الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
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Abstract
Infection with Brucella melitensis is one of the most important causes of abortion in small and large ruminants and also causes severe systemic disease in exposed animals. Although, World Health Organization recommendation on the use of combination of Doxycycline and Streptomycin or Doxycycline and Rifampicin but replace infection cases increased in last years. However, the treatment for this disease, is it vital to discover novel antibiotics and antimicrobial agents such as nano antimicrobial and nano antibiotics. Therefor this current study aimed to evaluate the effect of some antibiotics, metal oxide nanoparticles, and some plant extracts as chemotherapeutic agents for treatment of Brucella melitensis infected animals to appley the most efficient plant extract in soft white manufacture cheese.
The obtained results can be summarized as following
1- Twelve Brucella cultures were isolated from raw milk and infected animal tissue (fetal spleen and liver, suppra mammary, lymph node, and stomach content). According to morphological, biochemical and serological properties these isolates were identified as Brucella melitensis biovar 3.
2- Both of Doxycycline and Streptomycin loaded gelatin nanoparticles (GNPs) as well as zinc oxide nanoparticles were produced by a modified nanoprecipitation method in order to use for infected animal treatment.
• These polymeric nanoparticles were characterized using Dynamic light scattering, Transmission electron microscope, Encapsulation efficiency, X-ray diffraction, zeta potential and cytotoxicity assay.
• The particles size of gelatin NPs loaded with Doxycycline and Streptomycin ranged from 130 to 690 and 63.7 to 507.5 nm of intensity from 3.5 to 87.7% and 9.9 to 79.7%, respectively. These particles were monodispersed spherical nanoparticles whereas the GNPs loaded with Doxycycline were larger than that loaded with Streptomycin. The encapsulation efficiency of gelatin NPs carrier loaded with Doxycycline and Streptomycin was 89.32 ± 3.22% and 81.74 ± 1.15%, respectively.
• ZnONPS were spherical shape with smooth surface in small size about 50 n m with ZP value -4.45 mv as highly unstable. ZnONPs up to 10 µ/ ml was not toxic on Mouse normal liver cell line (BNL) recorded the cell viability and inhibition of 93.2 and 6.8 %, respectively. Whereas it reduced the viability to 20.5% at 100 µg/ ml ZnONPS. The IC50 value of ZnONPs on BNL cells was 45.38 µg/ ml.
3- Effect of antibiotics as chemotherapeutics treatment on animal tissue
• Doxycycline and Streptomycin combination in the traditional form displayed a highly significant reducing estimated means of Brucella CFU/gm (log10) at p < 0.01.
• The ability of a polymer-based gelatin nano particles as a nano-carrier in a combination therapy (Doxycycline and Streptomycin), and the efficacy of treatment compared with the traditional form of the same combination of drugs against Brucella melitensis filed strain were attempted and the outcomes of this trail are displayed a significant reduction of the log of Brucella C.F.U/gm in tissue at p < 0.05 three days after the end of the treatment regium which consisted of four doses with one day a part between doses for infected treated group if compared by the infected non-treated control group . To explore the effect of fabricated nano formula and the anti-biotics used in each treatment on animals’ tissue. Liver (A.S.T& A.L.T) and kidney (Creatinine& Urea) function tests were estimated as well as spleen, liver body weight ratio for each animal under investigation were taken and the result can be concluded in the bellowing points.
• Doxycycline and Streptomycin in traditional combination, Aminase aspartate transferase enzyme (AST) exhibits a significant elevation at p < 0.05 in infected non- treated groups 3 and 14 days post the treatment. Also, there was a significant increasing at p < 0.05 in liver and spleen body weight ratio in the infected non-treated group.14 days after the end of the treatment.
• Three- and fourteen-days post 4 shoots of gelatin nano particles carrier loaded with Doxycycline and streptomycin treatment (AST) exhibits a significant elevation at p < 0.05 in infected non-treated and infected treated groups, there was a significant increasing in liver/body weight ratio in infected non-treated and infected-treated groups 3 ,14 days after the end of the treatment and a significant increasing p < 0.05 in spleen / body weight.
