Spiroplasma Species, Groups, and Subgroups from North American Tabanidae

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. Received: 23 April 1997 / Accepted: 31 May 1997     

Spiralin: Major membrane protein specific for subgroup I-1 Spiroplasmas

Preparations of spiralin from membranes ofSpiroplasma citri, strain C189, purified by sequential solubilization with detergents followed by agarose-suspension electrophoresis induced rabbit antibodies that were largely specific forSpiroplasma citri Group I-1 spiroplasmas, as demonstrated by metabolic inhibition (MI), growth inhibition (GI), and deformation (DF) tests. By contrast, antibodies againstS. citri whole-membrane protein preparations reacted broadly with representative type cultures of seven subgroups of theS. citri complex. Neither antimembrane nor antispiralin sera reacted withS. floricola, S. mirum, or Group IV, (VI), (VII), or (VIII) spiroplasmas. Minor cross-reactions in MI and DF tests between antispiralin serum and Subgroup I-2 and I-3 antigens may have represented shared epitopes in a set of homologous membrane proteins of the three spiroplasmas, or antibodies against highly antigenic traces of other common membrane proteins in the purified spiralin preparations. The unique antigenic properties of spiralin, the most abundant protein in theS. citri membrane, explain in part the unique profiles shown by this spiroplasma species in comparative taxonomic serological tests.     

Direct isolation in cell-free medium of a spiroplasma fromHaemaphysalis leporispalustris (Acari: Ixodidae) in Maryland

A spiroplasma isolate, was obtained from rabbit ticks (Haemaphysalis leporispalustris) taken from cottontail rabbits in Maryland by inoculation of tick suspensions into SP-4 medium. The isolate was indistinguishable from an experimental vertebrate pathogen (suckling mouse cataract agent spiroplasma) when tested with other plant and tick spiroplasmas in growth inhibition, deformation, and metabolism inhibition tests. The isolated organism had a pathogenic profile for suckling rats and embryonated chicken eggs that differed significantly from that of other suckling mouse cataract agent strains. This is the first report of a direct spiroplasma isolation from ticks in cell-free medium, and confirms the specific association of spiroplasmas of the suckling mouse cataract agent serogroup with rabbit ticks.     

Spiroplasmas from coleopterous insects: New ecological dimensions

The genusSpiroplasma (helical wall-less prokaryotes) is a recently described group of microorganisms that cause disease in plants, arthropods, and experimentally, in vertebrates. Two spiroplasmas from beetles have now been discovered in a search for microorganisms suitable for biological control of economically important coleopterous insects. Colorado potato beetles (CPB) infected with spiroplasma were commonly found on potato and other solanaceous plants in Maryland. Although this spiroplasma occurred in high concentration in gut fluids and sputum, it could not be cultivated in conventional spiroplasma media. However, another spiroplasma (CN-5 and related strains) reported here to occur commonly in association with larvae and adults of the green June beetle,Cotinus nitida, could be cultivated readily in the SM-1 formulation and several other conventional spiroplasma media. The CN-5 spiroplasma was serologically distinct from representative members of all 8 major groups now recognized. Thus, it represents a ninth major spiroplasma serogroup (IX), and can be considered to be an unnamed species. The CPB spiroplasma is apparently maintained in plant surface-insect gut cycles, but details of maintenance of the CN-5 spiroplasma are incompletely understood. Isolation of CN-5 spiroplasma from soil in which host larvae had fed suggests that transmission of this agent may occur in the soil. Both CN-5 and CPB spiroplasmas exhibited unusually active translational motility in natural fluids, and CN-5 organisms exhibited such motility in culture media. Although we have no evidence that either spiroplasma is pathogenic to its usual host, the pathogenicity of spiroplasmas to many hosts, including the beetle,Melolontha melolontha, suggests possible application for biological control.     

