Electron microscopic study of ileum of mice infected experimentally with Salmonella hadar

Recently, Salmonella hadar has been isolated and identified from goat in Iraq. The purpose of the present study was to examine ultrastructural changes in the ileum epithelial cells of BALB/c mice experimentally infected with S. hadar. Mice were used as follows: Group A: 20 mice inoculated orally with phosphate buffer saline and considered as a control group. Group B: 20 mice inoculated orally with (100ID) by drenching the mice about 1 ml of the bacterial suspension which contain (1.5×10 cells) of Salmonella hadar and the ileum epithelial cells were examined by transmission electron microscopy at 24, 48, 72, 96 and 120 hours after infection. The ultra structural changes seen in the ileum of infected mouse at 24 hours were disorganization of the microvilli with severe cytoplasmic vacuolization, enlargement of the mitochondria and presence of intracellular Salmonella. Changes at 48 hours post infection, were detachments of many microvilli especially at the site of bacterial entry. Similar changes were observed after 72 hours but more severe; there was marked dilatation and proliferation of the endoplasmic reticulum with cytoplasmic vacuolization of the infected enterocytes. After 96 hours there were severe cytoplasmic vacuolization with accumulation of the bacteria within phagosomes and there was marked damage to the microvilli of the ileum. After 120 hours there was hypertrophy of goblet cell and thickening of the nuclear membrane and there was several Salmonella containing vacuoles.


Introduction
Globally, salmonellosis has remained one of the three most common meat associated diseases in human (1) and the disease caused by Salmonellae organisms is the most common and important zoonotic diseases (2).
Salmonella hadar is now one of the five most frequently isolated serotypes in human and animals (3)(4)(5) it had been isolated firstly in the early 1950 s , from a stool sample of a subject with gastro-enteritis and fever (6).
Salmonella enterica serovars cause a variety of diseases ranging from self-limiting gastroenteritis to severe systemic infections.Virulence of these facultative intracellular pathogens is dependent on their ability to invade and replicate within non-phagocytic cells (7,8).
Considerable attention has been given to the role of bacterial adherence in colonization of mucous membranes and in the pathogenesis of many infections.Both scanning and transmission electron microscopies (SEM and TEM) have been used widely in the study of intestinal colonization by Salmonellae (9,10).
Mikula et al. (11) found that post oral infection of calves with S. typhimurium 4/5 strain at a dose (1×10 6 C.F.U./ml), appear in discontinuous and irregular of the brush border membrane of jejunal enterocyte.
Yass (12) study the pathogenesis of S. typhimurium infection in calves by using transmission electron microscopy, he found that the early ultra structural changes observed in microvilli of small intestine were characterized by local derangement with slight swelling of the proximal end in addition to the presence of many bacteria either associated or adherent to the microvilli, and this Intracellular Salmonella was usually intact and enclosed by a membrane.Complete disappearance of the microvilli was observed after 5 days.

Bacterial strain
Salmonella hadar was isolated from Iraqi goats, diagnosed and confirmed according to (Standard Operating Procedure ''SOP'' (13) and serotyped in the national salmonella center in Iraq.

Estimating the infectious dose (ID)
Five colonies of S. hadar 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 then the sediment washed three times with phosphate buffer solution (PBS) (pH=7.2) and re-suspended with 1 ml of PBS, then tenfold dilution (10 -1 , 10 -2 , 10 -3 , 10 - 4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 and 10 -10 ) were done.The viable count of the bacteria in each diluent was made according to method of Miles and Misra (14) and selected the diluents which had these concentrations for drenching the mice: (1.5×10 5 cells), (1.5×10 6 cells), (1.5×10 7 cells), (1.5×10 8 cells), (1.5×10 9 cells), (1.5×10 10 cells) and (1.5×10 11 cells).Forty eight mice were divided into eight groups, (each group contain 6 mice) seven groups of mice drenched orally with one of the calculated diluents (C.F.U./ml) above diluted with 1 ml PBS while the last group (6 mice) served as control and drenched with 1 ml PBS (pH=7.2).All groups were observed for 30 days to calculate the live and dead mice, the infective dose (ID) was estimated by choosing the group of mice which showed clinical signs resemble to those in Salmonellosis with no mortality after ensure that by culture.This dose was calculated according to (15,16).

Laboratory animals
Total numbers (40) mice (BALB/c) of both genders with age range (6 -8) weeks old which adapted for two weeks before start of the experiment, all animals had negative fecal bacteriological culture for salmonella, then divided into two groups: Group (A): 20 mice inoculated orally with phosphate buffer saline and considered as a control group.Group (B): 20 mice inoculated orally with (100ID) by drenching the mice about 1 ml of the bacterial suspension which contain (1.5×10 9 cells) of Salmonella hadar (15).

Solutions
Phosphate buffer saline (PBS, pH=7.2): was prepared according to (17).2.5 % Glutaraldehyde: was prepared by adding 2.5 ml of absolute formalin to 97.5 ml of PBS (pH=7.2) and used for electron microscope specimens.

Transmission electron microscope (TEM)
At the specified time of each experiment, the mouse was anesthetized by using the diethyl ether by inhalation.The abdomen was quickly opened, and then the ileum was removed as quickly as possible and drained then according to (18) the ileal tissue pieces (size approximately 1-2 mm 3 ) of the experimental and control mice were fixed in 3% glutaraldehyde in phosphate buffer saline (pH= 7.2) at 5 ˚C for 6 hours and subsequently post-fixed/block stained in aqueous 1% osmium tetroxide (OsO 4 ) for 6 hours at room temperature.The fixed tissues were embedded in epoxy resin and ultra-thin sections (approximately <0.1 μm in thickness) were cut using glass knife with an ultramicrotome.Ultra-thin sections obtained on 3 mm diameter copper grids, were stained with uranyl acetate and lead citrate stains for contrast and examined under Philips (CM-10) transmission electron microscope in (College of Medicine, Al-Nahern University).

