More than 1,000 Spiroplasma isolates have been obtained from horse flies and deer flies (Diptera:Tabanidae) in the United States and Canada. However, the spiroplasma biota of Central America is poorly known. In August of 1995 and 1998, 13 isolates were obtained in 14 attempts from horse flies of a single species, Poeciloderas quadripunctatus, taken in the Costa Rican highlands (1,100–2,000 m). The majority of the “isolates” proved to be mixtures of two or more Spiroplasma species, but after filter cloning, single strains emerged that were designated as representatives of the 13 accessions. Six distinct spiroplasma serogroups were identified from these isolations. Three of the strains are putative new species with no serological relationship to any other Spiroplasma species. A fourth strain is a putative new species that may be distantly related to S. helicoides, a southeastern U.S. species. These four strains are accorded herein status as representatives of new serogroups: strain BARC 4886 (group XXXV); strain BARC 4900 (group XXXVI); strain BARC 4908 (group XXXVII); and GSU5450 (group XXXVIII). A fifth Spiroplasma species was very closely related to S. lineolae, known previously only from the Georgia (U.S.) coast. The sixth was most closely related to subgroup VIII-3, known from Texas and the southeastern U.S. Discovery of six spiroplasma species in only 13 attempted isolations reflects diversity seldom equaled in southeast Georgia, and never elsewhere in the U.S. These results are consistent with a hypothesis that spiroplasma diversity increases from north (Nova Scotia) to south (Georgia and Costa Rica). The discovery of significant affinity between some spiroplasmas of the southeastern U.S. and the Costa Rican highlands was unexpected, but may reflect a climatically complex Pleistocene history.

Wall-less prokaryotes from guts of five insect species (a corn root maggot [Plecia sp.: Diptera: Bibionidae, strain PS-1], a syrphid fly [Diptera: Syrphidae, strain YJS], two tabanid flies [Tabanus catenatus, strain TAC, andChrysops discalis, strain DF-2], and a vespid wasp [Monobia quadridens, strain MQ-3]) were characterized. The strains grew at 23°–32°C in conventional mycoplasma media containing 10% (vol/vol) serum, or in serum-free mycoplasma medium with or without fatty-acid-Tween-80 supplements. No helical forms were noted in dark-field microscopy, and electron micrographs of thin sections of the strains showed a single membrane. Two strains (DF and MQ-3) cross reacted with antiserum againstAcholeplasma florum. Although the other three strains did not react with antisera to establishedAcholeplasma orMycoplasma species, they were otherwise characteristic of acholeplasmas. This study, the first to demonstrate acholeplasmas from Arthropoda, in conjunction with previous isolations from plant surfaces, suggests that insects may constitute an important reservoir for acholeplasmas.

Mycoplasma pneumoniae is a non-invasive pathogen which colonizes the mucosal surface of the respiratory tract (1-3). The resultant pr imary atypical pneumonia occurs most frequently in children and young adults (4-6), but the incidence of disease is probably underestimated because the symptoms are relatively mild and diagnostic methods are suboptimal (6, 7). Infection with M. pneumoniae is often accompanied by (a) the appearance of cold agglutinins in the patient 's serum (8, 9); (b) a rise in complement fixation (CF) a titer (8, 10); and (c) the product ion of ant ibody that inhibits metabolic processes of the pathogen (8, 11, 12). Still, the role of the immune response during M. pneumoniae infection is not understood. Studies performed with intranasally infected hamsters demonstrated immunoglobul in-producing cells in association with pu lmonary infiltrates (13). Similar experiments cannot be done with human tissue because of the infrequency of mortali ty caused by mycoplasma pneumonia. However, some pertinent observations have been made with h u m a n patients and volunteers and with the experimental hamster model. Prior infection with virulent M. pneumoniae provides some protection against subsequent disease in both hamsters and humans (14-18). It is not known whether this protective effect is due to cellular or humoral immuni ty or both. Other reports suggest that humoral immuni ty may affect disease expression. For example, immunodeficient patients with B cell dysfunction are more severely ill with M. pneumoniae infection than normal patients, but show no evidence of radiographic pneumonia (19). The most commonly used serological test for M. pneumoniae is a CF assay that uses a lipid hapten extracted from the organism as the target antigen (9, 20). However, lipids are rarely good immunogens unless complexed to protein (21). To evaluate whether specific M. pneumoniae proteins are immunogenie during natural infection, radioimmunoprecipi ta t ion (RIP) was used to examine infected h u m a n and hamster sera. This paper reports the identification of two predominant proteinaceous immu-

