الفهرس 
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Abstract
Campylobacter is the leading cause of zoonotic enteric infections and emergence of antibiotic-resistant strains worldwide. Poultry is the major reservoir for human campylobacteriosis via ingestion of contaminated meat. Therefore, it was necessary to understand the pathogenesis mechanism following chronic campylobacter infection in vivo (mice model) and the role of rosemary essential oils as a natural flavonoids alleviating and improving negative changes resulting from campylobacter infection.
In this study, thermotolerant Campylobacter jejuni and Campylobacter coli was identified using PCR in cloacal samples (56) from 3 broiler farms in Assiut Governorate, as well as intestinal and liver samples (23 sample each) from chicken carcasses from 6 farms in Assiut, Sohag and Minya Governorates.
Antibiotic resistance of the isolated thermotolerant campylobacter strains (C. jejuni and C. coli) against quinolones, tetracycline, macrolide, Penicillin, and streptomycin was evaluated. Antibiotic resistance genes (tet O & gyra A) of thermotolerant campylobacter strains were detected..
Gas chromatography/mass spectrometry (GC/MS) was conducted for rosemary essential oil (REO), where two different commercial samples of rosemary oil were purchased to recognize their composition and active compounds.
Experimentally, BALB/c mice model was inoculated by oral gavage of thermotolerant campylobacter infection to identify the health effect on different body systems and the possible role of REO to alleviate campylobacter implications.. .Immunity of mice were examined in small and large intestine lysate for the presence of proinflammatory cytokines including tumor necrosis factor alpha (TNF-α), gamma interferon (IFN- γ), Interleukin 5 (IL-5), Interleukin 3 (IL-13) Interleukin 6 (IL-6) and Interleukin 17 (IL-17). Histopathology of small and large intestine, Peyer’s patches, spleen and liver as well as imunhistochemisty for liver and spleen using anti-PEACAM-1, anti- HSP70 and anti-C-MYC antibodies were performed.
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The results of this study revealed the following:
The polymerase chain reaction test confirmed Campylobacter jejuni and Campylobacter coli were 27.65% and 34.04%, respectively. The isolates of Campylobacter jejuni and Campylobacter coli were detected in the following percentages (27.58%, 27.58%), (22.22%, 66.66%), (33.33%, 22.22%) of cloacal swabs, intestinal and liver contents, respectively.
Concerning the fluoroquinolones group, the highest percentage of C. jejuni resistance was recorded for Enrofloxacin (100%) followed by Norfloxacin and Ciprofloxacin (69.23%). In addition, 100% of the strains were resistant for tetracyclines group (tetracycline and doxycycline), penicillin group (ampicillin and amoxicillin) and for streptomycin. For Macrolides group 84.61% and 92.30% of C. jejuni strains were resistance for erythromycin and spiramycin, respectively.
The lowest percentage of resistant strains (15.38%) was recorded for colistin
The percentage of antibiotic resistance of C. coli for fluoroquinolones group was 93.75% resistant strains for each norfloxacin, nalidixic acid and enrofloxacin while, 87.5 % of the strains were resistant for ciprofloxacin. However, 100% of the strains were resistant for both tetracycline and doxycycline, as well as for ampicillin, amoxicillin and streptomycin. For macrolides group 100% and 81.25 % of the strains were strains for erythromycin and spiramycin, respectively. While, for Colistin the lowest percentage was achieved (12.5%).
Our results showed that gyrA gene could be detected in the antibiotic-resistant campylobacter while, Campylobacter spp. were negative for the tetO gene.
The total percentage of bioactive compounds value of rosemary oil sample (A) was 8.79%, represented by Methyl 6,9,12,15-octadecatetraenoate (0.303%), 6-Chloroctanoic acid, chloromethyl ester (0.708%), 7,10,13-Hexadecatrienoic acid, methyl ester (0.46%), 7,10-Hexadecadienoic acid, methyl ester (0.340%), 91215Octadecatrienoic acid methyl ester (2.31%), 9,12-Octadecadienoic acid (Z,Z)-, methyl ester (0.764%), 9-Hexadecenoic acid, methyl ester (0.24%), Hexadecanoic acid methyl ester (1.217%), Methyl 4,7,10,13-hexadecatetraenoate (1.740%) and Octadecanoic acid methyl ester (0.70%). However, for
REO samples (B) the bioactive compounds was 63.02% including, 1,8-Cineole (14.732%), 14-.BETA.-PREGNA (8.810%), Alyl Cyclohexyl carbonate (4.304%), Bicyclo[3.1.1]heptane, 6,6-dimethyl-2- methylene-, (1S)- (2.259%), Camphene (0.824%), Decane (0.243%), D-Limonene (1.349%) 1-Bromo-Dodecane (0.268%), Heptadecyl 2,2,3,3,4,4,4-heptafluorobutanoate (4.801%), n-Undecane (0.506%), Pentafluoropropionic acid, tetradecyl ester (1.715%), Tetradecane (7.810%).
