INVESTIGATION OF MYCOPLASMA SYNOVIAE SEROPREVALENCE IN BROILER BREEDER FARMS IN SOUTH BAČKA REGION

Mycoplasma synoviae is known to cause respiratory disorders, syno-vitis, subclinical infections, air sacculitis and eggshell apex abnormalities in domestic poultry worldwide. Th e aim of this study was to determine M. synoviae seroprevalence in 5 diff erent broiler breeder farms in South Bačka from 2014 to 2017. A total of 1511 samples were tested using commercial indirect enzyme linked immunosorbent assay (ELISA) for detection of antibodies in the blood sera. In this study, the seroprevalence of 25.21% was found and 47 (40.87%) fl ocks out of 115 tested were positive to M.synoviae . Seroprevalence varied between 31.02% in 2015 and 16.78% in 2016. Flock prevalence ranged from 31.03% in 2014 to 55.88% in 2015. Th ese results suggest that M. synoviae infection is present in broiler breeder farms in South Bačka, and that is necessary to conduct further research, systematic monitoring and to improve biosecurity measures on broiler breeder farms.


INTRODUCTION
Mycoplasma synoviae (Ms) is one of the most important pathogens in domestic poultry worldwide, including breeders, broilers and layers (Vardaman et al., 1973;Kleven and Ferguson -Noeln, 2008). Ms infections in poultry have always been regarded as less important than infections with Mycoplasma gallisepticum, but during the last decade, the importance of infections with Ms have been highlighted in several researches and there is an increased consciousness to produce Ms free poultry (Feberwee et al., 2008;Landman, 2014). In chickens, Ms infections have been associated with the respiratory disorders, synovitis, subclinical infections, air sacculitis (Vardaman et al., 1973;Macowan et al., 1984), as well as eggshell apex abnormalities and egg production losses (Landman and Feberwee, 2001;Van Beek et al., 2002;Landman and Feberwee, 2004;Feberwee et al., 2007).
Mycoplasma synoviae can cause signifi cant economic losses due to decrease of egg production rate, growth and hatchability rates, poor eggshell quality, and condemnation of carcasses at slaughter because of air sacculitis and arthritis (Fiorentin et al., 2003;Kleven 2003a;Peebles et al., 2011). An additional contribution to the importance of Ms infection in domestic poultry is brought by the high seroprevalence of Ms in countries with developed poultry industry (Feberwee et al., 2008;Feberwee and Landman, 2012) and its ability to interact with other pathogens such as Newcastle disease virus and infectious bronchitis virus (Kleven et al., 1972;Hopkins and Yoder, 1982;Feberwee et al., 2009). Reduction in egg production of 5 to 10 %, a reduction in hatchability of 5 to 7 % and more than 5 % increased mortality in the young chickens were reported in infected breeder fl ocks (Stipkovits and Kempf, 1996). Broiler fl ocks from the seropositive broiler breeders showed higher rate of mortality from air sacculitis (King et al., 1973;Macowan et al., 1984). Th e economic impact of Ms infection is mainly focused on increased condemnation of carcasses due to air sacculitis (Sentíes-Cué et al., 2005). Reduced weight gain and poorer feed conversion are also the result of Ms infection in broilers (King et al., 1973). Mycoplasma synoviae can be transmitted horizontally and vertically (Jordan, 1975). Vertical transmission is considered to be a major way of spreading of Ms in domestic chickens, therefore the most effi cient method of control would be regular monitoring and elimination of positive breeder fl ocks (Kleven and Ferguson-Noeln, 2008;Lockaby et al., 1998). Although various live and attenuated Ms vaccines have been developed and used worldwide (Morrow et al., 1998), the Ms vaccine is not yet available in Serbia.
In Serbia, serological monitoring of Ms is still on voluntary basis and data about seroprevalence of Ms in poultry farms are lacking. It is necessary to identify Ms seropositive farms, to assess the epidemiological risks in diff erent poultry categories and to improve biosecurity measures on farms. Th e aim of this study was to investigate Ms seroprevalence on diff erent broiler breeder farms and to perceive the epidemiological situation of Ms infection in South Bačka Region.

MATERIAL AND METHODS
A total of 1511 blood samples were taken from 115 broiler breeder fl ocks from 5 diff erent farms in South Bačka region. Th e samples were taken from 2014 to 2017, following the voluntary M. synoviae control program established by Scientifi c Veterinary Institute "Novi Sad". Blood samples were aseptically taken from non-vaccinated broiler breeders that had no clinical symptoms.
Sera were tested for the presence of antibodies to M. synoviae by a commercial ELISA test kit (IDEXX 99-06728, IDEXX Laboratories, Westbrook, ME) according to manufacturer's instructions. Briefl y, 100 μl of each diluted serum sample (1:500) were added to the wells previously coated with Ms antigen. Undiluted positive and negative controls were tested in duplicate wells. Samples were incubated at 18-26 °C for 30 minutes. Aft er washing the plates with distilled water 3 to 5 times, 100 μl of conjugate (Goat antichicken antibodies: Horseradish peroxidase conjugate) were added to each well and incubated at 18-26 °C for 30 minutes. Plates were washed again 3 to 5 times with distilled water and 100 μl of TMB (tetramethyl benzidine) substrate were dispensed into each well of the plate. Th e substrate solution was incubated at 18-26 °C for 15 minutes. Th e reaction was quenched with 100 μl of stopping solution. Th e absorbance was measured at 650 nm. Serum samples with S/P values greater than 0.5 (titer greater than 1076) were considered positive.

RESULTS
Th e obtained results are presented in Table 1 and Table 2. In general, 381 samples were found positive for the presence of anti-Ms antibodies, with the overall prevalence of 25.21%. Th e highest -31.02% and the lowest -16.78% seroprevalence was found in 2015 and 2016, respectively (Table 1). It was found that 47 (40.87%) out of the 115 tested fl ocks were positive. Positive fl ock rates in diff erent years ranged between 31.03% and 55.88% (Table 2).  Since Ms can be transmitted vertically and horizontally (Stipkovits and Kempf, 1996), it can cause infection in broilers with increased mortality, feed conversion and condemnation. (Xue et al., 2017;Moreira et al., 2015). Feberwee et al. (2008) reported lower seroprevalence in meat rearing breeder stock (6%) than in meat-type grandparents stock (10%), due to reduced risk of vertical transmission by elimination of Ms-positive fl ocks. Authors also found higher seroprevalence in meat production breeder stock (35%) than in meat rearing breeder stock. Th e accuracy of the estimated seroprevalence could have been infl uenced by frequency and number of birds sampled per fl ock, which was diff erent for diff erent poultry categories (Feberwee et al., 2008). In order to establish reliable Ms control and prevention program sampling methods, sample size and poultry categories must be determined in advance. High prevalence of Ms infection was found in Portuguese breeder farms, so the authors suggested that culling Ms-positive fl ocks with such a high prevalence is not an option (

CONCLUSION
It is evident that Ms is present on broiler breeder farms in South Bačka. In the absence of systematic monitoring of Ms infection in Serbia, further studies need to be done to obtain an overall picture of Ms prevalence in our country. Serological monitoring of Ms infection plays an important role in the prevention and control of this infection in poultry. Besides, it is also important to improve biosecurity measures on farms and to import broiler breeder chickens only from the farms that conduct vaccination against Ms. It is recommendable to introduce vaccination against Ms in all broiler breeder farms in Serbia, as one of the most signifi cant prevention measures.