Introduction
The disappearance of bee colonies is caused by the use of insecticides in intensive agriculture, pollution, misapplication of beekeeping techniques and, especially, by bee colony pathology (Genersch, Reference Genersch2010; Moritz et al., Reference Moritz, Miranda, Fries, Conte, Neuman and Paxon2010; Soroker et al., Reference Soroker, Hetzroni, Yakobson, David, David, Voet, Slabezki, Efrat, Levski, Kamer, Klinberg, Zioni, Inbar and Chejanovsky2010; Vandame & Palacio, Reference Vandame and Palacio2010; Anderson et al., Reference Anderson, Sheehan, Eckholm, Mott and Hoffman-DeGrandi2011; Hamdi et al., Reference Hamdi, Balloi, Essanaa, Crotti, Gonella, Raddadi, Ricci, Boudabous, Borin, Manino, Bandi, Alma, Daffonchio and Cherif2011; Koch & Schmid-Hempel, Reference Koch and Schmid-Hempel2011).
Due to the organizational structure of the bee colony, disease transmission is rapid, especially through infected food or via the nurse worker bees that feed the brood bees of the colony (Feigenbaum & Naug, Reference Feigenbaum and Naug2010) and also by robber bees. According to Fuselli et al. (Reference Fuselli, Garcia, Susana, Eguaras and Fritz2008), the maximum quantity of spores of Paenibacillus larvae for colonies with American foulbrood disease is in the medial and posterior intestine, the transmission of these spores being affected by the nurse worker bees that feed the larvae.
For this reason, the use of prebiotics and probiotics in bees is of particular importance in preventing and combating disease.
Prebiotics create intestinal environmental conditions that favour the development of useful microorganisms. Maintaining the balance of the intestinal flora is a major factor that keeps the intestine functioning normally, any imbalance easily causing illness.
Acidifying substances can specifically act against the potentially pathogenic microorganisms, which colonize the intestinal wall epithelium and cause a state of transit finally leading to their elimination along with undigested residues (Stoica et al., Reference Stoica, Stoica and Pană1999; Stef, Reference Stef2003).
The antimicrobial effect of organic acids on various microorganisms is based on several mechanisms. One of the main effects of organic acids is the decrease of the pH in feed (sugar syrup) within the digestive tract, thus creating unfavorable conditions for the development of potentially pathogenic microorganisms (Fuselli et al., Reference Fuselli, Garcia, Susana, Eguaras and Fritz2008; Forsgren et al., Reference Forsgren, Olofsson, Vasquez and Fries2009).
Probiotic bacteria are known to be promoters of host body defense mechanisms. In addition to the effects that probiotics have upon the defense mechanisms of the intestine, which is characterized by stabilization of local micro-flora, probiotic bacteria have been identified as being responsible for triggering a humoral immune response, thus creating an intestinal immunological barrier (Evens & Lopez, Reference Evens and Lopez2004; Olofsson & Vasquez, Reference Olofsson and Vasquez2008; Corcionivoschi & Drinceanu, Reference Corcionivoschi and Drinceanu2009).
Probiotics have the ability to shape the immune system through their physiological action on the intestine level. Once in the intestine, they interact with intestinal cells, triggering an immune response due to the fact that intestinal cells produce a series of immune-simulator molecules when stimulated by bacteria (Corcionivoschi & Drinceanu, Reference Corcionivoschi and Drinceanu2009).
The choice of these two groups of substances took into account their mode of action, the prebiotics (lactic and acetic acids) acting at the intestinal level by reducing the number of pathogenic micro-organisms while the probiotics (Enterobiotics and Enterolactis Plus) favour the growth of micro-organisms beneficial to the organism.
The choice of the two probiotic products (Enterobiotics and Enterolactis Plus) was not arbitrary. They contain Lactobacillus acidophilus LA-14, Bifidobacterium lactis BI-04 and Lactobacillus casei. Species of Lactobacillus and Bifidobacterium were identified by Olofsson & Vasquez (Reference Olofsson and Vasquez2008) in the stomachs of healthy worker bees.
Kacániová et al. (Reference Kacániová, Pavlicová, Hascik, Kociubinski, Kazovická, Sudzina, Sudzinová and Fikselová2009) noted that the digestive tract of adult bees contains a higher proportion of anaerobic than aerobic bacteria. They isolated coliforms, enterococci, staphylococci, Bacillus sp. and Pseudomonas sp. Kacániová et al. (Reference Kacániová, Chlebo, Kopernický and Trakovická2004) had not found Lactobacillus in the bee digestive tract. Martinson et al. (Reference Martinson, Danforth, Minckley, Rueppell, Tingek and Moran2011) showed that the digestive tracts of honey bees (Apis mellifera) and of bumblebees host different bacterial microfloral species.
