Revised serological classification of spiroplasmas,new provisional groups,and recommendations for serotyping of isolates

Five new spiroplasma strains were analyzed in reciprocal growth inhibition, metabolism inhibition (MI), and deformation (DF) serological tests. New provisional groups from the waspMonobia quadridens (VII) and the syrphid flyEristalis arbustorum (VIII) were added to the existing classification. Three serovars—represented by the LB-12 green leaf bug spiroplassignificantly with subgroup I-4 and with each other. These new groups and serovars bring the number of recognized spiroplasma serovars to 16. Serological distinctiveness of a sixth group (VI) fromIxodes ticks was confirmed. Simple DF and/or MI procedures are described for typing new isolates.     

Differentiation of group VIII Spiroplasma strains with sequences of the 16S-23S rDNA intergenic spacer region

Spiroplasma species (Mollicutes: Spiroplasmataceae) are associated with a wide variety of insects, and serology has classified this genus into 34 groups, 3 with subgroups. The 16S rRNA gene has been used for phylogenetic analysis of spiroplasmas, but this approach is uninformative for group VIII because the serologically distinct subgroups generally have similarity coefficients >0.990. Therefore, we investigated the utility of the 16S-23S rRNA spacer region as a means to differentiate closely related subgroups or strains. We generated intergenic sequences and detailed serological profiles for 8 group VIII Spiroplasma strains. Sequence analyses using Maximum Parsimony, Neighbor Joining, and Maximum Likelihood placed the strains into 2 clades. One clade consisted of strains BARC 2649 and GSU5367. The other clade was divided into clusters containing representatives of the 3 designated group VIII subgroups (EA-1, DF-1, and TAAS-1) and 3 previously unclassified strains. The stability of the positions of the strains in various analytical models and the ability to provide robust support for groupings tentatively supported by serology indicates that the 16S-23S intergenic rDNA sequence will prove useful in intragroup analysis of group VIII spiroplasmas.     

Simplified media for spiroplasmas associated with tabanid flies

Traditionally, isolation, maintenance, and testing of Spiroplasma species (Mollicutes: Entomoplasmatales) from horse flies (Tabanus spp.) and deer flies (Chrysops spp.) (Diptera: Tabanidae) have been accomplished in the complex M1D medium. A relatively inexpensive, simplified medium for tabanid spiroplasmas could expedite procedures that require large quantities of growth medium. Nine strains of spiroplasmas, eight from tabanids and one from mosquitoes, were cultured in three simplified broth media, R2, R8-1, and C-3G, and in M1D. There was no significant difference in the rate of spiroplasma growth in M1D and the three simplified media. R2 medium supported the growth of tabanid spiroplasmas more consistently and with better morphology through 10 subcultures than did the other simplified media. Primary isolations were made in R2 medium from tabanids collected (i) in Georgia, U.S.A., with 10 isolations from 10 flies and (ii) in coastal Costa Rica, with isolation rates of 70% (28/40) and 73% (27/37), respectively, for R2 and M1D. Of the seven group VIII field isolates from Costa Rica, four were capable of sustained growth in R2, and three were triply cloned in this simplified medium. These results suggest that the simplified medium R2 is suitable for many procedures with tabanid spiroplasmas.     

Spiroplasmas: serological grouping of strains associated with plants and insects.

Spiroplasma strains from plant and arthropod hosts, and from surfaces of flowers, were classified into three serological groups (designated I, II, and III) based on results from growth-inhibition tests. No significant cross reactions were observed among groups. The groupings were confirmed by ring-interface precipitin and microprecipitin tests, using membrane preparations as test antigens, and by organism-deformation tests. Serogroup I contained three subgroups: subgroup A (Spiroplasma citri strains Maroc R8A2 and C189), subgroup B (strain AS 576 and closely related strains from honeybee or flowers), and subgroup C (corn stunt spiroplasma strains). Serogroup II contained strains 23-6 and 27-31 isolated from flowers of the tulip tree (Liriodendron tulipifera L.) growing in Maryland. Serogroup III contained strains SR 3 and SR 9 isolated from flowers of the tulip growing in Connecticut. The subgroups of serogroup I were based on organism deformation, microprecipitin, and ring-interface precipitin tests. The data are consistent with the hypothesis that the three serogroups represent no less than three distinct spiroplasma species.     

