The prevalence of the honeybee brood pathogens Ascosphaera apis,Paenibacillus larvae and Melissococcus plutonius in Spanish apiaries determined with a new multiplex PCR assay

The microorganisms Ascosphaera apis, Paenibacillus larvae and Melissococcus plutonius are the three most important pathogens that affect honeybee brood. The aim of the present study was to evaluate the prevalence of these pathogens in honeybee colonies and to elucidate their role in the honeybee colony losses in Spain. In order to get it, a multiplex polymerase chain reaction (PCR) assay was developed to simultaneously amplify the16S ribosomal ribonucleic acid (rRNA) gene of P. larvae and M. plutonius, and the 5.8S rRNA gene of A. apis. The multiplex PCR assay provides a quick and specific tool that successfully detected the three infectious pathogens (P. larvae, M. plutonius and A. apis) in brood and adult honeybee samples without the need for microbiological culture. This technique was then used to evaluate the prevalence of these pathogens in Spanish honeybee colonies in 2006 and 2007, revealing our results a low prevalence of these pathogens in most of the geographic areas studied.     

Molecular epidemiology and population structure of the honey bee brood pathogen Melissococcus plutonius

Melissococcus plutonius is the causative agent of European foulbrood (EFB), which is a serious brood disease of the European honey bee (Apis mellifera). EFB remains a threat because of a poor understanding of disease epidemiology. We used a recently published multi-locus sequence typing method to characterise 206 M. plutonius isolates recovered from outbreaks in England and Wales over the course of 2 years. We detected 15 different sequence types (STs), which were resolved by eBURST and phylogenetic analysis into three clonal complexes (CCs) 3, 12 and 13. Single and double locus variants within CC3 were the most abundant and widespread genotypes, accounting for 85% of the cases. In contrast, CCs 12 and 13 were rarer and predominantly found in geographical regions of high sampling intensity, consistent with a more recent introduction and localised spread. K-function analysis and interpoint distance tests revealed significant geographical clustering in five common STs, but pointed to different dispersal patterns between STs. We noted that CCs appeared to vary in pathogenicity and that infection caused by the more pathogenic variants is more likely to lead to honey bee colony destruction, as opposed to treatment. The importance of these findings for improving our understanding of disease aetiology and control are discussed.     

The occurrence of Melissococcus plutonius in healthy colonies of Apis mellifera and the efficacy of European foulbrood control measures

European foulbrood (EFB) persists in England and Wales despite current treatment methods, all of which include feeding honey bee colonies with the antibiotic oxytetracycline (OTC). A large-scale field experiment was conducted to monitor a husbandry-based method, using comb replacement (known as Shook swarm), as a drug free EFB control option. The understanding of EFB epidemiology is limited, with little information on the presence of Melissococcus plutonius in disease free colonies. Additional samples were collected from diseased and disease free apiaries to identify symptomless infection. EFB reoccurrence was not significantly different between OTC and husbandry methods and real-time PCR data demonstrated that fewer Shook swarm treated colonies contained M. plutonius carryover to the Spring following treatment. Asymptomatic colonies from diseased apiaries showed an increased risk of testing positive for M. plutonius compared to asymptomatic colonies from disease free apiaries. The probability of a sample being symptomatic increased when a greater quantity of M. plutonius was detected in adult bees and larvae. The possibility of treating EFB as an apiary disease rather than a colony disease and the implications of a control strategy without antibiotics are discussed.     

Geographical distribution and molecular detection of Nosema ceranae from indigenous honey bees of Saudi Arabia

The aim of the study was to detect the infection level of honey bees with Nosema apis and/or Nosema ceranae using microscopic and molecular analysis from indigenous honeybee race of eight Saudi Arabian geographical regions. A detailed survey was conducted and fifty apiaries were chosen at random from these locations. Infection level was determined both by microscope and Multiplex-PCR and data were analyzed using bioinformatics tools and phylogenetic analysis. Result showed that N. ceranae was the only species infecting indigenous honeybee colonies in Saudi Arabia. As determined by microscope, Nosema spores were found to be in 20.59% of total samples colonies, while 58% of the samples evaluated by PCR were found to be positive for N. ceranae, with the highest prevalence in Al-Bahah, a tropical wet and dry climatic region, whereas low prevalence was found in the regions with hot arid climate. Honeybees from all eight locations surveyed were positive for N. ceranae. This is the first report about the N. ceranae detection, contamination level and distribution pattern in Saudi Arabia.     

