Nguyen Pham Thao Nhi, Nguyen Khanh Thuan, Nguyen Phuc Khanh, Nguyen Thanh Lam* Tran Duy Thanh, Pham Trang Thanh Nguyen 

1. Introduction

Mycoplasma synoviae infection most frequently occurs as a subclinical upper respiratory infection. It may cause air sac lesions when combined with Newcastle disease, infectious bronchitis, or both. M. synoviae may also become systemic and results in infectious synovitis, an acute to chronic infectious disease of chickens and turkeys, involving primarily the synovial membranes of joints and tendon sheaths producing an exudative synovitis, tenovaginitis, or bursitis(McMullin 2020).

2.  Aetiology

2.1 Bacteria characteristics

The avian pathogen M. synoviae is a member of the class Mollicutes, a group of bacteria that are characterised by their very small size, lack of cell wall, complex nutritional requirements and ability to persist in their hosts and establish chronic infections. M. synoviae strains appear to have varying tissue tropisms and virulence, although these characteristics may depend on the route of infection. M. synoviae has 2 major phase and size variable antigens, MSPA and MSPB. MSPA has been shown to be a hemagglutinin, but MSPB has not been linked with any function so far. Cultures of the hemagglutinin negative phenotype expressed truncated versions of MSPB and were less pathogenic than hemagglutination positive culture. Sequence variation of a 12 amino acid sialoreceptor binding motif within MSPA is believed to be responsible for differences observed between M. synoviae strains in their capacity to adhere to the host cells. MSPA and MSPB are expressed as a prolipoprotein, variable lipoprotein hemagglutinin (VlhA), from a single gene named vlhA, but the product is then cleaved to form MSPB and MSPA. M. synoviae vlhA has a high degree of identity with the vlhA 4.10 (pMGA1.7 gene) of M. gallisepticum which provides a possible explanation for occasional cross‐reactivity between these organisms in serological testing. There is only a single complete copy of the vlhA gene associated with a promoter region in the  M. synoviae genome, but there are a large number of incomplete copies (pseudogenes) present in a cluster adjacent to the complete vlhA copy. Variability in expression of vlhA is thought to be controlled by homologous recombination events between the complete copy of vlhA and pseudogenes (McMullin 2020).

2.2 Classification

Currently, analysis of the conserved domain (approximately 400 bp of the 5′ end) of the vlhA gene by direct nucleotide sequencing or other techniques is frequently used for identification of MS strains. Three separate studies have found that multilocus sequence typing analysis was superior to vlhA‐based genotyping techniques although the set of genes found suitable for strain identification were totally different in these studies. Also, comparison of individual genes in all these studies found that the single copy conserved 5′ end of the vlhA gene provided the highest discrimination power amongst all genes examined. Analysis of the whole or partial genomic sequences of MS strains has allowed for the development of rapid strain identification techniques that can differentiate between live vaccines and field strains (McMullin 2020).

3. History 

M. synoviae colonies were first observed as satellites adjacent to Micrococcus colonies by Chalquest and Fabricant, who identified the requirement for nicotinamide adenine dinucleotide (NAD). It was designated as serotype S by Dierks et al. Olson et al. proposed the name M. synoviae, which was subsequently confirmed as a separate species. The complete genome sequence of at least 2 strains of M. synoviae has been published, whereas incomplete genome sequences of multiple other strains are also available.

4. Epidemiology

4.1 Geographic distribution

Infectious synovitis was observed primarily in growing birds of 4–12 weeks of age in broiler‐growing regions of the United States during the 1950s and 1960s. Since the 1970s the synovitis form has been less frequently observed in chickens in the United States, but the respiratory form has been seen more frequently. Infection without apparent clinical signs is not unusual. M. synoviae infection occurs frequently in multi‐age commercial layers. Infectious synovitis usually appears in turkeys when they are 10–20 weeks old. M. synoviae is worldwide in distribution. 

