American foulbrood and African honey bees (Hymenoptera: Apidae)

We have taken samples of honey from individual beekeepers (N = 64), and of domestic (N = 35) and imported honey (N = 15) retailed in supermarkets in several sub-Saharan countries and cultivated these samples for Paenibacillus larvae subsp. larvae Heyndrickx et al. causing American foulbrood in honey bee colonies. The results are compared with samples of similar backgrounds and treated the same way but collected in Sweden (N = 35). No P. larvae subsp. larvae spores were found in any honey produced in Africa south of the Sahara although honey imported into this region frequently contains the pathogen. Swedish honey frequently contains P. larvae subsp. larvae spores although the general level of visibly infected bee colonies is low (roughly 0.5%). The results suggest that large parts of Africa may be free from American foulbrood. Behavioral studies (hygienic behavior) on Apis mellifera subsp. scutellata Lepeletier in Zimbabwe suggest that hygienic behavior of African bees could influence the apparent low level, or even absence of American foulbrood in large parts of Africa.     

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.     

The mite Varroa jacobsoni does not transmit American foulbrood from infected to healthy colonies

The present study was conducted to determine whether Varroa jacobsoni can transmit American foulbrood (AFB), caused by the bacterium Paenibacillus larvae to healthy colonies by the surface transport of spores. Five two-storey Langstroth colonies of Apis mellifera ligustica were infested by placing a sealed brood comb, with 10% Varroa prevalence, between the central brood combs of each colony. Two months later the colonies were inoculated with P. larvae by adding brood comb pieces with clinical signs of AFB (45±5 scales per colony). After 60 days the brood area was completely uncapped by means of dissecting needles and tweezers, separating the Varroa mites from the larvae and the collected mites were introduced at a rate of 51 per colony into four recipient hives placed in an isolated apiary. Twenty female Varroa specimens were separated at random and observed by SEM. Paenibacillus larvae spores were found on the dorsal shield surface and on idiosomal setae. All colonies died after 4–5 months due to a high incidence of varroosis. No clinical AFB symptoms or P. larvae spores were observed in microscopic preparations. It is concluded that Varroa jacobsoni does not transmit AFB from infected to healthy colonies; it does, however transport P. larvae spores on its surface.     

Distribution of <Emphasis Type="Italic">Paenibacillus larvae</Emphasis> Spores Among Adult Honey Bees (<Emphasis Type="Italic">Apis mellifera</Emphasis>) and the Relationship with Clinical Symptoms of American Foulbrood

Knowledge of the distribution of Paenibacillus larvae spores, the causative agent of American foulbrood (AFB), among individual adult honey bees is crucial for determining the appropriate number of adult bees to include in apiary composite samples when screening for diseased colonies. To study spore distribution at the individual bee level, 500 honey bees were collected from different parts of eight clinically diseased colonies and individually analyzed for P. larvae. From the brood chamber and from the super, bees were randomly collected and individually put in Eppendorf vials. The samples were frozen as soon as possible after collection. Concurrently with sampling, each colony was visually inspected for clinical symptoms of AFB. The number of clinically diseased cells in the colony was visually estimated. All samples were cultured in the laboratory for P. larvae. The results demonstrate that the spores are not randomly distributed among the bees; some bees have much higher spore loads than others. It is also clear that as the proportion of contaminated bees increase, the number of spores from each positive bee also increases. The data also demonstrated a relationship between the number of clinically diseased cells and the proportion of positive bees in individual colonies. This relationship was used to develop a mathematical formula for estimating the minimum number of bees in a sample to detect clinical disease. The formula takes into account the size of the apiary and the degree of certainty with which one aims to discover clinical symptoms. Calculations using the formula suggest that adult bee samples at the colony level will detect light AFB infections with a high probability. However, the skewed spore distribution of the adult bees makes composite sampling at the apiary level more problematic, if the aim of the sampling is to locate lightly infected individual colonies within apiaries. The results suggest that false-negative culturing results from composite samples of adult bees from individual colonies with clinical symptoms of AFB are highly improbable. However, if single colonies have light infections in large apiaries, the dilution effect from uncontaminated bees from healthy colonies on the positive bees from diseased colonies may yield false-negative results at the apiary level.     