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.     

Difference in virulence of Mycobacterium avium isolates sharing indistinguishable DNA fingerprint determined in murine model of lung infection

Background

Opportunistic Mycobacterium avium typically causes disease in immunocompromised patients and in some groups of apparently healthy individuals. The high virulence of some bacterial lineages increases the disease risk. High-resolution molecular genotyping studies of M. avium clinical isolates demonstrated that some genotype patterns were more prevalent than others, suggesting that close genetic relatedness of these successful isolates sharing a similar genotype could determine similar biological properties associated with high virulence.

Methods and Findings

In this study, we aimed to compare the virulence and pathogenic properties of two epidemiologically unrelated M. avium isolates sharing an indistinguishable DNA fingerprint in a well-characterized model of pulmonary infection in mice, resistant or susceptible to mycobacteria. The mice, C57BL/6 wild- type or IFN-gamma gene disrupted (GKO), respectively, were intratracheally infected with two isolates, H27 (human blood isolate) and P104 (pig lymph node isolate), and the lungs were examined for bacterial loads, histopathology and cytokine gene expression. The obtained data demonstrated significant differences in the virulence properties of these strains. Although the H27 strain grew significantly faster than P104 in the early stage of infection, this bacterium induced protective immunity that started to reduce bacterial numbers in the wild- type mice, whereas the P104 strain established a chronic infection. In the GKO mice, both strains were capable of causing a chronic infection, associated with higher bacterial burdens and severe lung pathology, in a similar manner.

Conclusions/Significance

The results demonstrated that the studied isolates differed in the pathogenic properties although were indistinguishable by actually widely used genotyping techniques demonstrating that the genotype similarity does not predict similarity in virulence of M. avium isolates.     

Dermatophyte and non dermatophyte fungi in Riyadh City,Saudi Arabia

BackgroundDermatophytes are a scientific label for a group of three genera (Microsporum, Epidermophyton and Trichophyton) of fungus that causes skin disease in animals and humans. Conventional methods for identification of these fungi are rapid and simple but are not accurate comparing to molecular methods.ObjectiveThis study aimed to isolate human pathogenic dermatophytes which cause dermatophytosis in Riyadh City, Saudi Arabia and to identify these fungi by using conventional and molecular methods.MethodsThe study was conducted in Medical Complex, Riyadh and King Saud University. Samples of infected skin, hairs and nails were collected from 112 patients. Diagnosis of skin infections, direct microscopic test, isolation and identification of dermatophytes by conventional and molecular methods were carried out.ResultsThe results indicated that the tinea capitis infection had the highest prevalence among the patients (22.3%) while Tinea barbae had the lowest. In this study the identified dermatophyte isolates belong to nine species as Trichophyton violaceum, Trichophyton verrucosum, Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton schoenleinii, Trichophyton concentricum, Microsporum canis, Microsporum audouinii and Epidermophyton floccosum which cause skin infections were isolated during this study. Non dermatophyte isolates included 5 isolates from Aspergillus spp. 4 isolates from Acremonium potronii and 15 isolates from Candida spp. M. canis were the most common species (25% of isolated dermatophytes). Out of the 52 dermatophyte isolates identified by conventional methods, there were 45 isolates identified by the molecular method.ConclusionsThe results concluded that approximately M. canis caused a quarter of dermatophyte cases, tinea capitis infection was prevalent and the molecular method was more accurate than conventional methods.     

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.     

