Three different routes of EHEC O157:H7 infection were used to establish EHEC broiler model

Abstract

In order to study the prevention and control EHEC disease measures in poultry, the infection process and development of this disease and the pathological changes of various organs were to be observed. In this study, chickens were infected with different doses of enterohemorrhagic Escherichia coli (EHEC) O157:H7 using different routes of administration to establish EHEC broiler model. A total of 195 14-day-old broilers were randomly divided into 13 groups: including control group, Enema-drip groups (10¹⁰, 10¹¹, 10¹², 10¹³ CFUs E. coli O157:H7), gavage groups (P.O) (10¹¹, 10¹², 10¹³, 10¹⁴ CFUs E. coli O157:H7), and intraperitoneal injection group (I.P.) (10⁸, 10⁹, 10¹⁰, 10¹¹ CFUs E. coli O157:H7). Escherichia coli (E. coli) was given using enema-drip, gavage or intraperitoneal infection. Then the feed intake, weight changes, stool and clinical symptoms of the chicks were recorded during the experiment. 7 d after E. coli infection, blood was collected from the jugular vein and serological tests were carried out. The liver, spleen, and colon of the chicks were extracted to get the organ index, bacteria load, and their histopathological changes. After infection with E. coli, some chicks feces were green or red watery stool, sometimes accompanied by foam, and the material to weight ratio of broilers in I.P. group increased significantly (P < 0.05), the 10⁸ CFUs group were 1.3 times as large as control group. Three modeling methods can result in abnormal serum lipid metabolism and liver function indexes (increase of AST, TBA, T-Bil and TC level; decrease of ALB, TG, and TP level). Infection of chicks with O157:H7 by all 3 methods resulted in its detection in the liver, spleen, and colon. Three modeling methods significantly decreased liver index, and inflammatory cell infiltration and hyperemia were observed in liver. The spleen index in E. coli broilers by gavage and enema-drip was significantly decreased, splenic hyperemia and periarteriolar hyalinosis were observed. The spleen was enlarged with purplish-black spheroids in I.P. group broilers, and the spleen histological changes was more serious. The colon villi of broilers in gavage and enema-drip groups were thinner, more prone to rupture, intestinal lamina propria hyperemia, and inflammatory cell infiltration. Moreover, the number of goblet cells in the mucosal epithelium increased. E. coli O157:H7 can induce liver, spleen and intestinal damage and reduce growth performance of chicks. By comparing these 3 methods, we found that chicks infected with O157:H7 by gavage had more severe liver and intestinal damage, the enema-drip method caused most serious intestinal damage, and I.P. method significantly damaged the liver and spleen of chickens.

INTRODUCTION

Enterohemorrhagic Escherichia coli O157:H7 (EHEC O157:H7) is a typical strain of enterohemorrhagic E. coli, which is known to cause hemorrhagic intestinal inflammation, hemolytic uremic syndrome, thrombocytopenic purpura and other diseases in animals (Meng et al. 2011; Sun et al. 2022). EHEC O157:H7 is a zoonotic bacteria, and the main route of infection is digestive tract infection (Lim et al. 2010). E. coli O157: H7 has been detected in available poultry in the United States, China and Turkey (Dipineto et al. 2006; Kalin et al. 2012; Lu et al. 2015; Mitchell et al. 2015). Undercooked animal products are the most common route people get E. coli (Tarr 1995). Until 2022, world production of fresh or frozen chicken has reached 123 Million (from Food and Agriculture Organization of the United Nations) and chicken have become essential animal food on people's table. Therefore, the spread of E. coli through chickens can become a public health problem that endangers people's health (Doane et al. 2007; Lim et al. 2010; Ferens and Hovde 2011). However, there was few research about the effects of E. coli O157:H7 on broilers, and studies on different modes of O157:H7 infection on broilers were even rare.

E. coli not only poses a health threat, but also causes great economic loss to the poultry industry. Research shows that bacterial intestinal diseases can reduce poultry production performance (Wang et al. 2023), which is the main cause of huge economic losses (Guabiraba and Schouler 2015). At present, APEC O78 is the most widely studied strain of E. coli in poultry. There's only a handful of studies on EHEC in poultry, and the pathogenicity and pathogenesis of E. coli O157:H7 on chicks have not been clarified. Therefore, in order to better study the effect of E. coli O157:H7 on broilers, we used 3 E. coli infection modes with enema, gavage, and intraperitoneal injection to observe the pathological changes of liver, spleen, and colon of the chicks. This study lays an experimental foundation for the prevention and treatment of diseases caused by E. coli O157:H7.