Distribution of Paenibacillus larvae spores inside honey bee colonies and its relevance for diagnosis

One of the most important factors affecting the development of honey bee colonies is infectious diseases such as American foulbrood (AFB) caused by the spore forming Gram-positive bacterium Paenibacillus larvae. Colony inspections for AFB clinical symptoms are time consuming. Moreover, diseased cells in the early stages of the infection may easily be overlooked. In this study, we investigated whether it is possible to determine the sanitary status of a colony based on analyses of different materials collected from the hive. We analysed 237 bee samples and 67 honey samples originating from 71 colonies situated in 13 apiaries with clinical AFB occurrences. We tested whether a difference in spore load among bees inside the whole hive exists and which sample material related to its location inside the hive was the most appropriate for an early AFB diagnosis based on the culture method. Results indicated that diagnostics based on analysis of honey samples and bees collected at the hive entrance are of limited value as only 86% and 83%, respectively, of samples from AFB-symptomatic colonies were positive. Analysis of bee samples collected from the brood nest, honey chamber, and edge frame allowed the detection of all colonies showing AFB clinical symptoms. Microbiological analysis showed that more than one quarter of samples collected from colonies without AFB clinical symptoms were positive for P. larvae. Based on these results, we recommend investigating colonies by testing bee samples from the brood nest, edge frame or honey chamber for P. larvae spores.     

Diverse origins of tetracycline resistance in the honey bee bacterial pathogen Paenibacillus larvae

Paenibacillus larvae is the causative agent of the important honey bee larval disease American Foulbrood (AFB). This pathogen has been treated in bee colonies by a single registered antibiotic, oxytetracycline (OTC), for fifty years. Recently, widespread resistance to OTC has been reported. In this study, the degree of antibiotic resistance was contrasted with DNA sequence variation for 125 P. larvae isolates collected in North America. Resistance was uncorrelated with bacterial haplotype, suggesting either that resistance has evolved multiple times in P. larvae or that resistance involves recent horizontal transfer via a non-genomic (e.g., plasmid or conjugal transposon) route. The recency of OTC resistance in P. larvae across this broad survey area underscores the need to manage foulbrood infections carefully and to monitor populations for resistance.     

Evaluation of the shaking technique for the economic management of American foulbrood disease of honey bees (Hymenoptera: Apidae)

Shaking is a nonantibiotic management technique for the bacterial disease American foulbrood (AFB) (Paenibacillus larvae sensu Genersch et al.), in which infected nesting comb is destroyed and the adult honey bees, Apis mellifera L. (Hymenoptera: Apidae), are transferred onto uncontaminated nesting material. We hypothesized that colonies shaken onto frames of uninfected drawn comb would have similar reductions in AFB symptoms and bacterial spore loads than those shaken onto frames of foundation, but they would attain higher levels of production. We observed that colonies shaken onto drawn comb, or a combination of foundation and drawn comb, exhibited light transitory AFB infections, whereas colonies shaken onto frames containing only foundation failed to exhibit clinical symptoms. Furthermore, concentrations of P. larvae spores in honey and adult worker bees sampled from colonies shaken onto all comb and foundation treatments declined over time and were undetectable in adult bee samples 3 mo after shaking. In contrast, colonies that were reestablished on the original infected comb remained heavily infected resulting in consistently high levels of spores, and eventually, their death. In a subsequent experiment, production of colonies shaken onto foundation was compared with that of colonies established from package (bulk) bees or that of overwintered colonies. Economic analysis proved shaking to be 24% more profitable than using package bees. These results suggest that shaking bees onto frames of foundation in the spring is a feasible option for managing AFB in commercial beekeeping operations where antibiotic use is undesirable or prohibited.     

