Is Mycoplasma synoviae outrunning Mycoplasma gallisepticum? A viewpoint from the Netherlands

Abstract

Mycoplasma gallisepticum and M. synoviae are the most relevant mycoplasma species for commercial poultry from the clinical and economic point of view. Although the importance of M. gallisepticum was recognized many decades ago, the relevance of M. synoviae has been a matter of debate. Until the turn of the century, only the respiratory and synovitis forms of the disease were reported, while the majority of infections were subclinical. Since the year 2000 M. synoviae strains with oviduct tropism, able to induce eggshell apex abnormalities and egg drops, have been encountered worldwide. A decreasing incidence of M. gallisepticum has been observed, at least in breeding stock, in countries with control and eradication programmes for this Mycoplasma species. In contrast, the sero-prevalence of M. synoviae is much higher, especially in layer flocks, and in most continents exceeds 70%. Given the emergence of virulent M. synoviae strains with oviduct tropism, its ability to also induce joint and respiratory disease, to act synergistically with other pathogens as well as its much higher sero-prevalence, it seems that M. synoviae is outrunning M. gallisepticum, at least in countries with control and eradication programmes for the latter. This stresses the need to update M. synoviae prevention and control strategies. Thus, in January 2013, the Dutch poultry industry implemented a mandatory control and eradication programme for M. synoviae at all levels of poultry farming with the exception of broilers.

Introduction

Organisms that were later identified as “pleuropneumonia-like organisms” (Smith et al., 1948) were first isolated from chickens with “coryza of slow onset” in 1935 (Nelson, 1936a, b, c, d, 1939). Pleuropneumonia-like organisms were subsequently classed as Mycoplasma spp. and “coryza of slow onset” was redefined as chronic respiratory disease, which is caused by Mycoplasma gallisepticum (Markham & Wong, 1952; Van Roekel & Olesiuk, 1953). Mycoplasma synoviae was first reported in 1954 by Olson et al. (1954) and by Wills (1954) as an unidentified agent causing synovitis in chickens affecting primarily the synovial and bursal membranes. However, the true aetiology of infectious synovitis was not known until 1960, when Lecce (1960) demonstrated a mycoplasma growing as satellites to staphylococcus colonies on agar plates. This was confirmed by Chalquest & Fabricant (1960), who isolated a mycoplasma that was distinct from M. gallisepticum from birds with infectious synovitis, which was named M. synoviae (Olson et al., 1964). Much later in 1982 it was confirmed as a distinct Mycoplasma species (Jordan et al., 1982). Besides causing joint lesions, M. synoviae strains able to induce airsacculitis (Kleven et al., 1972) and egg shell apex abnormalities have been identified (Feberwee et al., 2009).

In general the mycoplasmas that infect animals are host specific, have an affinity for mucosal surfaces and lack a cell wall. The lack of a cell wall renders the organisms insensitive to antimicrobials that affect cell wall synthesis. However, they are more sensitive to environmental factors than those bacteria with a cell wall. Mycoplasmas have complex nutritional requirements and colonies are recognizable by their typical fried-egg morphology after culture on agar. Currently approximately 25 species (mostly belonging to the genus Mycoplasma but including some in the genera Acholeplasma and Ureaplasma) have been identified in birds, of which some 14 occur in chickens and turkeys. M. gallisepticum and M. synoviae are the most relevant for commercial chickens from a clinical and an economical point of view (Kleven, 2008).

In this opinion piece the relevance of M. synoviae compared with M. gallisepticum is discussed in view of the increasing clinical and economic relevance of the former.

Clinical and economic impact

M. gallisepticum can cause chronic respiratory disease and significant downgrading of carcasses. Additionally it can be responsible for decreased growth and egg production, and reduced hatchability rates. The severity of clinical signs may vary considerably between strains. The occurrence of other respiratory viral or bacterial agents (e.g. Newcastle disease virus, infectious bronchitis virus and Escherichia coli), immunosuppression and/or adverse climate and housing conditions may significantly worsen the clinical expression of the disease (Gross, 1961, 1990; Fabricant & Levine, 1962; Kleven, 1998).

