ABSTRACT. Of the PhD thesis:,, Etiopathogenic, clinical and therapeutic investigations in American foulbrood in honey bees

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1 ABSTRACT Of the PhD thesis:,, Etiopathogenic, clinical and therapeutic investigations in American foulbrood in honey bees Key words: bees, American foulbrood, Paenibacillus larvae, hydro alcoholic plant extracts, propolis tincture Bees are considered biologic resources with vital importance. By plant and trees in spontaneous and cultivated pollinating which is finalized with fecundation and seeds, vegetables and fruit formation, bees have an essential role in hundreds of thousands species of the world vegetation perpetuating and surviving. Meanwhile, bees have an important role in producing honey, pollen, wax, venom, royal jelly, drone bee larvae and propolis, used in food and pharmaceutical industry. Developing their activities at different heights beside the soil and interfering the pollutant element especially from air, water and plants (industrial and road particulate matters, pesticides, radioactive particles) which have a negative influence upon their life and activity, bees are considered sensors of environment pollution. Apiculture, in Romania, is one of the oldest occupations of the local populations and it developed on this land in special favourable natural conditions assured by country placing in the Carpatho-Danubian-pontic space, land with exceptional conditions regarding climate, relief and vegetation that determined year by year the bee colonies development and obtaining along millenniums important apiary products. American foulbrood is an infectious disease which affects the brood of honey bees (Apis mellifera) and other bees in Apis genre, its death appearing after cell capping. It is one of the most serious, old and spread at the world level bee disease, being known as bees pest or rotten brood. The first official diagnosis of this disease is dated from The disease is produced by Paenibacillus larvae bacterium and it is characterized by an intense proteolysis of the entire diseased larva, by colour, consistence, volume and odour of larva and their death in the stage of capped brood. The etiologic agent is a Gram positive bacillus. The resistant form of bacterium is the spore. The consequences of American foulbrood apparition and evolution in an apiary are very serious; with important loses of biologic material and a major economic impact. Without measures against the disease, the affected bee colonies are not cured themselves. The treatment of American foulbrood diseased bee colonies is made with sulphonamide and antibiotics, which are unfortunately found in the apiary products. Antibiotics do not have 1

2 any action upon these germ spores, while the vegetative forms, depend on the bacterial strain, are sensible to sulfathiazole, streptomycin, oxytetracycline, penicillin and tylosin. Due to the fact that within European Union countries is forbidden the commerce with honey bees and other apiary products which have antibiotics and chemotherapy residues used in the treatment against American foulbrood, the motivation of the present paper was to offer a viable alternative to the classic medication, based upon a solid knowledge of the etiologic agent. The research had as objectives the studies regarding the therapy of American foulbrood disease with natural alternative ways represented by the hydro alcoholic extracts of medicinal plants and propolis tincture, completed by investigations of highlighting some detection methods of the etiologic agent Paenibacillus larvae based upon the noticeable clinic aspects, classic (bacterioscopic and bacteriologic) and modern (detection of the specific genome with identifying of the bacterial genotype by molecular biology techniques PCR - Polymerase Chain Reaction) laboratory exams, to a fast intervention with adequate therapeutic ways into the affected apiary. The PhD thesis called: Etiopathogenic, clinical and therapeutic investigations in American foulbrood in honey bees is structured in two parts, the first one the bibliographic study and the second one personal researches. The PhD thesis consists in 7 chapters with 150 figures, 43 tables and 206 references. The bibliographic study is structured in a chapter with 10 subchapters, with 9 figures and 3 tables. Chapter I consists in data regarding the importance in beekeeping, the history in Romanian beekeeping, the history of American foulbrood and also of the apparition and evolution of the disease. It is done a description of the etiologic agent Paenibacillus larvae highlighting the morphologic features, the cultivation conditions, its cultural features and sensitivity beside environmental factors. There are described aspects about pathogeneity, pathogenesis and clinical manifestations. This chapter also consists in up-to-date notions regarding the used diagnosis techniques, and American foulbrood prophylaxis. The personal researches are structured in 6 chapters (with 141 figures and 40 tables) regarding the aim of the researches, materials and methods, conclusions and recommendations. In chapter II the objectives of our researches were: to identify the medicinal plant species and also propolis, which contain antimicrobial principles; easy extraction of the active principles from medicinal plants and propolis using usual solvents and techniques; physical and chemical analyze of the hydro alcoholic extracts from medicinal plants and propolis tincturea having in view the interested qualitative and quantitative determination. 2