A study on the possible utilization of immunodiffusion and immunofluorescence techniques as the diagnostic methods for American foulbrood of honeybees (Apis mellifera)

Four types of antisera were obtained from rabbits hyperimmunized with either spores or vegetative rods from two strains of the American foulbrood pathogen, Bacillus larvae. The specificity and sensitivity of these antisera were tested with immunofluorescence and immunodiffusion methods. No cross-reactions were observed between the antisera and other different species of Bacillus or different genera of bacteria. The specificity was not found between the antisera and two strains of B. larvae although stronger fluorescent intensity was observed between the antiserum and its corresponding strain of antigen in the immunofluorescence tests. Eight samples of 1- to 2-day-old larvae, 3- to 4-day-old larvae, decayed tissue, and dry remain, collected from eight infected colonies, were tested against antisera by the immunofluorescence and the immunodiffusion methods. The results indicated that both methods are sensitive and specific for making diagnosis of field samples of American foulbrood of honey bees.     

Prevalence of honeybee viruses in the Czech Republic and coinfections with other honeybee disease

Six bee viruses, which occur in Apis mellifera, were monitored in the Czech Republic between 2006 and 2009. Samples of larvae and pupae collected from hives where American foulbrood was detected were screened for bee viruses and in the 125 samples of larvae, there was no confirmed case of a larva infected with both American foulbrood and a bee virus. Of 145 samples infected with the protozoan Nosema apis, there were 23 cases of coinfections with the BQCV virus, 18 with the DWV virus and 11 with the ABPV virus. All coinfections with three or four viruses were also statistically significant apart from the one between ABPV with CBPV and DWV. The PCA ordination diagram indicates that BQCV occurs mainly with Nosema apis and DWV mainly with ABPV.     

Regional distribution of Paenibacillus larvae subspecies larvae, the causative organism of American foulbrood, in honey bee colonies of the Western United States

We examined honey bee, Apis mellifera L., colonies pollinating almonds in California during February 2003 for Paenibacillus larvae subsp. Larvae, the causative organism of the virulent brood disease American foulbrood. Colonies originating from the Rocky Mountain area and California had significantly higher numbers (P < 0.05) of bacterial colony-forming units (CFUs) (408 and 324 per 30 adult bees, respectively) than colonies from the upper Midwest (1.28). Colonies from the northwestern, central, and southwestern United States had intermediate CFU or bacterial colony levels. Operations positive for P. larvae larvae were relatively uniform at approximately 70-80%, and no regional significant differences were found. Percentages of colonies with high CFUs (> or = 400 per 30 bees) differed significantly, with those from the Rocky Mountain region having 8.73% compared with those of the upper Midwest with 0%. The significance of CFU levels was evaluated by inoculating healthy colonies with diseased immatures and sampling adult bees. The number of CFUs detected per diseased immature was conservatively estimated to be approximately 399 CFUs per 30 adult bees. We defined this spore level as 1 disease equivalent. Based on this, 3.86% colonies in our survey had 1 or more disease equivalent number of P. larvae larvae CFUs. Operations with high P. larvae larvae spore levels in their colonies will likely observe American foulbrood if prophylaxis is not practiced diligently.     

Nosema ceranae, a new parasite in Thai honeybees

Adult workers of Apis cerana, Apis florea and Apis mellifera from colonies heavily infected with Nosema ceranae were selected for molecular analyses of the parasite. PCR-specific 16S rRNA primers were designed, cloned, sequenced and compared to GenBank entries. The sequenced products corresponded to N. ceranae. We then infected A. cerana with N. ceranae spores isolated from A. florea workers. Newly emerged bees from healthy colonies were fed 10,000, 20,000 and 40,000 spores/bee. There were significant dosage dependent differences in bee infection and survival rates. The ratio of infected cells to non-infected cells increased at 6, 10 and 14 d post infection. In addition, hypopharyngeal glands of bees from the control group had significantly higher protein concentrations than infected groups. Bees infected with 40,000 spores/bee had the lowest protein concentrations. Thus, N. ceranae isolated from A. florea is capable of infecting another bee species, impairing hypopharyngeal gland protein production and reducing bee survival in A. cerana.     

