AN ESTIMATE OF THERAPEUTIC AND IMMUNE STIMULATING PROPERTIES OF BEE PRODUCTS

UNIVERSITY OF AGRICULTURA SCIENCES AND VETERINARY MEDICINE CLUJ-NAPOCA PhD SCHOOL FACULTY OF VETERINARY MEDICINE Rindt Iulia Krisztina AN ESTIMATE OF THERAPEUTIC AND IMMUNE STIMULATING PROPERTIES OF BEE PRODUCTS SUMMARY OF THE PhD THESIS SCIENTIFIC COOORDINATOR: Prof. Dr. Marina Spînu Cluj-Napoca 2011 XII

SUMMARY Resistance to antibiotics represents a highly expanded phenomenon that obliges the investigators to seek for efficacious therapy, especially for infectious diseases. The antibacterial drugs used in hospitals raise numerous problems, without succeeding to proficiently control the infection and restore the patient s health, be it human or animal, and also raise the costs of the treatment. In veterinary medicine there is a further aspect to be considered, that is the presence of antibiotic residues in food products of animal origin. The final result of the consumption of such food leads to a closed vicious circle represented by the increase in numbers of multi drug resistant bacteria. Honey is within this framework an available and many times efficient as an antimicrobial drug alternative, with beneficial side effects on the consumer s health. Numerous bacteria show sensitivity to honey, thus even methycilin resistant Staphylococcus aureus, an extremely aggressive bacteria encountered in both human and veterinary medicine. Therefore, the scientific proves for the efficacy of honey and other bee products against bacteria of medical significance could indicate a substantial progress (Ghisalberti 1979; Zumla and Lulat 1989). The more and more pronounced interest concerning the antimicrobial properties of various types of honey and the acceptance on its therapeutic potential made a link between traditional and alternative medicine (Beretta et al., 2005). Both the honedew honey and the seem to be a viable alternative in the fight against various pathogenic agents, by their antibacterial, antifungal and antiviral properties. At this time, the and honey are a subject of research, these natural compound offering a more biological alternative to synthetic drugs. The bovine carry a peculiar socio-economic importance by their products and various uses to the populaţion. Mastites are some of the most harmful diseases to these species especially in milking animals, directly influencing the milk production, welfare and human health. The treatment of these conditions is a major problem due to the increased costs and induction of resistance to antibiotics induced by injudicious treatments. Bee products show a complex composition, and besides their nutritious value they have important healing properties in various tissues as well as immune stimulating effects. Synthetic drugs became more and more inefficient facing the very high adaptability of pathogenic bacteria. As opposed to these, bee products could provide an efficient alternative. Their potential immune modulating use adds to the therapeutic value of honey and. Within this framework described in the first part of the thesis (chapters I IV), the researches entitled : An estimate of therapeutic and immune stimulating properties of bee products aimed to study the in vitro effects of certain bee products (honey, honeydew honey and ) against bacteria isolated from the milk of cows with sub clinical or clinical mastites, in particular against Staphylococcus aureus as well as an estimate of the immune modulating capacities of these compounds on the cell mediated innate and adaptive immune responses. The evaluation of the efficacy of these bee products based on their composition and concentration could allow a selection of such products based on their antibacteria XIII

