Journal of Babylon University/Pure and Applied Sciences/ No.(8)/ Vol.(21): 2013

Pathogenesis of in mice

Hayder S.Obayes College of Vet. Medicine Al-Qasim Green University Frias GAbd college of science Babylon university Abstract: Providencia rettgeri strains were isolated from urinary tract infections (UTIs) patients and identified according to morphological and biochemical tests, furthermore, the identification was confirmed using the Hi 25 system. The crude lipopolysaccharide (LPS) was extracted from P.rettgeri isolate by sonication with ethylene diamine tetraacetic acid (EDTA) and then with enzymic digestion, then partially purified by an ion exchange chromatography .It have been 7 revealed that the lethal dose 50% (LD50) of P.rettgeri was about 5.62 × 10 cell/mouse . The microscopic examination of stained tissue sections of organs obtained from mice injected with sub lethal doses of P.rettgeri and LPS, showed sever changes in kidney and bladder induced by bacterial suspension than LPS, while sever changes were shown in spleen ,liver , and intestine caused by LPS compared with control animals. In general, these histopathological changes included the congestion, hemorrhage, migration and infiltration of inflammatory cells, tissue necrosis and oedema but in spleen there were hypertrophy of white bulb and congestion of red bulb . Key words: Pathogenesis , LPS, Providencia, histopathogenesis,mice.