Enterobiotics and Enterolactis Plus are pharmaceutical products used in treatment of humans (both adults and children) for the re-establishment of intestinal equilibrium, being able within a short time to supplant the complete intestinal bacterial flora. If present in honey, such products would enhance rather than diminish its value.
Springtime stimulatory feeding is used in order to stimulate hive functions and to encourage the queen to start laying eggs as early as possible so that bee colonies can best develop and capitalize on the results of foraging from entomophilous plants (Bura & Pătruică, Reference Bura and Pătruică2003). After the cleansing flight, beekeepers may supplement the sugar syrup with apicultural stimulators that positively influence the development of bee colonies and exert a positive effect on the bees’ health status (Pătruică et al., Reference Pătruică, Bogdan and Bura2011a). In this study, during the stimulatory feeding process, sugar syrup was supplemented with prebiotics and/or probiotics in order to determine whether these supplements reduce the total number of potentially pathogenic bacteria and intestinal colonization with beneficial bacteria.
Materials and methods
The biological material used in this study was formed of 110 Apis mellifera carpatica bee colonies, housed in multistage hives. Bee colonies were divided into 11 experimental treatments, each made up of ten colonies of equal strength (six combs occupied with bees) and with queens of the same age.
Between 25th March and 15th April 2011, the bee colonies were fed sugar syrup with added acidifying substances (either lactic or acetic acid) and/or probiotic products (Enterobiotics or Enterolactis Plus) in different doses as shown in table 1.
Table 1. Experimental treatment scheme.
Each colony was fed 1.4l of sugar syrup on a weekly basis. The 1:1 (1kg sugar per litre water) feed contained the pre- or probiotic products mentioned above in different concentrations. The pH of the sugar syrup was adjusted to between 3 and 4 for the experimental treatments and to 6.5 for the control (table 1.) The sugar syrup was administered directly into the honeycomb and was consumed by the bees over the course of a week. The bee colonies were positioned in the same location and maintained according to identical procedures.
The choice of a 21-day feeding period was correlated to the duration of metamorphosis of the worker bees (which is 21 days) with the result that, at the collection point of samples for analysis, there should be recently emerged bees and nurse bees in the hive which have had the benefit of being fed with pre- and probiotic supplements.
On 15th April, samples of recently emerged worker bees were taken from each of the treated colonies for microbiological examination. Bee samples were collected in sterile tubes, with a sample consisting of ten bees from each colony. Samples were conveyed to the laboratory within 30min of collection.
Once there, the bee intestines were transferred aseptically into sterile tubes containing distilled water, using fine forceps and gas flaming tube necks. The biological material was collected in separate tubes from each bee colony. Serial dilutions of 10−1, 10−2 and 10−3 were then made from the contents of these tubes, and then each dilution was used for seeding ten nutrient agar poured Petri plates. These were thermostatically incubated for 24 hours at 37 °C. The choice of incubation temperature for the controls was made taking into account the optimum temperature for larval development (32–36 °C), the range of temperature within which a bee colony is optimally active and the body temperature of the bee, which is very dependent upon the environmental temperature and on the bee's metabolic rate. Thoracic temperature may reach values between 38.8 and 41.2 °C (Stabentheiner et al., Reference Stabentheiner, Vollmann, Kovac and Crailsheim2003).
Based on how bacterial colonies developed, it was decided to use counts from the samples at 10−2 dilution.
Gram-stained smears were made from each sample and were microscopically examined. Counting of the bacterial colonies was performed using the Nitech LKB 2002 apparatus.
For the bee samples that were fed sugar syrup with added probiotic products (Enterolactis Plus or Enterobiotics), the MRS agar culture Petri plates (pH 6.5) Merck (Merck Chemical, Bucharest, Romania) were used. Identification of bacterial cultures was achieved using, as a control sample, very small amounts of the Enterolactis and Enterobiotics material used in the experimental treatments.
Samples from the experimental variants 5, 6, 7, 8, 9 and 10 were incubated in anaerobic atmosphere generating bags (P and Anaerocult Anaerocult A, Merck), and then incubated in an anaerobic cabinet.
The total number of microbe analyses was repeated twice for each experimental variant. Dunnett's test was performed using the IBM SPSS Statistics, version 19, ANOVA package to compare the total number of microbes present in the intestine of bees fed with prebiotics and probiotic products.
