Biocontrol potential of Apilactobacillus kunkeei EIR/BG-1 against infectious diseases in honey bees (Apis mellifera L.).

The significant reduction of honey bee colonies due to the various infectious agents highlights the need for the development of new alternatives and integrated management strategies to keep a hive strong and healthy. The main purpose of this study was to develop an environmentally and friendly microbial feed supplements to prevent honey bee mortality and keep the bee colony population healthy and productive. For this aim, Apilactobacillus kunkeei EIR/BG-1 isolated from honey bee gut microbiota was evaluated for its preventive role against American Foulbrood disease and nosemosis. To test the ability of the strain EIR/BG-1 for suppressing Paenibacillus larvae growth under in vitro conditions, the agar well diffusion method was used and viable cells of the strain EIR/BG-1 inhibited the growth of P. larvae with an efficient inhibition zone (24 ± 0.8 mm) similar to tetracycline antibiotic (30 µg). To determine the preventive role of the strain EIR/BG-1 on infection progression, its viable cells were applied against nosemosis in a laboratory experimental setting. Our results showed that prophylactic supplementation of Al. kunkeei EIR/BG-1 (106 cfu/bee) significantly reduced the spore load (66 ± 6.1%). Besides, gene expression of antimicrobial peptides in gut tissue has been up-regulated and infected midgut epithelium integrity and peritrophic membrane production were improved. In conclusion, our findings suggest that prophylactic supplementation of Al. kunkeei EIR/BG-1 as a natural strategy may enhance the honey bee's response when challenged by pathogens. Field applications towards gaining a better understanding of its biocontrol role will be the main goal of our future researches.

Plant-based supplement containing B-complex vitamins can improve bee health and increase colony performance.

It is common knowledge that nutritive stress resulting from decreased diversity and quality of food, pollution of food sources and beekeeping errors may lead to increased susceptibility of bees to pathogens and pesticides. The dearth of adequate food is frequently compensated with supplements. Thus, this research was aimed to study the effects of the plant-based supplement B + on colony strength (assessed according to open and sealed brood area, honey and pollen/bee bread reserves, and the number of adult bees). In addition, Nosema ceranae spores and viruses were quantified and the level of infestation with Varroa destructor assessed. The experiment was conducted in late summer and early spring. In colonies which were given B + in feed a significant increase (p < 0.05) in the parameters of colony strength were noticed in comparison to the control (colonies fed on sugar syrup). Moreover, it was proven that the bees from these colonies had significantly lower (p < 0.05) N. ceranae spore counts, and acute bee paralysis, deformed wing and sacbrood virus loads. Our results suggest that the addition of B + supplement to the colonies provide them with nutrients, contribute to their strengthening, might prevent nutritive stress and increase the success of bees in combating pathogens.

Greater wax moth Galleria mellonella (Lepidoptera: Pyralidae) as a resistant model host for Nosema pyrausta (Microsporidia: Nosematidae).

Galleria mellonella fed 3 million Nosema pyrausta spores per larva showed 0 and 5% infestation rate at 30 °C and 24 °C, respectively. N. pyrausta virulence did not increase after passage through G. mellonella for three generations. When larvae were pretreated with phenylthiourea, Bacillus thuringiensis or combination of both, infection rates were 11%, 15% and 22%, respectively. Injection of untreated and potassium hydroxide-primed spores resulted in approximately 10% and 50% infection, respectively. G. mellonella is resistant to high dosages of N. pyrausta spores, serving as a prospective model of insect resistance to microsporidia, while host immunosuppression and/or spore activation increases success of the pathogen.

Protein nutrition governs within-host race of honey bee pathogens.

Multiple infections are common in honey bees, Apis mellifera, but the possible role of nutrition in this regard is poorly understood. Microsporidian infections, which are promoted by protein-fed, can negatively correlate with virus infections, but the role of protein nutrition for the microsporidian-virus interface is unknown. Here, we challenged naturally deformed wing virus - B (DWV-B) infected adult honey bee workers fed with or without pollen ( = protein) in hoarding cages, with the microsporidian Nosema ceranae. Bee mortality was recorded for 14 days and N. ceranae spore loads and DWV-B titers were quantified. Amongst the groups inoculated with N. ceranae, more spores were counted in protein-fed bees. However, N. ceranae infected bees without protein-diet had reduced longevity compared to all other groups. N. ceranae infection had no effect on protein-fed bee's longevity, whereas bees supplied only with sugar-water showed reduced survival. Our data also support that protein-feeding can have a significant negative impact on virus infections in insects. The negative correlation between N. ceranae spore loads and DWV-B titers was stronger expressed in protein-fed hosts. Proteins not only enhance survival of infected hosts, but also significantly shape the microsporidian-virus interface, probably due to increased spore production and enhanced host immunity.

Antifungal activity of the essential oil obtained from Cryptocarya alba against infection in honey bees by Nosema ceranae.