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ﻋزﻟـت ﺟرﺛوﻣـﺔ Providencia rettgeri ﻣـن اﻟﻣرﺿـﻰ اﻟﻣـﺻﺎﺑﯾن ﺑﺎﻟﺗﻬـﺎب اﻟﻣـﺳﺎﻟك اﻟﺑوﻟﯾـﺔ وﺷﺧـﺻت ﻋﻠـﻰ وﻓـق اﻻﺧﺗﺑـﺎرات اﻟﻣظﻬرﯾـﺔ واﻟﻛﯾﻣوﺣﯾوﯾﺔ وﺑﺎﺳﺗﻌﻣﺎل ﻧظﺎم ﯾﺣﺗوي ﻋﻠﻰ 25 اﺧﺗﺑﺎر ﻣﺟﻬز ﻣن ﺷرﻛﺔ Himedia ﺧﺎص ﺑﺎﻟﻌﺎﺋﻠﺔ اﻟﻣﻌوﯾـﺔ (Hi25 E system) .ﺗم اﺳﺗﺧﻼص ﻋدﯾد اﻟﺳﻛرﯾد أﻟﺷﺣﻣﻲ (LPS) ﻣن ﻋزﻟﺔ ﻟﺟرﺛوﻣﺔ Providencia rettgeri ﺑطرﯾﻘـﺔ اﻟﺗﻛـﺳﯾر ﺑـﺎﻷﻣواج ﻓـوق اﻟـﺻوﺗﯾﺔ ﻣـﻊ اﻻﺛﯾﻠـﯾن- ﺛﻧـﺎﺋﻲ اﻷﻣـﯾن- ﺛﻼﺛـﻲ ﺣـﺎﻣض أﻟﺧﻠﯾـك (EDTA) وﻣـن ﺛـم اﻟﻬـﺿم اﻹﻧزﯾﻣـﻲ ،و ﺗﻣـت ﺗﻧﻘﯾﺗـﻪ ﺟزﺋﯾـﺎ ًﺑﺎﺳـﺗﺧدام ﻛروﻣـﺎﺗوﻏراف 7 اﻟﺗﺑﺎدل أﻻﯾوﻧﻲ ، ﻛﻣـﺎ ﺣـددت اﻟﺟرﻋـﺔ اﻟﻣﻬﻠﻛـﺔ اﻟﻧـﺻﻔﯾﺔ ﻟﻠﻔﺋـران LD50 ﻟﺟرﺛوﻣـﺔ Providencia rettgeri وﻛﺎﻧـت ﺣـواﻟﻲ 10 × 5.62 ﺧﻠﯾــﺔ/ ﻟﻛــل ﻓــﺄرة.وﺟــد ﻣــن ﺧــﻼل دراﺳــﺔ اﻟﻣﻘــﺎطﻊ اﻟﻧــﺳﺟﯾﺔ ﻷﻋــﺿﺎء اﻟﻔﺋــران اﻟﻣﺣﻘوﻧــﺔ ﺑــﺎﻟﺗراﻛﯾز ﺗﺣــت اﻟﻣﻣﯾﺗــﺔ (Sublethal) ﻣــن ﻛــل ﻣــن اﻟﻌـﺎﻟق اﻟﺟرﺛــوﻣﻲ وﻣــﺳﺗﺧﻠص ﻋدﯾــد اﻟــﺳﻛرﯾد أﻟــﺷﺣﻣﻲ اﻟﻣﻧﻘــﻰ، إن اﻟﻌــﺎﻟق اﻟﺟرﺛــوﻣﻲ ﻗــد اﺣــدث ﺗﻐﯾﯾــرات ﺣــﺎدة ﻓــﻲ اﻟﻛﻠﯾــﺔ واﻟﻣﺛﺎﻧــﺔ ﻣﻘﺎرﻧــﺔ ﺑﻌدﯾــد اﻟــﺳﻛرﯾد أﻟــﺷﺣﻣﻲ ،ﻓــﻲ ﺣــﯾن أن ﻋدﯾــد اﻟــﺳﻛرﯾد أﻟــﺷﺣﻣﻲ ﻗــد ﺳــﺑب ﺗﻐﯾــرات ﺣــﺎدة ﻓــﻲ أﻧــﺳﺟﺔ اﻟﻛﺑــد واﻟطﺣــﺎل واﻷﻣﻌــﺎء ﻣﻘﺎرﻧــﺔ ﻣــﻊ ﺣﯾواﻧـــﺎت اﻟـــﺳﯾطرة.ﺗﻣﺛﻠـــت اﻟﺗﻐﯾـــرات اﻟﻣرﺿـــﯾﺔ اﻟﻧـــﺳﺟﯾﺔ ﺑـــﺻورة ﻋﺎﻣـــﺔ ﺑﺣـــدوث اﻻﺣﺗﻘـــﺎن اﻟـــدﻣوي واﻟﻧـــزف اﻟـــدﻣوي وﻫﺟـــرة وارﺗـــﺷﺎح اﻟﺧﻼﯾـــﺎ اﻻﻟﺗﻬﺎﺑﯾﺔ وﺗﻧﺧر اﻟﺧﻼﯾﺎ ووﺟود اﻟوذﻣﺔ، أﻣﺎ ﻓﻲ اﻟطﺣﺎل ﻓﻘد ﻛﺎن ﻫﻧﺎﻟك ﺗﺿﺧم ﻓﻲ اﻟﻠب اﻷﺑﯾض واﺣﺗﻘﺎن ﻓﻲ اﻟﻠب اﻷﺣﻣر. Introduction: The genus Providencia consists of eight species : P. alcalifaciens, P.heimbachae, P.rettgeri, P. rustigianii, P. stuartii , P. vermicola, P. sneebia and P.burhodogranariea (Galac and Lazzaro,2011 ; OʼHara et al.,2000). The urinary tract of the compromised or catheterized patient is the most common site of P. rettgeri and P. stuartii infections, the rise in importance of these two species is associated with their tendency to cause nosocomial infections and with their marked resistance to numerous antibiotics (Jones and Mobley,1987; Shiroto et al.,2005). P. rettgeri is one of Providencia species that is known to cause gastrointestinal tract infections (Müller,1986).Or traveller's diarrhea ( Yoh et al.,2005 ).Less common infectious syndromes, especially in immunocompromised patients, include surgical site infection, soft tissue infection, burn site infection, (particularly ventilator-associated), intravascular device infection, ocular infection,