Results
The ultrastructural examination of the brush border of the absorptive columnar epithelial cells that cover the villi of the control mice (group A) consisted of long, closely packed regular microvilli.The cytoplasmic region appeared normal with no ultrastructural changes (Fig. 1).
The ultrastructural changes which obeserved in the ileum of infected mice in group (B) at 24 hours post infection were disorganized with sever disruption of the M cell and disproportional of the microvilli with severe cytoplasmic vacuolization, dilation of endoplasmic reticulum, enlargement of the mitochondria and presence of intracellular Salmonella (Figs. 2 and 3).The ultrastructural changes at 48 hours post infection were detachments of many microvilli especially at the site of microorganism entry with vacuolar changes in the cytoplasm of the enterocytes (Fig. 4).The ultrastructural changes in the ileum of group (B) mice that killed after 72 hours post infection were the same with that observed previously but was more severe, there was marked dilatation and proliferation of the endoplasmic reticulum with cytoplasmic vacuolization of the infected enterocytes; in other sections the endoplasmic reticulum appeared to have dilated phagocytic vacuoles with lysosomes in addition to presence of many ribosomes detached and dispersed singly in the lamina properia and S. hadar is present in both an enterocytes and M cells (Figs. 5, 6 and 7).
At 96 hours post infection, there were severe cytoplasmic vacuolization with accumulation of the bacteria within phagosomes and there was marked damage to the microvilli of the ileum of group (B) mouse at the same time (Fig. 8).
Finally the ultrastructural changes in the ileum of group (B) mouse that killed after 120 hours post infection revealed hypertrophy of goblet cells, dilatation of endoplasmic reticulum, severe cytoplasmic vacuolization, thickening of the nuclear membrane and there were several Salmonella containing vacuoles (SCV) (Figs. 9, 10 and 11).

Discussion
The early ultrastructural changes observed in the microvilli by transmission electron microscope in the mice of group A which infected with S. hadar were characterized by local derangement with little swelling on the proximal end.One of the interesting findings is the ballooning of the microvilli and the appearance of this ultrastructural change may be due to the attachment of S. hadar to their membrane.While, when the infection was became more advanced, the microvilli became short, more effacement from epithelial cells in some location; these observations in the microvilli were similar to that mentions by (19) who studied the ultrastructural changes of ileum microvilli of rabbit infected with S. typhimurium, and also was similar to that recorded by (12).The effacement of the brush border of the enterocytes of the microvilli by S. hadar resulted in the loss of brush border enzymes (amino oligo peptidase, aspartate amino peptidase, dipeptidyl amino peptidase, carboxy peptidase and γ glutamyl transferase which hydrolyze small peptides that are formed in the intestinal lumen by the action of proteases to free amino acids during the process of absorption), carrier protein and surface area as mentions by (20).The shortening and loss of microvilli is in accordance with decreased alkaline phosphatase activity, this enzyme is located in the plasma membrane of the microvilli and is considered as a measurement of the digestive-absorptive surface (21).
The fragmentation and vesiculation of microvilli may be due to increases in cytoplasmic Ca 2+ concentration which lead to actin filaments of the microvillus core cytoskeleton to be broken into short filament as mentioned by (22).The ultrastructural changes was observed in the epithelial cells were probably due to the presence of many intracellular Salmonella that produced cytotoxin inside the cells; these observations is similar to that observed in ileum of the infected calves with S. typhimurium (12) and also with that found in the ileum of swine which infected with S. typhimurium DT104 (23).The loss of microvilli reduced the intestinal absorption surface, this together with the functional deficiency and destruction of many epithelial cells, may be resulted in decrease absorption and caused gastroenteritis and diarrhea.The ultrastructural changes observed in the cytoplasm of the enterocytes which manifested by vacuolization of cytoplasm, displacement of organelles and swollen mitochondria, these changes in the host cells were occurred as a result of the effect of S. hadar endotoxin that is part of the outer membrane or metabolic substances released from the bacteria as mentioned by (24).
Finally this study concluded that S.hadar has high ability to invade and established colonization in the intestine of mice, it is an invasive strain.

Fig. 3 :
Fig. 3: TEM.Ileum, mouse (group B) at 24 hours post infection with S. hadar, shows disorganization and severe disruption of the M-cell (black arrow), also there is cytoplasmic vacuolization (V) and presence of Salmonella containing vacuole that contain two Salmonella (white arrow).(Uranyl acetate & Lead citrate) X 41000.

Fig. 4 :
Fig. 4: TEM.Ileum, mouse (group B) at 48 hours post infection with S. hadar, shows bacterial attachment and invasion lead to loss of microvilli (white arrow) and vacuolation spaces (V), many intracellular invading bacteria (B) located in the lamina properia (black arrow), cell with apoptotic feature appear in field containing apoptotic bodies cell.(Uranyl acetate & Lead citrate) ((X 24500).

Fig. 5 :
Fig. 5: TEM.Ileum, mouse (group B) at 72 hours post infection with S. hadar, shows an invading Salmonella hadar located in both an enterocytes and M-cells (black arrow) with detachment of the some microvilli (white arrow).(Uranyl acetate & Lead citrate) X 16000.

Fig. 7 :
Fig. 7: TEM.Ileum, mouse (group B) at 72 hours post infection with S. hadar, shows dilation of the endoplasmic reticulum (black arrow), presence of phagocytic vacuole that contain many ribosomes (white arrow) in addition to presence of many lysosomes (L) which detached and dispersed singly in the lamina properia.(Uranyl acetate & Lead citrate) X 3400.