Avian mycoplasmosis in poultry treatment

18/09/2022

Control
Several vaccines are available for chickens (inactivated, ts-11, 6/85, and F strain). In general, vaccination does not prevent infection, but helps to reduce clinical signs and losses in chicken egg production. There are no good vaccines available to control Mycoplasma in turkeys. In fact, the F strain vaccine can be very pathogenic for turkeys. Ensure that birds are supplied from a Mycoplasma-free operation and test new birds before introducing to the existing flock. Protect the flock from wild birds, since they can carry M. gallisepticum. Incubation of eggs from Mycoplasma-negative flocks must be completely separated from eggs derived from positive flocks.

18/09/2022

Treatment
Mycoplasma infections can be treated with antibiotics to alleviate clinical symptoms. Avoid antibiotics that target the bacterial cell wall, as Mycoplasma do not have one. Tylosin, tilmicosin, and tiamulin are useful to reduce the mycoplasma load in the flock. However, antibiotic therapy cannot completely eliminate mycoplasma from the flock, which will remain Mycoplamsa-positive.

18/09/2022

Differential Diagnoses
Viral respiratory diseases. Infectious bronchitis, low pathogenic avian influenza, Newcastle, infectious coryza, avian cholera. In turkeys, chlamydiosis, respiratory cryptosporidiosis, low pathogenic avian influenza, Newcastle, aspergillosis, and Ornithobacterium rhinotracheale (ORT).

18/09/2022

Diagnosis
PCR and serology are the preferred techniques for diagnosis. Due to the slow transmission of the organism and the time necessary to develop an immune response, serology is not useful before 5 weeks of age. In adult birds, necropsy findings include: mucus and congestion in sinuses and trachea, airsacculitis , and in cases of secondary bacterial contamination, caseous exudate in the air sacs and abdominal cavity In pipped embryos, the presence of caseous exudate in the thoracic air sacs strongly correlates with the infection of the breeders with M. gallisepticum. Infections with M. synoviae will show creamy/grey exudate surrounding the synovial membrane of the tendon sheets and joints

18/09/2022

Transmission
The main portals of entry are the conjunctiva and the respiratory epithelium. Infected breeders transmit the infection to the eggs, contaminating the embryos. Horizontal transmission occurs after contaminated embryos hatch. Fomites are an important mechanism for horizontal transmission. It is important to note that wild songbirds can also be infected with M. gallisepticum and serve as reservoirs for the organism. Because previously infected birds will remain carriers of the organism even in the absence of clinical signs, this disease is impossible to eliminate in facilities that do not practice "all-in all-out."

18/09/2022

Occurrence
M. gallisepticum is distributed worldwide, affecting chickens, turkeys, ducks, pheasants, quail, and partridges. M. synoviae, which affects chickens and turkeys, is also widely distributed. M. meleagridis is only present in turkeys. The incubation period is slow and highly variable (1-3 weeks). Dissemination within the flock is also slow; thus in general, mycoplasmosis occurs in birds older than 4-5 weeks of age. The appearance of clinical signs seems to be related to stress factors. For example, it is common for layers to be infected at 6-10 weeks of age, but clinical signs may not appear until s*xual maturity and egg production (after 18 weeks of age). The disease is most common in layer farms because animals remain carriers of the organism, even after treatment.

18/09/2022

Clinical signs
Typical respiratory signs, including conjunctivitis, facial edema, tracheal rales, coughing, and nasal discharge, are commonly seen in birds with mycoplasmosis. Sinusitis is especially common in turkeys. Nostrils often appear dirty due to feed particles adhering to the mucus on the nostril surface. M. synoviae tends to produce a more benign infection, but it can become systemic, resulting in synovitis affecting joints and tendon sheets. Inflamed and uneven hocks may be seen; severe cases will result in lameness. Mortality is usually low in uncomplicated cases, especially in mature animals. Co-infection of lesions with other agents (e.g. E. coli) can result in more severe clinical signs and increased mortality. In laying birds, egg production may be affected, especially in unvaccinated flocks. Infection of embryos results in reduced hatchability, particularly due to increased numbers of pipped embryos that are unable to complete the hatching process.