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meningitis, osteomyelitis ,sepsis and intra-abdominal infection (Ladds et al.,1996; Pinto et al.,1999; Koreishi et al.,2006 ; Zuluaga et al.,2006 ; Russo and Johnson,2008 ; Cho et al.,2010) . Most P. rettgeri strains exhibit pathogenic properties similar to those of P. stuartii. P. rettgeri UTIs in catheterized and otherwise compromised patients are also difficult to treat owing to multiple antibiotic resistance (Manos and Belas ,2006).Virulence factors that were responsible for the pathogenicity of Providencia spp. that have been investigated include LPS, cellular adherence or biofilm formation, the production of fimbriae, sidrophores production, ß-lactamase production, and urease production (OʼHara et al.,2000). Endotoxins, also called lipopolysaccharides (LPS) is the major component of the outer membrane of the cell wall of Gram-negative , which forms its outer leaflet linked to the phospholipids inner leaflet by hydrophobic interactions,the LPS is considered as a virulence factor of , including Providencia (Penner et al.,1992).The lipopolysaccharide (LPS), as in other members of the family Enterobacteriaceae , consists of three domains, an endotoxic glycolipid (lipid A), an O-polysaccharide (O-PS) chain or O-antigen, and an intervening core oligosaccharide (OS) region ,the O-antigen is the major surface antigen, and its serological O specificity, in contrast to that of other Gram-negative bacteria (Aquilini et al.,2010). The lipid A is the most conserved part of endotoxin and is responsible for most of the biological activities of endotoxin, i.e. its toxicity (Vaara and Nurminen ,1999). Bacterial lipopolysaccharide play a critical role in the initiation of the proinflammatory events that contribute to the pathogenesis of the sepsis and the pathophysiologic mechanism(s) responsible for this illness are thought to result from the noncytotoxic interaction of LPS with mammalian host inflammatory mediator cells monocytes/macrophages constitutes a major mechanism responsible for innate immune response to Gram-negative infection(Luchi and Morrison ,2000). Locally little studies about the Providencia especially the virulence determinant like LPS .The aims of this study were partial purification of LPS and study of its pathological and histopathological defects in mice.

Materials and method: Bacterial strain: P. rettgeri strains were isolated from UTIs by culturing urine sample on MacConkey agar ,after 24 hr of incubation at 37 C, the suspicious colonies which were non lactose fermentative and were pale on MacConkey agar, traditional biochemical tests were used for final identification of bacterial isolates,and the confirmed identifications to species level were also carried out by using Hi25 E system (Enterobacteriacea identification system, Himedia,India) (Macfaddin ,2000). Isolation of LPS: An isolate of P. rettgeri from a patient with UTI were cultivated for extraction. Endotoxin extraction was carried out according to method of Mirzaei et al., (2011) with some modification as follow: P. rettgei strain was grown in brain heart infusion broth flask and then incubated at 37˚C for 24hr in shaker incubator (150 rpm). After incubation period suspension cultures were transferred to the test tubes and centrifuged for 30 min in 2500 to 3000 rpm. Then, supernatant was discarded and 2 ml of ethyl alcohol 95% was added to the test tube possessing sediment and mixed well by vortex till the bacteria sediment and alcohol were mixed properly and

- ٢٧٨٦ - Journal of Babylon University/Pure and Applied Sciences/ No.(8)/ Vol.(21): 2013 sediment was separated from the bottom of the test tube. After mixing, the test tube was centrifuged for 10 min in 2000 rpm. The supernatant was discarded and sediment was washed with 2 ml alcohol and again vortexed. The procedure was repeated three times as explained previously. Finally, the sediment was mixed with alcohol and centrifuged for 10 min in 2000 rpm. The supernatant was discarded and sediment was dried by placing the tube under the hood till the rest of alcohol was evaporated. The dried bacterium was re-suspended by 1 ml EDTA 10% and then it was sonicated for 2 minutes . 1 ml of saturated methanol/chloroform (1: 2 ratios) was added to bacterium- EDTA solution. The tube lid was covered by paraffin and shaked for 2 h ,following 10 min centrifuging in 2000 rpm. Three layers were formed; methanol, left biomass including cell lysate which was discarded and chloroform top to button respectively. Chloroform and methanol layers were separated and poured in new tubes and permit to evaporate completely. The dried pellet was LPS bacterium.