and/or immunostimulating qualities for therapeutic use, by the development of adapted schemes in combination (or not) with allopatic medicine. These objectives were followed within the research conducted between years 2006 and 2011, and the results are presented in the eight chapters of personal researches of the thesis (chapters V-XII), comprising the purpose, objectives and activities. In chapter V, entitled Characterization of the studied bee products, the honeys and to be investigates were tested for their qualitative and classified based on origin composition and concentration. Several samples of Trasylvanian honey and honedew honey were tested out of which three polifloral and one honedew honey were further investigated. The majority of the studied samples were local and kindly provided by the Bee keeping discipline of the University (USAMV), but some comercial samples were tested too. There were differences between polifloral and honeydew honey as total polyphenol content and antioxidant activity, which could support their diffrentiated biological activity. In chapter VI entitled The immunological characterisation of bacterial mastites in cows the 64 study cases were described, out of which 12 were healthy and the rest of 52 were with mastites of various gravityies (subclinical or clinical). To estimate the humoral reactivity total Ig concentrations and CIC levels were measured. An estimate of the CIC level is important indicating the immune depurative capacity of the body. The CIC levels in mastitic cows were increased when compared to the same parameters in the healthy animals, but there was no statistical significance of the differences. The resulst lso indicated mean circulating total Ig levels vclose to physiological ones, still the individual values were unevenly distributed. In Chapter VII Microbiological investigations the research was carried out on 87 milk samples collected from mastiotic cows within Cluj county. Depending on bacteriology and morphology the bacteria were sorted and characterized. Since the highest percentage was represented by the genus Staphylococcus spp. (32%) the isolated strains were identified by use of API STAPH 20. Out of the 29 strains positively identified by API STAPH 20, 19 were from the Staphylococcus genus, a much higher percentage when compared to the literature. Testing the sensitivity of the studied strains to antibiotics was presented in Chapter VIII: Monitoring the antibacterial resistence of the isolated Staphylococcus strains. The irrational and excessive use of antibiotics on farms led to the appearance of resistance. Therefore, the results on antibiotic resistance have to be obtained prior to any treatment (Gentilini şi col., 2002; Luthje şi Schwarz, 2006). The obtained results indicated a highly increased resistance to several antibiotics, most frequently used in practice. Total resistance or partially resistant colonies were present. XIV

Microbial resistance in staphylococcal strains Table 1 Sample ENF OB SXT OT AMC P AMP CR 7 2 3 1 5 7 8 R 10 2 7 4 2 3 2 One can think that the antibiotics that are inefficient (ie., enrofloxacine and amoxyclav) were the most frequently and incorrectly used to treat diseases of bovine, which led to an increased resistance to these antibiotics in the isolated bovine strains. This hypothesis is being supported by the increased sensitivity to cloxacilline, a quite rare antibiotic in bovine therapy. Therefore one would recommend the antibiogram prior to start any kind of antibiotic treatment. Table 2 MIC, MBC and bactericidal index for the studied honey types against ten staphylococcal strains Staphylo coccus aureus strains Honey dew honey MBC/ MIC Polyfl oral honey MBC/ MIC Propo lis MIC MBC MIC MBC MIC MBC T1 4% 4% 1 2% 2% 1 1% 1% 1 T2 2% 2% 1 2% 2% 1 1% 1% 1 T3 2% 2% 1 2% 2% 1 1% 1% 1 T4 4% 4% 1 2% 2% 1 2% 2% 1 T5 2% 2% 1 1% 1% 1 0.5% 0.5% 1 T6 4% 4% 1 2% 2% 1 2% 2% 1 T7 4% 4% 1 2% 2% 1 2% 2% 1 T8 2% 2% 1 2% 2% 1 1% 1% 1 T9 2% 2% 1 1% 1% 1 0.5% 0.5% 1 T10 2% 2% 1 1% 1% 1 0.5% 0.5% 1 MBC/ MIC In Chapter IX, entitled The evaluation of antimicrobial efficacy of different types of honey used in vitro on the isolated Staphylococcus strains, the sensitivity of the studied bacterial strains was investigated as a response to different honeys. For this evaluation a mixture called artificial honey was used, containing all four types of glucids as the natural honey. The properties of honeydew honey are similar to those of the polyfloral honey. Nevertheless, especially due to its richness in minerals and bioflavnoids the honeydew honey has an increased antioxidant, antimicrobial and anti-cancer activity (Al-Mamary et al., 2002; Wei and Zhirong, 2003). There are numerous active compounds in any biological sample and it is relatively difficult to quantify every one of them. The antibacterial activity of bee products is apparently dependant more on strain, MIC and MBC being diminished for certain strains as compared to those obtained for XV