The Mycoplasma lipophilum cluster (Weisburg et al. 1989) in the hominis group of the mollicutes is re-evaluated in this work to update the phylogenetic framework for classification of species within the genus Mycoplasma. Therefore, sequences of the 16S rRNA gene were determined from previously described species, and 11 were found to be closely related to the M. lipophilum cluster. A selection of members of the other hitherto defined clusters of the hominis group was included for phylogenetic analysis, revealing that the classical M. lipophilum cluster could be re-organized into two clusters, namely the M. lipophilum cluster and the Mycoplasma bovis cluster. The former was found to contain two species, while the latter contained 20 species. The two clusters were closely related, sharing an ancestral branch with the Mycoplasma synoviae cluster. Furthermore, the M. bovis cluster could be divided into subclusters. Interestingly, two species, Mycoplasma equigenitalium and Mycoplasma elephantis, formed a distinct and early branch of the M. lipophilum, M. bovis and M. synoviae clusters. This entity was termed the M. equigenitalium cluster. The clusters and subclusters could be verified by using neighbour-joining and maximum-likelihood analyses on a variety of data sets, bootstrap calculations, secondary structure analysis and signature nucleotides. Therefore, the new 16S rDNA data presented in this work were used to re-evaluate the M. lipophilum cluster, leading to the definition of two additional clusters. At present, the mollicutes belonging to the hominis group can be classified into ten evolutionary lineages.

The fastidious nature of the mollicutes (mycoplasmas), their lack of a classic bacterial cell wall, and their very small genome, make phylogenetic placements of new species in this enlarging group of prokaryotes an important and valuable aid in their classification. In this report we have determined the phylogeny of the Mycoplasma hominis cluster of the hominis group. The 16S rDNA sequences from several previously described Mycoplasma species were determined and ten species were found to belong to the M. hominis cluster. With almost complete sequences available, the phylogenetic analysis revealed that the M. hominis cluster currently comprises 19 species, forming a distinct clade as judged from branch lengths, bootstrap percentage values, nucleotide signature analysis, and structural elements in the 16S rRNA molecule. The 16S rRNA gene sequences of species in the M. hominis cluster were found to be > or = 94% similar and the range within which similarities can be used in the classification of new species is discussed. Members of the M. hominis cluster all share a major biochemical property of M. hominis, in that they hydrolyse arginine and are incapable of fermenting glucose. This consistency in phenotypic pattern has not been found in any of the other phylogenetic clusters of the hominis group. Two species, the non-cultivable agent of Grey Lung disease in rodents (tentatively named 'Candidatus Mycoplasma ravipulmonis') and the avian species Mycoplasma gypis strain B1/T1T, were regarded as close relatives to the M. hominis cluster, but are clearly separated from the species of this cluster. Both species formed early branches of the M. hominis cluster and should be regarded as individual lines containing one species.

The 16S rRNA gene sequences of Mycoplasma cavipharyngis and Mycoplasma fastidiosum have been determined. Phylogenetic analysis showed that these species formed a new cluster within the so-called pneumoniae group of the mollicutes (class Mollicutes). This cluster will be referred to as the M. fastidiosum cluster. Interestingly, the M. fastidiosum cluster formed a sister lineage to the haemotrophic bacteria, Eperythrozoon spp. and Haemobartonella spp. The two latter genera, formerly classified as rickettsias, formed a stable phylogenetic entity in the tree as judged from branch lengths, bootstrap values and sequence signatures. Thus, the members of the M. fastidiosum cluster are the closest known relatives to the haemotrophic bacteria. Our data strongly support that the haemotrophic bacteria should be reclassified to reflect their actual phylogenetic affiliation.