Bacteria in the gut of Japanese honeybee, Apis cerana japonica, and their antagonistic effect against Paenibacillus larvae, the causal agent of American foulbrood

We assessed the complexity of bacterial communities occurring in the digestive tract of the Japanese honeybee, Apis cerana japonica, using histological and 16S rRNA gene sequence analyzes. Both Gram-positive and -negative bacteria were observed, and the number of gut bacteria was higher in old larvae compared with young larvae. A total of 35 clones were obtained by a culture-dependent method, and 16S rRNA gene sequence analysis revealed that the bacterial population in the gut of Japanese honeybee was diverse, including the phyla firmicutes, actinobacteria, and alpha-, beta-, and gammaproteobacteria. Further investigation by in vitro inhibition assays was carried out to determine the ability of an isolate to inhibit Paenibacillus larvae, the causal agent of American foulbrood. Out of 35 isolates, seven showed strong inhibitory activity against P. larvae. Most of the antagonistic bacteria belonged to Bacillus species, suggesting that the bacterial isolates obtained in this study appear to be potential candidates for the biological control of P. larvae.     

European foulbrood in honey bees

European foulbrood (EFB) is a severe bacterial brood disease caused by the Gram-positive bacterium Melissocccus plutonius. The disease has a worldwide distribution and is an increasing problem in some areas. Although the causative agent of EFB was described almost a century ago, many basic aspects of its pathogenesis are still unknown. This review presents both historical results and recent molecular data to synthesize present knowledge of this enigmatic honey bee disease.     

Molecular diagnosis of begomovirus associated with Chilli leaf curl disease in Jeddah,Saudi Arabia

Chilli (Capsicum annum L.) is well known as ‘wonder spice’. This is a very valuable cash crop grown as a vegetable globally. Chilli leaf curl disease is a major threat and global concern for the cultivation of Chilli by farmers and growers. In this work, the molecular diagnosis, genetic diversity, phylogenetic relationship, and begomovirus association with Chilli leaf curl disease have been discussed. The infected leaves were randomly harvested from the Chilli field, at Jeddah, Saudi Arabia. A group of begomovirus vector, whiteflies were also observed on the Chilli crop and infected weeds growing in the neighboring field. The begomovirus was confirmed by coat protein gene specific primer, dot blot hybridization, sequencing and sequence analysis. The full coat protein gene was found to have 774 nucleotides. The nucleotide sequences analysis shared the highest identity with Tomato yellow leaf curl virus reported earlier infecting tomato from Saudi Arabia, and the lowest identity was observed with Tomato yellow leaf curl virus Oman isolate. The overall sequence identity ranged from more than ninety percent among the analyzed sequences. The phylogenetic relationship analysis formed the major three clusters and showed the closed clustering with Tomato yellow leaf curl virus isolates. The natural spread of the Tomato yellow leaf curl virus on the Chilli crop from other crops poses an important and serious threat to Chili cultivation in the Kingdom of Saudi Arabia. Based on the literature review and current evidence, this is the first report of leaf curl disease of Chilli from Saudi Arabia.     

Morphological,biochemical and molecular identification of petroleum hydrocarbons biodegradation bacteria isolated from oil polluted soil in Dhahran,Saud Arabia

Accumulation of petroleum hydrocarbon residual considered a major environmental problem in the kingdom of Saudi Arabia cause of intensive efforts for oil detecting. Until now, In situ biodegradation considered the most effective method for petroleum hydrocarbon residual biodegradation. The aim of this study is isolation and identification biodegradable capability bacteria from contaminated sites in Khurais oil field, Dhahran, Saud Arabia via Different morphological and biochemical and molecular methods. Furthermore, degradation level in contaminated liquid medium and soil were evaluated. Three bacterial strains were selected from petroleum-contaminated soils of Khurais oil field depending on their capacity to grow in the existence of hydrocarbon components and identified according to morphological, biochemical. Interestingly, 16S rDNA sequencing fingerprinting results confirmed our bacterial identification as Bacillus subtilis, Pseudomonas aeruginosa and Bacillus cereu. Phyllogenetic tree was constructed and genetic similarity was calculated according to alignments results. Biodegradation patterns for different three isolates were reflected varied degradation ability for three isolates regarding incubation time. Different features were studied for three biodegrading bacterial strains and identified as Bacillus subtilis, Pseudomonas aeruginosa and Bacillus cereus. Remarkable degradation rate % patterns for hydrocarbons residual were recorded for all three isolates with varied.     