other strains. The variability connected to the strain can be influenced by an intrinsic susceptibility to the tested bee products. This study revealed certain differences between the tested bee products activities. The level of bacterial growth inhibition, established by the micro dilution technique depends on the product more than on the bacterial strain. The bactericidal effect as expressed by calculation of the bactericidal index indicated the usefulness of polyfloral and honeydew honeys in fighting against persistent infections, stopping the bacterial growth at the end of the treatment (cidal effect). The results of this study encouraged the continuation of the research in evaluating the chemical and biological activities of honeys in Romania especially of honeydew honey. There is a substantial potential in the use of honeydew honey in the clinical environment, therefore local honeys are very important. The in vitro antibacterial effect of certain alcoholic extracts were described in chapter X entitled The evaluation of antibacterial efficacy of used in vitro on the isolated staphylococcus strains. The alcoholic is renown for its antibacterial activity, this property being ensured by its content in flavonoids. It is thought that these flavonoids act on the microbial wall, changing its permeability. Within this research the antibacterial effect of of various origins and concentrations was studied on different Staphylococcus spp. strains isolated from mastitic milk by use of radial diffusion against a control (70º alcohol). Reading the inhibition areas the effects of various concentrations and types were compared. According to the obtained data, the highest concentration was attributed to the 10% extract. Concentrations of 1 and 5% acted less, with similar values for all types. Inhibition areas for the increasing concentrations (10, 20, 30 and 40%) did not differ significantly, as shown in fig.1. The concentration with the strongest effects was that of 10%, while an increase in concentration did not augment the effect. The most sensitive to the antibacterial effect of the extracts were Staphylococcus sciuri strains and the less sensitive those of Staphylococcus lentus and Stahylococcus simulans. Table 3 Mean inhibition zones on studied Staphylococcus strains Type of Staphylococc us 1% 5% 10% 20% 30% 40% Alcohol Sciuri 9.76 13.18 15.41 15.05 13.86 14.75 0 Aureus 7.62 9.5 10.62 11.37 11.12 11.12 0 Xylosus 4.75 9.16 10.63 11.8 12 11.48 0.4 Lentus 5.27 7.69 9.12 8.56 9 7.89 1 Haemolyticus 8.87 11 12 11.62 12.12 11.75 0 Lugdunensis 7.5 8.25 9.75 8.75 8.5 8.5 0 Simulans 7.33 7.5 8.25 8.5 9 8.75 0 Intermedius 7.75 11.25 13.25 12 12.75 12.75 0 XVI

Fig. 1. Zones of inhibition of EAP (PP1 and PP2) with concentrations of 1, 5, 10, 20, 30, 40% in the presence of alcohol as a witness, on the strain of Staph. Xylosus The alcohol did not exert any effect on neither of the strains, therefore the antibacterial effect encountered for the extracts was due to the composition of the not the diluents. Unfortunately there are no methods available still, to standardize the extracts its composition being highly variable depending on the origin, environmental conditions, harvesting period and local flora. Chapter XI includes the Evaluation of the synergistic effect of the 10% extract and antibiotics used for the antibiogram. The problems caused during therapy due to the resistance to antibiotics of some bacterial strains from mastitic milk led to the idea of experiencing the in vitro effects of the combined and antibiotic treatment to monitor potential synergistic activities. Since the previous experiments indicated as showing maximal effects the 10% extract, this was the one used in combination with the antibiotics. Table 4 Mean inhibition titers red in the antibiogram (mm) -antibiotic +EAP 10% Tip/type OB+ ENF+ OT+ AMC+ SXT+EAP Staph. EAP EAP EAP EAP P+ EAP S. xylosus 25.33 37.33 26.66 CR 10.99 35.33 30.13 S. lentus 29 38 31 CR 9.5 40 40 Table 4 indicated the inhibition diameters for S. xylosus and S. lentus some of the most resistant staphylococcal strains. XVII

