A strain of Melissococcus pluton cultivable on chemically defined media

Abstract A Brazilian strain of Melissococcus pluton multiplied well under anaerobic conditions on chemically defined media buffered with potassium phosphate. Thymine, xanthine, pyridoxal and one or more other vitamins, CO₂, glucose, methionine and possibly other amino acids, were essential. Other strains multiplied only slightly and could not be subcultivated on the media. The Brazil strain is the least related serologically to all known strains of M. pluton and is the least fastiduous when cultivated on other media. It has a DNA base composition of 31.4 G + C mol%, whereas others tested have been 29.0 and 30.2 G + C mol%.     

Geographically diverse Australian isolates of Melissococcus pluton exhibit minimal genotypic diversity by restriction endonuclease analysis

Melissococcus pluton, the causative agent of European foulbrood is an economically significant disease of honey bees (Apis mellifera) across most regions of the world and is prevalent throughout most states of Australia. 49 Isolates of M. pluton recovered from diseased colonies or honey samples in New South Wales, Queensland, South Australia, Tasmania and Victoria were compared using SDS-PAGE, Western immunoblotting and restriction endonuclease analyses. DNA profiles of all 49 geographically diverse isolates showed remarkably similar AluI profiles although four isolates (one each from Queensland, South Australia, New South Wales and Victoria) displayed minor profile variations compared to AluI patterns of all other isolates. DNA from a subset of the 49 Australian and three isolates from the United Kingdom were digested separately with the restriction endonucleases CfoI, RsaI and DraI. Restriction endonuclease fragment patterns generated using these enzymes were also similar although minor variations were noted. SDS-PAGE of whole cell proteins from 13 of the 49 isolates from different states of Australia, including the four isolates which displayed minor profile variations (AluI) produced indistinguishable patterns. Major immunoreactive proteins of approximate molecular masses of 21, 24, 28, 30, 36, 40, 44, 56, 60, 71, 79 and 95 kDa were observed in immunoblots of whole cell lysates of 22 of the 49 isolates and reacted with rabbit hyperimmune antibodies raised against M. pluton whole cells. Neither SDS-PAGE or immunoblotting was capable of distinguishing differences between geographically diverse isolates of M. pluton. Collectively these data confirm that Australian isolates of M. pluton are genetically homogeneous and that this species may be clonal. Plasmid DNA was not detected in whole cell DNA profiles of any isolate resolved using agarose gel electrophoresis.     

Taxonomic studies on 'Streptococcuspluton'

Strains of ' Streptococcus pluton ' isolated from honeybee larvae with European foul-brood from widely separate parts of the world represent a relatively homogeneous taxon; all are related serologically. Added cysteine or cystine make a variety of otherwise unsuitable nitrogen sources satisfactory for all strains, which all have the same fastidious culture requirements. Test strains lack respiratory quinones and possess a Group A peptidoglycan based upon lysine, primarily straight-chain and cyclopropane-ring fatty acids, and a DNA base composition of 29–30 G + C mol %. Results obtained indicate that the strains presently designated ' Strep. pluton ' should constitute the nucleus of a new genus.     

Tyramine functions as a toxin in honey bee larvae during Varroa-transmitted infection by Melissococcus pluton

From wounds of honey bee pupae, caused by the mite Varroa destructor, coccoid bacteria were isolated and identified as Melissococcus pluton. The bacterial isolate was grown anaerobically in sorbitol medium to produce a toxic compound that was purified on XAD columns, gelfiltration and preparative HPLC. The toxic agent was identified by GC-MS and FTICR-MS as tyramine. The toxicity of the isolated tyramine was tested by a novel mobility test using the protozoon Stylonychia lemnae. A concentration of 0.2 mg/ml led to immediate inhibition of mobility. In addition the toxicity was studied on honey bee larvae by feeding tyramine/water mixtures added to the larval jelly. The lethal dosis of tyramine on 4-5 days old bee larvae was determined as 0.3 mg/larvae when added as a volume of 20 microl to the larval food in brood cells. Several other biogenic amines, such as phenylethylamine, histamine, spermine, cadaverine, putrescine and trimethylamine, were tested as their hydrochloric salts for comparison and were found to be inhibitory in the Stylonychia mobility test at similar concentrations. A quantitative hemolysis test with human red blood cells revealed that tyramine and histamine showed the highest membranolytic activity, followed by the phenylethylamine, trimethylamine and spermine, while the linear diamines, cadaverine and putrescine, showed a significantly lower hemolysis when calculated on a molar amine basis. The results indicate that tyramine which is a characteristic amine produced by M. pluton in culture, is the causative agent of the observed toxic symptoms in bee larvae. Thus this disease, known as European foulbrood, is possibly an infection transmitted by the Varroa destructor mite.     