Although both M. gallisepticum and M. synoviae are relevant to the poultry industry, the overall impact of M. gallisepticum has been regarded as much higher than that of M. synoviae (Goren, 1978; Van Eck et al., 1980; Mohammed et al., 1987). Nevertheless, the clinical and economic relevance of M. synoviae seems to be increasing, considering the number of publications on this mycoplasma species, the worldwide emergence of strains affecting the eggshell quality and egg production and the emergence in some countries of arthropathic and amyloidogenic strains (Table 1). The higher sero-prevalence of M. synoviae compared with M. gallisepticum in countries with a well-developed poultry industry (Feberwee et al., 2008; Feberwee & Landman, 2012) and its ability to interact with other pathogens such as Newcastle disease virus and infectious bronchitis virus (Kleven et al., 1972; Hopkins & Yoder, 1982; Feberwee et al., 2009) further contribute to the significance of this Mycoplasma species in commercial poultry. Raviv et al. (2007) claimed a role for M. synoviae as a complicating factor in the egg peritonitis syndrome of laying hens. However, this claim is open to question because the numbers of hens with egg peritonitis syndrome in their various experimental groups were very low and a statistical significant difference (P < 0.05) between birds infected with E. coli alone and those exposed to both E. coli and M. synoviae was not found. Significant difference (P < 0.05) was reported only between the negative control group and birds with the double infection. Moreover, both Vandekerchove et al. (2004), and Landman et al. (2013), showed that E. coli strains involved in egg peritonitis syndrome are primary pathogens.

Table 1. Worldwide sero-prevalence of M. synoviae in layers and the occurrence of M. synoviae pathology in layers and broilers.

Continent	Country	Sero-prevalence in layers (%)	EAA	IS	AS	References
Europe	the Netherlands	73	+	+		(Landman & Feberwee, 2001; Feberwee et al., 2008, 2009)
	Belgium		+^a	+		(Landman & Feberwee, 2001)
	Germany	95^b	+	+		(Hinz & Luders, 1969a, b; Kohn et al., 2009; Ranck et al., 2010)
	Denmark		+^a
	UK	78	+		+	(MacOwan et al., 1982; Hagan et al., 2004; Strugnell et al., 2011)
	France		+	+		(Bouchardon, 2012)
	Italy		+	+		(Catania et al., 2010)
Asia	Japan		+^a	+	+	(Sato, 1996)
	China		+^a	+
	Philippines	30				(Sato, 1996)
	Korea		+^a		+	(Sato, 1996)
Africa	South Africa			+	+	(Buys, 1976)
Oceania	Australia	69	+^c	+	+	(Gilchrist & Cottew, 1974; Morrow et al., 1990; Gole et al., 2012)
America	USA	84	+^a	+	+	(King et al., 1973; Mohammed et al., 1986; Kang et al., 2002)
	Colombia		+^d

EAA, eggshell apex abnormalities; IS, infectious synovitis; AS, airsacculitis. ^aC. Morrow, personal communication, 2009. ^bDetermined by polymerase chain reaction. ^cStatistically significant association between the occurrence of M. synoviae antibodies and eggshell translucency, shell breaking strength, percentage shell reflectivity and shell deformation, but not with egg weight, egg shell weight, percentage egg shell or shell thickness (Gole et al., 2012). ^dR. Delgado, personal communication, 2013.

Although the economic impact resulting from the trade limitations of M. synoviae-infected flocks free of clinical disease is generally disregarded, this should also be considered as a factor of economic relevance.