MATERIALS AND METHODS

Bacteria

The E. coli O157:H7 strain was originally obtained from Guangdong Microbial Culture Collection Center (GDMCC NO:1.1869, Guangdong Microbial Culture Collection Center Guangdong, China).

Animals and Experimental Design

A total of 195 one-day-old Spotted Chicken (one type of broiler) (Foshan Nanhai Poultry Corporation, Foshan, China) were randomly divided into 13 groups, including control group, Enema-drip group (10¹⁰ CFUs, 10¹¹ CFUs, 10¹² CFUs, 10¹³ CFUs E. coli O157:H7), Gavage (P.O) group (10¹¹ CFUs, 10¹² CFUs, 10¹³ CFUs, 10¹⁴ CFUs E. coli O157:H7), and intraperitoneal injection (I.P.) group (10⁸ CFUs, 10⁹ CFUs, 10¹⁰ CFUs, 10¹¹ CFUs E. coli O157:H7). At 14 d of age, chickens were infected with different doses of E. coli O157:H7 by gavage, Enema-drip, or intraperitoneal injection, respectively. Broilers had free access to food and water. Feed intake, body weight, clinical symptoms and feces of broilers were recorded during the experiment. Seven days after E. coli infection, blood samples were obtained from jugular veins and used for serological tests. Then, broilers sacrificed by CO₂ inhalation, and the liver, spleen, and colon of the chicks were extracted. The organ index of the liver and spleen was calculated. The bacteria load and histopathological changes were examined in the liver, spleen, and colon.

All broilers were raised with ordinary feeding, and feed ingredients were shown in Table 1. All animal work was carried out in accordance with the Guidelines for the Care and Use of Laboratory Animals formulated by the Ministry of Science and Technology of the People's Republic of China (Approval number: 2006-398) and approved by the Laboratory Animal Management Committee of Foshan University.

Table 1.

Feed composition.

Ingredient	Content (%)	Ingredient	Content (%)
Crude protein≥	19	Crude ash≤	11
Coarse fibre≤	5	Calcium	0.6-1.5
Total phosphorus≥	0.5	Methionine≥	0.34
Sodium chloride	0.30-1.0	Hydration≤	14

The feed-to-gain ratio of broilers was calculated. Material to weight ratio = (Feed intake/weight gain) / (control group feed intake/control group weight gain). Organ index = (organ weight/broiler weight) × 100%.

Serological Test

Blood was static for 1 h, and then centrifuged at 3,500 r/min for 10 min to collect serum. The serum samples were harvested for measurement of albumin (ALB, 148322010), alkaline phosphatase (ALP, 140323004), aspartate aminotransferase (AST, 140223007), alanine aminotransferase (ALT, 140123007), total bilirubin (T-BilirV, 140623005), total cholesterol (TC, 2285099), triglyceride (TG, 141723003), serum total protein (TP, 140821003) and total bile acid (TBA, 143223006) following the instructions on the commercial kits. The kits and biochemical analyzer (BS-240VET) were provided by Shenzhen Mindray Bio-Medical Electronics Co., Ltd (Mindray, Shenzhen, China).

Bacterial Load

Bacterial load of liver, spleen and colon were detected. 0.05 g of tissues (liver, spleen, and colon) were homogenized with 1 mL of sterilized water. About 100 μL of the homogenate tissue suspension was coated on Macconkey medium, then the medium were cultured in an incubator at 37 °C for 12 h to observe the growth of bacteria. The growth and quantity of bacteria were evaluated by scoring method, with 0 score - no bacterial growth. 1 point - Small amount of bacterial growth. 2 points - More growing bacteria, but can be divided into separate colonies. 3 points - The colony in the plate is densely packed, and no obvious colony is visible to the naked eye. The bacterial growth of 5 chickens was measured and added to the final score.