Purification of LPS by an ion exchange chromatography: This was carried out according to methods of Mannel and Mayer(1978) , Luchi and Morrison (2000) and Perdomo and Montero (2006) with some modification as follow: The gel was prepared by homogenizing it with Tris-HCl (pH=8) then settled and excess of buffer was removed. This step was repeated several times The gel was carefully poured into di-ethyl amino ethyl cellulose (DEAE-cellulose) column (20×2) cm (Pharmacia) down the wall to avoid accumulation of air bubble into it, the gel was left for few time to settle, the column outlet was opened .Later the packing gel was equilibrated with Tris buffer pH 8 (Mannel and Mayer,1978) . Nucleic acids were removed by reconstitution of the LPS enriched extracts to 10 mg/ml in Tris buffer (pH 8.0), with MgSO4 and CaCl2 and digestion with RNase (0.4 mg/ml) and DNase (20 mg/ml) by incubation at 37°C overnight. Contaminating protein was then removed by the addition of proteinase K (20 mg/ml) Tris (pH 8.0), followed by heating at 60°C for 1 hour and then incubation overnight at 37°C (Luchi and Morrison ,2000). Enzyme-digested LPS samples dissolved in Tris buffer were applied to the column and were eluted with a buffer with NaCl (0.2M) using a flow rate of 9ml/hr , 1ml for each fraction was collected by using separated test tube for each fraction and the absorbencies were measured at 206nm by spectrometer, and the peak fractions that showed endotoxin activity were collected and concentrated by dialysis against deionized D.W. and alcohol precipitation and finally the pellet was air dried (Perdomo and Montero ,2006). Bioassay of endotoxin (Pyrogenicity): This was carried out according to method of Rezania et al.(2011) with some modification as follow: An amount of 0.03 mg of LPS was suspended in 10 ml normal saline, from this suspension 0.1 ml was injected within foot pad of three white rats. Rectal temperatures were measured with indwelling rectal thermostats and corded before injection and 4 hr after LPS administration. three rats were injected with sterile PBS as control. The rise in temperature was recorded as positive results. Determination of LD50 for Bacterial Suspension: LD50 for bacterial suspension was determined by using 25 white mice (20-25) g, which divided into five groups each group consist of five mice. Each group injected intraperitoneally in one of the following bacterial dilution (105,106, 107,108, and109) cell/ml.and the injected volume was 1ml for each mice. After 5 days LD50 was determined according to Reed and Muench (1938).

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Evaluation pathological effects of LPS: Effect of LPS were determined by using 25 white mice each (20-25) g, which divided into five groups each group consist of five mice .Each group was injected intraperitoneally in one of the following concentration (125,150, 175, 200 and 250) µg/ml which prepared in PBS solution and the injected volume was 0.5ml for each mice. Another group of five mice injected with 0.5ml of PBS as negative control. After two days, the killed mice were tested histopathologically. Histopathological Study : Mice injected with (108 cell/ml of bacterial suspension, 125 μg/0.5ml and PBS as negative control) were killed after two days, the animals were sacrificed and specimens were taken from the kidney, liver, spleen, intestine and bladder .The tissues sections were prepared according to Humason (1972).The histopathological changes were observed by Dr. Naema Al-Jabori under the magnification power 100 X of light microscope. Result: Endotoxin Bioassay: The bioassay of LPS was investigated by using three rats. These rats were injected in foot pad with 0.1 ml LPS (300 µg/ml).The parameters which was used for assessing LPS include : skin thickness , body temperature , color and edema. The difference in thickness of tested rats was between 5-6 mm compared with control rats which was between 1-1.4 mm , color change from pale color into red (erythema) was showed in all tested rats and was associated with edema .Temperature rise to more than 1˚C in injected rats and its ranged between 37-37.5 ˚C compared with temperature degrees of rats before injection which were ranging between 35.5-36 ˚C. Pathogenesis of bacterium: Median lethal dose (LD 50) for live bacteria: LD50 dose was evaluated through intraperitoneal route in balb/c mice. Five serial concentrations three mice for each dose of live bacteria were used and the results showed that the LD50 was 5.62 × 107 cell/mouse (Table 1 and figure 1). Table ( 1 ): LD50 value of live P.rettgeri bacteria.