M. synoviae was reported in Australia, South America, Asia, Europe and Africa, nevertheless, M. synoviae distribution appeared regional in most cases, and largescale outbreaks rarely occurred. In the past ten years, it seems that M. synoviae had taken over the role of  M. gallisepticum in commercial poultry. Apart from air sacculitis and synovitis, eggshell apex abnormalities (EAA) and egg drop syndrome resulting from M. synoviae infection have been encountered worldwide. Hence, there has been a growing emphasis on understanding the prevalence of M. synoviae, especially its subclinical infection. Many reports world with a varying degree of incidence in multi-age white feather chickens or turkeys. To date, there has been no data about M. synoviae infection in Chinese native chicken breeds, and the incidence and epidemiology of M. synoviae in China are poorly understood. Between 2010 and 2015, a disease, characteristic of infectious synovitis occurred in native-type chickens in China. The disease resulted in the loss of millions of chickens in Chinese poultry farms (Sun, Lin et al. 2017).

4.2 Susceptible hosts

Chickens and turkeys are the common natural hosts of M. synoviae. Ducks, geese, guinea fowl, pigeons, Japanese quail, pheasants, red‐legged partridge, ostriches, a lesser flamingo, pigeons, sparrows, and other wild birds have been found to be naturally infected. Pheasants and geese, ducks, and budgerigars are susceptible by artificial inoculation. 

Natural infection in chickens has been observed as early as 1 week, but acute infection is generally seen when chickens are 4–16 weeks old and turkeys are 10–24 weeks old. Acute infection occasionally occurs in adult chickens. Chronic infection follows the acute phase and may persist for the life of the flock. The chronic stage may be seen at any age and in some flocks may not be preceded by an acute infection (McMullin 2020).

4.3 Transmission

Lateral transmission occurs readily by direct contact. Birds are infected for life and remain carriers. In many respects, the spread appears to be similar to that of M. gallisepticum except that it is more rapid. However, slow‐spreading infections have been reported. Transmission occurs via the respiratory tract, and usually 100% of the birds become infected, although it is possible for only a few to develop clinical signs. Infection may also occur as a result of environmental contamination or fomites. Vertical transmission plays a major role in spread of M. synoviae in chickens and turkeys; however, several flocks hatched from infected breeders may remain free of infection. Experimental infection of broiler breeders resulted in M. synoviae infection in the trachea of day‐old progeny, infertile eggs, and dead‐in‐shell embryos 6–31 days postinoculation (Jan, Brenner et al. 1996). When commercial breeder flocks become infected during egg production, the egg transmission rate appears to be highest during the first 4–6 weeks after infection; transmission thereafter may cease, but infected flocks may shed at any time (McMullin 2020).

5. Pathogenesis

Infectious synovitis has been seen in 6‐day‐old chicks, suggesting that the incubation period can be relatively short in birds infected by egg transmission. The incubation period following contact exposure is generally 1–21 days. Antibodies may be detected before clinical disease becomes evident. In birds experimentally infected at 3–6 week of age, the incubation period varies from 2 to 20 days, depending on the route of administration. Intratracheal inoculation results in infection of the trachea and sinus as early as 4 days and readily spreads to contact birds. Air sac lesions are at a maximum 17–21 days after aerosol challenge. The incubation period also varies with titer and pathogenicity of the inoculum.

The route of M. synoviae infection may play a significant part in the resulting disease. Natural infection may occur vertically in ovo or horizontally by direct contact or airborne spread. Pathogenicity of M. synoviae strains generally involves attachment and colonization of the upper respiratory tract plus additional unidentified factors associated with systemic invasion and lesion production. 

M. synoviae isolated from air sac lesions are more apt to cause airsacculitis, whereas those isolated from synovia are more apt to produce synovitis. Airsacculitis is exacerbated by Newcastle disease infectious bronchitis vaccination or any respiratory infection. The severity of the airsacculitis depends on the virulence of the infectious bronchitis virus used in conjunction with M. synoviae. Air sac lesions are greatly enhanced by cold environmental temperatures. Infectious bursal disease causes immunosuppression in chickens and dual infection with M. synoviae results in more severe air sac lesions. Nervous signs with lesions of meningeal vasculitis have been seen in M. synoviae‐infected turkeys displaying severe synovitis (McMullin 2020).