The honeybee disease nosemosis type C is a serious problem since its causative agent, microsporidium Nosema ceranae, is widespread among adult honey bees. Some of the feasible alternative treatments that are used to control this disease are plant extracts. The aim of the present work was to evaluate the effects of essential oils of Chilean plant species, such as Cryptocarya alba, which is used against N. ceranae, and to identify and quantify the majority active compounds in the EO as well as their potential use for the control of nosemosis. Essential oils were obtained using the stripping steam technique with Clevenger equipment and were subsequently analyzed by Gas chromatography-mass spectrometry. Mortality was recorded daily over at least 8days as worker honeybees were exposed to a range of doses of EO dispersed in a sucrose solution. C. alba oil appears to be nontoxic to A. mellifera adults at the tested concentration (the same concentration inhibits the growth of N. ceranae), showing that this oil can be used for the treatment of nosemosis. EO effectiveness was demonstrated against N. ceranae by calculating the percentage of decrease in infected bees from untreated infected groups vs infected groups treated with EO or the reference drug fumagillin. It was determined that a dose of 4µg EO/bee was most effective in controlling N. ceranae development. We determined innocuous doses of C. alba essential oil for honeybees. We demonstrated the antifungal activity of C. alba EO at 4µg/bee against N. ceranae and compared it to its major monoterpenes, such as ß-phellandrene (20µg/bee), eucalyptol (20µg/bee) and α-terpineol (20µg/bee). The major compounds of C. alba EO, α-terpineol, eucalyptol and ß-phellandrene, had significant effects against Apis mellifera infection by N. ceranae, but the antifungal effect of the complete essential oil on N. ceranae was larger than the effect of α-terpineol, eucalyptol or ß- phellandrene separately, showing that C. alba oil may be a candidate for the treatment or prevention of nosemosis.

Fumagillin control of Nosema ceranae (Microsporidia:Nosematidae) infection in honey bee (Hymenoptera:Apidae) colonies in Argentina.

Information on the long­term consequences of Nosema ceranae to honey bee lifespan and effectiveness of Nosema control with fumagillin is scarce and not always consistent. Our objective in this study was to evaluate the effectiveness of the antibiotic fumagillin to control N. ceranae in hives in East­Central Argentina. Honey bee hives were assigned to 3 experimental treatments, a control group with un­treated hives, a preventive strategy group with hives treated monthly, and a monitoring strategy group with hives treated according to a N. ceranae threshold level. Apiaries were monitored monthly during Fall­Winter 2009 and 2010 and N. ceranae spore intensity and honey bee colony strength measures were estimated. Fumagillin­treated colonies had reduced N. ceranae spores load in 2010 compared to control colonies. However, there was no significant difference between treated and control groups for colony strength measures including adult bee population, bee brood availability, honey, or pollen. Fumagillin treatment reduced N. ceranae intensities but had little effect on colonies. The bee population during Winter was reduced in treated as well as in control colonies. Our results clarify that fumagillin treatment should be at least reviewed and that further research should be conducted to acquire a more complete perspective of Nosemosis disease.

Identification of candidate agents active against N. ceranae infection in honey bees: establishment of a medium throughput screening assay based on N. ceranae infected cultured cells.

Many flowering plants in both natural ecosytems and agriculture are dependent on insect pollination for fruit set and seed production. Managed honey bees (Apis mellifera) and wild bees are key pollinators providing this indispensable eco- and agrosystem service. Like all other organisms, bees are attacked by numerous pathogens and parasites. Nosema apis is a honey bee pathogenic microsporidium which is widely distributed in honey bee populations without causing much harm. Its congener Nosema ceranae was originally described as pathogen of the Eastern honey bee (Apis cerana) but jumped host from A. cerana to A. mellifera about 20 years ago and spilled over from A. mellifera to Bombus spp. quite recently. N. ceranae is now considered a deadly emerging parasite of both Western honey bees and bumblebees. Hence, novel and sustainable treatment strategies against N. ceranae are urgently needed to protect honey and wild bees. We here present the development of an in vitro medium throughput screening assay for the identification of candidate agents active against N. ceranae infections. This novel assay is based on our recently developed cell culture model for N. ceranae and coupled with an RT-PCR-ELISA protocol for quantification of N. ceranae in infected cells. The assay has been adapted to the 96-well microplate format to allow automated analysis. Several substances with known (fumagillin) or presumed (surfactin) or no (paromomycin) activity against N. ceranae were tested as well as substances for which no data concerning N. ceranae inhibition existed. While fumagillin and two nitroimidazoles (metronidazole, tinidazole) totally inhibited N. ceranae proliferation, all other test substances were inactive. In summary, the assay proved suitable for substance screening and demonstrated the activity of two synthetic antibiotics against N. ceranae.

Spore Loads May Not be Used Alone as a Direct Indicator of the Severity of Nosema ceranae Infection in Honey Bees Apis mellifera (Hymenoptera:Apidae).