Measurement of Histological Damage After E. coli Infection

Tissues (liver, spleen, and colon) of chicks were fixed in 4% paraformaldehyde for 24 h, dehydrated by alcohol gradient, treated with xylene, embedded in paraffin, sliced, expanded and dewaxed, and then stained by H&E. The slices were dehydrated by alcohol gradient, and sealed by resin. Finally, the histopathological characteristics and changes of broiler tissues were observed under a microscope (Bio-Rad). Five fields of at least 2 sections per animal and 3 animals per experimental group were evaluated. The liver tissue severity score was evaluated by referring to the Ishak score system to evaluate including hepatocyte necrosis (0 points - No necrosis. 1 point - Less than 50% necrosis. 2 points -50% to 75% necrosis), liver fibrosis (Score 0 - No cellulification. Score 1 mark - Only cellulification is present, but not serious. Score 2 - Mild cellulification, showing reticular fibers), liver inflammation (Score 0 - No inflammatory cell infiltration. Score 1 - Mild inflammatory cell invasion. Score 2 - Moderate inflammatory cell infiltration. Score 3 - Severe inflammatory cell infiltration) and degree of biliary tract injury (Score 0 - No injury. Score 1 - Minor bile duct injury. Score 2 - Severe bile duct injury), the most severe injury score was 9. Intestinal injury was mainly assessed for intestinal mucosal injury, with 0 score - No mucosal injury, 1 score - Partial loss of mucosal recess, 2 score - Complete loss of mucosal recess, 3 score - Mucosal epithelial ulceration with obvious inflammatory cells, and the most serious injury was 3 score. Spleen damage score is based on presence or absence of vascular hyaluronidosis (0 - No vascular hyaluronidosis. Score 1 - Hyalinosis of blood vessels is present), inflammatory cell infiltration (score 0 - No inflammatory cell infiltration. 1 - Mild inflammatory cell invasion. Score 2 - Moderate inflammatory cell infiltration) and fibrous exudation of the spleen (0 points - No exudation. 1 score - Cellulification is present), the most serious injury was 5 points. Pathological sections of the liver, spleen, and colon of at least 3 broilers were analyzed in each experimental condition.

Statistical Analysis

Quantitative data are expressed as the mean ± standard deviation. All statistical analyses and chart drawings were performed using GraphPad Prism 8 (GraphPad, San Diego, CA). Differences between groups were analyzed using a one-way non-parametric analysis of variance (ANOVA). Statistical significance was set at P < 0.05.

RESULTS

Clinical Symptoms of Broilers Infected With EHEC

There were no clinical symptoms in control group, and feces was similar to a cone, the color was light gray, and white urate was attached to the surface of the feces (Figure 1). After infection with E. coli O157:H7, there was no obvious abnormality in the clinical symptoms of the chicks, but some chicks feces were green or red watery stool, sometimes accompanied by foam.

Figure 1.

Observation of daily state and feces of broiler chickens. (A) Clinical symptoms of chicks with different infection modes. (B) Observation of fecal state of chicks with different infection modes.

Effects of E. coli on Feed to Weight Ratio and Organ Index of Broilers

As shown in Figure 2, compared with control group, the liver index decreased significantly (P < 0.05) after 3 routes of E. coli infection. The spleen index in Enema-drip group (10¹⁰ CFUs,10¹¹ CFUs) was lower than that of the control group (P < 0.05). Moreover, the ratio of intestinal weight to length increased (P < 0.05) after Enema-drip and gavage of E. coli. After 3 methods of E. coli infection, the material to weight ratio of broilers was increased, especially by intraperitoneal injection, which was about 1.3 times as much as that of the control group. The above results show that E. coli infection can cause liver, spleen and intestinal damage to chicks, and the feed required for each additional 1 kg of body weight is 1.2 to 1.3 times as that of the control group, which greatly increases the breeding cost. And compared with the other 2 methods, E. coli by intraperitoneal injection had the greatest effect on material to weight ratio and liver organ index of chicks.

Figure 2.

Effects of E. coli on weight ratio and organ index of broilers. (A) Liver index and (B) spleen index of broilers. (C) Material to weight ratio. (D) The intestinal weight to length ratio. *P < 0.05 vs. Control.

Effects of E. coli on Serological Enzymatic Activity Changes in Broilers

As shown in Table 2 and Table 3, compared with control group, T-Bil, AST, TBA and TC level obviously increased (P < 0.05), while ALB, TG and TP level significantly decreased (P < 0.05) in both P.O and Enema-drip group. In the intraperitoneal injection group, T-Bil and TBA level increased (P < 0.05), while ALT, ALB, TG, and TP were significantly decreased (P < 0.05). The above results indicated that E. coli infection with 3 methods could lead to the increase of AST, TBA, T-Bil, and TC in serum to varying degrees, and the significant decrease of ALB, TG, and TP level. The results suggested that the liver function and intestinal absorption function were abnormal. Furthermore, compared with other infection methods, Enema-drip had more significant effects on the above indexes.

Table 2.

Serological liver function enzyme activity changes.

