Macroscopical and Microscopical Changes of Body Organs after E. coli O157:H7 Inoculation in Puppies

ةينولوقلا ايشيريشلااب ةباصلاا دعب مسجلا ءاضعلا ةيرهجملاو ةينايعلا تاريغتلا O157:H7 ءارجلا يف


Introduction
Escherichia coli O157:H7 is a closely an etiology of diarrhea in dogs and puppies (1,2,3). dogs, bats and avian species ( 4,5,6). Transmission pathways of STEC were the direct contact between humans and pet animals as well as fecal and urine contamination (7,8). Diarrhoeal and nondiarrhoeal dogs of all ages may be a possible human-borne source of the STEC O157:H7 multidrug resistant (9). E. coli O157: H7 sources was obtained and analyzed in feedlots and farms in the northwest of the United States, 65 dog samples and 33 cat samples for the presence of E. ColiO157: H7, Results were negative and 3,1% tested positive for all cat samples. The possible source of the bacterium for the dogs on these farms may be the cattle via the rivers (10). E. coli has been isolated from environment (11), While (12) diagnosed the hemolytic uremic syndrome in a dog with signs of acute onset of anorexia, vomiting and hemorrhagic diarrhea, the results show that these signs were typical to the signs caused by shiga toxin producing E. coli (STEC).
Shiga toxin (Stx) producing E. coli (STEC) is responsible for bloody diarrhea (hemorrhagic colitis) and the hemolytic uremic syndrome (HUS) (13). Hemolytic uremic syndrome caused by STEC is characterized by low platelet count, hemolytic anemia, and kidney failure (14,15) . Escherichia coli O157:H7 were isolated from dogs fecal sample which has class 1 integrons and they conclude that the presence of class 1 integrons serving as reservoirs of antibiotic resistance genes (16). (17) isolated E. coli O157 with different H types from dogs, and their results showed That dogs can harbor pathogenic E. Coli O157 that do not correspond to Shiga toxinproducing strains and should not be underestimated. Pets can act as a reservoirs of STEC strains, a study of (18) was conducted to examine nine strains belonging to several serotypes which previously recovered from cats or dogs. The findings showed that animals could play a major role in human infection by STEC, which is likely to act as a vector for bovine strains in the human disease cycle and thus is essential to the health risk of owners and their families. potential sources of STEC O157:H7 transmissible to humans (9). It can also reported to cause diarrhea in dogs (19), Calves (20,21), sheep ( 22) and Buffalo (23).
The aim of this study was to explore the gross and pathological lesions in puppies at a different time post E. coli O157:H7 inoculation.

1.
Bacterial isolates: virulent E. coli O157:H7 isolates were obtained from University of Baghdad/ College of Veterinary Medicine /Department of internal and preventive medicine, these were isolated from diarrheic and nondiarrheic dogs found in Baghdad Province. These isolates confirmed in a previous study by (2) , which possess five virulence gene (rfbO157, flicH7, Stx1, Stx2 and eaeA), these genes confirmed by conventional and real time PCR by (1).

2.
Preparation of E. coli O157:H7 infective dose:To prepare the bacterial suspension of E. coli O157:H7 , the bacteria inoculated in 10 ml of brain heart infusion broth at 37 °C for 18 hours, then centrifuged in cold centrifuge at 8000 rpm (round per minute) for 15 minutes, the sediment washed three times with phosphate buffer saline (PBS) (pH=7.2) and re-suspended with 5 ml of PBS, then this suspension drenched once orally, which containing 5×10 7 CFU according to (24). The infective dose of bacteria was made according to (25). The infective dose was experimented on 3 puppies for insurance of the dose before using in experimental puppies.

Experimental animals:
Total number of (20) puppies, aged between (35-45) days, were adjusted two weeks before starting the pumping experiment in clean and disinfected cages, fecal crops for E. Coli O157: H7 was made to remove natural pathogens and carriers from inoculation. Ad libitum milk was fed, then gradually adapted to chicken meat, rice and clean water.. These dogs were divided randomly into two groups as follows: a. Infected group: Fifteen puppies were used as infected group and drenched 5 ml suspension containing (5×10 7 ) CFU of E. coli O157:H7 according to (24).
b. Control group: Five puppies were drenched orally by plastic syringe with PBS dose 5 ml.
4. Examination of the puppies:-All puppies were examined for the following:a. Daily observation of clinical signs: The puppies were daily monitored for presence or absence of abnormal clinical signs.
b.Fecal examination: Fecal swabs were taken twice weekly from all animals for confirming the presence or absence of E. coli O157:H7 .
Macroscopic examination were done for all animals.