The arthropathic character of M. synoviae, first known as the causative agent of infectious synovitis, has been well documented (Kleven et al., 1975; Landman & Feberwee, 2001; Kleven, 2008). Layer flocks infected with arthropathic strains may suffer losses due to growth retardation and culling of lame birds. The extent of these losses will largely depend on the number of affected birds, which generally ranges from 5 to 15% of the flock, but in some cases may exceed 75%. In brown layer rearing pullets infected intra-articularly with an arthopathic M. synoviae field isolate, 26% weight reduction was found. If the same field strain was given intravenously, the weight reduction was 16% (Landman & Feberwee, 2001). In infected breeder flocks Stipkovits & Kempf (1996) reported a reduction in egg production of 5 to 10%, a reduction in hatchability of 5 to 7% and a more than 5% increased mortality in the offspring, without obvious clinical signs. Other scientists, however, did not observe detrimental effects in M. synoviae-infected flocks (Goren, 1978; Van Eck et al., 1980), which may be explained by the variation of disease-inducing potential between strains (Lockaby et al., 1998; Narat et al., 1998; Kang et al., 2002; Landman & Feberwee, 2012).

In broiler chickens the economic impact of M. synoviae infections has been studied more extensively and revolves primarily around increased condemnation of carcasses due to the occurrence of airsacculitis seen in the respiratory form of the disease. The incidence of air sac lesions is greatly influenced by co-infection with respiratory viruses and bacteria (Kleven et al., 1972; Springer et al., 1974; Hopkins & Yoder, 1982), environmental factors (Yoder et al., 1977) and/or immunosuppression (Giambrone et al., 1977). The synergism between arthropathic and salpingotropic M. synoviae strains and other respiratory pathogens such as infectious bronchitis virus has also been documented (Landman & Feberwee, 2004; Feberwee & Landman, 2010). Reduced performance as determined by a reduced body weight and poorer feed conversion has also been reported in M. synoviae-infected broilers (Kleven et al., 1972; King et al., 1973; Goren, 1978).

Until 2000, the respiratory (King et al., 1973) and synovitis form of the disease dominated in the field (Morrow et al., 1990; Landman & Feberwee, 2001, 2012). However, from the year 2000 onwards, M. synoviae strains with oviduct tropism that were able to induce or were associated with eggshell apex abnormalities (EAA) and egg drops have been increasingly found (Feberwee et al., 2009; Catania et al., 2010; Ranck et al., 2010; Strugnell et al., 2011). EAA are characterized by an altered shell surface, shell thinning, increased translucency and the occurrence of cracks and breaks. The EAA are confined to a region extending approximately 2 cm from the apex of the egg and in most cases there is a very clear demarcation zone. The eggshell strength is severely diminished (up to one-half of that of non-affected eggs), which is in accordance with the translucency that is detectable macroscopically, particularly at candling. Scanning electron microscopy shows that the apical shell of eggs with EAA lack the mammillary knob layer and part of the palisade layer. At post-mortem examination no macroscopic abnormalities could be detected in birds producing eggs with EAA (Feberwee et al., 2009). The economic damage due to EAA occurs both at the farm and at the egg packing station. It is due to breakage of eggs, increased downgrading and an increase in labour costs due to the selection of EAA eggs and the extra cleaning of the facilities due to broken eggs.

Sero-prevalence of M. gallisepticum and M. synoviae

The sero-prevalence of M. gallisepticum is generally low in categories of poultry subjected to control and eradication programmes; for example, breeder flocks in Europe. In the Netherlands, the control and eradication of M. gallisepticum started in the mid-1960s. During the first decades, layers and meat turkeys were not included in this programme and infected flocks were not slaughtered because the M. gallisepticum prevalence was very high and therefore it was not economically viable. In this period, eggs were dipped in tylosin tartrate solution using negative pressure if they originated from M. gallisepticum-infected parent layer/broiler flocks or were injected with this antibiotic if it concerned eggs from infected grandparent stock. Once the M. gallisepticum prevalence was sufficiently reduced, slaughtering of infected breeder flocks started at the beginning of the 1990s. Layers and meat turkeys were added to the control and eradication programme in the years 2000 and 2001, respectively. Both types of poultry were subjected to improvement of biosecurity, while M. gallisepticum-free layer rearing flocks to be housed on infected multiple age farms were vaccinated. Practical “channelling” was implemented to maintain separation of uninfected flocks and eggs from those with infection. For example, this could include infected replacement layer pullets being diverted (channelled) to positive multiple-age layer farms, eggs from infected breeders being collected by hatcheries using separate vehicles, and so forth.