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.

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.     

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.     

Seasonality and relative abundance of Tabanidae (Diptera) captured on horses in the Pantanal, Brazil.

Once a month, from June 1992 to May 1993, collections of tabanids on horse were conducted in the Nhecolandia, Pantanal State of Mato Grosso do Sul, Brazil. Tabanid catches using hand nets were conducted from sunrise to sunset at grassland and cerrad?o (dense savanna) habitats. A total of 3,442 tabanids from 21 species,12 genera, and 3 subfamilies were collected. Although species abundance varied seasonally depending on habitat, no habitat specificity was observed for the most abundant species. In the grassland, 1,625 (47.2%) tabanids belonging to 19 species were collected, while 1,817 (52.8%) tabanids from 17 species were caught in the cerrad?o. The number of tabanid species varied from 7 during winter (July/August) to 15 in the spring (October). Tabanus importunus (56%) was the most abundant species, followed by T. occidentalis (8.2%), and T. claripennis (8.1%). The tabanid peak, in October, coincided with the beginning of the rainy season. The population peak of most species, including those with higher vector potential, suggests that the rainy season can be considered as the period of potentially higher risk of mechanical transmission of pathogens by tabanids to horses in the region.     

Phenotypic differentiation of bifidobacteria of human and animal origins.

The phenotypes of 153 strains belonging or related to the genus Bifidobacterium were studied. These organisms included 38 collection strains and 115 wild strains (41 strains of human origin, 56 strains of animal origin, and 18 strains obtained from rivers or sewage). Our phenotypic analysis revealed seven main groups that were subdivided into 20 subgroups. Seven subgroups contained no type or collection strain. Among the human strains, the type strains of Bifidobacterium pseudocatenulatum and B. catenulatum fell into group I, which contained the type strains of B. adolescentis (subgroup Ib), B. dentium (subgroup Ic), and B. angulatum (ungrouped). The type strain of B. breve belonged to subgroup IIIa1, and the type strains of B. infantis and B. longum fell into subgroup IIIb1. Group VII comprised only wild strains that were isolated from human infant feces. Among the animal strains, group II consisted mainly of bifidobacteria that were isolated from pig feces and contained the type strains of B. suis (subgroup IIb), B. thermophilum (subgroup IIf), B. choerinum, and B. boum (ungrouped). Wild strains belonging to group V were isolated from pig, calf, cow, and chicken feces; this included the type strains of B. animalis (subgroup Va), B. magnum (subgroup Vb), B. pseudolongum, and B. globosum (subgroup Vc). The strains of human origin (groups I, III, and VII) were well separated from the animal strains (groups II, IV, and V). It was not surprising that the wild strains isolated from surface water or sewage were distributed in the animal groups as well as the human groups. Thus, bifidobacteria can be considered to be successful indicators of human or animal fecal pollution when they are correctly classified. The acidification patterns were not adequate to differentiate Bifidobacterium species, as determined previously by Mitsuoka (Bifidobacteria Microflora 3:11-28, 1984) and Scardovi (p. 1418-1434, in P. H. A. Sneath, N. S. Mair, M. E. Sharpe, and J. G. Holt, ed., Bergey's Manual of Systematic Bacteriology, vol. 2, 1986). However, enzymatic tests furnished new taxonomic criteria for the genus.     

Antibiotic sensitivities in vitro of diverse spiroplasma strains associated with plants and insects

Fifteen spiroplasma strains, representing five serological subgroups classified in three distinct serogroups, and four strains unassigned to serogroups were examined for sensitivity to antibiotics. The data contribute to the characterization of spiroplasmas and enlarge comparisons between plant pathogenic strains and strains that are evidently a part of the normal epiphytic microflora.     