Cultivation,serology, ultrastructure,and virus-like particles of spiroplasma 277F

Spiroplasma 277F, a helical, motile mycoplasma from rabbit ticks in Montana, was cloned and cultivated in liquid and solidified spiroplasma or mycoplasma media. Serum was required, glucose was fermented, and digitonin inhibited growth. Colonies of spiroplasma 277F possessed granular centers and were surrounded by smaller, subsurface “satellite” colonies. Cloned agent 277F was antigenically distinct from the suckling mouse cataract agent, the corn stunt organism, andSpiroplasma citri by growth inhibition and deformation tests, but exhibited weak cross-reactivity withS. citri in the precipitin ring tests. Although current passage levels did not cause cataracts in neonatal rats, kill embryonated hen's eggs, or cause bovine mastitis, definitive tests must await the availability of fresh isolates. Ultrastructurally, 277F closely resembledS. citri but displayed a system of 3-nm threadlike filaments in the exterior layer of its membranous covering. Two phagelike entities, similar to viruses associated withS. citri, were present.     

Growth inhibition of phytopathogenic spiroplasmas by membrane-interactive antimicrobial peptides Novispirin T7 and Caerin 1.1

Spiroplasma kunkelii and Spiroplasma citri, both helical-shaped cell wall-less bacteria, are the causative agents of corn stunt disease and citrus stubborn disease, respectively. Plants exhibiting natural resistance to these phytopathogenic spiroplasmas are currently lacking. Engineering artificial plant resistance using antimicrobial peptides (AMPs) has been conceived as a new approach to control the agronomically important spiroplasmal diseases. In preparation for such task, the present study focused on screening of AMPs that have potentials to curb the growth of S. kunkelii and S. citri. Four AMPs, including Novispirin T7, Caerin 1.1, Tricholongin and Dhvar4, were selected for in vitro growth inhibition test. A liquid assay method was developed for quick qualitative and quantitative evaluations of the AMPs. Our results demonstrated that Novispirin T7 and Caerin 1.1 were able to inhibit the growth of both phytopathogenic spiroplasmas with the efficacy comparable to that of tetracycline. Cell deformations were observed in spiroplasma cultures treated with these two peptides, indicating interactions of the AMPs with the spiroplasma cell membranes. The minimum inhibitory concentrations (MICs) of the AMPs against S. kunkelii and S. citri were determined.     

Wall-less prokaryotes from fall flowers in central United States and Maryland

Four spiroplasma strains and eleven isolates tentatively identified as acholeplasmas were obtained from fall flowers in Colorado, Nebraska, Illinois, and Maryland. Although the acholeplasma isolates were heterogeneous, all showed antigenic sharing with a group of unnamed organisms (L1 and related strains) isolated in othe studies from flowers in Florida. The W20 and W24 isolates from Nebraska were partially related to the L1 group by DNA-DNA homology and polyacrylamide gel electrophoresis (PAGE) analyses. A Colorado spiroplasma (W13) was identifed as a new strain of group IV complex. Three spiroplasma strains from flowers in Maryland old fields represent a new serovar with closest affinity to subgroup I-4 and to the LB12 and N525 serovars of group I. Widespread occurrence of acholeplasmas on flowers in this study, and on plant surfaces in general, suggests that, like spiroplasmas they probably will be found to reside in arthropods.     

Scanning Electron Microscopy of the Influence of Fixation Vehicle Osmolality on Cell Morphology of Spiroplasma, Acholeplasma, and Mycoplasma spp. grown on Agar

Young Spiroplasma citri, corn stunt spiroplasma, and honey bee spiroplasma colonies fixed in 5% glutaraldehyde in M 199 cell culture medium with 0.25 M sucrose showed elongated mycelium-like cells which were sometimes branched or helical. In older colonies beaded chains and rounded bodies were formed. Fixation in 6 % glutaraldehyde in distilled water resulted in amorphous masses in which rounded bodies were present. The spiroplasma cells did not remain osmotically active after glutaraldehyde fixation. Acholeplasma laidlawii and Mycoplasma hyorhinis colonies fixed in glutaraldehyde with or without M 199 medium with 0.25 M sucrose showed little difference in cell morphology.     

