Mycoplasma auris sp. nov., Mycoplasma cottewii sp. nov., and Mycoplasma yeatsii sp. nov., new sterol-requiring mollicutes from the external ear canals of goats.

Three mycoplasma strains, designated GIHT (T = type strain), UIAT, and VIST, were isolated from the external ear canals of goats and were shown to be serologically distinct from each other and from previously described Acholeplasma, Entomoplasma, Mesoplasma, and Mycoplasma species. Using light and transmission electron microscopy, we showed that the cells of these organisms were small, pleomorphic, coccoid, nonmotile, and nonhelical and that each cell was surrounded by a single cytoplasmic membrane. There was no evidence of a cell wall, and the organisms grew freely in media containing penicillin at concentrations of 1,000 U/ml or more and thallous acetate (final concentration, 1:4,000) and produced the "fried-egg" morphology typical of most mollicutes. Growth occurred both aerobically and anaerobically (as determined by the GasPak method). The ability to catabolize glucose and mannose and the ability to hydrolyze arginine varied among the three strains. All three strains required sterol for growth, and none of the strains hydrolyzed urea. The guanine-plus-cytosine contents of the DNAs of strains UIAT, VIST, and GIHT were determined to be 26.9, 27.0, and 26.6 mol%, respectively. Our data indicate that the three strains represent new Mycoplasma species, for which we propose the names Mycoplasma auris, Mycoplasma cottewii, and Mycoplasma yeatsii. The type strain of M. auris is UIA (= ATCC 51348 = NCTC 11731), the type strain of M. cottewii is VIS (= ATCC 51347 = NCTC 11732), and the type strain of M. yeatsii is GIH (= ATCC 51346 = NCTC 11730).

DNA relatedness between field isolates of Mycoplasma F38 group, the agent of contagious caprine pleuropneumonia, and strains of Mycoplasma capricolum.

DNA-DNA hybridization experiments were carried out in order to clarify the taxonomic relationships between the F38 group of caprine mycoplasmas, the established etiologic agents of classical contagious caprine pleuropneumonia, and Mycoplasma capricolum, an organism associated with septicemia, arthritis, and mastitis in goats and sheep. The taxonomic status of the F38 group has been uncertain, principally because of the serological, genomic, and other properties which it shares with M. capricolum. Tritium-labeled DNAs from the M. capricolum type strain (California kid) and from prototype strain F38 were hybridized with unlabeled DNAs from these two strains and from four other isolates belonging to each group. The results showed consistent DNA relatedness values of about 70% between the F38 and M. capricolum groups, compared with levels of relatedness of about 90 and 85%, respectively, for the strains within each group. In addition, the results of comparisons of these 10 strains in which growth inhibition and immunofluorescence tests were used confirmed the previously reported serological relationships between the two groups and reinforced other observations concerning their shared genomic and cell membrane characteristics, indicating that there is a close taxonomic relationship. However, as the 70% DNA relatedness values between the M. capricolum and F38 groups also indicate a degree of genomic difference inconsistent with a relationship at the species level, we conclude that our findings support previous proposals for classification of the F38 group as a subspecies of M. capricolum.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunogenicity of Mycoplasma gallisepticum.

The serological response and protective immunity elicited in the chicken by the pathogenic Ap3AS strain and the moderately pathogenic 80083 strain of Mycoplasma gallisepticum and variants of strain 80083 attenuated by repeated passage in mycoplasma broth were investigated. Strain 80083 elicited a substantial serum antibody response after administration either in drinking water or by conjunctival sac instillation to 7-week-old SPF chickens. No vaccinated chickens developed air sac lesions when challenged by intra-abdominal (IA) injection with the virulent Ap3AS strain. Chickens vaccinated with strain 80083M (50 broth passages) showed only a weak serological response but were substantially protected when challenged 4 weeks after vaccination. Chickens vaccinated with 80083H (100 broth passages) were serologically negative 4 weeks after vaccination and developed severe air sac lesions after challenge. Thirty-seven-week-old hens vaccinated 6 months previously with strain 80083 had high serum antibody levels and were completely protected against IA challenge with the homologous strain. However, 4/6 showed mild air sac lesions when challenged intra-abdominally with strain Ap3AS. Another group showed high M. gallisepticum serum antibody levels 6 months after vaccination with strain Ap3AS but 4/6 and 2/6 showed mild lesions after IA challenge with strains Ap3AS or 80083, respectively. Strains 80083 or 80083M were administered by conjunctival sac instillation to susceptible 11-week-old commercial pullets at the time of fowl pox vaccination. The concurrent use of both vaccines had no apparent adverse effect on the health of the chickens. Similar protection against IA challenge with strain Ap3AS was produced with the M. gallisepticum vaccines whether used alone or in combination with fowl pox.

Virulence and transmissibility of Mycoplasma gallisepticum.