18/09/2022

Mycoplasmosis is caused by several species of pathogenic Mycoplasma bacteria. The most relevant species in Pennsylvania include M. gallisepticum, M. synoviae, and M. meleagridis. Most disinfectants are effective against Mycoplasma, but once inside the host, the organism is very persistent. This genus of bacteria lacks a cell wall, unlike most typical bacteria. Therefore, when considering antibiotic treatment, it is important to avoid those antibiotics which target the cell wall. The antibiotics tylosin, tilmicosin, and tiamulin are useful to reduce the Mycoplasma load in the flock and alleviate clinical symptoms.

10/06/2022

14/11/2021

Mycoplasma gallisepticum control
Mycoplasma gallisepticum (MG) infections are common within the poultry industry and can have a particularly damaging impact on commercial layer flocks. In table egg laying flocks, MG infection can result in significant egg production losses.
For decades, antibiotics have been used to treat MG infections and single live or inactivated vaccines have been used globally to prevent the disease. But Dr. Ferguson-Noel set out to investigate claims from field veterinarians that a combined vaccine approach offered better protection.

14/11/2021

Avian mycoplasmosis in poultry treatment
Mycoplasma gallisepticum is the most economically significant mycoplasma pathogen of poultry, and has a world-wide distribution. In common with other mycoplasmas, M. gallisepticum is minute in size with minimal genetic information and with a total lack of a bacterial cell wall. These properties are reflected in a high degree of interdependence between M. gallisepticum and the host animal, and in the fastidious nature of the organism in vitro. Strains of M. gallisepticum differ markedly with respect to important biological properties such as pathogenicity, infectivity, tissue tropism and transmissibility. In addition, phenotypic variation of major surface antigens occurs at high frequency, which is a probable explanation for chronic infection by M. gallisepticum despite a strong immune response. Infection with M. gallisepticum has a wide variety of clinical manifestations, but even in the absence of overt clinical signs, the economic impact may be significant. The most dramatic disease presentation of M. gallisepticum is chronic respiratory disease in meat-type birds, often as one of several aetiological agents in a multi-factorial disease complex. Transmission of M. gallisepticum in ovo from infected breeder birds to progeny is the major route of dissemination of the infection, and is the prime consideration for international trade. In most countries, control programmes for M. gallisepticum are based on maintaining commercial breeding stock free of infection. In instances where control of M. gallisepticum infection is not feasible, vaccination, especially with newly developed live M. gallisepticum vaccines, is being evaluated as an option. Major advances in diagnostic methods have been made in recent years. Control programmes have been based on serological methods, with screening for infection usually accomplished by the slide plate agglutination (SPA) test or by enzyme-linked immunosorbent assay. Further serological testing and/or demonstration of the presence of the organism must be used to confirm SPA suspected positive tests. In principle, detection of the presence of the M. gallisepticum organism can be by isolation of the organism or detection of the deoxyribonucleic acid by molecular methods. Polymerase chain reaction represents a rapid and sensitive alternative to traditional culture methods, which require time-consuming specialised techniques. The development of molecular typing methods affords new opportunities for epidemiological studies and identification of reservoirs of infection.

16/06/2021

Prevention of disease by ensuring birds are acquired from an Mg free source is the most effective approach to prevent introduction of disease into a flock. However biosecurity and sanitation are essential to prevent the spread of Mg into a susceptible flock. Biosecurity should keep wild birds away from the flock and visitor access to the flock should be restricted and strict sanitation procedures should be followed, with no equipment being shared with other bird owners, or thorough cleaning and disinfection applied. When a flock has been removed a thorough cleaning and disinfection should be carried out. If a flock has become infected then depopulation and an extended down time with thorough cleansing and disinfection should be instigated before repopulating from a clean source.

Mg is generally susceptible to a number of antibiotics, however antibiotic resistance is increasingly being reported and it is known that most antibiotics just suppress the clinical signs and do not eliminate the infection. Antibiotics that should be effective include the macrolides, tetracyclines, spectiniomycin, lincomycin and fluoroquinolones (Salami et al., 1992). However Gharaibeh and Al-Rashdan (2011) reported resistance to 8 antibiotics in three families of antibiotics and Gerchman et al. (2008) reported resistance to the fluoroquinolone enrofloxacin. Soaking of infected eggs in antibiotics may prevent the transfer of Mg infection in ovo, but it may reduce egg hatchability (Hall et al., 1963).