Adult honeybee's resistance against Paenibacillus larvae larvae, the causative agent of the American foulbrood.

American foulbrood is a widespread disease of honeybee larvae caused by the spore-forming bacterium Paenibacillus larvae subsp. larvae. Spores represent the infectious stage; when ingested by a larva they germinate in the midgut. The rod-shaped vegetative forms penetrate the larva's intestinal tissue and start multiplying rapidly, which finally kills the larva. Spores fed to adult honeybees, however, do not harm the bees. We investigated this phenomenon. Specifically, we studied the influence of the adult honeybee midgut on the vegetative growth and on the germination of spores of P. larvae larvae. We focused on two groups of adult workers that are likely to have large numbers of spores in their gastrointestinal tracts in infected colonies: middle-aged bees, which are known to remove or cannibalize dead larvae and clean brood cells, and winterbees, which do not have frequent chances to defecate. We found that midgut extract from winterbees and worker-aged bees of different colonies almost completely inhibited the growth of the vegetative stage of P. larvae larvae and suppressed the germination of spores. The inhibiting substance or substances from the adult midgut are very temperature stable: they still show about 60% of their growth-inhibiting capacity against this bacterium after 15 min at 125 degrees C. We established a method to test growth-inhibiting factors against P. larvae larvae in vitro.     

In vitro study of the antimicrobial activity of Brazilian propolis against Paenibacillus larvae

The honey bee disease American foulbrood (AFB) is a serious problem since its causative agent (Paenibacillus larvae) has become increasingly resistant to conventional antibiotics. The objective of this study was to investigate the in vitro activity of propolis collected from various states of Brazil against P. larvae. Propolis is derived from plant resins collected by honey bees (Apis mellifera) and is globally known for its antimicrobial properties and particularly valued in tropical regions. Tests on the activity of propolis against P. larvae were conducted both in Brazil and Minnesota, USA using two resistance assay methods that measured zones of growth inhibition due to treatment exposure. The propolis extracts from the various states of Brazil showed significant inhibition of P. larvae. Clear dose responses were found for individual propolis extracts, particularly between the concentrations of 1.7 and 0.12 mg propolis/treatment disk, but the source of the propolis, rather than the concentration, may be more influential in determining overall activity. Two of the three tested antibiotics (tylosin and terramycin) exhibited a greater level of inhibition compared to most of the Brazilian samples, which could be due to the low concentrations of active compounds present in the propolis extracts. Additionally, the majority of the Brazilian propolis samples were more effective than the few collected in MN, USA. Due to the evolution of resistance of P. larvae to conventional antibiotic treatments, this research is an important first step in identifying possible new active compounds to treat AFB in honey bee colonies.     

A PCR detection method for rapid identification of Paenibacillus larvae

American foulbrood is a disease of larval honeybees (Apis mellifera) caused by the bacterium Paenibacillus larvae. Over the years attempts have been made to develop a selective medium for the detection of P. larvae spores from honey samples. The most successful of these is a semiselective medium containing nalidixic acid and pipermedic acid. Although this medium allows the growth of P. larvae and prevents the growth of most other bacterial species, the false-positive colonies that grow on it prevent the rapid confirmation of the presence of P. larvae. Here we describe a PCR detection method which can be used on the colonies that grow on this semiselective medium and thereby allows the rapid confirmation of the presence of P. larvae. The PCR primers were designed on the basis of the 16S rRNA gene of P. larvae and selectively amplify a 973-bp amplicon. The PCR amplicon was confirmed as originating from P. larvae by sequencing in both directions. Detection was specific for P. larvae, and the primers did not hybridize with DNA from closely related bacterial species.     

