MYCOPLASMA SYNOVIAE INFECTION IN LAYERS: DIAGNOSIS AND CONTROL MEASURES – A REVIEW

Mycoplasmas are widespread bacteria in domestic and wild birds. Among the important species in laying hen, Mycoplasma gallisepticum and Mycoplasma synoviae, are considered as an emergent pathogen in the last few years worldwide, causing considerable economic losses as a result of falling eggs and the decrease in egg quality. Transmission of M. synoviae occurs horizontally, more rapidly in multi-age sites, and vertically, leading to a decline in hatchability in breeding farms. Th e interaction between M. synoviae and the host’s immune system explains the immunosuppression induced by this pathogen. Inside the cell, M. synoviae can escape the immune system by implementing several mechanisms. Subclinical respiratory infection is oft en associated to M. synoviae. However, severe disease may be observed in the presence of other factors (respiratory viruses, stressors). Th e emergence of a new form of clinical manifestation of disease associated to M. synoviae infection has been described since the 2000s. Eggshell apex abnormalities of the produced eggs, associated to high risk of cracks and breakage, is described. Th e diagnosis of M. synoviae infection is based on various tests, including serology, culture and biomolecular methods. Control is based on the acquisition of free mycoplasma birds, biosecurity, regular monitoring and vaccination. Management of other risk factors is essential.


INTRODUCTION
Mycoplasmosis are worldwide infections of domestic and wild birds. Historically, infections were described fi rstly in turkeys in 1926, then in chicken in 1936 (Nascimento et al., 2005;Kleven 2008). More than 120 species, isolated in mammals, birds, reptiles and fi shes, were determined in Mycoplasma genus. Currently, more than 20 species of mycoplasma are considered pathogenic in poultry (Nascimento et  Mycoplasmosis caused by M. synoviae was fi rstly described in turkeys in 1926 and in chicken in 1936 (Nascimento et al., 2005, Kleven 2008). M. synoviae is responsible of locomotor disorders, including arthritis, tendinitis and synovitis. However, contrarily to M. gallisepticum, M. synoviae may be incriminated in a subclinical respiratory infection, which can predispose birds to the chronic respiratory disease following interactions with other pathogens (Newcastle disease virus, infectious bronchitis virus, Ornithobacterium rhinotracheale and Escherichia coli).
In last decade, the situation of mycoplasma infections is characterized by the emergence of M. synoviae compared to M. gallisepticum and a more pronounced respiratory tropism (aerosacculitis) than the articular tropism, especially in chicken and secondarily in turkey (Khalifa et al., 2013). In laying hens, M. synoviae has been causing serious egg drop problems since the 2000s with alteration of the eggshell quality.

ECONOMIC IMPACT
Economic impact of Mycoplasma infection in layer hen fl ocks are well documented regarding to M. gallisepticum. Whereas, the economic signifi cance of M. synoviae has been a subject of debate for many years. While the increasing occurrence worldwide of arthropathic and amyloidogenic M. synoviae strains in poultry as well as strains that induce eggshell apex abnormalities (EAA) and egg production losses Feberwee, 2001, 2004;van Beek et al., 2002;Feberwee et al., 2007), has increased attentiveness of the clinical and economic impact of infection with this Mycoplasma. Prevalence of M. synoviae infection in layers in some countries is summarized in Table 1. Currently, it is demonstrated that M. synoviae infection causes severe economic losses due to vertical transmission of the germ, resulting in death of embryo, consequently a decrease in hatch rate, signifi cant post-hatch mortality, bacteria diff usion in the hatchery, and quality degradation of day-old chicks. Moreover, M. synoviae may induce transient immunosuppression, an increase in mortality of 1-4%, particularly in broiler chickens, a decrease of 5-10% in egg production rate, and a decrease of 5-7% in hatch rate (Mohammed et al., 1987;Stipkovits and Kempf 1996).
Mycoplasmosis due to M. synoviae occurred in layer hens fl ocks are associated to a decrease in the egg quality ( Table 2). Egg-production level can decrease from 86% to 79% aft er three weeks in 54 weeks old layer hens (Jeon et al., 2014). Losses in infected hens are estimated at eight eggs per hen compared to healthy fl ocks (Mohammed et al., 1987). Additional losses are mentioned in aff ected fl ocks due to therapeutic and prophylactic means (Ferguson-Noel and Williams 2015). Similar reports are observed in layer hens fl ocks in Tunisia. Indeed, a decrease of 5-20% in eggs production is mentioned, according to the farms registers. Th e prevalence of M. synoviae infections in Tunisian poultry industry remains relatively high. Overall sero-prevalence of 19% (23 fl ocks / 63 visited) of all types of production in the Cap-Bon region (north-eastern of Tunisia) is mentioned (Boussetta et al., 1997). Th e prevalence in layer hens fl ocks is estimated to 28.5% (6 fl ocks / 21 visited). Currently, it appears that this prevalence is higher according to fi eld fi ndings, in the absence of offi cial publications. Positive layers do not achieve the optimal weight during the breeding phase allowing them to lay eggs period. Consequently, the production level, the eggs size and the eggshell quality are signifi cantly aff ected later.
A new syndrome, called "Eggshell Apex Abnormality" (EAA), has been identifi ed since 2000 in broiler-type breeders and layer hen's fl ocks. Th is new form is due to infection by certain strains of M. synoviae that multiply in the hen's genital tract. Th e presence of such eggs type in layer's fl ocks in Tunisia has also been reported in many integrations.
Th e upper part of the eggshell appears translucent, thin and very fragile, so it is easy to break. Th e demarcation is clear between the normal and the aff ected parts of the shell (Feberwee et al., 2009;Jeon et al., 2014). Eggshell discoloration was also observed in positive fl ocks, which further increasing the decommissioning rate of eggs. Th is rate may rise from 2.6% to 8.3% (Jeon et al., 2014). However, other causes, such as infectious bronchitis virus (IBV) and egg drop syndrome'76 virus (EDS'76) may induce more than 25% of egg quality degradation. Eggs from infected fl ocks are smaller, with a low commercial value. Because of possible eggshell cracks presence, interior components contamination is reported. Th e penetration of potential pathogens into aff ected eggs has been accompanied with the increase of embryo mortality (Hunton 2005).