6. Clinical signs and pathology 

6.1. Clinical signs

Chickens: The first observable signs in a flock affected with infectious synovitis are pale comb, lameness, and retarded growth. As the disease progresses, feathers become ruffled and the comb shrinks. In some cases, the comb is bluish red. Swellings usually occur around joints and breast blisters may occur. Hock joints and foot pads are principally involved, but in some birds most joints are affected. Birds are occasionally found with a generalized infection but without apparent swelling of the joints. Birds become listless, dehydrated, and emaciated. Although birds are severely affected, many continue to eat and drink if placed near feed and water. Acute signs described above are followed by slow recovery; however, synovitis may persist for the life of the flock. In other instances, the acute phase is absent or not noticed, and only a few chronically infected birds are seen in a flock. Chondrodystrophy was noted in the opposite leg of chickens inoculated via the foot pad. This may have been caused by increased weight‐bearing stress on the leg opposite the affected leg. Outbreaks of M. synoviae in brown egg layers in the Netherlands were associated with amyloid arthropathy, which was reproduced experimentally.

Chickens affected by the respiratory form of M. synoviae may show slight rales in 4–6 days or may be asymptomatic. Progeny of M. synoviae ‐infected breeders may have increased air sac condemnations, reduced weight gains, and reduced feed efficiency. Experimental inoculation of hens with M. synoviae resulted in a detectable drop in egg production in 1 week postchallenge; by 2 weeks production dropped 18%, and by 4 weeks production returned to normal. With naturally occurring infection of adults, however, there may be little or no effect on egg production or egg quality, although instances of egg production losses in commercial layers have been observed. M. synoviae has been implicated as a contributing factor in the development of Escherichia coli peritonitis syndrome and associated mortality in commercial layers. M. synoviae has also been linked with abnormalities of the apical eggshell in a number of countries. Broiler breeder hens appear to be less susceptible to producing eggs with abnormalities after M. synoviae infection (McMullin 2020).

Turkeys: M. synoviae generally causes the same type of signs in turkeys as in chickens. Lameness may be the most prominent sign. Warm flocculent swellings of 1 or more joints of lame birds are usually found. Occasionally, there is enlargement of the sternal bursa. Severely affected birds lose weight. Respiratory signs are not usually observed in turkeys, but M. synoviae has been isolated from sinus exudates obtained from turkey flocks exhibiting a very low incidence of sinusitis, and from tracheas and choanal clefts of turkeys exhibiting increased mortality and pneumonia. Airsacculitis may at times occur in day old and older turkeys in M. synoviae ‐infected flocks. Rhoades described a synergistic effect of M. synoviae and M. meleagridis in producing sinusitis in turkeys (McMullin 2020).

6.2 Pathology

Gross pathology 

Chickens: In the early stages of the infectious synovitis form of the disease, chickens frequently have a viscous creamy to gray exudate involving synovial membranes of the tendon sheaths, joints, and keel bursa, and hepatosplenomegaly. Kidneys are usually swollen, mottled, and pale. As the disease progresses caseous exudate may be found involving tendon sheaths, joints, and extending into muscle and air sacs. Articular surfaces, particularly of the hock and shoulder joints, become variably thinned to pitted over time. Generally, no gross lesions are seen in the upper respiratory tract. In the respiratory form of the disease, airsacculitis may be present.

Turkeys: Swellings of the joints may not be as prominent as in chickens, but fibrinopurulent exudate is frequently present when the joints are opened. Lesions in the respiratory tract are variable (McMullin 2020).

Microscopic pathology

The histopathology of infectious synovitis in chickens and respiratory disease caused by MS in chickens and turkeys has been described. The joints, particularly of the foot and hock, have an infiltrate of heterophils and fibrin into joint spaces and along tendon sheaths. The synovial membranes are hyperplastic with villous formation and a diffuse to nodular subsynovial infiltrate of lymphocytes and macrophages. Cartilage surfaces, over time, become discolored, thinned, or pitted. Air sacs may have a mild lesion consisting of edema, capillary proliferation, and the accumulation of heterophils and necrotic debris on the surface, to more severe lesions with hyperplasia of epithelial cells, a diffuse infiltrate of mononuclear cells and caseous necrosis. Other lesions reported to be associated with infectious synovitis are: hyperplasia of the macrophage–monocyte system associated with the sheathed arteries of the spleen; lymphoid infiltrates in the heart, liver, and gizzard; and thymic and bursal atrophy.