噬菌体疗法及其在美洲幼虫腐臭病中的研究进展

美洲幼虫腐臭病是西方蜜蜂中最严重的细菌病之一,给养蜂业带来了严重的损失。幼虫芽胞杆菌是幼蜂感染美洲幼虫腐臭病的病原菌。由于抗生素产生的耐药性越来越严重,并且抗生素的使用会破坏宿主肠道菌群,使蜂群处于高危的环境中,因此迫切需要抗生素治疗的替代技术,而噬菌体在预防和控制细菌耐药性方面已显示出显著的优势。主要综述了噬菌体疗法、安全性及其在蜜蜂美洲幼虫腐臭病中的研究现状,介绍了噬菌体疗法在各类细菌病中的研究与应用,对今后噬菌体治疗蜜蜂细菌病研究方向进行了展望。     

Paenibacillus larvae larvae spores in honey samples from Uruguay: a nationwide survey

American foulbrood is a severe bacterial disease affecting larvae of the honeybee Apis mellifera and it is caused by Paenibacillus larvae larvae. The disease is present worldwide and cases have been reported in almost all the beekeeping regions of the five continents. During 2001 and 2002 we carried out a nationwide study to assess the presence and amount of P. l. larvae spores in honey samples from Uruguay, combining classic bacteriological, and molecular approaches. The distribution of P. l. larvae spores in honey of the whole country showed a clear pattern and may provide useful data for a control and prevention strategy of American foulbrood.     

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.     

Efficacy of strips coated with Metarhizium anisopliae for control of Varroa destructor (Acari: Varroidae) in honey bee colonies in Texas and Florida

Strips coated with conidia of Metarhizium anisopliae (Metschinkoff; Deuteromycetes: Hyphomycetes) to control the parasitic mite, Varroa destructor (Anderson and Trueman) in colonies of honey bees, Apis mellifera (Hymenoptera: Apidae) were compared against the miticide, tau-fluvalinate (Apistan) in field trials in Texas and Florida (USA). Apistan and the fungal treatments resulted in successful control of mite populations in both locations. At the end of the 42-day period of the experiment in Texas, the number of mites per bee was reduced by 69-fold in bee hives treated with Apistan and 25-fold in hives treated with the fungus; however mite infestations increased by 1.3-fold in the control bee hives. Similarly, the number of mites in sealed brood was 13-fold and 3.6-fold higher in the control bee hives than in those treated with Apistan and with the fungus, respectively. Like the miticide Apistan, the fungal treatments provided a significant reduction of mite populations at the end of the experimental period. The data from the broodless colonies treated with the fungus indicated that optimum mite control could be achieved when no brood is being produced, or when brood production is low, such as in the early spring or late fall. In established colonies in Florida, honey bee colony development did not increase under either Apistan or fungal treatments at the end of the experimental period, suggesting that other factors (queen health, food source, food availability) play some major role in the growth of bee colonies. Overall, microbial control of Varroa mites with fungal pathogens could be a useful component of an integrated pest management program for the honey bee industry.     

DNA extraction and PCR detection of <Emphasis Type="Italic">Paenibacillus larvae</Emphasis> spores from naturally contaminated honey and bees using spore-decoating and freeze-thawing techniques

Summary Paenibacillus larvae causes American foulbrood (AFB), a severe disease that affects the brood of honey bee Apis mellifera. AFB is worldwide distributed and causes great economic losses to beekeepers, but in many cases early diagnosis could help in its prevention and control. The aim of the present work was to design a reliable protocol for DNA extraction of P. larvae spores from naturally contaminated honey and adult bees. A novel method that includes a step of spore-decoating followed by an enzymatic spore disruption and DNA purification was developed. Also a freeze-thaw cycle protocol was tested and the results were compared. The DNA extracted was used as template for specific bacterial detection by amplification of a 16S rDNA fragment. Both methods allowed the direct detection by polymerase chain reaction (PCR) of P. larvae spores present in naturally contaminated material. The spore-decoating strategy was the most successful method for DNA extraction from spores, allowing specific and remarkably sensitive PCR detection of spores in all honey and bees tested samples. On the other hand freeze-thawing was only effective for detection of spores recovered from bees, and extensive damage to DNA affected detection by PCR. This work provides new strategies for spore DNA extraction and detection by PCR with high sensitivity, and brings an alternative tool for P. larvae detection in natural samples.