Genetic and antigenic characteristics of ApxIIA and ApxIIIA from Actinobacillus pleuropneumoniae serovars 2, 3, 4, 6, 8 and 15

Apx toxins produced by Actinobacillus pleuropneumoniae are essential components of new generation vaccines. In this study, apxIIA and apxIIIA genes of serovars 2, 3, 4, 6, 8 and 15 were cloned and sequenced. Amino acid sequences of ApxIIA proteins of serovars 2, 3, 4, 6, 8 and 15 were almost identical to those of serovars 1, 5, 7, 9 and 11–13. Immunoblot analysis showed that rApxIIA from serovars 2 and 15 reacts strongly with sera from animals infected with various serovars. Sequence analysis revealed that ApxIIIA proteins has two variants, one in strains of serovar 2 and the other in strains of serovars 3, 4, 6, 8 and 15. A mouse cross‐protection study showed that mice actively immunized with rApxIIIA/2 or rApxIIIA/15 are protected against challenge with A. pleuropneumoniae strains of serovars 3, 4, 6, 8, 15, and 2 expressing ApxIII/15 and ApxIII/2, respectively. Similarly, mice passively immunized with rabbit anti‐rApxIIIA/2 or anti‐rApxIIIA/15 sera were found to be protected against challenge with strains of serovars 2 and 15. Our study revealed antigenic and sequence similarities within ApxIIA and ApxIIIA proteins, which may help in the development of effective vaccines against disease caused by A. pleuropneumoniae.     

Population dynamics of a maternally-transmitted <Emphasis Type="Italic">Spiroplasma</Emphasis> infection in <Emphasis Type="Italic">Drosophila hydei</Emphasis>

A maternally-inherited spiroplasma endosymbiont of Drosophila hydei does not exert apparent phenotypes on both sexes of its host and is prevalent in natural populations of D. hydei. Our previous experiments using a laboratory stock of D. hydei revealed that low temperatures (such as 15°C and 18°C) dramatically lower the vertical transmission rates of this spiroplasma. Therefore, we hypothesized that, in temperate regions, the infection frequencies may decrease in cool seasons but increase in the summer season. To clarify the temporal population dynamics of the spiroplasma infection, D. hydei were collected from two Japanese populations in 2006–2008 from May to early August, representing the only period when a number of D. hydei are collectable in Japan, and examined for spiroplasma infection. Within each year, the frequency of spiroplasma infection fluctuated considerably in both populations. Consistent with our hypothesis, the infection frequency showed an increasing trend in both populations in 2007. However, the data in 2006 and 2008 did not show consistent patterns of increase. The population dynamics of spiroplasma infection may be affected but not critically determined by temperature. Moreover, despite the fluctuation within each year, the infection frequencies seemed to be stable across the years. The frequencies of spiroplasma infection in D. hydei populations may be stabilized by multiple factors. One of these factors may involve a context-dependent positive effect of spiroplasma on the fitness of D. hydei, as was recently observed in laboratory experiments.     

Characterization of Spiroplasmas by one- and two-dimensional protein analysis on polyacrylamide slab gels

Differences betweenSpiroplasma citri isolates were detected by one-dimensional electrophoresis of proteins on gradient polyacrylamide slab gels. Two-dimensional protein maps (electrofocusing followed by electrophoresis) showed a highly characteristic pattern for allS. citri isolates examined. Coanalysis of mixed protein samples from pairs ofS. citri strains revealed more than 150 comigrating proteins common to allS. citri isolates, but also a number of noncomigrating proteins. Some noncomigrating proteins were present in one isolate but not in another, while other proteins whose migrational properties were only slightly different from one isolate to the other (homologous proteins), were present in more than one isolate.S. citri isolates had many common and only a few homologous proteins. Comparisons ofS. citri with the corn stunt spiroplasma revealed few common proteins and a large number of homologous proteins. When comparingS. citri and the suckling mouse cataract spiroplasma, few common and homologous proteins were apparent. However, several of these common proteins were also shared by the corn stunt spiroplasma, suggesting that they may well represent genus-specific proteins. The data also offer additional evidence that the suckling mouse cataract spiroplasma differs significantly fromS. citri and corn stunt spiroplasmas and probably deserves a separate species designation.     