3 The used materials were represented by basil (Ocimum basilicum), dandelion (Taraxacum officinale), thyme (Thymus vulgaris), yarrow (Achillea millefolium) and raw propolis collected from bees. For the medicinal plants it was used all the aerial part, respective Herba basilicum, Herba taraxaci, Herba thymi and Herba millefolii. Using pharmaceutical ethyl alcohol as solvent in different concentration, there were obtained the hydro alcoholic extracts of basil, dandelion, thyme, yarrow and propolis tincture, by 10 days maceration process. The ratio between the vegetal product mass and the extraction solvent was 1:10 for the plant extracts, and the ratio between the raw propolis and the pharmaceutical ethyl alcohol 3:7 for the propolis tincture. Following the physical and chemical laboratory analyzes, the RP-HPLC (Reversed Phase- High Performance/pressure Liquid Chromatography) chromatographic ones, and GC- MS analyze (chromatograph gas coupled with mass spectrometer) of the volatile compounds separated by hydro distillation extraction carried out for the hydro alcoholic extracts of basil, thyme, dandelion, yarrow and propolis tincture it was noticed that these have a main content of volatile oils and total flavonoids with antibacterial potential. The volatile oil content from all the analyzed plant extracts is best represented in the hydro alcoholic extract of basil (0,30 %), followed by thyme (0,29 %), dandelion (0,18 %) and yarrow (0,13 %). Regarding the concentration in total flavonoids, the richest one was the propolis tincture (3,16 %), followed by the hydro alcoholic extracts of thyme (1,28 %), yarrow (0,27 %), basil (0,18 %) and dandelion (0,10 %). It was firstly necessary to achieve the isolation and identification of the bacterium in the suspect pathogen material to testing the antibacterial potential of the obtained hydro alcoholic extracts from plants and propolis tincture. In chapter III the objectives were the isolation, identification of a Paenibacillus larvae strain with the aid of the classic laboratory tests and the detection of the specific genome of this bacterium by molecular biology techniques PCR (Polymerase Chain Reaction), from pathologic samples from a suspicious apiary. To proving the quality of the obtained mentioned tests there were carried out in parallel with the working strain and on certified referential materials (MRC), respective the referential bacterial strains Paenibacillus larvae 43 Anses 2013 and LMG 9820 used as positive control samples, from the international accredited collections. To achieving the objective, there were done anatomopathologic, bacterioscopic, bacteriologic, specific biochemical tests, the immunochromatographic and conventional PCR technique tests. The working sample with capped brood called 222-RO was positive considered following the multiple specific laboratory tests for Paenibacillus larvae the certain diagnosis was American foulbrood disease. 3