The result of MIC was not satisfactory because of the turbidity caused by the essential oil, so we relied on the result of MBC. There was inhibition of 2 strains (C. coli and C. jejuni), the first one at concentrations of 1, 1.5 and 2%, while, the other strain was inhibited at concentrations of 1.5 and 2%.
Studying the effect of REO on the campylobacter fecal shedding indicated that after 2 weeks of treatment there was a reduction in the total campylobacter count (3.78 log 10 CFU/ gram fecal matter) in campylobacter REO-treated mice compared with the campylobacter-infected mice.
The expression of IL-5, IL-6, IL-13, IL-17, TNF-α and IFN- γ in the small and large intestine lysates in campylobacter -infected mice was significantly increased compared to control mice. However, treatment with REO significantly reduced expression of these cytokines in both small and large intestine in Camp. + REO-treated mice compared with the Camp.-infected mice.
The histological changes in small intestine sections stained by H&E stain revealed that campylobacter infection led to histological alterations of normal ileum architecture and the expression of hyperplasia increased significantly in Camp.-infected mice compared to control mice. Treatment with REO partially restored the normal histological structures of Ileum and reduced the expression of hyperplasia Camp. + REO-treated mice compared with the Camp. -infected mice.
Sections of small intestine sections stained by Alciens blue stain revealed that campylobacter infection led to decrease in in goblet cells which affect absorption and mucous secretion. However, treatment with REO showing more or less normal distribution of goblet cells in lamina probria and mucus gland.
Small intestine sections stained by Masson’s trichrome stain and Sirrus red stain revealed that campylobacter infection led to obvious apparent collagen deposition that indicated fibrosis in small intestine. Treatment with REO displayed decreased collagen fibers in mucosa and submucosa.
Large intestine sections stained by H&E stain revealed that campylobacter infection led to histological alterations of normal colon architecture, including pyro-apoptosis, necrosis of cryptic region, stratification of epithelium that affect the normal absorption and secretion of intestine. Treatment with REO restored the normal histological structures of colon including.
Large intestine sections stained by Masson’s trichrome stain and Sirrus red stain revealed that campylobacter infection led to obvious collagen deposition apparent that indicated fibrosis in large intestine. Treatment with REO significantly reduced expression of Collagen fiber in colon in the Camp. + REO-treated mice compared with the Camp. -infected mice and the expression of collagen fiber was nearly similar to that in the control group.
Peyer’s patches sections stained by H&E stain revealed that campylobacter infection led to alterations of patches of lymphoid tissue. Treatment with REO decrease of collagen fibers of Peyer’s patches sections stained by Masson’s trichrome and Sirrus red stain.
Spleen sections stained with H&E revealed that campylobacter infection led to increased thickness of capsule &focal area of lymphocytic necrosis and depletion of lymphocyte in white pulp and red pulp with sidrophages and abundant esinophilic cytoplasm with extra-capsular mass. Treatment with REO improved restoration of histological structures of white pulp & red pulp which is composed of splenic cords and sinusoids and enhanced an obvious repair of spleen architecture.
There was a significant increase in expression of collagen fiber in spleen sections stained by Masson’s trichrome and Sirrus red stain in Camp. -infected mice compared to the control mice. Treatment with REO significantly reduced expression of collagen fiber in spleen in Camp. + REO-treated mice compared with the Camp. -infected mice and that expression was similar to that found in the control group.
Spleen sections stained by anti-PEACAM-1antibody showing massive diffuse progressive histological alterations with strong & diffuse immunoreaction in periarterial lymphatic sheath of white pulp indicating neovascularization of the spleen. Treatment with REO significantly reduced expression of PEACAM-1antibody in spleen in the Camp. + REO-treated mice compared with the Camp. -infected mice with nearly similar expression to that found in the control group.
Spleen sections stained by anti- HSP70 antibody showing massive diffuse progressive histological alterations with strong & diffuse brown granules in periarterial lymphatic sheath of white pulp and diffuse throughout the red pulp compared to control group which indicate inflammation of the spleen. Treatment with REO significantly reduced the expression of antibody HSP70 in Camp. + REO-treated mice compared with the camp. -infected mice and that expression was similar to the level found in the control group.
Spleen sections stained with anti-C-MYC antibody showing massive diffuse progressive histological alterations with strong & diffuse brown granules in periarterial lymphatic sheath of white pulp and diffuse throughout the red pulp compared to control group which indicate proliferation of the spleen. Treatment with REO significantly reduced expression of C-MYC antibody in Camp. + REO-treated mice compared with the Camp. -infected mice and the expression was similar to that found in the control group.