Inactivated bacterins have proved to be efficacious in some cases in reducing respiratory signs and lesions in chickens and reducing egg production losses and transmission, however other studies have shown minimal or no effect (Levisohn and Kleven, 2000). Three live Mg vaccines are available in different countries in the world. These are the F strain; 6/85 and ts-11, although not all countries officially permit use of live vaccines (Levisohn and Kleven, 2000). Some vaccines are for use in chickens only while others can also be used in turkeys. Some debate continues about the use of live vaccines, as some reports exist of introduction of disease into flocks by using the vaccines, whilst others report that the wild strain is eliminated by use of the vaccine. Feberwee et al. (2006) reported reduction in Mg infection but insufficient to prevent spread of disease. Ideally flocks should be kept free of Mg.

Mg is an OIE listed disease even though it occurs worldwide. The EU Directive 2009/198 includes Mycoplasma gallisepticum and Mycoplasma meleagridis and relates to animal health conditions governing intra-community trade and imports from third countries of poultry and hatching eggs.

16/06/2021

Mycoplasma gallisepticum is believed to cost the worldwide poultry industry over US $780 million every year. Loss of egg production in the United States is believed to cost over US $120 million, without the cost of culling and restocking infected flocks to prevent further spread (Hennigana et al., 2011). Serious economic losses are also incurred through reduced growth production, carcass condemnations, and re****ed growth in juveniles. Also, chickens have been documented to lose about 16 eggs over their laying cycle of 45 weeks (Peebles et al., 2012).

Earlier Mohammed et al. (1987) estimated 127 million eggs were lost during an Mg infection in 1984 in Southern California, with a financial loss of US $7 million

16/06/2021

Mycoplasma gallisepticum is in the Class Mollicutes which are phenotypically distinguished from other bacteria by their minute size and total lack of a cell wall. They have a small genome size (996?422 bp for Rlow strain (Papazisi et al., 2003)) which accounts for their complex nutritional requirements and its obligate parasitic mode of life.

The primary habitats of mycoplasmas in general are the mucosal membranes of the respiratory tract, and/or the uroge***al tract, eyes, mammary glands and joints (Levisohn and Kleven, 2000). Mg is one of the species of mycoplasma that can cause acute and chronic diseases at multiple sites, but is usually seen as a parasite of the airways of affected avian species. Uncontrolled proliferation of the organism in susceptible birds causes severe inflammation of the mucosa of the sinuses and/or trachea, and infection extends to the lungs and air sacs (Browning et al., 2010).

Transfer of the organism from hen to progeny through the eggs is an important means of spread in poultry. It is therefore important to source poults or chicks from Mg free breeding stock. Introduction of older birds into a flock can present a significant risk of introducing Mg, especially if purchased from mixed sources or through markets. Any stress, including the social stress of mixing birds together can precipitate an apparent healthy but infected bird to start shedding the organism. Direct spread from bird to bird via the respiratory route can occur readily, as well as on fomites, but spread within a flock is generally slow with an incubation period of 6 to 21 days. It is thought that an infection may persists for 18 months or even longer, but survival outside of the host under farm conditions was thought to be unlikely to exceed a few days, although Mg’s ability to form a biofilm may mean they can survive longer (Chen et al., 2013). Biosecurity is important to prevent spread from flock to flock, or farm to farm, as the organism can be carried and transmitted through contaminated footwear, clothing or equipment. The role of wild birds as a reservoir of disease and their role in transmitting Mg has been highlighted since the reports of Mg in house finches (Ley et al., 1996). If a flock becomes infected a complete depopulation followed by clean out and sanitization of the premises is required to ensure the disease is eliminated.

Despite its small genome Mg is genetically quite variable as it possesses between 30 and 70 variant vlhA genes, most of which are translationally competent (Baseggio et al., 1996). These genes have probably been acquired by lateral gene transfer between Mycoplasma species, but only one gene appears to be transcribed at a time, so only a single variant of this lipoprotein is expressed on the cell surface at any one time (Browning et al., 2010). This gene expression may change after initial infection, suggesting it has an initial role in adherence, but then the antigenic variation helps the organism evade the host’s immune response (Browning et al., 2010). Several approaches to molecular epidemiological typing have been used to differentiate isolates. Feberwee et al. (2005) used amplified fragment length polymorphism (AFLP) and random amplified polymorphic DNA (RAPD) analysis to give five clusters of Mg. Sprygin et al. (2010) demonstrated that Russian Mg isolates clustered more closely to each other than to isolates from USA, Australia, China and Iran using partial sequencing of a pvpA gene fragment. Ghorashi et al. (2010) used a PCR of the vlhA gene and high-resolution melting curve analysis to differentiate 10 Mg strains which included the ability to differentiate three vaccine strains.

