Characterization of secreted proteases of Paenibacillus larvae, potential virulence factors involved in honeybee larval infection

Paenibacillus larvae is the causative agent of American Foulbrood (AFB), the most severe bacterial disease that affects honeybee larvae. AFB causes a significant decrease in the honeybee population affecting the beekeeping industry and agricultural production. After infection of larvae, P. larvae secretes proteases that could be involved in the pathogenicity. In the present article, we present the secretion of different proteases by P. larvae. Inhibition assays confirmed the presence of metalloproteases. Two different proteases patterns (PP1 and PP2) were identified in a collection of P. larvae isolates from different geographic origin. Forty nine percent of P. larvae isolates showed pattern PP1 while 51% exhibited pattern PP2. Most isolates belonging to genotype ERIC I - BOX A presented PP2, most isolates belonging to ERIC I - BOX C presented PP1 although relations were not significant. Isolates belonging to genotypes ERIC II and ERIC III presented PP2. No correlation was observed between the secreted proteases patterns and geographic distribution, since both patterns are widely distributed in Uruguay. According to exposure bioassays, isolates showing PP2 are more virulent than those showing PP1, suggesting that difference in pathogenicity could be related to the secretion of proteases.     

Antibacterial activity of water extracts and essential oils of various aromatic plants against Paenibacillus larvae, the causative agent of American Foulbrood

Vegetal water extracts, namely the water remaining after hydro-distillation and decoctions, and essential oils of 10 plant species were tested as inhibitors for the growth of Paenibacillus larvae, the causative agent of American Foulbrood. Achyrocline satureioides, Chenopodium ambrosioide, Eucalyptus cinerea, Gnaphalium gaudichaudianum, Lippia turbinata, Marrubium vulgare,Minthostachys verticillata, Origanum vulgare, Tagetes minuta and Thymus vulgaris were included in the study. The water remaining after hydro-distillation showed the highest antibacterial activities, the growth of almost all the P. larvae strains tested was inhibited by these extracts. Regarding the plants tested, E. cinerea and M. verticillata were the plant species with the highest biological activity with 100% efficacy (all its extracts inhibited the growth of all P. larvae strains). Essential oils were less active for the inhibition of P. larvae growth.     

Inhibition of the growth of Paenibacillus larvae, the causal agent of American foulbrood of honeybees, by selected strains of aerobic spore-forming bacteria isolated from apiarian sources

The bacterium Paenibacillus larvae, the causative agent of American foulbrood disease of honeybee larvae, occurs throughout the world and is found in many beekeeping areas of Argentina. The potential as biocontrol agents of antagonic aerobic spore-forming bacteria isolated from honey samples and other apiarian sources were evaluated. Each isolate was screened against one strain of Paenibacillus larvae (ATCC 9545) by using a perpendicular streak technique. Ten randomly selected bacterial strains from the group that showed the best antagonistic effect to P. larvae ATCC 9545 were selected for further study. These were identified as Bacillus subtilis (m351), B. pumilus (m350), B. licheniformis (m347), B. cereus (mv33), B. cereus (m387), B. cereus (m6c), B. megaterium (m404), Brevibacillus laterosporus (BLAT169), B. laterosporus (BLAT170), and B. laterosporus (BLAT171). The antagonistic strains were tested against 17 P. larvae strains from different geographical origins by means of a spot test in wells. The analysis of variance and posterior comparison of means by Tukey method (P < 0.01) showed that the best antagonists were B. megaterium (m404), B. licheniformis (m347), B. cereus (m6c), B. cereus (mv33), and B. cereus (m387).     