Bacterial Mortality Died Survived Died Survived Mortality ratio Percent% con.(cell/ml) Ratio Accumulation accumulation D

(D) (S) D+S D× 100

D+S

109 3/3 3 0 3 0 3/3 100

108 2/3 2 1 2 1 2/3 66.66

107 0/3 0 3 0 4 0/4 0

106 0/3 0 3 0 7 0/7 0

105 0/3 0 3 0 10 0/10 0

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Proportional 50 - Mortality below 50 percent = distance Mortality above 50 percent – Mortality below 50 percent 50 – 0 = = 0.75 66.66 - 0

Negative Negative logarithm of Proportional logarithm of = + concentration below 50 percent distance LD50 titer mortality = 0.75 + 7

7.75 LD = 10 = =100.7×107 = 5.62 × 107 cell/mouse 50

12

10

8 e u

l a v

n 6 o i t Survive a l

a Deid m 4 u c

c A 2

0 10^9 10^8 10^7 10^6 10^5 Bacterial concentration (cell/ml)

Figure ( 1 ): LD50 value of live P.rettgeri bacteria.

Histopathological changes: The microscopic examination of stained tissue sections of organs obtained from dead mice after 24-48 hr injected with sublethal doses of P.rettgeri and LPS, showed sever changes in kidney and bladder induced by bacterial suspension than LPS, while sever changes were shown in spleen ,liver , and intestine caused by LPS compared with control animals that injected with PBS as negative controls ( figures 2- A to 2-R).

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Figure (2-A) :Kidney of control mouse: 1: Normal glomerulus showing glomerulus lobulation (a) , Bowman space (b)and Bowman capsule (c). 2: Renal tubules.(400X, H&E stain)

Figure (2-B) :Kidney of mouse treated with live bacteria:1:Glomerulus showing hypercellularity due to infiltration with PMN and deposition of red fibrinoid material within glomerulus capillaries,2:Narrowing of Bowman space with deposition of fibrinoid substance within it (F).,3:Focal tubular necrosis. (400X, H&E stain).

Figure (2-C): Kidney of mouse treated with bacterial LPS:1.Glomerulus shows normal cellularity no infiltration with PMN. But there is deposition of red fibrinoid material within glomerulus capillaries.2.Narrowing of Bowman space with depositionof fibrinoid substancewithin it (F).3.Sever tubular necrosis involving all renal tubules.(400X, H&E stain).

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Figure (2-D):Bladder of control mouse:L:Lumen ,1:Surface transitional epithelium. 2: Lamina properia.3:Submucosa.4: Muscular layer.(400X, H&E stain).

Figure (2-E):Bladder of mouse treated with live bacteria: L: Lumen. 1: Surface epithelium showing sever necrosis. 2: Lamina proper showing edema and infiltration with polymorph nuclear leukocytes. 3:Sub mucosa shows edema and infiltration with polymorph nuclear leukocytes.4:Muscular layer shows edema only(400X,H&E stain).

Figure ( 2-F) :Bladder of mouse treated with bacterial LPS: L: Lumen. 1:Surface epithelium showing mild degree of necrosis. 2: Lamina proper showing edema only. 3: Sub mucosa shows edema only 4. Muscular layer shows mild degree of edema only. (400X, H&E stain).

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Figure (2-G): liver of control mouse:1:Central vein. 2: Normal hepatocytes forming cellular columns separated by sinusoids. (100X, H&E stain).

Figure (2-H):Liver of mouse given live bacteria:Focal liver necrosis reflected as absence of normal liver architecture (Disarraying) and hyperplasia of Kupffers cells (arrow). (100X, H&E stain).