The nucleotide sequences of the 16S rRNA genes from the type strains of four goat mycoplasmas, Mycoplasma adleri, Mycoplasma auris, Mycoplasma cottewii and Mycoplasma yeatsii, were determined by direct solid-phase DNA sequencing. Polymorphisms were found in two of the 16S rRNA gene sequences, showing the existence of two different rRNA operons. Three polymorphisms were found in M. adleri, and one was found in M. yeatsii. The sequence information was used for the construction of phylogenetic trees. M. adleri was included in the Mycoplasma lipophilum cluster within the hominis group. M. auris was comprised in the Mycoplasma hominis cluster of the hominis group. M. cottewii and M. yeatsii were found to be very closely related with only four nucleotide differences, and they grouped with Mycoplasma putrefaciens in the Mycoplasma mycoides cluster within the spiroplasma group. Sequencing of two field isolates of M. cottewii and M. yeatsii, geographically distant from the type strains, showed that the 16S rRNA gene from the field isolate of M. cottewii was identical to the one from the type strain. The field isolate of M. yeatsii had only two nucleotide differences to the type strain and these were present in only one of the two rRNA operons. Sequencing of the 16S rRNA genes from two unidentified mycoplasma isolates from Nepal indicated that they should both be regarded as M. auris strains.

Twenty-one triply cloned spiroplasma strains from the United States east of the Rocky Mountains, all isolated from tabanid (Diptera:Tabanidae) flies or serologically related to strains from tabanids, were compared reciprocally by spiroplasma deformation (DF) and metabolism inhibition (MI) serological tests. Many of the strains were also tested against 28 antisera representing known spiroplasma groups, subgroups, and putative groups isolated from nontabanid hosts. Relationships among strains were indicated by reciprocal cross-reactivity in both DF and MI tests. The strains were found to represent 11 recognized spiroplasma groups or subgroups. On the basis of serological, biochemical, and genomic data, strain BARC 1901 from Tabanus lineola appeared to represent a previously unrecognized candidate group. Strain BARC 2649, also from T. lineola, also appeared to represent a new group, but its morphology, arginine utilization, and some one-way serological crossing patterns suggested that it may be distantly related to group VIII spiroplasmas. Morphological, serological, and genomic data were used to place tabanid spiroplasma strains into three informal clusters. These are (i) groups IV (strain B31) and XXXI (strain HYOS-1); (ii) the three existing subgroups and a new candidate subgroup of group VIII represented by strain BARC 1357 plus ungrouped strain BARC 2649; and (iii) 14 strains, including EC-1 and TATS-1 (group XIV); strains TN-1 and TAAS-2 (group XVIII); strains TG-1, TASS-1, and BARC 4689 (group XXIII), strains TALS-2 (group XXVII), strain TABS-2 (group XXXII), and strains TAUS-1 and TABS-1 (group XXXIII) and ungrouped but closely related strains BARC 1901, BARC 2264 and BARC 2555. Analysis of tabanids from other geographic regions probably will substantially increase the number of known spiroplasma groups from this insect family.