Genetic and phylogenetic analysis of the honey bee sacbrood virus from jiangxi isolates

The high prevalence of honeybee viral diseases poses a severe threat to the health of honeybees and causes substantial economic losses worldwide. Sacbrood virus (SBV) is a single-strand RNA virus that infects honeybees at all life stages. The infection can shorten the lifespan of adult bees and is lethal to larvae. SBV is the major cause of honeybee losses in Asia. Genetic and phylogenetic analyses of SBV isolates from different areas have been previously conducted. However, the impact of Apis mellifera Linnaeus and Apis cerana Fabricius coexistence on the infection and phylogeny of SBV remains unknown. In this study, we collected A. cerana and A. mellifera samples from commercial apiaries, only A. cerana in mountainous region. SBV prevalence was evaluated in three commercial apiaries of Jinxi, Tonggu and Nanchang and two mountainous regions of Zixi and Yifeng. In our sampling location, we found a higher SBV prevalence in the mountainous regions than in commercial apiaries. Partial structural polyprotein coding sequences were sequenced and compared with other GenBank SBV isolates. Phylogenetic tree topologies showed that SBV isolates form two major groups based on their host specificity, and isolates from same country tend to cluster together in subclades, indicating that the host and geographic region has significant effects on SBV strain specificity.     

Microbiological and molecular identification of bacterial species isolated from nasal and oropharyngeal mucosa of fuel workers in Riyadh,Saudi Arabia

This study aimed to determine the bacterial species colonizing the nasal and oropharyngeal mucosa of fuel workers in Central Riyadh, Saudi Arabia on a microbiological and molecular level. Throat and nasal swab samples were obtained from 29 fuel station attendants in the period of time extending from March to May 2014 in Riyadh, Saudi Arabia. Microbiological identification techniques were utilized to identify the bacterial species isolated. Antibiotic sensitivity was assessed for each of the bacterial isolates. Molecular identification techniques based on PCR analysis of specific genomic sequences was conducted and was the basis on which phylogeny representation was done for 10 randomly selected samples of the isolates. Blood was drawn and a complete blood count was conducted to note the hematological indices for each of the study participants. Nineteen bacterial species were isolated from both the nasal cavity and the oropharynx including Streptococcus thoraltensis, alpha-hemolytic streptococci, Staphylococcus hominis, coagulase-negative staphylococci, Leuconostoc mesenteroides, Erysipelothrix rhusiopathiae and several others. We found 100% sensitivity of the isolates to ciprofloxacin, cefuroxime and gentamicin. Whereas cefotaxime and azithromycin posted sensitivities of 85.7% and 91.4%, respectively. Low sensitivities (<60% sensitivity) to the antibiotics ampicillin, erythromycin, clarithromycin and norfloxacin were observed. Ninety-seven percent similarity to the microbial bank species was noted when the isolates were compared to it. Most hematological indices recorded were within the normal range. In conclusion, exposure to toxic fumes and compounds within fuel products may be a contributing factor to bacterial colonization of the respiratory tract in fuel workers.     

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.     

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.     

Comparative Genome Analysis of Mycobacterium avium Revealed Genetic Diversity in Strains that Cause Pulmonary and Disseminated Disease