Results and discussion
Before inoculation, all puppies showed a normal body temperature, heart rates and respiration rates. Post inoculation, the peak of infected group increased after 24, 48 and 72 hours and returned to normal at 4th and 5th days as compared with control group (Table 1) ( Figure 1).
Post inoculation, the infected group showed an increase in heart rate after 24 , 48 , 72 hours in contrast to the control group which showed normal ranges within the five days Table(1) (Fig. 2). Also, Post inoculation the puppies showed an increase in the respiration rates, the infected group gave higher means of respiration rates during 24, 48 and 72 hours, while the Vol. control group didn't show any change in respiration rates (Table 1; Figure 3). The puppies were monitored daily for development of other clinical signs such as diarrhea, dehydration and other signs. Before inoculation, all twenty puppies showed normal clinical signs, while post inoculation , after 24hrs all 15 puppies of infected group showed diarrhea ( Figure 4) which persisted for 4 days post inoculation. Also puppies appeared dehydrated with varying degree ( Figure 5), 6 puppies showed congested mucous membrane, also, 4 puppies showed bloody urine after 3 days post infection.
The presented study showed that infected animals exhibit different signs after 24hrs of E. coli O157:H7 inoculation. These results are in agreement with those recorded by (24) who showed that on the second day after E inoculation, both dogs experienced acute watery or mucoid diarrhoea. Coli O157:H7 with slightly bloody stool have also been observed reducing appetite, nausea and vomiting. Dogs also exhibit drastic loss of weight and mild to extreme exhaustion, fatigue and lethargy. In addition, 14 inoculated EPEC pups have been confirmed to have pasty-toliquid diarrhea, without vomiting 72 hours after infecting (27). Also, (28) showed that beagle dogs had slight to severe diarrhea after inoculation with E. coli O157:H7. (29) concluded that both types of E. coli AEEC or ETEC could be considered a relevant cause of diarrhea in dogs and pups.

