance of Mycoplasma synoviae infection but this problem is and 45% respectively.The prevalence of MS was worse in the greater sizes of flocks.For this reason, recorded highest at above 60 wk (47.8%), whereas the prevalence was lowest (14.2%) at 10-20 weeks of age lower incidence observed in flocks has a size up to (p<0.05).Farms were separated in two different zones 30,000 birds in each farm.Serological investigation (foothills with less humidity compared with coastal showed the highest infection rate (57%) in large scale area).The prevalence of Mycoplamosis in Foothills flocks (>40,000 birds) in comparison (41.9%) to small was significantly (p<0.05)higher (41%) than coastal (up to 30,000 birds) flocks.Similar report was area (34.5%).No significant difference was seen in demonstrated by Dufour-Gesbert et al. (2006).flocks up to 30,000 population (41.9%), 30,000- In the present study seasonal variation for prevalence 40,000 (52%) and upper 40,000 (57%).
of MS infection was observed.The prevalence was highest (44%) in winter and lowest (34%) in summer which was in agreement with the result of Heleili et M. synoviae can be transmitted vertically and al.( 2011 Kempf, 1996).Results Hossain et al. (2010).It might be due to the influence showed that the prevalence of MS infection increased of cold weather.The statistical analysis by one way with the increase of age.Culling of M. synoviae-ANOVA method (F-test) showed no significant positive flocks reduces the risk of vertical variation between the prevalence of seasons.transmission of this mycoplasma to the offspring, Regional variation of prevalence of M. synoviae which is reflected in the low seroprevalence of M. was observed in the present study.The prevalence of synoviae in rearing breeder stock (14.2%).Similar Mycoplamosis in Foothills was significantly (p<0.05)report was demonstrated by Feberwee et al. (2008) higher (41%) than coastal area (34.5%).Density of flocks was higher in foothills than costal area, who reported lowest MS infection (6%) at rearing age.As the risk of vertical transmission is minimized by probably this factor influence in prevalence of MS in these regions.As in Hagan's study (2004), infection culling M. synoviae-infected flocks, horizontal was more frequent on sites with several houses.This transmission is expected to be the most important risk factor is also observed for other diseases or transmission route for infected rearing breeder flocks.(Goodwin, 1985).This study confirmed breeder flocks > 51 weeks of age (60% of positive the high prevalence of M. synoviae infection in broiler flocks).The prevalence study of Hagan et al. (2004), breeder farms in Iran.Furthermore, the results show which was based on the detection of M. synoviae that occurrence of M. synoviae have a significant antibodies in eggs, reported a prevalence of 78.6% in relationship with the age of flocks and zone of sampling.commercial layer flocks in East England.In another study (Mohammed et al., 1986), a M. synoviae prevalence of 87% was found in commercial layer The authors would like to appreciate the experts flocks in Southern California.The infection was and technicians of the Chief Veterinary Office of associated with older flocks that had been moulted or Mazandaran province for their help and cooperation.frequently medicated.The high prevalence and persistence of M. synoviae infections in layer stock can be explained by the frequent occurrence of Authors declare that they have no conflict of multiple age housing and lower biosecurity standards interest.in this sector (Stipkovits and Kempf, 1996;Kleven and Ferguson-Noel, 2008).
No relation between a special breed with M.