Virus-mediated change in clumping properties ofDrosophila SR spiroplasmas

Transovarially transmitted SR spiroplasmas inDrosophila cause an abnormal sex ratio (SR condition: male-specific killing) in the host fly progenies. A reaction known as clumping takes place between different SR spiroplasma strains in which spiroplasmas instantly form aggregates upon mixing of the two strains. Each strain of SR spiroplasma carries an associated virus that is lytic to certain other strains. When the virus, HIV, from the recently discovered non-male-killingDrosophila hydei spiroplasma (HIS) is injected into host flies carrying the SR spiroplasma ofD. nebulosa (NSR), the latter spiroplasmas either undergo complete lysis and disappear, or survive with decreased numbers and with an abnormal morphology, and are transmissible from generation to generation in host flies. The surviving spiroplasmas possess two viruses, the endogenous virus of thenebulosa spiroplasma, spv-1, and the newly introduced superinfecting virus, HIV. This combination leads to a change in the surface properties of the superinfected spiroplasmas that is manifested in their ability to form clumps with normalnebulosa spiroplasmas, but does not interfere with male killing. This change in spiroplasma phenotype is discussed in terms of host-phenotype modification by infecting viruses.     

Further studies on a spiroplasma isolated from coconut palms in Jamaica

The pathogenicity of a spiroplasma isolated from coconut palms was tested by (1) transmission experiments to palms and other plants susceptible to infection by mycoplasmas, using the suspected vector of lethal yellowing, Myndus crudus, and vectors of the agents of other yellows diseases and (2) enzyme-linked immunosorbent assay (ELISA) to detect spiroplasma antigens in diseased palm tissues. Results of both these tests were negative and, as earlier attempts to repeat the isolations from lethal yellowing diseased palms had also been unsucessful, it was concluded that this organism was not the causal agent of lethal yellowing disease. Further analysis by serological tests and by polyacrylamide gel electrophoresis (PAGE) of spiroplasma proteins confirmed that the coconut isolates were related to members of the Spiroplasma citri serogroup but were distinct from other strains tested.     

Tubuloside A,a phenylethanoid glycoside,alleviates diclofenac induced hepato-nephro oxidative injury via Nrf2/HO-1

The most prominent adverse effects of nonsteroidal anti-inflammatory drugs (NSAIDs) such as diclofenac (DF) are hepato-renal damage. Natural antioxidants can be preferred as an alternative and/or combination to improve this damage. This present study was conducted to evaluate the protective effect of Tubuloside A (TA) against diclofenac (DF)-induced hepato-renal damage. TA (1 mg/kg, ip) was administered to male Sprague–Dawley rats for 5 days, and DF (50 mg/kg, ip) was administered on Days 4 and 5. Plasma aspartate amino transferase, alanine amino transferase, alkaline phosphatase, blood urea nitrogen and creatinine were measured to evaluate liver and kidney functions. Additionally, oxidative stress parameters (malondialdehyde, glutathione, superoxide dismutase, catalase, and 8-oxo-7,8-dihydro-2′-deoxyguanosine) in blood, liver, and kidney tissues, changes in mRNA expression of genes involved in the Nrf2/HO-1 signalling pathway (Nrf2, HO-1, NQO-1, IL-6, iNOS, Cox-2, TNF-α, IL1-β and NFκB) and apoptotic process (Bcl-2, Cas-3 and Bax) in liver and kidney tissues were determined. Additionally, tissue sections were evaluated histopathologically. Biochemical, histopathological, and molecular results demonstrated the hepato-renal toxic effects of DF, and TA treatment protected the liver and kidney from DF-induced damage. This provides an explanation for the hepato-nephro damage caused by DF and offers new ideas and drug targets together with TA for the prevention and treatment of DF injury.     

Gene content and organization of an 85-kb DNA segment from the genome of the phytopathogenic mollicute Spiroplasma kunkelii

Spiroplasma kunkelii, the causative agent of corn stunt disease in maize ( Zea mays L.), is a helical, cell wall-less prokaryote assigned to the class Mollicutes. As part of a project to sequence the entire S. kunkelii genome, we analyzed an 85-kb DNA segment from the pathogenic strain CR2-3x. This genome segment contains 101 ORFs and two tRNA genes. The majority of the ORFs code for predicted proteins that can be assigned to respective clusters of orthologous groups (COGs). These COGs cover diverse functional categories including genetic information storage and processing, cellular processes, and metabolism. The most notable gene cluster in this genome segment is a super-operon capable of encoding 24 ribosomal proteins. The organization of genes in this operon reflects the unique evolutionary position of the spiroplasma. Gene duplications, domain rearrangements, and frameshift mutations in the segment are interpreted as indicators of phase variation in the spiroplasma. To our knowledge, this is the first analysis of a large genome segment from a plant pathogenic spiroplasma.Communicated by W. Goebel     

Spiroplasma species in the Costa Rican highlands: implications for biogeography and biodiversity

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.