Serological classification of spiroplasmas: current status

Data concerning serological classification of spiroplasmas are in good agreement, but slightly different numerical designations have been given to existing groups. It is proposed that a standardized system be adopted based on information developed mainly by the IRPCM working team on spiroplasmas. The type species (Spiroplasma citri) should be redefined to include only the agent of citrus stubborn disease (subgroup I-1). Six other subgroups, including three proposed by Bové et al. in this volume (I-5, I-6, and I-7), are members of the Group I complex. Because subgroups I-1, I-2, and I-3 (1) show significant reciprocal differences in DNA-DNA homology and two-dimensional electrophoretic protein profiles, (2) occupy exclusive habitats, (3) are each associated with important diseases, and (4) consist of clusters of very similar or identical strains, it is suggested that Latin binomials could be assigned to subgroups I-2 and I-3. It is proposed that those criteria could serve as general guidelines for consideration of subgroups for species status in the class Mollicutes. The I-4 subgroup is assigned an uncertain status, pending comparisons with the LB-12 (I-5), M55 (I-6), and N525 (I-7) subgroups. To previously described serogroups we add the CN-5 Cotinus beetle spiroplasma (IX), the AES-1 mosquito strain (X), and the MQ-4 Monobia strain (XI).     

A comparison of trapping methods for Tabanidae (Diptera) in North Queensland, Australia

The ability to monitor the abundance and diversity of tabanid flies over wide areas requires effective and low-cost surveillance methods. Such monitoring activities help to quantify the risk of transmission of pathogens by tabanids. Here we examine the effectiveness and practicality of two types of trap (canopy traps and Nzi traps) and two types of attractant (octenol and carbon dioxide) for monitoring tabanid flies in tropical Australia. The Nzi trap consistently caught more tabanids and more species of tabanids than the canopy trap. It was also more robust and therefore required less maintenance in remote locations. The use of attractants substantially increased capture rates, both of individuals and species, and traps using both attractants were consistently the most effective. However, in remote locations, where it is not possible to check traps frequently, the use of attractants may not be feasible. When attractants were not used, the canopy trap caught very few tabanids, but the Nzi trap remained effective enough to be useful as a monitoring device. In addition, the number of tabanid species caught by the Nzi traps remained high, and included those that were most abundant. We therefore conclude that, in this region, Nzi traps are preferable for tabanid monitoring and that attractants greatly improve their effectiveness. However, for longterm monitoring, especially in remote locations, Nzi traps without attractants are a satisfactory option.     

Proposed subgroups of spiroplasmas of high guanine plus cytosine content, group IV

The plant surface and insect-inhabiting spiroplasmas of group IV, unlike other spiroplasmas, have not been demonstrated to utilize arginine. They require cholesterol for growth, produce spots and films on some media, and do not hydrolize arbutin. Electrophoretic and serological comparisons of strains from North America and Europe indicate the existence of strain differences within group IV. This study provides evidence for the existence of three discrete subgroups, group IV-(1) represented by temperate American strains, group IV-(2) represented by subtropical American strain PPS1, and group IV-(3) represented by Mediterranean and French strains.     

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.     

Characterization and taxonomic status of tick spiroplasmas: a review

Three serologically distinct groups of spiroplasmas have been recovered from ticks. Spiroplasma mirum strains (from rabbit ticks, Haemaphysalis leporispalustris) and Y32 group (VI) spiroplasmas (from Ixodes pacificus) are the only spiroplasmas to have a clear association with these arthropods. Group (VI) spiroplasmas are distinguished by an unusual nonhelical morphology and their capacity to hemadsorb guinea pig erythrocytes. S. mirum strains are unique in their ability to induce cataracts or lethal brain infections in a number of young vertebrates and in their virulence for the chick embryo. The 277F spiroplasma, while initially recovered from a pool of rabbit ticks (H. leporispalustris), is related by certain serological and genetic properties to spiroplasmas in the S. citri complex (serogroup I). These relationships suggest that the 277F spiroplasma may not be a natural inhabitant of the rabbit tick.     