低温导致中华蜜蜂后翅翅脉的新变异

在封盖发育期,将中华蜜蜂Apis cerana cerana封盖子放入24℃低温环境中分别处理24、48、72 h,再恢复到正常的发育温度35℃至羽化,或将封盖子放在30℃低温中培养至羽化,发现羽化后的蜜蜂后翅翅脉发生明显变异。发现翅脉变异有3种类型,翅脉增加、翅脉突出和翅脉缺失。后翅翅脉增加的变异只发现一种,基室、缘室共有的径分脉与基室、盘室共有的中脉之间,新增径中横脉。新增的径中横脉发生的程度不同,有的在径分脉和中脉相对的位置相向伸出,有的形成完整的新脉,新增的径中横脉与原径中横脉相似,在末端均有"弱化点"。由于原有径中横脉较短,一直忽略其存在,这次径中横脉的确定,可以判定原来公认的"中脉分叉"应分别是径分脉和中脉延伸出来的两条翅脉。后翅突出的变异有2种,中脉向基室突出,径分脉向基室突出。翅脉突出的程度不同,有的略突出,有的明显突出,有的伸出翅脉较长。中脉突出均发生在中脉的转折点上,且此类翅脉变异,仅发生在雄蜂样本中,在工蜂样本中还未见。后翅翅脉缺失的变异有2种,发生在肘臀横脉和中脉。肘臀横脉"弱化点"处缺失。将中华蜜蜂雄蜂封盖子放入低温30℃恒温培养箱中发育,发现2个羽化的雄蜂样本发生肘臀横脉在"弱化点"处缺失。中脉向盘室伸出的部分发生缺失。翅脉缺失的程度不同,有的明显缩短,有的仅存痕迹,有的缺失大部分或完全缺失。中脉向盘室伸出的部分是东方蜜蜂Apis cerana区别于西方蜜蜂Apis mellifera的主要形态特征之一,如果东方蜜蜂这段翅脉全部缺失,此部位的形态与西方蜜蜂完全相同。这意味着这个翅脉作为区分东方蜜蜂和西方蜜蜂的翅脉特征将面临挑战。对蜜蜂种质资源鉴定,蜜蜂的系统发育和进化生物学等领域的研究将具有重要影响。温度影响翅脉发育的深入研究,为研究蜜蜂翅脉发育过程,揭示翅脉发育调控机制,以及蜜蜂发育的温度适应生理机制提供一个新思路。     

The possible role of Varroa destructor in the spreading of American foulbrood among apiaries

The aim of this investigation was to establish whether Varroa destructor can play a role in the transmission of Paenibacillus larvae larvae spores from infected to healthy bee colonies. Mites, collected from an Apis mellifera carnica colony heavily infected with American foulbrood and treated with Apistan, were suspended in distilled water and treated in three different ways:homogenizing, shaking and stirring, or sonication. The resulting fluid samples were transferred onto selective agar medium. All culture plates showed colonies that could be identified as P.l. larvae. In view of the numbers of spores they can carry, it is concluded that mites may transmit American foulbrood from infected to healthy bee colonies.     

The distribution of Paenibacillus larvae spores in adult bees and honey and larval mortality, following the addition of American foulbrood diseased brood or spore-contaminated honey in honey bee (Apis mellifera) colonies

Within colony transmission of Paenibacillus larvae spores was studied by giving spore-contaminated honey comb or comb containing 100 larvae killed by American foulbrood to five experimental colonies respectively. We registered the impact of the two treatments on P. larvae spore loads in adult bees and honey and on larval mortality by culturing for spores in samples of adult bees and honey, respectively, and by measuring larval survival. The results demonstrate a direct effect of treatment on spore levels in adult bees and honey as well as on larval mortality. Colonies treated with dead larvae showed immediate high spore levels in adult bee samples, while the colonies treated with contaminated honey showed a comparable spore load but the effect was delayed until the bees started to utilize the honey at the end of the flight season. During the winter there was a build up of spores in the adult bees, which may increase the risk for infection in spring. The results confirm that contaminated honey can act as an environmental reservoir of P. larvae spores and suggest that less spores may be needed in honey, compared to in diseased brood, to produce clinically diseased colonies. The spore load in adult bee samples was significantly related to larval mortality but the spore load of honey samples was not.     

Thermoregulation in mixed-species colonies of honeybees (Apis cerana and Apis mellifera)

Apis cerana and Apis mellifera normally display different strategies in cooling hive temperature, raising the question whether they would coordinate their efforts in to achieve stable thermoregulation in mixed colonies. The results show that the normal temperatures in the brood area in mixed colonies are more similar to those of pure A. cerana colonies than pure A. mellifera colonies. Under heat stress, A. cerana workers are more sensitive, and initiate fanning earlier than A. mellifera workers. In mixed colonies, the former become the main force for thermoregulation. When worker bees of both species were fanning together at the entrance, their own species-specific postures were adopted, but due to a significantly smaller number of A. mellifera workers engaged in fanning, the cooling efficiency of mixed colonies were closest to that of pure A. cerana colonies.     