6.3 Morbidity and mortality

Chickens: Morbidity in flocks with clinical synovitis varies from 2% to 75%, with 5%–15% being most usual. Respiratory involvement may be asymptomatic, but 90%–100% of the birds may be infected. Mortality is usually less than 1%, ranging up to 10%. Turkeys. Morbidity in infected flocks is usually low (1%–20%), but mortality from trampling and cannibalism may be significant.

Turkeys: Morbidity in infected flocks is usually low (1%–20%), but mortality from trampling and cannibalism may be significant.

7. Diagnosis

7.1 Differential diagnosis

A presumptive diagnosis may be made on the basis of lameness, breast blisters, and enlarged but compressible foot pads or hock joints containing exudates. Other bacteria that may cause synovitis or arthritis must be eliminated by bacteriologic procedures. These include (but are not limited to) Staphylococcus aureus, Escherichia coli, pasteurellae, and salmonellae may also be present as primary causes of synovitis. Fibrosis of metatarsal extensor or digital flexor tendons and lymphocytic infiltration of the myocardium associated with the viral arthritis agent help to differentiate it from M. synoviae. Serum from viral tenosynovitis‐infected chickens does not agglutinate M. synoviae antigen, but one must bear in mind that M. synoviae agglutinins may be present without obvious joint involvement. In cases with respiratory involvement, M. gallisepticum, Avibacterium paragallinarum, Pasteurella multocida, and other causes of respiratory disease should be eliminated (McMullin 2020).

7.2 Laboratory diagnosis

The focus on testing for M. synoviae is different depending on the type of flock. For commercial birds, the goals are to see if vaccination was successful, when the flock seroconverts naturally, or to see if a negative flock status is maintained. For all of these flocks, serological tests are more common to use. In parent stock flocks, because the goal is to remain negative, more sensitive and specific tests are recommended. As a result, PCR is becoming the favored test, as the time for detection is more rapid and more accurate. The common serological tests include serum plate agglutination test (SPA, RPA), hemagglutination inhibition (HI), and enzyme linked immunosorbent assay (ELISA), all of which measure M. synoviae-specific antibodies of different types. SPA detects IgM antibody, found 3–5 days after infection and can persist for up to 80 days. HI and ELISA detect IgG antibody, typically found 7–10 days after infection, which can persist for up to 6 months. All serological tests for M. synoviae may show a low level of false positive results. False positives are most commonly observed in young chicks and birds vaccinated with an oil-emulsion 2–4 weeks prior to the serum test. Serological tests should therefore only be utilized for screening purposes, and positive results must be followed by isolation or PCR testing for confirmation. Polymerase chain reaction (PCR) is becoming the preferred method to confirm M. synoviae infection in a flock. The test detects M. synoviae -specific DNA, which implies the M. synoviae organism was in the bird at the time of sampling. M. synoviae-specific PCR tests have high sensitivity and specificity. The test only takes a few hours to obtain a result and will detect M. synoviae infection before serological tests are able to pick up a positive. Due to this, many parent stock farms are now using PCR sampling for screening purposes. For this, it is important to sample a minimum of 25 birds. The best samples should be taken from the bird’s choanal cleft and/or synovial membranes. An additional benefit to PCR testing is that specific DNA probes have been developed to differentiate M. synoviae field and vaccine strains. Culturing M. synoviae is most successful from acutely affected birds, becoming more difficult as the infection progresses. Samples include affected respiratory organs (trachea, air sacs, lungs, and sinuses). Should the birds display lameness, the affected synovial membranes and any exudate can be sampled. Isolation of mycoplasma requires special culture media and technique taking several days for a result. Immunofluorescence tests on mycoplasma colonies is a rapid and reliable method of identification of M. synoviae.