Complete genome sequence of Melissococcus plutonius ATCC 35311

We report the first completely annotated genome sequence of Melissococcus plutonius ATCC 35311. M. plutonius is a one-genus, one-species bacterium and the etiological agent of European foulbrood of the honeybee. The genome sequence will provide new insights into the molecular mechanisms underlying its pathogenicity.     

Preliminary investigations into possible resistance to oxytetracycline in Melissococcus plutonius,a pathogen of honeybee larvae

AIMS: To investigate the occurrence of oxytetracycline (OTC) resistance in Melissococcus plutonius, which causes European foulbrood in honeybee colonies. METHODS AND RESULTS: Strains of M. plutonius were isolated from diseased colonies in England and Wales and tested for resistance to OTC. The minimum inhibitory concentration (MIC) of OTC was also determined for selected isolates. No resistance to the antibiotic was found in any isolate and the average MIC was found to be 3.9 microg ml-1. Melissococcus plutonius was found to be susceptible to both chlortetracycline and tetracycline. CONCLUSIONS: No resistance to OTC was found in M. plutonius. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated that OTC can continue to be used to treat European foulbrood and that resistance may not explain why some treatments fail.     

A PCR Detection Method for Rapid Identification of Melissococcus pluton in Honeybee Larvae

Melissococcus pluton is the causative agent of European foulbrood, a disease of honeybee larvae. This bacterium is particularly difficult to isolate because of its stringent growth requirements and competition from other bacteria. PCR was used selectively to amplify specific rRNA gene sequences of M. pluton from pure culture, from crude cell lysates, and directly from infected bee larvae. The PCR primers were designed from M. pluton 16S rRNA sequence data. The PCR products were visualized by agarose gel electrophoresis and confirmed as originating from M. pluton by sequencing in both directions. Detection was highly specific, and the probes did not hybridize with DNA from other bacterial species tested. This method enabled the rapid and specific detection and identification of M. pluton from pure cultures and infected bee larvae.     

Identification of Mutations Involved in the Requirement of Potassium for Growth of Typical Melissococcus plutonius Strains

Melissococcus plutonius is a fastidious honeybee pathogen, and the addition of KH₂PO₄ to culture medium is required for its growth. Using genome sequences and a newly developed vector, we showed that mutations in genes encoding Na⁺/H⁺ antiporter and cation-transporting ATPase are involved in the potassium requirement for growth.     

中华蜜蜂的欧洲幼虫腐臭病病原研究

采用中华蜜蜂Apis cerana cerana F.临床自然发病的欧洲幼虫腐臭病(European Foulbrood, EFB)幼虫,对其病原体进行了分离鉴定。结果表明：中华蜜蜂EFB的病原系蜂房蜜蜂球菌Melissococcus pluton。该菌为革兰氏阳性的兼性厌氧菌,形态学及染色特性、致病性试验、血清学试验和细菌DNA G+C mol%试验均证明中华蜜蜂和西方蜜蜂的EFB病原属于同属的蜂房蜜蜂球菌。生理生化特性结果与国外的早期研究结果相近似。该试验为不同地理位置、不同种寄主间蜂房蜜蜂球菌的遗传差异的研究,以及中华蜜蜂EFB病的防治学研究打下基础。     

A nested array of rRNA targeted probes for the detection and identification of enterococci by reverse hybridization

Complete 23S and almost complete 16S rRNA gene sequences were determined for the type strains of the validly described Enterococcus species, Melissococcus pluton and Tetragenococcus halophilus. A comprehensive set of rRNA targeted specific oligonucleotide hybridization probes was designed according to the multiple probe concept. In silico probe design and evaluation was performed using the respective tools of the ARB program package in combination with the ARB databases comprising the currently available 16S as well as 23S rRNA primary structures. The probes were optimized with respect to their application for reverse hybridization in microplate format. The target comprising 16S and 23S rDNA was amplified and labeled by PCR (polymerase chain reaction) using general primers targeting a wide spectrum of bacteria. Alternatively, amplification of two adjacent rDNA fragments of enterococci was performed by using specific primers. In vitro evaluation of the probe set was done including all Enterococcus type strains, and a selection of other representatives of the gram-positive bacteria with a low genomic DNA G+C content. The optimized probe set was used to analyze enriched drinking water samples as well as original samples from waste water treatment plants.     