Slaughtering of infected breeder flocks and inclusion of layers and meat turkeys in the programme has significantly further decreased the sero-incidence of M. gallisepticum, as evidenced by the results of sero-monitoring during the period 1999 to 2010: the number of breeder farms infected with M. gallisepticum decreased significantly from 1.4 to 0.2%, layer farms from 10.5 to 1.9% and meat turkey farms from 12.5 to 0.0% (Feberwee & Landman, 2012). This reduction indicates that the control and eradication of M. gallisepticum in the Netherlands has been successful.

Although research on the sero-prevalence of M. synoviae is limited, several studies have shown that the prevalence of M. synoviae in layer stock in various parts of the world is very high (Table 1), while published data on the prevalence of M. synoviae in other poultry categories are scarce. A recent Dutch study described for the first time the M. synoviae sero-prevalence in different categories of commercial poultry, and confirmed that it was high, especially in layer farms where it was 73%. In layer and broiler grandparent farms it was 0% and 10%, respectively. In layer and broiler parent farms, the sero-prevalence was 25 and 35%, respectively; in both broiler parent rearing and broiler farms it was 6%; in meat turkeys it was 16% (Feberwee et al., 2008).

Until recently, the voluntary control and eradication of M. synoviae in the Netherlands has been limited to grandparent stock; flocks are eliminated if infected. As the risk of vertical transmission is minimized by culling M. synoviae-positive grandparent stock, lateral transmission is expected to be the most important transmission route for parent farms in the Netherlands. Sero-positive layer parent flocks are not culled, and a high risk for vertical transmission of M. synoviae to rearing layer flocks was found after an odds ratio analysis in a study that showed a very high sero-prevalence of M. synoviae in rearing layers (69% of layer rearing farms sero-positive) (Ter Veen et al., 2012).

Control and eradication of M. gallisepticum and M. synoviae

Mycoplasma control and eradication is based on detection of infections, elimination of infected flocks and hygienic measures. When these measures are not economically sustainable then antibiotic treatment and vaccination programmes may be used (Stipkovits & Kempf, 1996).

Monitoring programmes for the detection of M. gallisepticum and M. synoviae infections in the Netherlands and elsewhere are mainly based on serological tests. Regular serological monitoring of commercial poultry is essential for the detection of an infection, provided that representative sample sizes and tests with appropriate sensitivity and specificity are used. The sero-monitoring of M. gallisepticum and M. synoviae in Dutch poultry is done using the rapid plate agglutination (RPA) test and the enzyme-linked immunosorbent assay (ELISA) as prescribed by the Office International des Epizooties (OIE) (Kleven & Bradbury, 2008). In the past, the haemagglutination inhibition test was also used. This choice is in agreement with the results of a previous study in which the specificity and sensitivity of these serological tests were compared with those of culture, polymerase chain reaction and various commercial ELISA kits. The combination of the M. gallisepticum and M. synoviae RPA with haemagglutination inhibition test was found to perform equally well as the tested ELISA kits (Feberwee et al., 2005).

The control and eradication of M. gallisepticum and M. synoviae is achieved amongst other measures by eliminating infected breeder flocks, which will stop the vertical and horizontal transmission. In addition, standardized biosecurity programmes are implemented to minimize risk of horizontal transmission (Stipkovits & Kempf, 1996; Whithear, 1996). In countries with a well-developed poultry industry the primary breeding stock is free of M. gallisepticum and M. synoviae and the commercial breeding stock is free of M. gallisepticum.