	T-Bil (μmol/L)	ALT (U/L)	AST (U/L)	TBA (μmol/L)
Control	11.31±1.08	5.41±0.38	211.45±2.55	1.81±0.11
P.O11	13.45±0.22	4.51±0.37*	257.91±1.45*	2.40±0.09
P.O12	18.08±0.28*	5.45±0.33	317.30±1.66*	2.85±0.60*
P.O13	15.27±0.63*	5.45±0.77	251.52±17.27*	4.70±0.13*
P.O14	14.00±1.50*	4.90±0.31	234.92±6.77*	3.30±1.24*
Enema10	21.63±1.65*	5.06±0.33	286.6±13.32*	3.56±0.05
Enema11	18.88±1.46*	5.12±0.51	268.60±16.08*	1.93±0.45
Enema12	16.84±0.23*	5.91±0.31	255.20±12.92*	2.06±0.15
Enema13	17.39±0.85*	5.52±0.17	262.53±23.2*	2.36±0.19
I.P8	19.38±2.25*	3.26±0.41*	226.49±22.96	3.82±3.30*
I.P9	19.85±2.88*	3.42±0.67*	219.69±15.58	3.56±1.44*
I.P10	19.73±2.20*	3.42±0.68*	213.75±7.19	3.31±1.34*
I.P11	19.97±1.86*	3.72±0.59*	213.82±4.45	3.37±1.03*

^⁎P < 0.05 vs. Control.

Table 3.

Serological lipid metabolism enzyme activity changes.

	ALB (g/L)	TG (mmol/L)	TP (g/L)	TC (mmol/L)
Control	16.98±0.47	0.47±0.08	58.68±1.02	3.65±0.09
P.O11	9.55±0.29*	0.27±0.04*	29.48±0.39*	4.73±0.04*
P.O12	11.41±0.28*	0.24±0.04*	32.70±0.56*	4.67±0.20*
P.O13	11.22±1.66*	0.25±0.08*	38.24±3.23*	4.60±0.45*
P.O14	11.98±1.69*	0.32±0.02*	42.76±2.60*	4.84±0.18*
Enema10	12.56±1.19	0.21±0.01*	41.03±8.91*	5.51±0.49*
Enema11	10.76±1.62*	0.30±0.03*	35.96±3.73*	5.39±0.50*
Enema12	12.06±0.28*	0.29±0.07*	35.15±3.73*	5.12±0.28*
Enema13	10.50±0.15*	0.25±0.04*	36.35±5.32*	5.37±0.44*
I.P8	12.49±0.98*	0.30±0.06*	31.91±2.95*	3.76±0.44
I.P9	11.84±0.77*	0.29±0.04*	30.68±3.82*	3.97±0.49
I.P10	11.61±0.41*	0.30±0.08*	30.95±3.45*	3.76±0.45
I.P11	11.31±0.57*	0.32±0.05*	29.54±3.06*	3.74±0.42

^⁎P < 0.05 vs. Control.

Effects of E. coli on Bacterial Load of Liver, Spleen and Intestinal in Broilers

Bacterial load is important indexes reflected bacterial colonization. The bacterial coating method was used to observe the effect of different infection methods on the bacterial colonization of E. coli in liver, spleen, and colon. Compared with control group, all 3 routes of E. coli infection obviously resulted E. coli colonization at the liver, spleen, and intestinal tract of chicks. For each route of E. coli infection, the number of bacterial colonization gradually increased with the increase of E. coli infection concentration (Figure 3, Table 4). Through comparative analysis, intraperitoneal injection is the best route to induce the most serious E. coli colonization of chicks.

Figure 3.

The measurement of E. coli colonization in chicks by the bacterial coating method. Tissues were homogenized and tissue suspension was coated on Macconkey medium at 37°C for 12 h to observe and count the growth of bacteria. (A) Only a small amount of bacterial growth is calculated as 1 point. (B) Bacterial growth less than one-third of the dish area is calculated as 2 points. (C) Bacterial growth density more than half of the dish area is calculated as 3 points.

Table 4.

The number of E. coli colonization at different tissues.

	Control	P.O11	P.O12	P.O13	P.O14	Enema10	Enema11	Enema12	Enema13	I.P8	I.P9	I.P10	I.P11
liver	0	5	6	9	10	5	7	8	10	9	12	12	13
spleen	0	3	4	7	6	5	4	7	8	8	9	10	11
colon	3	9	11	13	13	8	11	11	12	9	11	13	13

Five chicks were collected in each group, and their tissues (liver, spleen and colon) were tested for bacterial colonization. The number of E. coli colonization in Table 3 was the total number of E. coli colonization from 5 chicks.