TRANSMISSION
One of the ways in which poultry mycoplasma are disseminated is through egg transmission. Th is mode of transmission eased by the oviduct contamination is mainly observed for M. meleagridis and M. iowae. Whereas, the contamination of embryonated eggs with M. gallisepticum and M. synoviae is mainly due to the contiguity of the oviduct and contaminated air sacs (Kempf 1997; Dufour-Gesbert et al., 2006). Possible transmission via contaminated semen may also occur during artifi cial insemination in turkeys.
Transmission of M. gallisepticum and M. synoviae is mainly the consequence of the direct and close contact between animals, where bacteria may penetrate via respiratory and/or conjunctival pathways. Transmission can also occur through indirect contact, due to possible persistence of Mycoplasma for several days in the environment. Th e involvement of several types of animated (people, wild birds) and inanimate vectors (vehicles, food, water...) has been also established ( Figure 1). Once infected, birds may be able to carry asymptomatically the bacteria even throughout the production period. Furthermore, because of the extension of poultry farms and the concentration of large integrations with multiage fl ocks in restricted geographical areas, maintaining the free-status of fl ocks becomes very diffi cult.

PATHOGENICITY AND INTERACTION WITH OTHER PATHOGENS
M. synoviae is involved in several types of disorders: respiratory, articular and genital. In general, this bacteria causes subclinical respiratory infection (Kleven 2008  It is currently well established that mycoplasmas cause immunosuppression in infected birds. Indeed, these bacteria cause excessive release of pro-infl ammatory cytokines, inhibit phagocytosis, and aff ect the B-cells and T-cells functions (Stipkovits et al., 2012).
Several escape mechanisms to the host immune response were described in mycoplasmas to explain the existence of chronic infections and some therapeutic failures. Th ese include intracellular location, molecular mimicry (recognition of mycoplasmas surface epitopes as well as the self by the immune system) and antigenic variability (Bencina et al., 1994;Garcia et al., 1994;Kleven 1998;Nascimento et al., 2005). In