This study showed that campylobacter infection led to massive diffuse progressive histological alterations, dilated congested central vein and obviously increase necrotic area of the liver lead to depletion of hepatocytes and loss of normal arrangement of hepatocytes compared to control group. Dilatation of central vein as result of campylobacter invasion caused liver injury led to increase blood supply through dilated central vein which impaired T Cell (kuffer cell), adaptive immune response. Treatment with REO improved restoration of histological structures of liver, enhanced an obvious repair of liver architecture and improved immune response. Moreover,
a significant increase in the expression of collagen fiber in liver sections stained by Masson’s trichrome and Sirrus red stain in Camp. -infected mice compared with the control mice were confirmed. However, treatment with REO significantly reduced expression of collagen fiber in Camp.+ REO-treated mice compared with the Camp.-infected mice.
Liver sections stained by anti-PEACAM-1antibody showing massive diffuse progressive histological alterations, dilated congested central vein with strong & diffuse immunoreaction in central vein and between hepatocytes compared with the control group which indicate neovascularization of liver. Treatment with REO significantly reduced expression of PEACAM-1antibody in liver in Camp. + REO-treated mice compared with the Camp. -infected mice and similar that found in control group.
Liver sections stained by anti- HSP70 antibody showing massive diffuse progressive histological alterations, dilated congested central vein with strong and diffuse brown granules in central vein and between hepatocytes which indicate inflammation of liver cells. Nevertheless, treatment with REO significantly reduced the expression of HSP70 antibody in Camp. + REO-treated mice compared with the Camp. -infected mice.
Liver sections stained with anti-C-MYC antibody showing massive diffuse progressive histological alterations, dilated central vein with strong & diffuse immunoreaction in central vein and between hepatocytes which indicate proliferation of hepatocytes compared. However, treatment with REO significantly reduced the expression of C-MYC antibody in Camp. + REO-treated mice compared with the Camp. -infected mice.
It can be concluded that during campylobacter infection, the pathogen triggers an innate inflammatory reaction throughout increased production of and TNF-α and IFN- γ initiates a TH1-polarized adaptive immune response, indicating acute intestinal inflammation. However, chronic intestinal inflammation characterized by a TH2 atypical immune response, alongside the classical proinflammatory cytokines, such as TNF-α and IFN- γ, in the pathogenesis of UC, a complex network in which the TH2 cytokines through up regulation of IL-5 & IL-13. Furthermore, campylobacter stimulated secretion of the pro-inflammatory cytokine IL-6. IL-6 is also thought to provide a protective effect on epithelial barrier maintenance within the gut yet has been associated with the severity of inflammatory bowel diseases and increased up regulation of the pro-inflammatory cytokine IL-17. The inflammatory signals caused by campylobacter infection extended from primary sites of colonization (small & large intestine) to gut- associated lymphoid tissue (GALT) “Peyer’s patches”, spleen and liver. Alterations in the histological architectures including hyperplasia, decrease in goblet cells small intestine, pyro-apoptosis of colon, focal area of lymphocytic necrosis with decrease population of lymphocytes & increase the spindle cell population in Peyer’s patches, increased thickness of capsule &focal area of lymphocytic necrosis and depletion of lymphocyte in white pulp and red pulp with sidrophages and megakerocyte in spleen, dilated congested central vein, obviously increase necrotic area , loss of normal arrangement of hepatocytes; as its arrangement in circles instead of cord, increase number of Kuffer cell of the liver. Campylobacter infection caused up-regulation the level of PEACAM-1, C-MYC and HSP70 which is an indicator of neovascularization, proliferation and inflammation in liver and spleen, respectively.
Rosemary essential oil is a natural antioxidant product that plays vital protective roles during Campylobacter infection by the following mechanisms: repairing the histological architectures of small, large intestine, peyer’s patches, liver, spleen and lymph node. Decreasing the level of pro-inflammatory cytokines Th1,Th2 and Th3 in small and large intestine, Decreasing the level of collagen fibers small, large intestine, peyer’s patches, liver, spleen and lymph node. decreasing the level of PEACAM-1 which is an indicator of neovascularization in liver and spleen, reduction the level of C-MYC which is an indicator of proliferation in liver and spleen, decreasing the level of HSP70 which is an indicator of inflammation & injury in liver and spleen .
The study recommends rosemary essential oil for its health benefits as anti-campylobacter activity in vitro and in vivo. Also, rosemary essential oil acts as anti-inflammatory, anti-fibrotic and anti-proliferative agent during campylobacter infection. Rosemary essential oil is candidate for intervention strategies against thermophilic campylobacter infection and post-infectious sequelae, which result from the permissive barrier defect along the leaky gut. Thus, rosemary essential oil could be judged as a kind of potential drug with rather antimicrobial activities.As well as, rosemary essential oil is a cheap herb, an available and not toxic. So it could be applied in food preservation & industry.