Evaluation of DNA barcoding and identification of new haplomorphs in Canadian deerflies and horseflies

This paper reports the first tests of the suitability of the standardized mitochondrial cytochrome c oxidase subunit I (COI) barcoding system for the identification of Canadian deerflies and horseflies. Two additional mitochondrial molecular markers were used to determine whether unambiguous species recognition in tabanids can be achieved. Our 332 Canadian tabanid samples yielded 650 sequences from five genera and 42 species. Standard COI barcodes demonstrated a strong A + T bias (mean 68.1%), especially at third codon positions (mean 93.0%). Our preliminary test of this system showed that the standard COI barcode worked well for Canadian Tabanidae: the target DNA can be easily recovered from small amounts of insect tissue and aligned for all tabanid taxa. Each tabanid species possessed distinctive sets of COI haplotypes which discriminated well among species. Average conspecific Kimura two‐parameter (K2P) divergence (0.49%) was 12 times lower than the average divergence within species. Both the neighbour‐joining and the Bayesian methods produced trees with identical monophyletic species groups. Two species, Chrysops dawsoni Philip and Chrysops montanus Osten Sacken (Diptera: Tabanidae), showed relatively deep intraspecific sequence divergences (～10 times the average) for all three mitochondrial gene regions analysed. We suggest provisional differentiation of Ch. montanus into two haplotypes, namely, Ch. montanus haplomorph 1 and Ch. montanus haplomorph 2, both defined by their molecular sequences and by newly discovered differences in structural features near their ocelli.     

Meteorological effects on the daily activity patterns of tabanid biting flies in northern Queensland,Australia

Information on the daily activity patterns of tabanid flies is important in the development of strategies that decrease the risk of pathogens transmitted by them. In addition, this information is useful to maximize numbers of tabanids trapped during short‐term studies and to target feeding behavior studies of certain tabanid species to their times of peak activity. The current study examined the effects of various meteorological factors on the daily activity patterns of common tropical species of tabanids in north Queensland. Each species studied responded differently to weather factors. Tabanus townsvilli Ricardo (Diptera: Tabanidae) was most active during late morning and early afternoon, whereas Pseudotabanus silvester (Bergroth) and Tabanus pallipennis Macquart were most active in the late afternoon. Tabanus dorsobimaculatus Macquart was most active in the morning and early afternoon. Data on daily activity patterns of tabanid flies indicates that in an area such as Townsville, North Queensland, where several species of tabanid are present concurrently in high numbers, the overlapping periods of high activity for these species indicate a high risk of pathogen transmission for most of the day (10.00–19.00 hours). Similarly, because each species responds differently to weather variables, only extreme weather conditions are likely to inhibit activity of all species. These data also indicate that for maximal results, trapping and feeding behavior studies should be tailored to the preferred activity period of the species under investigation.     

Hyaluronidase in meningococcus]

A total of 204 meningococcus strains were tested for the presence of hyaluronidase, and 45.5% of the strains were found to contain it. Strains penetrating into the cerebrospinal fluid were the ones which largely produced the enzyme (in 83% of the cases). The enzyme was revealed only in 25.5% of the strains habituating on the nasopharyngeal mucosa. Hyaluronidase was mostly found in the meningococcus strains referred to the serological group A; strains of other serological groups and ungrouped strains produced the enzyme in 23.7% of the case only. There was no correlation between the capacity to form hyaluronidase and the virulence determinable in intraperitoneal infection of mice.     

Influence of five spiroplasma strains on growth rate and survival of Galleria mellonella (lepidoptera: Pyralidae) larvae

Infection of Galleria mellonella larvae with five spiroplasma strains caused increased mortality and decreased growth rates of larvae. Reductions in growth rate and survival were related to spiroplasma strain and multiplication rate of the spiroplasma in the larvae. Three strains, considered to be ephiphytic on flowers (23-6, SR-3, PPS1), proved highly pathogenic to G. mellonella larvae, whereas strains known to be pathogenic to plants (SC-27) or honeybees (G-1) had a lesser impact.