Mycobacterium avium complex (MAC) infection causes disseminated disease in immunocompromised hosts, such as human immunodeficiency virus (HIV)-positive patients, and pulmonary disease in persons without systemic immunosuppression, which has been increasing in many countries. In Japan, the incidence of pulmonary MAC disease caused by M. avium is about 7 times higher than that caused by M. intracellulare. To explore the bacterial factors that affect the pathological state of MAC disease caused by M. avium, we determined the complete genome sequence of the previously unreported M. avium subsp. hominissuis strain TH135 isolated from a HIV-negative patient with pulmonary MAC disease and compared it with the known genomic sequence of M. avium strain 104 derived from an acquired immunodeficiency syndrome patient with MAC disease. The genome of strain TH135 consists of a 4,951,217-bp circular chromosome with 4,636 coding sequences. Comparative analysis revealed that 4,012 genes are shared between the two strains, and strains TH135 and 104 have 624 and 1,108 unique genes, respectively. Many strain-specific regions including virulence-associated genes were found in genomes of both strains, and except for some regions, the G+C content in the specific regions was low compared with the mean G+C content of the corresponding chromosome. Screening of clinical isolates for genes located in the strain-specific regions revealed that the detection rates of strain TH135-specific genes were relatively high in specimens isolated from pulmonary MAC disease patients, while, those of strain 104-specific genes were relatively high in those from HIV-positive patients. Collectively, M. avium strains that cause pulmonary and disseminated disease possess genetically distinct features, and it suggests that the acquisition of specific genes during strain evolution has played an important role in the pathological manifestations of MAC disease.     

Identification of Tomato black ring virus from tomato plants grown in greenhouses in Saudi Arabia

A survey was conducted in Al-Kharj governorate, Riyadh region to identify viruses causing variety of virus-like symptoms on tomato plants. A total of 135 samples were collected from symptomatic tomato plants. Symptoms included mottling, deformation, necrosis of leaves and fruits. Eighteen viruses were tested by DAS-ELISA. Tomato black ring virus (TBRV) was the virus of concern as it was not detected in Saudi Arabia before and was detected in 52.6% of the collected samples in this study. RT-PCR was used to confirm detection of TBRV and to sequence the amplified products to determine molecular characteristics of this virus. In the host range test study that was performed using a purified isolate of TBRV, sixteen out of the twenty two tested plants showed symptoms. Brassica oleracea was not infected by this virus. Gel electrophoreses (2% agarose) yielded fragments of 978 bp of coat protein gene of TBRV. Nucleotide sequences of purified RT-PCR products for three TBRV Saudi isolates were deposited in the GenBank with the following accession numbers MT274656, MT274657, and MT274658. These isolates of TBRV indicated a close Phylogenetic relationship of (99–100%) among themselves and with five isolates from Poland (95–98%) but a distant relationship of 85% with isolates from England and Lithuania deposited in the GenBank. This is the first report for detection and molecular characterization of TBRV infecting tomato plants in Saudi Arabia.     

Purification and Characterization of a Novel Anti-<Emphasis Type="Italic">Campylobacter</Emphasis> Bacteriocin Produced by <Emphasis Type="Italic">Lactobacillus curvatus</Emphasis> DN317

The lactic acid bacteria (LAB) microbiota of Saudi chicken ceca was determined. From 60 samples, 204 isolates of lactic acid bacteria were obtained. Three isolates produced antimicrobial activities against Campylobacter jejuni, Listeria monocytogenes, and Bacillus subtilis. The isolate DN317, which had the highest activity against Campylobacter jejuni ATCC 33560, was identified as Lactobacillus curvatus (GenBank accession numbers: KX353849 and KX353850). Full inhibitory activity was observed after a 2-h incubation with the supernatant at pH values between 4 and 8. Only 16% of the activity was conserved after a treatment at 121 °C for 15 min. The use of proteinase K, pepsin, chymotrypsin, trypsin, papain, and lysozyme drastically reduced the antimicrobial activity. However, lipase, catalase, and lysozyme had no effect on this activity. The active peptide produced by Lactobacillus curvatus DN317 was purified by precipitation with an 80% saturated ammonium sulfate solution, and two steps of reversed phase HPLC on a C18 column. The molecular weight of this peptide was 4448 Da as determined by MALDI-ToF. N-terminal sequence analysis using Edman degradation revealed 47 amino acid residues (UniProt Knowledgebase accession number C0HK82) revealing homology with the amino acid sequences of sakacin P and curvaticin L442. The antimicrobial activity of the bacteriocin, namely curvaticin DN317, was found to be bacteriostatic against Campylobacter jejuni ATCC 33560. The use of microbial antagonism by LAB is one of the best ways to control microorganisms safely in foods. This result constitutes a reasonable advance in the antimicrobial field because of its potential applications in food technology.