Fig. 2 Inhibitory effect of and antibiotics demonstrated synergy against Staphylococcus lentus Following the readings of the antibiogram for both antibiotics and the combinations of these antibiotics with the (EAP 10%), it could be observed that the enhanced the activity of all antibiotics. It could be also seen that the inhibition area resembled a champagne cork, reuniting the antibiotic inhibition and the inhibition areas. These results could be of importance for setting up treatment protocols involving both the antibiotics and extracts and obtaining combined products.. The has stimulating activity on the antibacterial effect of conventional drugs but it cannot replace them. The treatment has to be individual, based on all the influencing factors and based on the bacterial strain. The evaluation of the biological effects of bee products cannot be performed outside the framework of the immune system, especially of that of the cell mediated immunity. Testing the and polyfloral honeys or honeydew honey on phagocitosis and blast transformation were described in chapter XII. The in vitro blast transformation and carbon particle inclusion tests, could offer circumstances identical to those from the inside of the body and allow the evaluation of the effects of certain compound that could have biological influence, by maintaining health and stimulating the defense potential. In this chapter the immune status of the studied animals is monitored where the influence of bee products in order to find modalities to stimulate the immune system of the animals towards pathogenic agents. In order to observe the prolyferative response of lymphocytes subsequent to stimulation with bee products the blast transformation test was performed according to the protocol proposed by Spînu Marina et al. (1995). This technique allowed the calculation of stimulation indices based on the readings of OD units by spectrophotometer, for each experimental variant. XVIII

The method used whole blood and 12 experimental variants, each in duplicate as follows: PHA, LPS, ALC 70, PS1, PS2, PP1, PP2, MM, MP, MA, MB, where PS and P are samples and MM, MP are honey samples.. All the results were processed statistically to obtain mean values and standard deviations, and were compared for the statistical significance of the differences. The blast transformation test results indicated an immune modulating potential for certain bee products tested individually and compared to the spontaneous, untreated control, the alcohol treated control, cool 70% and the culture treated with artificial honey. In the healthy animal group (farm A), the mean values of the stimulation indices varied between -193.49 and 54.18, depending on the product added to the culture; - Relatively similar values were obtained for the control,, PHA, LPS, Alcohol, PS1, PS2, PP1 and PP2, the difference between the variant lacking statistical significance (p> 0,05); - In the variant treated with PHA, a maximal value of the stimulation index was present (IS=54.18%), close to the one induced by LPS (IS=53.40%); - The treatment of whole blood cultures with alcohol caused a stimulation almost identical with that of the untreated control (49.40% and 49.29% respectively); - For the treated samples (PS1, PS2, PP1, PP2), the results were not different statistically significantly (p> 0.05) from those of the controls; - In contrast, the honey samples acted less stimulating and sometimes inhibiting for the artificial honey (-3.95%); similarly in this case the standard deviation was extremly high, pleading for a large dispersion of the individual values; the maximal inhibiting effect can be observed for the artificial honey, a control for the tested honey samples. Some of the variants were stimulating when compared to the alcohol, but not with the spontaneous control. Fig.3. Immunomodulatory ability of tested bee products Jucu group XIX