Sun angle time windows for absconding by the dwarf honeybee, Apis florea

In Asia, the red dwarf honeybee, Apis florea, is notorious for its absconding habit. Interestingly, such colonies show a bimodal frequency distribution about a noonday lull throughout the year. Because slight errors in reading the relative position of the sun near its zenith results in very large orientation errors in the waggle dances of other honeybees in the tropics, we postulated that the frequency distribution of absconding in the red dwarf honeybee relative to local clock time could be explained in similar fashion. The frequency distribution of absconding by the red dwarf honeybee with respect to time was found to be bimodal with a pronounced lull at noonday, which in turn is related to the altitude angle of the sun. So, these bees largely avoid flying off between 12:00 h and 13:00 h on the one hand and that their preferred departure angle of the sun is between 55° and 65° on the other. Given the difficulties of taking an accurate reading of the sun at angles ±6° of the sun's zenith (resulting in a 1 h loss around noon) and the 2 h required to reach consensus over the final direction to be flown, the bees are simply left with two time windows, morning and afternoon, in which to abscond and, indeed some 90% of the red dwarf honeybee colonies do so. The noonday lull is not associated with high temperatures for any given day. Absconding is not inhibited by high temperatures.     

Chemical composition and antimicrobial activity of Citrus essences on honeybee bacterial pathogen <Emphasis Type="Italic">Paenibacillus larvae</Emphasis>, the causal agent of American foulbrood

Antimicrobial properties and chemical composition of four citrus fruit essential oils to control Paenibacillus larvae, the causal agent of American foulbrood disease (AFB) were determined. This honeybee larvae disease occurs throughout the world and is found in many beekeeping areas of Argentina. Citrus fruit essential oils tested were those from grapefruit (Citrus paradisi), sweet orange (Citrus sinensis), mandarin (Citrus nobilis) and lemon (Citrus limon). The components of the essential oils were identified by SPME-GC/MS analysis. The antimicrobial activity of the oils against P. larvae were determined by the broth microdilution method. Two way ANOVA tests for minimum inhibitory concentrations (MICs) data and minimal bactericide concentrations (MBCs) data, indicated significant differences between the strains and the oils tested. The antimicrobial assays showed that the oil of C. paradisi inhibited the bacterial strains at the lowest concentrations tested, MICs and MBCs averages of 385.0 mg/l and 770.0 mg/l, respectively. This property could be attributed to the kind and percentage of the volatile components of the oil, like limonene (69.9%) and myrcene (9.6%). The use of essential oils or their specific volatile components individually against pests related to food provision may represent an alternative scope for the control of this serious disease because it does not leave toxic chemical residues in honey nor in its by products.     

Identification of Paenibacillus larvae to the subspecies level: an obstacle for AFB diagnosis

This study was initially aimed at developing a PCR-test to differentiate between the pathogenic agent of American foulbrood (Paenibacillus larvae subsp. larvae) and powdery-scale disease (P. larvae subsp. pulvifaciens) of the honeybee. The test was based on the "insert of clone 9" (iC9), referring to a cloned 1.9 kB HaeIII fragment that occurs only in the P. larvae subsp. larvae reference strains and possibly correlates with American foulbrood virulence. It was shown that an iC9-based PCR-test discriminates between the BCCM/LMG reference strains of both subspecies. However, the screening of 179 Belgian field strains revealed five isolates that gave no iC9-based amplicon, thus rather resembling to P. larvae subsp. pulvifaciens. In addition, they all produced acid from mannitol, a characteristic previously assigned to the pulvifaciens subspecies. Because the reference strains gave conflicting data, this carbohydrate acidification was not conclusive. Therefore, the exact taxonomic position of the five retained strains was determined by a polyphasic approach using SDS-PAGE, AFLP, and ERIC-based PCR. Four iC9-negative field strains could be identified as P. larvae subsp. larvae; the taxonomic position of the fifth field strain remained ambiguous. The latter was provisionally classified as a subspecies pulvifaciens strain on the basis of SDS-PAGE. The present paper demonstrates the existence of field strains that do not fit well in the subdivision of the species P. larvae into two subspecies. Knowing that only one of both subspecies represents the pathogenic agent of AFB, this is a serious obstacle for the diagnosis of this honeybee disease.     