Figure (2-I) :Liver of mouse given bacterial LPS: 1: focliver necrosis reflected by absence of normal liver architecture (Disarraying).2: Congestion of portal vein (PV)and central veins(CV). (100X, H&E stain).

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Figure (2-J) :Spleen of control mouse architecture:White bulb and central artery (arrow1) and normal red bulb (arrow2). (40X, H&E stain).

Figure (2-K) :Spleen of mouse given live bacteria:Congestion of the red bulb with PMN infiltration (arrows). (400X, H&E stain).

Figure (2-L) :Spleen of mouse given bacterial LPS: Congestion of red bulb(arrows). (400X, H&E stain).

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Figure (2-M): Small intestine of control mouse: Finger like projections intothe lumen( arrow L) representing intestinal villi (arrow V) covered with normal intestinal simple columnar epithelium with Goblet cells. (100X, H&Estain).

L V

Figure (2-N): Small intestine of mouse given live bacteria:There is partial necrosis of the intestinal villi with complete necrosis of surface epithelium; L:lumen, V: villi. (100X, H&E stain).

L

V

Figure (2-O):Small intestine of mouse given bacterial LPS: There is severe and widespread necrosis of the intestinal villi with complete necrosis of surface epithelium; L: lumen, V: villi.(100X, H&E stain).

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Figure (2-P) :Large intestine of control mouse :L: lumen, Nc: normal colonic crypts. (100X, H&E stain).

Figure (2-Q):Large intestine of mouse given live bacteria: There is partial necrosis of colonic crypts, L: lumen, N: necrosis, Nc: normal crypts.(100X, H&E stain).

Figure (2-R) Large intestine of mouse given bacterial LPS :There is complete necrosis of the mucosa (N), L: lumen.(100X, H&E stain).

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Discussion: The bioassay of LPS was investigated by using three rats. These rats were injected in foot pad with 0.1 ml LPS (300 µg/ml).The parameters which was used for assessing LPS include : skin thickness , body temperature , color and edema . The difference in thickness of tested rats was between 5-6 mm .Temperature rise to more than 1˚C in injected rats and its ranged between 37-37.5 ˚C. This result was compatible with Al-Salamy (2005) study who found that ,the difference in thickness of tested rats was between 3.7-4.5mm compared with control rats that was between 1- 1.6 mm and temperature rise to more than 1C˚in injected rats and its ranged between 38-39C˚. In other study LPS extract of Salmonella typhimurium revealed increasing of rat body temperatures (1 to 2°C) in both 100 and 150 µg/kg dosages after 2 h treatment (Mirzaei et al.,2011). Sirakarn et al. (2010) suggested that when lipopolysaccharide (LPS, 50 µg/kg) injected intra-muscularly into male Wistar rats ,the maximum increase in rectal temperature was reached at 4 h (1.84°C) giving the maximum mean rectal temperature of 38.44°C after which there was a decrease. The acute phase response, as evoked by lipopolysaccharide (LPS), is characterized by fever (Koga et al.,1994;Schiffelholz et al.,2001 ). IL-1ß is responsible for most of the systemic activity attributed to IL-1, including fever, activation of phagocytes, and production of acute phase proteins (Kandathil et al.,2005). Infusion of recombinant IL-2 into humans induces fever that starts 3–4 h after injection ( Mier et al.,1988). Interleukin-6 is involved in modulating aspects of both innate and adaptive immunity via its ability to induce fever, B-cell Differentiation and corresponding Immunoglobulin production, T-cell activation, and enhanced proinflammatory responses of neutrophils (Biffl et al., 1996). The LD50 of P.rettgeri was 5.62 × 10^7 cell/mouse (Table 1and figure 1).The mice were observed for 1 week. Deaths among mice usually occurred within 48 h. Previous study recorded different value for LD50 of P.rettgeri suspension which were ranging from 1.5-4.4 x10^4 depended on strain (Penner and Whiteley,1978).