Shedding of E. coli O157:H7:
The fecal swabs were taken twice weekly from all animals. These swabs were cultured on EMB, MacConkey , Chrom and CT-SMAC agars , then the isolates were serotyped by latex agglutination test. The shedding patterns of E. coli O157:H7 in feces were started after 24hrs. From inoculation and ceased within two weeks (day 14th) ( Table  3). The internal organs of puppies infected orally with E. coli O157:H7 which examined at these times (24hrs, 48hrs, 72hrs, 6 , 12, 24 and 36 days post infection), the 2 puppies were killed in each time showed the following:-No gross lesions in the intestine, kidney, lung and spleen of the puppies at 24, 48 and 72 hours after inoculation.
While at days 6, 12, 24, 36, the intestine showed congestion and hemorrhage. Also the kidneys were swollen and showed varying levels of congestion. The lung showed sever hemorrhages at day 12 post infection, also the spleen showed enlargement at day 24 post infection ( Figures 6 , 7, 8, 9).  (Figures:14, 15). At 6 day post infection the infiltration of inflammatory cells became more prominent with severe congestion of blood vessels in kidney, intestine, liver, brain, spleen (Figures:16, 17,18,19,20,21,22 ). At day 12 post infection, there was mucus attachment in the epithelium of intestine, congestion of blood vessels, necrosis, with infiltration of the inflammatory cells in the examined organs (Figures:23, 24). The most important event at day 24 post infection is the presence of regeneration in the intestine which became more prominent at day 36 post infection in examined organs, while the other organs showed a mild regeneration espicially at day 36 (figures: 25,26,27). This study showed different histopathological changes in different organs at different times 24hrs, 48hrs, 72hrs, 6days, 12days, 24days and 36days. The inflammatory reaction in the intestine in the current study may indicate that this strain can survive in gastrointestinal environment, attachment, colonization of intestinal epithelial cells and stimulate the phagocytic cells to produce pro inflammatory cytokines that were responsible for inflammatory reaction, this investigation is greed with (32) who explained that STEC can survive in GIT environment and they colonized in the intestinal tissues .
The current study showed moderate pathological changes in the kidney during 24hrs post infection, this result may indicate that the local isolated strain of E. coli O157:H7 can survive and colonize the intestine and disseminate to main target organs especially the kidney post secret the toxins during these period through overcome the cellular and humoral innate immune system. This observation is in agreement with (33) who found that E. coli produced StcE that play an important role in regulating classical complement pathway via destroying serpinC1 esterase which are encoded on PO17 plasmid.
The result of this study revealed neutrophils infiltration in the kidney, this result may indicate that Stxs may reach the kidney from the intestine and stimulate production of IL 8 that attracted the neutrophils to injury site, this evidence is agreed with (34) who found that the inflammatory reaction induced impair function of intestinal barrier that facilitated the cross of Stxs from intestinal lumen to sub mucosa and systemically disseminated.
The pathological changes increased in the intensity with progressive time, at 48hr post infection, it was reported severe lesions in the intestine characterized by the presence of bacterial colonies in clear round spaces in the epithelial layer of the large intestine with focal erosion of epithelial cells and inflammatory cells infiltration in the lamina properia, this result may indicate development of attaching and effacing lesions which are the main intestinal lesions inducing by E. coli O157:H7 particularly intimin eaeA positive strain, this observation is in consistency with result of PCR assay in the current study, also the above idea agrees with (35) who demonstrated that eae positive E. coli strain can form AE lesions on epithelial cells of the intestine. The eae positive STEC O157:H7 induced AE lesion may be due to their possessing locus of enterocyte effacement (LEE) which encodes a type III secretion apparatu, outer membrane protein called intimin (eae ) (E. coli attaching and effacing protein) and translocated intimin receptor(TIR) (36), in addition to effector protein, this protein transported by secretion apparatus, a syringe molecule extended from inside the bacterial cells through inner and outer membrane to the cytoplasm of the host cells (37). this protein(TIR) acts as a receptor for intimin. TIR and other protein can cause reorganization of cytoskeletal of intestinal epithelial cells lead to accumulated these cytoskeletal protein under the bacteria which were intracellular proliferation and produced Stxs in the AE lesion (38 ; 39). This lesion can protect the bacteria from phagocytic cells and remove the bacteria by diarrhea (40).
The present finding demonstrated that the severe lesions in examined organs were recorded during six to 12 days post infection, the severity of the lesions induced by E. coli O157:H7 was dependent on virulence factors of these pathogens particularly Stxs, however, the severity of the lesions in intestine coincides with marked inflammatory reaction in the intestine, this result may support an idea said that, the inflammatory reaction play roles in exaggerated cytotoxic effects of shiga toxin produced by these bacteria, this idea agrees with (41) reported that Stxs can promote production of IL8 that facilitated pavementing neutrophils to endothelial cells.
At this time, the severe lesions in the intestine are coincident with severe diarrhea expressed by infected animals in the present study, this result is Vol. in agreement with that of (42) who revealed that E. coli O157:H7 can induced severe diarrhea and renal failure in infected host (human and animals).
Overproduction of mucin in the intestine at 12 days post infection, may be indicate a defense mechanism against bacterial infection. This result is compatible with (43;44) who showed that mucus secretion is considered one of intestinal barrier against pathogen infection , in addition to acetate in which inhibited transmission of shiga toxin to the tissue from the lumen of infected intestine. Normally mucin can prevent imbalance between intestinal epithelial cells and E. coli and other commensal bacteria (45;46). This results showed a severe hemorrhagic inflammation in the lung. These result may indicate that in the dogs the pulmonary vascular endothelial cells are rich with Stxs receptors (47). (48) mentioned that there are two receptors for shiga toxin include neutral glycolipid globotriosylceramide GB3 and globotetraosylceramide Gb4. (49) demonstrated Stxs receptors in the certain cells including enterocytes, renal, brain and lung endothelial cells as well as platelets and RBCs. However, hemorrhagic pneumonia in the dog experimentally infected with E. coli O157:H7 was considered the first observation in this study.
The intestinal lesions in the current study are in agreement with clinical signs that revealed certain infected experimental dogs, showed bloody or no bloody diarrhea, this observation is in consistency with (50) who recorded that the lesion of AE was not frequently associated with bloody diarrhea and HUS in humans and they considered EHEC eae positive strain are a risk for HUS, also these results are in agreement with observation of (51) who demonstrated that, E. coli serotype, O157:H7, a non invasive pathogen, can either induce sporadic or outbreaks of diarrhea associated with hemorrhagic colitis and HUS in human. Stxs can bind to vascular endothelial cells of GIT causing destroying these cells (52).
Thrombus formation in the blood vessels of kidney may be due to bacterial LPS and Stxs that may activate platelets and cause damage endothelial cells with inducing deposition of fibrin networks as discussed by (49).
At 24 days post infection, the result investigated that the body immune system started to eradicate the bacterial infection, in this period, low intensity of pathological lesions were recorded, and regeneration processes were initiated in the intestine, this may be due to activate innate immune cells, this idea is in consistency with (53) who recorded that activated innate immunity was considered one important defense mechanism against bacterial infection.
This eradication mechanism was discussed by many researchers like (54) who found that immune responses against bacterial colonization such as E. coli started through interaction between bacterial antigen with immune receptors which expressed by intestinal epithelium such as TLRs and activating these receptors leads to recruitment of neutrophils and other phagocytic cells to site of infection and eliminate the bacterial infection, in addition to adapt immune reaction that provided other protective mechanism against invading pathogens.

Conclusion
E. coli O157:H7 can causing different signs such as diarrhea, dehydration and other related signs. The shiga toxin can cause damage in many body organs like liver, spleen, kidney, lung, brain and intestine, also, the histopathological examination showed that the regeneration of tissue especially intestine started after 24days post infection, while the other organs showed regeneration at 36days post infection.