The virulence of 4 low passage strains of Mycoplasma gallisepticum obtained from different sources within Australia was studied by experimental infection of chickens. Strain Ap3AS, originally isolated from the air sac of a broiler chicken, produced severe air sac lesions following injection into the abdominal air sacs of 2-week or 3-week-old chickens, and adult hens. Strain 80083 which was isolated from a clinically normal broiler breeder hen was also capable of producing gross air sac lesions following intra-abdominal (IA) injection, although it did so less consistently than strain Ap3AS. Strain 82078 isolated from a layer hen and strain QXO which was isolated from a turkey were also moderately pathogenic in terms of the incidence and severity of lesions elicited following IA injection. Strains Ap3AS and 80083 both caused a substantial loss of egg production over a 5 week period after IA infection of 27-week-old hens. Neither strain Ap3AS nor 80083 caused gross lesions or loss of egg production when administered alone into the upper respiratory tract. However, when inoculated into the conjunctival sac in combination with the Vic S strain of infectious bronchitis virus (IBV) strains Ap3AS and 80083 produced identical clinical signs of conjunctivitis. The mean numbers of M. gallisepticum in tracheal washings were significantly higher 2 weeks after infection in the group receiving strain 80083 in combination with IBV than in the group infected with strain Ap3AS and IBV (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Protection against enzootic pneumonia of pigs: intraperitoneal inoculation with live LKR strain of Mycoplasma hyopneumoniae.

Pigs obtained from a mycoplasma-free piggery were randomised into 4 groups of 9. Groups 1 and 2 were injected by the intraperitoneal route with liquid culture of the LKR strain of Mycoplasma hyopneumoniae. Group 1 was injected once and group 2 twice. Group 3 was made up of pigs inoculated by the intranasal route with the virulent Beaufort strain of M. hyopneumoniae; they served as the source of infection for the challenge. Group 4 were uninfected, uninjected controls. Six weeks after the last injection, groups from 1 to 4 were placed in contact. Seven of the pigs in the 1-dose group and 6 in the 2-dose group were free of lesions at necropsy 6 weeks after challenge. Of the two pigs with lesions in the 1-dose group one had only a small lesion but the other had extensive lesions; it had not shown an antibody response after injection of culture. The lesions in the 3 pigs in the 2-dose group were all small. All 9 control pigs had lesions which varied from medium to large in size. The difference in the incidence of pneumonia between the injected and control groups was significant (P less than 0.05) and the proportion of severely affected pigs in the vaccinated groups was significantly lower (P greater than 0.01). There was no difference between those given one dose of vaccine and those receiving 2 doses.

Early serological responses to Mycoplasma hyopneumoniae infection.

A complement fixation (CF) test for Mycoplasma hyopneumoniae antibody, using the complement dilution method, will detect infection in inoculated pigs at an early stage. The interval between inoculation and the first positive CF response--defined as 4.6 u of complement fixed--was determined for four separate methods of inoculation and for pigs with artificially induced pneumonia. Sera from two groups were tested by the enzyme-linked immunosorbent assay (ELISA). By CF, the i.v. group showed positive titers at 9.8 +/- 0.4 (mean +/- SE) days. All pigs in this group had positive titers, most of them at the maximum level of 31 u fixed. The i.p. group first showed positive titers at 13.3 +/- 1.0; 17 of 70 had no CF response. The s.c. group first showed titers at 21.1 +/- 2.6; 7 of 24 did not respond. Intranasal inoculation first produced a CF response at 14.3 +/- 0.9, and all pigs had positive titers. Pigs in contact with those with induced pneumonia first showed titers at a mean of 27.2 days (SE +/- 1.3), and all pigs exhibited a CF response. There was no significant difference between the CF test and ELISA results.

Taxonomy of the Mycoplasma mycoides cluster.

Some of the mycoplasmas found in diseases of ruminants, Mycoplasma capricolum, M. mycoides subsp. capri (PG3), bovine group 7 (PG50), strain F38 and strain Y goat show various degrees of relationship to M. mycoides subsp. mycoides (PG1) and to one another, such that they are referred to as the M. mycoides cluster. Studies using serology, DNA homology, isoenzyme analysis and two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) of proteins have been carried out on the type and reference strains of these organisms and some representative strains in an effort to resolve this cluster into well-defined taxa. An ad hoc committee set up by the Subcommittee on the Taxonomy of the Mollicutes examined the known data, but found that it led to conflicting opinions on the classification of the unspeciated strains, partly because of the lack of guidelines on the relative weight to be given to each criterion, and partly because insufficient strains of each member of the cluster had been examined. Although classifications were proposed, total agreement was not achieved on any one proposal, and the committee believes that decisions on the taxonomy would have to await the results of repeat tests, in which several representative strains of each of the six groups would be included with the type strain.

Overview of mycoplasmoses in sheep and goats.

Over the past few years the role of Mycoplasma F38 in contagious caprine pleuropneumonia has been confirmed in Sudan as well as in Kenya, and further disease outbreaks involving the subspecies of M. mycoides have also occurred in goats. Recent work on the epidemiology of some of the mycoplasmoses of goats has established that feeding of contaminated colostrum is a significant method of transmission of disease to kids. Standard tests for freedom from mycoplasma infection are urgently needed to assist regulatory authorities to permit movement of sheep, goats and cattle within and between countries, and there are indications that serological tests may not always be adequate. Measures to improve this situation are discussed.