Differential gene expression of the honey bees Apis mellifera and A. cerana induced by Varroa destructor infection

Varroa destructor mite is currently the most serious threat to the world bee industry. Differences in mite tolerance are reported between two honey bee species Apis mellifera and Apis cerana. Differential gene expression of two honey bee species induced by V. destructor infection was investigated by constructing two suppression subtractive hybridization (SSH) libraries, as first steps toward elucidating molecular mechanisms of Varroa tolerance. From the SSH libraries, we obtained 289 high quality sequences which clustered into 132 unique sequences grouped in 26 contigs and 106 singlets where 49 consisted in A. cerana subtracted library and 83 in A. mellifera. Using BLAST, we found that 85% sequences had counterpart known genes whereas 15% were undescribed. A Gene Ontology analysis classified 51 unique sequences into different functional categories. Eight of these differentially expressed genes, representative of different regulation patterns, were confirmed by qRT-PCR. Upon the mite induction, the differentially expressed genes from both bee species were different, except hex 110 gene, which was up-regulated in A. cerana but down-regulated in A. mellifera, and Npy-r gene, which was down-regulated in both species. In general, most of the differential expression genes were involved in metabolic processes and nerve signaling. The results provide information on the molecular response of these two bee species to Varroa infection.     

Bacterial diversity in worker adults of Apis mellifera capensis and Apis mellifera scutellata (Insecta: Hymenoptera) assessed using 16S rRNA sequences

High-fidelity PCR of 16S rRNA sequences was used to identify bacteria associated with worker adults of the honeybee subspecies Apis mellifera capensis and Apis mellifera scutellata. An expected approximately 1.5-kb DNA band, representing almost the entire length of the 16S rRNA gene, was amplified from both subspecies and cloned. Ten unique sequences were obtained: one sequence each clustered with Bifidobacterium (Gram-positive eubacteria), Lactobacillus (Gram-positive eubacteria), and Gluconacetobacter (Gram-negative alpha-proteobacteria); two sequences each clustered with Simonsiella (beta-proteobacteria) and Serratia (gamma-proteobacteria); and three sequences each clustered with Bartonella (alpha-proteobacteria). Although the sequences relating to these six bacterial genera initially were obtained from either A. m. capensis or A. m. scutellata or both, newly designed honeybee-specific 16S rRNA primers subsequently amplified all sequences from all individual workers of both subspecies. Attempts to amplify these sequences from eggs have failed. However, the wsp primers designed to amplify Wolbachia DNA from arthropods, including these bees, consistently produced a 0.6-kb DNA band from individual eggs, indicating that amplifiable bacterial DNA was present. Hence, the 10 bacteria could have been acquired orally from workers or from other substrates. This screening of 16S rRNA sequences from A. m. capensis and A. m. scutellata found sequences related to Lactobacillus and Bifidobacterium which previously had been identified from other honeybee subspecies, as well as sequences related to Bartonella, Gluconacetobacter, Simonsiella/Neisseria, and Serratia, which have not been identified previously from honeybees.     

Detection of Paenibacillus larvae subspecies larvae spores in naturally infected bee larvae and artificially contaminated honey by PCR

American foulbrood (AFB), a severe bacterial disease of honeybee brood, has recently been found in Uruguayan apiaries. Detection of the causative agent, Paenibacillus larvae subspecies larvae, is a very important concern in order to prevent disease dissemination and decrease of honey production. Since spores are the infective forms of this pathogen, in the present work we report the use of polymerase chain reaction (PCR) to detect P. l. subsp. larvae spores from in vitro cultures, larvae with clinical symptoms and experimentally contaminated honey. The set of primers was designed based on the published P. l. subsp. larvae 16S rRNA gene. Using this approach we could amplify the pathogen DNA and obtain a great sensitivity and a notable specificity. Detection limit for spore suspension was a 10^–2 dilution of template DNA obtained from 32 spores, as determined by plate count. For artificially contaminated honey, we could detect the PCR product at a 10^–3 dilution of template DNA obtained from 170 spores. In addition, when PCR conditions were set to improve specificity, we were able to amplify P. l. subsp. larvae DNA selectively and no cross-reactions were observed with a variety of related bacterial species, including P. l. subsp. pulvifaciens. Since spore detection is very important to confirm the presence of the disease, this method provides a reliable diagnosis of AFB from infected larvae and contaminated honey in a few hours.     

