الفهرس 
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Abstract
In this thesis, we investigate some practical aspects of AFB diagnosis and control. We described a molecular technique for early diagnosis of the American foulbrood disease before clinical symptoms appearance and evaluated the honeybee lactic acid bacteria as a control agent of American foulbrood disease.
I. Early Diagnosis of AFB:
P. l. larvae AFB bacterial pathogen was isolated, identified and characterized using some morphological, microscopical and biochemical tests.
The experiment was set up in apiary yard of the Apiculture Research Department, Plant Protection Research Institute, Agricultural Research Center, Dokki, Egypt, during the summer 2011. A total of 5 honeybee Colonies were used, with 2 colonies left untreated and used as control. Treated colony was given a honey comb, heavily loaded by P. l. larvae spores previously isolated and cultured artificially in lab. Honey and Adult honeybee workers were sampled and cultured on J-agar for P. l. larvae weekly detection until appearance of the disease clinical symptoms. One of the treated colonies developed clinical disease symptoms on the third week post infection and for the other two colonies; clinical symptoms appeared on the fourth week.
The results of the total samples cultured from adult honeybee workers, all over the four weeks post infection, gave 90.9% positive AFB detection results while honey samples gave only 81.8%.
P. l. larvae were also detected from Adult bees and honey samples using KAT-PCR that has a PCR amplicon of size 550 bp, only found in P. l. larvae strains; used for AFB detection.
All honey and honeybee samples have shown positive PCR results except only one honey sample, showed negative result (P. l. larvae not detected). So honeybee samples gave 100% positive PCR AFB detection results while honey samples gave only 90.9%.
This PCR assay provides a specific detection for P. l. larvae from week 1 post infection even if there is no clinical symptoms appeared in a colony. The technique can be directly used to detect presence or absence of P. l. larvae spores in honeybee samples and contaminated honeys. Isolation and identification of the probiotic lactic acid bacteria (LAB):
Our study aimed to assess the gut of honeybee adults, PCR and direct sequencing results revealed the presence of seven lactic acid bacteria. Five anaerobic lactic acid bacterial strains belong to Lactobacillus kunkeei species, one aerobic LAB is closely related to Lactobacillus plantarum species and the last one is anaerobic, it is close to a strain of species Fructobacillus fructosus.
Lactic acid bacteria inhibition assays on agar plates were investigated, to evaluate the effects of the extracted honeybee LAB on P. l. larvae growth in vitro. Our results demonstrated a strong inhibitory effect of the combined honeybee LAB flora. The combination of all seven Lactic acid bacteria (LAB) phylotypes resulted in total inhibition (no visible growth) of the investigated P. l. larvae strain. Four Lab phylotypes (Lab 2, Lab 3, Lab 4 and Lab 11) individually, completely inhibited the represented P. l. larvae genotype. The results also demonstrate that not all LAB inhibit P. larvae, as the Lab 1 and Lab 6 strains showed only partial inhibition of the P. l. larvae by giving 1.9 and 1.5 cm inhibition zones, respectively whereas there was no growth inhibition detected by the isolate Lab 16. We additionally evaluated the results by comparing the antibacterial activity of the extracted honeybee LAB, Oxytetracycline (terramycine) and royal jelly, it was found that inhibitory effect of honeybee LAB against the P. l. larvae bacteria is clearly stronger than the Oxytetracycline (terramycine) and royal jelly by giving either total inhibition activity or clearly larger inhibitory zones.
Thus, our results strongly suggest that the probiotic LAB bacteria linked to the honeybee stomach have important implications for honeybee pathology in general and for AFB tolerance in particular.
We have provided the first information about a novel nonpathogenic LAB bacteria in the honeybee gut of Apis mellifera found in Egypt.