The control and eradication of M. gallisepticum is based on European Union Council Directive 2009/158/EC (EU, 2009) and European Commission Decision 2011/214/EU (EU, 2011), which state that the trade of offspring from M. gallisepticum-positive flocks is not allowed within the European Union. In order to execute this directive, the Dutch Commodity Board for Poultry and Eggs implemented a compulsory M. gallisepticum monitoring programme for breeding stock (Productschap voor Pluimvee en Eieren, 2012, 2013). M. gallisepticum infection is detected as early as possible (at 5 to 10% prevalence with 95% confidence) by monitoring flocks regularly and collecting a representative number of blood samples (30 to 60) per house (Cannon & Roe, 1982; De Wit, 2000). If a flock is M. gallisepticum-positive it is slaughtered, and thus the source for vertical and horizontal transmission is eliminated. In 2001, based on national legislation, the M. gallisepticum control and eradication programme was further expanded to layer stock. In addition to compulsory sero-monitoring of all layer flocks, compulsory vaccination was implemented for rearing layer flocks free of M. gallisepticum to be housed on infected multiple-age layer farms.

Vaccination against M. gallisepticum has proved to be a useful tool to control M. gallisepticum infections on multiple-age farms. Inactivated M. gallisepticum vaccines tend to reduce the infection pressure on a farm after long-term use (Kleven, 1985; Whithear, 1996). The beneficial use of live vaccines results from the replacement of field strains by the vaccine strain (Whithear, 1996; Kleven et al., 1998; Turner & Kleven, 1998; Barbour et al., 2000). More recent studies showed that although M. gallisepticum vaccination does not prevent colonization and horizontal transmission of the challenge strain, it significantly reduced its shedding (Feberwee et al., 2006a, b).

M. synoviae control and eradication in the Netherlands has been voluntary until recently. Awareness of the increasing clinical and economic relevance of M. synoviae due to the emergence of strains able to induce infectious synovitis and EAA has prompted the Dutch poultry industry to implement a mandatory control and eradication programme by decree (Productschap voor Pluimvee en Eieren, 2012, 2013) for all categories of poultry farming except broilers. This programme, launched in January 2013, is based on the current successful control and eradication programme for M. gallisepticum. Indeed, the sero-monitoring programme that is used for M. gallisepticum is followed, although culling of infected flocks is not performed for M. synoviae, except for grandparent stock where culling is voluntary. Vaccination against M. synoviae of rearing layer flocks free of M. synoviae to be housed on M. synoviae-positive multiple-age farms is not advised currently. This policy may change if forthcoming experimental studies show that such vaccination can help to reduce the shedding of challenge strains and if tests are readily available to differentiate field from vaccine strains. Also, with declining sero-incidence, culling of all breeding stock infected with M. synoviae may eventually be performed, as occurs with M. gallisepticum-infected flocks.

The current Dutch M. gallisepticum and M. synoviae control and eradication programmes are outlined in Table 2.

Table 2. Dutch M. gallisepticum and M. synoviae control and eradication programmes performed by GD—Animal Health Service: sampling frequency for sero-monitoring, sample size, detectable sero-prevalence, tests, interpretation, confirmation analysis and control measures taken per poultry category in case of infection.

							Control measures
Poultry category	Sampling age	Sample size per house^a	Detectable sero-prevalence (%)^a	Tests	Criterion for a positive sample	Confirmation analysis (maximum 10 positive samples)^b	M. gallisepticum	M. synoviae
Breeder chickens	End of rearing^c	30 to 60^d	≥10 to ≥5	GPS: combination ELISA Mg/Ms^e. PS: RPA Mg^f, RPA Ms^f	ELISA-positive Agglutination at serum dilution 1:2	ELISA Mg^g, ELISA Ms^h, PCR^i	Slaughter all birds on the farm	Voluntarily slaughter all birds on the farm (GPS only)
	GPS at age of 22 weeks and every 8 weeks
	PS at ages of 22 and 30 weeks and every 12 weeks
Layer chickens	End of rearing	24	≥12	RPA Mg, RPA Ms	Agglutination at serum dilution 1:2	ELISA Mg, ELISA Ms	“Channelling”^j	None
	End of lay	10	≥26	RPA Mg, RPA Ms		ELISA Mg, ELISA Ms	Vaccination^k	None
Meat turkeys	End of growing period	24	≥12	RPA Mg, RPA Ms	Agglutination, undiluted serum	ELISA Mg, ELISA Ms	None	None