Two small heat shock protein genes in Apis cerana cerana: characterization,regulation, and developmental expression

In the present study, we identified and characterized two small heat shock protein genes from Apis cerana cerana, named AccHsp24.2 and AccHsp23.0. An alignment analysis showed that AccHsp24.2 and AccHsp23.0 share high similarity with other members of the α-crystallin/sHSP family, all of which contain the conserved α-crystallin domain. The recombinant AccHsp24.2 and AccHsp23.0 proteins were shown to have molecular chaperone activity by the malate dehydrogenase thermal aggregation assay. Three heat shock elements were detected in the 5′-flanking region of AccHsp24.2 and eleven in AccHsp23.0, and two Drosophila Broad-Complex genes for ecdysone steroid response sites were found in each of the genes. The presence of these elements suggests that the expression of these genes might be regulated by heat shock and ecdysone, which was confirmed by quantitative RT-PCR (RT-qPCR). The results revealed that the expression of the two genes could be induced by cold shock (4 °C) and heat shock (37 °C and 43 °C) in an analogous manner, and AccHsp24.2 was more susceptible than AccHsp23.0. In addition, the expression of the two genes was induced by high concentrations of ecdysone in vitro and in vivo. The accumulation of AccHsp24.2 and AccHsp23.0 mRNA was also detected in different developmental stages and tissues. In spite of the differential expression at the same stage, these genes shared similar developmental patterns, suggesting that they are regulated by similar mechanisms.     

Diversity of Bacteria and Archaea from a landfill in Chandigarh,India as revealed by culture-dependent and culture-independent molecular approaches

The bacterial community structure of a municipal landfill in Chandigarh, India was analysed by culture-dependent as well as culture-independent molecular approaches, and archaeal structure by the latter method. Samples were collected in two phases from the surface and a depth of 0.91 m in June, 2004 and from 0.91 m, 1.52 m and 1.68 m in May, 2005. After serial dilutions, samples were plated onto tryptic soy agar (TSA), plate count agar (PCA), tryptic soy broth agar (TSBA) and TSBA100 (TSBA diluted 100 times and solidified with agarose), and incubated aerobically at 30 °C. The number of bacteria (CFU) on different media ranged between 9.4 × 10⁵ g⁻¹ (on PCA) and 1.9 × 10⁷ g⁻¹ (on TSA) (wet weight). The numbers of bacteria enumerated from plates incubated anaerobically (anaerobic agar and reinforced clostridial agar) were 2.1 × 10⁷ and 1.7 × 10⁶ g⁻¹, respectively. Of the 468 isolated and purified bacteria (183 in the first phase and 285 in the second phase), 135 were characterised using phenotypic characteristics as well as 16S rRNA gene sequence analysis. It was found that members of the phylum Firmicutes were overwhelmingly predominant (86.6%) in the landfill, followed by Actinobacteria (9.6%) and Proteobacteria (3.7%). Among the Firmicutes, at least 17 species from the single genus Bacillus were the most abundant inhabitants of the landfill. Detailed polyphasic characterisation of many of these isolates led to the discovery of a novel genus Paenisporosarcina (and the species P. quisquiliarum), a novel species of Microbacterium, M. immunditiarum, and reclassification of Sporosarcina macmurdoensis, Pelagibacillus goriensis, Bacillus silvestris, Bacillus insolitus, Bacillus psychrotolerans and Bacillus psychrodurans. Culture-independent analysis of two 16S rRNA gene libraries also revealed that the phylum Firmicutes was the predominant group in this community. The diversity of Archaea was found to be limited mainly to members of two orders: Methanosarcinales and Methanomicrobiales of the phylum Euryarchaeota. When these results were compared to those reported earlier on similar studies, it was found that irrespective of differences in composition of municipal solid waste (especially compostable organic matter and paper) and climate, the members of bacterial and archaeal communities in landfills of many countries remained broadly similar.