THE TYPE SERIES OF 'SINEMYS'WUERHOENSIS, A PROBLEMATIC TURTLE FROM THE LOWER CRETACEOUS OF CHINA, INCLUDES AT LEAST THREE TAXA

Abstract:  We re-examine the type series of ' Sinemys ' wuerhoensis Yeh (at least 20 specimens, including several shells and skulls on three slabs of matrix and one isolated skull) from the Early Cretaceous Tugulu Group of China. Our study shows that the type series of ' S. ' wuerhoensis is actually a chimera made up of at least three distinct taxa. The holotype of this taxon should be assigned to the basal eucryptodire genus Xinjiangchelys Yeh. As there are no characters that distinguish ' S. ' wuerhoensis from Xinjiangchelys species, we consider it to be a nomen dubium . This new assignment of ' S. ' wuerhoensis expands the temporal range of Xinjiangchelys from the Late Jurassic into the Early Cretaceous in Asia. The majority of the paratypes of ' S. ' wuerhoensis (several shells in dorsal and ventral aspect and skulls) are referred to the basal eucryptodire genus Ordosemys Brinkman and Peng. We establish a new name for these specimens, Ordosemys brinkmania sp. nov. One additional specimen in the type series of ' S. ' wuerhoensis , a skull, is referred to cf. Pantrionychia Joyce, Parham and Gauthier indet.     

The Problematic Paralbunea Hu and Tao, 1996: Homonymy, Generic Nom. Nov., and Correct Taxonomic Placement

The fossil genus Paralbunea Hu and Tao, described as an anomuran albuneid, is a junior homonym of Paralbunea Serène and a possible synonym of either the brachyuran raninid Ranilia H. Milne Edwards, 1837, or Cosmonotus Adams and White, 1848. The type series of P. taipeiensis Hu and Tao is composed of at least two species belonging to two raninid genera and appears (in part) assignable to the genus Ranilia and (in part) to Cosmonotus grayi Adams and White, 1848. However, due to both limited availability and poor quality of the holotype of Paralbunea taipeiensis , and destruction of the paratypes, this taxon cannot be unequivocally synonymized with any particular raninid taxon and a new name is proposed to replace Hu and Tao's preoccupied generic name. Caution is urged when describing purported albuneid fossils, due to strong convergence with raninid crabs, which are far more common in the fossil record.     

A rapid PCR based method to distinguish between Lactococcus and Enterococcus

Phenotypic characterisation of Lactococcus and Enterococcus species remains unreliable as strains of both genera have been isolated which do not conform to the traditional criteria for separation of these genera. A bank of 131 isolates was phenotypically characterised by three methods: (a) traditional broth tests, (b) API Rapid ID 32 Strep and (c) BBL Crystal ID kits. Differences in genus designation between commercial kits were evident for 12 strains (9%), while 7 strains (5%) remained unidentified by either kit. Published 16S rRNA sequences were aligned and used to design genus-specific primers which, when used in separate PCR reactions, were capable of distinguishing all type strains of Lactococcus and Enterococcus. These primers did not react with known species of Streptococcus, Pediococcus, Lactobacillus, Leuconostoc or Tetragenococcus. Isolates which could not be identified by phenotype were assigned to either genus on the basis of the gene primers.     

Differential resistance across paternal genotypes of honey bee brood to the pathogenic bacterium Melissococcus plutonius

Melissococcus plutonius is a pathogenic bacterium affecting immature stages of the western honey bee (Apis mellifera) and leads to European foulbrood (EFB) disease. Despite EFB outbreaks increasing in frequency in several countries in recent decades, there is little knowledge on the epidemiology of M. plutonius or on the defence mechanisms of honey bees against this pathogen. Mating of honey bee queens with multiple males (polyandry) can be such a mechanism, as it has been shown to be beneficial to colony health and fitness. It is hypothesized that a high level of polyandry was selected for in response to pathogen pressure to maximize the probability that at least some patrilines among nestmates in a colony possess a high degree of resistance to specific pathogens, ultimately protecting colonies against infections. We show that M. plutonius infection provokes differential mortality among patrilines of immature honey bee workers. Such differences indicate a genetic origin of resistance against this pathogen—supporting the polyandry hypothesis—and open up avenues to improve control of EFB disease via selective breeding.     