Honey production, in the bee colonies given the supplementary feed incorporating prebiotic and probiotic addititives, was measured, by weighing, in the periods 5–11 May (for rape flower honey) and 27–28 May (for acacia flower honey).
Results
Acidification of stimulating food given to the bee colonies during spring time caused a significant reduction (P < 0.001) in the number of microbes in the intestines of bees, until acidification reached the pH range 3–4, compared with the control group (6800microbes ml−1 in acid lactic (2ml) treatment group; 900 microbes ml−1 in acid lactic (2.5ml) treatment group; 1700microbes ml−1 in acid acetic (30ml) treatment group; 2500microbes ml−1 versus 43,100 microbes acid acetic (20ml) treatment group per ml in the control group). The smallest number of microbes was found in the intestines of the bees that were fed sugar syrup of pH 3 (acid lactic 2.5ml treatment group) (table 2).
Table 2. Mean and standard deviation values of the total number of microbes present in the intestine of bees fed with prebiotics and probiotic products (n = 220).
*** P < 0.001
M, control group; EG1, acid lactic (2ml) treatment group; EG2, acid lactic (2.5ml) treatment group; EG3, acetic acid (20ml) treatment group; EG4, acetic acid (30ml) treatment group; EG5, Enterobiotics (1.25g) treatment group; EG6, Enterobiotics (2.5g) treatment group; EG7, Enterolactis Plus (1.2g) treatment group; EG8, Enterolactis Plus (2.4g) treatment group; EG9, acid lactic (2.5) and Enterobiotics (2.5g) treatment group; EG10, acid lactic (2.5) and Enterolactis Plus (2.4g) treatment group.
Adding probiotic products, Enterobiotics (Lactobacillus Acidophilus LA-14 and Bifidobacterium lactis BI-04) or Enterolactis Plus (Lactobacillus casei), to the sugar syrup given to the bee colonies in spring resulted in a statistically significant decrease (P < 0.001) of the total number of potentially pathogenic (table 2) (14,400microbes ml−1 in Enterobiotics (1.25g) treatment group; 26,900 microbes ml−1 in Enterobiotics (2.5g) treatment group; 19,700bacteria ml−1 in Enterolactis Plus (1.2g) treatment group; 15,100microbes ml−1 versus 43,100 microbes Enterolactis Plus (2.4g) treatment group per ml in the control group) (fig. 1) and also of the intestinal colonization with beneficial bacteria (fig. 2).
Fig. 1. The number of microbes present in the intestine of bees fed with prebiotics and probiotic products. M, control group; EG1, acid lactic (2ml) treatment group; EG2, acid lactic (2.5ml) treatment group; EG3, acetic acid (20ml) treatment group; EG4, acetic acid (30ml) treatment group; EG5, Enterobiotics (1.25g) treatment group; EG6, Enterobiotics (2.5g) treatment group; EG7, Enterolactis Plus (1.2g) treatment group; EG8, Enterolactis Plus (2.4g) treatment group; EG9, acid lactic (2.5) and Enterobiotics (2.5g) treatment group; EG10, acid lactic (2.5) and Enterolactis Plus (2.4g) treatment group.
Fig. 2. The number of benefical bacteria present present in the intestines of bees fed with prebiotics and probiotic products. EG5, Enterobiotics (1.25g) treatment group; EG6, Enterobiotics (2.5g) treatment group; EG7, Enterolactis Plus (1.2g) treatment group; EG8, Enterolactis Plus (2.4g) treatment group; EG9, acid lactic (2.5) and Enterobiotics (2.5g) treatment group; EG10, acid lactic (2.5) and Enterolactis Plus (2.4g) treatment group.
In the case of lactic acid combination with one of the two probiotic products (Enterobiotics or Enterolactis Plus), the total number of microbes was reduced to 2300microbes ml−1 in acid lactic (2.5ml) and Enterobiotics (2.5g) treatment group and 2700microbes ml−1 in acid lactic (2.5ml) and Enterobiotic Plus (2.4g) treatment group (fig. 1) with significant differences in comparison with the control group (table 2).
The cultures seeded from bees fed with the probiotic product Enterobiotics developed colonies of beneficial bacteria (Lactobacillus acidophilus and Bifidobacterium lactis) showing 900 bacteria ml−1 in Enterobiotics (1.25g) treatment group and 1100 bacteria ml−1 in Enterobiotics (2.5g) treatment group, and those seeded with Enterolactis Plus developed 580 Lactobacillus casei bacterial colonies per ml in Enterolactis Plus (1.2g) treatment group and 1200 bacteria ml−1 in Enterolactis Plus (2.4g) treatment group (fig. 2).