8. Treatment

M. synoviae is susceptible in vitro to several antibiotics, including chlortetracycline, danofloxacin, enrofloxacin, lincomycin, oxytetracycline, spectinomycin, spiromycin, tetracycline, tiamulin, tilmicosin, aivlosin, and tylosin  Soluble lincomycin‐spectinomycin (2g/gallon of drinking water)  and tiamulin in the drinking water (0.006%–0.025%) have been shown to be effective in preventing clinical signs. Generally, suitable medication is of value inpreventing airsacculitis or synovitis, but treatment of existing lesions is less effective. Antibiotic medication is not thought to eliminate M. synoviae infection from the flock, but reports have been somewhat variable, maybe because of differences in treatment and M. synoviae strains.

In contrast to M. gallisepticum, M. synoviae isolates appear to be resistant to erythromycin. High‐level resistance to erythromycin and tylosin developed rapidly after low level exposure in vitro, but enrofloxacin resistance developed more gradually. No resistance to tiamulin or oxytetracycline was shown.

Treatment of eggs with antibiotics such as tylosin by egg dipping, or egg inoculation with tylosin and gentamycin, or heat treatment of hatching eggs has been used in breeding flocks to prevent egg transmission of M. synoviae. Exposure of breeders before the onset of egg production with virulent M. synoviae will reduce egg transmission. This should only be used in flocks in which infection will almost certainly occur (McMullin 2020).

List of VEMEDIM’products support for Mycoplasma synoviae infection, click on the product name to have further detail information

No.	Product’s name	Composition	Image
1	Licocin	Lincomycin Sulate 100 mg
2	Lincoseptryl	Lincomycin 80 mg Sulfamethoxazole 80 mg
3	OTC 20%LA	Oxytetracycline 200 mg
4	Vime- Pikacin	Spiramycin adipate 170 000 IU Kanamycin  50 mg Dexamethasone 0.5 ml
5	Genta - Tylo	Tylosin Tartrate 150 mg Gentamycin Sulfate 60 mg Dexamethasone  1 mg

9. Control and prevention of mycoplasma

9.1 Control and prevention

M. synoviae is egg transmitted and the most effective method of control is to select chickens or turkeys from M. synoviae‐free flocks. In countries where primary breeding stocks are free of infection, M. synoviae‐free sources of replacement breeding stocks should be available. Effective biosecurity measures should be used to prevent introduction of the infection.

Outbreaks of M. synoviae infection in broilers can often be traced to a specific breeder flock. By the time the infected breeder flock is found, egg transmission may be low or no longer of clinical significance. The decision to slaughter infected parent breeder flocks is often made on an economic basis. If such flocks are kept for egg production, progeny should be hatched separately and isolated from M. synoviae‐free flocks. Antibiotic treatment of breeders is not effective in eliminating M. synoviae infection, although the level of egg transmission may be reduced (McMullin 2020).

9.2 Vaccination

An inactivated, oil emulsion bacterin has been commercially available, but its role in the control of M. synoviae has not been adequately studied. It is commonly believed that these vaccines induce a strong humoral antibody response, but this may not be necessarily correlated with protection against infection. A live temperature sensitive M. synoviae vaccine strain, M. synoviae ‐H, was selected by mutagenesis of a field isolate from Australia. Its safety and efficacy have been established under laboratory and field conditions. Vaccine doses of 4.8×105 cfu/mL were protective; protective immunity was detected after 3–4 weeks post vaccination and persisted for at least 40 weeks. The vaccine has been shown to be effective in reducing apical eggshell abnormalities caused by M. synoviae infection. The temperature‐sensitivity phenotype of the M. synoviae ‐H vaccine is believed to be mediated by a point mutation in its GTP‐binding protein Obg, although factors other than the temperature‐sensitive phenotype appear to be involved in the attenuation of the M. synoviae ‐H vaccine strain. This vaccine has received wide use in Australia and many major poultry‐producing countries but registration in the United States is pending. The safety and efficacy of M. synoviae‐H vaccine have also been assessed in turkeys (McMullin 2020).

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