GPS, grandparent stock; Mg, M. gallisepticum; Ms, M. synoviae; PS, parent stock. ^aAt given sample size and sero-prevalence there is a 95% chance to find at least one positive serum sample (Johnson et al., 2004). ^bResults of M. gallisepticum examinations are sent to the farmer, the veterinarian, the hatchery, the Commodity Board for Poultry and Eggs and the Dutch Early Warning System (DEAS). The DEAS sends information on disease outbreaks to all Dutch poultry veterinary practices, while M. synoviae results are sent only to the farmer. ^cSampling of replacement cockerels for M. gallisepticum is compulsory; for sampling size see footnote d. ^dOne per cent of the flock must be sampled with a minimum sample size of 30 and a maximum of 60, except for M. synoviae serology of PS where the sample size is always 30 per flock. A flock is defined as a group of commercial chickens or turkeys (≥250 birds) of the same age, and for turkeys also of the same gender, housed together (flocks <250 birds are regarded as game birds and are not sampled). ^eIDEXX Mg/Ms antibody test (IDEXX Laboratories Inc., Westbrook, Maine, USA). ^fAntigen from Soleil sarl (Cantenay-Épinard, France). ^gIDEXX Mg antibody test. ^hIDEXX Ms antibody test. ⁱPolymerase chain reaction (PCR) is performed on 60 trachea swabs per flock as described elsewhere (Raviv & Kleven, 2009) and is compulsory for M. gallisepticum and is voluntary forM. synoviae. ^j M. gallisepticum-positive rearing flocks are housed on M. gallisepticum-positive layer farms. ^kVaccination of M. gallisepticum-free rearing flocks with inactivated oil–emulsion vaccine to be housed on M. gallisepticum-positive multiple-age layer farms. Vaccinated flocks with positive M. gallisepticum serology at the end of lay are considered M. gallisepticum infected.

In situations where freedom from M. gallisepticum and M. synoviae by elimination of infected flocks is not economically attainable, medication and vaccination are the best alternatives. Both can contribute to a reduction of the clinical signs and economic losses. The major drawback of medication is that it will not enable complete elimination of the infection (Ley, 2008). Although a live commercial M. synoviae vaccine is available, its use has been limited because the economic impact of M. synoviae infections has been a matter of debate until recently. In contrast, vaccination against M. gallisepticum is common practice in many areas of the world and has shown to be beneficial to protect against clinical disease and the reduction of shedding, as was mentioned earlier.

Despite the beneficial effects of live mycoplasma vaccines, they may jeopardize control and eradication programmes as long as tests that differentiate field from vaccine strains, and thus differentiate vaccinated infected flocks from vaccinated non-infected flocks, are lacking.

Concluding Remarks

The relevance of M. synoviae as a poultry pathogen has been a matter of debate for many decades, but this is clearly changing, as evidenced by the number of manuscripts documenting the occurrence and detrimental effects of arthropathic and amyloidogenic strains, and salpingotropic strains that also induce EAA and production losses. In addition, studies documenting the high worldwide prevalence of M. synoviae and its ability to interact with other respiratory pathogens stress the need for an update on its prevention and control. Although M. synoviae control and eradication programmes have been operating for a long time in a number of countries such as the USA and the UK, the Dutch poultry industry appears to be the first to implement a mandatory control and an eradication programme for M. synoviae at all levels of the poultry industry except for broilers. This programme was launched in January 2013.