Nosema ceranae Fries et al., 1996, a microsporidian parasite recently transferred from Asian honey bees Apis cerana F., 1793, to European honey bees Apis mellifera L., 1758, has been suspected as one of the major culprits of the worldwide honey bee colony losses. Spore load is a commonly used criterion to describe the intensity of Nosema infection. In this study, by providing Nosema-infected bees with sterilized pollen, we confirmed that pollen feeding increased the spore loads of honey bees by several times either in the presence or absence of a queen. By changing the amount of pollen consumed by bees in cages, we showed that spore loads increased with an increase in pollen consumption. Nosema infections decrease honey bee longevity and transcription of vitellogenin, either with or without pollen feeding. However, the reduction of pollen consumption had a greater impact on honey bee longevity and vitellogenin level than the increase of spore counts caused by pollen feeding. These results indicate that spore loads may not be used alone as a direct indicator of the severity of N. ceranae infection in honey bees.

Screening alternative therapies to control Nosemosis type C in honey bee (Apis mellifera iberiensis) colonies.

Nosemosis type C caused by the microsporidium Nosema ceranae is one of the most widespread of the adult honey bee diseases, and due to its detrimental effects on both strength and productivity of honey bee colonies, an appropriate control of this disease is advisable. Fumagillin is the only veterinary medicament recommended by the World Organization for Animal Health (OIE) to suppress infections by Nosema, but the use of this antibiotic is prohibited in the European Union and few alternatives are available at present to control the disease. In the present study three therapeutic agents (Nosestat®, Phenyl salicylate and Vitafeed Gold®) have been tested to control N. ceranae infection in honey bee colonies, and have been compared to the use of fumagillin. None of the products tested was effective against Nosema under our experimental conditions. Low consumption of the different doses of treatments may have had a strong influence on the results obtained, highlighting the importance of this issue and emphasizing that this should be evaluated in studies to test therapeutic treatments of honey bee colonies.

A preliminary investigation of alternatives to fumagillin for the treatment of Loma salmonae infection in rainbow trout.

The effects of the following six treatments against Loma salmonae, a microsporidian gill pathogen, were examined in rainbow trout: fumagillin (high dose), pyrimethamine + sulphaquinoxaline, albendazole, amprolium, fumagillin (low dose), and metronidazole. The fish were infected by mouth and the treatments were administered at intervals for a period of several weeks. The results were assessed on the basis of (1) delay in the formation of xenomas, and (2) the number of xenomas per gill arch. The first five treatments, in descending order of efficacy, delayed the formation of xenomas (P<0.01), but metronidazole had no such effect. Fumagillin (high or low dose) and albendazole both reduced the number of xenomas present 10 weeks after infection (P<0.01), but the other three treatments did not do so. From these results, both fumagillin and albendazole appeared to be of potential value in controlling L. salmonae infection in trout.

Efficacy of the fumagillin analog TNP-470 for Nucleospora salmonis and Loma salmonae infections in chinook salmon Oncorhynchus tshawytscha.

Oral treatment with fumagillin is effective for controlling various microsporean and myxosporean infections in fish. We tested a synthetic analog of fumagillin, TNP-470 (Takeda Chemical Industries), for its efficacy against 2 microsporean pathogens of salmon: Loma salmonae and Nucleospora salmonis. Chinook salmon Oncorhynchus tshawytscha were experimentally infected with either L. salmonae (per os) or N. salmonis (intraperitoneal, i.p., injection) and held in fresh water at 15 degrees C. Fish were then divided into 3 replicate groups: untreated or treated orally at 1.0 mg or at 0.1 mg drug kg-1 fish d-1. With L. salmonae, the high dose fish had 0.32 xenomas mm-2 of gill tissue compared to controls at 24.5 xenomas per mm2. With N. salmonis infections, untreated fish exhibited 100% infection, showed prominent clinical signs (e.g. renal swelling, anaemia), and high mortality. In contrast, fish treated at 1.0 mg kg-1 showed no clinical signs, and 16% of those treated at 0.1 mg kg-1 showed only mild gross pathological changes. With the treated groups, over 50% of the fish exhibited extremely light infections, even with high dose treatments, but no mortalities were attributed to N. salmonis infections. Uninfected fish treated at 1.0 mg drug kg-1 fish d-1 for 5 wk appeared clinically normal and showed no reduction in growth. However, about half of these fish exhibited atrophy of the renal interstitial hematopoietic tissue.

Mortality in captive wild-caught horned puffin chicks (Fratercula corniculata).

Sixteen horned puffin (Fratercula corniculata) and six parakeet auklet (Cyclorrhynchus psittacula) chicks of various prefledging ages were caught in Alaska and transported to the North Carolina Zoological Park (USA) in August 1995. Six of the 16 puffin chicks died within a 5-day period beginning 2 days after their arrival into quarantine at the zoo. The birds that died were collected at a young age, weighed 45.4-65.7 g, and had been fed a diet of thawed frozen ocean silversides (Atherinidae) that was not supplemented with vitamins. Clinical signs were nonspecific, and gross necropsies, insecticide toxicology screens, and bacterial cultures were unremarkable. Microscopic examination of tissues from five of the six birds showed myocardial necrosis and degeneration suggestive of vitamin E deficiency and intestinal protozoa resembling Microsporidia. The mortality pattern and histopathologic lesions observed in this case support the use of selective age capture and vitamin supplementation for wild alcid chick collection.