DIAGNOSIS
Field diagnosis of M. synoviae infection is diffi cult because of the nonspecifi c clinical signs, lesions and the numerous similar diseases. Th at is why, laboratory investigations are very important to confi rm clinical suspicion. Rapid detection of infection is demanded to prevent spread and reduce economic losses. Several direct and indirect diagnostic methods are available (Table 3).   Th e fi rst defence barrier is the application of the all-in-all-out band, associated to good biosecurity and monitoring program. However, in a multi-age system, layers fl ocks are usually infected by M. gallisepticum and M. synoviae in most regions of the world, which presents a potential risk of infection transmission to broiler fl ocks.
Th e fi rst step in controlling Mycoplasma infections is the acquisition of fertile eggs and Mycoplasma-free birds. Treatment of hatching eggs by heating (46°C for 12-14 hours), or more eff ectively, by injecting antibiotics, either by an in-ovo injector or by dipping into an antibiotic solution, are diff erent methods used for the eradication of the infection in grandparents (Nascimento and Nascimento 1994;Stipkovits and Kempf 1996).
National certifi cation programs have contributed to the control of Mycoplasma infections in many countries, such as the United States (USA 1997), Brazil (Villa, 1998) (in Nascimento et al., 2005) and France (Offi cial Hygienic and Sanitary Control) (Stipkovits and Kempf 1996). Monitoring is carried out by serological methods such as SPA, ELISA and/or HI. In Tunisia, the control of M. gallisepticum, M. synoviae and M. meleagridis is also based on an OHSC using the SPA technique. Mycoplasma detection is frequently confi rmed by PCR (Nascimento et al., 1994;Nascimento et al., 1998).
Mycoplasma monitoring is targeting breeding and commercial laying farms. Analyses are performed on a number of animals, chosen randomly, that vary according to the incidence of infection. Two samples are taken from breeding animals in Tunisia, between 10-12 weeks and between 20-24 weeks, on 2.5% of the total eff ective for M. gallisepticum and 5% for M. synoviae.
Antibiotic treatments can be administered in contaminated environments as a preventive measure, especially during stress period, or as part of a curative treatment. Th e administration of antibiotic as preventive tool is very common in several countries, including Tunisia. Several antibiotic molecules are used; these include macrolides (tylosin, tylvalosin, tiamulin, tilmycosin), tetracyclines (oxytetcracycline, doxicycline) and aminosides (spectinomycin) (Bébéar and Kempf 2005); Kreizinger et al., 2017). Tetracyclines, due to their relatively low cost, are primary antibiotics in the treatment of avian mycoplasmosis.
Th e implemented programs vary widely across countries, regions and farms. However, the emergence of antimicrobial resistance in M. synoviae limits the use of this control approach. Th erefore, the study of the sensitivity profi les of fi eld-isolated strains to antibiotic molecules is a fundamental step towards improving the eff ectiveness of medical protocols. However, although the treatments reduce signifi cantly clinical signs, mycoplasma may be further isolated aft er cessation of antibiotic administration, when animals are infected by resistant strains (Reinhardt et al., 2005;Carrou et al., 2006).
Vaccination against M. synoviae is performed especially in breeders and layers to prevent clinical signs and bacteria spread. Currently there are two commercialized live attenuated vaccines available against M. synoviae: the temperature sensitive MS-H vaccine strain and the NAD independent MS1 vaccine strain (Kreizinger et al., 2018). Interaction of these vaccines with other respiratory pathogens is documented. Indeed, M. synoviae vaccine strains can modify AIV replication and immune responses. Furthermore, live vaccines can act as a complicating factor during respiratory co-infection in layers, which may subsequently lead to vaccination strategies advance against poultry respiratory pathogens, in general (Umar et al., 2017).
Th Diff erentiation between vaccine and wild strain was investigated using nested PCR. Th is technique allows the presence of an adenine in a nucleotide at position 468 of the oppF-1 gene of the vaccine strain of M. synoviae-H. Indeed, the above-mentioned authors show the exclusivity of this mutation in the vaccine strain, compared to wild M. synoviae strains isolated in Australia (Zhu et al., 2017). Recently, diff erentiation between M. synoviae vaccine and fi eld strains was performed with indirect ELISA based on OppF-C gene (Kordafshari et al., 2019). Furthermore, a melt-curve and agarose gel-based mismatch amplifi cation mutation assays (MAMA) was recently provided to discriminate the MS1 vaccine strain from the MS-H vaccine strain and wildtype M. synoviae isolates (Kreizinger et al., 2018). However, these assays are limited by the available facilities and the cost.

CONCLUSION
Despite the lack of offi cial data on the incidence of M. synoviae and its economic impact in layers, it appears that this mycoplasma is gaining increasing interest around the world. Although M. synoviae cause usually a subclinical respiratory infection, it is responsible for several articular and genital disorders. Moreover, the interaction between fi eld and vaccine strains of M. synoviae and other viral and bacterial infections increase usually the severity of clinical signs and lesions, and consequently the economic losses. In layer hens, M. synoviae infection has been accompanied since the beginning of 2000s by a new form, characterized by EAA eggs with a very fragile apex shell. Signifi cant withdrawing rates for aff ected eggs, justifi es the importance of implementing appropriate control measures. In this sense, control of M. synoviae infection in layers should be based on an integrated approach involving biosecurity, vaccination, and regular surveillance. Th ese should be well performed to limit the problem of mycoplasmas persistence in infected fl ocks, especially in multi-age integrations, and minimize economic losses.