16/06/2021

Mycoplasma gallisepticum infections vary from asymptomatic to severe depending on the infecting strain and other factors. More severe infections are seen when the birds are infected concurrently with other bacterial or viral pathogens, including Escherichia coli, Newcastle disease virus or infectious bronchitis virus. Infected birds initially develop respiratory signs that may include rales, coughing, sneezing, nasal discharges and dyspnea. Turkeys may present with more severe clinical signs which includes swelling of the infraorbital sinus. Conjunctivitis with a frothy ocular exudate occurs more frequently in turkeys, but does also occur in chickens. The route of exposure and the infectious dose of Mg, in addition to environmental and stress factors such as temperature and ammonia concentration, age and type of birds are factors that influence the course of the disease (In: Levisohn and Kleven, 2000). Production is lower in infected flocks, with decreased weight gain, feed efficiency and egg production. In chickens with uncomplicated infections the morbidity rate is high and the mortality rate low; however more severe disease occurs when the birds are co-infected with other pathogens. Mortality rates in turkeys are generally higher than in chickens. Mg has been implicated in salpingitis and other pathologies of the reproductive system, but it is not clear if this is the main or sole cause of reduction in egg production (Nunoya et al., 1997) which is particularly noticeable at times of peak lay.

16/06/2021

ist of Symptoms/Signs
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Digestive Signs / Anorexia, loss or decreased appetite, not nursing, off feed Sign
General Signs / Lack of growth or weight gain, re****ed, stunted growth Sign
General Signs / Lameness, stiffness, stilted gait in birds Sign
General Signs / Orbital, periorbital, periocular, conjunctival swelling, eyeball mass Sign
General Signs / Swelling of the limbs, legs, foot, feet, in birds Sign
General Signs / Torticollis, twisted neck Sign
General Signs / Underweight, poor condition, thin, emaciated, unthriftiness, ill thrift Sign
General Signs / Weight loss Sign
Musculoskeletal Signs / Abnormal curvature, angulation, deviation of legs, limbs, feet of birds Sign
Nervous Signs / Dullness, depression, lethargy, depressed, lethargic, listless Sign
Ophthalmology Signs / Chemosis, conjunctival, scleral edema, swelling Sign
Ophthalmology Signs / Conjunctival, scleral, injection, abnormal vasculature Sign
Ophthalmology Signs / Conjunctival, scleral, redness Sign
Ophthalmology Signs / Lacrimation, tearing, serous ocular discharge, watery eyes Sign
Ophthalmology Signs / Purulent discharge from eye Sign
Reproductive Signs / Decreased, dropping, egg production Sign
Respiratory Signs / Abnormal lung or pleural sounds, rales, crackles, wheezes, friction rubs Sign
Respiratory Signs / Coughing, coughs Sign
Respiratory Signs / Dyspnea, difficult, open mouth breathing, grunt, gasping Sign
Respiratory Signs / Increased respiratory rate, polypnea, tachypnea, hyperpnea Sign
Respiratory Signs / Mucoid nasal discharge, serous, watery Sign
Respiratory Signs / Purulent nasal discharge

16/06/2021

Pathology
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The gross lesions of the respiratory tract may be mild and only consist of excess mucus or catarrhal exudate in the nares, trachea and lungs and oedema in the airsac walls. Caseous exudate may appear later in the airsacs or attached to their walls. Dilation of the infraorbital sinuses, particularly in turkeys, may be initially caused by mucus which may then be replaced by caseous material. In disease exacerbated by other pathogens, the lesions are more severe, pericarditis and perihepatitis may accompany the lesions of the airsacs and upper respiratory tract (Blaxland et al., 1982).

Encephalopathy can occur particularly in turkeys, but usually no gross lesions are visible. In salpingitis cases, caseous exudate occurs in the oviduct.