Effects of E. coli Infection With 3 Routes on Liver Histopathological Changes in Broilers

As shown in Figure 4, compared with the control group, there were no obvious changes in liver ocular vision after autopsy in Enema-drip group, serous exudates were observed in blood vessels at low magnification, and inflammatory cells were observed around blood vessels at high magnification. The color of liver with necropsy was darker in the group of gavage vascular congestion and serous exudation were observed at low magnification, and the blood vessels were full of red blood cells and surrounding inflammatory cell infiltration were observed at high magnification. In the intraperitoneal injection group, the liver was brown-green, the liver lobular structure was not obvious at low magnification, and there was intravascular congestion and inflammatory cell infiltration in the surrounding liver tissue. Moreover, serous exudation was observed at high power. Combined with quantitative analysis of histological damage of liver (Figure 7A), we found that intraperitoneal injection caused the most serious damage to liver, then followed by gavage groups.

Figure 4.

Effects of E. coli infection with 3 routes on liver histological changes in broilers. Liver necropsy and histological changes were observed in (A) gavage group, (B) Enema-drip group, and (C) intraperitoneal injection group broilers. Red triangle represents red blood cells. Yellow triangle represents inflammatory cell infiltration.

Figure 7.

Severity of histological injury after E. coli infection. (A) Liver severity score. (B) Spleen severity score. (C) Colon severity score. Different letters on the shoulder mark represent significant differences (P < 0.05), the same letter on the shoulder indicates no difference (P > 0.05).

Effects of E. coli Infection With 3 routes on Spleen Histopathological Changes in Broilers

As shown in Figure 5, compared with the blank group, spleen volume of chickens in the gavage group and the Enema-drip group was smaller, and splenic hyperemia and periarteriolar hyalinosis were observed under microscope. The spleen was enlarged with purplish-black spheroids, and some of them had grayish white necrotic spots under necropsy in the intraperitoneal injection group. At low magnification, hyperemia was observed, and hyalinization of splenic arterioles was observed at high magnification. The spleen histological changes caused by intraperitoneal injection was more serious. Combined with quantitative analysis of histological damage of spleen (Figure 7B), among the 3 infection routes, only gavage group had greater damage to the spleen, which was statistically significant compared with the other 2 ways.

Figure 5.

Effects of E. coli infection with 3 routes on spleen histological changes in broilers. (A) The liver of gavage group. (B) The liver of Enema-drip group. (C) The liver of intraperitoneal injection group. Red triangle represents red blood cells. Yellow triangle represents hyalinization of splenic arterioles.

Effects of E. coli Infection With 3 Routes on Colon Histopathological Changes in Broilers

As shown in Figure 6, compared with the control group, the villi in the colon of the chicks with 3 methods all became thinner and more likely to break. At the same time, the intestinal lamina propria was congested, inflammatory cells were infiltrated, and the number of goblet cells in the mucosal epithelium increased, resulting in enhanced secretion function, a large amount of mucus and shed epithelium on the mucosal surface. Combined with quantitative analysis of histological damage of spleen (Figure 7C), the colonic damage of broilers infected with O157:H7 by gavage and enema-drip was similar, while the colonic damage of E. coli infected by intraperitoneal injection was the least, which had statistical significance compared with the other 2 groups.

Figure 6.

Effects of E. coli infection with 3 routes on colon histological changes in broilers. Colon necropsy and histological changes were observed in (A) gavage, (B) Enema-drip, and (C) intraperitoneal injection group broilers. Red triangle indicates serous exudation. Blue triangle indicates a rupture of the intestinal villi. Yellow triangle indicates infiltration of inflammatory cells.

DISCUSSION

At present, for broilers, most clinical studies have focused on avian pathogenic E. coli (O78 E. coli), whose model construction is relatively mature (Ma et al. 2020; Tarabees et al. 2020), which can cause avian colibacillosis, mainly manifested as depression, decreased appetite, coarse feathers, wasting, etc. Escherichia coli infection will harm the respiratory system, digestive system and immune system of birds, and eventually cause avian sepsis (Zhang et al., 2014; Ali et al. 2020; Usman et al. 2022).

The literature shows that the O157: H7 detection rate of broilers is lower than that of ruminants such as cattle and sheep (Ferens and Hovde 2011), and the infected broilers are important threats to public safety. In this study, E. coli was administered in 3 different routes to establish EHEC model in broilers with the daily manifestations of chicks and pathological changes as indexes. Through comparing the damage caused by 3 different routes of infection with EHEC, to provide the basis for the study of E. coli disease mechanism and drug development.