4- Usage of ZnONPs as an antibacterial agent against Brucella pathogen displayed a highly significant reducing estimated means of Brucella CFU/mg (log10) at p < 0.05 post 4 doses of treatment taken one day apart for infected treated group if compared with infected non-treated control group 3 days post the end of treatment. Fabricated ZnONPs treatment animals exhibited a significant increasing p < 0.05 in liver / body weight ratio in the case of the infected non-treated group and infected treated group 3 days post the end of the treatment and in the infected non-treated group 14 days after the end of the treatment. Likewise, there was a significant elevation in (AST) enzyme was observed at p < 0.05 in non-infected non- treated group 3,14 days after the end of the treatment. All animals under investigation that reserved by ZnONPs treatment displayed a significant elevation at p < 0.05 in creatinine level.
5- Effect of different concentration of ZnONPs on the growth of B. melitensis bv.3 ranged from 1- 10 mg / ml
In Tryptone soya broth (Med.2), five ZnONPs concentrations were tested for their antibacterial activity against B. melitensis. A gradual decrease in B. melitensis count (CFU/ml) was noticed at 5 mg / ml ZnONPS. Increasing ZnONPs up to 7 mg / ml resulting in complete inhibition of B. melitensis growth.
• Three concentrations of ZnONPs were investigated as antibacterial agent against B. melitensis in contemned raw milk during 24 h at 4C°. Results demonstrated that increased ZnONPs concentrations up to 8 mg/ml cell viability significantly decreased to 5.41, 5.39, and 5.40 Log CFU as compared to control (5.84, 5.83, and 5.73 Log CFU) with reduction rates 0.43, 0.44, and 0.33 after incubation periods. High determination coefficient R2 of 0.93 was recorded after 1 & 3 h of incubation period.
6- Aqueous and ethanolic extracts of 12 medicinal plants were tested to detect their antibacterial activity against B. melitensis using agar well diffusion method. Ethanolic extract were more efficient than aqueous plant extract in inhibiting the growth of tested strain. Highest inhibitory effect was obtained by Nigella sativa ethanolic extract followed by Petroselinum crispum aqueous extract and Ocimum basilicum ethanolic extract with inhibition zone diameter of 25, 23 and 22 nm, respectively. The corresponding figures of activity index for Doxycycline and Streptomycin were 1.13 & 1.56, 1.4 & 1.43 and 1.0 & 1.37, respectively.
7- Minimum inhibitory concentration (MIC) value of most efficient plant extracts Nigella sativa, Petroselinum crispum, and Ocimum basilicum were 20, 83 and 166 mg / ml, respectively. These results indicated that Nigella sativa ethanolic extract was more efficient on B. melitensis growth than Petroselinum crispum, and Ocimum basilicum. Moreover, these extracts have a bacteriostatic effect on B. melitensis growth.
8- The results of Gas chromatography GC-MASS analysis of Nigella sativa ethanolic extract showed that 42 different compounds were detected. The presence of some major compounds from them is scientific evidence in support the use of this plant medicinal preparation. The association of these compounds in this variety of Nigella sativa could justify the antioxidant, anticancer and antimicrobial activity.
9- Application of Nigella sativa ethanolic extract as antibacterial additive during white soft cheese preparation and storge for 4 weeks at 4°C. Two white soft cheese treatments were inoculated with B. melitensis bv.3, one of them was hold as a control group (IC), while the other one was fortified by addition of Nigella sativa ethanolic extract (2%) in order to detect their effect on survive ability of B. melitensis bv.3 during the manufacture and storage of cheese. Results indicated that B. melitensis bv.3 was significantly affected by the cold storage temperature and the count was reduced significantly in both groups. Although that, Nigella sativa ethanolic extract (2%) still have antibacterial effect against B. melitensis bv.3 as the viable count was significantly decreased with greater reduction percentages from production of cheese till the end of storage period than the control group. To insure the applicability of cheese fortification with Nigella sativa ethanolic extract (2%), non-inoculated cheese samples were prepared for sensory evaluation during storage. The fortified cheese had slightly lower significant results than control group but still accepted to the consumer with high overall acceptability.