Diagnosis
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Clinical signs, post mortem and histological lesions are not pathognomic for Mg. Isolation of the causative organism is the definitive confirmation of infection, but this requires mycoplasma culture media and incubation of cultures for three to four weeks. Specificity of the diagnostic tests does present some difficulties as birds are often infected with more than one mycoplasma species, sometimes as many as five Mycoplasma species have been detected in one sample; and many of these are considered to be non-pathogenic mycoplasmas, which may give a false negative or false positive result. Surveillance for clinical signs and lesions of mycoplasma infection must be ongoing. Testing should be carried out on a statistical representative number of birds in a flock and birds should be sampled at random from each part of the flock.

Serological detection of antibodies is recognised as a way of monitoring flocks and detection of disease, but this usually also requires confirmation by other diagnostic methods along with veterinary diagnosis of clinical signs in the flock. The haemagglutination-inhibition (HI) test is rarely used now, but is still a prescribed test listed in the OIE terrestrial manual. More commonly used is the rapid/serum plate agglutination test or rapid slide agglutination test. This is a flock test as the test does give some false reactions. The early guidelines were that 60 serum samples should be taken per house and a flock was only considered positive when more than 15% of the undiluted samples were positive or more than 3% with a titre of more than 1:8 (Intervet information sheet). However different manufacturers now make this test and the sensitivity and specificity may vary with between manufacturers and different batches of the antigen. The test is simple to perform, but manufacturer’s instructions should be followed and appropriate positive and negative control serum tested. Essentially the antigen and serum to be tested should be allowed to warm to 20-25°C. Equal volumes of the serum and the antigen are mixed for two minutes and any agglutination observed recorded as positive. Dilutions of sera should be tested to resolve any doubts about a positive test result. Only fresh sera should be tested.

More recently several manufacturers are producing ELISA tests and these are designed for laboratory use. These are generally a flock based test and users should be aware that some differences in test performance and batches do occur.

A species-specific confirmatory serological test using a Western blot/immunoblot method has been developed (Welchman et al., 2013).

Antigen detection using molecular tests have been developed, they are mainly polymerase chain reaction (PCR) based tests. A PCR method for detecting Mg is given in the OIE Manual (OIE, 2012), but many other methods have been published as referenced in Kleven (2008). A PCR method using the 16S rDNA gene followed by the use of denaturing gradient gel electrophoresis has been able to detect and identify the majority of Mycoplasma species including Mg. This test is sensitive and will identify all of the Mycoplasma species that affects avian species in one test and will also detect and identify mixed infections (McAuliffe et al., 2005).

Samples to be tested can be swabs from live birds or swabs/tissues from carcases. Tracheal, choanal, or air sacs swabs are tested. The same samples can be used for mycoplasma culture.

No single medium formulation has been accepted as optimum for growth of Mg. Mg ferments glucose and requires 10-15% horse or swine serum, and a yeast source to provide nutrients (Kleven, 2008). Broth culture is usually more sensitive than agar but both agar and broth are inoculated at the same time and incubated at 37°C ideally for growth on agar with an additional 5-10% CO2 in a humid environment. Bradbury (1998a) describes methods to recover mycoplasma s from birds and gives details of suitable media formulations. Mg will ferment glucose producing an acid pH in the broth media and typical “fried egg” colonies can be observed on agar using a microscope under low magnification (approx 35X magnification). Mixed cultures are often obtained and identification need to be confirmed either by molecular methods or using a specific Mg antiserum in a growth inhibition test (Poveda and Nicholas, 1998) or using immuno-fluorescence antibody test (Bradbury 1998b).

16/06/2021

Four Mycoplasma species are recognised as pathogens of avian hosts, although more than 23 different Mycoplasma species have been recovered from birds. Mycoplasma gallisepticum is the pathogen addressed in this datasheet, but Mycoplasma synoviae may be seen in chickens and turkeys in association with synovitis and/or airsacculitis; Mycoplasma iowae may occur in several hosts but it is normally associated with mortality of turkey embryos but can give rise to joint and bone abnormalities and occasional airsacculitis; Mycoplasma meleagridis is usually found in turkeys causing airsacculitis, poor growth and skeletal abnormalities in progeny, and it has been associated with poor hatchability. M. gallisepticum causes chronic respiratory disease of domestic poultry, especially in the presence of management stresses and/or other respiratory pathogens. Disease is characterised by coryza, conjunctivitis, sneezing, and by sinusitis, particularly in turkeys and game birds. It can result in loss of production and downgrading of meat-type birds, and loss of egg production.