Research shows that avian pathogenic E. coli could increase liver organ index and infiltration of liver inflammatory cells in chicks (Wei et al. 2023). Moreover, avian pathogenic E. coli O78 could cause liver inflammation and damage in laying hens (Wang et al. 2023). In this experiment, compared with the control group, the liver index of chicks infected with E. coli was significantly decreased. Meanwhile, the serum biochemical detection showed that AST, TBA, T-Bil and TC were increased to varying degrees (P < 0.05), while ALB, TG, and TP were significantly decreased (P < 0.05), which suggested that O157:H7 can induce abnormal serum lipid metabolism and liver function indexes. The histopathological results showed that EHEC could also cause liver damage in chicks, with inflammatory cells around blood vessels. The results of bacterial loading showed that the content of E. coli load in liver was higher as the concentration of E. coli infection increases, which may be due to the rich blood flow in the liver to make E. coli more likely to colonization. These results indicate that EHEC O157:H7 can cause liver damage in chicks.

It's reported that colibacillosis can cause enlargement, necrosis and inflammation of chicken spleen (Chu 2022). Avian pathogenic E. coli could induce inflammation and injury of poultry spleen (Bagheri et al., 2023). In this experiment, compared with control group, the spleen organ index of the chickens infected with E. coli by gavage and enema-drip was significantly decreased. But the spleen organ index of the I.P. group chickens was increased, the spleen volume was enlarged, gray and white necrosis points were observed. All 3 methods can cause hyalinoid transformation of splenic arterioles. These results indicate that EHEC O157:H7 can also cause spleen damage in chicks.

O157:H7 will seriously affect the growth performance of broilers (He et al. 2022), in addition, it will lead to the reduction of intestinal villi/crypt ratio, resulting in cecum inflammation and damage (Xiang et al. 2022). Research shows that O157: H7 can continue to infect young broilers and keep them detoxicated. After different strains infect 1-day-old chicks, E. coli will colonize the liver, spleen and intestines of chicks. However, after the 19th d of infection, no E. coli was found in the liver and spleen of chicks, but some strains can still be detected in the intestines after 47 d of infection (La Ragione et al. 2005). In this experiment, we found that broilers of 14 d of age were infected with different concentrations of O157: H7 through 3 routes, and none of the broilers died. Combining Ragione's research and comparing it with mammals, we thought that O157: H7 is not as damaging to broilers as it is to mammals (Ferens and Hovde 2011). Our results found that the material to weight ratio of E. coli infected chicks was about 1.3 times as much as that of the control group, indicating that E. coli O157:H7 induced intestinal damage, and intestinal absorption capacity of nutrients decreased. In addition, we observed chick feces and found that the shape of normal chick feces was similar to a cone, the color was light gray, and white urate was attached to the surface of the feces. After infection with EHEC, some chicks' feces were green or red watery stool, sometimes accompanied by foam. The histopathological results showed that colon villi atrophied and intestinal wall became thinner, accompanied by catarrhal inflammation. These results indicate that the intestinal tract of chicks infected with E. coli O157:H7 was damaged.

Based on the histopathological results, we compared these 3 infection routes and found that the liver, spleen and colon of broiler chickens were significantly damaged when infected with E. coli in the route of gavage. After the infection of E. coli in broiler chickens by enema-drip, the damage to colon was serious, and the damage to liver and spleen was lightest compared with the other 2 routes. The infection of E. coli in broiler chickens by intraperitoneal injection has the most serious effect on the liver, and the high dose also causes damage to the spleen, and the damage to the intestine is less. Therefore, we speculate whether O157:H7 lacks the ability to penetrate the intestinal wall, which may be our next research direction.

CONCLUSION

EHEC O157:H7 infection resulted in liver, spleen and intestinal damage. E. coli by intraperitoneal injection had the greatest effect on material to weight ratio and liver organ index of chicks, and it was the best route to induce liver and spleen damage. Enema-drip method had more significant effects on colon damage. Gavage methods can cause liver, spleen and colon damage, and it is the best way to establish O157:H7 infected broiler colibacillosis.

In this study, we used 3 different modes of infection to establish a E. coli O157:H7 infection chick model. This study observed the development of the EHEC disease more accurately, to provide basis for study the prevention and control measures, and also lay a foundation for the development of drugs against this serotype bacteria. These 3 infection routes have their own advantages, and the experimenters can choose the appropriate infection route according to their own needs.