Temperature Ranges,Growth Optima,and Growth Rates of Spiroplasma (Spiroplasmataceae,class Mollicutes) Species

A new method was developed for determination of the doubling times of spiroplasmas. In this procedure, the time required for medium acidification of tubes in tenfold dilution series was recorded. Sixty-four spiroplasma strains, representing 24 groups and 11 subgroups, were studied. Eight strains representing putative new groups were also included in the study. Doubling times at 5, 10, 15, 20, 25, 30, 32, 37, 41, and 43°C were determined. The range of temperatures for spiroplasma growth was 5°–41°C. Twenty-three spiroplasmas had optima of 30°C, 29 had optima of 32°C, and 13 had optima of 37°C. The fastest growing spiroplasma was the MQ-4 strain (group XI), with a doubling time at optimal temperature of 0.6 h. The slowest was the Jamaican corn stunt strain B655 (subgroup I-3), with an optimal doubling time of 36.7 h. Spiroplasma strain B31 (group IV) had the widest range (5°–41°C), while the DW-1 strain and some subgroup I-3 strains had the narrowest, growing only at 25° and 30°C. Some spiroplasmas grew well at 41°C, but none grew at 43°C. The ability of spiroplasmas to withstand a wide range of temperatures may reflect the conditions to which they are exposed in nature, including the temperatures of the insect, tick, and/or plant hosts in which they are carried and the plant surfaces from which they may be acquired by arthropods.     

Spiroplasmas are the causal agents of citrus little-leaf disease

A spiroplasma isolated from citrus with little-leaf disease was grown in a cell-free medium and injected into leafhoppers (Euscelis plebejus) Injected leafhoppers, but not those fed on infected plants, transmitted the spiroplasma to white clover (Trifolium repens cv. S100) and sweet orange (Citrus sinensis cv. Valencia). Infected clover plants were severely stunted; infected sweet orange plants showed typical symptoms of citrus little-leaf disease. The spiroplasma was detected in clover and sweet orange plants by electron microscopy; the helical morphology of the organisms was most easily recognizable in sections 150–200 nm thick. The organism was re-isolated in cell-free media both from infected plants and from injected E. plebejus. The original isolate and those re-isolated from experimentally infected clover and sweet orange appeared by morphological, cultural, biochemical and serological criteria to be identical to each other and to the R8-A2 (type) and C-189 strains of Spiroplasma citri. Serological tests and electrophoretic analysis of protein preparations indicated no relationship to Acholeplasma laidlawii, although this organism survived for at least 10 wk after injection into E. plebejus. Our results show that the causal agent of little-leaf disease is related to S. citri.     

Infectivity of beetle spiroplasmas for new host species

Five beetle spiroplasmas, the Colorado potato beetlespiroplasma (CPBS, strain LD-1), the Cantharis carolinusspiroplasma (CCBS, strain CC-1), the Ellychnia corrusca fireflyspiroplasma (FS, strain EC-1), the Diabrotica undecimpunctatacorn rootworm spiroplasma (CRS, strain DU-1), and the Spiroplasmafloricola fall flower spiroplasma (FFS), all associated withbeetles, were fed to beetles (Maladera matrida and Carpophilushumeralis) and mosquitoes (Aedes aegypti and Culex pipiens). CPBSand CCBS were also injected into M. matrida. Attempts to recoverspiroplasmas from regurgitates and hemolymph were conducted 1–10days after their introduction. After day 1, orally administeredspiroplasmas could not be recovered from M. matrida beetles;however, at 2–5 days, four out of five spiroplasmas wererecovered from adult C. humeralis. Injected spiroplasmas survivedin the hemolymph of M. matrida beetles for a relatively longperiod (at least 22 days). All five spiroplasmas were recoveredfrom mosquitoes 1 day post feeding, but only two (CCBS and CRS)survived for five or more days. The results show short andvariable persistence in orally challenged non-host insects, withgeneral failure to pass the gut barrier. Such evidence should beconsidered when attempting to use these microbes in biocontrolprograms.