Phagocytosis is presented in table 5. Phagocytosis in time in healthy animals on farm A (IS%) Table 5 Control alcohol 70 PP2 MP 1/1v MA 1/1v ln0- ln45 ln45- ln60 ln0- ln45 ln45- ln60 ln0- ln45 ln45- ln60 ln0- ln45 ln45- ln60 ln0- ln45 ln45- ln60-0.034 0.047 0.041-0.050-0.122 0.210-0.242 0.101-0.132 0.072-0.095-0.067-0.137 0.019-0.093 0.109 0.089-0.182-0.562 0.940 0.016 0.096-0.016 0.127 0.023 0.008-0.053 0.096-0.189 0.180 med -0.038 0.025-0.037 0.032-0.064 0.109-0.069 0.005-0.294 0.397 stdev 0.056 0.083 0.091 0.089 0.077 0.101 0.166 0.162 0.233 0.473 The results of the spontaneous in vitro engulfment of carbon particles, as in the control variant, with no additives, mirrors the influence of the incubation temperature, with no other interventions as alcohol, vegetal extracts, vaccines, etc.) ( Hilton et al., 2002). The cells of the non specific immune system could be in this way monitored functionally and their reacting potential could be measured for the case of bacterial aggression or other stresfull factors, by expressing it as optical density units or ln of the values red (Vanmiert, 1991). Chapter XIII summarizes the data on the research concerning Evaluation of the influence of different types of and honeys on the immune function in mastitic cows. The values of blast transformation indices were presented for cows with subclinical and clinical mastitis. The data indicated that the in vitro effects of the tested bee products highly depend on the source and content in active principles. Another factor influencing the blast transformation index is the type of raising unit, with all its components, including the genetics of the herd. Thus, if in the animals with subclinical mastitis the effects of the were similar to those encountered for healthy animals, with maximal activity for PP2, in the latest case the most efficient was pp1, with a statistically significant difference for pp1 (p<0.05), the strongest activity was red for the honeydew honey and the poorest for the artificial honey. For the mastitic cows, the effects of the were much stronger (farms C and D) and those of the honey weaker. On the farm F the situation is reversed with spectacular activity of the honey, depending on its type, statistically supported (p<0.01). Relatively similar values were described for the other three groups with mastitis (E, F şi D) (fig. 54-56) if it is being considered that the blast transformation indices were calculated depending on the culture medium control and not the invertase control. The negative indices are relative, the correction being done when the control is being replaced with the glucose content of the invertase sample. The persistent negative indices are due to the partial hemolysis that interferes with the reading. XX

CHAP.XIV. GENERAL CONCLUSIONS Staphylococcus spp. was present in all the studied milk. These data were concordant to those in literature, proving that the pathology in mastitis is similar depending on technology The total resistance to antibiotics or presence of resistant colonies could be connected to their frequent inadequate use for the therapy of mastitis. Thus, the effects are maximal by use of a more obscure antibiotic such as cloxacilline as opposed to enrofloxacine, with high resistance A resistance percent as high as 65.6 in some of the tested strains indicated besides inappropriate treatment schemes the permanent character of such strains in the tested animals and farms Out of the types used in combination with the antibiotics, the maximal synergistic activity was measured for the polifloral one, increasing the efficacy of most antibiotics. These results recommend this kind of for further studies on composition associated with biological effects The comparative study of the efficacy of various concentrations of proved maximal effects for the 10% dilution, and decreasing effect with increasing concentration as opposed to the hypothesis increased amount, increased biological activity for the. The obtained results were encouraging from the clinical point of view, in combination with the antibiotics being considered as a future therapy In order to use alcoholic extract of in therapy procedures to type the compound should be found The analysis of the results obtained by performing the immunological tests (phagocytosis and blast transformation) with and honey indicated that the results were highly dependant on the health status of the animal. Propolis showed beneficial effects as opposed to the honey in healthy animals, while in mastitic animals the effect was highly inhibiting, more pronounced for honey than for the. The effects of bee products on adaptive cell mediated immunity is reversed when compared to the innate response. Thus the inhibits while the honey stimulates blast transformation. Similarly, the health of the animal is an influencing factor. RECCOMENDATIONS In order to avoid resistance to antibiotics in cases of mastitis, the identification of the pathogen and compulsory antibiogram to initiate the treatment, by parallel use of antibiotics and on the condition of finding an appropriate method to preserve the aqueous extract or attenuate the irritating effect of the alcoholic one Use of or honey extracts as immune modulators has to be rigorously adapted to the tape of product, its origin and chemical composition The bee products could be of better use once standardized, therefore further research is indicated XXI