The difference in the LD50 values of bacterial suspension may be due to different of strains used and the potential virulence factors like outer membrane proteins, LPS and other virulence factors . Another virulence factor like emetic toxin might be produced by P. rettgeri strains.Furthermore, most of the P. rettgeri strains showed invasive activity in Caco-2 cells (Yoh et al.,2005). The death of mice when injected with high concentration of P.rettgeri may due to LPS because endotoxin elicits a wide variety of pathophysiological effects, such as endotoxin shock, tissue injury, and death (Ogikubo et al.,2004 ). which induce release of large amount of inflammetry cytokines like IL-1 and α-TNF which causes tissue damage , endotoximia and finally shock and death( Luchi and Morrison,2000). The microscopic examination of stained tissue sections showed that the histopathological defects of kidney of mice injected with live bacteria included infiltration of PMN into Glomerulus and deposition of red fibrinoid material within glomerulus capillaries. Narrowing of Bowman space with deposition of fibrinoid substance within it and tubular necrosis.these defects were similar to LPS effect except that there is no infilteration of PMN into Glomerulus. The bladder histopathological defects of bacteria suspension were sever necrosis. Lamina proper showing edema and infiltration with polymorph leukocytes. Sub mucosa shows edema and infiltration with polymorph leukocytes. Muscular layer shows edema only, while LPS defects were less degree than bacterial suspension and without infilteration of PMN. This result was converged with study of Lloyd et

- ٢٧٩٦ - Journal of Babylon University/Pure and Applied Sciences/ No.(8)/ Vol.(21): 2013 al.,(2009) who demonstrated that focal infiltration of inflammatory cells,in the pelvis of kidney and moderate, widespread edema and minimal inflammatory infiltrate in bladder. Recruitment of neutrophils, shown to be essential for clearance of bacteria from the bladder and kidneys (Haraoka et al.,1999).The presence of pyelonephritis, or inflammation of the renal pelvis, was determined based upon the number of inflammatory foci (neutrophils) present in the renal pelvis, and the severity of inflammation was determined by the number of neutrophils in the tissues directly surrounding the pelvis (peripelvic region) (Lloyd et al.,2009). The liver histopathological defects of bacterial suspension include focal liver necrosis , absence of normal liver architecture hyperplasia of Kupffer cells and Cytoplasmic vaculation . in addition to above LPS causes congestion of portal vein (PV)and central veins(CV). The liver histopathological defects result from transfer of bacteria from bile sac to liver (Ishikawa et al., (1999).while LPS causes liver sinusoids damage as a result of super oxide free radicals released by LPS activation (Yokoyama et al., 1998). The histopathological defects of bacteria in spleen included congestion of the red bulb with PMN infiltration while LPS defects were white pulp hypertrophy and congestion of red bulb. The enlargement of the spleen may be accompanied by increased cellular proliferation or mitogenic activity of LPS for B-cells (Girard et al.,1981 ; Swierzko et al.,2000). The defects caused by LPS were severe and widespread necrosis of the small intestinal villi with complete necrosis of surface epithelium ,in large intestine there was a complete necrosis of the mucosa ,while incomplete necrosis of villi and mucosa induced by live bacteria. These results agree with other studies about gram negative bacteria such as AbdalHussain (2006) on Plesiomonas shigelloides who pointed that the defect caused by this bacterial suspension were necrosis of epithelium ,infiltration of inflammatory cells , hemorrhage and Oedema. The histopathological defects identified by Al-Muslemawi (2007) for C. freundii cells and LPS partially purified from it, in liver, spleen , lung and kidney which included the congestion, hemorrhage, migration and infiltration of inflammatory cells, tissue necrosis and oedema but in spleen there were hyperplasia in white pulp and increase of Megkaryocytes number.This may due to enterotoxins which cause changes in surface epithelium of intestines (Schmitt et al., 1999). Histology of adult rabbit small intestinal loops inoculated with P. alcalifaciens revealed bacterial attachment to, penetration of, and microulcer formation on the surface epithelium and hyperemia, edema, and polymorphonuclear cell infiltration of lamina propria (Albert et al.,1992).

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