4 American foulbrood could be diagnosed by clinical exam, upon the anatomopathologic alterations of diseased capped brood, the general layout of the comb with brood and the pathognomonic signs. Specific laboratory have done to confirm the diagnostic. The positive bacterioscopic exam achieved by Gram coloration method, with the clear exposure of bacterium morphology and its spores with the characteristic placing as brittle scales, and also the clinic diagnosis, were sufficient elements to a certain diagnose in American foulbrood. The biochemical tests done on 222-Ro working strain lead to the certain diagnostic, the working strain is Paenibacillus larvae. The identification by biochemical profiles of Paenibacillus larvae bacterium consecutive its culture and isolation on different enrichment and selective mediums represents the gold method. The conventional PCR (Polymerase Chain Reaction) proved to be a performing technique comparatively the classic microbiologic technique, having a similar sensitivity, meanwhile being superior to the bacteriologic techniques due to the high speed detection possibility of the specific genome of Paenibacillus larvae. The molecular biology technique was used to identify the Paenibacillus larvae bacterium from the 222-RO working strain (comb with suspicious capped brood) and other bacterial cultures. To achieve a diagnostic of certainty and to prove the higher sensitivity of the method, beside the mentioned samples, there were tested two related from the DNA characteristic point of view bacterial strains, Paenibacillus alvei and Brevibacillus laterosporus, as negative control samples. Analyzing the cultural characters of the isolated strain Paenibacillus larvae 222-Ro, the identified biochemical characters and their traceability to the reference strains P. larvae ANSES 43 and LMG 9820, corroborated with the specific genome detection by molecular biology techniques, it was established that the studied strain genotype is ERIC I. In chapter IV, the objective was the comparative testing in laboratory conditions of antibacterial potential of hydro alcoholic extracts of basil (Ocimum basilicum), thyme ( Thymus vulgaris), yarrow (Achillea millefolium), dandelion (Taraxacum officinale) and propolis tincture upon Paenibacillus larvae bacterium, by their qualitative and quantitative action on,,in vitro cultures. To achieving the mentioned objectives, there were carried out 1/32 water distilled dilutions from the plant hydro alcoholic extracts and propolis tincture, to see if applied on a bacterium culture they inhibit the development of the studied P. larvae. Parallel, it was tested the P. larvae strain sensitivity to oxytetracycline, a large range used antibiotic in treating against American foulbrood by micro tablets antibiogram method. 4

5 The obtained results highlighted the fact that the propolis tincture had the strongest antibacterial action, followed decreasingly by the hydro alcoholic extracts of basil, dandelion, thyme and yarrow. The propolis tincture (inhibition area 8 mm 1/32 dilution) and the hydro alcoholic extracts of basil (inhibition area 7 mm 1/32 dilution) and dandelion (inhibition area 6 mm 1/32 dilution) inhibited the development of bacterial cultures of Paenibacillus larvae to 1/32 dilution, proving a bactericide effect. The hydro alcoholic extract of thyme and yarrow inhibited the development of the bacterial cultures only to 1/2 dilutions, non significant result to be involved in American Foulbrood therapy. Following the carrying out of the oxytetracycline micro tablets antibiogram, the inhibition areas were 0 mm, so the studied Paenibacillus larvae 222-Ro strain is resistant to this antibiotic, the result confirms that the practice against American foulbrood disease with oxytetracycline is not efficient any more, along the years appearing resistant bacterial strains. In chapter V, the objectives of our researches consisted in the fact that the hydro alcoholic extracts of basil, dandelion and propolis tincture which have proved their bactericide effect upon the Paenibacillus larvae on bacterial cultures,, in vitro to 1/32 dilutions, were introduced in the bees food in sugar syrup and sugar paste to establish the optimum administering way and the optimum concentrations. The obtained results emphasized the fact that the sugar syrup administering form is the most indicated due to the fast ingestion. The optimum concentration of hydro alcoholic extracts of basil and dandelion proved to be 4%, having a 100% consumption range without adverse effects. The optimum concentration of propolis tincture administered in sugar syrup was 3%, with a 100% consumption range, without adverse effects. In chapter VI the objectives of our researches were: highlighting the clinical manifestations in the diseased families, the clinical testing of the plant hydro alcoholic extracts and propolis tincture on American foulbrood diseased colonies, stating a biological method against American foulbrood by the artificial swarm process and its applying in the diseased apiary. The researches were made during August, in a stationary apiary consisting in 180 bee colonies in vertical Dadant beehives on 10 frames, nearby town of Nehoiu, Buzau District. There were carried out clinical investigations of the bee families to find out the pathognomonic signs and specific anatomopathologic modifications. There were identified 15 bee colonies with clinical signs of American foulbrood to represent the experimental and control groups. 5