For the sugar syrup added to the two product groups, the experimental treatment 9 developed colonies showing 400 bacteria ml−1 (Lactobacillus acidophilus and Bifidobacterium lactis) with experimental treatment yielding 300 bacteria ml−1 (Lactobacillus casei) (fig. 2).
It has been established that bee colonies given supplemental early spring feeds of syrup with a pH of between 3 and 4 showed very good development, registering an average 13% better brood production than colonies fed with plain sugar syrup (Pătruică et al., Reference Pătruică, Bogdan, Bura, Bănătean Dunea and Gâltofet2011b,Reference Pătruică, Bogdan, Bura and Popovicic).
Colonies fed with probiotic supplemented feed showed between 10.67% and 20.34% more brood growth than the control group (Pătruică et al., Reference Pătruică, Bogdan, Bura and Popovici2011d,Reference Pătruică, Bogdan, Bura and Popovicie).
The colonies of bees studied showed a production of rape flower and acacia honey between 16.43% and 45.96% greater than the control group, the best results being obtained by EG6 and EG9.
Discussion
Reducing the total number of bacteria in the intestines of bees is a major prerequisite for ensuring good health status with implications for the development of colonies and also their productive potential. It has been found that the bee colonies stimulated in early spring with sugar syrup with a pH between 3 and 4 showed very good development, producing on average 13% more brood bees compared with colonies fed only with sugar syrup (Pătruică et al., Reference Pătruică, Bogdan, Bura, Bănătean Dunea and Gâltofet2011b,Reference Pătruică, Bogdan, Bura and Popovicic). The increasing number of worker bees is closely related to a superior utilization of nectar resources, resulting in higher honey production (Bura & Pătruică, Reference Bura and Pătruică2003). This aspect is closely followed by beekeepers who wish to obtain significant honey yields (Pătruică et al., 2006).
Probiotic bacteria (Lactobacillus acidophilus LA-14, BI-04 Bifidobacterium and Lactobacillus lactis casei) consumed by the bees together with sugar syrup (Enterobiotics 1.25g treatment group; Enterobiotics 2.5g treatment group; Enterolactis Plus 1.2g treatment group and Enterolactis Plus 2.5g treatment group) came to populate their intestines and led to a significant decrease (P < 0.001) of the total number of potentially pathogenic bacteria compared with the control group, possibly as a result of specific defense mechanisms of the intestine (table 2). The population of the bee digestive tract with beneficent bacterial species has very important implications for the improvement of bee health (Koch & Schmid-Hempel, Reference Koch and Schmid-Hempel2011). Bee health is seriously affected when a disturbance arises in the normal balance of beneficent microflora (Hamdi et al., Reference Hamdi, Balloi, Essanaa, Crotti, Gonella, Raddadi, Ricci, Boudabous, Borin, Manino, Bandi, Alma, Daffonchio and Cherif2011). Porrini et al. (Reference Porrini, Audisio, Sabaté, Ibarguren, Medici, Sarlo, Garrido and Eguaras2010) showed that extracted metabolites from Bacillus and Enterococcus strains which had been isolated from bee intestines have inhibitory effects on the development of Nosema ceranae.
The complementary effect of the acidifying substances and of the studied probiotic products created a statistically significant decrease (P < 0.001) in the total number of microbes in the two variants studied (EG9 and EG10) and the colonisation of the intestines with beneficial bacteria contained in the probiotics products (Enterobiotics and Enterolactis Plus), due to the combined effects of the two groups of additives. In these groups, a lower incidence of beneficial bacteria colonisation compared with groups 5, 6, 7 and 8 which had no lactic acid in their sugar syrup (table 2) can be noticed.
In conclusion, prebiotic and probiotic use for stimulating feeding of bee colonies in early spring, in the studied doses, is a method of maintaining the good health status of bee colonies, resulting from the specific action that these additives have on the bee intestine. The enhancement of the state of health was correlated with a more vigorous development of the bee colonies and higher yields of honey.
Acknowledgements
This work was co-financed by the European Social Fund through the Sectoral Operational Programme Human Resources Development 2007–2013, project number POSDRU/89/1.5/S/63258, ‘Postdoctoral school for zootechnical biodiversity and food biotechnology based on the eco-economy and the bio-economy required by eco-san-genesys’.
We, the authors, have no financial or commercial link with the company providing the prebiotics and probiotics.