Standard antisera produced in ponies for the identification of bovine mycoplasmas: comparative growth-inhibition results from six laboratories.

Antisera to 10 mycoplasma species of bovine origin were produced in 10 ponies and were distributed for evaluation in growth-inhibition tests at 6 laboratories in Australia, England, Denmark, France, and the United States. Except for a few failures with some antigens produced at the 6 laboratories, the antisera induced large zones of growth inhibition in homologous, but not heterologous, systems. These antisera may be useful as standard reagents for the identification of the bovine mycoplasmas.

Growth characteristics of large- and small-colony types of Mycoplasma mycoides subsp. mycoides on 5% sheep blood agar.

Mycoplasma mycoides subsp. mycoides of the large-colony (LC) type was isolated in pure culture on 5% sheep blood agar plates inoculated with lung specimens from a 4-month-old Toggenburg goat. The growth characteristics of this isolate, of four known LC types, and of five known small-colony (SC) types of M. mycoides subsp. mycoides were compared on 5% sheep blood agar at 2, 5, and 7 days. The SC types were not visible at 2 days and did not grow larger than 0.1 mm, whereas the LC types were visible in 2 days and increased in diameter over 7 days to between 0.4 and 0.7 mm. These results indicate that growth on 5% sheep blood agar can be used as an additional marker in differentiating LC and SC types of M. mycoides subsp. mycoides.

Mycoplasmas and mites in the ears of clinically normal goats.

Mycoplasmas were detected in the external ear canal of goats by swabbing and culture. Up to 10(8) colony forming units were recovered from single swabs. The resulting cultures were usually mixtures of mycoplasmas containing up to 5 species. The species present in sequential swabs varied. Pathogenic species (M.agalactiae, M.capricolum, M.mycoides subsp. capri, M.mycoides subsp. mycoides of the large colony (LC) type, M.putrefaciens) were isolated from the ears and in addition 3 untyped mycoplasmas G, U and V were often present. The same mycoplasmas were found in large numbers in the mites Psoroptes cuniculi and Raillietia caprae which were sometimes present in the external ear canal. The role of the mycoplasmas in the external ear canal as a source of infection and disease and of the mites in the spread of infection requires further elucidation.

Pathogenicity of the subspecies mycoides of Mycoplasma mycoides for cattle, sheep and goats.

Recent work has shown that strains classified as M. mycoides subsp. mycoides may be separated into 2 types according to their growth rate and their behaviour in certain biochemical tests. The large colony (LC) types, most of which are from goats, are pathogenic for sheep and goats but apparently not for cattle. The small colony (SC) types include the classical contagious bovine pleuropneumonia (CBPP) strains from cattle and four strains from goats. These SC types are potentially pathogenic for cattle, sheep and goats. Strains of M. mycoides subsp. mycoides from CBPP differ in their virulence in cattle. The degree of virulence is correlated with the quantity of galactan produced in cultures of the organism, suggesting an important role for galactan in pathogenicity. This is consistent with the production by galactan of physiological effects in calves and in the enhancement of infection in cattle given galactan at the same time as cultures of the organism. Contagious caprine pleuropneumonia (CCPP) can be produced experimentally in goats using cultures of M. mycoides subsp. capri. Whether the glucan produced in such cultures is a factor in pathogenicity of this organism has not been determined. Hydrogen peroxide demonstrated in tracheal organ cultures of M. mycoides subsp. capri may contribute to its pathogenicity.

Isolation of Mycoplasma hyopneumoniae from lesions in experimentally infected pigs.

Pigs aged 6 to 9 weeks from enzootic pneumonia-free herds were inoculated intranasally with a suspension of pneumonic lung containing Mycoplasma hyopneumoniae or were placed in contact with such inoculated pigs. All the inoculated pigs had gross lesions of enzootic pneumonia when killed 27 to 42 days after inoculation. The culture methods described enabled M. hyopneumoniae to be isolated from all 29 inoculated pigs. Of 45 pigs in contact with inoculated pigs 35 had gross lesions of enzootic pneumonia when killed 28 to 71 days later and M. hyopneumoniae was isolated from 33. Another 9 had lesions, detected only microscopically, and M. hyopneumoniae was recovered from 3 of these when killed 75 to 98 days after contact began. In a separate experiment M. hyopneumoniae isolated from experimentally infected pigs, and adapted to the culture medium after 6 passages, caused gross lesions of enzootic pneumonia in 1 of 4 pigs inoculated intranasally.

Subdivision of Mycoplasma mycoides subsp. mycoides from cattle and goats into two types.

Some strains of Mycoplasma mycoides subsp. mycoides, mostly isolated from goats, grow to greater turbidity in broth and form larger colonies on solid medium than does the type strain, PG1. These strains also digest casein, liquefy inspissated serum actively and survive longer at 45 degrees C and are referred to as LC (large colony) strains. Strains more closely resembling PG1 have been called SC (small colony) strains. The SC strains comprise those from contagious bovine pleuropneumonia (CBPP) and some from goats. One LC strain was isolated from a steer; all others have come from goats. Differentiation of M. mycoides subsp. mycoides into 2 types on the basis of the characteristics described may be relevant to their role in CBPP.