Mycoplasma pneumoniae is a common etiologic agent of lower respiratory tract infections in humans. However,ithasbeenreportedpreviouslythattheorganismhasoccasionallybeenisolatedfromsitesotherthan the oropharynx and respiratory tract. We report the isolation of 24 strains ofM. pneumoniaefrom urogenital specimens obtained from 22 female patients. Most isolates were of cervical origin from patients attending several local gynecological clinics over a 2-year period. Strains were also isolated from the urethra of one of three healthy male sexual partners of female patients positive for the organism. Single serum specimens obtainedfromthreefemalepatientsandthreedifferentmalesexualpartnersshowedantibodylevelssuggestive of either recent respiratory infection or genital tract colonization with M. pneumoniae. Although there is no apparent definitive explanation for the localized outbreak of the organism at these unusual sites, the possible transfer through sexual and/or orogenital contact remains the most likely mode of transmission. The occur- rence of an organism with obvious pathogenicity for human epithelial tissue in the urogenital tract suggests such transfer could play a role in genital tract infection. Mycoplasma pneumoniaeis one of the commonest etiologic agentsoflowerrespiratorytractinfectionsinhumans,account- ing for somewhere between 15 and 20% of all cases of pneu- monia (3, 7). The organism can clearly be associated with a wide range of mild to serious extrapulmonary complications following respiratory disease in adults, children, and infants. However, the most frequent clinical manifestations observed with the organism involve tracheobronchitis and pharyngitis, with almost 20% of infections being asymptomatic (3). In an earlier study in Canada (6), we isolated M. pneumoniae from

A mycoplasma cultured from synovial fluid specimens from a patient with pneumonia and subsequent polyarthritiswasidentifiedinitiallyasMycoplasmapneumoniae.Inretrospectivestudies,theculturewasshown also to containMycoplasma genitalium. In this paper, the laboratory techniques employed in the identification and separation of the two species are presented, and evidence to implicate postinfectious autoimmunity is provided. An increasing number of reports ofM. genitaliumin human tissue sites and difficulties in isolation and identification of the organism in the clinical laboratory suggest the need for more extensive application of rapid and specific detection systems for bothM. genitaliumandM. pneumoniaein the clinical laboratory. Mycoplasma genitaliumwasfirst isolated from the urogenital tracts of two patients with nongonococcal urethritis more than a decade ago (30). While subsequent experimental challenge studies with M. genitalium in primates suggested invasive and pathogenic qualities and an active role in urogenital tract dis- ease (22, 32), repeated cultural studies failed to confirm this associationinhumangenitalinfections.Itwasonlyrecently,by using gene amplification techniques (PCR), that two indepen- dent research groups have provided more significant evidence of involvement ofM. genitaliumin human urethritis (9, 10). Although earlier studies ofM. genitaliumclearly established itasadistinctspecies,itwasapparentthattheorganismshared

This chapter focuses on determination of cholesterol and polyoxyethylene sorbitan growth requirements of mollicutes. Recent reports have established that cholesterol requirements vary within certain phylogenetic groupings of the class Mollicutes, tests to measure the growth response to cholesterol and to polyoxyethylene sorbitan are still part of the recommended techniques in the minimum standards for description of new species within the class. Cholesterol requirements are best determined by a quantitative comparison of growth occurring in serum-free media containing various supplements of fatty acids, albumin, and various concentrations of solubilized cholesterol. Members of the Mycoplasmataceae and various organisms assigned to the Entomoplasmataceae, Spiroplasmataceae, and Anaeroplasmataceae show minimal or negligible growth in serum-free media, but enhanced growth in the presence of increasing amounts of cholesterol. A newly described group of mollicutes has no growth requirement for serum or cholesterol, but shows sustained growth in serum-free media containing low concentrations of Tween 80.

A test is described that is useful for characterizing mollicutes in terms of the ability to maintain growth in medium containing 15 to 20% fetal bovine serum or in serum-free media with or without 0.04% Tween 80 (polyoxyethylene sorbitan). Representative Acholeplasma species maintained growth in serum-free medium, and about half of the strains tested grew well in Tween 80-supplemented medium. Representative Mycoplasma and Entomoplasma species did not maintain growth in either serum-free medium alone or when Tween 80 was added. Spiroplasma species and group representatives generally failed to sustain growth in serum-free medium with or without Tween 80, but at least four of the spiroplasmas tested maintained growth in serum-free medium. The representative Mesoplasma species grew in serum-free media only when Tween 80 was added, as did Mycoplasma lactucae. Although the test has obvious determinative uses for members of the class Mollicutes, it does not supplant the conventional methodology for assaying the cholesterol requirements of these organisms.