Mycoplasma gallisepticum and Mycoplasma synoviae are on the World Organisation for Animal Health (OIE) list of economically important diseases and infections are notifiable to them. The EU Directive 2009/198 includes Mycoplasma gallisepticum and Mycoplasma meleagridis and relates to animal health conditions governing intra-Community trade and imports from third countries of poultry and hatching eggs.

Mycoplasma gallisepticum (Mg) is the most economically significant mycoplasma pathogen of poultry and has a world-wide distribution (Levisohn and Kleven, 2000). Infection with Mg may manifest in different ways but chronic respiratory disease (CRD) and downgrading of carcasses in meat-type birds is probably the most severe forms. Mg is often one of the aetiological agents in a multi-factorial disease complex, which may include respiratory viruses, Escherichia coli, Haemophilusparagallinarum and other bacteria. Loss of egg production in laying birds may occur and is usually most marked at peak laying times. Conjunctivitis and sinusitis may occur with severe infections causing inflammation of the tissues around the eyes resulting in a swollen distorted face. Mg is transmitted both vertically from hen to progeny through the egg (in ovo), through the semen of infected roosters; and horizontally by the respiratory route. Mg has been reported in wild birds, particularly as a cause of conjunctivitis in house finches (Carpodacus mexicanus) in North America where the disease emerged in 1994 (Ley et al., 2006).

Antimicrobial treatment may reduce the impact of the disease, but can not be relied upon to eliminate the disease. Good biosecurity and obtaining birds from Mg free stock is a good way of preventing diseases. Some live attenuated vaccines are available, but some questions about their effectiveness and ability to cause disease still need to be addressed.

The Mycoplasma species that occur in avian host species are not zoonotic.

04/06/2021

This chapter focuses on the mycoplasmas of animals. The mycoplasmas, which are members of the class Mollicutes and the order Mycoplasmatales, are the smallest free-living organisms. Unlike bacteria, they have no cell wall but are bounded by a membrane. This explains their remarkable pleomorphism. Most of the mycoplasmas require sterol for growth. The chapter presents the taxonomy of class Mollicutes. Clinical observations, laboratory studies, and the results of controlled research have demonstrated causal relationships to disease for several mycoplasmas. Pathogenic mycoplasmas have a predilection for serous surfaces—such as the thoracic, abdominal, and articular cavities of cattle and swine and the air sacs of poultry—where they localize and persist protected from antibody and therapeutic agents by the fibrinous tissue reactions that characterize mycoplasmosis. They cause pneumonia, arthritis, and serositis in swine and mastitis, arthritis, and pneumonia in cattle, sheep, and goats. The significance of mycoplasmas in diseases of the ge***al tract is not clear. The clinical signs of mycoplasmosis are not distinctive. The isolation and further cultivation for the identification of most mycoplasmas from clinical materials is not difficult if a suitable, well-prepared medium is used. The chapter further reviews the laboratory procedures and isolation techniques employed for mycoplasmas.

Avian mycoplasmas are primarily associated with respiratory diseases of chickens and turkeys, althou

04/06/2021

This chapter focuses on the mycoplasmas of animals. The mycoplasmas, which are members of the class Mollicutes and the order Mycoplasmatales, are the smallest free-living organisms. Unlike bacteria, they have no cell wall but are bounded by a membrane. This explains their remarkable pleomorphism. Most of the mycoplasmas require sterol for growth. The chapter presents the taxonomy of class Mollicutes. Clinical observations, laboratory studies, and the results of controlled research have demonstrated causal relationships to disease for several mycoplasmas. Pathogenic mycoplasmas have a predilection for serous surfaces—such as the thoracic, abdominal, and articular cavities of cattle and swine and the air sacs of poultry—where they localize and persist protected from antibody and therapeutic agents by the fibrinous tissue reactions that characterize mycoplasmosis. They cause pneumonia, arthritis, and serositis in swine and mastitis, arthritis, and pneumonia in cattle, sheep, and goats. The significance of mycoplasmas in diseases of the ge***al tract is not clear. The clinical signs of mycoplasmosis are not distinctive. The isolation and further cultivation for the identification of most mycoplasmas from clinical materials is not difficult if a suitable, well-prepared medium is used. The chapter further reviews the laboratory procedures and isolation techniques employed for mycoplasmas.