Bivalency and epitope specificity of a high-affinity IgG3 monoclonal antibody to the Streptococcus group A carbohydrate antigen. Molecular modeling of a Fv fragment

The binding of Strep 9, a mouse monoclonal antibody (mAb) of the IgG3 subclass directed against the cell-wall polysaccharide of Group A Streptococcus (GAS), has been characterized. The intact antibody and proteolytic fragments of Strep 9 bind differently to GAS: the intact mAb and F(ab)2' have greater affinity for the carbohydrate epitope than the monomeric Fab or F(ab)'. A mode of binding in which Strep 9 binds bivalently to portions of the polysaccharide on adjacent chains on GAS is proposed. A competitive ELISA protocol using a panel of carbohydrate inhibitors shows that the branched trisaccharide, beta-D-GlcpNAc-(1-->3)-[alpha-L-Rhap-(1-->2)]-alpha-L-Rhap, and an extended surface are key components of the epitope recognized by Strep 9. Microcalorimetry measurements with the mAb and two synthetic haptens, a tetrasaccharide and a hexasaccharide, show enthalpy-entropy compensation as seen in other oligosaccharide-protein interactions. Molecular modeling of the antibody variable region by homology modeling techniques indicates a groove-shaped combining site that can readily accommodate extended surfaces. Visual docking of an oligosaccharide corresponding to the cell-wall polysaccharide into the site provides a putative model for the complex, in which a heptasaccharide unit occupies the site and the GlcpNAc residues of two adjacent branched trisaccharide units occupy binding pockets within the groove-shaped binding site.     

A craniometric study of the black and white Colobus Illiger 1811 (Primates: Ceropithecoidea).

This study examines the craniometry of Black and White Colobus monkeys using 1072 specimens representing all the recognized subspecies (after Rahm, '70) of the genus. Seventy-six measurements were taken on each individual, and examined using canonical variates analysis and clustering by Ward's Error Sum method. The assumptions of the analytical techniques are shown to be met, and the results demonstrated to be stable. Examination of the populations for statistical difference and taxonomic distinctiveness using a multivariate extension of Mayr's Rule indicates that the taxonomy presented by Rahm ('70) is essentially correct, except that the subspecies of guereza across the northern part of Central Africa should be lumped into a single group--C. g. occidentalis--and the subspecies of montane angolan colobus in Eastern Zaire should all be lumped into C. a. ruwenzorii. The systematic patterns of the genus illustrate the whole range of the process of speciation, from barely distinct subspecies, to subspecies almost as distinct as allopatric species of the genus, and on the fully sympatric species Three major zoogeographic areas may be delineated: an East African area dominated by the effects of the Rift Valley, with a large number of subspecies isolated in forest islands; a Central African area with little subspeciation and sympatric overlap of the major species of Colobus; and a West African area with a clinal pattern of distribution of subspecies, and secondary intergradation. The arbitrary nature of Mary's Rule is also apparent. Lastly, the CVA indicated major differences across the genus to be located in the teeth and jaws, suggesting diet might be an important distinguishing feature in Colobus.     

Streptococcus caprinus sp.nov., a tannin-resistant ruminal bacterium from feral goats

An undescribed bacterium capable of clearing tannic acid-protein complexes has been isolated from ruminal contents of feral goats browsing tannin-rich Acacia species. The bacterium is a Gram-positive facultative anaerobe, characterized as a Streptococcus , but DNA-DNA hybridization and 16S rDNA sequencing show that it is distinct from the common ruminal species Strep. bovis. We propose the name Streptococcus caprinus for this species. The type strain is Strep. caprinus 2.3, Australian Collection of Microorganisms (ACM) 3969. The bacterium grows in media containing at least 2.5% w/v tannic acid or condensed tannin and produces zones of clearing around colonies on nutrient agar plates with added tannic acid. Streptococcus caprinus is not a major inhabitant of domestic livestock, but is found in feral goats browsing tannin-rich Acacia species, at a population of up to 2 times 10⁶ cfu ml^-1 of rumen fluid.     

'Lysobacter enzymogenes ssp. cookii ' Christensen 1978 should be recognized as an independent species, Lysobacter cookii sp. nov.

' Lysobacter enzymogenes ssp. cookii ' was proposed by Christensen and Cook in 1978; however, this subspecies name has not been cited in the Approved Lists of Bacterial Names and therefore the nomenclature has not been validated. In our genetic approach to clarify the relationships of the designated type strain of ' L. enzymogenes ssp. cookii ' PAGU 1119 (GenBank accession number ATCC29488 ) within the genus Lysobacter revealed that the strain was closely related to Lysobacter capsici YC5194 ^T (99.4%) rather than L. enzymogenes DSM2043 ^T (97.2%). The value for whole genome DNA–DNA relatedness between strain PAGU 1119 and L. enzymogenes DSM 2043^T or L. capsici YC5194 ^T was 20.7–26.1% or 60.9–62.0%, respectively. Although PAGU 1119 and L. capsici YC5194 ^T showed relatively high DNA relationships, the fatty acid profiles and some phenotypic characteristics were different, and we concluded that PAGU 1119 should be placed in a new species. We therefore propose a new species with the name Lysobacter cookii sp. nov. The type strain is PAGU 1119^T ( ATCC29488 ).