6 There were administered hydro alcoholic extracts of basil, dandelion and propolis tincture as sugar syrup with 4 %, respectively 3% concentration. These concentrations were established after testing on bees in laboratory conditions, which showed a 100% consumption range, without adverse effects. Each family received 2,5l of syrup, administered in 0,5l dose, in 3-5 days time, depending on consumption. The control group received only simple sugar syrup, 2,5lper family, in the same dose and time interval. There were used external feeders, located under the beehive top. All the 15 families benefit before the experiments of transferring in new hives and there were eliminated the frames with more than 30% diseased brood per frame, having as aim the infectious pressure decreasing, conformingly the general sanitary veterinary conduit for such cases. There were also replaces the queens with new younger ones, bred in the same season, from a health apiary, to intensify the egg laying for increasing the bees population. After transferring, there were counted the cells with diseased brood in the colony, with the aid of Netz frame, by their identification after the following features: colour, holes, cell caps reducing, and the wet-oiled layout of the caps. The counting was made in 7 days and also 21 days time after the administering of the hydro alcoholic extracts of basil, dandelion and propolis tincture, including in the control groups. Based on the obtained results, it was calculated the decreasing rate of infection (efficaciousness), express as percentage, individually per each colony. It was also calculated the average of the decreasing rate of infection per each group. The statistical processing of the data was made by ANOVA and T test. On a 3- bee families group it was applied the biologic method against American foulbrood by artificial swarm process. This consisted in eliminating of all the built combs and keeping the bees in a process like the natural swarming. The diseased capped cells affected by American foulbrood counting where the biologic method of artificial swarm was applied, with the aid of Netz frame, was achieved before applying the method, in 21 days time, respectively 42 days time after they were moved in beehives with artificial combs. The establishing of the time intervals were made taking into account that the metamorphosis period (from egg to adult) in working bees is 21 days and the bees benefit in the new hive only by artificial combs. The clinical investigations upon the bees colonies in the studied apiary highlight many types of modifications specified to American foulbrood disease: modified behaviour of adult bees, smell of joiner s hot glue or rotted, the appearance of the combs with diseased brood and diseased capped larva. Based upon the anatomo pathologic modifications and the identified pathognomonic signs the clinic diagnostic was American foulbrood. 6

7 The results obtained in 21 days after the 3% propolis tincture sugar syrup administering on an experimental group of 3 bee colonies highlights an average efficaciousness of 91,72%, the differences comparatively the control group being very significant (p < 0,001). The results obtained in 21 days after the 4% basil (Ocimum basilicum) hydro alcoholic extract sugar syrup administering on the experimental group of 3 bee colonies highlights an average efficaciousness of 82,31%, the differences comparatively the control group being very significant (p < 0,001). It was proved that in apiary conditions, the propolis tincture and basil (Ocimum basilicum) hydro alcoholic extract administered in sugar syrup in 3%, respectively 4% have a bactericide effect upon the Paenibacillus larvae. Regarding the biologic method against American foulbrood by artificial swarm process was used, the results in 21 days, respectively 42 days time after the applying the method were not conclusive, due to the fact that the tested bee colonies do not built the artificial combs in the hive even they were fed with sugar syrup, similar the control group. Meanwhile, it was noticed the migration of the adult bees in the experimental group from their hive to the neighboured hives, being recorded an increasingly depopulation along the 42 days. We consider that the biologic method by the artificial swarm process could not be applied against American foulbrood in Romanian climate and flora conditions, taking into considerations the fact that this disease appear in our country preponderantly during July-August, to the end of the active apiary season. Chapter VII: General conclusions and recommendations The PhD thesis is finalized with 24 general conclusions and 9 recommendations with main importance to specialists and beekeepers. 7